DOI: 10.5061/dryad.h18931zhf
title: Evolution of Poecilimon jonicus group (Orthoptera: Tettigoniidae): A history linked to the Aegean Neogene paleogeography

description: 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.
description: 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 10<sup>6</sup>
 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 1x10<sup>3</sup> 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.
 <i>Nucleic acids research, 40(W1)</i>,
 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. <i>PLoS computational biology, 15(4)</i>,
 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. <i>Molecular
 Biology and Evolution, 27</i>, 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.  <em>Systematic Biology</em><i>, 67(5)</i>, 901, doi: 10.1093/sysbio/syy032. Ronquist, F., & Huelsenbeck, J. P. (2003). MrBayes version 3.0: Bayesian phylogenetic inference under mixed models. <i>Bioinformatics, 19 (12)</i>, 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”. <i>Bioinformatics, 22(21)</i>, 2688–2690. Tamura, K., Battistuzzi F. U., Billing-Ross, P., Murillo, O., Filipski, A., & Kumar, S. (2012). Estimating Divergence Times in Large Molecular Phylogenies. <i>Proceedings of the National Academy of Sciences, 109</i>, 19333–19338. Tamura, K., Qiqing, T., & Kumar, S. (2018). Theoretical Foundation of the RelTime Method for Estimating Divergence Times from Variable Evolutionary Rates. <i>Molecular Biology and Evolution, 35</i>, 1770–1782
description: 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/).