Results of tank experiment.

<p>(a) Chl <i>a</i> concentration per algae (in black) and (b) algae numbers (in gray), of <i>S. pistillata</i> subjected to six different treatments (chilling the tank water to 21°C, hitting the tank water to 29–30°C, feeding once a day with <i>Artemia nauplius</i>, filtration of sea water via 0.2 µm mash creating conditions of starvation, High PFD of ∼135 µmol quanta cm⁻² s⁻¹ and Low PFD of ∼20 µmol quanta cm⁻² s⁻¹), T0 represent a measurement taken on the day the fragments were plucked, “Ctrl” is the control and “Trans” are the fraq1agments from transplantation experiment. n = 5 fragments per each treatment, control treatment includes 10 fragments. Bars represent standard deviation.</p

Algae numbers and chlorophyll concentration.

<p>Algae numbers (black) and Chl <i>a</i> concentration per algae (gray) for <i>Symbiodinium sp.</i> within <i>S. pistillata</i> (a) Collected at 5 m depth during May and October (b) collected at 60 m, September and October represent bleached and partially bleached fragments; March and May are <u>not bleached</u>. Scales of both y-axes in figures (a) and (b) are similar for ease of comparison. n = 5 per each month in each depth, bars represent standard deviation.</p

Experiment setup.

<p>Black squares represent the two water tables, holding the eight experiment tanks and experiment systems.</p

Environmental gradients in the north Red Sea water column.

<p>The environmental parameters (a) temperature, (b) Chl <i>a</i>, (c) total organic nitrogen and (d) logarithm of PAR in the Gulf of Eilat. Parameters are plotted along a 140 m depth gradient, during March (red), May (green), August (blue) and November (brown). Dotted line highlights 60 m depth.</p

Photosynthesis and respiration rates for <i>S. pistillata</i> from 60 m.

<p>Chl <i>a</i> fluorescence yield (a) and O₂ production/consumption (b, c), plotted to light intensity (FI curve and PI curve, respectfully) for <i>S. pistillata</i> from 60 m. October and September represent bleached and partially bleached colonies, March and May are <u>not bleached</u>. A shallow corals plot is added for comparison. n = 5 fragments from different colonies per each line. Legend for the three plots is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084968#pone-0084968-g004" target="_blank">Figure 4(b)</a>. Bars represent standard deviation. (b) O₂ production/consumption plotted to light intensity (PI curve). Red rectangle includes PFD that is ecologically relevant for 60 m colonies and is enlarged in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084968#pone-0084968-g004" target="_blank">Fig. 4(c)</a>. (C) An enlargement of the low PFD area in the PI curve, relevant for 60 m. For better appreciation of the results, only selected fragments are presented.</p

Net daily O₂ production/consumption (µmol O₂ cm⁻² Day⁻¹) for shallow and mesophotic <i>S. pistillata</i> corals.

<p>Net daily O₂ production/consumption (µmol O₂ cm⁻² Day⁻¹) for shallow and mesophotic <i>S. pistillata</i> corals.</p

Common photosynthetic physiological parameters, presented for deep (60 m) bleached and unbleached and shallow (5 m) <i>S. pistillata</i> corals.

<p>Common photosynthetic physiological parameters, presented for deep (60 m) bleached and unbleached and shallow (5 m) <i>S. pistillata</i> corals.</p

Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

<div><p>We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent <i>Kaupichthys</i> eels (<i>Kaupichthys hyoproroides and Kaupichthys n</i>. <i>sp</i>.<i>)</i>, two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.</p></div

<i>Kaupichthys hyoproroides</i> collected in Bahamas and used in this study.

<p>A) White light; B) Green fluorescence.</p

Excitation/emission spectra of Chlopsid FP I and UnaG.

<p>Excitation/emission spectra of Chlopsid FP I and UnaG.</p