4.1. Two genetically divergent species
Both molecular markers studied in this work (the mitochondrial COI and
nuclear 18S gene) distinguished two congruent groups, therefore there
was strong evidence for recognising two genetically divergent species
within C. verrucosa sensu lato populations from the Antarctic
Peninsula: C. verrucosa sp. A and C. verrucosa sp. B .
Nuclear and mitochondrial genes evolve independently because they differ
in the mode of inheritance, ploidy, amount of recombination, introns
number, mutation rate, repair mechanisms and effective population size
(Hill, 2015). Thus, studying only one type of marker, can lead to a
systematic bias in the inference of evolutionary processes (Ballard &
Whitlock, 2004). Such is the case of cichlid fish radiations in lakes of
Africa, where different speciation patterns were addressed using only
one type of marker (Seehausen et al., 2003). In this work both markers
showed the same speciation pattern, which implies that both molecules
were impacted in the same way by the same evolutionary mechanism, thus
making strong the evidence for the mutually isolated gene pools.
Within the results obtained here, for some particular specimens no
sequences were obtained of COI, while 18S nuclear gene could be
sequenced. In this case, the phylogeny constructed with 18S resolved
basal branches that comprised clades that would present deeper genetic
divergence with the rest of the animals, than the divergence amongC. verrucosa sp. A and sp. B (see Fig. 2). Nuclear genes
usually evolve at a slower rate compared to mitochondrial ones (Allio,
Donega, Galtier, & Nabholz, 2017; Havird & Sloan, 2016). Furthermore,
fast substitution rate and gene rearrangements were described for
ascidians mitochondrial genome, and have been proposed to cause
difficulties in standard polymerase chain reaction (PCR), because of
mutations on the primer site (Delsuc et al., 2018; Denoeud et al., 2010;
Gissi et al., 2010). Hereafter, the basal branches obtained in this
study with 18S sequences, composed by individuals from Burdwood Bank/MPA
Namucurá, Scotia Sea, Weddell Sea and Potter Cove (stations located in
the tip of the Antarctic Peninsula and South America, see Fig. 3), could
represent more distant species within C. verrucosa sensu lato,and be constituted by individuals in which the COI primer binding site
has been mutated or rearranged.
Following with the effort of distinguishing genetic differentiated
species, widely-adopted molecular markers, such as COI and 18S, are
helpful to characterize unstudied groups (Hebert, Cywinska, Ball, &
deWaard, 2003). Here, applying ABGD method in COI and 18S a barcode-gap,
with no intermediate values, was found in the frequency distribution of
the genetic differences between individuals of the putative C.
verrucosa. This gap is observed when the divergence between organisms
that belong to the same species is smaller than the divergence among
organisms that belong to different species (Puillandre et al., 2012).
Moreover, a robust approach for species delimitation is to compare
genetic distances with related undisputed species pairs, given that the
nucleotide substitution rate is quite homogeneous at interspecific level
(Griggio et al., 2014; Held, 2003). In this study, the genetic distance
between C. verrucosa sp. A and sp. B was
>10.20% for COI, and >3% for 18S. The COI
nucleotide divergence among ascidian species from the same genera range
from 8.7% to 21.7% (Hirose, Oka, & Hirose, 2009; Nydam & Harrison,
2007; Pérez-Portela & Turon, 2008), and between species within
Styelidae family range from 10.8% to 16.5% (Lacoursière-Roussel et
al., 2012; Reem et al., 2017). Regarding 18S gene, it has been found 0 -
0.58% nucleotide divergence among samples from genera Diplosoma .
Furthermore, it has been described smaller divergence among the suborder
of Diplosoma (Phlebobranchia) than among the suborder
Stolidobranchia, in which C. verrucosa is described (Yokobori,
Kurabayashi, Neilan, Maruyama, & Hirose, 2006). Bock, Macisaac, and
Cristescu (2012) found larger divergences (2.3 - 10.1%) in 18S gene
among putative cryptic species of Botryllus schlosseri ; important
discussion exists on the definition of cryptic species within B.schlosseri but no study has confirmed yet a genetic and/or
morphological evidence, therefore B. schlosseri is
currently being treated as a species complex (Lejeusne, Bock,
Therriault, MacIsaac, & Cristescu, 2011; Nydam, Giesbrecht, &
Stephenson, 2017b; Reem et al., 2017; Yund, Collins, & Johnson, 2015).
On the other hand, Nydam and Harrison (2010) studied five nuclear loci
in order to unravel the existence of two distinct forms ofCiona
intestinalis, and found that Type A vs. Type B (recently defined by
Brunetti et al. (2015) as two morphological and genetically different
species) divergences range from 3.5 to 12.4 %. All this indicates that,
within the samples studied here, the genetic differentiation was similar
to those found in other species in the same family and other ascidians
species pairs, therefore we can define two genetic divergent species
based on mitochondrial as well as nuclear evidence.