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In a few species (mostly marine), such cells acquire pigment prior to chromatophore migra- tion and the actual migration can be observed and documented. But in cypriniform and most other freshwater fishes, pigment is not present in chromatophores until after the cells reach their ultimate destination. For a specific species and developmental stage, pigmental variation in general or specific areas is largely a function of the number of chromatophores exhibiting pigment rather than a difference in chromatophore distribution. Chromatophores without pigment cannot contri- bute to the visible pattern. In addition, pigment in chromatophores can be variously displayed from tight, contracted spots, resulting in a relatively light appearance, to widely expanded, reticular networks, resulting in a dark or more strongly pigmented appearance. Differences in environmental conditions and food can signifi- cantly affect the presence and displayed form of pigmentation. Accordingly, researchers must be aware that pigmentation of cultured specimens can appear quite different from that of field-collected material. Pigmentation often changes considerably as larvae and early juveniles grow. Most of the change is due to increased numbers and distri- bution of chromatophores. Observable pigmen- tation might also be lost from certain areas through loss of pigment in chromatophores, loss of chromatophores themselves, or, in the case of subsurface or internal chromatophores, by growth and increased opacity of overlying tissues. Peritoneal melanophore pigmentation is an obvious character for later stages of some larvae, but in late metalarvae and especially juveniles, dark peritoneal pigmentation can be obscured by overlying muscle or membranes with silvery iridophores (this silvery pigment often dissipates over time in formalin preserva- tive, but is usually retained in alcohol). If internal melanophore pigmentation is obscured by overlying tissues, it can be observed by selec- tive dissection or careful clearing of specimens. Osteology When externally visible characters fail to segregate species conclusively, osteological characters may come to the rescue. While whole-specimen clearing and cartilage- and bone-staining techniques are relatively simple (see Methods), they require much time (a few days, mostly waiting) and a fair amount of attention (monitoring progress and changing fluids). Soft (longwave) X-ray techniques (Tucker and Laroche 1984) may be faster and easier, especially when examining many speci- mens, but they require appropriate X-ray equip- ment and a darkroom. Dunn (1983,1984) reviewed use of skeletal structures and the utility of developmental osteo- logy in taxonomic studies. Among the first bones to ossify are those associated with feed- ing, respiration, and orientation (e.g., jaws, bones of the branchial region, cleithrum, and otoliths). The axial skeleton follows with for- mation of vertebrae and associated bones. Once the axial skeleton is sufficiently established, median- and pelvic-fin supports form, and fins develop. Presence, number, position, and shape of certain bones in many parts of the skeleton can have diagnostic value, even for closely related species. Use of osteological characters for identification of fish larvae has received little attention, but its potential value is great, particularly for confirmation of questionable identities and for species in which external characters are diagnostically inadequate. 15