a workflow diagram describing the procedure of sequence retrieval , alignment , tree reconstruction , and sorting can be found in supplementary figure s6 ( supplementary material online ) . ( 2011 ) were used as query in a blastp search against protein sets from complete genomes and est datasets ( see supplementary table s3 , supplementary material online for the complete list of taxa included in the analysis ) . cdhit ( li and godzik 2006 ) was used to reduce redundancy within each protein dataset prior to blast in order to facilitate the subsequent tree interpretation by removing recent paralogs ( clustering threshold : 90% identity ) . the blast output was then parsed with a stringent e - value threshold of 1e-20 to minimize the inclusion of paralogs and hits were collected for each c. velia protein and multiple fasta files created . to prevent the inclusion of several closely related prokaryotic species , only the three best hits in each prokaryotic group mafft - linsi ( katoh et al . 2005 ) was used for aligning sequences and trimal ( capella - gutirrez et al . 2009 ) for selecting aligned positions , with sites containing more than 10% of gaps removed . raxml 7.2.8 ( stamatakis 2006 ) was used to reconstruct trees , with the lg substitution matrix  +  4  +  f evolutionary model and 100 bootstrap replicates . this approach resulted in 2,143 trees containing at least five species ( including c. velia ) . the pre - sorting of these trees was first done automatically with a text - parsing perl script used in chan , reyes - prieto , et al . ( 2011 ) , with the initial condition that c. velia be monophyletic with members of plants ( red algae , green algae , streptophytes , and/or glaucophytes ) and/or members of secondary plastid - bearing lineages of alveolates , stramenopiles , rhizaria , haptophytes and cryptophytes , and/or cyanobacteria ( supplementary table s3 , supplementary material online ) . an arbitrary bootstrap threshold of 80% was applied to restrict the sorting to trees with moderate to high statistical support . we also extended the condition to include the plastid - lacking stramenopiles ( oomycetes , blastocystis ) , alveolates ( ciliates ) , and rhizaria ( reticulomyxa filosa , gromia sphaerica , and paracercomonas longicauda ) to account for the prediction that endosymbioses might have occurred in their common ancestors , but found no additional trees . then , we manually scanned each tree for topologies consistent with egts and discarded the ones that did not contain at least c. velia , red and green algal representatives , and an outgroup . we used prokaryotic lineages as outgroup when possible , or alternatively members of animals , fungi , or amoebozoa . we also discarded trees with 10 taxa to reduce potential phylogenetic artifacts associated with poor taxon sampling ( which ultimately did not contribute to the differences between our results and those of woehle et al .  ) . in parallel , we also evaluated the extent of land plant contamination by pooling the trees showing c. velia nested within monocotyledons ( bootstrap support 80% ) . finally , we monitored the alveolate and prokaryotic signals from the remaining 1,781 trees by searching for exclusive monophyletic grouping including c. velia and apicomplexans , dinoflagellates and/or ciliates , and c. velia and prokaryotes ( bootstrap support    80% ) . supplementary tables s1s3 , figures s1s6 and supplementary materials are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .
the transition from endosymbiont to organelle in eukaryotic cells involves the transfer of significant numbers of genes to the host genomes , a process known as endosymbiotic gene transfer ( egt ) . in the case of plastid organelles , egts have been shown to leave a footprint in the nuclear genome that can be indicative of ancient photosynthetic activity in present - day plastid - lacking organisms , or even hint at the existence of cryptic plastids . here 
 , we evaluated the impact of egt on eukaryote genomes by reanalyzing the recently published est dataset for chromera velia , an interesting test case of a photosynthetic alga closely related to apicomplexan parasites . 
 previously , 513 genes were reported to originate from red and green algae in a 1:1 ratio . 
 in contrast , by manually inspecting newly generated trees indicating putative algal ancestry , we recovered only 51 genes congruent with egt , of which 23 and 9 were of red and green algal origin , respectively , whereas 19 were ambiguous regarding the algal provenance . 
 our approach also uncovered 109 genes that branched within a monocot angiosperm clade , most likely representing a contamination . 
 we emphasize the lack of congruence and the subjectivity resulting from independent phylogenomic screens for egt , which appear to call for extreme caution when drawing conclusions for major evolutionary events .