DIZON ET AL.: GENETIC STOCKS AND STATISTICAL POWER ColCOFl Rep., Vol. 35, 1994 INTRASPECIFIC STRUCTURE OF THE NORTHERN RIGHT WHALE DOLPHIN (LISSODELPHIS BOREALIS): THE POWER OF AN ANALYSIS OF MOLECULAR VARIATION FOR DIFFERENTIATING GENETIC STOCKS ANDREW E IIIZON, CARRIE A LEDUC', AND R GENE LEDUC Southwest Fishenes Science Center National Manne Fishenes Service, NOAA PO Box271 La Jolla, California 92038 ABSTRACT con concordancia geogrifica. Aun mis, la prueba de The northern right whale dolphin (Lissodelphis bore- Mantel, que examina correspondencia aparejada entre alis) has experienced very high levels of fishery-induced distancias geogrificas y genkticas entre las muestras, tam- mortality in international high-seas, large-scale driftnet poco detect6 evidencia de aislamiento asociado a1 dis- fisheries, from about 38"N to 46"N, and 171"E to 151"W. tanciamiento espacial entre 10s individuos. Debido a que Assessing the impact of these mortalities is difficult, how- datos negativos como 10s mencionados antes se usan para ever, because of the possible existence of a coastal pop- hacer decisiones en le manejo de las pesquerias, hicimos ulation off California and the Pacific Northwest that is un anilisis de potencia estadistica; determinamos la pro- separate from offshore populations. To obtain quantita- babilidad de que un reconocimiento de tamaiio similar tive measures of reproductive isolation between putative produzca resultados estadisticamente significativos con populations, a portion of the control region of the mito- niveles de divergencia hipotkticos per0 plausibles entre chondrial DNA (mtDNA) genome was sequenced in 65 stocks (suponiendo diferenciacibn,entre kstos). A partir geographically dispersed individuals, then analyzed in a del anilisis, se estinia que la fiecuencia de producir errores nested ANOVA format. No evidence of geographically del tipo I1 o beta es de alrededor 10')/0. concordant population structuring was apparent. In addi- tion, a Mantel test, examining painvise correspondence INTRODUCTION between geographic and genetic distances among sam- Although the northern right whale dolphin (Lissodelphis ples, failed to detect any evidence of isolation by dis- boreulis) is the most common cetacean species in the tance. Because negative data such as these are often used bycatch of the high-seas, large-scale driftnet fishery in in management decisions, a power analysis was conducted the North Pacific (Hobbs and Jones 1993), its intraspe- to determine the probability that a survey of comparable cific (stock) structure is unknown. A separate coastal size would yield statistically significant results under a population off California and the Pacific Northwest may hypothetical but likely level of divergence between "bona exist, but there are insufficient data to allow a rigorous fide" stocks. The analysis yielded an estimate of the test of this hypothesis even though the species has been rate of making a type I1 or beta error of about 10%. the focus of considerable biological effort and expense. Yet it is axiomatic that knowledge of the intraspecific RESUMEN (stock) structure is a critical component in determining El delfin lis0 Lissodelphis boreulic ha sufrido niveles de the status and subsequent management of species im- niortalidad muy alta en alta mar (alrededor de 38"N a pacted by human activities; stock, not species, is the unit 46"N y 117"E a 151"W) inducidos por una pesqueria a on which management parameters such as optimal gran escala que usa redes de deriva. Es dificil evaluar el sustainable population and allowable biological removal impact0 de esta mortalidad debio a que posiblemente estimates are based. Both the Marine Mammal Protection existen poblaciones costeras frente a California y el Act and the Endangered Species Act direct management Pacific0 Noroeste que estin separadas de las poblaciones efforts at taxa below the species level. The concern of de mar adentro. Con el fin de cuantificar el aislamiento the responsible manager is that if stock structure is ar- reproductivo entre las supuestas poblaciones se obtuvo bitrarily subdivided too finely, population estimates la secuencia de una porcibn de la regi6n central del and allowable removal levels may be so small that local genoma del ADN de la niitocondria (ADNmt) en 65 fisheries may be unnecessarily closed down. On the other individuos procedentes de varias regiones; estos datos hand, if a species is too coarsely subdivided, evolution- se analizaron usando un anilisis de varianza anidado. No arily significant local populations may be depleted or se encontri, evidencia de una estructura de la poblaci6n destroyed. Knowledge of stock structure comes from four classes of information: distribution, demography, morphology, 'Current address: Sequana Therapeutics, 11099 North Torrey Pines Road, and genetics (Waples 1991a, b; Dizon et al. 1992). What LaJolld, CA 92037 61 DIZON ET AL.: GENETIC STOCKS AND STATISTICAL POWER CalCOFl Rep., Vol. 35, 1994 Northern Right Whale Dolphin I * 80-91 POP sightings *% 0 sequenced samples IP I I I I I 220- 00 - ZCO- 03 - lly)oo- - woo- - woo- - Figure 1. High seas sightings (stars) of northern right whale dolphins (Lissodelphis borealis). Data from the National Marine Mammal Laboratory’s Platform of Opportunity Program (POP), 1980-91. Circles are sequenced sample collection locations. few data are available from these sources for L. borealis recombination effects (Giles et al. 1980). These prop- are summarized in Leatherwood and Walker (1979), erties greatly simplifjr data analysis, and the relatively high Baird and Stacey (1991), and Ferrero et al. (1993). Save rate of evolution (Moritz et al. 1987; Hoelzel et al. 1991) for some distributional data that indicate an area of allows resolution of closely related forms. Analyses of reduced density separating the potential coastal popula- mtDNA, primarily restriction fragment length poly- tion and the offshore one (figure 1), there is no infor- morphism analyses, but also including direct sequenc- mation in the literature regarding whether L. borealis ing, have been extensively used in the last several years exists as a single, panmictic population or is subdivided to study intraspecific structure of marine mammals (e.g., into two or more breeding populations. Although the see Baker et al. 1985, 1990, 1993; Lint et al. 1990; Dizon tendency of delphinids to subdivide into coastal and off- et al. 1991; Rose1 1992; Dowling and Brown 1993; shore populations is well known (see Perrin et al. 1985; Hoelzel et al. 1993). Dizon et al., in press), the stock structure of this species Here we report on the results of a study examining is an open question. the genetic diversity within and among three putative About 6,700 driftnet operations were observed be- populations of L. borealis sampled from locations span- tween 1989 and 1990 (Hobbs and Jones 1993), and bio- ning their known range. We conduct a power analysis logical samples from the cetacean bycatch were collected. to estimate the probability of making a type I1 or beta These, as well as samples collected off the U.S. west coast error-i.e., incorrectly accepting the null hypothesis that from beach-cast animals, cross-bow biopsies of bow- the population is not subdivided into stocks. From a con- riding animals, and fishery mortalities were used in a servation standpoint, the costs of making such an error molecular analysis of mtDNA. are perhaps higher than the costs of making a type I Analyses based on molecular information derive their error-incorrectly rejecting the null hypothesis-because power from two properties (Vigilant et al. 1991): (1) the of the potential for destroying an undetected but evo- evolution of the molecule is dominated by selectively lutionarily significant population component of the neutral substitutions, and (2) the changes occur at a rea- species. Using genetic dlversity studles fi-om other species, sonably constant rate, at least within fairly closely related we attempt to estimate the minimum genetic differences taxa. The mtDNA molecule has the features of neutral that separate closely related but “bona fide” stocks, and and rate-constant mutation (Moritz et al. 1987). In addi- we use that estimate to determine the statistical power tion, it is haploid and maternally inherited, eliminating of our current study. 62 DIZON ET AL.: GENETIC STOCKS AND STATISTICAL POWER CalCOFl Rep., Vol. 35, 1994 MATERIALS AND METHODS We performed sequencing reactions on the light-strand Fifty-two individuals from the bycatch of the high- DNA only, using standard conditions and reagents as seas driftnet fisheries were obtained by National Marine supplied with the Sequenase, Version 2.0 sequencing kit Fisheries Service (NMFS) observers from the National (United States Biochemical Corporation) and 5 pCi of Marine Mammal Laboratory (NMML). Of these, 16 [a-35S]dATF?Two internal primers, producing overlap- were from west of the dateline (figure 1). The majority ping sequences, allowed us to routinely sequence about of samples obtained from NMML were clustered to the 500 bp’s, about 80% of the amplicon. The sequencing east of the dateline but west of 155”Wlatitude; of those, reactants were then electrophoresed on 6% polyacryl- we sequenced 36. Note that the classification of animals amide wedge gels (0.4 to 0.8 mm) from 2 to 6 hours east and west of the dateline into separate populations and read from autoradiographs exposed to the vacuum- is arbitrary and not supported by any significant hiatus dried gels. The individual sequences