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Phylogeography and reproductive variation of

the poecilogonous polychaete Boccardia
proboscidea (Annelida: Spionidae) along the
West Coast of North America

ARTICLE in EVOLUTION & DEVELOPMENT · NOVEMBER 2011

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Phylogeography and reproductive variation of the poecilogonous
polychaete Boccardia proboscidea (Annelida: Spionidae) along the
West Coast of North America
Fernanda X. Oyarzun,a,b Andrew R. Mahon,c,d Billie J. Swalla,a,b and Kenneth M. Halanychb,c,∗
a
Department of Biology, University of Washington, Seattle, WA, 98195, USA
b
Friday Harbor Laboratories, University of Washington, Friday Harbor, WA, 98250, USA
c
Department of Biological Sciences, Auburn University, Auburn, AL, 36849, USA
d
Department of Biology, Institute for Great Lakes Research, Central Michigan University, Mt. Pleasant, MI, 48859, USA
∗
Author for correspondence (email: ken@auburn.edu)
SUMMARY The ability to produce more than one kind
of offspring, or poecilogony, is a striking example of repro-
ductive variability. Traditionally, larval nutrition has been clas-
sified as a dichotomy: if offspring obtain nutrition from their
mothers (lecithotrophy), there is lower fecundity and greater
chance of offspring survival than when they get their nutrition
from plankton (planktotrophy). The polychaete Boccardia pro-
boscidea (Spionidae) produces both types of embryos using
three different reproductive strategies. In this study, we ex-
amined the roles of genetic history and phenotypic plasticity
on explaining natural variation in B. proboscidea along the
Pacific coast of the United States using two genetic mito-
chondrial markers, 16S rDNA and Cyt b, and common gar-
den experiments. These data show a single North American
INTRODUCTION
Poecilogony, the ability to produce more than one kind of off-
spring (Giard 1905), is a striking example of sustained repro-
ductive polymorphism. Some poecilogonous species have dif-
ferent individuals within a population that produce offspring
with distinct developmental modes (Simon 1968; Tsutsumi
and Kikushi 1984; Schulze et al. 2000). In other cases, in-
dividuals within a species can have different developmental
modes as a result of mothers’ variable response to specific en-
vironmental conditions, known as developmental plasticity
(Krug 1998; Mackay and Gibson 1999; Krug et al. 2007). In
a third kind of poecilogony, a mother produces embryos with
more than one kind of development within a single clutch.
In most known examples of poecilogony, variability in ma-
ternal food provisioning to embryos or juveniles mediates
differences between alternative developmental modes (Bragg
1956, 1964; Gibson and Chia 1995; Chia et al. 1996; Gib-
son 1997; Gibson and Gibson 2004; Botello and Krug 2006;
Pernet and McArthur 2006).

C 2011 Wiley Periodicals, Inc.
EVOLUTION & DEVELOPMENT 13:6, 489–503 (2011)
DOI: 10.1111/j.1525-142X.2011.00506.x
West Coast network that is structured, geographically, by the
well-documented biogeographic break near Point Concep-
tion, California. The southern group within this network cov-
ers a smaller range, but has larger haplotype diversity, than
the northern group. Some individuals differing in reproduc-
tive type had the same haplotype, indicating independence
of these features; however, differences between laboratory
and field data suggest additional geographic variation within
one of the reproductive types. Females from higher latitudes
provide offspring with larger supplies of extra embryonic nu-
trition than females from southern latitudes. Results herein
suggest that both genetic history and developmental plastic-
ity are playing a role in the maintenance of this reproductive
polymorphism.
Poecilogonous species can also differ in proportions of re-
productive modes among populations and along a latitudinal
gradient. For example, Ellingson and Krug (2006) showed
that proportions of planktrotrophic versus lecithotrophic
larvae varied in populations of poecilogonous gastropods
(Alderia modesta and A. willowi) along the California coast.
This species can also exhibit developmental plasticity in re-
sponse to environmental variables (Krug 1998; Ellingson and
Krug 2006). For example, planktotrophy tends to occur in
summer, but lecithotrophy is more common in winter and
spring. Also, starvation and changes from field to laboratory
conditions can facilitate switches in developmental mode.
Likewise, the spionid annelid Streblospio benedicti produces
lecithotrophs in introduced California populations, but na-
tive East Coast populations produce mostly planktotrophic
larvae (Levin 1984). Although individuals cannot change
their reproductive type, interbreeding experiments and cy-
tochrome c oxidase subunit I (CO1) analyses have supported
the hypothesis that these females belong to the same species
(Levin and Creed 1986; Schulze et al. 2000).
489
490 EVOLUTION & DEVELOPMENT Vol. 13, No. 6, November–December 2011
In the spionid annelid Boccardia proboscidea Hartman,
1940, reproductive mode also varies with geographic loca-
tion. Boccardia proboscidea occurs along the West Coast of
North America, in Japan, and by recent introductions in
southern Australia, Hawai’i, and South Africa (Blake and
Kudenov 1978; Petch 1989; Bailey-Brock 2000; Sato-Okoshi
2000; Simon et al. 2009). In California south of Point Con-
ception, three reproductive types among females have been
found. Type I females produce capsules with only plank-
totrophic larvae (Hartman 1940, 1941; Gibson 1997; Gibson
et al. 1999). Type II females produce egg capsules with ap-
proximately 85% planktotrophs and 15% of nurse eggs, which
are eggs that never develop and serve as food for develop-
ing offspring (Gibson et al. 1999). Type III females produce
broods with three kinds of eggs within the same capsule:
(1) nurse eggs, (2) eggs that develop as planktotrophs that
remain small until they hatch as swimming larvae, and (3)
adelphophages that eat both nurse eggs and planktotrophs
until they hatch as large advanced larvae or postlarval ju-
veniles (Gibson et al. 1999). As mothers can open capsules
and liberate larva, maternal behavior can influence relative
percentages of adelphophage to planktotroph survival in
Type III individuals (Oyarzun and Strathmann 2011). Al-
though all three reproductive modes occur in Califor-
nia south of Point Conception, only the Type III mode
(adelphophages, planktotrophs, and nurse eggs in the same
capsule) has been reported in Canada, Washington, Oregon,
and in other locations in which this species is found, such as
Japan, Hawai’i, and Australia (Petch 1989; Sato-Okoshi and
Okoshi 1997; Gibson et al. 1999; Bailey-Brock 2000; Sato-
Okoshi 2000). In South Africa, two developmental modes
have been reported, Type I and Type III (Simon et al. 2009).
Note that South African, Hawaiian, and Australian popula-
tions are all introduced. Table 1 compares select reproductive
traits for several natural and introduced populations.
Using random amplification of polymorphic DNA
(RAPD) markers on B. proboscidea from Vancouver Island
and La Jolla, California, Gibson et al. (1999) concluded that
mothers of Type I and Type III belong to the same species.
These reproductive types also hybridize in laboratory condi-
tions and the reproductive biology of F1 females was similar
to that of the mother (Gibson et al. 1999). In addition, within
Type III reproductive mode, intracapsular sibling cannibal-
ism and maternal behavior can potentially modify propor-
tions of which larval mode survives to be released (Oyarzun
and Strathmann 2011). Whether B. proboscidea populations
with different reproductive modes have been genetically con-
tiguous over time, and whether the described reproductive
plasticity significantly influences populations dynamics is not
known. Invertebrate developmental polymorphisms are un-
common in the marine environment. Understanding the ge-
netic structure of populations with different developmental
modes and the additional levels of developmental plasticity
present in this species might shed light on mechanisms that
allow the evolution and persistence of variable reproductive
strategies.
In this study, we explore genetic differences between and
within populations of B. proboscidea along the West Coast of
North America. Our goal is to test how variation observed in
B. proboscidea’s developmental mode relates to genetic his-
tory and developmental plasticity by (1) examining phylogeo-
graphic structure of B. proboscidea using two mitochondrial
markers (16S rRNA and cytochrome b [Cyt b]) and (2) con-
ducting common garden experiments with females coming
from two populations located near the extremes of its natu-
ral distribution. We hypothesized that if members of different
reproductive modes cannot interbred, then genetic structure
will be associated with developmental mode. Alternatively,
structure could be lacking or present for other reasons such
as geography. The use of common garden experiments al-
lowed exploration of variation observed in developmental
modes so that we could begin to understand whether ob-
served differences are ecological in nature, or representative
of different localities.
MATERIALS AND METHODS
Collection of specimens
Boccardia proboscidea was collected from along the West Coast
of North America between 2005 and 2007 (Fig. 1). Previ-
ous studies (see below) described B. proboscidea ranging from
Alaska to Panama; however, established populations were found
only between False Bay, Washington and La Jolla, California
in our surveys. Individuals south of southern California have
been reported in the literature in five specific cases. Four cases
in Mexico: Rosarito (32.33◦ N, 117.07◦ W), Descanso (32.23◦ N,
116.97◦ W), Ensenada (31.53◦ N, 116.58◦ W; collected by Velero
Expedition in 1940; Petch 1995), and in Bahı́a Concepción
(26.25◦ N, 111.8◦ W; Salazar-Vallejo 1985). The southernmost
report was collected in 1971 in Paitilla, Panama (9◦ N, 78.5◦ W;
Fauchald 1977). However, Petch (1995) revised the Panamanian
specimen and concluded that it probably belonged to a different
species. We extensively searched the Mexican Coast, examined
collections, and contacted local researchers but did not find
B. proboscidea along the Pacific coast of Mexico. Nor did we
find B. proboscidea in Paitilla Bay, Panama, or its surround-
ing coast. Boccardia proboscidea had been found near Victoria
on Vancouver Island (Gibson et al. 1999), but to our knowl-
edge, no other record of this species further north of Vancou-
ver Island has been recorded. Boccardia proboscidea has not
been found to date in any of the sampling efforts performed
in Alaska under the umbrella of the Natural Geography In
Shore Areas (NaGISA; Héloı̈se Chenelot, personal communi-
cation). Therefore, the only abundant populations found and
sampled for this study were along the West Coast of the United
States.
Oyarzun et al. Poecilogonous spionid evolution 491

Table 1. Summary of reproductive traits reported for populations of Boccardia proboscidea. Reproductive types reported
for each population are indicated following the nomenclature of Gibson et al. (1999): Type I produce capsules with only
planktotrophic larvae, Type II produce egg capsules with 85% planktotrophs and 15% nurse eggs, and Type III produce
egg capsules with planktotrophs, adelphophages, and nurse eggs. A = adelphophagic larvae; P = planktotrophic larvae;
n.e. = nurse eggs; [F] = field data; [L] laboratory data

Capsule Egg No. of No. of No. of No. of

Reproductive size size caps per eggs per larvae nurse eggs
Population type (μm) (μm) brood caps per caps per caps Reference

Vancouver Island III - - “many” - - - Sato-Okoshi (2000)

[F]
Vancouver Island III - - - - - - Gibson (1999) [F],
Gibson and
Gibson (2004) [L]
CA, Caspar I and III - - >5 >50 Type I:40 max - Hartman (1940)
Type III:
1or 2 A
CA, Moss Landing, III 1150 × 700 150 14–38 104–131 5.4 A; 1.5 P - Woodwick (1977) [L]
Ballona Creek, Morro
Bay
CA, Moss Landing I - 100 - - - - Woodwick (1977) [L]
CA, Harbor Testing I - - 76∗ 149 - - King (1976) [L]
Laboratory
CA, Alamitos Bay (San I 900 × 570 96 76* 149 - - King (1976) [L]
Gabriel river)
CA, La Jolla I and III Varies 120–150 >20 20–80 - - Hartman (1941)[F]
according
to female
size
I - 95 42 - 52 0.6 Gibson (1997) [L]
II - 93 47 - 39 6.7 Gibson (1997) [L]
III - 109 43 - 5 34.4 Gibson (1997) [L]
Japan III 1200 × 800 120–150 - - 0–5 A; 1–9 P 30–120 Sato-Okoshi (2000)[F]
n.e.
Australia, Werribee Farm III - - - - - - Blake and Kudenov
(1981) [F]
Australia, Werribee 15E III - - - 65–115∗ 6–9∗ 40–120† Petch (1989) [F]
Australia, Werribee 145W III - - - - 6–10∗ 40–120† Petch (1989) [F]
Australia, Lake Borrie III - - - - - - Petch (1989) [F]
Australia, Murtcaim III - - - - 4–11∗ 40–120† Petch (1989) [F]
Australia, Gunnamatta III - - - - 2–6∗ 40–120† Petch (1989) [F]
∗ Maximum and minimum average extrapolated from the month to month values shown in the graph of the study. The exact values were not shown

in Petch (1989).
† Maximum and minimum average extrapolated from the month to month values shown in the graph of the study. The graph showed data from

four sites combined (Werribee 15E, Werribie 145W, Murtcaim and Gunnamatta). The exact values were not shown in Petch (1989).

For molecular analyses, specimens were collected in the
field and then extracted from their tubes under a dissecting
scope in the laboratory. The distribution of B. proboscidea
was patchy and worms were found in diverse habitats (sed-
iment, rocks, logs). Sampling was performed with a core of
5 cm in diameter in distant areas within each site to avoid collect-
ing individuals that were closely related and in an effort to have
a wide representation of the haplotypes present in each popula-
tion. In the laboratory, worm tubes were separated from the core
samples. When females were found with larval capsules in the
tube, the type of reproduction (Type I, II, or III) was determined
by observing capsules’ contents. After confirmation of species
identity and reproductive mode, samples were preserved in 95–
100% ethanol for molecular analyses or fixed in 10% formalin
and then transferred to 70% ethanol for voucher specimens.
Vouchers were deposited at the LA county museum (voucher
numbers LACM-AHF Poly 2790 through LACM-AHF Poly
2798).
492 EVOLUTION & DEVELOPMENT Vol. 13, No. 6, November–December 2011

Lat Long Rep. Site Vancouver Island

type
48.490° 123.066 III False Bay low
48.483° 123.071 III False Bay high
48.452° 122.962 III Cattle Point

45.56° 123.91 III Garibaldi

43.333° 124.376 III Sunset Bay

43.304° 124.398 III South Cove
area
geographic

explored

40.807° 124.159 III Eureka

35.372° 120.861 III Coleman Point Conception

in

1940
th
ra

35.347° 120.844 III Morro Bay

is
ng

1985
e repo

33.747° 118.118 I and III Alamitos Bay

33.729° 118.082 I and III Simple Green
r te

32.869° 117.254 I and III La Jolla

d

n stu
i

th dy
el
ite
rat
ure
1971

Fig. 1. Geographic range of Boccardia proboscidea along the West Coast of North America. Reported geographic range (gray line),
region explored in this study (black lines), and the enlarged region of the map that shows the site where B. proboscidea was found
and collected for the DNA analyses and the reproductive types found in that population. Reproductive types found at each site
are indicated. White circles indicate the sites outside the United States where B. proboscidea has been reported and collected before
this study, from north to south: Vancouver Island (numerous times), Rosarito (1940), Descanso (1940), Ensenada (1940), Bahia
Concepcion (1985), and Paitilla Bay in Panama (1971). We were unable to find B. proboscidea in Mexico or Panama. US localities
where B. proboscidea has been collected and reported in the literature are not shown in the map at the left, but it has been collected
extensively.

Molecular analyses Beckman CEQ8000 Genetic Analysis System (Beckman Coul-

Genomic DNA was extracted with the DNeasy Tissue
Kit (Qiagen Inc., Valencia, CA, USA). Portions of the mi-
tochondrial 16S rDNA and Cyt b genes were amplified
with standard polymerase chain reaction (PCR). We chose
these markers based on their informative nature between an-
nelid populations in previous studies (Schulze et al. 2000;
Dahlgren et al. 2001; Halanych and Janosik 2006; Struck et
al. 2007). We used the 16S rDNA primers (Palumbi 1996)
16SarL (5 -CGCCTGTTTATCAAAAACAT-3 ) and 16SbrH
(5 -CCGGTCTGAACTCAGATCACGT-3 ) and the following
cycling conditions: initial denaturation 94◦ C, 3 min; 40 cycles
of denaturation 94◦ C, 30 s; annealing 47◦ C, 30 s; extension
72◦ C, 1 min; final extension 72◦ C, 7 min. We also used the
Boore and Brown (2000) primer Cytb424F (5 -GGWTAYGTW
YTWCCWTGRGGWCARAT-3 ) and designed a new primer
Cytb-bp-876R (5 -RAAWARRAAGTATCAYTCAGG-3 ). Cy-
cling conditions for Cyt b were identical to 16S rDNA ex-
cept for the annealing temperature, which was 49◦ C. Result-
ing PCR products were verified by 1X sodium borate agarose
gel electrophoresis and purified either by QIAquick R
PCR
Purification Kit (Qiagen Inc.) or size selected on agarose
gels and purified with QIAquick R
Gel Extraction Kit (Qia-
gen Inc.). Products were bidirectionally sequenced with either
an ABI 3100 genetic analyzer (Applied Biosystems, Carlsbad,
CA, USA) at the Comparative Genomics Center of the De-
partment of Biology at the University of Washington or a

R ter) housed in KMH’s laboratory at Auburn University, Al-
abama. Sequencher 4.1. 4 (Gene Code Corp., Ann Arbor,
MI, USA) was used to assemble the two complementary
strands for each gene. GenBank accession numbers are given in
Tables 2 and 3.
Phylogeographic analyses
Sequence alignments were compiled with Clustal X (Thomp-
son et al. 1997) and manually corrected with MacClade 4.05
(Maddison and Maddison 2002). We translated Cyt b sequences
using the Drosophila mitochondrial code to verify the reading
frame. Alignments are available in Supplemental Information.
As the project progressed, we noticed that Cyt b data possessed
much more variation between individuals than 16S rDNA data.
Therefore, to maximize phylogeographic signal relative to the
cost of data collection, we focused on obtaining Cyt b data for
more individuals rather than obtaining both gene regions for
fewer individuals. Results for each gene separately and com-
bined data are congruent. However, we focus on the Cyt b
dataset as it provided greater resolution and included more
individuals.
We constructed parsimony networks for each marker to ex-
plore the recent history of B. proboscidea using TCS version 1.21
(Clement et al. 2000). We treated indels as missing data, used
default settings and assumptions, and tested branch connections
Oyarzun et al. Poecilogonous spionid evolution 493

Table 2. GenBank accession numbers and geographic distribution of 16S rDNA haplotypes of Boccardia proboscidea.
Localities: fbh (False Bay high), fbl (False Bay low), cat (Cattle Point), gar (Garibaldi), sun (Sunset Bay), scov (South
Cove), eur (Eureka), col (Coleman), mor (Morro Bay), ala (Alamitos Bay), sg (Simple Green), lj (La Jolla)

Haplotype Accession number fbh fbl cat gar sun scov eur col mor ala sg lj Total

A FJ434486 2 - - - 2 1 5 - 1 1 2 - 14
B JN600634 - - - - 1 - - - - - - - 1
C JN600628 - - - - - - - - - - 2 - 2
D FJ972543 - - - - - - - - - 9 2 3 14
E JN600629 - - - - - - - - - 1 - - 1
F JN600630 - - - - - - - - - 1 - - 1
G FJ972541 - - - - - - - - - 1 - - 1
H FJ972546 - - - - - - - - - 1 - - 1
I JN600631 - - - - - - - - - 1 - - 1
J FJ972547 - - - - - - - - - - 1 2 3
K FJ972544 8 9 6 11 5 3 2 3 6 1 - 1 55
L JN600632 - - - - - - - - - 2 - 1 3
M FJ972545 - 1 - - - - - - - - - - 1
N JN600633 - - - - - - - - - - - 1 1

between haplotypes at the 95% connection limit unless other-
wise noted. Reticulations were resolved following Crandall et al.
(1994).
Haplotype diversity (h) and its sampling variance were cal-
culated with Arlequin 3.11 (Schneider et al. 2000). We also cal-
culated nucleotide diversity (π) with DNASP 5.0 (Rozas et al.
2003). We conducted a hierarchical AMOVA, regional pairwise
φST comparisons based on haplotypic frequencies and estimates
of gene flow in Arlequin 3.11. Data were split into hierarchical
levels of northern and southern clades, among localities, and
within localities. To test for an excess of rare polymorphisms in
B. proboscidea populations, we performed Tajima’s D (Tajima
1989) and Fu and Li’s Fs (Fu and Li 1993) neutrality tests
using DNASP 5.0. We performed mismatch analyses (Rogers
and Harpending 1992; Rogers 1995) in DNASP by comparing
observed versus expected 16S rDNA and Cyt b haplotypes to
determine whether B. proboscidea has experienced population
expansion through its history.
Common garden experiments and field data
For two populations, False Bay, Washington in the north, and
Alamitos Bay, California in the south (Fig. 1), we collected 10
sediment samples in the intertidal from patches of B. proboscidea
with a 5-cm core pushed into 4-cm depth that were immediately
fixed in 10% formalin. Similar quantities of sediment were sam-
pled for collection of live specimens. All samples were collected
during late spring or early summer, given that seasonal varia-
tion has been reported in the literature in other poecilogonous
species (Levin and Huggett 1990). In the lab under a dissecting
scope, we separated 10 “mothers + capsules” of the observed
reproductive modes, Type I and Type III, from the fixed sam-
ples. For each capsule, we counted the number of nurse eggs (if
there were any), number of embryos, and developmental stage of
embryos as number of setigers. These data from field individuals
were subsequently used for comparison to laboratory individu-
als so that plasticity in laboratory experiments could be assessed
relative to field populations.
We reared live worms in common garden conditions using
methods modified from Gibson et al. (1999) to test if the re-
productive variation observed within and between these two
populations was the result of phenotypic plasticity. Worms were
kept in pairs in 50-ml glass beakers with filtered seawater (FSW)
and 10 ml of sediment. Each beaker was treated as an inde-
pendent unit and placed inside a 300-ml container with FSW
and aeration. Water was changed three times weekly, sediment
cleaned and replenished as needed, and adults fed with a 50:50
mix of Gerber baby-food cereal and ZoPlan (Advanced Zoo-
plankton Diet, Two Little Fishies, Inc., Miami Gardens, FL,
USA). Worms were reared at 20◦ C and on a 16:8 light/dark
cycle. We used only capsules that had larvae early in develop-
ment, to allow comparison in number of nurse eggs and embryos
between common garden and field capsules. We define “larvae
early in development” as stages including nonfeeding embryos
and presegmented larvae just starting to feed on pieces of nurse
eggs. Thus, the number of nurse eggs should have not been sig-
nificantly altered by consumption. Females actively brooded
developing embryos and larvae. They cleaned and ventilated
the capsules and liberated embryos from the capsules; as doc-
umented by King (1976) and Oyarzun and Strathmann (2011),
the larvae did not liberate themselves. Females were kept with
food ad libitum and checked daily for liberation of embryos or
larvae.
Analyses of data from field and common
garden experiments
We compared reproductive traits among field and common gar-
den conditions within reproductive modes. For Type I reproduc-
tive mode, we performed t-tests on capsule number and average
494 EVOLUTION & DEVELOPMENT Vol. 13, No. 6, November–December 2011

Table 3. Accession numbers and geographic distribution of Cyt b haplotypes of Boccardia proboscidea. Localities: fbh
(False Bay high), fbl (False Bay low), cat (Cattle Point), gar (Garibaldi), sun (Sunset Bay), scov (south cove), eur
(Eureka), col (Coleman), mor (Morro Bay), ala (Alamitos Bay), sg (Simple Green), lj (La Jolla)

Haplotype Accession number fbh fbl cat gar sun scov eur col mor ala sg lj Total

1 FJ972550 9 6 - 3 1 6 1 7 13 1 1 1 49
2 JN600636 - - - - - - - 1 - - - - 1
3 JN600637 - - - - 2 1 - 1 - - - - 4
4 FJ434483 1 9 4 1 2 5 1 2 2 - - - 27
5 FJ972548 2 1 - - 8 5 9 1 1 2 3 - 32
6 JN600638 - - 1 - - - - - - - - - 1
7 JN713132 - 1 - - - - - - - - - - 1
8 JN600639 - - - - - - - - - - 1 - 1
9 FJ972554 - - - - - - - - - 1 - - 1
10 FJ972552 - - - - - - - - - 1 - 2 3
11 FJ972551 - - - - - - - - - 3 - 5 8
12 FJ972555 - - - - - - - - - 1 - - 1
13 JN600640 - - - - - - - - - - - 1 1
14 FJ972556 - - - - - - - - - - - 1 1
15 JN600635 - - - - - - - - - - - 1 1
16 FJ972558 - - - - - - - - - 1 - - 1
17 JN600641 - - - - - 1 - - - - - - 1
18 FJ972553 - - - - - - - - - 1 1 - 2
19 FJ972557 - - - - - - - - - 1 - - 1
20 JN600642 - - - - - - - - - - 1 - 1
21 FJ972569 - - - - - - - - - - 1 1 2
22 FJ972565 - - - - - - - - - 2 - 2 4
23 FJ972564 - - - - - - - - - 1 - - 1
24 FJ972562 - - - - - - - - - - 2 2 4
25 FJ972560 - - - - - - - - - 1 - - 1
26 FJ972559 - - - - - - - - - 1 - - 1
27 JN600643 - - - - - - - 1 - - - - 1
28 FJ972568 - - - - - - - - - - - 1 1
29 JN642642 - - - - - - - - - - 1 - 1
30 FJ972566 - - - - - - - - - 1 - - 1
31 FJ972567 - - - - - - - - - - - 1 1
32 FJ972563 - - - - 1 - - - - - - 1 2
33 JN600644 - - - - - - - - - - 1 - 1
34 JN600645 - - - - - - - - - - - 1 1

number of embryos per capsule using condition (field and lab)
as the main factor (geographic origin is not a factor as Type I are
not found in False Bay, Washington). For Type III reproductive
mode, we performed two-way analysis of variances (ANOVAs)
on capsule number, average number of embryos per capsule,
and average number of nurse eggs per capsule, using condition
(field or lab) and geographic origin (Alamitos Bay, California or
False Bay, Washington) as the main factors. In all cases, we per-
formed analyses on the average number of embryos and nurse
eggs per female, calculated from 10 capsules for each female.
Females of similar, intermediate sizes were purposely selected to
avoid allometric effects, but if differences in reproductive traits
were significant, we performed a linear regression and correla-
tion analyses to confirm that the pattern was not driven by a
few larger than average females. Normality was checked with
Shapiro–Wilk W (Shapiro and Wilk 1965) test and homogene-
ity of variance was checked with Bartlett’s test (Snedecor and
Cochran 1989). All analyses were done with JMP 8.0.1. (SAS
Institute 1989).
We tested for differences in brooding time (defined as time
between deposition of capsules to time of liberation of embryos)
in laboratory conditions using a one-way ANOVA among fe-
males: Type I from Alamitos Bay, Type III from Alamitos Bay,
and Type III from False Bay. Normality and homogeneity of
variance was tested as above. Females were purposely selected
of similar intermediate size to avoid allometric effects. A random
subset of females was measured as setiger number; length was on
average 56.4 ± 1.8 segments and was not significantly different
among treatments (geographic locality: t-test = 0.68, df = 20, P
= 0.506; reproductive type: t-test = 1.43, df = 20, P = 0.168).
Oyarzun et al. Poecilogonous spionid evolution 495

RESULTS TCS analyses of either the 16S rDNA or combined dataset

Phylogeographic analyses
The Cyt b dataset consisted of 309 bp for 160 B. proboscidea
individuals with 73 variable positions and the 16S rDNA
dataset consisted of 386 bp for 99 individuals with 23 vari-
able positions. We had both genes (combined data) for 78 B.
proboscidea individuals that included 695 bp with 82 variable
positions. For each sampled locality, Tables 2 and 3 provide
a summary of the number of individuals sampled and the
haplotypes observed.
that used the 95% connection limit yielded a single network
that indicated geographical structure along the West Coast
of North America (Fig. 2). Within the network, a southern
group was observed that covers a small geographic range and
has large haplotype diversity. A second northern group cov-
ers a larger geographic range and has less haplotype diversity.
In contrast, Cyt b split these two groups into separate south-
ern and northern networks. However, we treated our sampled
B. proboscidea as a single species because the split into two
networks was not strongly supported (a 94% connection limit

16S r DNA Cyt b

I
A
1

III I I III
B C D 3
2

I I
III E F G H I J I III
K L 4 5

N
M
6 7

False Bay high

False Bay low
Cattle Point
8
Garibaldi
9
Sunset Bay
South Cove I I 12

Eureka 10 11 13

Coleman Point Conception

Morro Bay 15
Alamitos Bay 14 16 17 18 19
Simple Green 20 III
21 I III I
La Jolla
26 25
22 23 24
28
27 I
30 31 32
29

34
33

Fig. 2. 16S rDNA and Cyt b gene genealogy for Boccardia proboscidea. Statistical parsimony network analysis was calculated with
the 95% connection limit for 16S rDNA and 94% for Cyt b in the program TCS (when using 95% connection limit for Cyt b the
network broke in the long branch). Haplotypes are labeled with letters in 16S rDNA and numbers in Cyt b. The output of the program
was modified to show the geographical origin of the individuals used in this study. Each small square represents an individual, and
the color of the square represents the geographic origin of the individual as shown in the map. Geographical locations that are close
to each other did not show significant differences and are grouped in one color to show the general trend. Individuals that share the
same sequence are grouped by haplotypes as calculated by the TCS analysis. White circles represent missing or extinct haplotype.
Populations surveyed at Alamitos Bay, Simple Green, and La Jolla have both reproductive modes, Type I and Type III; reproductive
type of the individuals is indicated where reproductive mode was observed. In populations north of Point Conception, only Type III
reproductive mode was found; so the reproductive type is not specifically indicated to add clarity to the figure.
496 EVOLUTION & DEVELOPMENT Vol. 13, No. 6, November–December 2011

Table 4. Pairwise φST between collection localities. Cyt b values are below the diagonal and 16S rDNA above

Site fbh fbl cat gar sun scov eur col mor ala sg lj

fbh - 0.07 0.04 0.12 0.02 −0.20 0.35 −0.09 −0.13 0.15 0.40 0.18
fbl 0.24 - −0.06 0.01 −0.06 0.08 0.61 −0.18 0.01 0.18 0.47 0.21
cat 0.68 0.15 - 0.00 −0.01 0.11 0.64 0.00 −0.02 0.12 0.37 0.13
gar −0.12 0.07 0.71 - 0.07 0.27 0.73 0.00 0.07 0.19 0.50 0.23
sun 0.19 0.19 0.29 0.13 - −0.12 0.00 −0.15 −0.02 0.05 0.04 0.02
scov 0.06 0.03 0.17 −0.03 0.00 - 0.20 −0.09 −0.20 0.03 0.20 0.02
eur 0.50 0.44 0.74 0.57 −0.01 0.13 - 0.54 0.42 0.06 0.26 0.08
col −0.03 0.09 0.24 −0.14 0.08 0.00 0.24 - −0.17 0.02 0.22 −0.01
mor −0.05 0.28 0.72 −0.14 0.28 0.12 0.60 −0.01 - 0.12 0.35 0.13
ala 0.57 0.60 0.53 0.49 0.48 0.52 0.53 0.49 0.62 - 0.02 −0.08
sg 0.46 0.50 0.40 0.33 0.33 0.39 0.40 0.35 0.52 0.00 - −0.02
lj 0.54 0.58 0.49 0.45 0.48 0.52 0.51 0.48 0.58 0.02 0.06 -

Bold indicates P < 0.05.

Table 5. Hierarchical AMOVAs for Cyt b and 16S rDNA tistically different using an analysis of molecular variance,
for the north (above Point Conception) versus south (below AMOVA, and genetic diversity statistics. AMOVA results
Point Conception) regions of the North American confirmed genetic separation above and below Point Concep-
populations of Boccardia proboscidea tion (Table 5). In Cyt b, most variation (59%, P = 0.009) was
detected between northern and southern regions, with 39%
Percentage of (P < 0.0001) of the observed variation within sites, and only
variation φ statistic P-value 2% (P = 0.007) attributable to that between sites within re-
gions. In the 16S rDNA dataset, 76% (P = 0.028) of variation
Cyt b was detected within sites, with 28% (P < 0.0001) among re-
Among regions 59.23 φCT = 0.59 0.009 gions. Variation between sites within regions was reported
Among sites within regions 2.18 φSC = 0.05 0.007 as negative and not significant. Nucleotide (π) and haplo-
Within sites 38.59 φST = 0.61 <0.0001 type (h) diversity (Table 6) was higher in the southern region
16S rDNA
for both markers consistent with the TCS results. Tajima’s
Among regions 27.74 φCT = 0.28 <0.0001
Among sites within regions − 3.94 φSC = −0.05 0.46 D and Fu and Li’s D were negative but not significant for
Within sites 76.20 φST = 0.24 0.028 both mitochondrial markers (Table 6). Mismatch distribu-
tion was generally unimodal (Fig. 3) suggesting past popu-
lation expansion, but Cyt b displayed a bimodal distribution
for southern localities.
resulted in only a single network for Cyt b; Fig. 2) and because
there were shared haplotypes between southern and north-
ern populations. Furthermore, some haplotypes (haplotype Common garden experiments and field data
“D” for 16S rDNA and haplotype “5” for Cyt b) include both Figure 4 and Table 7 show results from field collections
Type I and Type III individuals suggesting that reproductive and common garden experiment on life-history traits of B.
differences do not correlate with genetic divergence between proboscidea. Females of Type III coming from Washington
populations. brood their offspring for significantly more days than fe-
Pairwise φST (Table 4) performed on Cyt b data indicate males of Type I in common garden conditions. However,
that worms collected at sites south of Point Conception (ala, Type III females that come from California brood their off-
lj, and sg) were genetically indistinct from each other, but spring for an intermediate amount of time (Fig. 4). In Type
different from the northern sites (fbh, fbl, cat, sun, scov, III reproductive mode, there was no significant difference in
eur, col, mor). Although 16S rDNA pairwise φST showed number of capsules and average number of embryos per cap-
fewer significant differences between localities than Cyt b, sule produced by females from the two locations nor from
the same north-south pattern was supported. Then, we de- females coming from the field versus common garden condi-
fined northern (fbh, fbl, cat, sun, scov, eur, col, mor) and tions. However, in the case of average number of nurse eggs
southern (ala, lj, and sg) geographic regions to test if the per capsule, there was a significant effect of site, but not be-
populations north and south of Point Conception are sta- tween females coming from the field versus common garden
Oyarzun et al. Poecilogonous spionid evolution 497

Table 6. Genetic diversity statistics for Boccardia proboscidea, using two genetic markers, for northern and southern
regions (above and below Point Conception). N refers to number of individuals, H is the number of haplotypes, π refers to
nucleotide diversity, and h is haplotype diversity

Regions Sites Marker N H π h Tajima’s Fu and Li’s

Northern fbh, fbl, cat, gar, sun, scov, eur, col, mor Cyt b 110 10 0.007 0.709 −1.61 −0.56
16S rDNA 66 4 0.001 0.332 −1.08 −1.84
Southern ala, sg, lj Cyt b 50 24 0.017 0.939 −1.96 −1.96
16S rDNA 33 12 0.004 0.807 −1.26 −1.02

(Table 7, Fig. 4). The relationship of female size and average
number of nurse eggs produced in these two populations was
not significant (R2 = 0.21, P = 0.18) and did not drive the
pattern observed. Type I reproductive mode females from
the lab deposited a greater number of capsules than females
coming from the field. There was also an apparent trend
for Type I and Type III females to produce capsules with a
greater number of embryos in the laboratory than in field
conditions, but the difference was significant only for the ef-
fect test of Type III (Table 7, Fig. 4). Females Type I and
Type III were never observed to change their reproductive
mode.
types. Supporting previous evidence, our data show that B.
proboscidea is a true poecilogonous species, and that there
are some interesting reproductive differences in the two pop-
ulations toward the extreme of their distribution. In com-
mon garden conditions, the brooding period between Type
I and Type III from California was not significantly differ-
ent, but Type III from higher latitudes brood, on average,
for longer periods than the Type I of California. In addition,
also in common garden conditions, females from higher lat-
itudes provided their offspring with higher amounts of ex-
tra embryonic nutrition per capsule, suggesting some local
adaptation.

DISCUSSION Genetic history

Both genetic history and developmental plasticity appear to
have a role in shaping observed patterns in mode of larval
development in the spionid annelid B. proboscidea. Type III
reproductive mode occurs throughout the species’ range in
North America, and, although population structure exists,
both reproductive modes shared some of the same haplo-
Genetic differences observed in B. proboscidea suggest that
the original source populations existed in the south (where
there is greater haplotype diversity) and that only some hap-
lotypes have colonized northern latitudes. Although some
of those haplotypes are present in both reproductive types,
only Type III has been found in northern latitudes. There
are two alternative explanations for this: either Type I larvae

Cyt B 16S
Northern clade Northern clade
0.6
0.4
Frequency

0.4

0.2
0.2

10 20 30 5 10 15 20

0.20 Southern clade 0.4

Southern clade
Frequency

0.15 0.3

0.10 0.2

0.05 0.1
Fig. 3. Mismatch distribution for Boccardia
proboscidea pooled by northern and south-
ern regions. Expected (continuous line)
5 10 15 20 25 5 10 15 20 and observed (dotted line) frequencies are
Pairwise differences Pairwise differences shown.
498 EVOLUTION & DEVELOPMENT Vol. 13, No. 6, November–December 2011

America, Type III might have extended to northern latitudes

B
18 via rafting, given that B. proboscidea frequently establishes
AB its tubes in logs by burrowing into wood. However, Type I
brooding time

14
could also have been transported as an adult via the same
(# days)

10 A
mechanism, indicating that larval dispersal might not be the
6 sole explanation.
2
In addition, the area with a putative genetic break in B.
proboscidea coincides with the California Transition Zone at
Point Conception. Point Conception has been recognized as
B
30 n.s.
an important biogeographic break in distributions of marine
species, from algae to barnacles to fishes (Addicott 1966;
# capsules

A
20 Valentine 1966; Briggs 1974; Newman 1979; Doyle 1985;
Valentine and Jablonski 1993). Change in community struc-
10 ture at Point Conception appears as a gradual transition
in the relative dominance of some species rather than by a
n.s.B sharp limit in the distribution of most species (Blanchette
50 A et al. 2008). However, the role of Point Conception in phylo-
mean # embryos
per capsule

geographic structuring—as our study suggests—has proven

30 to be more controversial. Although some studies testing for
intraspecific genetic differences in this region have failed to
10 C C n.s. C C detect such a pattern (Sarver and Foltz 1993; Van Syoc 1994;
Ford and Mitton 1995; Beauchamp and Powers 1996; Ed-
mands et al. 1996; Hellberg 1996), supporting evidence for
B
mean # nurse eggs

a phylogeographic barrier can be found in a benthic marine

per capsule

70 A gastropod, Nucella emarginata (Marko 1998, 2004), the cope-

50
30
10 NA
Alamitos Alamitos
Bay Bay
Type I Type III
Fig. 4. Average life-history data for Boccardia proboscidea of
two populations, Alamitos Bay and False Bay. Two reproduc-
tive modes are shown: Type I (only planktotrophs) and Type III
(planktotrophs, adelphophages, and nurse eggs). Darker gray
indicates data from laboratory conditions; lighter gray indicates
data from females obtained in the field. Letters indicate sig-
nificant differences. Bars indicate standard error. Absent bars
indicate standard error of zero. NA = not applicable.
cannot reach northern latitudes or Type I larvae are unable
to successfully colonize and survive new habitats in northern
latitudes. Type III has been a successful colonizer in other
regions (Australia, Hawai’i) where worms are presumed to
have been introduced via oyster aquaculture (Hutchings and
Turvey 1984; Bailey-Brock 2000). Movement of adults might
favor the Type III reproductive mode given that colonizing
individuals with extended parental care (or lecithotrophy,
or both) are able to establish populations by recruiting lo-
cally (Strathmann 1974). Along the West Coast of North
pod Tigriopsis californicus (Edmands 2001), the intertidal
fish Xiphister (Hickerson and Cunningham 2005), and the
clingfish Gobiesox maeandricus (Hickerson and Ross 2001).
In these studies, haplotypic diversity, heterozygosity, or both,
were also greater in southern populations. Our study sup-
False ports the hypothesis that physical processes around Point
Bay Conception might be partially blocking gene flow. Alterna-
tively, these results could reflect that B. proboscidea popula-
tions were originally restricted south of Point Conception,
California and that its range has recently expanded to the
north.
Developmental plasticity
Females, herein, showed variation in brooding time among
populations even under the same environmental conditions
in a common garden design. Type III females from north-
ern latitudes took care of their broods for longer periods
than Type III from California, although the difference was
not statistically significant. The incubation period for Type
III from southern California was intermediate between Type
I from southern California and Type III from Washington,
which were significantly different. Northern Type III females
deposited a larger number of nurse eggs in each capsule.
In additional experiments, females have been shown to be
able to extend or shorten their brooding time in response
to environmental temperature that is not exclusively related
to developmental time (Oyarzun and Strathmann 2011).
Oyarzun et al. Poecilogonous spionid evolution 499

Table 7. Results of t-test, one- and two-way analyses of variance for populations of Boccardia proboscidea. Comparisons
of life-history traits in: (1) common garden conditions versus field conditions and (2) females Type I from Alamitos Bay,
Type III from Alamitos, and Type III from False Bay. For brooding time, only laboratory data were available

Two-way ANOVA

Comparison One-way ANOVA or t-test Site Field lab Site versus field lab

Brooding time among females df = 2, F = 10 - - -

(1) Type I CA P < 0.001*
(2) Type III CA
(3) Type III WA
Type I no. of capsules df = 27, t = 2.60 - - -
(1) Field versus lab P = 0.015*
Type I average no. of embryos df = 19, t = 0.73 - - -
(1) Field versus lab P = 0.472
Type III no. of capsules - df = 1, F = 0.21 df = 1, F = 0.38 df = 1, F = 0.41
(1) Field versus lab P = 0.649 P = 0.543 P = 0.523
(2) CA versus WA
Type III average no. of embryos - df = 1, F = 0.28 df = 1, F = 6.60 df = 1, F = 0.025
(1) Field versus lab P = 0.60 P = 0.0179* P = 0.876
(2) CA versus WA
Type III average no. of nurse eggs - df = 1, F = 5.46 df = 1, F = 1.02 df = 1, F = 0.09
(1) Field versus lab P = 0.030* P = 0.320 P = 0.770
(2) CA versus WA
* Differences significant at P < 0.05.

Females liberated younger larvae at higher temperatures.
Then, in each reproductive event and according to environ-
mental factors, variation in brooding time observed in our
study can potentially be increased by the mother’s decision to
open capsules later. Consequently, population origin of the
mother, as well as environmental factors the mother is expe-
riencing, can potentially modify the developmental state at
which embryos are hatched, the resulting number of offspring
and the length of their planktonic period (Oyarzun and
Strathmann 2011). This flexibility, mediated by the mother’s
opening capsules behavior, could potentially allow females
to respond quickly to changes in environmental conditions
such as the onset of anoxia or destabilization of the substrate.
However, production of two types of offspring in the same
capsule increases conflict among siblings for resources and
between siblings and parent for her optimal distribution of
resources.
Variation in Type III females in nurse eggs per capsule can
potentially be a mechanism by which the mother can regu-
late rates of sibling cannibalism (Kamel et al. 2010; Oyarzun
and Strathmann 2011) especially when brooding times vary.
Planktotrophs will survive longer when more nurse eggs are
present. Adelphophages have not been observed to select for
nurse eggs or siblings within capsules, but eat what is closer to
their mouth, so the probability of being cannibalized will go
down the more nurse eggs are present in the capsule. Oyarzun
and Strathmann (2011) showed that 73% of females liberate
at least some planktotrophs in laboratory conditions. Fe-
males have been observed to liberate viable planktotrophs
from brood capsules as late as 27 days at 11◦ C and 12 days
at 20◦ C after capsule deposition. Geographic variation ob-
served in the present study agrees with this generalization
based on interspecies comparisons that a larger investment
per embryo and higher levels of parental protection occur
at higher latitudes (Thomson 1878; Thorson 1950; Pearse
et al. 1991). In B. proboscidea, selection for longer incuba-
tion periods at higher latitudes might have resulted from a
combination of slower developmental rates (in laboratory
conditions, embryos develop twice as fast at 30◦ C than at
11◦ C) and increased mortality during slower development.
Further experimentation in this system could give us further
insight into the costs of extended parental care and resolution
of conflict within families.
Poecilogony and habitat
In marine organisms, most documented cases of poecilo-
gony occur in annelids and opistobranch gastropods that
live in mud flats (Chia et al. 1996). This environment could
favor production of more than one kind of offspring as
a form of bet hedging. The action of tides, waves and
the activity of the organisms that live in mud flats create
500 EVOLUTION & DEVELOPMENT Vol. 13, No. 6, November–December 2011
microhabitats in which variation in grain size, sediment com-
position, oxygen concentrations, salinity, and organic con-
tent can vary considerably and change abruptly (Chuwen et
al. 2009).
In a constant environment, poecilogony should not be
sustained over time, as evolutionary forces would select one
mode of reproduction over the other, or divergence between
populations should eventually result in a speciation event
(Vance 1973; Christiansen and Fenchel 1979). However, in
the last 40 years, empirical and theoretical work has ex-
panded our understanding of evolution to include the com-
plexity and fluctuating nature of the environment (Levins
1968; Bull 1987; Cohen and Motro 1989; Meyers and Bull
2002). Along with many different documented cases of phe-
notypic plasticity and variation, documented cases of re-
productive polymorphisms have demonstrated that many
species, as Meyers and Bull (2002) pointed out, “fight change
with change.” From desert annual plants that produce seeds
with different germination times (Cohen 1966), to the phage
λ that alternates stochastically between lysogenic and ly-
sis cycles (Ptashne 1992), bet-hedging strategies are more
widespread in nature than previously thought (Crean and
Marshall 2009). Models have supported the hypothesis that
bet-hedging strategies might have evolved as adaptations
to the nonpredictable variability of species’ environments
(Capinera 1979; Philippi and Seger 1989; Mittler 1996),
as it reduces the variation in the fitness of the offspring
among generations by increasing the variation within gen-
erations (Seger and Brockmann 1987; Crean and Marshall
2009).
Petch (1989) documented that density of Australian B.
proboscidea populations fluctuated in response to variation
of organic material. Although the reproductive season is
mainly controlled by day length, it was extended at high
food levels (Petch 1989). Populations seemed to avoid in-
terspecific competition by increased tolerance to sewage
waste. In North American populations that inhabit False Bay
(Fig. 1), B. proboscidea occurs at high densities in the upper
most intertidal zone, where few other macrofaunal inverte-
brate species coexist. Seasonal deposition of Ulva sp. has
been observed to cover extensive areas of this zone each
summer making the sediment anoxic and negatively impact-
ing B. proboscidea populations (personal observation). In
addition, seasonally changing levels of sand, sediment de-
position in between barnacles, and availability of logs can
create frequent patch extinctions and colonization events
(Oyarzun unpublished, Pernet personal communication). Fi-
nally, the temperature of sediment 2–3 cm below the sur-
face where B. proboscidea inhabits has been recorded to
vary between 13 and 29◦ C during a low tide series dur-
ing summer time (Oyarzun 2010). Interestingly, Helmuth et
al. (2006) reported that temperature stress can be higher at
high latitudes where low tides coincide with mid-day high
temperature, implying a latitudinal gradient in temperature
stress.
In our study, we found Type III developmental mode in
all the populations we sampled along B. proboscidea’s geo-
graphic range. This developmental mode was predominant
north of Point Conception in a variety of habitats from rocks,
to sediment to logs, including the very high intertidal of
higher latitudes that has soaring levels of temperature stress
through the reproductive season. The ubiquity of Type III
suggests a higher ability to succeed in a variety of habitats
than Type I. So, avoiding phylogenetic confounding factors,
we show an example in which within a species the bet-hedging
reproductive strategy is more successful in terms of abun-
dance and extent of geographic range, than the none bet-
hedging strategy. Success of the Type III reproductive mode
might depend on a balance between fine-scale temporal and
spatial variation within the local population predominantly
maintained by the adephophagic larvae and larger scale dis-
persal events among habitats along the coast provided by the
planktotrophic larvae.
Future directions
Exploration of population dynamics of both reproductive
modes, Type I and Type III, where they coexist, as in
Alamitos Bay (Fig. 1) deserves further effort. Levin and
Hugget (1990) observed abundance, recruitment, size struc-
ture, and reproductive output of two populations of the
poecilogonous species S. benedicti (Polychaeta: Spionidae)
that differed in egg size and larval feeding. Larvae and ju-
veniles of the lecithotrophic population had a survival ad-
vantage, but planktotrophs had a fecundity advantage. The
result was nearly the same for eventual recruitment. However,
identification based on morphology of juveniles that are the
product of each reproductive mode is not possible in B. pro-
boscidea. A comparison with Boccardia columbiana Berkeley,
1927 could be informative at higher latitudes where Type I
is not found. Boccardia columbiana resembles B. proboscidea
in size, morphology, habitat, and range (Woodwick 1963)
and reproduces only with planktotrophic larvae, like Type
I of B. proboscidea (Oyarzun unpublished). Although this
species coexists with B. proboscidea in southern California,
it is not as ubiquitous, suggesting differences in population
dynamics. Finally, in order to understand implications of
this reproductive polymorphism, testing is needed to deter-
mine whether planktotrophic larvae of Type III reproductive
mode are actually playing a role in the population dynamics
by serving as an occasional dispersing mechanism. Further
investigation of the local adaptation of B. proboscidea, pop-
ulation dynamics, and the degree of plasticity encountered
in each population might bring further insight into environ-
mental conditions that favor the evolution of life histories
with extended parental care.
Oyarzun et al.
ACKNOWLEDGEMENTS
We thank Richard Strathmann for valuable contributions on the de-
sign and discussion of this research. Bruno Pernet kindly collected
specimens from California and processed samples in his laboratory.
Richard Emlet and Rachel Collins allowed use of their labs. Thanks
to Jesus Angel de Leon Gonzalez, Rolando Bastida-Zavala, Euge-
nio Carpizo, Pablo Hernandez, Nuria Mendez, Veronica Rodriguez-
Villanueva, Sergio Salazar-Vallejos, and Viviane Solis for checking
databases, museum collections, and sharing information of location
of Boccardia proboscidea in Mexico and to Paulo Paiva for infor-
mation of spionids in Panama. Federico Brown, Kellen Andrilenas,
Maria Jose Bravo, Stevan Springer, Tuna Kuyucu, Martha Groom,
Caroline Faria, Amer Dahmash, Paul Bourdeau, and the FHL Evo-
devo class of 2005 helped at different stages of this research. Assis-
tance from Friday Harbor Laboratories Staff, the Department of
Biology and the Comparative Genomic Center of the University of
Washington and to the Naos Island Laboratory of the Smithsonian
Tropical Research Institute is acknowledged. This work was funded
in part by the Short Term Fellowship of the Smithsonian Trop-
ical Research Institute, Friday Harbor Laboratories, NSF grants
OCE0217304 and OCE0623102 to R. Strathmann, the NSF Worm-
Net Grant EAR-0120646 to Ken Halanych. This work represents
contributions 83 and 5 to the Auburn University (AU) Marine Bi-
ology Program and Molette Biology Laboratory for Environmental
and Climate Change Studies, respectively.
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Supporting Information
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Fig. S1. Alignments of 16S rDNA nucleotide data
Fig. S2. Alignments of Cyt b nucleotide data
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