Cryptic species complexes are groups of closely related species that are difficult or impossible to distinguish by morphological traits. These complexes are known from a wide variety of arthropods and are common among the well-studied, medically-important insects. For example, many of the anopheline vectors of malaria parasites are members of cryptic species complexes. Complexes typically include both vector and non-vector species, and two or more member species are often found sympatrically. Until the late 1950, only two such Anopheles complexes were known, the A. gambiae complex from Africa and the A. maculipennis complex from Europe. Today, dozens of Anopheles cryptic species complexes are recognized, and accumulating evidence suggests that most important malaria vectors are likely to be members of such complexes. A variety of methods have been developed for identifying the species of individual specimens from these complexes, although until recently only those based on species-specific allozymes and polytene chromosome inversions were widely used. The limitations inherent in these methods have been circumvented with DNA-based procedures, which are especially useful because both sexes and all developmental stages can be identified, and DNA can be recovered from samples stored by a wide variety of simple methods. Several DNA-based identification techniques have been developed, including hybridization assays based on species-specific repeat sequences, and diagnostic PCR fragments produced either by the use of random PCR primers or by amplifying DNA with primers based on known species-specific sequences. In this review we discuss the relative marks of different methods of cryptic species identification, with emphasis on the use of ribosomal DNA as a target for species-diagnostic PCR assays.
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