High‐throughput species identification: from DNA isolation to bioinformatics

Ichthyoplankton collections provide a valuable means to study fish life histories. However, these collections are greatly underutilized, as larval fishes are frequently not identified to species due to their small size and limited morphological development. Currently, there is an effort underway to make species identification more readily available across a broad range of taxa through the sequencing of a standard gene. This effort requires the development of new methodologies to both rapidly produce and analyse large numbers of sequences. The methodology presented in this paper addresses these issues with a focus on the larvae of large pelagic fish species. All steps of the methodology are targeted towards high‐throughput identification using small amounts of tissue. To accomplish this, DNA isolation was automated on a liquid‐handling robot using magnetic bead technologies. Polymerase chain reaction and a unidirectional sequencing reaction followed standard protocols with all template cleanup and transferring also automated. Manual pipetting was thus reduced to a minimum. A character‐based bioinformatics program was developed to handle the large sequence output. This program incorporates base‐call quality scores in two types of sample to voucher sequence comparisons and provides suggested identifications and sequence information in an easily interpreted spreadsheet format. This technique when applied to tuna and billfish larvae collected in the Straits of Florida had an 89% success rate. A single species (Thunnus atlanticus) was found to dominate the catch of tuna larvae, while billfish larvae were more evenly divided between two species (Makaira nigricans and Istiophorus platypterus).     

Growth variation in larval Makaira nigricans

The Atlantic blue marlin Makaira nigricans larvae were collected from Exuma Sound, Bahamas and the Straits of Florida over three summers (2000–2002). Sagittal otoliths were extracted and read under light microscopy to determine relationships between standard length ( L _S) and age for larvae from each year and location. Otolith growth trajectories were significantly different between locations: after the first 5–6 days of life, larvae from Exuma Sound grew significantly faster than larvae from the Straits of Florida. Exponential regression coefficients were similar among years for Exuma Sound larvae (mean instantaneous growth rate, G _L = 0·125), but differed between years for larvae from the Straits of Florida ( G _L = 0·086–0·089). Differences in larval growth rates between locations resulted in a 4–6 mm difference in L _S by day 15 of larval life. These differences in growth appeared to be unrelated to mean ambient water temperatures, and may have been caused by location‐specific differences in prey composition or availability. Alternatively, population‐specific differences in maternal condition may have contributed to these differences in early larval growth.     

Habitat and food partitioning of billfishes (Xiphioidei)

Species composition and food habits of four istiophorid billfishes were investigated and compared in three different tropical areas of the eastern North Pacific Ocean by longline operations from September to November 2004. Sailfish Istiophorus platypterus, shortbill spearfish Tetrapturus angustirostris and blue marlin Makaira nigricans had specific habitat preferences and mainly occurred in the near‐continent area (13–16° N; 103–107° W), the open‐ocean area (16–18° N; 118–134° W) and the near‐equator area (5° N; 104–120° W). Small (<140 cm in lower jaw–fork length) striped marlin Kajikia audax mainly occurred in the near‐continent area; however, large (≥140 cm) individuals occurred throughout all three areas. Prey compositions of large K. audax in the three areas were different from one other reflecting the prey availability in each area. In the open‐ocean area, molid fishes were dominant in mass for both large K. audax (49%) and T. angustirostris (73%), and large K. audax also fed on ostraciid (33%) and scombrid fishes (15%). In the near‐continent area, tetraodontid fishes were dominant for large and small I. platypterus (54, 57%), and both large and small K. audax also fed on tetraodontid fishes (3, 12%). Large K. audax in this area fed mainly on scombrid fishes (86%). These results indicate that large K. audax show overlaps but little segregations of its prey with other billfishes. In the near‐equator area, stomach contents of large K. audax and M. nigricans were few and billfish prey items were thought to be scarce.     

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME,SINGLE-COPY NUCLEAR DNA,AND MITOCHONDRIAL DNA MARKERS

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θ_O) ranged from 0.00 to 0.15, with a mean of 0.08. The θ_O values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θ_O = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04–0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates.     

Development and characterization of 11 novel microsatellite loci for the roundscale spearfish Tetrapturus georgii and their cross‐species amplification among other istiophorid species

Eleven novel polymorphic microsatellite loci were developed and characterized for the recently validated roundscale spearfish Tetrapturus georgii. Characterization of these markers, based on 35 roundscale spearfish from the western North Atlantic, revealed two to 21 alleles per locus with an average expected heterozygosity (H_E) of 0·09–0·94, and all loci conformed to Hardy–Weinberg expectations. Cross‐amplification of these 11 loci against all other eight known istiophorid species indicates promising prospects for the utility of these markers for istiophorids in general.