Molecular r-evolution: the SSU rRNA

Instead I would like to consider in more detail another kind of phylogeny that has re-placed the above classical phenotype-based approach: the phylogenies inferred by compar-ing sequences of DNA or amino acids, i.e. molecular systematics, which have revolution-ized our understanding of evolutionary relationships. In 1965, Zuckerkandl and Pauling [Zuckerkandl and Pauling 1965] argued that collating of informative molecules would per-mit the evaluation of evolutionary relatedness. They were obviously right and since that time molecular phylogeny has been regarded as the tool of choice for reconstructing evolu-tionary histories –this is particularly true for protists where the interpretation of

morpho-Figure 1-ch.1. Haeckel’s three kingdoms.

From [Haeckel 1866].

Figure 2-ch.1. Copeland’s four kingdoms.

From [Copeland 1938].

Figure 3-ch.1. Whittaker’s five kingdoms.

From [Whittaker 1969].

The first molecular works aimed at determining the evolutionary relationships among eukaryotes date back to the mid eighties and principally depended on the small subunit ribosomal RNA (SSU rRNA) [Sogin et al. 1986; Friedman et al. 1987; Sogin et al. 1989;

Woese et al. 1990; Sogin 1991], although the large subunit (LSU rRNA), to a lesser extent, also contributed to phylogenetic reconstruction (e.g., [Perasso et al. 1989]). These pioneer studies were all characterized by a handful of deeply diverging protist lineages (e.g., Giardia, Trichomonas, Microsporidia), progressively emerging from the distant prokaryotic root, and followed by a densely branched “crown” nesting most eukaryotic diversity (Fig-ure 4-ch.1). From these early molecular analyses, evolutionists drew the following principal features:

1) As a result of the huge genetic diversity in SSU rRNA, the deep eukaryotic branches seemed to exceed the depth of branching within the entire prokaryotic world [Sogin et al. 1986].

2) Consequently, eukaryotes became distinct very early in the history of life, and were thought to be likely as old as the eubacteria and archaebacteria [Sogin et al. 1989].

3) The lowermost lineages of the eukaryotic tree were usually simple, most of which liv-ing parasitically within animal hosts and, importantly, lackliv-ing organelles, in particular the mitochondrion [Friedman et al. 1987]. This notion of primitive amitochondrial eu-karyotes (the “Archezoa” hypothesis [Cavalier-Smith 1989]) was in fact being discussed prior to the publication of molecular phylogenies that wrongly supported its validity. It postulated that mitochondria-lacking eukaryotes had diverged before the acquisition of mitochondria through endosymbiosis and had evolved under anaerobic conditions. So when the first SSU rRNA trees including such organisms came out, specifically showing a deeper branching than any previously known eukaryotic sequences [Friedman et al.

1987; Sogin 1989; Sogin et al. 1989], the general consensus converged towards the postu-late that these amitochondrial lineages were genuinely primitive, relicts of an ancient world devoid of oxygen (Figure 4-ch.1).

4) The apical part of the SSU rRNA tree, the so-called crown, contained major clades that branched near a common point, as if their divergence occurred nearly simultane-ously [Sogin 1991]. Here were included, among others, the animals, fungi, plants, and diverse protist lineages that now form the alveolate grouping. Because the branching pattern among these groups could not be resolved, it was suggested that they origi-nated in a massive radiation [Knoll 1992]. This lack of a clear order of divergence

eira et al. 2000], leading some to propose the “big-bang” hypothesis [Philippe et al.

2000a] which postulated that most eukaryotic phyla emerged in a relative short period of time, thus not enough phylogenetic signal could accumulate in the sequences.

Figure 4-ch.1. A typical SSU rRNA tree of eukaryotes, as it was being published in the mid-nineties.

Plastid-bearing lineages are indicated in colors approximating their respective pigmentation. From [Embley and Martin 2006].

In the nineties, as more and more species were being sequenced, intermediate groups appeared in between the Archezoa members and the eukaryotic crown. Similarly to the amitochondrial species, these newcomers were characterized by a high rate of evolution producing long branches in phylogenetic reconstructions. A classical example are the Fo-raminifera whose both SSU and LSU rRNA showed a mid-position in the tree [Pawlowski et al. 1994; Pawlowski et al. 1996].

Interestingly enough, this view of the eukaryotic tree relied almost entirely on a single molecular marker (the SSU rRNA, although as mentioned above a few others started to be used), and the gene trees were very much interpreted as the organismal tree. Unfortu-nately this marker proved to be highly mutationally saturated at the eukaryotic level, with very variable evolutionary rates between species [Philippe and Laurent 1998]. Because this characteristic was shown to be prone to the Long Branch Attraction (LBA) artifact, in which two distant species with fast evolving sequences are erroneously clustered together [Felsenstein 1978], the SSU rRNA topology became highly suspicious [Embley and Hirt

1998; Philippe and Laurent 1998]. Furthermore, two other important requirements for accu-rate phylogenetic inferences were not respected: the availability of a well sampled diversity of species and the use of appropriate tree reconstruction methods [Hendy and Penny 1989;

Lecointre et al. 1993; Huelsenbeck 1997; Brinkmann et al. 2005]. Indeed the taxon sam-pling at the beginning of molecular systematics was rather sparse, with often a single repre-sentative per major lineage, which increased the sensitivity of LBA by leaving unbroken the basal long branches. Likewise, simplistic approaches for inferring phylogenies (distances computed or parsimony) together with the use of unrealistically simplified models of evolu-tion were a serious brake for the resoluevolu-tion of the eukaryotic tree.

So the situation in the late nineties was a tree of eukaryotes very much based on a single molecular marker, with recognized shortcomings in its capability for being able to infer phylogenetic relationships at deep taxonomic levels.