Members of the leiognathid subfamily Gazzinae, comprising approximately two-thirds of ponyfish species, are sexually dimorphic with regard to features of the light organ system (LOS). In Gazzinae, the circumesophageal light organ (LO) of males is enlarged and varies in shape compared with similarly sized conspecific females. In association with male species-specific translucent external patches on the head and flank, these sexually dimorphic LO features are hypothesized to be correlated with species-specific luminescence displays. Anatomical differences in LO shape, volume, and orientation, and its association with the gas bladder and other internal structures that function in light emission, are compared to observations of luminescence displays for every major lineage within Leiognathidae. We reconstruct the character evolution of both internal and external morphological features of the LOS to investigate the evolution of LO sexual dimorphism and morphology. Both internal and external sexual dimorphism in the ponyfish LOs were recovered as most likely to have evolved in the common ancestor of Leiognathidae, and likelihood-based correlation analyses indicate that the evolution of internal and external dimorphism in males is statistically correlated. Magnetic resonance imaging technology was applied to examine the unique internal LOs of ponyfishes in situ, which provides a new metric (LO index) for comparison of LO structure across lineages.

Museum fish collections possess a wealth of anatomical and morphological data that are essential for documenting and understanding biodiversity. Obtaining access to specimens for research, however, is not always practical and frequently conflicts with the need to maintain the physical integrity of specimens and the collection as a whole. Non-invasive three-dimensional (3D) digital imaging therefore serves a critical role in facilitating the digitization of these specimens for anatomical and morphological analysis as well as facilitating an efficient method for online storage and sharing of this imaging data. Here we describe the development of the Digital Fish Library (DFL, http://www.digitalfishlibrary.org ), an online digital archive of high-resolution, high-contrast, magnetic resonance imaging (MRI) scans of the soft tissue anatomy of an array of fishes preserved in the Marine Vertebrate Collection of Scripps Institution of Oceanography. We have imaged and uploaded MRI data for over 300 marine and freshwater species, developed a data archival and retrieval system with a web-based image analysis and visualization tool, and integrated these into the public DFL website to disseminate data and associated metadata freely over the web. We show that MRI is a rapid and powerful method for accurately depicting the in-situ soft-tissue anatomy of preserved fishes in sufficient detail for large-scale comparative digital morphology. However these 3D volumetric data require a sophisticated computational and archival infrastructure in order to be broadly accessible to researchers and educators.