the strains that were the subject of this study and their source collections are listed in table1 . the nutrient utilization data presented in table1 are sorted in the same order as the 16s plastid rdna tree . the names in the authority column conform to the convention given in algaebase.org ( guiry and guiry 2014 ) . the phenotypic responses were scored as  for no utilization , ( + ) for doubtful utilization and + for positive utilization . the nutrient source is labeled a ) enteric / nonfermenter and b ) gram positive . phc is the abbreviation for p - n - p - phosphorylcholine . for a.  protothecoides ( marked with an   in table1 ) , incubations were for 48  h at 28c . sag 263 - 2 ( marked with a # in table1 ) was determined not to be uni - algal . the two resulting isolates were therefore isolated and split into two unique strains , 263 - 2.1 and 263 - 2.2 . approximately 1  ng of extracted genomic dna was used as template for the pcr reactions . primers were designed to anneal to regions of the 16s rrna and 23s rrna genes conserved across a wide variety of organisms . oligonucleotides used for the 16s rdnas were designed to amplify a region of 1,431  bp of plastidic dna and included a forward primer with the sequence 5-agagtttgatcctggctcag-3 and a reverse primer with the sequence 5-ggctaccttgttacgacttc-3 , annealing to nucleotides 122583 to 122602 and 124033 to 124014 , respectively in genbank accession no . oligonucleotides used for the 23s rdnas were designed to amplify a region of 530  bp of plastidic dna and included a forward primer with the sequence 5-tgttgaagaatgagccggcgac-3 and a reverse primer with the sequence 5-cagtgagctattacgcactc-3 , annealing to nucleotides 637 to 658 and 1182 to 1163 , respectively , in genbank accession no . algal cultures were incubated aerobically for 5  d at 28c , essentially as described by pore ( 1985 ) , on medium containing 2% glucose , or 2% glucose supplemented with 4  g    l yeast extract . nutrient utilization for each strain was assessed using the microbial bbl crystal identification systems ( becton dickinson , franklin lakes , nj , usa ) as described previously ( roesler et al.2006 ) . both the enteric / nonfermenter i d kit and the gram positive i d kit from crystal i d systems were employed to establish utilization profiles . prototheca sample panels were incubated for 48  h at 37c in a 70% humidified chamber . auxenochlorella sample panels were incubated for 48  h at 28c in a 70% humidified chamber . both 16s and 23s plastid rdna sequence data were used to build phylogenetic trees by the maximum - likelihood method . the multiple alignment algorithm used was clustal w while tree generation was performed with the program phyml , with a.  protothecoides selected as an outgroup to root the tree . parameters were gtr+g+i mode of sequence evolution search with 1,000 bootstraps ; other tree generation methods tested included bayesian , parsimony and neighbor joining , all of which yielded essentially identical clading patterns . the phylogenetic trees generated for both 16s ( fig.1 ) and 23s ( fig.2 ) rdna share a very similar structure , with only slight differences within clade e. the bootstrap value for each branch length demonstrates the strength of each branch point ; the branches separating each clade have support of 97 or higher . these bootstrap values demonstrate the robustness of this tree , which had the same clading structure regardless of the tree building method used ; very similar trees were generated using the bayesian , parsimony and neighbor joining methods . a 16s plastid rdna phylogenetic tree generated by the maximum - likelihood method . the 16s rdna plastid sequences from the 24 algal strains form five distinct clades , regardless of the tree generation algorithm used ; the maximum - likelihood tree is displayed in figure1 along with associated bootstrap values . the five clades ( a  e ) , are composed of : ( a ) auxenochlorella protothecoides ;  ( b )  a  single  prototheca wickerhamii isolate ( c ) a mixture of p.  wickerhamii and p.  moriformis isolates ; ( d ) p.  ulmea and p.  stagnora ; ( e ) primarily p.  zopfii isolates with two p.  moriformis , as well as p.  kruegani and p.  blaschkaea . 23s plastid rdna tree generated by the maximum - likelihood method . like the 16s rdna plastid sequences the 23s rdna plastid sequences form five distinct clades regardless of the tree generation algorithm used . the five clades are identical in makeup to those described for the 16s clades . using pairwise comparisons between individuals in each clade , the 16s rdna data show that clade a and b share 93.9% sequence similarity ( utex 250 and sag 263 - 11 ) ; clades b and c ( sag 263 - 11 and atcc 16529 ) share 86.8% sequence similarity ; clades c and d ( atcc 16529 and cbs 605.66 ) share 67.9% sequence similarity ; clades c and e ( atcc 16529 and sag 43 - 80 ) share 71.6% sequence similarity ; clades d and e ( cbs 605.66 and sag 43 - 80 ) share 76.1% sequence similarity . the auxanographic data ( table1 ) for these algal strains do not entirely conform to either the current taxonomic groupings or the molecular data presented here . the clades described in the phylogenetic trees group the majority of the trehalose consuming algae together ( clade c ) , with the exception of p.  wickerhamii sag 263 - 11 , which was unable to use trehalose . furthermore , a.  protothecoides utex 250 ( clade a ) also was able to use trehalose , and was only able to grow at the lower temperature of 28c . kruegani utex 329 and p.  stagnora atcc 16528 showed no growth on any of the alternative carbon sources , and p.  zopfii sag 263.7 only grew when proline was used as a carbon source . furthermore , a number of organisms in clades d ( p.  stagnora cbs 605.66 , p.  ulmea atcc 50112 , p.  sp . sag 263 - 2.2 ) and e ( p. stagnora utex 1442 , p.  moriformis atcc 50081 , p.  zopfii 43 - 80 ) only grew on arginine as an alternative carbon source , while others in clade e could use a number of different carbon sources ( fructose , glycerol , lysine and proline ) . the finding that the specificity of the strains with respect to the utilization of different fixed carbon sources is not strictly correlated with the clading results , is interesting since some of these strains show close phylogenetic relations ( see for example p.  zopfii sag 263.7 and p.  kruegani utex 329 ) . the molecular genetic data provide a robust phylogeny of the different prototheca strains based on both 16s and 23s plastid rdna sequences . this finding reinforces the conclusions of previous work ( ueno et al.2003 ) using 18s nuclear rdna and 16s plastidic rdna sequences . however , the data presented here are more extensive than previous studies because a larger number of taxa were examined and we included 23s plastid rdna information . the same strains that we used for the phylogenetic analyses were analyzed for nutrient utilization , and there were a number of cases in which the nutrient utilization profile was not consistent with the phylogenetic grouping of the organism . while nutrient utilization studies are relatively easy to perform and have been widely used to classify prototheca isolates in the past , our work clearly shows that inferences concerning phylogenetic relationships based on nutrient utilization are problematic . as an example , p.  stagnora atcc 16528 ( clade d ; figs.1 and 2 ) shows the exact same auxanographic fingerprint as p.  kruegani utex 329 ( clade e ; figs.1 and 2 ) and yet molecular genetic characterization shows these strains to be quite disparate . trehalose utilization was previously used to distinguish p.  wickerhamii from all other prototheca species . while others have successfully grown p.  wickerhamii sag 263 - 11 , one of the most well - characterized strains , on trehalose ( pore 1985 , ueno et al.2005 ) , in this study the growth of p.  wickerhamii sag 263 - 11 was not supported by trehalose ( under either of the temperature conditions used ) . this is surprising since the strain previously tested ( ueno et al.2005 ) is the same as ours based on identical 16s and 23s rdna sequences ( genbank : aj245645 and knauf and hachtel 2002 ) . it is possible that the strain lost its ability to utilize this sugar during long years of culturing in the absence of trehalose ; this could be a feature associated with many nutrient uptake and assimilation systems . furthermore , some of the features associated with nutrient utilization can be acquired as a consequence of lateral gene transfer and would not reflect the phylogeny of the organism . overall , our results are cautionary and suggest that basing strain classification solely on auxanographic features , which has been done in the past , can be misleading , while sequence - based phylogenies , particularly when utilizing multiple gene sequences , provide stronger evidence for evolutionary relationships . the naming of the different species within the genus prototheca has been driven largely by morphological and auxanographic characteristics over the past 120  years . as observed , based on the 16s and 23s plastid rdna phylogenetic trees that we generated , the current taxonomy forms paraphyletic groups . among two of the five clades , p. moriformis isolates share near identical 16s and 23s plastid rdna sequences with p.  wickerhamii isolates in clade c and p.  zopfii isolates in clade e. similarly p.  wickerhamii isolates are spread among clades b and c. furthermore , p.  wickerhamii sag 263 - 11 appears to be more closely related to a.  protothecoides utex 250 than to the p.  wickerhamii isolates in clade c , which includes strains designated as both p.  wickerhamii and p.  moriformis . based on the molecular genetic data presented here , the strains named p.  moriformis , p.  wickerhamii and p.  stagnora are part of paraphyletic groups that should be resolved in future studies .