Total-evidence data matrix for TJSP-2024-0003.R1 - Rogue sawflies: Rare late Eocene amber fossils provide new calibration points for dating the evolution of Tenthredinoidea (Insecta: Hymenoptera)

Abstract: True sawflies (Tenthredinoidea) have a substantial fossil record but are rarely encountered in Eocene ambers. Here we describe three new taxa from late Eocene ambers in this superfamily. †<i>Rovnotaxonus aristovi</i> n. gen. &amp; sp. is the first true sawfly reported from Rovno amber. Based primarily on characters in the fore wing venation, we assign the new taxon to Tenthredinidae: Allantinae: Allantini. We also describe two new species of Diprionidae from Baltic amber: †<i>Eodiprion pectinatus </i>n. sp. from a female specimen and †<i>Monodiprion gladius</i> n. gen. &amp; sp. from a male previously reported as †<i>Eodiprion </i>sp. by Schedl (2008); we redescribe †<i>Eodiprion </i>Schedl, 2007 and provide emended diagnoses of the genus and of †<i>Eodiprion groehni</i> Schedl, 2007. We integrate all these fossils as well as †<i>Sambia</i> Vilhelmsen &amp; Engel, 2012 (Tenthredinidae: Tenthredininae) previously described from Baltic amber in a combined data set assembled from previously published morphological and molecular data sets. We analyze the combined data set in a Bayesian framework and implement RoguePlots to evaluate the positions of the fossils. The diprionid fossils are unequivocally placed inside Diprionidae, in a polytomy with extant members of Diprioninae; the Monocteninae, the other subfamily currently recognized in the family, is not retrieved as monophyletic. †<i>Rovnotaxonus</i> is placed inside Allantinae and shares some characters with <i>Taxonus</i>. †<i>Sambia</i> is placed near the base of Tenthredininae. The evaluation of the phylogenetic position of the fossils treated here will make them available for future dating analyses of Tenthredinoidea, helping to further elucidate the evolutionary history of this significant lineage of herbivorous insects. Possible reasons for the comparatively low abundance of true sawflies in late Eocene ambers are discussed.Methods: We explored phylogenetic placement of the three fossils by including them in the most comprehensive total-evidence phylogeny (TEP) of the Tenthredinidae published so far. Our total-evidence analysis was based on previously published morphological and molecular phylogenies which had significant overlap in taxon sampling. Despite this and to maximize overlap in taxon sampling, it was necessary to create taxon chimeras between the morphological and molecular data (Supplementary Material Table 1). Morphological data from the three fossils were scored into the matrix of Vilhelmsen (2015) in Mesquite 3.70 (Maddison and Maddison, 2021). The morphological dataset was then combined with the molecular dataset of Malm &amp; Nyman (2015). Specifically, we used molecular data from the ’12 + CAD3 + GLN3_hym’ dataset (Appendix S3 in Malm &amp; Nyman 2015), which includes data from nine protein-coding genes (eight nuclear, one mitochondrial), including fragments of: CAD, gelsolin (GLN), glycogen synthase (GS), isocitrate dehydrogenase (IDH), sodium-potassium adenosine triphosphatase (NAK), phosphogluconate dehydrogenase (PGD), POL, and triose-phosphate isomerase (TPI), and a fragment of the mitochondrial COI. For details of sequence generation, alignment and partitioning readers are referred to Malm &amp; Nyman (2015). Our final matrix included 191 taxa, 5164 base pairs of molecular data from nine protein-coding genes and 146 morphological characters; the dataset can be downloaded from https://doi.org./xx.xxxx/xx.figshare.xxxxxxxx.For the Bayesian inference of the TEP dataset we used the same parameters as Malm &amp; Nyman (2015) with the addition that the morphological partition was analyzed under the Mk + G (Gamma) model (Lewis, 2001) and the analysis was run for 80 million generations. <i>Urocerus gigas</i> (Siricidae) was set as the outgroup. Computation for the TEP using MrBayes (Ronquist <i>et al.</i>, 2012b) was performed on the National Life Science Supercomputing Center - Computerome 2.0 (www.computerome.dk). We used R/RoguePlots (Klopfstein &amp; Spasojevic 2019; R Core Team 2021) to explore phylogenetic placement of the three fossil taxa in all output trees of the Bayesian analysis. The online blog by Mario Coiro (https://mariocoiro.blog/2020/12/02/how-torepresent-uncertainty-in-phylogenies-rogueplots-to-the-rescue/) was also useful when implementing RoguePlots. Statistical support for placements of the three fossil taxa was summarized using a majority-rule consensus tree and the post burn-in combined runs from the Bayesian analyses.