Towards a framework for the evolutionary genomics of Kinetoplastids: what kind of data and how much?

The current status of kinetoplastids phylogeny and evolution is discussed in view of the recent progresses on genomics. Some ideas on a potential framework for the evolutionary genomics of kinetoplastids are presented.

Editorial

In this journal, the concept of species are presented by Wendy Gibson [1] and Hooman Momen [2] while Michel Tibayrenc [3] reviews the evolution and variability of Trypanosoma cruzi. Veronique Hannaert and collaborators [4] provide a comprehensive review on the evolution of energy metabolism in Kinetoplastida and Austin Hughes and Helen Piontkivska [5] discuss the controversial issue of the monophyletic versus paraphyletic status of the genus Trypanosoma. The latter authors concluded that the markers used to date failed to provide a consistent answer about the origin of trypanosomes. The arguments for the use of more protein-based sequences and a larger number of species are admirable. However, some important questions remain: which genes and how many are needed? Which other species should be selected and how many should be explored?

Generally, a limited number of genes available for a large number of taxa is sufficient for most phylogenetic studies. However, in order to resolve the relationships among closely related species and strains, and to better understand their origin, the availability of a set of evolutionary informative genes would certainly be useful. The ribosomal (r)RNA genes (18S, 5S, SL) still dominate the field, while only a few informative protein-coding genes have been described. The identification and choice of individual genes or gene families is sometimes difficult and less cost-effective when compared with high throughput sequencing initiatives [6]. This observation makes us to reflect about the need to discover new candidate genes for the study of kinetoplastid evolution. According to Podlipaev (2000, 2001) [7,8], from the 10 trypanosomatid genera [9], those (six) associated with insects have so far received little attention or have been "neglected" compared to species with an impact on human health. The same is true for all bodonids.

Still, Trypanosomatina flagellates are in the forefront among kinetoplastids, since several species (T. cruzi, T. brucei and Leishmania major) will have their genomes completely sequenced within the next few months. While the recent start of genome projects for L. braziliensis, L. infantum, T. vivax and T. congolense is very welcome, it is worth noticing that all the above mentioned species belong to only two (Trypanosoma and Leishmania) of the 20 kinetoplastid genera, showing the stark under-representation of the other genera in the sequencing initiative.

A casual search in the Entrez division of NCBI showed that although virtually omnipresent in nature, leptomonads and phytomonads have only dozens of entries, a mere trickle when compared with 139667 and 29777 entries for Trypanosoma and Leishmania, respectively (see Table 1). It is clear from the pattern of data available that efforts should be focused on obtaining more sequences from the "non-Leishmania" and "non-Trypanosoma" kinetoplastids. Centering on one "representative" or "model" species for each of the underepresented genera Bodo, Trypanoplasma and Cryptobia would certainly be both worthwile and rewarding.

Table 1

Survey of kinetoplastid sequences available in the Genbank.

	Nucleotides	Proteins
Trypanosomatina
Trypanosoma	139667	3695
Leishmania	29777	2945
Crithidia	163	206
Herpetomas	137	15
Phytomonas	112	12
Leptomonas	55	23
Blastocrithidia	12	7
Wallaceina	10	3
Endotrypanum	9	5
Bodonina
Bodo	64	12
Trypanoplasma	22	24
Cryptobia	14	5
Dimastigella	9	1
Rhynchomonas	6	1
Perkinsiella	6	0
Rhynchobodo	3	0
Cruzella	3	0
Ichthyobodo	2	0
Parabodo	2	0
Procryptobia	1	0

Having a genome fully sequenced is an expensive initiative (although the cost per base is decreasing annually, rendering the whole genome sequencing efforts increasingly cost-effective), then centering the resources on EST (expressed sequence tags) projects to explore the transcriptome of some species would be certainly helpful for the comparative "transcriptomics" of kinetoplastids. It has been demonstrated that EST projects are relatively cheap when compared with whole genome shotgun strategies, with the advantage that a kinetoplastid EST project would provide with the coding sequences that would be of great help for evolutionary genomics. As an example, it would be particularly interesting to know more about the evolution and polymorphism of kinetoplastid-specific genes associated with metabolic pathways or even markers for typing. On the other hand, a potential disadvantage of the EST approach would be that one could not unequivocally evaluate the gene loss and genome structure because only a part of the genome would be surveyed. However, studies on gene and protein content, orthologs and paralogs would be perfectly allowed. Another advantage of having the transcriptome of other trypanosomatids sequenced would be the usefulness of such sequences for the annotation of the T. cruzi, L. major and T. brucei genomes. The sequencing consortia for these three species have been reporting a high number (sometimes >70 %) of orphan genes during the annotation process [10,11]. New data from other trypanosomatids may help to discover which of them are real "orphans" and/or species-specific genes, and which ones are genes confined to Kinetoplastida.

An important issue towards the exploration of the transcriptome of kinetoplastids outside Trypanosomatina is the choice of "model species". These "models" would be used to get insights into the evolution of bizarre structures and processes so far mostly described in Trypanosoma and less so in Leishmania. One proposal could be the study of the evolution of VSGs, as well as complex proteins involved in RNA editing or trans-splicing. A comparative approach has recently proved to be useful for our understanding of how the complex kinetoplast DNA network may have evolved [12]. The "models" should be members of major kinetoplastid clades in the current phylogenetic trees [13] that grow in reasonable densities in simple culture media, preferably axenic. Bodo saltans may be a good model, because it branches off on the border between bodonids and trypanosomatids, and is an ecologically important free-living species of potentially world-wide distribution. Its sole disadvantage is the growth with feeder bacteria. As far as we know, the only bodonid that grows in an axenic medium is Trypanoplasma borreli, a parasite of marine and freshwater fish, another potential candidate. The final proposed candidate is Diplonema papillatum, since it grows axenically in simple media to high densities. Moreover, it would be an excellent outgroup for kinetoplastids, since it represents the most related group and likely shares with them numerous peculiarities.

Inferring from the recently unveiled diversity of extremely small eukaryotes [14], one would predict that a great diversity of kinetoplastids is still to be discovered. Perhaps an enthusiastic overestimate, some discuss the existence of a great trypanosomatid biodiversity in insects alone [15]. Recent surveys have found new species in ocean depths [16,17] or unexpected niches [18]. One of the approaches used for the discovery of these new eukaryote taxa is culture-independent-survey by PCR (ciPCR), which has also helped to double the number of prokaryotic phyla [19]. This approach would be particularly interesting in a search for difficult-to-cultivate Kinetoplastida. For such a survey, a number of known conserved markers/genes would be needed for the design of PCR-based assays and, unfortunately, that is not possible with the limited data currently available. There may be some requirements for choosing the markers since according to Charlebois and collaborators [20] protein-coding regions are usually less universal, more difficult to PCR-amplify and often shorter and less information-rich than the rRNA genes. Possible discrepancies between the rRNA- and protein-based trees can be avoided by choosing informative and unbiased genes and by constructing trees on the basis of concatenated alignments. Whole-genome trees are becoming popular given the availability of a large number of bacterial genomes in the databases, however, for the near future, it is not going to be an option for non-pathogenic kinetoplastids and in general for eukaryotes because of the size of their genomes. New technologies for sequencing large genomes and cheaper costs per base could, in the future, facilitate that approach even for protozoans.

It seems that with the available genomics approaches, there are now more chances to map the real diversity of kinetoplastids in nature, and explore their fascinating biology, evolution and "roots".