4.4. Incongruent mitochondrial/nuclear pattern
An interesting pattern was observed when species delimitation was
performed with both markers: two individuals were not assigned to the
same group for both genes (we re-sequenced both genes and obtained
unchanged results for both individuals). On one hand individual 291, was
assigned to C. verrucosa sp. A in 18S species
delimitation, but to C. verrucosa sp. B for COI; on the
other hand, individual 116, was assigned to C. verrucosasp. B in 18S species delimitation, but to C. verrucosasp. A for COI. This means that only for these two specimens the
nuclear and mitochondrial genes were addressing different speciation
patterns. Apparent incongruence between phylogenies from 18S and COI
sequences was addressed already in ascidians by other authors
(Pérez-Portela, Bishop, Davis & Turon, 2009; Stach & Turbeville,
2002). Even though in 18S sequences 70 polymorphic sites were found,
only a single site was dividing the sequences into two groups that were
congruent with mitochondrial and morphological inference, then other 69
variable sites were explaining intraspecific variability, or difference
among the species (A and B) with four basal groups (see results section
3.3. and discussion section 4.1.). While C. verrucosa sp.
A was characterized by a thymine in the unique variable site of 18S
sequences, C. verrucosa sp. B was characterized by a
cytosine. Specimen 291 presented in this variable site two overlapped
peaks (thymine and cytosine). This can be explained under two different
assumptions, on the one hand, it can be expected that C.
verrucosa sp. A is characterized by a thymine in the unique
variable site, but at the same time presents a low-frequency second
allele; this allele carries a cytosine in the variable site, which would
be predominant in the C. verrucosa sp. B . Thus, the
individual 291 would be heterozygous. On top of this, it is also
possible that with the primers that we used to sequence 18S we sampled
by PCR only a proportion of the allele population, and missed a group of
molecules (null allele) that represents, for example, a heterozygote
group of individuals. On the other hand, specimen 291 can be interpreted
as a hybrid among C. verrucosa sp. A and sp. B .
In order to explain the crossed pattern of these two specimens, some
hypotheses can be considered: a) introgression, b) ancestral
polymorphism with incomplete lineage sorting or c) convergent mutation.
Hybridization and introgression patterns were already proposed in
ascidians (Bouchemousse, Liautard-Haag, Bierne & Viard, 2016; Nydam et
al., 2017). Broadcast spawners present a reproductive system that has a
higher probability of introgression/hybridization than others. Whilst
artificial cross-fertilization among Ciona intestinalis Type A
and B was demonstrated, hybrids remained infertile (Caputi et al., 2007;
Sato et al., 2014). Once hybridization may occur, backcrossing with the
parental types is more likely since genomes would be more similar than
between species (Edmands, 2002). In this study, specimen 291 may
represent the small population of hybrids among C. verrucosasp. A and sp. B , while specimen 116 could be the result of
an event of introgression, in which it might inherit allele A at COI
from one parent and allele B at 18S from the other. In the same way,
there is evidence of introgression within C. intestinalis Type A
and B, species that show high genetic divergence (12.4%) at COI (Nydam
& Harrison, 2011; Nydam et al., 2017). These cryptic species, newly
elevated to species status: C. intestinalis and C. robusta(Brunetti et al., 2015) show historical rates of gene flow, with almost
complete absence of contemporary hybridization (Bouchemousse et al.,
2016; Roux, Tsagkogeorga, Bierne, & Galtier, 2013). This evidence
supports the hypothesis of introgression within C. verrucosa sp.
A and sp. B which presented, in this work, 10% genetic
divergence at COI. Under those circumstances, it would be of interest to
experimentally test hybridization among C. verrucosa sp. Aand sp. B . Ancestral polymorphism with incomplete lineage sorting
(ILS) is another hypothesis that must be discussed under these results,
it is difficult to distinguish this evolutionary process from
introgression since both produce similar patterns. Discrepancies between
trees inferred with mitochondrial and nuclear genes can be explained by
these non-exclusive evolutionary hypotheses (Zhou et al., 2017).
Nevertheless, bearing in mind the high genetic divergence among C.
verrucosa sp. A and sp. B , ILS is unlikely under neutral
evolution (Pamilo & Nei, 1988), since the loss of polymorphism and the
fixation of an allele are positively correlated with time since
divergence (Maddison, 1997; Wendel & Doyle, 1998). There is
circumstantial evidence that ascidians may be characterized by an
elevated rate of molecular evolution (Delsuc et al., 2006). Therefore, a
convergent mutation can also explain that individual 116 presents allele
B in 18S, but being assigned to C. verrucosa sp. A for
COI, given that the thymine mutates to a cytosine in the unique variable
site.