Ascidian mitogenomics: comparison of evolutionary rates in closely related taxa provides evidence of ongoing speciation events

Abstract

Ascidians are a fascinating group of filter-feeding marine chordates characterized by rapid evolution of both sequences and structure of their nuclear and mitochondrial genomes. Moreover, they include several model organisms used to investigate complex biological processes in chordates. To study the evolutionary dynamics of ascidians at short phylogenetic distances, we sequenced 13 new mitogenomes and analyzed them, together with 15 other available mitogenomes, using a novel approach involving detailed whole-mitogenome comparisons of conspecific and congeneric pairs. The evolutionary rate was quite homogeneous at both intraspecific and congeneric level, and the lowest congeneric rates were found in cryptic (morphologically undistinguishable) and in morphologically very similar species pairs. Moreover, congeneric nonsynonymous rates (dN) were up to two orders of magnitude higher than in intraspecies pairs. Overall, a clear-cut gap sets apart conspecific from congeneric pairs. These evolutionary peculiarities allowed easily identifying an extraordinary intraspecific variability in the model ascidian Botryllus schlosseri, where most pairs show a dN value between that observed at intraspecies and congeneric level, yet consistently lower than that of the Ciona intestinalis cryptic species pair. These data suggest ongoing speciation events producing genetically distinct B. schlosseri entities. Remarkably, these ongoing speciation events were undetectable by the cox1 barcode fragment, demonstrating that, at low phylogenetic distances, the whole mitogenome has a higher resolving power than cox1. Our study shows that whole-mitogenome comparative analyses, performed on a suitable sample of congeneric and intraspecies pairs, may allow detecting not only cryptic species but also ongoing speciation events.

Keywords: ascidian; evolutionary rate; mitochondrial genome; species identification.

Fig. 1.—

Uncorrected distances (in %) of 11 intraspecies comparisons, calculated on the various functional regions of the mitochondrial genome. Note the different scale used in the two panels. (a) The four Botryllus schlosseri intraspecies pairs involving at least one of the two VE and TR Italian specimens (Bs_Ita pairs). (b) The EA-sc6ab B. schlosseri pair and all other intraspecies comparisons. P12: first and second codon positions; P3: third codon position. The analyzed mitogenomes are listed in table 2, together with specimen abbreviation and source data.

Fig. 2.—

Mean and standard deviation of the nonsynonymous substitution rates (dN) inferred for: (a) intraspecies and (b) congeneric ascidian pairs. Red: Botryllus schlosseri comparisons; blue: comparisons between cryptic or morphologically very similar species pairs. The number of pairs examined for each genus is reported in brackets, if >1. The analyzed mitogenomes are listed in table 1. dN rates were calculated with PAML v4.4b package (Yang 1997) according to Goldman’s codon substitution model (Goldman and Yang 1994).

Fig. 3.—

Saturation plot of the first plus second codon positions (P12) of the 13 mitochondrial protein-genes, drawn for all congeneric and intraspecies ascidian pairs. Continuous violet arrowhead lines: gap between intraspecies and all congeneric distances. Dotted violet arrowhead lines: gap between intraspecies and congeneric distances excluding the cryptic Ciona intestinalis and the Botrylloides leachii–Botrylloides nigrum species pairs. The “x = y” line represents the situation where the number of inferred substitutions is equal to the number of observed differences.

Fig. 4.—

ML phylogenetic tree of Botryllus schlosseri calculated on the cox1 barcode-fragment (PhyML: TIM3 + G model). Numbers at nodes indicate bootstrap support on 100 replicates; dots point to reliable nodes with bootstrap >70. The distinct clades, previously identified by Bock et al. (2012), are reported in different colors. Arrow points to the only highly supported large subclade within clade A. The number of GenEMBL cox1 entries corresponding to a given haplotype is reported in square brackets, according to supplementary table S2, Supplementary Material online.

Fig. 5.—

Mitochondrial genome organization of the four Botryllus schlosseri specimens, with size of the NCR. Black boxes: NCRs > 20 bp; gray boxes: NCRs > 20 bp containing a tRNA-like structure. Negative numbers indicate gene overlaps. NCRs with equal/different length in the four specimens are shown above and below the gene order diagram, respectively. Large black arrows: large NCR differences due to real indels. White arrows: large NCR differences due to the inclusion of a short sequence in a NCR or in a gene region, depending on the specimen. tRNAs are named by the transported amino acid (see also Materials and Methods). 8, atp8; Ga, tRNA-Gly(AGR); Gg, tRNA-Gly(GGN); Lu, tRNA-Leu(UUR); Lc, tRNA-Leu(CUN); Mc, tRNA-Met(CAU); Mu, tRNA-Met(UAU); Sa, tRNA-Ser(AGY); Su, tRNA-Ser(UCN).

Fig. 6.—

Secondary structures of trnK, trnL(UUR), and three tRNA-like structures (K2, K3, and L2), together with the overall tRNA nucleotide substitution pattern within Botryllus schlosseri. (a) Secondary structure of trnK and the tRNA-like structures K2 and K3. (b) Secondary structure of trnL(UUR) and the tRNA-like structure L2. (c) Distribution of nucleotide substitutions in the different structural elements of the B. schlosseri tRNAs. Numbers indicate average and standard deviation of the pairwise nucleotide differences (in percentage), over all 24 tRNAs and in five B. schlosseri pairwise comparisons (the EA-sc6ab pair was excluded due to the almost identity between the tRNA sequences). Gray background: sites with nucleotide substitutions (upper case) or indels (lower case) in at least one of the six intraspecies B. schlosseri pairs. Borders: sites with uncommon features for functional tRNAs. “x”: nucleotide mispairing in at least one of the four specimens, due to noncompensatory substitutions.