Nested PCR detection of microscopic life-stages of laminarian macroalgae and comparison with adult forms along intertidal height gradients

Abstract

Recruitment of kelp species (Laminari- ales) to rocky marine habitats relies on the prior establishment of microscopic life-stages (zoospores, gametophytes, gametes and microscopic sporo- phytes). Unlike macroscopic sporophyte stages of kelp, microscopic life-stages have proved difficult to detect and identify in their natural habitat. Using a species-specific PCR-based assay, we developed a method to detect putative microscopic life-stages of 2 northeast Pacific kelp species, Nereocystis luetkeana (Mertens) Postels et Ruprecht and Hedophyllum ses- sile (C. Agardh) Setchell, from rocky intertidal sub- strate samples. Species-specific primers were evalu- ated for specificity and sensitivity. Comparisons of nested and unnested PCR were performed for both species. Nested PCR was used to screen for target species present on rocks obtained along an intertidal height gradient (0.0 to 3.5 m Lowest Low Water (LLW)) from 2 sites at monthly intervals in summer 2005. Vertical distribution patterns of the putative microscopic life-stages and the macroscopic sporo- phyte form were compared at both sites. Putative microscopic life-stages of N. luetkeana were detected at both sites and displayed a widespread vertical dis- tribution with no clear spatial or temporal relation- ship. In comparison, H. sessile putative microscopic life-stage distribution was much more spatially restricted, showing a similar distribution to the macroscopic sporophyte. This study demonstrates the sensitivity, specificity and validity of utilizing molecu- lar techniques to describe the distribution of micro- scopic life-stages of certain kelp species.