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COMMENTARY MobileGeneticElements2:1,59–62;January/February2012;G2012LandesBioscience Phylogenomic approaches underestimate eukaryotic gene transfer Jan O. Andersson DepartmentofCellandMolecularBiology;ScienceforLifeLaboratory;UppsalaUniversity;Uppsala,Sweden Phylogenomic approaches have shown candidates are typically reported from that eukaryotes acquire genes via eukaryotic genome projects,4,5 whereas gene transfer. However, there are two directed studies suggest gene transfer to fundamental problems for most of these be important in the adaptation process of analyses; only transfers from prokaryotes eukaryotes.6-8 This is an intriguing incon- areanalyzedandthescreeningprocedures gruity. To understand these differences © 2012 Laappliend assudme theat gense tran sfeBr is rarie oit issusefucl to iconesider nthe asscumpetions . for eukaryotes. Directed studies of the applied in the screening procedures in the impact of gene transfer on diverse phylogenomic approaches used in genome eukaryoticlineagesproduceamuchmore projects (referred to as ‘traditional phylo- complex picture. Many gene families are genomicapproaches’herein)andhowwell affected by multiple transfer events from they match the knowledge we currently prokaryotes to eukaryotes, and transfers have from more directed studies of Do not distribute. between eukaryotic lineages are routinely eukaryote gene transfer. Here I will argue detected. This suggests that the assump- that the match is really poor leading to a tionsappliedintraditionalphylogenomic high number of false negatives. approaches are too naïve and result in Studies of gene transfers in eukaryotes many false negatives. This issue was using phylogenomic methods typically recently addressed by identifying and identify eukaryotic proteins with high analyzing the evolutionary history of sequence similarity to a prokaryotic pro- 49 patchily distributed proteins shared tein, but with no or significantly weaker betweenDictyostelium and bacteria. The similarity to any eukaryotic protein.4,5 vast majority of these gene families This is indeed a strong indication of a showed strong indications of gene trans- gene transfer event. The problem is that fers, both between and within the three thesetraditionalphylogenomicapproaches domains of life. However, only one of only identifies protein families in which a these was previously reported as a gene single transfer has occurred between a transfer candidate using a traditional prokaryote and a eukaryote, which prob- phylogenomic approach. This clearly ably is a rarity. Comparative genomics illustratesthatmore realisticassumptions studies of prokaryotes have shown that are urgently needed in genome-wide most protein families are patchily distri- studies of eukaryotic gene transfer. buted;theyareabsentfromasmallorlarge Keywords: protists, phylogenetics, lateral fraction of the genomes (Fig.1).9 These gene transfer, horizontal gene transfer, genes are distributed via gene transfer and genome evolution, adaptation, Transfer of genetic material between provide diversifying functions and niche diversification different organismal lineages is important adaptation to the recipients. Eukaryote Submitted: 01/20/12 in prokaryote evolution. Studies of single genome evolution, on the other hand, has gene families as well as phylogenomic been viewed as mainly influenced by Revised: 02/06/12 studies in the last decade have shown genome expansion and a few major endo- Accepted: 02/08/12 that also eukaryotes are affected by this symbiotic events. However, there are data http://dx.doi.org/10.4161/mge.19668 evolutionary mechanism.1-3 However, the suggesting that gene transfer of patchily importanceoftheprocessisstilluncertain; distributed proteins is important for the Correspondenceto:JanO.Andersson; Email:[email protected] only modest numbers of gene transfer diversificationprocessalsoforeukaryotes.1-3 www.landesbioscience.com MobileGeneticElements 59 Figure1.Patchilydistributedproteinsaredistributedviagenetransfer.(A)Comparativegenomicsofprokaryoteshaveidentifiedthreelooselydefined groupsofgenefamiliesbasedontheirfrequenciesingenomes:extendedcore,characterandaccessorygenes.9Coregenesencodesharedfunction betweenorganisms,charactergenesfunctionsthatdistinguishmajorgroupsandaccessorygenesfunctionsuniquetoafeworganisms.Theevolutionary modediffersbetweenthegroups.Coregenesareverticallyinheritedandusedfororganismalphylogenies,whereascharacterandaccessorygenes probablyaremoreinfluencedbygenetransfer;noneofthegroupsshouldbeviewedasrepresentativesoftheevolutionofthewholegenome. Theevolutionoftheaccessoryandcharactergeneswerestudiedbyidentifying49patchilydistributedproteinfamiliespresentinthecellularslimemold Dictyosteliumdiscoideumandbacteria.8(B)Themaximumlikelihoodphylogenyofaconservedhypotheticalproteinidentifiedinthestudy.Eukaryotes areshownincolorandprokaryotesinblack.Distantlyrelatedeukaryotesarefoundintermixedwithprokaryoticsequences,suggestiveofmultiple transferevents.8Thefigureisadaptedfromreferences8and9. © 2012 Landes Bioscience. Here I will review some recent findings studies have suggested that gene transfer approachtosearchforgenesofalgalorigin of adaptation by gene acquisition in contributedtothesimilaritiesbetweenthe in the genome of Monosiga brevicollis, a eukaryotes obtained by the usage of groups.10,11 phagotrophicunicellular choanoflagellate.6 phylogenomic approaches for the study Richards and coworkers studied this They reconstructed phylogenetic trees for ofgenetransfersbetweenspecificeukaryo- phenomenonfurther.7Theycouldidentify all genes in the genome. Using realistic tic groups,6,7 and one attempt to use a dozens of gene transfers between the filtering criteria they were able to identify Do not distribute. novel approach to study patchily distri- groups using a wide range of genomes 103 genes with strong support for algal buted proteins.8 With an increased under- from both groups together with clustering origin, mostly from haptophytes, diatoms standing of the evolutionary dynamics of and phylogenetic methods. Interestingly, and green algae. This could be the result all classes of gene families (Fig.1) we can all transfers except one were reported of repeated transfer of genes from food; apply realistic assumptions to large-scale to have occurred in the direction from choanoflagellates feed on bacteria and studies of eukaryotic gene transfer. fungi to oomycetes. Many of the trans- other eukaryotes. Alternatively, or rather ferred genes encode secreted decomposing in addition, the genes could have been Directed Studies Identify Gene enzymes and were specifically acquired by introducedfromapastalgalendosymbiont Sharing Leading to Adaptation plant-tissue colonizing oomycetes. These in the lineage leading to Monosiga.6 resultsshow thatoomycetemostlikelyare Interestingly, a quarter of the identified Many of the most devastating diseases in more recent plant pathogens than fungi genes appeared to first have been trans- plants are caused by fungi or oomycetes. andthattransferofgeneticmaterialfroma ferred from bacteria to a eukaryotic alga, These are two distantly related groups of distantly related eukaryotic group have and then secondarilytochoanoflagellates.6 eukaryotes that have similar lifestyles. played an important role in evolution of However, such a bacterial origin would Bothfeedby osmotrophy. The cells secret their pathogenic lifestyle.7 These fascin- not have been detected using a typical enzymes that decompose organic matter atingresultswouldnothavebeenobtained phylogenomicapproachsincethestrongest and the metabolites are imported into the with a traditional phylogenomic approach sequence similarity would be to algal gene cell.Thesimilarityinlifestylebetweenthe in which genes with strong sequence of bacterial origin. Functions in amino groups is an example of convergent similarities to other eukaryotes would acid and carbohydrate metabolism domi- evolution. Fungi are more closely related have been assumed to be present in the nated among the gene transfer candidates, to animals than to oomycetes, whereas common eukaryotic ancestor. indicatingthatthesechoanoflagellateshave diatoms, a group of photosynthetic algae, Studies of gene transfer are indeed able adapted by acquisition of algal genes that are a sister group to oomycetes. Absence to shed light on the diversification process expand their metabolic repertoire. of phagotrophy has been assumed to be of eukaryotes. Animals and fungi are both a barrier to gene transfer. Indeed, the members of Opisthokonta. No photo- A Novel Approach to Study oomycete and fungi genomes are not synthetic member has been identified in Patchily Distributed Proteins among the genomes for which traditional this group. Choanoflagellates are a group phylogenomic studies have indicated a of free-living microbial eukaryotes which The two examples outline above test well- significant role of gene transfer in eukar- aretheclosestrelativestoanimals.Sunand defined hypotheses about gene transfers yotes. Nevertheless, targeted evolutionary coworkers used a directed phylogenomic by using directed phylogenomic methods 60 MobileGeneticElements Volume2Issue1 in combination with careful filtering both free-living heterotrophs that can be as gene transfer candidates.4 This conser- and interpretation of the results. They found in soil and they both undergo cell vativeapproachisunlikelytopickupfalse are very powerful to characterize the role differentiation under certain conditions. positives, but will be very prone to false of gene transfers between distantly related However, they are distantly related negatives.Genesacquiredviagenetransfer eukaryotic groups in the adaptation of eukaryotes classified within two different in two or more different eukaryotes are eukaryotes. However, one disadvantage supergroups: Amoebozoa and Excavata. excluded, as are any genes without is that the approach relies on existing There exist at least two alternative sufficient sampling among prokaryotes knowledgeofthebiologyoftheorganisms; plausible explanations for this striking to be included in Pfam. Similarly, the if only transfers between two organismal gene-sharing pattern. These genes were N. gruberi gene set was screened with groups are addressed important contribu- present in the common ancestor of the similarity searches, and genes with signi- tions from other groups may be missed in Dictyostelium and Naegleria and distri- ficant similarity only to prokaryotes were such phylogenomic approaches. In addi- buted in eukaryotes strictly by vertical considered as gene transfer candidates.5 tion, these kinds of studies can only inheritance.8Inlineagesthathavedifferent Again, gene families with repeated estimate minimal number of transfers lifestyles (i.e., parasites) the genes have transfers are missed in the screen and between the groups analyzed; the number become obsolete and lost over evolution- eukaryote-to-eukaryote transfers are not of false negatives may be large. To ary time. Alternatively, the genes have evenconsidered.Thetruenumberofgene circumvent these problems I applied an been distributed via gene transfer in more families in these microbial eukaryotes are alternative approach to study these issues. recent evolutionary timescales providing likelymuch larger than hasbeenreported. I first identified patchily distributed selective advantage to the recipient These discrepancies should not be ©protei ns 2becaus0e thes1e are 2expecte dLto alineagnes. Phdylogeneetic ansalyses wBere peir- osurprsising cfromiaebiolongical vciewpeoint. . be enriched with gene transfer events formed on all protein families to distin- Microbesliveinsteadilychangingenviron- (Fig.1), instead of screening for unexpec- guish between these alternatives. The ments. Ecosystems are inhabited by dis- ted sequence similarities. Then I per- results were striking. The vast majority tantly related organisms which have formed phylogenetic analyses for each of the phylogenetic trees showed strong adapted to its specific condition. The identified gene family to evaluate whether indicationsoflateralgenetransferbetween spread of patchily distributed genes are the patchy distribution was a consequence prokaryotes and eukaryotes and within part of this adaptation process, and there Do not distribute. of gene transfer, or differential loss in the eukaryotes.8 Figure1B shows an example is no reason to assume that microbial eukaryotic domain.8 of an individual gene tree. The exact eukaryotes do not take part of this flux The soil-dwelling cellular slime mold details of the transfer events could in of genetic material (Fig.1).1,2,6-8 For D. discoideum was selected in the case many cases not be traced, because the example, if a gene provide the ability to study for two reasons: an active research density of organismal sampling was too utilize a carbon compound present in the community have produced a high quality low. Nevertheless, there are no strong environment it is likely to spread to annotation of the genome sequence indications that any of the proteins have different microbes in the environment (http://dictybase.org/),andonly18poten- evolved solely via vertical inheritance previously lacking this ability (provided tial gene transfers were reported in the and gene loss; gene transfer has likely that there are mechanisms in action). The original publication.8 I identified 49 affected all patchily distributed genes assumption that a gene has a vertical protein families in the Dictyostelium families identified in the analysis to some eukaryotic history is violated as soon as genome which were shared with at least extent.8 two eukaryotic lineages inhabit a similar one prokaryotic species, but only a environment and acquire their copy of limited number of other eukaryotes and Traditional Phylogenomic Studies a particular gene family independently prokaryotes (Fig.1). The evolutionary have Drastically Underestimated during the adaptation process. The tradi- history of these patchily distributed the Amount of Gene Transfer tional phylogenomic approaches will fail families were analyzed further.8 For seven to identify members of such protein of the families there were no eukaryotic Only a single protein among the 49 familiesasgenetransfercandidatesbecause sequences except the Dictyostelium identified as patchily distributed was they assume that vertical inheritance is sequences. The remaining 42 families among the 18 gene transfer candidates thenormforallproteinfamilieswithgene contained sequences from one or more in the original D. discoideum genome transfer events as very rare exceptions. eukaryotic species outside the Dictyo- publication,4 and very few were among However,thisisprobablyonlythecasefor stelium genus. The closest relative with a the 184 lateral gene transfer candidates universal core genes, and certainly not completelysequencedgenome,thehuman reported from N. gruberi.5 This may be for patchily distributed proteins (Fig.1).8 parasite E. histolytica, was represented surprising, but is logical if the details of Traditional phylogenomic approaches in only two families. In contrast, the the methods applied are considered. probably only have scratched the surface amoeboflagellate Naegleria gruberi had a Dictyostelium genes with significant of the gene transfer events and thereby representative in 25 of the families. similarity to a bacterial-specific Pfam drastically underestimated the impact Dictyostelium and Naegleria are having domain and only present in Dictyo- of the process on eukaryotic genome somewhat overlapping lifestyles, they are steliumamongeukaryoteswereconsidered evolution. www.landesbioscience.com MobileGeneticElements 61 References 6. 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