BULLETIN OF MARINE SCIENCE, 71(2): 1003–1017, 2002 EVOLUTION OF THE GILLS IN THE OCTOPODIFORMES Richard E. Young and Michael Vecchione ABSTRACT The gills of cirrate octopods are known to be unusual with terms ‘half orange’ and ‘sepioid’ commonly used to describe them. The structure and relationships of these gills to other cephalopod gills have not been adequately investigated. In this paper we investi- gate the evolution of the gills of cirrates and of octopodiforms in general. Octopodiform gills differ from the primitive cephalopod gill, as exemplified by the gills of Nautilus and decapodiforms, by the presence of septa along the axes of the primary and secondary lamellae. The septa apparently constrain the respiratory surfaces to form tree-like folds rather than the fan-like folds of other cephalopods. In members of the Vampyromorpha, the sister taxon of the Octopoda, gills have a peculiar circulation pattern that seems to be a unique adaptation to its deep-sea habitat. The arrangement of blood vessels in the cirrates involves the repositioning of the primary efferent vessels deep within the gill. In addi- tion, an axial anastomosis of superficial afferent vessels resulted in an appearance similar to a decapodiform gill but with afferent rather than efferent vessels on the ‘top’ of the gill. This, combined with the lack of a branchial canal and the presence of bilaterally sym- metrical lamellae, has resulted in the appearance of a ‘sepioid’ gill. The ‘half-orange’ gill appears to result from a foreshortening and rotation of the gill to give the impression of a nearly radial arrangement of equal-sized primary lamellae rather than the typical serial arrangement of primary lamellae that decrease in size distally. Apparently, the adapta- tions of the octopodiform gill resulted from a need to increase the efficiency of oxygen uptake. We suggest that a major factor in the evolution of the Octopodiformes was the adaptation to a habitat low in oxygen. The gills of cephalopods are unique in their mechanisms of blood flow. Not only are accessory hearts involved, but contractile vessels and muscular movements of the gill itself help. Many cephalopods are extremely active animals capable of rapid and pro- longed swimming. Many others, however, especially those in the deep sea, are lethargic and have gelatinous consistency. The nature of the gills can reveal much about the life styles of cephalopods and provide insight into how they evolved. In this paper, we exam- ine the basic structure of the gills to better understand how they evolved. Gills are difficult to describe because their dorsal and ventral aspects vary between species, a result of differences in gill attachments to the mantle wall. For simplicity, there- fore, we describe the gill based on the way it is usually illustrated (e.g., Naef, 1921, 1923) with the free surface of the gill on the ‘top’ and the attached surface on the ‘bottom’. The basic structure of the gills of the more common cephalopods has been known for many years (Cuvier, 1817; Tilesius, 1801; Joubin, 1885; Griffin, 1900; Schäfer, 1904; Isgrove, 1909; Thompsett, 1939). The structure of the Sepia gill was examined with the transmis- sion electron microscope by Schipp et al. (1979) and they summarized the structure of the gill and the blood flow through it as follows (Fig. 1). The gill has a series of primary lamellae that extend at right angles to the gill axis forming inner and outer demibranchs. Each primary lamella is folded in a fanlike pattern to form secondary lamellae that extend at right angles to the axis of the primary lamella. The secondary lamellae are free folds bound only at their tops and bottoms. These secondary lamellae are, in turn, folded in a fanlike pattern to form tertiary lamellae. The directions of folding of the secondary and tertiary lamellae are at right angles to one another. The major afferent vessel passes down 1003 1004 BULLETIN OF MARINE SCIENCE, VOL. 71, NO. 2, 2002 Figure 1. Diagram of the blood flow and folding patterns of respiratory surfaces of Sepia. On the right is a cut through the gill showing a primary lamella of each demibranch. On the left is a blowup of a small section of the primary lamella to show the fanlike folding of both the secondary lamellae and tertiary lamellae. Compare this with the similar pattern seen in the oegopsid, Histioteuthis hoylei (Fig. 6). v - vessel. Arrows indicate direction of blood flow. Drawing slightly modified from Schipp, et al., 1979. the axis of the gill just above the branchial gland and sends branching vessels up the surface of the external ‘ridge’ of the secondary lamellae. The primary efferent vessel also runs along the gill axis but on the top of the gill. It is fed by secondary efferent vessels that run along the top surface of the secondary lamellae. The latter vessels drain blood from the secondary lamellae via vessels that extend directly downward into the interior of the lamella when each lamellar fold passes beneath one of these secondary efferent ves- sels. Although the gills of Sepia differ considerably in general appearance, the basic orga- nization presented here is found also in Nautilus (e.g., Griffin, 1900). The structure of the gill and the blood flow in Octopus has been reviewed by Wells and Wells (1982). The basic structure of the Octopus gill is presented in Figure 2. Some fundamental differences exist between Octopus and Sepia gills. The afferent blood flow of Octopus is similar to that of the Sepia, although the vessels running up the external surface of the secondary lamellae (i.e., from the base to the top of the lamella) tend to anastomose with those of the opposite side at the ‘top’ of the gill (Fig. 6B,C). The efferent flow differs in that each secondary efferent vessel does not run along the free surface of the primary lamella but is buried in the middle of this lamella. The type of folding of the respiratory surfaces is presented in the Results To understand the evolution of the gill, several key taxa were examined in detail. Most important of these is Vampyroteuthis, a phylogenetic relic, whose gill structure was un- known. Also important are the cirrate octopods whose gill structure is poorly understood. Cirrates generally are thought to have two types of gills: ‘half-orange’ gills, a name that reflects their general appearance, and ‘sepioid’ gills a name that reflects their resem- blance to sepiid gills. The basic differences in cirrate gills, however, have never been adequately addressed. Although some of the basic differences in gill structure between Sepia and Octopus have long been known, no one previously has attempted to examine how they evolved, which, of course, depends on first having a resolved phylogeny. Also, little use has been YOUND AND VECCHIONE: GILLS IN THE OCTOPODIFORMES 1005 made of gill characteristics in phylogenetic studies of cephalopods. Young and Vecchione (1996) used 25 characters to examine the phylogenetic relationships among neocoleoid (sensu Haas, 1997) cephalopods. Many, however, could not be polarized and the result- ing analysis found only 16 characters useful. Their study resolved four basic nodes. The first node separated the Decapodiformes from the Octopodiformes. The decapodiform node had a support index of 1 (a single unambiguous character change supported the node). The octopodiform node had a support index of 3 (three unambiguous character changes supported the node) which is not strong support. The support for the octopod node was high (support index = 8) but support for the two subgroups within the Octopoda was about the same as for the octopodiform node. Support for most aspects of the tree, therefore, was not strong and the phylogeny presented cannot be considered firmly estab- lished. In spite of the fact that more recent studies on molecular phylogeny tend to sup- port these nodes (e.g., Carlini and Graves, 1999), further morphological analysis through the use of additional characters is needed. Here we derive characters mostly from the basic differences described above for Sepia and Octopus and examine them in represen- tatives of 17 cephalopod families. MATERIALS AND METHODS We injected the gills of 10 specimens of Vampyroteuthis, captured by trawls from the fishery training vessel HOKUSEI MARU from Hokkaido University in Japan, with squid ink taken from re- cently captured ommastrephid squids. Squid ink is easy to inject and doesn’t diffuse out of the vessels with time as can be the case with some commercial inks. We also injected the gills of Sthenoteuthis oualaniensis and Histioteuthis hoylei. All the above cephalopods were captured in Hawaiian waters in open nets that fished from the surface to approximately 800 m depth. Gills were subsequently examined in the laboratory via dissection under a stereomicroscope. Histological methods would have been helpful in confirming some observations but time constraints prevented their use. These and several other key species, described below, were examined in greatest detail. One of these, Graneledone verrucosa, was chosen because of the large size of the octopus and, therefore, large gills. This octopus was taken at 40°N, 71°W at a depth of 600–800 m. Once we had identified characters and character states, we were then able to survey a broad assortment of species. Gill characters have not been fully utilized in phylogenetic studies for several reasons. Gills are usually fragile and strongly affected by deformation during fixation. Poorly fixed gills reveal little useful information. Unlike vertebrate gills, cephalopod gills and their blood vessels are highly muscular and the state of contraction of the gill or the blood vessels can also greatly affect the appearance of the gills. Five gill characters extracted from this study were added to the data base of Young and Vecchione (1996).