Experimental design and statistical rigor in phylogenomics of horizontal and endosymbiotic gene transfer

Abstract

A growing number of phylogenomic investigations from diverse eukaryotes are examining conflicts among gene trees as evidence of horizontal gene transfer. If multiple foreign genes from the same eukaryotic lineage are found in a given genome, it is increasingly interpreted as concerted gene transfers during a cryptic endosymbiosis in the organism's evolutionary past, also known as "endosymbiotic gene transfer" or EGT. A number of provocative hypotheses of lost or serially replaced endosymbionts have been advanced; to date, however, these inferences largely have been post-hoc interpretations of genomic-wide conflicts among gene trees. With data sets as large and complex as eukaryotic genome sequences, it is critical to examine alternative explanations for intra-genome phylogenetic conflicts, particularly how much conflicting signal is expected from directional biases and statistical noise. The availability of genome-level data both permits and necessitates phylogenomics that test explicit, a priori predictions of horizontal gene transfer, using rigorous statistical methods and clearly defined experimental controls.

Figure 1

Endosymbiotic gene transfer and other potential explanations for finding "algal" genes in any given genome. A. Depicts the complexity of genome remodeling in typical secondary endosymbioses, particularly the massive transfer of genes from the primary plastid alga to its new host cell nucleus. Most genes from the original primary (cyanobacterial) plastid (1P) endosymbiont already made their way into the primary host nucleus (1Nu) via EGT. These genes are transferred, in turn, to the secondary host's nucleus (2Nu), if they are essential for plastid function. Some genes still present in the primary plastid genome could be transferred directly to the secondary host nucleus. Genes from the primary alga's nucleus that are not related to plastid function can be transferred to the secondary host, either replacing original homologs or adding novel functions to the host's metabolism. In addition, for as long as the secondary host was or remains phagotrophic, many additional "algal" genes could accumulate by more typical HGT from prey items. Finally, an unknown fraction of genes in the secondary host's genome are recovered with algal clades because of phylogenetic, tree-building artefacts. For more extensive reviews of eukaryotic EGT/HGT, see references [25,63]. B. Expectation if the secondary plastid is lost during the subsequent evolution of the algal genome shown in panel A. "Algal" genes directly related to plastid function are likely to accumulate null mutations and be lost, but many that were adapted to functions unrelated to photosynthesis should remain under strong purifying selection and be retained in the genome. These genes could represent a "footprint" of the past endosymbiosis, if they provide a significantly stronger phylogenetic signal than is expected from other known sources of tree-building conflicts, such as common HGT or phylogenetic artefacts. C. Panel shows a nucleus containing a comparable number of genes that cluster with algal sequences in phylogenetic analyses, but in this case most represent phylogenetic artefacts and none are from EGT. It is critical that investigations of EGT be designed to test explicitly among alternative, plausible explanations for the presence "algal" genes.

Figure 2

Testing specific predictions of competing hypotheses to explain conflicting gene trees. A. Patterned after an explicit test of the chromalveolate model of plastid evolution [43], the two hypothetical scenarios show mutually exclusive predictions about "algal" genes in a heterotrophic protist's genome based on two alternative evolutionary hypotheses. The upper scenario of a more ancient endosymbiosis predicts that genes from the secondary endosymbiont, those unrelated to plastid function, should be shared between the aplastidic heterotroph and its algal sister taxon. The lower scenario of a later, taxon-specific plastid origin, predicts that "algal" genes from the heterotroph are products of common HGT or phylogenetic artefacts and, therefore, should not be shared with the photosynthetic neighbor relative to a negative control (C^-). The control is a taxon generally agreed to be unrelated, phylogenetically or through endosymbiosis, to either the host or endosymbiont lineages. If the shared phylogenetic signal from the putative endosymbiont is not significantly greater than from the control group, then there is no objective basis for advancing EGT as an explanation for apparent "algal" genes in the heterotroph's genome. B. EGT versus HGT in a heterotrophic taxon. In this case, a rigorous test could be whether there are significantly more "algal" genes in the organism of interest than in phagotrophic control taxa with no presumed history of EGT. If there is not a significantly greater signal of HGT from the presumed endosymbiont in the target genome than in the control taxa, then algal genes are consistent with HGT or phylogenetic artefacts and EGT is not supported. C. The same approach could be used to test whether repetitive HGT is a superior hypothesis to phylogenetic artefacts by examining control taxa that should have had little to no opportunity to take up DNA from the organism in question, based on their presumed ecological and evolutionary histories. If it is biologically unreasonable to expect common products of HGT in the control genome (C^-), and there is comparable signal present as in the target genome (T), then HGT does not rise above the null hypothesis of signal from statistical biases and/or noise across genome-level data.