Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns

Abstract

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

Figure 1. Maximum likelihood (ML) consensus tree from concatenated (rrs + gyrB + rpoB + rpoD) sequences of nectar-inhabiting Pseudomonas isolates.

Bootstrap percentages from ML analysis are shown above lines, and clades with Bayesian posterior probabilities ≥0.9 are indicated by thick brown lines. The small phylogram is included to illustrate branch length heterogeneity (scale bar = 0.1 nucleotide substitutions per site). The geographic origin of isolates is shown on the leaves: Mediterranean, filled squares; South African, empty squares. Plant hosts and their corresponding families are also indicated, with abbreviations for the latter shown in parentheses. Insect pollinators associated to each plant species are listed in Table S4. Abbreviations for plant species names: AA: Adhatoda andromeda; AO: Ajuga ophrydis; CA, Convolvulus althaeoides; CD: Cycnium adonense; DC: Disa crassicornis; ED: Eriosema distinctum; EG: Echium gaditanum; FL: Fritillaria lusitanica; GI: Gladiolus illyricus; MG: Moraea graminicola; NP: Narcissus papyraceus; OR, Orobanche ramosa; PW, Protea welwitschii; RC, Ruellia cordata. Abbreviations for plant families: Aca, Acanthaceae; Ama, Amaryllidaceae; Bor, Boraginaceae; Con, Convolvulaceae; Fab, Fabaceae; Iri, Iridaceae; Lam, Lamiaceae; Lil: Liliaceae; Orc, Orchidaceae; Oro, Orobanchaceae; Pro, Proteaceae.

Figure 2. Unrooted neighbour-joining (NJ) consensus tree, based on phylogenetic analysis of concatenated (rrs + gyrB + rpoB + rpoD) sequences, displaying the relationships of nectar-inhabiting isolates (shown as red filled circles [nectar group (NG) 1], blue filled triangles [NG 2] or violet filled squares [NG 3]) and reference strains of Pseudomonas ‘sensu stricto’ species (shown as numbers, see below).

Nectar isolates belonging to subgroup NG 2′ are shown within a dotted circle. Evolutionary distances were computed using the Jukes-Cantor method and are in the units of number of base substitutions per site. Cellvibrio japonicum Ueda107^T was used as an outgroup. A complete version of this figure is provided as supporting information (Figure S3). Numeric codes for reference strains shown in the tree: 1, P. aeruginosa DSM 50071^T; 2, P. agarici LMG 2112^T; 3, P.alcaliphila AL15-21^T; 4, P. anguilliseptica NCIMB 1949^T; 5, P. asplenii ATCC 23835^T; 6, P. azotoformans IAM 1603^T; 7, P. cannabina CFBP 2341^T; 8, P. chlororaphis DSM 50083^T; 9, P. corrugata ATCC 29736^T; 10, P. extremorientalis KMM 3447^T; 11, P. flavescens B62^T; 12, P. fluorescens IAM 12022^T; 13, P. fragi ATCC 4973^T; 14, P. gessardii CIP 105469^T; 15, P. guineae LMG 24016^T; 16, P. indica IMT37^T; 17, P. jessenii CIP 105274^T; 18, P. koreensis KACC 10848^T; 19, P. lutea OK2^T; 20, P. mediterranea CFBP 5447^T; 21, P. oleovorans IAM 1508^T; 22, P. oryzihabitans IAM 1568^T; 23, P. pachastrellae KMM 330^T; 24, P. palleroniana CFBP 4389^T; 25, P. pertucinogena IFO 14163^T; 26, P. poae DSM 14936^T; 27, P. psychrotolerans C36^T; 28, P. putida IAM 1236^T; 29, P. rhizosphaerae IH5^T; 30, P. straminea IAM 1598^T; 31, P. stutzeri ATCC 17588^T; 32, P. syringae NCPPB 281^T; 33, P. tolaasii LMG 2342^T; 34, P. xanthomarina KMM 1447^T.