3 | Results
We successfully amplified and sequenced 174 individuals of 67 distinct
species. Phylogenetic analyses of the resulting COI sequences
revealed high diversity and a number of cryptic species within all
groups, but especially within Lobata, which had not been evident from
previous 18S rDNA sequence analyses. COI and 18Ssequence fragments were deposited in GenBank with accession numbers
(xxx-xxx) (Table S1). Taxon-optimized primer sequences are indicated in
Figure 1.
Analysis of multiple isolates of Bolinopsis suggested that the
genus is polyphyletic. In addition, we confirmed morphological
differences of distinct genera with COI sequence fragments, such
as Deiopea , Kiyohimea , and Eurhamphaea , that were
unresolved with the 18S fragment, and found support for subtle
morphological differences between undescribed species within the generaLampocteis and Bathocyroe .
3.1 | Primer Design and Combinations
Successful primer pairs varied by taxon (Table S1, Figure 1, and
branches of Figure 2). Primer combinations amplified a range of fragment
lengths (Figure 1), so we trimmed the ends of the alignments used to
generate phylogenies to exclude missing data. A few sets of primers such
as F259/1060R worked well for many species, and reverse primer R866 was
successful for many species of cydippids (Figure 2, starred primers).
However, amplification of many genera such as Euplokamis,
Ocyropsis, Beroe, and Lampea required customized primers. Some
primers designed for one species also worked for other closely related
taxa, such as Bfor259F, which was specific to B. forskalii but
also amplified most Beroids. For several species, multiple combinations
of primer pairs successfully amplified the COI fragment (Figure
1, 2).
3.2 | Phylogenetics and Species Delimitation
The COI alignment included 174 sequences representing 67 MOTUs
(molecular operational taxonomic units). It was trimmed to a 765
base-pair (bp) fragment and we used a GTR+i+Γ model for phylogenetic
analyses based on jModelTest results (Figure 2). Mitochondrial loci in
ctenophores are generally rich in the bases adenine (A) and thymine (T)
(Pett et al. 2011). Average base composition for all ctenophores
sequenced were ~ 50% A, 21% T, 16% C, and 13% G, and
the vast majority of changes were at the third codon position.
In order to examine saturation between and within taxa, we plotted the
proportion of transitions and transversions versus GTR distance among
all sequences (Figure 3a). For ctenophores sequenced, we found that the
proportion of transitions was greater than that of transversions, which
is in contrast to the general rule (for mammal mitochondrial DNA), where
transversions are often two-fold more common than transitions (Xia et
al. 1996). Transitions were saturated for species that were more than
~25% distinct and transversions were saturated for
species that were more than ~30% distinct. We saw the
same pattern for the other mitochondrial loci that we could align
without the presence of stop codons including; COII, COIII, CytB,
ND4 and ND5 (Figure S1). In these genes as well, transversions
were more common than transitions and there were high levels of
saturation.
The high levels of saturation between distantly related species (Figure
3a) were reflected in the poor resolution of deeper level relationships
amongst taxa in the Bayesian and maximum likelihood phylogeny trees.
Phylogenetic relationships for COI among all the ctenophores were
therefore portrayed in an unrooted tree without support values (Figure
2). In order to illustrate levels of diversity within the phylum, we
also plotted within and between pairwise GTR distances for all
individuals sequenced (Figure 3b). In comparison to other phyla,
distances between MOTUs were relatively large, ranging between from
0–43%, and showed four peaks. The first peak represented
within-species variability, around 0-4% GTR, although we had few
within-species samples for this estimate. The second small peak centered
at 10% GTR distance, was represented by closely-related species
complexes. The two largest peaks were represented by the majority of our
sequencing efforts and occurred at ~17% (within group;
pink, blue, or green) and 30% GTR distance among different orders
(gray) of ctenophores (Figure 3b).
The 18S alignment of the Lobata included 82 sequences for 29
MOTUs. This was trimmed to 1780 bp and had a TrN+i+Γ model determined
with jModelTest for phylogenetic analyses. The 18S fragment was
very conserved and included only 47 parsimony-informative sites within
all sequence data for the Lobata and phylogenetic analyses showed little
differentiation within and even between many genera (Fig 4a). Posterior
probabilities and bootstrap values were relatively poor and unresolved
for most relationships among taxa due to low resolution among species
and genera (Figure 4a). Distinguishing taxa using the COIfragment limited to the Lobata was more successful since the marker is
more variable than 18S. The COI fragment had 199 parsimony
informative sites between 27 MOTUs (Figure 4b). Although deeper level
relationships amongst more distantly related taxa were still not well
supported and some saturation was evident (Figure 3a), analyses revealed
high support for several cryptic species complexes and new MOTUs.
Conversely, other species were revealed to be truly cosmopolitan with
worldwide or at least ocean-basin wide distributions (Figure 4b).