Invertebrate Biology 127(1): 45–64.
r 2008, The Authors
Journal compilation r 2008, The American Microscopical Society, Inc.
DOI: 10.1111/j.1744-7410.2007.00104.x
The Polydora cornuta complex (Annelida: Polychaeta) contains populations that are
reproductively isolated and genetically distinct
Stanley A. Rice,a,1 Stephen Karl,2 and Katherine A. Rice1
1
2
Department of Biology, University of Tampa, Tampa, Florida 33606, USA
The Hawaii Institute of Marine Biology, University of Hawaii at Manoa, Kaneohe 96744, Hawaii, USA
Abstract. We have collected evidence that the nominal species, Polydora cornuta BOSC 1802,
contains at least three separate species in North America. Specimens of P. cornuta were collected in California, Florida, and Maine, raised in the laboratory, and assessed for reproductive compatibility, genetic similarity, gamete characteristics, and developmental rates.
Reproductive crosses between each combination of sex and population revealed variable levels of hybridization at the level of fertilization. Percent fertilization was very low for all combinations (0–7%) except for California females crossed with Florida males (42%). In all
interpopulation crosses, fertilized eggs arrested in cleavage and no viable larvae were produced. All pairwise comparisons of the studied populations showed significant differences in
multiple reproductive traits. Mitochondrial cytochrome oxidase subunit I DNA sequences
revealed large differences between Florida and California worms with a maximum likelihood
genetic distance of d 5 0.860, while Florida and Maine worms were d 5 0.806, and California
and Maine d 5 0.156. California and New Zealand worms were very similar genetically
(d 5 0.010). These data strongly suggest that populations of P. cornuta in North America
comprise a cryptic species complex composed of at least three distinct lineages.
Additional key words: cryptic species, COI gene sequences, reproduction, morphology
Sibling species have been identified in nearly all
major groups of marine animals (see reviews by
Knowlton 1993, 2000; Palumbi 1994). Close examination of widespread populations of so-called ‘‘cosmopolitan’’ species has revealed that multiple cryptic
species are often present (Mayr 1948; Grassle &
Grassle 1974; Ayala 1975; Clark 1977; Reish 1977;
Guerin & Kerambrun 1984; Hoagland & Robertson
1988). Morphological differences between these
cryptic species may be subtle or absent, leading to
difficulties with identification (Åkesson 1984). Alternatively, morphological variation may be substantial
both within and between populations, leading to similar taxonomic problems (Rice 1991). The discovery
and characterization of cryptic species complexes
thus sometimes requires going beyond morphological features to include behavioral, ecological, genetic,
and reproductive attributes. In particular, the use of
reproductive characteristics in distinguishing between closely related species has long been a valu-
a
Author for correspondence.
E-mail: [email protected]
able tool in evolutionary biology and systematics
(Smith 1958; Mayr 1963; Åkesson 1977; Fauchald
1977; Wilson 1991; Gamenick et al. 1998; Blake &
Arnofsky 1999; Rouse & Pleijel 2006).
Polychaetes hold significant promise for investigations of reproductive isolation and speciation. This
taxon has been estimated to include between 9000
(Rouse & Pleijel 2001) and 13,000 (Beesley et al.
2000) species, with many more undescribed species
awaiting discovery. Within marine communities,
polychaetes often are the predominant macrofaunal
component and can be found from the deep sea to the
intertidal zone. Greatest abundances are usually reported in soft sediments extending from the subtidal
benthos to the continental slope (Blake 1996). Despite being a diverse and ancient group, our understanding of the evolution of the Polychaeta is
rudimentary (Rouse & Pleijel 2006). Several genera
of polychaetes comprise unresolved networks of
closely related taxon complexes. These include Capitella (Grassle & Grassle 1976), Eulalia (Bonse et al.
1996), Hediste (Rohner et al. 1997), Malacoceros
(Guerin & Kerambrun 1984), Microphthalmus (Westheide & Rieger 1987), Platynereis (Pfannenstiel &
46
Grunig 1984), Prionospio (Mackie 1984), Sabella
(Andrew & Ward 1997), Ophryotrocha (Åkesson
1978, 1984; Dahlgren et al. 2001), and Petitia (Soosten et al. 1998). Closer examination of sibling species
complexes has helped to establish relationships
among genetic divergence, morphological change,
and reproductive incompatibility associated with
speciation (Palumbi 1994).
The Polydora complex in the family Spionidae is a
closely related group of at least 115 species in nine
genera: Amphipolydora, Boccardia, Boccardiella, Carazziella, Dipolydora, Polydora, Polydorella, Pseudopolydora, and Tripolydora. These genera are thought
to constitute a monophyletic group (Blake & Arnofsky 1999; Blake 2006) and share a modified fifth
chaetiger (chaetae-bearing segment; Fig. 2A) with
specialized chaetae used in tube construction and
maintenance. Polydora cornuta BOSC 1802 (formerly
Polydora ligni, WEBSTER 1897 see Blake & Maciolek
1987) is one of the most widely distributed species in
the complex (Grassle & Grassle 1974; Rice & Simon
1980). Members of P. cornuta will settle on almost
any substrate, after which they form thin tubes of
sediment. They occur in most of the world’s oceans,
excluding polar regions, often in dense populations.
Populations of P. cornuta have been reported in parts
of California (Rice 1975; Light 1978; Blake 1996),
Washington State (Ferner & Jumars 1999), New England (Webster & Benedict 1884; Mortensen 1945;
Dean 1965; Grassle & Grassle 1974), North Carolina
(Day 1973; Levin & Creed 1984), throughout western
Europe (Soderstrom 1920; Hartman 1959; Rasmussen 1973; Ramberg & Schram 1982; Mustaquim
1986), the Mediterranean (Cinar et al. 2005), the Caribbean, South America (Foster 1971), Taiwan
(Radashevsky & Hsieh 2000), southeastern Australia
(Blake & Kudenov 1978, as P. ligni), and New Zealand (Read & Gordon 1991). A rigorous morphological investigation of global populations of P. cornuta
(Radashevsky 2005) concluded that insufficient
differentiation has occurred to warrant unique species status for any of the geographically separated
populations.
It seems unlikely, however, that these geographically isolated, conspecific populations of P. cornuta
are homogenously interconnected by migration and
gene flow for a variety of reasons. First, even for
temperate and tropical organisms with highly mobile,
pelagic adults, populations in separate ocean basins
commonly are isolated from each other and generally
have been so for a considerable amount of time (e.g.,
bigeye tuna: Bremmer et al. 1998; swordfish: Rosel &
Block 1995; sea turtles: Bowen et al. 1992, 1997; Karl
et al. 1992; trumpet fish: Bowen et al. 2001). Second,
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vol. 127, no. 1, winter 2008
Rice, Karl, & Rice
even within ocean basins, species with mobile larvae
and/or adults often are differentiated between the
northern and southern hemispheres and often even
divided again into eastern and western subpopulations in a given ocean within hemispheres (general:
Mayr 1954; prawns: Duda & Palumbi 1999; sea urchins: McCartney et al. 2000; copepods: Lee 2000;
polychaetes: Breton et al. 2003). Third, previous
studies of similar, closely related species of polychaete worms, Streblospio benedicti WEBSTER 1879
and Streblospio gynobranchiata RICE & LEVIN 1998
revealed considerable divergence not only between
morphologically similar species but also between
geographically proximate populations within North
America (Schulze et al. 2000). In this report, we present data from reproductive crosses among three
populations of P. cornuta along with associated mitochondrial gene sequences that demonstrate reproductive isolation and substantial genetic divergence
characteristics of separate species. Using traditional
adult morphology as well as developmental rates and
reproductive morphology, we suggest avenues for
identifying individuals and populations within this
species complex.
Methods
Source populations
Specimens of Polydora cornuta used in this study
were collected from four sites:
(1) Tampa Bay, FL: Planktonic larvae were collected from several different locations within Tampa Bay
at several different times between 2001 and 2005. Most
collections were made in the late winter or early spring
(January–March) because planktonic larvae are most
abundant during this period. The primary collecting
sites included the south side of the Gandy Bridge located in the middle of Old Tampa Bay (271530 700 N,
821320 600 W) and the public boat ramp on Bunces Pass
in Fort DeSoto Park near the mouth of Tampa Bay
(271380 4900 N, 821430 0.500 W). Samples were collected using a 35-mm mesh plankton net and returned to the
laboratory, where they were transferred to glass jars
and provided with aeration and food until the larvae
settled and formed tubes on the bottom of the containers. Adult and juvenile worms were removed from
these containers and transferred into Petri dish cultures for experiments. These Petri dish cultures were
monitored for planktonic larvae that, when observed,
were isolated and raised in the laboratory for experimental crosses and other purposes.
(2) Anaheim Bay, CA: Two separate collections
were made by Dr. B. Pernet from intertidal mud flats
Polydora cornuta: cryptic species
in Anaheim Bay (331430 4900 N, 1181050 0100 W), in May
and June 2005. Mud samples were sieved through a
0.5-mm sieve at the site and returned to the lab at
California State University, Long Beach, where specimens were separated from other worms, held in the
laboratory for a few days, and shipped overnight to
Tampa. Upon arrival, the specimens were isolated
and placed into Petri dish cultures for experiments.
(3) Lowes Cove, ME: Two separate collections
were made by Dr. S. Lindsay from Lowes Cove
near the Darling Marine Laboratory (431560 0500 N,
691340 4100 W). The first collection was made in June
2001 and the second in June 2003. Intertidal mud
cores were collected at the site and returned to the lab
at the University of Maine, Orono, where they were
sieved and specimens were separated from other
worms before overnight shipping to Tampa. Upon
arrival, specimens were isolated and placed into Petri
dish cultures for experiments.
(4) Auckland, New Zealand: A sample of ethanolpreserved specimens was obtained for genetic analysis. These specimens were collected in 2002 by I. Paterson from mud tubes on oyster shell at two sites on
North Island north of Auckland, preserved, and
transported to North America.
Culture procedures
Adult worms were maintained as separate populations in physically separated portions of the laboratory. Stock cultures of adult worms were kept in
100 25 mm plastic Petri dishes with 10–50 worms
per dish. Isolated males and females were maintained
in 12-well and six-well tissue culture dishes (Corning
Cell Wells, Corning, NY, USA) for gamete studies
and experimental crosses. All adult cultures were fed
every 2–3 d along with water changes. Adult food
consisted of natural sediment from the mouth of
Tampa Bay that had been blended in a blender and
frozen at 201C for at least 4 weeks, followed by a
second blending before use. Sediment was added to
culture containers with a plastic pipette and water
was changed after gently washing loose sediment and
fecal matter out of the dishes. In addition to sediment, 10–30 drops of cultured algae, Tetraselmis and
Nanochloropsis (both from Florida Aqua Farms,
Dade City, FL, USA), were added to the adult cultures depending on the size of the dish and the density
of adult worms. Seawater was collected from Bunces
Pass near the mouth of Tampa Bay, transported to
the laboratory at the University of Tampa, and filtered through Qualitative P5 filter paper (Fisher Scientific, Pittsburg, PA, USA). Filtered seawater was
adjusted to 25% salinity with deionized water and
47
stored in Nalgene carboys. Swimming larvae were
collected from adult cultures by pouring the water
from the adult culture dish through a 35-mm screen
(separate screens were used for each population to
prevent contamination) and gently washing the larvae into a clean 100 25 mm Petri dish. Larvae were
fed and water was changed every 1–2 d. Larval food
consisted of cultured Tetraselmis and Nanochloropsis
supplemented with diluted Rotirich (Florida Aqua
Farms). Larval cultures were maintained on an orbital shaker (Daigger, Vernon Hills, IL, USA) rotating at 44 rpm to keep the larvae and food in
suspension. In larval growth and metamorphosis
studies, cultures were provided with a final concentration of 6.5 106 cells mL1 of phytoplankton. As
larvae grew and began to settle on the bottom of the
dishes, they were removed from the orbital shaker,
provided with adult food (sediment), and maintained
as above for adult cultures. The laboratory temperature was maintained as close as possible to a range
of 221–251C.
Experimental crosses
Selected juvenile worms were transferred from larval cultures into 12-well tissue culture dishes as they
approached metamorphosis (began to crawl on the
bottom of the dish or build mud tubes). One worm
was placed into each well and maintained until sexual
maturity. At maturity, female worms were transferred into six-well tissue culture dishes, one worm
per well, and monitored daily for egg capsule production by inverting the dish on the stage of a dissecting microscope and viewing the worm through
the bottom of the dish. Male worms were transferred
into 60-mm Petri dishes and monitored for spermatophore production. Spermatophores were collected
daily from the male culture dishes of each of the three
populations. Female worms were observed daily until
egg capsules were deposited and determined to be
entirely unfertile (confirming that the females contained no stored sperm). Females in six-well dishes
were then given spermatophores by hand from another population using a fine-tip glass pipette.
Following spermatophore transfer, the worms
were examined daily for egg capsule production.
When new egg capsules were deposited, a sample of
capsules was removed from the parental tube and
examined on a compound microscope for number of
developing and non-developing eggs. An egg was
considered to be unfertilized if no polar bodies were
visible and there were no signs of cell division. A
minimum of 300 eggs or embryos was counted for
each group of egg capsules deposited by each female
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vol. 127, no. 1, winter 2008
48
worm. The mean percent fertilization and standard
deviations (SD) were calculated for interpopulation
crosses and intrapopulation crosses. Each interpopulation cross was repeated with a fresh batch of
spermatophores following each deposition of egg
capsules for a target total of two or three separate
spawnings. Following the last interpopulation
spermatophore transfer and subsequent egg capsule
deposition, each female was given spermatophores
from her population of origin and monitored for new
egg capsules. If an individual female worm failed to
produce fertilized eggs following this intrapopulation
cross, she was excluded from the data set. All combinations of male/female crosses were attempted for
the three populations for which we had living specimens (California, Florida, Maine).
DNA extraction and sequencing
Whole adult live and ethanol preserved worms
were used for DNA extraction. Live worms were selected from the field-collected samples or from F1
generations produced in laboratory cultures. Total
DNA was extracted using a modified CTAB method,
followed by extraction with chloroform/isoamyl alcohol and DNA precipitation in absolute ethanol
(Shulze et al. 2000). Universal cytochrome oxidase
subunit I (COI) primers (Folmer et al. 1994) were
used to amplify an initial fragment of the COI gene,
which was then cloned and sequenced for use in
P. cornuta population-specific primer production
(sequences are available from S.K.). The target sequence was PCR amplified using the P. cornuta
population-specific primers. Both strands were sequenced using an ABI Big Dye cycle sequencing
reaction kit according to the manufacturer’s recommendations (Perkin-Elmer, Norwalk, CT, USA), and
a Perkin-Elmer ABI 310 Automated Sequencer.
Reproductive and adult morphology
In order to test the reliability of gametogenic segment position as population markers, we determined
the first and last gametogenic segments plus total
segments in a sample of laboratory-cultured individual worms over time. Data were collected at 1–2week intervals over a period of 63 d (Maine) or 81 d
(Florida) for individual worms raised in isolation in
six-well tissue culture dishes. Only two of the three
populations (Florida and Maine) were included in
this experiment because the California population
was not available at that time. For these measurements, individual worms were removed from their
tubes, placed in cold (41C) isotonic MgCl2, quickly
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Rice, Karl, & Rice
examined under a dissecting microscope for first and
last gametogenic chaetiger and total chaetigers at 35–
40 , and returned to their culture container.
Growth rates were calculated in chaetigers per day
for each individual worm and compared between
populations. Specimens for scanning electron microscope (SEM) studies were fixed in either 10% buffered formalin or 2.5% glutaraldehyde in 0.2 mol L1
PO4 plus 0.14 mol L1 NaCl buffer, postfixed in 2%
OsO4 in 0.15 mol L1 NaHCO3, and dehydrated in
ethanol to acetone for critical point drying. Dried
specimens were mounted and coated with gold–palladium and viewed on a Novascan 30 SEM (Zeiss,
Thornwood, NY, USA).
Time to metamorphosis was determined for Florida, California, and Maine populations in the laboratory. Pairs of reproductive adults were placed in
50 10 mm plastic Petri dishes and checked for released larvae daily. Daily collections of released larvae
were pooled and placed in 100 25 mm plastic Petri
dishes, fed, and maintained as above. Larvae that
were released from these adult cultures on Tuesdays
through Fridays were checked daily for signs of metamorphosis (worms crawling on the bottom of the dish
with palps held forward or inside tubes built on the
bottom). Larvae collected on Mondays were not included in the data analysis because their time of release
could not be determined with accuracy (larvae were
not routinely collected on Saturdays and Sundays).
Size at metamorphosis was determined for the
Florida, Maine, and California populations. Worms
from the ‘‘time to metamorphosis’’ cultures (above)
were removed at first sign of adult behavior (crawling
on the dish bottom or tube formation), relaxed in
cold (41C) isotonic MgCl2, and counted for number
of chaetigers using a dissecting microscope. The
smallest size at metamorphosis and the mean size
were compared between populations. Size at sexual
maturity was determined for the Florida, Maine, and
California populations. Juvenile worms from the larval cultures were isolated after metamorphosis and
placed in 12-well tissue culture dishes, one worm per
well. Worms were fed adult food (phytoplankton
plus freeze-thawed sediment) and water was changed
every 2–3 d. The sex of each worm was determined by
inverting the culture dish on the stage of a dissecting
microscope and viewing the worm inside its tube
through the clear bottom of the dish. Worms containing gametes (males had milky white gamete-bearing segments while females had orange to white
spherical eggs inside the coelom of gamete-bearing
segments) were examined for distribution of gametes
and total length in chaetigers. The time of and size at
sexual maturity were compared between populations.
Polydora cornuta: cryptic species
Unfertilized egg diameter and sperm length were
determined for each of the three populations. Unfertilized eggs were removed from egg capsules and
spread onto a microscope slide. Eggs were measured
to the nearest 0.1 mm along their longest dimension
(Polydora eggs are slightly oval) at 400 on a compound microscope using a calibrated ocular micrometer. Spermatophores were collected from male
worms, broken open with fine dissecting needles on
a microscope slide, and examined at 1000 under oil
immersion on an Olympus BH2 microscope (Olympus, New Hyde Park, NY, USA) equipped with
differential interference contrast optics. Sperm heads
(acrosome1nucleus1middlepiece) were measured
for at least 50 sperm to the nearest 0.1 mm using a
calibrated ocular micrometer.
Data analysis
Statistics for population comparisons were computed using StatView 5.0.1 (SAS Institute Inc., Cary,
NC, USA). A single-factor analysis of variance
(ANOVA) was run for each population character
measured and pairwise significance tested using Fisher’s protected least significant difference method as
modified in StatView to use unequal as well as equal
sample sizes.
DNA sequences were proofed and aligned using
Sequencher (ver. 4.1; Gene Codes Co., Ann Arbor,
MI, USA) and using the protein open reading frame
as an aid. Using Modeltest v3.06 (Posada & Crandall
1998), the Akaike information criterion indicated
that the best-fit model of evolution was TIM1G
with proportion of invariable sites equal to 0.7314,
and base frequencies of A: 0.3487, C: 0.1991, G:
0.1102, and T: 0.3421. Phylogenetic analysis was performed in PAUP 4.0b10 (Swofford 1998) using a
neighbor-joining algorithm and mid-point rooting
(Saitou & Nei 1987), with maximum likelihood distances estimated using the optimal model identified
with Modeltest. Statistical support for the nodes was
estimated with 100 non-parametric bootstrap replicates (Felsenstein 1985).
Results
Reproductive biology
The sexes were separate in all populations studied.
On rare occasions, an individual with both eggs and
sperm in coelomic cavities was observed in laboratory cultures. No hermaphrodites were encountered
in field-collected adult worms. Males and females
produced and deposited (females) or released (males)
49
gametes without regard to the presence of the opposite sex. Males produced spermatophores that were
composed of a central packet of aligned sperm surrounded by a meshwork of microvilli derived from
the enlarged segmental nephridia. The shape of
spermatophores was consistent among populations
of Polydora cornuta (sensu latu) and different from
those of other species outside the P. cornuta complex.
Isolated males in laboratory cultures routinely released numerous spermatophores into their culture
containers. These spermatophores were stable for up
to 24 h at room temperature (231C) or could be maintained for 2–3 d at 41C. In either case, active sperm
were observed when these spermatophores were
opened. The sizes of the spermatophores produced
increased as the males grew but the general shape of
the spermatophores remained constant. Females
picked up spermatophores with their palps and transported them toward the opening of their tube.
Spermatophores commonly broke open during transport along the ciliated grooves of the female’s palps,
releasing the sperm in an active cloud that was observed to flow into the female’s tube along with the
respiratory current. These sperm were stored inside
paired segmental seminal receptacles of the female or
inside the segmental nephridia through processes that
have yet to be fully described. A single spermatophore transfer provided enough sperm to support reproductive activities and produce fertilized eggs for
up to 7 weeks (seven to eight spawning events).
Isolated females produced a decreasing number of
fertilized eggs as stored sperm were used up. As this
happened, unfertilized eggs were consumed by developing larvae producing larger larvae with each successive brood (S.A. Rice, unpubl. data). Egg capsules
were deposited inside the female’s tube regardless of
whether the female was in a mixed male1female culture or in isolation. Development of embryos from
fertilization to three-chaetiger nectochaetes took
about 4–5 d, at which time the female released the
larvae from the capsules. The release process was directly observed on several occasions and involved the
female worm turning her ventral surface toward the
egg capsules (she is usually in a position with her
dorsal surface toward the egg capsules) and slowly
moving down the egg string applying her mouth area
to the capsules and opening each capsule in turn.
Rapid contractions of the female’s body then expelled the larvae from the tube. To test the ability
of larvae to release themselves from the egg capsules,
we isolated 63 capsules from Maine females and 86
capsules from Florida females. These capsules were
maintained in Petri dishes with regular water changes
and observed for released larvae. In the Maine
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50
Rice, Karl, & Rice
capsules, 63% of the capsules remained unopened
after 6 d while 58% of the Florida capsules remained
unopened after 10 d. The larvae in the unopened capsules all died. Several individuals of P. cornuta were
maintained in the laboratory for over 12 months and
were still reproductively active.
Experimental crosses
Previous results in our laboratory indicate that the
Florida populations of P. cornuta are reproductively
compatible at least from Fort Pierce to the Florida
panhandle (S.A. Rice, unpubl. data). The following
results are based on the Tampa Bay (Gandy Bridge)
population as representative of Florida.
The results of the experimental crosses among the
three geographically isolated populations of P. cornuta present a clear picture of reproductive isolation
(Table 1). With one exception, all reciprocal interpopulation crosses resulted in means of o8% fertilization. The only interpopulation cross to display
substantial interpopulation fertilization success was
California females crossed to Florida males (Table
1). Of the three California females that were successfully crossed to Florida males, the percent of hybrid
embryos ranged from a low of 31% to a high of 53%
with a mean of 42% fertilization. The reciprocal
cross of California males and Florida females had a
mean percent fertilization of only 4.2, indicating a
strong asymmetry in reproductive isolation between
these two populations. Even though the percent fertilization was high when California females were
crossed with Florida males, these hybrid embryos
did not develop beyond the early cleavage stages.
In fact, when these hybrid embryos were examined
several days after they were deposited inside egg capsules, they were indistinguishable from unfertilized
eggs as the blastomeres had fused and development
had completely ceased. Following the interpopulation crosses, each female was given spermatophores
from her native population to ensure her fertility and
the percent fertilization of subsequent spawnings
were recorded. The percent fertilization averaged
nearly 100% in these intrapopulation crosses for
each population, except the California females (Table 1) and these embryos developed normally producing fertile adult worms.
Genetic differentiation
We sequenced a total of 32 individuals (six
from Maine, four from Gandy Bridge, Tampa
Bay, Florida, eight from Fort DeSoto, Tampa Bay,
Florida, ten from Orange County, California, and
four from New Zealand; GenBank accession numbers EF525280–EF525291). We were not able to sequence the entire fragment from all individuals and
so the final data set includes 1012 nucleotides (nt) for
Fort DeSoto (Tampa Bay) and California, 748 nt for
Maine, 731 nt for New Zealand, and 653 nt for Gandy Bridge (Tampa Bay) individuals with unsequenced regions coded as missing data. The number
of haplotypes and genetic distances, therefore, represent a minimum. Excluding these regions had little to
no effect on our results. In total we resolved 12 haplotypes. Overall, within-location variation was low
(B1.0%) and only a single haplotype was seen in
the ten individuals from California. Among major
geographic locations, however, divergence was generally quite large (e.g., d 5 0.86070.046 for Florida
Table 1. Results of laboratory crosses between males and females from three different populations. Each experimental
cross used a unique female worm. Spermatophores from males may have been used in more than one cross. CA, California; FL, Florida; ME, Maine.
Female
Male
Experimental
CA
ME
CA
FL
ME
FL
crosses
ME
CA
FL
CA
FL
ME
Control crosses
CA
CA
FL
FL
ME
ME
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vol. 127, no. 1, winter 2008
# Crosses
(# of females)
# Spermatophore
transfers per cross
% Fertilization7SD
# Eggs or embryos
counted
12
8
3
10
12
5
1–4
1–3
1–3
2–3
1–4
3
2.675.1
1.1710.0
42.0710.7
4.278.4
7.079.0
0.0
9106
3265
2839
6484
4549
1320
11
6
6
1
1
1
63.4729.0
100.0
98.971.3
3813
1136
3069
Polydora cornuta: cryptic species
51
vs. California/New Zealand; Table 2). The phylogenetic analysis resulted in three clearly defined and
well-supported groups (Fig. 1). There was very little
difference between the Gandy Bridge samples and the
Fort DeSoto (Florida) samples and they were combined for analysis (as ‘‘Tampa Bay’’ in Fig. 1). This is
similarly true for the New Zealand and California
haplotypes. The Maine haplotypes appear to be more
closely related to the New Zealand/California group
than to the Florida group. All these groups were well
supported as is indicated by a bootstrap support of
94–100 for all major nodes in the tree (Fig. 1).
Adult morphology
We did not undertake an extensive morphological
investigation of adult worms during this study because our focus was on reproductive characters and
genetics. Extensive morphological comparisons
among reproductively isolated populations of P. cornuta have been published previously (Rice 1991), and
we have continued to search for distinctive characters
that might allow for identification of the Florida
form of P. cornuta. One such character involves the
capillary chaetae on the modified fifth chaetiger of
adult worms. Typically, the chaetae of the fifth chaeTable 2. Maximum-likelihood estimated genetic distance
and reproductive compatibility in Polydora cornuta. Florida, California, and New Zealand populations were pooled
for these comparisons because of high genetic similarity.
Reproductive compatibility refers to production of viable
larvae (full), no larvae (none) or signs of early development, followed by developmental arrest (partial).
Group
Distance
Within clade
Maine
0.003a
Florida
0.01270.003
California/
0.01070.010
New Zealand
Between clade
Maine versus 0.15670.012
California/
New Zealand
Florida versus 0.80670.028
Maine
Florida versus 0.86070.046
California/
New Zealand
a
Reproductive compatibility
Full, reciprocal
Full, reciprocal
Full, reciprocalb
None, reciprocalb
None, reciprocal
Partial,
asymmetricalb
A single comparison for the two observed haplotypes.
Crossing results are for California individuals only
because New Zealand individuals were only available as
preserved specimens.
b
Fig. 1. Neighbor-joining tree representing phylogenetic
relationships among the 12 mtDNA COI haplotypes
observed in this study. Numbers above branches
represent bootstrap support for each node in 100
replicates. Numbers in parentheses indicate the number
of individuals per haplotype, where greater than one.
COI, cytochrome oxidase subunit I.
tiger in adult worms are composed of a row of heavy
spines, each accompanied by a single brush-tip companion seta (Fig. 2). In most specimens from Florida,
additional capillary chaetae are present in an inferior
ventral fascicle and in a superior dorsal fascicle
(Fig. 2B). The morphology of the fifth chaetiger
spines and companion chaetae is quite variable between individuals from all populations. Further, the
spines and especially the companion chaetae are subject to wear because these chaetae are used in tube
construction and maintenance. The wear patterns of
the spines make the chaetae more blunt distally and
may reduce the size or the shape of the subdistal
tooth. The companion chaetae emerge from the body
wall at the posterior of the segment as pointed capillaries (Fig. 2C). As these companion chaetae wear,
they take on the classic ‘‘brush tip’’ appearance supposedly characteristic of P. cornuta. The presence of
these ‘‘brush tip’’ chaetae is thus not a species-specific
morphological character but rather a wear pattern
of an otherwise common chaetal type in the genus
Polydora.
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52
Rice, Karl, & Rice
Fig. 2. Polydora cornuta, SEM, Tampa Bay, FL. A. Lateral view of the anterior end showing palps (p) and fifth setiger (f).
Scale bar, 0.1 mm. B. Close-up of the fifth chaetiger showing dorsal superior (ds) and ventral inferior (vi) capillary
chaetae. Scale bar, 4 mm. C. Fifth chaetiger spines (s) and companion chaetae (cc) showing wear pattern on companion
chaetae from the newest (left) to the oldest (right). Scale bar, 4 mm. D. Closer view of wear pattern of companion chaetae
with the oldest in upper right and the newest at lower left. Scale bar, 4 mm.
Larval growth, metamorphosis, and sexual maturity
We determined for each of the three populations
the time to metamorphosis, size in chaetigers at
metamorphosis, and time to and size at sexual maturity. To eliminate variation due to laboratory culture
conditions, all three populations were tested for time
to metamorphosis during the same time period (July–
August 2005). During this time, all larval cultures
were maintained and observed using consistent procedures as outlined above (‘‘Methods’’). Figure 3A
shows the population means of time to metamorphosis. Florida worms reached metamorphosis in the
shortest time with the least variation between cultures. Maine larvae took significantly longer
(p 5 0.001) to reach metamorphosis than Florida
worms, with California larvae intermediate between
the other two populations. The mean size of larvae at
metamorphosis was variable among populations
(Fig. 3B). The Florida larvae not only reached meta-
Invertebrate Biology
vol. 127, no. 1, winter 2008
morphosis in a shorter time than the other populations but were significantly smaller in size at metamorphosis than Maine and California worms
(po0.0001). Maine and California worms were also
significantly different from each other in size at metamorphosis (p 5 0.017).
Following metamorphosis of larvae in laboratory
cultures, individual worms were isolated and monitored for first appearance of gametes and time to
and size at sexual maturity. The mean time to sexual
maturity for female worms from each population is
presented in Fig. 4. The California females took significantly longer than females from the other two
populations to reach sexual maturity (po0.0001).
The same was true for California males relative to
males of the other two populations (Fig. 4). Based on
these same samples of worms, the sex ratio in each
population was estimated to be 1:1. The size of
worms at sexual maturity is presented in Table 3.
Male worms from California were significantly larger
Polydora cornuta: cryptic species
53
Fig. 4. Time to sexual maturity (d) as determined by first
appearance of gametes for males and females from each
population in the laboratory. For females and males, the
California (CA) population took significantly longer to
reach sexual maturity than the Florida (FML) and Maine
(ME) populations (po0.0001). The FL and ME populations
were not significantly different for males (p 5 0.31) or
females (p 5 0.46). Error bars are one standard deviation
around the mean, sample size above bars.
cantly shorter time (Florida, po0.01; Maine,
p 5 0.01) and at a significantly smaller size than
females (Florida, p 5 0.049; Maine, po0.001).
Fig. 3. Time to metamorphosis in days and size at
metamorphosis (in chaetigers) for each population raised
in the laboratory. A. Mean time to metamorphosis for
cultures of larvae from individual females raised from
release to metamorphosis. Time to metamorphosis
represents a minimum value because not all larvae settled
at the same time. In pairwise comparisons, only the FL and
ME values are significantly different (p 5 0.01). B. Mean
size at metamorphosis in chaetigers for each population
raised in the laboratory. In pairwise comparisons, the FL
population was significantly smaller (po0.0001) than CA
and ME. CA and ME were significantly different
(p 5 0.17). Error bars are one standard deviation around
the mean, sample size above bars. CA, California; FL,
Florida; ME, Maine.
than males of the other two populations at sexual
maturity while females were not significantly different between populations. Small sample sizes and
large SD may have prevented detection of significant
differences between some populations.
The mean time to sexual maturity (in days, Fig. 4)
and the size at sexual maturity (in chaetigers, Table 3)
were compared between males and females within
each population. California males and females were
not significantly different in either time to sexual
maturity (p 5 0.96) or size at sexual maturity
(p 5 0.78). In both the Florida and the Maine populations, males reached sexual maturity in a signifi-
Gamete distributions
The location of the first gamete-bearing segment
(first gametogenic chaetiger) was significantly different in worms from the Maine and California populations when compared with Florida worms (Table
4). On average, the location of the first gametogenic
chaetiger in both males and females from Florida was
more anterior than in the other two populations.
California and Maine populations were not significantly different from each other. It should be noted
that worms examined from the Maine population
were on average significantly larger than those from
the other two populations (Table 5). The mean sizes
of worms from the California and Florida populations were not significantly different (Table 5).
The effect of the total body size on the location of
the first gametogenic chaetiger has been questioned by
some workers (see ‘‘Discussion’’) but relevant data are
scarce. To address this issue, we conducted repeated
measurements on individual worms from the Florida
and Maine populations as they grew in laboratory
culture over a period of months. Our results indicate
that the location of the first gametogenic chaetiger is
quite stable for individual worms over time. Figure 5
shows the results by population and sex for groups of
8–12 worms examined at regular intervals for 6–81 d.
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Rice, Karl, & Rice
Table 3. Mean size of females and males (in total chaetigers) at sexual maturity for each population raised in the laboratory. ‘‘Between-population significance’’ column refers to comparisons between populations by sex (CA females vs.
FL females vs. ME females, and same for males). Samples of the same sex with the same-letter designation in ‘‘betweenpopulation significance’’ column are not significantly different (p 0.05). ‘‘Within-population significance’’ column
compares sexes within populations (CA females vs. CA males, etc.). Paired samples from the same population with the
same number designation are not significantly different (p 0.05). CA, California; FL, Florida; ME, Maine.
Population (size)
Size in chaetigers
Between-population significance
Within-population significance
CA females (7)
FL females (11)
ME females (8)
CA males (11)
FL males (9)
ME males (11)
48.8675.15
43.8278.45
44.6374.00
48.2773.55
37.0075.17
36.9173.59
a
a
a
b
c
c
1
2
4
1
3
5
The location of the first gametogenic chaetiger in both
sexes and both populations remained essentially constant over the entire period of the experiment. Growth
rates calculated as chaetigers per day for individual
worms were not significantly different between sexes
(Florida, p 5 0.247; Maine, p 5 0.865) and were combined for comparison between populations. Individual variation in growth rate was higher in Maine worms
(0.33670.247, mean7SD) than in Florida worms
(0.35870.15, mean7SD), and the two populations
were not significantly different (p 5 0.747). These
rates represent adult growth and do not include the
rapid addition of chaetigers that occurs in juveniles
following metamorphosis.
Gamete morphology
The diameter of unfertilized eggs has been used as
a distinguishing population (or species) characteristic
Table 4. Mean location of the first gamete-bearing chaetiger by population and sex. ‘‘Significance’’ column refers
to comparisons between populations within each sex (not
between males and females). Samples of the same sex that
share the same-letter designation in the ‘‘significance’’ column are not significantly different (p .05) based on Fisher’s protected least significant difference (Fisher PLSD)
method. CA, California; FL, Florida; ME, Maine.
Population
(sample size)
1st gametogenic
chaetiger
Significance
CA females (20)
FL females (35)
ME females (23)
14.2070.70
13.2071.02
14.2270.74
a
b
a
CA males (27)
FL males (53)
ME males (21)
13.6370.97
12.0670.60
13.7670.70
a
b
a
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vol. 127, no. 1, winter 2008
among spionid and other polychaetes. As with the
data above on location of the first gametogenic chaetiger, egg diameter alone may not be sufficient to distinguish between individuals but population means
can be informative. Figure 6A presents the mean unfertilized egg diameter for three populations of P.
cornuta raised in the laboratory. Because the unfertilized eggs of all populations are slightly elliptical in
shape, the measurements represent the longest dimension of each egg. The mean diameter of unfertilized eggs from Maine females was significantly larger
than the other two populations (p 5 0.0001). California and Florida egg diameters were not significantly
different (p 5 0.55).
Figure 6B presents the population mean size of
sperm heads from all three populations. The sperm
heads of worms from all three populations are elongate and cylindrical. Sperm head length was significantly different in all pairwise comparisons between
Table 5. Mean size in total chaetigers for males and females of Polydora cornuta used in gamete distribution analysis (Table 4). Significance was tested between populations
by sex (CA females vs. FL females vs. ME females, same
for males). Samples of the same sex that have the same-letter designation in the ‘‘significance’’ column are not significantly different (p 0.05). Pairwise significance based on
Fisher’s protected least significant difference (Fisher
PLSD) method. CA, California; FL, Florida; ME, Maine.
Population (sample size)
Size in chaetigers
Significance
CA females (20)
FL females (31)
ME females (23)
46.3074.66
43.68710.04
51.5778.65
a
a
b
CA males (27)
FL males (51)
ME males (21)
42.9375.68
41.8678.19
52.3877.77
a
a
b
Polydora cornuta: cryptic species
55
Fig. 5. Location of the first gametogenic chaetiger, last gametogenic chaetiger, and total chaetigers versus time for Florida
females (A, n 5 9), Florida males (B, n 5 8), Maine females (C, n 5 12), and Maine males (D, n 5 9). Error bars are one
standard deviation around the mean.
populations. California worms had the shortest
sperm heads while Maine sperm heads were the longest. Sperm tails were not measured due to the difficulty in determining accurate length when the sperm
are alive and the tails are moving.
Fecundity
A sample of 12 isolated females from each of the
Maine and Florida cultures was observed daily for
90 d. Each spawning was recorded and the number of
egg capsules deposited was recorded, along with the
number of eggs or larvae per capsule (based on a
sample of ten capsules per spawning). The frequency
of spawning was not significantly different between
populations but the number of egg capsules deposited and the number of eggs or larvae per capsule were
significantly different (Table 6). Florida females produced significantly more egg capsules but fewer eggs
per capsule compared with Maine females. Based on
the mean values presented in Table 6, a Maine female
could produce 31,716 eggs in 90 d while a Florida female could produce 26,204 eggs over the same time
period under laboratory conditions.
Discussion
The relationship between reproductive isolation
and morphological or genetic differentiation is complex and highly variable among taxonomic groups
(Avise 1994). This complexity is compounded when
populations display high degrees of polymorphism
for taxonomically important characteristics or when
taxa display pronounced morphological conservation over long periods of separation. In marine species, the discovery of morphological or genetic
differences between populations is rarely corroborated with experiments aimed at assessing levels of
Invertebrate Biology
vol. 127, no. 1, winter 2008
56
Rice, Karl, & Rice
Fig. 6. Mean unfertilized egg diameter and sperm head
length by population. A. Unfertilized egg diameter in
micrometers. The ME population is significantly different
(po0.0001) from both CA and FL. CA and FL are not
significantly different (p 5 0.55) based on pairwise
comparisons (Fisher PLSD). B. Sperm head length
(acrosome1nucleus1middlepiece) by population based
on mature live sperm removed from spermatophores. All
pairwise
comparisons
are
significantly
different
(po0.0001) based on Fisher PLSD. CA, California; FL,
Florida; ME, Maine; Fisher PLSD, Fisher’s protected least
significant difference method. Error bars are one standard
deviation around the mean, sample size above bars.
reproductive isolation (Palumbi 1994; Knowlton
2000). Traditionally, species have been defined and
described based on specific morphological differences
(large or small) between populations without regard
to genetics or reproductive biology (Wiley 1981).
More recently, the opposite practice of measuring
genetic differentiation without accompanying morphological or reproductive assessment has become
more common (e.g., Westheide & Hass-Cordes
2001).
Previous research aimed at characterizing closely
related polychaete species has encompassed various
combinations of: (a) measurement of adult morphology (Rice 1991; Blake 1996); (b) biogeography and
ecology (Blake & Kudenov 1978; Gamenick et al.
1998; Radashevsky 2005); (c) reproductive biology
(Blake 1969, 2006; Åkesson 1977; Rice 1981; Levin &
Creed 1984; Pfannenstiel & Grunig 1984; Pernet
1999); and (d) genetics (Rice & Simon 1980; Mustaquim 1988; Weinberg et al. 1990; Robotti et al.
1991; Gibson et al. 1999; Schmidt & Westheide
1999; Westheide & Hass-Cordes 2001). In terms of
documentation of speciation, reproductive incompatibility and significant genetic differentiation between populations strongly support separate species
status. Morphological differences and additional reproductive characteristics can then be used in a practical sense to distinguish between these species. One
of the best-documented polychaete sibling species
complexes is Capitella (Grassle & Grassle 1974,
1976; Grassle 1984; Grassle et al. 1987). At least ten
sibling species are thought to exist in a currently unresolved complex of closely related but clearly differentiated taxa. All show morphological similarity
although significant differences exist between some
species in adult morphology (Wu et al. 1991) as well
as reproductive compatibility (Gamenick et al. 1998),
life-history characteristics (Pearson & Pearson 1991;
Chia et al. 1996; Willcox & Nickell 1998), allozyme
allele frequencies (Wu et al. 1991), and gamete morphology (Eckelbarger & Grassle 1982, 1987). A comprehensive assessment of Capitella molecular genetics
is needed to sort out the phylogeny of this complex
and to assess the evolutionary history of species
differences. Many of the species in the genus Ophryotrocha show little morphological differentiation but
have been shown to be reproductively isolated in
laboratory cross-breeding experiments (Åkesson
Table 6. Fecundity of Florida (FL) and Maine (ME) females under laboratory conditions. Values represent means7one
standard deviation followed by sample size. Pairwise p-values based on Fisher’s protected least significant difference
(Fisher PLSD) method.
Population
FL
ME
Significance (p)
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vol. 127, no. 1, winter 2008
Days between spawning
(sample size)
# Egg capsules
(sample size)
Eggs/larvae per
capsule (sample size)
5.0671.703 (96)
4.7271.236 (176)
0.0592
34.9276.998 (95)
32.3178.283 (181)
0.0094
42.19714.396 (177)
57.48724.350 (297)
o0.0001
Polydora cornuta: cryptic species
1984). Recent molecular studies of 18 strains of Ophryotrocha are consistent with these reproductive barriers and
have helped to establish an objective phylogeny of the
complex and to shed some light on the evolution of reproductive modes (Dahlgren et al. 2001).
The Polydora cornuta complex of species is among
the most widespread taxa of polychaetes, with populations reported throughout most of the world’s
oceans, excluding the polar regions (see the review
by Radashevsky 2005). Variation in morphology and
reproduction within and between populations of P.
cornuta (as Polydora ligni before 1987) has been documented in numerous studies. Rasmussen (1973)
went as far as to propose synonomy for P. cornuta
and Polydora ciliata JOHNSTON 1838 from Denmark
based on variation and overlap in taxonomic characters. Rice & Simon (1980) assessed morphological
variation and reproductive characteristics for several
Florida populations of P. cornuta plus one population from California. They reported significant differences between populations in allozyme frequencies,
reproductive compatibility, and physiological tolerance to environmental variables. In further studies of
geographical variation among populations of P. cornuta, Rice (1991) reported no viable offspring in laboratory crosses between Florida (Tampa Bay) worms
and morphologically indistinguishable worms from
California (Orange County) and North Carolina. Interestingly, California and North Carolina worms
were interfertile in laboratory crosses, suggesting
that these geographically separated populations
may have shared a more recent common ancestor.
The present crossbreeding results support the biological species status of geographically separated
populations of P. cornuta (sensu lato) including
new samples from Maine and additional samples
from California. The reproductive crosses suggest a
biological species break between Florida and Maine,
between Florida and California, and between Maine
and California. These species breaks are further supported by the genetic data, suggesting that there are
at least three reproductively and genetically distinct
species of ‘‘P. cornuta’’ in North America. To our
knowledge, no other reports of crossbreeding experiments between geographically separated populations of the P. cornuta complex have been
published. The reproductive compatibility of populations of P. cornuta from Europe, New Zealand,
South America, and the Western Pacific with those
from North America remains unknown but might
shed some light on the origin and spread of this species complex.
The mechanism resulting in reproductive isolation
between the populations studied remains to be fully
57
explained. In the nereidid polychaete, Neanthes
acuminata (EHLERS 1868), changes in chromosome
number and morphology have been suggested to account for rapid divergence between laboratory and
field populations (Weinberg et al. 1990, 1992). Rice
(1980) suggested that a species-specific recognition
signal was carried on the exterior of spermatophores
that are covered with a mesh coat of microvilli derived from the male nephridium. Such a signal has yet
to be identified in Polydora; however, the ability to
discriminate chemical signals in the environment has
been demonstrated in Boccardia and Pseudopolydora
by Ferner & Jumars (1999) and in Dipolydora by
Lindsay et al. (2004). Further, olfactory cues have
been shown to be sufficient for population discrimination in N. acuminata (Sutton et al. 2005). Attempts
at in vitro fertilization in P. cornuta have not been
successful (S.A. Rice, unpubl. data), suggesting that
specific physiological conditions are necessary for
fertilization to occur or that sperm must undergo
capacitation within storage organs before fertilization. Likewise, there is no simple relationship between reproductive compatibility and genetic
differentiation. As noted below, California and
Maine individuals were more closely related than
Florida and California individuals by a factor of
about five based on comparative genetic distances
(Table 2). Nonetheless, where California and Maine
individuals were completely reproductively incompatible, crosses between Florida and California individuals produced zygotes (albeit non-viable ones).
There clearly are different degrees of reproductive
incompatibility that have not been fully explored. It
would be particularly instructive to perform breeding
trials between the closely related New Zealand and
California individuals and to determine the genetic
relatedness of California and North Carolina individuals that have previously been shown to be reproductively compatible (Rice 1991).
Previous genetic studies of closely related Polydora
species have mostly involved comparative allozyme
frequencies. Rice & Simon (1980) reported one genetically distinct population of P. cornuta among
four Florida populations tested. Mustaquim (1988)
compared populations of P. ciliata from nine locations in England and Wales for allozyme frequencies
and found them to be significantly different between
the shell-boring form and non-boring form. Polydora
cornuta (as P. ligni) was similar in allele frequencies
to the non-boring form of P. ciliata but significantly
different from the boring form. Manchenko &
Radashevsky (1993) reported a similar study on Polydora cf. ciliata and Polydora limicola ANNENKOVA
1934 from Japan using allozyme frequencies and
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58
found significant differences between these two taxa,
concluding that P. cf. ciliata and P. limicola were
distinct species in Japan.
The results of comparisons of gene sequences
among reproductively isolated populations of P. cornuta are reported here for the first time. Samples from
the Maine population are more similar to the California population than with Florida worms (Table 2
and Fig. 1) even though Maine and California are the
most geographically distant North American populations sampled in this study. Additional samples
from the mid-Atlantic coast, especially Charleston,
SC, the type locality for P. cornuta, and the northwestern Gulf of Mexico might help clarify this pattern. The New Zealand worms were very similar to
the California worms in mtDNA sequence. This
might be a consequence of a species introduction.
Other spionid polychaetes such as Boccardia proboscidea HARTMAN 1940, thought to be native to California, have become established in Australia (Blake
& Kudenov 1978). Even though P. cornuta from California and New Zealand are closely related, seven of
the 19 variable nucleotide sites found between California and New Zealand worms are geographic location specific, indicating that these two populations
can be identified genetically. The genetic similarity
between California and New Zealand worms suggests that mitochondrial haplotypes may be useful
in tracking the origins of introduced populations
(Carlton & Geller 1993). Even though we do not
have the specific details of past introductions, as
more worldwide populations are characterized genetically, a pattern of spread of P. cornuta will likely
emerge from sequence comparisons. Of particular interest would be the recently described populations
from Taiwan (Radashevsky & Hsieh 2000) and the
eastern Mediterranean (Cinar et al. 2005). It is clear
that the three North American populations of P. cornuta reported on here are reproductively isolated and
genetically distinct, suggesting that they should be
conferred separate species status. The basis of a practical method for distinguishing between these separate species remains problematic.
Mustaquim (1986) used SEM to assess morphological variation in three species of Polydora (P. ciliata, P. cornuta [as P. ligni], and P. limicola) from
British coastal waters and reported significant variation in most traditional characters within and between species. In a similar study, Rice (1991) reported
considerable variation in most taxonomically important morphological characteristics within three populations of P. cornuta. Based on population means,
the Florida worms differed from California and
North Carolina worms in the length of the caruncle,
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Rice, Karl, & Rice
the percent of individuals with two or more lateral
teeth on fifth chaetiger major spines, the presence of
capillary chaetae on the modified fifth chaetiger, and
the presence of capillary neurochaetae posterior to
chaetiger six.
The presence of capillary chaetae on the fifth chaetiger either above (dorsal superior) or below (ventral
inferior) the major spines in Florida specimens is reported again in this study. Blake & Kudenov (1978)
reported a ventral fascicle of unilimbate capillary
chaetae on the fifth chaetiger of all Australian specimens (as P. ligni), and Kudenov (1982) suggested
that the Australian population may represent an undescribed species. Radashevsky (2005) reported that
dorsal and ventral capillary chaetae on the fifth chaetiger were always absent in adult specimens that he
examined. These dorsal and ventral capillary chaetae
on the fifth chaetiger remain one of the few morphological features that distinguish the Florida populations from most others. Even though an individual
Florida specimen may not have these chaetae, they
are present in 75% of Florida specimens as reported
by Rice (1991). The long-standing diagnostic chaetal
character in P. cornuta, the brush-tip companion chaeta on the fifth chaetiger, is reported here to be a wear
pattern rather than a distinct chaetal type (Fig. 2).
Even recent morphological reviews (Radashevsky
2005) have continued to refer to these companion
chaetae as ‘‘unique’’ to P. cornuta even though the
wear pattern is clearly visible in the published micrographs. These worn companion chaetae should no
longer be considered diagnostic for P. cornuta.
Radashevsky (2005) extended the known range of
P. cornuta to include several locations in the western
Pacific, South America, and the Gulf Coast of Mexico. Based on samples collected by Radashevsky
along with museum samples from many locations
(unfortunately, none from Florida or the Northern
Gulf of Mexico), he concluded that P. cornuta constitutes a single species worldwide. This conclusion is
not supported by the present results where we have
reported essentially complete reproductive isolation
(in terms of viable offspring) and significant genetic
differentiation between widely separated populations
within North America. Further, the lack of consistent morphological differences between individuals
from distant populations should not be taken to indicate same species status especially in light of reproductive isolation and genetic differentiation.
Radashevsky (2005) described reproduction and larval development based on samples of P. cornuta collected in Brazil. These Brazilian worms differed from
North American populations in some reproductive
characteristics. For example, the minimum size of
Polydora cornuta: cryptic species
Brazilian larvae at metamorphosis was 18 chaetigers
while the mean size of Florida larvae at metamorphosis was about 1471.5 chaetigers and the smallest
was 11 chaetigers. Radashevsky (2005) reported the
smallest Brazilian males to have 22 chaetigers while
females had 30 chaetigers at sexual maturity. Florida
worms in the present study had a minimum size for
males at 26 chaetigers and females at 27 chaetigers.
Culture conditions may have been different for the
Brazilian worms compared with the Florida worms.
However, even under identical culture conditions, the
Florida population displayed a significantly shorter
mean time to metamorphosis than the Maine population. Cross-breeding and genetic analyses of the
Brazilian worms were not reported but are needed
to determine the relationships among Brazilian and
North American populations.
The differences between North American populations in size at sexual maturity and time to sexual
maturity after metamorphosis under identical culture
conditions were unexpected. The Maine (coldest) and
Florida (warmest) populations reached sexual maturity in both males and females in a significantly shorter time than the California (intermediate)
population. Likewise, size at sexual maturity was
smaller for Maine and Florida males than for California males (females were not significantly different). Anger et al. (1986) conducted extensive
development and growth studies in the laboratory
with specimens of P. cornuta (as P. ligni), P. ciliata,
and Pygospio elegans CLAPAREDE 1870 collected from
Helgoland (North Sea). They reported that the developmental rate in P. elegans (a cold-adapted species) was faster than either species of Polydora at low
temperatures (61–81C) but slower at higher temperatures (181C). At intermediate temperatures (121C), all
three species had similar growth rates. Interestingly,
the reported growth curves for P. cornuta and P. ciliata diverged at low temperatures (61–81C), perhaps
indicating inherent species-level adaptations in these
closely related taxa. A more complete analysis of
North American population growth rates and attainment of sexual maturity might include experiments
on multiple populations cultured simultaneously
over a range of temperatures.
Reproductive morphology, including gamete distributions within adult worms as well as gamete
structure and size, have been used to distinguish between closely related species (Eckelbarger & Grassle
1987; Rice & Levin 1998; Schulze et al. 2000). The
mean location of the first gamete-bearing chaetiger is
significantly different among North American populations of P. cornuta. Rice (1991) reported significantly different gamete distributions for both male
59
and female worms from Florida when compared with
worms from California and North Carolina. In the
present report, Florida male and female worms had
significantly different gamete distributions than California and Maine worms. In both reports, the Florida worms had gametes in more anterior chaetigers
than the other populations. This particular characteristic has been criticized on the assumption that the
most anterior gamete-bearing chaetiger changes as
individual worms grow. Our data suggest that this is
not the case, because the location of the first gametebearing chaetiger did not change in individual worms
over a growth period of 60–80 d in the laboratory
(Fig. 5). The mean location of the first gamete-bearing chaetiger thus remains a diagnostic character for
Florida P. cornuta, even in worms of different size.
Sperm and egg sizes have been reported to vary
among populations of P. cornuta (Rice & Simon
1980). In the present study, the mean unfertilized
egg diameter was significantly larger in the Maine
population than in Florida and California samples
while the Florida population was significantly different from both California and Maine samples in mean
sperm head length. Because all the worms in the current study were raised under identical temperature
and nutritional conditions, these differences in gamete morphology are likely not due to nutritional
state. Blake (1969) reported the egg diameter for
Maine P. cornuta (as P. ligni) to be 120 mm, somewhat larger than the mean value of 105 mm reported
here for specimens collected from the same area 34
years later. Rice & Simon (1980) also reported similar
egg diameters between Florida and California specimens raised in the laboratory but reported some
Florida populations with significantly smaller mean
egg diameters. When sample sizes are large enough
and nutritional conditions can be accounted for,
mean unfertilized egg diameter can be a useful character for distinguishing between species and is likely
under genetic control (Levin et al. 1991).
Sperm morphology in spionid polychaetes has
been reviewed by Blake & Arnofsky (1999) with a
summary of differences between species, while sperm
ultrastructure in P. cornuta has been described by
Rice (1981). In comparing sperm morphology, especially sperm head length, care must be taken to ensure that the sperm being measured are mature and
that they have not changed dimensions due to preservation or observational conditions. In the present
study, we report sperm dimensions for only live mature sperm freshly removed from spermatophores.
The source and state of sperm are often not reported
(especially in reviews), leading to variations that may
be spurious. We found the mean sperm head length
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vol. 127, no. 1, winter 2008
60
(acrosome1nucleus1middlepiece) to be significantly
different between all three populations examined.
Like unfertilized egg diameter, the mean sperm
head length may be a useful character for distinguishing between species.
Polydora cornuta has been characterized as an ‘‘opportunistic’’ species (Grassle & Grassle 1976), meaning that populations can be quickly established in
benthic habitats that are disturbed, polluted, or otherwise low in species diversity. This type of life-history
strategy is well adapted for natural and anthropogenic spread of populations as has apparently
repeatedly happened (Cinar et al. 2005). Few laboratory reports, however, are available documenting the
potential fecundity of P. cornuta or its variant populations. Gudmundsson (1985) summarized life-history patterns in five populations of spionid polychaetes
including P. ciliata (but not P. cornuta) and found
high variability between species in maximum number
of offspring per reproductive effort. In P. ciliata, the
maximum offspring per brood was reported to be
2200 with a spawning period of February to June in
North East England. In the present study, Florida
individuals of P. cornuta produced significantly more
egg capsules per brood than Maine worms but Maine
worms produced significantly more eggs per capsule.
Using maximum values for number of capsules per
brood and number of eggs per capsule, Maine females
could produce a maximum of 3318 eggs per reproductive effort while Florida females could produce
2372 eggs per brood under identical conditions of
temperature and nutritional state. With a spawning
period in Florida spanning most of the calendar year,
an introduction of Maine individuals of P. cornuta
into Tampa Bay could potentially result in displacement of the endemic population.
The historical zoogeography of populations of P.
cornuta in North America is unknown. Some have
suggested that P. cornuta, along with other polychaete species, were introduced into California along
with transplanted oysters from the Chesapeake Bay
region in the early 1900s (Carlton 1989; Cohen &
Carlton 1995). Previous data (Rice 1991) showing
reproductive compatibility between California and
North Carolina populations support this scenario.
Others have suggested that P. cornuta originated in
the Pacific Ocean and spread from there to its current
distribution (J.A. Blake pers. comm.). This scenario
is based on the occurrence of P. cornuta’s closest
morphological congeners (P. ciliata, Polydora cirrosa RIOJA 1943 and Polydora nuchalis WOODWICK
1953) in the Pacific and the fact that far more species of polydorids are found in the Pacific compared
with the Atlantic. A widespread sampling of P. cor-
Invertebrate Biology
vol. 127, no. 1, winter 2008
Rice, Karl, & Rice
nuta populations in the Pacific, followed by cross
breeding and genetic analyses might help to settle
this issue.
For practical purposes, it remains difficult to distinguish the species in the P. cornuta complex. Traditional morphological characteristics fail to
differentiate between individuals but population statistics and reproductive morphology can be used to
identify samples of individuals from a single location.
The mean location of the first gametogenic chaetiger
in both males and females has proven to be a consistent character distinguishing Florida populations
from Atlantic coast and Pacific coast populations.
This characteristic is independent of worm size as
demonstrated first by Rice (1991) and in the present
study. The mean length of the mature sperm head is
significantly different in Florida populations when
compared with the other populations studied. The
remaining reproductive characters reported in this
study show a mixed assemblage of relationships, but
taken together, can help to differentiate between species. The most reliable method for identifying individuals of P. cornuta would be to attempt laboratory
crosses of the specimen with individuals from known
populations. This method requires live material and
substantial effort but would not be unprecedented as
this is how many species of Ophryotrocha are identified (Åkesson 1978, 1984). Alternatively, identification of individual worms could be accomplished
through mtDNA COI gene sequences. This method
can be used with live or ethanol-preserved specimens
but is time consuming and costly. Nevertheless, COI
sequences and 18S rDNA sequences have previously
been shown to be effective tools for the identification
of individual invertebrates (Hare et al. 2000; Larsen
et al. 2005).
Regardless of the obstacles to identification, it is
incumbent upon researchers to use whatever means
available to investigate and characterize cryptic species complexes whenever they are encountered. Not
only is this necessary for a comprehensive assessment
of diversity within a taxonomic group, but studies of
cryptic species offer excellent opportunities for exploration of the mechanisms and rates of speciation
because these complexes are often in the early stages
of the speciation process.
Acknowledgments. This work was supported in part by
a grant from the National Science Foundation (DEB
0317890) to K.A.R. and S.A.R. and by a Delo Grant
from the University of Tampa to S.A.R. We thank Bruno
Pernet, Ian G. Paterson, and Sara Lindsey for collecting
and shipping samples from distant locations and Cecilia
Puchulutegui for laboratory work. We also thank James
Polydora cornuta: cryptic species
A. Blake, and Sara Lindsey for thoughtful reviews of an
earlier version of this manuscript.
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