CHAPTER TWO Getting under the Skin A CALL FOR SPECIMEN-BASED RESEARCH ON THE INTERNAL ANATOMY OF BIRDS* Helen F. James Abstract. Study of the comparative internal anatomy scans enable the production of detailed computer of birds is undergoing a renaissance, spurred by models of the bones for biometric and biome- technological and methodological advances. Our chanical studies. Online repositories of morpho- ability to image the soft anatomy and bones in 3D logical image files can make internal anatomy using x-ray computed tomography (CT), magnetic widely accessible. Avian skeletal and anatomical resonance imaging (MRI), and optical surface collections are far less comprehensive than tradi- imaging has opened the door to a wide range of tional study skin collections, yet they represent a analyses using avian skeletal and anatomical spec- wealth of relatively unexplored phenotypic varia- imens. For anatomical specimens, simple staining tion in birds. The purpose of this chapter is to techniques that enhance the contrast between dif- review and encourage the use of these techniques ferent soft tissues, and at the same time raise the in the study of avian phenotypes, emphasizing the opacity of soft tissues to x-rays, enable the simul- various specimen types that can be used as well taneous 3D visualization of skeletal and soft tis- as the deeper understanding of the ecological and sue anatomy. Image processing software further behavioral context of the phenotype that emerges allows anatomical features such as individual from such studies. muscles to be segregated and measured on a com- puter monitor, without necessitating dissection of Key Words: anatomical specimens, avian paleontol- the anatomical specimen. Perfusion techniques ogy, computed tomography, functional anatomy, can allow the vascular or respiratory system to be geometric morphometrics, skeletons, spirit col- similarly imaged. For the skeleton, CT and optical lections, 3D imaging. * James, H. F. 2017. Getting under the skin: a call for specimen-based research on the internal anatomy of birds. Pp. 11–22 in M. S. Webster (editor), The Extended Specimen: Emerging Frontiers in Collections-based Ornithological Research. Studies in Avian Biology (no. 50), CRC Press, Boca Raton, FL. 11 dvances in instrumentation and meth- In consonance with the theme of this volume, ods for studying vertebrate morphology I omit discussion of several important research Aoffer exciting new ways to reveal and areas that admittedly make good use of skeletal analyze internal phenotypic variation in birds. and anatomical collections, but that do not aim Emblematic of these advances are the captivat- specifically to understand the phenotype in an ing 3D visualizations of internal anatomy that evolutionary or ecological context. Thus, I discuss have recently appeared in a diversity of journals avian paleontology but not zooarchaeology, and I (e.g., Figure 2.1). The primary tools employed to omit ancient biomolecules such as the gene frag- create those images are computed tomography ments and proteins that are often preserved in skel- (CT), magnetic resonance imaging (MRI), and etal and sometimes anatomical specimens. Good laser or other optical surface scanning technolo- reviews are available on these topics, such as Wiley gies, combined with software programs for cre- et al. (Chapter 6, this volume) for stable isotope ating 3D images from the scans (Chatham and analysis of avian museum specimens, Wood and Blackband 2001, Rosset et al. 2004, Goldman De Pietri (2015) for emerging paleo-ornithological 2007, Marshall and Stutz 2012). These approaches, techniques including the study of ancient bio- together with a suite of other techniques, some molecules, and McCormack et al. (Chapter 9, old and some new, have opened a frontier in our this volume) for genomic approaches that utilize ability to see and study the insides of birds. specimens. My objective in this chapter is to spur orni- thologists to adopt these techniques, to incorpo- AvIAN ANATOMICAL AND rate internal anatomical traits more frequently SKELETAL COLLECTIONS in their study designs, and to collect the ana- tomical specimens that make these stud- Traditional museum study skin collections lie at ies possible. The chapter touches on topics in the heart of our knowledge about the species and avian biology to which studies of skeletal and taxonomy of birds, their geographic distributions, anatomical specimens principally contribute, plumages, and other external traits. The major sci- and highlights emerging techniques in imag- entific collections of birds were built up primarily ing, data gathering, and data analysis that can from the 1880s through the 1960s (Winker 1996, facilitate research using those collections. I also Livezey 2003), a period when much ornithological offer brief comments on the readiness of avian research effort was devoted to establishing basic skeletal and anatomical collections to fulfill this information about the systematics and biogeogra- research mission. phy of the world’s birds. The avian study skin was Downloaded by [Smithsonian Institution Libraries], [Helen James] at 10:21 11 October 2017 Figure 2.1. Visualization of the cranial vasculature of an American Flamingo (Phoenicopterus ruber), created by injecting a latex/barium medium into the vasculature and producing a CT scan. (From Holliday et al. 2006.) 12 STudies IN Avian Biology NO. 50 Webster adopted early on as a common unit of comparison which are anything other than a study skin. It for this research, a unit that was also conveniently almost goes without saying that the phenotypic fungible for curators engaged in trades with other variation that lies beneath the skin of birds has not institutions. When, in the 1960s and 1970s, the been studied to nearly the same extent as has the attention of ornithologists turned increasingly to variation observable in study skins, particularly ecology, the study skin was still the specimen of when it comes to intrageneric and intraspecific choice because it preserved external traits of birds patterns. Thus, specimens that capture the inter- that could be compared with those of birds stud- nal anatomy of birds can be considered as part of ied in the field. the “extended specimen” that is the focus of this A consequence of this love for the beautiful book (see Chapter 1, this volume). study skin is that collectors of birds have preserved far fewer skeletal and anatomical specimens than COMPARATIvE ANATOMY skins. As an example, at the National Museum of Natural History (NMNH; Washington, DC), the The comparative study of avian anatomy, tra- avian holdings fall out as roughly 84% study skins ditionally based on dissection and histology, representing 75% of the world’s species, but only has fallen into a relatively quiet period in recent 11% skeletons representing 54% of the species and decades (Livezey 2003), although certainly some 5% anatomical specimens representing 44% of the notable work has continued to appear (e.g., Moreno species; eggs and nests are excluded from these and Carrascal 1993, Patak and Baldwin 1998, calculations. The skeletal and anatomical holdings Maxwell and Larsson 2007). The rise in interest in at NMNH are the most comprehensive anywhere, avian ecology and behavior, and the supplanting yet they continue to lag far behind the study skin of comparative anatomy with molecular genetics collection, even though, in recent decades, the in the field of systematics, must partly account for curators at NMNH have placed a priority on clos- this. The result has been that fluid-preserved avian ing those gaps (Figure 2.2). The emphasis on study specimens have by and large been languishing in skins for alpha taxonomy is further highlighted their jars, and curators have received very few by the composition of avian type collections. requests for their use. Yet this situation is begin- Again using the NMNH as an example, 3,971 type ning to reverse itself, and anatomical specimens specimens are in the bird collection, only five of are coming back in demand thanks to exciting 70 60 50 40 30 ousands of specimens 20 Downloaded by [Smithsonian Institution Libraries], [Helen James] at 10:21 11 October 2017 10 0 18201830184018501860187018801890190019101920193019401950196019701980199020002010 Decade Skins Eggs Nests Skeletons Anatomicals Figure 2.2. Bird specimens added to the scientific collection of the National Museum of Natural History, Smithsonian Institution, by decade and specimen type. The 2010 time bin represents the first half of the decade. Getting under the Skin 13 developments in imaging and computer-based fluid-preserved anatomical specimens of small analytical methods. animals with iodine or iodine potassium iodide Some recent examples of this trend include (I2KI) to enhance the contrast between soft tis- studies of the vascular system of the head of sues, such as individual muscles and organs, in flamingoes that revealed a previously unknown CT scans. A mineral stain like iodine also raises paralingual sinus that may be part of the filter the opacity of soft tissues to x-rays, making them feeding mechanism (Holliday et al. 2006), a study similar enough to bone in opacity that bones and of the anatomy of the knee joint of ostriches soft anatomy can be visualized in a single CT scan. that detailed the skeletomuscular system associ- This simple but exciting insight about the advan- ated with their unusual second patella (Chadwick tages of iodine-based staining facilitates simul- et al. 2014), and a study