Phylogenetic species tree of the Diplogastridae
Here we present a first comprehensive molecular phylogeny of beetle-associated diplogastrid nematodes providing a framework for the investigation of nematode-beetle interactions and the interplay of ecological factors and nematode evolution. The Diplogastridae include more than 300 species of free-living or beetle-associated nematodes, which are grouped in 28 genera [19, 20]. They are a monophyletic clade placed within the paraphyletic rhabditids . The sister group to the diplogastrids is formed by the eurhabditids, which encompass Caenorhabditis, Oscheius and others .
For the purpose of assessing phylogenetic relationships, we choose representative species of the diplogastrid genera for molecular analyses. Phylogenetic species trees can be derived from single gene trees if speciation events are well separated in time. In general, however, this premise cannot be assumed and true species trees may differ from gene trees [31, 32]. Thus, to expand the dataset, multiple genes are needed for a robust phylogenetic reconstruction. Phylogenetic species trees from multi-gene data can be inferred in two different ways. They can either be constructed from the consensus of separately generated gene trees or from an alignment of concatenated multiple gene sequences is used to infer the species tree. The latter was shown to perform better in yielding more accurate trees in a test situation . Here, we applied both procedures, but chose to focus on the concatenation approach since the phylogenetic information content of the single gene sequences was limited.
A molecular phylogeny of beetle-associated diplogastrid nematodes
Although the topologies of the phylogenetic trees produced by the different algorithms were not always congruent, four fundamental features can be extracted (Figure 2). Firstly, species of the same genus always clustered together, as shown by the genera Pristionchus, Koerneria, and Diplogasteroides, for which two geographically distinct species each were included in the analysis. The two divergent species of Koerneria displayed the highest intra-genus distance, whereas Pristionchus and Diplogasteroides species from Europe and North America or Japan, respectively, grouped together more closely. This feature is consistent with the morphological separation of the two subclades within Koerneria, as already discussed by Fürst von Lieven . Secondly, terminal branches were exceedingly long while branches separating internal nodes with low support values were very short or collapsed completely. Although most beetle-associated diplogastrid genera were represented in the analyses, increased taxon sampling including non-beetle associated diplogastrids, might resolve some of those internal nodes. Overall, however, this indicates rapid initial diversification and speciation events separated by short divergence times in relation to the persistence of the extant taxa. Genealogies of individual genes may contribute to misleading species tree topologies if polymorphisms in ancestral populations of high effective population sizes are incompletely sorted during speciation events in close succession . The long persistence of the extant genera resulted in accumulation of multiple substitutions in terminal branches. Terminal taxa, therefore, can be expected to be rich in homoplasic characters due to parallel sequence evolution or evolutionary convergence. The resulting long-branch attraction effects interfere with the clear resolution of internal nodes. Thirdly, several monophyletic clades with high support from bootstrapping or from posterior probabilities became evident from the comparison of various trees. The first clade encompassed Diplogasteroides with Pseudodiplogasteroides as its putative sister genus, Diplogastrellus, and Rhabditidoides. The second clade included Mononchoides, Neodiplogaster and Tylopharynx. The third group combined Acrostichus and Diplogasteriana. The inclusion of Micoletzkya and Pristionchus in this group occurs with posterior probabilities of ≥ 0.8 and thus remains unresolved. The taxa Myctolaimus and Oigolaimella could not be linked firmly to any of the above clades. Fourthly, the genus Koerneria, which was in close relationship to Pristionchus in previous studies, assumes a basal position to all diplogastrid nematodes in this molecular analysis [19, 20]. This topology is also indicated in the molecular phylogeny of Kiontke et al. .
Comparison of molecular and morphological phylogenies
There is a fairly good agreement between the phylogeny of Diplogastridae as proposed by Sudhaus and Fürst von Lieven based on apomorphic morphological characters and the molecular phylogeny presented here . This example strongly supports the fact that morphological and molecular phylogenies should - under ideal conditions - result in similar findings. If morphological characters are well chosen and molecular characters are informative and substantial, homoplasy should be reduced, thus resulting in similar phylogenies.
The only major discrepancy in the case of the Diplogastridae is the genus diverging first from the stem species of Diplogastridae (see above). While in the morphological tree this position is taken by Pseudodiplogasteroides, in the molecular tree it is occupied by Koerneria. As a consequence, the molecular analysis places Pseudodiplogasteroides as the sister taxon to Diplogasteroides. The same well-supported clade also unexpectedly harbours the genus Diplogastrellus, which appears to be closer to Acrostichus in a yet unresolved relationship following Sudhaus and Fürst von Lieven . The latter, on the other hand, is the sister taxon to Diplogasteriana in the molecular tree.
Although positioned distinctively on the molecular phylogenetic tree, Pristionchus and Koerneria share a number of morphological characters, such as a dimorphism in the buccal cavity, the shape and arrangement of denticles, overall body shape, and a prominent ripping of the cuticle . These features could have evolved in two different ways. First, the shared characters may have been present in the stem species followed by subsequent loss in genera other than Pristionchus and Koerneria. This scenario is, however, unlikely since multiple independent character losses were required. The second, more likely possibility assumes parallel or convergent evolution. Given the long terminal branches separating the genera, independent convergent gain of advantageous characters is easily conceivable.
Nematode-beetle associations: Coevolution or host switching?
Is there a correlation between the rapid ancestral diversification of the diplogastrid lineages and their subsequent evolution with the phylogeny of their coleopteran hosts? The beetles appeared around 285 million years ago (mya) followed by radiations of suborders [34–36]. The minimum age of the Scarabaeoidea and the Chrysomeloidea, which are major hosts for Diplogastridae, were both estimated to 150 mya, whereas their last common ancestor lived more than 236 mya . Dieterich et al. estimated the divergence of the Pristionchus pacificus from the eurhabditid genus Caenorhabditis to a time range of 280-430 mya . The ancestral radiations of Diplogastridae thus occurred in a time period that overlaps with the diversification of beetles into major lineages. It is therefore conceivable that the initial radiation of the Coleoptera had influence on the diversification of diplogastrids by providing new ecological niches, to which the nematodes were adapting. If these interactions persisted for a long time, coevolution of the nematodes with the beetles should have shaped the evolutionary pattern of the diplogastrids and its traces should be detectable in phylogenetic tree topologies. Inspecting the monophyletic diplogastrid clades, such as Mononchoides, Tylopharynx, and Neodiplogaster for these features will provide clues to resolve this issue. Mononchoides is associated with scarab beetles, curculionids, and Silphidae, Tylopharynx is associated with scarabs, and Neodiplogaster with curculionid, buprestid and cerambycid beetles. Likewise, in a second monophyletic group, Diplogasteroides and Diplogastrellus occur on curculionids and cerambycids, Pseudodiplogasteroides on cerambycids, and Rhabditidoides on Scarabaeidae. Following the comprehensive beetle phylogeny provided by Hunt et al. the Cucujiformia, which encompass cerambycid, and chrysomelid beetles, originated 236 mya, well separated from Scarabaeoidea . Thus, members of the same monophyletic diplogastrid clade or even the same genus are associated with beetles of ancient divergence, refuting the possibility of a long-term coevolutionary relationship. Together, these data support host switching rather than coevolution as mechanism to explain the observed patterns of diplogastrid - beetle associations.
A case of recent host switching of a European species to an American host is given by Pristionchus uniformis [13, 17]. Pristionchus dauer larvae are generally found on scarab beetles but Pristionchus uniformis also infests at high frequencies the Colorado potato beetle, a chrysomelid beetle, both in Europe and in North America [12, 13]. The nematode populations on the two hosts species are genetically undistinguishable indicating recent and repeating events of host changes. The evolution and population genetics are currently under investigation (Isabella d'Anna, W.E.M. and R.J.S., unpublished observations).
Phylogeny: A further mark for genome evolution
Sequencing the whole genome of an organism results in data that can only be interpreted in the context of a robust phylogenetic framework, including other related taxa. The genome of P. pacificus has recently been sequenced and the analysis of its genetic composition identified homologous nematode-specific genes, but also a remarkable number of features not shared with C. elegans or other available nematode genomes . Firstly, the P. pacificus genome contains 23,500 predicted protein-coding genes, a substantial amount of which does not show sequence similarities to other metazoan genes. Secondly, the P. pacificus genome contains a massive expansion of genes predicted to be involved in the detoxification of xenobiotics, such as cytochrome P450 enzymes and ABC transporters. Thirdly, a conspicuous set of genes encoding cellulases or glycosylhydrolases is apparent, which has no counterpart in other nematodes but is most similar to corresponding genes in Dictyostelium or other microbes. The acquisition of these and several other genes could only be explained by horizontal gene transfer. With phylogenetic information on the taxa involved, as presented here for subclades of the Diplogastridae the analyses of horizontal gene transfer events in diplogastrid genomie evolution can be studied in greater detail in the future.