A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits

Abstract

Background: The nematode Caenorhabditis elegans is a major laboratory model in biology. Only ten Caenorhabditis species were available in culture at the onset of this study. Many of them, like C. elegans, were mostly isolated from artificial compost heaps, and their more natural habitat was unknown.

Results: Caenorhabditis nematodes were found to be proliferating in rotten fruits, flowers and stems. By collecting a large worldwide set of such samples, 16 new Caenorhabditis species were discovered. We performed mating tests to establish biological species status and found some instances of semi-fertile or sterile hybrid progeny. We established barcodes for all species using ITS2 rDNA sequences. By obtaining sequence data for two rRNA and nine protein-coding genes, we determined the likely phylogenetic relationships among the 26 species in culture. The new species are part of two well-resolved sister clades that we call the Elegans super-group and the Drosophilae super-group. We further scored phenotypic characters such as reproductive mode, mating behavior and male tail morphology, and discuss their congruence with the phylogeny. A small space between rays 2 and 3 evolved once in the stem species of the Elegans super-group; a narrow fan and spiral copulation evolved once in the stem species of C. angaria, C. sp. 8 and C. sp. 12. Several other character changes occurred convergently. For example, hermaphroditism evolved three times independently in C. elegans, C. briggsae and C. sp. 11. Several species can co-occur in the same location or even the same fruit. At the global level, some species have a cosmopolitan distribution: C. briggsae is particularly widespread, while C. elegans and C. remanei are found mostly or exclusively in temperate regions, and C. brenneri and C. sp. 11 exclusively in tropical zones. Other species have limited distributions, for example C. sp. 5 appears to be restricted to China, C. sp. 7 to West Africa and C. sp. 8 to the Eastern United States.

Conclusions: Caenorhabditis are "fruit worms", not soil nematodes. The 16 new species provide a resource and their phylogeny offers a framework for further studies into the evolution of genomic and phenotypic characters.

Figure 1

Rotting substrates from which Caenorhabditis species were isolated. Examples of sampled plant parts from which Caenorhabditis isolates could be successfully isolated. Most pictures illustrate the rotting sample on the ground, while a few others show the corresponding non-rotten plant in the same location. See additional files 7 and 8 for identifications and further sampling. First column: banana pseudo-stems, cabbage leaves. Second column: rotting flowers (mixed flowers, torch ginger, Hibiscus flower). Third column: rotting wild fruits (figs, chestnut, cucumber (Cucumis), unidentified). Fourth column: rotting cultivated fruits (cocoa, apple, pineapple, tomato). Pictures: MAF, except the Hibiscus flower (MVR) and the torch ginger flower (yielding JU1373), courtesy of V. Robert.

Figure 2

Our current best hypothesis for the phylogenetic relationships of all Caenorhabditis in culture and convergent evolution of hermaphroditism. Depicted is result of the maximum likelihood bootstrap analysis. Numbers on branches show, respectively, the support values for 100 bootstrap repeats in the ML analysis in percent, the posterior probabilities from the Bayesian inference analysis (blue), and the support values for 2000 jackknife repeats in a weighted maximum parsimony analysis in percent. * Weighted maximum parsimony analysis favors a position of C. sp. 15 as the sister species of the Elegans group with 60% support. ** Bayesian inference favors C. sp. 20 to form the sister species of the Angaria and Drosophilae groups with a clade credibility value of 96, the lowest in this analysis. Three species (in red) reproduce as self-fertilizing hermaphrodites with rare males, whereas all other species (in blue) are gonochoristic with females and males at approximately equal proportions. Hermaphroditism evolved convergently in all three lineages.

Figure 3

Likelihood phylogram for RNA polymerase II genes. The species relationships follow the phylogeny in Fig. 2. A general time-reversible model was used to estimate branch lengths (GTR+Γ+I, parameters estimated from the data).

Figure 4

World distribution of new Caenorhabditis species discovered since 2005. Based on the strains listed in Additional File 8 and [54]. Squares: Drosophilae super-group species. Circles: Elegans super-group species.

Figure 5

Evolution of spicule shape. For each Caenorhabditis species, a drawing of the spicule is shown in right lateral view with the dorsal side to the right (the spicules of C. drosophilae and C. sp. 2 and of C. angaria and C. sp. 12 are identical and are shown for only one species). Three features of the spicule are distinguished, each with two alternative character states: the overall shape (orange boxes), the curvature of the spicule blade (pink boxes) and the shape of the spicule tip (blue boxes). The character states for each species are indicated by filled or empty colored boxes above the images.

Figure 6

Evolution of male tail characters. Drawings of the male tail in ventral view are shown above the Caenorhabditis phylogeny. The male tail possesses a cuticular fan around the cloaca. Nine pairs of sensory rays are embedded in the fan. Differences between species are found in the shape of the anterior margin and the terminal end of the fan, in the arrangement of the rays and in the shape of the precloacal lip (cf. [12]). Seven characters of the male tail with two character states each are mapped onto the tree. The mating position is included as an eighth character. The spiral mating position is found only in the Angaria group (C. angaria, C. sp. 12 and C. sp. 8). It is correlated with a particularly narrow fan (compare the images). Male tails are largely identical in all species of the Elegans group, in C. drosophilae and C. sp. 2 and in C. angaria and C. sp. 12.

Figure 7

Graphic representation of differences in the ITS2 region between and within Caenorhabditis species as branch lengths of a tree for 50 Caenorhabditis strains. Branch length was determined by maximum parsimony (see Methods). With one exception (* C. sp. 8), the differences between strains of the same species (blue boxes) are smaller than the smallest differences between the two most closely related species pairs (orange boxes). In all cases, the differences separating any pair of species is much greater than the differences separating strains of the corresponding species. **The differences between C. remanei strains are larger than the differences seen within other species. Recently, however, hybrid breakdown has been observed in matings between strain VX0088 from China and several strains from Ohio, congruent with the long ITS2 branch of the Chinese isolates (Asher Cutter and Alivia Dey, pers. comm).

Figure 8

Discovery rate of Caenorhabditis species. The number of Caenorhabditis species is plotted cumulatively by year of description or discovery (if known). As of 2010, there were 38 Caenorhabditis species; this is a maximum number, since 6 of the 20 described species are not very well known and are potentially synonymous with other species [11]. The 16 species reported in this study were discovered between 2005 and 2010. The rate of discovery has increased since sampling efforts have focused on rotting fruit and other decaying plant material. The shape of this curve suggests that only a fraction of Caenorhabditis species is presently known.