Recombination Does Not Hinder Formation or Detection of Ecological Species of Synechococcus Inhabiting a Hot Spring Cyanobacterial Mat

Abstract

Recent studies of bacterial speciation have claimed to support the biological species concept-that reduced recombination is required for bacterial populations to diverge into species. This conclusion has been reached from the discovery that ecologically distinct clades show lower rates of recombination than that which occurs among closest relatives. However, these previous studies did not attempt to determine whether the more-rapidly recombining close relatives within the clades studied may also have diversified ecologically, without benefit of sexual isolation. Here we have measured the impact of recombination on ecological diversification within and between two ecologically distinct clades (A and B') of Synechococcus in a hot spring microbial mat in Yellowstone National Park, using a cultivation-free, multi-locus approach. Bacterial artificial chromosome (BAC) libraries were constructed from mat samples collected at 60°C and 65°C. Analysis of multiple linked loci near Synechococcus 16S rRNA genes showed little evidence of recombination between the A and B' lineages, but a record of recombination was apparent within each lineage. Recombination and mutation rates within each lineage were of similar magnitude, but recombination had a somewhat greater impact on sequence diversity than mutation, as also seen in many other bacteria and archaea. Despite recombination within the A and B' lineages, there was evidence of ecological diversification within each lineage. The algorithm Ecotype Simulation identified sequence clusters consistent with ecologically distinct populations (ecotypes), and several hypothesized ecotypes were distinct in their habitat associations and in their adaptations to different microenvironments. We conclude that sexual isolation is more likely to follow ecological divergence than to precede it. Thus, an ecology-based model of speciation appears more appropriate than the biological species concept for bacterial and archaeal diversification.

Keywords: Ecotype Simulation; Synechococcus; cyanobacteria; ecotype; multi-locus sequence typing; population genetics; recombination; speciation.

Figure 1

Positional analysis relative to the Synechococcus strain A genome of jointly recruited syntenous (red line joins end sequences) and jointly recruited non-syntenous (blue) end sequences of 60°C BAC clones that are (A) recruited by this genome, and (B) contain an A-like Synechococcus 16S rRNA region, and (C) have anti-normal long end-sequence mate pairs. In (C) percent nucleotide identity of recruited sequences with genomic homologs is also plotted and lines connect mate-pairs.

Figure 2

A-like Synechococcus phylogenies based on maximum likelihood analysis of (A) 7 concatenated BAC loci and (B) BAC-associated rbsK sequences. Putative ecotypes (PEs) demarcated by Ecotype Simulation are indicated by brackets or vertical bars adjacent to each tree. STs within PEs are colored according to the MLSA phylogeny shown in A and colors are maintained in both phylogenies (color correspondence also maintained in Supplemental Data Presentation Figure 14). The number of variants belonging to dominant and subdominant sequence types (STs) is indicated in parentheses. Stars, colored coded according to gene (see inset star legend), demarcate recombination events interpreted from SNP patterns confirmed (closed stars) or not confirmed (open stars) by Clonal Frame analysis. Clade splitting events between rbsK and the concatenated phylogeny, where grouped variants within rbsK are split apart into separate PEs in the concatenated phylogeny, are indicated by dashed (clade splitting event of rbsK PE1) and solid arrows (clade splitting event of rbsK PE2). Non-syntenous STs are shaded in gray and STs that contained a combination of sequences that were syntenous and non-syntenous are indicated by an asterisk; syntenous STs are not shaded and are not annotated by an asterisk. Shared SNP pattern between STs of two distinct PEs indicated by a bidirectional arrow colored according to gene (yellow = lepB). Bootstrap values are provided for major nodes. Reference genome indicated by “SynA”; Genbank accession number CP000239.

Figure 3

rbsK vertical distributions in the 63–65°C Mushroom Spring microbial mat of dominant rbsK and psaA variants associated with Synechococcus putative ecotypes (PE) demarcated from multi-locus sequence analyses (MLSA) by Ecotype Simulation, shown as colored solid lines. (A) rbsK distributions of two MLSA PEs that are demarcated as a single PE in rbsK analysis. (B,C) rbsK (solid line) and psaA (dotted line) distributions of two MLSA PEs connected through genomes of representative isolates. Bars represent standard error (n = 3). PE colors correspond across panels.

Figure 4

eBURST population snapshot of A-like Synechococcus BACs for the 7-locus analysis shown in Figure 2A. Clonal complexes enclosed by solid black lines. PE demarcation from Ecotype Simulation analysis are overlaid, using different colors corresponding to Figure 2A to represent distinct PEs. STs are represented by numbers and those colored in gray belong to PEs demarcated from a single sequence with a unique ST. Reference genome indicated by “SynA” next to ST10.

Figure 5

Single nucleotide polymorphism patterning of A-like Synechococcus BACs grouped around the same dominant variant (also consensus sequence) by Ecotype Simulation and eBURST. Variants detected only by eBURST (blue), only by Ecotype Simulation (red) or by both (purple) are compared to the shared dominant variant (consensus sequence). (A) Dominant variant ST1 in PEA7 and clonal complex A7-I and (B) subdominant variant ST6 in PE7 and clonal complex A7-III. STs correspond with STs in Figure 2A.