Five of the Pristionchus species found on scarab beetles in the US do not occur in Europe
To determine if P. pacificus and other species of the genus Pristionchus are associated with scarab beetles in the United States, we analyzed beetles from Massachusetts, New York, Ohio, Iowa, Kansas, Texas, and Nebraska. The scarab beetle fauna differs between North America and Europe. For example, the cockchafer Melolontha melolontha and the dung beetle Geotrupes stercorosus that host the predominant European Pristionchus species are not known from North America. However, the subfamily Melolonthinae, which includes the june beetles and chafers, contains more than 500 species in North America, some of which are serious pests [12]. We used the same sampling strategies in Europe and the United States and collected scarab beetles from the US that are comparable with those obtained in our European study. However, we have not been able to sample identical species on both continents. In total, we investigated 1241 beetles of more than 15 genera yielding 285 Pristionchus isolates (Fig. 1, Table 1). SSU sequence analysis and mating experiments of the 285 isolates revealed that they fall into seven species, two of which were known to us (Fig. 2). We obtained two strains of P. entomophagus (both from beetles from Ohio), a hermaphroditic species that is one of the most common Pristionchus species in Europe [8]. We also found P. pacificus, but in no more than three beetle samples, one from Ohio, one from Nebraska and one from Massachusetts. Surprisingly, the remaining 280 (98%) isolates represent five Pristionchus species, which had up to then neither been found by beetle sampling in Western Europe [8] nor by previous soil sampling in the United States [13].
Nematode species of the genus Pristionchus have little diagnostic morphological characters and usually show overlapping ranges of morphometric values [14,8]. Prior to our own studies on P. pacificus, only one Pristionchus species, P. aerivorus, was described for North America by Cobb in 1916 [9,15]. All previously obtained strains of P. pacificus from North America result from soil samples taken by various researchers [8,13]. It is important to note that all of these soil-derived P. pacificus strains represent random findings within a sampled locality. P. pacificus was a rare nematode in these soil samples and multiple isolates from the same soil sample are not available.
Morphometric analysis revealed that the most common species in our analysis is very similar to P. aerivorus (Table 2). Thus, we have classified this species as P. aerivorus. The four other species are described as novel species and we provide morphological measurements, molecular SSU sequence data, the results of mating experiments and species designations for all of them (Fig. 2, Table 2, see species diagnosis below). The reference strains of all species are available as living cultures and frozen stocks in our laboratory and can be provided to other researchers upon request. Also, type material has been delivered to museums. Together, these five species account for the majority of isolates from scarab beetles (Table 1). Specifically, we obtained P. aerivorus (85 isolates), P. pseudaerivorus n. sp. (71 isolates), P. marianneae n. sp. (45 isolates), P. pauli n. sp. (35 isolates) and P. americanus n. sp. (1 isolate) in the US scarab beetle survey. In the following, we provide the diagnoses for the four novel Pristionchus species:
Pristionchus marianneae n. sp
Diagnostic character: The species is characterized by a 471 bp sequence of a 1 kb fragment of the small subunit ribosomal RNA gene (SSU) that was amplified by PCR with the primers shown in Materials and Methods. This SSU sequence is distinct from the SSU sequence of all other Pristionchus species, but is identical between all isolates of P. marianneae. The sequence is shown in Fig. 2.
GenBank accession code: DQ419901
A second diagnostic character is provided by mating experiments. P. marianneae males mate with females of other isolates but not with females/hermaphrodites of other species.
Morphological measurements: see Table 2
Type host and locality: On Popilia japonica (Coleoptera: Scarabaeidae) near Geneva, New York, United States of America.
Etymology: R. J. S and M. H. want to dedicate this species to their mothers.
Frozen strain number: RS5108
Holotype: One male permanent slide No: SMNK-NEMA-T0022 (Natural History Museum Karlsruhe, Germany)
Paratypes: One male and one female permanent slide No: SMNK-NEMA-T0023; One male and one female permanent slide No: SMNK-NEMA-T0024
Pristionchus pauli n. sp
Diagnostic character: The species is characterized by a 471 bp sequence of a 1 kb fragment of the small subunit ribosomal RNA gene (SSU) that was amplified by PCR with the primers shown in Materials and Methods. This SSU sequence is distinct from the SSU sequence of all other Pristionchus species, but is identical between all isolates of P. pauli. The sequence is shown in Fig. 2.
GenBank accession code: DQ419900
A second diagnostic character is provided by mating experiments. P. pauli males mate with females of other isolates but not with females/hermaphrodites of other species.
Morphological measurements: see Table 2
Type host and locality: On Lichnanthe vulpina (Coleoptera: Scarabaeidae) in Carver, Massachusetts, United States of America
Etymology: M. H. wants to dedicate this species to Dr. Paul S. Robbins, an outstanding beetle expert and true friend.
Frozen strain number: RS5130
Holotype: One male permanent slide No: SMNK-NEMA-T0025 (Natural History Museum Karlsruhe, Germany)
Paratypes: One male and one female permanent slide No: SMNK-NEMA-T0026; One male and one female permanent slide No: SMNK-NEMA-T0027
Pristionchus americanus n. sp
Diagnostic character: The species is characterized by a 471 bp sequence of a 1 kb fragment of the small subunit ribosomal RNA gene (SSU) that was amplified by PCR with the primers shown in Materials and Methods. This SSU sequence is distinct from the SSU sequence of all other Pristionchus species. The sequence is shown in Fig. 2.
GenBank accession code: DQ419904
P. americanus males do not mate successfully with females/hermaphrodites of other species.
Morphological measurements: see Table 2
Type host and locality: On Polyphylla fullo (Coleoptera: Scarabaeidae) in Centerville, Massachusetts, United States of America
Etymology: This species has been found only in the United States of America so far. Frozen strain number: RS5140
Holotype: One male permanent slide No: SMNK-NEMA-T0019 (Natural History Museum Karlsruhe, Germany)
Paratypes: One male and one female permanent slide No: SMNK-NEMA-T0020; One male and one female permanent slide No: SMNK-NEMA-T0021
Pristionchus pseudaerivorus n. sp
Diagnostic character: The species is characterized by a 472 bp sequence of a 1 kb fragment of the small subunit ribosomal RNA gene (SSU) that was amplified by PCR with the primers shown in Materials and Methods. This SSU sequence is distinct from the SSU sequence of all other Pristionchus species, but is identical between all isolates of P. pseudaerivorus. The sequence is shown in Fig. 2.
GenBank accession code: DQ419902
Morphological measurements: see Table 2
Type host and locality: On Phyllophaga sp. (Coleoptera: Scarabaeidae) near Lincoln, Nebraska, United States of America.
Etymology: The species is very similar in respect to morphological measurements and molecular sequence to P. aerivorus. Mating experiments revealed that crosses between P. pseudaerivorus and P. aerivorus can result in sterile females.
Frozen strain number: RS5139
Holotype: One male permanent slide No: SMNK-NEMA-T0028 (Natural History Museum Karlsruhe, Germany)
Paratypes: One male and one female permanent slide No: SMNK-NEMA-T0029; One male and one female permanent slide No: SMNK-NEMA-T0030
Pristionchus species sampled on American scarab beetles are predominantly gonochoristic
The result of the US survey indicates a striking difference in the mode of reproduction of North American and European beetle associated Pristionchus species. P. aerivorus, P. pseudaerivorus n. sp., P. pauli n. sp., P. marianneae n. sp. and P. americanus n. sp. are gonochoristic (male/female) species and account for 98% of the isolates from scarab beetles (Fig. 3). In contrast, more than 60% of the isolates from scarab beetles in Western Europe belong to two hermaphroditic species, P. maupasi and P. entomophagus, respectively (Fig. 3) [8]. Thus, North America and Europe differ in the Pristionchus species associated with scarab beetles and in the mode of reproduction of these nematodes.
The Pristionchus species from the US form a phylogenetic clade together with P. maupasi
To determine if the Pristionchus biogeography would match species phylogeny we incorporated the North American species into the phylogenetic framework of the genus Pristionchus with a representative of the closely related genus Koerneria as outgroup (Fig. 4). Interestingly, P. aerivorus, P. pseudaerivorus n. sp., P. pauli n. sp., P. marianneae n. sp. and P. americanus n. sp. form a single clade and are much more closely related to one another than the species found in Europe. P. aerivorus, P. pseudaerivorus n. sp. and P. americanus n. sp. are the most closely related species and differ in only one out of 471 base pairs in their SSU sequence (Fig. 2). Surprisingly, the cockchafer-associated hermaphroditic species P. maupasi from Europe belongs to the same clade and carries an SSU sequence identical to the P. aerivorus sequence (Figs. 2, 4). A more detailed analysis including several ribosomal protein encoding nuclear genes distinguishes the two species and indicates that P. aerivorus and P. pseudaerivorus n. sp. are the two most closely related species and that P. maupasi is the sister taxon to the P. aerivorus/P. pseudaerivorus n. sp. species pair (Fig. 5). Specifically, P. aerivorus and P. pseudaerivorus n. sp. differ in 19 nucleotides of the concatenated sequences of the rpl-26, rpl-28 and rps-14 genes. In contrast, P. maupasi and P. aerivorus differ in 31 nucleotides and P. maupasi and P. pseudaerivorus n. sp. differ in 33 nucleotides of the corresponding sequences (Fig. 5). P. americanus n. sp. and P. marianneae n. sp. show even more nucleotide differences to the other species (Fig. 5). Thus, the investigated genes allow us to clearly distinguish these four species.
P. aerivorus and P. pseudaerivorus n. sp. follow Haldane's rule
The unusually high DNA sequence similarity between P. aerivorus and P. pseudaerivorus n. sp. suggests that they result from a recent speciation event. Therefore, these two species might still possess the potential to mate and form hybrid offspring. To obtain support for this hypothesis, we carried out cross-species mating experiments and found a strong case of Haldane's rule (Fig. 6). Haldane's rule states that in hybrids between diverging species the sterile absent or underrepresented offspring is of the heterogametic sex [11]. We found F1 hybrids in 12 of 24 crosses between P. aerivorus and P. pseudaerivorus n. sp. and all of the F1 hybrid animals were phenotypically female (Fig. 6A). Specifically, F1 hybrids were obtained after reciprocal crosses of P. pseudaerivorus n. sp. from Texas with four strains of P. aerivorus. In contrast, P. pseudaerivorus n. sp. from Nebraska produced F1 hybrids only when males were used for mating with P. aerivorus females. Another P. pseudaerivorus n. sp. strain from Lincoln, Nebraska (LNE) did not produce F1 hybrids with any of the P. aerivorus strains. Thus, the occurrence of species hybrids is highly strain-specific and does not correlate with the geographic origin of the strains used in the analysis.
The primary signal in P. pacificus and C. elegans sex determination is the ratio of the set of X chromosomes to autosomes [1]. Animals with two X chromosomes develop as females (XX), whereas animals with a single X chromosome develop as males (XO). Theoretically, the absence of males among the F1 hybrid animals could result from the death of the putative XO animals or the sexual transformation of these animals into females. The latter scenario was observed in crosses of Caenorhabditis remanei with Caenorhabditis briggsae [16].
Additional support for the results of the laboratory crosses came from genotyped Pristionchus nematodes from wild scarab beetles. We found two cases of hybrids between P. aerivorus and P. pseudaerivorus n. sp. emerging from individual beetles (Fig. 6B). Genotyping of other Pristionchus individuals from the same two beetles revealed that they carried both P. aerivorus and P. pseudaerivorus n. sp., suggesting that the observed hybrids are the result of cross-species fertilization (Fig. 6B). However, both hybrid animals were isolated more than a week after the beetle was placed on the petri dish. Therefore, it remains unknown if the observed hybrids came from the generation that lived on the beetle or whether it originated from fertilization in the laboratory. Taken together, P. aerivorus and P. pseudaerivorus n. sp. co-exist on the same beetles in nature, they can form hybrids and laboratory-derived F1 hybrids follow Haldane's rule. In addition, both species have a similar geographic distribution in our sampling.
The Colorado potato beetle hosts the same Pristionchus species in North America and Europe
Finally, we wanted to know if Pristionchus nematodes occur in association with the CPB Leptinotarsa decemlineata in the United States. Our analysis of CPB in Europe had indicated that one species, P. uniformis, infests these beetles with high specificity [8]. The CPB is a pest species that originated in Mexico, where it extended its range of host plants from the wild solanaceae buffalo bur (Solanum rostratum) to include the potato (Solanum tuberosum). It migrated further north to the United States and was first described in Colorado in 1811. Spreading towards the East it reached the Atlantic coast in 1874 and was first reported in Europe in 1877 [17,18].
We collected CPB from two different locations in Ohio and New York State and found Pristionchus nematodes with an infestation rate of up to 60% (Table 1). Sequence analysis and mating experiments revealed that all of these isolates belong to P. uniformis, the same species that is found on European CPB. Thus, in contrast to scarab beetles, the CPB hosts the same (and only one) Pristionchus species on both continents, P. uniformis. Given the recent invasion of the CPB to Europe we speculate that P. uniformis was introduced to Europe together with its beetle vector, the CPB, at the end of the 19th century.
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