Molecular species identification of newly hatched Hawaiian amphidromous gobioid larvae

This report describes a method for the determination of species identity of newly hatched larvae of five sympatric Hawaiian amphidromous gobioids (Len tipes concolor, Sicyopterus stimpsoni, Awaous guamensis, Stenogobius hawaii ensis, and Eleotris sandwichensis). Polymerase chain reaction (PCR) was use d to amplify a homologous section of the cytochrome b (Cyt b) region of the mitochondrial genome (mtDNA) from adults of all five species. The resultin g PCR-amplified DNA was subjected to restriction fragment length polymorphi sm (RFLP) analysis producing species-specific restriction patterns. PCR pro ducts from the five species were sequenced to substantiate correct amplific ation, restriction site locations, and fragment sizes. The sequence data we re also used to construct a phylogenetic tree. Individual, newly hatched, w ild-caught larvae of amphidromous gobioids of unknown species affinity were sorted into six morphotypes based on physical characteristics. These typed larvae and those from two species that spawned in captivity were subjected to the same molecular analysis as the adults. The RFLP results from adults and larvae were compared, allowing larval morphotypes to be assigned to th e appropriate species. These comparisons permitted construction of an ident ification key to the newly hatched larvae of these species based solely on physical characteristics for use in future field studies.