Table 1.
Scoring schemes of different nucleotide versus nucleotide search algorithms from the NCBI blast pages.
Table 2.
Performance of blast searches in a badly sampled group using the COI-5P sequence of Cryptomonas curvata strain CCAC 0080 as a query (570 nt): rankings and statistics of the discontiguous megablast search.
Table 3.
K2P and K2P and GTR+I+ distances computed by maximum likelihood in Cryptomonas curvata CCAC 0080 COI-5P versus cryptophyte and non-cryptophyte sequences.
Table 4.
Results of the tests for substitution saturation.
Figure 1.
Examples for pairwise alignments of the COI-5P query sequence of Cryptomonas curvata strain CCAC 0080 with the Guillardia theta COI-5P region using the discontiguous megablast algorithm.
A – Discontiguous megablast inserted two gaps into the alignment at high gap costs. B – Manual modifications of the query sequence to test the impact of gaps on ranking. Top row: Shifting the G (red) by three positions downstream resulted in disappearance of both gaps with one mismatch. Bottom row: Deletion of a G and insertion of a T three nucleotides downstream resulted in a perfect match in this short DNA stretch. C – Result of a discontiguous megablast search using the modified query sequence with an exact matching four-nucleotide string.
Table 5.
The impact of gap costs in a saturated DNA barcode marker: discontiguous megablast search using a manually modified Cryptomonas curvata CCAC 0080 COI-5P sequence as a query.
Figure 2.
Unrooted maximum likelihood trees of partial nuclear LSU rDNA sequences of the Chroomonas clade and of the genus Cryptomonas.
For both, phylogenetic tree construction and for computing distances, the same data sets with unalignable positions being pruned have been used. Evolutionary models: GTR+I+; support values: maximum likelihood/posterior probabilities, support of 100%/1.0 as bold lines; scale bar = expected substitutions per site. A – The Chroomonas clade included three different genera. In rooted phylogenies, the genus Chroomonas was paraphyletic with the genus Hemiselmis being nested within Chroomonas [56]. Only the genus Hemiselmis has been subject to a previous revision using an integrated taxonomy approach, thus, assignment of Hemiselmis species corresponded to tree topology [50]. New sequences labeled in bold face, sequences used for blast queries in green (see text and Table 5). 45 taxa, 920 positions. B – Partial LSU rDNA phylogeny of the genus Cryptomonas. Species designations according to previous revisions. Turquoise branches: intraspecific distances in distance classes 4 and 5; red branches: interspecific distances in class 2, green branches: interspecific distances in class 3 (see Fig. 4 and text). 64 taxa, 975 positions; new sequences in bold face, taxon label in green: sequence has been used for blast searches (see text).
Table 6.
Performance of megablast searches (default settings) using cryptophyte 5′-partial nuclear LSU rDNA as query sequences.
Figure 3.
Frequency distibutions of genetic distances in the Chroomonas clade under p-distances and three different evolutionary models.
The distances have been computed from 5′-partial nuclear LSU rDNA using the same data set as in Fig. 2A. The simpler the evolutionary model, the smaller the range of genetic distances due to an underestimation of the number of mutations. The positions of putative barcode gaps changed with evolutionary models (arrows). Sizes of the distance classes in steps of 0.005 with closed lower and open upper limits: class 1 = [0.000–0.005[, class 58 = [0.285–0.290[. A – The frequency distribution of p-distances does not show gaps. B – K2P distances with a gap in distance class 10 [0.0450–0.0500[(1). C – TIM2+I+. D – GTR+I+
. (2) Gaps in the complex evolutionary models TIM2+I+
and GTR+I+
in the distance classes 3 [0.0100–0.0150[, 7 [0.0300–0.0350[and 12 [0.0550–0.0600[.
Figure 4.
Genetic distances in the Chroomonas clade under different distance measures (ordinate) plotted against GTR+I+ (abscissa).
Figure 5.
Frequency distributions of intra- and interspecific distances in the genus Cryptomonas in an unmodified and in a modified data set.
The distances have been computed using the GTR+I+ model. A – Frequency distribution of the unmodified data set. In Cryptomonas borealis taxa with long branches have been lumped to one species. Distance class 3 [0.02–0.03[contained only two genetic distances, labeled in green in Fig. 2B. B – Frequency distribution after duplicating the sequences of the strains M1634 and M2089 four times each to examine a hypothetically better taxon sampling in these two species. The putative blurred barcode gap filled up.
Figure 6.
Results of the GMYC analysis: Bayesian tree inferred from the Cryptomonas data set under the assumption of a molecular clock (top) and the corresponding lineage-through-time (LTT) plot (bottom).
Due to a lack of cryptophyte fossil record the tree could not be calibrated, thus branch lengths and abscissa of the LTT plot represent only relative time scales. The vertical red and turquoise lines in the LTT plot demarcate the two thresholds predicted by the multiple threshold model with speciation to the left and coalescence events to the right [63]. The red and turquoise branches in the tree indicate clusters of species identified by the two thresholds, respectively. The bars to the right of the tree’s terminal nodes represent the species predicted by the multiple threshold GMYC model (1), by GMYC with a single threshold (2) and according to previous revisions using a combination of multiple molecular markers and morphology [33] [20]. Black bars represent singletons, gray bars unrevised putative species. Clade PyrX has not been merged to one species in the previous revisions due to considerable divergence in internal transcribed spacers 2, whereas the single strain of Cryptomonas gyropyrenoidosa has been described due to a unique set of morphological characters. The ordinate of the LTT plot has been logarithmized. 0.000 in the abscissa represents present. Cbo, Cryptomonas borealis; Cco, Cryptomonas commutata; Ccu, Cryptomonas curvata; Cer,Cryptomonas erosa; Cgy, Cryptomonas gyropyrenoidosa; Clo, Cryptomonas loricata; Clu, Cryptomonas lundii; Cma, Cryptomonas marssonii; Cob, Cryptomonas obovoidea; Cov, Cryptomonas ovata; Cpa, Cryptomonas paramaecium; Cph, Cryptomonas phaseolus; Cpy, Cryptomonas pyrenoidifera; Cte, Cryptomonas tetrapyrenoidosa.