Marine Biology (2001) 138: 113±123 Ó Springer-Verlag 2001

G. Hendler á L. U. Tran

Reproductive biology of a deep-sea brittle star Amphiura carchara

(Echinodermata: Ophiuroidea)

Received: 14 February 2000 / Accepted: 3 July 2000

Abstract New information on the brooding reproduc-

tion of Amphiura carchara, and previous studies of sev-
Introduction
eral deep-sea congeners, contradict the prevailing notion
Ophiuroids contribute a signi®cant portion of biomass
that direct development is exceptional among deep-sea
in many marine environments, and in the deep sea can
echinoderms. Over 500 specimens of A. carchara from
far outnumber other components of the benthic me-
2,850 to 4,100-m depths o€ the coast of California,
gafauna and macrofauna (Sanders and Hessler 1969;
USA, were studied. The species was found to be go-
Gage and Tyler 1991). They constitute the most diverse
nochoric, although most brooding species of ophiuroids
class of living echinoderms; approximately 2,000 species
are hermaphroditic. Females each brooded up to 72
are recognized (Hendler et al. 1995). It has been sug-
embryos at a time, with up to 10 in a single bursa. The
gested that there are relatively few species of ophiuroids
embryos of individual adults were often at di€erent
in deep-sea habitats (Sanders and Hessler 1969). How-
stages of development, but those brooded in a single
ever, the conclusion was based on a restricted sampling
bursa tended to be at the same stage. Thus, the species
program in a region with relatively low ophiuroid
has characteristics that breach the distinctions between
diversity. Worldwide, a large proportion of ophiuroid
sequential and simultaneous brooding exhibited by
species are bathyal and abyssal forms. We estimate that
shallow-water ophiuroids. The embryos developed from
37% are shallow-water species restricted to depths less
yolky eggs that are large for an ophiuroid, with the mean
than 100 m, 39% occur between 100 and 1,000 m, and
largest oocytes 0.45 mm, and ranging to 1.28 mm in
24% live at depths exceeding 1,000 m (based on bathy-
diameter. Almost all late-stage embryos were positioned
metric records for 1,512 species in key monographic
with their mouth and arms pressed against the wall of
references: Clark 1915; Mortensen 1936; Clark and
the bursa, possibly to facilitate the uptake of nutrients
Rowe 1971; Clark and Courtman-Stock 1976; Paterson
from the parent. Thus, A. carchara may be matro-
1985; Maluf 1988; Hendler et al. 1995).
trophic. The largest embryos examined had a rudimen-
Despite their prevalence, abundance, and diversity,
tary disk skeleton, and arms with four joints and a ter-
the mode of larval development has been documented
minal plate. Emerging juveniles probably di€er in disk
for only 4% of all ophiuroid species, and documentation
diameter. The species appears to brood year round, al-
of development in the many deep-sea species is partic-
though di€erences in gonad size, the incidence of
ularly scanty (Hendler 1975; Gage and Tyler 1991). In
brooding, and the relative numbers of early develop-
most cases, the mode of reproduction of deep-sea
mental stages in summer and winter samples indicated
ophiuroids has been inferred from the approximate re-
that there are seasonal trends in reproduction.
lationship between egg size and type of development
(Schoener 1972; Hendler 1975, 1991; Gage and Tyler
1991). Generally, ophiuroids with small eggs have
Communicated by M. H. Horn, Fullerton planktotrophic larvae, those with moderate-sized eggs
have lecithotrophic larvae, and those with large eggs
G. Hendler (&) á L. U. Tran
Natural History Museum of Los Angeles County, brood embryos and release crawl-away young. Unfor-
900 Exposition Boulevard, Los Angeles, tunately, the sizes of eggs in the three developmental
California 90007, USA groupings constitute a continuum that confounds accu-
Fax: +1-213-7462999 rate predictions of reproductive mode solely based on
e-mail: hendler@nhm.org egg diameter (Hendler 1991; Sewell and Young 1997).
114
The reproductive behavior of deep-sea ophiuroids
has not been observed in situ. Although the planktonic
ophioplutei of a few eurybathic polar species and deep-
sea species have been described, they have not been
reared to metamorphosis in the laboratory (Thorson
1934, 1946; Ursin 1960; Tyler and Gage 1982; Tyler and
Fenaux 1994). Even so it is claimed, based on their
``probable'' developmental modes, that most deep-sea
ophiuroid species release pelagic larvae (Pearse 1994;
Pearse and Bosch 1994).
Therefore, we used a rare opportunity to investigate
the reproductive biology of a deep-sea ophiuroid that
was a€orded by recently collected specimens of Amphi-
ura carchara H.L. Clark. The species is known from
Baja California, Mexico, to Alaska, across the Bering
Sea, and from Sagami Bay and Suruga Gulf, Japan
(Clark 1911, 1913; Djakonov 1954; Baranova 1957;
Fujita et al. 1997). It has been found at bathyal-abyssal
depths and previously reported from 1,123 to 3,608 m,
except at the northern limit of its range in Alaska, where
it may occur at depths as shallow as 110 m (Clark 1911;
Madsen 1951). In northern California, A. carchara is
one of the most abundant megafaunal species between
2,000 and 3,000 m depth (Nybakken et al. 1998). The
reason for its restricted bathymetric distribution is not
known, and its biology has not previously been exam-
ined (Summers and Nybakken 2000).
In this study we recognized that A. carchara is a
direct-developing species because we found embryos in
the bursae (brood sacs) of adult individuals. With adult
specimens of A. carchara at our disposal from several
large collections, we studied its reproductive system,
gametes, embryos, and body size to describe its mode of
reproduction in more detail than has been possible for
most deep-sea ophiuroids (references in Gage and Tyler
1991). We analyzed data on the incidence of brooding,
and the number and development of embryos and
gonads in individuals from several samples, to appraise
potential di€erences in reproductive biology within and
between populations. We also made observations on
embryonic ontogenesis, incidence of embryos of di€er-
ent developmental stages, and appearance and position
of embryos in brooders to shed light on the maturity and
size of emergent juveniles, transfer of nutrient from
parent to embryos, and reproductive periodicity.
Materials and methods
Source of specimens
Specimens of A. carchara were collected from an abyssal site west
of Point Conception, California, by Scripps Institution of Ocean-
ography (see Lauerman et al. 1996 and references therein). Addi-
tional material was collected by the Moss Landing Marine
Laboratory from the continental slope o€ the Farallon Islands,
west-southwest of San Francisco, California (see Nybakken et al.
1998; Summers and Nybakken 2000; and references in both).
Ophiuroids separated from trawl catch were preserved in 10%
bu€ered formalin and transferred to 70% ethanol. They were
deposited in the echinoderm collection of the Natural History
Museum of Los Angeles County (LACM). In the results below,
each of the three collections studied (see Table 1) is referred to by
the month it was collected.
Treatment of specimens
Specimens were sexed by opening the dorsal body wall to expose
the gonads for microscopic examination. Severely damaged indi-
viduals, and those parasitized by ascothoracicans and myzostomes,
were set aside. The oral frame diameter (OFD) of the other spec-
imens was measured using a Wild M-5 stereomicroscope equipped
with an ocular micrometer. Measurements were precise to 0.1 mm.
The OFD was used as an index of body size, instead of disk di-
ameter (dd), because the disks of many specimens were deformed or
damaged during sampling and preservation. OFD equals the dis-
tance from the distal edge of the madreporite across the mouth to
the proximal edge of the ®rst ventral arm plate. The regression of
OFD on the dd of intact specimens was expressed in the equation
OFD ˆ 0.89 + 0.304(dd) (n ˆ 84, r2 ˆ 0.608).
Twenty-seven individuals from the June sample were inspected
histologically to evaluate whether their sex had been determined
accurately by visual inspection. Their disks were decalci®ed in
Bouin's ®xative and embedded in paran, and sections were cut at
5 lm thickness and stained with hematoxylin and eosin.
All the individuals in the June sample were dissected (Table 2).
For each one, the numbers of gonads were counted, the length of
each gonad was recorded, and the largest oocyte in each ovary was
measured. The arrangement of the gonads and bursae in each
brooding female was drawn using a camera lucida, and the num-
bers of embryos per bursa were counted. The stage of development
of every advanced embryo was assessed in terms of the number of
its arm joints and terminal arm plate (e.g. in stage 3 + t embryos,
the arms bear a terminal plate and 3 joints), and its disk diameter.
The greatest length and width of less developed, stage 0 embryos,
which lack disk and arm rudiments, were measured along and
perpendicular to the body axis.
The size and sex of all the specimens in the July and February
samples were ascertained. The specimens were then sorted by eye
into subjectively determined groups of large, medium, and small
individuals. Ten specimens were selected arbitrarily from each
group and dissected (Table 2). The number and length of their
gonads were determined, and the stage and size of embryos in the
brooding females were recorded. The position of embryos was
examined in the June, July and February samples. Counts were
made of the number of embryos in each dissected female. It was
noted whether the mouth of each embryo touched the bursal wall,
or faced inward toward the bursal lumen.
Preservation can a€ect the dimensions of soft tissue structures
that were measured, such as gonads and gametes. Some published
descriptions of ophiuroid oocyte sizes have been based on fresh
tissue and others, particularly involving deep-sea species, on pre-
served material (see references in Hendler 1991). In a population of
the ophiuroid Amphioplus abditus (Verrill), the mean diameters of
freshly spawned oocytes and preserved oocytes were similar
(Hendler 1973). The eggs of 3 of 13 echinoid species studied ap-
preciably expanded or contracted during preservation, but it was
deemed unlikely that such variation biased intraspeci®c temporal
and spatial comparisons of egg size (Lessios 1987).
Table 1 Station data for three collections of Amphiura carchara
used in the present study
Site Location Depth (m) Date of
collection
Pt. Conception 34°49¢N, 123°07¢W 4,100 25 Jun 1989
Farallon Is. 37°37.50¢N, 2,900±3,000 30 Jul 1991
123°29.98¢W
Farallon Is. 37°39.47¢N, 2,850±2,990 27 Feb 1992
123°30.19¢W

Table 2 Sexual characteristics of Amphiura carchara individuals from the three collections used in the present study: sex ratio, incidence of
brooding, and fecundity. M Male, B brooding female, NB non-brooding female
Date and locality Sex ratio Number, sex, and
of collection (male:female) of brooding condition
specimens examined of dissected specimens females examined)
25 Jun 1989 Pt. Conception 0.61:1 (n = 77) 25M 23B 17NB
30 Jul 1991 Farallon Is. 0.37:1 (n = 210) 6M 11B 12NB
27 Feb 1992 Farallon Is. 0.77:1 (n = 239) 15M 14B 1NB
Specimens of A. carchara in the collection of the National
Museum of Natural History (USNM) were studied by examining
their open bursal slits, without dissection, for evidence of embryos.
The estimated incidence of brooding based on USNM specimens is
conservative, because in many cases the contents of the bursae were
not visible.
Statistical analysis
The signi®cance of the di€erences between samples was tested with
a single-factor ANOVA (Zar 1984), followed by a Games and
Howell multiple comparison of means (StatView 1998). Normality
and the homogeneity of variances of samples were examined using
Kolmogorov-Smirnov and Bartlett's tests, respectively (Zar 1984).
Data were treated using logarithmic or square-root transformation,
as appropriate, if the assumptions of normality and homogeneity of
variances were not met, and ANOVA tests were carried out when
normality and equal variances resulted. Non-transformed data are
presented in the text. In cases where data transformation did not
correct for heteroscedasticity, a non-parametric Mann±Whitney
test or Kruskal-Wallis one-way ANOVA by ranks was used to
evaluate di€erences among samples, followed by a non-parametric
analogue to the Tukey's multiple comparison test appropriate for
unequal sample sizes (Zar 1984). Deviations from expected fre-
quencies in the sex ratio and in the orientation of embryos in the
bursae were tested by calculating chi-square goodness of ®t (Zar
1984). A two-tailed Student's t test was used to test for a linear
relationship between the dependent and independent variables in
simple linear regressions, and to test the hypothesis that the pop-
ulation correlation coecient equals zero. The signi®cance level
used in all tests was P ˆ 0.05.
Results
Geographic and bathymetric distribution
Previous records of the species in the eastern Paci®c were
limited to Alaska, Washington and Mexico (Clark 1911,
1913). The specimens used in the present study included
the ®rst A. carchara studied from California waters, and
those collected o€ Pt. Conception, California, at
4,100 m, represented a new depth record for the species
(Table 1).
Body size and sex
The individuals studied ranged from 2.0 to 4.6 mm OFD
(=4.3 to 9.6 mm dd), which is somewhat larger than the
maximum size (8 mm dd) previously recorded for the
species (Clark 1911). With the exception of a single
teratological hermaphroditic individual, the 526 speci-
115
Percentage of females brooding Mean number
embryos (n = number of of embryos per
brooding female
58% (n = 40) 8.1 (SD = 8.5, n = 23)
52% (n = 153) 15.9 (SD = 15.1, n = 11)
74% (n = 135) 22.1 (SD = 19.4, n = 14)
mens examined were identi®able as male or female on
the basis of gonadal morphology (Table 2, Fig. 1A, C).
The number of females exceeded that of the males in the
three samples, and for February and July samples the
hypothesis that the sex ratio was 1:1 could be rejected
(chi-square test, P < 0.05, df ˆ 1: June v2 ˆ 1.877, July
v2 ˆ 43.886, February v2 ˆ 4.021). The hermaphrodite
was from the June sample. It was 9.1 mm dd, had sep-
arate ovaries with yolky oocytes and testes with sper-
matozoa, and was brooding one embryo.
The slight di€erence between the mean OFD of males
(x ˆ 3:03 mm, SD ˆ 0.64, n ˆ 46) and females
(x ˆ 3:10 mm, SD ˆ 0.36, n ˆ 78) for the three samples
combined, was not statistically signi®cant (Mann±
Whitney, Zc ˆ )1.291, P ˆ 0.1969), and the size-fre-
quency distributions of the males and females were alike
(Fig. 2). Similarly, the OFD of the brooding females
(x ˆ 3:08 mm, SD ˆ 0.36, n ˆ 48) and the non-brood-
ing females (x ˆ 3:12 mm, SD ˆ 0.36, n ˆ 30) were not
signi®cantly di€erent (ANOVA, F ˆ 0.208, df ˆ 1,76,
P ˆ 0.6495), and their size-frequency distributions co-
incided (Fig. 2). The OFD of the females did not di€er
signi®cantly among the three samples (ANOVA,
F ˆ 1.515, df ˆ 2,75, P ˆ 0.2265), although there were
signi®cant di€erences in the body sizes of the males
(Kruskal-Wallis test, Hc ˆ 18.368, P < 0.0001) with the
OFD values for June > February > July (Tukey type
test, P ˆ 0.05).
Characteristics of the gonads, gametes, and bursae
The testes and ovaries were ``sausage-shaped'' gonadal
tubules (Fig. 1A, C). They were not branched although
the distal tips of some testes were slightly bifurcated.
There were typically one or more gonads attached to the
genital plate, which lay atop the portion of the arm
enclosed within the disk, and one or more gonads at-
tached to the genital scales, which lay within the inter-
radius. Each of the ten bursae within the disk opened
into a bursal slit at the base of an arm (Fig. 1A, C).
Some bursae swelled dorsally and covered the gonads,
and expanded into the disk interradius, depending on
the number and size of the embryos that they contained
(Fig. 1F).
In preserved specimens the testes were uniformly
whitish (Fig. 1A, B). The ovaries contained large, yolky,
pale yellow or orange-brown oocytes and smaller, more
116
translucent oocytes (Fig. 1C±E). In histological prepa-
rations the oocytes were generally eosinophilic; very few
of the smallest were basophilic. They were typically as-
sociated with small follicle or somatic cells, and eosin-
ophilic haemal ¯uid ®lled the gaps between the oocytes.
Larger oocytes had a meshlike vesicular ooplasm, and
smaller oocytes had a denser ooplasm. The intensity of
stain in the sectioned oocytes was not related to the size
of the cells. The females in all three samples had ovaries
containing oocytes of mixed sizes. The mean maximum
diameter, based on the largest oocyte measured in every
ovary, was 0.45 mm (SD ˆ 0.15, n ˆ 1,744). Fourteen
percent of the largest oocytes were 0.5 mm or more in
diameter, and the largest measured was 1.28 mm.
The numbers of ovaries in the brooding (x ˆ 22:7/
ind, SD ˆ 7.9, n ˆ 48) and the non-brooding females

b were each based on the combined data for the three
Fig. 1A±G Amphiura carchara. A, C, E, F show dissected individuals
with the wall of the disk and the stomach removed. A Disk of male,
dorsal view; TE testis. B Histological section of testes; BU bursa, SL
spermatogenic layer, SP spermatozoa in lumen of testis. C Disk of
non-brooding female, dorsal view; BU bursa, OV ovary. D
Histological section of ovary; SC somatic cell, HF haemal ¯uid, LO
large yolky oocyte with meshlike vesicular ooplasm, NU nucleus, SO
smaller yolky oocyte with dense ooplasm. E Ovaries within dissected
disk, containing a mixture of opaque (arrowhead) and translucent
(double arrowhead) oocytes. F Disk of brooding female, dorsal view,
showing bursae packed with late-stage embryos. Mouths of embryos
(arrows) are pressed against walls of bursae. G Embryos removed
from bursae; 1 stage 0, 2 stage t, 3 stage t + 1, 4 stage t + 4. Scale
bars: A, C, F ˆ 2 mm; B, D ˆ 100 lm; E ˆ 1 mm; G ˆ 0.5 mm
(x ˆ 21:9/ind, SD ˆ 14.1, n ˆ 30) were not signi®cantly
di€erent (ANOVA, F ˆ 0.082, df ˆ 1,76, P ˆ 0.7757),
although the ovaries of brooders (x ˆ 0:80 mm,
SD ˆ 0.29, n ˆ 1,086) were signi®cantly longer than
those of non-brooders (x ˆ 0:71 mm, SD ˆ 0.33,
n ˆ 658) (Mann±Whitney test, Zc ˆ )7.021, P <
0.0001). The mean length of the ovaries (x ˆ 0:77 mm,
SD ˆ 0.30, n ˆ 1,744) exceeded that of the testes
(x ˆ 0:49 mm, SD ˆ 0.20, n ˆ 1,585) (Mann±Whitney
test, Zc ˆ )27.918, P < 0.0001). The trends in gonad
numbers and length shown in the preceding tests, which
Fig. 2 Amphiura carchara. Size-frequency distributions. Above Males
(n ˆ 46) and females (n ˆ 78) in the combined June 1989 (Pt.
Conception), July 1991 and February 1992 (Farallon Islands) samples.
Below Females brooding embryos (n ˆ 48) and those lacking embryos
(n ˆ 30) in the three samples
117
samples, match the trends in separate statistical tests for
the February, June, and July samples. The gonadal tu-
bules in males were more numerous than in females in
the February (ANOVA, F ˆ 5.557, df ˆ 1,28, P ˆ
0.0256) and June samples (ANOVA, F ˆ 43.240,
df ˆ 1,63, P<0.0001); however, the di€erence in gonad
number between the sexes in the July sample was not
statistically signi®cant (ANOVA, F ˆ 0.097, df ˆ 1,27,
P ˆ 0.7574) (see descriptive statistics of gonad length in
Table 3).
There was not a signi®cant linear relationship be-
tween body size (OFD) and the number of gonads in
females or males, as determined using the combined data
from the three samples (Student's t-test: females,
t ˆ 0.670, P ˆ 0.5048; males, t ˆ 0.535, P ˆ 0.5955).
The gonads, however, were signi®cantly larger with in-
creasing body size in females (Student's t-test, t ˆ 9.866,
P ˆ 0.0001) and males (Student's t-test, t ˆ 11.538,
P ˆ 0.0001). These trends were consistent with an in-
crease in gonad length but not in gonad number as in-
dividuals grow.
The mean numbers and mean lengths of the gonads
di€ered signi®cantly among individuals in the three
samples (Table 3). The ovaries and testes were signi®-
cantly longer in February than in July samples, and
longer in June than in July samples. However, the
greater testis length in June and February in comparison
to July may have been a function of the signi®cantly
smaller body size of the individuals in the July sample.
The ovaries were signi®cantly longer and individuals had
more testes and ovaries in February than in June. There
were more testes per individual in February than in July,
and more ovaries per individual in July than in June
samples.
Brooding: incidence and ¯uctuations
The percentage of females that were brooding embryos
varied among the summer and winter samples (Table 2).
There were embryos in 1±10 bursae (x ˆ 5:1, SD ˆ 3.2,
n ˆ 48) within each brooding individual. The number of
bursae occupied by embryos in each brooder di€ered
signi®cantly among samples (ANOVA, F ˆ 5.770, df ˆ
2,45, P ˆ 0.0059). There was a signi®cantly lower
number of gravid bursae in June than in February
(Games and Howell test, P ˆ 0.05).
The numbers of embryos per bursa ranged from 1 to
10 (x ˆ 2:6, SD ˆ 1.9, n ˆ 241), and the number of
embryos per brooding female ranged from 1 to 72
(x ˆ 14:0, SD ˆ 15.0, n ˆ 48). There was not a signi®-
cant linear relationship between the size of the brooding
females and the number of embryos that they brooded,
considering the data combined from all three samples
(Student's t-test, t ˆ 1.297, P ˆ 0.2010). There were,
however, signi®cant di€erences in the numbers of em-
bryos per brooder among the three samples (see Table 2;
ANOVA, F ˆ 5.003, df ˆ 2,45, P ˆ 0.0109), among
118

Table 3 The mean gonad length and mean number of gonads per
individual of Amphiura carchara, compared among samples from
o€ Pt. Conception (1989) and the Farallon Islands (1991, 1992).
Values for mean ‹ SD (number) are shown in the upper rows. The
lower rows summarize the results of single-factor ANOVA and
Kruskall-Wallis tests of gonad numbers and size in the three
samples, and multiple comparison tests between pairs of samples.
Brackets signify that the signi®cantly smaller body size of males
may be the cause of the signi®cantly smaller testis length in the July
sample. Di€erences in the numbers of testes per individual were
tested with a Games and Howell test; other comparisons were
tested with a non-parametric Tukey type test. Comparisons that
were statistically signi®cant at P = 0.05 are denoted with an
asterisk

Testis length (mm) No. of testes Ovary length (mm) No. of ovaries

Jun 1989 0.506 ‹ 0.206 (733) 29.320 ‹ 11.632 (25) 0.765 ‹ 0.276 (614) 15.375 ‹ 5.052 (40)
Jul 1991 0.373 ‹ 0.124 (170) 28.333 ‹ 7.581 (6) 0.704 ‹ 0.299 (702) 30.522 ‹ 12.116 (23)
Feb 1992 0.496 ‹ 0.197 (682) 45.467 ‹ 18.894 (15) 0.867 ‹ 0.327 (428) 28.533 ‹ 5.604 (15)
ANOVA P = 0.0032
F = 6.596, df = 2,43
Kruskal-Wallis P < 0.0001 P = 0.0001 P = 0.0001
Hc = 74.487 Hc = 71.309 Hc = 44.365
Feb vs Jun * * *
Feb vs Jul [*] * *
Jun vs Jul [*] * *

which only the ®gures for February and June were sig-
ni®cantly di€erent (Games and Howell test, P ˆ 0.05).
Brooding specimens of A. carchara were detected at
seven of the nine stations represented among specimens
in the National Museum of Natural History (USNM).
They occurred in collections from Sagami Bay, Japan
(USNM catalog number 25993; October 1906; from
1,123 m depth), Bering Sea, Aleutian Islands (USNM
26213; May 1906; 3,230 m), Shumagin Islands, Alaska
(USNM 26053; May 1906; 3,609 m), Kodiak Island,
Alaska (USNM 26115 and 27017; August 1890;
1,271 m. USNM 33639; August 1890; 1,261 m), Wash-
ington, USA (USNM 26114; June 1889; 1,390 m),
northern Baja California, Mexico (USNM 39297; Jan-
uary 1889; 1,800 m. USNM 32377; March 1911;
1,994 m).
arm plates, and lacked adult features such as oral pa-
pillae on the jaws and ventral interradial disk plates.
Almost all of the 655 embryos examined had ®ve
arms. There was a low incidence of abnormal embryos,
but no evidence of normal embryos ingesting small or
abnormal siblings. One two-armed, stage t embryo was
found in one female, two three-armed, stage 1 + t and
four, four-armed stage 1 + t embryos in another fe-
male, and one six-armed, stage t + 3 embryo in a third
individual. In every case the brooding female was ®ve-
armed, and the other late-stage embryos in the brood
had ®ve arms.
The length of the stage 0 embryos and the disk di-
ameter of the more advanced embryos ranged from 0.3
to 1.3 mm (x ˆ 0:80, SD ˆ 0.12, n ˆ 655) (Fig. 3). The
correlation between the length and width of the stage 0
embryos di€ered signi®cantly from zero (Student's

Characteristics of embryonic developmental stages

In the earliest (``stage 0'') embryos, rudimentary arms

had not yet developed (Fig. 1G). The smallest embryos
were polygonal, touching each other or nearly so, and
were anchored in position in the bursa by microscopic
®laments. Larger embryos were irregular in form and
closely packed within the bursa. Their shape appeared to
be molded by the embryos against which they pressed as
they grew. The presumptive dorsal ossicles of the adult
disk were the most obvious skeletal elements that
formed over the central yolk mass. They corresponded
to the ®ve primary plates that develop in planktotrophic
larvae, but were not typically of uniform size or precisely
®ve in number. The presumptive ossicles of the jaws and
terminal arm plates arose before the arms developed.
Older embryos developed a thick, blunt terminal Fig. 3 Amphiura carchara. Box plots comparing the body size of
plate at the arm tip and up to four arm joints (Fig. 1F, embryos at each developmental stage. Size (mm) is presented in terms
G). The 1 + t and 2 + t embryos had an opaque, yolky of body length for stage 0 embryos, and as disk diameter for more
mass in the disk, presumably localized in the digestive advanced stages. Horizontal lines denote the 10th, 25th, 50th
(median), 75th, and 90th percentiles for body size, and values above
tract. The 3 + t and 4 + t embryos were translucent, the 90th percentile and below the 10th percentile are plotted
and had major skeletal ossicles of the disk and arms. separately. The numbers of embryos at each stage are: 0, 149; t, 24;
They, however, had only rudimentary dorsal and ventral 1 + t, 91; 2 + t, 209; 3 + t, 131; 4 + t, 51
 119

t-test, t ˆ 1.978, P ˆ 0.05). However, it is so weak

(r ˆ 0.176), presumably because of the irregular shapes
of the embryos, that the size of stage 0 embryos should
not be compared with the disk diameter of later devel-
opmental stages.
The ®ve advanced developmental stages fell into three
groups of di€erent body sizes (t <1 + t ˆ 2 + t <3
+ t ˆ 4 + t) (Tukey type test, P ˆ 0.05). The mean
disk diameters of the embryos (excluding stage 0 speci-
mens) varied slightly but signi®cantly among the three
samples (June, x ˆ 0:86, SD ˆ 0.11, n ˆ 169; July,
x ˆ 0:81, SD ˆ 0.09, n ˆ 70; February, x ˆ 0:82,
SD ˆ 0.11, n ˆ 267) (ANOVA, F ˆ 8.455, df ˆ 2,503,
P ˆ 0.0002). Notably, the percentage of embryos at
various stages of development contrasted markedly
among samples. The embryos in the July sample con-
sisted predominantly of early stages, and those in the
June and February samples consisted largely of later
stages (Fig. 4).
Individuals brooded from one to four di€erent de-
velopmental stages although most contained embryos at
only 1 stage of development (Fig. 5). The number of
stages in any given bursa of an individual was even more
uniform, with one stage clearly predominant (Fig. 5).
In each sample most embryos at stages t to 4 + t
were oriented with their mouth and the ventral surface
of the arms pressed against the wall of the bursa. Even in
the most advanced embryos the arms were extended to
their full length, and not coiled dorsal to the disk. A
total of 410 embryos were oriented facing outward in the
three samples combined, and only 26 had their ventral
surface directed toward the lumen of the bursa. The
hypothesis that the ratio of outward- and inward-facing
embryos was 1:1 could be rejected (chi-square test,
P < 0.001, df ˆ 1, v2 ˆ 338).

E€ects of parasites on reproduction

Internal parasites were found in only 6 of the 124 dis-

sected specimens. Two female and one male A. carchara
in the June sample were parasitized by individuals of
Ascothorax sp., an ascothoracican ``barnacle'', one of
which was hyperparasitized by a cryptoniscid isopod.
Two female and one male A. carchara in the February
sample were parasitized by individuals of Protomyzo-
stomum sp., a myzostome annelid. The females harboring
myzostomes were brooding embryos, but those parasi- Fig. 4 Amphiura carchara. Histograms showing the relative abun-
tized by ascothoracican crustaceans lacked embryos. dance of six developmental stages in June 1989, o€ Pt. Conception,
and in July 1991, and February 1992, o€ the Farallon Islands. The
numbers of embryos examined are: June, 172; July, 174; February,
309
Discussion

Relationship of brooding to bathymetric
distribution and body size
The brooding reproduction in A. carchara is of special
interest in light of the perception that there are ``very few
observations of brooding in deep-sea echinoderms''
(Gage and Tyler 1991: 311), and of the recent proposal
that ``pelagic development prevails in polar and deep-sea
echinoderms'' Pearse (1994: 33). However, the conclu-
sions of those authors do not represent accurately the
information that has been published on ophiuroid re-
production. In the cosmopolitan genus Amphiura alone,
120
Fig. 5 Amphiura carchara. Histograms showing the number of
developmental stages occurring in single bursae (above), compared
to the number of stages occurring in individual brooding females
(below). The plots represent 243 bursae from 48 brooding individuals,
combining data for samples from o€ Pt. Conception (1989) and the
Farallon Islands (1991, 1992). Bursae without embryos were not
tallied
17 brooding species can now be recognized (Hendler
1991; present report). Five of them occur at depths
>200 m, and four others, including A. carchara, at
depths >1000 m. Six of the nine deep-sea species live in
polar waters. The only two Amphiura species known to
have planktonic larvae are restricted to the continental
shelf in boreal-warm temperate waters (see refs. in
Hendler 1991). Thus, it seems premature to conclude
that, at least among the ophiuroids, brooding behavior
is exceptional in deep-sea and polar environments. The
incidence of brooding reproduction has not yet been
fully or adequately assessed, either among shallow-water
or deep-sea ophiuroids (Hendler 1975, 1991). As noted
in the introduction, the mode of reproduction has been
documented for only 4% of all ophiuroid species, nearly
one-half of which are relatively little known, deep-sea
forms.
The body size of A. carchara was comparable to that
of most brooding ophiuroids, which usually have a disk
diameter of 10 mm or less and are typically smaller than
broadcast spawners (Hendler 1975: Fig. 1). The arms of
A. carchara adults are only 4±5 times greater in length
than its disk diameter. The species is small in compari-
son to confamilials that have disk:arm ratios of 1:10 and
more, and can burrow deeply in soft sediments (Hendler
et al. 1995). The short arms of A. carchara adults suggest
that they live at or near the surface of the sediment.
Two groups of brooding ophiuroids have been
characterized, some producing discrete, sequential
clutches of embryos (``sequential brooders'') and others
brooding more than one cohort simultaneously (``si-
multaneous brooders'') (Hendler 1991). A. carchara in-
dividuals brooded up to four di€erent developmental
stages, which is indicative of simultaneous brooding.
Most of them, however, brood embryos of only one
developmental stage at a time, and the range of devel-
opmental stages within a single bursa was even narrower
than that within an individual, a situation suggesting
sequential brooding. Apparently, individuals stagger the
production of groups of embryos, although the devel-
opmental stage of the cohorts may overlap to a degree.
Thus, the regulation of reproduction shown by
A. carchara is intermediate between the pure ``sequen-
tial'' and ``simultaneous'' patterns.
Size of eggs, embryos, and juveniles
The largest oocytes in the ovaries of A. carchara, with a
mean diameter of 0.45 mm and which ranged up to
1.24 mm in diameter, are large in comparison with other
ophiuroid eggs, which are generally from 0.1 to ap-
proximately 1.0 mm in diameter (Hendler 1991). Previ-
ously, only two ophiuroid species were known to have
eggs with a maximum diameter 1.0 mm: Astrothorax
waitei (Benham), an exceptionally large brooding form
with 0.5 to 1.0-mm oocytes, and Astrospartus mediter-
raneus (Risso), a basket star, which has an unknown
mode of development and 1.5-mm oocytes reported
(references in Hendler 1991).
Recent research suggests that much of the nutrient
and mass of lecithotrophic echinoderm eggs are not used
during larval development, and might serve to enhance
the survival of postlarvae (Emlet and Hoegh-Guldberg
1997; Hoegh-Guldberg and Emlet 1997; Bosch and
Slattery 1999). In addition, large body size may be ad-
vantageous for the juveniles released by brooding
ophiuroids (Hendler 1975). For echinoids, larger young
have been shown to grow more rapidly and have higher
survivorship than smaller siblings (Miller and Emlet
1999). Therefore, one does not expect the size range of
the later embryonic stages of A. carchara (Fig. 3) to
suggest that young leaving the parent are of di€erent
sizes or that they may di€er in their reserves of nutrients.
In the brooding antarctic species, Ophiacantha vivi-
para Ljungman, which has 0.6-mm diameter eggs, ju-
veniles as small as 1.5 mm dd cling to adult individuals,
yet embryos as large as 2.5 mm dd occur within their
bursae. On that basis, Ludwig (1899) concluded that the
young do not emerge from the parent's bursa at a ®xed

size. Unfortunately, comparable data are not available
for other brooding ophiuroids with large eggs (Hendler
1991).
In a viviparous ophiuroid species that has small eggs,
Amphipholis squamata (Delle Chiaje), the correlation
between the stage of development and embryonic body
size changes much as in A. carchara. Early stages have a
wider range of disk diameters than later stages. It is not
yet known whether the largest embryos of A. carchara,
with four arm joints, could develop still further before
leaving the parent. They have fewer arm joints than the
embryos of several other brooding Amphiura species
(Hendler 1991). However, it is not known whether the
brooded juveniles leave the parents at a range of stages
and sizes (Hendler 1975). Notably, in the asteroid
Patiriella vivipara (Dartnall), which has small eggs and
intraovarian development, juveniles are born at a wide
range of sizes, from 1.4 to 5.0-mm diameter (Byrne 1996;
Prestedge 1998). In another brooding asteroid with large
eggs, Neosmilaster georgianus (Sladen), there is less
variability in the size of early embryos than in juveniles
just prior to emergence (Bosch and Slattery 1999). Both
asteroid species exhibit adelphophagy (brood cannibal-
ism), which may account to some extent for the variable
size of advanced embryos.
The transparency of the bodies of the late-stage
embryos of A. carchara suggests that yolk reserves in the
gut are greatly diminished or fully exhausted before
the young are released. The position of embryos within
the bursae is of interest in that regard, because it sug-
gests that the developing embryos could take up nutri-
ents secreted by the bursal wall through their mouth and
tube feet. In support of that idea are observations that
the brooded embryos of Amphipholis squamata and
Ophiolepis paucispina (Say) apply their mouth and par-
tially everted stomach to sinuses in the bursal wall that
contain PAS-positive contents (Walker and Lesser 1989;
Byrne 1994). Embryos of other brooding ophiuroids, for
example Ophionereis olivacea H.L. Clark, maintain the
same position in relation to the bursal wall (Byrne 1991).
There is still no proof that the PAS-positive material is
of nutritional value to the embryos. Identical associa-
tions between embryos and the bursal wall in several
species, however, suggest that the relationship is a
physiological necessity and that the possibility of
matrotrophy (sensu Frick 1998) in brooding ophiuroids
merits further study.
Relationships between brooding and sex
The preponderance of separate male and female indi-
viduals, veri®ed by histological examination, indicates
that A. carchara is gonochoric. Hermaphroditism is ru-
led out by the equivalent size-frequency distribution of
males and females, and by the absence of transitional
hermaphroditic individuals.
Most ophiuroid species are gonochoric although
teratological hermaphrodites are found among popula-
121
tions of some species. The occurrence of a single tera-
tological hermaphrodite among the 526 specimens of
A. carchara that were examined, an incidence of <1%,
is consistent with the low incidence of abnormal her-
maphroditism among other ophiuroid species (Hendler
1991). Over 40 ophiuroid species, however, are known to
have stable hermaphroditism. At least 37 of the her-
maphroditic species are brooders (Hendler 1991).
Among the approximately 70 species of brooding
ophiuroids only 13 species, including A. carchara, are
known to be gonochoric (Hendler 1991; present report).
The seemingly strong association between brooding and
hermaphroditism among ophiuroids, ®rst noted by
Mortensen (1920), remains an enigma (Hendler 1975,
1991). Perhaps information regarding the sexuality of
additional brooding species will invalidate the ostensible
trend, or point to a phylogenetic or ecological explana-
tion.
The mean number of gonads in A. carchara falls
within the range (10±500) reported for other brooding
and broadcast-spawning Amphiuridae (Hendler 1991).
It is not known whether the greater number of testes
than ovaries in individuals of A. carchara is typical,
because the relative numbers of gonads in male and fe-
male ophiuroids have not been documented in other
species. The ®gures for the numbers of gonads per in-
dividual suggest that male A. carchara produce more
gonadal tissue than females. That disparity, however, is
presumably contravened to a degree by the signi®cantly
greater length of the ovaries than the testes. The sex
ratio of A. carchara departs from unity in favor of fe-
males, and the di€erence was statistically signi®cant for
the Farallon Island population; a similar situation ob-
tains in a number of ophiuroid species (Hendler 1991).
The two other deep-sea ophiuroids that have been ex-
amined, which are broadcast-spawners, have equal
numbers of males and females (Tyler 1986). The signif-
icance of the unequal sex ratio in A. carchara is not
known, and additional data are needed to determine
whether it is typical for brooding species.
Brooding season
It is not known whether brooding ophiuroids have an
extended period of gamete release compared to broad-
cast spawners. Some, however, may have an extended
brooding season, and several months can elapse between
their spawning and the release of young (Hendler 1991).
The presence of embryos in a majority of female
A. carchara in the June, July, and February samples
indicates that brooding occurs in the summer and in the
winter in waters o€ California. The possibility that the
species broods year round is supported by the presence
of embryos in A. carchara in January, March, May,
June, August and October in specimens from USNM
collections.
Data in the present report, based on only three
samples collected in three di€erent years at two sites, are
122
insucient to characterize reproductive seasonality
fully. Considering only samples from o€ the Farallons, it
seems noteworthy that the testes and ovary length
measurements in July are signi®cantly smaller than in
February. The numbers of testes are also signi®cantly
lower in July than in February. In addition, the number
of early developmental stages is higher in July than in
February, and the percentage of brooding females is
higher in February than in July. The numbers of
embryos per individual would also be expected to
change if reproduction were seasonal. Embryo numbers,
however, are similar among the three samples, and only
the February and June data di€er signi®cantly in that
regard.
Thus, the trends suggest elevated reproductive activ-
ity in the summer o€ the Farallons. A decrease in gonad
size and an increase in the number of early develop-
mental stages are consistent with spawning at that time,
which presumably could result in the higher incidence of
brooding and the higher number of advanced embryos
in the winter. The ovary lengths and the number of
ovaries, however, di€er signi®cantly between the June
(Pt. Conception) and July (Farallon) samples. Thus, the
trends in the data for gonad length and number do not
show a clear, consistent seasonal pattern when the
results for the two localities are considered together.
Additional data are needed to determine whether this
situation re¯ects sampling error, non-seasonal repro-
duction, or di€erences in reproduction at contrasting
latitudes and depths.
Acknowledgements We are indebted to individuals who collected
and provided the ophiuroids used in this project including K. Smith
(Scripps Institution of Oceanography), A. Summers and J. Ny-
bakken (Moss Landing Marine Laboratory), and their colleagues.
It is also a pleasure to thank L. Cooper (Southern California
Coastal Water Research Project) for assistance with histology,
K. Friedmann for translation of German text, P. Gordon for
translation of Russian text, M. Grygier (Lake Biwa Museum) for
identi®cation of parasites, D. Pawson and C. Ahearn (National
Museum of Natural History) for access to specimens in their care,
and J. Dearborn (University of Maine), J. Pechenik (Tufts Uni-
versity), F. Nishida (LACM), and two anonymous reviewers for
comments on the manuscript.
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