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grow are often characteristic at the species level. Used with caution, preferably in com- bination with other characters, and with an awareness of both intra- and inter-regional variation, chromatophore distribution and patterns for many fishes are among the most useful characters available for identification. However, in some instances, differences are so subtle or variation so great that use of pigmentation is impractical and may be misleading. In cypriniform and most other fishes, chromatophores other than melanophores have not been sufficiently studied for identification purposes. Such chromato- phores are typically neither as numerous nor as obvious as melanophores and their pig- ments are difficult to preserve. In contrast, melanin, the amino acid breakdown product responsible for the dark, typically black, appearance of melanophores (Lagler et al. 1977), remains relatively stable in preserved specimens. However, melanin is subject to fading and bleaching if specimens are stored or studied extensively in bright light for long periods of time, stored in highly alkaline pre- servatives, or subjected to changing concen- trations of preservative fluids. To minimize the latter effects, as well as shrinkage and deformation, dilute formalin solutions (3-5%, unbuffered or buffered to near neutral) are strongly recommended over alcohol solutions as storage media. Most of the following dis- cussion refers to chromatophores in general, but in this manual and others for freshwater species in North America, pigmentation typi- cally refers to that of melanophores. According to Orton (1953), pigment cells originate in the neural crest region (dorsal portion of body and tail) and migrate in amoeboid fashion in waves to their eventual position. The first wave of chromatophores occurs late in the embryonic period or early in the larval period and establishes a rela- tively fIXed basic or primary pattern of chromatophore distribution. In a few species (mostly marine), such cells acquire pigment prior to chromatophore migration and the actual migration can be observed and docu- mented. 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 is largely a function of number of chromatophores exhibiting pig- ment, either in general or in specific areas, and not a change in chromatophore pattern or distribution. Lack of pigment in chroma- tophores of a particular an area, precludes that portion of the visible pattern. In addi- tion, pigment in chromatophores can be var- iously displayed from tight, contracted spots, resulting in a relatively light appearance, to widely expanded, reticular networks, resulting in a dark or more strongly pigmented appear- ance in the affected area. Differences in environmental conditions and food can signi- ficantly affect the presence and displayed form of pigmentation. Accordingly, research- ers must be aware that pigmentation of cul- tured 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 distribution of chromatophores. Observable pigmentation might also be lost from certain areas through loss of pigment in chromato- phores, 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 tissues with silvery iridophores (this silvery pigment often dissip.ates over time in preservative). If internal melano- phore pigmentation is obscured by overlying tissues, it can be observed by selective 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 chang- ing fluids). Soft (longwave) X-ray techniques (Tucker and Laroche 1984) may be faster and 14