Two main mechanisms might account for the unexpected phylogenetic relationships among class 4 HDACs and the anomalous phyletic distribution of the eukaryotic ones: (i) ancient gene duplication followed by differential gene loss or (ii) horizontal gene transfer (HGT).
Let us first examine the anomalous phyletic distribution of some eukaryotic class 4 HDACs in the light of the 'gene duplication-gene loss' hypothesis (Figure ). As both the mixed-group type and the eukaryotic-group type occur (often separately, sometimes together) in a wide range of eukaryotes, the duplication event postulated to have given rise to these two types of genes must have occurred early in eukaryotic evolution. The presence of two types of class 4 HDACs would thus be the ancestral situation of most or all eukaryotes. The punctate distribution we see today would be due to loss, at a high rate, of one or the other type of class 4 HDAC. As the mixed-group class 4 HDACs of eukaryotes appear more closely related to eubacterial HDACs than to the eukaryotic-group HDACs, we must even consider the possibility that the duplication event occurred before the eukaryotes and eubacteria diverged (Figure ). The presence of two types of class 4 HDACs would be the ancestral situation for both eubacteria and eukaryotes. The transition to the present-day situation would have involved not only a high rate of differential gene loss in eukaryotes, but also the loss of one of the paralogues in eubacteria, probably at an early stage of eubacterial evolution.
Evolutionary history of class 4 HDACs. Two possible evolutionary scenarios are represented. E, eukaryotic-group HDAC; M, mixed-group HDAC. Dotted lines indicate that the gene is not present in all eukaryotic species (see text for details).
One main problem with this view is that both types of class 4 HDAC genes must have coexisted in the ancestors of lineages (e.g. metazoans and viridiplantae) where some descendants have one type of HDAC and other descendants have the other type. We would expect many of these organisms to still possess both genes, but as a rule, this is not so (Figure ). We found both gene types in only three eukaryotic species, as opposed to 37 species possessing only one gene. As more genomes are sequenced, more will probably be found to contain both genes, but the presence of a single gene in most genomes studied to date does not support the notion that the two categories of class 4 HDACs represent two paralogous groups that originated early in eukaryotic evolution. In addition, the model of ancient gene duplication followed by differential gene loss fails to fully explain some of our observations, such as the strongly supported separation of two diatom HDACs, those of Thalassiosira pseudonana and Phaeodactylum tricornutum, within the mixed group (Figure ). Much more complicated scenarios are therefore required, making this model less plausible and not especially parsimonious.
The other main possibility is HGT, the transmission of genetic material from one species to another (Figure ). HGT is a widespread and important phenomenon in prokaryotes. It is one of the driving forces of genome evolution in both archaea and eubacteria [13
]. Over the past few years, it has become increasingly clear that HGT has had an impact on eukaryote evolution also, at least in the case of unicellular and/or parasitic eukaryotes [19
], yet the occurrence and the importance of HGT in organisms such as land plants and animals is less obvious and very controversial. Although claims have been made for HGT in multicellular organisms, only very few cases have been clearly demonstrated, and these mainly concern eukaryote-eukaryote and/or host-parasite gene transfer [15
]. The main criteria used in the aforementioned publications to detect HGT are unexpected phyletic distribution, differential presence or absence in closely related species, and incongruent phylogenetic trees [15
]. Our data meet all these criteria (see Figures and ), and are therefore very suggestive of the occurrence of HGTs having shaped the evolutionary history of the class 4 HDACs.
Although our data do not allow a firm determination of the direction of these putative HGTs (the identity of donors and recipients remains unknown), we favour the hypothesis that transfer occurred from prokaryotes to eukaryotes, and that the eukaryotic-group members are the 'original' eukaryotic HDACs and the mixed-group members are the 'transferred' HDACS. In support of this view, class 4 HDACs are found in many diverse eubacterial species representative of most major eubacterial lineages (Figure ). To imagine that a class 4 HDAC was present in early eubacterial evolution (and subsequently transferred a few times to eukaryotes) is a more parsimonious mechanism than to postulate that the different prokaryotic class 4 HDACs were acquired from eukaryotes by numerous independent HGTs.
An important feature of the putative prokaryote-eukaryote HGTs is that most of them are probably ancient, as indicated by the species ranges covered by the monophyletic groups distinguished among the mixed-group eukaryotic HDACs (Figure ). For example, the existence of the aforementioned monophyletic group comprising all nine metazoan sequences indicates that the transferred gene was already present in the last common ancestor of these animals, i.e. in that of most or all animals. This means that the recipient of the putative HGT was not a present-day complex metazoan but an ancient, probably much more simple (maybe unicellular) ancestor. This is important, as gene transfers from prokaryotes to eukaryotes with sequestered germ lines, such as most present-day animals, appear to be very rare [23
]; almost all other putative HGTs we have detected concern unicellular eukaryotes. A possible exception concerns the second HDAC sequence found in the genome of the cnidarian Nematostella vectensis
, which forms a monophyletic group with the sequences of Cytophaga hutchinsonii
and Psychrobacter cryhalolentis
(Figure , orange circle). Although we cannot rule out contamination of the genomic data from which these sequences were obtained, this might be indicative of a much more recent HGT involving a complex multicellular organism.
Besides these putative eubacterium-eukaryote transfers, there is also the possibility of at least one eukaryote-eukaryote HGT. This is suggested by the existence of a monophyletic group including very distant eukaryotic species (Figure , yellow circle): the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (chromalveolata), the red alga Cyanidioschyon merolae, and the green alga Ostreococcus tauri (plantae). The green alga sequence is more closely related to those of the evolutionarily very distant diatoms than to those of any other viridiplantae. We suggest that this association may be the result of eukaryote-eukaryote HGTs between these phytoplanctonic species.
Lastly, we note that in most lineages only a single HDAC is found (Figures and ), yet two different proteins are found in the diatoms Thalassiosira pseudonana
and Phaeodactylum tricornutum
, the cnidarian Nematostella vectensis
, and the green alga Ostreococcus tauri
. In all these cases, both proteins belong to the mixed group and are not closely related, suggesting independent HGTs. The existence of eukaryotes with only mixed-group HDACs (and thus lacking a eukaryotic-group member) suggests that gene transfer was sometimes followed by functional replacement of the 'original' eukaryotic gene by the transferred one. The only eukaryotes to possess both a mixed-group and a eukaryotic-group protein are the green alga Chlamydomonas reinhardtii
and two animals (Strongylocentrotus purpuratus
and Locusta migratoria
) (Figures and ). Similar multiple replacements have been reported for the eukaryotic translation elongation factor 1α [21
]. Whether these replacements are due solely to chance or have selective advantages [33
] is an open question that awaits functional and biochemical characterization of the proteins and still broader sampling of eukaryotic HDAC genes.