.lounuil ,:fShcllthh Research. Vol. 20. No. 1. 453-157. 2001. A MOLECULAR ASSAY IDENTIFIES MORPHOLOGICAL CHARACTERS USEFUL FOR DISTINGUISHING THE SIBLING SPECIES LITTORINA SCUTULATA AND L. PLENA PAUL A. HOHENLOHE' * AND ELIZABETH G. BOULDING" ^ Friday Harbor Laboratories. University oj Wasiiini>ton. 620 University Rd. Friday Harbor. Washington 98250; 'Department of Zoology. University of Giielph, Gnelpli, Ontario NIG 2W I. Canada ABSTRACT Sibling specie.s Litwrina sculidciui and L. plena are difficult to distinguish in the field. Here we present a new molecular liiul and use it lo evaluate the discrete and quantitative morphological characters that have been proposed as diagnostic. We collected 38.5 snails of both species from 1 1 sites in Washington state and used restriction enzyme digestion of a PCR-amplified, 480 bp fragment of the mitochondrial cytochrome b gene to distinguish the species. This new molecular assay produces species-specific restriction fragment patterns that correspond with identification of males by penis morphology. To evaluate the usefulness of morphological characters, we scored three discrete shell characters (presence of basal band, presence of basal ridge, and size of checker pattern) as well as tentacle coloration. The four discrete characters differ significantly between the two species, though none is completely diagnostic. Tentacle coloration is the most reliable character and may be combined with the shell characters for successful identifi- cation. The two species also differ significantly in overall size and in three out of five size-independent shell shape measurements, with L scunilata having larger, taller-spired shells with narrower apertures. However, shell shape does not separate the species well because of intraspecific variation, and it is unlikely to be useful for species identification. Further analysis suggests that at least some of this intraspecific variation is genetic rather than environmental. The distributions of the two species overlap broadly in Washington, though only L plena was found in exposed outer coast habilals, contrary to previous work. KEY WORDS: LiUonna scuhilahi. L. plena, species identification, sibling species, molecular systematics INTRODUCTION The taxoiiomic history of the Littorina scundata species com- plex, a group of sympatric intertidal prosobranch gastropods in the Northeastern Pacific, has been complicated by morphological siniilarity across species and variation within species. Currently two sibling species are recognized, L. scitntUita [.sen.sii siricta) Gould 1849 and L plena Gould 1849, which are distinguished on the basis of reproductive characters, including penis, pallial ovi- duct, and egg capsule morphology (Murray 1979, Mastro et al. 1982, Reid 19961. These characters, however, are difficult to use for the non-destructive field identification that is necessary for many ecological studies. Reproductive characters cannot be used for juveniles, and we have found the dissection necessary for ex- amining pallial oviduct morphology to be difficult, especially in small specimens. Other diagnostic morphological characters have been proposed. Three discrete shell characters have been described: a pale basal band (Murray 1982, Reid 1996, Chow 1987, Rugh 1997) and a narrow basal ridge (Rugh 1997), both found more often in L plena, and the pattern of checkers on many shells, which tend to be smaller in L plena than in L. scutulata (Reid 1996, Rugh 1997). Rugh (1997) was able to use these three shell characters alone to correctly identify 17 male specimens of both species from southern California. Reid (1996) described differences in tentacle color- ation: L scunilata individuals tend to have "transverse black bands and flecks," while L. plena tend to have a "broad, unbroken black stripe with transverse flecks, or all black." Murray (1982) de- scribed a set of discriminant functions of four quantitative shell measurements that correctly classified 961 of specimens. Further principal component analysis by Murray (1982) showed L sciitii- *Corresponding author. E-mail: hohenlo(a'u. washington.edu lata shells to be generally taller with narrower spire angles and shorter aperture openings relative to shell height. Chow (1987) combined three quantitative shell measurements with number of whorls, presence of a basal band, and presence of tesselation in another discriminant function analysis. This analysis correctly classified 92% of specimens, but only when using snails from one habitat; combining specimens from different habitats introduced too much intraspecific variation to allow correct classification. Chow (1987) also found L. scutulata shells to be larger, narrower, and less likely to have a basal band, which agreed with past work. Other characters used with varying success to distinguish these species include spiral sculpture on the shell and radular characters (Reid 1996, Mastro et al. 1982). Mastro et al. (1982) found eight polymorphic allozyme loci at which the two species differ in their allele frequencies. However, none of these loci was diagnostic. No other molecular studies to date have identified a reliable molecular character at a polymor- phic locus that distinguishes the two species. The previous studies of moiphological differences used speci- mens that were positively identified using reproductive characters, thus excluding pre-reproductive animals. Here we present a mo- lecular technique for identifying individuals of all ages using mi- tochondrial DNA. We use this tool to evaluate the reliability of characters that can be observed on intact animals: the three discrete shell characters described above, tentacle coloration, and quanti- tative shell shape differences. MATERIALS AND METHODS We collected 385 snails of both species from 1 1 areas around Puget Sound and the outer coast of Washington state in January 1998 (see Fig. 1) and kept them alive until DNA extraction. Ani- mals of all sizes, including juveniles, from within a randomly chosen, small (-1 m"^) area of rocky shore habitat were collected. Individuals anesthetized in 1% MsCU seawater solution were 453