(Received 1 November 2005; accepted 20 September 2006) ABSTRACT

Transcription

1 A NEW POECILOGONOUS SPECIES OF SEA SLUG (OPISTHOBRANCHIA: SACOGLOSSA) FROM CALIFORNIA: COMPARISON WITH THE PLANKTOTROPHIC CONGENER ALDERIA MODESTA (LOVE N, 1844) PATRICK J. KRUG 1, RYAN A. ELLINGSON 1,RONBURTON 2 AND A NGEL VALDE S 3 1 Department of Biological Sciences, California State University, Los Angeles, CA , USA; 2 Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA , USA; 3 Natural History Museum of Los Angeles County, 900 Exposition Blvd., Los Angeles, CA 90007, USA (Received 1 November 2005; accepted 20 September 2006) ABSTRACT Cryptic species are increasingly recognized as commonplace among marine gastropods, especially in taxa such as shell-less opisthobranchs that lack many discrete taxonomic characters. Most cases of poecilogony, the presence of variable larval development within a single species, have historically turned out to represent cryptic species, with each possessing a single canalized type of development. One well-characterized example of poecilogony was attributed to the sacoglossan opisthobranch Alderia modesta; in southern California, slugs resembling this member of a monotypic genus produce both long-lived, planktotrophic and short-lived, lecithotrophic larvae. Paradoxically, however, A. modesta is exclusively planktotrophic everywhere else in the northern Pacific and Atlantic Oceans. A recently completed molecular study found that slugs from poecilogonous populations south of Bodega Harbor, California, comprise an evolutionarily distinct lineage separate from northern, strictly planktotrophic slugs. We now describe the southern species as A. willowi n. sp., based on differences in morphology of the dorsum and radula, characteristics of the egg mass, larval development mode and nuclear and mitochondrial genetic markers. A DNA barcode is provided, based on 27 fixed differences in the cytochrome c oxidase subunit I gene that can reliably differentiate Pacific specimens of Alderia species. Genetic and morphological data are concordant with developmental evidence, confirming that A. willowi is a true case of poecilogony. An improved understanding of the ecological differences between these sister taxa may shed light on the selective pressures that drove the evolution of lecithotrophy in the southern species. INTRODUCTION Many marine invertebrates are capable of widespread dispersal as larvae, resulting in species with cosmopolitan distributions and panmictic populations (Palumbi, 1994, 1995; Caley et al., 1996; Bohonak, 1999; Pechenik, 1999). However, molecular analyses often reveal deep genetic divides within morphospecies, and between clades occupying different regions or even adjacent microhabitats (Knowlton, 1993; Johannesson, Rolan-Alvarez & Ekendahl, 1995; Lee, 2000; Dawson & Jacobs, 2001; McGovern & Hellberg, 2003; Caudill & Bucklin, 2004; Fukami et al., 2004; Lee & O Foighil, 2004). Divergence in the sea may frequently occur without morphological diversification, impairing attempts by conventional taxonomy to catalogue marine biodiversity (May, 1994; Knowlton, 2000; Collin, 2005). This can result from a shortage of discrete characters in some taxa, or phenotypic plasticity of key traits such as sponge spicules (Klautau et al., 1999), bryozoan spines (Schwaninger, 1999) or shells and radulae of gastropods (Padilla, 1998; Simison & Lindberg, 1999; Marko, Palmer & Vermeij, 2003). When evolutionarily independent lineages are also reproductively isolated, they can be considered biological species (Mayr, 1942, 1963; Burton, 1990; Knowlton et al., 1992; Ganz & Burton, 1995; Knowlton et al., 1997; Lee, 2000). Along the Pacific coast of North America, numerous cryptic species have been Correspondence: P. J. Krug; pkrug@calstatela.edu uncovered among shelled gastropods (Murphy, 1978; Mastro, Chow & Hedgecock, 1982; Palmer, Gayron & Woodruff, 1990; Collins et al., 1996; Marko & Vermeij, 1999; Marko, Palmer & Vermeij, 2003). Phylogeographic studies of opisthobranch sea slugs have lagged behind those of marine snails, due to the excellent fossil record and shell characters afforded by the latter. The frequency of widespread species and shortage of inflexible characters in opisthobranchs suggest that cryptic species may be common in this group (Hirano & Hirano, 1991; Morrow, Thorpe & Piction, 1992; Clark, 1994; Sisson, 2002). Development mode is generally a canalized trait among invertebrates, and is often used as a basis for differentiating opisthobranch species (Miles & Clark, 2002). However, opisthobranchs exhibit a higher frequency of poecilogony, the presence of variable larval development modes within a species, than any taxon other than spionid polychaetes (Giard, 1905; West, Harrigan & Pierce, 1984; Bouchet, 1989; Clark, 1994; Chia, Gibson & Qian, 1996; Krug, 1998). Thus, like morphological traits, development mode may be an unreliable basis for taxonomic distinction in sea slugs. The estuarine sacoglossan A. modesta (Love n, 1844) belongs to a monotypic genus (Gascoigne, 1976). Formerly given its own family Alderiidae, the genus Alderia is now included within the Limapontiidae in a clade of cerata-bearing sacoglossans with sabot-shaped radulae (Gascoigne, 1985; Jensen, 1996). One of the most widely distributed species of this group, A. modesta is found on both sides of the temperate north Atlantic (Engel, Journal of Molluscan Studies (2007) 73: Advance Access Publication: 9 January 2007 # The Author Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. doi: /mollus/eyl025

2 P. J. KRUG ET AL. Geerts & van Regteren Altena, 1940; Hartog, 1959; Bleakney & Bailey, 1967; Clark, 1975; Vader, 1981), and in the north Pacific from western Russia (Chernyshev & Chaban, 2005) to Alaska (Goddard & Foster, 2002), and south to San Diego, California (Trowbridge, 1993, 2002; Krug, 1998). All northern Pacific and Atlantic populations studied to date were exclusively planktotrophic (Engel, Geerts & van Regteren Altena, 1940; Hand & Steinberg, 1955; Seelemann, 1967; Gibson & Chia, 1994). Bleakney (1988) found no differences in the radulae and penial stylets of specimens from the north Atlantic and north Pacific, but did not examine Pacific specimens south of Washington. However, only in California do slugs classified as A. modesta exhibit poecilogony, producing both planktotrophic and lecithotrophic veliger larvae (Krug, 1998). A combination of developmental and molecular evidence indicated that poecilogonous populations in California comprise a cryptic species south of Bodega Harbor, California, U.S.A. Analysis of mitochondrial DNA indicated the two lineages likely diverged in the Piocene (Ellingson & Krug, in press). Here, we describe the poecilogonous species and provide diagnostic characters to differentiate it from its planktotrophic congener A. modesta, based on differences in: (1) morphology of the dorsum and radula of adult specimens; (2) egg mass morphology and larval development mode; (3) allozymes and mtdna sequences. MATERIALS AND METHODS Collection and developmental typing of organisms Pacific specimens of Alderia were collected on mudflats from patches of the host algae Vaucheria species (Abbott & Hollenberg, 1976), over a 3800-km stretch of the northeastern Pacific coast ranging from San Diego, California to Kodiak Island, Alaska (Table 1). Individual adult slugs were placed in petri dishes of seawater overnight for egg-mass deposition. Development mode was typed by egg size and characteristics of veliger larvae (Krug, 1998); typed adults were frozen at 2808C prior to allozyme and DNA-sequence analysis. Wet weights were measured within a day of collection by blotting slugs dry with a paper towel and weighing to the nearest 0.1 mg. Atlantic specimens (n ¼ 13) and one egg mass were collected by T. Van den Neuker from the Paardenschor mudflat on the Scheldt estuary, Doel, Belgium ( N, E) in August 2005, and preserved in ethanol. Western Pacific specimens (n ¼ 3) were collected by Alexei Chernyshev and Andrei Shukalyuk in Amurskii Bay, Sea of Japan, Russia ( N, E) in June 2004, and preserved in ethanol. The genus Alderia was previously monotypic, and no closely related genera occur in the temperate eastern Pacific. Within the family Limapontiidae, Jensen (1996) united the genera Limapontia, Ercolania and Alderia based upon their sabot-shaped teeth; for outgroup comparisons, L. depressa was collected in August 2005 at the Ardfrey experimental oyster pond in County Galway, Republic of Ireland ( N, E). Table 1. Field sites and sampling dates for Alderia species. Location Latitude, Longitude Date sampled Doel, Belgium N, E 8/05 Kodiak, Alaska, N, W 6/05 U.S.A. Vancouver, British Columbia, Canada Tillamook, Oregon, U.S.A N, W 7/ N, W 3/05 Coos Bay, Oregon N, W 7/00 a,10/01, 2/05 Humboldt N, W 2/04 Bodega Harbor N, W 9/03, 9/04 Walker Creek N, W 9/04 Cow Landing N, W 9/04 South Tomales Bay N, W 9/03, 9/04 San Francisco Bay N, W 9/03 9/04 (5x) Morro Bay N, W 9/02 Santa Barbara N, W 7/99 a,2/00 San Pedro N, W 1/04 8/04 (4x) Newport Bay N, W 6/03 8/04 (6x) San Diego N, W monthly 96-98; 7/99 a, 8/03 8/04 (3x) Unless otherwise indicated, all sites are in California, U.S.A. a Collections used for allozyme electrophoresis. Bay and Bataquitos estuary in San Diego). For morphometric analyses, the width of the leading tooth was measured at a point one-third of the way from the tip of the tooth to the base of the cusp; the ratio of width to cusp length was then calculated at this point. To test for differences in radular morphology, ratios of southern and northern slugs were compared using a nonparametric Mann Whitney U-test, as the data were not normally distributed. Allozyme electrophoresis Specimens from San Diego and Santa Barbara, California, and Coos Bay, Oregon, were homogenized in 20 ml of grinding buffer and 2 ml of the resulting mixture loaded onto a polyacrylamide gel. The gel was run for 6.5 h and stained for phosphogluco-isomerase (PGI) enzyme activity. Allozymes were classified by measuring the distance travelled into the gel and normalizing to the most common allele in slugs from southern populations; standards of rare alleles were run on subsequent gels for consistent identification. A total of 11 alleles were distinguishable. Results were analysed by log-likelihood ratio tests to determine whether genotypic proportions within each population were in Hardy Weinberg equilibrium (Zar, 1984). Radular examination Individual specimens that had been previously typed by external morphology were placed in 10% NaOH for 1 week to remove all soft tissues. Radulae were washed in distilled water and then ethanol, prior to mounting for scanning electron microscopy (SEM). Radulae were examined from three to four individuals collected at each of three northern sites (Tillamook, Oregon; Netarts Bay, Oregon; Bodega Bay, California) and three southern sites in California (south Tomales Bay, Morro DNA sequence analysis Polymerase chain reactions amplified portions of the mitochondrial cytochrome c oxidase subunit I (COI) and 16S rrna genes, using primers LCO1490 and HCO2198 (Folmer et al., 1994) for COI and 16Sar-5 0 and 16Sbr-3 0 (Palumbi, 1996) for 16S. For a full description of molecular methods see Ellingson & Krug (in press). Sequences were obtained for a 480-base pair (bp) segment of COI from 233 Pacific specimens typed for development mode and morphology, and from seven Atlantic 30

3 NEW POECILOGONOUS SACOGLOSSAN specimens. Fixed differences between species were determined in Mega 3.0 (Kumar, Tamura & Nei, 2004) using COI data from all northeastern Pacific specimens; differences were assembled into a DNA barcode for differentiating the two taxa (Hebert et al., 2003a; Hebert, Ratnasingham & de Waard, 2003b; Blaxter, 2004). A 450-bp portion of the 16S gene was sequenced from a subset of Pacific (n ¼ 32) and Atlantic (n ¼ 7) specimens. A model of secondary structure was used to align 16S sequences, to maintain base-pairing interactions in stem regions (Lydeard et al., 2000; Medina & Walsh, 2000; Valde s, 2003). Genetic distances were estimated with the Tamura Nei model of sequence evolution, based on Modeltest 3.7 results (Tamura & Nei, 1993; Posada & Crandall, 1998). Sequences were deposited in GenBank (accession numbers DQ DQ364426). The COI and 16S datasets were combined for phylogenetic analyses after a partition-homogeneity test in PAUP 4.0 revealed no conflict between the two loci (Cunningham, 1997; Wiens, 1998; Swofford, 2001). Parsimony analysis was done in PAUP, saving a maximum of 5000 trees and obtaining bootstrap values (1000 replicates) with the fast stepwise-addition option for computational efficiency. For Bayesian analysis, the TrN þ G model of sequence evolution (a ¼ ) was applied to the COI data and the HKY þ I þ G model (a ¼ ) to the 16S data, based on the Akaike Information Criterion implemented in Modeltest 3.7 (Posada & Crandall, 1998). Bayesian analysis utilized the Metropolis-coupling Markov Chain Monte Carlo method as implemented in MrBayes (Ronquist & Huelsenbeck, 2003). The analysis was run for 3,000,000 generations saving a tree every 1000 generations; a consensus tree was generated in PAUP after a burn-in of 300 trees. SYSTEMATIC DESCRIPTION Family Limapontiidae Gray, 1847 Genus Alderia Allman, 1846 Alderia modesta (Lovén, 1844) (Figs 1A, B, 2A) Alderia amphibia Thompson, 1844: 250 (nomen nudum ). Stiliger modestus Lovén, 1844: 49 (Bohusla n, Sweden). Alderia modesta Love n, 1845: 8. Canthopsis harvardiensis Agassiz, 1850: 191 (nomen nudum ). Alderia harvardiensis Gould, 1870: , pl. 16, figs Alderia scaldiana Nyst, 1855: 435, figs 1, 2. Alderia uda Marcus & Marcus, 1956: 8 17, figs Nomenclatural remarks: Alderia amphibia was reported for the first time by Thompson (1844) who mentioned the new genus and species discovered by Allman in Ireland, but included no description, thus it is a nomen nudum. The same year, Lovén (1844) described the species Stiliger modestus based on specimens collected from Bohusla n, Sweden. Subsequently, Allman (1845) provided a description of his new genus Alderia, which was based on specimens collected in southern Ireland in 1842; these findings were presented at the York meeting of the British Association for the Advancement of Science, which were published in The original description of the name Alderia by Allman (1845) mentioned no species names. The species name A. amphibia appeared for the second time in the same volume, in a report by Alder & Hancock (1845), with species authorship of Allman, but again no description was included. Lovén (1845) became aware of Allman s discoveries and realized that both of them had described the same species, but considered that Allman was correct in placing this Figure 1. External morphology of adults and egg masses of Alderia species. A, B. Alderia modesta. A. Adult morphology of A. modesta collected from Bodega Harbor, California. Dorsum is smooth and squared off near the cephalic lobes. Dark pigment is speckled over the surface of dorsum and cerata, on top of yellow background coloration. Scale bar ¼ 1 mm. B. Planktotrophic spawn of A. modesta, showing characteristic spiral arrangement of ova within the egg mass. Scale bar ¼ 1 mm. C, D. A. willowi, n. sp. C. Adult morphology of A. willowi n. sp., collected from San Pedro, California. Dorsum tapers towards cephalic lobes and is raised into a hump, down which runs a stripe of yellow background colour. Dark pigment evenly covers most of the dorsal surface and cerata. Scale bar ¼ 200 mm. D. Planktotrophic and lecithotrophic spawn of A. willowi n. sp., showing haphazard arrangement of ova within the egg mass, and difference in egg and capsule diameters for the two development modes. Scale bar ¼ 1 mm. 31

4 P. J. KRUG ET AL. Figure 2. A, B. Alderia modesta. A. Complete radula of A. modesta (Netarts Bay, Oregon) showing ascending and descending limbs, and ascus containing many discarded teeth. Scale bar ¼ 100 mm. B. Leading tooth of A. modesta, which narrows towards the pointed tip. Scale bar ¼ 10 mm. C, D. A. willowi, n. sp. C. Complete radula of A. willowi n. sp., including ascending and descending limbs, and small ascus. Scale bar ¼ 100 mm. D. Leading tooth of A. willowi n. sp., which widens before narrowing towards the blunted tip. Scale bar ¼ 10 mm. organism in a new genus, rather than in Stiliger (Ehrenberg, 1831). Therefore, he proposed that the valid name for the species should be A. modesta. This opinion was subsequently corroborated by Allman (1846). Because S. modestus was the species name first assigned to the genus Alderia, it constitutes the type species by subsequent monotypy. Material examined: 4 specimens, Bodega Harbor, California ( N, W), collected on 1 September 2004 (LACM ). Description: Body elliptical, lacking rhinophores, with rounded lobes on either side of head; visible eyespots behind lobes. A groove separates body from foot, which tapers posteriorly; foot margin wider than body in some specimens, but not visible beneath cerata in others. Anus a tube on dorsal side of body, opening above posterior end of tail on midline. Cerata cylindrical, with occasional white spots, each ceras containing one blood vessel and digestive diverticulum. No heart or pericardium present; circulation affected by rhythmic pulsing of cerata on alternating sides of body, creating a flow of hemocoel through lateral and pedal sinuses that encircle the main visceral mass (Evans, 1953). Arrangement and shape of cerata variable. Oviducal pore located on right side below cerata. Dorsum smooth and flat (Fig. 1A). Background colour yellow, with fine brown pigment spots covering dorsum and cerata but not anteriormost region of cephalic lobes (Fig. 1A). Recently-fed animals have green colour due to Vaucheria chloroplasts retained in digestive diverticula, which extensively ramify throughout body including cerata, foot and penis (Krug, in press). For 6 surveyed populations, mean wet weight of adults ranged from mg SE (Coos Bay, Oregon) to mg SE (Walker Creek, California), with an overall mean weight of mg SE (n ¼ 231, range ¼ mg). Penis ends in a stylet used in hypodermic insemination, with sperm transfer occurring anywhere on body of the recipient. Egg mass and development: Ova visible through thin areas of the foot in ramifying extensions of the reproductive system. Egg mass a gelatinous cylinder with one end attached to the substrate, and with eggs arranged in a characteristic spiral (Fig. 1B). Mean egg number per clutch was (n ¼ 15) for slugs from Bodega Harbor, California in October 2005 (Krug, in press). Uncleaved ova mm in diameter, each enclosed within a spherical capsule 120 mm in diameter; development strictly planktotrophic, with larvae maturing and hatching in days SD at 258C (Krug, in press). Larvae grow for about 30 days, after which settlement is cued by host algae Vaucheria spp. (Seelemann, 1967). Distribution: Previously reported from both coasts of the north Atlantic and north Pacific. (1) European populations were reported from Scandinavia, the Gulf of Finland, the German coast of the Baltic, the United Kingdom, the south and west coasts of Ireland, the Netherlands, Belgium and Normandy, France (Engel, Geerts & van Regteren Altena, 1940, and references therein; Hartog & Swennen, 1952; Pruvot-Fol, 1954; Bleakney, 1988). There is an unconfirmed record from the Adriatic Sea, but it is unclear whether the specimen illustrated (Pruvot-Fol, 1954: Fig. 76a) was collected in the Adriatic Sea or the Atlantic Ocean, but there have been no subsequent reports of this species from the Mediterranean. (2) Western Atlantic populations were reported from Nova Scotia, Newfoundland, New Brunswick, New Jersey, Massachusetts, Maryland and Virginia (Hand & Steinberg, 1955; Marcus, 1972; Clark, 1975; Clark, Jensen & Stirts, 1990; Bleakney, 1988; and see below records of Alderia harvardiensis Gould, 1870). The species was reported from Brazil under the synonym A. uda (Marcus, 1972). (3) Eastern Pacific populations expressing planktotrophic development were reported from Alaska south to Elkhorn Slough, California (Hand & Steinberg, 1955; Bleakney, 1988; Trowbridge, 1993; Goddard & Foster, 2002). We have collected A. modesta only as far south as San Francisco Bay, California, where its abundance varies seasonally. (4) Western Pacific specimens occur in Peter the Great Bay, Russia, in the Sea of Japan (Chernyshev & Chaban, 2005). Molecular analysis of three Russian specimens confirmed that they represent A. modesta (Ellingson & Krug, in press). 32

5 NEW POECILOGONOUS SACOGLOSSAN Remarks: Two species names have been introduced for the western Atlantic records of Alderia modesta. Agassiz (1850) mentioned the name Canthopsis harvardiensis referring to specimens observed in brackish water at Cambridge, Massachusetts, but included no species description and therefore this is a nomen nudum. This name was used later by Stimpson (1853) referring to specimens collected in Grand Manan, Canada; he added that good colour drawings of the species were in Agassiz s possession, but again Stimpson included no description of the species. Finally, Gould (1870) provided a full description of the species including references to Agassiz s and Stimpson s papers and drawings of the living animals. Gould transferred the species to the genus Alderia but commented that the western Atlantic species is different from A. modesta in the number and shape of cerata and the darker colour. Later, Hand & Steinberg (1955) examined preserved specimens and considered that A. harvardiensis is a synonym of A. modesta. Our examination of Gould s drawings confirms this synonymy. The second species is A. uda, originally described by Marcus & Marcus (1956) from Brazil as different from both A. modesta and A. harvardiensis. Subsequently, Marcus (1972) examined specimens of A. modesta from Chesapeake Bay and compared them with the original description of A. uda, concluding that these two species are synonyms. A revision of Marcus and Marcus original description reveals that the external and internal features of A. uda fit within the variability described for A. modesta and therefore, we agree with Marcus (1972) conclusion. Another enigmatic synonym of A. modesta is A. scaldiana, which was originally described by Nyst (1855) based on specimens collected in salt marshes in northern Belgium. The species was later considered extinct due to habitat destruction (Raeymaekers, 1895), and subsequent records of Alderia from Belgium were assigned to A. modesta. Later, Adam & Leloup (1939) synonymized A. scaldiana with A. modesta. Alderia comosa A. Costa, 1867 was a possible distinct species described from Naples, Italy. Odhner (in Franc, 1968) introduced the new genus Alderella for this species. However, a review of redescriptions of the species (Vayssie re, 1913; Pruvot-Fol, 1954) suggested that this is probably a member of the genus Ercolania (Trinchese, 1872). Finally, A. nigra Baba, 1937 is another related species originally described from Amasuka, Japan. Years later, Baba (1968) studied additional specimens and concluded that there are significant differences between A. nigra and A. modesta including the morphology of the rhinophores, the arrangement of the cerata and particularly the nonterminal position of the anus in A. nigra. Therefore, Baba (1968) introduced the new genus Alderiopsis for A. nigra, which he placed in the family Alderiidae. There are no recent records of this genus, thus its phylogenetic placement and affinity to Alderia remain uncertain. Prior to the present study, the only species convincingly belonging to the genus Alderia was A. modesta. The published anatomy of A. nigra (Marcus & Marcus, 1956; Baba, 1968) does not fit within the diagnosis of Alderia (Hand & Steinberg, 1955; Gascoigne, 1976, 1985). Gascoigne (1976) maintained the family Alderiidae to reflect the absence of a heart and the unusual reproductive features of A. modesta (Evans, 1953), interpreted as primitive (basal) character states. Jensen (1996) dissolved the family by moving the genus Alderia into the Limapontiidae. However, the morphological simplification of the Alderia body plan may confound phylogenetic analyses, as reflected in its alternative placement as basal (Gascoigne) or derived (Jensen) by different authorities. The widespread A. modesta was originally described from the Atlantic, but comparison with preserved specimens from Europe revealed little morphological variation between Atlantic and Pacific populations. Examination of preserved specimens from The Netherlands and Belgium revealed no differences in the external morphology or penial stylet compared to specimens from Bodega Harbor, California and Tillamook, Oregon; all share the typical speckled appearance and smooth dorsum. Bleakney (1988) found no internal or radular differences between Atlantic and Pacific slugs from many populations. Only planktotrophy has been reported from Atlantic specimens, consistent with our findings from the Pacific: all specimens that fit the morphological description of A. modesta are exclusively planktotrophic. The characteristic spiral arrangement of ova inside the egg mass is plesiomorphic in limapontiid slugs (Jensen, 1996). Alderia willowi n. sp. (Figs 1C, D, 2B) Types. Holotype: Cabrillo Aquarium s wetland, San Pedro, California ( N, W), collected on 18 July 2005 (LACM 2034). Paratypes: 5 specimens, Cabrillo Aquarium s wetland, San Pedro, California ( N, W), collected on 18 July 2005 (LACM 2035). Etymology: The species name derives from several sources: (1) because the cerata droop over the edge of the body on large specimens, resembling a willow tree; (2) an homage to the first author s grandmother, who always sang him a song that starts,... so I ask each weeping willow... ; and (3) a tribute to the character of Willow from the TV show Buffy the Vampire Slayer, who (as played by Alyson Hannigan) embodied the idea of sexual flexibility, in recognition of the variable reproductive modes in Alderia. Description: Body elliptical, lacking rhinophores, with rounded lobes on either side of head; visible eyespots behind lobes. Groove separates body from foot, which tapers posteriorly; foot margin typically wider than body in most specimens, rarely covered by cerata. Anus a tube on dorsal side of body, opening above posterior end of tail on midline. Cerata cylindrical, with occasional white spots; each ceras contains one blood vessel and digestive diverticulum. No heart or pericardium present; circulation affected by rhythmic pulsing of cerata on alternating sides of body. Oviducal pore located on right side below cerata. Dorsum and cerata covered by extensive patches of brown pigment over a yellow base coloration. Dorsum raised into a hump down which runs a band of yellow background colour, splitting the otherwise solid patches of brown pigmentation down the midline (Fig. 1C). Recently-fed animals have a green colour due to Vaucheria chloroplasts retained in digestive diverticula, which ramify throughout body including cerata and foot. For 6 surveyed populations, mean wet weight of adults ranged from mg SE (San Francisco Bay) to mg SE (south Tomales Bay), with an overall mean weight of mg SE (n ¼ 192, range ¼ mg). Penis ends in a stylet used in hypodermic insemination, with sperm transfer occurring anywhere on body of the recipient. Egg mass and development: Both planktotrophic and lecithotrophic development expressed within a given population, and in the laboratory by an individual (Krug, 1998, in press). Egg mass a gelatinous cylinder attached to the substrate at one end. Each egg enclosed within an individual capsule, one ovum per capsule; capsules arranged haphazardly within the egg mass instead of in a spiral, for both development modes (Fig. 1D). Lecithotrophic eggs mm diameter within mm diameter spherical capsules, developing in 5-6 days at 258C into mm larvae competent to settle immediately upon hatching (Krug, 1998, 2001). Planktotrophic eggs mm within mm capsules, developing within the egg mass for three days and hatching as mm precompetent larvae (Krug, 1998). Planktotrophic larvae mature over a 32-day pelagic period, growing to the size 33

6 P. J. KRUG ET AL. of newly hatched lecithotrophic larvae before settling in response to chemical cues from the host alga Vaucheria longicaulis (Krug & Zimmer, 2000, 2004). Animals produce approximately one egg mass per day when well fed, of either development mode. Lecithotrophic egg masses contain 10-fold fewer eggs than planktotrophic egg masses ( versus eggs per clutch; Krug, 1998). Distribution: Extending to Baja California, Mexico, but the southern range limit is presently unknown. We have consistently collected A. willowi in Tomales Bay, California, where it can occur in sympatry with A. modesta; only once in 3 years was it collected in Bodega Harbor, California, making this the northern range limit of the species. Remarks: Although studies on A. modesta were previously conducted in Elkhorn Slough and San Francisco Bay, California (Hand & Steinberg, 1955), the southern congener A. willowi went undetected prior to its discovery in San Diego, California (Krug, 1998). Morphological differences are subtle, but the shape and coloration of the dorsum are reliable traits for distinguishing the sibling species. Size is also a useful feature, as at most sites and times of year, the southern A. willowi is smaller than A. modesta, even where they co-occur on the same algal patches (Ellingson & Krug, in press). In most other respects the species are very similar, except for the critical differences in larval development. The poecilogonous A. willowi is presently the only known animal that can switch development modes within the lifetime of an individual; lecithotrophic specimens switch to planktotrophy upon starvation, and following collection from the field during summer months (Krug, 1998, in press). Lecithotrophic larvae express a dispersal dimorphism in which a variable percentage of hatchlings from each egg mass undergo spontaneous metamorphosis prior to, or within 1-2 days of hatching; their remaining siblings disperse until selectively induced to settle by species-specific chemical cues produced by the host alga (Krug & Manzi, 1999; Krug, 2001). This species thus exhibits an unprecedented degree of life-history variation, with a single genome able to produce long-term dispersing planktotrophic larvae, short-lived pelagic lecithotrophic larvae that settle in response to the host algae and nondispersing larvae that metamorphose within the egg mass or shortly after hatching (Botello & Krug, 2006). Competent, 32-day-old planktotrophic larvae are similar to newly hatched lecithotrophic larvae in size, sinking and swimming speed and behavioural response to physical and chemical cues (Krug & Zimmer, 2000, 2004). RESULTS Differentiating northern and southern slugs by development, morphology and allozymes Poecilogony was first described for specimens in the genus Alderia in San Diego, California (Krug, 1998). South of Bodega Harbor, California, most adults produce lecithotrophic larvae from May to September, whereas a variable percentage of adults lay planktotrophic eggs from October to April (Ellingson & Krug, in press). Slugs from Oregon are exclusively planktotrophic, and significantly larger than southern specimens. Allozyme comparisons were performed for two southern sites with poecilogony (San Diego and Santa Barbara, California) and one northern site where only planktotrophy was expressed (Coos Bay, Oregon). At the highly polymorphic PGI locus, each of the three populations was in Hardy Weinberg equilibrium (likelihood ratio test: P. 0.27), but different alleles were nearly fixed in Californian versus Oregonian populations (Table 2). The predominant allele in Oregon slugs was rare or absent in southern California, and vice versa. Given the ambiguity in resolving allozymes, closely migrating bands may have been confused Table 2. Allozyme frequencies at the PGI locus in three populations of Alderia species. Allele Coos Bay, Oregon Santa Barbara San Diego N Allelic designations are in the order of relative decreasing anodal mobility. with the major allele from distant populations in reciprocal comparisons. The evidence indicated a lack of gene flow between northern and southern slugs, and together with developmental differences, prompted a detailed assessment of whether this was a cryptic species complex. Fifteen sites in the eastern Pacific were surveyed from 1999 to 2005, spanning 3800 km of coastline from San Diego, California to Kodiak Island, Alaska. All specimens from Bodega Harbor northward were planktotrophic, as in prior studies, and were morphologically consistent with previous descriptions of Alderia modesta. In contrast, populations from Tomales Bay southward were predominantly lecithotrophic in summer and expressed both planktotrophic and lecithotrophic development in winter. Differences in external morphology were also noted between adult slugs across the break at Bodega Harbor. Slugs from Bodega northward exhibited a smooth dorsal surface, with mottled brown spots evenly dispersed over a yellow background; the coloration extends over the surface of the cerata as well as the flattened dorsum (Fig. 1A). Slugs south of Tomales Bay had a characteristically raised dorsum, giving them a humped appearance, and instead of spots, their brown pigment fused into continuous patches covering the dorsum and cerata (Fig. 1B). In southern specimens, the brown colouring is split by a band of yellow background coloration running down the midline of the dorsal hump. Lecithotrophic egg masses were exclusively produced by slugs having the southern-type morphology. Both morphotypes co-occurred in Tomales Bay, California. Differences were also noted between the planktotrophic egg masses laid by the two types of slugs. Northern slugs deposited clutches with eggs arranged in an internal spiral, coiled within the outer covering of the egg mass (Fig. 1C). This matches other descriptions of the egg masses of A. modesta from northern latitudes (Engel, Geerts & van Regteren Altena, 1940; Hand & Steinberg, 1955; Chernyshev & Chaban, 2005). In contrast, both planktotrophic and lecithotrophic egg masses of southern slugs lacked this characteristic spiral, with eggs haphazardly packed inside the egg mass (Fig. 1D). Radular morphology Limapontiid slugs with sabot-shaped teeth feed on filamentous algae such as Vaucheria and Cladophora. We compared radulae of multiple individuals using SEM from three northern and three southern populations, to test for consistent differences. No differences were noted between radulae of northern slugs and previously published SEM images of A. modesta radulae from northern Pacific and Atlantic populations (Fig. 2A, B; Bleakney, 1988). The ascus contained discarded teeth in 34

7 NEW POECILOGONOUS SACOGLOSSAN Table 3. Alderia species. DNA barcode for differentiating Alderia modesta from its southern congener, A. willowi n.sp. Site A. modesta C T A A T A G A T C A G T A T T A T C G G A C G C A C A. willowi T C T T A T A G G T G T C T C C T A T A A T T A T C T Data are 27 fixed nucleotide differences in the mitochondrial COI gene between Pacific specimens of A. modesta (n ¼ 84 haplotypes) and A. willowi new species (n ¼ 62 haplotypes). Positions are given relative to the Drosophila yakuba COI gene sequence (GenBank Accession X03240). a loose sac (Fig. 2A), rather than in an organized ribbon as in Hermaea. Evans (1953) argued the lack of organization in the ascus was an apomorphy of Alderia, distinguishing it from more basal taxa that have the plesiomorphic spiral arrangement of used teeth. There was no difference in the mean length of the leading radular tooth from A. modesta ( mm; range: mm; n ¼ 12) versus lecithotrophic specimens of A. willowi ( mm; range: mm; n ¼ 12). However, the teeth of A. willowi were consistently different in shape than the teeth of A. modesta. Based on the ratio of tooth width-to-length, the leading tooth of southern slugs was significantly wider near the tip (Fig. 2, and results of a Mann Whitney U-test, Z ¼ 2.44, P, 0.05). The radular teeth of A. willowi curved to a blunt tip (Fig. 2B), whereas those of A. modesta narrowed to a more pointed end (Fig. 2D). The consistency of these differences suggests that the two species may feed on strains or species of Vaucheria differing in some structural element of the algal cell wall. Molecular phylogenetic analysis In a molecular study of specimens from the northeastern Pacific, the two Alderia species formed exclusive clades that were 18 24% divergent (Tamura Nei distance) at the COI locus (Ellingson & Krug, in press). The clades were a mean 2.9% divergent at the more conserved 16S locus. Such divergence is typical for interspecific comparisons among other sacoglossans (R. Ellingson and P. Krug, unpubl.). The genetic divide was concordant with morphological and developmental differences across the Bodega Harbor breakpoint. Out of 146 COI haplotypes of Pacific origin, there were fixed differences between the two species at 27 positions (Table 3). These differences comprise a DNA barcode that can be used to differentiate the sibling species in the Pacific. Atlantic specimens had different fixed differences, but were not included in the analysis due to the low number of specimens available. Within A. modesta, Atlantic and Pacific specimens formed two exclusive clades (mean divergence ¼ 11.0% for COI, 1.0% for 16S) (Fig. 3). The genetic divergence between Atlantic and Pacific A. modesta is consistent with prolonged allopatry, and molecular clock estimates suggest the two clades were isolated by the onset of Pleistocene glaciation (Ellingson & Krug, in press). were observed to switch from lecithotrophy to planktotrophy in the laboratory (Ellingson & Krug, in press; Krug, in press). Thus, A. willowi expresses alternative developmental pathways within a single species, and is a true case of poecilogony. This species is distinguishable from its northern congener A. modesta by morphology of the dorsal surface and radula, egg mass characteristics, developmental flexibility and genetic differences in nuclear and mitochondrial markers. There was no evidence of hybridization in Tomales Bay where Alderia spp. co-occur, and breeding studies suggest these comprise good biological species (Ellingson & Krug, in press). The present study and Bleakney (1988) found that populations of A. modesta from the Atlantic and Pacific have not diverged in morphology or development. The Atlantic stock was likely founded by a Pacific ancestor during the trans- Arctic exchange, which began about 3.5 million years ago (Ma) when the Bering Strait formed (Reid, 1990). In this asymmetric invasion, 261 species of Pacific molluscs colonized the north Atlantic, whereas only 34 Atlantic molluscs moved into the north Pacific (Vermeij, 1991). Based on two mitochondrial markers, Atlantic and Pacific A. modesta are substantially divergent; trans-arctic gene flow was likely interrupted during Pleistocene glaciations and, as hypothesized by Bleakney (1988), does not occur at present. The lack of suitable Arctic habitat for estuarine taxa may account for the lack of gene flow between ocean basins, despite the high dispersal potential realized by planktotrophic larvae of Alderia species within the Pacific. DISCUSSION Most putative cases of poecilogony yield to molecular investigations, revealing cryptic species differing in development (Hoagland & Robertson, 1988; Chia, Gibson & Qian, 1996). The null expectation was that specimens of Alderia producing planktotrophic larvae would be genetically similar throughout the northeastern Pacific, but distinct from slugs producing lecithotrophic larvae in southern California. Instead, southern populations comprised a cryptic species herein named A. willowi. Haplotypes were shared by specimens of A. willowi that expressed different development modes, and individuals Figure 3. Alderia species. Bayesian tree showing phylogenetic relationships among populations of A. modesta and A. willowi n. sp., based on combined COI and 16S mtdna sequence data. Individuals of A. modesta from the north Atlantic are indicated by the white bar, whereas Pacific specimens of both species are indicated with shaded bars. Bolded branches had 100% bootstrap support by maximum parsimony and.90% posterior probabilities in Bayesian analysis. Scale bar indicates a branch length corresponding to five character-state changes. 35

8 P. J. KRUG ET AL. Molecular clock estimates suggest the split between Atlantic and Pacific A. modesta occurred about 1.7 Ma, whereas the sister species of Alderia diverged over 4 Ma, suggesting a Pacific origin for the genus (Ellingson & Krug, in press). Ecological differences between northern versus southern California likely led to speciation and the evolution of poecilogony in A. willowi, whereas the similar but isolated environments of the north Pacific and Atlantic have resulted in morphological and developmental stasis in A. modesta (e.g. Schneider et al., 1999). Populations of A. modesta in different ocean basins may constitute a single species, but the genetic divergence between Atlantic and Pacific specimens is comparable to interspecific distances between many molluscan species pairs (Hebert, Ratnasingham & de Waard, 2003b). Further sampling in the Atlantic and breeding studies should clarify whether Atlantic and Pacific A. modesta also comprise cryptic species. Planktotrophy is presumably ancestral in Alderia, being plesiomorphic in the Limapontiidae ( Jensen, 1996). The evolution of seasonal lecithotrophy in A. willowi is intriguing as it may be the only species with pelagic lecithotrophic larvae in the family, which is strongly biased towards planktotrophy (Jensen, 1996, 2001). Among the eight genera in this family, there is one species with encapsulated metamorphosis (Costasiella ocellifera; Miles & Clark, 2002) and one with ametamorphic development bypassing the veliger stage (Limapontia senestra, formerly Acteonia cocksi; Chia, 1971). Type 2 development (¼lecithotrophic; Thompson, 1967) was reported for Costasiella nonatoi from Bermuda (Clark & Jensen, 1981). However, specimens of C. nonatoi recently collected in Bermuda produced planktotrophic larvae (Ellingson & Krug, unpubl.). Clark & Jensen (1981) may have attributed type 2 development to this species because the veligershaveeyespotsathatching, which is rare but not unprecedented among planktotrophic larvae of opisthobranchs (Goddard, 2001). Lecithotrophic development is also particularly rare among opisthobranchs from the eastern Pacific, with only six out of 126 species exhibiting pelagic lecithotrophy (Goddard, 2004). The rarity of lecithotrophy in the Limapontiidae worldwide, and among opisthobranchs from the northeastern Pacific, suggests that A. willowi evolved under strong selective pressure against obligate planktotrophy as its sole development mode. An improved understanding of the ecological differences between Alderia species may shed light on the selective forces that drove the evolution of lecithotrophy in A. willowi, and on the factors that maintain the present-day range limits of the sister species. ACKNOWLEDGEMENTS For critical logistical support we thank D. and O. Tilton, and T. and S. Kreines. Specimens of Alderia modesta were generously provided by T. Van den Neuker and R. Dekker from Europe, and Alexei Chernyshev and Andrei Shukalyuk from the western Pacific. We thank L. Angeloni for help with allozyme gels, N. Smolensky for mating trials, S. Millen for SEMs of penial stylets and specimens from Vancouver, D. Willette and G. Botello for assistance with collections, S. Escorza-Trevino and E. Torres for access to laboratory facilities, and two reviewers for comments that greatly improved the manuscript. Access to sites was provided by I. Kay (Natural Reserve Office, University of California, San Diego), K. Brown and P. Connors (Bodega Marine Reserve), B. Shelton (Newport Bay Ecological Reserve) and M. Schaadt (San Pedro). This study was supported by awards from the National Science Foundation (OCE and HRD ) to P.J.K. and by a Lerner-Gray award to R.A.E. 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