Light Capture and Pigment Diversity in Marine and Freshwater Cryptophytes1
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J. Phycol. *, ***–*** (2019) © 2018 Phycological Society of America DOI: 10.1111/jpy.12816 LIGHT CAPTURE AND PIGMENT DIVERSITY IN MARINE AND FRESHWATER CRYPTOPHYTES1 Brady R. Cunningham School of the Earth, Ocean & Environment, University of South Carolina, Columbia, South Carolina 29208, USA Matthew J. Greenwold Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA Eric M. Lachenmyer, Kristin M. Heidenreich, Abigail C. Davis School of the Earth, Ocean & Environment, University of South Carolina, Columbia, South Carolina 29208, USA Jeffry L. Dudycha Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA and Tammi L. Richardson 2 School of the Earth, Ocean & Environment, University of South Carolina, Columbia, South Carolina 29208, USA Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA Phenotypic traits associated with light capture the PBPs. Pigment data showed evidence of trade- + and phylogenetic relationships were characterized in offs in investments in PBPs vs. chlorophylls (a c2). 34 strains of diversely pigmented marine and Key index words: absorption; LSU rDNA; phycobilins; freshwater cryptophytes. Nuclear SSU and partial phycobiliproteins; phylogeny; pigments; PUR; spec- LSU rDNA sequence data from 33 of these strains tral irradiance plus an additional 66 strains produced a con- catenated rooted maximum likelihood tree that Abbreviations: CTAB, cetyltrimethyl ammonium classified the strains into 7 distinct clades. Molecular bromide; PBP, phycobiliprotein and phenotypic data together support: (i) the reclassification of Cryptomonas irregularis NIES 698 to the genus Rhodomonas, (ii) revision of phy- Cryptophytes are a widespread yet poorly charac- cobiliprotein (PBP) diversity within the genus terized group of unicellular eukaryotic algae that Hemiselmis to include cryptophyte phycocyanin (Cr- inhabit ponds, lakes, estuaries, and oceans (Klave- PC) 569, (iii) the inclusion of previously unidentified ness 1988, Hoef-Emden 2008, Hoef-Emden and strain CCMP 2293 into the genus Falcomonas, even Archibald 2016). Cryptophytes originated via sec- though it contains cryptophyte phycoerythrin 545 (Cr- ondary endosymbiosis (engulfment of a red algal PE 545), and (iv) the inclusion of previously cell by an unknown eukaryote), which led to the unidentified strain CCMP 3175, which contains Cr-PE evolution of pigments that are unique among algae 545, in a clade with PC-containing Chroomonas (Archibald and Keeling 2002, Bhattacharya et al. species. A discriminant analysis-based model of 2004, Gould et al. 2008). Cryptophytes are thus group membership correctly predicted 70.6% of the complex with respect to their genetic and cellular À clades using three traits: PBP concentration · cell 1, structure, and are distinctive in their pigmentation the wavelength of PBP maximal absorption, and and the organization of these pigments within the habitat. Non-PBP pigments (alloxanthin, chl-a, chl-c2, plastids (Gould et al. 2007). a-carotene) did not contribute significantly to group Visually, cryptophytes vary markedly in color classification, indicating the potential plasticity of (Fig. S1 in the Supporting Information). These vari- these pigments and the evolutionary conservation of ations arise from differences in the type of phyco- biliprotein (PBP) produced by the cell. Cryptophyte strains contain a single type of phycoerythrin or phycocyanin, but not both (Hill and Rowan 1989). 1 Received 18 September 2017. Accepted 22 October 2018. First Eight types of cryptophyte PBPs have been defined: Published Online 22 November 2018. 2Author for correspondence: e-mail [email protected]. three cryptophyte phycoerythrins (Cr-PE) (Cr-PE Editorial Responsibility: J. Collier (Associate Editor) 545, Cr-PE 555, and Cr-PE 566) and five cryptophyte [Correction added on March 22, 2019, after first online publica- phycocyanins (Cr-PC) (Cr-PC 569, Cr-PC 577, Cr-PC tion: Figures 4 and 5 updated and Hanusia replaced by Hemiselmis 615, Cr-PC 630, and Cr-PC 645), all of which are in two spots in Discussion section] 1 2 BRADY R. CUNNINGHAM ET AL. named according to the approximate wavelength of scanned from 400 to 800 nm at 1 nm intervals. Spectra were maximum absorption of the PBP (Hill and Rowan scatter-corrected by subtracting the average absorbance 1989, Hoef-Emden and Archibald 2016). In part, between 730 and 750 nm from the spectrum (Shibata 1958). Scattering was minimized by ensuring that each filter was taxonomic classification to genus level is related to fully loaded with material (Roesler 1998). Log10 absorbance the type of PBP. For example, all known Rhodomonas values were then converted to chl-a -specific absorption coeffi- species contain Cr-PE 545. However, PBP type can- cients (aChl) according to: not be used solely to classify cryptophytes to genus (Hill and Rowan 1989). Strains containing Cr-PE 2:303 Á AðkÞ aChlðkÞ¼ ; ð1Þ 545, for example, may belong to one of several gen- L Á b Á N era including Rhodomonas, Geminigera, Guillardia,or k = = Proteomonas (Hoef-Emden 2008, Hoef-Emden and where A( ) the wavelength-dependent absorbance, L the optical path length of the particles on the filter (i.e., sample Archibald 2016). Researchers now usually combine volume divided by clearance area of the filter), N = the con- information on strain-specific PBP type with cellular centration of chl-a in the culture, and b = the path length ultrastructure and molecular data to classify crypto- amplification factor. Because the filters had a high particle phytes to genus and species (Hill and Rowan 1989, load, a b correction factor of 2.0 was used (Roesler 1998). Photosynthetically usable radiation. Photosynthetically usable Hoef-Emden and Melkonian 2003, Hoef-Emden À À 2008, Hoef-Emden and Archibald 2016). radiation (in lmol photons Á m 2 Á s 1) was calculated for We measured phenotypic traits related to light each cryptophyte strain as: Z absorption (PBP type, PBP concentrations, non-PBP 200 pigment concentrations, whole cell absorption spec- PUR ¼ PARðkÞAðkÞdk; ð2Þ tra, photosynthetically usable radiation (PUR), and 400 cell volume) for 34 strains of cryptophytes. These traits where the weighting function AðkÞ represents the probability allowed us to assess aspects of phenotypic diversity that a photon with a given wavelength is absorbed by the cell related to light capture. We constructed molecular (Morel 1978). It is derived from the absorption spectrum aChl(k), measured as described above, normalized to its maxi- phylogenies using SSU and LSU rDNA data from 33 of Chl our strains and sequences downloaded from the mum absorption (a max) in the full spectrum light environ- ment of the culture incubators. A flame spectrometer (Ocean National Center for Biotechnology Information Optics, Dunedin, FL, USA) was used to measure the spectral (NCBI) for 66 others. Then, recognizing that light distribution of available irradiance in the incubators (400– absorption likely has genetic roots, we used discrimi- 700 nm at 1 nm intervals) just outside of culture containers. nant analysis to determine which phenotypic traits best PBP analysis by spectroscopy. Cryptophyte PBP concentra- predicted clade membership. Our results illustrate the tions were determined using the freeze/thaw centrifugation remarkable genetic and phenotypic diversity even method of Lawrenz et al. (2011). Aliquots (40–50 mL) of mid- within this subset of cryptophytes, and provide new exponential phase culture of each strain were sampled and then centrifuged at 2,054g for 10 min. The resulting supernatant was information about links between phylogenetic and decanted and the pellet re-suspended in 0.1 M phosphate buf- functional diversification in this group of microalgae. fer (pH = 6) and homogenized using a vortexer. Samples were then placed at À20°C for 2 h until frozen. Once frozen, samples were moved to 5°C to thaw for 24 h. Thawed samples were cen- METHODS trifuged at 10,870g for 5 min to remove excess cellular debris. Cryptophyte cultures. Cryptophyte strains were acquired The absorbance of each sample extract was measured from 400 from six culture repositories (details in Table S1 in the Sup- to 750 nm at 1-nm intervals with a Shimadzu UV-VIS 2450 dual porting Information). All were grown in batch culture on a beam spectrophotometer using a 1-cm quartz glass cuvette À À 12:12 h light:dark cycle at 30 lmol photons Á m 2 Á s 1 with against a phosphate buffer blank. Data were scatter-corrected by illumination provided from the side by Daylight Deluxe Arc- subtracting absorbance at 750 nm from the maximum PBP absorption peak (Lawrenz et al. 2011). PBP concentration (c; tic White 32-watt fluorescent lamps (Philips, Inc. NJ). All cul- À â 1 tures were grown in 250 mL Pyrex flasks with no more than pg · cell ) was calculated according to: 150 mL of culture in each and were gently swirled by hand 12 each day. Growth temperatures and culture media were as A Vbuffer 10 c ¼ Â MW Â Â ð3Þ described in Table S1. All stock cultures were transferred to e Á d Vsample N fresh culture media while in the exponential phase. Samples for phenotypic characterization were taken in triplicate from where A = absorbance of sample, e = the extinction coeffi- À À a single culture in mid-exponential phase as determined by cient for Cr-PE (5.67 9 105 L Á mol 1 Á cm 1; MacColl et al. À À cell counts. Means and standard deviations, therefore, repre- 1976) or Cr-PC (5.7 9 105 L Á mol 1 Á cm 1; MacColl et al. sent analytical variability, i.e., are technical replicates, but do 1973) d = path length of the cuvette in cm, MW = molecular = = not represent any possible within-strain variability under the weight of Cr-PE 45,000 Da or Cr-PC 50,000 Da, Vbuffer same culture conditions. and V = volume of buffer and sample, respectively, N is sample À Light absorption. Absorbance spectra for each cryptophyte the concentration of cells (cells Á L 1), and 1012 is a conver- À strain were acquired with a Shimadzu dual-beam UV/VIS sion factor to convert the result to pg · cell 1.