Inferring the evolutionary history of IncP-1 plasmids despite incongruence among backbone gene trees

Abstract

Plasmids of the incompatibility group IncP-1 can transfer and replicate in many genera of the Proteobacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental remediation. Although it is well understood that the accessory genes are transferred horizontally between plasmids, recent studies have also provided examples of recombination in the backbone genes of IncP-1 plasmids. As a consequence, phylogeny estimation based on backbone genes is expected to produce conflicting gene tree topologies. The main goal of this study was therefore to infer the evolutionary history of IncP-1 plasmids in the presence of both vertical and horizontal gene transfer. This was achieved by quantifying the incongruence among gene trees and attributing it to known causes such as 1) phylogenetic uncertainty, 2) coalescent stochasticity, and 3) horizontal inheritance. Topologies of gene trees exhibited more incongruence than could be attributed to phylogenetic uncertainty alone. Species-tree estimation using a Bayesian framework that takes coalescent stochasticity into account was well supported, but it differed slightly from the maximum-likelihood tree estimated by concatenation of backbone genes. After removal of the gene that demonstrated a signal of intergroup recombination, the concatenated tree was congruent with the species-tree estimate, which itself was robust to inclusion/exclusion of the recombinant gene. Thus, in spite of horizontal gene exchange both within and among IncP-1 subgroups, the backbone genome of these IncP-1 plasmids retains a detectable vertical evolutionary history.

Fig. 1.

Genetic map of a typical IncP-1 plasmid showing the different functional modules: region involved in initiating replication, composed of origin of replication (oriV), and replication initiation gene (trfA); trb, involved in mating bridge formation during conjugation; tra, involved in DNA processing for transfer during conjugation; ctl, also called central control region, is composed of regulatory genes and involved in maintaining plasmid stability; and accessory regions are composed of host-beneficial genes. Genes that were included in this study are colored dark gray. Numerals inside the circle indicate tree topologies that were shared by several genes (topologies 1–4) or unique to one gene (U); topologies were inferred in this study by maximum likelihood (fig. 2).

Fig. 2.

Cladograms showing four topologies produced by 21 gene trees. (A) Topology 1: supported by 46% of gene trees, namely, those of trfA2, trbA, trbC, trbG, traG, traH, traI, kfrA, kfrB, kfrC, korB, korA, and kleE. (B) Topology 2: supported by gene trees of trbD, trbK, and traJ. (C) Topology 3: supported by gene trees of trbF, trbI, and traE. (D) Topology 4: supported by gene trees of korC and klcA. Trees were rooted using IncP-1γ as outgroup.

Fig. 3.

ML tree of concatenated data estimated from a partitioned analysis based on codon position. Nodal support is shown as nonparametric ML bootstrap values. The tree was rooted using IncP-1γ as outgroup.

Fig. 4.

Congruence test for trbB. (A) ML tree for trbB, ML_trbB, (B) ML tree constrained to fit concatenated tree, ML_Hyp, (C) null distribution and test statistic δ (ln L[ML_hyp] − ln L[ML_trbB]) = 651.6. P value of obtaining a test statistic higher than 651.6 is <0.01. (D–F) Re-evaluated difference between ML_trbB and ML_hyp after removing 14 plasmids (from top to bottom in panel A: two IncP-1β plasmids pB3 and pAOVO02; all six IncP-1ε plasmids pAKD16, pAKD25, pEMT3, pHH128, pHH3414, and pKJK5; the IncP-1δ plasmid pAKD4; and IncP-1β plasmids pB4, pB12, pA1, pRSB222, and pYS1).

Fig. 5.

(A) Species tree estimated as maximum clade credibility tree by *BEAST. (B) Species tree estimated as maximum clade credibility tree by *BEAST after removal of recombinant gene traE. Nodal support is shown as posterior probabilities.