I Nematology

BRILL Nematology 15 (2013) 109-123 brili.com/nemy

Diploscapter formicidae sp. n. (: Diploscapteridae), from the Prolasius advenus (: Formicidae) in New Zealand

Zeng Qi ZHAO ^'*, Kerrie A. DAVIES ^, Evan C. BRENTON-RULE ^, Julien GRANGIER ^, Monica A.M. GRUBER ^, Robin M. GIBLIN-DAVIS "^ and Philip J. LESTER ^ ' Landcare Research, Private Bag 92170, Auckland Mail Centre, Auckland 1142, New Zealand ^Australian Centre for Evolutionary Biology and , School of Agriculture, Eood and Wine, University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA 5064, Australia ^ Centre for Biodiversity and Restoration Ecology, Victoria University of Wellington, P.O. Box 600, Wellington 6140, New Zealand '^ Fort Lauderdale Research and Education Center, University of Elorida-IFAS, 3205 College Avenue, Fort Lauderdale, FL 33314-7719, USA Received: 8 March 2012; revised: 5 May 2012 Accepted for publication: 5 May 2012; available online: 10 July 2012

Summary - formicidae sp. n. was collected from the ant Prolasius advenus and its nests in native beech forests of the South Island, New Zealand. This is a new host record for the and the first report of an ant associate from the southern hemisphere. Diploscapter formicidae sp. n. appears to be native to New Zealand. No males were found from collections from 16 nests, in agreement with previously published data on the other members of this genus, suggesting that males are absent or very rare. The adult females have bilateral symmetry of the head, characteristic dorsal and ventral projections of the putative cheilostom with paired hook-like structures or hamuli, expansive membranous lateral lip flaps or laciniae, gymnostom and stegostom with parallel walls, a swollen procorpus, large terminal bulb with a strong valve, paired ovaries with medial vulva, and a short conoid tail with slender pointed or spicate tip. Scanning electron micrographs of the structure of the head confirmed that the lateral laciniae with finger-like tines or filopodia are moveable (alternately covering and exposing the mouth). These lateral lip flaps are located posterior to the stoma, but anterior to the pore-like amphidial openings. The anterior margin of the cheilostom possesses apomorphic lateral bell-shaped projections and the hamuli are broader and less pointed than other species that have been examined. Molecular phylogeny of near full-length small subunit, D2/D3 expansion segments of the large subunit rRNA gene and heat shock protein 90 (Hsp90) gene showed that D. formicidae sp. n. is monophyletic with the Diploscapter species and isolates available in GenBank, but is on an independent trajectory supporting separate species status. Keywords - description, distribution, molecular, morphology, morphometrics, new host, new species, phylogeny, .

Nematodes of the genus Diploscapter Cobb, 1913 are 1983). However, recent work with SEM confirms that usually thermotolerant, free-living bacterial-feeding soil the bilateral head symmetry involves 6 lips (each with a found world-wide in compost, sewage and labial sensillum), with 3 (1 small subdorsal -|- 1 elongate agricultural soil (Gibbs et al, 2005). The genus also lateral 'lacinia' -I- 1 small subventral) lips being present contains species that are facultative parasites of human on each side of the extensive and diagnostic dorsal and and other (Chandler, 1938; Morimoto et al, ventral projections (= hamuli) of the putative cheilostom 2006), and has been investigated as a potential carrier (Fig. 6A in Abolafia & Peña-Santiago, 2007). The genus of bacteria pathogenic to humans (Gibbs et al, 2005). currently contains 15 putative species (Sudhaus, 2011): Morphologically, Diploscapter is an interesting genus, D. angolaensis Siddiqi, 1998; D. cannae Rahm, 1928; having a head with bilateral symmetry and only 4 apparent D. comutus (Siddiqi, 1998) Sudhaus, 2011; D. coronatus lips, in contrast to the usual 3 or 6 of rhabditids (Andrássy, (Cobb, 1893) Cobb, 1913; D. cylindricus Rahm, 1929;

* Corresponding author, e-mail: zhaozOlandcareresearch.co.nz

© Koninklijke Brill NV, Leiden, 2013 DOi: 10.1163/156854112X649828 Z.Q. Zhao st al.

D. indicus Tahseen, Siddiqi & Rowe, 2002; D. lycostoma laboratory at ca 60% RH, 20°C and a 16:8 h light:dark Volk, 1950; D. libycus Fenso, 1938; D. nodifer MiVa&lcïc, light cycle. Colony sizes ranged from approximately 200 1953; D. orientalis Kannan, 1960; D. pachys Steiner, to 2000 workers with from 1 to several queens. Colonies 1942; D. rhizophilus Rahm, 1928/29; D. striatus Siddiqi, were all kept in polyethylene boxes (22 x 14 x 7 cm) 1998; D. tentaculatus (Carter, 1859) Sudhaus, 2011; and with Fluon® (ethylene tetrafiuoroethylene) coated walls D. tokobaevi Lemzina & Gagarin, 1994. However, the to prevent ant escape. Within each box, a nest-tube was status of several of these is unclear in the absence of provided to shelter the queens and juvenile stages. These males, molecular and SEM studies. Some species are very nest-tubes were 14.5 x 1.5 cm, contained moist cotton widely distributed. For example, there are descriptions wool, and were covered in tin-foil to eliminate light. of putative D. coronatus from Ethiopia, India, Iran, All colonies were fed with 1 freshly killed mealworm Japan, The Netherlands, Paraguay, Spain, the USA and and 7 ml of a 20% honey/water solution 3 times per Venezuela (Loof, 1964; Andrássy, 1983; Bongers, 1988; week. These colonies were maintained separately in the Abebe et al, 1998; Tahseen et al, 2002; Gibbs et al, laboratory for up to a year prior to ant dissection and 2005; Morimoto et al, 2006; Abolafia & Peña-Santiago, nematode extraction. 2007; Shokoohi & Abolafia, 2011). Free-living nematode stages were extracted from the Recently, collections of a new species of Diploscapter nesting material of P advenus. Nematodes were fre- were made from the formicine ant Prolasius advenus Fr. quently observed in or around wet cotton wool. To extract Smith, 1962 from nests in a native beech {Nothofagus nematodes, some nest material, including cotton wool, spp.) forest of the South Island, New Zealand. Prolasius was taken from nest-tubes within the nest boxes. The advenus inhabits native forests, nesting in soil under nest material was transferred into water or half strength stones, in or under rotting logs, and occasionally in Ringer's solution and left for about 1 min for the ne- trees (Shattuck, 1999; Don, 2007). Colonies may contain matodes to emerge. The second method of nematode ex- many thousands of workers and frequently contain many traction was to dissect parasitic stages from the head and queens. It is a dominant generalist forager that competes gaster of using a Nikon SMZ1500 microscope in a with invasive wasps (Grangier & Lester, 2011), foraging medium of water or half strength Ringer's solution. For and scavenging for small and tending scale morphological study, some nematodes were mounted in for honeydew. Food exchange (trophallaxis) takes water and examined immediately. Others were killed and place between workers and larvae. Prolasius advenus is fixed using hot 4% formaldehyde. All nematodes were endemic to New Zealand (Don, 2007). All the other 18 processed in glycerin and mounted on glass slides, as Prolasius species are found in Australia, of which 1 is also described by Southey (1986) and modified by Davies & present in Papua New Guinea (Shattuck, 1999). Giblin-Davis (2004). Diploscapter lycostoma is commonly found associated Drawings were made using interference contrast mi- with ants, especially in their detritus piles. It has been croscopy with a camera lucida (Nikon, Eclipse 90i)- Mea- recorded from Europe and the USA (Volk, 1950; Carta surements were made from material mounted in glyc- et al, 2006). This is the first report of a species of erin using NIS-Elements Basic Research (Nikon, Ver- Diploscapter from ant nests in the southern hemisphere sion 3.0). Head diam. was measured at the most anterior and hence its morphology and some DNA sequences part. Maximum body diam. was measured at mid-body. were compared with published descriptions of the genus Body length was measured along the curved mid-line. and species from the northern hemisphere. Diploscapter A camera (Nikon Camera Head DS-Fil) attached to the formicidae sp. n. is described and illustrated with line art microscope was used to take a series of digital images of drawings and SEM photographs. the specimens. For scanning electron microscopy (SEM), nematodes Materials and methods fixed in formalin were washed in 3 changes of water puri- fied by reverse osmosis (RO). Then, they were dehydrated NEMATODE COLLECTION through an ethanol series, with 20-30 min in each stage (30, 70, 80, 90 and 95% ethanol, and then 2 changes of Prolasius advenus colonies were collected from within 100% ethanol). After this, the nematodes were transferred Nothofagus forests of the northern South Island of New to microporous specimen capsules with 8 /xm membrane, Zealand, in March and April 2010, and maintained in a using Critical Point Dryer Balzers CPD 030 for critical

110 Nematology Diploscapter formicidae sp. n. from New Zealand ants point drying. Stubs were coated with gold using Neo- Twelve SSU and Hsp90 published sequences from Coater MP-19020NCTER and viewed using a Benchtop GenBank were included in our phylogenetic analysis, SEM (NeoScope) JCM-5000. respectively. Nematode species and GenBank accession numbers are listed for each taxon in the phylogenet- ic trees, if known. DNA sequences were aligned in MOLECULAR METHODS ClustalX2 (Larkin et al, 2007) using default parameter A modified nematode DNA extraction method of Zheng values and we used ModelTest 3.04 (Posada & Crandall, 1998) and PAUP*4.0bl0 (Swofford, 1998) to select the et al (2002) was used. Total genomic DNA from a sin- best model using Akaike Information Criterion (AIC). gle living nematode was extracted using lysis buffer con- A Bayesian tree was obtained using MrBayes 3.1.2 (Ron- taining proteinase K (Williams et al, 1992). Tubes con- quist & Huelsenbeck, 2003). We ran 4 MCMC chains for taining nematodes with 20 ß\ lysis buffer were stored at 10^ generations under the best-fit model (GTR -I-1 + T) -80°C for at least 30 min before DNA extraction. DNA for SSU, and (GTR -|- G) for Hsp90. Prior distributions extractions were stored at —4°C until used as template for were as follows: revmatpr = dirichlet (1, 2, 1, 1, 2, 1), PCR amplification. Primers used for rDNA small subunit shapepr = exponential (5), brlenspr = unconstrained: ex- (SSU) were from Holterman et al (2006): first SSU frag- ponential (10). We started analysis from a random topol- ment forward primer 1096F, 5'-GGTAATTCTGGAGCTA ogy and used a temperature of 0.2, and a 'burnin' of 10% ATAC-3' and reverse primer 1912R, 5'-TTTACGGTCAG of trees. Multiple runs were performed, and the result- AACTAGGG-3' and the second fragment forward primer ing perimeter files were inspected for chain convergence 1813F, 5'-CTGCGTGAGAGGTGAAAT-3' and reverse in Tracer 1.4 (Rambaut & Drummond, 2007). The SSU 2646R, 5'-GCTACCTTGTTACGACTTTT-3', respective- tree was rooted using sequences of myriophila ly. Primers for rDNA D2/D3 expansion segments of the Poinar, 1986 and the Hsp90 tree was rooted using se- large subunit (LSU) were from Nunn (1992): forward quences of Acrobeloides amurensis Truskova, 1971 and primer D2A, 5'-ACAAGTACCGTGAGGGAAAGT-3', Cephalobus cubaensis Steiner, 1935, respectively. and reverse primer D3B, 5'-TGCGAAGGAACCAGCTA CTA-3'. Primers used for Hsp90 were designed by the au- thors: first Hsp90 fragment forward primer DC-65F, 5'- Results GCACCAAGGCTTTCATGGAG-3' and reverse primer DC-965R, 5'-CAGATGGGCTTCGTCTTGTT-3' and the Diploscapter formicidae* sp. n. second fragment forward primer DC-907F, 5'-GCAGAA (Figs 1-3) GGAAAAGAAGGAAGG-3' and reverse DC-1536R, 5'- AAACACGGCGGACGTAGCTG-3', respectively. The 20 /xl PCR reactions contained 10 /xl Go Tag® Green Mas- MEASUREMENTS ter Mix (Promega), 1 /xl (0.05 ßM) each forward and re- See Table 1. verse primer and 2 /xl of DNA template. The thermal cy- cling programme was as follows: denaturation at 95°C for DESCRIPTION 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55 °C for 30 s and extension at 72°C for Female 45 s. A final extension was performed at 72°C for 10 min. Small nematodes. Body straight to arcuate to C-shaped PCR products were purified by Wizard® SV Gel and PCR when heat-relaxed, with max. diam. at mid-body. Cuti- Clean-Up System (Promega Corporation). Purified PCR cle clearly annulated (annule width — ca 1 /xm wide) in products were sequenced using Big Dye TM Termina- head region, apparently smooth on rest of body, but en- tor Cycle Sequencing Ready reaction Mix v3.1 Kit. Cy- larged annules observed on tail of a few specimens. With cle sequencing products were cleaned using 96-well-plate SEM, annules are apparent but they are not raised. Small, ethanol precipitation and analysed on an ABI 3100 Avant pore-like amphid openings at base of lateral flaps, with Genetic Analyser (Applied Biosystems). The quality of SEM seen to be raised, papilliform. Lateral fields broad, each sequence was confirmed by inspection of sequence ca 2 ßm wide, ribbon-like with 4 lines, arranged as 2 trace files. The sequences were deposited in GenBank and the accession numbers are JQ838254 (SSU), JQ838255 ' Species name is derived from the family name of the phoretic (LSU) and JQ838256 (Hsp90). host ants, the Formicidae.

Vol. 15(1), 2013 111 Z.ß.Z/iao et al.

Fig. 1. Diploscapter formicidae sp. n. AU in lateral view. A: Habitus of adult female; B: Anterior of female; C: Vulva and posterior ovary showing spermatheca containing sperm; D: Tail showing characteristic stepped shape of rectum. (Scale bars: A = 100 ^m; B-D = 50 ßm.)

112 Nematology Diploscapter formicidae sp. n. from New Zealand ants

Fig. 2. Diploscapter formicidae sp. n. All of Uving adult female. In lateral view, except for ventral views of head and vulva. A-D: Ventral views of head at different focal depths; E, F: Lateral views of head at different focal depths, lateral labial sensillum arrowed; G: Stoma, amphid opening arrowed; H: Vulva in ventral view; I: Lateral field. tube-like structures, 1 on each side of field, ending ca atal hamuli, lateral lips present as large flaps or membra- 4 /xm anterior to anus. SEM showing fields bisected by nous laciniae, extending out from between stoma and am- annules. Head with bilateral symmetry. Six lips, paired phid for ca 1 head diam., fringed with finger-like projec- sub-dorsal and sub-ventral lips present as small bumps tions or tines equal in length to half width of lip, when straddling dorsally and ventrally projecting cheilostom- extended forward, each seen to bear a centrally situated.

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Vac-High PC-Std. 10 kV x 8000 001688 Vac-High PC-Std. 10 kV x 7000

Fig. 3. Diploscapter formicidae sp. n. Scanning electron micrographs. A: En face view of head showing hamuli and laciniae with fllopod-hke tines; B: Head and stoma; C: Vulva; D: Tail region showing lateral lines and anus. pore-like, lateral labial sensillum, apparently moveable tom ca 1.5 head diam. long. Stegostom lacking glottoid with ability to cover mouth or be retracted to expose apparatus or teeth. Pharyngeal collar extending anteriorly mouth. Cheilostom heavily cuticularised with dorsal and to ca one third length of gymnostom -f- stegostom. Secre- ventral bifid claw-like hamuli projecting ca 1 head diam. tory/excretory pore opening posterior to nerve ring near from centre of stoma, each projection being ca twice as level of valve of terminal bulb, hemizonid immediately long as wide at distal edge and evaginated and creased anterior to pore but at different focal plane. Anterior pha- for ca half its distal length. Both hamuli sloping gently ryngeal corpus slightly swollen, with pseudo-valve, not posteriorly at ca 30°. SEM semi-en face view showing set off from procorpus, approximately same length as isth- apomorphic lateral bell-shaped protuberances on anterior mus and terminal bulb combined, isthmus cylindroid, ter- edge of cheilostom. Gymnostom and stegostom with par- minal bulb with prominent valve. Body diam. 2.5 times allel walls, long, tubular, ca 2 head diam. long. Gymnos- metacorpus diam. Nerve ring situated anterior to terminal

114 Nematology Diploscapter formicidae sp. n. from New Zealand ants

Table 1. Morphometrics of glycerin-mounted females of anus as a depressed slit. Pore-like phasmid on lateral side Diploscapter formicidae sp. n. from Prolasius advenus in New at level of anal opening (SEM). Tail conoid, straight, 3-4 Zealand. All measurements are in /¿m and in the form: mean ± ABD long, tip slender, pointed. s.d. (range). Juvenile Character Holotype Paratypes Extracted from ant heads. Second-stage juvenile (J2) n 16 stage based upon size of gonad primordium (2 or 3 cells). L 421 432 ± 21 Different stages apparently with different numbers of (394-487) lateral lines. SEM showing 1 lateral line on J3 and J4 13.8 13.1 ±0.7 a stages. Free-living juveniles collected from nest detritus (12.0-14.4) were J3 and J4. No dauers observed. b 3.7 4.0 ± 0.2 (3.8-4.1) 9.1 9.3 ±1.3 TYPE HOST AND LOCALITY (8.2-12.5) 3.3 3.3 ±0.4 Prolasius advenus nests collected from St Arnaud, (2.5-4.0) Tasman, South Island, New Zealand (41.8207°S; V 57.5 56.5 ± 2.3 172.8544°E) in March and April 2010, by Julien Grangier. (53.0-60.8) Juveniles dissected from the heads of worker ants from St. Max body diam. 30 33 ± 2.5 Arnaud, South Island. (28-36) Stoma diam. 3.1 3.5 ± 0.5 TYPE MATERIAL (2.8-4.6) Stoma length 22 22.6 ±1.5 Holotype female (slide no. NNCNZ 276) and 16 pa- (20.0-24.8) ratype females (slide Nos NNCNZ 3036-3043 depos- Anterior pharynx length 34 44.7 ± 2.5 ited at the National Nematode Collection, New Zealand (42-49) (NNCNZ). One paratype male and 1 paratype female de- Posterior pharynx length 56 45.6 ± 4.3 posited in, respectively, the ANIC (Australian National (39-56) Collection), CSIRO Entomology, Canberra, Aus- 110 Excretory pore from anterior end 103 ± 5.5 tralia, and the USDA Collection, Baltimore USA. (95-110) Anterior ovary length 104 100 ± 19 (70-131) DIAGNOSIS AND RELATIONSHIPS Posterior ovary length 98 87 ±11 Diploscapter formicidae sp. n. is characterised by (64-108) Anal body diam. 14.2 13.1 ±0.7 having membranous lateral lips (laciniae) with finger- (12.0-14.4) like projections or tines equal in length to half diam. Tail length 46 47.3 ± 6.7 of the lip, heavily cuticularised putative cheilostom with (37-59) lateral, anteriorly pointed, bell-shaped projections and dorsal and ventral bifid claw-like hamuli projecting ca 1 head diam. from the centre of the stoma, each hamulus bulb. Intestine with broad lumen posterior to bulb, nar- being ca twice as long as wide at the distal edge and rowing sharply ca 15 /xm posterior to bulb, with obvi- evaginated and creased for ca half its distal length, both ous lumen lining. Deirids not seen. Reproductive system hamuli sloping gently posteriorly at ca 30°, a long tubular didelphic, amphidelphic, outstretched, frequently refiexed gymnostom -I- stegostom without teeth, weak cuticular at distal end. Anterior ovary on right side of intestine, pos- annulations, excretory pore opening at the anterior end terior ovary on left. Oocytes arranged in single file at distal of the terminal bulb, swollen procorpus with metacorpus end of ovary. Spermatheca apparently containing sperm, not offset, vulva near mid-body length, uterus containing not offset. Vulva located just posterior to mid-length, 7-8 sperm but males absent, and a short tail with a spicate tip. anal body diam. (ABD) anterior to anus. Vulval lips not It is distinguished by its head and stomatal morphology, or slightly raised, vulva a plain slit, vagina perpendicular biogeography, and its status as a distinct species is to body wall. Rectum a simple tube with a characteristic corroborated by molecular data from sequencing of 18S kink or bend posterior to end of intestine, SEM showing and Hsp90.

Vol. 15(1), 2013 115 Z.Q. Zhao et 3.1

Morphologically, the new species is most similar to D. are included, with a posterior probability of 100%. From lycostoma. In size, D. formicidae sp. n. (394-487 /xm) is the results of the BLAST search, D. formicidae sp. n. was smaller than D. libycus and D. lycostoma (respectively, also close to all 4 Diploscapter with available sequences, 650 and 519-546 ßm long) (Penso, 1938; Wahab, 1962). with 99% of bases identical for the 18S sequences, and It is bigger than D. angolaensis, D. indicus and D. striatus 97% identical bases when compared to the only D2/D3 (respectively, 270-340, 220-249 and 270-350 /xm) (Sid- sequence available in GenBank. Diploscapter formicidae diqi, 1998; Tahseen et al, 2002). By having a relatively sp. n. can clearly be differentiated from all 4 SSU se- slim body, D. formicidae sp. n. has a larger ratio a than D. quences of Diploscapter available in GenBank including angolaensis, D. coronatus, D. nodifer and D. tokobaevi 1 from D. coronatus (9 differences in 1591 bp). 18S se- (12.0-14.4 vs 4.4-7.1, 5.8-9.6, 5.3, and 5.7-6.8) (Mihel- quences from the work on D. lycostoma reported by Carta cic, 1953; Abebe et al, 1998; Siddiqi, 1998; Lemzina & et al (2006) were not available. Gagarin, 1994). It is separated from D. lycostoma by ratio The consensus tree inferred from Hsp90 (Fig. 5) shows b (3.8-4.1 vs 6.3-6.5), i.e., has a relatively longer length that Díplo.icapter is monophyletic when all ten Hsp90 from head to basal bulb. In D. formicidae sp. n., the pro- sequences of Diploscapter spp. available in GenBank are corpus and metacorpus of the pharynx are not offset, and included, with a posterior probability of 100%. A BLAST the isthmus is only slightly narrower than the metacorpus. search using sequences of Hsp90 confirmed that D. In D. libycus, D. striatus and D. orientalis, the isthmus formicidae sp. n. is part of the Diploscapter clade. It is distinctly narrower than the procorpus (Penso, 1938; also suggested that, with <90% homology (86-90%) Siddiqi, 1998; Kannan, 1960, respectively). In having a between Hsp90 sequences from D. formicidae sp. n. and shorter tail, it differs from D. tokobaevi (c = 8.2-12.5 vs D. lycostoma, and between D. formicidae sp. n. and D. 5.7-6.8) (Lemzina & Gagarin, 1994). Again, tail propor- coronatus, the New Zealand Diploscapter is on a non- tions differentiate it from D. angolaensis and D. tokobaevi coalescing independent trajectory. This is because these (d = 2.5-4.0 V.Î 5.0-7.4 and 5.0-6.5, respectively) (Sid- sequence differences exceed the intraspecific variability diqi, 1998; Lemzina & Gagarin, 1994, respectively) and of about 5% for this locus seen in other closely related the latter has a filiform tail tip. The vulva is more ante- species (Vovlas et al, 2011). rior in D. formicidae sp. n. than in D. nodifer (V = 53- 61 vs 70-85) (Mihelcic, 1953). While the length of the stoma in D. formicidae sp. n. overlaps that of all species BIOLOGICAL CHARACTERISTICS AND DISTRIBUTION of Diploscapter, its cylindrical shape separates it from D. No males of D. formicidae sp. n. were recovered in orientalis, in which the stoma is concave (Kannan, 1960). our samples or cultures. Maupas (1900) described D. Diploscapter formicidae sp. n. has a tiny conoid tail lycostoma as an autogamous protrandrous hermaphrodite with a spicate tip when observed with the SEM. In this, and Wahab (1962) stated that males are very rare. Markin it is similar to D. comutus (Siddiqi, 1998) Sudhaus, & McCoy (1968) did not find any males in their cultures 2011, and seems to differ from all described species of of D. lycostoma and they were not inducible at high Diploscapter where it is longer and more attenuated. In (29.4-35°C) temperatures. Here, no males were found addition, it is similar to D. cylindricus Rahm, 1929. These although a spermatheca apparently containing sperm was 3 species (D. cylindricus, D. comutus and D. formicidae present in adult females, suggesting that D. formicidae sp. sp. n.) all have a wide isthmus (from Rahm's comments, n. is a hermaphrodite. Morphologically, it is difficult to in D. cylindricus it is very wide) and tails with similar determine a species of rhabditid in the absence of males, morphology. as the morphology of the male is considered extremely Sudhaus (in Köhler, 2012) noted that juveniles of D. informative for systematics and phylogeny (Kiontke et al, coronatus do not nictate but that it is common in those 2011). of D. lycostoma. Nictation was observed in D. formicidae This nematode is probably native to New Zealand as sp. n. it was collected from P. advenus, a native New Zealand ant, and has been found from samples throughout the study sites in the northern South Island and the south- MOLECULAR PHYLOGENETIC RELATIONSHIPS em North Island. These study sites are in relatively undis- The consensus tree inferred from SSU (Fig. 4) shows turbed habitats with few or no other exotic ants. The Ar- that Diploscapter is monophyletic when all 4 SSU se- gentine ant, Linepithema humile Mayr, 1868 (formerly quences of Diploscapter spp. available in the GenBank Iridomyrmex humilis Mayr), has recently entered New

116 Nematology Diploscapter formicidae sp. n. from New Zealand ants

FJ040432 Heterorhabditis megidis

100 l>— M92036S Heterorhabditis zealandica

FJ040428 Heterorhabditis bacteríophora

AF083026 sonorae

f AY602180 Caenorhabditis sp. 100 AF083024 sp. SB208

100 EU196002 Protorhabditis sp.

100 . Diploscapter formicidae sp. n.

100 EU196003 Diploscapter sp. JU359

AF083009 Diploscapter sp. PS1897

100

U81586 DSU81586 Diploscapter sp.

AY593921 Diploscapter coronatus

U81588 RMU81588 Rhabditis ntyriophila 0.05 Fig. 4. Bayesian phylogenetic tree inferred from SSU gene DNA sequences. Posterior probabilities greater than 50% are given on appropriate clades. Nematode species, GenBank accession numbers and locations are listed for each taxon, if known.

Zealand (Green, 1990) and is a known host of D. lycos- indicate considerable variability both within and between toma (Markin & McCoy, 1968). However, Diploscapter species. Tahseen et al (2002) examined 6 populations has not been recovered from Argentine ants in the New of D. coronatus from different states in India, and from Zealand field sites (unpubl. data). This further suggests Idaho in the USA, and included 1 in culture, and found as that D. formicidae sp. n. is native and widespread in New much variability within the latter as between the various Zealand. collections. Species of Diploscapter have been described on the basis of size, form of the corpus and isthmus, Discussion shape of the labial hooks, form of the lip fiaps, number of oocytes in each ovary, position of the excretory pore, Various morphological characters, measurements and position of the vulva, form of the cuticular annulations, morphometrics, including some from SEM studies, for and presence/absence of males (Siddiqi, 1998). Where nominal Diploscapter species are given in Table 2. These males are rare or absent, it is not practical to do mating

Vol. 15(1), 2013 117 Z.Q. Zhao et al

EF467041 Diploscapter lycostoma

EF467040 Diploscapter lycostoma

r DQ 340379 Diploscapter lycostoma 100 100

'— DQ 340380 Diploscapter lycostoma 100

— EF467042 Diploscapter lycostoma

84

DQ 340381 Diploscapter lycostoma

91 DQ 340383 Diploscapter lycostoma

64 99 DQ 340382 Diploscapter lycostoma

Diploscapter formicidae sp. n.

99 EF 467043 Diploscapter coronatus

100

'— EF 467044 Diploscapter coronatus

DQ 340377 Acrobeloides amurensis

100

DQ 340378 Cep/ia/obus cubaensis 0.09 Fig. 5. Bayesian phylogenetic tree inferred from Hsp90 gene DNA sequences. Posterior probabilities greater than 50% are given on appropriate clades. Nematode species, GenBank accession numbers and locations are listed for each taxon, if known. experiments to test for biological species. As early as studies using TEM are needed to help confirm the nature 1942, Steiner (1942) suggested that D. coronatus is a of the lips and stoma of Diploscapter. It will be necessary species complex. Thus, the variability and overlap of to do extensive global re-sampling and examination of values makes it difficult to differentiate species in this the material of the nominal species and to do both SEM genus. and molecular studies in order to determine where the Recent investigations (Abebe et al, 1998; Carta et al, species boundaries lie. In addition, using LM, Siddiqi 2006; Morimoto et al, 2006; Abolafia & Peña-Santiago, (1998) described the genus Carinoscapter Siddiqi, 1998 2007) indicate that the use of SEM is vital for detection on the basis of apparent inner frontal spines on the hamuli. of differences in the structure of the lips and cheilostom, In D. formicidae sp. n., these are apparent at some focal which may be apomorphic. In addition, ultrastructure levels with LM, but SEM study showed that they are an

118 Nematology Diploscapter formicidae .vp. n. from New Zealand ants

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Vol. 15(1), 2013 119 Z.O.Z/îao et al. artefact as the hamuli have a smooth surface. Sudhaus hooks on the hamuli appears to vary (wide and flattened (2011) synonymised Carinoscapter with Diploscapter. in D. formicidae sp. n., pick-like in D. coronatus and D. SEM studies and molecular analysis are therefore needed indicus; and bluntly rounded in D. pachys). In our speci- to confirm the status of this nematode. mens, the anterior surface of the hamuli is smooth, but in With SEM, the lateral lines of D. formicidae sp. n. D. coronatus it is rugose (Abebe et al, 1998). Differences can be seen to be areolated, i.e., crossed by the annules. in the shape of the outer margins of the stoma opening, This is also seen in the micrographs of D. coronatus en face, were noted between D. formicidae sp. n. and D. published by Abolafia & Peña-Santiago (2007), but not in lycostoma (Carta et al, 2006), but in the absence of other those of Abebe et al (1998). Thus, this character appears studies it is not known if these differences are significant to be variable. Similarly, the degree of annulation and or not. Thus, more SEM studies of isolates characterised the positions on the body in which annules are most by sequencing and light microscopy are needed to estab- prominent varies within 1 putative species (Abebe et al, lish the reliability of the head morphology for differenti- 1998; Morimoto et al, 2006). These differences could ating Diploscapter species. reflect differences in nutrition, or may be due to the It is likely that the position of the phasmids will different methods used to prepare the nematodes for SEM be an important character for species differentiation in studies. Thus, annule morphology does not appear to be a Diploscapter. However, these sensory organs can be dif- character useful for species differentiation. ficult to find, even using SEM. Their position varies from Carta et al (2006) examined 2 genetically distinguish- the level of the anus to mid-tail. Typically, Diploscapter able populations of D. lycostoma. Morphological differ- is a saprophagous genus (Gibbs et al, 2005). As they are ences were noted between the 2 morphospecies, includ- associated with detritus heaps, this is the probable origin ing the shape of the cheilostom opening, size of the pha- of their development in ant nests. It has been argued that, rynx, the ratio body diam./diam. of metacorpus, the go- because they destroy the pharyngeal glands of ants, the ju- nad length, and position of the phasmid on the tail. In veniles of D. lycostoma are parasitic (Markin & McCoy, comparing the written description of the morphology, as 1968). However, Köhler (2012) argues ihai Diploscapter seen with SEM, of the nematode Carta et al (2006) de- associated with ants are internally phoretic. He was able scribed with D. formicidae sp. n., it appears that there are to demonstrate that they can be spread from ant to ant by differences in the face views of the lateral cheilostomatal trophallaxis, a behaviour undertaken by Prolasius work- projections. They were centrally incurved in Carta's spec- ers, i.e., they could easily spread nematodes horizontally. imens, but not here, and Carta's specimens apparently did Poinar (2003) suggested that D. lycostoma was normally not have the anterio-lateral bell-shaped projections seen free-living and that dauers enter ants when environmen- in the New Zealand ones. In addition, the tail of the ne- tal conditions are unsuitable for the nematode {e.g., low matodes collected from New Zealand usually lacked deep relative humidity). He suggested that a nematode would annulations. Unfortunately, the only studies with en face stay in an ant until the ant died, and would then feed SEMs of Diploscapter are those of Carta et al (2006), on microbes in the decomposing cadaver (necromeny). Abolafia & Peña-Santiago (2007), Shokoohi & Abolafia However, Markin & McCoy (1968) noted that uninfested (2011) and those presented herein. SEM shows that the Argentine ants in contact with infested detritus picked membranous laciniae of D. formicidae sp. n. are fringed up infective juveniles within 24 h, and continued so to with finger-like projections or tines (Fig. 3A, B). In this, do. Worker ants depositing refuse in the detritus could it differs from D. coronatus, in which the tines are den- accidentally pick up juveniles on their mouthparts, and tate. However, Morimoto et al. (2006) found that a pu- these could then move to the pharyngeal glands. Poinar tative D. coronatus isolated from a human patient had (2003) suggested the nematodes would invade larvae, but tines similar to those of D. formicidae sp. n. The num- Markin & McCoy (1968) did not find any in larvae or pu- ber of tines per lacinia also seems to vary. From published pae of Linepithema humile, further supporting an inter- micrographs, D. coronatus may have 19 tines (Abebe et nal commensal association. They have not yet been found al, 1998), 16-18 (Tahseen et al, 2002), 12 (Morimoto in larvae or pupae of P. advenus (unpubl. data). Infec- et al, 2006), 11 (Abolafia & Peña-Santiago, 2007) or 13 tive juveniles of D. formicidae sp. n. were observed nic- (Shokoohi & Abolafia, 2011). Diploscapter indicus has tating, behaviour which suggests host-seeking (Campbell 16-18 (Tahseen et al, 2002) and we observed 17-19 per & Gaugler, 1993). Köhler (2012) noted Diploscapter sp. lacinia in D. formicidae sp. n. In addition, the shape of the nictating, and found evidence that they entered ants more

120 Nematology Diploscapter formicidae sp. n. from New Zealand ants readily under dry conditions, leaving them again when to examine a potential role between reproductive mode the environment was more moist. He also noted that their and successful dispersal and colonisation. phoretic behaviour was not insect-specific, as some in- Relatively few nematodes have been found parasitising vaded Tenebrio beetles. Putative isolates of D. lycostoma ants (Poinar, 2003). This is surprising given that ants have have been reported from rotten wood, soil, old cowpats been studied by entomologists for many years, and would and a range of beetles and in earthworms (Sachs, 1950; seem to suggest that this group is not widely Volk, 1950; Kömer, 1954). parasitised or associated with nematodes. However, most Phylogenetically, Diploscapter is an interesting genus collectors either dry out their ant specimens or preserve (see Sudhaus & Fitch, 2001, van Megen et al, 2009). An- them in alcohol. Both alcohol and desiccation make small drássy (1983) put it into the Diploscapteridae (with only nematodes difficult to differentiate from other tissues, the one genus) within the Rhabditoidea. The phylogenetic and this means that only very large nematodes, like analysis of Sudhaus & Fitch (2001), using sequences of mermithids, would be observed in subsequent dissections. SSU rDNA, suggested that Diploscapter species were de- Recent work using termites as a focal sampling group for rived from within a paraphyletic P roto rhabditis group, elucidating insect-associated biodiversity in a tropical rain and were most closely related to a species of the Xylo- forest demonstrated much higher estimates of nematode cola group of Protorhabditis. Diploscapter may be sister association (commensal and parasitism) than would have to Caenorhabditis. From a taxon-dense phylogeny based been predicted from the literature when live termites on sequences of SSU and LSU and examining the evo- were dissected from transects (Giblin-Davis & Kanzaki, lution of vulva development in rhabditids, Kiontke et unpubl. obs.; Powers et al, 2009). In the work on al (2007) found that Diploscapter formed a clade with Prolasius reported here, both a Diploscapter species and Protorhabditis, which was sister to Caenorhabditis. The a hexatylinid (unpubl. data) were found. It seems unlikely BLAST search carried out in this work, using 18S and that we have stumbled on an unusual situation, suggesting D2/D3 sequences from the nematode collected in New that nematodes may indeed commonly associate with Zealand, broadly supported the findings of Sudhaus and ants, and that these insects may prove to be a new Fitch (2001). While having close molecular sequence sim- and exciting source of previously overlooked nematode ilarity to the Diploscapter species and the Protorhabditis biodiversity. whose sequences are in GenBank, this nematode was not close to Caenorhabditis. Morphologically, Diploscapter, Protorhabditis and Caenorhabditis are widely divergent Acknowledgements (Andrássy, 1983), but species oi Protorhabditis are known associates of scolytid, cerambycid and scarabaeid beetles We thank Mr Joe Tang, The Ministry of Agriculture (Andrássy, 1983) and Caenorhabditis are known asso- and Forestry, New Zealand, for designing primers for the ciates of a variety of invertebrates (Kiontke et al, 2011). Hsp90 sequences. This research was supported by the Using SSU sequences. Carta et al (2006) could not sep- Ministry for Science and Innovation through the Defining arate 2 morphospecies of D. lycostoma in Bayesian anal- New Zealand's Land Biota programme. Initial work in the yses, but they were resolved in analyses using Hsp90 se- collection of ants and nematode discovery was supported quences. These genetic differences were also reñected in by a Royal Society of New Zealand Marsden Grant to subtle morphological differences. Further molecular work PJL. should be undertaken to learn more about the genetic dif- ferences between the isolate of Diploscapter collected in References New Zealand and those of the northern hemisphere. Such work would shed light on questions of genetic drift due to isolation vs changes associated with host selection. Abebe, E., Karegar, A., Nabil, H. & De Ley, P. (1998). Recent work with the model invertebrate-associated ne- A redescription and ultrastructural study of Diploscapter matode genera Caenorhabditis and Pristionchus suggests coronatus (Cobb, 1983) Cobb, 1913 from Ethiopia and Iran. Russian Journal of Nematology 6, 17-22. that hermaphroditism may be a character trait tied to those Abolafia, J. & Peña-Santiago, R. (2007). Nematodes of the order species that exhibit greater cosmopolitanism than their Rhabditida from Andalucia Oriental, Spain. The genera Pro- gonochoristic congeners (Herrmann et al, 2010; Kiontke torhabditis (Osche, 1952) Dougherty, 1953 and Diploscapter et al, 2011). Diploscapter is yet another candidate genus Cobb, 1913, with description of P. spiculocrestata sp. n. and

Vol. 15(1), 2013 121 Z.ß.

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