A NEW POECILOGONOUS SPECIES OF SEA SLUG
(OPISTHOBRANCHIA: SACOGLOSSA) FROM CALIFORNIA:
COMPARISON WITH THE PLANKTOTROPHIC CONGENER
ALDERIA MODESTA (LOVÉN, 1844)
PATRICK J. KRUG 1 , RYAN A. ELLINGSON 1 , RON BURTON 2
AND ÁNGEL VALDÉS 3
1
Department of Biological Sciences, California State University, Los Angeles, CA 90032-8201, USA;
Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 90093-0202, USA;
3
Natural History Museum of Los Angeles County, 900 Exposition Blvd., Los Angeles, CA 90007, USA
2
(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; e-mail: [email protected]
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 (Lovén, 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: 29–38. Advance Access Publication: 9 January 2007
# The Author 2007. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved.
doi:10.1093/mollus/eyl025
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.
Table 1. Field sites and sampling dates for Alderia species.
Location
Latitude, Longitude
Date sampled
Doel, Belgium
518190 N, 48160 E
Kodiak, Alaska,
578350 50 N, 1528280 17 W
8/05
00
00
00
00
00
00
6/05
U.S.A.
Vancouver, British
498180 21 N, 1238050 32 W
7/03
Columbia,
Canada
Tillamook, Oregon,
458260 55 N, 1238500 57 W
3/05
U.S.A.
Coos Bay, Oregon
00
00
438210 48 N, 1248110 45 W
0
00
0
00
Humboldt
40844 31 N, 124812 59 W
Bodega Harbor
388180 58 N, 1238030 24 W
Walker Creek
388130 46 N, 1228540 56 W
Cow Landing
388110 02 N, 1228540 40 W
South Tomales Bay
388060 53 N, 1228510 08 W
San Francisco Bay
378520 57 N, 1228310 09 W
Morro Bay
358200 39 N, 1208500 35 W
Santa Barbara
348240 01 N, 1198320 07 W
0
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
0
00
San Pedro
33842 48 N, 118817 02 W
Newport Bay
338370 16 N, 1178530 32 W
San Diego
328470 36 N, 1178130 47 W
00
00
00
00
7/00a, 10/01, 2/05
2/04
9/03, 9/04
9/04
9/04
9/03, 9/04
9/03– 9/04 (5x)
9/02
7/99a, 2/00
1/04– 8/04 (4x)
6/03– 8/04 (6x)
monthly 96-98; 7/99a,
8/03– 8/04 (3x)
MATERIALS AND METHODS
Unless otherwise indicated, all sites are in California, U.S.A.
a
Collections used for allozyme electrophoresis.
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
(518190 N, 48160 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 (438090 N, 1318540 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 (538150 N, 088560 E).
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
DNA sequence analysis
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
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-50 and 16Sbr-30 (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
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
30
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;
Valdés, 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
DQ364252– 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 0.1397) was
applied to the COI data and the HKY þ I þ G model
(a ¼ 0.2932) 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 3.1.1 (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 (Bohuslän, Sweden).
Alderia modesta—Lovén, 1845: 8.
Canthopsis harvardiensis Agassiz, 1850: 191 (nomen nudum ).
Alderia harvardiensis—Gould, 1870: 254-255, pl. 16, figs 226–228.
Alderia scaldiana Nyst, 1855: 435, figs 1, 2.
Alderia uda Marcus & Marcus, 1956: 8 –17, figs 8 – 23.
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 Bohuslän, 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 1845. 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
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.
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 463 + 117
(n ¼ 15) for slugs from Bodega Harbor, California in October
2005 (Krug, in press). Uncleaved ova 64 – 72 mm in diameter,
each enclosed within a spherical capsule 120 mm in diameter;
development strictly planktotrophic, with larvae maturing and
hatching in 4.7 + 1.2 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).
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 0examined:
4 specimens,
Bodega Harbor, California
0
00
(388180 58 N, 1238030 24 W), collected on 1 September 2004
(LACM 172506).
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 23.1 + 1.2 mg
SE (Coos Bay, Oregon) to 5.5 + 0.5 mg SE (Walker Creek,
California), with an overall mean weight of 11.2 + 0.5 mg SE
(n ¼ 231, range ¼ 1.4 – 36.4 mg). Penis ends in a stylet used in
hypodermic insemination, with sperm transfer occurring anywhere on body of the recipient.
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).
Egg mass and development: Ova visible through thin areas of the
foot in ramifying extensions of the reproductive system. Egg
32
NEW POECILOGONOUS SACOGLOSSAN
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).
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 (Vayssiére, 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
Alderia willowi n. sp.
(Figs 1C, D, 2B)
Types. Holotype: Cabrillo
Aquarium’s
wetland, San Pedro,
00
00
California (338420 48 N, 1188170 02 W), collected on 18 July
2005 (LACM 2034). Paratypes: 5 specimens, Cabrillo
00
Aquarium’s
wetland, San Pedro, California (338420 48 N,
00
0
118817 02 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 5.8 + 0.5 mg SE
(San Francisco Bay) to 0.9 + 0.1 mg SE (south Tomales Bay),
with an overall mean weight of 2.8 + 0.2 mg SE (n ¼ 192,
range ¼ 0.3–15.5 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
105 + 5 mm
diameter
within
247 + 31 mm diameter spherical capsules, developing in 5-6
days at 258C into 186 + 9 mm larvae competent to settle
immediately upon hatching (Krug, 1998, 2001). Planktotrophic
eggs 68 + 4 mm within 121 + 12 mm capsules, developing
within the egg mass for three days and hatching as
116 + 8 mm precompetent larvae (Krug, 1998). Planktotrophic
larvae mature over a 32-day pelagic period, growing to the size
33
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 (32 + 12 versus 311 + 134 eggs per
clutch; Krug, 1998).
Table 2. Allozyme frequencies at the PGI locus in three populations of
Alderia species.
Allele
Coos Bay, Oregon
0.54
0.008
Santa Barbara
0.002
0.71
0.79
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.
0.002
0.008
0.01
0.82
0.87
0.014
0.937
0.93
0.012
0.006
0.037
0.054
0.873
1.0
0.056
0.963
1.09
0.008
0.012
1.14
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).
0.01
0.018
1.18
2N
San Diego
0.012
128
84
514
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).
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
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
NEW POECILOGONOUS SACOGLOSSAN
Table 3. Alderia species. DNA barcode for differentiating Alderia modesta from its southern congener, A. willowi n.sp.
Site
1
1
1
1
1
2
2
2
2
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
5
6
6
5
5
6
7
7
0
4
8
8
3
7
9
3
5
7
7
7
9
0
3
3
4
6
7
8
1
1
4
7
6
5
8
8
4
3
6
5
3
7
0
7
2
3
5
6
8
2
5
7
3
1
4
3
9
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).
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 transArctic 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.
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 (113 + 15 mm; range: 98–
129 mm; n ¼ 12) versus lecithotrophic specimens of A. willowi
(112 + 15 mm; range: 92–122 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).
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
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 veligers have eyespots at hatching, 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.
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