Tettigettalna Puissant 2010

4.1 | Phylogeny of Tettigettalna

With this study, we used an extended set of mitochondrial sequences and obtained the first nuclear data to investigate the evolution of the Mediterranean genus Tettigettalna. These small-sized and colour-cryptic cicadas occur at fairly low density, being very difficult to sample without the use of acoustic location. Hence, and because females are mute and seldom seen, the dataset is composed of males only. The inclusion of nuclear sequences is particularly relevant to corroborate patterns found with mitochondrial data and discard potential bias in male dispersal. As expected, nuclear EF-1α had much lower diversity than mitochondrial genes and lacked resolution in Tettigettalna, though without conflicting with mitochondrial phylogenies.

Regarding the order of lineage splitting among Tettigettalna, our study gives support to T. josei as sister to the remaining extant members of the genus, consistent with morphological and acoustic differentiation observed for this species (Mendes et al., 2014). Nonetheless, the other two phylogenetic scenarios in the species coalescent tree (i.e subclades T. josei – Core Tettigettalna and T. afroamissa – T. josei) have some degree of support (8%–9%). Species T. afroamissa and T. argentata are among the most genetically distant taxa in the phylogeny, even though they share a similar calling song pattern. Tettigettalna has no parallel with any other cicada genus occurring in Iberia, being the most species rich, with species defined mainly by song characterization (Puissant & Sueur, 2010). Such diversity of songs within Tettigettalna has evolved without significant morphological divergence, not even at genitalia morphology, often a key trait to distinguish between closely related species, as it may prevent successful mating of heterospecific couples (Knowles et al., 2016; Langerhans et al., 2016). A few song operational taxonomic units within Tettigettaln a remain polyphyletic with the new sequence data as in Nunes, Mendes, Marabuto, et al. (2014) and Costa et al. (2017), namely the widespread T. argentata with cryptic T. mariae and T. aneabi, which present partially overlapping distribution ranges (Nunes, Mendes, Quartau, et al., 2014). Insect songs are genetically inherited but the genes ruling them remain poorly known, in particular for cicadas (Fujisawa et al., 2018; Sueur, 2006; Xu & Shaw, 2019, 2021). Neutral genetic markers fail to differentiate among such close species, either because divergence among their songs is too recent for complete lineage sorting or episodes of introgression on secondary contact have eroded such divergence, or very likely both. Similar cases of polyphyly among cryptic species of Cicadettinni have been detected in Cicadetta of Italy and Greece (Hertach et al., 2015, 2016; Wade et al., 2015), and in Kikihia and Maoricicada of New Zealand (Buckley et al., 2006; Marshall et al., 2008), where molecular phylogeny failed to recover some acoustically defined taxa.

Only a few nuclear genes have been sequenced thus far to investigate phylogenetic relationships among cicadas worldwide, being EF-1α the most extensively used and informative (Arensburger, Buckley, et al., 2004; Banker et al., 2017; Buckley & Simon, 2007; Hill et al., 2021; Lee & Hill, 2010; Marshall et al., 2008, 2016, 2018; Owen et al., 2017; Price et al., 2019; Simon et al., 2019). Nuclear genes have been shown as insufficient to resolve the phylogeny of cryptic cicada species where hybridization is suspected, even when multiple genes were used (Banker et al., 2017; Buckley & Simon, 2007; Wade et al., 2015). A genomic approach and a fine-scale sampling at contact zones would be preferable to overcome single-gene shortcomings and will certainly help to disentangle introgression events and incomplete lineage sorting among T. argentata, T. mariae and T. aneabi.

Another incongruence between acoustics and genetics found within the T. helianthemi ssp. remains unexplained, as T. h. galantei Type II, though sharing the same calling song with T. h. galantei Type I, is remarkably different at mitochondrial data. T. h. galantei Type II has a parapatric distribution with Type I (they were not overlapping) with no obvious breaks in habitat features to justify such level of genetic divergence. Amplification of nuclear mitochondrial DNA sequences (NUMTs) could be a reason for this pattern of divergence i.e, mutations at the primer biding sites that would bias amplification towards nuclear copies of COI at this particular taxon (Song et al., 2008). This seems unlikely since the same phylogenetic pattern was recovered for all three independently amplified fragments of mitochondrial DNA (COI-LEP, COI-CTL and ATP). Further studies with phylogenomic data should bring some light to the roots to this mismatch.