Schematic diagram of the experimental set-up. Water was fed into the inner compartment of the experimental tank through perforated airline tubing. It then passed to the outer compartment through many small holes and was drained by a siphon. The cuttlefish (black) was placed in the inner compartment.

Ultrasound images illustrating the spatial relationships between organs in a non-dissected cuttlefish. The dorsal mantle is always at the top of the image, and the ventral mantle is always at the bottom. Non-cardiovascular organs are abbreviated with lower case letters. (A) Transverse section through the anterior mantle. (B) Midsagittal section showing organs from planes 1 and 2 of Fig. 1 (see supplementary material, Movie 1). The head is towards the right. (C) Transverse section through the branch point and the efferent branchial vessels (plane 3, Fig. 1; also see supplementary material, Movie 2). (D) An oblique transverse section through the ventricle. Organs visible on the right are posterior to those on the left. This roughly corresponds to plane 4 in Fig. 1. Nidamental glands (ng) are present only in females. AU, auricle; AVC, anterior vena cava; BP, branch point; cf, collar flap; dg, digestive gland; EBV, efferent branchial vessel; f, funnel; int, shared course of the intestine and ink sac duct; LVC, lateral venae cavae; m, mantle; V, ventricle. All scale bars are 2 cm.

Phase shift between the maximum contraction of the ventricle (arbitrarily set at 0°) and the branch point (open circles; BP in Fig. 1), the lateral venae cavae (grey circles; LVC in Fig. 1), the branchial hearts (black circles; BH in Fig. 1) and the efferent branchial vessels (hatched circles, EBV in Fig. 1). Time proceeds clockwise. Each concentric circumference shows the averaged data for one cuttlefish. NS indicates averages calculated from raw data that were not significantly similar (significance only determined if there were more than two data points. There were never more than four data points). Because data on the efferent branchial vessel and ventricle were not collected simultaneously, contraction phase of the efferent branchial vessel was calculated from its average phase shift from the average phase shift of the branch point. We obtained no data on the branchial heart for the cuttlefish of innermost circumference.

(A) Schematic representation of the valve when open. Blood travels from the anterior vena cava (AVC) into the lateral venae cavae (LVC). When blood pressure rises in the LVC relative to the AVC, the valve closes. (B) Blue tracing medium in the AVC (circled). (C) Once tracing medium was pushed from the AVC (circled) into the branch point, it could not be pushed back into the AVC. Scale bar, 1 cm. (D) Close to the lateral wall, the valve spanned the whole vessel (×6 magnification; scale bar, 0.5 mm). (E) Mid-sagittally a natural split occurred in the valve tissue. We verified that it was not an artifact through analysis of successive serial sections. The distal end of the larger portion of the valve tissue was reinforced by a polysaccharide-rich thickening (×6 magnification; scale bar, 0.5 mm). (F) The muscular, small side of the valve, indicated by a broken box in E. Muscle cells stained red (×60 magnification; scale bar, 50 μm). AVC, anterior vena cava; BP, branch point; LVC, lateral venae cavae; PAV, posterior azygos vein.

Phase shifts between the contraction cycle of the mantle and the contraction cycle of Point A on the anterior vena cava (AVC). Full expansion of the mantle is arbitrarily set at 0° and time proceeds clockwise. The heavy black line starts at the full contraction of Point A (AVC) and ends at the full expansion of Point A. The heavy grey line starts at full mantle contraction and ends at full mantle expansion. Each concentric circumference represents the averaged data of a different cuttlefish.