DOI: 10.5061/dryad.73n5tb362
title: Data from: Independent evolution of atypical sperm morphology in a passerine bird

description: Spermatozoa exhibit striking morphological variation across the animal
 kingdom. In passerine birds, sperm exhibit considerable variation in size,
 yet the basic sperm phenotype is highly conserved; sperm are filiform, the
 head is corkscrew-shaped, and the midpiece is elongated and twisted around
 the flagellum. A significant departure from this typical sperm morphology
 has been reported in the sister species, the Eurasian bullfinch (Pyrrhula
 pyrrhula) and Azores bullfinch (P. murina). Here, we report a second
 evolutionary shift in passerine sperm phenotype in the nominate subspecies
 of the red-browed finch (Neochmia temporalis temporalis); sperm are
 non-filiform, with an ellipsoid head, and an extremely short midpiece
 restricted to the nuclear-axoneme junction. Additionally, we show that the
 sperm phenotype of the red-browed finch is similar to the putatively
 neotenous sperm described in the two bullfinch species. Using whole-genome
 data, we found no evidence that the unusual sperm phenotype of the
 red-browed finch is associated with reduced genetic variation or a
 population bottleneck. In contrast, using data on relative testes size, we
 find some support for the hypothesis that relaxed post-copulatory sexual
 selection, via a lack of sperm competition, may, at least in part, explain
 the unusual sperm of the red-browed finch. We also discuss the possible
 roles of mutation, genetic drift, and genetic hitchhiking, in the
 evolutionary origins and maintenance of neotenous sperm phenotypes.
 Finally, we suggest that these dramatic evolutionary shifts in sperm
 phenotype warrant further investigation and highlight the need for a
 greater understanding of the developmental and genomic basis of sperm
 phenotype.
description: # Independent evolution of atypical sperm morphology in a passerine bird
 Authors: Melissah Rowe¹*, Daniel M. Hooper², Antje Hofgaard³, Laura L.
 Hurley⁴, Callum S. McDiarmid⁴, Ioanna Pataraia¹, Jan T. Lifjeld⁵, and
 Simon C. Griffith⁴ 1 Department of Animal Ecology, Netherlands Institute
 of Ecology (NIOO‐KNAW), 6700 AB, Wageningen, The Netherlands. 2 Institute
 for Comparative Genomics and Richard Gilder Graduate School, American
 Museum of Natural History, New York, NY 10024, USA. 3 Electron
 Microscopical Unit for Biological Sciences, Department of Molecular
 Biosciences, University of Oslo, 0316 Oslo, Norway. 4 School of Natural
 Sciences, Macquarie University, Sydney, NSW 2109, Australia. 5 Natural
 History Museum, University of Oslo, 0318 Oslo, Norway. * Author for
 correspondence: [m.rowe@nioo.knaw.nl](mailto:m.rowe@nioo.knaw.nl) In this
 study, we gather raw data on sperm size to investigate sperm morphology
 (sperm length) in the Australian Estrildidae grassfinch, the red-browed
 finch (Neochmia temporalis temporalis) and Lesser red-browed finch
 (Neochmia temporalis minor). Our results show that the red-browed finch
 has highly atypical sperm morphology. [Access this dataset on
 Dryad](https://doi.org/10.5061/dryad.73n5tb362) ## Description of the data
 and file structure ### Sperm size data Sperm size data were obtained using
 using SpermSizer v1.6.6 (McDiarmid et al. 2021). *SpermSizeData.csv* -
 data on sperm size for the Red-browed finch and Lesser red-browed finch.
 Dataset includes the following variables: * Species = species common name
 * Male ID = a unique identifier for each male bird * Sampling location =
 location of the sampled indiviual, includes town, state, and country
 information. * wild/captive = whether the bird was wild or captive
 (captive born) * Sampling date = date of sampling in forat DD/MM/YYYY *
 Sperm head length = average length of the sperm head in µm * Sperm
 midpiece length = average length of the sperm midpiece in µm * Sperm
 flagellum length = average length of the sperm flagellum in µm * Total
 sperm length = average length of the entire sperm cell in µm * Standard
 deviation of total sperm length = the standard deviation of length measure
 in µm * Number of sperm cells measured = total number of unique sperm
 cells measured to obtain sperm size data For the red-browed finch, we
 obtained measures for the length of the sperm head (including the
 acrosome, nucleus, and midpiece, as it was not possible to distinguish the
 midpiece using brightfield microscopy) and flagellum. In these cases,
 midpiece length is represented in the data as NAs. For the lesser
 red-browed finch, we obtained measures for the length of the sperm head
 (acrosome and nucleus), midpiece length, and flagellum length. For both
 species, total sperm length was calculated as the sum of head and
 flagellum lengths. ### Sperm swimming speed data (folder) Sperm swimming
 speed data were collected from captive male red-browed finches (maintained
 at the Netherlands Institute of Ecology, NIOO-KNAW) by
 computer-assisted-sperm-analysis (CASA) using the Sperm Class Analyzer ®
 (SCA, Microptic, Barcelona). All buffers and equipment were preheated to
 40°C (avian body temperature). Sperm recordings were captured at 50 frames
 per second using negative phase contrast microscopy at 100x magnification
 with a Basler acA1300-200uc camera connected to a Nikon E200 microscope.
 All recordings were completed within 1-minute of sample loading. All
 recordings were later visually examined, and any tracking or detection
 errors (e.g. detection of cell debris) were deleted manually. We
 considered sperm immotile when with VCL &lt; 20.0 μm/s (based on analysis
 of dead sperm videoed using the same capture settings to identify the
 kinematic profile of drifting sperm). From these analyses, we obtained
 data on curvilinear velocity (VCL), straight-line velocity (VSL), average
 path velocity (VAP) of the sperm cells. *SpermSwimmingSpeedData.zip* -
 folder containing sperm swimming speed data for 6 male red-browed finches
 provided as individual .csv files. Each file includes the following
 variables: * Field = Field of view during CASA analysis * Sperm number =
 individual ID for each cell identified by CASA. * Head X = X coordinate
 for identified cell * Head Y = Y coordinate for identified cell * First
 Frames = frame number, between 0 - 50, identifies which frame the cell is
 identified in * Area = size in pixels of identified cell * Elongation =
 sperm elongation (Length-Width)/(Length+Width) * VCL = curvilinear
 velocity (m/s). Time-averaged velocity of a sperm head along its actual
 curvilinear path, as perceived in two dimensions in the microscope. * VSL
 = straight-line (rectilinear) velocity (µm/s). Time-averaged velocity of a
 sperm head along the straight line between its first detected position and
 its last. * VAP = average path velocity (µm/s). Time-averaged velocity of
 a sperm head along its average path. This path is computed by smoothing
 the curvilinear trajectory according to algorithms in the SCA Motility
 module. * STR = straightness. Linearity of the average path, VSL/VAP. *
 LIN = linearity. The linearity of a curvilinear path, VSL/VCL. * WOB =
 wobble. A measure of oscillation of the actual path about the average
 path, VAP/VCL. * ALH = amplitude of lateral head displacement (µm).
 Magnitude of lateral displacement of a sperm head about its average path.
 * BCF = beat-cross frequency (Hz). The average rate at which the
 curvilinear path crosses the average path. * Velocity = Classification of
 velocity into cateogries static, slow, medium, and rapid. static = &lt; 20
 µm/s, slow = 20-50 µm/s, medium = 51-100 µm/s, rapid = &gt; 100 µm/s. *
 Progression = Classification of sperm motility as 'motile' or
 'progressive' based on default settings. Sperm is classified as
 progressive when motile AND when STR &gt; 70. * IsHyperactive =
 Classification as hyperactive 'True' or 'False'. Based
 on default parameters on CASA. Not considered in avian sperm motility
 analysis. * IsMucusPenetration = Classification as Mucus Penetration
 'True' or 'False'. Based on default parameters on
 CASA. Not considered in avian sperm motility analysis. * IsCircular =
 Classification as circular track 'True' or 'False'.
 Based on default parameters on CASA. Not considered in avian sperm
 motility analysis. * IsRoundCell = Classification as Round Cell
 'True' or 'False'. Based on default parameters on
 CASA. Not considered in avian sperm motility analysis. * IsValid =
 Classification as valid track 'True' or 'False'. Based
 on default parameters on CASA. Not considered in avian sperm motility
 analysis. * IsSperm = Classification as sperm cell 'True' or
 'False'. Based on default parameters on CASA. Not considered in
 avian sperm motility analysis. ### SEM images Scanning electron microscopy
 (SEM) was used to image sperm cells from the red-browed finch and the
 lesser red-browed finch, as well as the Crimson finch and Double-barred
 finch. Sperm were adhered to a glass coverslip, dehydrated using a grade
 ethanol series and critical point dried. Coverslips were sputter-coated
 with 4-6 nm platinum using a Cressington 308R coating system, and sperm
 cells imaged using a Hitachi S-4800 Field Emission Scanning Electron
 Microscope operated at 5.0 kV. *SEM_images_for_other_finches.zip* contains
 SEM images of sperm from the Lesser red-browed finch, Crimson finch, and
 Double-barred finch. Includes images presented in Rowe et al. and
 additional images obtaind during the study.
 *SEM_images_for_Red-browed_finch.zip* contains SEM images of sperm from
 the Red-browed finch. Includes images presented in Rowe et al. and
 additional images obtaind during the study. ### TEM images Transmision
 electron microscopy (TEM) was used to image sperm cells from the
 red-browed finch. Fixed sperm samples were washed 3x in 1x PHEM, postfixed
 with 1% OsO4 + ferricyanide followed by 3 washes in 1x PHEM. Samples were
 then stained with 1% UA and finally washed another 3x with 1xPHEM. Samples
 were then dehydrated in a graded ethanol series at room temp followed by
 infiltration with a EPON (without DMP30): ETOH 1:1, rotating over 2 days
 at room temperature. Subsequently, samples were further infiltrated with
 100% EPON (with 1.5% DMP-30) and incubated overnight (rotating) at room
 temperature. Following infiltration, samples were placed in moulds and
 polymerized over two days at 60°C. Finally, ultrathin sections (80 nm)
 were obtained with a Leica ultramicrotome equipped with a diatome diamond
 knife and observed using a JEOL 1400plus transmission electron microscope
 operated at 120 kV. *TEM_images_for_Red-browed_finch.zip* contains TEM
 images of sperm cells from red-browed finch. Includes images presented in
 Rowe et al. and additional images obtained during the study. ### Testis
 mass and Body mass data Body mass (grams) was measured using an electronic
 balance (+/- 0.01 mg). The left testis was snap frozen immediately after
 collection, and we later thawed the left testis and measured testis length
 and width (+/- 0.01 mm) using digital callipers. From these dimensions we
 calculated the mass of the left testis, estimated combined testes mass by
 doubling this value, and estimated relative testes mass for each male
 (i.e., as testes mass/body mass x 100).
 *Red-browed_finch_testis_and_body_mass_data.csv* contains testis mass and
 body mass data for the five males examined in this study. Of these five
 males, only four were reproductively active at the time of sampling, and
 thus only these four males were used in our estimation of average relative
 testes mass. ### Phylogenetic tree
 *RAxML_bipartitions.240612.wg.raxml.10k_thin.tre* phylogentic tree file
 used in the analyis in Rowe et al. ### Digital images of sperm Digital
 images of sperm from the red-browed finch and lesser red-browed finch
 captured using brightfield microscopy. Images were captured using various
 camera and microscope systems. Raw images are provided for all males
 sampled during the study; However, not all sperm cells imaged could be
 measured to obtain data on sperm length. *LesserRedBrowedFinchSperm.zip*
 compressed file containing raw images of sperm cells from the lesser
 red-browed finch. *RedBrowedFinchSperm.zip* compressed file containing raw
 images of sperm cells from the red-browed finch. ## Code/Software For the
 red-browed finch, we tested for differences in sperm size (sperm head
 length, flagellum length, and total sperm length) among the different
 populations sampled using an ANOVA (see below) using R v4.4.4.1 (R Core
 Team 20204) and R studio (Posit Team 2023). Differences among populations?
 m1&lt;-aov(DATA$SpermTotalLength~~DATA$Population) summary(m1)
 m2&lt;-aov(DATA$SpermFlagellumLength~~DATA$Population) summary(m2)
 m3&lt;-aov(DATA$SpermHeadLength~DATA$Population) summary(m3) R Core Team.
 2024. R: A Language and Environment for Statistical Computing. R
 Foundation for Statistical Computing, Vienna, Austria. Posit team. 2023.
 RStudio: Integrated Development for R. Posit Software, PBC, Boston, MA. ##
 Version changes **2-July-2025**: added additional raw data associated with
 the project. Added *SpermSwimmingSpeedData.zip* containing folder
 containing sperm swimming speed data for 6 male red-browed finches
 provided as individual .csv files. Added *SEM images for other finches*
 and *SEM images for Red-browed finches* folders containing raw SEM images,
 including files included in Rowe et al. and additional images obtained
 during the study. Added *TEM images for Red-browed finch* folder
 containing TEM images of sperm cells from red-browed finch. Includes
 images presented in Rowe et al. and additional images obtaind during the
 study Added *Red-browed finch testis and body mass data.csv* Added *RAxML
 bipartitions.240612.wg.raxml.10k_thin.tre* phylogentic tree file used in
 the analysis in Rowe et al. Added *LesserRedBrowedFinchSperm.zip* and
 *RedBrowedFinchSperm.zip* containing raw digital images of sperm.