Small brains: Body shape constrains tissue allocation to the central nervous system in ant-mimicking spiders.

DATA & CODE FILE OVERVIEW
This data repository consists of 1) Supplementary material S1 (PDF), 2) Dataset S1 (excel spreadsheet), 3) R project files, 4) Analyze 7.5 files, 5) Segmentation files, 6) Tiff stacks & log files (raw data) and 7) this README document.

1) Supplementary material S1 (PDF)
Supplementary material text:
- Section I. Extended methods - A detailed description of the methodology used for the sample preparation for microCT scanning and the segmentation of microCT scans.
- Section II. Equation to convert pixel size to volume for the relevant structures - description and equation of how pixel count was calculated as a volume for all analysed anatomical structures used in the study.
- Section III. Equations used for quantifying the central nervous system (CNS) and venom gland volume relative to total prosoma volume, the relative sizes of the CNS and venom glands as a proportion of the total prosoma volume and the relative sizes of the smaller structures within the CNS as a proportion of the entire CNS volume. Includes a Table with abbreviations used in the equations.
Supplementary Figures:
- Figure S1. A plot showing the bootstrap segmentation repeatability estimates of measurements for the PNSOG, venom glands and the arcuate body across the two study species.
Supplementary Tables:
- Table S1: Minimum, maximum, mean, and sample standard deviation (±) values for total body length (mm), prosoma length (mm), and prosoma/width ratios for all specimens.
- Table S2: Absolute minimum (mm3), maximum (mm3), mean (mm3), and sample standard deviation (±) values, for the volumes of the 10 internal anatomical structures measured, the visual neuropils collectively and the total CNS volume for all specimens.
- Table S3: Log2 ratios of percentage volumes of internal structures of the CNS for M. smaragdina and A. hariola, relative to the total central nervous system volume (TCV).
- Table S4: Proportional sizes (minimum, maximum, mean, and sample standard deviation (±)), relative to the TCV (mm3), for the measured 9 internal anatomical structures included as components of the CNS, as well as the visual neuropils, for all specimens.

2) Dataset S1 (excel spreadsheet)
Segmentation data (Tab 1):
Specimen data, including collection data, specimen measurements, raw volumetric data and calculations of volumes for all specimens.

Column A: Specimen # = unique specimen identification number used in analysis.
Column B: Species = species of specimen.
Column C: Sex = sex of specimen.
Column D: Collection location = location where the specimen was collected.
Column E: GPS co-ordinates = GPS co-ordinates for the location where the specimen was collected.
Column F: Collection date = the date the specimen was collected.
Column G: Dissection date = the date of specimen dissection for preperation for microCT scanning.
Column H: Prosoma length (mm) = the specimens prosoma length in millimetres.
Column I: Prosoma width (mm) = the specimens prosoma width in millimetres.
Column J: Prosoma width/length ratio = the specimens prosoma width to length ratio (i.e., prosoma width divided by prosoma length).
Column K: Total body length (mm): the specimens total body length from posterior of prosoma (not including chelicerae) to the anterior of the abdomen (not including spinnerets) in millimetres.
Column L: Whole-body mass (WBM) (g) = the specimens whole-body mass in grams.
Column M: Voxel size - pixel size (cubed) = value calculated from the reported pixelsize in the tiff stack log file (i.e., pixelsize cubed).
Column N: Protocerebral neuropils (PN) and the subesophageal ganglion (SOG) (i.e., PNSOG) (voxel count) = the voxel count for the PNSOG taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column O: PNSOG (mm3) = the volume of the PNSOG in millimetres cubed (i.e., voxel size (column M) times voxel count (column N).
Column P: Arcuate body (voxel count) = the voxel count for the arcuate body taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column Q: Arcuate body (mm3) = the volume of the arcuate body in millimetres cubed (i.e., voxel size (column M) times voxel count (column P).
Column R: Mushroom bodies (voxel count) = the voxel count for the mushroom bodies taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column S: Mushroom bodies (mm3) = the volume of the mushroom bodies in millimetres cubed (i.e., voxel size (column M) times voxel count (column R).
Column T: Mushroom body bridge (voxel count) = the voxel count for the mushroom body bridge taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column U: Mushroom bodies (mm3) = the volume of the mushroom body bridge in millimetres cubed (i.e., voxel size (column M) times voxel count (column T).
Column V: AL2/PL2 (voxel count) = the voxel count for the 2nd order visual neuropils of the anterior/posterior lateral eyes taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column W: AL2/PL2 (mm3) = the volume of the 2nd order visual neuropils of the anterior/posterior lateral eyes in millimetres cubed (i.e., voxel size (column M) times voxel count (column V).
Column X: AM1 (voxel count) = the voxel count for the 1st order visual neuropils of the anterior median eyes taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column Y: AM1 (mm3) = the volume of the 1st order visual neuropils of the anterior median eyes in millimetres cubed (i.e., voxel size (column M) times voxel count (column X).
Column Z: AM2 (voxel count) = the voxel count for the 2nd order visual neuropils of the anterior median eyes taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column AA: AM2 (mm3) = the volume of the 2nd order visual neuropils of the anterior median eyes in millimetres cubed (i.e., voxel size (column M) times voxel count (column Z).
Column AB: PL1 (voxel count) = the voxel count for the 1st order visual neuropils of the posterior lateral eyes taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column AC: PL1 (mm3) = the volume of the 1st order visual neuropils of the posterior lateral eyes in millimetres cubed (i.e., voxel size (column M) times voxel count (column AB).
Column AD: AL1 (voxel count) = the voxel count for the 1st order visual neuropils of the anterior lateral eyes taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column AE: AL1 (mm3) = the volume of the 1st order visual neuropils of the anterior lateral eyes in millimetres cubed (i.e., voxel size (column M) times voxel count (column AD).
Column AF: Visual neuropils (mm3) = the volume of all visual neuropils added together (5) in milimetres cubed (i.e., column W + column Y + column AA + column AC + column AE).
Column AG: Venom glands (voxel count) = the voxel count for the venom glands taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column AH: Venom glands (mm3) = the volume of the venom glands in millimetres cubed (i.e., voxel size (column M) times voxel count (column AG).
Column AI: Total CNS volume (mm3) (TCV) = the summed volume of all components of the CNS in milimetres cubed (i.e., column O + column Q + column S + column U + column W + column Y + column AA + column AC + column AE).
Column AJ: PNSOG / TCV = calculated proportion of the PNSOG volume relative to the total CNS volume (i.e., column O divided by column AI).
Column AK: AB / TCV = calculated proportion of the arcuate body volume relative to the total CNS volume (i.e., column Q divided by column AI).
Column AL: MB / TCV = calculated proportion of the mushroom bodies volume relative to the total CNS volume (i.e., column S divided by column AI).
Column AM: MBbr / TCV = calculated proportion of the mushroom body bridge volume relative to the total CNS volume (i.e., column U divided by column AI).
Column AN: (AL2/PL2) / TCV = calculated proportion of the 2nd order visual neuropils of the anterior/posterior lateral eyes volume relative to the total CNS volume (i.e., column W divided by column AI).
Column AO: AM1 / TCV = calculated proportion of the 1st order visual neuropils of the anterior median eyes volume relative to the total CNS volume (i.e., column Y divided by column AI).
Column AP: AM2 / TCV = calculated proportion of the 2nd order visual neuropils of the anterior median eyes volume relative to the total CNS volume (i.e., column AA divided by column AI).
Column AQ: PL1 / TCV = calculated proportion of the 1st order visual neuropils of the posterior lateral eyes volume relative to the total CNS volume (i.e., column AC divided by column AI).
Column AR: AL1 / TCV = calculated proportion of the 1st order visual neuropils of the anterior lateral eyes volume relative to the total CNS volume (i.e., column AE divided by column AI).
Column AS: VN / TCV = calculated proportion of all the visual neuropils summed volume relative to the total CNS volume (i.e., column AF divided by column AI).
Column AT: Prosoma volume (voxel count) = the voxel count for the total prosoma taken from the volume and statistics function in ITK-snap based on the segmention analysis.
Column AU: Total prosoma volume (mm3) (TPV) = the volume of the total prosoma in millimetres cubed (i.e., voxel size (column M) times voxel count (column AT).
Column AV: TCV / TPV = calculated proportion of the total CNS volume relative to the total CNS volume (i.e., column AI divided by column AU).
Column AW: VG / TPV = calculated proportion of the venom gland volume relative to the total CNS volume (i.e., column AH divided by column AU).
Column AX: Log TCV = the log of the total CNS volume (i.e., LOG(AI)).
Column AY: Log VG volume = the log of the venom gland volume (i.e., LOG(AH)).
Column AZ: Log Mass = the log of the whole-body mass in grams (i.e., LOG(L)).

SD calculations (Tab 2):
Copy of Tab 1 with the addition of standard deviation calculations for the reported data found in Supplementary Tables S1, S2 & S4.

Repeatability analysis (Tab 3):
Voxel counts of the ventral nerve cord, venom glands and arcuate body as determined by two independent assessors to determine the repeatability of the segmentation.

Column A: Specimen # = unique specimen identification number used in analysis.
Column B: Species = species of specimen.
Column C: voxel count of the ventral nerve cord assessed by analyser 1 (data used in the reported anaylses).
Column D: voxel count of the ventral nerve cord assessed by analyser 2 (data not used in the reported anaylses).
Column E: voxel count of the venom glands assessed by analyser 1 (data used in the reported anaylses).
Column F: voxel count of the venom glands cord assessed by analyser 2 (data not used in the reported anaylses).
Column G: voxel count of the arcuate body assessed by analyser 1 (data used in the reported anaylses).
Column H: voxel count of the arcuate body assessed by analyser 2 (data not used in the reported anaylses).
Column I: notes regarding the segmentation.

3) R project files:
The R project files zipped folder contains the scripts used for all analyses used in the study and creating Figures 3 & 4.
Analytic scripts:
1) Allometric scaling analysis (i.e., allometry.R) - test of common slopes and differences in elevation for log CNS and venom gland volume against log body mass (including creating Figure 3).
2) Statistical analysis of of differences between components of the CNS (i.e., brain-structure.R) as relative proportions of the total CNS volume for both study species (including creating Figure 4).

4) Analyze 7.5 files:
- Analyze files were created by importing the tiff stacks produced by the microCT scanning into ImageJ and then exporting them in the Analyze 7.5 format. The Analyze 7.5 files were then imported into the ITK-snap software for segmentation and volumetric analysis.

5) Segmentation files:
- The segmentation files contain the segmentation data of the CNS, venom glands and the prosoma for all specimens used in the analyses.

6) Tiff stacks & log files - raw data produced from the microCT scanning used to create the Analyze 7.5 files in ImageJ.

7) This README file.

REFERENCES
Sombke, A., Lipke, E., Michalik, P., Uhl, G. and Harzsch, S., 2015. Potential and limitations of X‐Ray micro‐computed tomography in arthropod neuroanatomy: A methodological and comparative survey. Journal of Comparative Neurology, 523(8), pp.1281-1295.

Yushkevich, P.A., Piven, J., Hazlett, H.C., Smith, R.G., Ho, S., Gee, J.C. and Gerig, G., 2006. User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. Neuroimage, 31(3), pp.1116-1128.

