Data supplement for "Flagellum tapering and midpiece volume in songbird spermatozoa" Emily R. A. Cramer, Gaute Grønstøl, Jan T. Lifjeld Paper abstract: In contrast to numerous studies on spermatozoa length, relatively little work focuses on the width of spermatozoa, and particularly the width of the midpiece and flagellum. In flagellated spermatozoa, the flagellum provides forward thrust while energy may be provided via mitochondria in the midpiece and/or through glycolysis along the flagellum itself. Longer flagella may be able to provide greater thrust but may also require stronger structural features and more or larger mitochondria to supply sufficient energy. Here we use SEM imaging to investigate the ultrastructure of spermatozoa from 55 passerine species in 26 taxonomic families in the Passerides infraorder. Our data confirm the qualitative observation that the flagellum tapers along its length, and we show that longer flagella are wider at the neck. This pattern is similar to mammals, and likely reflects the need for longer cells to be stronger against shearing forces. We further estimate the volume of the mitochondrial helix and show that it correlates well with midpiece length, supporting the use of midpiece length as a proxy for mitochondrial volume, at least in between-species studies where midpiece length is highly variable. These results provide important context for understanding the evolutionary correlations among different sperm cell components and dimensions. Description of data supplement: We provide the stitched, 4000 x magnification SEM images used in the main dataset, code used for statistical analysis, and measurements taken from all three image types (4k [i.e., the main data set], Single Images [a lower- and variable- resolution dataset where each cell was imaged in a single image], and background cells [a high- and variable- resolution dataset where measured cells were in the background of images taken for another purpose]). Image files are named with Genus_species_NHM-BI Accession Number. FlagellumStructureCode.R is the code for statistical analysis. ColumnHeaders.csv contains a detailed description of each column for all the data files. MaleIDKey.csv provides connections among the different naming conventions used in other data files (since we occasionally used an SEM sample number as the male identity, or accession number, or the original (often blinded) name of the photo). LM_data_speciesMeans.csv: contains a subset, and calculation of flagellum length, from the light microscopy dataset made by Omotoriogun et al 2020, https://doi.org/10.1016/j.zool.2020.125770. Species mean segment lengths from light microscopy. FlagMinorMtDiam_4k.csv: The main dataset, measures of the flagellum width and the minor axis of the mitochondrial helix, plus other explanatory variables. One row (measurement pair) for each measureable location along each cell. FirstTailMsmt_4k.csv: The main dataset, a single measure of the width of the flagellum at the start of the tail (i.e., just after the end of the midpiece), plus other explanatory variables. MajorAxisData_4k.csv: The main dataset, measures of the major axis of the mitochondrial helix, plus other explanatory variables. One row (measurement) for each measureable location along the cell. MtHelixVolume_4k.csv: The main dataset, estimated mitochondrial volume; one row per cell. Plus other explanatory variables. Gyres_4k.csv: The main dataset, distance among successive gyres (wraps) of the mitochondrial helix. One row for each measureable gyre. Plus explanatory variables. HeadDiameters_4k.csv: The main dataset, a single measure of the diameter of the nucleus. FlagMinorMtDiam_SingleImages.csv: From the single images dataset, measures of the flagellum width and the minor axis of the mitochondrial helix, plus other explanatory variables. One row (measurement pair) for each measureable location along each cell. Gyres_SingleImages.csv: The single images dataset, distance among successive gyres (wraps) of the mitochondrial helix. One row for each measureable gyre. Plus explanatory variables. BackgroundCellsMeasurements.csv: The background images dataset, containing the diameters of flagellum, and major and minor axes of the mitochondrial helix, for cells in the background of images used by Støstad et al. 2018 https://doi.org/10.1111/evo.13555. One row per measured cell. Note that some images contained more than one background cell, and thus may have multiple rows per image. BirdTreeoutput.nex contains our download from birdtree.org of 1000 trees from the Hackett backbone.