Material and methods
Studied speciesnext section
The following species were studied:
Lomisoidea Bouvier, 1895
Lomisidae Bouvier, 1895
Lomis hirta (Lamarck, 1818) (North coast of Tasmania near Penguin: 41°6’1.469’’ S, 146°3’23.711’’ W)
Aegloidea Dana, 1852
Aeglidae Dana, 1852
Aegla cholchol Jara & Palacios, 1999 (Cholchol river, Chile: 38°36’5.56’’ S, 72°52’25.36’’ W)
Chirostyloidea Ortmann, 1892
Kiwaidae MacPherson, Jones & Segonzac, 2005
Kiwa puravida Thurber, Jones & Schnabel 2011 (deep sea off Costa Rica: 8°55.8’ N 84°18.8’ W)
A full list of the studied specimens (which are catalogued and stored in the Zoological Collection of the University of Rostock) and a description of each of the methods used are provided in Supporting Information Table S1.
The acrylic casting resin Mercox 2-CL (Ladd Research, Williston, VT) or the polyurethane-based casting resin PU4ii (vasQtec, Zurich, Switzerland) was injected into the heart of defrosted specimens (K. puravida) or specimens previously killed using CO2 -saturated water (A. cholchol) or water mixed with 1 g / 100 mL clove powder (L. hirta). In a first step, PU4ii was mixed with an x-ray opaque additive (sublimated iodine or Tetric EvoFlow® composite (Ivoclar Vivadent, Eilwangen, Germany) dissolved in 2-butanone) while Mercox was used unadulterated (see S1). In a next step, the resin was mixed with the appropriate catalyst and placed in a 5 ml syringe (Luer Lock Solo, Braun, Melsungen, Germany) just before use. The resin in the syringe was injected via an injection cannula (diameter 0.3-0.6 mm, Sterican, Braun, Melsungen, Germany) through the carapace into the heart and the specimens were left for several minutes to allow the resin to polymerize and temper.
Fixation and dehydration
For MicroCT specimens (injected and non-injected) were fixed in Dubosc’s fluid or with saline (30-35 PSU) Bouin’s fluid or glutaraldehyde (4%) for several days, washed, stained with 1% alcoholic or aqueous iodine, washed and then critical point (EMITECH K850, UK) or freeze (UniCryo MC2L, UniEquip, Munich, Germany) dried.
Microcomputer tomography (MicroCT)
Dried specimens were mounted with hot glue on a specimen holder. X-ray imaging was performed with a
- • Phoenix Nanotom-180 (Phoenix|x-ray, GE Sensing & Inspection Technologies) high-resolution MicroCT system.
- • Xradia MicroXCT-200 X-ray imaging system (Carl Zeiss X-ray Microscopy Inc., Pleasanton, USA).
For detailed parameters and details of the process see Keiler et al. (2015b).
For all 3D reconstructions, the software Imaris 6.4.1 and 7.0.0 (Bitplane) was used (for details of the process see Keiler et al., 2015a).
All figure plates were arranged using Corel Graphics Suite X3 (Corel, Ottawa). Bitmap images were embedded into Corel Draw X3 files and digitally edited with Corel PhotoPaint X3.