Phylogeny and fitness of Vibrio fischeri from the light organs of Euprymna scolopes in two Oahu, Hawaii populations

Abstract

The evolutionary relationship among Vibrio fischeri isolates obtained from the light organs of Euprymna scolopes collected around Oahu, Hawaii, were examined in this study. Phylogenetic reconstructions based on a concatenation of fragments of four housekeeping loci (recA, mdh, katA, pyrC) identified one monophyletic group ('Group-A') of V. fischeri from Oahu. Group-A V. fischeri strains could also be identified by a single DNA fingerprint type. V. fischeri strains with this fingerprint type had been observed to be at a significantly higher abundance than other strains in the light organs of adult squid collected from Maunalua Bay, Oahu, in 2005. We hypothesized that these previous observations might be related to a growth/survival advantage of the Group-A strains in the Maunalua Bay environments. Competition experiments between Group-A strains and non-Group-A strains demonstrated an advantage of the former in colonizing juvenile Maunalua Bay hosts. Growth and survival assays in Maunalua Bay seawater microcosms revealed a reduced fitness of Group-A strains relative to non-Group-A strains. From these results, we hypothesize that there may exist trade-offs between growth in the light organ and in seawater environments for local V. fischeri strains from Oahu. Alternatively, Group-A V. fischeri may represent an example of rapid, evolutionarily significant, specialization of a horizontally transmitted symbiont to a local host population.

Figure 1

Geographic distribution of the V. fischeri strains used in this study. The majority of these strains were sampled from locations in the Pacific Ocean, specifically the Hawaiian Island of Oahu (inset). The shapes next to strain names denote the strains' ecological habitat: Filled black circle, planktonic (seawater); triangle or square or star, light-organ symbiont. The triangle, square and star fill colors represent host organisms as follows: White triangle, C. gloriamarus; black triangle, M. japonicus; white square, E. scolopes; gray square, E. tasmanica; black square, E. morsei; white star, S. affinis; black star, S. robusta. The bars in the main figure and inset represent 2000 and 20 km, respectively. The background continent and Oahu outlines were taken from maps made available freely at http://d-maps.com/.

Figure 2

V. fischeri VfRep-PCR type-I strains form a monophyletic group within a phylogenetic reconstruction of global V. fischeri. A consensus network inferred from 6000 phylograms produced by ClonalFrame analyses of all taxa using a concatenation of four loci: recA, mdh, katA and pyrC. ClonalFrame was used to infer genealogical relationships from posterior distributions of 2000 samples from three independent runs (3 × 2000=6000 total samples). This aggregate ClonalFrame posterior distribution was used by SplitsTree to build a consensus network. For this network, reticulate relationships found in at least 20% of the sample may be visualized by parallel branches in parallelograms; nodes supported by a posterior probability greater than 95% (as summarized by ClonalFrame) are at the terminus of red-colored branches. The root (dashed red branch with arrow) has been truncated to expand the branches of the main V. fischeri group; the root contains both outgroup taxa: V. salmonicida LFI1238 and V. logei SA6. VfRep-PCR patterns have been mapped onto E. scolopes symbionts with colors: identically colored strains all share the same VfRep-PCR pattern (or type) (Supplementary Table S1): Blue, type-I (boxed); orange, type-II; green, type-III. The strains within the dotted box have been designated as ‘Group-A'. The black bar is equivalent to a branch length of 0.1 substitutions per site.

Figure 3

Dual-strain, juvenile E. scolopes experiments competing single V. fischeri ‘Group-A' strains against either (a) MB14A3 or (b) MB15A4. In both graphs, each row represents a single experiment, summarized by the box on the left as follows: First line, strains' identities; second line, initial mean concentration of each strain in c.f.u./ml (the s.d. was less than 50% of this mean value for all experiments); third line, duration of inoculation. All experiments were concluded at 48 h after hatching and involved 9–15 juvenile squid born from mating adults collected from Maunalua Bay, Hawaii. The unfilled circles represent individual squid that were found to contain only one of the strains (that is, the other strain was below the limit of detection—approximately 500 times less abundant than the strain present). The unfilled circles are arbitrarily positioned at 1.25 and −1.25 units on each axis. Each filled circle represents an individual squid that contained both strains, and is positioned on the graph as the logarithm of the RCI (logRCI) for the two strains. Filled circles with a logRCI value greater than 1.25 or less than −1.25 are placed either to the right or to the left of these values (and unfilled circles), respectively, because of graphical limitations.

Figure 4

Single-strain V. fischeri inoculations of juvenile squid demonstrated no clear difference between the levels of symbiosis initiation by Group-A and non-Group-A V. fischeri. Each bar represents the mean c.f.u./squid of 10–15 squid inoculated with a single isolate of V. fischeri and sacrificed at 24, 48 or 72 h after inoculation. The error bars represent 95% confidence intervals. The histogram bars relate to V. fischeri strains as follows: Non-'Group-A' V. fischeri strains are represented by the three black bars (left, ES114; center, MB14A3; right, MB15A4), whereas ‘Group-A' V. fischeri strains are represented by the three white bars (left, ES213; center, MB11B1; right, KB2B1).

Figure 5

Single-strain V. fischeri growth experiments in microcosms filled with either (a) 0.22-μm-filtered or (b) unfiltered fresh seawater from Maunalua Bay, Hawaii. In both graphs, each bar represents the mean c.f.u./ml of three biological replicates of a single strain, with error bars indicating the s.e.m. The histogram bars relate to V. fischeri strains as follows: Non-'Group-A' V. fischeri are represented by the three black bars at each time point (left, ES114; center, MB14A3; right, MB15A4), whereas ‘Group-A' V. fischeri are represented by the white bars at each time point (left, ES213; center, MB11B1; right, KB2B1). In the unfiltered seawater data (b), the mean abundance values that are indicated at 3 c.f.u. ml⁻¹ for the 48- to 96-h time points were below this limit of detection.