The Aegean archipelago is among the largest on Earth with astonishing biodiversity within Europe. Its territory underwent a massive geotectonic transformation in Neogene that resulted in multitude of changes in land-sea configuration and disintegrated the formerly united Aegean land to a complicated mainland-archipelago system. Therefore, it represents an excellent laboratory for studying evolution of terrestrial fauna. In the present study we use a model group of flightless bush-crickets with annual reproduction cycle – Poecilimon jonicus species group – to trace correlation of lineage diversification with the known paleogeographic events in the Aegean area. The group belongs to the hyperdiverse genus Poecilimon and has a disjunct distribution along the Hellenic arc from southwestern Anatolia through Crete to the western Balkans and the Apennines. To test our hypothesis, we inferred phylogenetic relationships of the P. jonicus group sensu lato using a nuclear fragment covering two spacers of the ribosomal cistron (ITS1+ITS2). To study intra-group phylogeny we compared mitochondrial phylogenies based on two matrices– (1) a concatenated ND2 and COI dataset of 1656 bp and (2) a 16S rRNA+12S rRNA dataset of 1835 bp. As a second step, we estimated divergence times applying Bayesian approach with BEAST and a relative rate framework with RelTime on the mitochondrial matrices. We compare trees calibrated based on evolutionary rates and tectonic events and discuss radiation scenarios in concordance with known paleogeographic events in the Aegean area. Our results revealed robust phylogeny of the Poecilimon jonicus group and confirmed a strong link between its evolution and the Aegean paleogeography. The phylogenetic relationships of the group supported reconsideration of its systematics.

DNA sequences used in the study are deposited in Genbank. Maximum likelihood (ML) analyses were conducted using RAxML (Stamatakis 2006) on the T-rex online platform (http://www.trex.uqam.ca/index.php?action=raxml&project=trex) (Boc et al. 2012). Support of nodes was acquired with 1000 bootstrap resampling. Bayesian inference (BI) analyses were accomplished in Mr. Bayes version 3.2.5 (Ronquist and Huelsenbeck 2003; Ronquist et al. 2005) with four simulations of Markov chains and 4 x 10⁶ generations saving each 100th tree. The stationary distribution of the MCMC parameters was checked with Tracer ver. 1.7.1 (Rambaut et al. 2018). The first 25% of trees were excluded as burnin. Results were visualized in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

To estimate divergence times for the ND2+COI phylogeny we followed two calibration strategies. First, we calibrated the BEAST analysis using a substitution rate of 0.0177 site/Ma for COI (Papadopoulou et al. 2010). Second, we conducted BEAST analyses for the ND2+COI dataset setting the divergence of P. cretensis to 5.6±0.4.

BEAST analyses were run using a Yule speciation process and MCMC chain for 100x106 generations sampling every 1x10³ generations in BEAST 2.5.2 (Bouckaert et al. 2019). BEAST was run on the CIPRES Science Gateway web server (Miller et al. 2010). Convergence to stationary and the effective sample size of model parameters were checked via Tracer ver. 1.7.1 (Rambaut et al. 2018). Maximum clade credibility trees were established with TreeAnnotator (Rambaut and Drummond 2002–2013) to summarize the output, discarding the initial 10% of the trees as burn-in.

Alternatively, the RelTime method, implemented in Mega X, was applied on the ND2+COI dataset to infer a timetree (Tamura et al. 2012, 2018). Tree topology was set according to our BI and ML results. One constraint at the branching off of P. cretensis was set within a range between 5.2 and 6.0 Ma.  See the article for details.

Boc, A., Diallo, A. B., & Makarenkov, V. (2012). T-REX: a web server for inferring, validating and visualizing phylogenetic trees and networks. Nucleic acids research, 40(W1), W573-W579.

Bouckaert R., Vaughan, T.G., Barido-Sottani, J., Duchêne, S., Fourment, M., Gavryushkina, A., et al. (2019). BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS computational biology, 15(4), e1006650.

Miller, M.A., Pfeiffer, W. & Schwartz, T. (2010). “Creating the CIPRES Science Gateway for inference of large phylogenetic trees" in Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, pp. 1–8.

Papadopoulou, A., Anastasiou, I., & Vogler, A. P. (2010). Revisiting the insect molecular clock: the Mid-Aegean Trench calibration. Molecular Biology and Evolution, 27, 1659–1672.

Rambaut, A., & Drummond, A. J. (2002–2019). TreeAnnotator. Ver.2.5.2 [Computer software and manual]. Available via http://beast.bio.ed.ac.uk/treeannotator.

Rambaut, A., Drummond, A. J., Xie, D., Baele, G., & Suchard M. A. (2018). Posterior summarisation in Bayesian phylogenetics using Tracer 1.7.  Systematic Biology, 67(5), 901, doi: 10.1093/sysbio/syy032.

Ronquist, F., & Huelsenbeck, J. P. (2003). MrBayes version 3.0: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19 (12), 1572–1574.

Ronquist, F., Huelsenbeck, J. P., & van der Mark, P. (2005). MrBayes 3.1 manual. Draft 5/26/2005. Available at http://mrbayes.csit.fsu.edu/manual.php

Stamatakis, A. (2006). “RAxML-VI-HPC: Maximum Likelihood-based Phylogenetic Analyses with Thousands of Taxa and Mixed Models”. Bioinformatics, 22(21), 2688–2690.

Tamura, K., Battistuzzi F. U., Billing-Ross, P., Murillo, O., Filipski, A., & Kumar, S. (2012). Estimating Divergence Times in Large Molecular Phylogenies. Proceedings of the National Academy of Sciences, 109, 19333–19338.

Tamura, K., Qiqing, T., & Kumar, S. (2018). Theoretical Foundation of the RelTime Method for Estimating Divergence Times from Variable Evolutionary Rates. Molecular Biology and Evolution, 35, 1770–1782

Files are named after the corresponding figure numbers in the article. All files could be opened with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).