Isolation of nematodes
We collected different beetles at adult stage using sweeping nets, blacklight traps and pitfall traps baited with dung. The beetles were transferred to the lab alive, sacrificed by cutting them in half transversely, and put on NGM agar plates (6 cm diameter) seeded with 300 μl of the slowly growing E. coli strain OP50. The plates were checked daily using a Zeiss Stemi 2000 dissecting scope over a period of one to three weeks for emerging and reproducing nematodes. From the emerging nematodes we produced isogenic lines by transferring single gravid females or hermaphrodites to new plates. To see whether the isolated nematodes belonged to gonochoristic or hermaphroditic species, virgin larvae were singled out onto plates. The presence of offspring indicated that they represent a hermaphroditic species.
Emerging nematodes were determined to family level with a Zeiss Stemi 2000 dissecting scope and to genus level with a Zeiss Axioplan 2 microscope using the key by Sudhaus and Fürst von Lieven [9]. For determination several worms were transferred onto microscopic slides covered with a 0.5 mm thick layer of 5% agar and either immobilized by heating the slide over an open flame to about 60°C for a few seconds or anesthesized with sodium azide. Within the genus Pristionchus many species can not be identified by morphological methods. We therefore chose to use molecular tools and mating experiments with reference strains to distinguish the different species.
We have originally isolated up to three isogenic female lines per beetle and have processed them for sequence analysis. In nearly all cases, these isogenic female lines had identical SSU sequences. The exceptions were the observed hybrids between P. aerivorus and P. pseudaerivorus n. sp. described above, and two cases where isogenic female lines of P. aerivorus and P. pseudaerivorus or P. aerivorus and P. marianneae were derived from the same beetle. When the SSU sequences of different isogenic female lines from the same beetle were identical, only one isolate was further considered. Throughout the text (and in the Tables and Figures), we consider only one of these identical isolates per beetle. In particular, the numbers and frequencies provided in Table 1 and Figure 3 consider only one of these isolates. In Table 1, beetle individuals from the different sampling sites are pooled. There was little variation in incidence of Pristionchus species on beetles among sampling sites.
Molecular species identification
Molecular species identification was done using the small subunit rRNA gene (SSU) as described before [8]. In short, genomic DNA from single nematodes was prepared using the NaOH digestion procedure described by Floyd et al. [23]. A single worm was transferred to 20 μl of 0.25 M NaOH, incubated overnight at 25°C and heated to 99°C for 3 min before 4 μl of 1M HC1, 10 μl of 0.5 M Tris-HCl (pH 8.0) and 5 μl of 2% Triton X-100 were added. The mixture was heated to 99°C for 3 min, frozen at -20°C and reheated at 99°C for further 3 min. Two microliters of this extract were used for subsequent polymerase chain reaction (PCR).
A 1 kb fragment of the SSU was amplified by PCR using the primers SSU18A (5'-AAAGATTAAGCCATGCATG-3') and SSU26R (5'- CATTCTTGGCAAATGCTTTCG-3') [23,24]. The reactions were performed in 25 μl of 1× PCR buffer (Amersham Biosciences, Freiburg, Germany) containing 2.5 mM of MgCl2, 0.16 mM of each deoxynucleoside triphosphate, 0.5 μM of each primer, 2 μl of the lysate, and 2 units of Taq DNA polymerase (Amersham). The reactions were started by initial denaturation at 95°C for 2 min in a PTC-200 (MJ Research, Biozym, Hess. Oldendorf, Germany) or T gradient (Biometra, Göttingen, Germany) thermocycler, followed by 35 to 40 cycles of denaturation at 95°C for 15 sec, primer annealing at 50°C for 15 sec, and extension at 72°C for 2 min. A final incubation step at 72°C for 7 min concluded the reaction. PCR products were purified by the Qiagen PCR product gel extraction kit (Qiagen, Hilden, Germany). Approximately 500 bp of the 5'-terminal end were sequenced using the primer SSU9R (5'-AGCTGGAATTACCGCGGCTG-3') and the Big Dye terminator protocol (Applied Biosystems, Darmstadt, Germany).
Sequences were aligned manually using the Seqpup 0.6f software for Macintosh [25]. The substitution model for the reconstruction of phylogenetic relationships was selected by the hierarchical likelihood ratio test as implemented in the Modeltest 3.7 software [26]. The selected substitution model corresponds to the Kimura 2-parameter model [27] with Ts/Tv = 1.4727, equal base frequencies, and a γ-correction with shape parameter α = 0.2381.
Phylogenetic analysis
Phylogenetic trees were determined using the heuristic search algorithm under the maximum likehood (ML) criterion using PAUP*4.0bl0 program [28]. Trees were rooted by the Koerneria sp. sequence as outgroup. Neighbour joining (NJ, [29]) and maximum parsimony (MP) trees were drawn by the same program. Alignment gaps were eliminated from the analysis. The topological stability of the trees was assessed by 1000 bootstrap replications [30].
Mating experiments
To confirm the species identification by the molecular sequence of a novel isolate we performed mating experiments with the reference strain of the respective species (see below for definitions). Five virgin females were put on a plate with a small spot of OP50 together with five males of the reference strain of a certain species. On a second plate we picked the opposite sexes of the two strains to test for reciprocity. If there was no offspring after one week, the experiments were repeated two more times. If fertile offspring occurred we considered the two strains to belong to the same species.
Isolate and strain definitions
We use the following definitions to distinguish "isolates" and "strains". An isolate is an isogenic female line, which is derived from a beetle sample and subjected to molecular and experimental analysis. After species identification we established one isolate per species and location as a strain. The strains are permanently cultured in the lab, have a strain number and are also kept as frozen stocks. For each new species designated by molecular sequence analysis and mating experiments, one strain was defined as a reference strain (see below).
Assigning names to reference strains
According to the designation of reference strains, the 285 isolates obtained from American scarab beetle material fell into seven Pristionchus species, two of which were already cultured in our lab (P. pacificus, reference strain PS312 and P. entomophagus ref. strain RS0144). The other five could not be identified. We found the morphometric data of the most common of the unidentified species to coincide with the description of the only North American Pristionchus species listed in the catalog provided by Sudhaus and Fürst von Lieven [9], namely P. aerivorus. The other four species are novel and are described based on morphometric analysis, SSU sequence analysis and mating experiments (Fig. 2, Table 2 and species diagnoses). In total, the following eight Pristionchus species were obtained from scarab beetles (seven species) and the Colorado potato beetle (one species) in North America:
Pristionchus entomophagus (Steiner, 1929) (ref. str. RS0144)
Pristionchus pacificus Sommer, Carta, Kim & Sternberg, 1996 (ref. str. PS312)
Pristionchus uniformis Fedorko & Stanuszek, 1971 (ref. str. RS0141)
Pristionchus aerivorus (Cobb in Merrill & Ford, 1916) (ref. str. RS5106)
Pristionchus pseudaerivorus n. sp. Herrmann, Mayer & Sommer 2006 (ref. str. RS5139)
Pristionchus marianneae n. sp. Herrmann, Mayer & Sommer 2006 (ref. str. RS5108)
Pristionchus pauli n. sp. Herrmann, Mayer & Sommer 2006 (ref. str. RS5130)
Pristionchus americanus n. sp. Herrmann, Mayer & Sommer 2006 (ref. str. RS5140)
Comparison of ribosomal proteins
In order to obtain nuclear protein coding genes to distinguish P. americanus n. sp. from its sister species P. aerivorus, P. pseudaerivorus n. sp. and P. maupasi we isolated total RNA from 50 to 100 μg of nematodes using the TRIZOL reagent (Invitrogen). The RNA was reverse transcribed into cDNA with the help of the Omniscript reverse transcriptase kit (Qiagen, Hilden, Germany) and the primer RH5620 (5'-GAAGATCTAGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'). Based on EST data from SL1-transspliced genes from European Pristionchus species (unpublished data) we designed generic RT-PCR primers for Pristionchus ribosomal protein genes (WM8220, 5'-TCGACAACGACAGAAAGAAGA-3', rpl-26, sense; WM8221, 5'-ACGGAGTCRTCGCTGTRCTTGC-3', rpl-26, antisense; WM8263, 5'-CCGTCAGCGYGGMATCCAGAAG-3', rpl-28, sense; WM8264, 5'-GCTGGASGGAGCGGAGRAGCTG-3', rpl-28, antisense; WM8114, 5'-GCYCAYATYTTCGCYTCTTTCAA-3', rps-14, sense; WM8113, 5'-GGRGTCTTNGTTCTRGTTCCTC-3', rps-14, antisense) and used them to synthesize the complete transcript in two overlapping fragments by PCR. The SL1-specific primer BJ234 (5'-GGTTTAATTACCCAAGTTTGAG-3') was used with the antisense primers to obtain the 5' part of the transcripts and the combination of RH5620 and the sense primers to obtain the 3' parts. The PCR was performed with the help of the HotStar Taq DNA polymerase kit (Qiagen) including the Q solution in the reaction mix. Conditions were initial activation of the enzyme at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 30 sec, primer annealing at 50°C for 30 sec, and primer extention at 72°C for 3 min. The reaction was completed by an incubation at 72°C for 10 min. PCR fragments were gel purified using the Wizard SV gel purification kit (Promega) and sequenced directly using the PCR primers.
Authors' contributions
MH carried out all of the field work and the generation of isogenic female lines and crosses. WEM generated isogenic female lines and did all of the molecular analysis. RJS designed and discussed the experiments with MH and WEM and wrote, together with MH and WEM the manuscript.
Acknowledgements
We thank Paul Robbins and Antony Shelton at the Geneva Campus of Cornell University, the „Team Scarab“ Brett Ratcliffe, Mary Liz Jameson and Matt Paulsen at the University of Nebraska, Michael Klein and Casey Hoy for their help. We thank H. Haussmann for maintaining of Pristionchus strains and Dr. R. Hong and M. Riebesell for critically reading this manuscript.
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