DNA evidence for global dispersal and probable endemicity of protozoa

Abstract

Background: It is much debated whether microbes are easily dispersed globally or whether they, like many macro-organisms, have historical biogeographies. The ubiquitous dispersal hypothesis states that microbes are so numerous and so easily dispersed worldwide that all should be globally distributed and found wherever growing conditions suit them. This has been broadly upheld for protists (microbial eukaryotes) by most morphological and some molecular analyses. However, morphology and most previously used evolutionary markers evolve too slowly to test this important hypothesis adequately.

Results: Here we use a fast-evolving marker (ITS1 rDNA) to map global diversity and distribution of three different clades of cercomonad Protozoa (Eocercomonas and Paracercomonas: phylum Cercozoa) by sequencing multiple environmental gene libraries constructed from 47-80 globally-dispersed samples per group. Even with this enhanced resolution, identical ITS sequences (ITS-types) were retrieved from widely separated sites and on all continents for several genotypes, implying relatively rapid global dispersal. Some identical ITS-types were even recovered from both marine and non-marine samples, habitats that generally harbour significantly different protist communities. Conversely, other ITS-types had either patchy or restricted distributions.

Conclusion: Our results strongly suggest that geographic dispersal in macro-organisms and microbes is not fundamentally different: some taxa show restricted and/or patchy distributions while others are clearly cosmopolitan. These results are concordant with the 'moderate endemicity model' of microbial biogeography. Rare or continentally endemic microbes may be ecologically significant and potentially of conservational concern. We also demonstrate that strains with identical 18S but different ITS1 rDNA sequences can differ significantly in terms of morphological and important physiological characteristics, providing strong additional support for global protist biodiversity being significantly higher than previously thought.

Figure 1

Comparisons of evolutionary changes in the 18S and the ITS1 sequence. Left tree (black): Maximum likelihood (ML) phylogeny of cercomonad 18S rDNA sequences calculated from an alignment of 15 sequences and 213 nucleotide positions from the hypervariable V4 region. Right tree (grey): ML phylogeny of corresponding ITS1 rDNA (213 positions). Branch lengths and scale bars standardised to illustrate the much greater evolutionary rate of ITS1 than even the most hypervariable 18S rDNA region. Equivalent parsimony analyses using gaps as a 5th character (not shown) showed a parsimony score of 367 (minimum changes) for ITS1 and 120 for the V4 18S rDNA region. Cultures used for ecological and phenotypic character experiments (Table 2) marked by circles.

Figure 2

Phylogeny of cercomonads. BioNJ bootstrap tree (1000 replicates; corrected for Γ & I) of a subset of known cercomonad lineages, representing all known main cercomonad clades and genera (Cercomonas, Eocercomonas, and Paracercomonas) as defined by Karpov et al. [14]. The composition and phylogenetic position of the groups α, β, and γ forming the basis of this study are shown. A group-specific primer set was designed for each of these groups (see Methods).

Figure 3

Global provenance, biogeography and phylogeny of cercomonads. (A) World map showing major animal biogeographical regions. (B-D) 18S-ITS1 rDNA phylogenies of groups α (278 nucleotide positions), β (414 positions), and γ (335 positions). See Methods for details of each sequence dataset. Coloured discs correspond to hoops on map. Grey arrowheads indicate sequences found in only one biogeographical region. Square black brackets indicate 18S-genotype boundaries. X and Z (bracketed) indicates a possible endemic genotypic radiation. Branches marked a, f, and h in (B) are discussed in relation to additional sampling and ITS-type-specific primer probing. Branches without coloured discs originate from cultures. Sampling range differs among the three groups (Table 1). Key to bootstraps (ML/parsimony) given to right of scale. Bootstraps shown only when equal to or exceeding the value stated, although precise values are shown for clades marked X and Z, as these are discussed in the text as potentially geographically-restricted clades. Branch labels underlined and in bold refer to culture-derived sequences, including those used in the culture experiments. NB: in some cases a single branch corresponds to >1 culture or both environmental and culture-derived sequences. The long branch for 11.7E is not shown in (D) for the sake of clarity. It was not detected in any environmental library.

Figure 4

Shared alignment characters supporting putative geographically restricted clades X and Z (boxed in red). (A) Paracercomonas group α tree; (B) Paracercomonas group β tree (taken from Fig. 3). Characters at specific alignment positions are coloured black when 0.8 of positions agree; otherwise they are unshaded. Unique clade-specific alignment characters are illustrated with triangles across the bottom of the alignments. Shared sequence motifs in a hyper-variable section are illustrated with an oval across the bottom. Sequence position numbers across the top refer to DB-1002-6 and P101 PANAMA.