EVOLUTION OF POECILOGONY FROM PLANKTOTROPHY:...

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� 2006 The Society for the Study of Evolution. All rights reserved.

Evolution, 60(11), 2006, pp. 2293–2310

EVOLUTION OF POECILOGONY FROM PLANKTOTROPHY: CRYPTIC SPECIATION,PHYLOGEOGRAPHY, AND LARVAL DEVELOPMENT IN THE GASTROPOD

GENUS ALDERIA

RYAN A. ELLINGSON AND PATRICK J. KRUG1

Department of Biological Sciences, California State University, 5151 State University Drive, Los Angeles, California 90032-82011E-mail: [email protected]

Abstract. Poecilogony, a rare phenomenon in marine invertebrates, occurs when alternative larval morphs differingin dispersal potential or trophic mode are produced from a single genome. Because both poecilogony and crypticspecies are prevalent among sea slugs in the suborder Sacoglossa (Gastropoda: Opisthobranchia), molecular data areneeded to confirm cases of variable development and to place them in a phylogenetic context. The nominal speciesAlderia modesta produces long-lived, feeding larvae throughout the North Atlantic and Pacific, but in California canalso produce short-lived larvae that metamorphose without feeding. We collected morphological, developmental, andmolecular data for Alderia from 17 sites spanning the eastern and western Pacific and North Atlantic. Estuaries southof Bodega Harbor, California, contained a cryptic species (hereafter Alderia sp.) with variable development, sister tothe strictly planktotrophic A. modesta. The smaller Alderia sp. seasonally toggled between planktotrophy and lecith-otrophy, with some individuals differing in development but sharing mitochondrial DNA haplotypes. The siblingspecies overlapped in Tomales Bay, California, but showed no evidence of hybridization; laboratory mating trialssuggest postzygotic isolation has arisen. Intra- and interspecific divergence times were estimated using a molecularclock calibrated with geminate sacoglossans. Speciation occurred about 4.1 million years ago during a major marineradiation in the eastern Pacific, when large inland embayments in California may have isolated ancestral populations.Atlantic and Pacific A. modesta diverged about 1.7 million years ago, suggesting trans-Arctic gene flow was interruptedby Pleistocene glaciation. Both Alderia species showed evidence of late Pleistocene population expansion, but thesouthern Alderia sp. likely experienced a more pronounced bottleneck. Reduced body size may have incurred selectionagainst obligate planktotrophy in Alderia sp. by limiting fecundity in the face of high larval mortality rates in warmmonths. Alternatively, poecilogony may be an adaptive response to seasonal opening of estuaries, facilitating dispersalby long-lived larvae. An improved understanding of the forces controlling seasonal shifts in development in Alderiasp. may yield insight into the evolutionary forces promoting transitions to nonfeeding larvae.

Key words. Cryptic species, larval development, lecithotrophy, phylogeography, planktotrophy, poecilogony, sacog-lossan, trans-Arctic exchange.

Received March 9, 2006. Accepted July 20, 2006.

Marine ecosystems have fewer morphologically distin-guishable species than terrestrial habitats, and many marinespecies have cosmopolitan distributions (May 1994). Thismay result from the considerable dispersal ability of ocean-dwelling organisms, since pelagic adults and planktonic lar-vae face few geographical obstacles to large-scale transport(Strathmann 1990; Palumbi 1992; Pechenik 1999). However,molecular studies of marine organisms frequently uncoversubdivided populations and cryptic species, suggesting ge-netic divergence may commonly occur at small spatial scalesor with minimal morphological diversification (Palmer et al.1990; Palumbi 1992; Knowlton 1993, 2000; Lee 2000; Daw-son and Jacobs 2001; McGovern and Hellberg 2003; Lee andO Foighil 2004; Tarjuelo et al. 2004; Collin 2005; Meyer etal. 2005). Where biological lines are sharply drawn in whatappears to be an easily traversed oceanic milieu, we may gaininsight into the forces that promote divergence, maintainboundaries between species, and drive the adaptive evolutionof marine life histories.

Development mode strongly affects patterns of phylo-geography and macroevolution in marine invertebrates(Arndt and Smith 1998; Collin 2000; Jeffery and Emlet2003). Planktotrophic species produce feeding larvae withhigh dispersal potential, resulting in panmictic populationsand extended ranges (Caley et al. 1996; Todd 1998; Bohonak1999; Pechenik 1999). An abbreviated planktonic periodtends to decrease a species’ range and geological longevity

while increasing local adaptation and the likelihood of spe-ciation events (Hansen 1978; Vermeij 1982; Jablonski 1986;Palumbi 1995; Sanford et al. 2003). Understanding transi-tions from feeding to nonfeeding modes of larval develop-ment is an outstanding goal of evolutionary biology (Strath-mann 1978; Raff 1987; Hadfield et al. 1995; Havenhand1995; Hart et al. 1997; Duda and Palumbi 1999; Hart 2000;McEdward 2000; Jeffery et al. 2003). However, the selectiveforces that drive the evolution of nonfeeding developmentremain opaque, partly for lack of robust phylogenies pairedwith relevant ecological and developmental data for the spe-cies involved (Havenhand 1995; Wray 1995; McEdward2000). Small body size has been hypothesized to favor brood-ing or direct development (Strathmann and Strathmann 1982;Byrne and Cerra 1996), but there is little theory accountingfor why pelagic lecithotrophy should be more adaptive thanplanktotrophy (Chia 1974).

Insight into life-history evolution may come from organ-isms that exhibit poecilogony, a stable reproductive poly-morphism in which larvae of a single species can developby alternative pathways (Giard 1905; Chia et al. 1996). Mo-lecular data often reveal that putative cases of poecilogonyare cryptic species differing in development, however (Hoag-land and Robertson 1988; Bouchet 1989). Convincing ex-amples of variable development are limited to polychaeteworms in the family Spionidae (Levin 1984; Gibson et al.1999; Morgan et al. 1999; Schultze et al. 2000; Gibson and

2294 R. A. ELLINGSON AND P. J. KRUG

TABLE 1. Populations of Alderia modesta sampled for development mode, morphology, and genetic analyses. Slugs from Russia, Belgium,Vancouver, and Humbolt were obtained as ethanol-preserved specimens. Unless otherwise noted, all populations are in California.

Location Latitude, longitude Date sampled

Doel, Belgium (DB) 51�19�N, 4�16�E Aug. 2005Amurskii Bay, Russia (RS) 43�09�N, 131�54�E June 2004Kodiak, Alaska (AK) 57�35�50�N, 152�28�17�W June 2005Vancouver, British Columbia, Canada (BC) 49�18�21�N, 123�05�32�W July 2003Tillamook, Oregon (TI) 45�26�55�N, 123�50�57�W March 2005Coos Bay, Oregon (CB) 43�21�48�N, 124�11�45�W July 2000, October 2001, February 2005Humboldt (HU) 40�44�31�N, 124�12�59�W February 2004Bodega Harbor (BH) 38�18�58�N, 123�03�24�W September 2003, September 2004Walker Creek (WC) 38�13�46�N, 122�54�56�W September 2004Cow Landing (CL) 38�11�02�N, 122�54�40�W September 2004South Tomales Bay (TB) 38�06�53�N, 122�51�08�W September 2003, September 2004San Francisco Bay (SF) 37�52�57�N, 122�31�09�W September 2003–August 2004 (5�)Morro Bay (MB) 35�20�39�N, 120�50�35�W September 2002Santa Barbara (SB) 34�24�01�N, 119�32�07�W July 1999, February 2000Los Angeles Harbor (LA) 33�42�48�N, 118�17�02�W January 2004–August 2004 (4�)Newport Bay (NB) 33�37�16�N, 117�53�32�W June 2003–August 2004 (6�)San Diego (SD) 32�47�36�N, 117�13�47�W monthly 1996–1998; July 1999–August 2004 (4�)

Gibson 2004) and herbivorous sea slugs in the gastropodsuborder Sacoglossa (West et al. 1984; Clark 1994; Krug1998). The adaptive value of expressing multiple dispersalstrategies from one genotype is well documented for adultmorphs of terrestrial insects (Denno et al. 1980; Harrison1980; Langellotto and Denno 2001); it is therefore perplexingthat poecilogony is so rare among marine invertebrates.Equally puzzling are the phylogenetic constraints that maylimit this phenomenon to polychaetes and opisthobranch mol-luscs.

Sacoglossan sea slugs are suctorial feeders that specializeon coenocytic or large-celled, filamentous algae (Jensen1983, 1996; Trowbridge 2002). Adults feed, mate, and de-posit eggs on their algal host and are generally unable toswitch hosts after settlement (Jensen 1989; Trowbridge 1991;Trowbridge and Todd 2000). Furthermore, planktonic larvaepreferentially metamorphose in response to host-specificchemical cues (West et al. 1984; Krug and Manzi 1999; Trow-bridge and Todd 2000; Krug 2001). Host acceptance via se-lective metamorphosis may thus determine both assortativemating and ecological specialization in these marine herbi-vores, as in some insect host races (Feder et al. 1994; Caillaudand Via 2000). In addition to their host specificity, sacog-lossans display a high degree of reproductive plasticity, withmany independent transitions to nonfeeding larval develop-ment and several documented cases of poecilogony (West etal. 1984; Clark 1994; Jensen 1996; Krug 1998). Such de-velopmental flexibility may facilitate studies of the selectivepressures behind, and consequences of, alternative larvalmorphs (Krug and Zimmer 2004).

The sacoglossan Alderia modesta is found in temperateestuaries throughout the Northern Hemisphere with its ob-ligate hosts, yellow-green algae in the genus Vaucheria (Har-tog 1959; Trowbridge 1993). Atlantic populations of themonotypic genus Alderia are planktotrophic, as are Pacificpopulations from Monterey, California, north to Canada, andfrom Russia (Hand and Steinberg 1955; Hartog 1959; See-lemann 1967; Gibson and Chia 1995; Chernyshev and Cha-ban 2005). However, the nominal taxon A. modesta exhibitspoecilogony in southern California, where adults produce ei-

ther planktotrophic larvae with a month-long maturation pe-riod, or lecithotrophic larvae competent to settle upon orbefore hatching (Krug 1998, 2001). In this life-history trade-off, lecithotrophy limits adult fecundity by an order of mag-nitude and doubles benthic development time, but greatlyreduces the planktonic period and hence the mortality rateof larvae. Although prior evidence indicated this was a caseof variable development, the northern limit of poecilogonywas not established, nor was the evolutionary relationshipbetween strictly planktotrophic and southern, poecilogonouspopulations delineated.

The geographically restricted expression of poecilogony inan otherwise planktotrophic taxon provided the opportunityto explore an evolutionary origin of variable development.We used molecular, developmental, and morphological char-acters to assess the global phylogeography of the genus Al-deria. Sequences from two mitochondrial loci were used totest for genetic breaks between strictly planktotrophic versuspoecilogonous populations, and to examine population struc-ture in the eastern Pacific. A molecular clock was calibratedwith geminate sacoglossans to date major divergence eventsbetween and within lineages. Our data support a model ofcryptic speciation via peripheral isolation, accompanied bya rare life-history shift to environmentally cued changes indevelopment mode. We discuss features of adult and larvalecology that may have favored seasonal lecithotrophy insouthern estuaries.

MATERIALS AND METHODS

Study System and Population Sampling

The genus Alderia was described from Ireland (Thompson1844; Allman 1845), and the sole recognized species, A. mo-desta, from Sweden (Loven 1844). Specimens superficiallyresembling A. modesta were collected between 1999 and 2005from 15 sites along the northeastern Pacific coast of the Unit-ed States and Canada (Table 1). Slugs were collected at lowtide from exposed patches of Vaucheria species, examinedmorphologically under a dissecting microscope, and placedindividually in petri dishes of seawater for egg mass depo-

2295EVOLUTION OF VARIABLE LARVAL DEVELOPMENT

sition. Development mode was typed one to two days lateraccording to egg size within the first deposited clutch (Krug1998). Typed adults were frozen at �80�C or stored in 95%ethanol prior to DNA extraction. Specimens were collectedand preserved from the western Pacific in Amurskii Bay, Seaof Japan, Russia, in June 2004, and from the Atlantic in theScheldt estuary, Doel, Belgium, in August 2005 by col-leagues (see Acknowledgments).

No closely related genus exists in the temperate easternPacific, and the phylogenetic position of Alderia is contro-versial (Evans 1953; Gascoigne 1976; Jensen 1996). The At-lantic genus Limapontia was united with Alderia in the familyLimapontiidae based on distinctively sabot-shaped radularteeth (Jensen 1996), and was closely related to Alderia in apreliminary molecular phylogeny of the Limapontioidea (R.A. Ellingson and P. J. Krug, unpubl. data). For outgroupcomparisons, L. depressa was therefore collected from Gal-way Bay, Republic of Ireland (53�13�N, 08�54�E) in August2005.

DNA Extraction, Amplification, and Sequencing

Genomic DNA was extracted from whole frozen slugs us-ing the QIAamp DNA Mini Kit (Qiagen, Inc., Valencia, CA)and stored in extraction buffer at �20�C prior to amplifica-tion. Polymerase chain reactions (PCR) were used to amplifya 710-base pair (bp) fragment of the mitochondrial cyto-chrome oxidase subunit I (COI) gene, using primersLCO1490 and HCO2198 (Folmer et al. 1994), and a 480-bpfragment of the mitochondrial 16S rRNA (large ribosomalsubunit) gene using primers 16Sar-5� and 16Sbr-3� (Palumbi1996). A negative control (no template) was included in eachrun. Amplification used a reaction volume of 50 �l containing1� Promega (Madion, WI) PCR buffer (10 mM Tris-HCl pH9.0, 50 mM KCl, 0.1% Triton X-100), 2.5 mM MgCl2, 0.2mM dNTPs, 0.2 �M of each primer, 1 U Promega Taq DNAPolymerase and 50–200 ng of template DNA. To amplify theCOI fragment, the following thermal cycler profile was used:denaturation at 94�C for 2 min followed by 35 cycles of (94�Cfor 30 sec, 40�C for 45 sec, and 72�C for 60 sec). For 16S,the thermal cycler profile began with denaturation at 94�Cfor 2 min followed by 40 cycles of (94�C for 30 sec, 50�Cfor 30 sec, and 72�C for 60 sec), followed by an extensionstep at 72�C for 7 min.

Polymerase chain reaction products were visualized byelectrophoresis on a 1% agarose gel, and were purified withthe Wizard SV Gel and PCR Clean-Up System (Promega).Both strands of purified products were directly cycle-se-quenced in both directions using the amplification primersand Big Dye Terminator 3.1 Cycle Sequencing chemistry,and electrophoresed on an ABI 3100 Avant Capillary Se-quencer (Applied Biosystems, Foster City, CA). Sequencingboth strands and resequencing of unique haplotypes revealedno evidence of PCR error. Sequences used for this study havebeen deposited in GenBank (accession numbers DQ364252–DQ364426).

Phylogenetic Analyses

To determine phylogenetic relationships and populationstructure, COI sequences were amplified from specimens of

Alderia from the eastern Pacific (n � 233), western Pacific(n � 3), and Atlantic (n � 7). Suitable sequence data wasobtained from all specimens for 480 bp of COI, correspond-ing to positions 139 to 618 of the complete Aplysia californicaCOI gene (GenBank accession number AY569552), andaligned with SeqScape 2.1.1 software (Applied Biosystems).A 450-bp fragment of the 16S gene was sequenced from asubset of specimens (n � 49), resulting in 12 haplotypes fromA. modesta (six each from the Atlantic and Pacific), and eighthaplotypes from Alderia sp. (see Results). All 16S sequenceswere aligned in ClustalX 1.83 (Thompson et al. 1997), withadjustments made by eye using three models of gastropod16S secondary structure (Lydeard et al. 2000; Medina andWalsh 2000; Valdes 2003). Amino acid substitutions, meanbase composition, and transition/transversion (s/v) ratio weredetermined in Mega 3.0 (Kumar et al. 2004).

A neighbor-joining (NJ) tree of all eastern Pacific COIhaplotypes was constructed using PAUP* 4.0b10 (Swofford2001). For NJ analysis, the Tamura-Nei model of sequenceevolution was used (Tamura and Nei 1993), and robustnessof the topology was assessed with 2000 bootstrap replicates.Subsequent analyses included Atlantic and western Pacificspecimens, and used both COI and 16S sequence data from49 individuals to determine (1) interspecific relationships and(2) intraspecific divergence between ocean basins. A parti-tion-homogeneity test as implemented in PAUP* indicatedno significant conflict between COI and 16S datasets; wetherefore performed Bayesian and parsimony analyses on thecombined data (Cunningham 1997; Wiens 1998). For Bayes-ian analysis, COI and 16S datasets were partitioned so thata separate substitution model could be applied to each. Thebest-fit model for the COI dataset selected using the Akaikeinformation criterion as implemented in Modeltest 3.7 (Po-sada and Crandall 1998) was TrN � , with gamma distri-bution shape parameter of 0.1397. For 16S, the best-fit modelwas HKY � I � , with gamma shape 0.2932. Bayesiananalysis used the metropolis-coupling Markov chain MonteCarlo method as implemented in MrBayes 3.1.1 (Ronquistand Huelsenbeck 2003). The analysis was run for 3,000,000generations with a tree saved every 1000 generations; a con-sensus tree was generated in PAUP* after a burn-in periodof 300. For parsimony analysis, the maximum number of treessaved was limited to 5000 due to computational time con-straints, and bootstrap values (1000 replicates) were obtainedusing the ‘‘fast’’ stepwise-addition option.

Within-Clade Genetic Structure and Expansion Estimates

Genetic structures of the two major COI clades from theeastern Pacific were characterized in Arlequin 2.000 (Schnei-der et al. 2000). Haplotype diversity (H), nucleotide diversity(), and mismatch distributions were calculated for eachclade, hereafter referred to as A. modesta and Alderia sp. (seeResults). The proportion of genetic variation partitionedamong estuaries was estimated by analysis of molecular var-iance (AMOVA; Excoffier et al. 1992), using both Slatkin’s(1991, 1995) linearized FST and �ST values. Sites withinTomales Bay (south Tomales Bay, Cow Landing, and WalkerCreek) were not significantly different in preliminary AMO-VA analysis and, being located in the same estuary, were


grouped together for subsequent analyses. Based on Model-test results, the best-fit model of sequence evolution for eachclade was used to calculate �ST distance matrices and (A.modesta, Tamura-Nei; Alderia sp., Tamura three-parameter)(Tamura 1992; Tamura and Nei 1993). Tajima’s D and Fu’sFS were calculated by comparing observed values against anull distribution based on 1000 simulations to determine sig-nificance levels for each test statistic. Tajima’s D comparesthe number of segregating sites with the number of pairwisedifferences in a population, and should be near zero for anequilibrium population; negative values indicate a selectivesweep or population expansion. Large negative values of Fu’sFS suggest population expansion (Fu 1997; Excoffier andSchneider 1999).

Parameters � (time since expansion, expressed in units ofmutation rate), 0 (population size before expansion), and 1(population size after expansion) were estimated from mis-match distributions using a generalized nonlinear least-squares approach (Schneider and Excoffier 1999). Confidenceintervals were determined by a parametric bootstrap proce-dure, assuming a sudden expansion model. The validity ofthe model was tested using the sum of square deviations(SSD) between observed and expected mismatch distribu-tions as a test statistic. The SSD distribution was generatedvia coalescent simulations of expansion using the estimatedparameters �, 0 and 1; the P-value was the proportion oftimes the difference between simulated and expected mis-match distribution exceeded the observed SSD (Schneiderand Excoffier 1999). To express expansion time in years,values of � were multiplied by the mutational time scale(generation time/2u, where u is the number of substitutionsper year for the fragment of interest) (Rogers 1995; Excoffierand Schneider 1999). The substitution rate per nucleotide sitewas calculated from our COI data, and then multiplied bythe length of the COI fragment used in mismatch analyses,yielding a rate of 2.62 � 10�5 substitutions/yr. Based onlarval period and laboratory rearing data, we estimated gen-eration times (egg-to-egg period) of two months for A. mo-desta, versus one month for Alderia sp. (averaging the gen-eration time from a lecithotrophic larva, 2.5 weeks, and aplanktotrophic larva, 5.5 weeks).

Divergence Estimates

We calibrated a molecular clock using the only well-es-tablished geminate sacoglossans. Specimens of the CaribbeanElysia (� Tridachia) crispata were collected December 2004from Bocas del Toro, Panama, with permission of the Pan-amanian government; preserved specimens of E. (� Trida-chiella) diomedea from the eastern Pacific were obtained fromthe Los Angeles County Museum of Natural History. Anygeminates may have been isolated prior to final closure ofthe Panamanian seaway (Knowlton and Weigt 1998; Marko2002). However, E. crispata and E. diomedea live in shallowmangrove fringe, probably the last habitat sundered by theIsthmus; we therefore used a minimum divergence time of3.1 million years ago (MYA) for rate calibration (Coates andObando 1996). Plots of s/v ratio against genetic distanceindicated third positions of COI were saturating at distancesgreater than 15% (mean ratio � 1.5); substitution rate was

thus determined for the first codon position of COI, for whichlog-likelihood ratio tests could not reject molecular clockassumptions. Based on Modeltest, the TrNef (Tamura-Nei,equal base frequencies) model of sequence evolution wasused to calculate first position distances (Tamura and Nei1993; Huelsenbeck and Crandall 1997; Posada and Crandall1998). To correct for intraclade variance, net nucleotide di-vergences were calculated as �AB(net) � �AB � 0.5(�A � �B),where �A and �B are mean intraclade distances, and �AB isthe mean interclade distance (Nei and Li 1979; Edwards andBeerli 2000). The rate determined with E. crispata–E. di-omedea was used to estimate divergence times for (1) A.modesta versus Alderia sp., and (2) Pacific versus Atlanticpopulations of A. modesta.

Copulatory Behavior and Reproductive Isolation

Reproductive isolation between slugs was assessed bymonitoring mating behavior and egg mass production be-tween pairs in the laboratory. Because field-caught Alderiastore sperm for weeks, virgin slugs were reared by individ-ually metamorphosing lecithotrophic larvae of Alderia sp.from San Diego parents on filaments of Vaucheria longicau-lis. Virgin slugs were raised in isolation for three to fourweeks, by which time all were reproductively mature; controlslugs raised in pairs began reproducing within two weeks ofmetamorphosis. Virgin A. modesta were not available, dueto difficulties in culturing their long-lived planktotrophic lar-vae. Laboratory-reared virgins (n � 7) were paired with in-dividual specimens of the northern clade from Bodega Har-bor, and observed under a dissecting microscope over 6 h.The members of each pair were then isolated and monitoredfor egg production for 48 h. Controls were paired virginAlderia sp. put together at the same time as the northern-southern pairs.

Size and Fecundity Measurements

For wet weight determinations, freshly collected slugswere first blotted dry with a paper towel and weighed to thenearest 0.1 mg, then returned to individual dishes for egglaying. Size differences between northern and southern spe-cies (see Results), and between sites within each species,were tested with a nested ANOVA using site as a randomeffect nested within species as a fixed effect (Sokal and Rohlf1995). For fecundity measurements, the first egg mass de-posited by each preweighed adult was maintained in a petridish with 4 ml of 0.22 �m filtered seawater until hatching(three days for planktotrophic, six days for lecithotrophiceggs), and larvae per clutch were counted under a dissectingmicroscope. Relationship between size and fecundity wasdetermined by regressing egg number for the first clutch de-posited after collection on adult weight (Krug 2001). Re-gressions were run for three populations of A. modesta andfor two populations of Alderia sp. (one for each larval morph).Mean fecundity was measured for an additional two popu-lations of A. modesta and five populations of Alderia sp.

RESULTS

Cryptic Species and Variable Development in Alderia

In a survey of estuaries along the northeastern Pacificcoastline, specimens of A. modesta from Bodega Harbor, Cal-


FIG. 1. Seasonal distribution of development modes exhibited by sea slugs in the genus Alderia along the northeastern Pacific coast.Data are the proportion of adult slugs at each site that produced planktotrophic (black) or lecithotrophic (white) larvae, with sample sizegiven on each pie graph. Sampling dates are in Table 1; summer sampling was conducted from June through September, winter samplingfrom November through February.

ifornia, northward laid only planktotrophic eggs; in contrast,south of Bodega, slugs produced mostly lecithotrophicclutches in summer and both development modes in winter(Fig. 1). Concordant morphological differences were evident:slugs from Bodega north had a smooth yellow dorsum dottedwith brown speckles, whereas south of Tomales Bay, mosthad a dark background color with a raised dorsal hump, splitdown the midline by a band of yellow (Krug et al. 2007).

Both morphotypes were present within Tomales Bay. Inplanktotrophic egg masses from northern slugs, eggs werecoiled into a spiral, a plesiomorphic character in the super-family Limapontioidea. In contrast, both planktotrophic andlecithotrophic clutches of southern slugs lacked any spiral,with eggs haphazardly arranged inside the egg mass (Kruget al. 2007).

To determine whether there were genetic differences across


the Bodega Harbor breakpoint, a portion of the mitochondrialCOI gene was sequenced from slugs previously scored fordevelopment mode of their larvae. The mean percent basecomposition was 37T:23A:21C:19G. This fragment washighly polymorphic, yielding 146 unique haplotypes with 155variable positions (21 first, 2 second, 132 third codon posi-tion). Nearly all substitutions were synonymous, exceptingfour haplotypes that had one conservative amino acid sub-stitution compared to all others, each at a different site.

A neighbor-joining (NJ) tree of eastern Pacific COI hap-lotypes showed a genetic divide between populations northand south of Bodega Harbor, concordant with developmentaland morphological differences (Fig. 2). The COI haplotypesformed two reciprocally monophyletic clades with 100%bootstrap support. Intraclade divergence was a maximum of5% but the two clades were 18–24% different from each other(Tamura-Nei distance), exceeding the interspecific diver-gence between many sacoglossans (R. A. Ellingson and P.J. Krug, unpubl. data). Further, there were fixed differencesbetween clades at 27 positions (Krug et al. 2007). Based ondifferences in morphology, development, and DNA sequencedata, the southern clade represents a cryptic species hereafterreferred to as Alderia sp.

Within Alderia sp. there was no concordance between de-velopment and tree topology, and 12 different haplotypeswere shared by specimens that produced different types oflarvae (Fig. 2). The southern Alderia sp. is thus poecilogon-ous, expressing alternative larval developmental modes with-in a single evolutionarily independent lineage. There weretwo weakly supported intraspecific clades that did not cor-relate with geographic origin of haplotypes. Northern pop-ulations were morphologically consistent with the originaldescription of A. modesta, which is planktotrophic in allknown Atlantic and Pacific populations.

The more broadly distributed A. modesta had higher hap-lotype and nucleotide diversities and more segregating sitesthan its geographically restricted congener (Table 2). Thenorthern species A. modesta also had a significantly highermean number of differences between haplotypes (Fig. 3 andresults of a permutation test, P � 0.0005). In contrast, Alderiasp. had fewer pairwise differences and a common haplotypeshared throughout its range (Fig. 3). Tajima’s D and Fu’s FSwere significantly negative for both species, suggesting re-cent expansion (Table 3), and a model of sudden populationexpansion could not be rejected for either species (A. modesta,P � 0.89; Alderia sp., P � 0.95). Parameters � and 0, es-timated from mismatch distributions, suggested A. modestabegan expanding at an earlier date and from a population oflarger size (Table 3). The mutational time scale calculatedfor each species placed these expansions at the end of thePleistocene.

Phylogeography of Alderia spp

Based on FST values, a significant percentage of the totalgenetic variance was distributed among estuaries in Alderiasp. (AMOVA: P � 0.05). All significant pairwise FST com-parisons involved Morro Bay, the most geographically iso-lated site (Table 4). Only Morro Bay contained multiple pri-vate haplotypes shared by four or more individuals. In Alderia

sp., no among-estuary comparisons were significant based on�ST. No population structure was evident in Pacific A. mo-desta by either FST or �ST.

We subsequently included specimens of A. modesta fromthe Atlantic and western Pacific in a broader phylogeographicanalysis. In addition to COI, a 450-bp fragment of the moreconserved 16S gene was sequenced from a subset of speci-mens. In both Bayesian and parsimony trees based on com-bined COI and 16S sequence data, A. modesta from the NorthPacific and Atlantic formed a clade that was reciprocallymonophyletic with Alderia sp. (Fig. 4). The sister species ofAlderia were a mean 20.6% divergent in COI (Tamura-Neidistance) and 3.1% in 16S (uncorrected distance). Within A.modesta, Pacific and Atlantic populations were also recip-rocally monophyletic and substantially divergent (11.0% inCOI, 1.3% in 16S). Three haplotypes from the western Pacific(Sea of Japan) fell within the larger A. modesta clade, butdid not group together. Within Alderia sp., bootstrap valuesand posterior probabilities supported a clade composed pri-marily of haplotypes from southern California (Fig. 4).

A geminate pair of elysiid sacoglossans yielded a rate of1.57% change per million years for the first codon positionof COI. Based on this rate, Alderia species diverged about4.1 MYA, in the Miocene, whereas Atlantic and Pacific pop-ulations of A. modesta diverged about 1.7 MYA, in the earlyPleistocene.

Overlap Zone

To refine the range limits of Alderia spp., five sites weresampled between San Francisco Bay and Bodega Harbor in2003–2004 (Fig. 5). Individuals were typed by morphology,development, and mitochondrial DNA analysis. In both sum-mers, only A. modesta was found in Bodega Harbor, and thesouthern end of Tomales Bay contained only Alderia sp. Thetwo species co-existed in central (Cow Landing site) andnorthern (Walker Creek site) Tomales Bay (Fig. 5). A fewA. modesta were found in San Francisco Bay in December2003 and August 2004. Genetic analysis confirmed that allslugs (n � 80) from mixed populations were correctly typedby morphology (haplotypes included in Fig. 2). There wasno evidence of mitochondrial DNA introgression in eitherdirection where the two species coexisted. The host alga atthe south Tomales Bay site (nominally V. longicaulis) wassimilar in appearance to Vaucheria from southern California,forming dense, discrete patches with a mosslike texture. Thealgae in San Francisco, the rest of Tomales Bay, and BodegaHarbor had a more filamentous morphology not typicallyobserved in southern California.

Copulatory Behavior and Reproductive Isolation

To assess reproductive isolation between the sibling spe-cies, laboratory-reared virgin Alderia sp. (n � 7) were pairedwith individual A. modesta from Bodega and observed for 6h. All Bodega slugs reacted to contact with a virgin Alderiasp. by immediately extending the penis; at least three wereobserved to inseminate a virgin, based on flow of spermthrough the translucent penis. All virgin slugs extended theirpenis upon contact with a Bodega slug and one aberrantlydischarged sperm into the seawater, but none were observed


FIG. 2. Phylogenetic relationships among slugs in the genus Alderia from the northeastern Pacific, based on COI sequences. A neighbor-joining (NJ) tree was derived from all nucleotide sites; branches with bootstrap support �70 are bold. Unmarked haplotypes were obtainedfrom adults producing planktotrophic larvae, those labeled with a dark circle from adults producing lecithotrophic larvae, and thosemarked with a triangle were shared by adults that produced larvae differing in development mode.


TABLE 2. Molecular diversity in Alderia species from the northeastern Pacific. Sequence data for COI were collected for A. modestaand its cryptic congener, Alderia sp., and analyzed in Arlequin 2.000. ‘‘Unique’’ haplotypes were sampled only once in the study.Haplotype and nucleotide diversities are given � variance.

A. modesta Alderia sp.

n (no. of individuals) 104 129k (no. of haplotypes) 84 62% unique haplotypes 94.0% 74.2%No. of segregating sites (S) 103 72Haplotype diversity (H) 0.9860 � 0.0062 0.9119 � 0.0217Nucleotide diversity () 0.0209 � 0.0107 0.0141 � 0.0074Mean no. of pairwise differences (95% CI) 9.68 (5.86–12.29) 6.66 (3.29–10.28)

to inseminate their partner even after prolonged contact (�10min). Only one of seven inseminated virgins produced eggmasses over the next 48 h, whereas all paired control virginsreciprocally inseminated each other and produced fertilizedclutches. One former virgin produced two fertilized clutchesthat developed normally; however, it was subsequently foundthat Alderia sp. can self-fertilize, leaving the parentage ofoffspring from the virgin slug ambiguous (N. Smolensky andP. J. Krug, unpubl. data).

Size and Fecundity

Overall, specimens of A. modesta were significantly largerthan Alderia sp. (Fig. 6, Table 5). This size difference initiallydrew attention to rare individuals of A. modesta in San Fran-cisco Bay. Within each species, there was highly significantvariation in size between sites (Table 5). Specimens fromCoos Bay, Oregon were larger than slugs from all Californiansites (Fig. 6, and results of a post-hoc Scheffe test: P � 0.05).At Cow Landing, specimens of A. modesta were significantlylarger than sympatric Alderia sp. (post-hoc Scheffe test: P �0.05). Size did not consistently discriminate between species,however; A. modesta from Walker Creek 2004 were not sig-nificantly larger than Alderia sp. from that or any other site(Fig. 6). Interannual differences were found at San Francisco,where the mean size in Aug 2004 was significantly largerthan in Aug 2003 (post-hoc Scheffe test: P � 0.05). WithinAlderia sp., there was no increase in size with latitude, andthe smallest specimens came from south Tomales Bay.

Fecundity (eggs per clutch) was positively related to sizein both species and, in Alderia sp., for both developmentmodes (Table 6). The mean fecundity of A. modesta rangedfrom 267 � 34 to 702 � 98 planktotrophic eggs per clutch(n � 5 populations), whereas specimens of Alderia sp. pro-duced 166 � 15 and 311 � 25 planktotrophic eggs per clutchin two populations. The mean number of lecithotrophic eggsper clutch ranged from 32 � 2 to 325 � 71 (n � 5 popu-lations), but the maximum value was atypically high; four ofthe five populations had a mean fecundity of less than 120lecithotrophic eggs per clutch.

DISCUSSION

Cryptic Speciation and Phylogeography in Alderia

Based on molecular, morphological, and developmentaldifferences, most specimens previously regarded as A. mo-desta south of Bodega Harbor, California, comprised a crypticspecies (Dayrat 2005; DeSalle et al. 2005). The sibling spe-

cies can be differentiated by fixed nucleotide differences inthe COI gene, allozymes at the PGI locus, and dorsal mor-phology (Krug et al. 2007). Whereas A. modesta is exclu-sively planktotrophic, the poecilogonous Alderia sp. express-es either planktotrophy or lecithotrophy depending on theseason; specimens differing in development cannot be dis-tinguished by any other criteria, share COI haplotypes, andswitch development mode under laboratory conditions (Krug1998, 2007).

The Californian endemic Alderia sp., likely evolved at thesoutheastern range limit of a broadly distributed ancestor. Anemerging paradigm for macroevolution of intertidal gastro-pods is speciation via peripheral isolation. Intertidal organ-isms are effectively confined to one-dimensional strips ofshoreline, and may be susceptible to transient isolationcaused by climate change (Valentine and Jablonski 1983;Reid 1990). For instance, Pleistocene conditions forced manytaxa into refugia in the Southern California Bight, followedby Holocene expansion northward (Hellberg 1994; Marko1998; Dawson et al. 2001; Hellberg et al. 2001; Marko et al.2003; Jacobs et al. 2004). However, in no previous case hasa major life-history shift been potentially linked to transientisolation at the edge of a species’ distribution in the easternPacific. As lecithotrophy is expressed by the southern speciesprimarily in summer months, it is likely an adaptation towarm conditions. The data for Alderia sp. are consistent withspeciation by peripheral isolation from a planktotrophic an-cestor. Lecithotrophy is an apomorphy of the southern spe-cies, as planktotrophy is pleisiomorphic in the Limapontiidae(Jensen 1996). Whereas A. modesta retained the ancestraldevelopment mode, Alderia sp. incorporated an extraordinarydegree of flexibility into its life history, seasonally expressingalternative larval morphs and bet-hedging dispersal strategies(Krug 2001).

In the absence of paleontological data for shell-less mol-luscs, molecular methods may permit reconstruction of theirevolutionary history. A molecular clock, calibrated with gem-inate sacoglossans, suggested a coalescence time of 4.1 MYAfor A. modesta–Alderia sp. In the late Miocene and earlyPliocene, coastal geomorphology and high productivity inthe eastern Pacific contributed to radiations of many marinetaxa (Jacobs et al. 2004). High sea level also created isolatedestuaries in the Central Valley of California, providing op-portunities for allopatric speciation among wetland organ-isms such as Alderia (Hall 2002; Jacobs et al. 2004).

Pleistocene climate change likely severed trans-Arcticgene flow in A. modesta. The molluscan fauna of the North


FIG. 3. Histogram showing the distribution of pairwise differences between COI haplotypes for (A) Alderia modesta, ranging fromKodiak, Alaska, south to San Francisco, California, and (B) Alderia sp., ranging from Walker Creek, California, south to San Diego.

TABLE 3. Comparative estimated population expansion parametersfor Alderia species, based on mismatch distributions of COI se-quences from the northeastern Pacific. Time since expansion, �, isgiven in units of the mutational time scale, or as t in 1000 years(KY; Excoffier and Schneider 1999). Calculations were based on u� 2.62 � 10�5 substitutions per year and generation times of twomonths (A. modesta) versus one month (Alderia sp.). Raw valuesof 0 were divided by 2u to estimate population size at expansion(given as no. of individuals), and after expansion ( 1, given as 1000individuals). The 95% confidence intervals around t, 0 and 1 aregiven in the corresponding units. *P � 0.05; ***P � 0.0001.

A. modesta Alderia sp.

Tajima’s D �1.67* �1.58*Fu’s FS �24.49*** �24.92***� 11.76 11.23t (KY) 37 1895% CI 18–59 6–27 0 (individuals) 24,000 4095% CI 0–72,000 0–100,000 1 (1000 individuals) 405 20795% CI 220–1710 100–830

Atlantic and Pacific evolved independently for about 100million years until the opening of the Bering Strait, as earlyas 5.5 MYA or as recently as 3.1 MYA (Marincovich andGladenkov 2001). Formation of the Strait initiated a trans-Arctic migration lasting until the late Pliocene. In this asym-metric invasion, molluscs of Pacific origin disproportionatelycolonized the North Atlantic by an 8:1 ratio (Vermeij 1991).Species that participated in the trans-Arctic exchange showeither genetic differences suggesting prolonged isolation(Grant and Stahl 1988; McDonald and Koehn 1988; Zaslav-skaya et al. 1992; Collins et al. 1996), or low intraspecificvariation indicating recent gene flow (Graves and Dizon1989; Palumbi and Wilson 1990; Palumbi and Kessing 1991).A divergence time of 1.7 MYA for Atlantic and Pacific A.modesta suggests early Pleistocene glaciation removed suit-able habitat from the Arctic, and conditions have since re-mained unfavorable for larval transport between basins. At-lantic and Pacific slugs have not diverged in morphology ordevelopment, perhaps due to ecological similarity of theirhabitats (Schneider et al. 1999); however, they represent evo-lutionarily independent lineages separated by Quaternary cli-mate change. Speciation predated the trans-Arctic exchange,indicating a Pacific origin for the genus Alderia.


TABLE 4. Pairwise differences among estuaries in Alderia sp. Data are Slatkin’s linearized FST values, with sample size given inparentheses. Nucleotide diversity � variance is given on the diagonal. Significant pairwise differences were determined by AMOVA inArlequin with 10,000 permutations of the data. *P � 0.05, **P � 0.01.

Tomales Bay(n � 29)

San Francisco(n � 32)

Morro Bay(n � 26)

Los Angeles(n � 14)

Newport Bay(n � 14)

San Diego(n � 14)

TB 0.013 � 0.007SF �0.000 0.015 � 0.008MB 0.037* 0.036* 0.012 � 0.006LA 0.024 0.010 0.082** 0.018 � 0.010NB �0.002 0.001 0.017 0.049 0.014 � 0.008SD �0.008 �0.011 0.034 0.019 �0.014 0.015 � 0.008

Both species show evidence of recent population expan-sion, the parameters of which were estimated from mismatchdata. Assuming Atlantic and Pacific A. modesta diverged 1.7MYA, we derived a substitution rate of 5.47% per site permillion years for COI, or u � 2.62 � 10�5 substitutions peryear for a 480-bp fragment. The average generation time ofA. modesta was estimated as twice that of Alderia sp., dueto obligate planktotrophy and colder temperatures during lar-val maturation. Dividing their respective generation times by2u gave a mutational time scale of 3180 for A. modesta and1580 for Alderia sp.; multiplying by � converts this parameterto years since expansion (Rogers 1995). From these esti-mates, Alderia sp. began expanding about 18,000 years ago(95% CI: 6000–27,000), whereas A. modesta expanded about37,000 years ago (95% CI: 18,000–59,000). Values of 0 werealso higher for A. modesta ( 0 � 24,000 slugs) than Alderiasp. ( 0 � 40 slugs), but the confidence intervals for both weretoo broad (95% CI: 0–100,000) to draw conclusions aboutpre-expansion population sizes. However, the higher haplo-type diversity and mean number of pairwise differences inA. modesta are also consistent with a larger pre-expansionpopulation. The ability of long-lived larvae to disperse acrossthe Pacific may have allowed the cold-adapted A. modesta tomigrate between viable patches during glacial maxima, es-caping population crashes associated with regional glaciation(Warner et al. 1982; Marko 2004; Hickerson and Cunningham2005).

In contrast, low sea levels and a concomitant loss of es-tuarine habitat across central California may have restrictedAlderia sp. to refugia in southern California throughout thePleistocene; reduced nucleotide and haplotype diversities, thesingle common COI haplotype, and estimates of 0 all suggestAlderia sp. experienced a more dramatic bottleneck than itscongener (Grant and Bowen 1998; Caudill and Bucklin2004). A selective sweep, the alternative basis for reduceddiversity, is unlikely given the predominance of silent sub-stitutions in our COI dataset. The expansion of Alderia sp.dates to the beginning of the Holocene, when higher sea levelsmay have allowed it to recolonize estuaries as they reformedacross central California, eventually coming into secondarycontact with its sister species (e.g., Marko 1998).

Both Alderia species are specialists on Vaucheria species,which grow only in protected back bays separated by unin-habitable stretches of open coast. The fragmented nature oftheir estuarine habitat was expected to inhibit gene flow.However, no phylogeographic structure was evident in A.modesta within the Pacific, despite its considerable range;

this strictly planktotrophic species was panmictic in thenortheastern Pacific, and haplotypes from the Sea of Japangrouped within the eastern Pacific clade. The dispersal po-tential of long-lived, feeding larvae is evidently realized inA. modesta, genetically homogenizing distant estuaries. With-in Alderia sp., FST results indicated reduced gene flow be-tween Morro Bay and three other estuaries. This could resultfrom the geographically isolated nature of Morro Bay, andalso from the limited dispersal potential of lecithotrophiclarvae produced by Alderia sp. for much of the year (Krug2001; Krug and Zimmer 2000, 2004). Although �ST analysisof the COI data found no phylogeographic structure in Alderiasp., a clade with primarily southern distribution was evidentin phylogenetic studies combining COI and 16S data.

Present Distribution of the Sister Species

Historical processes do not explain the current biogeog-raphy of Alderia. The range endpoints of Alderia species fallneither at Point Conception, a major faunal transition zone,nor in central California, where some coastal taxa have phy-logeographic breaks (Valentine 1966; Burton 1998; Wares etal. 2001; Dawson 2001; Sotka et al. 2004). The northern limitof Alderia sp. was Bodega, and A. modesta was rare southof Tomales Bay. Evidence from diverse taxa suggests geneflow may be interrupted between Bodega and sites to thesouth of Point Reyes, California. In the supralittoral copepodTigriopus californicus, a northern clade with reduced geneticdiversity ranges from Alaska to Bodega, suggesting T. cal-ifornicus expanded from refugia south of Point Reyes (Ed-mands 2001). The intertidal crab Petrolisthes cinctipes has agenetic discontinuity between Bodega and the mouth of To-males Bay, only 10 km distant (R. Toonen and R. Grosberg,unpubl. data). In the urchin Strongylocentrotus franciscanus,only Bodega is differentiated from other populations (Mobergand Burton 2000). The concordant break at Bodega acrossdiverse taxa may reflect (1) historical processes such as con-tracting ranges during glaciation events; (2) differences inthe abiotic environment imposing divergent selection on ei-ther side of Bodega, affecting the distribution and frequencyof linked loci; (3) unusual hydrographic features that affectpatterns of dispersal around the mouth of Tomales Bay; orsome combination thereof. Further study of the factors con-trolling the demographics of Alderia species around PointReyes may shed light on why this area produces phylogeo-graphic patterns in diverse invertebrates.

Although larvae of Alderia species can disperse hundreds


FIG. 4. Bayesian tree showing phylogenetic relationships among Alderia species from the Pacific and Atlantic, based on combined COIand 16S mtDNA haplotypes. Bayesian posterior probabilities (above branches), and parsimony bootstrap values based on 1000 replicates(below branches), are shown for clades that received �80% support in both analyses. Haplotypes from the western Pacific are indicatedwith asterisks. Collection sites: Doel, Belgium (DB); Sea of Japan, Russia (RS); Kodiak Island, Alaska (AK); Vancouver, British Columbia(BC); Coos Bay, Oregon (CB); Bodega Harbor, California (BH); Tomales Bay (TB); San Francisco (SF); Morro Bay (MB); Los Angeles(LA); Newport Bay (NB); and San Diego (SD).

of km, they were partitioned over spatial scales of less than15 km in Tomales Bay. Distributions of Alderia species mayreflect those of the host algae if larvae only settle in areaswhere a preferred species of Vaucheria grows. Proximal pop-ulations can remain differentiated due to larval habitat choicebehavior, an emerging mechanism by which genotypes seg-regate without extrinsic barriers to gene flow (Appelbaum etal. 2002; Gilg and Hilbish 2000, 2003a,b; Hilbish et al. 2003;Baird et al. 2003; Bierne et al. 2003). The sister species could

also express divergent host-specific adaptations limiting theirability to use different Vaucheria species but few studies havetested whether local coadaptation occurs in marine herbivoresand host algae (Sotka 2003; Sotka et al. 2003). Alternatively,a gradient in the physical environment may limit where Al-deria species live if they differ in physiological tolerance(Theison 1978; Koehn et al. 1980; Schmidt and Rand 2001;Bierne et al. 2002; Brind’Amour et al. 2002; Riginos et al.2002).


FIG. 5. Demography of Alderia species in a 100-km overlap zone. Data are the proportion of A. modesta (hatched bars) versus Alderiasp. expressing planktotrophic (black) or lecithotrophic (white) development. Sample size is given below each pie graph.

There was no evidence of hybridization or introgressionwhere Alderia species were sympatric, yet both species at-tempted to inseminate each other in laboratory crosses. Thereis no premating isolation between Alderia species; slugs mateyear-round via hypodermic insemination, injecting spermanywhere on the recipient’s body (Hand and Steinberg 1955;Angeloni 2003). Divergence in sperm chemoattractants orgamete recognition proteins may promote prezygotic isola-tion (Vacquier 1998; Lyon and Vacquier 1999; Swanson andVacquier 2002; Zigler et al. 2003; Riffell et al. 2004), butthis has not been tested in a marine organism with internalfertilization. Postzygotic isolation between Alderia specieswas implicated by the failure of most laboratory crosses de-

spite insemination, and could be a pleiotropic by-product ofthe different genetic programs that direct larval developmentin the sister species.

Evolution of Poecilogony

Transitions between planktotrophy and lecithotrophy haveoccurred frequently in the evolutionary history of many taxaand can substantially alter the population structure and mac-roevolutionary fate of a lineage (Raff 1987; Jeffery and Swal-la 1992; Hadfield et al. 1995; Hart et al. 1997; Duda andPalumbi 1999; Hart 2000; McEdward 2000; Jeffery et al.2003, Collin 2004). However, for any given species the se-


FIG. 6. Interspecific versus intraspecific differences in body weight for Alderia species. Data are mean wet weights � SE. All sites arein California except Coos Bay, Oregon.

TABLE 5. Results of a nested ANOVA on weights of Alderia sp. and A. modesta from sites in California and Oregon, with site as arandom effect nested within species as a fixed effect.

Effect Type df SS MS F P

Intercept fixed 1 16,493.25 16,493.25 24.50 0.0005Site(species) random 10 7011.50 701.15 42.16 0.0000Species fixed 1 6015.15 6015.15 8.93 0.0136Error 318 5288.61 16.63

lective forces that favored the evolution of nonfeeding larvaeremain unclear. Due to the rarity of poecilogony, studiescontrasting different development modes usually employ in-terspecific comparisons (e.g., Villinski et al. 2002), whichmay be confounded by other differences between species. Asa case of variable development, Alderia sp. should be a modelsystem with which to study developmental evolution fromecological and biochemical perspectives.

There are few confirmed examples of poecilogony amongmarine invertebrates, despite the adaptive value of dispersalpolymorphisms (Giesel 1976; Denno et al. 1980; Harrison1980; McPeek and Holt 1992; Toonen and Pawlik 1994; Chiaet al. 1996; Hopper 1999; Langellotto and Denno 2001). Mostclaims of poecilogony were resolved as cryptic species dif-fering in development upon detailed molecular or morpho-logical study (Hoagland and Robertson 1988; Hirano andHirano 1992; Sisson 2002). In molluscs, multiple larval typeswithin a species have been confirmed by breeding studies ormolecular data for three sacoglossans: Elysia chlorotica(West et al. 1984), Alderia sp. (Krug 1998; present study),and Costasiella ocellifera (R. A. Ellingson and P. J. Krug,unpubl. data). Among polychaetes, examples include Boc-

cardia proboscidea (Gibson et al. 1999; Gibson and Gibson2004), Streblospio benedicti (Levin 1984; Schulze et al.2000), and Pygospio elegans (Morgan et al. 1999). Identifyingthe selective forces favoring poecilogony, and the phyloge-netic constraints that limit it to a few taxa, may substantiallyadvance our understanding of marine life-history evolution.

In southern California, slugs produced mostly lecithotroph-ic clutches from May through September, with a varyingproportion expressing planktotrophy the rest of the year. Fur-thermore, some adults switch from lecithotrophic to plank-totrophic clutch production upon transport to the laboratory(Krug 2007). Development in Alderia sp. is thus to someextent phenotypically plastic. Populations of the poecilogon-ous polychaete S. benedicti did not change development sea-sonally, nor did individuals ever change the trophic mode oftheir offspring (Levin and Huggett 1990; Levin and Creed1991; Levin and Bridges 1994). The adaptive value of sea-sonal polyphenism has been studied for morphological char-acters (Brakefield and French 1999; Nijhout 1999), diapausestrategies (Shapiro 1976), and sex determination (Conoverand Heins 1987), but has not been described for larval de-velopment in a marine animal.


TABLE 6. Linear regressions of the effect of weight fecundity in Alderia modesta and Alderia sp.

Species Population Development F-ratio r2 P Regression equation

A. modestaBodega planktotrophic F1,18 � 9.07 0.34 0.008 y � 0.007x � 5.45San Francisco planktotrophic F1,9 � 9.86 0.52 0.01 y � 35.25x � 101.33Tillamook planktotrophic F1,18 � 7.86 0.50 0.02 y � 0.435x � 0.01

Alderia sp.San Francisco planktotrophic F1,18 � 18.03 0.50 0.0005 y � 0.003x � 0.19San Diego lecithotrophic F1,27 � 4.53 0.14 0.04 y � 0.01x � 1.74

The same factors that favored lecithotrophy in the ancestorof Alderia sp. could favor its present-day expression duringwarm months. Understanding the ecology of Alderia sp. maythus pinpoint the selective pressures behind the evolution ofnonfeeding development. Lecithotrophy could be an adap-tation to decrease larval mortality, either from offshore ad-vection during upwelling or from a seasonal reduction inphytoplankton abundance. However, the transition away fromplanktotrophy occurs in May and June in southern California,after the peak in upwelling and when phytoplankton abun-dances are high (Bakun and Nelson 1977; Reid et al. 1985;P. J. Krug, unpubl. data). The environmental parameter bestcorrelated with lecithotrophy is freshwater input from winterrain and spring snowmelt. Historically, estuaries in southernCalifornia were closed by sand berms when runoff ceased insummer and fall; lecithotrophy may thus be an adaptive re-sponse to seasonal habitat closure. Runoff could then flushplanktotrophic larvae out of back bays and allow alongshoretransport in winter and spring, facilitating dispersal. Alter-natively, lecithotrophy could allow exploitation of stableadult food resources if Vaucheria is less seasonal in the south,which could be tested with monthly field surveys of hostalgal abundance.

Size and fecundity differences between Alderia speciessuggest another hypothesis for the evolution of poecilogony,integrating biochemical with ecological constraints. Thesouthern species Alderia sp. is smaller than its congener evenwhere they coexist, and produces fewer larvae. Due to highplanktonic mortality (Morgan 1995), there may be a thresholdclutch size below which no long-lived larvae are likely tosurvive, leading to selection against obligate planktotrophyin Alderia sp. If Vaucheria species are of lower nutritionalvalue during the summer in southern waters, Alderia sp. maybe unable to produce enough planktotrophic offspring to off-set larval mortality. The adult energy budget and odds oflarval survivorship could thus interactively drive evolutionof development mode. Quantifying the caloric content ofVaucheria spp. should indicate whether host value fluctuateswith latitude or over the course of a year. Presently beingdescribed (Krug et al. 2007), Alderia sp. is an excellent modelorganism with which to explore the factors controlling ex-pression of alternative developmental pathways, providinginsight into how adult and larval ecology interactively shapelife-history evolution.

ACKNOWLEDGMENTS

Key logistical support was provided by D. and O. Tiltonand T. and S. Kreines. For help with collections, we thankD. Willette, N. Smolensky, G. Botello, V. Rodriguez, and S.

Millen. Special thanks to T. Van Der Knocker and R. Dekkerfor Atlantic specimens, and A. Chernyshev and A. Shukalyukfor western Pacific specimens. We thank S. Escorza-Trevinoand E. Torres for advice and access to laboratory facilities.Access to field sites was provided by I. Kay (Natural ReserveOffice of the University of California, San Diego), K. Brownand P. Connors (Bodega Marine Reserve), B. Shelton (UpperNewport Bay Ecological Reserve), and M. Schaadt (Los An-geles Harbor). Helpful comments on earlier drafts were pro-vided by R. Collin, D. Eernisse, M. Hellberg, D. Jacobs, R.Kelly, C. Meyer, D. O Foighil, A. Valdez, and J. Wares. Thisstudy was supported by awards from the National ScienceFoundation (OCE 02-42272 and HRD 03-17772) to PJK, andby a Lerner-Grey award to RAE.

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