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Full text of "Malacologia"

2001 
EDITORIAL BOARD 



J. A. ALLEN 

Marine Biological Station 
Millport, United Kingdom 
¡alien ©udcf.gla.ac. uk 

E. E. BINDER 

Museum d'Histoire Naturelle 

Geneve, Switzerland 

P. BOUCHET 

Muséum National d'Histoire Naturelle 

Paris, France 

bouchet@cimrs1.mnhn.fr 

P. CALOW 

University of Sheffield 
United Kingdom 

R. CAMERON 

Sheffield 

United Kingdom 

R.Cameron@sheffield.ac.uk 

J. G. CARTER 

University of North Carolina 

Chapel Hill. U.S.A. 

MARYVONNE CHARRIER 

Universite de Rennes 

France 

Maryvonne. Charrier @ univ-rennes 1 . fr 

R. H. COWIE 
University of Hawaii 
Honolulu. HL. U.S.A. 

A. H. CLARKE, Jr. 
Portland. Texas, U.S.A. 

B. С CLARKE 
University of Nottingham 
United Kingdom 

R. DILLON 

College of Charleston 

SC, U.S.A. 

C. J. DUNCAN 
University of Liverpool 
United Kingdom 

D.J. EERNISSE 
California State University 
Fullerton, U.S.A. 

E. GITTENBERGER 
Rijksmuseum van Natuurlijke Historie 
Leiden, Netherlands 

sbu2eg @ rulsfb. leidenuniv. de 

F. GIUSTI 

Universita di Siena, Italy 
giustif@unisi.it 



A. N. GOLIKOV 
Zoological Institute 
St. Petersburg, Russia 

S.J.GOULD 
Harvard University 
Cambridge, Mass., U.S.A. 

A. V. GROSSU 
Universitatea Bucuresti 
Romania 

T. HABE 

Tokai University 

Shimizu, Japan 

R. HANLON 

Marine Biological Laboratory 

Woods Hole, Mass.. U.S.A. 

G. HASZPRUNAR 

Zoologische Staatssammlung Muenchen 

Muenchen, Germany 

haszi@zi.biologie.uni-muenchen.de 

J. M. HEALY 

University of Queensland 

Australia 

jhealy@ zoology, uq. edu. au 



D. M. HILLIS 
University of Texas 
Austin, U.S.A. 



K. E. HOAGLAND 

Council for Undergraduate Research 

Washington. DC, U.S.A. 

Elaine @ cur org 



MCZ 
LIBRARY 

FEB 1 4 zmz 



B. HUBENDICK 
Naturhistoriska Museet 
Göteborg. Sweden 

S. HUNT 
Lancashire 
United Kingdom 

R. JANSSEN 

Forschungsinstitut Senckenberg, 
Frankfurt am Main, Germany 

M.S.JOHNSON 
University of Western Australia 
Nedlands, WA, Australia 
msj@cyllene. uwa. edu.au 

R. N. KILBURN 
Natal Museum 
Pietermaritzburg, South Africa 



M. A. KLAPPENBACH 

Museo Nacional de Historia Natural 

Montevideo, Uruguay 



. HAf^VARD 
UNlVi£R3JTY 



J. KNUDSEN 

Zoologisk Institut Museum 

Kobenhavn. Denmark 

C. LYDEARD 
University of Alabama 
Tuscaloosa. U.S.A. 
clydeard @ biology, as.ua. edu 

C. MEIER-BROOK 
Tropenmedizinisches Institut 
Tubingen. Germany 

H. K. MIENIS 

Hebrew University of Jerusalem 

Israel 

J. E. MORTON 
The University 
Auckland. New Zealand 

J. J. MURRAY, Jr. 
University of Virginia 
Charlottesville. U.S.A. 

R. NATARAJAN 
Marine Biological Station 
Porto Novo, India 

DIARMAIDO'FOIGHIL 
University of Michigan 
Ann Arbor, U.S.A. 

J. OKLAND 
University of Oslo 
Norway 

T. OKUTANI 
University of Fisheries 
Tokyo. Japan 

W. L. PARAENSE 

Instituto Oswalde Cruz, Rio de Janeiro 

Brazil 

J. J. PARODIZ 
Carnegie Museum 
Pittsburgh. U.S.A. 

R. PIPE 

Plymouth Marine Laboratory 
Devon, United Kingdom 
RKPI @ wpo. nerc. ac. uk 

J. P. POINTIER 

Ecole Pratique des Hautes Etudes 
Perpignan Cedex, France 
pointier® gala, univ-perp. fr 



Ol Z. Y 

Academia Sínica 

Qingdao. People's Republic of China 

D. G. REÍD 

The Natural History Museum 

London. United Kingdom 

S. G. SEGERSTRÂLE 
Institute of Marine Research 
Helsinki. Finland 

A. STANCZYKOWSKA 
Siedice. Poland 

F. STARMÜHLNER 

Zoologisches Institut der Universität 

Wien, Austria 

Y. I. STAROBOGATOV 
Zoological Institute 
St. Petersburg. Russia 

J. STUARDO 
Universidad de Chile 
Valparaiso 

С THIRIOT 

University P. et M. Curie 

Villefranche-sur-Mer, France 

thiriot@obs-vlfr.fr 

S. TILLIER 

Museum National d'Histoire Naturelle 

Paris, France 

J.A.M. VAN DEN BIGGELAAR 
University of Utrecht 
The Netherlands 

N. H. VERDONK 
Rijksuniversiteit 
Utrecht, Netherlands 

H. WÄGELE 

Ruhr-Universität Bochum 

Germany 

Heike. Waegele @ ruhr-uni-bochum. de 

ANDERS WAREN 

Swedish Museum of Natural History 

Stockholm. Sweden 

B. R. WILSON 

Dept. Conservation and Land Management 

Kallaroo, Western Australia 

H. ZEISSLER 
Leipzig, Germany 



W. F. PONDER 
Australian Museum 
Sydney 



A. ZILCH 

Forschungsinstitut Senckenberg 

Frankfurt am Main. Germany 



MALACOLOGIA, 2002, 44(1): 1-15 

SMALL-SCALE MUSSEL SETTLEMENT PATTERNS WITHIN MORPHOLOGICALLY 
DISTINCT SUBSTRATA AT NINETY MILE BEACH, NORTHERN NEW ZEALAND 

Andrea 0. Alfaro^ & Andrew G. Jeffs^ 

ABSTRACT 

Microscale settlement patterns of juveniles of the mussel Perna canaliculus were investigated 
within drift material at Ninety Mile Beach, northern New Zealand. Size- and site-specific selec- 
tivity on various morphologically distinct algal and hydroid species were identified within drift ma- 
terial and corroborated in laboratory experiments with similar artificial substrata. Mussel spat 
densities were greater within fine-branching natural (-28-57%) and artificial materials 
(-13-20%) compared to medium- and coarse-branching natural (-7-8%) and artificial 
(-2-3%) materials. Size-frequency distributions of mussel spat within natural and artificial ma- 
terials suggested a relationship of increasing mussel size with decreased branching of substrata. 
Field and laboratory investigations indicated higher settlement of 1.5-2.0 mm mussel size 
classes in coarse-branching substrata, whereas fine-branching substrata had greater settlement 
of mussels within the <0.5 mm size class. Mussel settlement comparisons within node and inter- 
node areas of all substrata in the field and in the laboratory indicated a strong preference of set- 
tlement in node areas over inter-node areas. The microscale settlement patterns observed in this 
study are argued to be indicative of a life strategy to maximize juvenile mussel survival within the 
dynamic environment of drift material in oceanic currents, before the potential arrival and re-set- 
tlement to rocky coastal areas. The present study is the first to elucidate settlement patterns of 
Perna canaliculus on drift material that washes up on Ninety Mile Beach, where >70 tonnes/year 
of this material is collected and supplied to the New Zealand aquaculture industry to seed mus- 
sel farms. 

Key words: mussels, small-scale settlement patterns, micro-habitat selection, size-frequency 
distribution, drift algae. 



INTRODUCTION 

The complexity and diversity of planktonic 
larval settlement patterns has received a 
great deal of attention within various temporal 
and spatial scales (Butman & Grassle, 1992; 
Bourget & Harvey, 1998). Physical and bio- 
logical factors have been investigated as po- 
tential contributors to passive and active set- 
tlement outcomes (Butman, 1987; Butman & 
Grassle, 1992; Grassle et al., 1992). For ma- 
rine invertebrates, such as mussels, larval 
settlement on filamentous substrata repre- 
sents an important intermediate step to the 
eventual recruitment to the adult population 
(Bayne, 1964; Davies, 1974; Highsmith, 
1985; King et al., 1990; Harvey et al., 1993; 
Hunt & Scheibling, 1996). However, the dy- 
namics of these interactions are not well un- 
derstood for most broadcast spawners 
(Dame, 1996). 



Bayne (1 964) first demonstrated that larvae 
of the mussel Mytilus edulis tended to settle 
on filamentous algae (primary settlement) and 
later moved to established adult mussel beds 
as secondary settlers (1 -2 mm) through a 
bysso-pelagic phase. Bysso-pelagic drifting 
has since been shown to be a common 
means of dispersal among pelagic larvae and 
juveniles (Lane et al., 1985; Martel, 1993; 
Martel et al., 1994; Buchanan & Babcock, 
1997). Buchanan & Babcock (1997) also 
found a size-specific settlement pattern for 
the New Zealand green-lipped mussel Perna 
canaliculus on various intertidal algae, hy- 
droids, and adult mussel beds at Piha Beach, 
North Island, New Zealand. In their study, 
Buchanan & Babcock (1997) observed that 
mussels <0.5 mm (primary settlers) were 
more abundant in attached filamentous algae 
and a hydroid species, whereas secondary 
settlers (>0.5 mm) were numerous in coarser- 



^Corresponding author. School of Environmental and Marine Sciences, The University of Auckland, Auckland, New Zealand; 

a.alfaro@auckland.ac.nz 

^National Institute of Water & Atmospheric Research Ltd., P. O. Box 109695, Newmarket, Auckland, New Zealand; 

a.jeffs@nlwa.crl.nz 



1 



ALFARO & JEFFS 



branching algae and the adjacent ¡ntertidal 
mussel bed. Elsewhere in northern New 
Zealand, at Ninety Mile Beach (NMB), early 
juveniles or spat of Perna canaliculus a\so are 
found associated with floating algae and hy- 
droids. Large quantities (>70 t/y) of spat, at- 
tached to this drift material, are used to seed 
the extensive mussel farms in New Zealand, 
after collection from the surf zone (Jeffs et al., 
2000). Nowhere else in New Zealand but 
along the 90 km stretch of NMB. are such vast 
amounts of wild spat concentrated in one 
place on detached material that can be con- 
veniently and economically harvested to sup- 
ply the mussel industry (Jeffs et al., 2000). 
Distinctive patches of filamentous substrata 
that arhve at the beach (wash-up events) may 
vary in substratum abundance and density, 
and spat size-frequency distribution (Hick- 
man, 1982). A single wash-up at NMB may 
contain up to 70 tonnes of substratum and 
spat material (C. Hensley, personal communi- 
cation). These filamentous substrata may de- 
rive from intertidal to deep subtidal regions in 
particular areas along NMB, and mussel set- 
tlement onto these substrata may take place 
before or after detachment of the alga or hy- 
droid. Regardless of the timing of settlement, 
algal and hydroid materials appear to be es- 
sential to the transport of mussel spat, as they 
may not only provide a means for long- 
distance dispersal, but also an alternative 
habitat to maintain or increase physiological 
processes (Highsmith, 1985; Schneider & 
Mann, 1 991 a). If a size-specific mussel settle- 
ment pattern occurs on the filamentous sub- 
strata found within a wash-up event at NMB, it 
is likely that substratum selection is the pre- 
dominant force behind mussel settlement pat- 
terns, rather than random physical encoun- 
ters along this particularly high energy portion 
of the New Zealand coastline. The persis- 
tence of size-specific settlement patterns of 
mussels on drift material, while exposed to 
the energetic hydrodynamic regimes often en- 
countered in the ocean, suggests a complex 
life history strategy. The aim of the present 
work is to elucidate microscale (cm) settle- 
ment patterns on the substrata found in wash- 
up events that occur along the 90 km length of 
NMB. Thus, characterization of substratum 
types, and size- and site- specific settlement 
patterns upon various drift substrata, may 
provide a first step to understanding spat-sub- 
stratum interactions at various spatial and 
temporal scales. 



METHODS AND MATERIALS 
Study Site and Terminology 

Samples of Perna canaliculus spat and as- 
sociated substrata were collected from the 
surf zone at various locations along Ninety 
Mile Beach (NMB), Northland, New Zealand 
(Fig. 1). Each sample was collected from dis- 
tinct wash-up events on 17 October 1998, 22 
December 1998, and 16 May 1999. Wash-up 
samples were collected by net from the surf 
along the beach. Once collected, all samples 
were immediately frozen for later laboratory 
analyses at the University of Auckland, Auck- 
land, New Zealand. 

Recruitment is an operational word that 
does reflect a unique and specific biological 
event (Hunt & Scheibling, 1997). There is no 
standard size nor age at which mussels are 
defined to be settling or recruiting. Bayne 
(1964) suggests that primary settlers are 
mussels <0.5 mm in shell length, and sec- 
ondary settlers are mussels between 0.5-2.0 
mm in shell length. Because mussels can, 
and often do re-settle even at shell lengths of 
up to 25 mm, it is difficult to suggest a cut-off 
line between settlement and recruitment. The 
mussels that were found attached to algae 
and hydroids in our experiments are likely to 
have used the drifting substrata as an inter- 
mediate settlement step before recruiting to 
the adult mussel beds in suitable rocky sub- 
strata. We use the term settlement in this 
study, because the majority of the mussels we 
encountered in the drift samples were <5 mm 
in shell length. 

Natural Substrata 

As a first step to investigate small-scale set- 
tlement patterns of mussel spat on various 
drifting natural substrata, two 100-g wet 
weight subsamples were taken from each of 
three wash-up samples. The original wash-up 
samples were between 1 -5 t (wet weight) of 
very homogeneous substratum and mussel 
wash-up material. All distinctive algal and hy- 
droid substrata within each subsample were 
separated and grouped into four morphologi- 
cally distinctive categories, with algal cate- 
gories based on the level or degree of branch- 
ing. The categories were: coarse-branching 
algae (Osmundaria colensoi, Carpophyllum 
angustifolium, and Rhodymenia dichotama); 
medium-branching algae (Melanthalia ab- 



SMALL-SCALE MUSSEL SETTLEMENT 



Cape Reinga 



Scott Point 



-35° OO'S- 



NEW ZEALAND Í ^^ 




FIG. 1 . Location of study site at Ninety Mile Beach, Northland, New Zealand. Rocky intertidal areas are found 
at Tonatona Beach, The Bluff, and Scott Point. Spatfall collection sites on 17 October 1998, 22 December 
1998, and 16 May 1999 are marked (x) along the beach. 



scissa, Laurencia thyrsifera, Pterocladia lu- 
cida, P. capillacea, Gigartina marginifera, G. 
alveata, and Pactiymenia lusoria); fine- 
branching algae {Cfiampia laingii, Plocamium 
costatum, Haliptilon roseum, and Corallina of- 
ficinalis); and hydroids {Amphisbetia bispi- 
nosa, Dictyocladium moniliferum, Craterit- 
heca insignis, Aglaophenia acanthocarpa, 



and Lytocarpia incisa). The distribution of 
these algae and hydroids varies from below 
the low water mark of spring tides to deep 
subtidal, with the exception of Gigartina sp. 
and Pachymenia lusoria, which also can be 
found in the low intertidal zone (Morton & 
Miller, 1968; Adams, 1994). All algal species 
found in the samples were red algae, except 



ALFARO & JEFFS 



Carpophyllum angustifolium. which is a brown 
alga (Adams, 1994) and constituted <5% of 
each sample. The remaining material (shells, 
wood fragments, and other debris) did not 
contain attached mussels and was discarded. 
Mussels within each algal and hydroid sub- 
stratum type were removed by vigorous agita- 
tion in water, and the removal of remaining 
mussels was achieved with forceps 
(Buchanan & Babcock, 1997). Mussels within 
five different size-classes (<0.49, 0.5-0.99, 
1.0-1.49, 1.5-1.99, >2.0 mm in length) were 
separated with a set of sieves of appropriate 
sizes. Previous research had verified this 
technique as an effective means of reliably 
sorting mussel material (A. Alfaro, unpub- 
lished data). The mussels were then dried to 
constant weight in an oven at 80°C. The num- 
ber of mussels within each size class was ini- 
tially determined by counting under the micro- 
scope. The number of dried mussels of each 
size class per unit volume was calculated, 
and volume subsequently was used to esti- 
mate numbers of mussels. Several test sam- 
ples were processed initially to ensure the ac- 
curacy of the methodology. The surface area 
of flattened algal and hydroid samples was 
determined from video-computer images 
using NIH Image 1 .61 software for the Macin- 
tosh (Harvey et al., 1993). All algae and hy- 
droids were then dried in an oven at 80°C and 
weighed. Percent data sets of natural sub- 
stratum abundance and mussel densities 



were arcsine transformed to normalize the 
data. 

An additional 20 samples of -25 g wet 
weight were taken from each wash-up sample 
to determine the number of mussels attached 
to nodes and internodes of each of the four 
types of substrata (Fig. 2a). A node area was 
defined as a 1 cm^ area including at least one 
axil, and an inter-node area as a 1 cm^ area 
without an axil (Fig. 2b). Node and internode 
areas were randomly chosen from a large set 
of suitable areas. All mussels within node and 
internode areas on each substratum sample 
were removed with forceps and counted. The 
exact area occupied by each substratum 
within the 1 cm^ region was determined from 
image analyses using NIH Image 1.61 soft- 
ware. The total number of mussels per unit 
area of node and internode was calculated for 
each sample of algal and hydroid substrata. 

Artificial Substrata 

In order to further examine the settlement 
dynamics of mussel spat onto morphologically 
distinct substrata, settling patterns onto artifi- 
cial substrata of different shapes were moni- 
tored in the laboratory. Five to six 10-cm-long 
plastic aquarium plants of six different types 
were placed in separate seawater tanks of 1 .5 
liters. The artificial substrata were standard 
aquatic plant replicas and, for consistency, 
also were classified as coarse-branching 



Node 




FIG. 2, (A) Diagram depicting node and internode areas on a filamentous substratum. (B) Close-up of node 
and inter-node area (1 cm^). 



SMALL-SCALE MUSSEL SETTLEMENT 



{Myriophyllum verticillatum, Ceratophyllum 
demersum, and Cabomba aquatica), medium- 
branching {Elodea densa, Elodeasp., and Ro- 
íala indica), and fine-branching (Vallisneria 
americana, V. spiralis, and Ceratopteris thalic- 
troides) substrata to facilitate comparisons 
with the natural substratum experiments. Al- 
though the aquatic plants were not identical to 
the algae found in the wash-up samples, the 
surface area and degree of branching were 
very similar to the natural substrata, and the 
degrees of branching were distinctive among 
the three experimental groups. Mussel spat 
were collected from intertidal habitats at NMB, 
detached from their original algal substrata, 
and placed in a water tank with seawater on 
the same day. The density of mussels of dif- 
ferent sizes was adjusted to be similar among 
size classes. From this mixture, subsamples 
of about 500 mussels, representing all sizes 
within 0.5 to 3.0 mm in length, were separated 
using a standard plankton splitter. The mussel 
subsamples were then placed in each of the 
50 tanks containing artificial substrata. Each 
tank was aerated to ensure constant resus- 
pension of unattached mussels. The water 
temperature was maintained similar to the col- 
lection site at 1 5°C, and no food was added to 
the tanks during these short-term experi- 
ments. The light regime was set to mimic out- 
door conditions. After two days, mussel abun- 
dance, size-frequency distribution, site of 



mussel re-settlement, and substratum area 
were determined with the procedures outlined 
for natural substrata. Percent mussel spat 
densities were arcsine transformed to normal- 
ize the data. 

RESULTS 
Natural Substrata 

Substratum characterization in wash-up 
samples revealed overall differences in the 
total abundance of the four different settle- 
ment substrata, possibly due to seasonal 
changes in algal and hydroid productivity. The 
total amount of algal and hydroid material (± 
SE) from the original 100 g subsample for the 
three wash-up samples was 96 ± 2.3% in Oc- 
tober 1998, 31 ± 5.2% in December 1998, 
and 64 ± 3.5% in May 1999. However, the 
proportional algal and hydroid substratum 
comparisons among replicates and wash-up 
samples indicated that coarse-branching 
algae were more abundant than any other 
substratum for all three wash-up events (Fig. 
3). The mean percent area (± SE) of coarse- 
branching algae was 69.1 ± 2.7%, followed 
by medium-branching algae (14.1 ± 1.4%), 
hydroids (12.1 ± 1.4%), and fine-branching 
algae (4.6 ± 1 .2%). A two-way ANOVA of per- 
cent substratum among wash-up events (arc- 
sine transformed data) showed no statistical 




■ 17-Oct-98 
D22-Dec-98 
1 6-May-99 1 






Coarse-branching Mediurn-branching 
algae algae 



Fine-branching 
algae 



Hydroids 



Substratum Type 



FIG. 3. Percent area of four natural substratum types among three spatfall events. Non-significant Tukey 
tests for substratum differences are shown (*). 



ALFARO & JEFFS 



significance among wash-up events (ANOVA; 
F,2i2) = 0-35, p > 0.05), but a significant dif- 
ference among substratum types (ANOVA; 
F,3 ,2) = 217.33, p < 0.05). The interaction be- 
tween wash-up and substratum type was not 
statistically significant (ANOVA; F^g ^2) = 1 •^'^ - 
p > 0.05). Tukey's painwise compansons of 
means failed to find significant differences be- 
tween medium-branching algae and hydroids 
(Tukey test; p = 0.799). 

The total number of mussels differed 
greatly among samples of different wash-up 
events. The first sample, collected on 17 Oc- 
tober 1998, yielded mussel numbers ranging 
from 12 to 3,866 mussels/cm^ of substratum, 
and a mean (± SE) of 658.7 ± 230.7 mus- 
sels/cm^ of substratum. The second wash-up 
sample of 22 December 1998 contained very 
few mussels, ranging from to 12 
mussels/cm^ of substratum and a mean (± 
SE) of 2.6 - 0.7 mussels/cm^ of substratum. 
Finally, the third sample of 1 6 May 1 999 had a 
range of 9 to 1 ,632 mussels/cm of substra- 
tum and a mean (± SE) of 255.2 ± 123.5 
mussels/cm^ of substratum. While the ab- 
solute number of mussels among the three 
wash-up samples was very different, the per- 
cent of mussels within each substratum type 
was similar for all wash-up samples (Fig. 4). 
The mean percent of mussels between repli- 



cates and among wash-up events indicates 
that hydroid substrata consistently accumu- 
lated the greatest number of mussels com- 
pared to all other substrata (Fig. 4). The mean 
percent of mussels/cm^ (± SE) among sub- 
strata was 57 ± 4, 28 ± 2, 8 ± 2% and 7 ± 
2% mussels/cm^ for hydroids, fine-branching, 
medium-branching, and coarse-branching 
algae, respectively. Results from a two-way 
ANOVA on the arcsine transformed data, with 
wash-up event and substratum type as fac- 
tors, indicated that there was no statistically 
significant difference among wash-up events 
(ANOVA; F(2 щ = 0-57, p > 0.05) and interac- 
tion (ANOVÁ; F,g^2) = 4.41, p > 0.05), al- 
though there was a significant difference 
among substratum types (ANOVA; F^g ^^^ = 
419.97, p < 0.05). Tukey's test comparisons 
failed to find significant differences between 
coarse- and medium-branching substrata 
(Tukey test; p = 0.714). 

Results from the size-frequency distribution 
of mussels within each substratum type re- 
vealed a relationship between increasing 
mussel size and decreased degree of branch- 
ing of substratum. Coarse-branching algae 
had a greater percent of large mussels 
(1.5-1.99 mm in length), whereas fine- 
branching algae and hydroids had the great- 
est percent of small mussels (<0.5 mm in 



70 



60 



|B19-Oct-98 
lD22-Dec-98 
016-May-99 




шъ 





Coarse-branching 
Algae 



Medium-branching 
Algae 



Fine-branching 
Algae 



Hydroids 



Substratum Type 



FIG. 4. Mussel densities within four natural substrate types and three events samples. Non-significant Tukey 
tests between substrates are shown (*). 



SMALL-SCALE MUSSEL SETTLEMENT 



100 



80 



60 



40 



Coarse-branching Algae 



1 аж'^т 




<0.49 



0.5-0.99 



m 



1.0-1.49 



1.5-1.99 



100 
80 
60 
40 
20 




Medium-branching Algae 



iia rL il H 



<0.49 



0.5-0.99 



1 .0-1 .49 



1.5-1.99 



Fine-branching Algae 




wTm 



-Ä. 



<0.49 



0.5-0.99 



1 .0-1 .49 



1.5-1.99 



100 
80 
60 
40 
20 



Hydroids 




<0.49 



0.5-0.99 1.0-1.49 1.5-1.99 

Mussel Size Class (mm) 



■ 17-Oct-98 
G22-Dec-98 
И16-Мау-99 



_à^ 



>2.0 



■ 17-Oct-98 
D22-Dec-98 
И16-Мау-99 



>2.0 



■ 17-Oct-98 
П22-Оес-98 
И16-Мау-99 



>2.0 



■ 17-Oct-98 
D22-Dec-98 
И16-Мау-99 



>2.0 



FIG. 5. Size-frequency distribution of mussels within five size classes and three spatfall events for four nat- 
ural substratum types. Painwise comparisons (Tukey test) were performed for substrata that showed non-sig- 
nificant interactions (coarse-branching algae, medium-branching algae, and hydroids) in an overall ANOVA 
test and are shown (*). 



length) (Fig. 5, Table 1). Two-way ANOVA's, 
with mussel size class and wash-up event as 
treatment, were performed for each substra- 
tum (Table 1). 

Results from mussel density comparisons 
between node and inter-node areas within 



each of four natural substrata indicate that 
mussels were more abundant in node areas 
in all substratum types (Fig. 6). Furthermore, 
the relationship between increasing mussel 
abundance and decreased degree of substra- 
tum branching was generally apparent in 



ALFARO & JEFFS 

TABLE 1 . Mean (± SE) percent mussel density within each of five size classes for four natural 
substratum types. Two-way ANOVAs are shown for each substratum type. Data were arcsine 
transformed for statistical analyses and back transformed to obtain the means. 







Mussel Size 






ANOVA 










Class 


Мряп -+- SE 












Substratum Type 


(mm) 


%/cm^ 


Source 


df 


F 


pa = 


0.05 


Coarse-branching 


<0.49 


5.62 ± 3.50 


Sample 


2 


0.20 


0.819 


ns 


Algae 




0.5-0.99 


6.74 * 3.10 


Size 


4 


17.63 


0.000 








1.0-1.49 


23.31 : 6.30 


Sample x Size 


8 


2.33 


0.075 


ns 






1.5-1.99 


52.63 ± 3.34 


Error 


15 












>2.0 


2.75 ± 3.97 












Medium-branching 


<0.49 


15.15 - 4.94 


Sample 


2 


0.10 


0.906 


ns 


Algae 




0.5-0.99 


14.77 ± 3.64 


Size 


4 


10.44 


0.000 








1.0-1.49 


27.35 r 2.37 


Sample x Size 


8 


3.52 


0.017 








1.5-1.99 


31.85 i 4.23 


Error 


15 












>2.0 


2.57 ± 4.35 












Fine-branching 


<0.49 


50.48 ± 1.26 


Sample 


2 


1.08 


0.365 


ns 


Algae 




0.5-0.99 


30.57 r 1.46 


Size 


4 


167.10 


0.000 








1.0-1.49 


15.57 - 1.10 


Sample x Size 


8 


2.23 


0.086 


ns 






1.5-1.99 


1.76 ±2.28 


Error 


15 












>2.0 


0.17 ± 1.76 












Hydroids 




<0.49 


42.81 i 0.96 


Sample 


2 


0.11 


0.894 


ns 






0.5-0.99 


29.27 ± 1.15 


Size 


4 


173.34 


0.000 








1.0-1.49 


17.60 ±0.71 


Sample x Size 


8 


1.82 


0.150 


ns 






1.5-1.99 


9.50 ± 2.22 


Error 


15 












>2.0 


0.05 ± 0.48 












10ПП ^ 




















1¿UU 




















.¿Г" 














T 








M 

E 


■ Nodes 






u 

^ 1000 


Internodes 












■ 








w 










(0 






















3 














^^^1 








2 800 














^^B 








> 














^^^^ 








XI 














^^H 










с 














^^H 










■:í 600 








, 






^^1 










^•w*^ 








^^^^^H 






^^^^^1 


ШША 






>. 








^^^^H 






^^^H 


^P 














^^^^^H 






^^^^^1 


^^P 






« 








^^^^1 






^^^H 








с 400 








^^^^1 






^^^1 








о 








^^^^^H 






^^^^^1 








о 








■ 






^^1 


^^P 






щ 














^^1 


^^Ш 






«я 200 








^^^^1 






^^^1 


yÍWMW/ 






(Я 

3 










■ 






■ 


■ 










^^^^^шттгтТптгл 


^k 






■ 


Éi 






Coarse-branching 


Medium-branching 


Fine-branching 




Hydr 


oids 








/ 


Mgae 




Algae 


Algae 

















Substratum Type 

FIG. 6. Mussel densities within node and internode areas within four natural substratum types (n = 20). 



these samples as well (Fig. 6). However, no 
difference between coarse- and medium- 
branching algae was observed (Fig. 6). The 
mean number of mussels within node areas 
were 4.7 ± 0.9, 52.4 ± 9.5, 557.1 ± 33.9, and 
1,055.0 ± 41.5 individuals/cm^, and within in- 



ternode areas were (0.4 ± 0.3, 23.7 ± 6.0, 
61.8 ± 13.9, and 581.2 ± 145.6 individuals/ 
cm^) for coarse-, medium-, and fine-branch- 
ing, and hydroid substrata, respectively. Al- 
though size classes were not differentiated in 
this part of the study the fact that the pattern 



SMALL-SCALE MUSSEL SETTLEMENT 



is similar for all substratum types (which con- 
tained different predominate mussel size 
classes) suggests that mussels of all size 
classes were more abundant within node 
areas. In samples with low total mussel abun- 
dances, clumps of mussels within node areas 
were often clearly visible. As the total density 
of the sample increased, the clumps of mus- 
sels extended from internodes along the 
branches, and in some cases, the entire algal 
or hydroid substratum was covered with mus- 
sels. A two-way ANOVA, with site of attach- 
ment (node versus internode) and substratum 
type, showed statistical significance for site of 
attachment (ANOVA; F^^ 4^21 = 248.74, p < 
0.05) and substratum type (ANOVA; F(^4J2) = 
562.03, p < 0.05). A statistically significant in- 
teraction (ANOVA; F ,34^2) = 72.54, p < 0.05) 
reflected the inability to differentiate settle- 
ment patterns between coarse- and medium- 
branching algae. 

Artificial Substrata 

While the absolute number of mussels that 
settled on the artificial substrata was much 
lower than the numbers recorded from the 
natural material found in wash-up samples, 
the general trend among morphologically dis- 
tinct groups was consistent with the natural 
substrata (Fig. 7). Mussel settlement within 



coarse-branching artificial substrata ranged 
from to 2 mussels/cm^ of substratum and 
had a mean of 0.21 ± 0.05 mussels/cm^ of 
substratum. Medium-branching material had 
a settlement range from to 6 mussels/cm^ 
and a mean of 0.38 ± 0.09 mussels/cm^ of 
substratum. Finally, fine-branching material 
had a range of to 25 mussels/cm^ of sub- 
stratum and had a mean settlement of 3.23 ± 
0.45 mussels/cm^ of substratum. Three one- 
way ANOVAs, with artificial plant type as treat- 
ment, were run on the arcsine transformed 
data to test for settlement differences among 
plastic aquarium plants within each of the 
three experimental branching groups. The re- 
sults of these tests showed no significant dif- 
ferences among any of the groups (ANOVA; 
F,2 15) = 0.02, p > 0.05, ANOVA; F(2 15) = 0.07, 
p > 0.05, and ANOVA; F(2^5) = 0.17, p > 0.05 
respectively for coarse-, medium-, and fine- 
branching substrata) (Fig. 7). However, a one- 
way ANOVA comparing mussel settlement on 
artificial substrata with different branching de- 
grees found significant settlement differences 
among substrata (ANOVA; F(2,5i) = 80.17, p < 
0.05) (Fig. 7). A one-way ANOVA comparing 
all coarse- and medium-branching substrata, 
regardless of plant species, resulted in non- 
significant substratum effects (ANOVA; F,^ ^^. 
= 1.06, p> 0.05). 
Frequency distribution results from the arti- 




Coarse-branching substrate 



Medium-branching substrate 
Substratum Type 



Fine-brancliing substrate 



FIG. 7. Mussel densities within three experimental group containing artificial substrata. A significant ANOVA 
test between coarse- and medium-branching algal substrata only are shown (*) (n = 6). See text for further 
explanations. 



10 



ALFARO & JEFFS 




<0.49 



0.5-0.99 



1.0-1.49 



1.5-1.99 



>2.0 



E 
О 

"55 

с 
о 
о 

Ъ 

О) 
(Л 

3 




<0.49 0.5-0.99 1.0-1.49 1.5-1.99 >2.0 

Mussel Size Class (mm) 

FIG. 8. Size-frequency distribution of mussels within five size classes and three experimental groups of arti- 
ficial substrata. Painwise comparisons (Tukey test) were performed for substrates that showed non-signifi- 
cant interactions (coarse- and medium-branching substrata) in an overall ANOVA test and are shown (*). 



ficial substratunn experiments also indicated 
greater settlement on fine-branching material 
than coarse- and medium-branching material 
(Fig. 8). Generally, fine-branching substrata 
had a higher percentage of < 0.99 mm mussel 



size class, whereas coarse- and medium- 
branching substrata had a similarly high per- 
centage of > 1.5 mm mussels, and very little 
to no settlement of < 1.5 sized mussels (Fig. 
8). Results from statistical analyses, including 



SMALL-SCALE MUSSEL SETTLEMENT 



11 



two-way ANOVAs for each experimental 
group (degree of branching and mussel size 
as treatment), are shown in Table 2. 

Settlement comparisons between nodes 
and internodes areas within artificial substrata 
also resulted in greater mussel densities in 
node areas than in internode areas (Fig. 9). 
Again, no settlement differences between 
coarse- and medium-branching substrata 
were observed, but fine-branching substrata 
did contain greater settlement than the other 



two substrata (Fig. 9). Mussels often were ob- 
served extruding their foot to test surrounding 
substrata. In some cases, mussels were ob- 
served moving from the tip of an artificial plant 
to the nearest node. This migratory behavior 
sometimes resulted in clumping of mussels. A 
two-way ANOVA, with site of attachment 
(node versus internode) and substratum type, 
showed significant differences between node 
and internode attachment sites (ANOVA; 
F,2 102) = "13.40, p < 0.05) among artificial sub- 



TABLE 2. Mean (± SE) percent mussel density within each of five size classes for three artificial 
substratum types. Two-way ANOVAs are shown for each substratum type. Data were arcsine 
transformed for statistical analyses and back transformed to obtain the means. 











Mussel Size 
Class 
(mm) 


Mean ± SE 
%/cm^ 




ANOVA 






5 

s 




Substratum Type 


Source 


df 


F 


эа = 0.0> 




Coarse-branching 


<0.49 


0.00 ± 


0.00 


Substrate 


2 


0.00 0.998 


n 




Substrata 


0.5-0.99 


0.00 ± 


0.00 


Size 


4 


20.61 0.000 








1.0-1.49 


0.00 ± 


0.00 


Sample x Size 


8 


0.12 0.998 


ns 






1.5-1.99 


0.29 ± 


0.06 


Error 


75 












>2.0 


3.66 ± 


0.09 














Medium-branching 


<0.49 


0.01 ± 


0.01 


Substrate 


2 


0.24 0.786 


ns 




Substrata 


0.5-0.99 


0.01 ± 


0.01 


Size 


4 


15.79 0.000 








1.0-1.49 


0.05 ± 


0.03 


Sample x Size 


8 


0.33 0.953 


ns 






1.5-1.99 


0.46 ± 


0.07 


Error 


75 












>2.0 


5.23 ± 


0.13 














Fine-branching 


<0.49 


47.26 ± 


0.30 


Substrate 


2 


3.14 0.049 






Substrata 


0.5-0.99 


18.79 ± 


0.54 


Size 


4 


41.75 0.000 








1.0-1.49 


2.65 ± 


0.15 


Sample x Size 


8 


5.34 0.000 








1.5-1.99 


2.15 ± 


0.23 


Error 


75 












>2.0 


1.44 ± 


0.09 












- 




ifi 






















1 \j 






















E 
о 




■ Nodes 


14 
12 
10 




D Intemodes 


















(Л 
(0 










> 






■Б 

с 


8 




















■«^ 






















с 


6 




















0) 
Q 


4 
















Т 




(0 
(0 

3 


2 





1 r 






■ 










s 


1 








шш^ 


1 1 







FIG 



Coarse-branching substratum Medium-branching substratum Fine-branching substratum 

Mussel Size Class (mm) 

9. Mussel densities within node and inter-node areas within three artificial substratum types (n = 6). 



12 



ALFARO & JEFFS 



Stratum types (ANOVA; F2102J = 41.60, p < 
0.05), and interaction (AÑOVA; F(2io2) = 
11.00, p< 0.05). 



DISCUSSION 

One of the great interests in the field of in- 
vertebrate ecology is the question of how ini- 
tial settlement patterns affect the distribution 
of adult broadcast spawners, such as mus- 
sels. While oceanic-current dispersal of lar- 
vae and juveniles is a passive means of trans- 
port, some degree of habitat choice is 
exercised by individuals that encounter and 
attach to drift material. This initial settlement 
process on various morphologically distinct 
drift materials may be of significance to the 
survival and successful resettlement of juve- 
niles to the adult population. Thus, elucidation 
of microscale settlement patterns of mussel 
spat on natural drift substrata may enhance 
our understanding of the process of spat 
transport and arrival to coastal areas. 

Substratum characterization within three 
wash-up events at NMB indicated that 
coarse-branching algal substrata consistently 
comprised the majority of settlement sub- 
strata found associated with wash-up events. 
Medium-branching algae, fine-branching 
algae, and hydroids were less abundant. Drift 
algal material may originate from rocky inter- 
tidal and subtidal areas that may have been 
detached from their natural substrata by 
storms or strong ocean currents. These mate- 
rials may become aggregated by local 
océanographie conditions. However, the ma- 
jority of the material (mostly red algae) is sub- 
tidal in origin {Osmundaria colensoi, Car- 
pophyllum angustifolium. and Rhodymenia 
dichotoma : Aúarus, 1994; Steneck & Dethier, 
1994), indicating the importance of subtidal 
sources. The large amount of subtidal mater- 
ial in NMB wash-up samples also may be a re- 
sult of the scarcity of rocky intertidal areas 
that could provide intertidal algal sources 
along the beach (Fig. 1). Furthermore, none 
of the intertidal areas at or near NMB contain 
the same species of algae found washed-up 
at NMB in enough abundance to indicate a 
likely source (A. Alfaro, personal observation). 
The often fresh and intact nature of the mate- 
rial that arrives at the beach suggests that the 
subtidal material comes from rocks situated 
just offshore (<35 m water depth) of three 
rocky outcrops along the beach (Tauroa Point, 
The Bluff, and Scott Point). Indeed, algal beds 



have been observed off Tauroa Point (S. 
Hooker, unpublished), and extensive rocky 
substrata, likely to harbor algal material, have 
been noted by local fishers off Scott Point. 
Furthermore, spawning peaks of intertidal and 
subtidal mussels (< 35 m water depth), as well 
as algal seasonal cycles, may strongly influ- 
ence the abundance, dispersal and coloniza- 
tion potential of spat within temporal and spa- 
tial scales (Alfaro et al., in review). However, 
more samples collected throughout the year 
are necessary to clarify these patterns. 

Quantification of mussel spat among algal 
and hydroid substrata indicated that a greater 
number of spat settle on hydroids, followed by 
fine-branching algae; whereas fewer spat set- 
tle on medium- and coarse-branching algae. 
Laboraton/ settlement experiments with artifi- 
cial substrata also resulted in greater settle- 
ment on fine-branching material over coarse- 
and medium-branching substrata. Buchanan 
& Babcock (1997) found a similar size- 
frequency distribution of mussel spat on inter- 
tidal algae at Piha, North Island, New 
Zealand, in their study, the authors found that 
primary settlement of mussels (< 0.4999 mm 
in length) was mostly on the hydroid Amphis- 
betia blspinosa, and fine- and medium- 
branching algae, whereas larger mussels in 
the dispersal (0.5-5.4999 mm in length) and 
stable (> 5.0 mm in length) stages of their 
life history tended to resettle onto coarser- 
branching algae and the adult mussel bed. By 
contrast, the present study included consider- 
ation of a wider range of free-drifting algal- 
branching types (coarser red and brown 
algae) that are likely of mostly subtidal origin. 
Nonetheless, the size-frequency distribution 
of mussel spat on natural (in the field at NMB) 
and artificial (in the lab) substrata strongly 
corroborates observations at Piha by Bu- 
chanan & Babcock (1997). The field experi- 
ments at NMB indicated that the percent of 
mussel spat among the four algal and hydroid 
substrata was similar for all three wash-up 
samples and may indicate that settlement pat- 
terns are not affected by absolute variations in 
substratum abundance. Furthermore, mussel 
spat will settle predominantly on less abun- 
dant natural material even when existing spat 
densities may be quite high ( -3,800 individu- 
als/cm^ on hydroid material). This strong se- 
lectivity has been observed elsewhere (But- 
man, 1987; Schneider & Mann, 1991 a, b; 
Butman & Grassle, 1 992; Grassle et al., 1 992; 
Harvey et al., 1993; Buchanan & Babcock, 
1997). Schneider & Mann (1991a) found that 



SMALL-SCALE MUSSEL SETTLEMENT 



13 



there was a strong selectivity of epifaunal in- 
vertebrates to macroalgae with varying de- 
grees of branching. These authors concluded 
that the relationship between invertebrate 
species and algal morphology benefited the 
associated invertebrates in terms of food 
source and living space provisions. It is un- 
clear as to which different ecological proper- 
ties may be provided by different substrata 
(such as coarse-branching algae versus hy- 
droids) to the associated mussel spat, but it is 
likely that the relationship is based primarily 
on attaining a structurally secure place to in- 
habit. Indeed, results from the artificial sub- 
stratum experiments suggested that mussels 
will preferentially settle onto filamentous sub- 
strata on the basis of their physical shape. 
While these experiments with artificial sub- 
strata do not rule out the possibility that chem- 
ical cues exuded by natural algae affect mus- 
sel settlement patterns, they do support the 
idea that substratum morphology alone 
strongly influences settlement of different- 
sized mussels. The observed movement of 
mussels from the tips of artificial aquarium 
plants to node areas suggest that attraction 
cues among mussels may exist, as well as 
substratum selectivity. 

The size-frequency distribution of mussel 
spat on the various natural substrata within 
NMB wash-up samples (Fig. 5), and artificial 
substrata (Fig. 8), shows a general inverse re- 
lationship between spat size and degree of 
substratum branching. Thus, larger mussels 
(1.5-2.0 mm) appear to settle predominantly 
on coarse-branching substrata, whereas 
smaller mussels (< 0.5 mm) preferentially set- 
tle on fine-branching material (Figs. 5, 8). 
Buchanan & Babcock (1997) suggested that 
such size-specific settlement on morphologi- 
cally distinct substrata in the intertidal zone 
may be largely a result of recolonization of 
natural substrata. Our results suggest that the 
selection process also may take place in sub- 
tidal habitats and in the water column, possi- 
bly within accumulated patches of drift algae 
and hydroids. Furthermore, this size-specific 
selection persists even after the mussels 
have been transported large distances by dy- 
namic océanographie conditions evident at 
NMB. The possibility exists that the observed 
relationship between spat size and substra- 
tum type is a result of differential growth rates 
that allow the less abundant spat on coarse- 
and medium-branching algae to attain a 
larger size due to lower crowding effect. How- 
ever, the fact that all substratum types and 



mussel spat size classes were consistently 
represented in all three NMB wash-up sam- 
ples suggests that differential growth rates of 
spat within patches of drift material is unlikely. 
Furthermore, artificial substratum experi- 
ments resulted in similar trends after a period 
of only two days, which was not long enough 
to note size differences due to growth. 

A size-specific settlement pattern may rep- 
resent a choice for scaled physical stability, 
where, for example, larger mussels may re- 
quire larger morphologically stable substrata, 
such as the coarse- and medium-branching 
algae. However, smaller-scale selectivity also 
may contribute to stability requirements of 
mussel spat. Comparisons of settlement den- 
sities between node and inter-node areas 
within four different natural substrata (Fig. 6), 
and three artificial plant substrata (Fig. 9), in- 
dicate that node settlement is preferred by 
mussels settling in all types of substrata. 
Micro-scale selectivity has been demon- 
strated in laboratory flume experiments (Har- 
vey et al, 1993; Harvey & Bourget, 1995; 
Bourget & Harvey, 1998). Bourget & Harvey 
(1998) found that recruits of nine marine in- 
vertebrates, including six bivalve species, 
were more abundant in nodes as compared to 
internodes, and that the rate of recruitment in- 
dicated that passive deposition alone was not 
sufficient to obtain such an outcome. The au- 
thors ruled out the influence of differential ero- 
sion and mortality on higher settlement pat- 
terns for nodes over linear areas. Evidence 
has been accumulating that points to behav- 
ioral responses to small-scale (<1 cm) sub- 
stratum irregularities, such as pits, grooves, 
and depressions, as determinants in mi- 
croscale settlement preferences (Bourget et 
al., 1994; Nellis & Bourget, 1996; Bourget & 
Harvey, 1998). While chemical cues between 
spat and substratum, and among individual 
mussels, cannot be excluded as factors in the 
site-settlement preference experiments re- 
ported here, it is likely that morphology is the 
driving force to the strong habitat selectivity 
observed in this study. 

The large volume of wash-up material (up 
to 70,000 t/y; С Hensley, personal communi- 
cation) that can be collected only along NMB 
suggests that settlement on drift material may 
be an important part of the life history strategy 
for Perna canaliculus. Indeed, floating algal 
clumps have been found to provide alterna- 
tive habitats and transport of invertebrates 
(Schneiders Mann, 1991a; Highsmith, 1985; 
Bologna & Heck, 1999). Micro-scale selectiv- 



14 



ALFARO & JEFFS 



ity of mussel spat of different sizes onto mor- 
phologically distinctive substratum types may 
reflect a delicate level of organization within 
the transitional environment of drift material. 
Transport by drift material may be crucial to 
the invasion and retention of mussel spat onto 
new intertidal and subtidal sites (Highsmith, 
1985). Pulses of new mussel settlements 
have been observed at Scott Point, NMB after 
large accumulations of drift material on the 
rocky shore (C. Hensley, personal communi- 
cation). It is possible that entire rocky shore 
mussel communities may depend on the peri- 
odic arrival of spat from drift material. In the 
case of NMB, where rocky habitats constitute 
a very small portion of the coastal area, it ap- 
pears that most wash-up events may arrive 
on the sandy beach where mussels have little 
chance of survival, unless collected by spat 
collectors and transported to mussel farms. 
Additional research on substratum availability 
and the dynamics of océanographie transport 
of drift material at NMB is critical to further un- 
derstanding of mussel dispersal, colonization, 
and maintenance of adult populations at vari- 
ous temporal and spatial scales. 



AKNOWLEDGMENTS 

We thank С and R. Hensley, K. Campbell, 
and D. Tasker for their invaluable assistance 
with sample collections in the field. We are 
grateful for the technical support of A. Turner, 
I. MacDonald, V. Ward, and D. Todd. Algal 
identifications were done by W. Nelson, and 
S. O'Shea conducted the hydroid identifica- 
tions. The Geology Department at the Univer- 
sity of Auckland provided lab space and 
equipment for the size-frequency analyses. 
We thank K. Campbell and two anonymous 
reviewers for their comments on the manu- 
script. 



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SMALL-SCALE MUSSEL SETTLEMENT 



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MARTEL, A. 1993, Dispersal and recruitment of 
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nearshore area in west-central Lake Erie: the sig- 
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3-12. 

MARTEL, A., A. MATHIEU, S. FINDLAY, S. NEP- 
SZY & J. LEACH, 1994, Daily settlement rates of 
the zebra mussel, Dreissena polymorphe, on an 



artificial substrate correlate with veliger abun- 
dance. Canadian Journal of Fisheries and 
Aquatic Sciences, 51 : 856-861 . 

MORTON, J. & M. MILLER, 1968, The New 
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NELLIS, P & E. BOURGET 1996, Influence of 
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SCHNEIDER, F. & K. MANN, 1991a, Species spe- 
cific relationship of invertebrates to vegetation in 
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145: 101-117. 

SCHNEIDER, F. & K. MANN, 1991b, Species spe- 
cific relationship of invertebrates to vegetation in 
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Revised ms. accepted 29 March 2001 



MALACOLOGIA, 2002, 44(1): 17-22 



DETECTION OF PHENOLOXIDASE (PO) IN HEMOCYTES OF THE CLAM 

VENUS ANTIQUA 

Luis A. Mercado\ Sergio H. Marshall & Gloria M. Arenas^ 

Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias 
Básicas y Matemáticas, Universidad Católica de Valparaíso, Chile 



ABSTRACT 

The prophenoloxidase (proPO) system, particularly studied in Arthropoda, is one of the im- 
portant mechanisms of non-self recognition in the immunological response of some inverte- 
brates. Under natural conditions, the activation of the system involves the stimulation of serine 
proteases by the presence of LPS and ß-1 ,3 glycans on the surface of microorganisms. It results 
in the triggering of proPO, which in turn generates peptides responsible of opsonizing and lytic 
actions. We report here the preliminary analysis of this type of response in hemocytes of the bi- 
valve mollusc Venus antigua, both in intact cells and in crude lysates. Our strategy was first to 
monitor PO activity in whole hemocytes. Second, to confirm its presence in hemocyte lysate su- 
pernatant (HLS) by Dot and Western blot analysis using polyclonal antibodies against tyrosinase, 
the commercially available form of PO. Third, to measure its enzymatic activity in HLS by com- 
paring the oxidation of the substrate L-DOPA in the presence and absence of known activators 
or inhibitors. In this report, we demonstrate PO activity in granular cell subpopulations. In HLS, 
we quantitate the specific activity of the enzyme by spectromethomertric methods. Finally, im- 
munological characterization of HLS suggests the existence of two PO-like polypeptides, one of 
45 kDa and a second one slightly higher. 

Key words: mollusc immunity, clam hemocyte, PO activity, immunodetection. 



INTRODUCTION 

The proPO system, best studied in arthro- 
pods, corresponds to a complex of he- 
molymph enzymes and associated protein 
factors that activate the phenoloxidase (PO) 
cascade generating small peptides, which act 
as mediators of the host immune response 
(Rattcliffe, 1991; Kwon etal., 1997; Perazzolo 
& Barracco, 1 997). The system has been best 
characterized in crustaceans and other se- 
lected invertebrates (Johansson & Söderhäll, 
1989; Iwanaga et al., 1992; Hoffmann & 
Reichhart, 1997). The proPO system has also 
been described in such molluscs as Bio- 
phalaria glabrata (De Aragao & Bacila, 1976), 
Mytilus edulis (Coles & Pipe, 1994; Ren- 
wrantz et al., 1996), and Perna viridis (Asokan 
et al., 1997). Notwithstanding, the marine bi- 
valve Perna perna did not show PO activity 
(Barracco etal., 1999). 

The proPO system is primarily activated by 
polysaccharides from the microorganisms 



surface, of which ß-1,3-glucans from fungal 
cell wall and LPS-peptidoglycan complexes 
are considered to be the most classical acti- 
vators (Unestam & Söderhäll,) 1977; Char- 
alambidis et al., 1996). In arthropods, the 
proPO has been reported as a 76 kDa mole- 
cule (Aspan & Söderhäll, 1991), which is 
turned into an active form (PO) by ppA, a ser- 
ine protease of 36 kDa (Aspan et al., 1990). 
Other endogenous proteinases activate 
proPO in a way that could very well represent 
an invertebrate counterpart of the vertebrate 
complement system (Hernández-López et al., 
1 996). Once PO is formed, a number of meta- 
bolic pathways are activated oxidazing L- 
DOPA to melanin as the final common end- 
product (Nappi et al., 1995; Marmaras et al., 
1 996). A great variety of molecules have been 
described during these activated intermediate 
steps. Among them, aglutinines (Iwanaga et 
al., 1998; Gollas-Galván et al., 1999), adhe- 
sive proteins (Söderhäll et al., 1990), and 
quiñones (Ratcliffe, 1991). 



Present Address: Departamento de Bioquímica y Biología IVIolecular, Facultad de Veterinaria, Universidad Santiago de 
Compostela, Lugo, Spain. 

Address correspondence to: Instituto de Biología, Universidad Católica de Valparaíso, Avenida Brasil 2950, Casilla 4059, 
Valparaíso. Chile; garenas@ucv.cl 

17 



18 



MERCADO ETAL. 



Considering that the proPO system is well 
established in arthopods and insects and that 
there is some controversy in molluscs, we de- 
cided to verify if this relevant innate immune 
response could be assayed in another marine 
bivalve. In this report using the clam Venus 
antigua as model system, we demonstrate its 
activity. Moreover, by introducing immunolog- 
ical detection as an innovative tool, we also 
demonstrate its presence and potential evolu- 
tionary conservation. 



MATERIALS AND METHODS 
Recovery of Hemolymph 

Twelve adult specimens of Venus antiqua 
were collected from natural grounds in the 
coast of the V Region in Chile. Hemolymph 
was drained from the posterior adductor mus- 
cle of each individual (2.0 ml) using sterile 
plastic seringes (Friebel & Renwrantz, 1995). 
Resulting hemolymph was pooled in a 
polypropylene tube and kept at 4°C. 



Recovery of Hemocytes 

The hemolymph was centrifuged in a clini- 
cal centrifuge at 300 x g for 5 min at 4°C. The 
plasma was stored at 4°C until processed and 
the cellular pellet carefully resuspended in ca- 
codylate buffer (10 mM sodium cacodylate 
buffer (NCB): 0.45 M NaCI, 20 mM CaCl2, 10 
mM MgClj, pH 7.4) and intact hemocytes 
were washed twice and resuspended in the 
same buffer. 



Hemocyte Lysate Supernatant (HLS) 

Hemocytes in 800 ¡x\ of NCB buffer were 
fully homogenized in a glass-plungered ho- 
mogenator. The homogenate was centrifuged 
at 35.000 X g (Sorvall RC-5B centrifuge; HB-4 
rotor) for 20 min at 4°C and the pellet dis- 
carded. The supernatant constitutes HLS, the 
source for PO evaluation. 



Cytochemistry 

Intact hemolymph diluted 2:1 in NCB buffer 
(pH 7.4) buffer were fixed with 5% formalde- 
hyde on a polylysine-treated (0.1%) slide. 
After 15 min, slides were exposed to L-DOPA 



(0.1% in NCB 150 mM NaCI) for 12 h at room 
temperature in the dark (Renwrantz et al., 
1996). In control samples, L-DOPA was om- 
mitted. 



PO Activity in HLS 

Triplicates of 50 |.il of HLS were incubated 
with inducers, either 50 |д1 of trypsin (1 mg/ml) 
or SDS (1 mg/ml), for 5 min at 20°C. Then, 50 
ul of L-DOPA (3 mg/ml), with or without 1 mM 
CaClj, a PO cofactor, were added and further 
incubated for 30 min. In paralel, samples con- 
taining either inducer were supplemented with 
50 |.il of 0.2 mM phenilthiourea (PTU) as a PO 
Inhibitor to rule out endogenous peroxidase 
activity as well as to determine the real PO 
specific activity. Reactions were ended by di- 
lutions with deionized water to a final volume 
of 1.0 ml. PO activity was determined spec- 
trophotomethcally by measuring oxidation of 
the L-DOPA into a DOPA-chrome detected at 
490 nm. The specific activity of the enzyme 
(S.A.) is expressed as the ratio of the ab- 
sorbance value (Abs) per time per amount of 
protein. S.A. = Abs x min"^ x mg prot"^ (Smith 
& Söderhäll, 1991; Perazzolo & Barracco, 
1997). Final S.A. was expressed as average 
values ± SD, according to the t-test for inde- 
pendent samples (p < 0.05) (Statistica 5.0). 



Immunological Tests 

Polyclonal monospecific anti-tyrosinase 
(commercial mushrom PO, Sigma) were 
elicited as previously described (Mercado & 
Arenas, 1997) and used in all immunological 
detections. Protein concentration was deter- 
mined by the method of Bradford (1 976) using 
Bovine Serum Albumin (BSA) as a standard. 

Dot Blot Analysis 

5 |ig of HLS and plasma; 1 [ig of tyrosinase 
(Sigma) and horseradish peroxidase (HRP) 
(Sigma) were spotted onto nitrocellulose 
membranes and incubated for 2 h at room 
temperature with anti-tyrosinase antibody 
(1 :3,000) in TBST buffer (0.05% Tween 20 in 
TBS, pH 7.4), in the presence of 3% BSA and 
0.2% sodium azide. Second antibody was 
rabbit anti mouse IgG-alkaline phosphatase 
(PA) (1 :30,000). After 1 h incubation the mem- 
brane was developed with. BCN-NBT Fast 
(Sigma) (Dyson, 1991). 



DETECTION OF PHENOLOXIDASE IN HEMOCYTES OF THE CLAM 



19 



Western Blot Analysis 

20 |ig of HLS resolved Into a 12% SDS- 
PAGE (Laemnnii, 1970) was electrotransfered 
to an immobilon-P membrane (PVDF, Milli- 
pore) (Towbin et al., 1979) and processed as 
described above. Non-specific sites were 
blocked with 3.0% BSA in TEST. 



RESULTS 




FIG. 2. Qualitative assay of PO activity in V. antique 
hemolymph. (1) activated HLS, (2) activated HLS in 
the presence of 0.2 mM PTU, (3) non activated 
plasma, (4) 10 mM Na* cacodilate buffer. 



The existence of the proPO activating sys- 
tem is demonstrated in hemocytes of the clam 
Venus antigua. 58-60% of intact granular he- 
mocytes showed a positive PO reaction by 
the conversion of the initial substrate to L- 
DOPA into a dark end product (Fig. 1A), as 
opposed to the 1 00% absence in the negative 
controls (Fig. 1B). In HLS fractions, pigment 
formation was also indicative of PO activity, 
although a slight positive reaction is detected 
in plasma exposed to L-DOPA. Nontheless, 
there is no dark precipitate in the presence of 
the enzyme inhibitor PTU (Fig. 2). 

In enzymatic activity assays, proPO in HLS 
was activated by both inducers trypsin and 
SDS, with a slighter higher value for the latter 
in the presence of calcium (Fig. 3). 

Dot blot analysis demonstrate the speci- 
ficity of our antityrosinase antibody (Fig. 4). 
The polyclonal antibody showed no cross-re- 
activity with either HRP or the negative con- 
trol, while an intense reaction is seen in the 
HLS sample in contrast to a feeble one in 
plasma. 

Finally, Western blot shows in HLS, two im- 
munoreactive bands: a very intensive one 
with a mobility of approximately 45 kDa, and a 
less reactive slightly higher molecular weight 
band, which correlates with the stained profile 
of the corresponding gel (Figs. 5: lanes 3 and 
2, respectively). 



DISCUSSION 

The presence of the PO system among dif- 
ferent groups of invertebrates is under dis- 
cussion. In arthropods, it has been described 
exclusively in the hemocytes of some cray- 
fish, such as Penaeus californiensis and P. 
paulensis (Hernández-López et al., 1996; 
Perazzolo & Barracco, 1997), or solely in the 
plasma of insects Bombyx mori ana Manduca 
sexta (Ashida et al., 1983; Saul et al., 1987). 
In bivalve molluscs, Renwrantz et al. (1996) 
described PO activity in hemocytes of Mytilus 
edulis, whereas in Perna viridis Asokan et al. 
(1997) found it in both plasma and hemo- 
cytes. 

Our study in clams supports the putative 
existence of PO activity in marine inverte- 
brates. We demonstrate that a high percent- 
age of bulk granular hemocytes display the 
activity (Fig. 1). Moreover, statistically signifi- 
cant spectrophotometric assays measuring 
the oxidation of L-DOPA to DOPA-chrome 
seem to be good indicators of PO activity in 
HLS from Venus antigua (Fig. 3). The basal 
activity detected in plasma (Fig. 2), might be 
interpreted as a reflection of the physiological 
stage of the specimens analyzed. Finally, our 
novel immunological approach suggest the 
existence of two size-specific polypeptides 




FIG. 1. A, Hemocytes subpopulations from Venus antiqua with a positive PO oxidizing reaction. Monolayer 
incubated with L-DOPA as substrate, 1.000 x. B, Hemocytes incubated in the absence of substrate. 



20 



MERCADO ETAL. 



Assay 


Ft) activity in HbS. 




Abs 4Wiim (min ' \ mg pro<^). 


Control 


0.064+ 0.016 


Trypan - C'a"* * 


0.133^0.033 


SDS C'a^ * 


0.277 + 0.104 


Trypsin ** 


0.018+0.012 


SDS** 


0.037 + 0.009 


Tr>pan 0.2шМРТи 


0.006 + 0.002 


SDS 0.2 m M PTII 


0.007 + 0.001 




FIG. 4. PO immunodetection in Dot blot. (1) acti- 
vated HLS and (2) non activated plasma, (3) com- 
mercial tyrosinase, (4) HRP. Each dot contains 1 |ng 
of protein. 



The enzymatic activity values aie expressed as average j_ SD. 

* The substrate ЬТЮРА was added in the presence оГЮшМ СаОг. 

** The substrate L ЕЮРА was Ca' fiée added. 

FIG. 3. Differential effect of Trypsin, SDS, Ca^^ and PTU over PO activity in HLS. Spectrophotometric quan- 
titation of PO specific activity. 

also resulted in two proteins with relative mol- 
ecular weights of 381 ± 13.7 and 316 ± 11.1 
kDa, respectively (Renwrantz et al., 1996). 
This might very well mean that the two Venus 
antiqua polypeptides could represent sub- 
units of a larger enzyme. 

In spite of the fact that the PO values re- 
ported for HLS of Venus antiqua are low com- 
pared to other species, we think they are real. 
PO activity fully responds to serine proteases, 
such as trypsin (Fig. 3), in agreement with 
other reports (Nellaiappan & Sugumaran, 
1996). Additionally, the same figure summa- 
rizes the expected activating effect of SDS 
(Moore & Flurkey, 1990). Also, for both cases, 
the inducing effect of Ca^"" as a co-factor, as 
well as the fully inhibitory action of PTU (Sug- 
umaran et al., 1988). Further studies tending 
to optimize the measurement of PO activity in 
Venus antiqua or in other mollusc model sys- 
tem might contribute to asses the observa- 
tions reported here. 

The specific recognition of two polypeptides 
in hemocytes of Venus antiqua using antibod- 
ies elicited agaist a commercial enzyme of 
mushrom origin clearly suggests the exis- 
tence of common epitopes of evolutionary 
conserved enzymes, situation expected for in- 
nate immunological responses. 



kDa 

205 
97 
66 

45 

29 



i 



kPa 

66— 

45— 

29— 



2 3 






45 



FIG. 5. SDS/PAGE characterization of HLS and 
Western blot analysis. (1) HLS, 50 цд (10% gel) 
Coomassie blue stained, (2) HLS, 20 цд (12% 
stained gel). (3) Western blot of a parallel gel shown 
in lane 2. Reactive bands indicated by slim arrows. 
Relative molecular weights in kDa indicated on the 
left of each lane. 



SUMMARY AND CONCLUSIONS 



(50 and 45 kDa), although different from those 
of crayfish molecule (Aspan & Söderhäll, 
1991), are highly conserved and presumably 
responsible for the detected PO activity in 
Venus antiqua. In Mytilus edulis, the native 
enzyme characterized in non dénaturant gels 



The PO activating system was studied in 
hemocytes of the bivalve mollusc Venus anti- 
qua. Qualitative assays were used to demon- 
strate the formation of dark precipitates in in- 
tact hemocytes and quantitative assays of 
enzyme activity measured by oxidation of L- 



DETECTION OF PHENOLOXIDASE IN HEMOCYTES OF THE CLAM 



21 



DOPA to DOPA-chrome formation. Immuno- 
logical detection was incorporated in order to 
characterize the putative polypeptides in- 
volved in the activation process in mollusc. 
We conclude that the PO system is present 
mostly in hemocytes of Venus antiqua. The 
putative polypeptides recognized by antibod- 
ies elicited to a mushrom enzyme, suggest a 
high degree of evolutionary conservation. Al- 
though they do not match the expected 
molecular weights described for crayfish, they 
might correspond to subunits of the native en- 
zyme described in Mytillus edulis. In sum- 
mary, our results tend to indicate a structured 
innate immune response active in a species 
of marine bivalve. 



ACKNOWLEDGMENTS 

The present work was financed by the Di- 
rección de Investigación, Vice Rectoría de In- 
vestigación y Estudios Avanzados, Universi- 
dad Católica de Valparaíso, Chile. 



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Revised ms accepted 25 March 2001 



MALACOLOGIA, 2002, 44(1): 23-31 

A SURVEY OF GENETIC VARIATION AT ALLOZYME LOCI AMONG GONIOBASIS 
POPULATIONS INHABITING ATLANTIC DRAINAGES OF THE CAROLINAS 

Robert T. Dillon, Jr. & Andrew J. Reed 

Department of Biology, College of Charleston, Charleston, South Carolina 29424, U.S.A.; 

dillonr@cofc.edu 

ABSTRACT 

We estimated gene frequencies at eight polymorphic enzyme loci in 12 populations of Go- 
niobasis Uom Atlantic drainages of North Carolina, South Carolina, and Georgia. Our sample in- 
cluded four populations of G. próxima from the piedmont, five populations of G. catenaria cate- 
naria, one population of G. catenaria postellii, and two populations of G. catenaria dislocata from 
the coastal plain. The fit to Hardy-Weinberg expectation was good within all populations of both 
species (F|g = 0.042 próxima, 0.035 catenaria), while levels of interpopulation divergence were 
high (Fg.^ = 0.461 próxima, 0.564 catenaria). In spite of slightly overlapping geographic distribu- 
tions, and instances of striking similarity in shell morphology between the two species, G. próx- 
ima and G. catenaria were genetically distinct (average values of Nei's D near 1 .0), with no evi- 
dence of hybridization. Although their ranges are fragmented into numerous isolated and 
genetically distinct populations, both species remain broadly recognizable across states, 
drainages, and physiographic regions. The relationship between G. catenaria posteilii and seven 
other nominal species and subspecies of Goniobasis sometime recognized from Atlantic 
drainages of Georgia is called into question. 

Key words: electrophoresis, isozymes, freshwater, Carolina, Georgia, gastropods, pleuroceri- 
dae, Eiimia. 



INTRODUCTION 

Freshwater snails of the family Pleuroceri- 
dae are abundant, widespread, and Important 
elements of the grazing and shredding com- 
munity in lotie ecosystems throughout the 
southeastern United States (Power et al., 
1988; McCormick & Stevenson, 1991; Hill et 
a!., 1995; Huryn et al., 1995; reviews by Fem- 
inella & Hawkins, 1995; Dillon, 2000). Their di- 
verse and often Isolated populations, easily 
sampled year around, have made pleuro- 
cerids attractive models for the study of diver- 
gence and speciation (Chambers 1980, 1982; 
Dillon 1989, 1991; Bianchi et al., 1994; Dillon 
& Lydeard, 1998; Lydeard et al., 1997; Holz- 
nagel & Lydeard, 2000). Yet the pleurocerid 
fauna of much of the southeastern United 
States remains obscure and poorly docu- 
mented, and their systematic relationships 
unclear 

Goodrich (1942) listed three pleurocerid 
genera, 15 species, and 26 subspecies in- 
habiting Atlantic drainages from New England 
to Texas. Four of these taxa will be treated in 
the present work: Goniobasis próxima (Say, 
1825) from the "highlands of North and South 



Carolina"; G. catenaria dislocata (Ravenel, 
1834) inhabiting "headstreams" in Virginia, 
east-central North Carolina, and South Car- 
olina; G. catenaria catenaria (Say, 1 822) from 
"springs of eastern South Carolina"; and G. 
catenaria postellii (Lea, 1858) from the "Al- 
tamaha, Ogeechee, and Canoochee Rivers" 
of Georgia. Goniobasis catenaria and G. 
próxima were two of the first four species of 
this diverse North American genus to reach 
formal description. 

Although altering the generic name to 
"Eiimia," Burch (1982) and Burch & Totten- 
ham (1980) generally adopted Goodrich's un- 
derstanding of the pleurocerid fauna of North 
America as outlined above. But the system- 
atic relationships among the Georgia repre- 
sentatives of this group have also been re- 
viewed by Clench & Turner (1956), Krieger 
(1977), Chambers (1990), and Mihalchik 
(1998). Specific nomina applied to popula- 
tions from Georgia Atlantic drainages have in- 
cluded Goniobasis bentoniensis {Lea, 1862), 
G. boykiniana viennaensis (Lea, 1862), G. 
mutabilis (Lea, 1862), G. mutabilis timida 
(Goodrich, 1942), G. timida (Goodrich, fide 
Mihalchik, 1998), G. postellii {Lea, 1858), and 



23 



24 



DILLON & REED 



G. suturalis (Haldeman, 1840). All these 
names would be junior synonyms of G. cate- 
naria, to the extent that G. catenaria extends 
into Georgia, as suggested by Goodrich. 

Between 1986 and 1995, Dillon & Keferl 
(2000) surveyed 629 aquatic sites distributed 
evenly throughout South Carolina, document- 
ing approximately 30 pleurocerid populations 
at 44 sites. They found ten populations of G. 
próxima in small streams in or near the Ap- 
palachian foothills, confirming Goodrich's 
(1942) report. Ten populations of G. catenaria 
catenaria were found at 26 sites in streams 
and rivers of varying size through the South 
Carolina midlands, expanding the known 
range of that species substantially from that 
suggested by Goodrich. The disjunct distribu- 
tion of these populations suggested to Dillon 
& Keferl that this species may have been 
highly impacted by agricultural siltation and 
impoundment. Dillon & Keferl also reported 
six G. catenaria dislocata populations at eight 
sites in small streams of the coastal plain. 

Goniobasis catenaria populations were oc- 
casionally found well into the South Carolina 
piedmont, closely neighboring populations of 
G. próxima in upstate tributaries of the 
Broad/Santee and Catawba/Santee. And the 
similarity in shell morphology between G. 
próxima and G. catenaria dislocata of the 
coastal plain was striking. The shells of both 
species were identically shaped and marked 
by a single spiral carina. Goniobasis catenaria 
dislocata shells differed from G. próxima only 
by the presence of faint axial costae near the 
apex, disappearing after the first 2-3 whorls. 
Populations of both species inhabit small, 
rapidly flowing streams, G. catenaria dislo- 
cata generally being found in those rather un- 
usual regions of the coastal plain (often bor- 
dering large rivers) with slope and marl 
exposure. But whether the gross shell similar- 
ity of G. próxima and G. catenaria dislocata 
might reflect a genuine genetic relationship, 
or might be the result of convergence, Dillon & 
Keferl could not determine. 

The genetics of North Carolina and Virginia 
populations of G. próxima have been the sub- 
ject of intensive study for 20 years (Dillon & 
Davis, 1980; Dillon, 1984a; Stiven & Kreiser, 
1994). Intrapopulation variation seems to be 
unusually low, and interpopulation divergence 
high, as might be expected from populations 
of such a poorly mobile animal isolated in 
small streams of the piedmont and mountains 
(Dillon, 1984b). Despite the occurrence of 
fixed differences at multiple enzyme loci, how- 



ever, transplants and artificial introductions 
have revealed no evidence of reproductive 
isolation among G. próxima populations (Dil- 
lon, 1986, 1988a). 

The purposes of the present work are 
threefold. First we compare the levels of intra- 
and interpopulation genetic variance ob- 
served in G. catenaria to the better-known G. 
próxima. Second, we evaluate the genetic 
distinctiveness of G. próxima and G. cate- 
naria, paying particular attention to the possi- 
bility of hybridization. Third, we examine evi- 
dence that populations of G. catenaria 
dislocata might be extralimital G. próxima, 
rather than G. catenaria with shell morphol- 
ogy convergent on G. próxima. 



METHODS 

We sampled five populations of G. cate- 
naria catenaria, two populations of G. cate- 
naria dislocata from eastern South Carolina, 
and one G. catenaria postellii from Georgia. 
The four populations of G. próxima we sam- 
pled included Yad from northwestern North 
Carolina, which we have previously compared 
to other populations of Goniobasis (as Yad in 
Dillon & Davis, 1980; Yad1 in Dillon, 1984b, 
1988b) and which served as a control for the 
present investigation. Our Georgia site was 
identical to "Station X" of Krieger & Burbanck 
(1976) and YELD of Krieger (1977), inhabited 
by a population identified as "G. suturalis" in 
the former publication and "G. boykiniana vi- 
ennaensiä' in the latter Population CIrk of G. 
catenaria and population Bull of G. próxima 
were sampled from tributaries of the Broad 
River separated by only about 15 km through 
water The sites at which we collected all 12 
populations may be located in Figure 1. Lo- 
cality data for all sites are provided in the Ap- 
pendix. Voucher specimens have been de- 
posited in the Academy of Natural Sciences of 
Philadelphia. 

At each site we collected at least 30 indi- 
vidual snails, which were cracked and stored 
in Tns-phosphate 7.4 tissue buffer at -70°C 
upon return. Allozyme variation was resolved 
from whole-animal homogenates by horizon- 
tal starch gel electrophoresis, using equip- 
ment and techniques as previously described 
(Dillon, 1985, 1992; Dillon & Lydeard, 1998). 
We initially screened ten enzyme systems 
and six buffers by requiring that polymor- 
phism interpretable as the product of codomi- 
nant Mendelian alleles be expressed in a 



GOA//Oß/4S/S POPULATION GENETICS 



25 



Pee Dee 




Santee 



Altamaha 



FIG. 1. The study area, showing primary drainages and sample sites. Circles indicate G. catenaria, and 
squares G. próxima. 



comparison of G. próxima population Yad, G. 
catenaria postellii popu\at\on Yel, and G. cate- 
naria dislocata population Srp. 

Ultirnately, we compared all populations on 
the basis of eight loci, most resolved on two 
buffer systems, as follows. The AP6 buffer of 
Clayton & Tretiak (1972) was used to resolve 
mannose-phosphate isomerase (MPI, EC 
5.3.1.8), 6-phosphogluconate dehydroge- 
nase (6PGD, EC 1.1.1.44) and isocitrate de- 
hydrogenase (IDHF and IDHS, EC 1 .1 .1 .42 - 
cathodal and anodal loci, respectively). A 
TEB8 buffer (Shaw & Prasad 1970) was used 
for esterases (EST1, EC 3.1. 1.2 -the strong, 
slow locus only), xanthine dehydrogenase 
(XDH, EC 1.2.1.37), MPI, and IDHF. The 



Poulik (1 957) buffer was used for octopine de- 
hydrogenase (ODH, EC 1.5.1.11), glucose- 
phosphate isomerase (GPI, EC 5.3.1.9) and 
EST1. 

Chambers (1980) demonstrated that inher- 
itance of allozyme phenotype is Mendelian at 
the 6PGD locus in G. floridensis. Dillon (1986) 
verified Mendelian inheritance at GPI, ODH, 
and EST1 by mother-offspring analysis in G. 
próxima. The designations of putative alleles 
used for population Yad were retained from 
the system of Dillon (1984b) for GPI, MPI, 
ODH, EST1 and XDH. For those loci which 
had not previously been examined for popula- 
tion Yad (6PGD, ISDHF ISDHS), the most 
common Vad allele was named "100." Then 



26 



DILLON & REED 



alleles in all other populations were named 
according to the mobility of their allozymes in 
millimeters relative to the Vad standard. 
Data were initially analyzed using BIOSYS- 

1 (Release 1.7; Swofford & Selander, 1981). 
Genotype frequencies at all polymorphic loci 
were tested for conformance to Hardy-Wein- 
berg expectation within populations using chi- 
square statistics, with Yates correction in 2 x 

2 cases, pooling rare classes as required. 
Mean F-statistics (Whght, 1978) were calcu- 
lated across loci for the four populations of G. 
próxima and the eight populations of G. cate- 
naria separately. Values of Nei's (1978) unbi- 
ased genetic similarity and distance were cal- 
culated pairwise over all populations, as well 
as the chord distance of Cavalli-Sforza & Ed- 
wards (1967). The matrix of chord distances, 
which are Pythagorean in Euclidean space 
(Wright, 1 978), was input to STATISTICA (Re- 
lease 5.0, StatSoft, Inc.) and clustered using 
the method of unweighted pair-group averag- 
ing (UPGMA). 



RESULTS 

Example shells are shown in Figure 2. Typ- 
ical G. catenaria bore shells with both spiral 
costae (or cords) and axial costae (or ribs) in- 
tersecting to form nodules. The shells of G. 
próxima had no axial costae and only a single 
spiral costa (or carina) becoming obsolete 
with age. Typical G. catenaria shells also 
tended to be wider per unit length than G. 
próxima. The shells of the Yel population, 
nominally G. catenaria postellii, did not differ 
noticeably from those of typical G. catenaria 



catenaria. But as noted by Dillon & Keferl 
(2000), the shells of G. catenaria dislocata 
were narrower and lacked both spiral and 
axial costae on later whorls, rendering them 
more similar to G. próxima than typical G. 
catenaria (Dillon & Keferl 2000). 

For allozyme analysis, sample sizes aver- 
aged across loci ranged from N = 29 at West 
to N = 43 at Vaci (Appendix). Sample sizes at 
all 12 X 8 loci examined were greater than 30, 
except N = 29 for Bull (XDH), N = 26 for Bull 
(EST1), N = 14 for West (IDH), N = 20 for 
Morg, (IDH), and N = 26 for Morg (GPI, XDH). 

Allele frequencies are given in Table 1. 
Over all observations, a total of 26 loci were 
polymorphic by the 95% criterion, a value 
somewhat higher than has been observed in 
comparable surveys of pleurocerid population 
genetics previously published (Chambers, 
1980; Dillon & Davis, 1980; Dillon, 1984b; Dil- 
lon & Lydeard, 1998). Fits to Hardy-Weinberg 
expectation were excellent within popula- 
tions. Chi-square tests uncovered no signifi- 
cant differences between observed genotypic 
frequencies and those expected from Hardy- 
Weinberg equilibrium in any case. The values 
of F|g for both G. próxima and G. catenaria av- 
eraged over eight loci were negligible (Table 
2). 

As might be expected from their highly iso- 
lated population structure, divergence among 
the four G. próxima populations was high (Fg^ 
= 0.461; Table 2). Table 1 shows fixed (or 
nearly fixed) differences at a minimum of one 
locus between all pairs of G. próxima, except 
Morg Bull. Divergence among the eight G. 
catenaria was not as striking upon first exam- 
ination, the tho of populations from the middle 






4^ 




FIG. 2. Example shells from the four taxa studied. From left, Goniobasis catenaria catenaria (СЩ, G. cate- 
naria dislocata {Srp), G. próxima {Bull), and G. catenaria postellii ( Yel). The standard length of the G. cate- 
naria catenaria shell is 1 .75 cm, the remaining shells are figured at the same scale. 



GONIOBASIS POPULATION GENETICS 



27 



TABLE 1. Allele frequencies at 8 enzyme loci in 12 populations of Goniobasis Uom southern Atlantic 
drainages of the United States. 





allele 




G. próxima 










G. catenaria 








Locus 


Yad 


West 


Morg 


Bull 


CIrk 


Uwh 


inch 


Cola 


Sant 


Srp 


McC 


Yel 


EST1 


100 


0.167 


0.191 


0.513 






0.219 


0.188 














103 


0.802 


0.794 


0.474 


0.885 


1.000 


0.734 


0.813 


1.000 


1.000 


0.679 


0.789 


0.524 




106 


0.031 


0.015 


0.013 


0.115 




0.047 








0.321 


0.211 


0.012 




107 
























0.463 


GPI 


98 


















0.014 


0.976 








100 










1.000 


1.000 


1.000 


1.000 


0.986 


0.024 


0.162 


1.000 




102 


1.000 


1.000 


1.000 


1.000 




















105 






















0.784 






110 






















0.054 




IDHF 


93 

97 

99 

100 

103 


1.000 


1.000 


0.125 

0.750 
0.050 


1,000 


0.953 


0.016 






0.319 










105 






0.075 




0.047 


0.984 


1.000 


1.000 


0.681 


1.000 


1.000 


1.000 


IDHS 


100 


1.000 


1.000 


1.000 


1.000 




















104 










1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


MPI 


95 


0.833 




0.200 


0.750 




















100 


0.167 


1.000 


0.800 


0.250 


1.000 


1.000 


1.000 


1.000 


1.000 


0.989 


1.000 


1.000 




103 




















0.011 






ODH 


106 
109F 
111 
113F 


0.738 
0.125 

0.138 


1.000 


0.145 
0.855 


1.000 














0.118 


0.431 




114 










1.000 


0.969 


1.000 


1.000 


1.000 


1.000 


0.882 


0.569 




118 












0.031 














6PGD 


100 
105 
110 


1.000 


0.882 
0.118 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


0.522 
0.422 
0.056 


XDH 


97 




1.000 


0.731 


0.603 








0.015 


0.875 


0.390 








98 


1.000 




0.269 


0.397 


1.000 


1.000 


1.000 


0.985 


0.125 


0.610 


1.000 


0.568 




100 
























0.432 



TABLE 2. Values of Wright's (1978) F-statistics 
averaged over eight loci for four populations of G. 
próxima and eight populations of G. catenaria. 



G. próxima 



G. catenaria 



F,s 
F,T 
F.. 



0.042 
0.484 
0.461 



0.035 
0.580 
0.564 



of the Study area {Uwh -inch -Cola) being 
genetically Indistinguishable. But the south- 
ern populations were nnore divergent. The Yel 
population of G. catenaria postellii had un- 
usual alleles in high frequency at three loci 
(EST1 , 6PGD, XDH), and both the Srp popu- 
lation of G. catenaria dislocata and the McC 
population of G. catenaria catenaria were 
strikingly distinct at the GPI locus. Population 
CIrk was distinguished by a unique IDHF al- 
lele. Consequently, the mean value of F for 



the eight G. catenaria populations over the 
three-state region was 0.564, similar to that 
observed for G. próxima. 

Nei's statistics among all populations are 
presented in Table 3, and the result of the 
и PGM A cluster analysis based on interpopu- 
lation chord distances is shown in Figure 3. 
Each G. catenaria dislocata population was 
more similar to its neighboring G. catenaria 
catenaria population (I = 0.86, 0.89) than the 
pair of dislocata were to each other (I = 0.81 ). 
This tends to support the hypothesis that the 
two populations of G. catenaria dislocata 
have lost shell sculpture independently. 

Reinforcing the impression left from the F- 
statistics, Figure 3 suggests that the levels of 
interpopulation divergence within G. próxima 
and G. catenaria are comparably high. The 
four G. próxima populations are nevertheless 
quite distinct from the eight G. catenaria pop- 
ulations, sharing no alleles at three loci (GPI, 



28 



DILLON & REED 



TABLE 3. Nei's (1978) statistics among pairs of Gon/obas/s populations. Unbiased genetic iden- 
tity is shown below \he diagonal, and unbiased genetic distance above. 







Yel 


McC 


Srp Sant Cola 


inch 


Uwh 


Clrk 


Bull 


Morg West 


Yad 


Yel 


— 


0.22 


0.29 0.21 0.11 


0.11 


0.10 


0.27 


1.48 


1.21 1.24 


1.34 


McC 


0.81 


— 


0.15 0.24 0.10 


0.10 


0.11 


0.24 


1.08 


0.96 1.06 


0.92 


Srp 


0.75 


0.86 


- 0.21 0.17 


0.17 


0.17 


0.33 


1.08 


0.91 0.95 


1.10 


Sant 


0.81 


0.78 


0.81 - 0.12 


0.13 


0.13 


0.21 


0.96 


0.80 0.73 


1.23 


Cola 


0.90 


0.91 


0.84 0.89 


0.00 


0.01 


0.12 


1.08 


0.98 1.05 


0.93 


inch 


0.90 


0.91 


0.84 0.88 1.00 


— 


0.00 


0.13 


1.13 


0.96 1.08 


0.94 


Uwh 


0.90 


0.90 


0.84 0.88 0.99 


1.00 


— 


0.13 


1.14 


0.95 1.09 


0.95 


Clrk 


0.76 


0.79 


0.72 0.81 0.89 


0.88 


0.88 


— 


1.07 


1.00 1.05 


0.92 


Bull 


0.23 


0.34 


0.34 0.38 0.34 


0.32 


0.32 


0.34 


— 


0.09 0.28 


0.16 


Morg 


0.30 


0.38 


0.40 0.45 0.38 


0.38 


0.39 


0.37 


0.91 


0.15 


0.26 


West 


0.29 


0.35 


0.39 0.48 0.35 


0.34 


0.34 


0.35 


0.76 


0.86 


0.28 


Yad 


0.26 


0.40 


0.33 0.29 0.40 


0.40 


0.39 


0.40 


0.85 


0.77 0.76 


~ 


■ 




' 






. r 


— n ' ■ : ■ ■ 


Yad 
Morg 












■ 














: 1 








G. próxima 


















^ 








Bull 














































West 






: 


: 






























Clrk 






































Uwh 






























Lnch 






























Cola 






































McC 






: 












• 












- 












Sant 
Yel 


G. catenan 
1 G. catenar 














a dislocata 




















a postellii 





0.7 0.6 0.5 0.4 0.3 0.2 0.1 

Cavalli-Sforza & Edwards Chord Distance 



0.0 



FIG. 3. UPGMA Cluster analysis of the 12 study populations, based on a matrix of genetic distances calcu- 
lated using the chord method of Cavalli-Sforza & Edwards (1967). 



ODH, IDHS) and almost connpletely distinct at 
IDHF. Table 1 shows no evidence of hy- 
bridization between the species, in the 
Broad/Santee drainage between С/л/с and Bull 
or elsewhere. 



DISCUSSION 

Although inhabiting a more moderate cli- 
mate and a less mountainous environment, 
the population genetic structure of G. cate- 
naria does indeed resemble that of the better- 
studied G. próxima. The similarity of the G. 
catenaria populations inhabiting the Pee Dee 
drainage {Uwh and Lnch) and the (apparently 
isolated) Santee populations (particularly 
Cola) was unexpectedly high. But the overall 
values of Wright's F-statistics and most val- 
ues of Nei's genetic distances among G. cate- 



naria populations were comparable to values 
obtained from G. próxima. 

Values of genetic similarity and distance 
weakly suggest that our two dislocata popula- 
tions may have independently lost shell sculp- 
turing, Sanf deriving from Cola and Srp from 
McC. But as indicated in Figure 3 and Table 3, 
the two putative source G. catenaria catenaria 
populations, Cola and McC, are more similar 
to each other than either is to its G. catenaria 
dislocata neighbor. Sant and Srp do share a 
unique allele (GPI98) not found in any other 
population, and are also more similar to each 
other at the XDH locus than either is to G. 
catenaria catenaria. Additional data will be re- 
quired before the origin of G. catenaria dislo- 
cata can be proposed with any confidence. 

It is clear, in any case, that G. catenaria dis- 
locata populations are not extralimital G. próx- 
ima, nor do they bear more genetic similarity 



GONIOBASIS POPULATION GENETICS 



29 



to G. próxima than to other G. catenaria of 
more typical shell morphology. We found no 
evidence of hybridization between the two 
species, in the upper Santee tributaries where 
they are closely neighboring or elsewhere. 
Goniobasis próxima and G. catenaria are 
quite distinct. 

As might be predicted from its geographic 
situation on the periphery of the study area, 
population Yel was the most genetically dis- 
tinctive of the populations here identified as 
G. catenaria (Fig. 3). But the level of diver- 
gence displayed clearly does not warrent the 
removal of this population to another specific 
name. Population West is more different from 
other G. próxima than population Yel is from 
other G. catenaria. Goodrich's (1942) sugges- 
tion that Goniobasis populations from this re- 
gion of Georgia be referred to G. catenaria as 
a subspecies postellii, rather than distin- 
guished as a separate species, would seem 
to have considerable merit. 

Based on a comparison of his freshly col- 
lected shells to shells in the University of 
Michigan collections, Krieger (1977) identified 
Georgia populations of Goniobasis from the 
Atlantic drainages as G. postellii in the 
Oconee River, G. suturalis (= G. mutabilis) in 
the Yellow River above Porterdale, and G. 
boykiniana viennaensis in the Apalachee 
River and in the Yellow/Ocmulgee River 
below Porterdale. Mihalcik (1998) identified a 
population of Goniobasis from Snapping 
Shoals on the South River, a tributary of the 
Ocmulgee 20 km west of Porterdale, as G. 
mutabilis. She identified a population from an 
Ocmulgee tributary downstream about 350 
km from Porterdale as G. tímida, and sug- 
gested that a third population from the Atlantic 
tributaries of Georgia, Rocky Creek of the 
Oconee drainage, might be an undescribed 
"Species C." Mihalcik referred Goniobasis 
populations from the Savannah River to G. 
bentoniensis. 

Neither Krieger nor Mihalchik examined 
populations from South Carolina, nor did ei- 
ther researcher consider the possibility that 
Georgia Goniobasis might be referable to the 
older name G. catenaria. Resolution of the 
longstanding systematic confusion regarding 
the Goniobasis of Georgia would seem a fer- 
tile direction for future studies. 



ACKNOWLEDGMENTS 

We thank Mr. Kevin Swift for help with the 
field work. This research was supported by 



grants from the Biology Department, College 
of Charleston. 



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Revised ms. accepted 25 March 2001 



APPENDIX 

Identification and locality data for study popu- 
lations. The sample sizes given are averaged 
over the 8 enzyme loci reported in Table 1 . 

Bull-G. próxima (N = 31). Bullock's Creek at 
SC 889 bridge (Boheler Road), 10.9 km 
NNW of York, York Co., South Carolina. 
8ri9'W, 35°05'N. Same as site 20p of 
Dillon & Keferl (2000). 

CIrk-G. catenaria catenaria (N = 32). Clarks 
Fork Creek at SC 55 bridge, 16.1 km NW 
of York, York Co., South Carolina. 
81°24'W, 35°05'N. Same as site 17c of 
Dillon & Keferl (2000). 



GONIOBASIS POPULATION GENETICS 



31 



Cola-G. catenaria catenaria (N = 34). Big 
Cedar Creek at SC 59 bridge (Wildflower 
Road), 21.3 km N of Forest Acres, Rich- 
land Co., South Carolina. 81°06'W, 
34°1 1 'N. Same as site 23c of Dillon & Ke- 
ferl (2000). 

Lncti-G. catenaria catenaria (N = 32). 
Lynches River at SC 265 bridge, 3.8 km 
SW of Jefferson, Chesterfield/Lancaster 
Cos., South Carolina. 80°26'W, 34°38'N. 
Same as site 40c of Dillon & Keferl 
(2000). 

McC-G. catenaria catenaria (N = 38). 
Stevens Creek at SC 23 bridge, 24.6 km 
WSW of Edgefield, Edgefield/McCormick 
Cos., South Carolina. 82°11 'W, 33°44'N. 
Same as site 4c of Dillon & Keferl (2000). 

Morg-G. próxima (N = 33). Small tributary of 
Clear Creek at bridge 50 m N of U.S. 64, 
5 km S of Morganton, Burke Co., North 
Carolina. 81°45'W, 35°40'N. 

Sant-G. catenaria dislocata (N = 36). Head 
of Chapel Branch, Santee, Orangeburg 
Co., South Carolina 80°29'W, 33°30'N. 



Same as site 28d of Dillon & Keferl 

(2000). 
Srp-G. catenaria dislocata (N = 39). Lower 

Three-Runs Ck at SC 70 bridge, 9 km S 

of Snelling, Barnwell Co., South Carolina. 

81°27'W, 33°11'N. Same as site 6d of 

Dillon & Keferl (2000). 
Uwti — G. catenaria catenaria (N = 32). 

Uwharrie River at NC 109 bridge, 1 km 

NW of Uwharrie, Montgomery Co., North 

Carolina. 80°01'W, 35°26'N. 
West-G. próxima (N = 29). Small tributary of 

the Chauga River at SC 1 96 bridge, 1 km 

W of Mountain Rest, Oconee Co., South 

Carolina. 83°10'W, 34°52'N. 
Yad-G. próxima (N = 43). Naked Creek at 

NC 1154 bridge, 5.2 km N of Ferguson, 

Wilkes Co., NC. 81°22'W, 36°09'N. 

Same as Yadk oi Dillon & Davis (1980), 

Vad/of Dillon (1984). 
Yel—G. catenaria postellii (N = 40). Yellow 

River below dam at Porterdale, Newton 

Co., Georgia. 83°53'W, 33°34'N. 



MALACOLOGIA, 2002, 44(1): 33-46 

SPATIAL DISTRIBUTION, DENSITY AND LIFE HISTORY IN FOUR ALBINARIA 
SPECIES (GASTROPODA, PULMONATA, CLAUSILIIDAE) 

Sinos Giokas^ & Moysis Mylonas^ 



ABSTRACT 

In this study, we analysed field sequential data on density, mortality, and spatial distribution of 
four Albinaria species. We also recorded and compared a series of life-history traits, including 
the onset and end of aestivation, copulation, oviposition, and hatching. Our aim was to obtain 
more information on the ecology of Albinaria, to reveal possible life-history patterns, and to un- 
derstand the association of life-history characteristics with both extrinsic (density independent) 
and intrinsic (density dependent) factors and microevolutionary processes. The onset of aesti- 
vation period occurs always in April and is independent of the end of rainfall, whereas awaken- 
ing and copulation is actually synchronous and occurs after the first autumn rains. The duration 
of the oviposition and hatching period is short, but oviposition can be prolonged under favourable 
climatic conditions. Density is high, but its fluctuations were not related to climatic or time factors. 
Any density dependent pattern resulting from competition was not detected. Higher mortality did 
not coincide with aestivation, and usually relatively high mortality results after hatching. Juveniles 
usually exhibit higher mortality than adults. Spatial distribution of these rock-dwelling snails is al- 
ways highly contagious. Clustering behaviour was probably influenced by the substratum, mo- 
saic or uniform, and the occurrence of crevices. Predictions about the population dynamics of 
these iteroparous, long-lived species were not possible because of sampling inadequacies 
and/or because fluctuations of population structure, density, mortality and spatial distribution 
were random. 

Key words: Albinaria, Greece, life history, density, mortality, spatial distribution. 



INTRODUCTION 

Population dynamics is affected, either di- 
rectly or indirectly, by such density-indepen- 
dent factors as climate, and/or such density- 
dependent factors as prédation, disease and 
competition. Life-history studies are essential 
for understanding the role of these factors and 
the action of natural selection (Stearns, 1 992). 
However, in land snails the influence of these 
factors is often abstruse or inadequately un- 
derstood. Though regulated life cycles are not 
likely to be distinguishable from random den- 
sity fluctuations (May, 1975; Cook, 1990), sev- 
eral studies indicate the effect of environmen- 
tal conditions on abundance (Baur & Raboud, 
1988; Heller & Dolev, 1994; Lazaridou-Dimitri- 
adou & Sgardelis, 1997), or the influence of 
intraspecific crowding on density, via its direct 
or indirect affect on individual fecundity, sur- 
vival, locomotion, adult size, and growth rate 
(Yom-Tov, 1972; Cameron & Carter, 1979; 
Dan & Bailey, 1982; Tilling, 1985; B. Baur, 
1988; Staikou & Lazaridou-Dimithadou, 1989; 



A. Baur & B. Baur, 1992). Resource or inter- 
ference competition is another controversial 
subject in land snails (Pearce, 1997). 

Albinaria is a pulmonale genus distributed 
around the northeastern coasts of the Mediter- 
ranean, exhibiting a high degree of morpho- 
logical and molecular differentiation. More 
than 90, in some cases dubious (Douris et al., 
1995, 1998a, b), nominal species have been 
described (Nordsieck, 1999). Ecological stud- 
ies of Albinaria populations can be very infor- 
mative about evolutionary processes in this re- 
gion, which has a complex palaeogeographic 
and climatic history (Paepe, 1986; Anasta- 
sakis & Dermitzakis, 1990; Westeway, 1994), 
because the high species number does not 
appear to be associated with proportionate 
ecological differentiation (Gittenberger, 1991). 
However, although the biogeography and sys- 
tematics of Albinaria have been well consid- 
ered (for a review, see Giokas, 2000), and Al- 
binaria species constitute about 15% of the 
total Greek land-snail species, studies of their 
life history are lacking. Similarly, very few data 



Corresponding author — Zoological Museum, Department of Biology, University of Athens, GR-15784, Athens, Greece; 
slnosg@biol.uoa.gr 
Natural History Museum, Department of Biology, University of Crete, GR-71409, Iraklelon, Greece; director@nhmc.uch.gr 

33 



34 



GIOKAS & MYLONAS 



are available (Warburg, 1972; A. Baur, 1990; 
B. Baur & A. Baur, 1990; 1991, 1992; Heller & 
Dolev, 1994) on the biology and life history of 
the Clausiliidae. 

Albinaria snails live on limestone substrata 
(Schilthuizen, 1994;Giokas, 1996). They feed 
on the microflora, that is, lichens and 
bryophytes. They are active only during the 
wet season, which in southern Greece is from 
late October through the end of April. Eggs 
are laid in clutches of about 5-7 eggs, gener- 
ally shortly after the beginning of the wet sea- 
son. Dunng the intervening dry periods, juve- 
niles and adults aestivate on the rock 
surfaces, in rock crevices, and occasionally 
on shrubs, or under stones. Especially during 
aestivation, aggregates are often formed, 
sometimes including many tens of individuals. 
Albinaria live about seven years. The devel- 
opment from a juvenile (juveniles do not have 
a lip or well-formed internal lamellae) to a fully 
grown snail takes two and occasionally three 
wet seasons. Shell development precedes 
maturation, which occurs during the last dry 
season after enlargement of genitalia size. 
Copulation then takes place during the first 
weeks of autumn rains. Population densities 
can sometimes be very high, in spite of mor- 
tality caused by desiccation, especially during 
aestivation, or of occasionally heavy préda- 
tion by larvae of the beetle family Drilidae 
(Schilthuizen et al., 1 994), or by typical preda- 
tors of snails, such as rodents and birds. 

This is the first comparative field study of 
both qualitative and quantitative life history 
characteristics for Albinaria. We analysed se- 
quential density, mortality, and spatial distri- 
bution data of four well-defined Albinaria 
species. We also recorded and compared a 
series of life-history traits, including the onset 
and end of aestivation, copulation, oviposi- 
tion, hatching, and aggregation. Our aim was 
to obtain more information on the ecology of 
Albinaria. to reveal possible life-history pat- 
terns and to understand the association of 
life-history characteristics with both extrinsic 
(density independent) and intrinsic (density 
dependent) factors and microevolutionary 
processes. 



MATERIALS AND METHODS 
Species Studied 

Four different Albinaria species, with each 
one population per species, were studied 



(Fig. 1). These were A. caerulea (Deshayes, 
1 835)- at Vravrona, Attiki, on the Acropolis hill 
beside the archaelogical site, A. turrlta (Pfeif- 
fer, 1850) - Kea island, N. W. Kyklades, 
around the Monastery of Panagia Kastriani, 
A. ci/sco/oA( Pfeiffer, 1846) - Aigina Island, Ar- 
gosaronikos Gulf, 5 km east from Agios Nek- 
tarios Monastery, and A. v'o/Y/?//(Rossmassler, 
1 836) - at Parori, Lakonia, near Sparti, at the 
entrance of the gorge. 

Habitat and Bioclimatic Characteristics of the 
Studied Sites 

Table 1 shows the main bioclimatic 
(Mavrommatis, 1978) and habitat characteris- 
tics of the studied sites. Albinaria caerulea, A. 
turrlta and A. discolor face the same biocli- 
matic conditions, which generally can be 
characterised as arid. However, A. uo/f/i// lives 
in a more humid habitat and the dominant 
vegetation type at Parori is maquis and not 
phrygana as in the other areas. Aigina, though 
arid, has a mixed vegetation (phrygana and 
maquis). Other climatic data with unambigu- 
ous seasonality (temperature, precipitation, 
humidity) are not presented because they 
were uncorrelated with the examined life his- 
tory parameters (see Results). All the above- 
studied populations are very localized and re- 
stricted to the sampled areas, which can be 
as small as that of Parori. Though the sub- 
strata at all sites is calcareous, sites are dif- 
ferentiated by the presence and density of 
crevices and calcareous plates, and the con- 
tinuous or mosaic allocation of rock bulks. 

Sampling Procedure 

We adopted the quadrat random sampling 
procedure (Krebs, 1989; Chalmers & Parker, 
1986; Williams, 1987). Within each site, sam- 
ples were taken every month for A. caerulea 
(April 1992-April 1994) and on a seasonal 
basis for A. volthii (April 1992-August 1994), 
A. turrlta (August 1992-April 1994), and A. 
discalar (Ju\\/ 1992-Apnl 1994). The size of 
the sampling quadrats (50 cm x 50 cm) was 
determined after preliminary sampling work 
(Krebs, 1989). A 20% confidence limit was 
chosen (Elliot, 1 971 ; Hayek & Buzas, 1 997) to 
set the number of sampling quadrats for each 
studied species. The number of quadrats on 
each sampling occasion was 15 in Vravrona, 
and 20 in Parori, Kea and Aigina. During aes- 
tivation we sampled once. All samplings were 



LIFE HISTORY IN ALBINARIA 



35 





^-•J>Vtavronc\ '-' ' ^ 

■ p a '^ t'.'.4 




FIG. 1. The distribution of the genus Albinaria (shaded area) and the studied sites. 



done on non-rainy days, at approxinnately 
9.00 a.m. In each quadrat, all the adult and ju- 
venile specimens, both alive and dead, were 
recorded; we counted only the dead speci- 
mens still attached to the substratum and not 
the empty shells fallen to the ground. Dead 
specimens were removed after each sam- 
pling. 

One month before the start and the end of 
the aestivation period and one month after 
that period we were visiting the sampling sites 
more often (every ten days at Vravrona) in 
order to record more precisely alterations of 
associated life-history traits (aestivation onset 
and ending, copulatory activity, oviposition, 
hatching). 

Statistical Analysis 

Besides descriptive statistics, we used 
ANOVA and Tukey HSD tests in order to 
analyse density fluctuations for the alive and 
dead specimens (adults and juveniles). We 
used log-transformed values. We examined 
ratio (adults/juveniles mortality) differences 



with contingency table analysis (G-test, 
Fisher exact test) (Zar, 1984). Additionally, we 
used time-series analysis (Wilkinson, 1989), 
in order to investigate possible autocorrela- 
tion and cross-correlation patterns for the 
density series of alive and dead specimens 
(adults and juveniles). 

Spatial distribution was estimated using: (a) 
Green's index of dispersion Gl = (s^/m) - 1/(n 
- 1), (m = mean number of individuals, s^ = 
variance, and n = total number of individuals 
in sample), and (b) Taylor's Power Law (s^ = 
am'^ (a and bare population parameters) (El- 
liot, 1 971 ; Ludwig & Reynolds, 1 988; Hayek & 
Buzas, 1997). Parameter b, in Taylor's Power 
Law, can vary from zero to infinity. When b > 
1 the distribution is contagious, when b = 1 the 
distribution is random and when b < 1 the dis- 
tribution is uniform. 

Finally, we used MANOVA and multiple re- 
gression analysis in order to examine possi- 
ble effects of climatic (precipitation, humidity, 
temperature) and time (month, season, year) 
factors to density, mortality and spatial distri- 
bution. 



36 



GIOKAS & MYLONAS 



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LIFE HISTORY IN ALBINARIA 



37 



RESULTS 

Aestivation, Awakening, Copulation, Oviposi- 
tion, Hatching, Activity 

Aestivation always began in April. However, 
at the глоге arid sites of Vravrona and Kea 
aestivation began earlier (first week of April) 
than at Aigina and Parori (last week of April). 
Specifically, at Vravrona, on average for the 
three sampling years, in the middle of April 
77.4% of adults had formed epiphragm, while 
at the end of April 100% of them were fully 
aestivating. The onset of the aestivation pe- 
riod was always independent of the end of the 
rainfall period. It is notable that aestivation, 
both in the arid springs of 1992 and on the 
much more humid and rainy of years 1993 
and 1994, started approximately at the same 
time (April) for all species. 

Awakening and copulation occurred only 
after the first autumn rain (in 1992 in the mid- 
dle of October and in 1993 in early Novem- 
ber). However, destruction of the epiphragm 
started in early October. More precisely at 
Vravrona, whereas in mid-September we did 
not observe any specimens with fragmenting 
epiphragm, in the beginning of October, on 
average for the two years, 86% of adults had 
an epiphragm with some holes, and at the end 
of October all specimens had no trace of 
epiphragm. Copulation is actually immediate 
and synchronous, and it is the main activity of 
Albinaria specimens after awakening. Copu- 
lation period lasts for about a week on aver- 
age. 

Oviposition starts about 20 days after cop- 
ulation and stops after ten days, but at the 
more humid site of Parori continued almost 
until the end of the wet period. Eggs were usu- 
ally laid in crevices or under stones in 
clutches of about five to eight eggs. Hatching 
occurred approximately two to three weeks 
after oviposition. 

During the wet period, on the sampling 
days, only a portion of the population was ac- 
tive, feeding on the microflora. However, we 
did not estimate that portion. 

Density 

Table 2 shows the mean densities (speci- 
mens per 0.25 m^) of the live specimens of the 
four studied species, and the significant differ- 
ences (between the sequential samples of 
each species) are indicated. Figure 2 shows 
density fluctuations during sampling. Autocor- 



TABLE 2. Mean density of alive specimens (speci- 
mens/0.25 m^) of the studied Albinaria species. 
Asterisks indicate significant (at a = 0.05) density 
differences between sequential samples of each 
population. 



total 



adults juveniles 



A. 


caerulea 


14.1 ± 


2.1 


9.1 ±2.1* 


5.0 ± 0.7* 


A. 


turrita 


8.4 ± 


1.5* 


4.4 ± 0.7* 


4.0 ± 1* 


A. 


discolor 


8.6 ± 


1.5 


6.9 ± 1.2 


1.7 ±0.5* 


A. 


voithii 


9.4 ± 


1.5* 


5.0 ± 0.9* 


4.4 ± 0.8* 



relation analysis showed that these were ran- 
dom series. It is notable that mean densities 
can be quite high. Higher densities were usu- 
ally observed during the aestivation period. 
However, in neither species, the differences 
between sequential samples that were sug- 
gested by the Tukey HSD test could be attrib- 
uted (MANOVA, Multiple regression) to cli- 
matic (temperature, precipitation, humidity) 
and time (year, season, month) sources of 
variation, and consequently are not pre- 
sented. 

Significant mean density differences (adults 
plus juveniles) were not found among the four 
studied populations (ANOVA, p = 0.05). Like- 
wise, the mean densities of the adult speci- 
mens did not differ significantly (ANOVA, p = 
0.09), but mean densities of juveniles differed 
significantly (ANOVA, p = 0.003), and the 
Tukey HSD test indicated significant differ- 
ences between juveniles at Aigina with those 
at Vravrona and Parori. 

Mortality 

Mean densities of dead specimens (speci- 
mens per 0.25 m^) of the four studied species, 
and significant differences (between the se- 
quential samples of each species) are shown 
in Table 3; Figure 3 shows density fluctua- 
tions during sampling. Adults and juveniles 
usually exhibit high mortality after hatching 
period; however, differences between se- 
quential samples were not caused by climatic 
and time sources of variation (MANOVA, Mul- 
tiple regression). 

Significant differences between the mean 
densities of dead individuals (adults plus ju- 
veniles) were not found among the four stud- 
ied populations (ANOVA, p = 0.05). Mean 
densities of adult dead specimens differed 
(ANOVA, p = 0.015), and the Tukey HSD test 
showed differences between adults at Aigina 
and those at Kea and Parori. Densities of 



38 



GIOKAS & MYLONAS 



Jnnü^ünüi 



months 

A tumta 



Aug Ocl Jan Apr Jen Apr 




Jul Od Jen Apr Jan Apr 



Apr Jul Dec Mar Jun Dec Mar Aug 



FIG. 2. Density fluctuations of alive specimens for the studied Albinaria species (in bars black stands for 
adults and white for juveniles). 



LIFE HISTORY IN ALBINARIA 



39 



S 1.8 

i 



] П jJn 



AMONDJ FMANDJ FMA 



month» 

A tunita 



I 0.6 



Aufl Oct Jan А|у Jan A()r 



jiiiL 

,qq, Jul Od Jan A«» Jan Apr 



I 1.5 



üOiün 




Apr Jul Dec Mar Jun Dec Mar Aug 



FIG. 3. Density fluctuations of dead specimens for the studied Albinaria species (In bars black stands for 
adults and white for juveniles). 



40 



GIOKAS & MYLONAS 



dead juveniles (ANOVA; p = 0.016) also dif- 
fered significantly, and the Tukey H.S.D. test 
indicated significant differences between Kea 
and Parori. 

Mean mortality ratio (dead specimens/ 
(dead + living specimens)) can vary signifi- 
cantly (p < 0.0001) between sequential sam- 
ples (Table 3, Fig. 4). Juvenile mortality (the 
ratio of dead juveniles to dead -i- living juve- 
niles) was significantly greater (p < 0.05) than 
adult mortality (dead adults/dead + living 
adults), across time, for A. caerulea and A. 
voithii and greater, however not significantly 
for A. discolor. In A. turrita. juvenile and adult 
mortality did not differ significantly, even 
though juvenile mortality was lower than that 
of adults. 

Spatial Distribution 

Spatial distribution was always contagious 
(TalDle 4), according to the parameters a and 
b of Taylor's Power Law and Green's Index, 
except in the case of living adults of A. turrita. 
Values of b were (except for A. turrita) high 
(above 2). Higher values of b (for total and 
adults) were estimated for A. discolor and A. 
coerulea. For juveniles, A. coerulea had the 
lower b value. Adults were more aggregated 
than juveniles, except for A. turrita. For dead 
specimens, values of b were more or less of 
the same order. However, comparison of 
slopes b is not always justified, because bean 
be affected by scale changes (Hayek & 
Buzas, 1997). 

DISCUSSION 

The start of the aestivation period, regard- 
less of site, year or species, was not corre- 

TABLE 3. Mean density of dead specimens (speci- 
mens/0.25 m^) of the studied Albinaria species. 
Asterisks indicate significant (at a = 0.05) density 
differences between sequential samples of each 
species. In parentheses mean mortality ratio (dead 
specimens/dead + living specimens). 







total 


adults 


juveniles 


A. 


coerulea 


1.4 ± 0.2 


0.7 r 0.1 


0.6 i 0.1* 






(9%) 


(7.1%) 


(10.7%) 


A. 


turrita 


0.5 ± 0.1 


0.3 ± 0.1 


0.2 л 0.1 






(5.6%) 


(6.4%) 


(4.8%) 


A. 


discolor 


1.8 ±0.5* 


1.3 - 0.4* 


0.5 t 0.1 






(17.3%) 


(15.8%) 


(22.7%) 


A. 


voithii 


1.2 ± 0.3* 


0.4 - 0.2* 


0.8 Í 0.1* 






(11.3%) 


(7.4%) 


(15.4%) 



lated directly to certain climatic conditions 
(rain, temperature and humidity). Even 
though it is not possible to separate geo- 
graphical, species and population effects, 
probably this implies that aestivation onset in 
Albinaria is relatively independent from these 
fluctuating environmental factors, and the 
possible cardinal action of an unknown intrin- 
sic physiological mechanism possibly associ- 
ated with day-length perception. However, the 
end of the aestivation period and the onset of 
copulation require rainfall (yet, not before the 
end of September). Heller & Dolev (1994) re- 
ported similar observations for the clausiliid 
Cristataria genezarethana. These life-history 
characteristics have been observed for sev- 
eral other (32) Albinaria populations belong- 
ing to 13 species sampled from 1990 to 1995 
throughout Greece (Giokas, 1996). Addition- 
ally, laboratory experiments with 25 Albinaria 
populations belonging to 12 species (among 
them the studied species of A. coerulea, A. 
turrita, A. discolor and A. voithii) proved that 
awakening is actually synchronous (on aver- 
age 90% of snails awake within five hours) 
and that copulation is the main activity of Albi- 
naria specimens for the first 36 hours after 
awakening (Giokas, 1996). According to 
Heller & Dolev (1994) and Lazahdou-DJmitri- 
adou & Sgardelis (1997), the rapid awakening 
response of aestivating snails to autumn rains 
is advantageous in seasonally dry habitats, 
which prevail in Mediterranean climate condi- 
tions. In that way, snails avoid adverse effects 
during unfavourable conditions. Several Albi- 
naria species, for example, those belonging to 
the grisea group, which aestlvate under 
stones or deep in crevices and have a thin, 
transparent shell, awake later (in late Novem- 
ber) (Giokas, 1996), and Giokas et al. (2000), 
support that awakening is partly associated 
with aestivation behaviour and morphological 
features, for example, thin shell. 

Oviposition and hatching stopped after the 
first month of the wet period at the more arid 
sites of Vravrona, Kea and Aigina. However, 
at the more humid and inland Parori site, 
oviposition and hatching continued through- 
out the active period, though mating was re- 
stricted to its beginning. Likewise, according 
to Lazahdou-Dimithadou & Sgardelis (1997), 
coupling and oviposition are more or less con- 
temporaneous within populations of land 
snails living in coastal habitats, where the 
suitable period is relatively short, whereas 
breeding lasts longer for the inland species. 
Perhaps egg laying period can be prolonged 



LIFE HISTORY IN ALBINARIA 



41 





montfis 
A voithii 




- П I J Ifc 



FIG. 4. Fluctuations of mortality ratio (dead specimens/(dead + living specimens)) for the studied populations 
of Albinaria (black bars stand for total, hatched bars for adults, and white bars for juveniles). 



42 



GIOKAS & MYLONAS 



+1 



+1 



+1 



T- en 00 'í CO r^ CD 

a)(X)a>coh~Ln-r^co 

II II II II II II II II 



CO 


C\J 
CD 


CM 


о 


о 


о 



+ 1 



+ 1 



+ 



t^ Ю -^ 'i t^ 

CVJCMr^OvJ'íOOO'í- 
ООООООООСХЭОО 

CDCNJCDC\JT-^CVJC>T^ 

II II II II II II II II 



■^ ■.- Ю r- ■>- -^ 

C0Or^LnOC4JCI)Ln 

II II II II II II II II 



CD CT) 00 CM t^ Ю CT) 

юсмст)ОГ-~сд1ПО 

COOCÛCOOOOO-r-;CT) 

II II II II II II II II 
со^азх)пзхзпзхз 



00-*00-'--'-'d-COCO 
CDCDCnCOt^CMO^-- 
■^COi-r^-^OI^O 



о CM ^ о 



га ^ Ш jn 



+ 1 о 

int^OO xiCTl-^OiCM 
Or^LT) ^'Г-Г^СОт- 

■^coc\jr~-CMcûcqo 

ÖCM-T^i-cbcMÖCM 

Il II II II II II II II 
raj^raj^ra^rajD 



о 


со 


о 


CM 


II 


II 


II 


II 


ra 


JD 


го 


n 




со 




CD 
CO 




о 




О 




+1 




+ 1 



LIFE HISTORY IN ALBINARIA 



43 



under favourable conditions for tine survival of 
the juveniles. Giokas (1996) reports that Albi- 
naria specimens collected during aestivation 
laid eggs during captivity in the laboratory 
without copulation (among them A. caerulea 
and A. voithii), suggesting that sperm or fer- 
tilised eggs can be retained as in other land 
snails (Duncan, 1975; Tompa, 1984). 

Mean values of density (Table 2) are high 
and comparable with those reported for other 
clausillids. For example, Vestía elata has 400 
specimens/m^, with a maturation period of 
five years (Piechocki, 1982), and Balea per- 
versa has 70 specimens/m^, with a matura- 
tion period of two years (B. Baur & A. Baur, 
1992). The Israeli Alopiinae land snail Crista- 
taha genezarethana (Heller & Dolev, 1994) 
has a mean density of 150-200 individu- 
als/m^. Heller & Dolev (1994) attribute those 
high densities to the presence of crevices, the 
absence of predators and competitive spe- 
cies, and to the snails' small size. Additionally, 
they consider that low copulation frequencies, 
as a response to lack of food, and growth in- 
hibition (11 years to mature), as a response to 
high population density through limited activ- 
ity (prevention of simultaneous demand for 
food, lichens with extremely low growth rate, 
in quantities that the rock surface may not be 
able to supply) or competitive action of adult 
specimens, may regulate population size. 
However, studies of resource and interfer- 
ence competition in land snails have resulted 
to argumentative outcomes (Pearce, 1997). 
These particular density-dependent mecha- 
nisms suggested by Heller & Dolev (1994) 
were not proved to act in Albinaria, in which 
(Schilthuizen, 1994; Giokas, 1996): copula- 
tion is synchronised, maturation comes after 
1 .5-2 years, Albinaria specimens are of the 
same size (on average 18 mm height) as 
Cristataha genezarethana, have the same 
diet preferences and similar activity pattern, 
and food (lichens and bryophytes) was al- 
ways abundant (however only a portion of 
specimens was active simultaneously). Addi- 
tionally, in neither case, at any time lag, higher 
mortality as a consequence of high density 
was observed. Therefore, in Albinaria intra- 
population competition for food and space or 
interference competition that would influence 
survival and abundance is still ambiguous 
(but of course needs further field and labora- 
tory examination). 

The abundance of the four studied Albinaria 
species fluctuated almost randomly through- 
out the sampling period, exhibiting no de- 



tectable trend, despite the stable oscillations 
of the environmental parameters. Lazaridou- 
Dimithadou & Sgardelis (1997) have reported 
a similar pattern for Bradybaena fruticum and 
Eobania vermiculata. Though Lazaridou-Dim- 
itriadou & Sgardelis (1 997) also demonstrated 
several examples of predictable phenological 
oscillation patterns, they claimed that the lat- 
ter are more evident for semelparous and 
short-lived species. Albinaria species are 
iteroparous and relative long-lived, as are 
Bradybaena fruticum and Eobania vermicu- 
lata, and population fluctuations could not be 
attributed directly to extrinsic environmental 
conditions. However, the role of intrinsic fac- 
tors remained unclear, in part because regu- 
lated life cycles are often difficult to distinguish 
from random density fluctuations (May, 1975; 
Cook, 1990), or because our sampling tech- 
niques failed to estimate with accuracy popu- 
lation dynamics. Density estimates of several 
Albinaria populations (Giokas, 1996) con- 
firmed the established notion (Lazaridou-Dim- 
ithadou & Sgardelis 1997) that population 
densities of different snail species may differ 
considerably, and that even populations of the 
same species do not always exhibit a peak 
density at the same time of the year. 

Mean estimated mortality percentage was 
generally low (Table 3) yet higher than the es- 
timation of 5% for Cristataha genezarethana 
(Heller & Dolev, 1994) and the estimation of 
2.5% for marked aestivating specimens of A. 
discolor (Giokas et al., 2000). However, 
higher mortality did not coincide with aestiva- 
tion period (Fig. 4). Besides sampling defi- 
ciencies, we can suggest that these rock- 
dwelling snails do not suffer considerably 
during the adverse hot and dry period, taking 
into account the tendency of Albinaria speci- 
mens to form clusters near deep and narrow 
crevices, the thickness and the white colour of 
the shell, and the protective function of the 
clausilium. However, high mortality, especially 
of adult specimens, was usually reported after 
hatching (Fig. 4). Probably this indicates that 
a major mortality cause is weakening after 
parental investment. Lazaridou-Dimitriadou 
(1995) came to a similar conclusion for Heli- 
cella pappi. Juveniles also exhibit high mor- 
tality after hatching, probably because of low 
intolerance. 

Albinaria turhta and A. d/sco/orjuveniles did 
not exhibit significantly higher mortality than 
adults, even though A. discolor juveniles 
show higher mortality than adults. Heller and 
Dolev (1994) state the same for Cristataha 



44 



GIOKAS & MYLONAS 



genezarethana, whereas A. Baur (1990) re- 
ports higher mortality for the juveniles of 
Balea perversa. That was unexpected, be- 
cause Albinarla juveniles lack some internal 
shell morphological features (lamellae and 
door like clausilium) that may prevent desic- 
cation (Gittenberger & Schilthuizen, 1996), 
even though their significance is disputed 
(Arad et al., 1995; Giokas, et al., 2000). Per- 
haps aggregation and crevice-dwelling can 
be in some cases important for juvenile sur- 
vival, even when juvenile mortality is higher 
than adult mortality (A. caerulea and A. 
voithii). 

The contagious spatial distribution of the 
four studied Albinaria species conformed to 
the general attitude of the xero-thermophilic 
species (Lazahdou-Dimitriadou, 1981). This 
behaviour seems to be important for rock- 
dwelling snails (Arad et al., 1995), and espe- 
cially for Clausiliidae (Heller & Dolev, 1994; 
Arad et al., 1995), and Albinaria (Warburg, 
1972; Kemperman & Gittenberger, 1988; 
Schilthuizen & Lombaerts, 1994), as in this 
way loss of water and prédation is inhibited. 
However, the fluctuations and differences of 
the contagious spatial distribution are not eas- 
ily explainable given the problems that con- 
cern the sampling procedure and the indices 
used. Nevertheless, higher contagious distri- 
bution could possibly be attributed to the frag- 
mentation of the biotopes and the presence of 
crevices. Parameter b of Taylor's Power Law 
had higher values on Aigina and Vravrona. 
Rocks on the site of Aigina are scattered, and 
on Vravrona crevices are very abundant. On 
the contrary, on Kea and Parori the suitable 
and favourable rock habitats are united, and 
there is a relative lack of crevices. 

Unfortunately, the relatively instant picture, 
on which we had to rely, as in most short-term 
life-history studies, does not allow us to fore- 
cast, because on investigating the effect of in- 
trinsic and extrinsic factors we have to con- 
sider several aspects that prevail or change in 
time and space. Consequently, some popula- 
tion characteristics, such as density, mortality, 
and spatial distribution, cannot be predicted 
per se, and we must be skeptical about fore- 
casting. 



ACKNOWLEDGMENTS 

We are grateful to M. Lazahdou-Dimitri- 
adou, from the Aristotle University of Thessa- 
loniki, for her helpful comments, to the editor 



and two anonymous referees for their con- 
structive suggestions, and to Stephen 
Roberts for grammatical suggestions. 



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Revised ms accepted 10 June 2001 



MALACOLOGIA, 2002, 44(1): 47-105 

THE EASTERN PACIFIC RECENT SPECIES OF THE CORBULIDAE (BIVALVIA) 

Eugene V. Coan 

Department of Invertebrate Zoology & Geology, ' California Academy of Sciences, Golden Gate 
Park, San Francisco, California 94118-4599, U.S.A.; gene.coan@sierraclub.org 

ABSTRACT 

There are 18 Recent species of the Corbulidae in the eastern Pacific, of which one has been 
introduced from the northwestern Pacific. Division of Corbula into additional genera is premature 
without new characters and a formal cladistic analysis. Seven subgenera are utilized, with six 
species remaining in Corbula, s. I. Three new species are described: C. (Caryocorbula) otra, С. 
(Varocorbula) groves!, and Corbula (s. I.) colimensis. One neotype and 14 lectotypes are desig- 
nated. The distributions and habitats of the species are documented, along with their fossil oc- 
currences and the relationships to other Recent and fossil species. 

Key words: Corbulidae, Corbula, eastern Pacific, western Atlantic. 



INTRODUCTION 
Previous Treatments 

Deshayes (1845-1848: 212-237, pis. 20, 
21) described and illustrated the anatomy 
of Corbula gibba (Olivi, 1792) (as "C. stri- 
ata" Fleming, 1828, ex Walker ms, one of 
its synonyms) and Lentidium mediterraneum 
(Costa, 1829). White (1942) described but did 
not figure the pericardial area of C. gibba, and 
Yonge (1946) discussed the anatomy of С 
gibba (without reference to Deshayes' earlier 
treatment). Morton (1986) discussed the biol- 
ogy and functional morphology of С crassa 
Reeve, 1843, at the same time questioning 
whether the Corbulidae are properly placed in 
the Myoidea. However, the only molecular 
phylogenies thus far published that have in- 
cluded a corbulid closely ally Corbula to Mya 
(Adamkewicz et al., 1997; Steiner & Hammer, 
2000). 

Important early papers describing a number 
of species of Corbula at the same time are 
those of G. B. Sowerby I (1833), Hinds 
(1843), and С В. Adams (1852a, b). Reeve 
(1843-1844) published the only comprehen- 
sive monograph on Corbula. Tryon (1869) 
listed the then-known species. Lamy (1926) 
discussed the species of Lamarck, and then 
gave synonymies for many of the known 
species (Lamy, 1941). 

Dall (1898, 1900) discussed the genera of 



the family in connection with his review of the 
fossil species of the eastern United States. 
Vokes (1945) and Keen (1969b) also covered 
the genera of the family. Zhuang & Cai (1 983) 
treated the species of China, and Habe (1 977: 
280-284) those of Japan. 

Anderson (1994, 1996) discussed the 
species of the Neogene of the Dominican Re- 
public, and showed that the eastern Pacific 
corbulids now average larger than those of 
the western Atlantic (Anderson, 2001). 

McLean (1942) discussed sculptural differ- 
ences between very inequivalve and less in- 
equivalve species of Corbula. Bacesco et al. 
(1957) and Gomoiu (1965) treated popula- 
tions of Lentidium mediterraneum (Costa, 
1829), and Hrs-Brenko (1981) those of Cor- 
bula gibba. Lewy & Samtleben (1979), de 
Cauwer (1985), Morton (1986), Anderson 
(1992), Harper (1994), and Kardon (1998) 
discussed prédation of corbulids and the role 
of the thick layer of conchiolin in combatting it. 

A preliminary outline of the results of the 
present study is given in Coan & Skoglund 
(2001). 

Format 

In the following treatment, each valid taxon 
is followed by a synonymy, information on 
type specimens and type localities, notes on 
distribution and habitat, and an additional dis- 
cussion. 



'Mailing address: 891 San Jude Avenue, Palo Alto, California, 94306-2640, U.S.A.; also Research Associate, Santa Barbara 
Museum of Natural History and Los Angeles County Museum of Natural History. 

47 



48 



COAN 



The synonymies include all major accounts 
about the species, but not most minor men- 
tions in the literature. The entries are 
arranged in chronological order under each 
species name, with changes in generic allo- 
cation from the previous entry, if any, and 
other notes given in brackets. 

The distributional information is based on 
Recent specimens I have examined, except 
as noted. Fossil occurrences are taken from 
the literature, except as noted. 

References are provided in the Literature 
Cited for all works and taxa mentioned. 

Abbreviations 

The following abbreviations are used in the 
text: AMNH, American Museum of Natural 
History, New York, New York, USA; ANSP, 
Academy of Natural Sciences of Philadelphia, 
Philadelphia, Pennsylvania, USA; BMNH, 
British Museum (Natural History) collection, 
The Natural History Museum, London, Eng- 
land; BMSM, Bailey-Matthews Shell Museum, 
Sanibel, Florida, USA; CAS, California Acad- 
emy of Sciences, San Francisco, California, 
USA; ICZN, International Commission on Zo- 
ological Nomenclature; LACM, Natural His- 
tory Museum of Los Angeles County, Califor- 
nia, USA; MCZ, Museum of Comparative 
Zoology, Harvard University, Cambridge, 
Massachusetts, USA; SBMNH, Santa Bar- 
bara Museum of Natural History, Santa Bar- 
bara, California, USA; UCMP, University of 
California Museum of Paleontology, Berkeley, 
California, USA; UMML, University of Miami 
Marine Laboratory, Rosenstiel School of Ma- 
rine and Atmospheric Sciences, Miami, 
Florida, USA; and USNM, United States Na- 
tional Museum collection, National Museum 
of Natural History, Smithsonian Institution, 
Washington, DC, USA. 

The eastern Pacific Corbulidae in the pri- 
vate collections of Carol С Skoglund, 
Phoenix, Arizona, USA; and Kirstie L. Kaiser, 
Puerto Vallarta, Jalisco, México, were also ex- 
amined. 



other shapes. All corbulids are inequivalve, 
with the right valve larger than the left, which 
fits into it, overlapping most conspicuously 
posteroventrally. Most eastern Pacific species 
are only slightly inequivalve, and only three 
very inequivalve. Some species may become 
thick-shelled as adults, whereas others are 
never greatly thickened. The anterior end is 
rounded in all species, but more broadly in 
some and more sharply in others. The shape 
of the posterior end is more characteristic of 
each. In some, it may be extended by a short, 
shelly "spout" (e.g., Figs. 2, 3), but this may be 
present in only some specimens; other 
species never have a spout. A useful charac- 
ter is the nature of the division between the 
central and posterior slopes. In some, it is de- 
lineated by a carina, a ridge, a change in 
angle, and/or a change in sculpture; in others, 
is it scarcely set off. Similarly, a narrow, elon- 
gate escutcheon of different degrees of 
prominence may be present; it is generally 
widest and more evident in the right valve. 

Sculpture, while variable in some species, 
can nonetheless be of diagnostic value. Com- 
marginal sculpture predominates, but radial 
ribs are present in some taxa. Color provides 
a useful character for several species. 

In some taxa, the hinge plate is broad, in 
others narrow. In general, the conspicuous 
tooth in the right valve is too variable in shape 
to be a useful diagnostic tool. The resilifer in 
the right valve may be visible on the hinge 
plate, or recessed beneath it. The chon- 
drophore in the left valve may be very project- 
ing or only slightly projecting, and it may be 
conspicuously divided into sections in some. 
At its posterior margin, a small tooth may be 
present, which varies in prominence among 
species. (It articulates on the posterior side of 
a small tooth in the resilifer of the right valve.) 
The palliai line, and its sinus, if present, have 
informative characters, as shown in the draw- 
ings (Figs. 40-57). A small sinus is evident in 
some taxa, not in others; in some, a small 
posterior extension is visible at the pos- 
teroventral corner of the palliai line (e.g., Fig. 
40). 



Morphological Characters 



In spite of the morphological plasticity of 
corbulids, there are a number of characters 
that are useful in distinguishing among the 
species. Some of these characters are sum- 
marized in Table 1 . 

Overall shape is a useful criterion, with 
some species oval, some trigonal, and some 



SYSTEMATIC ACCOUNT 
Family Corbulidae Lamarck, 1818 

Lamarck, 1818: 493, as "corbulidées" (ac- 
cepted under ICZN Code, 1999: Art. 
11.7.2) 



EASTERN PACIFIC CORBULIDAE 



49 



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50 



COAN 



Two Recent subfamilies are current recog- 
nized, the Corbulinae and the Lentidiinae 
Vokes. 1945 (pp. 6, 23-24), the latter con- 
taining only the living genus Lentidium, which 
is briefly discussed below. 

Subfamily Corbulinae Lamarck, 1818 
Genus Corbula Bruguière, 1797 

Corbula Bruguière, 1797: pi. 230, genus with- 
out named species (ICZN Code, 1999: 
Art. 12.2.7); description by Lamarck 
(1 799: 89); original list by Lamarck (1 801 : 
237) (ICZN Code. 1999: Art. 67.2.2). 
Type species (subsequent designation of 
Schmidt, 1818: 77, 177): Corbula sulcata 
Lamarck, 1801: 137 (species based on 
Bruguière, 1797: pi. 230, fig. la-c). Re- 
cent; west Africa. 
Not to be confused with Corbula Röding, 
1798: 184-185, the type species of 
which (subsequent designation of Winck- 
worth, 1930: 15) is Corbula anómala 
Röding, 1798, a junior synonym of Venus 
deflorata Linnaeus, 1758: 687. Corbula 
Röding is thus a synonym of Asaphis 
Modeer, 1793 (pp. 176, 182), a member 
of the Psammobiidae (Keen, 1969a: 633; 
Willan, 1993:5-6) 
Aloides Megerle von Mühlfeld, 1811: 67 
Type species (monotypy): A. guineensis 
Megerle von Mühlfeld, 1811: 67; = Cor- 
bula sulcata Lamarck, 1801, making 
Aloides an objective synonym of Corbula. 
The genus Corbula Bruguière was long a 
source of nomenclatural confusion, and a 
number of authors, lacking key pieces of liter- 
ature as well as the modern nomenclatural 
rules, attempted to sort out the taxonomic tan- 
gle (Vokes, 1945; J. Q. Burch, 1960). The for- 
mula given above represents the consensus 
under the current rules. 

Corbula sulcata, which occurs from Mauri- 
tania to Angola, is large, heavy, and sube- 
quivalve. It has heavy commarginal sculpture 
in the right valve and finer commarginal sculp- 
ture in the left valve. Its posterior end has two 
radial ridges, one between the central and 
posterior slopes and another defining a broad 
escutcheon. The hinge is very heavy. None of 
the Recent species in the New World is 
closely similar to the type species of Corbula. 
There are a bewildering array of specific 
and generic taxa in this subfamily, with char- 
acter sets that do not covary. Many of the gen- 
era were established based mostly on single 
characters, such as Anisocorbula I rédale. 



1930 (p. 404), for an elongate species with a 
sharp posterior keel, Solidicorbula Habe, 
1949 (p. 2), for a thick-shelled species, and 
Minlcorbula Habe, 1977 (p. 282), for a small- 
sized species. Subsequent authors have at- 
tempted to shoehorn every species into vari- 
ous subgenera, no matter how uncomfortable 
the fit. Efforts to make a meaningful arrange- 
ment of genera and subgenera will be fraught 
with inconsistencies until additional charac- 
ters become available and are rationally eval- 
uated. In light of this, elevating subgenera to 
genera, as many authors have done (includ- 
ing by Coan et al., 2000: 478-480), is prema- 
ture. 

Here, a conservative effort has been made 
to group similar species under some of the 
named subgenera and leaving several 
species in Corbula, s. I., but even these 
groupings should not be given much weight 
until a full-scale revision of the family is un- 
dertaken. 

Grant & Gale (1931 ) placed Corbula luteola 
and С porcella in Corbula (Lentidium), and 
the first assignment was followed by some 
other authors. The type species by monotypy 
of Lentidium Cristofori & Jan, 1832 (De- 
scrizione, p. 9; Disposito, p. [ii]; Conchylia ter- 
restha, p. 8; Mantissa, p. 4), is Tellina mediter- 
ránea Costa, 1 829 (pp. 1 4, 26-27, 1 31 , pi. 1 , 
fig. 6), which is small, thin, shiny, translucent, 
and shaped like a Tellina. It is sufficiently dif- 
ferent from other corbulids that it has been 
placed in a separate subfamily, the Lentidi- 
inae Vokes, 1945 (p. 23). No eastern Pacific 
species is similar. 

Lamy (1941 ) placed Corbula fragilis and С 
luteola in Corbula (Corbulomya). Corbulomya 
Nyst, 1845 (p. 59), has as its type species 
(subsequent designation of Herrmannsen, 
1847: 308) Corbula complánala J. Sowerby, 
1822 (p. 86, pi. 362, figs. 7, 8), from the upper 
Pliocene of England. This genus is now re- 
garded as a synonym of Lentidium (Vokes, 
1 945: 23, 26; Gilbert & van de Poel, 1 966: 58; 
Keen, 1969b: 696). 

The eastern Pacific Corbulidae include the 
following two species (or species complexes) 
of formidable morphological diversity, which 
cannot be fully resolved without new lines of 
evidence not available in the current study: 

(1 ) Corbula (Caryocorbula) nasuta is by far 
the most common species in collections, and 
it occurs in enormous populations on soft- 
bottoms in shallow-water. Individuals of this 
species can attain a fairly large size, but most 
populations contain small individuals. AI- 



EASTERN PACIFIC CORBULIDAE 



51 



though there is a wide range in morphologies, 
there is as yet no reliable evidence that more 
than one species is present, and specimens 
of intermediate morphologies can be found. 

(2) Corbula biradiata, while not having as 
much morphological plasticity as C. nasuta, 
varies considerably with respect to the posi- 
tion of its beaks, the strength of its commar- 
ginal sculpture, and its color. 

Corbulids are not only abundant, but there 
are many collection lots in alcohol or with 
dried animals. Thus, the group would be a 
prime candidate for biochemical genetic stud- 
ies, not only to test for possible additional di- 
versity at the species level but also to under- 
stand phylogenetic relationships in the family 
as a whole. 

Subgenus (Caryocorbula) Gardner, 1926 

Caryocorbula Gardner, 1 926: 46 

Type species (original designation): Corbula 
alabamiensis Lea, 1833 (reference 
below, under C. nasuta Discussion). 
Eocene, eastern United States. 

Serracorbula Olsson, 1961 : 433 

Type species (original designation): S. tu- 
maca Olsson, 1961, = Corbula nasuta 
G. B. Sowerby 1, 1833 (references below) 

Small to moderate in size, subequivalve, 
subequilateral, with a strong to weak ridge be- 
tween central and posterior slopes. Sculpture 
of strong to weak commarginal ribs, similar on 
both valves. Specimens of some species with 
"marginal" ribs along the ventral and dorsal 
margins; these are further discussed below. 

I include five eastern Pacific species in this 
subgenus. Other authors have placed addi- 
tional species here as well. 

Lamy (1941) placed Corbula nasuta in Cor- 
bula (Cuneocorbula). Cuneocorbula Coss- 
mann, 1886 (p. 49 [reprint: 37]), has as its 
type species (subsequent designation by Dall, 
1898: 836) Corbula biangulata Deshayes, 
1857 (p. 231, pi. 13, figs. 19-23).^ This 
species, from the Paleoceno of France, is 
thin, elongate, and birostrate. 



Gilbert & van de Poe! (1966: 55) renamed this 
species Cuneocorbula pelseneeri on the grounds 
that it was a junior homonym of "Corbula biangulata 
Sowerby, 1833," but there is no such senior 
homonym. They must have misread either Corbula 
bicarinata G. B. Sowerby I, 1833, or C. biradiata 
G. B. Sowerby I, 1833. 



Corbula (Caryocorbula) amethystina 

(Olsson, 1961) 

Figures 1 , 39 

Caryocorbula (Caryocorbula) amethystina 
Olsson, 1961 

Olsson, 1961:431, 548, pi. 75, fig. 1-1c; 
Keen, 1971: 262, 264, fig. 674 [Corbula 
(Caryocorbula)] 

Type Materials & Localities 

ANSP 218902, holotype, pair; length, 27.6 
mm; height, 18.1 mm; width, 13.8 mm (Fig. 1). 
"Tortutilla" [? = Isla Tortolita, Panamá Pro- 
vince], Panamá (8.8°N); H. В. Johnson, 1958. 
In the plate explanation, Olsson gives the lo- 
cality as [Isla] "Taboguilla," but both the text 
and label with the specimen say "Tortutilla". 
UMML30.11391, paratype, right valve; Puerto 
Mensabé, Los Santos Province, Panamá. 
UMML 30.11368, paratype, left valve; San 
Carlos, Panamá Province, Panamá. UMML 
30.11389, paratypes, right valve, left valve; 
San Carlos, Panamá Province, Panamá. 
UMML 30.11390, paratypes, 3 right valves, 2 
left valves; El Lagartillo, Panamá Province, 
Panamá. UMML 30.11388, paratype, right 
valve; Isla Gibraleón, Archipiélago de las Per- 
las, Panamá. UMML 30.11326, paratype, left 
valve; Punta Cocos, Isla del Rey, Archipiélago 
de las Perlas, Panamá. UMML 30.11390, 
paratype, left valve; Puerto Callo, Manabí 
Province, Ecuador. UMML 30.11388, para- 
type, right valve; Santa Elena, Guayas Pro- 
vince, Ecuador. 



Description 

Ovate-trigonal, thick; right valve slightly 
larger than left; posterior end longer (beaks 
37-43% from anterior end); anterior end 
rounded; posterior end pointed, slightly ex- 
tended by a spout in some specimens; poste- 
rior slope set off from central slope by a mod- 
erately sharp ridge that extends to the ventral 
margin. Escutcheon defined by a low ridge. 

Beaks with fine commarginal sculpture. 
Central slope with moderate commarginal ribs 
and much finer commarginal striae. Exterior 
color light to dark purple; interior color purple 
around margins, brown in some. Hinge plate 
broad; right valve with a large tooth; left valve 
with a broad, slightly projecting chondrophore 
and a small tooth. Posterior end of palliai line 
with small posterior extension (Fig. 39). 



52 



COAN 




FIG. 1. Corbula amythestina, holotype; ANSP 218902; length, 27.6 mm. FIG. 2. C. nasuta. Lectotype of C. 
nasuta; BMNH 1966565/1; length, 17.6 mm. 



EASTERN PACIFIC CORBULIDAE 



53 



Length to 30.8 mm (SBMNH 345487; Playas 
[de Villamil], Guayas Province, Ecuador). 

Distribution 

Mazatlán, SInaloa (23.2°N) (UCMP E.8424; 
CAS 121790, 121793; LACM 152689, 
152690, 63-11.78; SBMNH 126540), and La 
Paz, Baja California Sur (24.2°N) (CAS 
121786; SBMNH 345503 ["Punta Coyote," 
probably one of the two In this vicinity]), Méx- 
ico, to Playas [de Villamil], Guayas Province, 
Ecuador (2.6°S) (SBMNH 345487). It has 
been found living from the Intertldal zone to 82 
m (mean, 17.2 m; n = 27), on sand bottoms. I 
have seen 67 eastern Pacific lots. Including 
the types. A single lot labeled as having come 
■from the western Atlantic at Islas Los Roques 
(as "Las Rochas Is."), Venezuela (BMSM 
15008), seems Improbable (R. Cipriani, e- 
mail, 30 Nov. 2000) and probably represents 
a labeling error. 

Discussion 

Corbula amythestina merits comparison 
with C. dominicensis Gabb, 1873b (p. 247), 
from the Miocene of the Dominican Republic 
(concerning the latter: Anderson, 1996: 
14-15, pi. 1, figs. 9, 12, pi. 2, figs. 1, 2, 4, 5). 

Corbula (Caryocorbula) nasuta G. B. 
Sowerby I, 1833 
Figures 2-7, 40 

Corbula nasuta G. B. Sowerby I, 1833 

G. B. Sowerby I, 1833: 35; Reeve, 1843: 
pi. 1, fig. 1; d'Orbigny, 1845: 571; Car- 
penter, 1857a: 183, 228, 300; 1864b: 537 
[1872 reprint: 23]; Tryon, 1869: 65; Lamy, 
1941: 233 [Corbula (Cuneocorbula)]; 
Hertlein & Strong, 1950: 240, 252, pi. 2, 
fig. 9 [Aloidis (Caryocorbula)]; Hertlein & 
Strong, 1955: 205-206; Soot-Ryen, 
1957: 11; Keen, 1958: 209, fig. 527 [Cor- 
bula (Caryocorbula)]; Olsson, 1961: 
429-430, 548, pi. 75, fig. 3-3e [Cary- 
ocorbula (Caryocorbula)]; Keen, 1971: 
265-266, fig. 677 [Corbula (Caryocor- 
bula)]; Gemmell et al., 1987: 59, figs. 72, 
73 [as Corbula cf. nasuta] 

Corbula nuciformis G. B. Sowerby I, 1833 
G. B. Sowerby I, 1833: 35; Reeve, 1843 
pi. 2, fig. 9; Carpenter, 1857a: 183, 300 
1864b: 537, 668 [1872 reprint: 23, 154] 



Tryon, 1869: 65; Lamy, 1941: 234 [Cor- 
bula (Cuneocorbula)]; Olsson, 1961: 
430-431, 548, 549, pi. 75, figs. 7, 8; pi. 
76, fig. 7 [Caryocorbula (Caryocorbula)]; 
Keen, 1971: 265-266, fig. 678 [Corbula 
(Caryocorbula)] 

First revision herein 

[NON Corbula nuciformis, auctt., which = C. 
obesa] 

Hertlein & Strong, 1950: 241, pi. 3, fig. 1 
[Aloidis (Caryocorbula)]; Keen, 1958: 

209, fig. 528 [Corbula (Caryocorbula)] 
[not to be confused with Corbulolumna nuci- 

formis yokes, 1945: 9-11, pi. 2, figs. 5-8, 
from the Cretaceous of Lebanon.] 
Corbula fragilis Hinds, 1843 

Hinds, 1843: 56; Reeve, 1844: pi. 3, fig. 
13; Hinds, 1845: 68, pi. 20, fig. 11; Car- 
penter, 1857a: 207, 300; Tryon, 1869: 
64; Lamy, 1941: 240 [Corbula (Corbu- 
lomya)]; Hertlein & Strong, 1950: 243- 
244 [Aloidis (Tenuicorbula)]; Keen, 1958: 

210, 211, fig. 537; Olsson, 1961: 430 [as 
a synonym of Caryocorbula nasuta]; 
Keen, 1966:268, pi. 4, fig. 3 

Corbula alba Phlllppi, 1846 

Phllippi, 1846: 19; Carpenter, 1857a: 
224, 244; 1857b: 534, 547 [as a synonym 
of Corbula bicarinata]; Tryon, 1869: 63 

Corbula pustulosa Carpenter, 1 857 

Carpenter, 1857a: 244, 300 [nomen 
nudum]; 1857b: 22-23; 1864a: 368 
[1872 reprint: 204]; 1864b: 553 [1872 
reprint: 39]; Tryon, 1869: 65; Lamy, 1941: 
143; Hertlein & Strong, 1950: 240 [as a 
synonym of Aloidis (Caryocorbula) na- 
suta]; Palmer, 1951: 13; Brann, 1962:29, 
pi. 4, fig. 32; Keen, 1968: 402, pi. 56, fig. 
25 [as a synonym of Corbula nasuta] 

Serracorbula tumaca Olsson, 1961 

Olsson, 1961: 433, 549, pi. 76, fig. 4-4d; 
Keen, 1971: 268-269, fig. 690 [as Cor- 
bula (Serracorbula)] 

Type Materials & Localities 

Corbula nasuta-BMNH 1966565/1, lecto- 
type liere designated, open pair, the speci- 
men closest to the original length measure- 
ment; length, 17.6 mm; height, 11.5 mm; 
width, 12.0 mm (Fig. 2). BMNH 1966565/2-4, 
paralectotypes: closed pair, length, 18.4 mm; 
closed pair labeled "4", length, 1 6.0 mm; open 
pair, length 15.3 mm, perhaps the specimen 
figured by Reeve (1843)."Xlpixipl" [Jipijapa; 
Puerto de Cayo], Manabí Province, Ecuador 



54 



COAN 




FIGS. 3, 4. Corbula nasuta. FIG. 3. Lectotype of C. nuciformis; BMNH 1966566/1; length, 13.4 mm. FIG. 4a. 
Lectotype of C. fragilis; BMNH 1966234/1; length, 7.3 mm. FIG. 4b. Paralectotype of C. fragilis; BMNH 
1966234/2; length, 6.4 mm. 



(1.3°S); 10 fms. [18 m], sandy mud; Hugh 
Cuming. G. B. Sowerby I (1833) also reported 
some small specimens, tentatively assigned 
to this species, from the Golfo de Nicoya, 
Puntarenas Province, Costa Rica. 

Corbula nuciformis- BMNH 1 966566/1 , 
lectotype here designated, open pair, la- 



beled "6", probably the specimen figured by 
Reeve (1843); length, 13.4 mm; height, 8.4 
mm; width, 9.4 mm (Fig. 3). ANSP 50875, 
possible paralectotypes, 2 small, closed pairs. 
"Real Llejos" [Rio Realejo; Corinto], Chinan- 
dega Province, Nicaragua (12.5°N), 6 fms. [11 
m], sandy mud; Hugh Cuming. G. B. Sowerby 



EASTERN PACIFIC CORBULIDAE 



55 



I (1833) also cited fossil material from near 
Guayaquil, Guayas Province, Ecuador. 

Corbula fragilis- 1 966234/1 , lectotype 
here designated, right valve; lengthi, 7.3 mm; 
hieighit, 4.3 mm; width, 1.9 mm (Fig. 4a). 
BMNH 1966234/2, paralectotype, left valve; 
length, 6.4 mm (Fig. 4b). A larger specimen 
cited by Hinds (1843) is not in the BMNH. Ve- 
ragua[s] Province, Panamá (approximately 
7.7°N), 18 fms. [33 m]; Edward Belcher. 

Corbula a/ba- Presumably lost. The origi- 
nal specimen measured 13.0 mm in length, 
8.2 mm in height, and 7.6 mm in width. 
Mazatlán, Sinaloa, México (32.2°N); Kinder- 
man. 

Corbula pustulosa-BMNH 1857.6.4.77, 
lectotype (Keen, 1968: 402), closed pair 
mounted on a glass slide; length, 4.2 mm; 
height, 3.2 mm; width, approximately 2.0 mm 
(difficult to measure because of being glued to 
the slide) (Fig. 5). USNM 715647, paralecto- 
type, left valve (glued to a glass slide); length, 
3.4 mm. Mazatlán, Sinaloa, México (32.2°N); 
Frederick Reigen. 

Serracorbula tumaca- AHSP 218948, lec- 
totype here designated, right valve; length, 
12.4 mm; height, 8.4 mm; width, 5.4 mm (Fig. 
6a). Of the material that had been in the ANSP 
lot labeled "holotype," this valve comes clos- 
est to the holotype measurements in Olsson's 
text, but it does not match any of his illustra- 
tions labeled "holotype." Olsson's fig. 4, 4b, 
and 4c corresponds to a paralectotype pair 
measuring 12.0 mm in length (ANSP 405291) 
(Fig. 6b). His fig. 4d is a paralectotype left 
valve measuring 11.8 mm in length (also in 
ANSP 405291 ). The left valve shown in his fig. 
4a was not present in the lot. Tumaco, Nariño 
Province, Colombia (1.8°N). The ANSP lot 
also contained a paralectotype right valve 
from San Miguel, Isla del Rey, Archipiélago de 
las Perlas, Panamá (now ANSP 405292). No 
type material was located in the Olsson col- 
lection in the UMML. 

Description 

Ovate-elongate, heavy; right valve slightly 
larger than left; longer posteriorly to slightly 
longer anteriorly (beaks 34-51% from ante- 
rior end). Posterior end somewhat pointed 
ventrally, sometimes extended by a spout. 
Posterior slope set off by a well-defined ridge, 
often with a slight radial sulcus just anterior to 
it. Ventral margin sometimes growing medially 
to form an inflated, flattened ventral surface. 
Margins, particularly the ventral margin. 



sometimes with "marginal"^ ribs, resulting in a 
serrate edge. Escutcheon defined by a ridge, 
more evident in right valve. 

Beaks relatively smooth, with fine, pustules. 
Sculpture on central and posterior slopes of 
closely spaced moderate commarginal ribs 
and fine, radially arrayed pustules. 

Periostracum tan. External color white; in- 
ternal color white, but yellow or tan in some 
specimens. Hinge plate heavy; right valve 
with a large tooth; left valve with a broad chon- 
drophore, projecting in some, less in others; 
tooth small (Fig. 40). Length to 18.4 mm (a 
paralectotype of C. nasuta). 

Distribution 

Isla Natividad, Baja California Sur (27.9°N) 
(SBMNH 345488); Bahía Magdalena, Baja 
California Sur (24.5°N) (CAS 121538), into 
and throughout the Golfo de California to its 
head at Puerto Peñasco, Sonora (31.4°N) 
(UCMP E8431; CAS 115324, 121917, 
121919, 122072; SBMNH 21350, 116514, 
119426; USNM 212789, 707996; Skoglund 
Collection; and many other lots), México, to 
Callao, Lima Province, Perú (12.1 °S) (LACM 
35-1 52.1 , 35-1 53.1 , 35-1 77.2); Isla del Coco, 
Costa Rica (SBMNH 345489, 345490, 
345491; LACM 38-180.2); Isla Santa Cruz, 
Islas Galápagos, Ecuador (LACM 38-193.13); 
from the intertidal zone to 152 m (mean, 28.7 
m; n = 328), on mud or sand. (A single valve 
with an indicated depth of 384 m -LACM 35- 
177.2 -is probably the result of drift from 
shallower water.) I have seen 875 eastern Pa- 
cific lots, including the types. Two lots labeled 
as having come from "Monterey, California" 
(USNM 21480, 58346), probably represent la- 
beling errors, and one of them may be the 
source of the Monterey record of C. fragilis by 
Dall (1921: 53) and Oldroyd (1925: 203). 

This species has also been recorded in 
beds of Pleistocene age at Puerto Peñasco, 
Sonora (Hertlein & Emerson, 1956: 165), and 
at Bahía Magdalena, Baja California Sur (Jor- 
dan & Hertlein, 1936: 111 , as "C porcella" and 
as "C. fragili^'; CAS Loc. 754), México, in 
beds of Pliocene age of the Loreto Basin, 



^These ribs appear along the outer ventral surface 
near the shell margins (Fig. 6a, b). Olsson (1961) 
referred only to "marginal serrations". They are not 
radial ribs, because they do not radiate from the 
beaks. I settled on the term "marginal ribs" because 
they run laterally along the shell margin. 



56 



COAN 




FIGS. 5-7. Corbula nasuta. FIG. 5. Lectotype of C. pustulosa; BMNH 1857.6.4.77; length, 4.2 mm. FIG. 6a. 
Lectotype of Serracorbula fumaca; ANSP 218948; length, 12.4 mm. FIG. 6b. Paralectotype of S. tumaca; 
ANSP 405291 ; length, 12.0 mm. FIG. 7. SBMNH 345490; Bahía Chatham, Isla del Coco, Costa Rica; 46-69 
m; largest specimen length, 5.0 mm. 



EASTERN PACIFIC CORBULIDAE 



57 



Baja California Sur, México (Piazza & Robba, 
1998: 238, 248, 260, as "C. nuciformisf'), and 
in the middle to late Pliocene Canoa Forma- 
tion at Punta Blanca, Manabí Province, 
Ecuador (Pilsbry & Olsson, 1941: 11, 75). 
G. B. Sowerby I (1833) cited fossil material 
of unknown age from Guayaquil, Guayas 
Province, Ecuador. 

Discussion 

Corbula nasuta is the most variable of the 
eastern Pacific taxa. When small (< 10 mm), 
specimens are thin shelled, with conspicuous 
fine pustulose radial sculpture. This is the 
morphology named C. fragilis. As specimens 
grow, the ventral margin may soon become 
flattened, yielding the short, inflated morphol- 
ogy that was named С nuciformis and С pus- 
tulosa. The size at which material thickens 
and forms a flattened base varies greatly 
among populations, and this can occur in 
specimens as small as 5 mm. Corbula nasuta 
typically becomes rostrate posteriorly, and a 
short spout may be added in some speci- 
mens. 

In some large specimens, marginal ribs 
may be developed at the margins, especially 
the ventral margin. This is the form that was 
named Serracorbula tumaca. The type lots of 
both C. nasuta and C. nuciformis also contain 
specimens with such ribs. 

I believe that Corbula alba belongs here as 
a synonym in that Phiiippi's description 
speaks of the posterior slope being acute and 
subrostrate, and because the two European 
Mio-Pliocene species with which he com- 
pared С alba— С. revoluta (Brocchi, 1814: 
516, 685, pi. 12, fig. 6 -orginally described as 
Tellina) and the closely related C. carinata Du- 
jardin, 1837 (p. 257)- are much more similar 
to C. nasuta than to С bicarinata, with which 
it was previously synonymized. 

Some material from the northwestern coast 
of South America is shorter, more rounded, 
without much of a posterior sulcus setting off 
the posterior end, and it has finer sculpture 
than material from further north. A similar mor- 
phology is seen in material from Isla del Coco, 
Costa Rica (lots cited in the Distribution), 
which is uniformly small, rounded, and 
smooth (Fig. 7). This might be regarded as a 
separable species or subspecies, but given 
the variability of this species (or species com- 
plex) as a whole, it is premature to bestow ad- 
ditional names. 

Corbula nasuta is most similar to the later- 



named western Atlantic С swiftiana С В. 
Adams, 1852c (pp. 236-237), which has 
been reported from Massachusetts to Ar- 
gentina. Given the variability of these two 
taxa, I doubt that the two can be told apart on 
a morphological basis, and perhaps they 
should be synonymized until more evidence is 
available. Synonyms in the western Atlantic 
may include: С kjoerianaC. B. Adams, 1852c 
(p. 237), C. barrattiana С В. Adams, 1852c 
(pp. 237-238), C. chittyana С В. Adams, 
1852c (p. 238), C. fulva С В. Adams, 1852c 
(pp. 240-241), C. caribaea d'Orbigny, 1853^ 
(p. 284 [Spanish ed., p. 323], pi. 27, figs. 5-8), 
C. lavalleana d'Orbigny, 1853 (p. 284 [Span- 
ish ed., p. 323], pi. 27, figs. 9-12), and C. 
uruguayensis Marshall, 1928 (p. 5, pi. 4, figs. 
7-9). Records of С nasuta from the western 
Atlantic, such as those of Dall (1889b: 70, pi. 
2, fig. 6-6c, as C. nasuta "Say") and Haas 
(1953: 204), were presumably based on С 
swiftiana. 

Records of С nasuta from Australia 
(Angas, 1867: 913, 1878: 869; Hedley, 1918: 
M31) were based on specimens of С coxi 
Pilsbry, 1897 (pp. 363-364, pi. 9, figs. 1-3). 

Dautzenberg (1912: 99) reported Corbula 
nasuta Sowerby from several localities in 
west Africa. This African species was also de- 
scribed as C. lyrata E. A. Smith, 1872 (p. 729, 
pi. 75, fig. 2), a junior homonym of С lyrata J. 
de С. Sowerby, 1840 (expl. to pi. 21), and 
Smith's species was renamed С dautzenberg 
Lamy, 1941 (pp. 235-236), the name cur- 
rently in use. 

Corbula nasuta Sowerby, 1833 (17 May), is 
not to be confused with Corbula nasuta Con- 
rad, 1833 (3 Sept.) (p. 38; unpublished pi. 19, 
fig. 4 [not "pi. 20, fig. 2," as stated in text]), 
from the Middle Eocene Claiborne Formation 
of Alabama, for which the name Corbula al- 
abamiensis Lea, 1833 (Dec.) (p. 45, pi. 1, fig. 
12), is now used (Palmer & Brann, 1965: 74). 
Corbula nasuta Conrad was later reported by 
Conrad (1 857: 1 61 , pi. 1 9, fig. 4) from the Ter- 



"Dall (1889b: 18), followed by Aguayo (1943: 38), 
maintained that the entire Atlas to d'Orbigny's 
monograph on the mollusks of Cuba appeared in 
1842, with the French text on the bivalves appear- 
ing between 1847 and 1853. Aguayo also thought 
that the entire Spanish text might have appeared in 
1845; Keen (1971: 1006) dated the Spanish text as 
1846. However, there is no evidence that any of the 
bivalves were published, in text or plates, and in ei- 
ther edition, before 1853, when citations to the 
species begin to appear in other works. 



58 



COAN 



tiary of western Texas, and this Texas mater- 
ial was later named С conradi by Dall (1898: 
842).^ 

Small specimens of this species commonly 
occur in shallow water with С marmorata. 
These species may most easily be distin- 
guished as follows: 





nasuta 




marmorata 


Posterior end 


tapered, 




subquadrate, 




pointed 




pointed pos- 




posterio 


riy 


teroventrally 


Exterior color 


white 




mottled, with 
purplish 
blotches near 
beaks 


Interior color 


white 




magenta, espe- 
cially around 
margins 



The complex of nasuta-Wke taxa has been 
vastly overnamed in the Pliocene and 
Miocene of the western Atlantic region. A par- 
tial list of taxa that belong in this complex in- 
cludes: С {Cuneocorbula) sarda Dall, 1898 
(pp. 847-848: 1900: pi. 36, fig. 14); C. (C.) 
whitfieldi DaW, 1898 (p. 849; 1900: pi. 36, fig. 
18); С {Caryocorbula) whitfieldi stika Gard- 
ner, 1928 (pp. 232-233, pi. 35, figs. 8, 9); and 
C. (C.) whitfieldi boyntoni Gardner, 1928 (p. 
233, pi. 35, figs. 10-13), from the Miocene of 
Florida; С (Cuneocorbula) sericea DaW, 1898 
(pp. 848-849; 1900: pi. 36, fig. 8) [synonyms: 
C. (C.) cercadica Maury, 1917: 396-397 [= 
232-233], pi. 65 [= 39], figs. 16, 17; С (С.) 
caimitica Maury, 1917: 395 [= 233], pi. 65 [= 
39], figs. 18, 19] (concerning: Anderson, 
1996: 15-17, pi. 2, figs. 7-21); С (С.) hele- 
nae Maury, 1912 (p. 62, pi. 9, fig. 25) [syn- 
onyms: С (С.) smithiana Maury, 1912: 63, pi. 
9, figs. 29, 30; С (С.) caribaea pergrata 
Maury, 1925: 255 [= 103], pi. 20, fig. 8; С (С.) 
daphnis Maury, 1925: 256 [= 104], pi. 20, figs. 
10-11] (concerning: Jung, 1969: 407-409, pi. 
38, figs. 12, 13, pi. 39, figs. 1-9), from the 
Miocene and Pliocene of the Caribbean; 
Caryocorbula (Caryocorbula) prenasuta Ols- 
son, 1964 (p. 70, pi. 9, fig. 10), from the 
Miocene of Ecuador; С oropendula dolicha 
Woodring, 1982 (p. 712, pi. 119, figs. 4-6), 
from the Miocene of Panama; Corbula (Cu- 
neocorbula) swiftiana harrisii Dall, 1898 (p. 



^Corbula conradi Dall is not the renaming of a 
homonym, as assumed by Boss et al. (1968: 88), 
but rather a new species based on Conrad's mate- 
rial (USNM 9899). 



855), from the Miocene of Texas; and С inae- 
qualis Say, 1824 (p. 153, pi. 13, fig. 2), from 
the Pliocene of Maryland and Virginia [syn- 
onym: С inaequalis mansfieldi Richards, 
1947: 32, pi. 11, figs. 27, 31] (concerning: 
Campbell, 1 993: 48, pi. 21 , fig. 1 91 ). 

Corbula (Caryocorbula) otra Coan, 
new species 
Figures 8, 41 

Corbula ovulata, auctt., in part, non G. В. 
Sowerby I, 1833 

G. В. Sowerby I, 1833: 35-36 [material 
cited from Mazatlán, México, and Corinto, 
Nicaragua]; Hanley, 1 843: 47, 4 (pi. expl.), 
pi. 10, fig. 52; 1856: 344; Reeve, 1843 
[material cited from Mazatlán, México, 
and Corinto, Nicaragua]; C. B. Adams, 
1852a: 522-523 [1852b: 298-299] [in 
part; the large pair cited from Isla Taboga, 
Panamá]; Carpenter, 1857b: 23 [speci- 
men cited from Mazatlán]; Lamy, 1941: 
127-128; Hertlein & Strong, 1950: 241, 
252, pi. 2, fig. 11 [Aloidis (Caryocorbula)]; 
Hertlein & Strong, 1 955: 206; Keen, 1 958: 
209, fig. 530 [Corbula (Caryocorbula)]; 
Olsson, 1961: 428-429, 548, pi. 75, fig. 
2c [Caryocorbula (Caryocorbula)]; Keen, 
1971: 265, 266, fig. 680 [lower right fig. 
only] [Corbula (Caryocorbula)] 

Type Material & Locality 

SBMNH 345493, holotype, pair; length, 
22.8 mm; height, 13.6 mm; width, 12.0 mm, 
including portion of dried soft parts (Figs. 8, 
41). SBMNH 345494, paratypes; open pair, 
length, 23.3 mm; closed pair, length, 21.0 
mm; right valve, length, 21 .5 mm. Manzanillo, 
Colima, México (19.1 °N, 104. 3°W); 30-45 m; 
Carl & Laura Shy; exSkoglund Collection. 

Description 

Ovate-elongate; heavy; right valve slightly 
larger than left; longer posteriorly (beaks at 
40-43% from anterior end). Anterior end 
rounded; posterior end tapered, slightly up- 
turned posteriorly, often extended by a short 
spout. Posterior slope set off by a rounded 
ridge, becoming less evident ventrally. Es- 
cutcheon weakly defined by a low ridge, most 
evident in right valve. 

Beaks smooth; most of surface covered by 
well-spaced, rounded moderate commarginal 



EASTERN PACIFIC CORBULIDAE 



59 




FIG. 8. Corbula otra, holotype; SBMNH 345493; length, 22.8 mm. FIG. 9. C. ovulate, lectotype; BMNH 
1967946/1; length, 26.7 mm. 



60 



COAN 



ribs and very fine commarginal striae. Poste- 
rior slope with much less conspicuous ribs. 
Ventral and dorsal margins with marginal ribs 
in some specimens, such as in the holotype. 
Periostracum light tan. Exterior color white 
on anterior and posterior ends and ventrally, 
purple to pink medially; beaks white. Interior 
white to purple. Hinge plate heavy; right valve 
with large tooth; left valve with chondrophore 
not very extended and a small tooth. Posterior 
end of palliai line with short posterior extension 
(Fig. 41 ). Length to 26.1 mm (Bahía Chamela, 
Jalisco, México; Skoglund Collection). 

Distribution 

Isla Carmen, Baja California Sur (26.0°N) 
(CAS 121884), and Guaymas, Sonora 
(27.9°N) (UCMP E.8425, CAS 121883), Méx- 
ico, to La Libertad, Guayas Province, Ecuador 
(2.2°S) (SBMNH 109827). A lot in the USNM 
21479 labeled as "Monterey, California," is un- 
doubtedly a labeling error. A few valves num- 
bered with a locality in the Islas Galápagos, 
Ecuador, now isolated as CAS 121890, are 
thought to represent numbering errors on a 
few shells that had been a large lot from Cor- 
into, Nicaragua. Recorded depths of live col- 
lected material are from the intertidal zone to 
55 m (mean, 17.9 m; n = 31), on mud or sand. 
I have seen 107 lots. 

Etymology 

The species name is the Spanish word for 
"other." 

Discussion 

This new species is closest to С ovulata, 
and their distributions overtap from Costa 
Rica to Ecuador. Corbula ovulata is more 
elongate, even as a juvenile, more produced 
posteriorly, and it is always white in color. The 
radial rib between the central and posterior 
slope is more pronounced, and the commar- 
ginal sculpture is denser and more evenly dis- 
tributed. 

Corbula (Carycorbula) ovulata 

G. B. Sowerby I, 1833 

Figures 9, 42 

Corbula ovulata, G. B. Sowerby I, 1833 

G. B. Sowerby I, 1833: 35-36; Hanley, 
1843; 47 [in part; not the fig.]; Reeve, 



1 843; pi. 1 , fig. 7 [in part]; d'Orbigny, 1 845; 
571-572; С В. Adams, 1852a; 522-523 
[1852b; 298-299] [in part]; Carpenter, 
1857a; 183, 228, 244, 280, 300; 1857b; 
23 [in part]; 1864a; 368 [1872 reprint; 
204]; 1864b; 537, 668 [1872 reprint; 23, 
154]; Tryon, 1869; 65; Lamy, 1941; 
127-128 [in part]; Hertlein & Strong, 
1950; 241 [Aloidis (Caryocorbula)] [in 
part; not the fig.]; Hertlein & Strong, 1 955; 
206; Keen, 1958; 209 [Corbula {Cary- 
ocorbula)] [in part; not the fig.]; Olsson, 
1961; 428-429, 548; pi. 75, fig. 2-2b 
[Caryocorbula (Caryocorbula)] [in part; 
not fig. 2c]; Keen, 1 971 ; 265-266, fig. 680 
[Corbula (Caryocorbula)] [in part; not 
lower right fig.] 

Type Material & Locality 

BMNH 1967946/1, lectotype here desig- 
nated, open pair, the largest specimen, that 
figured by Reeve (1843); length, 26.7 mm; 
height, 15.1 mm; width, 12.1 mm (Fig. 9). 
BMNH 1967946/2-3, paralectotypes, two 
other pairs; closed pair, length, 24.3 mm; 
open pair, length, 24.1 mm. "Caraccas" [Bahía 
de Caráques], Manabí Province, Ecuador 
(0.6°S). A collective depth of 7-1 7 fms. [1 3-31 
m] was given for ail the original material. 

Five localities were originally mentioned. 
The two northernmost -Mazatlán, Sinaloa, 
México, and "Real Llejos" [ = Rio Realejos; 
Corinto], Chinendega Province, Nicaragua - 
specifically refer to the species ["beautiful pink 
color"] here described as Corbula otra and are 
not represented by material in the BMNH col- 
lection, nor is material present from the Golfo 
de Montijo, Veraguas Province, Panamá, or 
from the other cited Ecuadorian locality - 
"Xipixapi" [Jipijapa; Puerto de Cayo], Manabí 
Province. Without examining type material, 
Hertlein & Strong (1950; 241) "designated" 
the latter as the type locality, but this is now 
superceded by the locality of the lectotype 
designated here (ICZN Code, 1999; Art. 
76.2). 

Description 

Ovate-elongate, heavy; right valve slightly 
larger than left; equilateral to longer posteri- 
orly (40-50% from anterior end); anterior end 
rounded; posterior end produced, extended 
by a spout in large specimens. Posterior slope 
set off from central slope by a very low ridge 



EASTERN PACIFIC CORBULIDAE 



61 



that disappears ventrally. Escutcheon well de- 
fined by a ridge. 

Beaks snnooth; most of surface with closely 
spaced, moderate commarginal ribs and very 
fine commarginal striae. Ventral margin with 
marginal ribs in some material. Escutcheon 
smooth. 

Periostracum tan. Exterior white; interior 
with brown patches and brown marginal color 
in large specimens. Hinge plate heavy; right 
valve with a large tooth; left valve with a 
broad, not very projecting chondrophore and 
a small tooth. Posterior end of palliai line with 
posterior extension (Fig. 42). Length to 29.2 
mm (Skoglund Collection; Playas, Guayas 
Province, Ecuador). 

Distribution 

Bahía Juanilla, Guanacaste Province, 
Costa Rica (10.9°N) (LACM 72-13.30), to 
Cabo Blanco, Piura Province, Perú (4.3°S) 
(CAS 121777), from the intertidal zone to 55 
m (mean, 8.2 m; n = 13), on sand bottoms. I 
have seen 58 lots, including the types. 

Reported on the Pleistocene third terrace 
on the Peninsula de Santa Elena (Hoffstetter, 
1 948: 81 ) and, with question, as a subfossil at 
Laguna de Salinas (Hoffstetter, 1952: 44), 
both in Guayas Province, Ecuador. Also re- 
ported in the early Pliocene Jama Formation 
at Puerto Jama, Manabi Province, Ecuador 
(Pilsbry & Olsson, 1941: 11, 75), in the 
Pliocene at Rio la Vaca and Rio Blanca, 
Burica Peninsula, Puntarenas Province, 
Costa Rica (Olsson, 1942: 171, 172), and in 
the Miocene Tumbes Formation at Zorritos, 
Tumbes Province, Perú (Olsson, 1932: 140). 

Discussion 
See under С otra. 

Corbula (Caryocorbula) porcella Dal I, 1916 
Figures 10, 43 

Corbula porcella Dal 1 , 1 9 1 6 

Dall, 1916a: 41 [nomen nudum]; Dall, 
1916b: 415-416; Oldroyd, 1925: 204; 
Grant & Gale, 1931: 422 [Corbula {Len- 
tidium)]; Lamy, 1941: 242 [as C. "porcel- 
//o"]; Hertlein & Strong, 1950: 242, 252, pi. 
2, figs. 13, 15 [Aloidis (Caryocorbula)]; 
Keen, 1958: 210-211, fig. 531 [Corbula 
(Caryocorbula)]; Olsson, 1961: 430, 549, 
pi. 76, fig. 8 [Caryocorbula (Caryocor- 



bula)]; Keen, 1971: 265-266, fig. 681 
[Corbula (Caryocorbula)]; Coan et al., 
2000: 478, pi. 102 [Caryocorbula] 
Corbula fragilisHmós, auctt., non Hinds, 1843 
Dickerson, 1922: 563; Grant & Gale, 
1931: 422 [Corbula (Lentidium)] 

Type Material & Locality 

USNM 97039, lectotype here designated, 

open pair; length, 7.9 mm; height, 5.2 mm; 
width, 4.9 mm (Fig. 10). USNM 880652, para- 
lectotypes, 1 closed pair, 6 right valves, 8 left 
valves, and assorted fragments. USFC Stn. 
2838, off the east side of Isla Cedros, Baja 
California, México (28.2°N, 115.2°W), 44fms. 
[99 m], green mud; May 5, 1888. 

Description 

Shell ovate-subquadrate, of moderate 
thickness for size; right valve slightly more in- 
flated than left; beaks closer to anterior end 
(32-34% from anterior end). Anterior end 
rounded. Posterior end almost vertically trun- 
cate. Posterior slope separated from central 
slope by a fairly strong radial ridge. Es- 
cutcheon sharply defined by a ridge. 

Beaks smooth. Central slope with moder- 
ate, irregular commarginal ribs, lamellar on 
posterior slope; posterior end also with radial 
rows of pustules in many specimens. 

Color white exteriorly and interiorly. Hinge 
plate broad; right valve with a narrow tooth; 
left valve a narrow, non-projecting chon- 
drophore and a small tooth (Fig. 43). Length 
to 10.2 mm (MCZ 260684; S of Laguna San 
Ignacio, Baja California Sur). 

Distribution & Habitat 

Esteros Bay, San Luis Obispo County, Cal- 
ifornia (35.4°N) (USNM 207636), to Punta 
Magdalena, Baja California Sur, México 
(24.6°N) (LACM 71-13.5), in 27 to 210 m 
(mean, 80.3 m; n = 55), on mud and sand. I 
have seen 68 lots, including the types. 

The record of С porcella from Bahía Bal- 
lena, Puntarenas Province, Costa Rica, by 
Hertlein & Strong (1950: 242) was based on 
specimens of C. nasuta (CAS 121941). 

Also recorded in the late Pleistocene Miller- 
ton Formation at Tómales Bay, Marin County, 
California (Dickerson, 1922: 563, as "C. frag- 
///s"-CAS Loc. 561; Valentine, 1961: 390, as 
"C. frap/V/s"; R. G. Johnson, 1962: 115-120; J. 



62 



COAN 




FIG. 10. Corbula porcella. lectotype; USNM 97039; length, 7.9 mm. FIG. 11. C, esmeralda. FIG. 11a. Lecto- 
type; ANSP 218903; length, 20.6 mm. FIG. 11b. Paralectotype; ANSP 403198; length, 21.9 mm. 



EASTERN PACIFIC CORBULIDAE 



63 



E. Johnson, 1987: 116, as both С porcella 
and "C fragiüä'). The record of this species in 
the Pleistocene at Bahía Magdalena, Baja 
California Sur, México (Loe. 754; Jordan, 
1936: 111), was based instead on specimens 
of C. nasuta. 

Discussion 

Corbula procella may be distinguished from 
C. luteola, which is sympatric but in shallower 
water, in that the latter is more elongate and 
longer anteriorly, is frequently colored, has 
finer sculpture, is less inflated, has a less pro- 
nounced rib separating the central slope from 
the posterior slope, and has a less sharply de- 
fined escutcheon. 

Hertlein & Strong (1950), followed by Soot- 
Ryen(1957:11),^Keen(1958, 1971), and Ols- 
son (1961), suggested a relationship of С 
porcella to the Panamic C. obesa Hinds, 
1843. However, С obesa is very distinct, at- 
taining a larger size, being very inflated, hav- 
ing a proportionately more inflated right valve, 
having radial ribs on its umbones, in lacking 
an escutcheon, in being colored interiorly, and 
in having a heavier periostracum. 

Specimens of the highly variable C. nasuta 
account for Panamic records of С porcella. 
They differ as follows: 



С nasuta 

more pointed poster- 
oventral corner, often 
set off by a shallow 
radial sulcus just 
anterior to it 

radial ridge fairly sharp 
all the way to ventral 
nnargin 

tinted yellow to brown 

escutcheon indistinct 

sometime with a poste- 
rior spout 



С porcella 
even, truncate poste- 
rior end 



radial ridge becoming 

indistinct near ventral 

margin 
white 
eschtcheon defined by 

a ridge 
never with a posterior 

spout 



Subgenus (Hexacorbula) Olsson, 1932 

Hexacorbula 0\ssor\, 1932: 140 

Type species (original designation): Corbula 
hexacyma Brown & Pilsbry, 1913, = С 
gatunensis Toula, 1909 (references in 



^Oddly Soot-Ryen (1957) made C. obesa Hinds, 
1843, a junior synonym of С porcella Dall, 1916b, 
in reporting the latter from Panamá. His specimen 
was probably one of the variable C. nasuta. 



Discussion below). Middle Miocene, 
Panamá. 

Medium-sized, heavy, ovate to ovate-elon- 
gate, subequivalve, subequilateral; sculpture 
of strong commarginal undulations. 

Woodring (1 982: 71 3) and Anderson (1 996: 
12) suggested that Hexacorbula is very simi- 
lar in sculpture to Bothrocorbula Gabb, 1873a 
(p. 274, pi. 10, fig. 3, 3a); its type species, by 
monotypy, is Corbula vimlnea Guppy, 1866a 
(pp. 293, 295, pi. 18, fig. 11), from the middle 
Miocene of Jamaica and the Dominican Re- 
public. Bothrocorbula differs from Hexa- 
corbula in having a lunular pit. 

Corbula (Hexacorbula) esmeralda 
(Olsson, 1961) 
Figures 11, 44 

Caryocorbula (Hexacorbula) esmeralda Ols- 
son, 1961 

Olsson, 1961: 432-433, 549, pi. 76, fig. 
3-3c; Keen, 1971: 266-267, fig. 683 
[Corbula (Hexacorbula)] 

Type Material & Locality 

ANSP 218903, lectotype here desig- 
nated, right valve, the specimen shown in 
Olsson's fig. 3a, c; length, 20.6 mm; height, 
12.4 mm; width, 5.0 mm (Fig. 11a). ANSP 
403198, paralectotypes-left valve, shown in 
Olsson's fig. 3b, length, 21.9 mm; left valve, 
shown in Olsson's fig. 3; length, 20.6 mm 
(similar to holotype in size and shape, but 
they are not a pair) (Fig. lib); right valve, not 
figured in Olsson (1961), length, 20.3 mm. 
UMML 30.11326, paralectotypes, 3 right 
valves, 9 left valves. Esmeraldas, Esmeraldas 
Province, Ecuador (1 .0°N); 20 ft. [5 m]; Axel A. 
Olsson, 1958. 

Olsson (1961) illustrated an unmatched 
pair of valves as the "holotype", necessitating 
a lectotype selection. 

Description 

Shell ovate-elongate, thin in small speci- 
mens to very thick in large specimens; sube- 
quivalve; longer posteriorly (beaks at 33-39% 
from anterior end). Central slope with a broad 
radial sulcus. Posterior slope set off from cen- 
tral slope by a relatively sharp ridge. Es- 
cutcheon set off by a ridge. 

Beaks relatively smooth, with fine commar- 
ginal ribs and still finer radial rows of pustules. 



64 



COAN 



Most of surface with broad commarginal un- 
dulations and finer commarginal ribs. Poste- 
rior slope with only fine commarginal sculp- 
ture. 

Color white exteriorly and interiorly. Right 
valve with a large tooth; left valve with a broad 
chondrophore, which is not very projecting, 
and a small tooth (Fig. 44). Length to 22.5 mm 
(2.5 km S of river at Esmeraldas; Skoglund 
Collection). 

Distribution 

Esmeraldas, Esmeraldas Province (1.0°N) 
(type locality), to Chone, Bahía de Caráquez, 
Manabí Province (0.6°S) (USNM 709895), 
Ecuador, in 5-43 m (mean, 23.8; n = 4); no 
bottom types have been recorded. I have 
seen only 6 lots, including the types. 

Discussion 

Corbula esmeralda differs from the middle 
Miocene С (/-/.) gatunensis Tou\a, 1909 (p. 
733, pi. 27, fig. 12), in being more elongate 
and in having its posterior end less set off by 
a sulcus. In addition to С hexacyma Brown & 
Pilsbry, 1913 (p. 518, pi. 26, fig. 4), Woodring 
(1982: 714) listed C. (Carycorbula) buenavis- 
tana F. Hodson, in F Hodson & H. Hodson, 
1931 (p. 24, pi. 8, fig. 6, pi. 12, figs. 8-13), 
from the Miocene of Venezeula as a synonym 
of H. gatunensis. 

Corbula (H.) cruziana Olsson, 1 932 (p. 141 , 
pi. 3, fig. 5, pi. 4, fig. 9), from the early Mio- 
cene of Perú and Panamá is apparently the 
oldest member of this subgenus. 

Subgenus (Juliacorbula) 
Olsson & Harbison, 1953 

Juliacorbula Olsson & Harbison, 1953: 148- 

149 
Type species (original desigation): Corbula 
cubaniana d'Orbigny, 1853; = С knoxi- 
ana С В. Adams, 1852c; = С aequivalvis 
Philippi, 1836 (references in Discussion 
below). Recent, western Atlantic. 
Shell small to medium in size, heavy, sube- 
quivalve; beaks just posterior to midline; pos- 
terior end truncate; with a strong ridge be- 
tween central and posterior slopes and 
another outlining the escutcheon. Sculpture of 
strong commarginal ribs in both valves. 

After saying that their new genus contained 
"several" species, Olsson & Harbison (1953) 



referred only three to it: the type species, С 
scutata Gardner, 1944 (reference below), and 
the eastern Pacific С biradiata; the latter was 
perhaps an error for C. bicarinata, which is 
very similar if not identical to the type species. 

Corbula {Juliacorbula) bicarinata 

G. B. Sowerby I, 1833 

Figures 12, 13, 45 

Corbula bicarinata G. B. Sowerby I, 1833 
G. B. Sowerby I, 1833: 35; Hanley, 1843: 
46, 6, pi. 12, fig. 31; 1856: 344; Reeve, 
1844: pi. 3, fig. 23; d'Orbigny, 1845: 571; 
С В. Adams, 1852a: 521 [1852b: 297]; 
Carpenter, 1857a: 183, 224, 228, 244, 
280, 281, 300; 1857b: 21-22, 547; 
1864a: 368 [1872 reprint: 204]; 1864b: 
537 [1872 reprint: 23]; Tryon, 1869: 63; 
Lamy, 1941: 128-129; Hertlein & Strong, 
1950: 238 [Aloidls {Caryocorbula)]; 
Keen, 1958: 208-209, fig. 523 [Corbula 
(Caryocorbula)]; Olsson, 1961: 436, 548, 
pi. 75, fig. 6 -6b [Juliacorbula]; Keen, 
1971: 266-268, fig. 684 [Corbula {Julia- 
corbula)]; Gemmell et al., 1987: 60 

Type Material & Locality 

BMNH 1966567/1, lectotype here desig- 
nated, closed pair, the specimen figured 
by Reeve (1844); length, 10.6 mm; height, 
8.5 mm; width, 6.6 mm (Fig. 12). BMNH 
1966567/2-3, paralectotypes, 2 open 
pairs, lengths, 10.9, 10.5 mm. BMNH 
1907.12.30.102, paralectotype, open pair, 
length 9.6 mm. Four localities in "Columbiae 
Occidentalis" [West Colombia] were given by 
G. B. Sowerby I -Panamá; "Real Llejos" [= 
Rio Realejos; Corinto], Chinendega Province, 
Nicaragua; and "Caraccas" [Bahía Cará- 
quez], Manabí Province, Ecuador; and Santa 
Elena, Guayas Province, Ecuador. Neither of 
the two lots in the BMNH collection has a spe- 
cific locality, so the type locality is here clar- 
ified (ICZN Code, Recommendation 76a) as 
being Playa Kobbe, Panamá Province, 
Panamá (8.9°N), where the species is com- 
mon (Skoglund Collection). A collective depth 
for the original material was given as 7-17 
fms. [13-31 m], in sandy mud; Hugh Cuming. 

Description 

Ovate-subquadrate, moderately heavy; 
right valve slightly larger than left; posterior 



EASTERN PACIFIC CORBULIDAE 



65 




FIGS. 12, 13. Corbula bicarinata. FIG. 12. Lectotype; BMNH 1966567/1; length, 10.6 mm. FIG. 13. CAS 
120689; San Felipe, Baja California, México; length, 11.2 mm. 



end longer (beaks at 38-40% from anterior 
end). Anterior end rounded; posterior end 
abruptly truncate. Posterior slope set off fronn 
central slope by a sharp ridge that becomes 
somewhat more rounded ventrally. Es- 
cutcheon set off by a similarly sharp ridge. 

Beaks relatively smooth. Most of surface 
with moderate, rounded commarginal ribs, 
which continue onto posterior slope, and very 
fine radial striae. Escutcheon with fine com- 
marginal ribs. 

Periostracum light tan. White exteriorly; in- 
terior white to yellowish. Hinge plate narrow; 
right valve with a large tooth; left valve with a 
narrow chondrophore and a very inconspicu- 
ous tooth (Figs. 13, 45). Length to 13.0 mm 



(Bahía Cholla, Puerto Peñasco, Sonora, Méx- 
ico; Skoglund Collection). 

Distribution 

Head of the Golfo de California at Puerto 
Peñasco, Sonora, México (31.4°N) (Skoglund 
Collection; UCMP B.6008, E.8416, E.8431; 
LACM 60-11.33, 62-22.28, 63-56.38; SBMNH 
113654), south to Zorritos, Tumbes Province, 
Perú (3.7°S) (UMML 30.11424); Isla Santa 
Maria, Islas Galápagos, Ecuador (LACM 32- 
2.4); from the intertidal zone on undersides of 
rocks to 110 m, in rubble (mean, 12.4 m; n = 
61). I have seen 257 lots, including the types. 

This species has been reported in the Pleis- 



66 



COAN 



tocene at Bahía Santa Inez and Isla Carmen 
(Hertlein, 1957: 63), and in the Pliocene on 
Isla Carmen (Emerson & Hertlein, 1 964: 341 ), 
Baja California Sur, México. 

Discussion 

Carpenter (1 857b: 547) and some other au- 
thors have synonymized С alba Philippi, 
1846, with this species. However, Philippi's 
description, together with shapes of the Euro- 
pean fossil species with which he compared 
it, indicate that C. alba should instead be re- 
garded as a synonym of С nasuta (see Dis- 
cussion under the latter). 

Olsson (1961) suggested that C. ira Dali, 
1908, described from Panamá, might be a 
synonym. Although superficially similar in 
shape, C. ira is longer and narrower posteri- 
orly, there is no nb defining the escutcheon, 
and the commarginal ribs are fewer and much 
more prominent. 

This species is similar to the western At- 
lantic type species of the subgenus, С ae- 
quivalvis Philippi, 1836 (pp. 227-228, pi. 7, 
fig. 4), which has been reported from Florida 
to Panamá. Synonyms of С aequivalvis in- 
clude C. knoxiana С В. Adams, 1852c (pp. 
238-239), and C. cuban/ana d'Orbigny, 1853 
(p. 283 [Spanish ed., p. 322], pi. 26, figs. 
51 -54). Corbula aequivalvis seems to have 
finer commarginal sculpture than С bicari- 
nata (based on comparison with CAS 130494; 
Salinas Papaya, near Ensenada, southwest 
coast, Puerto Rico). Corbula aequivalvis was 
discussed by Jung (1969: 410-411, pi. 39, 
figs. 11-15) and by Anderson (1996: 17-18, 
pi. 2, figs. 22-26), who compared it with 
closely related fossil taxa, including С knoxi- 
ana fossilis Pilsbry, 1922 (pp. 427, 435, pi. 46, 
fig. 14), from the Miocene of the Dominican 
Republic; C. aequivalvis stainforthi Rutsch, 
1942 (p. 124-125, pi. 3, figs. 8, 9), from the 
Miocene of Trinidad; and С scutata Gardner, 
1944 (pp. 140-141, pi. 23, figs. 16, 30-32), 
from the Plio-Pleistocene of Florida and North 
Carolina. 

Subgenus {Panamicorbula) Pilsbry, 1932 

Panamicorbula PWsbry, 1932: 105 

Type species (original designation): Pota- 
momya inflata С В. Adams, 1852a; = 
Corbula ventricosa A. Adams & Reeve, 
1850 (references below); tropical eastern 
Pacific, in mangrove swamps. 
Shell large, thin in most material to thick in 



largest specimens; subequivalve; beaks just 
anterior to midline; left valve with submarginal 
marginal ridges; with fine commarginal sculp- 
ture; chondrophore broad, conspicuously di- 
vided. 

С В. Adams (1852a, b) described his three 
synonymous taxa in the genus Potamomya 
J. de С. Sowerby, 1835 (p. 241). Its type 
species, by subsequent designation of Keen 
(1969b: 698), is Mya plana J. Sowerby, 1814 
(pp. 173-174, pi. 76, fig. 2), from the Eocene 
and Oligocène of Europe. Potamomya is now 
considered to be a synonym of Erodona Bosc, 
1 801 , ex Daudin ms (vol. 2:329-330, pi. 6, fig. 
2) (Keen, 1969b: 698). Erodona, placed in its 
own family, the Erodonidae, within the My- 
oidea, is still living in brackish waters on the 
east coasts of Central and South America. It 
has a projecting chondrophore in the left valve 
similar to that of Mya. Tryon (1869) placed 
Potamomya aequalis in Corbula (Azara). 
Azara d'Orbigny, 1 842 (p. 1 61 , pi. 7), is an ob- 
jective synonym of Erodona (Vokes, 1 945: 26; 
Keen, 1969b: 698). 

Corbula (Panamicorbula) ventricosa 

A. Adams & Reeve, 1850 

Figures 14-19, 46 

Corbula ventricosa A. Adams & Reeve, 1850 
A. Adams & Reeve, 1850: 83, pi. 13, fig. 
12; Carpenter, 1857a: 284, 300; Tryon, 
1869:66 

NOT С ventricosa A. Adams & Reeve, auctt. 
[= С colimensis Coan, n. sp.] 
Hertlein & Strong, 1950: 242-243, 251, 
pi. 2, figs. 3, 4; Keen, 1958: 210, 211, fig. 
532; Olsson, 1961: 428, 549, pi. 76, fig. 
9; Keen, 1971: 266-267, fig. 682 

Potamomya aequalis С В. Adams, 1852 

С. В. Adams, 1852a: 519-520, 547-548 
[1852b: 295-296, 323-324]; Carpenter, 
1857a: 280, 300; 1864a: 363 [1872 
reprint: 204]; Tryon, 1869: 67 [Corbula 
(Azara)]; Turner, 1956: 28, 128, pi. 19, 
figs. 5, 6 [Potamomya]; Keen, 1958: 210- 
211 , fig. 533 [Corbula (Panamicorbula)] 

Potamomya inflata C. B. Adams, 1852 

С В. Adams, 1852a: 520, 548 [1852b: 
296, 324]; Carpenter, 1857a: 280, 300; 
1864a: 363 [1872 reprint: 204] [as a ju- 
nior synonym of P. aequalis]; Tryon, 
1869: 67 [as a junior synonym of С ae- 
qualis]; Pilsbry, 1932: 105 [Corbula 
(Panamicorbula)]; Vokes, 1945: 9, 11- 
1 2, pi. 2, figs. 1 -4; Turner, 1 956: 56, 1 26, 
pi. 17, figs. 12, 13 [Potamomya]; Keen, 



EASTERN PACIFIC CORBULIDAE 



67 




FIGS. 14, 15. Corbula ventricosa. FIG. 14. Lectotype of C. ventricosa; BMNH 1967980/1; length, 22.0 mm. 
FIG. 15. Holotype of Potamomya aequalis; MCZ 186325; length, 19.4 mm. 



1958: 210-211, fig. 535 [Corbula {Pana- 
micorbula);]; Olsson, 1961: 434-435, 
549, pi. 76, fig. 1-1c [Panamicorbula]; 
Keen, 1971: 268-269, fig. 689 [Corbula 
(Panamicorbula)] 
Potamomya trigonalisC. B. Adams, 1852 
С В. Adams, 1852a: 520, 548 [1852b: 



296, 324]; Carpenter, 1857a: 280, 300; 
1864b: 363 [1872 reprint: 204] [as proba- 
ble synonym of P. aequalis]; Tryon, 1869: 
67 [as probable synonym of Corbula ae- 
qualis]; Turner, 1956: 93, 128 [Pota- 
momya; as "triagonalis^'], pi. 18, figs. 3, 4; 
Hoffstetter, 1952: 44, fig. 10 [Pana- 



68 



COAN 




FIGS. 16, 17. Corbula ventricosa. FIG. 16. Lectotype of Potamomya inflata: MCZ 186315; length, 17.2 mm. 
FIG. 17. Lectotype of P. trigonalis; MCZ 186314; length, 23.8 mm. 



micorbula]: Keen, 1958: 210-211, fig. 
536 [Corbula (Panamicorbula)] 
Corbula macdonaldi Dal I, 1912 

Dall, 1912: 3; Dall, 1925: 15, pi. 17, figs. 
1, 3 [catalogue nunnber misquoted as 



214358]; Olsson, 1961: 435 [as a syn- 
onynn of P. inflata] 
Panamicorbula cylindrica Morrison, 1946 
Morrison, 1946: 47, pi. 1, figs. 15, 17; 
Keen, 1958: 210-211, fig. 534 [Corbula 



EASTERN PACIFIC CORBULIDAE 



69 




FIGS. 18, 19. Corbula ventricosa. FIG. 18. Lectotype of Corbula macdonaldi; USNM 214353; length, 20.7 
mm. FIG. 19. Holotype of Panamicorbula cylindrica; USNM 542186; length, 13.3 mm. FIG. 20. Corbula 
amurensis; CAS 089104; Martinez, Contra Costa County, California; length, 16.4 mm. 



70 



COAN 



{Panamicorbula)]; Olsson, 1961: 435, 

549, pi. 76, fig. 2, 2a [Panamicorbula]: 

Keen, 1971: 267-268, fig. 688 [Corbula 

{Panamicorbula)] 
?Corbula ustulata Reeve, auctt., non Reeve, 

1844 

Menke, 1847: 191; Carpenter, 1857a: 

236; 1857b: 539 
[non Reeve, 1844 -reference in Discussion 

of next species] 

Type Materials & Localities 

Corbula ventricosa- BMNH 1967980/1, 
lectotype here designated, pair, the larger of 
two specimens, probably that figured by A. 
Adams & Reeve (1850); length, 22.0 mm; 
height, 16.8 mm; width, 13.6 mm (Fig. 14). 
BMNH 1967980/2, paralectotype, pair; 
length, 1 6.0 mm. "China Sea," but type local- 
ity here clarified as the mangrove swamp at 
Paitilla, near Panamá City, Panamá Province, 
Panamá (9.0°N), where this species is known 
to occur (ANSP 155409). 

Potamomya aequalls~MCZ 186325, holo- 
type, pair; length, 19.4 mm; height, 16.5 mm; 
width, 10.2 mm (Fig. 15). Mangrove thicket, 
2.5 miles [6.5 km] E of Panamá City, Panamá 
Province, Panamá (9.0°N); soft mud; С В. 
Adams;, 27 Nov. 1850-2 Jan. 1851. 

Potamomya inflata-MCZ 186315, lecto- 
type (Turner, 1956: 126), pair; length, 17.2 
mm; height, 13.8 mm; width, 12.4 mm (Fig. 
16). MCZ 186316, paralectotypes, 2 pairs 
Same locality as P. aequalis. 

Potamomya trIgonalls-MCZ 186314, lecto- 
type (Turner, 1956: 128), pair; length, 23.8 
mm; height, 19.8 mm; width, 13.6 mm (Fig. 
17). There are no paralectotypes in the MCZ. 
Same locality as P. aequalis. 

Corbula macdonaldl-USNM 214353, lec- 
totype here designated, the specimen fig- 
ured by Dall (1925), left valve; length, 20.5 
mm; height, 16.5 mm; width, 5.7 mm (Fig. 18). 
USNM 517479, paralectotype, right valve; 
length, 22.7 mm; and pair; length, 18.6 mm. 
"Loc. 5848; Pleistocene muck beds at Colon," 
but label says "Miraflores Locks," and Olsson 
(1961: 435) says "near Panama City," 
Panamá Province, Panamá (9.0°N). 

Panamicorbula су//псУл/са- USNM 542186, 
holotype, pair; length, 13.3 mm; height, 9.3 
mm; width, 6.3 mm (Fig. 19). USNM 542187, 
paratypes: pair, length, 12.5 mm; right valve, 
length, 20.6 mm; broken right valve, length, 
approximately 21.2 mm; left valve, length, 
19.3 mm. Rio Marina mangrove swamp. Isla 



San José, Archipiélago de las Perlas, Panamá 
(8.3°N); J. R E. Morrison, 19 February 1944. 

Description 

Shell variable in shape, from ovate to trigo- 
nal, from thin- to thick-shelled; right valve 
slightly larger than left. Posterior end slightly 
longer (beaks 46% from anterior end). Ante- 
rior end rounded; posterior end obliquely sub- 
truncate. Posterior slope set off from central 
slope by a low ridge, more pronounced in 
some specimens than in others. Escutcheon 
present in most specimens, set off by a slight 
ridge and a change in sculpture. 

Anterior and posterior slopes of juvenile 
portion of unworn specimens with fine, regu- 
lar commarginal ribs. Central and posterior 
slopes of larger specimens with fine, irregular 
commarginal sculpture and fine radial striae. 

Periostracum brown to greenish, generally 
eroded away. White exteriorly and interiorly. 
Right valve with a large, triangular tooth and 
elongate submarginal ridges resembling lat- 
eral teeth situated well away from cardinal 
tooth; left valve with a large, projecting, di- 
vided chondrophore, having a moderate tooth 
on its posterior end; anteroventral hinge mar- 
gin swollen into a small tooth medially. Palliai 
sinus absent (Fig. 46). Length to 35.0 mm 
(USNM 61 2203; Indian kitchen midden at Val- 
divia, Guayas Province, Ecuador). 

Distribution 

Medaño Blanco, N of Topolobampo, 
Sinaloa, México (25.7°N) (Skoglund Collec- 
tion), to Puerto Pizarro, Tumbes Province, 
Perú (3.5°S) (SBMNH 127876; UMML 
30.11329, 11338). All living material has been 
collected from intertidal mudflats, generally in 
mangrove swamps. I have seen 78 lots, in- 
cluding the types of the various synonyms. 

Parker's (1964: 162) west Mexican offshore 
records of С ventricosa were based on spec- 
imens of C. ira (MCZ 260660, 260668), C. na- 
suta (MCZ 253667), and C. porcella (MCZ 
260013, 260679, 260684). 

This species was recorded as a subfossil at 
Laguna de Salinas, Guayas Province, Ecua- 
dor (Hoffstetter, 1952: 44, fig. 10, as 'Tana- 
micorbula trigonalis"). 

Anderson (1996: 18-19, pi. 3, figs. 1-10) 
described С (P.) canae from the upper Mio- 
cene Cercado Formation of the Dominican 
Republic. It is more rostrate and acuminate 
than the Recent species. She also discussed 



EASTERN PACIFIC CORBULIDAE 



71 



one other possible new species from the 
same formation. 

Discussion 

Carpenter (1857a: 284, 300) first sug- 
gested that Corbula ventricosa came from the 
eastern Pacific. Hertlein & Strong (1950) also 
discussed the likelihood that the type material 
of С ventricosa actually came from the 
Panamic Province, rather than the "China 
Sea," as originally indicated.'' This lot did in- 
deed come from the eastern Pacific, but it is 
not the species Hertlein & Strong thought. 

Given the fact that С В. Adams described 
three synonymous taxa from a single station, 
subsequent workers might have exercised 
more caution in proposing additional names 
without a better understanding of the variabil- 
ity of this species of Corbula {Panamicorbula). 
Although material of this species remains un- 
common in most collections, because few 
workers have collected in mangrove swamps, 
abundant specimens from single stations 
at the Instituto Nacional de Biodiversidad 
(INBio) in Costa Rica clearly demonstrate that 
only a single taxon is present. The largest 
specimens tend to become trigonal. Speci- 
mens in which the ventral margin turns medi- 
ally at a smaller size become inflated and 
more cylindrical. 

Subgenus {Potamocorbula) Habe, 1955 

Potamocorbula Habe, 1 955: 272 

Type species (original designation): Corbula 

amurensis Schrenck, 1861. Recent, 

northwestern Pacific. 
Small sized, thin, subequivalve, smooth. 
Left valve with a more prominent radial ridge 
than the right valve. Left valve with a project- 
ing chondrophore. 

Corbula (Potamocorbula) amurensis 
Schrenck, 1861 
Figures 20, 47 

Corbula amurensis Schrenck, 1861 

Schrenck, 1861: columns 412-413; 
Schrenck, 1867: 584-586, pi. 25, figs. 

''Although the Samarang did not stop in the eastern 
Pacific, the earlier Su/prtur expedition, on which Ed- 
ward Belcher also served, did so and was probably 
the source of the eastern Pacific material mixed into 
the Samarang collection. This confusion is dis- 
cussed by Carpenter (1857a: 224; 1864b: 534) and 
Hertlein & Strong (1950: 241). 



5-8; Lamy, 1941: 247-248 [as Corbula 
(Erodona)]; Oyama, 1980: 116, pi. 55, 
figs. 6, 8, 10, 13 [as Potamocorbula]; 
Scarlato, 1981: 392-393, pi. figs. 
415-417, text fig. 14; Zhuang & Cai, 
1983: 65, fig. 12; Coan et al., 2000: 
479-480, pi. 102. 

Corbula amplexaA. Adams, 1862 
A.Adams, 1862:223-224 

Corbula frequens Yokoyama, 1 922 

Yokoyama, 1922: 123, pi. 6, figs. 16, 17 

Corbula pustulosa Yokoyama, 1 922 [non Car- 
penter, 1857] 

Yokoyama, 1922: 123-124, pi. 6, fig. 18 
[non Carpenter, 1857b: 22-23] 

Corbula sematensis Yokoyama, 1922 

Yokoyama, 1922: 124-125, pi. 6, fig. 19 
[not fig. 20, which is a Poromya (Oyama, 
1980: 120)] 

Corbula vladivostokensis Baiisch, 1929 
Bartsch, 1929: 133, pi. 2, figs. 1-7 

Corbula amurensis takatuayamaensis Ando, 
1965 
Ando, 1965:209, fig. 28 

Type Materials & Localities 

Under study in a separate project by an- 
other worker. 

Description 

Ovate, thin; right valve decidedly larger 
than left valve; beaks anterior to midline (ap- 
proximately 41% from anterior end); anterior 
end sharply rounded; posterior end sharply 
rounded. Posterior end not set off from central 
slope in right valve, but set off by an angle in 
left valve. Escutcheon not evident. 

Beaks smooth; rest of surface with low, ir- 
regular commarginal ribs. Periostracum tan. 
Shell white exteriorly and interiorly (Fig. 20). 

Hinge plate narrow; right valve with a nar- 
row tooth, attached to shell wall below hinge- 
line; left valve with a long, projecting chon- 
drophore that is conspicuously divided, and 
with a very small tooth on its posterior end; 
anteroventral hinge margin swollen into a low 
tooth medially. Palliai line with a small sinus 
(Fig. 47). Length to 19.7 mm (CAS 121534; 
Carquinez Strait, San Francisco Bay, Contra 
Costa County, California). 

Distribution 

This introduced species is thus far found 
only in San Francisco Bay, California, its ecol- 



72 



COAN 



оду there discussed by Carlton et al. (1990), 
Nichols et al. (1990), and Duda (1994). It oc- 
curs from the intertidal zone to 8 m, on mud, 
sand or clay. 

Discussion 

Another worker in a separate project is at- 
tempting to understand the taxonomy of the 
Asian species attributed to this subgenus. 
Corbula laevis Hinds, 1843 (p. 59), and C. us- 
tulata Reeve, 1844 (pi. 4, fig. 25), are earlier 
names for members of this species complex. 
Resolution of this group will require access 
not only to the type material of the nominal 
species but also suites of specimens from 
several Asian localities to fully understand 
variability and distributions. Material of the 
San Francisco Bay import shows significant 
variability in shape and thickness. For exam- 
ple, CAS 121 534 contains specimens that are 
ovate, subtrigonal, and ovate-elongate. 

A comparison between С (Potamocorbula) 
amurensis and С {Panamicorbula) ventri- 
cosa is instructive because they both inhabit 
brackish waters: 



С amurensis 

inequivalve 

ovate, ovate-elongate, 

to subtrigonal 
smooth, with fine 

posterior radiais 

in right valve 
length to 20 mm 
has a small palliai sinus 
division between central 

and posterior slopes 

evident only in left 

valve 
no lateral ridges on 

hinge 
tooth in right valve 

seated deeply under 

hinge plate 
chondrophore very 

projecting 



С ventricosa 
subequivalve 
subquadrate 

stronger commarginal 
sculpture 

length to 35 mm 
palliai sinus not evident 
division between central 

and posterior slopes 

in both valves 

lateral ridges in nght 

valve 
tooth less deeply seated 



less projecting 



Subgenus {Tenuicorbula) Olsson, 1932 

Tenulcorbula 0\sson, 1932: 141 

Type species (original designation): Corbula 

tenuis G. B. Sowerby I, 1833 (reference 

below). Recent, eastern Pacific. 

Thin, subequivalve, longer posteriorly. With 

a strong keel separating posterior and central 

slopes and another keel defining a lunule. 

Posterior end truncate. Sculpture of fine, 



raised commarginal ribs, strongest on poste- 
rior slope. 

Corbula ( Tenuicorbula) tenuis 

G. B. Sowerby I, 1833 

Figs. 21,22, 48 

Corbula tenuis G. B. Sowerby I, 1833 

G. B. Sowerby I, 1833: 36; Hanley, 1843: 
47; Reeve, 1843: pi. 2, fig. 13; С В. 
Adams, 1852a: 523-524 [1852b: 299- 
300]; Carpenter, 1857a: 183, 228, 244, 
280, 300; 1864a: 363 [1872 reprint: 204]; 
1864b: 537 [1872 reprint: 23]; Tryon, 
1869: 66; Lamy, 1941: 143-144; Vokes, 
1945: 9, 14-15, pi. 2, figs. 10, 11; Keen, 
1958: 211, fig. 538 [Corbula (Tenui- 
corbula)]; Olsson, 1961: 433-434, 550, 
pi. 77, fig. 3, 3a [Tenuicorbula]; Keen, 
1971: 268-270, fig. 691 [Corbula (Tenui- 
corbula)] 

Corbula glyptaU, 1930 

Li, 1930: 264, pi. 5, fig. 38, 38a; Pilsbry, 
1931 : 431 [as a synonym of С tenuis] 

Type Materials & Localities 

Corbula tenuis-BMNH 1966563, holotype, 
pair; length, 22.8 mm; height, 12.1 mm; width, 
10.2 mm (Fig. 21). Bay of [Golfo de] Montijo, 
Veraguas Province, Panamá (7.7°N); 12 fms. 
[22 m]; Hugh Cuming. 

Corbula g/yptà- AM N H 268094 [formerly 
Columbia University 22098], holotype, pair; 
length, 23.9 mm; height, 13.4 mm; width, 10.6 
mm (Fig. 22). Mouth of Rio Grande near La 
Boca, Panamá Province, Panamá (8.9°N); 
10-40 ft. [3-12 m]; D. F. MacDonald, 1907. 
As "Miocene," but actually Recent (Pilsbry, 
1931:428,431). 

Description 

Shell elongate-subquadrate, thin, sube- 
quivalve; longer posteriorly (beaks at 40% 
from anterior end). Posterior end slightly ta- 
pered ventrally, truncate, sharply turned dor- 
sally. Posterior slope set off from central slope 
by a carina. Escutcheon well defined by a 
sharp angle. 

Beaks, central and posterior slopes with 
fine, dense commarginal ribs, strongest on 
posterior slope. Escutcheon much smoother. 

Periostracum tan; white exteriorly and inte- 
riorly. 

Right valve with a long, narrow tooth. Left 
valve with a projecting chondrophore; tooth 



EASTERN PACIFIC CORBULIDAE 



73 




FIGS. 21, 22. Corbula tenuis. FIG. 21. Holotype of C. tenu/s; BMNH 1966563; length, 22.8 mm. FIG. 22. 
Holotype of C. fif/ypfa; AMNH 268094; length, 23.9 mm. 



not evident (Fig. 48). Length to 24.5 mnn (Ve- 
nado Beach, Pananná Province, Panamá; 
Skoglund Collection). 

Distribution 

Southeastern coast of Isla Tiburón, Sonora, 
México (28.9°N) (MCZ 319592), to Zorritos, 



Tumbes Province, Perú (3.7°S) (UMML 
30.11308, 11358, 11359, 11395, 11396, 
11397, 11401), from the intertidal zone to 73 
m (mean 12.9 m; n = 8), on mud or sand. I 
have seen 30 lots, including the types. 

Although Olsson (1932) mentioned a spec- 
imen from an old collection at Cornell Univer- 
sity labeled as having come from Mazatlán, 



74 



COAN 



Sinaloa, México, no specimens from north of 
Panamá have been collected in recent years 
other than the single specimen from the cen- 
tral Golfo de California in the MCZ cited here 
as the northern record (MCZ 31 9592). ^ An- 
other specimen in the MCZ (319593) from an 
old collection is labeled has having come from 
"Margarita Bay, California," but there is no 
such place in California or Baja California, 
and this specimen may instead have come 
from Panamá. The record by DuShane (1962: 
44) of С tenuis from Puertecitos, Baja Cali- 
fornia, México, cannot be verified, because 
the specimen has not been located in the 
AMNH (J. Cordero, e-mail, 1 7 January 2001 ), 
the present location of the bulk of the 
DuShane collection. 

This species has also been recorded on the 
Pleistocene third terrace. Peninsula de Santa 
Elena, Guayas Province, Ecuador (Hoffstet- 
ter, 1948:81). 

Discussion 

Corbula tenuis lupina Olsson (1932: 143, 
pi. 14, figs. 7, 10), described from the Mio- 
cene Tumbes Formation at Quebrada Tucillal, 
Zorhtos, Tumbes Province, Perú, was said to 
differ in being heavier, more narrowly elon- 
gate, and more coarsely sculptured. Jung 
(1965: 477, 620, pi. 62, figs. 8, 9) subse- 
quently reported this subspecies from upper 
middle Miocene beds on the Paraguaná 
Peninsula of Venezuela, and he described the 
similar Tenuicorbula melajoensis \rorr\ the late 
Miocene of Trinidad (Jung, 1 969: 413-414, pi. 
40, figs. 7-9); it was said to differ in having 
heavier sculpture. These fossil taxa merit 
comparison with C. acutirostra Spieker, 1 922 
(pp. 176-177, pi. 10, figs. 18, 19), described 
from the late fvliocene Zorhtos Formation of 
Perú. 

Subgenus {Varicorbula) Grant & Gale, 1931 

Varicorbula Grant & Gale, 1931: 420, foot- 
note 1 



®The voucher collection in the MCZ from Parker's 
(1964) study of the benthic fauna of the west Mexi- 
can coast was incomplete, and the labels with the 
remaining specimens do not always correspond to 
the publication. For example, the single specimen 
that now constitutes the northern record of this 
species was not cited in the report, and the only 
other extant Parker voucher lot labeled С tenuis 
(his station 1) contained a mixture of С nasuta 
(MCZ 253549) and С biradiata (MCZ 320412). 



Type species (original designation): Tellina 
g/bba Olivi, 1792: 101. Recent, Mediter- 
ranean. 
Shell of medium size, very inequivalve, with 
hght valve larger, higher, more inflated and 
more rostrate. Right valve with pronounced 
commarginal sculpture; left valve with sub- 
dued commarginal sculpture and often with 
sparse radial ribs. 

Corbula (Varicorbula) grovesi 

Coan, new species 
Figures 23, 49 

Type Materials & Locality 

LACM 2891, holotype, pair; length, 11.0 
mm; height, 10.1 mm; width, 6.7 mm (Figs. 
23, 50). LACM 2892, paratype, pair; length, 
11.0 mm; height, 9.0 mm; width, 6.0 mm. Sal 
Si Puedes Basin, S end of Isla San Lorenzo, 
Baja California Sur, México (28.7°N, 
113.0°W), in 732 m (LACM 67-135). 

Description 

Trigonal-ovate; right valve much larger than 
left; left valve fitting inside right valve, leaving 
a wide margin; subequilateral (beaks at 48% 
from anterior end). Anterior end rounded; pos- 
terior end narrowed, subtruncate, without a 
radial rib between the central and posterior 
slopes. Escutcheon present, but not defined 
by a rib. 

Beaks smooth. Right valve with dense, 
closely set commarginal ribs. Left valve with 
5-6 faint radial ribs; otherwise without sculp- 
ture. 

Periostracum very thin. White externally; in- 
ternally white to yellowish. Hinge plate broad; 
hght valve with a prominent tooth; left valve 
with a large, vertically projecting chon- 
drophore and a prominent, elongate tooth 
(Fig. 49). Length to 11.0 mm (holotype and 
paratype). 

Distribution 

Thus far known from only the type lot con- 
taining two pairs. 

Etymology 

This species is named for Lindsey T. 
Groves of the Natural History Museum of Los 



EASTERN PACIFIC CORBULIDAE 



75 




FIG. 23. Corbula grovesi, holotype; LACM 2891; length, 11.0 mm. 



Angeles County, who has helped with this and 
many other projects. 

Discussion 

A similar western Atlantic Recent species is 
C. operculata Philippi, 1848 (p. 13), which oc- 
curs from North Carolina to Brazil. Synonyms 
of C. operculata include С krebsiana C. B. 
Adams, 1852c (p. 234), C. disparilis d'Or- 
bigny, 1853 (p. 283 [Spanish ed., p. 322], pi. 
27, figs. 1-4), and C. philippii E. A. Smith, 
1885 (p. 33, pi. 7, fig. 4-4b [not "pi. 8," as 
stated in text]). A commensal foraminiferan of 



С operculata was discussed by Bock & 
Moore (1968). Corbula caloosae Dall, 1898 
(p. 853), 1900 (pi. 36, fig. 16), from the Plio- 
Pleistocene Caloossahatchee Formation of 
Florida, merits close comparison with С oper- 
culata. The two Recent species differ as fol- 
lows (based on a comparison with BMSM 
15009; W of Cape Romano, Collier County, 
Florida; and BMSM 15011; Boca Grande, Lee 
County, Florida): 



grovesi 

rounded posteriorly 
fine sculpture in left 
valve 



operculata 
truncate posteriorly 
heavier sculpture in left 
valve 



76 



COAN 



periostracum inconspic- periostracum shiny, light 

uous, transparent tan 

right valve smooth, with nght valve with moderate 
fine radial rays commarginal sculpture 

and still finer radial 
rays 
beaks less prominent beaks prominent 

Of Recent eastern Pacific species, C. 
grovesi\s closest to С obesa, differing in liav- 
ing a thin, transparent periostracum, being 
more inequivalve, in lacking radial ribs on the 
beaks, in having a smoother left valve with 
faint radial ribs, and in having a proportion- 
ately broader hinge plate in the left valve with 
larger teeth. 

Corbula grovesi differs from the European 
type species of the subgenus Varicorbula-C. 
gibba-\u being more triangular, less truncate 
posteriorly, and white in color. 

There are two Pliocene species of Corbula 
(Varicorbula) in western North America. Cor- 
bula gibbiformis Grant & Gale, 1931 (pp. 
420-421, 920, pi. 19, figs. 4-6), was de- 
scribed from the early Pliocene upper 
Etchegoin Formation (near lower boundary of 
San Joaquin Formation) at Southern Califor- 
nia Gas Company Well 1 -4 (Sec. 4, T. 28 S., 
R. 23 E.; approximately 35.5°N, 119.5°W), 
Kern County, California, at a depth of 3,951 - 
3,952 feet. Examination of the holotype of C. 
gibbiformis (SDMNH 172, right valve; length, 
about 13 mm; height, about 13 mm) demon- 
strates that it is very different from C. grovesi, 
with heavy sculpture, bulbous, projecting 
beaks, and more prominent radial sculpture in 
the left valve. This Pliocene species is also re- 
ported from the late Pliocene San Joaquin For- 
mation, Kettleman Hills, Kings County (Grant 
& Gale, 1931; Woodring, 1938: 55-56, pi. 6, 
figs. 8, 9; Woodring et al., 1941: opposite p. 
78), the Niguel Formation, Orange County 
(Vedder, 1960: 326), and the San Diego For- 
mation, San Diego County (Hertlein & Grant, 
1972: 323-324, pi. 57, figs. 3, 4), California, 
and northwestern Baja California (Rowland, 
1972:29, as C. "gibbiformis (Sowerby, 1833)"), 
and from the middle Pliocene Pico [and/or 
Saugus] Formations, Ventura County (Watts, 
in Grant & Gale, 1 931 ; Woodring et al., in Win- 
terer & Durham, 1962: 304-305), the Santa 
"Clara" [Clarita] Valley, "Ventura" [Los Ange- 
les] County (Grant & Gale, 1 931 ), and the East 
Coyote field, USGS Loc. 13873, Los Angeles 
Basin, Los Angeles County (Woodring, 1938; 
USNM 496103), California. 

Corbula {Varicorbula) grand 0\sson, 1942 
(p. 197 [=45], 238 [=86], pi. 15 [=2], figs. 8, 9), 



was described from the Pliocene Charco Azul 
Formation, Quebrada Peñitas, Burica Penin- 
sula, Costa Rica. The type material (PRI 
5505, 5506) is now missing (W. Alimón, e- 
mail, 11 August 2000). It has a more rostrate 
posterior end and heavier sculpture in the 
right valve than C. grovesi, and it has no radial 
sculpture on the left valve. 

The Pliocene species merit further compar- 
ison with several other species from New 
World Pliocene, Miocene, and Oligocène 
strata, including Corbula chowanensis Bailey, 
1977 (pp. 129-130), from the Pliocene of 
North Carolina, and С caloosaeDaW, 1898 (p. 
853; 1 900: pi. 36, fig. 1 6), from the Pliocene of 
Florida. Corbula bradleyi Nelson, 1870 (p. 
200), from the Miocene Tumbes Formation at 
Zorhtos, Tumbes Province, Perú, illustrated 
and discussed by Spieker (1922: 171-172, 
196, pi. 10, figs. 13, 14), was subsequently 
tentatively identified from the Pliocene Charco 
Azul Formation at Quebrada Melissa, Burica 
Peninsula, Panamá (Olsson, 1942: 197- 
198). Other taxa include: Corbula chipolana 
Gardner, 1928 (p. 229, pi. 34, figs. 13-17); С 
chipolana Carolina Richards, 1977 (p. 33, pi. 
11, figs. 34, 35); C. waltonensis Gardner, 
1928 (p. 229, pi. 34, figs. 18-22), and С wal- 
tonensis rubisiniana Mansfield, 1932 (pp. 
156-157, pi. 34, figs. 2-4), from the Miocene 
of Florida; C. sanctidominici Maury, 1925 (pp. 
98-99, pi. 19, fig. 2), from the upper Miocene 
of the Dominican Republic; C. vieta Guppy, 
1866b (pp. 580-581, 590, pi. 26, fig. 8), and 
C. islatrinitalis Maury, 1925 (p. 101, pi. 19, 
figs. 8-10), from the Miocene of Trinidad; С 
heterogena Dall, 1898, ex Guppy ms (p. 850; 
1900: pi. 36, fig. 15), from the Miocene of 
Panamá; С. carrizalana F. Hodson, in F Hod- 
son & H. Hodson, 1931 (p. 23-24, pi. 10, figs. 
4, 6-9), from the Miocene of Venezuela; C. 
ргелис/а Speiker, 1922 (p. 172, pi. 10, fig. 12), 
from the Miocene of Perú, and C. zuliana F. 
Hodson, in F Hodson & H. Hodson, 1931 (pp. 
22-23, pi. 10, figs. 1-3, 5), from the upper 
Oligocène of Venezeula. (For a discussion of 
some of these species: Woodring, 1982: 
715-717; Anderson, 1996: 20-22). It it likely 
that there are fewer species than there are 
names. 

Corbula (Varicorbula) obesa Hinds, 1843 
Figures 24, 25, 50 

Corbula obesa Hinds, 1843 

Hinds, 1843: 57 [1844 reprint: 230]; 
Reeve, 1 844: pi. 5, fig. 38 [in part; text but 



EASTERN PACIFIC CORBULIDAE 



77 




FIGS. 24, 25. Corbula obesa. FIG. 24. Neotype of C. obesa; SBMNH 345495; length, 12.7 mm. FIG. 25. Fig- 
ure of С obesa from Hinds (1845). 



not fig., which = С nasuta]; Hinds, 1845 
68, pi. 20, fig. 12; С В. Adams, 1852a 
522 [1852b: 298]; Carpenter, 1857a: 207 
280, 300; 1864a: 368 [1872 reprint: 204] 
1864b: 537, 668 [1872 reprint: 23, 154] 
Tryon, 1869: 65; Oldroyd, 1925: 202 [in 
part]; Lamy, 1941: 133; Keen, 1958: 209, 
fig. 529 [Corbula {Caryocorbula)]; Keen, 
1966: 268; Keen, 1971: 265-266, fig. 
679 
Corbula nuciformis G. B. Sowerby I, auctt., 
nonG. B. Sowerby I, 1833 
Hertlein & Strong, 1950: 241, 251, pi. 2, 
fig. 1; Keen, 1958: 209, fig. 528 



Type Materials & Localities 

Corbula obesa- SBMNH 345495, neotype 
here designated, pair; length, 12.7 mm; 
height, 11.0 mm; width, 9.0 mm, with dried 
soft parts (Figs. 24, 50). Mazatlán, Sinaloa, 
México (23.2°N, 106.4°W); 91-128 m; Don- 
ald R. Shasky, October 3, 1961; exSkoglund 
Collection. (Other material from this station in 
the Skoglund and museum collections has no 
type status.) Two localities were originally pro- 
vided for this species -San Bias, Nayarit, 
México (21.5°N), and Veragua[s] Province, 
Panamá (approximately 7.7°N); the collective 



78 



COAN 



depth distribution originally given was 22-33 
fms. [38-60 m], on mud; Edward Belcher. No 
material from either station has been located 
in the BMNH collection. 

Workers have guessed about the identity of 
this species. Reeve (1844) illustrated a spec- 
imen of С nasuta as C. obesa. Hertlein & 
Strong (1950), followed by Keen (1958), fig- 
ured the present species as C. nuciformis, 
which is here regarded as a synonym of C. 
nasuta. Keen (1958, 1971) speculated on a 
possible relationship between С porcella and 
С obesa, but they are not very similar (see 
Discussion under C. porcella). A neotype is 
therefore necessary to stabilize nomencla- 
ture. 

Hinds' description and subsequent figure 
(Fig. 25) are in accord with the neotype here 
designated, which is about twice as large as 
the originally measured specimen. Mazatlán 
is approximately 225 km north of San Bias, 
but this species occurs still further north. 

Description 

Shell ovate, very inflated, rotund; right valve 
much more inflated than left; equilateral 
(beaks at about 50% from anterior end). Pos- 
terior end narrow, subtruncate, only slightly 
extended by a spout. Posterior slope set off 
from central slope by a broadly rounded ridge. 
Escutcheon not evident. 

Beaks of both valves with commarginal and 
radial ribs. Right valve with heavy commar- 
ginal sculpture; left valve with much finer com- 
marginal sculpture. Periostracum heavy, light 
brown. Exterior surface white; interior surface 
white, suffused light brown; adductor muscle 
scars and palliai line stained brown in some. 
Right valve with a large tooth; left valve with a 
narrow, non-projecting chondrophore and a 
projecting ridge on its posterior end (Fig. 50). 
Length to 12.7 mm (neotype). 

Distribution 

Isla Espíritu Santo, Baja California Sur 
(25.5°N) (CAS 121638; LACM 60-6.27; 
SBMNH 996; Skoglund Collection), and 
Mazatlán, Sinaloa (23.2^^N) (SBMNH 129866, 
21358, 21362), México, to near Isla Coiba, 
Veraguas Province, Panamá (7.4"N) (Kaiser 
Collection); from 14-205 m depth (mean, 
100.9 m; n = 33), on mud bottoms. There is a 
lot in the CAS labeled as having been col- 
lected on Isla Cedros, Baja California 



(28.2°N) (CAS 121638), presumably in beach 
drift. This locality requires further verification. 
I have seen 38 lots. 

The record of C. obesa from Santa Catalina 
Island, California (Dall, 1921: 53), was based 
on specimens of C. nasuta (USNM 199001), 
and also these were probably from Isla Santa 
Catalina in the southern Golfo de California, a 
labeling error that has resulted in other mis- 
taken Californian records of Panamic taxa. 



Discussion 

Corbula grant! Olsson, 1942, from the 
Pliocene of Costa Rica, may be a synonym of 
C. obesa (reference in Discussion of С 
grovesi). Olsson's material was admittedly 
worn, and the radial sculpture on the beaks 
may not have been visible. Short of rediscov- 
ery of the now-missing type material, topo- 
typic specimes would be required to make a 
convincing case. 

This species has some resemblance to С 
patagónica d'Orbigny, 1845 (p. 570; 1847: pi. 
82, figs. 18-22), which occurs from Brazil to 
Argentina. Corbula patagónica attains a 
larger size, has a narrower, more produced 
posterior end, and has less bulbous beaks 
that lack the prominent radial sculpture pres- 
ent on those of С obesa. Corbula patagó- 
nica also has sparse radial ribs on its right 
valve. 



Corbula, S.I. 

I am hesitant to place any of the following 
species in named subgenera. Much more 
work must be done to demonstrate true lin- 
eages within this family. 

Corbula biradiata G. B. Sowerby I, 1833 
Fig. 26-31,51 

Corbula biradiata G. B. Sowerby I, 1833 

G. B. Sowerby I, 1833; 35; Hanley, 1843: 
47, 4, pi. 10, fig. 51; 1856; 344; Reeve, 
1843: pi. 1, fig. 3; d'Orbigny, 1845: 571; 
С В. Adams, 1852a: 521-522 [1852b: 
297-298]; Carpenter, 1857a: 183, 244, 
280, 300; 1857b: 22; 1864a: 368, 369 
[1872 reprint: 204, 205]; 1864b: 534, 537 
553, 637 [1872 reprint: 20, 23, 39, 123] 
Tryon, 1869: 63; Lamy, 1941: 134 
Hertlein & Strong, 1950: 238 [Aloidis 
{Caryocorbula)]; Keen, 1958: 208-209, 



EASTERN PACIFIC CORBULIDAE 



79 




FIGS. 26-28. Corbula biradiata. FIG. 26. Lectotype of C. biradiata; BMNH 1966564/1 ; length, 16.2 mm. FIG 
27. Holotype of С rubra; MCZ 186313; length, 12.5 mm. FIG. 28. Holotype of С ecuabula; ANSP 14486- 
length, 16.3 mm. 




FIGS. 29-31. Corbula biradiata. FIG. 29. Holotype of Juliacorbula e/enens/s; ANSP 218913; length, 17.0 
mm. FIG. 30. Paratypes of J. elenensis; UMML 30.11384; lengths, 11.5 mm, 9.4 mm. FIG. 31. LACM 34- 
318.2; Isla La Plata, Manabi Province, Ecuador; 13-18 m; length, 12.3 mm. 



fig. 524 [poor redrawing of Reeve's fig- 
ure] [Corbula (Caryocorbula)]; Olsson, 
1961: 437, 548, pi. 75, figs. 6-6b [Juli- 
acorbula]; Keen, 1971: 267-268, fig. 685 
[ Corbula ( Juliacorbula) ] 
Corbula rubra C. B. Adams, 1852 

С В. Adams, 1852a: 523, 548 [1852b: 
299, 324]; Carpenter, 1857a: 280, 300; 
1864a: 363 [1872 reprint: 204] [as a syn- 
onym of С biradiata]; 1864b: 553 [1872 



reprint: 39]; Turner, 1956: 82-83, 126, pi. 
17, figs. 8, 9 

Corbula polychroma Gould & Carpenter, 1 857 
Gould & Carpenter, 1857: 198-199; Car- 
penter, 1857a: 226, 228, 300; 1864a: 31 
[1872 reprint: 205] [as a synonym of С 
biradiata]; Carpenter, 1864b: 534, 553 
[1872 reprint: 20, 39]; Palmer, 1958: 117; 
1963: 318; R. I. Johnson, 1964: 129 

Corbula ecuabula Pilsbry & Olsson, 1941 



EASTERN PACIFIC CORBULIDAE 



81 



Pilsbry & Olsson, 1 941 : 75, 78, pi. 1 2, figs. 
3-5; Olsson, 1961 : 437 [Juliacorbula] 
Juliacorbula e/enens/s Olsson, 1961 

Olsson, 1961: 438, 550, pi. 77, fig. 5; 
Keen, 1971: 267, 268, fig. 686 [Corbula 
{Juliacorbula)] 

Type Materials & Localities 

Corbula biradiata-BMNH 1966564/1, lec- 
totype here designated, closed pair, num- 
bered "5" on shell, possibly the originally nnea- 
sured specimen and that figured by Reeve 
(1843); length, 16.2 mm; height, 11.1 mm; 
width, 8.7 mm (Fig. 26). BMNH 1966564/2, 
paralectotype, open pair, length 15.2 mm. 
BMNH 1966564/3, paralectotype, closed pair; 
length, 14.7 mm; BMNH 1966564/4, open 
pair; length, 14.1 mm. [Golfo de] Chiriquí, Ve- 
raguas Province, Panamá (8.0°N); Hugh 
Cuming. BMNH 1907.12.30.118, paralecto- 
type, open pair, that figured by Hanley (1 843); 
length, 9.5 mm. "Bay of Caraccas" [Bahía de 
Caráquez], Manabí Province, Ecuador; Hugh 
Cuming. A collective habitat of 3-6 fms. [5-11 
m], on mud and sand, was given for this 
species. 

Corbula rubra-MCZ 186313, holotype, 
pair; length, 12.5 mm; height, 4.6 mm; width, 
3.6 mm (Fig. 27). Panamá, presumably near 
Panamá City, Panamá Province (about 
9.0°N), Panamá; С. В. Adams, 27 Nov. 1850- 
2 Jan. 1851. 

Corbula polych roma- No\ located. The 
originally cited Gould collection specimens, 
stated to have come from Santa Barbara, Cal- 
ifornia, might be expected to be either in the 
MCZ or the USNM, but they have not been lo- 
cated. Carpenter (1864a:31) later said that 
these specimens probably instead came from 
either Acapuico, México, or Panamá. Speci- 
mens were also originally cited from the Cum- 
ing Collection obtained from the Golfo de Cal- 
ifornia, and these might be expected in the 
BMNH, but they have not been located. The 
originally stated measurements were: length, 
13.4 mm; height, 6.8 mm; width, 9.4 mm 
(height and width were probably reversed). 

Corbula ecuabula- ANSP 14486, holotype, 
right valve; length, 16.3 mm; height, 11 .5 mm; 
width, 4.2 mm (Fig. 28). ANSP 78998, 
paratype, left valve; length, 11.2 mm. Punta 
Blanca, Manabí Province, Ecuador (1.1 °S); 
Canoa Formation, middle to late Pliocene. 
UMML 30.11455, paratypes, 1 right valve, 2 
left valves; Puerto Callo, Manabí Province, 
Ecuador (1.3°S); Recent. 



Juliacorbula e/enens/s- ANSP 218913, 
holotype, right valve; length, 17.0 mm; height, 
12.0 mm; width, 4.4 mm (Fig. 29). UMML 
30.11341, paratypes, 5 right valves, 1 left 
valve; [Salinas], Santa Elena Peninsula, 
Guayas Province, Ecuador (2.2°S). UMML 
30.11384, paratypes, 5 right valves, 1 left 
valve (Fig. 30); Puerto Callo, Manabí 
Province, Ecuador (1.3°S). UMML 30.11456, 
paratype, 1 right valve; Zorritos, Tumbes 
Province, Perú (3.7°S) 

Description 

Ovate-elongate, thin to moderately heavy; 
right valve slightly larger than left; posterior 
end usually longer (beaks at 41 -48% from 
anterior end), but anterior end slightly longer 
in some specimens (beaks at 54-55% from 
anterior end). Anterior end rounded; posterior 
end narrowed, obliquely subtruncate, ext- 
ended by a small spout in some specimens; 
bluntly subtruncate in some specimens. Pos- 
terior slope set off from central slope by a 
fairly sharp ridge that continues to ventral 
margin. Escutcheon well defined by a ridge. 

Beaks, central and posterior slopes with 
moderate commarginal ribs and very fine ra- 
dial striae. Some material with only fine com- 
marginal striae. Escutcheon smooth. 

Exterior light tan to orange, with light radial 
color bands about a third of the way to ends in 
small specimens; ends purple. Interior red- 
dish-brown to purple, particularly around mar- 
gins, but some material nearly colorless. 
Hinge plate broad; right valve with a large 
tooth; left valve with a narrow chondrophore 
and a conspicuous tooth (Fig. 51). Length to 
20.8 mm (Búcaro, Los Santos Province, 
Panamá; UMML 30.11442). 

Distribution 

El Sólita, Laguna Ojo de Liebre [Scam- 
mons]. Baja California (27.8°N) (SBMNH 
31519); Bahía San Luis Gonzaga, Baja Cali- 
fornia (29.8°N) (LACM 40-36.5), and Isla San 
Jorge, Sonora (31.0°N) (Skoglund Collec- 
tion), México, to Punta Peña Mala, Piura 
Province, Perú (4.2°S) (UMML30.11449); Isla 
Santa Cruz (LACM 38-193.14) and Isla San 
Cristóbal (LACM 34-43.22), Islas Galápagos, 
Ecuador; from the intertidal zone to 57 m 
(mean, 7.3 m; n = 86), on mud or sand. I have 
seen 248 lots, including the types of the vari- 
ous synonyms. 



82 



COAN 



This species is reported in beds of Pleis- 
tocene age at Puerto Libertad, Sonora, Méx- 
ico (Stump, 1975: 182, 186, 193, 195), and 
from the Pleistocene Armuelles Formation, 
Burica Peninsula, Chiriqui Province, Panamá 
(Olsson, 1942; 162). It is also recorded from 
the middle to late Pliocene Canoa Formation 
at Punta Blanca, Manabí Province, Ecuador 
(Pilsbry & Olsson, 1 941 : 1 1 , 75, as both C. bi- 
radiata and C. ecuabula: UMML 30.1 1 380). 

Rutten (1 931 : 661 ) reported but did not fig- 
ure this species (as "cf.") from the Quaternary 
of Surinam, based on an unpublished thesis, 
a record that requires further vehfication. 



Discussion 

I have come to the conclusion that С 
ecuabula is inseparable from С biradiata. 
The key feature of Corbula ecuabula, which 
Pilsbry & Olsson (1941) and Olsson (1961) 
also reported from the Recent fauna, was that 
it is significantly longer anteriorly. Olsson de- 
scribed the beaks of С ecuabula as being in 
the "posterior third", but even in his type ma- 
terial, the beaks are only 4-5% behind the 
midline. Occasional specimens may be found 
throughout the distribution of С biradiata that 
have a slightly longer anterior end. 

I then concluded that Juliacorbula elenen- 
sis is also a synonym. This species was 
based on beach-drift material that is light in 
color, rounded, and with even commarginal 
sculpture. I have found similar material from 
the Golfo de California (LACM 78-30.12, from 
Bahía San Carlos, Sonora, and LACM 79- 
111.14, from Bahía de los Angeles, Baja Cali- 
fornia), and a specimen from Esmeraldas, 
Ecuador (UMML 30.11321), came to light with 
subdued sculpture in the left valve and "ele- 
nens/s"-like sculpture in the right valve. An off- 
shore specimen from Isla La Plata, Manabí 
Province, Ecuador (LACM 34-318.8), is thin, 
white, and almost equilateral (Fig. 31). 

In the fossil fauna, this Corbula biradiata 
merits comparison with С {Caryocorbula) 
urumacoensis F Hodson, in F. Hodson & H. 
Hodson, 1931 (pp. 25-26, pi. 12, figs. 1-7), 
and C. (C) democraciana F. Hodson, in F. 
Hodson & H. Hodson, 1 931 (pp. 26-27, pi. 1 1 , 
figs. 1-6), both described from the middle 
Miocene of Venezuela, and perhaps to С (С) 
retusa Gardner, 1944 (p. 140, pi. 23, figs. 33, 
34) [synonym: C. (C) соплас// Gardner, 1944: 
139-140, pi. 23, figs. 27, 28, пол Dali, 1898), 
from the Pliocene of Virginia and North Car- 



olina (concerning C. retusa: Campbell, 1993: 
47-48, pi. 21, fig. 190). 

Corbula colimensis Coan, new species 
Figures 32, 52 

Corbula ventricosaA. Adams & Reeve, auctt., 
non A. Adams & Reeve, 1850 
Hertlein & Strong, 1950: 242-243, pi. 2, 
figs. 3, 4 [Aloidis {Caryocorbula)]; Keen, 
1958: 210-211, fig. 532 [Corbula (Cary- 
ocorbula)]; Olsson, 1 961 : 428, 549, pi. 76, 
fig. 9 [Caryocorbula {Caryocorbula)]; 
Keen, 1971: 266-267, fig. 682 [Corbula 
{Caryocorbula)] 

Type Material & Locality 

SBMNH 345496, holotype, pair; length, 
13.7 mm; height, 9.5 mm; width, 7.7 mm 
(Figs. 32, 52). SBMNH 345497, paratypes, 3 
closed pairs; lengths, 14.0 mm, 13.4 mm, 3.1 
mm. Las Ventanas, Manzanillo, Colima, Méx- 
ico (19.0°N, 104.3°W), 42 m; Cari & Laury 
Shy, 1969-1970; exSkoglund Collection 

Description 

Shell ovate, thin to moderate in thickess; 
right valve slightly larger than left; posterior 
end longer (beaks at 32% from anterior end). 
Posterior end subtruncate, extended by a 
short spout in some specimens; posterior 
slope set off from central slope by a rounded 
ridge. Escutcheon faint, set off by a slight 
ridge in right valve. 

Beaks with fine commarginal ribs; most of 
surface with moderate commarginal ribs. Pe- 
riostracum light brown. Shell white exteriorly 
and interiorly. Hinge plate of moderate thick- 
ness; right valve with a large tooth; left valve 
with a chondrophore of medium width, which 
is not very projecting, and a moderate tooth. 
Palliai sinus with a somewhat sharp bend 
posteriorly (Fig. 53). Length to 14.0 mm (a 
paratype). 

Distribution 

Los Corchos, Nayarit (21.7°N) (MCZ 
260451), to Bahía Tangola Tangola, Oaxaca 
(15.8°N)(LACM34-240.5), México, in 29-112 
m (mean, 55.9 m; n = 7), on mud bottoms. 
This species is thus far known from 8 lots. The 
lot cited by Hertlein & Strong (1950) from off 



EASTERN PACIFIC CORBULIDAE 



83 




FIG. 32. Corbula colimensis, holotype; SBMNH 345496; length. 13.7 mm. FIG. 33. C. ira, lectotype; USNM 
122944; length, 11.4 mm. 



84 



COAN 



Bahía Tangola Tangola has not been located 
intheCASorAMNH. 

Etymology 

This species is named for the Mexican state 
of Colima. 



Periostracum tan. Exterior white; interior 
white, sometimes suffused with purple or 
brown. Hinge plate broad; right valve with a 
large tooth; left valve with a very small chon- 
drophore and a small tooth (Fig. 53). Length 
to 13.6 mm (USNM 810921; Cabo Lobos, 
Sonora, México). 



Discussion 



Distribution & Habitat 



Corbula colimensis is most similar in size, 
shape, and color to C. obesa, but the new 
species is equivalve, thinner, and less in- 
flated, and it has no radial sculpture. 

Corbula ira DaW, 1908 
Figures 33, 53 

Corbula (Cuneocorbula) ira Dali, 1908 

Dali, 1908: 423; Lamy, 1941: 233-234; 
Keen, 1 958: 210 [as a synonym of С ven- 
tricosa]; Olsson, 1961: 436, 549, pi. 76, 
fig. 5 [Juliacorbula]: Keen, 1971: 267- 
268. fig. 687 [Corbula (Juliacorbula)] 

Type Material & Locality 

USNM 122944, lectotype here desig- 
nated, right valve; length, 11.4 mm; height, 
8.6 mm; width, 3.0 mm (Fig. 33). USNM 
880651, paralectotypes, 1 left valve, length, 
11.8 mm (close in size to lectotype, but they 
are not a pair); right valve, length, 11.0 mm. 
Albatross Stn. 3355; Dall (1908), followed by 
other authors, reported this as "Gulf of 
Panamá," but the log and map (Townsend, 
1 901 : 412) place this station at 7.2°N, 80.9°W, 
just off Punta Manato, Peninsula de Azuero, 
Veraguas Province, Panamá; 182 fms. [333 
m], mud; Feb. 23, 1891. 

Description 

Ovate-subquadrate, moderately heavy; 
right valve slightly larger than left; beaks 
closer to anterior end (beaks at 30-34% from 
anterior end). Ventral surface sometimes flat- 
tened. Anterior end sharply rounded. Poste- 
rior end tapered, obliquely truncate. Posterior 
slope set off from central slope by a sharp 
ridge that runs almost to ventral margin. Es- 
cutcheon defined by a ridge. 

Beaks relatively smooth. Central slope with 
strong, rounded, shingle-like commarginal 
ribs and very fine radial ribs. Posterior slope 
with finer commarginal ribs. 



Cabo Lobos, Sonora (29.9'^N) (USNM 
212797; MCZ 260668), and Bahía San Luis 
Gonzaga, Baja California (29.8°N) (LACM 37- 
199.10, 40-36.6), México, to Callao, Lima 
Province, Perú (12.1°S) (LACM 35-153.2, 35- 
177.3); Isla del Coco, Costa Rica (SBMNH 
345530), from 15 to 388 m (mean, 98.4 m; 
n = 60), on sand and mud. I have seen 85 lots, 
including the types. 

Discussion 

This species can be distinguished from С 
marmorata of similar size in being more 
quadrate and elongate, more truncate poste- 
riorly, with a stronger radial rib, and commar- 
ginal ribs that are higher and narrower. It may 
also become ventrally flattened, as in C. na- 
suta, which is never true of С marmorata. 

Corbula luteola Carpenter, 1 864 
Figures 34, 35, 54 

Corbula luteola Carpenter, 1864 

Carpenter, 1 864b: 611 , 637 [1 872 reprint: 
97, 123]; 1865: 207; Tryon, 1869: 65; 
Arnold, 1903: 181, pi. 17, fig. 11 [a poor 
figure]; Oldroyd, 1925: 203; Grant & 
Gale, 1 931 : 421 -422, 920, pi. 1 9, figs. 2, 
7 [Corbula (Lentidium)]; Lamy, 1941: 
240-241 [Corbula (Corbuloyma)]; 
Hertlein & Strong, 1950: 239 [Aloidis 
(Caryocorbula)]; Keen, 1958: 209 [in 
part; not fig. 525, which = C. marmorata] 
[Corbula {Caryocorbula)]; Palmer, 1958: 
117-118, 340, pi. 15, fig. ^3-^8 [Corbula 
(Lentidium)]; Keen, 1971: 264 [in part; 
not fig. 675, which = С marmorata] [Cor- 
bula (Caryocorbula)]; Hertlein & Grant, 
1972: 324-325, pi. 55, figs. 1, 2, 5, 6, 15 
[Corbula (Lentidium)]; Coan et al., 2000: 
479, pi. 102 [Juliacorbula] 

Corbula luteola rosea Williamson, 1905, non 
T. Brown, 1843 (or earlier), non Reeve, 
1844 
Williamson, 1905: 120; Coan, 1989: 298 



EASTERN PACIFIC CORBULIDAE 



85 




FIGS. 34, 35. Corbula luteola. FIG. 34. Lectotype of С luteola; USNM 14897; length, 10.2 mm. FIG. 35. Holo- 
type of C. luteola rosea; LACM 1421 ; length, 7.0 mm. 



[as a synonym of C. luteola] [non Corbula 
rosea!. Brown, 1843 (or earlier): 105, pi. 
42, fig. 6; non Corbula rosea Reeve, 
1844: pi. 5, fig. 26] 

Type Materials & Localities 

Corbula luteola-USNM 14897, lectotype 
here designated, the largest pair, closest to 
Carpenter's (1865) measurennent; length, 
10.2 nnm; height, 6.8 mm; width, 4.5 mm (Fig. 
34). USNM 880650, paralectotypes: pair, 
length, 8.4 mm (the specimen figured by 
Palmer, 1958); right valve, length, 8.3 mm; left 
valve, length 5.9 mm; pair, length, 5.7 mm. 



San Pedro, Los Angeles County, California 
(33.7°N); James G. Cooper. (Evidently, at 
some point in the past, USNM 15668 had 
been combined into this lot, because this 
number is written on the back of the label.) 
USNM 73457, paralectotypes, 1 right valve, 1 
left valve; San Diego, San Diego County, Cal- 
ifornia (32.7°N); James G. Cooper. Lots in 
other collections from Cooper material are not 
types, because they were not studied by Car- 
penter. 

Corbula luteola rosea Williamson -LACM 
1421, holotype, pair; length, 7.0 mm; height 
4.6 mm; width, 2.3 mm (Fig. 35). Terminal Is- 
land, San Pedro, California (33.7°N); on an 



86 



COAN 



anemone in a rock pool on the old breakwa- 
ter; Martha Burton Williamson. The original 
description specified a single valve, but what 
is in the type lot is a matched pair of valves. 



Description 

Shell ovate, solid; right valve slightly larger 
than left; posterior end longer (approximately 
42-48% from anterior end). Posterior end 
vertically truncate. Posterior slope set off from 
central slope by a rounded ridge that be- 
comes obsolete ventrally. Escutcheon evident 
but not sharply defined. 

Beaks, central and posterior slopes with 
fine commarginal sculpture. 

Exterior white or pink, sometimes with pur- 
ple patches on either side of beaks. Interior 
white or tan. Hinge plate relatively narrow; 
right valve with a triangular tooth; left valve 
with relatively broad chondrophore and a 
small tooth (Fig. 54). Length to 10V.2 mm 
(holotype; CAS 121848; Point Loma, San 
Diego County, California). 



Distribution 

Monterey, Monterey County, California, 
U.S.A. (36.7°N) (CAS 121792), possibly a re- 
sult of larval settlement in an El Niño year; 
Topanga Creek, Los Angeles County, Cali- 
fornia (33.7°N) (LACM 68-192.21), south to 
Bahía Magdalena, Baja Californ Sur (CAS 
121791; USNM 217823; Skoglund Collec- 
tion); from the intertidal zone to 80 m (mean, 
16.1 m; n = 47), in rubble. I have seen 157 Re- 
cent lots, including the types. 

Records of this species from the Golfo de 
California were based on misidentified С 
marmorata, or, in the case of Parker (1964: 
161), С nasuta (MCZ 253667, 259948, 
260263) and С ira (MCZ 260703). 

This species has been widely reported as a 
fossil in California and Baja California. In the 
late Pleistocene, there are records from ter- 
races on Santa Rosa Island, Channel Islands, 
Santa Barbara County (A. G. Smith, in Orr, 
1960: 1117, A. G. Smith, 1968: 22); Playa del 
Rey (Willett, 1937: 391), the Baldwin Hills (B. 
L. Burch, 1947: 9), terraces in the Palos 
Verdes Hills (Chace, 1966: 169; Mahncovich, 
1976: 20), San Pedro (DeLong, 1941: oppo- 
site p. 244; Valentine, 1961: 375, 376, 377), 
Huntington Beach (Valentine, 1959: 54), Los 
Angeles County; Newport Bay, Orange 
County (Kanakoff & Emerson, 1959: 22); San 



Diego, San Diego County (Dall, 1878a: 11, 
1878b: 27; Emerson & Chace, 1959: 338; 
Valentine, 1961: 357; Valentine & Meade, 
1961: 10; Kern et al., 1971: 333), California; 
northwestern Baja California (Valentine, 
1957: 297; Valentine & Rowland, 1969: 518); 
Bahía San Quintín (Dall, in Orcutt, 1921: 24; 
Jordan, 1926: 245; Manger, 1934: 293), Baja 
California; Laguna San Ignacio (Hertlein, 
1934: 64), Bahía San Bartolomé [Turtle Bay] 
(Emerson, 1980: 72; Emerson et al., 1981: 
111), and Bahía Magdalena (Jordan, 1936: 
111), Baja California Sur. Records of this 
species from the Pleistocene of the southern 
Golfo de California (Durham, 1950) were 
based on specimens of С marmorata (see 
under the latter). 

It has been recorded in strata of early Pleis- 
tocene age in Santa Monica (Woodring, in 
Hoots, 1931: 121; Valentine, 1956: 196; 
Rodda, 1957: 2484) and San Pedro (DeLong, 
1941: opposite p. 244), Los Angeles County, 
California. 

Cooper, in Watts (1897: 79) and Woodring, 
in Hoots (1931: 116) reported this species 
from late Pliocene strata in Los Angeles 
County, and Hertlein & Grant (1972: see syn- 
onymy) reported it from the late Pliocene San 
Diego Formation, San Diego County, Cali- 
fornia. There is a record in the early Pliocene 
Towsley Formation of Los Angeles County, 
California (Woodring, in Winterer & Durham, 
1962:304-305). 

There are also records from the late 
Miocene Santa Margarita Formation (Gale, in 
Preston, 1931: 15) of central California, the 
late Miocene Castaic Formation of southern 
California (Stanton, 1966: 23), from the early 
to middle Miocene Temblor Formation (Ade- 
goke, 1969: 153) of central California, and 
from the Miocene Isidro Formation near Bahía 
Magdalena, Baja California Sur (Beal, 1948: 
66). 

Discussion 

This species is most similar to С mar- 
morata, which accounts for Panamic records 
of C. luteola. The two differ as follows: 



С luteola 

oval 

larger (to 10.2 mm) 

beaks closer to midline 

solid color 

without a medial sulcus 

radial ridge becomes 

obscure ventrally 
chondrophore larger 



С marmorata 

pointed posteroventrally 

smaller (to 8.2 mm) 

longer posteriorly 

mottled color 

with a medial sulcus 

radial ridge strong to 

ventral margin 
chondrophore smaller 



EASTERN PACIFIC CORBULIDAE 



87 



hinge plate narrower at wider hinge plate 

same size 
finer, sharper sculpture heavier, more undulating 

sculpture 
beaks sculptured beaks less sculptured 

For comparison with the sympatric C. por- 
cella, see under the latter. 

Of the western Atlantic taxa, С. tuteóla is 
most similar to C. contracta Say, 1822 (p. 
312), differing from it in being more inflated, 
having a much more prominent ridge between 
the central and posterior slopes, and having 
more lamellar sculpture, which extends onto 
the beaks. 

Corbula marmorata Hinds, 1843 
Figures 36, 37, 55 

Corbula marmorata Hinds, 1843 

Hinds, 1843: 58 [1844 reprint: 231]; 
Reeve, 1844: pi. 5, fig. 39; Hinds, 1845: 
69, pi. 20, fig. 13; Carpenter, 1857a: 207, 
300; Tryon, 1869: 65; Lamy, 1941: 133- 
134; Hertlein & Strong, 1950: 239-240, 
252, pi. 2, fig. 1 7 [Aloidis (Caryocorbula)]; 
Keen, 1958: 209, fig. 526 [Corbula {Cary- 
ocorbula)]; Olsson, 1961: 431-432, 548, 
pi. 75, fig. 5 [Caryocorbula {Caryocor- 
bula)]; Keen, 1966: 268 [Corbula]; Keen, 
1971: 264-265, fig. 676 [Corbula (Cary- 
ocorbula)]; Gemmell et al., 1987: 58-59 
[Corbula] 

Corbula luteola Carpenter, auctt., non Car- 
penter, 1864 

Durham, 1950: 94, 170, pi. 25, figs. 15, 
16; Keen, 1958: 209, fig. 525 [Panamic 
records and the fig.]; Keen, 1971: 265, 
fig. 675 [Panamic records and the fig.] 

Type Material & Locality 

Not located in the BMNH collection (Keen, 
1 966). The description and Hinds' subsequent 
figure, however, are sufficiently clear and un- 
ambiguous that a neotype is not required. A 
copy of the figure from Hinds (1845) is given 
here (Fig. 36). The originally measured spec- 
imen was 4.2 mm in length, 2.8 mm in height, 
and 2.1 mm in width. Veragua[s] Province, 
Panamá (approximately 7.7°N); 26 fms. [48 
m], mud; Edward Belcher. 

Description 

Shell ovate-subquadrate, moderately 
heavy for size; right valve slightly larger than 
left; beaks in anterior third of shell (beaks 



28-36% from anterior end). Anterior end 
rounded. Shallow medial radial sulcus pres- 
ent. Posterior end truncate, pointed pos- 
teroventrally. Posterior slope set off from 
central slope by a radial ridge that runs to ven- 
tral margin. Escutcheon evident, narrow, 
strongest in right valve. 

Beaks relatively smooth; central slope with 
moderate, undulating commarginal ribs. 
Sculpture more lamellar on posterior slope. 
Exterior white, brown, or red, mottled with ma- 
genta or white, and with a magenta patch an- 
terior to beaks. Interior magenta, particularly 
around margins. Right valve with a triangular 
tooth; ligament under valve margin. Left valve 
with a narrow chondrophore; tooth small 
(Figs. 37, 55). Length to 8.2 mm (Bahía 
Cholla, Sonora, México; Skoglund Collec- 
tion). 

Distribution 

Bahía Magdalena, Baja California Sur 
(24.6°N) (CAS 121641; LACM 67-70.26), and 
throughout the Golfo de California to its head 
at Puerto Peñasco, Sonora (31.4°N) (UCMP 
E.8419; CAS 121646; Skoglund Collection), 
México, south to Callao, Lima Province, Perú 
(12.1°S) (LACM 35-153.3). This species also 
occurs in the western Atlantic, where speci- 
mens are sometimes labeled as C. blandiana 
C. B. Adams, 1852, which is a synonym of C. 
dietziana С В. Adams, 1852 (see Discussion 
under C. speciosa), as C. barrattiana С В. 
Adams, 1852, a synonym of С swiftiana С В. 
Adams. 1852 (see Discussion under C. na- 
suta), or C. contracta Say, 1822 (p. 312), a 
distinct, uncolored, more inflated, more heav- 
ily sculptured species that occurs as far north 
as New England. Corbula marmorata occurs 
from the intertidal zone to 137 m (mean, 19.9 
m; n = 115), in rubble and on the undersides 
of rocks. I have seen 299 eastern Pacific lots. 

The record of this species from the Galapa- 
gos Islands (Kaiser, 1997: 25) was based on 
a specimen of C. bicarinata (LACM 34-43). 

Also present in the Pleistocene at Bahía 
Santa Inez, Baja California Sur, México 
(Durham, 1950: 94, as "C. luteola"). 

Discussion 

Corbula marmorata sometimes occurs at 
the same stations as С speciosa. Small spec- 
imens of the latter are similar to С marmorata 
in having a mottled color pattern and a purple 
patch in front of the beaks. In specimens of 



COAN 




FIGS. 36, 37. Corbula marmorata. FIG. 36. Figure of C. marmorata from Hinds (1845). FIG. 37. SBMNH 
345499; Bahía Cholla, Sonora, México; length, 6.8 mm. FIG. 38. С. speciosa, lectotype; BMNH 1967945/1; 
length, 19.7 mm. 



EASTERN PACIFIC CORBULIDAE 



89 



similar size, С speciosa is more elongate; 
longer, broader, and more truncate posteri- 
orly; has a stronger rib separating the central 
from the posterior slope; has a more pro- 
nounced medial radial sulcus; and the pos- 
terodorsal margin is more elevated and 
flange-like. 

Species that are possibly ancestral to C. 
marmorata include: С engonata burnsii Dall, 
1898 (p. 847), C. (Caryocorbula) franc/ Gard- 
ner, 1928 (pp. 231-232, pi. 35, figs. 1-4), С 
(С.) wakullensis Gardner, 1928 (p. 232, pi. 35, 
figs. 5, 6), and С barrattiana leonensis Mans- 
field, 1932 (p. 160, pi. 33, figs. 1, 3), from the 
Miocene of Florida, and С cuneata Say, 1824 
(pp. 1 52-1 53, pi. 1 3, fig. 3), from the Pliocene 
of Maryland and Virginia. 

Corbula speciosa G. B. Sowerby I, 1833 
Figures 38, 56 

Corbula radiata G. B. Sowerby I, 1833, non 
Deshayes, 1824 

G. B. Sowerby I, 1833: 36; Hanley, 1843: 
47 

[non Deshayes, 1824: 58-59, pi. 9, figs. 
11, 12^] 

Corbula speciosa Reeve, 1 843 

Reeve, 1843 (Aug.): pi. 1, fig. 6; Hinds 
1843 (Nov.): 57 [1844 reprint: 230] 
Hinds, 1845: 68-69, pi. 20, figs. 7, 8 
Carpenter, 1857a: 207, 300; Tryon, 1869 
66; Lamy, 1941: 133; Hertlein & Strong 
1950: 237 [Aloidis (Aloidis)]; Keen, 1958 
208-209, fig. 522 [Corbula (Corbula)] 
Olsson, 1961: 438-439, 550, pi. 77, fig 
7-7c [Varicorbula]; Keen, 1971: 269- 
270, fig. 692 [Corbula (Varicorbula)] 

Type Materials & Localities 

Corbula rad/atà- Not located in the BMNH. 
The original measurements were: length, 8.9 
mm; height, 6.3 mm; width, 4.3 mm. Acapuico, 
Guerrero, México (16.9°N); Hugh Cuming. 

Corbula spec/osa- BMNH 1967945/1, lec- 
totype here designated, the largest speci- 
men and closest to Reeve's figure; length, 
19.7 mm; height, 15.4 mm; width, 11.6 mm 
(Fig. 38). BMNH 1967945/2, paralectotype, 
left valve; length, 18.4 mm. BMNH 1967945/3, 
paralectotype, left valve; length, 15.3 mm. 
Golfo de Nicoya, Costa Rica (approximately 
9.9°N); Edward Belcher. BMNH 1879.2.26.91, 



Not in Brocchi (1814), as Reeve (1843) claimed. 
Deshayes' species is a Cardiomya. 



pair; length, 17.8 mm. Panamá, 6 fms. [11 m], 
mud; Edward Belcher. The latter seems to be 
the specimen figured in Hinds (1845), but ma- 
terial from Panamá was not mentioned by 
Reeve (1843), who made the name available 
three months before Hinds. 

Description 

Shell subtrigonal as an adult, heavy; right 
valve much larger than left, very inflated. Pos- 
terior end truncate. Posterior slope set off 
from central slope by a rounded ridge. Es- 
cutcheon apparent in some specimens only. 

Juvenile shell set off by a remarkable 
change in growth direction, shape, sculpture, 
and color. Subquadrate juvenile shell flat- 
tened, with commarginal undulations, a me- 
dial radial sulcus, fine radial ribs, and a mot- 
tled color pattern. 

Right valve of adult with commarginal un- 
dulations; left valve with finer, lamellar, some- 
what oblique sculpture. Exterior color of red- 
dish-brown radial ribs. Periostracum light to 
dark brown. Interior white to yellowish, with 
red ribs visible ventrally. Right valve with a 
large tooth; left valve with a small, non-pro- 
jecting chondrophore, with the ligament con- 
fined to a small area medial to a large poste- 
rior cardinal (Fig. 56). Length to 20.6 mm 
(Bahía Tenacatita, Jalisco, México; LACM 34- 
146.14). 

Distribution 

San Felipe, Baja California (31 .0°N) (LACM 
40-279.3), and Isla Tiburón, Sonora (LACM 
36-80.45) (28.7°N), México, to Punta Utria, 
Choco Province, Colombia (6.0°N) (LACM 
35-63.18, 35-170.7); Isla Socorro, Islas Revil- 
lagigedo, México (LACM 34-5.17; Kaiser col- 
lection); from the intertidal zone to 125 m 
(mean, 40.2 m; n = 98), on sand or gravel, or 
in rubble. I have seen 137 lots, including the 
types. 

This species has been reported in late 
Miocene deposits of the Imperial Formation in 
Riverside County, California (Powell, 1988: 
16). 

Discussion 

This species is very similar and perhaps 
identical to the later-named western Atlantic 
C. dietziana С В. Adams, 1852c (pp. 235- 
236), which occurs from North Carolina to 
Brazil. Synonyms of С dietziana include С 



90 



COAN 



blandiana С. В. Adams, 1852c (pp. 234-235), 
and C. cymella Dall, 1881 (p. 115) (based on 
comparison with BMSM 15012; Boca Grande, 
Lee County, Florida). 

Small specimens of Corbula speciosa can 
be distinguished from C. ira, with which it 
sometimes occurs, by its more quadrate out- 
line, longer posterior end, mottled color, less 
prominent sculpture, and medial radial sulcus. 

Some authors have placed this species in 
the subgenus Varicorbula because it is in- 
equivalve, but I think that this allocation is pre- 
mature. Corbula speciosa differs from other 
species of Varicorbula in having a highly dif- 
ferentiated juvenile shell. Indeed, in several 
respects small specimens of С speciosa are 
closest to C. marmorata (for a comparison, 
see Discussion under C. marmorata). 

ADDITIONAL NOTES & RECORDS 

Corbula altirostris Li, 1930 (pp. 263-264, 
pi. 5, fig. 37), supposedly from an offshore 
outcrop of the Miocene Gatun Formation in 
the Bay of Panamá, was based on a Recent 
specimen of the mactrid Mulinia pallida 
(Broderip & Sowerby, 1829: 360, as Mactra) 
(Pilsbry, 1931:431). 

Carpenter (1857a: 300; 1860: 2) listed a 
Corbula boivinei, without authorship, from 
Central America. This is a nomen nudum. 

The record of "Corbula cf. collazica Maury," 
1920 (pp. 44-45, pi. 6, figs. 10, 11), suppos- 
edly from an offshore outcrop of the Gatun 
Formation in the Bay of Panamá (Li, 1930: 
263, pi. 5, fig. 36, 36a), was actually based on 
a Recent specimen of Corbula ovulata, ac- 
cording to Pilsbry (1931: 431). 

Corbula gibbosa Broderip & Sowerby, 1 829 
(p. 361), from the Arctic coast of Alaska, is a 
probable synonym of Mya truncata Linnaeus, 
1758 (p. 670) (Coan et al., 2000: 471). 

Corbula kelseyi Dall, 1916b (p. 416; Dall, 
1916a: 41, nomen nudum), was based on a 
worn specimen of Cumingia californica Con- 
rad, 1837: 234 (Coan & Scott, 1991; Coan et 
a!., 2000: 437, 478). 

The record of "Corbula cf. swiftiana С В. 
Adams," 1852c (pp. 236-237), supposedly 
from an offshore outcrop of the Gatun Forma- 
tion in the Bay of Panamá (Li, 1 930: 264, pi. 5, 
fig. 39), was based on a Recent specimen of 
the mactrid Mulinia pallida (Broderip & 
Sowerby, 1829) (Pilsbry, 1931: 431). 

Corbula tenuis Moody, 1916 (pp. 59, 62, pi. 
2, fig. 4a, 4b), non G. В. Sowerby I, 1 833, = С. 
binominata Hanna, 1924 (p. 163), from the 



Pliocene Fernando Formation in downtown 
Los Angeles, California [holotype: UCMP 
11087; Loc. 3030], is not a corbulid. Although 
broken and partially sediment- and glue-filled, 
the holotype seems to be a poromyid, closest 
and probably identical to the Recent Der- 
matomya mactrioides (Dall, 1889a: 448, as 
Poromya (Dermatomya)); Coan et al. (2000: 
572) treated this Dermatomya. Corbula bi- 
nominata was also later recorded from the 
Pliocene of the Los Angeles Basin by Soper & 
Grant (1932: 1060). 

NOTE ADDED IN PROOF 

A paper has recently appeared that sheds 
new light on the western Atlantic species of 
Corbula (Varicorbula) (Mikkelsen & Bieler, 
2001). Using Varicorbula as a full genus, they 
adopt the traditional early dates of d'Orbigny's 
species of Corbula, with the result that V. dis- 
paralis (d'Orbigny, 1842) is regarded as the 
valid name for the species closest to Corbula 
(Varicorbula) groves/ named here. A synonym 
that I had not noted, Corbula limatula Conrad, 
1846 (p. 25, pi. 1, fig. 2), would be the valid 
name if the dates of d'Orbigny are as late as I 
now think. They regard Corbula operculata 
Philippi, 1848, the name I used for this 
species, as a nomen dubium, because of the 
absence of type material. Clearly, reaching a 
definitive conclusion about the dates of the all- 
important d'Orbigny work should be a high pri- 
ority for workers on the western Atlantic fauna. 

CONRAD, T. Д., 1846, Descriptions of new species 
of fossil and Recent shells and corals. Proceed- 
ings of the Academy of Natural Sciences of 
Philadelphia, 3(1): 19-27, pi. 1 

MIKKELSEN, P. M. & R. BIELER, 2001, Varicorbula 
(Bivalvia: Corbulidae) of the western Atlantic: tax- 
onomy, anatomy, life habits, and distribution. The 
VeZ/gfer, 44(3): 271 -293 

ACKNOWLEDGMENTS 

I appreciated the help of the following cura- 
tors, other personnel and their institutions, 
who made specimens, literature, and informa- 
tion available: Warren D. Alimón, Paleonto- 
logical Research Institution, Ithaca, New York, 
USA; Laurie С Anderson, Louisiana State 
University, Baton Rouge, Louisiana, USA; 
Adam J. Baldinger & Kenneth J. Boss, Mu- 
seum of Comparative Zoology, Harvard Uni- 
versity, Cambridge, Massachusetts, USA; 
Warren Blow, Tyjuana Nickens, and Thomas 
Waller, National Museum of Natural History, 



EASTERN PACIFIC CORBULIDAE 



91 




39 





40 





41 






42 

FIGS. 39-42. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, palliai sinus, 
and adductor scars of right and left valves. Stippling in left valve indicates socket for tooth of right valve. 
Cross-hatching indicates areas of ligarnent attachment on chondrophore of left valve and visible areas of at- 
tachment on hinge margin of right valve. FIG. 39. C. amethystina; SBMNH 345487; Playas de Villimil, 
Guayas, Ecuador; trawled; length, 30.8 mm. FIG. 40. C. nasuta; SBMNH 345492, between Santa Cruz and 
Platinitos, Nayarit, México; 6-18 m; length, 15.9 mm. FIG. 41. C. otra, new species; SBMNH 345493, holo- 
type; Manazanillo, Colima, México; 30-45 m; length, 22.8 mm. FIG. 42. C. ovulata; SBMNH 345498; Playas 
de Villamil, Guayas, Ecuador; trawled; length, 29.2 mm. 



92 



COAN 




FIGS. 43-46. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallia! sinus, 
and adductor scars of right and left valves. FIG. 43. С porcella; CAS 1 42446; E end of Isla Cedros, Baja Cal- 
ifornia, México; 82 m; unpaired valves: right valve, 7.4 mm; left valve, 7.2 mm. FIG. 44. С esmeralda; ANSP 
218903, 403198, lectotype and paralectotype; Esmeraldas, Esmeraldas, Ecuador; lengths, 20.6 mm. FIG. 
45. C. bicarinata; CAS 120689; San Felipe, Baja California, México; length, 11.2 mm. FIG. 46. С ventricosa; 
CAS 120699; Panamá; length, 15.3 mm. 



EASTERN PACIFIC CORBULIDAE 



93 




47 




48 





49 





50 



FIGS. 47-50. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, palliai sinus, 
and adductor scars of right and left valves. FIG. 47. C. amurensls; CAS 089104; Martinez, Contra Costa 
County, California; length, 16.4 mm. FIG. 48. С tenuis; CAS 120702; IslaTaboga, Panamá, Panamá; length, 
19.1 mm. FIG. 49. C. grovesi; LACM 2891, holotype; S end of Isla San Lorenzo, Baja California Sur, Méx- 
ico; 732 m; length, 11.0 mm. FIG. 50. C. obesa; SBMNH 345495, neotype; Mazatlán, Sinaloa Mexico- 
length, 12.7 mm. 



94 



COAN 




51 





52 





53 






54 

FIGS 51 -54 Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, palliai sinus, 
and adductor scars of nght and left valves. FIG. 51. С biradiata; SBMNH 345500; Playa Kobbe, Panama, 
Panamá- length 16 8 mm. FIG. 52. С colimensis. new species; SBMNH 345496, holotype; Las Ventanas, 
Manzanillo México- length, 13.7 mm. FIG. 53. С ira: SBMNH 345501; Bahía Carazal, Colima, Mexico; 75 
m; length, 11.4 mm. FIG. 54. C. tuteóla; CAS 120696; Arch Rock, Corona del Mar, Orange County, Califor- 
nia; length, 10.4 mm. 



EASTERN PACIFIC CORBULIDAE 



95 




55 





56 




FIGS. 55, 56. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallia! sinus, 
and adductor scars of right and left valves. FIG. 55. С marmorata; CAS 120688; Mazatlán, Sinaloa, México; 
length, 5.8 mm. FIG. 56. С speciosa; CAS 120695; Bahía Santa Inez, Baja California Sur, México; 55-64 
m; length, 17.5 mm 



Washington, DC, USA; Yolanda Cannacho, In- 
stituto Nacional de Biodiversidad, Santo 
Domingo, Heredia, Costa Rica; James 
Cordeiro and Paula MIkkelsen, American Mu- 
seum of Natural History, New York, New York, 
USA; Thomas A. Deméré and N. S. Rugh, 
San Diego Natural History Museum, San 
Diego, California, USA; Charlene Fricker, 
Mark Kitson, and Gary Rosenberg, of the 
Academy of Natural Sciences, Philadelphia, 
Pennsylvania, USA; Lindsey T Groves and 
James H. McLean, Natural History Museum 
of Los Angeles County, Los Angeles, Califor- 
nia, USA; Elizabeth Kools, Department of In- 
vertebrate Zoology, California Academy of 
Sciences, Golden Gate Park, San Francisco, 
California, USA; Rafael La Perna, Université 
degle Studi di Bah, Bari, Italy; José Leal, Bai- 
ley-Matthews Shell Museum, Sanibel, 
Florida, USA; David R. Lindberg and Karen 
Wetmore, Museum of Paleontology, Univer- 
sity of California, Berkeley, California, USA; 
Joan Pickering and Kathie Way, The Natural 
History Museum, London, England, UK; Patri- 
cia Sadeghian and Paul Valentich Scott, 



Santa Barbara Museum of Natural History, 
Santa Barbara, California, USA; Nancy Voss, 
University of Miami, Miami, Flohda, USA. 
Carol C. Skoglund and Kirstie L. Kaiser gen- 
erously made available material or informa- 
tion from their collections. David Campbell, 
Roberto Cipriani, Juan Diaz, Carole M. Hertz, 
Alan R. Kabat, and Konstantin Lutaenko pro- 
vided some information, and Richard Petit 
supplied copies of scarce literature. Laurie С 
Anderson, Lindsey T. Groves, Carol C. 
Skoglund, and Paul Valentich Scott made 
many helpful comments on the manuscript. 
Sharon Williams helped to prepare the plates. 



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Revised ms. accepted 10 June 2001 



MALACOLOGIA, 2002, 44(1): 107-134 

GENUS PARVITHRACIA (BIVALVIA: THRACIIDAE) WITH DESCRIPTIONS 

OF A NEW SUBGENUS AND TWO NEW SPECIES FROM 

THE NORTHWESTERN PACIFIC 

Gennady M. Kamenev 

Institute of Marine Biology, Russian Academy of Sciences, Vladivostol< 690041 , Russia; 

inmarbio@mail.primorye. ru 



ABSTRACT 

A new subgenus, Pseudoasthenothaerus, of the genus Parvithracia Finlay, 1 927, and two new 
species, Parvithracia (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) sirenkoi, 
are described from the Pacific seas of Russia. Previously representatives of the genus Parvithra- 
cia, with the type species Parvithracia suteri Finlay, 1927, were found only off New Zealand. 
Study of thraciid genera showed that Japanese species assigned to the genus Asthenothaerus 
Carpenter, 1864, should be placed in the genus Parvithracia. For Asthenothaerus sematana 
(Yokoyama, 1922) and A. /saofa/(/7 Okutani, 1964, new combinations are suggested: Parvithra- 
cia (Pseudoasthenothaerus) sematana (Yokoyama, 1 922) and P. (Pseudoasthenothaerus) isao- 
fa/c;V (Okutani, 1964). The main morphological characteristics of the new subgenus are the pres- 
ence of conspicuous lateral teeth in both valves and a subumbonal plate for the attachment of 
the lithodesma that is supported by pillars. Expanded descriptions of P. suteh, P. (Pseudoas- 
thenothaerus) sematana. and P. (Pseudoasthenothaerus) isaotal<ii are given. 

Key words: Parvithracia, Thraciidae, Bivalvia, northwestern Pacific, morphology, anatomy. 



INTRODUCTION 

The bivalve family Thraciidae Stoliczka, 
1870, in the Pacific seas of Russia has long 
been represented by a sole genus Thracia 
Blainvllle, 1824 (Scarlato, 1981; Kafanov, 
1991). Recently, members of the genus Lam- 
peia MacGinitie, 1959, previously recorded 
from America (MacGinitie, 1959; Coan, 1990), 
were found in this region (Kamenev & Nad- 
tochy, 1998). Examination of thraciids col- 
lected by expeditions in the shelf and bathyal 
zones of the Pacific seas of Russia revealed 
two new species. Based on the shell mor- 
phology and anatomy, these species were as- 
signed to a new subgenus of the genus 
Parvittiracia Finlay, 1927, previously known 
only from the South Pacific (Finlay, 1927a, b; 
Powell, 1937, 1955, 1979). Study of the rep- 
resentatives of other genera of the Thraciidae 
showed that Japanese species previously as- 
signed to the genus Asthenothaerus Carpen- 
ter, 1864, should also be placed in this sub- 
genus. 

Diagnoses and descriptions of the genus 
Parvithracia and description of the type 
species given in the literature are brief, some- 
times inaccurate, and often illustrated only by 
schematic figures of Parvithracia sufer/ Finlay, 



1927 (Suter, 1913; Finlay, 1927a, b; Keen, 
1969; Powell, 1955, 1979). Thorough study of 
shell morphology of P. suteri and of north- 
western Pacific species has enabled an ex- 
panded and modified diagnosis of the genus 
and an improved description of P. suterHo be 
made. This paper provides new data on its 
shell morphology, and describes a new sub- 
genus of Parvithracia and four species, two of 
which are new. For Asthenothaerus sematana 
(Yokoyama, 1922) and A. /saotó/c// Okutani, 
1964, new combinations are suggested. 



MATERIALSAND METHODS 

In this study, I have used the material col- 
lected by numerous expeditions to the shelf 
and bathyal zones of Pacific seas of Russia 
from 1931 to 1995. Mollusks were fixed in 
70% ethanol or 4% formaldehyde. The mate- 
rial was stored in 70% ethanol and in the dry 
forminZIN, 1MB, and PRIFO. 

For comparison purposes, collections of the 
following taxa were used: Asthenothaerus 
diegensis (Dall, 1915) (USNM); A. hemphiilii 
Dall, 1866 (USNM); A. /saotó/c// Okutani, 1964 
(NSMT); A. sematana (Yokoyama, 1922) 
(NSMT, CAS); Eximiothracia concinna (Gould, 



107 



108 



KAMENEV 



1861) (NSMT); Lampeia adamsi (MacGlnlWe, 
1959) (UAM; MIMB); L triangula Kamenev & 
Nadtochy, 1998 (MIMB); L. posteroresecta 
Kamenev, 1998, in Kamenev & Nadtochy, 
1998 (MIMB); Thracia curta Conrad, 1837 
(USNM); T. myopsis Moller, 1842 (MIMB, 
ZIN); T. kakumana {Yokoyama, 1927) (MIMB, 
ZIN); T. seminuda Scarlato, 1981 (ZIN); T. 
septentrionalis Jefireys, 1872 (USNM; ZIN); 
Ttiracidora japónica Habe, 1952 (NSMT); 
Trigonothracia pusilla (Gould, 1861) (NSMT); 
Parvithracia suteri FInlay, 1 927 (MNZ and ma- 
terial of R. Asher (CIN)). Asttienothaerus isao- 
takii ano Lampeia species from Pacific seas of 
Russia were fixed and stored in 70% ethanol. 
All other material was stored dry. 

The gross anatomy of the new species has 
been described from whole mount speci- 
mens. 

Shell Measurements 

Figure 1 shows the position of the shell 
morphology measurements. Shell length (L), 
height (H), width of each valve (W) (not 
shown), anterior end length (A), maximal dis- 
tance from the posterior end to the top of pal- 
lia! sinus (S), and lithodesma length (LI) were 
measured for each valve. The ratios of these 
parameters to shell length (H/L, W/L, A/L, S/L, 
L1/L, respectively) were determined. The 



number of pillars (N) supporting the subum- 
bonal plate for lithodesma in each valve was 
also recorded. Shell measurements were 
made using a caliper and an ocular microme- 
ter with an accuracy of 0.1 mm. 

Statistics 

Statistical analysis of the material used a 
package of statistical programs STATISTICA 
(Borovikov & Borovikov, 1997) and Data 
Analysis Module of MS Excel 97. 

The calculated indices (H/L; W/L; A/L; S/L 
and L1/L) are less susceptible to change as 
compared with other measured parameters. 
Therefore the statistical analysis was per- 
formed using only these characteristics. All 
data was tested with a Kolmogorov test for 
their fit to a normal distribution. The distribu- 
tion of some indices was different from the 
norm. Therefore all analyses were performed 
on log^Q transformations of the original vari- 
ables. All indices for pairs of different valves of 
new species were compared using the Stu- 
dent (t) parametric test and one-way analysis 
of variance (ANOVA). A discriminant analysis 
was used to test the validity of the hypothesis 
of division of all studied specimens into two 
groups. 

Throughout this study, statistical signifi- 
cance was defined as P < 0.05. 




FIG. 1. Placement of shell measurements: L — shell 
length; H — height; A — anterior end length; LI — lith- 
odesma length; S — maximal distance from poste- 
rior shell margin to the top of palliai sinus. 



Morphological Terminology 

Subumbonal plate is a plate on the inner 
shell wall to which the internal ligament and 
lithodesma are attached. It is located ventral 
to the umbo and is partially or completely at- 
tached to the shell wall. Various specialists 
designate it either as a chondrophore (Ya- 
mamoto & Habe, 1959, for the genus Trigo- 
nothracia Yamamoto & Habe, 1959; Keen, 
1969, for the genus Lampeia; Xu, 1980, for 
the genus Trigonothracia), buttressed struc- 
ture (Coan, 1990; Kamenev & Nadtochy, 
1998, for the genus Lampeia), or resilifer 
(Coan et al., 2000, for the genus Lampeia)). 

Resilifer \s a depression or projection of the 
hinge plate not attached to the inner shell wall 
that serves for the attachment of the internal 
ligament. 

Abbreviations 

The following abbreviations are used in the 
paper: CAS -California Academy of Sei- 



THE BIVALVE GENUS PARVITHRACIA 



109 



enees, San Francisco; CIN-Cawthron Insti- 
tute, Nelson; 1MB -Institute of Marine Biol- 
ogy, Russian Academy of Sciences, Vladi- 
vostok; MIMB- Museum of the Institute of 
Marine Biology, Vladivostok; MNZ- Museum 
of New Zealand, Wellington; NIGNS -Na- 
tional Institute of Geological and Nuclear Sci- 
ences, Lower Hutt; NSMT- National Science 
Museum, Tokyo; PRIFO — Pacific Research 
Institute of Fisheries and Oceanography, 
Vladivostok; UAM- University of Alaska Mu- 
seum, Fairbanks; USNM- United States 
National Museum of Natural History, Smith- 
sonian Institute, Washington, D.C.; ZIN- 
Zoological Institute, Russian Academy of Sci- 
ences, St. -Petersburg. 



SYSTEMATICS 

Order Pholadomyoida Newell, 1965 

Superfamily Thracioidea Stoliczka, 1870 

Family Thraciidae Stoliczka, 1870 

Genus Parvithracia Finlay, 1927 

Type species: Montacuta triquetra Suter, 
1913: 915, non Verrill & Bush, 1898: 782, 783, 
pi. 91, fig. 3; = Parvithracia suteri Finlay, 
1927b: 529 

Diagnosis 

Shell small (< 11 mm), very thin and fragile 
to medium in thickness, ovate-elongate to 
subtrigonal, inequivalve; right valve larger and 
more inflated. Beaks central or posterior. Pos- 
terior end truncate, with a faint radial ridge. 
Periostracum thin, adherent, colorless or tan. 
Surface with conspicuous growth lines and 
very fine granules. Escutcheon present; 
lunule present in some. Ligament internal, 
supported by a strong lithodesma attached to 
elongate-trigonal subumbonal plate that is 
sometimes supported by a series of pillars. 
Hinge plate weak, with large, short anterior 
and more or less conspicuous, elongate pos- 
terior lateral teeth in right valve, and long, 
lamellate, anterior lateral tooth in left valve. 
Palliai line with deep palliai sinus of same 
shape and size in both valves, sometimes 
reaching midline. Palliai sinus not confluent 
with palliai line. 

Remarks 

Previously this genus included two species: 
P. suteri and Parvitliracia cuneata Powell, 



1937 (Powell, 1937, 1955, 1979). However, 
studies of Bruce A. Marshall (MNZ) have 
shown that P. cuneata belongs to a different 
genus (Bruce A. Marshall, personal communi- 
cation). 

Subgenus Parvithracia, s.s. 

Diagnosis 

Shell small (< 5 mm), thin, fragile, subtrigo- 
nal, inequivalve; right valve more inflated. 
Beaks slightly posterior. Posterior end trun- 
cate, with very faint radial ridge. Periostracum 
thin, adherent, colorless. Surface with faint 
growth lines and very fine granules. Es- 
cutcheon and lunule present. Ligament inter- 
nal, supported by a strong lithodesma, at- 
tached to elongate-trigonal subumbonal 
plate. Hinge plate weak, with large, high ante- 
rior and more or less conspicuous, elongate, 
posterior lateral teeth in right valve and long, 
lamellate, anterior lateral tooth in left valve; in- 
ternal part of anterodorsal and posterodorsal 
margins with long grooves. Palliai line with 
deep palliai sinus of same shape and size in 
both valves, reaching midline. Palliai sinus not 
confluent with palliai line. 

Parvithracia (Parvithracia) suten Finlay, 1927 
Figs. 2-10, Table 1 

Montacuta triquetra Suter, 1913: 915, pi. 
53, fig. 7a, non Verrill & Bush, 1898: 782, 783, 
pi. 91, fig. 3; Finlay, 1927a: 461. 

Parvithracia suteri F\n\a\/, 1927b: 529; Pow- 
ell, 1955: 45; Powell, 1979: 434, fig. 117. 

Type Material and Locality 

Lectotype (NIGNS TM 399) and 4 paralec- 
totypes (NIGNS TM 405-409) (Boreham, 
1959; Bruce A. Marshall, personal communi- 
cation). Port Pegasus, Stewart Island, New 
Zealand, 18 fathoms (approximately 33 m) 
(Suter, 1913; Powell, 1955, 1979). 

Material Examined 

1 lot (MNZ ex M.44790) from North Arm, 
Port Pegasus, Stewart Island, New Zealand 
(47° 1 1 'S, 1 67° 41 'E), 37-44 m, mud, 22 Feb- 
ruary 1972 (R/V "Acheron") (3 spec); 1 lot 
(CIN) from Tasman Bay, Nelson, New 
Zealand (40°35'S, 173°54'E), 60 m, 
mud/sand, Coll. Rod Asher, January 1997 (1 
damaged spec). 



110 



KAMENEV 




FIGS 2-10 Parvithracia (Parvithracia) suteri Finlay, 1927 (MNZ ex M,44790), North Arm, Port Pegasus, 
Stewart Island, New Zealand (47'^1 1 'S, 1 67°41 'E), 37-44 m. 2, 3: Left and right valves (bar = 1 тгл). 4: Gran- 
ules on the shell surtace (bar = 100 цт). 5: Hinge of the right valve (bar = 300 цт). 6: Hinge of the left valve 
(bar = 300 urn), 7: Dorsal view of teeth on the nght valve (bar = 100 цт). 8: Close-up of a left valve showing 
the subumbonal plate and the lateral tooth on the anterodorsal shell nnargin, ventral view (bar = 300 цт). 9; 
Close-up of a nght valve showing inner part of antero- and posterodorsal shell margins with a shallow groove 
(bar = 1 mm). 10: Ventral view of lithodesma (bar = 100 цт). 



Description 

Expanded and modified from Suter (191 3)- 
Exterior: Shell small (< 5.0 mm), subtngonal, 
high (H/L = 0.824-0.892), slightly inequivalve 



(right valve more inflated), inflated (W/L of 
nght valve 0.281-0.303; W/L of left valve 
0.235-0.250), slightly inequilateral, thin, 
solid. Surface v^/ith faint grov\/th lines and very 
dense fine granules. Periostracum dull, thin, 



THE BIVALVE GENUS PARVITHRACIA 



111 



adherent, colorless. Beaks small, high, pro- 
jecting considerably above dorsal margin, 
slightly posterior to midline (A/L = 0.531- 
0.588), somewhat sharp, orthogyrate. Ante- 
rior end narrow, rounded. Posterior end de- 
cidedly truncate, with a faint radial ridge ex- 
tending beaks to junction of posterior end with 
ventral margin. Anterodorsal margin straight 
or slightly convex, very steeply descending 
ventrally, smoothly transitioning to rounded 
anterior end. Ventral margin slightly curved 
(more curved in right valve). Posterodorsal 
margin straight or slightly convex, very 
steeply descending ventrally, forming a dis- 
tinct angle at transition to posterior end. Pos- 
terior end slightly curved, anteriorly directed, 
forming a rounded angle at transition to ven- 
tral margin. Lunule present only in left valve, 
narrow, well expressed along entire an- 
terodorsal margin, demarcated by a ridge. Es- 
cutcheon narrow, more expressed in left 
valve, demarcated by ridges extending along 
posterodorsal margin from beaks to posterior 
end. 

Interior: Right valve with anterior and pos- 
terior lateral teeth and long grooves on inner 
part of anterodorsal and posterodorsal mar- 
gins; left valve with anterior lateral tooth. In 
right valve, anterior lateral tooth very large, 
high, strongly projecting above inner part of 
anterodorsal margin, ventrally directed; pos- 
terior lateral tooth small, elongate, somewhat 
projecting or not projecting above inner part of 
posterodorsal margin, extending along pos- 
terodorsal margin; inner part of antero- and 
posterodorsal margins thickened, with a long, 
shallow groove more expressed in inner part 
of posterodorsal margin, running parallel to 
anterior and posterior dorsal slopes for almost 
their entire length. In left valve, anterior lateral 
tooth long, lamellate, noticeably projecting 
above inner part of anterodorsal margin; inner 
part of anterodorsal margin slightly concave 
between beak and tooth; inner part of pos- 
terodorsal margin straight. Subumbonal plate 
short, slightly elevated above inner shell sur- 
face, attached to shell wall, free along its 
anteroventral margin. Pillars absent. Litho- 
desma large (L1/L = 0.121-0.125), ovate- 
trapezoidal. Anterior adductor muscle scar 
large, elongate, kidney-shaped. Posterior ad- 
ductor scar small, rounded. Palliai sinus dis- 
tinct, of the same shape and size in both 
valves, deep, broad, rounded anteriorly, 
reaching midline (S/L = 0.455-0.5), anterior 
limit short of faint vertical line from beaks. 
Shell interior with faint radial striae. 



Variability 

Slight variations occur in shell shape, shell 
height, valve width, and position of the beaks 
(Table 1). There is also variation in the shape 
and size of the teeth, lithodesma shape, and 
palliai sinus length. 

Distribution and Habitat 

Parvithracia (P.) suteri occurs near New 
Zealand (Three Kings Islands; Cape Brett; 
Hen and Chickens Islands; Mayor Island; Tas- 
man Bay; Stewart Island, Port Pegasus; 
Bounty Islands) (Suter, 1913; Powell, 1955, 
1979), from 33 m (Stewart Island, Port Pega- 
sus) to 260 m (Three Kings Islands), on mud 
and sandy mud. 

Pseudoasthenothaerus Kamenev, 
new subgenus 

Type species: Pseudoasthenothaerus lukini 
Kamenev, new species 

Description 

Shell small (< 11 mm), ovate-elongate to 
ovate-trigonal, inequivalve; right valve larger, 
more inflated. Beaks central or posterior. Pos- 
terior end truncate, with faint radial ridge. Pe- 
riostracum thin, adherent, colorless or tan. 
Surface with conspicuous growth lines and 
very fine granules. Lunule absent. Es- 
cutcheon narrow, well expressed. Ligament 
internal, supported by a strong lithodesma, at- 
tached to elongate-trigonal subumbonal plate 
that extends obliquely posterior from beaks, 
free along its anteroventral margin, where it is 
supported by a series of pillars separated by 
shallow or deep pits. Hinge plate weak, with 
large, short anterior and more or less con- 
spicuous, elongate posterior lateral teeth in 
right valve and long, lamellate anterior lateral 
tooth in left valve. Palliai line with deep palliai 
sinus, of same shape and size in both valves, 
not reaching midline. Mantle lobes fused, with 
small pedal and three palliai apertures. 
Siphons long, separate. Ctenidia consisting of 
two demibranchs; demibranchs subequal to 
inner demibranch much larger than outer. 
Labial palps broadly triangular. Stomach very 
large, to right of visceral mass; much of stom- 
ach to right of visceral mass not surrounded 
by digestive diverticula. Style sac joined to 
mid gut. Intestine passing through heart. Si- 



112 



KAMENEV 



TABLE 1. Parvithracia (Parvithracia) sufer/ Finlay, 1927. Shell measurements (mm), indices and summary 
statistics of all characteristics: L — shell length; H — height; W — width; A — anterior end length; S — maximal 
distance from the posterior shell margin to the top of palliai sinus; L1 — lithodesma length. Numerator indi- 
cates shell measurements and indices for the right valve, denominator— for the left valve. NM — not mea- 
sured. 



Statistics 


L 


H 


W 


A 


S 


LI 


H/L 


W/L 


A/L 


S/L 


L1/L 


Depository 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


MNZ ex M. 44790 




3.4 


2.8 


0.8 


2.0 


1.7 




0.824 


0.235 


0.588 


0.500 








3.3 


2.8 


1.0 


1.9 


1.6 


NM 


0.848 


0.303 


0.576 


0.485 


NM 


MNZ ex M. 44790 




NM 


NM 


NM 


NM 


NM 




NM 


NM 


NM 


NM 








3.3 


2.9 


1.0 


1.9 


1.6 


0.4 


0.879 


0.303 


0.576 


0.485 


0.121 


MNZ ex M. 44790 




3.3 


2.9 


0.8 


1.9 


1.5 




0.879 


0.242 


0.576 


0.455 


0.121 






3.2 


2.8 


1.7 


JL6 


0_.9 


0.4 


0.875 


0.281 


0.531 


0.500 


0.125 


MNZ ex M . 44790 




3.2 


2.7 


1.7 


1.5 


0.8 




0.844 


0.296 


0.531 


0.469 


0.125 




Mean 


3.27 


2.83 


0.97 


1.83 


1.60 


0.4 


0.867 


0.275 


0.561 


0.480 


0.123 






3.30 


2.80 


0.80 


1.87 


1.57 




0.849 


0.258 


0.565 


0.475 


0.123 




SD 


0.06 


0.06 


0.06 


0.12 








0.017 


0.031 


0.026 


0.023 


0.003 






0.10 


0.10 





0.15 


0.12 




0.028 


0.033 


0.030 


0.023 


0.003 




SE 


0.03 


0.03 


0.03 


0.07 








0.010 


0.018 


0.015 


0.013 


0.002 






0.06 


0.06 





0.09 


0.07 




0.016 


0.019 


0.017 


0.013 


0.002 




Min 


3.2 


2.8 


0.9 


1.7 


1.6 


0.4 


0.848 


0.242 


0.531 


0.455 


0.121 






3.2 


2.7 


0.8 


1.7 


1.5 




0.824 


0.235 


0.531 


0.455 


0.121 




Max 


3.3 


2.9 


1.0 


1.9 


1.6 


0.4 


0.879 


0.303 


0.576 


0.500 


0.125 






3.4 


2.9 


0.8 


2.0 


1.7 




0.879 


0.296 


0.588 


0.500 


0.125 




n 


3 


3 


3 


3 


3 


2 


3 


3 


3 


3 


2 






3 


3 


3 


3 


3 




3 


3 


3 


3 


2 





multaneous hermaphrodite. Testes occupying 
ventral position in visceral mass above foot. 
Paired ovaries occupying posterior position In 
visceral mass. 

Remarks 

The major morphological characters of the 
genera recognized within the Thraclldae by 
various specialists are presented In Table 2. 
The new subgenus has a combination of mor- 
phological characteristics not seen In any ex- 
isting thraclld genus. Parvithracia (Pseudoas- 
thenotfiaerus) differs from all other genera In 
having anterior and posterior lateral teeth In 
the right valve and an anterior lateral tooth 
in the left valve, and a subumbonal plate sup- 
ported by pillars. 

On the basis of external and Internal shell 
morphology, as well as hinge structure, the 
new subgenus is most similar to Parvithracia. 
This subgenus differs from Parvithracia, s.S., 
In having pillars supporting the subumbonal 
plate, in lacking a groove on the Inner part of 
the dorsal shell margin, and In having a 
lunule. In my opinion, these differences are 
less substantial compared to the differences 
among genera within this family (Table 2). 



Therefore, Pseudoasthenothaerus is as- 
signed as a subgenus of Parvithracia, with 
which It Is most similar In shell morphology. 

On the basis of Internal shell morphology, 
hinge structure, and relative size of the 
lithodesma, this subgenus is also similar to 
Lampeia. Only Lampeia and Parvithracia 
(Pseudoasthenothaerus) have a subumbonal 
plate supported by pillars. However, unlike 
Parvithracia (Pseudoasthenothaerus), Lam- 
peia has only an anterior tooth in the right 
valve. Moreover, Parvithracia (Pseudoas- 
thenothaerus) differs from Lampeia In lacking 
a thick brown periostracum, lunule, and exter- 
nal ligament, and in having granules on the 
shell surface. 

In shell form, proportions and morphology, 
Parvithracia (Pseudoasthenothaerus) resem- 
bles Asthenothaerus. For this reason, Japa- 
nese malacologlsts assigned representa- 
tives of Parvithracia (Pseudoasthenothaerus) 
found off the coast of Japan to Astheno- 
thaerus. However, Asthenothaerus has no lat- 
eral teeth, the subumbonal plate is tightly at- 
tached to the shell wall, and the lithodesma is 
less massive, more angular, with long, sharp 
ends. Perhaps, Asthenothaerus huanghaien- 
sisXu, 1989, described from northern Huang- 



THE BIVALVE GENUS PARVITHRACIA 



113 



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114 



KAMENEV 



hai Sea (China) (Xu, 1989), should also be 
placed In Parvithracia (Pseudoastheno- 
thaerus). 

The subgenus name is derived from the 
similarity of external shell morphology of 
species of this subgenus to members of the 
genus Asthenothaerus. 

Parvithracia (Pseudoasthenothaerus) lukini 

Kamenev, new species 
Figs. 11-34, Tables 

Type Material and Locality 

Holotype (MIMB 4047), Evreinova Strait 
(Onekotan Island -Makanrushi Island), Kuril 
Islands, 100 m, silty sand, Coll. V. I. Lukin and 
S. I.Grebelny,26-VIII-1987(R/V"Tikhookean- 
sky"); paratypes (15): paratype (MIMB 4048) 
from the type locality; paratype (MIMB 4049), 
Fourth Kuril Strait (Onekotan Island -Para- 
mushir Island), Kuril Islands (49°38'5N, 
1 55°22'3E), 500 m, sandy silt. Coll. V. I. Lukin 
and S. I. Grebelny, 27-X-1 987 (R/V "Tikhooke- 
ansky"); paratype (ZIN 1), southern Sea of 
Okhotsk (51°35'6N, 15Г46Е), 418 m, silty 
sand, bottom water temperature of +1.53°C, 
salinity 33.62%o, Coll. P. V. Ushakov, 10-VII- 
1932 (R/V "Gagara"); paratype (MIMB 4050), 
west coast of Kamchatka, Sea of Okhotsk 
(52°20'N, 155°18'E), 142 m, sand + silt. Coll. 
V. A. Nadtochy, 10-V-1989 (R/V "Mys Dalny"); 
paratypes (4) (MIMB 4051), Black Cape, 
Medny Island, Commander Islands, Bering 
Sea (54°41 .2'N, 167°59.7'E), 1 50-200 m, silt, 
Coll. V I. Lukin, 16-IX-1973 (R/V "Rakyt- 
noye"); paratype (ZIN 2), Aniva Cape, 
Sakhalin Island, Sea of Okhotsk (45°58'N, 
143°46.3E), 207 m, sandy silt, bottom water 
temperature of -0.4°C, Coll. Z. I. Kobjakova, 
24-IX-1947 (R/V "Toporok"); paratype (ZIN 3), 
Svobodnogo Cape, Sakhalin Island, Sea of 
Okhotsk (46°47.5'N, 143°52.3'E), 187 m, 
sandy silt, bottom water temperature of 
-0.4°C, Coll. Z. I. Kobjakova, 4-IX-1947 (R/V 
"Toporok"); paratype (MIMB 4052), Morzovaja 
Rock, Kunashir Island, Kuril Islands, Sea of 
Okhotsk (44'^33'8N, 146°28'7E), 101 m, silty 
sand + gravel. Coll. V. I. Lukin and V. P. 
Pavluchkov, 23-VII-1987 (R/V "Tikhookean- 
sky"); paratypes (2) (MIMB 4053), Kunashir Is- 
land, Kuril Islands, Pacific Ocean (44°08'N, 
146°54'4E), 1 89 m, silty sand + gravel, Coll. V. 
I. Lukin and V. P Pavluchkov, 1 9-VII-1 987 (R/V 
"Tikhookeansky"); paratypes (2) (ZIN 4), Peter 
the Great Bay, Sea of Japan (42°32.2'N, 
131°13.5'E), 65 m, Coll. Tarasov, 2-IX-1932. 



Other Material Examined 

One left valve (ZIN 5) from Svobodnogo 
Cape, Sakhalin Island, Sea of Okhotsk 
(46°47.5'N, 143'=52.3'E), 187 m, sandy silt, 
bottom water temperature of -0.4°C, Coll. Z. 
I. Kobjakova, 4-IX-1947 (R/V "Toporok"); one 
left valve (MIMB 4054) from east coast of 
Sakhalin Island, Sea of Okhotsk, 280 m, silty 
sand. Coll. V. N. Koblikov, 5-VIII-1977 (R/V "8- 
452"); one left valve (ZIN 6) from Tatar Strait, 
Sakhalin Island, Sea of Japan (49°00.4'N, 
141°44.2'E), 99 m, silty sand + gravel + peb- 
ble, bottom water temperature of +2.3°C, Coll. 
Skalkin, 28-VIII-1949 (RA/ "Toporok"); one 
right valve (ZIN 7) from Pesherny Cape, Sea 
of Japan, 230-238 m, silt + sand, Coll. 
Ohromkin, 17-VII-1931 (R/V "Rossinante"); 
one specimen (ZIN 8) from Peter the Great 
Bay, Sea of Japan (42°32.2'N, 13Г13.5'Е), 
65 m. Coll. Tarasov, 2-IX-1932; one left valve 
(MIMB 4055) from Bolshoy Peles Island, 
Peter the Great Bay, Sea of Japan (42°31 '8N, 
131 °23'5E), 72 m, large-particle sand. Coll. V. 
V Gulbin, 19-IX-1995 (R/V "Akademik 
Oparin"); one specimen and left valve (MIMB 
4056) from Black Cape, Medny Island, Com- 
mander Islands, Bering Sea (54°41.2'N, 
167°59.7'E), 150-200 m, silt. Coll. V. I. Lukin, 
1 6-IX-1 973 (R/V "Rakytnoye"); one right valve 
(MIMB 4057) from Yodny Cape, Iturup Island, 
Kuril Islands, Pacific Ocean (44°35'5N, 
147°27'5E), 200 m, sandy silt. Coll. V. I. Lukin 
and S. I. Grebelny (R/V "Tikhookeansky"). 
Total of 2 specimens, 5 left, and 2 right valves. 

Description 

Exterior: Shell small (< 11 .0 mm), ovate-an- 
gular, high (H/L = 0.785-0.936), slightly in- 
equivalve (right valve slightly higher, more in- 
flated), moderately inflated (W/L of right valve 
0.185-0.273; W/L of left valve 0.185-0.267), 
inequilateral, thin, solid. Surface with conspic- 
uous growth lines and fine, very dense gran- 
ules. Periostracum dull, thin, adherent, color- 
less, yellowish, pinkish or light brown, with 
dark brown or black patches or crusts near 
beaks and on dorsal shell margin, extending 
into inner surface. Beaks small, moderately 
projecting above dorsal margin, posterior to 
midline (/VL = 0.585-0.765), slightly rounded, 
opisthogyrate. Anterior end rounded. Poste- 
rior end narrow, truncate, with faint radial 
ridge from beaks to ventral limit of posterior 
end. Anterodorsal margin straight or slightly 
convex, gently descending ventrally, smoothly 



THE BIVALVE GENUS PARVITHRACIA 



115 




FIGS. 11-24. Parvithracia (Pseudoasthenothaerus) /и/с/л/ Kamenev, new species. 11-15: Holotype (MIMB 
4047), Evreinova Strait (Onekotan Island - Makanrushi Island), Kuril Islands, 1 00 m, shell length 9.9 mm. 1 6: 
Lithodesma of holotype, ventral view (bar = 1 mm). 17-22: Paratypes (MIMB 4051), Black Cape, Medny Is- 
land, Commander Islands, Bering Sea (54°41.2'N, 167°59.7'E), 150-200 m. 17, 18: Left and right valves of 
a young specimen (bar = 1 mm). 19: Dorsal view of both valves of a young specimen (bar = 1 mm). 20, 21 : 
Ventral view of right and left valves showing subumbonal plate and teeth (bar = 1 mm). 22: Ventral view of 
lithodesma (bar = 1 mm). 23: Paratype (MIMB 4050), west coast of Kamchatka, Sea of Okhotsk (52°20'N, 
155°18'E), 142 m, ventral view of lithodesma (bar = 1 mm). 24: Paratype (MIMB 4053), Kunashir Island, Pa- 
cific Ocean (44°08'N, 146°54'4E), 189 m, close-up of right valve showing attachment of lithodesma (ventral 
view) to subumbonal plate and the tooth (bar = 1 mm). 



116 



KAMENEV 




FIGS. 25-32. Parvithracia (Pseudoasthenothaerus) lukini Katnenev, new species. FIGS. 25-28, 30. 
Paratypes (MIMB 4051), Black Cape, Medny Island, Commander Islands, Bering Sea (54°41.2'N, 
167^59. 7'E), 150-200 m. 25, 26; Dorsal view of teeth on right and left valves (bar = 1 mm). 27, 28: Hinge of 
right and left valves (bar = 1 mm). 29: Paratype (MIMB 4050), west coast of Kamchatka, Sea of Okhotsk 
(52"20'N, 155"18'E), 142 m, subumbonal plate with pillars (ventral view), nght valve (bar = 1 mm). 30: Sub- 
umbonal plate with pillars (ventral view), left valve (bar = 1 mm). 31, 32: Right valve with of specimen with 
reversed hinge (ventral view). Black Cape, Medny Island, Commander Islands, Bering Sea (54°41.2'N, 
1 67^^59. 7'E), 150-200 m (bar = 1 mm). 



THE BIVALVE GENUS PARVITHRACIA 



117 




FIG. 33. Parvithracia (Pseudoasthenothaerus) lukini. Organs of mantle cavity as seen from right side with 
right shell valve and mantle removed (shell length, 6.4 mm). AA, anterior adductor muscle; APR, anterior 
pedal retractor muscle; DD, digestive diverticula; E, exhalant siphon; EM, edge of mantle; F, foot; HG, hind 
gut; I, inhalant siphon; ID, inner demibranch; K, kidney; LP, labial palp; MC, mineralized concretions; OD, 
outer demibranch; PA, posterior adductor muscle; PPR, posterior pedal retractor muscle; SM, siphonal mus- 
culature. 



transitioning to rounded anterior end. Ventral 
nnargin slightly curved (more curved in right 
valve), sometimes straight in left valve. Pos- 
terodorsal margin slightly concave, rather 
steeply descending to form a smooth angle at 
posterior end. Posterior end slightly curved, 
almost vertical, only slightly turned anteriorly 
to form a rounded angle at ventral shell mar- 
gin. Escutcheon narrow, well defined, bor- 
dered by ridges along entire posterodorsal 
margin. 

Interior: Right valve with anterior and pos- 
terior lateral teeth; left valve with anterior lat- 
eral tooth. In right valve, anterior lateral tooth 
large, triangular, strongly projecting above 
inner part of anterodorsal margin, anteroven- 
trally directed; posterior lateral tooth small, 
elongate, only slightly projecting or not pro- 
jecting above inner part of posterodorsal mar- 
gin. In left valve, anterior lateral tooth long, 
lamellate, slightly projecting above inner part 
of anterodorsal margin; inner part of an- 



terodorsal margin straight between beak and 
tooth; inner part of posterodorsal margin 
straight. Subumbonal plate short, elevated 
above inner shell surface. Supporting pillars 
high, of varying thickness, generally of the 
same thickness throughout their individual 
lengths or becoming thinner ventrally; width 
and height of pillars, and width of pits between 
pillars decreasing posteriorly from anterodor- 
sal margin. Number of pillars from 2 to 7, 
usually equal in both valves. Lithodesma 
large (L1/L = 0.098-0.225), butterfly-shaped, 
sometimes trapezoidal, often asymmetrical. 
Anterior adductor muscle scar large, elon- 
gate, kidney-shaped. Posterior adductor scar 
small, rounded. Palliai sinus distinct, of the 
same shape and size in both valves, deep, 
rounded anteriorly, sometimes reaching mid- 
line (S/L = 0.395-0.54), anterior limit short of 
faint vertical line from beaks. Shell interior 
with faint radial striae. 
Anatomy: Gross morphology differs little 



118 



KAMENEV 




MG 



FIG. 34. Parvithracia (Pseudoasthenothaerus) lukini. Internal morphology as seen from right side with right 
shell valve, mantle, and ctenidium removed (shell length, 6.4 mm). A, anus; AA, anterior adductor muscle; 
APR, anterior pedal retractor muscle; DD, digestive diverticula; F, foot; H, heart; HG, hind gut; K, kidney; LP, 
labial palp; MC, mineralized concretions; MG, mid-gut; O, ovary; OE, oesophagus; PA, posterior adductor 
muscle; PPR, postenor pedal retractor muscle; S, stomach; SS, style sac; T, testis; VG, visceral ganglia. 



from previous descriptions of Thracia (Kiener, 
1834; Deshayes, 1846). Mantle very thin, ex- 
cept at ventral margin, which is slightly thick- 
ened, completely fused except at extensive 
siphonal and pedal gapes, and small, round 
aperture ventral to inhalant siphon. Siphons 
long; inhalant siphon longer, larger. Anterior 
adductor muscle large, elongate dorsoven- 
trally, slightly curved parallel to anterior end. 
Posterior adductor muscle smaller, almost 
round. Foot small, laterally compressed, pro- 
jecting anteroventrally, its shape in preserved 
specimens variable depending on degree of 
contraction. Anterior pedal retractor muscles 
ascending almost vertically from base of foot, 
passing dorsally above labial palps, attaching 
dorsal to anterior adductor muscle. Posterior 
pair of narrow pedal retractor muscles pass- 
ing on either side of anus as single muscle 
sheets from base of foot, attaching dorsal to 



posterior adductor muscle. Ctenidia thin, con- 
sisting of two demibranchs; inner demibranch 
much larger than outer. Paired labial palps 
long, elongate-thangular, of equal length. 
Mouth small, situated close to anterior adduc- 
tor muscle, at basal junction of inner and outer 
palps. Oesophagus long, straight, extending 
parallel to anterodorsal side of body, opening 
into anterodorsal part of stomach. Stomach 
very large, oval, lying along dorsoventral axis, 
on right side of visceral mass; stomach wall 
very close to dorsal and right sides of visceral 
mass; much of stomach on right side not sur- 
rounded by digestive diverticula (stomach 
contents may be visible through the translu- 
cent walls), with a combined style sac and mid 
gut. Combined style sac and mid gut leaving 
stomach at posteroventral border, passing 
ventrally into visceral mass. Mid-gut separate 
from style sac, forming large number of short 



THE BIVALVE GENUS PARVITHRACIA 



119 



loops extending anteriorly ventral to stomach 
almost as far as mouth, turning back along 
dorsal margin of foot to style sac. Hind gut 
thickened, turning dorsally between paired 
transparent ovaries, passing through heart 
and posteriorly along posterodorsal margin of 
body above kidney, partially circling posterior 
adductor muscle, terminating near exhalant 
siphon as an anal papilla free of attachments. 
Digestive diverticula surrounding oesophagus 
and greater part of stomach. Kidneys large, 
posterodorsal between posterior adductor 
muscle and ovaries, containing large number 
of mineralized concretions. Cerebral ganglia 
consisting of two small commissural bodies 
between mouth and anterior adductor muscle. 
Pedal ganglia large, lying at interface of foot 
and viscera. Visceral ganglia large, conspicu- 
ous, lying ventral to kidney, in contact with in- 
testine near anus. This species is a simulta- 
neous hermaphrodite. Testes occupying a 
ventral position in visceral mass dorsal to foot. 
Paired ovaries occupying a posterior position 
in visceral mass, appearing as transparent 
sacs with large eggs (up to 150-200 цт). 

Variability 

Shell shape and proportions vary markedly 
(Table 3). The shell shape varies from ovate- 
angular to rounded. Shell height and width, 
the position of the beaks, the degree of con- 
cavity of posterodorsal margin, and the rela- 
tive length of palliai sinus vary. The shape, 
length, and width of lithodesma is also 
markedly variable, but it is most often butter- 
fly-shaped. In young specimens, it is some- 
times trapezoidal. The number of pillars 
varies and, as a rule, increases with shell 
size; some of them bifurcate ventrally or are 
fused. The sizes and shapes of the lateral 
teeth in both valves, and the inclination of an- 
terior lateral tooth in right valve vary little. One 
specimen has the hinge reversed (Figs. 31, 
32); its left valve has two lateral teeth, the 
shape and size of which are identical to the 
teeth of right valve of other specimens exam- 
ined. The right valve possesses an anterior 
lamellate tooth analogous to the tooth of left 
valve of normal specimens. 



near the west coast of Kamchatka, near 
Sakhalin Island, and in the southern part of 
the sea (51°35'6N, 151°46E); near the Kuril 
Islands -in Evreinova and Fourth Kuril 
Straits, near Iturup and the Kunashir Islands; 
in the Sea of Japan -in Tatar Strait (near 
Sakhalin Island), near Pesherny Cape, and in 
Peter the Great Bay. 

In the Bering Sea, this species was ob- 
tained at depth 150-200 m on silt; in the Sea 
of Okhotsk -from 142 m (west coast of Kam- 
chatka) to 418 m (southern Sea of Okhotsk) 
on silty sand and sandy silt at a bottom tem- 
perature from -0.4°C (Svobodnogo and Aniva 
Capes, Sakhalin Island, depth 187 and 207 
m, respectively) to 1.53°C (southern Sea of 
Okhotsk, depth 418 m); near the Kuril Is- 
lands -from 100 m (Evreinova Strait) to 500 
m (Fourth Kuril Strait) on silty sand and sandy 
silt sometimes with some admixture of gravel; 
in the Sea of Japan -from 65 m (Peter the 
Great Bay) to 238 m (Pesherny Cape) on 
large-particle sand, silty sand and silt some- 
times with admixture of gravel and pebbles at 
a bottom temperature 2.3°C (Tatar Strait, 
Sakhalin Island, depth 99 m). 

Comparisons 

Parvithracia (Pseudoasthenothaerus) lukini 
is easily distinguished from Parvithracia 
(Pseudoasttienottiaerus) sematana and P. 
(Pseudoastlienotfiaerus) isaotal<ii in having a 
thicker, heavier shell (Table 4). Moreover, in 
contrast to P. (Pseudoasthenottiaerus) se- 
matana, this species has a higher, less elon- 
gate shell, with a highly elevated subumbonal 
plate and distinct, high pillars. Unlike P. 
(Pseudoasthenottiaerus) isaotakii, it has a 
more oval shell, with a considerably less ex- 
panded posterior end, and it has opisthogy- 
rous beaks. 

This species is most similar to Parvithracia 
(Pseudoasthenothaerus) sirenkoi; but differs 
in having a more elongate, oval, lower, and 
less inflated shell. The shell of Parvithracia 
(Pseudoasthenothaerus) lukini is more ex- 
panded anteriorly, the beaks are more poste- 
rior, the lateral teeth in both valves are 
smaller, and there is a deeper palliai sinus. 



Distribution and Habitat (Fig. 35) 

In the Bering Sea, P. (Pseudoastheno- 
thaerus) lukini occurs near Medny Island, 
Commander Islands; in the Sea of Okhotsk - 



Etymology 

The specific name honors Dr. Vladimir I. 
Lukin, a famous Russian researcher of the 
marine fauna of the Kuril Islands, who has col- 



120 



KAMENEV 



TABLE 3. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species. Shell measurernents (mm), 
indices and summary statistics of characters: L — shell length; H — height; W — width; A — anterior end length; 
S — maximal distance from the posterior shell margin to the top of palliai sinus; L1 — lithodesma length; N — 
number of pillars under subumbonal plate. Numerator indicates shell measurements and indices for the right 
valve, denominator — for the left valve. NM — not measured. 



Statistics 


L 


H 


W 


А 


S 


L1 


N 


H/L 


W/L 


PJL 


S/L 


L1/L 


Depository 




9.9 


8.0 


2.5 


6J 


4J 


1.7 


6 


0.808 


0.253 


0.677 


0.465 


0.172 


Holotype 
MIMB 




9.8 


7.8 


2.2 


6.5 


4.5 




6 


0.796 


0.224 


0.663 


0.459 


0.173 


4047 




9.7 


8.5 


L6 


LO 


4^ 


2.0 


4 


0.876 


0.268 


0.722 


0.443 


0.206 


Paratype 
MIMB 




9.7 


8.2 


2.4 


7.0 


4.0 




4 


0.845 


0.247 


0.722 


0.412 


0.206 


4049 




8J. 


6¿ 


L5 


57 


3.5 


1.4 


7 


0.802 


0.185 


0.704 


0.432 


0.173 


Paratype 
MIMB 




8.1 


6.4 


1.5 


5.7 


3.4 




7 


0.790 


0.185 


0.704 


0.420 


0.173 


4050 




7.9 


6.3 


1.8 


5.4 


3.4 


1.3 


5 


0.797 


0.228 


0.684 


0.430 


0.165 


Paratype 
MIMB 




7.9 


6.2 


1.8 


5.5 


3.2 




5 


0.785 


0.228 


0.696 


0.405 


0.165 


4053 




6.7 


5.8 


1.8 


4.1 


2.8 


NM 


4 


0.866 


0.269 


0.612 


0.418 


NM 


Paratype 
ZIN2 




6.7 


5.7 


1.7 


4.1 


2.8 




4 


0.851 


0.254 


0.612 


0.418 


NM 






6.5 


5.6 


L3 


4.2 


3.0 


1.0 


5 


0.862 


0.200 


0.646 


0.462 


0.154 


Paratype 
ZIN 1 




6.5 


5.6 


1.3 


4.4 


3.1 




4 


0.862 


0.200 


0.677 


0.477 


0.154 






6A 


5.5 


1.7 


4.0 


2.8 


1.2 


3 


0.859 


0.266 


0.625 


G.438 


0.188 


Paratype 
ZIN3 




6.4 


5.5 


1.7 


4.0 


2.8 




3 


0.859 


0.266 


0.625 


0.438 


0.188 






6.4 


5.3 


1.5 


4.2 


3.2 


1.2 


5 


0.828 


0.234 


0.656 


0.500 


0.188 


Paratype 
ZIN 4 




6.3 


5.2 


1.5 


4.3 


3.4 




5 


0.825 


0.238 


0.683 


0.540 


0.190 






аз 


5^ 


L5 


4.1 


2.8 


1.1 


4 


0.889 


0.238 


0.651 


0.444 


0.175 


Paratype 
MIMB 




6.3 


5.5 


1.5 


4.1 


2.8 




5 


0.873 


0.238 


0.651 


0.444 


0.175 


4051 




6.3 


5.4 


1.4 


4.0 


2.9 


1.0 


4 


0.857 


0.222 


0.635 


0.460 


0.159 


Paratype 
MIMB 




6.3 


5.3 


1.5 


4.0 


2.9 




4 


0.841 


0.238 


0.635 


0.460 


0.159 


4048 




5.8 


5.3 


1.3 


37 


2.7 


0.9 


4 


0.914 


0.224 


0.638 


0.466 


0.155 


Paratype 
MIMB 




5.8 


5.3 


1.3 


3.7 


2.7 




4 


0.914 


0.224 


0.638 


0.466 


0.155 


4051 




5.8 


4.8 


1.2 


4.3 


2.7 


1.0 


4 


0.828 


0.207 


0.741 


0.466 


0.172 


Paratype 
MIMB 




5.8 


4.7 


1.2 


4.3 


2.7 




4 


0.810 


0.207 


0.741 


0.466 


0.172 


4053 




5.3 


4.8 


1.1 


3.1 


2.4 


NM 


2 


0.906 


0.208 


0.585 


0.453 


NM 


Paratype 
MIMB 




5.3 


4.7 


1.0 


3.1 


2.4 




3 


0.887 


0.189 


0.585 


0.453 


NM 


4051 




4.7 


4.4 


1.0 


2.8 


2.2 


0.8 


2 


0.936 


0.213 


0.596 


0.468 


0.170 


Paratype 
MIMB 




4.7 


4.4 


1.1 


2.9 


2.2 




3 


0.936 


0.234 


0.617 


0.468 


0.170 


4051 




4.1 


ЗА 


0.8 


2.8 


1.7 


0.4 


3 


0.829 


0.195 


0.683 


0.415 


0.098 


Paratype 
MIMB 




4.1 


3.4 


0.8 


2.8 


1.7 




3 


0.829 


0.195 


0.683 


0.415 


0.098 


4052 




32 


2.9 


0.7 


2.0 


1.6 


0.5 


2 


0.906 


0.219 


0.625 


0.500 


0.156 


Paratype 
ZIN 4 




3.2 


2.8 


0.7 


2.0 


1.6 




2 


0.875 


0.219 


0.625 


0.500 


0.156 






11.0 


9.6 


3.0 


7.0 


4.5 


2.2 


7 


0.873 


0.273 


0.636 


0.409 


0.200 


MIMB 




NM 


NM 


NM 


NM 


NM 




NM 


NM 


NM 


NM 


NM 


NM 


4057 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


ZIN 5 




9.0 


7.8 


2.4 


5.4 


3.8 




6 


0.867 


0.267 


0.600 


0.422 


NM 






NM 


NM 


NM 


NM 


NM 


2.0 


NM 


NM 


NM 


NM 


NM 


NM 


MIMB 




8.9 


7.8 


2.0 


6.1 


4.0 




6 


0.876 


0.225 


0.685 


0.449 


0.225 


4054 




8.6 


7.1 


2.1 


6.2 


3.4 


1.5 


7 


0.826 


0.244 


0.721 


0.395 


0.174 


ZIN 8 




8.5 


7.0 


2.0 


6.5 


3.7 




7 


0.824 


0.235 


0.765 


0.435 


0.176 





THE BIVALVE GENUS PARVITHRACIA 



121 



TABLE 3. (Continued) 



H W 



L1 N 



H/L 



W/L 



A/L S/L L1/L Depository 



NM 


NM 


NM 


NM 


NM 


NM NM 


NM 


NM 


NM 


NM 


NM 


7.8 


6.8 


1.9 


5.7 


3.6 


5 


0.872 


0.244 


0.731 


0.462 


NM 


NM 


NM 


NM 


NM 


NM 


NM NM 


NM 


NM 


NM 


NM 


NM 





7.1 


6.1 


1.7 


4.3 


3.2 




5 


0.859 


0.239 


0.606 


0.451 


NM 




ел 


5JD 


L3 


4^ 


23 


NM 


4 


0.833 


0.217 


0.667 


0.483 


NM 




NM 


NM 


NM 


NM 


NM 




NM 


NM 


NM 


NM 


NM 


NM 


Mean 


6.77 


5.77 


1.57 


4.49 


3.02 


1.25 


— 


0.858 


0.230 


0.658 


0.450 


0.169 




6.87 


5.82 


1.58 


4.59 


3.07 






0.851 


0.228 


0.664 


0.450 


0.171 


SD 


2.00 


1.65 


0.64 


1.46 


0.82 


0.51 


— 


0.040 


0.027 


0.043 


0.029 


0.024 




1.78 


1.43 


0.48 


1.35 


0.74 






0.039 


0.023 


0.051 


0.032 


0.027 


SE 


0.46 


0.38 


0.15 


0.34 


0.19 


0.12 


— 


0.009 


0.006 


0.010 


0.007 


0.006 




0.39 


0.31 


0.10 


0.30 


0.16 






0.008 


0.005 


0.011 


0.007 


0.007 


Min 


3¿ 


23 


0^ 


2.0 


1.6 


0.40 


2 


0.797 


0.185 


0.585 


0.395 


0.098 




3.2 


2.8 


0.7 


2.0 


1.6 




2 


0.785 


0.185 


0.585 


0.405 


0.098 


Max 


11.0 


9.6 


3.0 


7.0 


4.6 


2.20 


7 


0.936 


0.273 


0.741 


0.500 


0.206 




9.8 


8.2 


2.4 


7.0 


4.5 




7 


0.936 


0.267 


0.765 


0.540 


0.225 


n 


19 


19 


19 


19 


19 


17 


19 


19 


19 


19 


19 


16 




21 


21 


21 


21 


21 




21 


21 


21 


21 


21 


16 



ZIN6 

MIMB 
4056 
ZIN7 



Russia 



Bering Sea 

Kamchatka 

^^* 

Commander Islands 




-, , ... лг Paramushir Is. 

Makanrushi Is._ (7 

1 ^» Onekotan Is. 



Pacific Ocean 



FIG. 35. Distribution of Parvithracia (Pseudoasthenothaerus) lukini{M type locality). 



122 



KAMENEV 



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CL 



THE BIVALVE GENUS PARVITHRACIA 



123 



lected many of the specimens examined in 
this paper. 

Parvithracia (Pseudoasthenothaerus) 

sirenkoi Kamenev, new species 

Figs. 36-54, Table 5 

Type Material and Locality 

Holotype (MIMB 4058), Iturup Island, Ku- 
ril Islands, Pacific Ocean (44°52'N, 149° 
27.7'E), 910-920 m, silty sand. Coll. B. I. 
Sirenko, 25-VII-1984 (FW "Odissey"); 
paratypes (10): paratypes (2) (MIMB 4059) 



from holotype locality; paratypes (2) (ZIN 1), 
Iturup Island, Kuril Islands, Pacific Ocean 
(44°46.8'N, 149°06.7'E), 880 m, silty sand, 
Coll. B. I. Sirenko, 26-VII-1984 (R/V "Odis- 
sey"); paratype (ZIN 2), Iturup Island, Kuril Is- 
lands, Pacific Ocean (44°02.6'N, 148°11'E), 
600 m, silty sand + gravel + pebbles. Coll. B. 
I. Sirenko, 22-IX-1984 (R/V "Odissey"); 
paratypes (4) (MIMB 4060), Kuril Islands, 
Coll. V. I. Lukin, 1987 (R/V "Tikhookeansky"); 
paratype (MIMB 4061), Yury Island, Kuril Is- 
lands, Pacific Ocean (43°10'N, 146°17'E), 
490 m, silty sand + gravel + pebbles, bottom 
water temperature of +2.33°C, Coll. V. I. Lukin 




FIGS. 36-48. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. 36-40: Holotype 
(MIMB 4058), Iturup Island, Kuril Islands, Pacific Ocean (44°52'N, 149°27.7'), 910-920 m, shell length 8.3 
mm. 41 -45: Paratype (MIMB 4060), Kuril Islands, shell length 8.0 mm. 46-48: Paratypes (MIMB 4059) from 
type locality. 46, 47: Right and left valves of a young specimen (bar = 1 mm). 48: Dorsal view of both valves 
of a young specimen (bar = 1 mm). 



124 



KAMENEV 




FIGS. 49-54. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. 49, 50: Paratype 
(MIMB 4060), Kuril Islands, hinge of the left and right valves (bar = 1 mm). 51 , 52: Paratypes (ZIN 2), Iturup 
Island, Kuril Islands, Pacific Ocean (44°02.6'N, 148°11 'E), 600 m. 51: Subumbonal plate with pillars (ventral 
view), left valve (bar = 1 mm). 52: Close-up of right valve showing attachment of lithodesma (ventral view) to 
subumbonal plate and teeth (bar = 1 mm). 53: Paratype (MIMB 4060), Kuril Islands, ventral view of lith- 
odesma (bar = 1 mm). 54: Paratype (ZIN 1), Iturup Island, Kuril Islands, Pacific Ocean (44°46.8'N, 
149°06.7'), 880 m, dorsal view of teeth on right valve (bar = 1 mm). 



and V. P. Pavluchkov, 
"Tikhookeansky"). 

Other Material Examined 



8-VII-1987 (RA/ 



Two right and one left valves (MIMB 4062) 
from type locality; one specimen (ZIN 3) from 
Iturup Island, Kuril Islands, Pacific Ocean 
(44'^59'N, 14847'E), 450 m, silty sand + peb- 
bles, Coll. B. I. Sirenko, 24-IX-1984 (R/V 
"Odissey"); one specimen (ZIN 4) from south- 
western Sea of Okhotsk (45°06'N, 
143°43.5'E), 188 m, silt, bottom water tem- 
perature of +0.5°C, Coll. Yu. I. Galkin, 25-VIII- 



1948 (R/V "Toporok"); one left valve (MIMB 
4063) from Morzovaja Rock, Kunashir Island, 
Kuril Islands, Sea of Okhotsk (44°33'8N, 
146°28'7E), 101 m, silty sand -i- pebbles, Coll. 
V. I. Lukin and V. P. Pavluchkov, 23-VII-1987 
(R/V "Tikhookeansky"). Total of 2 specimens, 
2 left, and 2 right valves. 

Description 

Exterior: Shell small (<8.9 mm), ovate-trig- 
onal, high (H/L = 0.84-0.963), slightly in- 
equivalve (right valve slightly higher, more 
inflated), inflated (W/L of right valve 0.211- 



THE BIVALVE GENUS PARVITHRACIA 



125 



0.313; W/L of left valve 0.237-0.293), inequi- 
lateral, thin, solid. Surface with conspicuous 
growth lines and fine, very dense granules. 
Periostracum dull, thin, adherent, colorless, 
pinkish or light brown, with dark brown or 
black patches or crusts near beaks and dorsal 
shell margin, extending into inner surface. 
Beaks small, high, projecting considerably 
above dorsal margin, posterior to midline (A/L 
= 0.568-0.72), slightly rounded, opisthogy- 
rate. Anterior end sharply rounded. Posterior 
end higher than anterior, decidedly truncate, 
with a faint radial ridge extending from beaks 
to ventral limit of posterior end. Anterodorsal 
margin straight or slightly convex, very 
steeply descending, smoothly curving to 
rounded anterior end. Ventral margin slightly 
curved (more curved in right valve). Pos- 
terodorsal margin straight or slightly concave, 
descending and forming a distinct angle with 
posterior end. Posterior end slightly curved, 
descending ventrally or slightly anteriorly, 
forming a rounded angle with ventral shell 
margin. Escutcheon wide, well defined, de- 
marcated by ridges extending along pos- 
terodorsal shell margin from beaks to poste- 
rior end. 

Interior: Right valve with anterior and pos- 
terior lateral teeth; left valve with anterior lat- 
eral tooth. In right valve, anterior lateral tooth 
large, high, triangular, projecting considerably 
above inner part of anterodorsal margin, ven- 
trally (sometimes anteroventrally) directed; 
right posterior lateral tooth small, elongate, 
slightly projecting above inner part of pos- 
terodorsal margin, extending along pos- 
terodorsal margin. In left valve, anterior lateral 
tooth long, lamellate, strongly projecting 
above inner part of anterodorsal margin; inner 
part of anterodorsal margin slightly concave 
between beak and tooth; inner part of pos- 
terodorsal margin slightly convex. Subum- 
bonal plate short, elevated above inner shell 
surface. Pillars supporting plate high, varying 
in thickness between each other, generally of 
same thickness throughout their individual 
lengths or becoming thinner ventrally; width 
and height of pillars, and width of pits between 
pillars decreasing posteriorly from anterodor- 
sal margin. Number of pillars from 2 to 6, 
usually equal in number in both valves. Lith- 
odesma large (L1/L = 0.067-0.225), butterfly- 
shaped, sometimes ovate-trapezoidal, often 
asymmetrical. Anterior adductor muscle scar 
large, elongate, kidney-shaped. Posterior ad- 
ductor scar small, rounded. Palliai sinus dis- 
tinct, of the same shape and size in both 



valves, deep, narrow and rounded anteriorly, 
not reaching midline (S/L = 0.2-0.447), ante- 
rior limit short of faint vertical line from beaks. 
Shell interior with dense, tiny pockmarks and 
faint radial striae. 

Anatomy: Internal morphology similar to 
that of P. (Pseudoasthenothaerus) lukini. 
Mantle very thin, except at ventral margin, 
which is slightly thickened, completely fused 
except for the small pedal and three palliai 
apertures. Siphons long; inhalant siphon 
longer, larger. Anterior adductor muscle large, 
elongate dorsoventrally, slightly curved paral- 
lel to anterior shell margin. Posterior adductor 
muscle smaller, almost round. Foot small, lat- 
erally compressed, projecting anteroventrally. 
Anterior pedal retractor muscles almost verti- 
cally from base of foot, passing above labial 
palps to attach dorsal to anterior adductor 
muscle. Posterior pair of narrow pedal retrac- 
tor muscles passing on either side of anus as 
a single muscle sheets from base of foot to at- 
tach dorsal to posterior adductor muscle. 
Ctenidia thin, consisting of two subequal 
demibranchs. Paired labial palps long, elon- 
gate-triangular, of equal length. Mouth small, 
close to anterior adductor muscle at basal 
junction of inner and outer palps. Oesopha- 
gus long, straight, extending parallel to an- 
terodorsal side of body, opening to anterodor- 
sal part of stomach. Stomach very large, oval, 
lying along dorsoventral axis, in right side of 
visceral mass; stomach wall located close to 
dorsal and right sides of visceral mass, much 
of stomach on right side not surrounded by di- 
gestive diverticula (stomach contents some- 
times visible through the translucent walls). 
Combined style sac and mid-gut leaving 
stomach at posteroventral border and passing 
ventrally into visceral mass. Hind gut thick, 
passing dorsally between paired, transparent 
ovaries, through heart, posteriorly along pos- 
terodorsal margin of body above kidney, 
passing over posterior adductor muscle, ter- 
minating near exhalant siphon. Anal papilla 
free. Digestive diverticula surrounding oe- 
sophagus and stomach. Kidney large, oc- 
cupying a posterodorsal position between 
posterior adductor muscle and ovaries. Vis- 
ceral ganglia large, conspicuous, lying below 
kidney, touching intestine near anus. This 
species is a simultaneous hermaphrodite. 
Testes occupying a ventral position in visceral 
mass dorsal to foot. Paired ovaries occupying 
a posterior position in visceral mass, appear- 
ing as transparent sacs with large eggs (up to 
150-200 |im). 



126 



KAMENEV 



Variability 

Shell shape and proportions change little 
with age. in young specimens (<6 mm), the 
shell is less inflated (Table 5). In adults, shell 
height, width, the position of beaks, and rela- 
tive length of the palliai sinus vary slightly. The 
sizes and shape of the lateral teeth in both 
valves, and the inclination of the anterior tooth 
in the right valve are fairly variable. The 
shape, length, and width of lithodesma can 
vary considerably, but it is generally butterfly- 
shaped. The number of pillars supporting the 
subumbonial plate varies and correlates with 
shell size. 



Distribution and Habitat (Fig. 55) 

South Kuril Islands: Yury Island (43°10'N, 
146°17'E), Kunashir Island (44°33'8N, 146° 
28'7E), Iturup Island (44°59'N, 148°47'E; 
44°52'N, 149°27.7'E; 44°46.8'N, 149°06.7'E; 
44°02.6'N, 148°11'E); southwestern Sea of 
Okhotsk (45°06'N, 143°43.5'E). 

This species was recorded off the South 
Kuril Islands at depth from 101 m (Kunashir 
Island) to 910-920 m (Iturup Island) on silty 
sand, sometimes with some pebbles and 
gravel; in the southwestern Sea of Okhotsk, it 
was present at a depth of 188 m, on silt, at a 
bottom water temperature of -i-0.5°C. 

Comparisons 

This species is easily distinguished from 
other species of this subgenus by its high, 
ovate-triangular shell with a narrow anterior 
end (Table 4). Moreover, P. (Pseudoastheno- 
thaerus) sirenkoi differs from P. (Pseudoas- 
thenothaerus) sematana and P. (Pseudoas- 
thenothaerus) isaotakii by its thicker, more 
solid shell with a larger lateral tooth. It is clos- 
est to P. (Pseudoasthenothaerus) lukini, dif- 
fering from it in having a higher, more inflated 
shell, higher and less posteriorly placed 
beaks, more steeply sloping anterodorsal and 
posterodorsal shell margins, a smaller apical 
angle, larger lateral teeth in both valves, the 
inner part of the posterodorsal margin convex 
in left valve, and a shallower palliai sinus. 
t\/lean values and variances of the indices 
characterizing the relative height (H/L) and 
width (W/L) of the shell, as well as the position 
of beaks (A/L), and the relative length of the 
palliai sinus (S/L) were significantly different 
in these species (Table 6). 



Discriminant analysis of all lots containing 
P. (Pseudoasthenothaerus) lukini and P. 
(Pseudoasthenothaerus) sirenkoi showed 
that these species differ significantly in a com- 
plex of parameters (Wilks Lambda = 0.3331, 
F-value = 1 5.31 34, df = 3, 23, Squared Maha- 
lanobis Distance = 8.1 063, p < 0.0001 ; Means 
of Cannonical variables: P (Pseudoastheno- 
thaerus) lukini = -1.21764; P. (Pseudoas- 
thenothaerus) sirenkoi = 1.52205). Off 30 
specimens analysed, 28 were accurately 
classified (93.33%), with two specimens mis- 
takenly classified as P. (Pseudoastheno- 
thaerus) lukini. The most significant indices 
for dividing all specimens into two species 
were S/L for right valves, and W/L, and A/L for 
left valves (Table 7). 

Etymology 

The specific name honors Dr. Boris I. 
Sirenko, Zoological Institute - St. Petersburg, 
who has collected almost all of the specimens 
of this species examined for this paper. 

Parvithracia (Pseudoasthenothaerus) 

sematana (Yokoyama, 1 922) 

Figs. 56-60, 65, Table 8 

Thracia sematana Yokoyama, 1922: 173, 
pi. 14, figs. 17, 18. 

Parvithracia sematana (Yokoyama, 1922), 
Oyama, 1973: 120, pi. 57, figs. 13, 14. 

Asthenothaerus sematensis (Yokoyama, 
1922), Ito, 1989:66, pi. 28, fig. 3. 

Asthenothaerus sematanus (Yokoyama, 
1922), Tsuchida & Kurozumi, 1996: 15, 16. 

Asthenothaerus sematana (Yokoyama, 
1922), Habe, 1977: 312; Higo et al., 1999: 
524; Okutani, 2000: 1039, but not pi. 517, fig. 
6, which is Trigonothracia pusilla 

Trigonothracia pusilla (Gould, 1861), Oku- 
tani, 2000: pi. 517, fig. 8 

Type Material and Locality 

Lectotype (CM 21507, left valve) and para- 
lectotype (CM 21508, right valve) (Oyama, 
1973). Shito, Semata, Ichihara City, Chiba 
Prefecture, central Honshu, Japan (Pleis- 
tocene) (Yokoyama, 1922; Higo et al., 1999). 

Material Examined 

1 lot (NSMT Mo 48820) from Jyogashima, 
Miura Peninsula, Japan, 73-85 m (3 right and 



THE BIVALVE GENUS PARVITHRACIA 



127 



TABLE 5. Parvithracia (Pseudoasthenothaerus) s/ren/(o/ Kamenev, new species. Shell measurements (mm), 
indices and summary statistics of all characteristics: L — shell length; H — height; W — width; A — anterior end 
length; S — maximal distance from the posterior shell margin to the top of palliai sinus; L1 — lithodesma 
length; N — number of pillars under the subumbonal plate. Numerator indicates shell measurements and 
indices for the right valve, denominator— for the left valve. NM — not measured. 



Statistics 



H W 



LI N H/L 



W/L 



A/L S/L L1/L Depository 





8.3 


7.3 


2.5 


5.5 


3.3 


1.3 


4 


0.876 


0.301 


0.663 


0.398 


0.155 


Holotype 
MIMB 




8.3 


7.2 


2.4 


5.3 


3.3 




4 


0.863 


0.289 


0.639 


0.398 


0.155 


4058 




8.0 


7.4 


2.4 


5.3 


3.3 


1.5 


5 


0.925 


0.300 


0.663 


0.413 


0.188 


Paratype 
MIMB 




8.0 


7.3 


2.3 


5.2 


3.3 




5 


0.913 


0.288 


0.650 


0.413 


0.188 


4060 




8.0 


7.7 


2.5 


5.0 


3.2 


1.8 


5 


0.963 


0.313 


0.625 


0.400 


0.225 


Paratype 
MIMB 




8.0 


7.4 


2.3 


5.0 


3.2 




NM 


0.925 


0.288 


0.625 


0.400 


0.225 


4060 




7.7 


7.3 


2.3 


4.8 


2.9 


1.5 


5 


0.948 


0.299 


0.623 


0.377 


0.195 


Paratype 
MIMB 




7.7 


7.2 


2.2 


4.8 


2.6 




4 


0.935 


0.286 


0.623 


0.338 


0.195 


4060 




7.4 


6.7 


2.0 


4.6 


3.1 


1.3 


4 


0.905 


0.270 


0.622 


0.419 


0.176 


Paratype 
MIMB 




7.4 


6.6 


2.0 


4.6 


2.8 




4 


0.892 


0.270 


0.622 


0.378 


0.176 


4060 




6.2 


5.3 


1.6 


3.9 


2.5 


1.0 


4 


0.855 


0.258 


0.629 


0.403 


0.161 


Paratype 
ZIN 2 




6.2 


5.3 


1.5 


3.9 


2.5 




4 


0.855 


0.242 


0.629 


0.403 


0.161 






5.4 


4.8 


1.3 


3.3 


2.3 


NM 


3 


0.889 


0.241 


0.611 


0.426 


NM 


Paratype 
ZIN 1 




5.4 


4.8 


1.3 


3.3 


2.2 




3 


0.889 


0.241 


0.611 


0.407 


NM 






5.4 


4.7 


1.3 


3.4 


2.3 


0.8 


2 


0.870 


0.241 


0.630 


0.426 


0.148 


Paratype 
MIMB 




5.4 


4.6 


1.3 


3.4 


2.3 




2 


0.852 


0.241 


0.630 


0.426 


0.148 


4059 




5.0 


4.3 


1.2 


3.2 


2.0 


0.8 


2 


0.860 


0.240 


0.640 


0.400 


0.160 


Paratype 
MIMB 




5.0 


4.2 


1.2 


3.2 


2.0 




2 


0.840 


0.240 


0.640 


0.400 


0.160 


4059 




4.5 


4.0 


1.1 


2.8 


0.9 


0.3 


3 


0.889 


0.244 


0.622 


0.200 


0.067 


Paratype 
ZIN 1 




4.5 


4.0 


1.1 


2.8 


0.9 




3 


0.889 


0.244 


0.622 


0.200 


0.067 






3.8 


3.6 


0.8 


2.2 


1.7 


0.6 


2 


0.947 


0.211 


0.579 


0.447 


0.158 


Paratype 
MIMB 




3.8 


3.6 


0.9 


2.2 


1.7 




2 


0.947 


0.237 


0.579 


0.447 


0.158 


4061 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


MIMB 




8.9 


8.0 


2.4 


5.2 


3.8 




6 


0.899 


0.270 


0.584 


0.427 


NM 


4063 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


MIMB 




8.2 


7.2 


2.4 


5.9 


3.1 




2 


0.878 


0.293 


0.720 


0.378 


NM 


4062 




6.6 


6.0 


1.8 


4.4 


2.5 


NM 


4 


0.909 


0.273 


0.667 


0.379 


NM 


MIMB 




NM 


NM 


NM 


NM 


NM 




NM 


NM 


NM 


NM 


NM 


NM 


4062 




5.8 


5.1 


1.4 


3.9 


2.1 


0.9 


3 


0.879 


0.241 


0.672 


0.362 


0.155 


ZIN3 




5.8 


5.1 


1.4 


3.9 


2.1 




3 


0.879 


0.241 


0.672 


0.362 


0.155 






3.8 


3.5 


0.8 


2.4 


1.5 


NM 


2 


0.921 


0.211 


0.632 


0.395 


NM 


MIMB 




NM 


NM 


NM 


NM 


NM 




NM 


NM 


NM 


NM 


NM 


NM 


4062 


Mean 


6.27 


5.68 


1.69 


3.95 


2.47 


1.13 


— 


0.904 


0.262 


0.630 


0.392 


0.168 






6.71 


5.99 


1.79 


4.22 


2.62 






0.890 


0.263 


0.628 


0.387 


0.168 




SD 


1.59 


1.53 


0.61 


1.04 


0.75 


0.48 


— 


0.034 


0.033 


0.030 


0.058 


0.041 






1.63 


1.51 


0.56 


1.07 


0.78 






0.031 


0.022 


0.037 


0.059 


0.041 




SE 


0.41 


0.39 


0.16 


0.27 


0.19 


0.14 


— 


0.009 


0.009 


0.008 


0.015 


0.012 






0.42 


0.39 


0.14 


0.28 


0.20 






0.008 


0.006 


0.010 


0.015 


0.012 




Min 


3.8 


3.5 


0.8 


2.2 


0.9 


0.30 


2 


0.855 


0.211 


0.568 


0.200 


0.067 






3.8 


3.6 


0.9 


2.2 


0.9 




2 


0.840 


0.237 


0.568 


0.200 


0.067 




Max 


8.3 


7.7 


2.5 


5.5 


3.5 


1.80 


5 


0.963 


0.313 


0.672 


0.447 


0.225 






8.9 


8.0 


2.4 


5.9 


3.8 




6 


0.947 


0.293 


0.720 


0.447 


0.225 




n 


15 


1_5 


15 


15 


15 


12 


15 


15 


15 


15 


15 


12 






15 


15 


15 


15 


15 




15 


15 


15 


15 


15 


12 





128 



KAMENEV 



140 



ISO" 



160" 



60 



Russia 



V 



Bering Sea 



c»o 



Sea of Okhotsk 



Sakhalin Is. 



/Kamchatka > 



50° 



China rviadivostok. 



Sea of Japan 




<T 



Iturup Is. 



Commander Islands 



50' 



^ Japa» >» \ Kunashirls. 



Pacific Ocean 



40 



150° 



160° 



FIG. 55. Distribution of Parvithracia (Pseudoasthenothaerus) sirenkoi (Ш type locality). 



TABLE 6. Results of comparison by pairs of mean values (Student (t) test) and variances (ANOVA) 
of indices of right and left valves of Parvithracia (Pseudoasthenothaerus) lukini and P. 
(Pseudoasthenothaerus) sirenl<oi: L — shell length; H — height; W — width; A — anterior end length; 
S — maximal distance from posterior margin to the top of palliai sinus; LI — lithodesma length. P.— 
probability that index values in P. (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) 
sirenkoi are drawn from the same population; n — number of valves of P. (Pseudoasthenothaerus) 
lukini and P. (Pseudoasthenothaerus) sirenkoi, respectively. 



Indices 




R 


ight valves 










Left valves 














ANOVA 












ANOVA 










t 


P 


F (1,26) 


P 


n 


t 


P 


F (1,25) 


P 


r 


^ 


H/L 


-3.59 


0.001 


9.93 


0.004 


19, 


15 


-3.09 


0.004 


9.13 


0.006 


21 


14 


W/L 


-3.16 


0.003 


11.36 


0.002 


19, 


15 


-2.09 


0.044 


18.48 


<0.001 


21 


15 


A/L 


2.16 


0.038 


6.74 


0.015 


19, 


15 


2.27 


0.030 


11.36 


0.002 


21 


15 


S/L 


3.82 


<0.001 


10.93 


0.003 


19, 


15 


2.81 


0.008 


12.86 


0.001 


21 


15 


L1/L 


0.13 


0.901 


0.02 


0.900 


16, 


12 


-0.76 


0.454 


<0.01 


0.989 


15, 


12 



THE BIVALVE GENUS PARVITHRACIA 



129 



TABLE 7. Results of discriminant analysis for the 
most significant characteristics for dividing all spec- 
imens into species of Parvithracia (Pseudoas- 
thenothaerus) lukini (17 spec.) and P. (Pseudo- 
asthenothaerus) sirenkoi (13 spec): L — shell 
length; W— width; A — anterior end length; S — 
maximal distance from the posterior shell margin to 
the top of palliai sinus. 

Significant Standard- 
charac- ized Wilks 

Valves teristics coefficient Lambda P 



Right 

Left 

Left 



S/L 
W/L 
A/L 



-0.7335 

0.5985 

-0.5970 



0.4930 0.015 
0.4331 0.003 
0.4228 0.021 



1 left valves); 1 lot (CAS 63354) from garni 
Bay, Honshu, Japan (2 right and 1 left valves). 
Total of 5 right and 2 left valves. 

Description 

Expanded from Yokoyama (1922)- Exte- 
rior: Shell small (<6.6 mm), elongate, ovate- 
elongate, moderately high (H/L = 0.656- 
0.776), slightly inequivalve (right valve slightly 



higher), moderately inflated (W/L = 0.212- 
0.283), inequilateral, thin, fragile, translucent, 
white. Surface with faint growth lines and very 
fine granules. Periostracum dull, thin, adher- 
ent, colorless. Beaks small, moderately pro- 
jecting above dorsal margin, posterior to mid- 
line (A/L = 0.618-0.655), slightly rounded, 
opisthogyrate. Anterior end narrowly rounded. 
Posterior end truncate, with a faint radial ridge 
extending from beaks to where posterior end 
meets ventral margin. Anterodorsal margin 
straight or slightly convex, smoothly descend- 
ing and smoothly transitioning to rounded an- 
terior end. Ventral margin slightly curved, 
sometimes straight in left valve. Posterodor- 
sal margin slightly concave, smoothly de- 
scending, forming a smooth angle with poste- 
rior end. Posterior end slightly curved, almost 
vertical, only slightly turned anteriorly to form 
a rounded angle where it meets ventral mar- 
gin. Escutcheon narrow, well expressed, de- 
marcated by ridges along posterodorsal mar- 
gin from beaks to posterior end. 

Interior: Right valve with anterior and pos- 
terior lateral teeth; left valve with anterior lat- 
eral tooth. In right valve, anterior lateral tooth 




FIGS. 56-64. Shells of Parvithracia species from Japan. 56-60. Parvithracia (Pseudoasthenothaerus) se- 
matana (Yokoyama, 1922) (NSMT Mo 48820), Jyogashima, Miura Peninsula, Japan, 73-85 m. 56, 57: Left 
valve, length 6.2 mm. 58, 59: Right valve, length 5.8 mm. 60: Subumbonal plate with pillars (ventral view), 
left valve (bar = 1 mm). 61 -64. Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964) (NSMT-Mo 
71463), Tosa Bay Japan, Pacific Ocean (33°05,6'N, 133°41,4'E), 800 m, shell length 7.6 mm. 



130 



KAMENEV 



large, triangular, projecting considerably 
above inner part of anterodorsal margin, an- 
teroventrally directed; posterior lateral tooth 
small, elongate, slightly projecting above 
inner part of posterodorsal margin, extending 
along posterodorsal margin. In left valve, an- 
terior lateral tooth long, lamellate, slightly pro- 
jecting above inner part of anterodorsal mar- 
gin; inner part of anterodorsal shell margin 
slightly concave between beak and tooth; 
inner part of posterodorsal margin slightly 
convex. Subumbonal plate short, slightly ele- 
vated above inner shell surface, almost com- 
pletely attached to shell wall, free along its an- 
teroventral margin. Pillars very short, 
sometimes lying on shell wall, but not sup- 
porting subumbonal plate. Number of pillars 
from 2 to 4. Anterior adductor muscle scar 
large, elongate, kidney-shaped. Posterior ad- 
ductor scar small, rounded. Pallia! sinus dis- 



tinct, of the same shape and size in both 
valves, deep, rounded anteriorly, not reaching 
to midline (S/L = 0.358-0.385), anterior limit 
short of faint vertical line from beaks. Shell in- 
terior with faint radial striae, noticeable along 
ventral shell margin. 

Variability 

Shell shape and proportions vary little 
(Table 8). Pillars supporting the subumbonal 
plate are sometimes difficult to see and count. 

Distribution and Habitat 

Parvithracia (Pseudoasthenothaerus) se- 
matana occurs off Japan: in the Pacific 
Ocean -in Otsushi Bay (Honshu) (Tsuchida & 
Kurozumi, 1996), near Boso Peninsula and 



TABLE 8. Parvithracia (Pseudoastiienothaerus) sematana (Yokoyama, 1922). Shell measurements 
(mm), indices and summary statistics of all characteristics: L — shell length; H — height; W — width; A — 
anterior end length; S — maximal distance from the posterior shell margin to the top of pallia! sinus; N — 
number of pillars under the subumbonal plate. Numerator indicates shell measurements and indices for 
the right valve, denominator — for the left valve. NM — not measured. 



Statistics 


L 


H 


W 


A 


S 


N 


H/L 


W/L 


A/L 


S/L 


Depository 
























Lectotype 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


CM 21507 




6.6 


4.8 


1.7 


NM 


NM 


NM 


0.727 


0.258 


NM 


NM 


Paralectotype 




6.1 


4.0 


1.3 


NM 


NM 


NM 


0.656 


0.213 


NM 


NM 


CM 21508 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 






NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NSMT Mo 48820 




6.2 


4.2 


1.5 


4.0 


2.3 


NM 


0.677 


0.242 


0.639 


0.371 






5.8 


4.5 


1.6 


3.8 


2.1 


NM 


0.776 


0.276 


0.655 


0.362 


NSMT Mo 48820 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 






5.5 


3.9 


1.4 


3.4 


2.1 


NM 


0.709 


0.255 


0.618 


0.382 


NSMT Mo 48820 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 






5.3 


3.9 


1.4 


3.4 


1.9 


NM 


0.736 


0.283 


0.642 


0.358 


NSMT Mo 48820 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 






5.2 


4.0 


1.1 


3.4 


2.0 


3 


0.769 


0.212 


0.654 


0.385 


CAS 63354 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 






5.2 


4.0 


1.1 


3.4 


2.0 


3 


0.769 


0.212 


0.654 


0.385 


CAS 63354 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 






NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


CAS 63354 




5.2 


3.9 


1.1 


3.4 


1.9 


3 


0.750 


0.212 


0.654 


0.365 




Mean 


5.52 


4.05 


1.33 


3.48 


2.02 


— 


0.736 


0.242 


0.645 


0.374 






6.00 


4.30 


1.43 


3.70 


2.10 




0.718 


0.237 


0.650 


0.368 




SD 


0.37 


0.23 


0.21 


0.18 


0.08 


— 


0.047 


0.034 


0.016 


0.013 






0.72 


0.46 


0.31 


0.42 


0.28 




0.037 


0.023 


0.006 


0.004 




SE 


0.15 


0.92 


0.08 


0.08 


0.04 


— 


0.019 


0.014 


0.007 


0.006 






0.42 


0.26 


0.18 


0.30 


0.20 




0.022 


0.013 


0.005 


0.003 




Min 


5.2 


3.9 


1.1 


3.4 


1.9 


3 


0.656 


0.212 


0.618 


0.358 






5.2 


3.9 


1.1 


3.4 


1.9 


3 


0.677 


0.212 


0.645 


0.365 




Max 


6.1 


4.5 


1.6 


3.8 


2.1 


3 


0.776 


0.283 


0.655 


0.385 






6.6 


4.8 


1.7 


4.0 


2.3 


3 


0.750 


0.258 


0.654 


0.371 




n 


6 


6 


6 


5 


5 


5 


6 


6 


5 


5 






3 


3 


3 


2 


2 


2 


3 


3 


2 


2 





THE BIVALVE GENUS PARVITHRACIA 



131 



southwards (Honshu) (Higo et al., 1999); in 
the Sea of Japan — near Oga Peninsula, off 
Honshu (Ito, 1989; Higo et al., 1999); in the 
Yellow Sea -off Kyushu (Higo et al., 1999). 
This species was recorded at a depth from 20 
m to 300 m, on the fine sand (Tsuchida & 
Kurozumi, 1996; Habe, 1977; Higo et al., 
1999). 

Comparisons 



attached to the shell wall and indistinct, short 
pillars (Table 4). 

Parvithracia (Pseudoasthenothaerus) 

isaotakii (Okutani, 1 964) 

Figs. 61 -64, Table 9 

Asthenothaerus isaotakii Okutani, 1964: 
84, 85, text fig. 6; Habe, 1 977: 31 2; Higo et al., 
1999: 524; Okutani, 2000: 1039, pl. 517, fig. 6 



This species is easily distinguished from 
other species of the subgenus in having a 
small, elongate, very thin translucent shell 
with the subumbonal plate almost completely 



Type Material and Locality 

Holotype (shell length 7.9 mm), Sagami 
Bay, Japan (35°05.35N, 139°18.65E), 550 m. 








H 



FIG. 65. The hinge and subumbonal plate of Parvithracia species from Japan by camera lucida. A, B. 
Parvittiracia (Pseudoastfienottiaerus) sematana (Yokoyama, 1922) (NSMT Mo 48820). A: Hinge of right 
valve. B: Hinge of left valve. C-F Parvittiracia (Pseudoastfienotfiaerus) isaotakii (Okutani, 1964) (NSMT-Mo 
71463). C: Hinge of right valve. D: Hinge of the left valve (inner part of anterodorsal shell margin broken). E: 
Close-up of right valve showing attachment of lithodesma (ventral view) to subumbonal plate. F: Subumbonal 
plate with internal ligament and pillars (ventral view), left valve. Bar = 1 mm. 



132 



KAMENEV 



TABLE 9. Parvithracia (Pseudoasthenothaerus) isaotaki i {OWuXani, 1964). Shell measurements (mm) and 
indices: L — shell length; H — height; W — width; A — anterior end length; S — maximal distance from the pos- 
terior shell margin to the top of palliai sinus; LI — lithodesma length; N — number of pillars under the subum- 
bonal plate. Numerator indicates shell measurements and indices for the right valve, denominator— for the 
left valve. ?— ventral margin of the left valve is partly broken. NM — not measured. 



L 


H 


w 


A 


S 


LI 


N 


H/L 


W/L 


A/L 


S/L 


L1/L 


Depository 


13 
7.5 


6^ 

6.1? 


1.6 

NM 


3.8 

4.0 


3.7 
3.7 


1.0 


6 
6 


0.855 
0.813? 


0.211 
NM 


0.500 
0.533 


0.487 
0.493 


0.132 
0.133 


NSMT 
Mo 71463 



Coll. T. Okutani, 27-IX-1960 (RA/ "Soyo- 
Maru") (Okutani, 1964). 

Material Examined 

1 lot (NSMT-Mo 71463) from Tosa Bay, 
Japan (33°05.6'N, 133°41.4'E), 800 m, 12- 
IX-1997 (RA/ "Kotaka-Maru") (1 spec). 



Description 

Expanded from Okutani (1964)- Exterior: 
Shell small (<7.9 mm), subovate, high (H/L = 
0.855), slightly inequivalve (right valve slightly 
longer and higher), moderately inflated (W/L = 
0.211), thin, fragile, ashy white under perio- 
stracum. Surface rough, with conspicuous 
growth lines and very fine granules. Perio- 
stracum dull, thin, adherent, sometimes peel- 
ing near beaks, dark grayish, sometimes with 
dark brown or black patches or crusts near 
beaks and dorsal shell margin. Beaks small, 
moderately projecting above dorsal margin, 
central or slightly posterior to midline (/V/L = 
0.5-0.533), slightly rounded, orthogyrate. An- 
terior end sharply rounded. Posterior end ex- 
panded, vertical, higher than anterior, decid- 
edly truncate, with a faint radial ridge 
extending from beaks to junction of posterior 
end with ventral margin. Anterodorsal margin 
straight, steeply descending, smoothly transi- 
tioning to rounded anterior end. Ventral mar- 
gin slightly curved. Posterodorsal margin 
straight, smoothly descending, forming a 
smooth, obtuse angle with posterior end. Pos- 
terior end straight or slightly curved, almost 
vertical, only slightly turned anteriorly to form 
a rounded angle where it meets ventral mar- 
gin. Escutcheon very narrow, well developed, 
demarcated by ridges extending along pos- 
terodorsal margin from beaks to posterior 
end. 

Interior: Right valve with anterior and pos- 
terior lateral teeth; left valve with anterior lat- 
eral tooth. In right valve, anterior lateral tooth 



large, tnangular, projecting considerably 
above inner part of anterodorsal margin, ven- 
trally directed; posterior lateral tooth small, 
elongate, slightly projecting above inner part 
of posterodorsal margin, extending along pos- 
terodorsal margin. In left valve, anterior lateral 
tooth long, lamellate, considerably projecting 
above inner part of anterodorsal margin; inner 
part of anterodorsal margin straight between 
beak and tooth; inner part of posterodorsal 
margin slightly convex. Subumbonal plate 
short, elevated above inner surface. Pillars 
supporting plate short, wide, partly fused, 
considerably tapering ventrally. Number of pil- 
lars in both valves 6. Lithodesma large (L1/L = 
0.1 33), curved. Anterior adductor muscle scar 
large, elongate, kidney-shaped. Posterior ad- 
ductor scar small, rounded. Palliai sinus dis- 
tinct, of the same shape and size in both 
valves, deep, narrow and rounded anteriorly, 
reaching midline (S/L = 0.487-0.493). Shell 
interior polished, with faint radial striae. 



Distribution and Habitat 

Parvithracia (Pseudoasthenothaerus) isao- 
takii occurs on the Pacific coast of Japan: 
Tosa Bay (33° 05.6'N, 133°41.4'E), 800 m; 
Sagami Bay (35°05.35'N, 139°18.65'E; 
35°09.9'N, 139°30.4'E), 550-700 m (Oku- 
tani, 1964); Sea of Enshu-Nada (34^25. 7'N, 
137°58.5'E), 620 m (Okutani, 1964). 



Comparisons 

In contrast to other species of the subgenus, 
P. (Pseudoasthenothaerus) isaotakii has a 
broader posterior end, and the beak is almost 
central and orthogyrate (Table 4). This species 
also differs from P. (Pseudoasthenothaerus) 
lukini and P. (Pseudoasthenothaerus) sirenkoi 
in having a thin, fragile shell, and a curved lith- 
odesma that is not butterfly-shaped. Parvithra- 
cia (Pseudoasthenothaerus) isaotakii is dis- 
tinguished from P. (Pseudoasthenothaerus) 



THE BIVALVE GENUS PARVITHRACIA 



133 



sematana, which also has a thin and fragile 
shell, in having a highly elevated subumbonal 
plate, with distinct, high pillars. 



ACKNOWLEDGMENTS 

I am very grateful to Dr. V. A. Nadtochy 
(PRIFO, Vladivostok) and Mrs. N. V. 
Kameneva (1MB, Vladivostok) for great help 
during work on this manuscript; to Drs. V. B. 
Durkina, L. N. Usheva, M. A. Vaschenko, and 
I. G. Syasina (1MB, Vladivostok) for consulta- 
tions and help during study of anatomy of the 
new species; to Dr. H. Saito (NSMT, Tokyo), 
Ms. R. N. Germon (USNM, Washington), Dr. 
N. R. Foster (UAM, Fairbanks), Mr. R. Asher 
(CIN, Cawtron) and Dr. K. A. Lutaenko (1MB, 
Vladivostok) for providing at my disposal 
specimens of different species of Thraciidae 
genera; to Drs. B. I. Sirenko, A. V. Martynov 
and all collaborators of the Marine Research 
Laboratory (ZIN, St.-Petersburg) for help dur- 
ing work with collection of bivalve molluscs at 
the ZIN; to Dr. B. A. Marshall (MNZ, Welling- 
ton) for consultations and sending material of 
the genus Parvithracia and reprints of papers; 
to Dr. E. V. Coan (Department of Invertebrate 
Zoology, CAS, San Francisco) for consulta- 
tions, comments on the manuscript, and 
sending reprints of papers; to Drs. T 
Kurozumi and E. Tsuchida (NHMI, Chiba), Dr. 
K. Amano (Joetsu University of Education, 
Joetsu) for sending reprints of papers; to Mr. 
E. V. Jakush (PRIFO, Vladivostok) and D. V. 
Fomin (1MB, Vladivostok) for help in work with 
the scanning microscope; to Mr. A. A. 
Omelyanenko (1MB, Vladivostok) for making 
photographs; to Ms. T N. Kaznova (1MB, 
Vladivostok) for translating the manuscript 
into English; Dr. George M. Davis for help in 
the publication of the manuscript; two anony- 
mous reviewers for comments on the manu- 
script. 

This research was partly supported by 
Grants 98-04-48279, 01-04-48010, and 00- 
15-97890 from the Russian Foundation for 
Basic Research. 



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KEEN, A. M., 1969, Family Thraciidae. Pp. 
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Revised ms accepted 8 July 2001 



MALACOLOGIA, 2002, 44(1): 135-143 

LOCALIZATION OF NADPH-DIAPHORASE-POSITIVE ELEMENTS 

IN THE INTESTINE OF THE MUSSEL CRENOMYTILUS GRAYANUS 

(MOLLUSCA: BIVALVIA) 

Anatoly A. Varaksin^*, Eugenia A. Pimenova^, Galina S. Varaksina^ & Lydia T. Frolova^ 

ABSTRACT 

By NADPH-diaphorase histochemistry (Hope & Vincent, 1989), localization of nicotinamide 
adenine dinucleotide phosphate diaphorase-positive (NADPH-d-positive) elements was studied 
in the direct and recurrent loops of the midgut and hindgut of the mussel Crenomytilus grayanus. 
Intraepithelial NADPH-d-positive cells were found in the intestinal groove and major typhlosole 
of the direct loop and in the recurrent loop of the midgut, as well as in the hindgut. The cells are 
fusiform and lie separately or in minor groups. Their apical processes are directed to the gut 
lumen, and the basal processes contact with the basiepithelial plexus. The latter, in turn, sends 
separate NADPH-d-positive fibers to make contacts with subepithelial plexus. Both plexuses are 
well developed in the intestinal groove and major typhlosole of the direct loop and in the recur- 
rent loop of the midgut, as well as in the hindgut. In the minor typhlosole and crystalline style sac, 
both plexuses are less developed. In the ciliary groove of the hindgut, the basiepithelial plexus 
is absent. Possible role of NADPH-d-positive elements in regulation of the digestion in bivalve 
mollusks is discussed. 

Key words: histochemistry, NADPH-diaphorase, basiepithelial plexus, subepithelial plexus, in- 
testine, Crenomytilus grayanus (Dunker), Mollusca. 



INTRODUCTION 

Nitric oxide (NO) acts as a transcellular 
messenger in the mamnnalian nervous sys- 
tem (Garthwaite & Boulton, 1995; Rand & Li, 
1995). NO, together with citrulline, is pro- 
duced by NO-synthase from L-arginine. For 
the reaction to proceed normally, O2, Ca^^, 
and nicotinamide adenine dinucleotide phos- 
phate (NADPH) are required (Bredt & Snyder, 
1990). In the course of the histochemical re- 
action, exogenous nitro blue tetrazolium is re- 
duced by NO-synthase to diformazan precipi- 
tate, which thereby marks cells containing the 
enzyme. At present, of all NADPH-dependent 
oxidoreductases, NADPH-diaphorase is only 
considered to retain activity after para- 
formaldehyde fixation (Matsumoto et al., 
1993). Histochemical activity of NADPH-di- 
aphorase is thus believed to be a topographi- 
cal marker of NO-synthase-containing struc- 
tures in tissues of both vertebrates (Hope et 
al., 1991; Dawson et al., 1991) and inverte- 
brates (Moroz & Gillette, 1995, 1996; Moroz 
etal., 1996). 

To date, NO-ergic element localization and 
functional role in the mammalian alimentary 



tract have been intensively studied (Schleiffer 
& Raul, 1997; Ruber et al., 1998). Among 
lower vertebrates, NO-synthase-containing 
neurons were found in the alimentary tract of 
the toad Bufo marinas (Li et al., 1992, 1993; 
Murphy et al., 1993); an agama lizard. Адата 
sp. (Knight & Burnstock, 1999); the rainbow 
trout. Salmo gairdneri (Li & Furness, 1993); 
the Atlantic cod, Gadus morhua; and the spiny 
dogfish, Squalus acanthias (Olsson & Karila, 
1 995). At the same time, NO-ergic element lo- 
calization and functional role in the inverte- 
brate digestive system are poorly studied. 
These elements are revealed in the cardiac 
stomach of the starfishes Marthasterias 
glacialis (Martinez et al., 1994) and Asterias 
rubens (Elphick & Melarange, 1998), and in 
the oesophagus of the opisthobranch Pleuro- 
branchaea californica (Moroz & Gillette, 
1996). To date, no data have been present on 
NO-ergic elements in the alimentary tract of 
bivalve mollusks. 

The present work focused on NADPH-d- 
positive element localization in the direct and 
recurrent loops of the midgut, and in the 
hindgut of the mussel Crenomytilus grayanus 
(Dunker). 



Institute of Marine Biology, Russian Academy of Sciences, Vladivostok 690041, Russia; inmarbio@mail.primorie.ru 
^Far East State University, Vladivostol< 690600, Russia 
"corresponding author 

135 



136 



VARAKSIN ETAL. 



MATERIALS & METHODS 

Samples were obtained from five adult 
mussels with shells 10.5-11.0 cm high and 
4.5-5.0 cm wide. Mollusks were collected in 
Amurskii Bay, Sea of Japan, in November 
1998 and kept in aerated aquaria for 2-3 
days. During that time, the mollusks were not 
fed. 

NADPH-d-positive elements in the intestine 
were revealed by a NADPH-diaphorase histo- 
chemistry method (Hope & Vincent, 1989). 
Fragments of the direct and recurrent midgut 
loops and of the hindgut, shown in Figure 1, 
were fixed in 4% paraformaldehyde in 0.1 M 
sodium phosphate buffer (pH 7.2) for 2 h at 
4°C. They were then rinsed in 3-4 changes of 
15% sucrose prepared in 0.05 M Tris-HCI 
buffer (pH 8.0) for 24 h at the same tempera- 
ture. Both transverse and longitudinal, 15 |im 
thick, cryostat sections were prepared and 
placed on to slides. 

NADPH-Diaphorase Histochemistry 

Sections were incubated in a medium con- 
taining 0.5 mM ß-NADPH, 0.5 mM nitro blue 
tetrazolium, and 0.3% Triton X-100 in 0.15 M 
Tris-HCI buffer (pH 8.0) for one h at 37°C. 
Then, they were rapidly washed twice in 
distilled water, dehydrated in an ethanol se- 
ries of increasing concentrations, and 
mounted in Dammar Resin. Control sections 



were incubated in the following media: (1) 
without ß-NADPH, (2) without nitro blue tetra- 
zolium. 



Drugs Used 

Nitro blue tetrazolium, ß-NADPH were sup- 
plied by Sigma Chemical Co, USA. 



Statistical Analysis 

Sections were observed and photographed 
using Olympus BH2-RFCA microscope (BHS 
model). NADPH-d-positive cells were drawn 
by means of a Carl Zeiss drawing tube at a 
total magnification of x400. Cell height and 
width were measured using a plotting scale. 
For each of the intestine parts studied, a total 
number of NADPH-d-positive cells was 
counted in ten transverse sections, and a 
mean and standard deviation per section was 
calculated. Statistical significance was tested 
by paired t-tests at the 0.05 confidence level. 



RESULTS 
Direct Loop of the Midgut 

The direct loop of the midgut consists of: 
the intestinal groove, the major and minor ty- 



hg rl 




FIG. 1 . Anatomy of the digestive system of Crenomytilus grayanus. Abbreviations: aa, anterior adductor; dg, 
digestive gland; dl, direct loop of the midgut; gi, gills; h, heart; hg, hindgut; ml, mouth lobes; oe, oesophagus; 
pa, posterior adductor; rl, recurrent loop of the midgut; s, stomach. Scale bar, 10 mm. 



NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 



137 



CSS 




FIG. 2. Schematic localization of NADPH-d-positive elements in the intestine of Crenomytilus grayanus. A. 
Direct loop of the midgut; B. Recurrent loop of the midgut; С Hindgut. Abbreviations: bep, basiepithelial 
NADPH-d-positive plexus; eg, ciliary groove; cs, crystalline style; ess, crystalline style sac; ct, connective tis- 
sues with separate muscular ceils; e, epithelium; gl, gut lumen; ic, intraepithelial NADPH-d-positive cells; ig, 
intestinal groove; sep, subepithelial NADPH-d-positive plexus; mat, major typhlosole; mit, minor typhlosole; 
rt, the region of the typhlosole, adjacent to the crystalline style sac. Scale bar, 1 mm. 



phlosoles, and the crystalline style sac (Fig. 
2A). Annong these structures, a considerable 
difference in intensity and pattern of NADPH- 
d-positive elements staining was observed. 

In the intestine groove wall, fairly developed 
basi- and subepithelial NADPH-d-positive 



plexuses were observed (Fig. ЗА). The 
basiepithelial plexus was dense and inten- 
sively stained, with the maximum thickness of 
13.7 ± 4.6 ¡am. In the intestine groove epithe- 
lium, there occur NADPH-d-positive cells av- 
eraging 2.6 ± 1.9 per section (Table 1). The 



138 



VARAKSIN ETAL 



gl 




v: v, 



^Щ. 



i л 



А 



Qm 






^'^ 

* 


е 






*. 


-С^г -->.' 


,Ьер^^^^ 


с 









/ 



D 



■i 






FIG. 3. NADPH-d-positive element localization in the direct loop of Crenomytilus grayanus midgut. A. In- 
testinal groove. B. Major typhlosole. С Crystalline style sac. D. Intraepithelial NADPH-d-positive cell in the 
major typhlosole. Abbreviations: f , NADPH-d-positive fibers in the connective tissue of the intestinal groove; 
for other abbreviations, see caption to Fig. 2. Scale bar, A, C, D, 100 цт; В, 150 цт. 



cells are located rnainly in the basal zone of 
the epithelium; they are fusiform, 10.9 ± 2.0 
|im high and 3.6 ± 0.9 |im wide. The subep- 
ithelial plexus represents a dense, thick (30.1 
± 8.2 |im), interlacement of NADPH-d-posi- 
tive fibers (Fig. ЗА). Both plexuses are con- 
nected to each other with separate thin fiber 



bundles. Occasional large, varicose fibers 
branch off from the subepithelial plexus to 
submerge to the adjacent connective tissue 
(Fig. ЗА). 

In the major typhlosole, the basiepithelial 
plexus 6.7 ± 2.8 fim thick has the appearance 
of a rather sparse, slightly branched interlace- 



NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 



139 



TABLE 1 . The average proportion of NADPH-d-positive cells to the total number of 
epithelial cells per section in the Crenomytilus grayanus intestine. 



Intestine region 



Direct loop of the midgut 

Intestine groove 

Major typhlosole 

Minor typhlosole 

Crystalline style sac 
Recurrent loop of the midgut 
Hindgut 



Total 


NADPH-d- 


NADPH-d- 


epithelial 


positive cell 


positive cell 


cell number 


number 


proportion (%) 


509 ± 152 


2.6 ± 1.9 


0.51 


1349 ± 457 


17.3 ± 10.4 


1.28 


1109 ±295 


— 


— 


585 ± 154 


— 


— 


1810 ±452 


15.1 ±4.8 


0.83 


1474 ± 244 


13.3 ±6.1 


0.92 



Values represent the mean ± the standard error of the mean. 



ment of fibers bearing varicosities (Fig. 3B). It 
is more prominent in the region of the ty- 
phlosole, adjacent to the crystalline style sac. 
In the major typhlosole epithelium, NADPH-d- 
positive cells also occur averaging 17.3 ± 
10.4 per section (Table 1). The cells are 
fusiform and lie diffusely, close to the gut 
lumen (Fig. SB). They are 14.9 ± 2.9 |im high 
and 5.7 ± 1.7 iim wide (Fig. 3D). In the minor 
typhlosole, the basiepithelial NADPH-d-posi- 
tive plexus is formed by thin fibers, most of 
which are longitudinally oriented. In the region 
of the minor typhlosole, adjacent to the crys- 
talline style sac, at the epithelium base, only 
rare thin fibers occurred, no NADPH-d-posi- 
tive cells being observed. In both typhlosoles, 
the subepithelial plexus represents an inter- 
lacement of weakly branched NADPH-d-pos- 
itive fibers bearing varicosities. The subep- 
ithelial layers of the major and minor 
typhlosoles are, respectively, 31.9 ± 10.7|im 
and 12.0 ± 7.2 |im thick. In the adjacent con- 
nective tissue, occasional NADPH-d-positive 
fibers occur. 

In the area of the crystalline style sac, the 
basiepithelial plexus represents a thin (4.2 ± 
1 .4 |im) interlacement of fibers bearing vari- 
cosities (Fig. 3C). In the subepithelial plexus, 
which is 5.2 ± 1.3 |im thick, the longitudinal 
orientation of NADPH-d-positive fibers pre- 
vails. In the adjacent connective tissue, occa- 
sional fibers beaded with varicosities occur. 



^ 



- 1 




fsepi^'^ • 




FIG. 4. NADPH-d-positive element localization in 
the recurrent loop of Crenomytilus grayanus 
midgut. Abbreviations are the same as in Fig. 2. 
Scale bar, 70 цт. 



Recurrent Loop of the Midgut 

In the recurrent loop of the midgut, NADPH- 
d-positive elements were found in the basal 
zone of the epithelium and in the underlying 
connective tissue (Fig. 28). The basiepithelial 



NADPH-d-positive plexus represents a 
dense, intensively stained nerve fiber inter- 
lacement 23.7 ± 4.7 |im thick (Fig. 4). In the 
epithelium of the loop occur NADPH-d-posi- 
tive cells averaging 15.1 ± 4.8 per section 
(Table 1). These lie separately or in groups of 



140 



VARAKSIN ETAL. 



2-3 cells (Fig. 4). The cells are fusiform, 17.3 
± 2.8 |im high, and 7.9 ± 1.3 цт wide. The 
subepithelial plexus is 43.2 ± 5.9 цт thick. It 
is formed by densely interlacing NADPH-d- 
positive fibers (Fig. 4). Occasional fibers bear- 
ing varicosities leave the plexus to enter the 
adjacent connective tissue. 

Hindgut 

In the hindgut, NADPH-d-positive elements 
were found in both layers (Fig. 2C). The 
basiepithelial plexus represents a dense, in- 
tensively stained NADPH-d-positive fibers in- 
terlacement 14.3 ± 3.7 |im thick. (Fig. 5A-D). 
The only exception is the ciliary groove re- 
gion, which lacks a basiepithelial plexus (Fig. 
5D). In the basal zone of the epithelium, sin- 
gle fusiform NADPH-d-positive cells are re- 
vealed (Fig. 5B, C) averaging 13.3 ± 6.1 per 
section (Table 1). These are 17.9 ± 4.6 |im 
high and 5.7 r 0.9 |im wide. The celts pos- 
sess apical processes directed to the gut 
lumen, their basal processes contacting the 
basiepithelial plexus (Fig. 5B, C). 

The subepithelial plexus is formed by an in- 
terlacement of NADPH-d-positive fibers, 
which are mainly longitudinally oriented (Fig. 
5A, D). Its thickness reaches 27.2 ± 11 .2 |im 
and 1 06.3 ± 1 1 .7 цт at the concave and con- 
vex sides of the gut, respectively. At the con- 
vex side of the gut, the plexus is denser than 
at the concave side. 

Neither control test revealed NADPH-d- 
positive elements in any of the С grayanus 
gut parts studied. 



DISCUSSION 

We revealed NADPH-d-positive cells lo- 
cated in the epithelium of the direct and re- 
current loops of the midgut, as well as in the 
hindgut of the mussel С grayanus. In many 
invertebrate groups, specialized cells were 
found in the epithelial lining of the alimentary 
tract that are not directly involved in the 
process of digestion and seemingly serve reg- 
ulatory functions (Punin, 2000). In bivalve 
mollusks, the system regulating gut function- 
ing includes both nerve and endocrine cells. 
By using methylene blue staining, nerve cells 
were revealed in the Anodonta cellensis 
midgut epithelium (Gilev, 1952). Electron mi- 
croscopy studies of Árctica islándica and 
Mytilus edulis gut epithelium revealed intraep- 
ithelial cells with a fairly developed rough en- 



doplasmic reticulum, the Golgi apparatus of 
dictyosomic type, numerous granules, and 
first-order processes. The latter formed pro- 
nounced basiepithelial plexus. Based on their 
morphology, these cells were considered 
nerve cells (Punin, 1981, 1989). At the same 
time, endocrine cells in the gut epithelium 
of bivalve mollusks are characterized by 
larger cytoplasmic granules and the lack of 
processes (Punin, 2000). From the above 
facts, we believe that intraepithelial NADPH- 
d-positive cells in the С grayanus gut are of 
nerve type. 

We revealed a dense, intensively stained 
plexus of NADPH-d-positive fibers in the 
basal zone of the mussel intestinal epithelium. 
A plexus of similar or even greater density and 
complexity was found in the subepithelial 
zone of the direct and recurrent midgut loops 
and of the hindgut. 

The bivalve alimentary tract is known to 
possess complicated systems comprised of 
the basi- and subepithelial nerve plexuses 
(Giusti, 1970; Punin, 1981, 1989). Based on 
electron microscopy data, both zones were 
shown to be to a certain degree autonomous 
(Punin & Konstantinova, 1988; Punin, 1989). 
The subepithelial plexus is assumed to be a 
peripheral component of the molluscan ner- 
vous system (Punin & Konstantinova, 1988). 

The basiepithelial plexus was earlier shown 
to consist of the processes of intraepithelial 
nerve cells (Punin, 1989). Nerve elements of 
the subepithelial plexus are also presumed to 
contribute to basiepithelial plexus formation. 
By light microscope studies of M. edulis 
(Punin & Konstantinova, 1988) and of A. is- 
lándica (Punin, 1981), processes were found 
that penetrated into the neighbouring connec- 
tive tissue. Terminals of nerve processes can 
penetrate into the basal zone of the intestine 
epithelium from the surrounding connective 
tissue, as in the case in the Tapes waltingii 
hindgut (Dougan & McLean, 1970) and in the 
M. galloprovincialis midgut (Giusti, 1970). We 
repeatedly found processes connecting the 
basi- and subepithelial plexuses. This sug- 
gests that both plexuses were structurally and 
functionally integrated, and the putatively N0- 
ergic system of the C. grayanus intestine pos- 
sess a complex multilevel structure. 

It is worth noting that the basiepithelial 
plexus is relatively well developed in all parts 
of the gut. However, it contains few intraep- 
ithelial cells of putative NO-ergic nature. Nu- 
merous NO-ergic fibers were revealed in the 
neuropils of the С grayanus gangWa, whereas 



NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 



141 






'"^^'é \kí ''^i3k ^V^ 

• \^ Vi' ' 



bep ... 






'л.' ». M 




gi. 



.^•í^^ 




'4S'.' 






D 



cg . 



s 



et 



FIG. 5. NADPH-d-positive element localization in the hindgut of Crenomytilus grayanus. A. Convex side of 
the gut. Scale bar, 150 цт. В, С. Intraepithelial NADPH-d-positive cells. Scale bar, 50|am. D. Concave side 
of the gut. Scale bar, 250 цт. The solid arrow shows the apical process; the dashed arrow, the basal process; 
for other abbreviations, see caption to Fig. 2. 



the nunnber of specifically stained perlkarya 
was relatively small there (Annlkova et al., 
2000). A sinnilar pattern of NO-positive stain- 
ing was observed in other gastropod and bi- 
valve mollusks and in echinoderms (Moroz & 
Gillette, 1996; Dyuizen et al., 1999). It is pro- 
posed that С grayanus NO-ergic neurons are 
not only capable of NO synthesis in the cell 
body but also possess a systenn of local nitric 
oxide synthesis within the processes (An- 



nikova et al., 2000). The presence of numer- 
ous, putatively NO-ergic fibers bearing vari- 
cosities in the С grayanus intestine supports 
this proposal. 

Thus, in the wall of the С grayanus intes- 
tine, there exists an integrated system com- 
prised of intraepithelial, putatively NO-ergic 
nerve cells and of basi- and subepithelial 
nerve plexuses, too, of putative NO-ergic na- 
ture. 



142 



VARAKSIN ETAL. 



To date, the role of NO in regulation of in- 
vertebrate digestion is poorly studied. It is 
shown that in the oesophagus of Lymnaea 
stagnalis, nitric oxide is capable of functioning 
as a neuromodulator (Elphick et al., 1994; 
Moroz et al., 1993). NO is capable of modu- 
lating the frequency of epitheliocyte cilia beat- 
ing, for instance, in the ciliary epithelium of the 
mammalian respiratory system (Jain et al., 
1993). In С. grayanus. NO-synthase activity is 
found to be high in the basiepithelial plexus of 
the intestine, the fibers of which establish di- 
rect contacts with ciliated epitheliocytes. We 
suppose that the NO role in controlling ep- 
itheliocyte locomotory activity consists in 
modulating the transport of water with food 
masses kept by epitheliocyte ciliary streaming 
and the transport of fecal masses through the 
intestine. However, in the epithelium of the 
crystalline style sac, where a highly intensive 
cilia beating is maintained, basiepithelial 
plexus is unexpectedly ordinary developed. 
Obviously, NO does not play a principal role in 
regulating ciliary activity of epitheliocytes in 
the С grayanus intestine. 

In the intestine of bivalve mollusks, epithe- 
liocytes secrete mucus, digestive enzymes, 
and protein-like substances of the crystalline 
style matrix (Owen, 1966; Reid, 1966; Giusti, 
1970). The secretory activity is especially 
prominent in epitheliocytes of the region of the 
major typhlosole, adjacent to the crystalline 
style sac (Frolova, 1989). In this segment, a 
lot of putatively NO-ergic cells (Table 1) were 
found and could be related to prominent basi- 
and subepithelial plexuses. On the contrary, 
putatively NO-ergic cells are absent, and 
basi- and subepithelial putatively NO-ergic 
plexuses are poorly developed in the minor ty- 
phlosole and crystalline style sac, which are 
characterized by a low secretory activity. 
Then, NO is likely to control epitheliocyte se- 
cretory activity in the mollusk intestine. 



ACKNOWLEDGMENT 

The research described in this publication 
was made possible in part by Award No. REC- 
003 of the U. S. Civilian Research & Develop- 
ment Foundation for the Independent States 
of the Former Soviet Union (CRDF). 



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Revised ms. accepted 30 May 2001 



MALACOLOGIA, 2002, 44(1): 145-151 

ORIGIN OF THE EXCRETORY CELLS IN THE DIGESTIVE GLAND OF THE LAND 

SNAIL HELIX LUCORUM 

Vasilis K. Dimithadis & Vasiliki Konstantinidou 

Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of 
Sciences, Arisrotle University Of Thessaloniki, Thessalonil<i 54006, Greece; 

vdimitr @ bio.auth.gr 

ABSTRACT 

The examination of the digestive gland cells of the land snail He/Zx lucorum with light and elec- 
tron microscopes after starvation and subsequent periods of feeding supports the hypothesis 
that the so-called excretory cells are digestive cells in a final step of their cell cycle, rather than 
a distinct cell type. Results supporting this hypothesis are: (1) the fact that the so-called diges- 
tive cells containing heterolysosomes and residual bodies or the so-called excretory cells con- 
taining excretory vacuoles were not found in significant numbers in the digestive gland epithe- 
lium at the same time; (2) the fact that the decrease in the number and size of the 
heterolysosomes implicated in the endocytotic uptake and digestion of nutrients was combined 
with an increase in the number and size of the excretory vacuoles responsible for the excretory 
processes, and vice versa; and (3) the fact that starvation caused the appearance of a large 
number of excretory vacuoles and the reduction of the heterolysosomes in the digestive gland 
epithelium to a minimum. 



INTRODUCTION 

The digestive gland (or midgut gland, or he- 
patopancreas or liver) is the largest organ in 
the body of terrestrial gastropods. It consists 
of two lobes communicating with the stomach 
via large ducts, which branch to form smaller 
ducts, ductules, and complex branched blind 
tubules. It is an organ dealing with elaboration 
of enzymes, absorption of nutrients, endocy- 
tosis of food substances, food storage, and 
excretion (Runham, 1975). 

In spite of the numerous studies on the 
morphology and physiology of the digestive 
gland cells, there are conflicting reports about 
the cell types that constitute the digestive 
gland epithelium of terrestrial gastropods. 
Even at the ultrastructural level, undoubted in- 
terpretation of the results is still difficult be- 
cause of the changes in the fine structure as- 
sociated with phases of activity. Most of the 
findings are consistent with the hypothesis 
that the digestive gland epithelium is com- 
posed of three cell types: digestive cells, cal- 
cium cells, and excretory cells (Fig. 1), sur- 
rounded by connective tissues, muscular 
layers, and haemocoelic spaces (Sumner, 
1 965; Dimithadis & Hondros, 1 992). The pres- 
ence of a fourth type, the thin cells, is docu- 
mented in many instances, but these cells 



are regarded as undifferentiated precursors of 
the other cell types (Sumner, 1965; Walker, 
1970). 

Digestive cells are the most frequent cell 
type found in the digestive gland of snails and 
slugs. They are usually columnar in shape, 
varied in size, and usually display microvilli. 
Digestive cells are characterized by numer- 
ous granules usually located in the apical por- 
tion of the cells consisting of secondary lyso- 
somes, the heterolysosomes, as well as by 
vacuoles with dense cores consisting residual 
bodies, named green and yellow granules, re- 
spectively, after light microscopic observa- 
tions in Helix and Agriolimax {Sumner, 1965; 
Walker, 1970, 1972), or apical granules and 
cisternae with electron-dense cores after 
electron microscopic observations in Helix 
(Dimithadis & Hondros, 1992; Dimithadis & 
Liosi, 1992). The second cell type of the di- 
gestive gland, the calcium cells, are charac- 
terized by their pyramidal shape and the pres- 
ence of calcium granules (Sumner, 1965; 
Dimithadis & Hondros, 1992). The third cell 
type, the excretory cells are larger in size than 
the digestive cells and are charactehzed by 
one or more large excretory vacuoles con- 
taining cores or amorphous mass of high 
electron density (Sumner, 1965; Dimithadis & 
Hondros, 1992). 



145 



146 



DIMITRIADIS & KONSTANTIN I DOU 




FIG. 1. Drawing of the three cell type regarded by 
many authors that form the digestive gland epithe- 
lium of terrestrial gastropods, based on the original 
in Dimitriadis & Hondros (1992). Abbreviations: CC, 
calcium cell: DC, digestive cell: EC, excretory cell: 
Ev, excretory vacuole: HI, heterolysosome; N, nu- 
cleus; Rb, residual body. 



In spite of the view that the excretory cells 
are a separate cell type, there are also results 
supporting the view that these cells are either 
digestive cells differentiated towards excre- 
tory cells or cells of the final developmental 
stage of the digestive cells. The present study 
was designed to answer the questions related 
to the structure and function of the excretory 
cells. 

A previous study (Dimitriadis & Hondros, 
1 992) showed that the digestive gland cells of 
Helix lucorum, even after 40 days of starva- 
tion or 37 days hibernation, exhibited in- 
creased number of large excretory vacuoles, 
as well as heterolysosomes compared to con- 
trols, and this increase was attributed to the 
accumulation of residual indigestible material 
inside the tubule cells reflecting decreased di- 
gestive and excretory function due to stan/a- 
tion and hibernation. Similar results were also 
reported in starved Helix aspersa Müller 
(Sumner, 1965; Porcel et al., 1996), in which 
prolonged starvation caused a significant de- 
crease in the percentage of the digestive cells 
and an increase of the excretory and narrow 
cells. In addition, the carbohydrate cytochem- 
istry of the content of these lysosomal struc- 
tures did not change after the same periods of 
starvation and hibernation compared to con- 
trols (Dimitriadis & Liosi, 1992). 

Reports from a variety of molluscs show the 
existence of different phases of cell activity, 
each with a distinct appearance, the digestive 
and fragmentation state being the most fre- 



quent (Morton, 1974, 1975, 1979). In the lat- 
ter reports, the length of the cell cycle is ap- 
proximately 24 hours. Because preliminary 
observations in the digestive gland of H. luco- 
rum showed a substantially longer cell cycle 
and less extensive cell alteration during this 
cycle compared to other molluscs, another 
purpose of the present study was to present 
indications related to the digestive rhythm of 
the digestive gland cells of H. lucorum. 



MATERIALS & METHODS 

The snails used in the experiments were 
collected from a natural habitat of Helix luco- 
rum (Gastropoda, Pulmonata, Helicidae) in 
the Logos region of Edessa, northern Greece. 
The shell diameter of the snails varied from 20 
to 40 mm, and the body weight was between 
14 and 20 g. 

The animals were kept in the laboratory 
under stable and controlled conditions of pho- 
topehod (L;D 13:11 h), temperature (22 ± 
2°C), and relative humidity (90 ± 5%). They 
were fed lettuce, which is the most assimi- 
lated food for H. lucorum (Staikou & Lazari- 
dou-Dimithadou, 1989). The controls were 
kept in the conditions described above, while 
others were starved for 14 days, being in 
identical conditions of photoperiod, tempera- 
ture, and humidity as the controls. During the 
starvation period, the animals developed a 
velum of mucus. After starvation, the animals 
were fed again with lettuce, and food was kept 
in the boxes during the whole experiment, 
while each of the animals was observed at 
subsequent periods. The snails started to 
move at about 1.5 h after feeding. Animals 
were examined after 0, 1 0, 22, 34, 45, 56, 65, 
75 h from the beginning of food ingestion. For 
each experimental stage, the animals began 
feeding at the same time and were actively 
feeding for the whole experimental period. 
Five actively feeding animals from each ex- 
perimental stage were examined under the 
light and electron microscope. 

For light and electron microscopic observa- 
tions, samples were fixed in 3% glutaralde- 
hyde, postfixed in 2% osmium tetroxide, de- 
hydrated, and embedded in Spurr's resin. 
Ultrathin sections were post-stained with 
uranyl acetate and lead citrate and examined 
under a JEOL 100B electron microscope op- 
erating at 80 KV. For light microscopic obser- 
vations, thick sections were stained with 1% 
toluidine blue. 



DIGESTIVE GLAND OF HELIX LUCORUM 



147 



RESULTS 

In the following paragraphs, the light and 
electron microscopic views of the digestive 
gland epitheliunn of snails Helix lucorum, 
which were starved for 14 days and then fed 
for 10, 22, 34, 45, 56, 65 and 75 h are de- 
scribed. 

At the beginning of food ingestion (aninnals 
after 14 days of starvation), as well as 10 h 
from the beginning of food ingestion, exami- 
nation of transverse sections of digestive 
tubules under the light (Fig. 2A) or the elec- 
tron microscope (Fig. 2B) show a large num- 
ber of excretory vacuoles in the form of large 
vacuoles with dense cores, similar to that de- 
scribed in the excretory cells of Helix lucorum. 
These large vacuoles, the accumulation of 
which in the digestive gland epithelium is at- 
tributed to starvation, reach usually 2-8 ¡am, 
while in certain cases reach 32 |im. However, 
cells containing heterolysosomes in the form 
of apical granules, which is a main character- 
istic of the digestive cells, are very few. The 
apical plasma membranes show limited 
pinocytotic phenomena at h, expressed by 
the formation of a moderate number of coated 
vesicles. However, at 10 h these phenomena 
become intensive and the formation of coated 
pinocytotic vesicles, and a network of micro- 
tubules and vesicles in the apical cytoplasm is 
apparent (not shown). The calcium cells lo- 
cated in the base of the epithelium (Fig. 2A) 
exhibit the usual morphology described for 
this cell type. 

The apical region of the cells show a large 
number of excretory vacuoles, as well as in- 
tensive pinocytotic phenomena 22 h from the 
beginning of food ingestion. Also, a small 
number of heterolysosomes is present in the 
tubule cells in the form of apical granules of 
moderate size (0.5-1 , 5 |im) filled with mater- 
ial with low electron density. After 34 h from 
the beginning of food ingestion, these het- 
erolysosomes are larger in size and numbers, 
reaching 1 -2 цт and are located near the 
apical border of the cells (Fig. 2C). The excre- 
tory vacuoles are now located adjacent to the 
apical region of the cells, being probably in a 
phase just before their excretion, while such 
vacuoles are present in the tubule lumen in 
certain cases (not shown). The ultrastructural 
observations show that micropinocytosis con- 
tinues at this stage. 

At the 45 h stage, a large number of het- 
erolysosomes, about 2 |im in size, as well as 
an increased number of excretory vacuoles 



are obvious in the apical and middle portion of 
digestive gland cells under the light micro- 
scope. The heterolysosomes 56 h after the 
beginning of food ingestion are increased 
very much in number, and their size usually 
reaches 4 цт (Fig. 2D). With the electron mi- 
croscope, the presence of recently formed 
large vacuoles with dense cores is apparent, 
while some of them are observed to fuse with 
each other (Fig. ЗА). The calcium cells do not 
show any obvious alteration in the number 
and location, comparing to the previous 
stages. 

The digestive tubules cells 65 h and 75 h 
from the beginning of food ingestion are char- 
acterized by an increased number of excre- 
tory vacuoles, the size of which being more 
evident at 75 h (Fig. 3B). The apical het- 
erolysosomes decrease in number, being al- 
most absent at 75 h (Fig. 3B). No any visible 
change in the number and location of the cal- 
cium cells was noted. 

Considering the digestive gland cells of the 
control animals, that is the snails fed with let- 
tuce and kept in identical photopehod, tem- 
perature, and humidity, their functional mor- 
phology resemble that already described for 
these snails examined under normal condi- 
tions. 



DISCUSSION 

There is a disagreement on the origin and 
the function of excretory cells in terrestrial 
gastropods. Fretter (1952), Billett & McGee- 
Russell (1955), Walker (1970), and Morton 
(1979) regarded these cells as differentiated 
digestive cells, whereas Thiele (1953) and 
Sumner (1965) suggested that they are de- 
generate calcium cells. In other studies, 
Abolins-Krogis (1961) found mucopolysac- 
charides, proteins, lipids, and small quantities 
of RNA inside the cisternae of excretory cells 
in Helix aspersa and suggested that these 
materials are implicated in shell repair. 

The results of the present study support the 
hypothesis that in the digestive gland, the so- 
called excretory cells are, simply, digestive 
cells in a final step of their cell cycle rather 
than a distinct cell type. The results showed 
that the so-called digestive cells containing 
heterolysosomes and residual bodies or the 
so-called excretory cells containing excretory 
vacuoles did not follow independent cell cy- 
cles, because they were not found in signifi- 
cant numbers at the same time. The increase 



148 



DIMITRIADIS & KONSTANTINIDOU 




A ^k'.°i^. 




è 



FIG. 2A, B. Beginning of food ingestion. Transverse sections of digestive tubules. Under the light (arrows) or 
the electron nnicroscope (asterisks), the digestive gland cells of the snail Helix lucorum show absence of het- 
erolysosomes, while abundance of large vacuoles with dense cores is noted. A number of calcium cells are 
located at the base of the epithelium. С 34 h from the beginning of food ingestion. Large numbers of het- 
erolysosomes (apical granules) are present at the apical portion of the digestive gland cell (asterisks). D. 45 
h from the beginning of food ingestion. Transverse sections of a digestive tubule. Under the light microscope, 
the cells contain large number of heterolysosomes (small arrows), as well as an increased number of ex- 
cretory vacuoles (large arrows). Abbreviations: CC, calcium cell; Lu, lumen; Mv, microvilli. Scale bars — A = 
15 цт, В = 1.5 цт, С = 1 цт, D = 16 |am. 



DIGESTIVE GLAND OF HELIX LUCORUM 



149 




FIG. ЗА. 56 h from the beginning of food ingestion. Under the electron microscope, the digestive gland cells 
show abundance of excretory vacuoles in the form of vacuoles containing dense cores (asteriks). The arrows 
show possible sites of fusion between adjacent vacuoles. B. 75 h from the beginning of food ingestion. Trans- 
verse section of a digestive tubule. The abundance of large vacuoles with dense cores (arrows) and the ab- 
sence of heterolysosomes are prominent in the digestive gland cells at this stage. Scale bars: A = 1 цт, В = 
25 цт 



in the number and size of the heterolyso- 
somes and residual bodies implicated in the 
endocytotic uptake and digestion of nutrients 
from the gland lumen and their digestion was 
combined with a decrease in the number and 
size of the excretory vacuoles responsible for 
the excretory processes in the epithelium, and 
vice versa. The cell changes observed in the 
digestive gland epithelium should be re- 
garded as different phases of cell activity of 
the same cell type, the digestive cells. 

Another result that supports the above hy- 
pothesis was the fact that in the digestive 
gland cells starvation caused the appearance 
of a great number of excretory vacuoles and 
the reduction of the heterolysosomes to a 
minimum (Fig. 2A). It is also supported by re- 
sults in starved Helix lucorum (Dimitriadis & 
Hondros, 1992) and Helix aspersa (Porcel 
et al., 1996), in which prolonged starvation 
caused a decrease in the percentage of the 
digestive cells compared to the increased per- 
centage of the excretory cells. 

According to the above approach, the phe- 



nomena related to the endocytotic and excre- 
tory processes reported in the present study 
could exhibit the following sequence: at the 
beginning of food ingestion (end of 14 days 
starvation period), the digestive gland epithe- 
lium consists only of cells with excretory vac- 
uoles formed during the previous cell cycle 
and were accumulated in the cells during star- 
vation (Fig. 4C). In the following periods, the 
excretory vacuoles are relocated adjacent 
to the apical border of the cells and finally 
are extruded from the cells, which could be 
conclude by the presence of such vacuoles 
in the tubule lumen. The digestive activities 
progress and initially increase in number and 
size the heterolysosomes, that is, the cell 
elements implicated in the function of endocy- 
tosis and lysosomic digestion of the endocy- 
tosed material (Fig. 4A) and later (approxi- 
mately at the 45 h), the residual bodies 
formed by further digestion and condensation 
of the content of the heterolysosomes (Fig. 
4B). Digestion proceeds, and there is a de- 
crease in the number and size of both het- 



150 



DIMITRIADIS & KONSTANTINIDOU 




FIG. 4. Drawing of the various stages that the digestive cells possibly undergo during the endocytotic and 
excretory processes in the digestive gland epithelium. A. As the digestive activity progresses, in the diges- 
tive cells initially increase in number and size the heterolysosomes (HI), the secondary lysosomes implicated 
in the function of endocytosis and lysosomic digestion of the endocytosed material. B. Intralysosomal diges- 
tion in the heterolysosomes and condensation of their content leads to the formation of large number of resid- 
ual bodies (Rb) or heterolysosomes condensed to residual bodies С Fusion of residual bodies (arrow) leads 
to the formation of large excretory vacuoles and to a decrease in the number and size of both heterolyso- 
somes and residual bodies. D. Cytoplasmic vessels containing excretory vacuoles, amongst other or- 
ganelles, are cut off from the apical region of digestive cells and are extruded to the lumen or the whole cell 
is degenerated and is extruded to the lumen. 



erolysosomes and residual bodies and an in- 
crease in number and size of the excretory 
vacuoles, which possibly arise after fusion of 
smaller residual bodies (Figs. ЗА, 4C). The 
excretory vacuoles are finally extruded into 
the tubule lumen as described above. 

The proposed sequence is supported by 
the results of Walker (1970) in Agriolimax 
reticulatus, in which the excretory cells were 
also regarded as the final step in the develop- 
ment of the digestive cells. The excretory cells 
in Agriolimax contained the digestive rem- 
nants of ingested material, which is absorbed 
by the former cells, degraded by lysosomal 
action and subsequently extruded to the 
lumen of the digestive gland. 

Reports show that in the land pulmonale 
Dereceras reticulatum (Walker, 1972; Triebs- 
korn & Florschutz, 1993), the food transit 
through the digestive tract takes 9-12 h, while 
in the freshwater pulmonales Lymnaea stag- 
nalis (L.) (Veldhuijzen, 1974) and Biophalaria 
grabrata (Say) (Florschutz & Becker, 1999) 
the digestive gland is emptied of food every 
60-110 min, discharging undigested parti- 
cles. In the absence of information, the latter 
authors postulated the existence of a rhythmic 
activity of the basommatophoran digestive 
gland cells. On the other hand, reports from a 
variety of different molluscs, from the phy- 
tophagous pulmonale Amphibola cerenata 



(Morton, 1974) to the zoophagous pulmonale 
Deroceras caruanae (Morton, 1979) and the 
microphage bivalve Geloina próxima Prime, 
1864 (Morton, 1975) showed the existence of 
different phases of cell activity, each with a 
distinct appearance, the digestive and frag- 
mentation state being the most frequent. In 
these reports, the length of the cell cycle is es- 
timated to be approximately 24 h. Although a 
different methodology would be required for 
studying cell turnover, the results of the pres- 
ent study display indications that in Helix lu- 
corum this cycle seems to be longer, reaching 
approximately 55 h. This could be concluded 
by the fact that at 0-10 h, as well as at 65-75 
h from the beginning of food ingestion, identi- 
cal morphological features were observed in 
the digestive gland cells. In addition, the mor- 
phological changes in the cell that accompany 
the cell cycle were not as extensive as in other 
gastropods and were related to the presence 
or absence of certain lysosomic structures, 
rather than extensive changes, for example in 
the thickness of the epithelium. 

By using light and electron microscope ob- 
servations the present study provides infor- 
mation about the cell types and the physiol- 
ogy of the digestive gland cells of H. lucorum. 
However, more studies, using for example 
histochemistry in cryosections and immuno- 
cytochemistry are needed for the better un- 



DIGESTIVE GLAND OF HELIX LUCORUM 



151 



derstanding of the fine structure and function 
of these cell types. 

LITERATURE CITED 

ABOLINS-KROGIS, A., 1 961 , The histochemistry of 
the hepatopancreas of Helix pomatia (L.) in rela- 
tion to the regeneration of the shell. Arkiv fur Zo- 
ologie, 13: 159-201. 

BILLETT, F. & S. M. McGEE-RUSSELL, 1955, The 
histochemical localization of ß-glucuronidase in 
the digestive gland of the Roman snail Helix po- 
matia (L.) Quarterly Journal of Microscopical Sci- 
ences, 96: 35-48. 

DIMITRIADIS, V. K. & D. HONDROS, 1992, Effect 
of starvation and hibernation on the fine structural 
morphology of digestive gland cells of the snail 
Helix lucorum. Malacologia, 24: 63-73. 

DIMITRIADIS, V. K. & M. LIOSI, 1992, Ultrastruc- 
tural localization of periodate-reactive complex 
carbohydrates and acid-alkaline phosphatases in 
digestive gland cells of fed and hibernated Helix 
/ucorum (Mollusca: Helicidae). Journal of Mollus- 
can Studies, 58: 233-243. 

FRETTER, v., 1952, Experiments with ^^P and ^^^1 
on species of Arion, Helix and Agriolimax. Quar- 
terly Journal of Microscopical Science, 93: 
133-146. 

FLORSCHUTZ, A. & W. BECKER, 1999, Gastroin- 
testinal transit and digestive rhythm in Bio- 
phalaria glabrata (Say.) Journal of Molluscan 
Studies, 65: 163-170. 

MORTON, B. S., 1974, The seasonal variation in 
the feeding and digestive cycle of Amphibola cer- 
enata (Martin 1784) (Gastropoda: Pulmonata). 
Forma et Function, 8: 175-190. 

MORTON, B. S., 1975, The diurnal rhythm and the 
feeding responses of the South East Asian man- 
grove bivalve, Geloina próxima Prime 1864. (Bi- 
valvea: Corbiculacea). Forma et Functio, 54: 
482-500. 

MORTON, B. S., 1979, The diurnal rhythm and the 
cycle of feeding and digestion in the slug Dere- 
ceras caruanae. Journal of Zoology, 187: 135- 
152. 



PORCEL, D., J. D., BUENO & A. ALMENDROS, 
1996, Alterations in the digestive gland and shell 
of the snail Helix aspersa Müller (Gastropoda, 
Pulmonata) after prolonged starvation. Compara- 
tive Biochemistry & Physiology, A [Physiology], 
115: 11-17. 

RUNHAM, N. W. 1975, Alimentary canal. Pp. 
53-105, in: FRETTER, V. and peake, j., eds., Pul- 
monates. Vol. 1. Functional anatomy and physi- 
ology. London, Academic Press. 

STAIKOU, A. & M. LAZARIDOU-DIMITRIADOU, 
1 989, Feeding experiments on and energy flux in 
a natural population of the edible snail Helix luco- 
rum L. (Gastropoda: Pulmonata: Stylommato- 
phora) in Greece. Malacologia, 31: 217-227. 

SUMNER, A. T, 1965, The cytology ad cytochem- 
istry of the digestive gland cells of Helix. Quar- 
terly Journal of Microscopical Science, 1 06: 1 73- 
192. 

THIELE, G., 1953, Vergleichende unterschuchun- 
gen über den Feinbau und die Funktion der mit- 
terdarmdruse einheimischer Gastopoden. Zeit- 
schrift für Zellforschung und Microscopische 
Anatomie, 38: 87-93. 

TRIEBSKORN, R. & A. FLORSCHÜTZ, 1993, 
Transport of uncontaminated and molluscicide- 
containing food in the digestive tract of the slug 
Dereceras reticulatum (Müller). Journal of Mol- 
luscan Studies, 59: 35-42. 

VELDHUIJZEN, J. P., 1974, Sorting and retention 
time of particles in the digestive gland of Lym- 
naea stagnalis (L). Netherlands Journal of Zool- 
ogy 24: 10-21. 

WALKER, G., 1970, The cytology cytochemistry 
and ultrastructure of the cell types found in the di- 
gestive gland of the slug Agriolimax reticulatus 
(Müller.). Protoplasma, 71: 91-109. 

WALKER, G., 1972, The digestive system of the 
slug Agriolimax reticulatus (MuWer.): Experiments 
on phagocytosis and nutrient absorption. Pro- 
ceedings of the Malacological Society London, 
40: 33-43. 

Revised ms. accepted 25 April 2001 



MALACOLOGIA, 2002, 44(1): 153-163 

INTERPOPULATION VARIATION IN LIFE-HISTORY TRAITS OF POMACEA 

CANALICULATA (GASTROPODA: AMPULLARIIDAE) IN SOUTHWESTERN 

BUENOS AIRES PROVINCE, ARGENTINA 

Pablo R. Martin & Alejandra L. Estebenet 

Universidad Nacional del Sur, Departamento de Biología, Bioquímica y Farmacia, San Juan 
670, 8000 Bahía Blanca, Argentina; pmartin@criba.edu.ar 

ABSTRACT 

The Argentinean apple snail Pomacea canaliculata is a freshwater gastropod with a high inter- 
population variation in shell shape, size and thickness. Previous experimental studies have shown 
that many life-history traits are highly dependent on rearing conditions. Three natural populations 
located in one same drainage basin and climatic regime showed marked differences in birth, ma- 
turity and maximum sizes. Most of this variation disappeared when newborns from each popula- 
tion were reared under homogeneous conditions in the laboratory, indicating its ecophenotypic ori- 
gin. However, a significant variation in reproductive, growth and survival patterns attributable to 
genetic differences among the source populations was still discernible among laboratory cohorts. 

The three sites studied represent a marked gradient in stability and food availability. Females 
from the most unstable and poorer site showed a faster prematurity growth and a higher ovipo- 
sition rate than those from the most stable and productive site. This higher oviposition rate was 
associated with bigger clutches and a higher mortality rate. The different patterns of survival and 
somatic and reproductive allocation in the three populations, being heritable and adaptively cor- 
related to different environmental conditions, could be considered as parts of different life-history 
strategies. 

Since the 1 980s, P. canaliculata has become a serious pest of paddy fields in most Southeast 
Asian countries. Nearby and recently isolated populations of P. canaliculata from the same basin 
showed genetically different life-history strategies, so that different populations of this pest could 
require different control programs. 

Key words: apple snail, life-history traits, ecophenotypic variation, genetic variation 



INTRODUCTION 

Pomacea canaliculata (Lamarck, 1822) is a 
freshwater prosobranch gastropod with a high 
polymorphism in shell shape, size and thick- 
ness (Cazzaniga, 1987). Its natural distribu- 
tion is basically tropical and subtropical, in- 
cluding the Amazonas and La Plata basins 
(Ihering, 1 91 9), though in the last two decades 
it has invaded most Southeastern Asia coun- 
tries, becoming a serious rice pest (Wada, 
1997). During an extensive survey in the 
southern limit of the native area of this species, 
the southernmost apple snail in the world 
(Martin et al., 2001), we obsen/ed a great in- 
terpopulation variation in maximum size, shell 
thickness and egg size, even at a small spatial 
scale and within the same drainage system. 

Experimental trials have shown that growth 
rates, longevity and age at maturity are highly 
dependent on temperature (Estebenet & Caz- 
zaniga, 1992), crowding (Cazzaniga & Es- 
tebenet, 1988; Tanaka et al., 1999), and food 



(Lacanilao, 1990; Estebenet, 1995; Albrecht 
et al., 1999; Lach et al., 2000). This evidence 
and the strong intersite environmental varia- 
tion (Martin et al., 2001 ) seems to support the 
hypothesis of an ecophenotypic origin of the 
interpopulation variation. Cazzaniga (1987) 
reported that shells from lentic, soft-bottom 
water bodies are generally bigger and lighter 
than those from hard-bottom running waters. 
However, a genetic basis of this interpopula- 
tion variation cannot be ruled out. 

The aims of this study were to analyze the 
variation in birth, maturity and maximum sizes 
among populations of P. canaliculata from 
three environmentally different sites belonging 
to the same drainage basin, and to determine 
if they were ecophenotypic or genetic in origin. 



STUDY AREA 

The study area lay between 36°40'S and 
37°35'S and 61°15'W and 63°10'W in south- 



153 



154 



MARTIN & ESTEBENET 



western Buenos Aires Province, Argentina. 
The climate of the region is temperate, with a 
mean annual temperature and amplitude of 
13.8°C and 15.1°C, respectively; mean an- 
nual rainfall is 733 mm, mainly concentrated 
in fall and spring. The rainfall is highly vari- 
able, with marked fluctuations between dry 
and wet years (Scian & Donnari, 1997). 

The three sites selected belong to the En- 
cadenadas del Oeste Basin (Fig. 1), a closed 
drainage system composed of a chain of six 
shallow lakes of increasing conductivity to- 
wards the west, from Alsina Lake (freshwater) 
to Epecuén Lake (hypersaline) and de la Sal 
Lake (temporary salt lake). Streams and 
creeks of pluvial hydrological regime, with 
scant and very variable water discharge, run 
northwards into the lakes from the Ventanía 
Mountains. 

Alsina Lake is a permanent, eutrophic lake, 
with a surface area of 1 05 km^, maximum and 
mean depths of 6.4 m and 2.8 m respectively 



and a shoreline length of 62 km. The bottom 
is composed of fine sand and mud. The dom- 
inant macrophytes during the present study 
were the emergent Typha spp. and Schoeno- 
plectus californicus and the submerged Pota- 
mogetón sp. and Myriophyllum elatinoides, 
sometimes heavily colonized by cyanophytes. 

Curamalal Stream is a permanent stream 
approximately 91 km long that discharges in- 
termittently into Alsina Lake. The bottom is 
composed of mud with minor areas of lime- 
stone bed. The macrophytes were the emer- 
gent Cyperus spp. and S. californicus, the 
submerged Cliara sp. and Potamogetón sp. 
and the floating-leaved Ludwigia sp. 

Cochicó Rivulet is a 20-km-long creek with 
intermittent flow along most of its course. It 
discharges in the salty Cochicó Lake (con- 
ductivity 11.3 mS.cm"^), and during high 
floods the Curamalal Stream overflows into it. 
The dominant substratum is mud with some 
areas of limestone bedrock and cobble. 




FIG. 1 . Map of the Encadenadas del Oeste basin showing the location (©) of the three study sites (from north 
to south Alsina Lake, Cochicó Rivulet and Curamalal Stream) (Inset: map of Argentina showing the study 
zone). 



LIFE-HISTORY VARIATION IN POMACEA 



155 



Emergent macrophytes were absent and only 
occasionally was the presence of scarce Lud- 
wigia sp., Potamogetón sp. and Enteromor- 
pha sp. detected. 



MATERIALS & METHODS 

The three sites were visited in February, 
July and October 1998 and February 1999. 
Two or three people searched for apple snails 
during up to four hours among the submerged 
vegetation, under stones, or buried in the sub- 
stratum; all snails larger than 20 mm were col- 
lected and their shell length from the apex to 
the basal extreme of the aperture was mea- 
sured. In October 1998 and February 1999 
copulating snails (i.e., couples in which male 
penis sheath was inserted into female palliai 
cavity) were collected and sized. The pink, 
aerial and conspicuous egg masses, de- 
posited on riparian or emergent plants, were 
obtained in March and October 1998 and 
maintained until hatching in the laboratory. 
Temperature, conductivity (mS.cm"^) and pH 
were determined in situ with a multimeter 
Horiba U-10. A subsurface water sample was 
collected at each site for further analyses. The 
concentrations of major ions and other limno- 
logical variables were measured as detailed 
in Martin etal. (2001). 

Progeny from at least five clutches from 
each site (March 1998) that hatched in a pe- 
riod of 72 hours were pooled and 100 new- 
born were randomly selected to generate 
three experimental cohorts. They were reared 
in sets of ten snails under homogeneous con- 
ditions (aereated tap water saturated with 
COgCa, temperature of 25 ± 3°C, permanent 
illumination and lettuce ad libitum). 

During the first two months the sets of ten 
snails were kept in 3 I aquaria and then trans- 
ferred to 15 I aquaria to avoid crowding (Gaz- 
zaniga & Estebenet, 1 988). When the first egg 
mass was observed in each 15 I aquarium, 
snails were sexed on the basis of shell shape 
(Cazzaniga, 1990; Estebenet, 1998) and 
color and appearance of the gonad (Andrews, 
1964), visible through the thin shell. One fe- 
male and one male were randomly selected, 
and reared again in 3 I aquaria. The eight re- 
maining snails of each aquarium were sacri- 
ficed and stored for later morphometric analy- 
sis, which will be published elsewhere. 

Water was changed weekly and survivor- 
ship, shell length and number of clutches and 



eggs were recorded. Dead snails were re- 
placed by mature snails of the same sex with 
a shell length larger than 30 mm, obtained in 
the wild and maintained in the lab under the 
same rearing conditions, to keep rearing con- 
ditions constant for the survivors, especially 
mating possibilities and density. The oviposi- 
tion and spawning rates were calculated as 
the ratio of life-time production of eggs or egg 
masses to the length of the spawning period 
(from first to last spawn). Clutch size was es- 
timated as the ratio of the life-time production 
of eggs to the life-time production of egg 
masses. 

Field egg masses obtained in March and 
October 1998 and one egg mass from each 
couple from each cohort were incubated in 
the lab. After hatching, groups of 30-50 new- 
born were dried (80°C, 48 h), weighed to the 
nearest 0.1 mg, and reweighed after ignition 
(600°C, 4 h) to obtain total and ash dry 
weights. Shell lengths of ten hatchlings from 
field spawns and fifteen from laboratory 
spawns were measured under a stereoscopic 
microscope to the nearest 0.01 mm. 

To test for differences among sites in repro- 
ductive variables one way Anovas were per- 
formed. Two way Anovas were used for length 
and age at death, since sexual dimorphism in 
these traits has been recorded (Estebenet & 
Cazzaniga, 1998). For newborn and imma- 
ture snails nested Anovas were performed on 
shell length with egg masses or aquaria as 
the nested factor. 



RESULTS 

The values of the physico-chemical vari- 
ables measured in Alsina and Curamalal sites 
fall within the range of the water bodies of the 
Southern Buenos Aires province classified as 
"habitable for snails" by Martin et al., (2001). 
The Cochicó, contrarily, showed marked 
fluctuations in conductivity (Fig. 2) and Na* 
concentration, reaching values out of the 
mentioned range. The variation in water tem- 
perature among the three sites was less than 
3°C during most part of the year, except in 
summer, when temperatures 7°C higher were 
measured in Cochicó. 

Maximum sizes differed markedly among 
the three populations. The mean shell length 
of the 15 larger snails on each sampling date 
is showed in Figure 3. Snails from Cochicó 
were generally smaller than those from Alsina 
and Curamalal. Despite the similarity in maxi- 



156 



MARTIN & ESTEBENET 



' I -A- Aisina 
С~ Curamalal 
— > ^ ^ I ♦Cochlea 



Э 

с 

8 '^ 




J F 

1998 



J J A S N D 

date 



F M 
1999 



FIG. 2. Seasonal variation in conductivity in the 
studied sites of Alsina Lake, Cochicó Rivulet and 
Curamalal Stream. 



5 40 




J F M A M J 


J A s N D J 


F M 


1998 


date 


1999 



FIG. 3. Seasonal variation in mean shell length of 
the fifteen larger snails in the studied sites of Alsina 
Lake, Cochicó Rivulet and Curamalal Stream (bars 
are 95% confidence intervals). 



mum sizes in spring, the size of the copulating 
snails was smaller in the Cochicó population 
than in the Curamalal population in October 
1998, and also in February 1999 (Fig. 4; tgg = 
3.07, p < 0.01 and t^^g = 6.03, p < 0.001, re- 
spectively). Couples were not observed in 
Alsina on those dates. Despite the highly sig- 
nificant variation in newborn shell length 
among clutches of the same site (F12 270 = 
19.52, P < 0.0001 and Fg ^^g = 35.33, P < 
0.0001, respectively), the intersite variation 
was highly significant both in March and Oc- 
tober 1998, Cochicó hatchlings being the 
smallest in both dates (Table 1). The new- 
borns from Cochicó were also lighter than 
those from Alsina and Curamalal, though dif- 
ferences were significant only in March 1998 









n Curamalal 


1 




40 




□ Cochicó 


































1 








E 


i 




F 




















30 
















^ 


















% 


















g 


















Ф 


















H 


20 
















Ф 


















1" 




















10 J 




1 ' 















October 1998 



February 1999 



FIG. 4. Mean shell length of copulating snails in the 
Cochicó Rivulet and Curamalal Stream sites in Oc- 
tober 1998 and February 1999 (bars are 95% con- 
fidence intervals). 

(Table 1). Egg and clutch sizes were also evi- 
dently smaller in Cochicó (pers. obs.). 

Growth, survivorship and oviposition pat- 
terns for the three laboratory cohorts are 
shown in Figure 5. The initial size differences 
disappeared before the age of two months 
(Table 2), suggesting a higher growth rate for 
the Cochicó cohort. Growth curves were very 
similar from two months of age onwards, and 
female shell length at first spawn was not sig- 
nificantly different (Table 2; one female from 
Cochicó and Alsina each died accidentally 
and one from Curamalal that never spawned 
was excluded from analysis). Length at death 
was significantly different between sexes 
(males smaller than females) but not among 
cohorts (Table 3). 

Mortality was null during the pre-reproduc- 
tive phase in the three cohorts but after the 
beginning of spawning the mortality rate in- 
creased steadily. It was highest in the Cochicó 
cohort, with a maximum longevity of 318 
days. The Alsina cohort showed the lowest 
mortality, with some snails surviving up to 650 
days. Differences in age at death (Table 3) 
were significant only between Cochicó 
(253.53 days) and Alsina cohorts (343.58 
days) (critical value for the SNK test = 71.33 
days, p < 0.05). The survivorship pattern of 
the Curamalal cohort was intermediate, with a 
mean survival time of 300.37 days and a max- 
imum longevity of 627 days. 

Although the age at first spawning and the 
duration of the spawning period in the Alsina 
cohort were higher than in the Cochicó and 
Curamalal ones, they were not significantly 
different (Table 2), probably due to the high 
variation in reproductive behavior in that со- 



LIFE-HISTORY VARIATION IN POMACEA 



157 



TABLE 1 . One-way Anova for length and dry weights of newborn from field egg masses (&: 
Nested Anova with four or five spawns (nested factor) per site and ten newborn per spawn). 
Underlined means are significantly different to the other two (SNK tests, p < 0.05). 





M.S. 


d.f. 


F 


P 




Means 




Variable 


Cochicó 


Curamalal 


Alsina 


March 1998 
















total weight (mg) 


0.966 


2, 16 


20.572 


0.000 


1.053 


1.808 


1.555 


ash weight (mg) 


0.411 


2, 16 


9.459 


0.002 


0.329 


0.824 


0.648 


shell length (mm)& 


2.414 


2, 12 


24.738 


0.000 


2.199 


2.628 


2.498 


October 1 998 
















total weight (mg) 


0.181 


2,9 


2.564 


0.131 


0.920 


1.236 


1.356 


ash weight (mg) 


0.060 


2,9 


2.070 


0.182 


0.524 


0.735 


0.768 


shell length (mm)& 


0.931 


2,9 


4.392 


0.047 


2.156 


2.388 


2.447 



hort (Fig. 6). The life-time production of eggs 
and clutches, and the spawning rate were 
not significantly different among cohorts, al- 
though the ovipositlon rate was significantly 
lower in Alsina females. On the other hand, 
the clutch size from the Cochicó cohort was 
the highest (Table 2). 

The total and ash dry weights of newborns 
were not significantly different among the lab- 
oratory cohorts (Table 2). The variation in 
newborn shell length among clutches of the 
same cohort was highly significant (F^5 252 = 
34.27, P < 0.0001 ) and greater than the inter- 
cohort variation (Table 2). Only for the 
Cochicó site there was a significant variation 
between newborn from egg masses laid in the 
field (March 1998) and their own progeny in 
the laboratory, both in length (nested Anova 
with five spawns -nested factor — per source 
and ten newborn per spawn: F^ s = 9.62, P < 
0.01 ) and weight (t^g = 3.97, p < Ó.001 for total 
dry weight and t^g = 8.25, p < 0.0001 for ash 
dry weight). 



DISCUSSION 

Pomacea canaliculata shows great life-his- 
tory interpopulation variation, related mainly 
to different thermal regimes (Estebenet & 
Cazzaniga, 1992). Though the three natural 
populations studied here are located in the 
same drainage basin and under the same cli- 
matic regime, they showed marked differ- 
ences in some life-history traits. The variation 
in maximum sizes among the sites indicates 
different survivorship or growth rates. On the 
other hand, at the beginning of the reproduc- 
tive season (early spring, Hylton-Scott, 1958) 
the size of copulating snails differed between 
the two stream populations, though the size 
distributions were very similar, suggesting dif- 



ferences in size or age at maturity. Interpopu- 
lation variation in the size of eggs, clutches 
and newborn was also observed. 

Most of the interpopulation variation in birth, 
maturity and maximum sizes disappeared 
when the snails were reared under homoge- 
neous conditions in the laboratory, indicating 
an ecophenotypic origin of the variation. How- 
ever, notwithstanding the elimination of envi- 
ronmental differences, some variation in re- 
productive, growth and survival patterns was 
still discernible and attributable to genetic dif- 
ferences among populations. Although the 
maternal and environmental effects on the 
newborn used to initiate the cohorts cannot be 
discarded, their influence seems to be irrele- 
vant. The effect of site conditions during em- 
bryo development was probably null, since 
very fresh clutches (easily recognizable by 
their moisture and bright pink color) were 
used. On the other hand, the differences in 
newborn size, partially attributable to mater- 
nal size or condition (see below) soon disap- 
peared under the highly favorable rearing 
conditions in the laboratory. 

The three sites represent a marked gradi- 
ent in stability and food availability. The 
Cochicó is an intermittent creek, with very 
marked seasonal fluctuations in water level, 
the lower section being the only one that 
never dries completely; however, high con- 
ductivity peaks caused by the rise in Cochicó 
Lake water levels affect this section. These 
peaks exceed the maximum values of con- 
ductivity recorded for sites inhabited by P. 
canaliculata in this area (2.89 mS.cm"\ 
Martin et al., 2001). Minimum discharge in 
Curamalal Stream is higher than in the 
Cochicó Rivulet, and the flow is continuous 
even during dry summers. Though the area of 
Alsina Lake is quite variable, water level fluc- 
tuations are comparatively slow and related 



158 



MARTIN & ESTEBENET 



Alsina 



80 
60 
40 
20 



□ egg production 
-^ shell length 
survirvor ship 




600 
500 
400 
300 
200 
100 



17 52 87 122 157 192 227 262 297 332 367 402 




17 52 87 122 157 192 227 262 297 332 367 402 

age (days) 

FIG. 5. Growth, survivorship and oviposition patterns for the three laboratory cohorts from Alsina Lake, 
Cochicó Rivulet and Curamalal Stream (bars are 95% confidence intervals for shell length). 



LIFE-HISTORY VARIATION IN POMACEA 



159 



TABLE 2. One-way Anova for growth and reproductive variables from laboratory cohorts. Underlined 
means are significantly different to the other two (SNK tests, p < 0.05). (#: Nested Anova with ten aquaria 
(nested factor) per cohort and ten snails per aquarium; &: nested Anova with six females (nested factor) 
per cohort and fifteen newborn per female; +: data from some females were lost). 





M.S. 


d.f. 


F 


P 




Means 




Variable 


Cochicó 


Curamalal 


Alsina 


Length at 17 d (mm) # 


27.883 


2,27 


13.354 


0.000 


5.07 


6.00 


5.11 


Length at 65 d (mm) # 


16.367 


2,27 


2.052 


0.148 


23.70 


23.99 


23.19 


Length at first spawn (mm) 


32.490 


2,24 


1.598 


0.223 


47.27 


47.82 


50.80 


Age at first spawn (days) 


2806.48 


2,24 


0.752 


0.482 


136.78 


164.55 


169.55 


Spawning period (days) 


11322.11 


2,24 


2.655 


0.091 


90.00 


92.78 


152.78 


Clutch size (eggs. clutch"'') 


25361.35 


2,24 


6.696 


0.005 


264.75 


204.29 


158.94 


Oviposition rate (eggs.wk"'') 


49543.56 


2,24 


4.576 


0.021 


375.10 


346.36 


234.66 


Spawning rate (clutches.wk"^) 


0.0191 


2,24 


0.748 


0.484 


1.47 


1.73 


1.48 


Life-time egg production 


126115.15 


2,24 


0.023 


0.977 


4554.22 


4407.22 


4641.45 


Life-time clutch production 


195.44 


2,24 


1.261 


0.302 


19.00 


22.45 


28.21 


Newborn shell length (mm) & 


0.960 


2, 15 


3.501 


0.056 


2.48 


2.46 


2.65 


Newborn total weight (mg) 


0.179 


2, 18+ 


2.333 


0.126 


1.37 


1.41 


1.67 


Newborn ash weight (mg) 


0.018 


2, 18+ 


0.618 


0.550 


0.78 


0.80 


0.88 



TABLE 3. Two-way Anova for growth and survival of males and females from labo- 
ratory cohorts. 



Variable 



Source 



M.S. 



d.f. 



Length at death (mm) 



Age at death (days) 



Site 


52.87 


2 


2.761 


0.073 


Sex 


222.80 


1 


11.633 


0.001 


Site X Sex 


17.56 


2 


0.917 


0.406 


Error 


19.15 


51 






Site 


38394.78 


2 


4.495 


0.016 


Sex 


10938.61 


1 


1.281 


0.263 


Site X Sex 


1224.78 


2 


0.143 


0.867 


Error 


8541.13 


51 







Curamalal 



Cochicó 



50 100 150 200 250 300 350 400 450 500 550 600 

age (days) 

FIG. 6. Spawning period duration of females from 
the three laboratory cohorts from Alsina Lake, 
Cochicó Rivulet and Curamalal Stream ranked by 
age at first spawn. 



гла1п1у to multiannual cycles of rainfall 
(lATASA, 1994; González Urlarte & Orloll, 
1998). On the other hand, the scarcity of 
aquatic macrophytes and riparian vegetation 
suggest that trophic constraints are stronger 
in the Cochicó Rivulet. In this region, water 
bodies inhabited by P. canaliculata generally 
are quite shallow, quiet and turbid, with low 
NaV(K* + Mg"""") ratios as compared to the un- 
inhabited sites (Martin et al., 2001). The 
Cochicó Rivulet is a less suitable habitat for 
these snails than the other two sites. 

The lower maximum and maturity sizes in 
Cochicó Rivulet were probably due to lower 
growth rates, although the detrimental influ- 
ence of recurrent disturbance on longevity 
cannot be ruled out. The low food availability, 
and not the temperature, was probably the 



160 



MARTIN & ESTEBENET 



growth limiting factor, because laboratory 
snails from this source showed the highest 
growth rates during most of the pre-reproduc- 
tive period, and summer water temperature 
was higher at this site. Minimum female re- 
productive sizes of approximately 25 mm 
have been recorded for P. canaliculata grow- 
ing under very different densities, food and 
temperature conditions (Martin, 1986; Es- 
tebenet & Cazzaniga, 1992; Tanaka et al., 
1999), age at first oviposition being hence 
highly dependent on growth rate. In this study, 
the first spawn of each cohort was observed 
at ages between 80 and 99 days, a very early 
age as compared to other cohorts at 25°C 
(Estebenet & Cazzaniga, 1992, 1998), but at 
39 mm shell length, indicating that maturity 
depends not only on size, but that a minimum 
age is also required. The slow growth at the 
Cochicó site and the lack of genetic interpop- 
ulational variation in age and size at maturity 
suggest that differences in size of field copu- 
lating snails are not due to an earlier matura- 
tion in Cochicó. In this population, the mini- 
mum age is probably attained before the 
minimum size, whereas in the Curamalal the 
minimum size is reached before the minimum 
age. 

Hatching in P. canaliculata occurs by me- 
chanical fracture of the calcareous egg shell, 
after consuming all the perivitelline egg re- 
serve, at which stage the length of the new- 
born equals the diameter of the egg (Es- 
tebenet & Cazzaniga, 1993; Heras et al., 
1998). Estebenet & Cazzaniga (1993) re- 
ported that the egg size variation among 
clutches from the same pond was greater 
than interpond variation. In the present study, 
on the contrary, the intersite variation was 
greater than intrasite variation. The size of 
newborn was the lowest in Cochicó Rivulet, 
probably limited by the small size of females, 
because newborns from laboratory reared, 
big-sized females from this source were big- 
ger and similar in size to those from the other 
two sources. Female size in the permanent 
ponds studied by Estebenet & Cazzaniga was 
greater than in Cochicó and probably was not 
a constraint for egg size. 

Under homogeneous conditions, snails 
from the three sites showed variation in re- 
productive, growth and survival patterns. Fe- 
males from Cochicó showed a genetically 
fixed tendency to lay a higher number of eggs 
per week and packed in bigger clutches than 
those from Alsina. This higher oviposition rate 
implies a higher reproductive allocation asso- 



ciated with a higher mortality rate, probably 
owing to a competence between somatic 
maintenance and reproduction only (Jokela, 
1 997), because growth was almost null during 
most of the reproductive period. 

The different patterns of survival, somatic 
and reproductive allocation in the three popu- 
lations could be considered as parts of differ- 
ent life-history strategies, because they 
showed a genetic component and are corre- 
lated in an adaptive way to different environ- 
mental conditions (Calow, 1978, 1983). Under 
the thermal regime of the study zone, P. 
canaliculata is iteroparous, with two or more 
reproductive periods limited to the warmer 
months (Estebenet & Cazzaniga, 1992, 
1993). Fast prematurity growth and a higher 
reproductive output, despite the detrimental 
effect of the latter on survivorship and future 
egg production, seems to be adaptive in the 
Cochicó Rivulet. At this site, where frequent 
and unpredictable disturbance events cause 
mass mortality episodes, probably only a few 
mature snails survive to reproduce in a sec- 
ond season. In contrast, in the Alsina Lake, 
where the frequency and magnitude of the 
disturbances are lower, a restrained repro- 
ductive allocation can led to more reproduc- 
tive seasons, thus maximizing the lifetime egg 
production. 

The adaptive value of early maturation and 
high reproductive effort depends on a lower 
survival of parents relative to their offspring 
(Calow, 1983). Adults of Pomacea spp. from 
ephemeral but more predictable water bodies 
aestivate (Burky, 1974; Kushlan, 1975; Don- 
nay & Beissinger, 1 993), but this behavior has 
not been described for P. canaliculata. Al- 
though water loss and susceptibility are prob- 
ably higher in small snails (Turner, 1 996), their 
chances of fitting in humid microhabitats are 
also higher. Pomacea canaliculata mobility 
and feeding are highly reduced when water 
depth is lower than shell diameter (Wada, 
1997), so post-drought survival is probably 
negatively related to size. Conductivity peaks 
and drought episodes shorter than the lapse 
between oviposition and hatching (about 17 
days in this region) will affect parent survival 
without harmful effects on their aerial eggs. In- 
deed, despite their normal nocturnal spawn- 
ing behavior (Schnorbach, 1995; Albrecht et 
al., 1996), P. canaliculata \ema\es exposed to 
air for several hours frequently spawn, even 
during light hours (Cazzaniga, 1981; pers. 
obs.), probably increasing the chances of 
leaving progeny. Scouring by floods into the 



LIFE-HISTORY VARIATION IN POMACEA 



161 



salty Cochicó Lake is probably a major source 
of catastrophic mortality, specially for big 
snails, less able to withstand fast flow and to 
find in-stream flow refuges of adequate size 
(Calow, 1983; Dussart, 1987; Johnson & 
Brown, 1997). On the other hand, clutches 
can endure submersion for several days with- 
out deleterious effects on the embryos (pers. 
obs.). 

Apart from local adaptation, genetic drift is 
another probable cause of interpopulation ge- 
netic variation. Encadenadas del Oeste basin 
was once part of an allocthonous river running 
through the present Vallimanca River into the 
Salado River (Fig. 1). The chain of lakes was 
originated in its paleo-valley when discharge 
diminished after the end of the last glacial age 
and climate became increasingly arid in the re- 
gion (Frenguelli, 1 945; Malagnino, 1 989). Fos- 
sil records of P. canaliculata are common in the 
Salado basin (Camacho, 1966; Dangavs & 
Blasi, 1992), and the species was probably 
widespread in it when subtropical conditions 
advanced to southern Buenos Aires Province 
(8-6 Kyr BP; Iriondo, 1994). Since then, local 
populations probably became increasingly 
isolated because of increasing habitat discon- 
tinuity. The P. canaliculata populations in the 
studied sites do not seem to have originated 
from independent arrivals from a genetically 
heterogeneous source, rendering founder ef- 
fects unimportant. 

The maintenance of genetic differentiation 
among the studied populations is noteworthy, 
taking into account that their isolation was not 
complete in the recent past. The three study 
sites secularly connected each other in most 
peaks of the multiyear rainfall cycle, espe- 
cially when Alsina and Cochicó lakes merge 
and both present freshwater conditions 
(lATASA, 1994; González Uriarte & Orioli, 
1998). However, at present the spread of P. 
canaliculata in this geographic region seems 
to be very slow, even within the same stream 
(Martin et al., 2001) and gene flow is probably 
strongly unidirectional, mostly due to down- 
stream drift. Since 1992, a roadway embank- 
ment with sluice gates divided Alsina and 
Cochicó lakes, probably increasing their iso- 
lation. On the other hand, population size in 
the Cochicó Rivulet was probably very small, 
due to the small size of the habitable section 
and low food availability. These factors proba- 
bly account for the persistence of the genetic 
differentiation provoked by the strong selec- 
tive pressures, even in the absence of total 
habitat discontinuity. 



Asian pest managers have showed deep 
concern about the specific identity of apple 
snails that invaded paddy fields (Wada, 1997; 
Cowie, in press). However, close and recently 
isolated populations of P. canaliculata from 
the same basin showed genetically different 
life-history strategies, so even populations 
from the same species could require different 
control measures. The situation is compli- 
cated by the fact that most Asian countries 
suffered repeated introductions of apple 
snails (Litsinger& Estaño, 1993; Wada, 1997; 
Cowie, in press), probably from distant geo- 
graphic regions, among which genetic in- 
traspecific variation is expected to be even 
greater. Moreover, since the early 1980s the 
control and culture practices in paddy fields 
have been exerting new and strong selective 
pressures that are probably already shaping 
the major features of Pomacea life history. 



ACKNOWLEDGMENTS 

We wish to express our gratitude to Natalia 
Pizani and Eduardo Albrecht for their assis- 
tance in field work. This study was funded with 
grants by CONICET ("Consejo Nacional de 
Investigaciones Científicas y Técnicas") and 
"Agencia Nacional de Promoción Científica y 
Técnica", Argentina. ALE is a researcher in 
CONICET 



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of long-lived bivalves. Pp. 179-196, in: в. streit, 
т. STADLER & с. M. LIVELY eds.. Evolutionary ecol- 
ogy of freshwater animals. Basel. 

KUSHLAN, J. A., 1975, Population changes of the 
apple snail, Fomacea paludosa, in the southern 
Everglades. The Nautilus, 89: 21 -23. 

LACANILAO, F., 1990, Reproduction of the golden 
apple snail (Ampullariidae): egg mass, hatching, 
and incubation. Philippine Journal of Science, 
119: 95-105. 

LACH, L., D. K. BRITTON, R. J. RUNDELL & R. H. 
COWIE, 2000, Food preference and reproductive 
plasticity in an invasive freshwater snail. Biologi- 
cal Invasions, 2: 279-288. 

LITSINGER, J. A. & D. B. ESTAÑO, 1993, Manage- 
ment of the golden apple snail Fomacea canali- 
culata (Lamarck) in rice. Crop Protection, 12: 
363-370. 

MALAGNINO, E. C, 1989, Evolución del sistema 
fluvial de la Provincia de Buenos Aires desde el 
Pleistocene hasta la actualidad. Actas de las Se- 
gundas Jornadas Geológicas Bonaerenses: 
201-211, Bahía Blanca. 

MARTÍN, P. R., A. L. ESTEBENET & N. J. CAZ- 
ZANIGA, 2001, Factors affecting the distribution 
of Fornácea canaliculata (Gastropoda: Ampullari- 



LIFE-HISTORY VARIATION IN POMACEA 



163 



idae) along its southernmost natural limit. Mala- 
cologia, 43: 13-23. 

MARTIN, S. M., 1986, Ciclo reproductivo de Am- 
pullaria canaliculata (Gastropoda; Ampullariidae) 
en el área rioplatense. Neotrópica, 32: 171 -181 . 

SCHNORBACH, H. J., 1 995, The golden apple snail 
(Pomacea canaliculata Lamarck) an increasingly 
important pest in rice, and methods of control with 
Bayluscid Pflanzenschutz-Nachrichten Bayer, 
48:313-346. 

SCIAN, B. & M. DONNARI, 1997, Aplicación del 
índice Z de Palmer para la comparación de se- 
quías en las regiones trigueras II, IV y V sur de 
Argentina. Revista de la Facultad de Agronomía, 
Universidad de Buenos Aires, 1 7: 41 -46. 

TANAKA, K., T WATANABE, H. HIGUCHI, K. 
MIYAMOTO, Y YUSA, T KIYONAGA, H. Kl- 
YOTA, Y SUZUKI & T WADA, 1999, Density de- 



pendent growth and reproduction of the apple 
snail, Pomacea canaliculata: a density manipula- 
tion experiment in a paddy field. Research in 
Population Ecology, 41: 253-262. 

TURNER, R. L., 1996, Use of stems of emergent 
plants for oviposition by the Florida applesnail, 
Pomacea paludosa, and implications for marsh 
management. Florida Scientist, 59: 34-49. 

WADA, T, 1997, Introduction of the apple snail Po- 
macea canaliculata and its impact on rice agri- 
culture. Proceedings of the International Work- 
shop on Biological Invasions of Ecosystems by 
Pests and Beneficial Organisms, pp. 170-180, 
Tsukuba. 

Revised ms. accepted 9 August 2001 



MALACOLOGIA, 2002, 44(1): 165-168 

CHROMOSOMES OF PISIDIUM COREANUM {В\УА1\/\А: VENEROIDA: 
CORBICULOIDEA: PISIDIIDAE), A KOREAN FRESHWATER CLAM 

Gab-Man ParkJ Tai-Soon Yong,^ Kyung-il Im,^ & John B. Burch^ 

ABSTRACT 

Polyploidy is reported as occurring in Pisidiidae, being found in Pisidium coreanum from 
Korea. The chromosome number is n = 95 and 2n = 1 90. Because of the large number of its chro- 
mosomes, P. coreanum is obviously a polyploid species. The basic chromosome number for the 
superfamily Corbiculoidea is X = 18 and X = 19. The polyploid condition found in P. coreanum 
probably originally resulted from a single event. Once the polyploid condition was reached, the 
stability of this number has been maintained. 

Key words: Pisidium coreanum, chromosome, Pisidiidae, polyploid, Korea. 



INTRODUCTION 

The bivalve superfamily Corbiculoidea has 
two families of special interest, Corbiculidae 
and Pisidiidae, both with wide geographical 
and ecological distributions (Clarke, 1973; 
Burch, 1975; Kuiper, 1987; Kwon & Park, 
1993). The Corbiculoidea are represented in 
Korea by six species of the Corbiculidae and 
two species of the Pisidiidae (Kwon et al., 
1993). The Corbiculidae are Asian in origin, 
but in relatively recent times they have been 
spread to many other parts of the world 
through human commerce. Particularly in 
Asia, corbiculid clams are an important food 
source, not only for wildlife, including fish, but 
also for humans. Sphaehid clams, on the other 
hand, are not economically important, other 
than providing an important link in various 
aquatic food chains. But from a biological 
standpoint, they are significant because of 
their peculiar lifestyles. They live in many dif- 
ferent habitats and environments, and some 
species seem to be nearly cosmopolitan 
(Burch, 1975). All the species presumably are 
hermaphroditic, with the same individuals pro- 
ducing both sperm and ova, although direct 
observations have been made for only a few 
species. Fertilization is internal, and the young 
are brooded in special gill chambers of the par- 
ent, in some species until the young are rela- 
tively large (Park & Kwon, 1 993). However, ex- 
actly how fertilization and development take 
place, that is, whether by cross- or self-fertil- 
ization, or by some type of parthenogenesis, is 



not known, nor is it known how polyploidy 
came about (i.e., are the species autopoly- 
ploids or allopolyploids, and in the later case 
segmental or genomic?). In the marine bivalve 
genus Lasaea, which shares many similarities 
with the corbiculoidean clams (Ó Foighil & 
Smith, 1995), polyploidy is also a common oc- 
currence, and here, at least in the polyploid 
clones, parthenogenetic development, trig- 
gered by autosperm (without syngamy) is the 
rule (Ó Foighil & Thiriot-Quiévreux, 1 991 ). Our 
preliminary observations on gametogenesis in 
sphaeriid clams suggest that it is likely that 
some, perhaps many, species of corbicu- 
loidean clams have similar reproductive 
strategies. 

Many species are known to be capable of 
propagation by self-fertilization when the 
more normal iDiparental mating behavior is 
prevented. In such groups, a large amount of 
polyploidy might be expected, but chromo- 
some surveys have shown that this is not the 
case. Natural polyploidy has also been ob- 
served in Argopecten purpuratus (Alvarez & 
Lozada, 1992), Sphaerium striatinum (Lee, 
1999), in Lasaea spp. (Thiriot-Quiévreux et 
al., 1988, 1989; Ó Foighil & Thiriot-Quiévreux, 
1999) and in Corbicula spp. (Park et al., 
2000). The first Corbiculoidea clams in which 
polyploidy was detected was Corbicula (Cor- 
biculina) leana (Okamoto & Arimoto, 1986). 
The chromosome numbers have been deter- 
mined for several other species belonging to 
the order Corbiculoidea (Table 1). In these, 
the chromosome number varies from n = 1 2 to 



Department of Parasitology, Yonsel University College of Medicine, Seoul 120-752, Korea; tsyong212@yumc.yonsei.ac.kr 
^Museum of Zoology and Department of Biology, College of Literature, Science and the Arts, and School of Natural Re- 
sources, University of Michigan, Ann Arbor, Michigan 48109, USA 

165 



166 



PARKETAL 



TABLE 1. Chromosome numbers in Corbiculoidea. 



Species 



Chromosome number 



References 



Corbicula fluminea 
С papyracea 
С leana 
С CO lo rata 
С japónica 
С. sandai 
"С. leana" 
Musculium securis 
Sphaehum corneum 
S. occidentale 
S. striatinum 
S. striatinum 
Pisidium casertanum 
P. casertanum 
P. coreanum 



54 (3n) 

54 (3n) 

54 (3n) 

38 (2n) 

38 (2n) 

36 (2n) 

24 

approx. 247 

36 (2n) 

approx. 209 

approx. 68-98 

approx. 152 

approx. 150, 180 

approx. 190 

190 (2n) 



Parketal., 2000 
Parketal., 2000 
Okamoto & Arimoto, 1986 
Parketal., 2000 
Okamoto & Arimoto, 1986 
Okamoto & Arimoto, 1986 
Nadamitsu & Kanai, 1978 
Burch etal., 1998 
Key!, 1956 
Burch etal., 1998 
Woods, 1931 
Lee, 1999 

Barsiene et al., 1996 
Burch etal., 1998 
Present study 



n = 76. The chromosome number of С flu- 
minea, С papyracea and С leana suggests 
that these species may be members of a 3n 
species that has been established by poly- 
ploid evolution. The chromosome numbers of 
Sphaeriidae have been reported for five 
species (Table 1). The chromosome numbers 
obtained in the Sphaeriidae are all vei7 large 
(over 150 mitotic chromosomes), except for 
the European S. corneum (2n = 36). This sug- 
gests that the large numbers of chromosomes 
in this family represent polyploids. 

This study presents the chromosome 
analysis of P. coreanum based on mitotic 
metaphase chromosomes. 



cell suspension was then pipetted by a micro- 
hematocrit capillary tube and dropped onto a 
clean slide glass pre-cooled to 4°C. The cells 
on the slide were air-dried and then stained 
for 10 min with 4% Giemsa (Gurr R66) solu- 
tion made up in 0.1 M phosphate buffer, pH 
7.0. The prepared slides were observed 
under an Olympus (BX50F-3) microscope 
with a 1 0OX (n.a. 1 .25) oil immersion objective 
and a 10X ocular Voucher specimens of the 
shells used in this investigation have been 
placed in the Department of Parasitology, 
Yonsei University College of Medicine, Korea. 

RESULTS 



MATERIALSAND METHODS 

Twenty-one specimens of P. coreanum 
were collected from a spring pond located in 
Bongmung-ri, Chunchon-city, Kangwon-do, 
Korea, from June to September 2000, and ex- 
amined shortly after collection. 

Chromosome preparations were made 
from gonadal tissues by the standard air-dry- 
ing method. The live specimens were set 
aside for one week in a petri-dish containing 
10 ml of distilled water with 0.3 ml of 0.05% 
colchicine solution. The treated tissues were 
dissected and minced with needles in a hypo- 
tonic 0.01% NaCI solution. Separated cells 
were collected by centhfugation at 930 g for 
10 min. These cells were fixed in freshly 
mixed modified Carney's fixative (three parts 
methyl alcohol and one part glacial acetic 
acid). The supernatant was replaced by fresh 
fixative. The centhfugation (930 g, 10 min) 
was repeated two more times. A drop of the 



The mean shell size determined in this 
study consisted of a measured shell length of 
4.2 mm, shell height of 4.1 mm, and shell 
breath of 2.0 mm. Twelve individuals were ex- 
amined for chromosomes. Eighteen cells 
clearly had 190 chromosomes. Chromosome 
numbers of n = 95 and 2n = 1 90 were counted 
(Fig. 1). Ninety-five bivalents were observed 
during late prophase (diakinesis) (Fig. 1A). 
The chromosome types of this species con- 
sisted of metacentric, submetacentric and te- 
locentric chromosomes as shown (Fig. IB). 
However, this chromosome figure was not 
sufficient for analyzing karyotypes, not only 
because of the large numbers but also the 
small size. The longest dimension of the 
largest metaphase bivalent was only 3.6 jum. 

DISCUSSION 

Polyploidy is the multiplication of the normal 
chromosome number of an organism. Be- 



CHROMOSOME OF PISIDIUM COREANUM 



167 




FIG. 1 . Chromosomes of Pisidium coreanum. A, diakinesis (n = 95). B, metaphase chromosomes (2n = 190) 
Scale bars indicate 5 |im. 



cause of the inability of a newly derived poly- 
ploid to breed with its sibling diploid it is usu- 
ally chromosomally sterile. Accordingly, poly- 
ploidy is a method, and the only clearly 
established one, for instantaneous speciation 
(Wagner et al., 1993). The occurrence of 
triploid chromosomes in three species of the 
genus Corbicula has been reported (Okamoto 
& Arimoto, 1986; Park et al., 2000) (Table 1). 
From the numbers found in the three species 
of Corbicula, each of these species is poly- 
ploid. The family Pisidiidae contains three sub- 
families, the Pisidiinae, Sphaeriinae and Eu- 
perinae. In the Sphaeiinae, four species has 
been studied, Sphaerium corneum (Keyl, 
1956), S. occidentale (Burch et al., 1998), S. 
stríatinum (Woods, 1931; Lee, 1999) and 
Musculium securis (Burch et al., 1998), and 
the mitotic chromosome numbers of these 
species have been reported ranging from 36 to 
approximately 152. In the Pisidiinae, the chro- 
mosome numbers of P. casertanum is 2n = ap- 
proximately 247 as counted by Burch et al. 
(1998). This study, the first report of polyploidy 
in Pisidiidae clams in Korea, determined that 
the chromosome number of P. coreanum is 2n 
= 190. Polyploidy is probably an important 
method of evolution in the cosmopolitan and 
common genus Pisidium, and may account for 
the morphological patterns that have made 
taxonomy so difficult. Most molluscan groups 
are generally conservative with regard to chro- 
mosomal change (Patterson, 1969). However, 



the chromosome numbers are very variable 
among Mytilidae and Pectinidae (Nakamura, 
1985). Also, the chromosome number within 
two families of the order Veneroidea, 18 to 
95 pairs of chromosomes, is not constant. 
The origin of polyploidy in the Pisidiidae is 
still speculative. In mollusks, Burch & Huber 
(1966) suggested that polyploidy came about 
in the African-Near Eastern Bulinus by hy- 
bridization, followed by a doubling of the chro- 
mosome number, that is, allopolyploidy was 
involved. Cytological evidence presented by 
Goldman et al. (1983) tends to support this 
conclusion. Certainly the ecological conditions 
where most of the ploidy states in Bulinus 
occur- the Ethiopian highlands, located near 
the equator - provide the opportune physical 
characteristics in which polyploid events might 
be expected to occur (Patterson & Burch, 
1988). 

Future studies of the chromosomes of other 
species of the Pisidiidae family are needed to 
document the variability of the chromosome 
number within this group. 

LITERATURE CITED 

ALVAREZ, S. & L. E. LOZADA, 1992, Spontaneous 
triploidy in Chilean scallop Argopecten purpura- 
ius(Lamark 1819) (Bivalvia, Pectinoidae). Inves- 
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BARSIENÉ, J., G. TAPIA & D. BARSYTE, 1996, 
Chromosomes of mollusks inhabiting some 



168 



PARKETAL 



mountain springs of eastern Spain. Journal of 
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96 pp. 

BURCH, J. B. & J. M. HUBER, 1966, Polyploidy in 
mollusks. Malacologia. 5: 41-43. 

BURCH, J. В., G. M. PARK & E. Y. CHUNG, 1998, 
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CLARKE, A. H., 1973, The freshwater mollusca of 
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KEYL, H. G., 1956, Beobachtungen über die :^- 
meiose der Muschel Sphaehum corneum. Chro- 
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KUIPER, J. G. J., 1987, Systematic rank, synonymy 
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KWON, O. K. & J. С PARK, 1993, Studies of the 
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idae). The Korean Journal of Malacology, 9: 
33-38. 

KWON, O. K., G. M. PARK & J. S. LEE, 1993, 
Coloured shells of Korea. Academy Publishing 
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LEE, T, 1999, Polyploidy and meiosis in the fresh- 
water clam Sphaehum striatinum (Lamark) and 
chromosome numbers in the Sphaeriidae (Bi- 
valvia, Veneroida). Cytologia Tokyo, 64: 247- 
252. 

NADAMITSU, S. & T KANAI, 1978, On the chro- 
mosomes of the three species in two families in 
freshwater Bivalvia. Bulletin Hiroshima Women's 
University. 10: 1-5. 

NAKAMURA, H. K., 1985, A review of molluscan cy- 
togenetic information based on the CISMOCH- 
Computerized Index System for Molluscan Chro- 
mosomes. Bivalvia, Polyplacophora and 
Cephalopoda. Venus, 44: 193-225. 

Ó FOIGHIL, D. & M. J. SMITH, 1995, Evolution of 
asexuality in the cosmopolitan marine clam 
Lasaea. Evolution, 49: 140-150. 

Ó FOIGHIL, D. & С THIRIOT-QUIÉVREUX, 1991, 



Ploidy and pronuclear interaction in northeastern 
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Sympatric Australian Lasaea species (Mollusca, 
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OKAMOTO, A. & B. ARIMOTO, 1986, Chromo- 
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(Corbiculina) leana (Bivalvia: Corbiculidae). 
Venus, Л5: 194-202. 

PARK, J. С & О. К. KWON, 1993, Studies on the 
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idae). The Korean Journal of Malacology, 9: 
33-38. 

PARK, G. M., T S. YONG., K. IM & E. Y CHUNG, 
2000, Karyotypes of three species of Corbicula 
(Bivalvia: Veneroida) in Korea. Journal of Shell- 
fish Research, 19: 979-982. 

PATTERSON, С M., 1969, Chromosomes of mol- 
luscs. Proceedings of the Symposium on Mol- 
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635-689. 

PATTERSON, С M. & В. BURCH, 1988, Chromo- 
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FRETTER & J. PEAKE, eds., Pulmonales, Vol. 2A, 
Systematics, evolution and ecology. Academic 
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540 pp. 

THIRIOT-OUIÉVREUX С, A. M. INSUA POMBO & 
R ALBERT, 1989, Polyploidie chez un bivalve in- 
cubant, Lasaea rubra (Montagu). Comptes Ren- 
dus de Séances d'Académie de Science, Pans, 
308: 115-120. 

THIRIOT-QUIÉVREUX C, J. SOYER, F. de 
BOVÉE & P ALBERT 1988, Unusual chromo- 
some complement in the brooding bivalve 
Lasaea consanguínea. Genética, 76: 143-151. 

WAGNER, R. R, M. R MAGUIRE & R. L 
STALLINGS, 1993, Variation in chromatin organi- 
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WOODS, R H. 1931, History of the germ cells in 
Sphaehum striatnum (Lam.). Journal of Morphol- 
ogy and Physiology, 51 : 545-595. 

Revised ms. accepted 15 August 2001 



f 



MALACOLOGIA 



International Journal of Malacology 



^ 



^4 









Vol. 44(1) 2002 



^i^A|4Í,4., 



Publication dates 

Vol. 34, No. 1-2 9 Sep. 1992 

Vol. 35, No. 1 14 Jul. 1993 

Vol. 35, No. 2 2 Dec. 1993 

Vol. 36, No. 1-2 8 Jan. 1995 

Vol. 37, No. 1 13 Nov. 1995 

Vol.37, No. 2 8 Mar. 1996 

Vol. 38, No. 1-2 17 Dec. 1996 

Vol. 39, No. 1-2 13 May 1998 

Vol.40, No. 1-2 17 Dec. 1998 

Vol.41, No. 1 22 Sep. 1999 

Vol.41, No. 2 31 Dec. 1999 

Vol.42, No. 1-2 18 Oct. 2000 

Vol.43, No. 1-2 20 Aug. 2001 



VOL. 44, NO. 1 MALACOLOGIA 2002 

CONTENTS 

ANDREA С ALFARO & ANDREW G. JEFFS 

Small-scale Mussel Settlement Patterns Within Morphologically Distinct 
Substrata at Ninety Mile Beach, Northern New Zealand 1 

EUGENE V. COAN 

The Eastern Pacific Recent Species of the Corbulidae (Bivalvia) 47 

ROBERT T DILLON, JR., & ANDREW J. REED 

A Survey of Genetic Variation at Allozyme Loci among Goniobasis 
Populations Inhabiting Atlantic Drainages of the Carolinas 23 

VASILIS K. DIMITRIADIS & VASILIKI KONSTANTINIDOU 

Origin of the Excretory Cells in the Digestive Gland of the Land Snail 

Helix lucorum 1 45 

SINOS GIOKAS & MOYSIS MYLONAS 

Spatial Distribution, Density and Life History in Four Albinaria Species 
(Gastropoda, Pulmonata, Clausiliidae) 33 

GENNADY M. KAMENEV 

Genus Parvithracia (Bivalvia: Thraciidae) with Descriptions of a New 
Subgenus and Two New Species from the Northwestern Pacific . 107 

PABLO R. MARTIN & ALEJANDRA L. ESTEBENET 

Interpopulation Variation in Life-history Traits of Pomacea canaliculata 
(Gastropoda: Ampullahidae) in Southwestern Buenos Aires Province, 
Argentina 1 53 

LUIS A. MERCADO, SERGIO H. MARSHALL, & GLORIA M. ARENAS 

Detection of Phenoloxidase (PO) in Hemocytes of the Clam Venus antiqua 17 

GAB-MAN PARK, TAI-SOON YONG, KYUNG-IL IM, & JOHN B. BURCH 

Chromosomes of Pisidium coreanum (Bivalvia: Veneroida: Corbiculoidea: 
Pisidiidae), a Korean Freshwater Clam 165 

ANATOLYA. VARAKSIN, EUGENIA A. PIMENOVA, CALINAS. VARAKSINA, & 

LYDIA T FROLOVA 

Localization of Nadph-diaphorase-positive Elements in the Intestine of the 
Mussel Crenomytilus grayanus (Mollusca: Bivalvia) 1 35 



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VOL. 44, NO. 1 MALACOLOGIA 2002 

CONTENTS 

ANDREA С ALFARO & ANDREW G. JEFFS 

Small-scale Mussel Settlement Patterns Within Morphologically Distinct 
Substrata at Ninety Mile Beach, Northern New Zealand 1 

LUIS A. MERCADO, SERGIO H. MARSHALL, & GLORIA M. ARENAS 

Detection of Phenoloxidase (PO) in Hemocytes of the Clam Venus antigua 17 

ROBERT T DILLON, JR., & ANDREW J. REED 

A Survey of Genetic Variation at Allozyme Loci among Goniobasis 
Populations Inhabiting Atlantic Drainages of the Carolinas 23 

SINOS GIOKAS & MOYSIS MYLONAS 

Spatial Distribution, Density and Life History in Four Albinaria Species 
(Gastropoda, Pulmonata, Clausiliidae) 33 

EUGENE VCOAN 

The Eastern Pacific Recent Species of the Corbulidae (Bivalvia) 47 

GENNADY M. KAMENEV 

Genus Parvithracia (Bivalvia: Thraciidae) with Descriptions of a New 
Subgenus and Two New Species from the Northwestern Pacific 107 

ANATOLY A. VARAKSIN, EUGENIA A. PIMENOVA, GALINA S. VARAKSINA, & 

LYDIA T FROLOVA 

Localization of Nadph-diaphorase-positive Elements in the Intestine of the 
Mussel Crenomytilus grayanus (Mollusca: Bivalvia) 135 

VASILIS K. DIMITRIADIS & VASILIKI KONSTANTINIDOU 

Origin of the Excretory Cells in the Digestive Gland of the Land Snail 

Helix lucorum 1 45 

PABLO R. MARTIN & ALEJANDRA L. ESTEBENET 

Interpopulation Variation in Life-history Traits of Pomacea canaliculata 
(Gastropoda: Ampullariidae) in Southwestern Buenos Aires Province, 
Argentina 1 53 

GAB-MAN PARK, TAI-SOON YONG, KYUNG-IL IM, & JOHN B. BURCH 

Chromosomes of Pisidium coreanum (Bivalvia: Veneroida: Corbiculoidea: 
Pisidiidae), a Korean Freshwater Clam 165 



2002 
EDITORIAL BOARD 



J.A.ALLEN 

Marine Biological Station 

Millport. United Kingdom 

jallen@udcf.gla.ac.uk 

E. E. BINDER 

Museum d'Histoire Naturelle 

Geneve. Switzerland 

P. BOUCHET 

Muséum National d'Histoire Naturelle 

Paris, France 

bouchet@cimrs 1 . mnhn. fr 

P. CALOW 

University of Sheffield 
United Kingdom 

R. CAMERON 
Stieffield 
United Kingdom 
R.Cameron @ sheffield.ac.uk 

J. G. CARTER 

University of North Carolina 

Chapel Hill. U.S.A. 

MARYVONNE CHARRIER 

Universite de Rennes 

France 

Maryvonne. Charrier @ univ-rennes 1 . fr 

R. H. COWIE 
University of Hawaii 
Honolulu. Hi. U.S.A. 

A. H. CLARKE, Jr. 
Portland, Texas. U.S.A. 

B. С CLARKE 
University of Nottingham 
United Kingdom 

R. DILLON 

College of Charleston 

SC. U.S.A. 

C.J. DUNCAN 
University of Liverpool 
United Kingdom 

D.J. EERNISSE 
California State University 
Fullerton. U.S.A. 

E. GITTENBERGER 
Rijksmuseum van Natuurlijke Historie 
Leiden, Netherlands 

sbu2eg @ rulsfb. leidenuniv. de 

F. GIUSTI 

Universita di Siena, Italy 
giustif@unisi.it 



A. N. GOLIKOV 
Zoological Institute 
St. Petersburg, Russia 



S.J. GOULD 




Harvard University 
Cambridge, Mass., U.S.A. 


|^^ICZ 


A. V. GROSSU 
Universitatea Bucuresti 


IDRARY 


Romania 
T. HABE 


SLP - 6 2002 


Tokai University 
Shimizu, Japan 

R. HANLON 

MPihna Rinlnniral 1 ahnrafnr 


HARVARD 
'j.N. VARSITY 



Woods Hole, Mass., U.S.A. 

G. HASZPRUNAR 

Zoologische Staatssammlung Muenchen 

Muenchen. Germany 

haszi @zi. biologie, uni-muenchen. de 

J. M. HEALY 

University of Queensland 

Australia 

jhealy @ zoology, uq. edu.au 

D. M. HILLIS 
University of Texas 
Austin, U.S.A. 

K. E. HOAGLAND 

Council for Undergraduate Research 

Washington. DC. U.S.A. 

Elaine@cur.org 

B. HUBENDICK 
Naturhistoriska Museet 
Göteborg, Sweden 

S. HUNT 
Lancashire 
United Kingdom 

R. JANSSEN 

Forschungsinstitut Senckenberg. 
Frankfurt am Main, Germany 

M. S. JOHNSON 

University of Western Australia 
Nedlands, WA, Australia 
msj@cyllene. uwa.edu.au 

R. N. KILBURN 
Natal Museum 
Pietermaritzburg. South Africa 

MA. KLAPPENBACH 

Museo Nacional de Historia Natural 

Montevideo, Uruguay 



J. KNUDSEN 

Zoologisk Institut Museum 

Kobenhavn. Denmark 

С LYDEARD 
University of Alabama 
Tuscaloosa. U.S.A. 
clydeard @ biology, as.ua. edu 

С MEIER-BROOK 
Tropenmedizinisches Institut 
Tubingen. Germany 

H. K. MIENIS 

Hebrew University of Jerusalem 

Israel 

J. E. MORTON 
The University 
Auckland. New Zealand 

J. J. MURRAY. Jr. 
University of Virginia 
Charlottesville. U.S.A. 

R. NATARAJAN 
Manne Biological Station 
Porto Novo. India 

DIARMAIDOTOIGHIL 
University of Michigan 
Ann Arbor U.S.A. 

J. OKLAND 
University of Oslo 
Norway 

T. OKUTANI 
University of Fisheries 
Tokyo. Japan 

W. L. PARAENSE 

Instituto Oswalde Cruz. Rio de Janeiro 

Brazil 

J. J. PARODIZ 
Carnegie Museum 
Pittsburgh. U.S.A. 

R. PIPE 

Plymouth Marine Laboratory 
Devon, United Kingdom 
RKPI @ wpo. nerc. ac. uk 

J. P. POINTIER 

Ecole Pratique des Hautes Etudes 
Perpignan Cedex, France 
pointier @ gala, univ-perp. fr 

W. F. PONDER 
Australian Museum 
Sydney 



01 Z. Y. 

Academia Sínica 

Qingdao. People s Republic of China 

D. G. REÍD 

The Natural History Museum 

London. United Kingdom 

S. G. SEGERSTRALE 
Institute of Marine Research 
Helsinki. Finland 

A. STANCZYKOWSKA 
Siedlce. Poland 

F. STARMÜHLNER 

Zoologisches Institut der Universität 

Wien, Austria 

Y. I. STAROBOGATOV 
Zoological Institute 
St. Petersburg, Russia 

J. STUARDO 
Universidad de Chile 
Valparaiso 

C.THIRIOT 

University P. et M. Curie 
Villefranche-sur-Mer France 
thiriot@obs-vlfr.fr 

S. TILLIER 

Museum National d'Histoire Naturelle 

Paris, France 

J. A.M. VAN DEN BIGGELAAR 
University of Utrecht 
The Netherlands 

N. H. VERDONK 
Rijksuniversiteit 
Utrecht. Netherlands 

H. WÄGELE 

Ruhr-Universität Bochum 

Germany 

Heike. Waegele @ ruhr-uni-bochum. de 

ANDERS WAREN 

Swedish Museum of Natural History 

Stockholm. Sweden 

B. R. WILSON 

Dept. Conservation and Land Management 
Kallaroo, Western Australia 

H. ZEISSLER 
Leipzig, Germany 

A. ZILCH 

Forschungsinstitut Senckenberg 

Frankfurt am Main. Germany 



MALACOLOGIA, 2002, 44(2): 175-222 

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 
(OCTOPODIDAE, CEPHALOPODA) 

Bent Muus 

Zoological Museum, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, 

Denmark; bbisballe @zmuc. ku. dk 

ABSTRACT 

Profound confusion among deep-water octopods of the genera Bathypolypus and Benthocto- 
pus stems from misidentifications of the Atlantic Bathypolypus arcticus (Presch, 1849), which is 
here shown to consist of at least two allopatric species— the nominal form is a truly arctic 
species; the other, Octopus ba/rd/'/ Verrill, 1873, is a boreal cold-water form, which is reinstated 
as a Bathypolypus. A\h\rä species, Bathypolypus pugniger, n. sp., of uncertain systematic posi- 
tion, occurs in a narrow belt along the southern limit of B. arcticus. 

Synonyms are discussed, and evidence is advanced that all records of Benthoctopus pisca- 
torum (Verrill, 1879) are erroneous and that the type specimen is identical with B. bairdii. 

Using an amended generic diagnosis, a critical survey of all Bathypolypus species is given. Six 
species, all Atlantic, are recognized. Distribution and habitats are given, and a key and tables 
provided to facilitate identifications. 

Key words: Bathypolypus, Benthoctopus, North Atlantic, Bathypolypodinae (Cephalopoda). 



INTRODUCTION 

The octopod genera Bathypolypus and 
Benthoctopus comprise deep-water species 
that have caused much taxonomic confusion 
since Victor Presch described the first species, 
Octopus arcticus, from southwest Greenland 
waters in 1849. 

The two genera were established in a rather 
casual way by Grimpe (1921), the respective 
type species being Bathypolypus arcticus 
(Presch, 1849) and Benthoctopus piscatorum 
(Verrill, 1879). Robson (1924b, 1927) defined 
the two genera and then treated all known 
species in his monograph on octopods (1 932). 

From Robson's work on B. arcticus, supple- 
mented by findings of Hoyle (1886), Pfeffer 
(1908), Joubin (1920), Grimpe (1933), Kon- 
dakov (1936), Adam (1939), Bruun (1945), 
Jaeckel (1958), Kumpf (1958), Macalaster 
(1976), Perez-Gandaras & Guerra (1978), 
Nesis (1987), and Voss (1988b), B. arcticus 
would appear to be a highly variable species 
with a huge geographic range in the North At- 
lantic. It seems in fact to be the most abun- 
dant bottom-living octopod on the upper half 
of the continental slopes from Florida to West 
Greenland, along both sides of the ridge be- 
tween Greenland and Scotland and from the 
Bay of Biscay to the Barents Sea, Svalbard, 
and even the Kara Sea. 



In a report on the cephalopods from the 
Danish Godthaab Expedition 1928 to Davis 
Strait and Baffin Bay (Muus, 1962), I dis- 
cussed specimens of B. arcticus from near 
the type locality. This species was easily iden- 
tified by means of the type material in ZMUC. 
However, I also described a new, rather abun- 
dant species, Bathypolypus proschi, which in 
spite of a superficial similarity diverged from 
B. arcticus, especially in eye size, ligula, 
radula, and spermatophores. 

When years later I inspected the Norwegian 
collections, I was baffled to find B. arcticus 
only in the arctic material and B. proschi, 
along the Norwegian West coast, labelled as 
B. arcticus. It dawned upon me that there are 
still serious identification problems, not only 
with B. arcticus but, as I would learn, also with 
other species of the genera Bathypolypus and 
Benthoctopus, and that a revision was badly 
needed. 

Robson (1932) expressed the need for 
much larger collections to settle the questions 
left by his revision. Fortunately, growing trawl 
and dredge activity along the continental 
slopes has procured substantial additional 
material from most parts of the North Atlantic. 
Thus, I have had over 600 specimens at my 
disposal and ample opportunity to inspect rel- 
evant type material and most of the speci- 
mens treated by previous authors to settle 



175 



176 



lUUS 



what seemed to be a case of confusion 
among sibling species. 



MATERIAL 

The main revision concerns North Atlantic 
material deposited in various museums as 
species placed in one or the other of the gen- 
era Bathypolypus and Benthoctopus. supple- 
mented with recently collected material from 
Faroese and Icelandic waters (the BIOFAR 
and BIOICE projects respectively) and from 
the deep-sea prawn fisheries off southwest- 
ern Greenland. The material is listed in Ap- 
pendix I. 



METHODS 

Measurements, counts and calculation of 
indices were performed according to the stan- 
dard for descriptive characters of octopods 
given by Roper & Voss (1983). The soft body 
and variable state of preservation, however, 
make many measurements of octopods sub- 
jective, and repeated measurements of the 
same specimens often gave results deviating 
5-10%. If different persons perform the mea- 
surements, the deviation may be even 
greater, which I noticed when I remeasured 
specimens treated by other authors. Meristic 
characters, such as number of suckers and 
lamellae copulatoriae of the hectocotylus, 
were often more reliable. Also the eye lens 
was useful, being a solid structure, and sup- 
plementing the dorsal mantle length as a 
standard of size. The lens was extracted with 
tweezers through a slit cut horizontally in the 
lower part of the eyeball. Remnants of the 
darkish primary cornea suspending the lens 
at "equator" was removed, and the diameter 
was measured gently with a calipers, or under 
a microscope. 

Due to the slightly subjective manner in 
which many measurements of octopods have 
to be conducted, all graphs and figures com- 
paring species are based on my own figures 
to minimize bias. Only in Figure 9, a few 
meristic data from Macalaster (1976) are in- 
cluded. 

For convenience, the standard measure- 
ments used in this work are presented below. 
In addition to conventional indices, I have 
added some new indices that might be useful 
in taxonomic work on other octopod genera. 

Note that as a slight deviation from stan- 



dards as given by Roper & Voss (1983), ligula 
and calamus length are measured from the 
center of last sucker, not from its rim. It is ac- 
curate and eases the use of calipers. Mantle 
length is always dorsal ML (midpoint between 
eyes to mantle apex). 



Measurements 


Indices 


AL; 


Arm length 


ALI: °o of ML 


CaL: 


Calamus length 


CaLI: % of LigL 


ED: 


Diameter of eye ball 


EDI: % of ML 


HcL: 


Hectocotylized arm length 


HcLI:%of ML 


HW: 


Head width 


HWI: % of ML 


LD: 


Lens diameter 


LDEDI: LD % of ED (Muus) 


LIgL: 


Lígula length 


LigLI: % of HcL 


ML: 


Mantle length 


MLTLI:%ofTL 


MW: 


Mantle width 


MWI: % of ML 
OAI:HcL%of 3, leftAL 


SD: 


Sucker diameter (largest) 


SDI: % of ML 

SDLDI: SD % of LD (Muus) 

SDEDI: SD % of ED (Muus) 


SpL: 


Spermatophore length 


SpLI: % of ML 


SpRL: 


Sperm reservoir length 


SpRI: % of SpL 


SpW: 
TL: 


Spermatophore width 
Total length 


SpWI:%ofSpL 


WD: 


Web depth 


WDI: °o of longest armpair 




(web sectors: A,B,C,D,E) 


WDMI: WD % of ML 



Counts 

LamC: Laminae copulatoriae LamCI: LamC % of SHcC (Muus) 
SHcC: Suckers on hectocotylized arm 

Acronyms of museum collections used: 

BMNH British Museum of Natural History, London, England 
IMNH Icelandic Museum of Natural History, Reykjavik. Iceland 
IRSNB Institut Royal des Sciences Naturelles de Belgique, 

Brussels, Belgium 
MNHN Muséum National D'Histoire Naturelle, Pans, France 
MNHT Museum of Natural History. Tórshavn, Faroe Islands, 

Denmark 
TMDZ Tromso Museum, Department of Zoology, Norway 
USNM National Museum of Natural History, Washington, DC, 

USA 
ZIASP Zoological Institute, Academy of Sciences, 

St. Petersburg, Russia 
ZMUB Zoological Museum University of Bergen, Nonway 
ZMUG Zoological Museum University of Copenhagen, Denmark 
ZMUG Zoological Museum University of Oslo, Norway 



REDESCRIPTION OF BATHYPOLYPUS 
ARCTICUS {PROSCH, 1849) 

The earliest recognition of octopodids in 
Greenland was by Fabricius (1780: 353). His 
Sepia octopodia is, however, at most a nomen 
dubium, because the meagre description fits 
any of the now known species, and no type 
material exists. Sepia groenlandica Dewhurst 
(1834: 263) is a nomen nudum, because no 
description was given, and Octopus granula- 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 177 



tus Möller (1843: 77) is both a nomen nudum 
and a junior homonym of O. granulatus 
Lamarck, 1798. 

The first direct reference to B. arcticus 
(under the name ''Octopus granulatus^') was in 
Reinhardt & Prosch's (1846) paper on the 
anatomy of Cirroteuthis mullen, in which 
some specimens were used for anatomical 
comparison, and it was stated for the first time 
that both species are devoid of an ink sac. 
The authors mentioned that they had many 
specimens of the Greenland species at their 
disposal "different in sex, age and develop- 
ment" [my translation]. 

In 1849, Prosch described Octopus arcticus 
based on some of the specimens mentioned 
in 1846, which were sent from west Green- 
land in the early 1840s. He did not designate 
a type specimen, and the lectotype, and two 
paralectotypes described below were identi- 
fied as prosch's original material and marked 
"types" about 1930 by the curator R. Spärck, 
among the specimens that were retained in 
the ZMUC. 

Material Examined 

ZMUC CEP-13: Lectotype of Bathypolypus 
arcticus (Prosch, 1849), called "holotype" in 
Kristensen & Knudsen (1983): Male, ML: 42 
mm. Label: Greenland, K. M. Jörgensen, Au- 
gust 26, 1841; CEP-14, paralectotype: Fe- 
male, ML: about 50 mm. Label: Greenland, 
K. M. Jörgensen, July 27, 1840; CEP-15, 
paralectotype: The dissected parts of a male 
used by Prosch (1849) for his drawings (his 
figs. 1-3). Depository: ZMUC. 

Further 120 specimens from arctic and sub- 
arctic North Atlantic as listed in Appendix I. 

Remarks on the Type Series 

The lectotype has been dissected and 
some measurements are not reliable. The 
funnel organ is lost, but was formerly present 



and clearly VV-shaped. Measurements and 
indices are given in Table 1 . 

Paralectotype CEP-14: Female, ML about 
50 mm. Has been dissected, and most mea- 
surements are unreliable. SD: 2-6 mm, ED: 
12 mm. Crop diverticulum present containing 
polychete bristles and crustacean remains. 
Gonads filled with eggs. 

Paralectotype CEP-15: The dissected parts 
of a male used by Prosch (1849) for his figs. 
1 -3. Hectocotylus abnormal: only left side of 
lígula developed, 14 laminae. Remnants of 
typical B. arcf/cus spermatophores (Fig. 2d). 

Material seen by Steenstrup (1856a) and 
probably by Prosch (1 849): Male, ML: 32 mm, 
ED: 9.5 mm, LD: 3.8 mm, SD: 2.7 mm. Ligula 
9 mm with 9 laminae. Funnel organ lost. Too 
fragile and flabby for measurements. Label: 
Holböl and Möller, Julianehâb (southwestern 
Greenland, probably 1840). 

Three females in a jar. They have all been 
dissected but agree in all recognizable char- 
acters with the lectotypes (sucker size, eye- 
balls, crop). I extracted the beak and radula 
from one of the specimens. (Fig. 5e) Label: 
Octopus arcticus, Greenland (probably 1840). 

Synonymy 

Octopus arcticus Prosch, 1849: 55, pi. 2, figs. 

1-3 
Octopus grönlandicus Dewhurst: Steenstrup 

1856a: 17, pi. 2, fig. 2; 1856b: 234, pi. 11, 

fig. 2; 1857:97, pi. 3, fig. 2 
Octopus piscatorum Verrill: Hoyle, 1886: 91 
Polypus piscatorum: Russell, 1922: 7, pi. 2, 

fig. 2 
Polypus faeroensis Russell, 1909: 446; 1922: 

5, pi. 1,fig. 1, pi. 2, figs. 4-6 
Bathypolypus arcticus: Robson, 1927: 251, 

figs, la, 2A; 1932 [in part]: 286, pi. 6, figs. 

1, 2, text-figs. 53-60; Kondakov, 1936: 

61 , figs. 1 , 2; Adam, 1939 [in part]: 9, figs. 

2-4; Bruun, 1945 [in part]: 6; Muus, 

1959: 224, figs. 109A, C, 115; 1962: 10, 

figs. 1, 2, 4c 



TABLE 1 . Measurements (mm) of the B. arcticus Lectotype. 



TL: 


165 






AL: 


1 


II 


III 


ÍV 


ML: 


42 


MLTLI: 


25 


r 


119 


112 


93 


114 


HW: 


27 


HWI: 


64 


1 


120 


113 


112 


110 


MW: 


(38) 


MWI: 


(90) 












ED: 


9 


EDI: 


21 












LD: 


5.6 


SOLDI: 


50 






WD: 






SD: 


2.8 


SDI: 


7 


A 


В 


С 


D 


E 


LigL: 


19 


LigLI: 


20 


33.5 


33 


30.5 


32 


29 


LamC: 


14 


SHcC: 


42 




38 


35 


35 





178 



MUUS 



Benthoctopus piscatorum: Robson, 1927: 

254, figs. 2B, 3; 1932: 224, figs. 31, 34, 

35 
Benthoctopus sasa/c/V Robson, 1927: 257, fig. 

8. 
Bathypolypus faeroensis:To\\, 1985: 598, figs. 

1,2 

Diagnosis 

Body egg-shaped, papiiiated with minute 
warts often in a stellate pattern on small light 
spots: head narrower than body; each eye 
with a verrucose supraocular cirrus; funnel 
organ double, a clear-cut VV: hectocotylus 
with about 40 suckers and a deeply exca- 
vated ligula with 10-16 laminae. Radula usu- 
ally with irregularly multicuspid, seldom ho- 
modont rachidians. Esophagus with crop 
diverticulum. Total length rarely over 200 mm. 

Description 

Skin and Colors: In freshly caught speci- 
mens, the skin is violet to purple strewn with 
lighter yellowish subcircular spots with minute 
warts, often surrounding a central slightly big- 
ger wart in concentric rings, as observed by 
Presch (1849). Ventral side paler, with few or 
no warts. Over each eye is an erectile stout 
and verrucose cirrus often with adjacent 
smaller protuberances. The cirrus may be 
about 10 mm long but often more or less re- 
tracted in preserved specimens. Color and 
sculpture patterns of the skin vary with state 
of preservation. In some specimens, the skin 
is smooth with no evident sculpture. This is 
often the case with preserved juvenile speci- 
mens (ML; < 30 mm.) 

Bodily Proportions: The mantle is ovoid in 
outline (MWI; 65-90) slightly constricted be- 
hind the eyes (Fig. 1). ML rarely over 60-70 
mm., TL rarely over 200 mm. The head is nar- 
rower than the mantle. Due to allometric 
growth, HWI decreases from 60-85 at ML 10, 
to 40-70 at ML 60. 

The eyeballs are not very prominent. They 
decrease in relative size with age, EDI being 
28-40 at ML 10, 20-33 at ML 60 (Fig. 4). The 
lens measures about 35% of the ED (LDEDI: 
30-40). 

The mantle aperture is 40-50% of the cir- 
cumference of the neck. The funnel is free of 
the mantle for about 50%. 

The funnel organ is VV-shaped. Typically its 
limbs appear as narrow swollen "ropes" that 



are easily detached from the funnel wall. They 
may vary in form (Fig. 18). 

The gills are reduced and have 6-7(8) gill 
filaments on each demibranch. 

The brachial complex is stout, with the arm 
order I. II. III. IV. The ML constitutes 25-35% 
of the TL, which leaves 65-75% to the 
brachial complex. Arm length Index; I 231, II 
218, III 202, IV 189 (mean of 12 specimens). 

The web extends along the arms almost to 
the tips. The web sectors A, B, С and D are 
subequal in most specimens (WDI; 33-34 at 
an average), E usually slightly shallower 
(WDI: mean 30). In a beautifully preserved fe- 
male with fully extended umbrella, the WDI 
was; A; 38, B; 46/46, C: 46/46, D; 46/43, E; 41 
(ML; 24 mm, M/K "Asterias", Svalbard, 
ZMUO). 

The suckers are bisehal, rather small and 
well spaced, SDI: 6.5-7.3-8.4 depending on 
degree of expansion. They are of the same 
sizes on all arms, and there is no sexual di- 
morphism. They number 80-90 on the dorsal 
arms, 60-70 on the ventral. 

The hectocotylized third right arm is some- 
what shorter than the corresponding left arm 
(OAI; 75-86-100) and carries about 40 suck- 
ers (Fig. 8). The number is individually con- 
stant throughout life (Fig. 23). The ligula was 
not described by Presch (1849) probably be- 
cause he had only one male with an abnormal 
hectocotylus at his disposal when he finally 
described B. arcticus (paralectotype CEP- 
15). But Steenstrup (1856a, b, 1857) pictured 
a male with hectocotylized arm and, based on 
five specimens, he stated that the arm carries 
41-43 suckers and ligula 13-17 transverse 
laminae. The ligula is a spoon-shaped pointed 
organ with inrolled curved sides (Fig. 1). 
The width is about 50-70% of the length. It 
has a central ridge and 11-16 (17) deep, well- 
separated laminae (variation shown in Fig. 9). 
The number of laminae is individually con- 
stant from the onset of maturity (Fig. 22). 
LigLI; 9-23, the observed variation also de- 
pending on state of sexual maturity (Fig. 3). 
Calamus is short and pointed, CaLI; approx. 
20. Spermatophoral groove well developed, 
the strong membrane curling the arm inwards 
in preserved specimens. Already at ML 16 
mm the ligula may be discernible as a 1.5 
mm-long undifferentiated tip of the third right 
arm. 

Female Organs: The ovary is large with big 
globular and heavily pigmented oviducal 
glands (Fig. 2c). Proximally, the united 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 179 




FIG. 1. Bathypolypus árcticas (Frosch, 1849). a: hectocotylus, ML: 40 mm; b: upper and lower beak, $ ML 
46 mm, "Godthaab" Stn. 87, ZMUC; c: cf ML 54 mm, South Greenland, Just & Vibe Stn. 45, ZMUC. 



oviducts are partly covered by the outer тегл- 
brane of the ovary; distally they are short, very 
stout and leaving the oviducal glands forming 
a right angle. In ripe females, the gonad oc- 
cupies 30-40% of the mantle cavity and is 
stuffed with 60-80 yellow or brownish eggs. 
Ripe eggs measure 16-18 mm in length. 
They are smooth, with fine longitudinal lines. 
The pointed end of each egg has a short stalk, 
and the stalks of all eggs are attached to the 
same small area of the ovary wall. 

Male Organs: The penis has a well-devel- 
oped diverticulum (Fig. 2b). Its size and shape 
very much depend on presence or absence of 



spermatophores, and it was not measured. 
Needham's sac was often distended by 3-6 
spermatophores, as in Figure 2b. The large 
brownish spermatophores are very character- 
istic (Fig. 2d; Presch 1849: fig. 2): the sperm 
reservoir occupies only about one third of the 
total length, and the oral end is a long, stout 
horn. The casing is very opaque, and details 
of the reservoir and middle piece could not be 
obtained. SpLI: 105-130, SpWI: approx. 18, 
SpRI: 26-32 (data from six males). 

Beaks and Radula: The beaks do not have 
distinctive features. The radula seems to be 
very variable. In most cases, the central teeth 



180 



MUUS 




FiG 2. Bathypolypus arcticus, a: digestive tract; ant. sal. gl.: anterior salivary glands; cae.; spiral caecum; 
dig gl : digestive or hepatic gland; post.sal.gl.: postenor salivary glands; stom.: stomach, b: male reproduc- 
tive organs- ace. gl.: accessory gland; needh.: Needham's sac containing npe spermatophores; pen.div.: 
penis diverticulum- sem.ves.: seminal vesicles; test.: testis; vas.def.: vas deferens, c: female reproductive or- 
gans; ov.gl.: oviducal gland; d: spermatophore. a,b and d from çf ML 43 mm, Ymer Island, East Greenland, 
1932, ZMUC; c: 9 ML 42 mm, BIOFAR Stn. 274, ZMUC. 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 81 




ML mm 



FIG. 3. Lígula length versus mantle length in B. arcticus. Open circles: juveniles. Maturity is reached at about 
ML 30 mm. Inserted lectotype of B. arcticus (encircled dot) and the "type" of B. faeroensis in ZMUC (dot in 
square). Juv.: y = -1 .90 + 0.225x, r^ = 0.81 ; ad.: y = -3.9 + 0.340x, r^ = 0.39. 



EDI 

бе- 



зо 



20 



10- 




o B.bairdii n=142 
Ü B.arcticus n=92 



FIG. 4. Eye diameter index versus mantle length in B. bairdii anä В. arcticus. No sex difference was found. 
Allometric growth is discernible, but correlation is weak; bairdii: у = 52.68 - 0.2204x, r^ = 0.249; arcticus: у = 
36.7-0.199x, r^:- 0.321. 



are clearly nnulticuspid (Fig. 5), with a seri- 
ation of 2-5 symmetrical (Fig. 5c) or asym- 
metrical teeth (Fig. 5b, d-f). In some speci- 
mens, the ectocones are rather delicate, 
uniform thorns (Fig. 5b); in others they form ir- 



regular rough knots. The ectocones may also 
be reduced to a certain lateral ruggedness of 
the rachis teeth even in presumed unworn 
parts of the radula. This may lead to quasi ho- 
modont rachidians, but completely homodont 



182 



MUUS 




FIG. 5. Radula and rachidians of В. arcticus: a and b: V ML 54 mm; c: V ML 37 mm; d: V ML 44 mm; e: $ 
ML approx. 50 mm; f and g: $ $ ML 33, 52 mm. Caught at various positions off northiem Iceland, except e: 
southwestern Greenland, probably 1840. Scale: 100 pm. 



rachidians are sometimes found. A striking ex- 
ample is demonstrated by two otherwise typi- 
cal arcticus females (Fig. 5f, g) taken at 
BIOICE Stn. 2751. One has multicuspid, the 
other one homodont rachidians. 



Digestive Tract: The esophagus has a well- 
developed crop diverticulum (Fig. 2a) on 
which the posterior salivary glands are loosely 
attached when in situ. The spiral caecum is 
reduced to at most one turn. The liver is large 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 83 



and bulbous, about the same size as the 
testis. There is no ink sac. 

Comparison with Prosch's Description 

The material examined fits well with the 
types and Prosch's description, which is sup- 
plemented by Steenstrup's (1856a, b, 1857) 
remarks and illustrations of the species. One 
serious discrepancy, however, is the state- 
ment by Presch that arcticus has no crop. This 
error of Presch is hard to explain unless one 
of the specimens dissected during the study 
of the anatomy of Cirroteuthis (Reinhardt & 
Presch, 1846) was В. bairdii, which is very 
common in West Greenland. I have however 
no evidence for this. 

An important point that the present investi- 
gation confirms is that the rachidians are usu- 
ally heterodont (Muus, 1962: 11, fig. 1A), 
though much more variable than I previously 
thought. 

Junior Synonyms and Supposed Synonyms 
of B. arcticus 

Three species which have contributed con- 
siderably to the prevailing taxonomic confu- 
sion are discussed below: Bathypolypus 
faeroensis (Russell, 1909), which was re- 
moved from the junior synonomy of B. arcticus 
by Toll (1985), and the two species that had 
been placed in Benthoctopus- sasai<ii (Rob- 
son, 1927) and piscatorum (Verrill, 1879). 

Bathypolypus faeroensis (Russell, 1909) 

Russell (1909) described Polypus faeroen- 
sis based on two males and a female trawled 
in the Faeroe Channel by the Fishery Cruiser 
"Goldseeker" Stn. 19a, 60°40'N, 4°50'W at 
1 030 m. August 24, 1 908. In 1 922, Russell re- 
peated his earlier description based on the 
same three specimens, adding illustrations of 
radula, ligula, a papillary area of the skin, and 
a good photograph of the whole animal. 



Russell stated that faeroensis is closely al- 
lied to the squat form that he knew as "bairdii 
= arcticus", from which it differs in certain well- 
defined ways: it has a better marked neck, a 
narrow head, and a shorter ligula, with more 
numerous laminae. The skin has a character- 
istic papulation with tiny warts arranged in a 
stellate pattern on light, circular spots. 

Bathypolypus faeroensis has been consid- 
ered a synonym of B. arcticus (Presch, 1849) 
by Robson (1932, with hesitation), Jaeckel 
(1958), Kumpf (1958), Roper et al. (1984), 
and Nesis (1987). Toll (1985) redeschbed B. 
faeroensis and reinstated it as a valid species 
based on a female specimen taken by FFS 
Walther Непл /ig in Denmark Strait, 67°21, 
5'N, 23°30'W, 480-485 m, September 9, 
1973. Because Toll was unable to trace the 
types of B. faeroensis, this specimen was 
designated neotype. 

In ZMUC I found a misplaced jar labelled 
"Polypus faeroensis, Type, 61°27'N, 1°47'W, 
1240 m, July 25, 1909. Russell det. Received 
from A. С Stephen, the Fishery Board of Scot- 
land, March 25, 1924." The specimen, a male 
with ML 34 mm, was caught a year after the 
specimens used for the description by Russell 
1 909 (and later 1 922). I am inclined to believe 
it was received as a donation to ZMUC, but it 
is unknown whether Russell or the ZMUC cu- 
rator (R. Spärck) is responsible for the "type" 
label. I found in BMNH a female specimen 
taken in the same haul as the above-men- 
tioned male. Because it had been misplaced, 
the ZMUC-type is not recorded in the ZMUC 
type list (Kristensen & Knudsen, 1983). 

The "type" in ZMUC might have been a bet- 
ter choice than the neotype designated by 
Toll, because it is a male, caught at the type 
locality and identified by the deschber. Table 2 
shows measurements of the specimen in 
ZMUC. 

The ZMUC "type" has a VV-funnel organ. 
Hectocotylized arm with 39 suckers. The buc- 
cal mass has been removed, so the radula 
and beaks are missing. The esophagus has a 



TABLE 2. Measurements (глт) of the B. faeroensis "t^pe" in ZMUC. 



TL: 


105 






AL: 


1 


II 


III 


IV 


ML: 


34 






r 


67 


64 


52 


56 


HW: 


20 


HWI: 


59 


1 


70 


62 


59 


57 


MW: 


27 


MWI: 


79 












ED: 


12 


EDI: 


35 












LD: 


3.7 


SDLDI: 


18.3 






WD 






SD: 


2.2 


SDI: 


6.5 


A 


В 


С 


D 


E 


LigL: 


7 


LigLI: 


13.5 


22 


22 


20 


22 


20 


LamC: 


11 


SHcC: 


42 




? 


25 


21 





184 



MUUS 



crop diverticulum. Needham's sac with four 
unripe spermatophores. SpL: 22 mm, SpRI 
31, of the characteristic arctlcus type (SpRI 
approx. 30; Fig. 2d). 

In all measurable characters, the ZMUC 
specimen agrees with the general descrip- 
tions by Russell (1909, 1922) and Toll (1985). 

Neither Russell nor Toll examined type ma- 
terial of B. arcticus. Both authors assumed 
that the "bairdii'-iorm is the true B. arctlcus. 
Ironically, however, the species described by 
Russell and the faeroensis specimen in 
ZMUC is the genuine Bathypolypus arcticus. 
and all the data on B. faeroensis fit measure- 
ments and indices as indicated in the present 
redescnption of that species. Toll's careful re- 
description of faeroensis might as well be 
a redescription of a female arcticus. The ho- 
modont rachidians pictured by Toll (his fig. 1 b) 
are unusual but not unknown in arcticus. Ho- 
modont rachidians were found in one of two 
females (Fig. 5f, g) caught in the same haul at 
68 OON, 20°40'W (BIOICE Stn. 2751). In all 
aspects. Toll's specimen is indistinguishable 
from B. arcticus. 

Benthoctopus sasal<ii Robson, 1 927 

Material examined: Types in BMNH: 
89.1 .24.33-34. A male and a female labelled: 
"Bentlioctopus sasakii Robson, HMS 'Triton' 
Stn. 9, Faroe Channel, 60'5'N, 6'21'W, 608 
fms. August 23, 1882". 

Male: ML about 32 mm, distorted and dis- 
sected. Hectocotylized arm 59 mm with 44 
suckers, LigL: 7.8 mm. Ligula with 12-15 in- 
distinct lamellae. SD: 2.3 mm, ED: 10 mm, 
SpL: 34 mm, SpRL: 9 mm. Funnel organ VV 
with the outer limbs somewhat shorter than 
the median limbs. Skin smooth. 

Female: TL: 100 mm, ML: 27, MW: 24, HW: 
20, SD: 2.3 mm, ED: 10 mm. Funnel organ 
fragmented. 

Robson (1927: 262, fig. 8) depicted the 
radula, which shows a more or less irregular 
seriation of multicuspid rachidians. Both spec- 



imens are typical of juvenile B. arcticus, as ev- 
idenced by the hectocotylus, the funnel organ 
and the indices: SDEDI: 23 and SpRI: 26 
(Table 9). They were furthermore trawled in 
arctic water masses at the same depth and lo- 
cality as B. faeroensis (= B. arcticus). Robson 
became doubtful about his sasal<ii and later 
(1932) treated it as a junior synonym of Ben- 
tiioctopus piscatorum (Verrill, 1879), which 
prompted me to reexamination of the type of 
piscatorum in USNM. 

Benthoctopus piscatorum (Verrill, 1879) 

Material examined: Holotype USNM 
574641. From western part of Le Have Bank 
off Nova Scotia, October 1879, 120 fms. (219 
m). Female, ML: 39. Measurements are give 
in Table 3. The holotype was a unique speci- 
men (Roper & Sweeney, 1978). 

Description: The skin is smooth, with no evi- 
dent sculpture. A much contracted cirrus de- 
tectable over the hght eye, no trace over the 
left eye. The funnel organ partly missing but of 
ba/rd// type (Fig. 18). Esophagus without crop 
diverticulum, only with a slight swelling. 
Number of suckers on each arm 65-75. 
Radula and jaws not available. 

Verhll's description (1879: 470) is not very 
exhaustive, as noted by Robson (1932). In 
Verhll's opinion, piscatorum was easily distin- 
guished from Octopus bairdii 'Ъу a more elon- 
gated body, longer arms, shorter web, lack of 
supraocular cirrus and by its smoothness." 

I believe that the type specimen of Octopus 
piscatorum is a somewhat aberrant specimen 
of Bathypolypus bairdii (Verrill, 1873), as is 
the case with his O. lentus and O. obesus 
(see below). None of the bodily proportions 
are distinctive. The only index that falls out- 
side the natural variation of bairdii is SDEDI: 
14 (Table 9), but this is compensated for by 
SDLDI: 28, which is a more reliable index, 
being based on the firm eye lense as standard 
for eye size (Fig. 16). 



TABLE 3. Measurernents of holotype of Bentiioctopus piscatorum. 



TL: 


153 






AL; 


1 


II 


III 


IV 


ML: 


39 






r 


93 


75 


82+ 


78 


HW; 


28 


HWI; 


71 


1 


98 


102 


77+ 


75+ 


MW: 


33 


MWI; 


85 






WD; 






ED; 


15 


EDI: 


38 


A 


В 


С 


D 


E 


LD: 


7.5 


SDLDI; 


28 


25 


16 


27 


24 


12? 


SD: 


2.1 


SDI; 


5.4 




32 


32 


25 





THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 85 



I believe that the smoothness of the body is 
due to contraction of all warts or flaccidity at 
the time of death, as seen in varying degrees 
in many preserved specimens of both bairdii 
and árcticas . Traces of a retracted cirrus over 
the right eye suggest that the smoothness 
over the left eye hides a potential cirrus. 

The identity with bairdii is further substanti- 
ated by the lack of a crop diverticulum. This 
contrasts with Robson's statement that pisca- 
torum has "a tolerably well-developed crop" 
(1932: 225) and his figure 31. However, Rob- 
son based this statement on dissections of B. 
Sasaki i {= árcticas) . 

In 1 981 , 1 discussed the Batiiypolypus-Ben- 
tlioctopus problems with Gilbert Voss, who 
also examined the type specimen of piscato- 
rum. He concurred with my opinion that Verrill 
(1879) described a somewhat atypical speci- 
men of Bathypolypus and consequently the 
genus Benttioctopus Gúmpe, 1921, would be 
indistinguishable. (Voss & Pearcy 1990). 

East Atlantic '^piscatorum" 

The true identity of piscatorum raises seri- 
ous doubts as to the identity of the many 
specimens attributed to piscatorum from the 
East Atlantic by Hoyle (1 886), Appellöf (1 893), 
Pfeffer (1908), Massy (1909), Russell (1922), 
Robson (1932), Grieg (1933), and Grimpe 
(1933). 

The first to record piscatorum from the 
Faroe Channel was Hoyle (1886). I examined 
his specimens in BMNH, and they are identi- 
cal with the specimens later described as 
Benthoctopus sasakiiby Robson (1927) and 
once again as piscatorum by Robson (1932). 
As argued above, the type specimens of 
sasakii are conspecific with arcticus. 

Appellöf (1893), inspired by Hoyle (1886), 
identified three very juvenile specimens from 
66°41'N, 6°59'E and 78°2'N, 9°25'E as pis- 
catorum. He does not give particulars, except 
that the body is smooth and that mantle and 
head of the male measure 14 mm. One of the 
specimens was caught with a juvenile male 
"Octopus lentus" (junior synonym of bairdii), 
and later identified by Grieg (1933) as pisca- 
torum. Because piscatorum specimens from 
the Svalbard-Barents Sea area are all caught 
in cold water (-0.9°-+0.8°C), I strongly sus- 
pect them to be smooth and partly juvenile 
specimens of arcticus sensu stricto. I dismiss 
the records as unreliable, although I have not 
seen the specimens. 

Pfeffer (1908) merely repeated the Ameri- 



can records of Verrill and the East Atlantic 
records of Hoyle (1886) and Appellöf (1893). 
He repeated Verrill's pictures of piscatorum 
and for unknown reasons synonymized Ben- 
tlioctopus ergasticus with piscatorum. As 
shown below, B. ergasticus is a distinct 
species. 

In conclusion, Pfeffer's records of piscato- 
rum are unreliable. 

Massy (1907) described Polypus normani, 
which she later, having consulted Pfeffer, de- 
cided was a piscatorum (Massy, 1909). It is a 
male, TL: 206 mm, trawled off Ireland at 
51°15'N, 11°47'W, 707-71 Ofms., September 
1907. 

I have not examined the type, but Massy's 
measurements and drawings (1909: pi. II, 
figs. 3, 4) allow the conclusion that it is not 
conspecific with either the type of Verrill's pis- 
catorum nor bairdii or arcticus: the hectocotyl- 
ized arm has 64 suckers, far beyond the num- 
ber found in ba/M/ (Fig. 8). OAI is 64.6, which 
is off the range of variation in bairdii and arcti- 
cus, in which OAI is about 80-90. SDI is 9.5, 
which means that the suckers are larger than 
in bairdii ana arcticus. The smoothness of the 
skin and the bodily proportions given by 
Massy are not distinctive, but the shortness of 
the hectocotylized arm and its relatively high 
number of suckers reminiscent of Bentfi- 
octopus ergasticus (P. Fischer & H. Fischer, 
1 892). Two males and a female of this species 
were caught in the same haul as normani. 
Still, B. normani may be a valid species. 
Massy was advised by Pfeffer who, as men- 
tioned above, considered ergasticus synony- 
mous with piscatorum. The ligula and cala- 
mus of normani is clearly juvenile (Massy 
1909: figs. 3, 4). Her figure was erroneously 
used by Nesis (1987: 318, fig. 84H) to illus- 
trate the calamus and ligula of piscatorum. 

Russell (1922) recorded three male and 
four female Polypus piscatorum taken in the 
Faeroe Channel in the same haul as his P. 
faeroensis ("Goldseeker" Stn. 19a). All seven 
specimens are juvenile. His measurements 
do not allow the species to be even tentatively 
identified. But if his measure "Posterior end to 
eye" is accepted as expression of the ML, the 
SDI of the largest female (ML: 32 mm) and the 
largest male (ML: 27 mm) is 9.4 and 7.4 re- 
spectively, beyond the sucker size of bairdii, 
but compatible with arcticus. The large female 
faeroensis (= arcticus) taken in the same haul 
(Russell, 1922: 6) has SDI: 8.3. The ligula of 
the largest male is depicted and shows a 6.5- 
mm-long typical juvenile arcticus ligula with 



186 



MUUS 



"half a dozen indistinct transverse ridges" 
(Russell, 1922: fig. 7). 

Like many of the earlier writers, Russell has 
attached undue weight to the smoothness of 
the skin of his " piscatorurri' specimens. I con- 
sider it indubitable that they are juvenile arcti- 
cus. as all the other specimens I have exam- 
ined from the arctic water masses in the depth 
of the Faeroe Channel (Fig. 19). 

Robson (1932) did not examine the type of 
piscatorum. He based his revision on three 
specimens, two of which are the specimens 
originally described as piscatorum by Hoyle 
(1886), later as sasakiib\/ Robson (1927). As 
argued above, sasakii is a junior synonym of 
arcticus. 

The only other specimen seen by Robson is 
a female (Robson's C30) surprisingly caught 
in the same haul as the "type" of faeroensis 
found in ZMUC: Faroe Channel 61°27'N, 
r47'W, 681 fms., July 25, 1909. I have ex- 
amined the specimen at BMNH: the body is 
ovoid, the skin smooth with no trace of warts. 
The funnel organ is not well preserved but 
shows VV, the inner limbs are however 
weakly joined anteriorly. Measurements are 
given in Table 4. 

Robson (1932: 225, fig. 35) shows the 
radula of this specimen. The rachidians are 
heterodont, showing irregular lateral cusps 
and a seriation of about four teeth. The esoph- 
agus has a well-developed crop diverticulum. 

The multicuspid rachis teeth, the presence 
of a crop diverticulum, as well as the mea- 
surements and indices in Table 4 show that 
Robson's piscatorum is really the smooth- 
skinned specimen of B. arcticus. 

In conclusion, Benthoctopus piscatorum 
sensu Verrill, 1 879, is a junior synonym of Ba- 
thypolypus bairdii, whereas B. piscatorum 
sensu Hoyle (1886), Russell (1922), and Rob- 
son (1932) is smooth-skinned and often juve- 
nile Batliypolypus arcticus. 

A large female octopod caught 1967 at a 
depth of 60 fathoms in Placenta Bay, New- 
foundland, and identified as Benthoctopus 
piscatorum by Aldrich & Lu (1968: table 1, 



figs. 1, 2) is undoubtedly misidentified and 
should be reexamined. With a TL of 362 mm, 
ML 89 mm and HWI of only 35, the specimen 
deviates considerably from both B. bairdii anä 
В. arcticus. The authors were aware of the in- 
consistency with Robson's description of pis- 
catorum but put it down to its poor state of 
preservation. Nixon (1991), studied the eggs 
of this specimen (as ''piscatorum" eggs). 

Revision of B. arcticus by Robson (1932) 

Robson (1932) was very uncertain about 
the large number of forms ascribed to the 
arcticus-bairdii complex. Though with hesita- 
tion, he concluded that faeroensis with the 
ovoid body and narrow head at the one ex- 
treme and the bairdii form with the saccular 
body and large ligula at the other were one 
and the same species, B. arcticus. 

Robson presented two excellent photos of 
the two forms (1932: pi. VI, figs. 1, 2), one of 
which is a "type"-specimen of B. arcticus Uom 
Greenland on loan from ZMUC. ! have tried in 
vain to identify the specimen in the Copen- 
hagen collections, but the photo is easily rec- 
ognizable as B. arcticus, sensu stricto. The 
other photo depicts a typical B. bairdii. the 
square-bodied boreal form, which is rein- 
stated as a distinct species below. 

Because the two species were confounded, 
Robson's text is of limited use. Besides the 
photo, he provided (1 932: 290-291 ) three fig- 
ures of a B. arcticus, sensu stricto, from the 
Barents Sea: outline of mantle (fig. 54), an oc- 
ular cirrus (fig. 55), and a funnel organ (fig. 
56). The radulae (fig. 58) are from B. bairdii 
specimens. 

Robson (1932) enforced the prevailing tax- 
onomic confusion by his erroneous identifica- 
tion of smooth skinned and juvenile B. arcti- 
cus as Benthoctopus piscatorum. 

Records of B. arcticus by Adam (1 939) 

Adam (1939: 6, table, figs. 2-4) records 
six juvenile specimens of B. arcticus from 



TABLE 4. Measurernents of Robson's Benttioctopus piscatorum. 



TL: 


185 






AL: 


1 


II 


III 


IV 


ML 


43 






r 


133 


130 


131 


130 


HW: 


34 


HWI: 


79 


1 


129 


136 


? 


134 


MW: 


48 


MWI: 


112 






WD: 






ED: 


14 


EDI: 


33 


A 


В 


С 


D 


E 


SD: 


3.3 


SDI: 


7.6 


30 


33 


38 


38 


30 






SDEDI: 


24 




35 


36 


39 





THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 87 



the Iceland-Faroe area (about 66°25'N, 
12°30'W). I reinspected the material on loan 
from IRSNB and found, that four of the speci- 
mens (Adam's specimens b and c) were iden- 
tical with arcticus as here redescribed. They 
all have the typical VV funnel organ and the 
diagnostic index SOLDI is: 53, 53, 53 and 59, 
respectively, which distinguishes arcticus 
from B. bairdii and B. pugniger, n. sp. (with 
SOLDI < 40). Two of the specimens are erro- 
neously identified as females (specimens b: 
ML 14 and 16 mm). They are juvenile male 
arcticus with undifferentiated ligulae of 1.5 
and 1 .9 mm. Adam's drawing of the radula of 
a female arcticus (p. 12, fig. 4, specimen c) 
shows a homodont rachidian reminiscent of 
the radula from "S. faeroensis" pictured by Toll 
(1985: 600, fig. 1) and my Figure 5g. It under- 
lines the fact that B. arcticus has a very vari- 
able radula. 

The remaining two specimens of the mate- 
rial treated by Adam (1939: specimens a, figs. 
2, 3), I consider to be B. pugniger, n. sp., and 
are discussed below. 



REINSTATEMENT AND REOESCRIPTION 

OF BATHYPOLYPUS BAIRDII 

(VERRILL, 1873) 

Material examined: about 500 specimens 
from boreal parts of the North Atlantic as listed 
in Appendix I. Types examined: Octopus 
bairdii Ует\\, 1873 (syntype, USNM 574638) 
Octopus piscatorum Verrill, 1879 (holotype, 
USNM 574641) Octopus lentus Verrill, 1880 
(holotype, USNM 34223) Octopus obesus 
Verrill, 1880 (holotype, USNM 382469). 

Synonymy 

Octopus ba/rc/// Verrill, 1873a: 5; 1873b: 394, 
figs. 76, 77; 1881: 107, pi. 2, fig. 4, pi. 4, 
fig. 1; 1882: 368, pi. 33, fig. 1,1a, pi. 34, 
figs. 5, 6, pi. 36, fig. 10, pi. 38, fig. 8, pi. 
49, fig. 4, 4a, pi. 51 , fig. 1,1a; Sars, 1 878: 
339, pi. 17, fig. 8, pi. 33, figs. 1-10; 
Kumpf, 1958 (passim) 

Octopus piscatorum У ernW, 1879: 470; 1882: 
377, pi. 36, figs. 1,2 

Octopus obesus Verrill, 1880: 137; 1882: 379, 
pi. 36, figs. 3, 4; Robson, 1932: 299; 
Kumpf, 1958 (passim). 

Octopus /enius Verrill, 1880: 138; 1881: 108, 
pi. 4, fig. 2; 1 882: 351 , 375, pi. 35, figs. 1 , 
2, pi. 51, fig. 2; Robson, 1932: 297; 
Kumpf, 1958 (passim) 



Octopus arcticus: Norman, 1890: 466; Joubin 

1920:32, pi. 7, figs. 4, 5 
Polypus arcticus: Pfeffer, 1908: 16, fig. 6 
Polypus lentus: Pfeffer, 1908: 17, figs. 7, 8 
Bathypolypus arcticus: Robson, 1932 [in 
part]: 286, figs. 53-60, pi. 6, figs. 1, 2 
Thiele, 1935: 992, fig. 890; Boone, 1938 
360, pi. 152; Bruun, 1945 [in part]: 6 
Kumpf, 1958: 1-135; Jaeckel, 1958: 563 
Cairns, 1976: 261; Macalaster, 1976 
Perez-Gandaras & Guerra, 1978: 201 
figs. 6-8; O'Oor & Macalaster, 1 983: 401 
fig. 24.1, 24.2; Roper et al., 1984: 222 
Nesis, 1987: 315, fig. 83B-E; Guerra, 
1992: 251, fig. 89 
Bathypolypus proschi Muus, 1962: 13, figs. 
2-4 



Diagnosis 

Square-bodied, with papillated skin; arms 
short; head broad, with large eyeballs, each 
with a supraocular cirrus; funnel organ dou- 
ble, pad-like; hectocotylized arm with about 
40 suckers; ligula very large deeply exca- 
vated with 8-12 prominent laminae. Radula 
with homodont central teeth. Esophagus with- 
out crop diverticulum. Total length rarely over 
200 mm. 



Description 

Skin and Colors: In newly caught specimens, 
the skin is violet to purple, often without any 
conspicuous spots or patterns, but sometimes 
speckled with small greyish spots. In pre- 
served specimens, the skin may be smooth, 
but more often the dorsal surface is papil- 
lated, especially in the antero-dorsal region. 
Single warts or aggregations occur, the latter 
sometimes in a stellate pattern, a number of 
small warts encircling a larger one, similar to 
B. arcticus. Over each eye is an erectile 
pointed cirrus with adjacent smaller protuber- 
ances. Erected It measures 5-10 mm. 

Bodily Proportions: Square-bodied, with 
broad head and huge eyeballs. HWI de- 
creases from 80-1 00 at ML 10 to 60-90 at ML 
60 mm. TL rarely over 200 mm. 

The eyeballs are very prominent. They de- 
crease in relative size from EDI 40-60 at ML 
1 0, to 30-45 at ML 60 (Fig. 4). The lens mea- 
sures on an average 41% of the ED (LOEOI: 
35-46). 

The mantle aperture is 36-42% of the cir- 



188 



lUUS 




FIG. 6. Bathypolypus bairdii {\/err\\\, 1873). a: habitus (after Vecchione et al., 1989); b: upper and lower beak; 
c: hectocotylus of o' ML 42 mm, Greenl. Fish. Invest. Stn. 5047, ZIVIUC. 



cumference of the mantle. The funnel is free 
of the mantle for about 50%. 

The funnel organ consists typically of two 
pad-like structures with a very variable ap- 
pearance (Fig. 18). They are never strictly VV- 
shaped, but may have an anterior indentation 
like a heart or be broken up into bars, sug- 
gesting an evolutionary past as VV-shaped 
organs. They are rather loosely connected 
with the funnel wall and easily lost. 

The gills are reduced and have 6-8 gill fila- 
ments on each demibranch. 

The arm order is I. II. III. IV. The ML consti- 
tutes 28-38% of the TL, which leaves 62- 
73% to the brachial complex. ALI: I 201, II 
184, III 174, IV 168 (mean of 21 specimens). 

The web extends along the arms. The web 
sectors B, С and D are subequal. WDI: A 32, 



В 35, С 36, D 35, E 27. WDMI: A: 54, B: 59, C: 
60, D: 59, E: 46 (mean of 21 specimens). 

The suckers are biserial, small and well 
spaced. SDI: 2.9-3.9-4.8 depending on de- 
gree of expansion. They are similar in size on 
all arms, and there is no sexual dimorphism. 

The hectocotylized third right arm is shorter 
than the left (OAI: mean, 88). Spermatophoral 
groove well developed. The number of suck- 
ers on the hectocotylized arm shows geo- 
graphical variation: the east American popula- 
tion (S of 45°N) has 26-40 suckers, whereas 
specimens from western Greenland and the 
eastern Atlantic have 35-49 (Fig. 8). The 
number is individually constant throughout life 
(Fig. 23). 

The ligula is a large spoon-shaped organ, 
which in ripe animals (ML > 30 mm) is often 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 89 




FIG. 7. B. bairdii. a: digestive tract; b: reproductive organs; c: spermatophore of cf ML 41 глт, Dana Stn. 
11648, ZMUC; d: female reproductive organs, ML 41 mm, Greenl. Fish. Invest. Stn. 5112; e: hectocotylus of 
juvenile cf ML 23 mm, Dana Stn. 2346, ZMUC. 



rather square, with almost parallel sides dis- 
tally ending with rounded flaps and a small 
pointed tip (Fig. 6). The width is 50-75% of the 
length. It has a central ridge and a number of 
deep and well-separated laminae. The num- 
ber Is individually constant from the onset of 
maturity. It has a total range of 7-13, but 
shows a geographical variation that is very ap- 
parent in the western Atlantic, the Newfound- 
land waters being a transitional area between 
the American population and the populations 



off Labrador and western Greenland (Fig. 9). 
Also, the size of the lígula in ripe animals 
shows geographical variation: at a ML > 30 
mm, LigLI in the eastern American population 
is 24-44, whereas in western Greenland and 
the rest of the North Atlantic, It is 18-38. The 
American population apparently reaches ma- 
turity at a ML of 25-35 mm, whereas in the rest 
of the Atlantic maturity is generally reached at 
ML 30-40 (Fig. 10). 
Calamus is short and pointed (CaLI approx. 



190 



lUUS 



1 

per 


cent 


30 


B.arcticus 
n=40 






10- 


1 












30 


B.bairdii 

E.Atl. 

n=40 








10- 


1 












30- 


B.b. 

W.GreenI 

n=61 








10- 


L 












30- 








10- 












B.b 

Am.<45°N 

n=23 



27 30 33 36 39 42 45 48 Suckers 

FIG. 8. The American population of B. bairdii (< 45 "N) deviates in mean number of suckers on the hecto- 
cotylus from the western Greenland population (P = 0.95); mean: 32.6, SE 2.6 versus 41 .4, SE 3.4. The east- 
ern Atlantic population differs only slightly from Greenland (mean: 42.7, SE 3.1). Inserted: arcticus, mean: 
39.7, SE 3.1. 



20). Already at ML 11 mm the ligula may be 
identified as a 1.2-mm-long undifferentiated 
tip of the third right arm. 

Female Organs: The ovary is large, with con- 
spicuous globular blackish oviducal glands 
(Fig. 7d). Ripe females with 60-85 yellowish 
eggs measuring 15-18 mm in length. The 
eggs are smooth with fine longitudinal lines. 

Male Organs: The penis with well-developed 
diverticulum (Fig. 7b). In Needham's sac was 



found up to six spermatophores. The sausage- 
shaped sperm reservoir of the spermatophore 
occupies about half of the total length, and the 
oral end is a slim, rather stiff and curved tube 
(Fig. 7c; Verrill, 1 882; pi. 36, fig. 1 0). The sper- 
matophore is glossy and opaque; the interior 
details are difficult to make out. SpLI; 60-90- 
115, SpWI about 19, SpRI 50-60. 

Jaws and Radula: The beaks do not present 
distinctive features. The radula is homodont, 
the rachidians having smooth concave sides 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 91 



I 

per cent 

50- 

40H 

30 

20H 

10 



30 
20 
10 

50 
40 
30 
20 
10 



Lab.-Greenl. 
n=92 




E.AtL 
n=47 



6 8 10 12 14 



Newf. 
n=65 



\ / 
<h- B.bairdii 



8 10 12 



Am.<45°N 
n=121 



B.pugniger n.sp 
n=17 



B.arcticus 
_ n=36 



6 8 10 12 



8 10 12 14 16LamC 



FIG. 9. Number of laminae copulatoriae. B. bairdii shows geographic variation with a highly significant dif- 
ference (P: > 0.99, z-test) in mean number between the American (< 45°N) and the western Greenland pop- 
ulation (mean: 10.12, SB 0.109 versus 8.38, SE 0,3). The Newfoundland area forms a transitional zone 
(mean: 9.56, SE 0.2). East Atlantic (mean: 9.89, SE 0.2) versus western Greenland shows a similar signifi- 
cant difference. Variation in B. pugniger, n. sp., and B. arcticus inserted for comparison. 



and never showing any sign of ectocones 
(Fig. 11; Verrill, 1882: pi. 49, fig. 4). 

Digestive Tract: The esophagus has no crop 
diverticulum, only a slight swelling where the 
posterior salivary glands are fastened when in 
situ (Fig. 7a). The spiral caecum is reduced to 
less than one turn. The liver is large, about the 
same size as the testis. 

Junior Synonyms of B. bairdii. 

Robson (1932) strongly suspected that Oc- 
topus lentusMemW, 1880, and O. obesus Ver- 
rill, 1880, were conspecific with the typical 
bairdii form, because he was not able to find 
critical differences. Not having seen the type 
specimens, however, he hesitated to place 
them in synonomy. 



In a master's thesis, Kumpf (1958) tried to 
sort out the Battiypolypus arcticus-bairdii- 
lentus-obesus complex. He had a large 
amount of material, 178 specimens, caught 
off the eastern American coast, including the 
types of Verrill's Octopus bairdii, O. lentus and 
0. obesus. In addition, he used measure- 
ments of seven "ß. arcticuä' specimens ex- 
amined by Gilbert Voss in the BMNH. 

Kumpf applied the standard of measure- 
ments of Robson (1927, 1932) and amplified 
by Pickford (1945, 1949). He thoroughly com- 
pared his material with the types of bairdii, 
lentus and obesus and concluded that these 
species are conspecific and that in spite of all 
variation, only one Battiypolypus species is 
represented in his material. 

I can confirm this part of his conclusion. The 
bulk of Kumpf s material stems from between 



192 



MUUS 



Lig 


1 
L 




® 






B.bairdii East America < 45° N 








n=54 


. 


y/^ 




30 






// 


. 


- 










20 


..'У 


у '. . 






- 


• 








10- 




о 






- 


о о -*^^ 

° °^§^o 
,^oo 


о 







10 



20 



30 



40 



50 



60 



70 ML mm 




70 ML mm 



FIG. 10. Ligula length versus mantle length in ß. bahdïi. Open circles: juveniles. Lower figure: pooled data 
for western Greenland and NE Atlantic (juv.: y = -3.34 + О.ЗЗЗх, r^ = 0.58: ad.: у = -3.20 + 0.505x, r^ = 0.66). 

Upper figure: Eastern America < 45''N (juv.: у = -0.49 + 0.248x, r^ = 0.39: ad.: у = 4.90 + 0.452x, r^ = 0.58). 

In eastern America, maturity is often reached already at ML 25 mm and ligula grows larger than in the North 
Atlantic. Dot in circle: syntype of ba/rcf/7 (USNM 574638). Dot in square: holotype of B. obesus (Verrill, 1882) 
(USNfH 382469). 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 93 




FIG. 11 . Radula and central teeth of B. bairdii. cf ML 55 mm., Davis Strait 65°12'N, 56°21 'W. Scale: 100 цт. 



30°N and 45°N, and I examined and remea- 
sured most of it, including the types of bairdii, 
obesus and lentus, during a visit to USNM in 
1975. The data are included in the present re- 
vision. 

Kumpf's main conclusion, however, that 
Verrill's species are junior synonyms of arcti- 
cus, is erroneous. The genuine arcticus is a 
more northerly species, not found off the east 
coast of USA (Fig. 20). Paradoxically, Kumpf 
never saw the species he purports to revise. 
To understand B. arcticus, he had to rely on 
the literature, especially on Robson's (1932) 
revision, with all its ambiguities and errors. 
Kumpf focused on the squat "ba/rd/7-type" as 
being the typical arcticus, thus missing the 
point that Verrill's bairdii \s a separate species. 

Kumpf (1958) was incorrect in stating that 
Verrill did not know of Prosch's arcticus. In a 



footnote describing bairdii, Verrill (1873a: 5) 
wrote that his species differed from O. groen- 
landicus Dewhurst, which has a smaller hec- 
tocotylus "with more numerous folds, and the 
basal part bears 41 -43 suckers". This is a ci- 
tation from Steenstrup's then well-known 
paper (1856a, or the translations of it: 1856b, 
1857), in which O. groenlandicus is syn- 
onymized with arcticus and in which the 
species and its hectocotylized arm are figured 
(see also Verrill, 1880: 138, footnote). 

The knowledge of West Atlantic Batliypoly- 
pus was much extended and elaborated upon 
by Macalaster (1976), who studied no less 
than 750 Battiypolypus specimens collected 
in Canadian waters from Georges Bank to the 
Labrador Sea. Macalaster was warned by me 
that "arcticus" covered more than one 
species, but through morphometric analyses 



194 



MUUS 



she showed convincingly that only one 
species occurs in Canadian waters and that it 
is identical with Kumpf's "B. arcticus'. Leaning 
on Kumpf's revision, she excusably put the 
wrong species in the title of her otherwise 
valuable contribution, instead of B. bairdil. 
This mistake was repeated by O'Dor & 
Macalaster (1983) and by Wood et al. (1998), 
which are in fact accounts of life cycle and 
breeding ecology, respectively, of B. bairdli. 
not of B. arcticus. as indicated in the titles. 

Macalaster's primary interest was not tax- 
onomy but the opportunity to study the life 
cycle, growth, and reproduction of Bathypoly- 
pus. including observations of live animals 
kept in aquaria. Of phncipal interest to the 
present revision, however, is Macalaster's 
comprehensive measurements, which sup- 
plement Kumpf's (1958) and my own Ameri- 
can data and which show that B. bairdil is 
commonly distributed along the upper part of 
the slopes from Flohda to the Labrador Sea 
and that the genuine B. arcticus seems to be 
absent. I included some of her data as a sup- 
plement to my own to demonstrate morpho- 
logical differences between the southern and 
northern populations of B. bairdii. 



Octopus piscatorum Verrill, 1 879 

A full account of this nominal species and 
description of the holotype is given above 
under the synonymy of B. arcticus, which has 
been the species most often mistaken for pis- 
catorum. It is here shown beyond reasonable 
doubt that O. piscatorum is a junior synonym 
of O. bairdil УеггШ, 1873. It is also shown that 
Robson's concept of piscatorum was based 
upon misidentified arcticus specimens from 
the Faroe Channel and that consequently 
characters of B. arcticus, for example, pres- 
ence of crop diverticulum, multicuspid radula, 
have crept into his diagnosis of piscatorum. 
This of course has repercussions on the con- 
cept of the genus Benthoctopus, of which pis- 
catorum is the type species. 



Bathypolypus proschi Muus, 1 962 

Holotype: Male TL 115 mm, "Dana" Stn. 
10018, 65 02'5"N, 56^^00'W, Davis Strait, 
730-740 m, trawl, July 19, 1956. Depository: 
ZMUC. 

The unanimous claim among earlier au- 
thors (exception: Sars, 1878) that bairdii is 



synonymous with arcticus led me to describe 
proschi as a new species, because it was ob- 
viously distinct from the type material of B. 
arcticus {Muus 1962: 18, table III). 

The large amount of material now available 
and examination of Verrill's types of bairdii 
made it evident that proschi is identical with 
the here-reinstated bairdii anä is a junior syn- 
onym of that species. It should however be 
pointed out that there are (subspecific?) mor- 
phological differences between the American 
and West Greenland populations (see below). 

Bathypolypus Species Recorded from 
Northwestern Spain 

A paper by Perez-Gandaras & Guerra 
(1978) recorded for the first time Bathypoly- 
pus hom 120-439 m off the Galician coast. Of 
22 specimens, 14 were identified as В spon- 
salls. three as В arcticus. another three (type 
A) with hesitation as B. proschi, while two de- 
fective specimens could not be assigned to 
any known species. 

The present revision shows that it is highly 
improbable that arcticus, a true arctic species 
(Fig. 20), would be found off Spain. The bo- 
real bairdii, formerly confused with arcticus, is 
a more likely candidate and could be ex- 
pected to have its southern East Atlantic limit 
somewhere in the Biscayan neighbourhood. 

The data for '^arcticus" given by Perez-Gan- 
daras & Guerra (Table 5) seem to support this 
expectation. The specimens concerned are 
two males, ML: 27-39 mm, and a female, ML: 
37 mm. 

With few probably insignificant deviations, 
the index values lie within the range of varia- 
tion of bairdii (Table 9) and exclude the other 
here recognized Bathypolypus species. Such 
important meristic characters as number of 
laminae copulatoriae (8-10) and suckers on 
the hectocotylized arm (31-35) confirm affin- 
ity to bairdii. One male specimen figured by 
Guerra (1992: 252, fig. 89) shows a typical 
bairdil iorm, apart from the ligula, which has a 
pointed tip different from the broad-tipped 
norm for bairdil known from eastern America 
and the Scotland-Greenland Ridge (Fig. 6). 

Some other characters described by Perez- 
Gandaras & Guerra (1992) are ambiguous. 
The rachidian (their fig. 7) is described as 
multicuspid on account of two very small 
cusps at the base. This is unusual in the 
northern populations of bairdii, which have 
smooth bases (Fig. 11). Further the sper- 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 95 



TABLE 5. Measurements (mm), indices and counts 
of three dubious "arcticuä' from tfie Gallean coast 
extracted from data of Perez-Gandaras & Guerra 
(1978). 



Sex 


male 


male 


female 


TL: 


78 


102 


113 


ML: 


27 


39 


42 


MLTLI: 


35 


33 


37 


HWI: 


85 


62 


62 


EDI: 


44 


36 


31 


SDI: 


5.2 


4.4 


5.7 


SDEDI: 


12 


12 


18 


ALI: 








1 


178 


179 


150 


II 


174 


179 


152 


III 


174 


179 


136 


IV 


167 


167 


131 


HcLI: 


130 


115 


— 


OAi: 


74 


64 


— 


LIgLI: 


20 


18 


- 


LamC: 


9-10 


8-9 


— 


SHcC: 


31 


35 


— 


SpLI: 


- 


144 


- 


SpRI: 


— 


31 


— 



matophore (their fig. 8) has a reservoir occu- 
pying only 31% of the spermatophore length, 
which among the Bathypolypus species is a 
value known only from arcticus (Table 9). 
Apart from the longer oral end, the sper- 
matophore has proportions similar to what is 
found in bairdii, for example, the characteris- 
tic light swelling seen in the cement gland por- 
tion. In arcticus, the oral end of the sper- 
matophore is a thick, opaque tube with no 
swelling where the cement gland is located 
(Fig. 2d). 

Unfortunately, funnel organ and digestive 
tract were not described. Until further material 
can be studied, I think the three specimens 
should be assigned to B. bairdii, but with 
reservation. 

"Type A", described by Perez-Gandaras & 
Guerra (1978: figs. 9-11) includes one female 
and two males, ML: 34-40 mm, that could not 
be assigned to a known species with any cer- 
tainty. The authors suggest B. proschi, but 
that species is now shown to be synonymous 
with bairdii. The specimens possess a pecu- 
liar supraocular cirrus, which is smooth, cylin- 
drical and raised at the anterior edge of a 
hemispherical wart (their fig. 9). The ligula 
(their fig. 10) is a simple spoon-shaped organ 
with midrib and 9-12 low laminae and a small 
pointed calamus, LigLI: 16-18. Compared 
with bairdii, it looks juvenile. Proportions of 



body, spermatophore and most indices (their 
table 4) are compatible with bairdii. The 
rachidians have two small denticles at their 
bases like the "arcticu^' specimens described 
above, and one radula is abnormal, having 
nine rows of teeth (their fig. 11). One male 
(Guerra, 1992: 254, fig. 90) has much smaller 
eyeballs (EDI: approx. 27, measured on the 
drawing) than indicated by Perez-Gandaras & 
Guerra (1978: table 4), in which EDI is given 
as 35-41, compatible with bairdii. 

I think "type A" has to retain its dubious po- 
sition until a larger series of specimens be- 
comes available. 

Bathypolypus pugniger, n. sp. 

Material examined: 31 specimens from the 
Iceland-Faroe area as listed in Appendix I. 

Holotype: Male, ML 32 mm, Icelandic Fish- 
ery Investigations, haul B5-78-44, 64°58'N, 
27°44'W, 860-870 m, March 14, 1978. Mea- 
surements: Table 5, specimen 470. Deposi- 
tory: IMNH 19990971. 

Paratypes: 9 males, 10 females as listed 
and measured in Tables 6 and 7. 

Synonymy 

Bathypolypus arcticus: Adam, 1939 [in 
part]: 9, figs. 2, 3 

Diagnosis 

Ovoid or square-bodied with papillated 
skin; arms very short, arm order lll:IV:ll:l or of 
subequal length; head broad, with large eyes, 
each with a supraocular erectile cirrus; hecto- 
cotylized arm with about 35 suckers; ligula 
globular fleshy and deeply excavated, with 
4-6 laminae; funnel organ double and pad- 
like. Radula with broad homodont central 
teeth; esophagus without crop diverticulum. 
Total length rarely over 150 mm. 

Description 

Skin and Colors: Freshly preserved, the skin 
is violet to purple dorsally, ventrally lighter and 
yellowish or brownish. The antero-dorsal re- 
gion more or less equally strewn with numer- 
ous small warts. Over each eye is a 3-7 mm- 
long single verrucose cirrus, which may be 
bifurcated, the anterior branch being shortest, 
or there are two closely set cirh. 



196 



MUUS 





FIG. 12. Holotype of Bathypolypus pugniger. n. sp., ö' ML 32 mm. Depository: IMNH 19990971. 



Bodily Proportions: Ovoid (Fig. 12) or more 
square-bodied, like bairdii {F\g. 13), with large 
eyeballs. MWI: males 67-92-106, females 73- 
96-118. HWI: males 73-85-100, females 48- 
79-96. TL rarely 150 mm, ML rarely over 50 
mm. MLTLI: 33-37.5-42. EDI for both sexes 
30-44-50, highest for young specimens. The 
lens measures on an average 37% of the eye 
diameter. The rough diagnostic index SDEDI, 
useful to discriminate arcticus and bairdii, 
shows pugniger to lie between the two 
species (Table 9). The corresponding relation 
between SD and LD is supposed to be more 
accurate, but still shows some overlap with 
young specimens of bairdii {F\g. 16). 

The mantle aperture is about 40% of the 
mantle circumference, and the funnel is free 
for about 50%. 

The funnel organ is a couple of pad-like 
structures sometimes heart-shaped but vari- 
able as in B. bairdii {F\g. 18). 

The gills are reduced and have 6-7 gill fila- 
ments on each demibranch. 

The arm length order is lll:IV:ll:l or sube- 
qual (Tables 6, 7). This is reverse from arm 
order of B. arcticus and B. bairdii. The ML 
constitutes 33-42% of the TL making B. pug- 



niger the most short-armed of the three 
species. ALI: I 145, II 150, III 155, IV 154 
(means in Tables 6, 7). 

The web extends along the arms. WDMI: A 
53, В 56, С 59, D 60, E 51 (mean of 18 spec- 
imens). In well-preserved specimens, web 
sectors С and D tend to be subequal. 

The suckers are biserial, small, and well 
spaced. Each arm with 65-75 suckers. They 
are of the same order of size on all arms, and 
there is no sexual dimorphism. SDI: 4.7-5. 
8-7.6. 

The hectocotylized third right arm is usually 
slightly shorter than the left opposite arm. 
OAI: 83-91-106. It has 31-35-45 suckers. 
Spermatophoral groove well developed. 

The ligula is fleshy, short, and broad, with 
firmly inrolled borders, giving it a globular ap- 
pearance as a clenched fist (Figs. 12, 13c). 
LigLI: 22-27-34 in 11 specimens > 25 mm ML. 
The ligula has a tiny pointed tip between the 
broadly rounded anterior flaps (Fig. 13a). The 
width is 80-100% of the length. The spoon 
has a central ridge and 4-6 deep, well-sepa- 
rated laminae (Figs. 9, 13a). Calamus is stout, 
CLI: 26-38-51. Already at a ML of 6 mm, the 
nectocotylized arm may be identified by the 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 97 




FIG. 13. Bathypolypus pugniger, n. sp. a: distal hectocotylus of young cf ML 18 mm. The "clenched" ligula 
was forced open to show the laminae; b; upper and lower beak of cf ML: 33 mm; c: paratype ZMUC CEP- 
17, cf ML 54 mm. Dana Stn. 16437, southwestern Greenland. 



0.8-mm-long, as-yet undifferentiated ligula 
(Table 6: no. 77). 

Female Organs: The ovary is large with 
blackish globular oviducal glands (Fig. 14a, 
b). Proximally the oviducts are joined, distally 
they are short, stout, leaving the oviducal 
glands forming right angles. Supposedly ripe 
eggs nneasure about 10 mm. They are 
smooth, with fine longitudinal lines. 

Male Organs: The penis with well-developed 
diverticulum (Fig. 14f). The spermatophores 
are of bairdii type, with sausage-shaped 
sperm reservoir occupying about 55% of the 
total length. SpLI: 60-100. 



Beaks and Radula: The beaks do not present 
distinctive features. Only four radulae have 
been investigated. The rachidians deviate 
considerably from both arcticus and bairdii 
(Fig. 15), whereas the lateral teeth show no 
distinct features. Basically, the rachidians are 
homodont, but they may be very broad with 
highly set or drooping shoulders (Fig. 15b, e, 
respectively) or just stocky and pointed ho- 
modont, with almost straight sides (Fig. 15c, 
f), but different from the slim, slightly concave 
homodont teeth of bairdii. 

Digestive Tract 

The esophagus has no crop diverticulum, 
but swells to double diameter from the point 



198 



MUUS 



TABLE 6. Measurements (mm) of male Bathypolypus pugniger. n. sp. (Note that in Tables 6 and 7 arm 
length is given as average of paired arms, except for males, where AL III represent the left arm only. Web 
depth for sectors B, С and D are likewise averages.) 



No 


77 


76 


75 


659 


653 


463 


648 


470 


453 


122 


TL: 


11 


30 


38 


51 


67 


76 


88 


90 


91 


146 


ML: 


6.2 


11 


15 


18 


25 


30 


33 


32 


36 


54 


HW: 


5.9 


10 


12 


15 


25 


22 


27 


31 


29 


45 


MW: 


5.5 


9.3 


12 


19 


28 


20 


34 


32 


33 


45 


ED: 


2.0 


4.5 


6.2 


10 


13 


11 


13 


16 


16 


22 


LD: 


1.0 


2.0 


— 


3.7 


5.0 


4.2 


5.5 


5.8 


6.0 


7.4 


SD: 


0.4 


0.7 


0.8 


1,2 


1.9 


1.5 


1.8 


2.0 


2.0 


2.5 


AL: 






















1 


8.5 


15 


17 


26 


36 


39 


40 


55 


50 


85 


II 


8.5 


15 


19 


26 


37 


41 


48 


55 


55 


87 


III 


8.5 


14 


19 


29 


37 


42 


45 


56 


53 


89 


IV 


9.0 


— 


19 


28 


40 


43 


44 


55 


55 


87 


HcL: 


9.0 


13 


16 


24 


34 


37 


38 


52 


50 


83 


LigL: 


0.8 


2.4 


3.1 


3.5 


10 


8.3 


13 


15 


13 


18 


LamC: 





4 


4 


5 


6 


5 


5 


6 


5 


6 


SHcC: 


35 


31 


32 


34 


31 


42 


34 


40 


40 


45 


WD 
























A 




3.8 


— 


— 


9.5 


14 


14 


14 


20 


17 


33 


В 




3.8 


— 


— 


11 


14 


15 


16 


19 


19 


35 


С 




3.9 


— 


— 


10 


15 


15 


16 


20 


20 


38 


D 




3.4 


— 


— 


10 


16 


15 


16 


20 


23 


40 


E 




3.0 


— 


— 


7.8 


14 


14 


10 


15 


18 


29 



TABLE 7. Measures of females attributed to B. pugniger, n. sp. 



No 


74 


649 


71 


455 


461 


466 


465 


456 


457 


464 


TL: 


38 


46 


55 


83 


с 90 


100 


97 


99 


103 


С115 


ML: 


15 


18 


23 


27 


с 32 


33 


36 


39 


40 


с 44 


HW: 


12 


16 


19 


26 


— 


30 


30 


33 


30 


21 


MW 


11 


17 


21 


31 


— 


39 


34 


35 


35 


34 


ED: 


6.6 


11 


10 


13 


15 


14 


14 


16 


16 


13 


LD: 


— 


4.0 


3.6 


5.5 


5.3 


— 


— 


5.8 


5.8 


5.0 


SD: 


0.8 


1.1 


1.1 


2.1 


2.0 


1.9 


1.9 


2.0 


2.2 


2.1 


AL: 






















1 


18 


26 


29 


47 


53 


60 


58 


50 


58 


64 


II 


19 


25 


32 


52 


56 


60 


66 


50 


55 


69 


III 


22 


28 


33 


55 


58 


68 


62 


50 


59 


64 


iv 


19 


26 


32 


55 


59 


60 


61 


52 


56 


65 


WD 






















A 


7 


10 


12 


19 


17 


19 


17 


21 


14 


21 


В 


6.5 


11 


13 


16 


20 


18 


18 


23 


21 


21 


С 


7.5 


10 


13 


19 


22 


24 


22 


26 


20 


22 


D 


8.5 


8.5 


12 


19 


23 


23 


22 


22 


23 


24 


E 


8 


8 


11 


20 


19 


19 


19 


17 


16 


20 



where the leaf-like second salivary glands are 
fastened and down to the stomach (Fig. 14c). 
The two ducts fronn the postenor salivary 
glands are separate for more than half of the 
distance to the buccal mass (Fig. 14c-e). In 
arcticus ana bairdii, the united excretory canal 
is relatively longer (Figs. 2a, 7a). When in situ, 
the very large stomach rests in a deep groove 



of the liver, which is almost bilobed. The spiral 
caecum is scarcely coiled. There is no ink sac. 

Etymology 

pugniger, derived from the Latin pugnas, a 
fist, alludes to the characteristic boxing glove 
appearance of lígula. 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 1 99 




FIG. 14. Bathypolypus pugniger, n. sp. a: reproductive organs of $ ML 40 mm; b: egg from same specimen, 
Stn. B5-77-46, IMNH; c-e: digestive tract; f: reproductive organs of cf ML 38 mm. A spermatophore is at- 
tached to the penis. MNHT, haul 25, July 19, 1979, now ZMUC. 



200 



MUUS 



f^^ 




FIG. 15. Radula and rachidians of В. pugniger, п. sp. а and b: о' ML 31 mm., BIOFAR Stn. 269; c: ö' ML 36 
mm, Iceland Fish. Invest., haul B5-77-46; d and e: cf ML 33 mm, BIOFAR Stn. 269; f: 9 ML 33 mm, Iceland 
Fish. Invest., haul B5-77-48. Scale; 100 цт. 



Synonyms of B. pugniger. n. sp. 

A revision of specimens identified by Adam 
(1939) as B. arcticus showed two specimens 
to be identical to B. pugniger, n. sp. They are 
juvenile, a male (ML: 22 mm) and a female 



(ML: 1 8 mm) caught in 1 938 off the east coast 
of Iceland (66'23'N, 12°53'W) in 200-250 m. 
Adam (1939: 10, specimens a, figs. 2, 3) fig- 
ures the reproduction organs of the male and 
the globose ligula with five distinct laminae 
typical of B. pugniger 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 201 

TABLE 8. Measurements (mm) of a male B. pugniger, n. sp., identified as B. arcticus by Adam 
(1939). Adam's 60-year-old measurements in parenthesis. 



TL: 


59 


(-) 




Indices 






Arm Length 






ML: 


22 


(25) 


MLTLI: 


37 


(-) 




right 




left 


HW: 


17 


(19) 


HWI: 


77 


(73) 


1 


32 (33) 


34 


(36) 


MW: 


20 


(18.5) 


MWI: 


91 


(71) 


II 


30 (33) 


35 


(36) 


ED: 


9.0 


(-) 


EDI: 


40 


(-) 


III 


28 (32) 


33 


(34) 


SD: 


1,3 


(1.3) 


SDI: 


5.9 


(5) 


IV 


(-) 


— 


(28+) 


LigL: 


6.2 


(6.5) 


LigLI: 


22 


(20) 











Table 8 shows measurements and indices 
of the male specimen. Comparison with 
Adam's measurements, which were done 
while the material was fresh, show consider- 
able preservational shrinkage. 

Recognition of B. pugniger as a 
New Species 

The first specimens seen were in fact iden- 
tified as slightly abnormal B. bairdii. The glo- 
bose ligula of the males at first believed to be 
an accidental deformity or a regeneration phe- 
nomenon turned out to be a regularly occur- 
ring stable structure with 4-6 laminae, thus 
deviating significantly from North and East At- 
lantic B. bairdii {F\g. 9). This character, in com- 
bination with a reverse arm order as compared 
with bairdii and arcticus, deviating rachidians 
(Fig. 15), and sucker and eye sizes (Fig. 16) 
justified establishment of a new species. 

As is usual in octopods, the hectocotylized 
males offer the best distinguishing characters. 
The females were only recognized after re- 
newed inspection of all material from the 



Faroe-Iceland area that I had previously iden- 
tified as B. bairdii. 

The search for females of pugniger was 
based on the assumption that, like in other 
Batliypolypus spec\es, there would be no sex- 
ual dimorphism. This means that bodily pro- 
portions, arm order and size of eyes and 
suckers should match pugniger rr\a\es of sim- 
ilar sizes. In this way, 12 females could be at- 
tributed to B. pugniger A circumstantial evi- 
dence for the plausibility of the result is the 
sex ratio of 39% females, close to the sex 
ratio found in bairdii {38% females; n = 235). 

Morphologically pugniger is very close to 
bairdii. As yet few characters distinguishing 
these species have been found. Among juve- 
niles and females mistaken identity between 
pugniger and bairdii \s possible, because the 
state of preservation can blur the differences 
in arm order and size of suckers and eyes. 

The rough diagnostic index SDEDI useful to 
discriminate between arcticus and bairdii, 
shows pugniger to lie between the two 
species (Table 9). The corresponding relation 
between SD and LD should be more accu- 



TABLE 9. Range of distinctive indices and counts of the Batiiypoiypus species based on material examined. 
The sparse data for valdiviae are extracted from text and drawings of Chun & Thiele (191 5), Robson (1 924b), 
and Sanchez & Moli (1984). Data for sponsa//s from the Mediterranean population. 





arcticus 


bairdii 


ergasticus 


pugniger 


sponsalis 


valdiviae 


CaLI 


15-25 


15-25 


30-40 


26-38-51 


33-42 


28-38 


EDI 


20-35 


30-55 


31-41-47 


30-44-50 


30-41-47 


40 


LamC 


11-16 


7-12 


7 (n = 4) 


4-5-6 


4-6-7 


4-5 


LigLI 


9-23 


18-44 


6-9-12 


22-27-32 


10-12-15 


15-18 


MLTLI 


24-29-35 


27-33-38 


13-17-23 


33-38-42 


16-22-26 


30 


OAI 


83-89-100 


71-88-104 


53-60-70 


83-91-106 


47-63-73 


75 


SDEDI 


18-26-34 


6-9-13 


9-11-13 


11-13-16 


4-6.5-8.5 


13 


SDI 


6-7-8.4 


3-4-5 


3.5-4-5.5 


4.7-5.1-6 


2.1-3-3.7 


5 


SDLDI 


50-70-100 


19-25-36 


22-25 (n = 2) 


32-35-40 


15-19-22 


— 


SHcC 


32-49 


36-49 


73-77-83 


31-35-45 


52-57-61 


45 


SpLI 


105-130 


60-90-115 


110-150 


90 (n = 3) 


50-70 


100 


SpRI 


26-32 


50-60 


50-60 


50-60 


50-60 


50 


Eye cirri 


present 


present 


absent 


present 


absent 


present 


Crop divert. 


present 


absent 


present 


absent 


absent 


? 



202 



lUUS 



rate, but still shows some overlap with bairdii. 
especially with youriger specimens (Fig. 16) 

In bodily proportions and hectocotylus, B. 
pugniger has a striking resemblance to the 
South African Bathypolypus valdiviae (Chun & 
Thiele, 1915), known from the Agulhas Bank 
and off the Namibian coast. 

With one exception from West Greenland 
(Fig. 13c, Table 6: no. 122), all specimens of 
pugniger are caught on the Faroe-Iceland 
plateau in warm Atlantic water. The zoogeo- 
graphical and ecological perspectives are dis- 
cussed below. 



HISTORICAL REVIEW AND 

PRESENT STATUS OF BATHYPOLYPUS 

AND BENTHOCTOPUS. 

In 1 921 , Grimpe erected two new genera in 
the subfamily Octopodinae to accommodate 
some characteristic octopods without ink sac: 
Octopus arcticus Prosch, 1849, was desig- 
nated type species of Bathypolypus. and O. 
piscatorum Verrill, 1879, type species of Ben- 
thoctopus. Grimpe just stated that the two 
species "erheblich verschieden" [differ con- 
siderably], but he did not otherwise define the 
genera. 

To accommodate two new species {Bath- 
ypolypus grimpei and Benthoctopus berryi), 
Robson (1924a, b) defined the genera this 
way: 

'bathypolypus Grimpe: Deepwater poly- 
pods with broad and long hectocotylus and 
unicuspidate rhachidian teeth. The skin is 
usually covered with warts and may be gelati- 
nous. There is no ink sac. Type: B. arcticus." 

"Benthoctopus Grimpe: Abyssal polypods 
with small hectocotylus, multicuspid rhachid- 
ian teeth and smooth skin. There is no ink sac. 
Type: B. piscatorum." 

Without discussing details, Grimpe (1925: 
100, footnote) declared himself in complete 
agreement with Robson's generic definitions. 

With small modifications these definitions 
have persisted (for example, Thiele, 1935; 
r\/langold & Portmann, 1989). Robson (1927) 
amended the generic definitions slightly and 
later (1928) erected a new subfamily, Bathy- 
polypodinae, comprising the two genera and 
simply defined as: "Octopods mainly abyssal 
in habitat and devoid of an ink sac". 

In his monograph, however, Robson (1929, 
1932) moved Benthoctopus with 14 nominal 
species back to the subfamily Octopodinae, 
while Bathypolypus with six nominal species 



was retained in Bathypolypodinae. With the 
characters of the subfamily (Robson, 1932: 
286), Bathypolypus was now defined as: 
"Abyssal octopodids devoid of an ink sac and 
in which the crop is usually reduced and 
sometimes absent. Eggs and vaginae are 
large and spermatophores large and few in 
number. The mantle aperture is very narrow 
and the general habit squat and short armed." 

Robson removed Benthoctopus to Octo- 
podinae because he felt that some of the 
species resembled ordinary forms of Octopus 
and that "were it not for the lack of the ink-sac 
one would place them in that genus" (Robson, 
1932: 51). On the other hand, Robson real- 
ized that some species have traits of both 
genera. 

The latter problem has caused some taxo- 
nomic confusion. After a redescription of 
Bathypolypus sponsalis, it prompted Wirz 
(1955: 146) to declare that the strict distinc- 
tion between the genera Benthoctopus and 
Bathypolypus made by Robson was unjusti- 
fied considering the small number of differ- 
ences. Thiele (1935) reunited the two genera 
in Bathypolypodinae. 

As presented by Robson, the two genera 
represent an array of species that in varying 
degrees demonstrate traits believed to reflect 
adaptation to benthic life in deep water-ab- 
sence of ink sac; reduction of gills, radula, and 
crop; increasing size of eggs and sper- 
matophores; elaboration of ligula; shortening 
of the arm complex; and funnel organ tending 
to be double. 

In general, the least specialized species, 
that is, those most similar to Octopus, seem to 
be accommodated in Benthoctopus, the most 
divergent in Bathypolypus. 

The present revision shows that most of the 
existing confusion derives from misidentifica- 
tions that have led to errors in the concept of 
the two type species and subsequent misun- 
derstandings in the generic definitions of Ba- 
thypolypus and Benthoctopus. 

The choice by Grimpe (1921) of Octopus 
piscatorum as type for Benthoctopus was es- 
pecially unlucky, because an examination of 
the type shows it to be identical with the here- 
reinstated Bathypolypus bairdii {Уеп\\\, 1873). 

Aware of this fact and in connection with the 
description of five new species of Benth- 
octopus from the Pacific, Voss & Pearcy 
(1990) plead for the preservation of the name 
Benthoctopus. which now includes 20 species 
worldwide (Sweeney & Roper, 1998). 

Voss (1 988a, b) restricted the subfamily Ba- 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 203 



I 

SD mm 
4- 



3- 



D 
D/ О 

/ B.arcticus 

^ n=33 




LDmm 



1 2 3 4 5 6 7 



9 10 11 



SDmm 
4- 



B.arcticus 



B.pugniger n.sp. 




B.bairdii 



LDmm 



3 4 



9 10 11 



FIG. 16. Sucker diameter versus lens diameter in arcticus, pugniger, n. sp., and bairdii. Reduced major axis 
regressions show that the relation differs among arcticus and bairdii and among arcticus and pugniger at the 
P = 0.01 level of significance. Among pugniger ano bairdii a\ P = > 0.1 < 0.05. Regression slopes with 95% 
confidence intervals and coefficient of determination: 0.8372 (0.7177-0.9766; 0.9766) for arcticus; 0.4112 
(0.3541-0.4774; 0.9025) for pugniger, and 0.2643 (0.2294-9.3045; 0.7921) for bairdii. The regression in- 
tercepts are 0.1614, 0.0641, and 0.0592 respectively and not considered different in pairwise comparison 
among species. 

Inserted in upper figure: data for the type of B. arcticus (crossed square), the "type" of B. faeroens/s found in 
ZMUC (star) and the holotype of Bentlioctopus piscatorum (cross in circle). 



204 



MUUS 



thypolypodinae to benthic octopods with bise- 
rial suckers and devoid of ink sac and pro- 
posed the following definition of Benthoctopus 
Grimpe, 1921; 

"Deepwater octopods of normal Octopus- 
like appearance with short to long arms, suck- 
ers biserial; hectocotylus Octopus-like, ligula 
slightly to moderately excavated with indis- 
tinct midrib, smooth or bearing low, often in- 
distinct rugae, never laminate; crop present, 
usually with diverticulum; ink sac absent; 
radula with strongly, seldom weakly, multicus- 
pid rachidian; body entirely smooth, papillae 
or ocular cirri absent. Type species Octopus 
piscatorum Verrill, 1879, by original designa- 
tion." 

If the genus Benthoctopus is to be pre- 
served, it would need a different type species 
to be designated by the International Com- 
mission on Zoological Nomenclature. A sensi- 
ble choice would be Benthoctopus januarii 
(Hoyle, 1885). This species is revised and 
thoroughly redeschbed by Toll (1981) based 
on a series of recently caught male and fe- 
male specimens. It conforms to the diagnosis 
of Benthoctopus suggested by Voss and fur- 
thermore has the advantage of being a wide- 
spread species in the Gulf of Mexico and off 
Brazil. 

The final decision and the plea to the Inter- 
national Commission on Zoological Nomen- 
clature, however, should be made by a reviser 
of the Benthoctopus species. 

The genus Bathypolypus should under any 
circumstances be preserved. Robson, being 
the first revisor of the two genera, acknowl- 
edged Octopus arcticus as type of a group of 
species characterized by large lamellated 
ligulae, as opposed to another group of 
species characterized by small Octopus-like 
ligulae, the present Benthoctopus group. To 
uphold the distinction between the two groups 
and to match the definition of Benthoctopus 
by Voss & Pearcy (1990). I propose the fol- 
lowing modification of Robson's definition of 
Bathypolypus in an attempt to make an oper- 
ational distinction between the two genera: 

Sai/iypo/ypus Grimpe, 1921. Type species: 
Octopus arcticus Prosch, 1849, by original 
designation. 

Deepwater octopods of normal Octopus- 
like appearance, with stout body and gener- 
ally with short arms; suckers biserial; hecto- 
cotylus with deeply excavated ligula bearing a 
number of well-defined laminae; crop, if pres- 
ent, seldom with diverticulum; ink sac absent; 
radula with homodont or weakly and irregu- 



larly multicuspid rachidians; skin usually with 
papillae; supraocular cirri often present. 



GENERAL SURVEY OF THE 
BATHYPOLYPUS SPECIES 

The elaborated hectocotylus, with a promi- 
nent, deeply excavated, laminated ligula, is 
the most distinctive character in the amended 
generic definition of Bathypolypus. In addi- 
tion, there are reductions in digestive tract, 
gills, radula, relative arm length, and the en- 
largement of eyes exhibited by the species in 
varying degrees. A tendency to develop some 
of these traits is found in Benthoctopus. The 
difference between the hectocotyli of the Ba- 
thypolypus and the Benthoctopus species 
provides sufficient substance to justify pre- 
serving the two genera, if only as a prelimi- 
nary measure. For the time being, our igno- 
rance regarding evolutionary sequence and 
weighting of derived characters in Octopodi- 
dae precludes serious phylogenetic consider- 
ations. 

Bathypolypus comprises six species in the 
most recent list of accepted cephalopod taxa 
(Sweeney & Roper, 1998); arcticus (Prosch, 
1849); faeroensis (Russell, 1909); proschi 
Muus, 1962; salebrosus (Sasaki, 1920); 
sponsalis (P. Fischer & H. Fischer, 1892); and 
valdiviae (Chun & Thiele, 1915). 

The species proschi and faeroensis are 
here shown to be synonyms of the reinstated 
bairdli and arcticus respectively, whereas 
pugniger is recognized as new. It remains, 
however, to reconsider the remaining three of 
the hitherto recognized species in the light of 
the amended generic definitions of Bathypoly- 
pus and Benthoctopus. Also, the position of 
Benthoctopus ergasticus (P. Fisher & H. 
Fischer, 1892) is reconsidered. 



Benthoctopus salebrosus 
(Sasaki, 1920), new comb. 

Po/ypussa/ebrosus Sasaki, 1920: 182; 1929: 
99, text-fig. 54, pi. 6, figs. 5, 6. 

Bathypolypus salebrosus: Robson, 1929: 41; 
1932: 302; Akimushkin, 1965: 134, fig. 
34; Nesis, 1987: 315, fig. 83A. 

Type and paratype: two females, USNM 

332969, TL: 153 mm, ML: 45 mm; USNM 

332970, TL: 77 mm, ML: 23 mm (not ex- 
amined). 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 205 



A characteristic, though not well-known 
species from the Okhotsk Sea and off the 
Japanese Pacific coast in 212-1070 m. 

Based on the female types, Robson (1929, 
1932) assigned salebrosus to Bathypolypus 
with hesitation, mostly on account of its short 
arms, rather deep web (33%), and because it 
is sculptured with closely set, well-defined, 
roundish warts. He found that the rachidians 
were weakly multicuspid, with at most one 
denticle on each side, and he established the 
absence of an ink sac. 

The hectocotylus was later described by 
Akimushkin (1965: fig. 34) and Nesis (1987: 
fig. 83A). The ligula is narrow, conical and 
pointed, LigLI about 14. The central groove is 
shallow and transversely striated with numer- 
ous indistinct rugae. Calamus is short and 
pointed. 

Benthoctopus salebrosus has no supraocu- 
lar cirri. The funnel organ is figured by Sasaki 
(1929: text-fig. 54) as a single W, the outer 
legs being the shortest. Gills not much re- 
duced, each demibranch with 9-10 filaments, 
the mantle aperture correspondingly moder- 
ate (B-C in Robson's terminology). 

The slim, striated ligula unknown to Robson 
is of a type seen in some Benthoctopus 
species but very different from the highly spe- 
cialized ligulae of Bathypolypus arcticus and 
B. bairdii. 

I conclude that salebrosus is best placed in 
the genus Benthoctopus due to the simple 
non-laminated ligula and the single funnel 
organ, irrespective of its warty skin, which is 
unusual among other species of that genus. 
No other character contradicts this opinion. 

Bathypolypus sponsalis 
(P Fischer & H. Fischer, 1892). 

Octopus sponsalis P. Fischer & H. Fischer, 
1892: 297, fig. A; Fischer & Joubin 1907: 
322, pi. 22, figs. 5-11 

Bathypolypus sponsalis: Robson, 1927: 252; 
1932: 300, figs. 61-63; Wirz, 1954: 139, 
figs. 1-5; 1955: 129, figs. 1-12; Adam, 
1960: 504, fig. 2; Mangold-Wirz, 1963: 
49; Perez-Gandaras & Guerra, 1978: 
195, figs. 2-5; Nesis, 1987: 315, fig. 831; 
Guerra, 1992: 249, figs. 88, 91. 

Material examined: Syntypes of Octopus 
sponsalis P. Fischer & H. Fischer, 1892, 
MNHN 571097: 4 males ML: 27-41 mm, 
Exp. du "Talisman", 332-1250 m, NW- 
Africa; in ZMUC, 8 males, ML: 33-49 mm 
and 3 females ML: 27-38 mm, Catalan 



Sea, western Mediterranean, June- 
September 1954 and June-September 
1955, 200-500 m; in BMNH: one female, 
ML: 36 mm, Exp. du "Talisman", July 12, 
1885. 

This species was originally caught off west- 
ern Sahara (22°N, 19°46'E) at the same lo- 
cality as B. ergasticus. It has since been re- 
ported as a common mesobenthic species in 
the western Mediterranean (Wirz, 1954, 
1955), the Aegean Sea (D'Onghia et al., 
1993), and off Portugal and Galicia (Perez- 
Gandaras & Guerra, 1978). 

Good pictures of the whole animal and the 
hectocotylus are presented by Fischer & 
Joubin (1907: pi. 22), radula and internal or- 
gans by Robson (1932: figs. 61 -63), and Wirz 
(1954: figs. 3-5; 1955: figs. 5-12). 

Robson (1927, 1932) placed sponsalis in 
Bathypolypus because of its apparent affini- 
ties to the arci/cus-g roup: a squat, relatively 
short-armed body with huge eyes, small suck- 
ers, double funnel organ, a reduced radula 
with homodont rhachidians, lack of crop, and 
a ligula that is deeply excavated, with 6-7 
laminae. 

The morphometries and major features of 
the life cycle of the Mediterranean population 
of sponsalis are well known (Wirz, 1955; 
Mangold-Wirz, 1963) based on about 600 
specimens caught at all times of the year. 
Table 9 gives the main distinctive indices 
based on my own measurements of speci- 
mens from the Catalan Sea. 

There are, however, some discrepancies 
between these data and descriptions of spec- 
imens from the type locality (Cape Verde Is- 
lands). Examination of the syntypes (MNHN 
571097) shows that the Mediterranean popu- 
lation deviates in several important traits: 

Two spermatophores extracted from one of 
the syntypes confirmed Robson's observation 
(1932) that the sperm reservoir is long, SpRI: 
65-70 (versus 50-60 in the Mediterranean). 
The short oral end has a distinct swelling in 
the middle, making it spindle-shaped, unlike 
any other spermatophores of the genus (Fig. 
17). The apical end is swollen compared with 
the smaller and more cylindrical reservoir in 
Mediterranean specimens. 

Calamus is very large and fleshy (Fischer & 
Joubin, 1907: pi. 22, fig. 6). In four syntypes, I 
found CaLI: 57-71 (Medit.: 33-42). The dif- 
ference was noted by Wirz (1955). 

The ligula is larger in the type series, LigLI: 
14-22 (n = 5) (Medit.: 10-15), and the num- 



206 



MUUS 





1 cm 

FIG. 17. Spermatophores of В. sponsalis. Left: syn- 
type, ML 41 mm., Cape Verde. Right: specimen 
from the Catalan Sea, western Mediterranean, ML 
44 mm. 



ber of suckers on the hectocotylus is low, 
SHcC: 44-50 (n = 4) (Medit.: 57-61). 

The type specimens have warts or pustules 
over the eyes and often on the antero-dorsal 
region. Adam (1960: fig. 2) found supraocular 
cirri, as well as a mixture of multifid and sim- 
ple warts sprinkled dorsally on two specimens 
from the Cape Verde Plateau. In contrast, the 
Mediterranean and Galician specimens have 
smooth skin without traces of warts. 

I conclude that the morphological devia- 
tions between populations may be rooted in 
subspecific variation or even represent unrec- 
ognized sibling species. Not least, the mor- 
phology of hectocotylus and spermatophores 
speak for the latter possibility. The Mediter- 
ranean population is well documented, but 
more specimens from the Cape Verde Pla- 
teau are needed. Regardless, the specimens 
hitherto identified as sponsalis clearly belong 
to the genus Bathypolypus. 

Bathypolypus valdiviae 
(Chun & Thiele, 1915) 

Polypus valdiviae Chun & Thiele, 1915: 485, 
text-figs 52, 53, pi. 80, figs. 1 -5 

Bathypolypus grimpei Robson, 1924a: 208; 
1924b: 663, text-figs. 37-41, pi. 2, fig. 10 



Bathypolypus valdiviae: Massy, 1927: 165; 
Robson, 1932: 303, figs. 64-68; San- 
chez & Moli, 1984: 19, fig. 18; Nesis, 
1987:315, fig. 83F-H 

Type: Z. M. Humboldt Univ. Berlin. Male, ML: 
approx. 30 mm (not examined). 

This species is known from the South 
Afncan Agulhas Bank and off the Namibian 
coast. 

Bathypolypus valdiviae has a remarkable 
resemblance to B. pugniger. n. sp, being a 
short-armed, big-eyed, warty species usually 
with supraocular cirri and having the same 
kind of characteristic hectocotylus, Ligula is 
globular and deeply excavated with 4-5 well 
developed laminae. LigLI: 15-18. The hecto- 
cotylized arm bears about 45 suckers, HcLI: 
approx. 75. The arms are of subequal length 
(Chun & Thiele, 1915: pi, 1, fig. 4; Robson, 
1924b: fig. 37, as "S. grimpei"). 

Also in other characters valdiviae shows 
affinity to pugniger and bairdii. The funnel 
organ is a pair of widely separated V-shaped 
pads (Robson, 1932: text-fig. 66); the rhachid- 
ians are homodont, but more pointed than the 
broad teeth in pugniger; ihe spermatophore is 
a true copy of bairdii and pugniger sper- 
matophores (Robson 1924b: text-figs. 39, 
41). 

I conclude that placement of valdiviae in 
Bathypolypus is justified. 

Bathypolypus ergasticus 
(P. Fischer & H. Fischer, 1892), new comb. 

Octopus ergasticus P. Fischer & H. Fischer, 
1 892: 298, fig. B; Fischer & Joubin, 1 907: 
325, fig, 2A, pi. 22, figs. 1-4 

Octopus profundícela Massy, 1907: 277, 

Polypus ergasticus: Massy, 1909: 7, pi. 1, 
figs. 1-3, pi. 2, fig. 1; 1913: 1; Robson, 
1924b: 668 

Benthoctopus ergasticus: Grimpe, 1 921 : 300; 
Robson, 1927: 255, figs. 4-6, 9; 1932: 
244, figs. 44, 45; Nesis, 1987: 320, fig. 
84E, F. 

Material examined: Syntypes of Octopus er- 
gasticus P. Fischer & H. Fischer 1892, 
MNHN 5811: 3 juv, males ML: approx. 
17-35 mm, Exp. du "Talisman" 1883, 830 
m, NW-Africa; in BMNH: syntypes of 
Polypus profundicola Massy, 1907: 3 
males, ML: 50-80 mm; 3 females, ML: 
57-61 mm. "Helga" stations 363, 365, 
369, 400, 477 and 489, approx. 51°25'N, 
11-12"W, 385-720 fath. One male er- 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 207 



gasticus, ML: 45 mm, NW-Africa (gift 
from Prof. Joubin). 

This characteristic species originally caught 
off western Sahara (22°24'N, 19°46'E, 860 
m) is well described by Fischer & Joubin 
(1907: pi. 22) and by Massy (1907, as O. pro- 
fundicola; 1909: pis. 1, 2) based on speci- 
mens caught off western Ireland. Robson has 
described reproductive organs, radula (1927: 
figs. 4-6, 9), and the digestive tract (1932: 
figs. 44, 45). 

The main indices of ergasticus are given in 
Table 9 based on my own measurements. Ad- 
ditional data in Massy (1909). 

Robson found it difficult to accommodate 
ergasticus in either Bathypolypus or Benth- 
octopus. He decided on the latter genus, how- 
ever, mainly because he, in contrast to Massy, 
found that the rachidians are multicuspid and 
because the esophagus has a crop diverticu- 
lum. As mentioned earlier, Robson confused 
the type species of the genus, Benthoctopus 
piscatorum, with specimens of Bathypolypus 
arcticus from the Faroe Channel. Multicuspid 
rhachidians and a crop diverticulum are found 
in the latter species and are thus not unique to 
Benthoctopus. 

Robson (1932: 247) admits that the hecto- 
cotylus of ergasticus is unlike those of other 
Benthoctopus species. The hectocotylized 
arm is short (OAI: 60) with a well-developed, 
deep, spoon-shaped ligula with about 7 
strong laminae (LigLI: 9). Calamus prominent 
(CaLI: 30-40). The hectocotylus has a strong 
likeness to that of sponsalis. Chun & Thiele 
(1915: 485) consider it obvious that ergasti- 
cus, sponsalis, and valdiviae are related due 
to the form of the hectocotylus. 

Another character showing affinities to the 
Bathypolypus species is the enormous sper- 
matophore. One specimen with a total length 
of 1 1 1 mm, had a sperm reservoir of 51 mm in 
length and 8 mm in width. Apart from the size, 
the proportions are very much like the sper- 
matophores of bairdii and pugniger {Robson, 
1927:255, fig. 4). 

The funnel organ is double, and in the spec- 
imens from Ireland consists of two very char- 
acteristic subquadratic or pentagonal pads 
(Fig. 18), a type unknown in Benthoctopus, 
but not far from the structures in bairdii and 
pugniger In the specimen from West Africa, 
the funnel organs each had a slight indenta- 
tion anteriorly, making them more heart- 
shaped. 

In the light of the amended generic diag- 



noses given, ergasticus is best accommo- 
dated in genus Bathypolypus. 

The genus Bathypolypus, as here recog- 
nized, thus includes only the following six At- 
lantic species: arcticus (Presch, 1849), bairdii 
(Verrill, 1873), pugniger, n. sp., sponsalis (P. 
Fischer & H. Fischer, 1892), valdiviae (Chun 
& Thiele, 1915), and ergasticus (P. Fischer & 
H. Fischer, 1892). The species salebrosus 
(Sasaki, 1920) is moved to Benthoctopus on 
account of the simple, non-laminated hecto- 
cotylus and single funnel organ. 



DISTRIBUTION OF BATHYPOLYPUS 

Bathypolypus arcticus, s. str., are confined 
to Nonwegian Sea Deep Water (NSW), 
whereas the squat, broad-headed species (B. 
bairdii, B. pugniger, n. sp.) are found in the 
warmer Atlantic water south of, or on top of, 
the Greenland-Scotland Ridge. 

This is brought out in Figure 19, which is 
based on the updated hydrographie and topo- 
graphic rewiew papers by Hansen (1985) and 
Westerberg (1990) in connection with the 
BIOFAR benthic projects. It is seen that sub- 
zero NSW fills the trough of the Faroe-Shet- 
land Canal 500-700 m under the north-going 
warm Atlantic Current. Almost barred in the 
south by the Wyville Thomson Ridge, the 
main flow of NSW is forced to make a bend 
round the southern end of the Faroe Plateau 
and flows through the Faroe Bank Canal to 
reach, and eventually slide down, the south- 
ern slopes of the Iceland-Faroe Ridge mixed 
with Atlantic water. 



Northern species - B. arcticus, B. bairdii, 
B. pugniger, n. sp. 

Bathypolypus arcticus 

Horizontal Distribution: Figure 19 clearly 
demonstrates the stenothermal arctic affinity 
of arcticus. Specimens caught outside NSW 
are invariably in places intermittently exposed 
to overflow of cold water, which on the Faroe- 
Iceland Ridge often is a mixture of NSW and 
Arctic Intermediate Water (AI) generated in 
the Iceland and Greenland seas. 

The arctic affinity of arcticus is confirmed by 
its wider distribution in the cold parts of the 
Barents Sea, off eastern Greenland, and in 
the cold threshold fiords of southwestern 



208 



MUUS 



B.bairdii 









B.pugniger 



00 OOÜÜO^ Wölö" 



B.arcticLis 



V V^<^^'^'i'^i^\\^-^ 



B.ergasticus 



00 0000 00 Oö 



B.sponsalis 



FIG. 18. Variability of funnel organ in five North Atlantic Bathypolypus species. The left pair in each row is 
considered typical of the species. 



Greenland, not influenced by the warm 
Irminger Current (Fig. 20). According to Kon- 
dakov (1936), arcticus occurs in the Kara Sea 
(78'01 'N, 105 27'W, 175 m, -0.64X). I have 
not had the opportunity to verify this identifi- 
cation, but Kondakov's drawing looks con- 
vincing. 

Bruun (1945: 8) in his treatise of Icelandic 
cephalopods, though following Robson's 



sensu lato concept of arcticus, is the only au- 
thor who noticed that the narrow-headed 
specimens occur off northern Iceland, 
whereas the "bairdii form" is found off the 
south and southeast coast of Iceland, that is 
south of the North Atlantic Ridge. 

The material of adult arcticus are too few to 
disclose possible geographic variation in 
meristic characters. 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 209 



1f W 9" 




FIG. 19. Distribution in Faroese waters of B. arcticus (squares), B. bairdii (dots) and B. pugniger, n. sp. 
(stars). Arctic deep water with negative temperatures darkly screened. In northwest occasional overflow of 
arctic water is indicated. 



Bathymetrical Distribution: Based on the 
present material, the depth range of arcticus 
is 37-565-1210 m. Only in the northernmost 
localities is the species caught in less than 
1 00 m. In the south, optimal low temperatures 
are found at greater depth, usually over 400 
m. Juveniles and sexually mature animals are 
evenly scattered at all depths. 

Batiiypolypus bairdii 

Horizontal Distribution: This species seems 
to prefer Atlantic water masses with tempera- 
tures in the range of 2-8°C (Figs. 19,21). Due 



to the warm Norwegian Current, Ьа/гсУ/7 ranges 
from the northern North Sea and Skagerrak 
along the Norwegian Coast to the southern 
parts of the Barents Sea. It is common on the 
southern slopes of the Iceland-Greenland 
Ridge, but is probably barred from southern 
Greenland by the eastern Greenland Polar 
Current. The southeastern Atlantic limit for 
bairdii seems to be northwestern Iberian wa- 
ters (Perez-Gandaras & Guerra 1978). 

In western Greenland, bairdii is found in 
water tempered by the warm Irminger Cur- 
rent, and it is common on the prawn trawling 
grounds and fishing banks south of the ridge 



210 



lUUS 




FIG. 20. Distribution of Bathypolypus arcticus (Prosch, 1849). Open circles: positions not precisely known. 



separating Davis Strait and Baffin Bay. There 
are fewer records along the Labrador coast, 
but the Newfoundland area seems to offer the 
species excellent conditions. The Labrador 
Current, with admixture of warmer Atlantic 
water, fills the deeper parts (> 100-200 m) of 
the Laurentian Channel with water that rather 
constantly holds 2-5 С (Brunei et a!., 1998). 
Macalaster (1976, as "ß. arcticus") records an 
average temperature of 4.3'C (SD: 1 .VC) for 
stations where the species was caught. 

The species was reported as far south as 
Fowey Rocks, Miami (Boone, 1938). Cairns 
(1976) showed bairdii to be the most abun- 
dant cephalopod in the Straits of Florida. 

Geographic variation: Bathypolypus bairdii is 
distributed in a many thousand km long, nar- 
row band largely following the upper 180- 
1000 m of the continental slopes of the North 
Atlantic (Fig. 21 ). As it probably is a rather sta- 
tionary animal and having non-pelagic young, 
a certain clinal geographic variation could be 
expected. In bodily proportions, there does 
not seem to be variation. But characters of 



the hectocotylus show statistically significant 
geographic variation. 

Populations south of 45°N have larger ligu- 
lae (Fig. 10), with more laminae (Fig. 9) but 
fewer suckers on the hectocotylized arms 
(Fig. 8) than populations from Labrador and 
western Greenland. As regards the number of 
laminae, the Newfoundland area acts as a 
transitionary zone (Fig. 9). The clinal variation 
may be due to genetic différencies, or it may 
be a response to different ecological or hydro- 
graphic regimes. 

Small but perceptible geographic variation 
exists between the population from Labrador 
and western Greenland and their eastern At- 
lantic fellows. The Cape Farewell area at the 
southern tip of Greenland seems to form a 
distributional gap (Fig. 21 ). The occurrence of 
B. arcticus in this area (Fig. 20) suggests that 
the eastern Greenland Polar Current, which 
sweeps the slopes causes the absence of 
bairdii. In the eastern Atlantic, the ligula has 
more laminae (Fig. 9) and the hectocotylus 
slightly fewer suckers than found in Labrador 
and western Greenland. The relative size of 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 21 1 




FIG. 21. Distribution of Bathypolypus ba/rd/7 (Verrill, 1873). Dots: specimens examined by the author, open 
circles: specimens treated by Kumpf (1958) or Macalaster (1976), but not examined by the author. 



lígula (LigLI) is the same for both populations 
(Fig. 10). 

The geographic variation is established for 
the nnales only, and it is most distinct in the 
western Atlantic population south of New- 
foundland. At the present state of knowledge I 
do not think a subspecific division is advis- 
able. The two major dividing areas, New- 
foundland and Cape Farewell, southern 
Greenland, have led to three slightly different 
populations. 

Bathymétrie Distribution: Kumpf (1958) states 
the depth range of his large western Atlantic 
material of bairdiHo be 20-350-1545 m. In 
the Strait of Florida, Cairns (1976) found the 
depth range to be 190-365-674 m. The 
southernmost record (29°45'N, 30°09'W) 
was also the deepest. Similar depth ranges 
were found off Greenland (maximum 1100 m) 
and in the East Atlantic (maximum 910 m). 

Juveniles and sexually mature animals are 
found at all depths, and there are no evidence 
of vertical migrations. 

Habitat and Biology: The vast majority of 
specimens are caught in trawling grounds on 
muddy or sand-mixed bottoms, which seem to 



be its natural habitat. The catches suggest a 
rather even dispersion. Undenwater photos 
from the western Greenland prawn grounds 
showed bairdii\r\ its natural surroundings. The 
animal was seen resting unprotected in an ev- 
idently self-made shallow depression with the 
arms neatly curled along its sides. The stom- 
ach contents of simultaneously caught speci- 
mens showed a variety of polychete bristles 
and crustacean remains, including Pandalus. 

Based on aquaria observations, O'Dor & 
Macalaster (1983) suggest that bairdii prac- 
tises a sit-and-wait feeding strategy, and they 
list food items demonstrating its omnivorous 
nature. 

Undenwater photos confirm the supposed 
feeding strategy. In the prawn trawling 
grounds, bairdii is surrounded by a rich food 
supply of roaming crustaceans, polychetes, 
and molluscs. 

Mortality must be very low. O'Dor & Macal- 
aster (1983) show that a three-year lifespan, 
including one reproduction period, is probable, 
but that a longer life cannot be ruled out. They 
estimate that due to the few eggs (at most 1 00) 
and even fewer hatchlings, about 4% of the lat- 
ter have to survive to replace the parents. 

To deposit and guard the eggs, the females 



212 



MUUS 



need a firm substratum. This may explain why 
I found a skewed sex ratio on the level trawl- 
ing grounds poor in shelters (38°o females, n 
= 235). Hiding egg-guarding females are less 
apt to be caught in a trawl. 

Brooding females may be found in rocks 
(Macalaster, 1976) or in cans and containers 
discharged from ships (my observation) and 
even in plastic bags (Bergstrom, fide Macal- 
aster, 1976). 

Wood et al. (1 998) observed mating behav- 
iour and brooding of B. ba/rd// (called "S. arctl- 
cus") from Fundy Bay, Canada. In aquaria, 
brooding of eggs lasted over 400 days (at 
6-1 0'C) or roughly one-third of the stipulated 
lifetime of bairdii. Hatchlings had a mantle 
length of 6 mm. 

Bathypolypus pugniger, n. sp. 

Horizontal Distribution: While arcticus and 
bairdii present a neat picture as distinct al- 
lopatric species, B. pugniger n. sp., brings 
new problems through its enigmatic system- 
atic position and peculiar distribution. The sin- 
gle find from western Greenland (Table 6: no. 
122) and the specimens from the Faroe-Ice- 
land Ridge are all caught in waters with posi- 
tive temperatures but in localities episodically 
exposed to overflow of arctic water from the 
realm of arcticus. I find this distribution sug- 
gestive and propose three different specula- 
tive explanations: 

(1) S. pugniger \s a stunted form of B. bairdii. 
Adult bairdii living on the Faroe-Iceland 

Ridge may endure periods of overflow with 
arctic water. Brooding females probably stay 
on. Is it possible that negative temperatures 
during embryonic development result in 
stunted development? If this is the case, why 
are there not transitional stages between 
bairdii and pugniger? 

(2) B. pugniger is a hybrid between bairdii 
and arcticus. 

The similarity in lifestyle, habitat, and mor- 
phology of the two species suggests that mat- 
ing behaviour may also be similar. Is it possi- 
ble that male arcticus in places with episodic 
overflow of arctic water extend their territory 
and seduce (rape?) female bairdii left behind 
and perhaps less resistant, chilled as they 
are? This and the previous suggestion could 
explain the peculiar distribution of pugniger \n 
a narrow zone bordering the arcticus habitat. 



(3) B. pugniger \s a valid species. 

It has been overlooked and confused with 
bairdii. The Bathypolypus species of the East 
Atlantic are not yet well known and more ma- 
terial will show a wider distribution of pug- 
niger 

The question can best be solved by molec- 
ular approaches, such as protein elec- 
trophoresis or DNA-sequencing using fresh 
material not previously preserved in formalin. 

Bathymétrie Distribution: The depth range of 
20 stations where B. pugniger was caught is 
200-610-1000 m. 

The Southern Species -ß. sponsalls, 
B. ergastlcus, B. valdlviae 

Bathypolypus sponsalls is an East Atlantic 
species, which replaces bairdii irom the Gali- 
cian coast to the Cape Verde Islands. It is 
widespread in the Mediterranean. It seems to 
prefer the same habitats as bairdii and has a 
similar bathymétrie distribution: 170-1250 m 
(P. Fischer & H. Fischer, 1892: Wirz, 1955; 
Perez-Gandaras & Guerra, 1978). Some evi- 
dence of upslope ontogenetic migration was 
found in the eastern Mediterranean by Vil- 
lanueva (1992). 

Bathypolypus ergastlcus occurs off south- 
western Ireland and the Cape Verde Islands 
but is not known from the Mediterranean. In 
its northern distribution, it overlaps bairdii ana 
further south sponsalls. However, ergastlcus 
prefers deeper water than its congeners: 
704-1350 m off Ireland (Massy, 1909), 932- 
1139 m off Cape Verde Islands (P. Fischer & 
H. Fischer, 1 892). The three species are para- 
patric. 

Bathypolypus valdlviae is the only repre- 
sentative of the genus known from the south- 
ern hemisphere. Off the Namibian coast and 
on the Agulhas Bank, the species does not 
seem to be rare on soft bottoms. Bathymétrie 
range: 500-900 m (Chun & Thiele, 1915; 
Massy, 1927; Roeleveld, 1974; Sanchez & 
Moli, 1984). 

REMARKS ON THE 
IDENTIFICATION OF SPECIES 

The application of bivahate ratios (indices) 
in multivariate statistical analysis is inadvis- 
able (Atchley et al., 1976), but ratios are use- 
ful in taxonomic work, such as in keys. They 
are also used here for comparison with earlier 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 21 3 



published data. Ratios based on variables 
with isometric or approximately isometric 
growth (e.g., SDI, LDI, OAI) are acceptable 
tools for identification of species and of 
course so are indices based on size-indepen- 
dent meristic characters. Allomethc growth, 
which may impair the usefulness of indices, is 
not prominent in the Bathypolypus species 
within the relevant size range of adolescent 
and adult animals (ML 20-60 mm). It is mod- 
est even where it can best be demonstrated 
(EDI, Fig. 8, HWI), and generally intraspecific 
inherent variability and artifacts far exceed 
and mask variation due to allomethc growth in 
these phenetically similar species. For pur- 
poses of species identification, the allometric 
variation was less than overall variation and 
was not calculated. Regrettably, interspecific 
overlap is often unavoidable (Table 9). The 
parameters are normally or approximately 
normally distributed, and the application of 
several indices tends to obliterate marginal 
values and leads to clearer dischmination of 
species, supplementing other, more tangible 
distinctive characters (funnel organ, crop, 
radula, sculpture, meristic characters). 

An important point that the present revision 
brought out is that the number of suckers on 
the hectocotylus is individually constant, even 
from the juvenile stage, and that the number 
of laminae is constant from the onset of matu- 
rity, irrespective of later increase in size (Figs. 
22, 23) Counts of suckers and laminae are 
good size-independent meristic characters to 
use in concert with other characters, even if 
cases of overlap exist (Fig. 9, Table 9). The 
relative constancy of SHcC was also demon- 
strated by Toll (1988), who used differences in 



SHcC as additional argument for taxonomic 
separation of Atlantic and Pacific Scaeurgus. 
In the radula, only the rachidians are dis- 
tinctive in the Bathypolypus species. They 
may be very uniform (bairdii, sponsalis), in 
others very variable {arcticus, pugniger, n. 
sp.). A striking example of similar variability is 
seen in Graneledone pacifica, in which the 
rachidians range from heterodont B8 seriation 
to simple homodont or even degenerate con- 
dition (Voss & Pearcy, 1990: 87, fig. 19). 



KEY TO THE NORTH ATLANTIC 
BATHYPOLYPUS SPECIES 

The majority of specimens may be identi- 
fied by outer distinctive features, supple- 
mented by inspection of the funnel organ (Fig. 
1 8). Additional help may be gained from Table 
9, which shows ranges of essential indices 
and counts. For juveniles, females, aberrant 
males, and less well-preserved specimens, 
however, inspection of radula, digestive tract, 
and spermatophores may be necessary. 

la. Mantle length usually more than 25% of 
total length. Arms with fewer than 100 
suckers. Length of hectocotylus at least 
70% of opposite (3, left) arm and with 
fewer than 50 suckers. Skin often warty 
and with a pointed cirrus over each eye .2 

lb. Mantle length usually less than 25% of 
total length. Arms with over 140 suckers. 
Length of hectocotylus at most 70% of op- 
posite arm and with over 50 suckers. Skin 
smooth, supraocular cirri absent 4 

2a. Body egg-shaped, with a constriction be- 
hind the head. Diameter of eyes less than 
33% of ML. Largest sucker diameter 



is- 
le 

14- 

12 

10 

8 

6- 



44 



Laminae 



Q a 

— в — 




e П 

DDG a a a 

• D D DD 

• • • 
•• • • •• • 

• •f»« ■•• •• • — • ••••• • 



D B. arcticus n = 35 
• B. bairdii n = 48 



>• •• 



• • • 



10 



20 



30 



40 



50 



60 



ML 



FIG. 22. In Bathypolypus species the number of laminae on ligula seems to be individually constant from the 
onset of maturity Regress, slopes: < +/- 0.023, r^: < 0.015 (data for bairdii Uorr\ western Greenland). 



214 



MUUS 



I 

Suckers hect. arm 

55 

B.arcticus 

50 
45 
40 
35 
30 



> 



50 
45H 
40 
35 



1 



,• • • • % 



B.bairdii (W.Greenl.) 



— ti**!** •• • • 

• tut ►••• • ••% • 



10 



20 



30 



40 



50 



ML mm 



FIG. 23. In Bathypolypus species, the number of suckers on the hectocotylized arm seems to be constant 
throughout life. Regress, slopes: < +/- 0.03, r^ < 0.02. 



6.5-8.5% of ML and 18-33% of eye di- 
ameter. Lígula with 11-16 laminae; funnel 
organ a clear-cut VV (Fig. 18); esophagus 
with prominent crop diverticulum; radula 
usually with heterodont rachidians (Fig. 
5); seminal reservoir occupying only 30% 
of the spermatophore length . . . .arcticus 

2b. Body squat; diameter of eye-balls 30- 
45% of ML; largest sucker diameter less 
than 6.5% of ML and at most 6-16% of 
eye diameter. Funnel organ pad-like, 
never a clear-cut VV. Ligula with less than 
13 laminae. Esophagus without crop di- 
verticulum; radula homodont; seminal 
reservoir occupying about 50% of sper- 
matophore length 3 

3a. Arm length order usually: 1.2.3.4. Ligula 
large, oblong, with 7-12 laminae; cala- 
mus 15-25% of ligula length; radula with 
long, slim homodont rachidians (Fig. 11) 
bairdii 

3b. Arm length order usually 3.4.2.1 or sube- 
qual. Ligula globular, fleshy, deeply hol- 
lowed, with 4-6 laminae; calamus 
26-51% of ligula length; radula variable 
but with broad homodont rachidians (Fig. 
15) pugniger, n. sp. 

4a. Hectocotylus with 70-85 suckers; other 
arms usually with over 200 suckers. Fun- 
nel organ almost square pads (Fig, 18). 
Esophagus with crop diverticulum. Sper- 
matophore very large, longer than ML .er- 
gasticus 



4b. Hectocotylus with 50-65 suckers; other 
arms with 140-200 suckers. Funnel organ 
VV (Fig. 18); esophagus without crop di- 
verticulum. Spermatophore shorter than 
ML sponsalis 



ACKNOWLEDGEMENTS 

I am indebted to the staff of the BIOFAR 
and BIOICE projects for access to the valu- 
able recent collections from the Greenland- 
Scotland ridge and the Faroe-Shetland Canal. 
Dr. С С. Lu kindly supplied me with speci- 
mens of B. bairdii from Canadian waters and 
Dr. Katharina Mangold blessed me (and 
ZMUC) with a collection of B. sponsalis from 
the Catalan Sea. With gratitude I have re- 
ceived advice and/or valuable information 
from Dr. G. Perez-Gandaras, Dr. K. Nesis, Dr. 
С F. E. Roper, and, not least, from the late Dr. 
G. Voss, with whom I had fruitful discussions. 
Finally, I wish to thank the curators of mol- 
luscs at the zoological museums in Bergen, 
Berlin, London, Oslo, and Washington for 
pleasant stays and help at various phases of 
my work. In ZMUC Stine Elle, Tom Schlotte 
and Gert Brovad offered me invaluable tech- 
nical assistance. My wife Kirsten Muus and 
two unknown but meticulous referees have 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 215 



improved the first version of the script in nu- 
merous ways. 

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Revised ms accepted 5 September 2001 



APPENDIX I 

Collection localities and specimens examined 
in this study. Sex and mantel length are 
stated. 

Bathypolypus bairdii (Verrill) East Atlantic 

BMNH: 

W of Ireland, May 9, 1896, 610-680 fms., 9 
ML: 55.-Off S Ireland, May 21, 1898, 
Norman coll., 2 9? ML: 47, one dis- 
torted.-60°57'N, 05°47'W, June 26, 
1909, 348 m, juv. ML: 8.-61°16'N, 
02°08'W, July 9, 1913, 630 m, juv. ML: 
8.-Udsire Hole, 58°58'N, 03°37'E, May 
15, 1912, 9 ML: 35.-71°50'N, 28°10'E, 
May 1, 1975, 180 fms., 9 ML: approx. 
50. 

MNHT 

Haul 46, 61 °25'N, 05°1 3'W, July 7, 1 979, 481 
m, d" ML: 23.-BIOFAR project: Stn. 117, 
62°00.7'N, 09°4.63'W, July 25, 1987, 
481 m, 9 ML: 16.-Stn. 124, 62°16.94'N, 
09°38.93'W, July 26, 1987, 600 m, cf 
ML: 35.-Stn. 158, 61°38'N, 05°38'W, 
May 7, 1988, 322 m, cf ML: 36.-Stn. 

419, 62°25'N, 10°38.17'W, June, 1, 
1989, 702 m, 2 cfcf ML: 40, 42.-Stn. 

420, 62°32'81N 10°27.57'W, June 1, 
1989, 597 m, cf ML: 33.-Stn. 482, 
61°01.94'N, 05°13.94'W, July 22, 1989, 
509 m, 9 ML: 24. 

IMNH: 

B5-77-?, 65°42'N, 27°53'W, February 24, 
1977, 750-820 m, 2 99 ML: 23, 33.- 
B5-77-48, 65°38'N, 29°27'W, March 23, 
1977, 450 m, c/ ML: 41, 9 ML: 33.-B5- 
77-49, 65°37'N, 29°32'W, March 23, 



218 



MUUS 



1977, 420-430 m, сГ ML: 41, 99 ML: 
21, 50.-B5-77-50, 63°38'N, 29°27'W, 
March 23, 1977, 450-460 m, 2 cfcf ML: 
35, 45, 2 99 ML: 24. 36.-B5-77-73, 
63°38'N, 26°14'W, March 27, 1977, 575 
m, 9 ML: 35.-B5-78-07, 63 48'N, 
27°00'W, March 10, 1978. 1110-1095 m, 
Cf ML: 63.-B5-78-22, 64°56'N, 
27°59'W, March 14, 1978, 970-1030 m, 
9 ML: 47.-DALB-1-80-13, 66°39'N, 
28=43'W, October 21, 1980, 415 m, 9 
ML: approx. 64.-B3-81-34, 65°22'N, 
32°37'W. February 25, 1981, 910 m, cf 
ML: 75.- BIOICE project: Stn. 2299, 
63°00'10N, 22 39'61W, September 10, 
1992, 775 m, juv. ML: 28.- Stn. 2346, 
63°23'N 12°88'W, May 6, 1993, 501 m, 
cf ML:39, 9 ML: 18. 

ZIASP: 

Nr. 25. near Spitsbergen, 215 m, cf ML: 35. 

ZMUB: 

Norwegian North-Atl. Exp. Stn. 290, 72°27'N, 
20°51 'E, July 7, 1878, 349 m, cf ML: 26, 
9 ML: 18.-0uter part of Laksefjord, Fin- 
mark (approx. 71 °N, 27°E), August 27, 
1900, 280 m, çf ML: 24, 9 ML: 39.-R.V. 
"Michael Sars": Stn. 56, 70°09'N, 
31 ^^00' E, May 21 , 1 901 , 200 m, juv. cf ML: 
23.-Stn. 108, 70°32'N, 18°17'E, June 
18, 1909, 300 m, juv. cf ML: 11. -Stn. 5, 
70°07'N, 30°53'E, June 4, 1914, 9 ML: 
approx. 44.-Stn. 28, 70°16'N, 32°20'E, 
June 24, 1914, 9 ML: approx. 30.-MS 
"Armauer Hansen" Stn. 2, outer part of 
Sognefjord (N of Bergen), March 10, 
1917, juv. cf ML: 15.-Salhus (N of 
Bergen), July 17, 1916, 400-500 m, cf 
ML: 55.-42837, Salhusfjord (N of Ber- 
gen), July 12, 1934, 2 cfcf ML: 40, 42.- 
Mangerfjord (N of Bergen), March 18, 
1931, 300-400 m, 2 cfcf ML: 36, 38.- 
53336, Kvinnheradsfjord, 59°59'50"N, 
05°54'E, December 7, 1956, 687-672 m, 
3 cfcf ML: 34-42. 3 99 ML: 22-36.- 
Hardangerfjord, NE of Varaldsoy, Stn. F 
103, November 8, 1957, 690 m, cf ML: 
40.-50281, Sognefjord, 6r03'N, 
06°25'E, May 3, 1966, 1238-1228 m, cf 
ML: 56, 3 99 ML: 28-47.- R.V. "G.O. 
Sars", S of Bear Island (Björnöya), August 
4, 1974, 500 m, cf ML: 74. 



ZMUG: 

Trondhjemsfjord, Norway, March 4, 1891, 
Storm leg., 9 ML: 26.- Trondhjemsfjord, 
September 19, 1934, 180-220 m, 9 ML: 
16.-Skager-rak, NNW of Skagen, July 

28, 1897, 210 fms., 2 cfcf ML: 29, 49.- 
Skagerrak, July 28, 1897, С G. Joh. Pe- 
tersen leg., 275 fms., 2 juv. ML: 7, 7.-Sk- 
agerrak, about 14 n.m. NW of Hirtshals, 
June21, 1911,313 m, cf ML: 41. -Trond- 
hjemsfjord, NonA/ay, N of Tatra, Septem- 
ber 19, 1934, 180-220 m, Stephensen 
leg., 9 ML: 15.-"Thor": Stn. 167, 
63°05'N, 20°07'W, July 14, 1903, 557 m, 
9 juv. ML: 9.-Stn. 223, 64°30'N, 
12°25'W, March 17, 1904, 535 m, cf ML: 
60. Stn. 99, 61°35'N, 9°35'W, May 22, 
1904, 900 m, cf ML: 38.- Stn. 274, Sk- 
agerrak, NW of Hirtshals, October 9, 
1904, 660 m, cf juv. ML: 23. Stn. 1074, 
Skagerrak, 4.5 n.m. S of Okso light- 
house. May 28, 1907, 480 m, cf ML: 43, 
9 ML: 45.-Stn. 1570, Skagerrak, 53 
n.m. N of Hanstholm, June 23, 1911, 
525-550 m, cf ML: 58, juv. 11. -"Dana": 
Stn. 6001, 63°33'N, 11°25'W, July 24, 
1938, 322 m, cf ML: 33.- Stn. 6004, 
63°06'N, 1 0°40'W, July 24, 1 938, 437 m, 
cf ML: 43.-Stn. 11643, 57°44'N, 
07°40'W, April 17, 1961, 440 m, 2 cfcf 
ML: 50, 55.-Stn. 13320, 58°10'N, 
04°22'E, March 12, 1965, 270 m, cf ML: 
42. -Stn. 15194, 57°35'N, 08°08'E, Sep- 
tember 19, 1969, 210 m, 2 cfcf ML: 27, 

29. Stn. ?, 61°03'N, 05°04'W, April 20, 
1988, 800-600 m, cf ML: 38. 

ZMUO: 

Oslofjord, off Filtvet lighthouse, April 26, 1910, 
100 fms., juv. ML: 13.-Oslofjord, Filtvet, 
May 3, 1966, 150-280 m, 9 ML: 61.- 
32291, Oslofjord, Torbjornskaer light- 
house, cf ML: 39.- Oslofjord, Drobak, cf 
ML: 43, 9 ML: 31 .-Oslofjord, Vestmedet, 
August 14, 1937 



Greenland 



ZMUG: 



"Ingolf": Stn. 28, 65°14'N, 55°42'W, July 1, 
1895, 420 fms., cf ML: 37.- Stn. 32, 
66^^35'N, 56°38'W, July 11, 1895, 2 99 
ML: 18, 62 + 4 juv.- Stn. 35, 65°16'N, 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 219 



55°05'W, July 1 8, 1 895, 682 m, 2 juv. ML: 
8, 9.-"Tjalfe": Stn. 337, 64°05'N, 
55°20'W, May 8, 1909, 1100 m, Ç ML: 
54.-Stn. 428, 63°54'N, 53°15'W, June 
8, 1909, 988 m, cf ML: 21. -"Rink": Stn. 
45, Bredefjord, SW Green!, (approx. 
60°49'N, 46°45'W), July 18, 1912, 
430-450 m, cf ML: 52.-"Dana": Stn. 
2346, 66°37'N, 56°37'W, June 22, 1925, 
450 m, 6 cfç^ ML: 16-68, 2 9Ç ML: 32, 
44, 3juv.-Stn. 2361, 68°08'N, 57°30'W, 
June 26, 1925, 398 m, 3 cf Cf ML: 46-55, 
9 ML: 54.-Stn. 10018, 65°02'N, 
56°00'W, July 19, 1957, 730-740 m, cf 
ML: 45 (type of B. proschi Muus, 1962).- 
Stn. 13662, 64°24'N, 52°57'W, July 27, 
1966, 450-510 m, cf ML: 32.-Greenl. 
Fish. Invest.: Stn. 4539, 64°19'N, 
52°55'W, June 4, 1971, 470-570 m, 29 
cfcf ML: 26-50, 10 $9 ML: 25-54.- 
Stn. 5043, 63°57'N, 52°21'W, June 18, 
1975, 300 m, cf ML: 33 + juv.-Stn. 5047, 
64°21'N, 52°58'W, June 24, 1975, 
550-580 m, 8 cf Cf ML: 15-49, 8 9 9 ML: 
24-42.-Stn. 5101, 66°34'N, 54°15'W, 
August 10, 1975, 335-340 m, cf ML: 
20.-St. 5112, 64°23'N, 52°58'W, August 
21, 1975, 450-510 m, 5 cfcf ML: 32-41, 
9 ML: 41. -Stn. 5206, 63°58'N, 
52°21'W, June 3, 1976, 300-310 m, cf 
ML: 38.-Stn. 5209, 63°58'N, 52°21'W, 
June 8, 1976, 300-308 m, cf ML: 34, 9 
ML: 46.-Stn. 5215, 64°21'N, 52°59'W, 
June 10, 1976, 510-520 m, 4 cfcf ML: 
29-45, 5 99 ML: 37-41. -Stn. 5384, 
64°21'N, 52°59'W, April 22, 1977, 485- 
510 m, cf ML: 44.-"Elias Kleist": Stn. 
791010/3, 67°57'N, 57°10'W, October 
10, 1979, 310-100 m, 9 ML: 47. 



American East Coast 



BMNH: 



Off Marthas Vineyard, Massachusetts, May 
21, 1898, 200-388 fms., cf ML: 35 (la- 
belled Octopus bairdii). 

USNM: 

34223, Le Have Bank, Nova Scotia, 120 fms., 
holotype of Octopus lentus Verrill, 1 880.- 
382469, fishing banks off Massachusetts, 
36 miles E of NE Light, Sable Island, from 
halibut stomach, holotype of Octopus 



obesus Verrill, 1880, cf ML: 44.-39943, 
off New Jersey, 39°49'30N, 71 °1 0' W, Au- 
gust 3, 1884, 420 fms., 9 ML: 51. -52979, 
off New Jersey, 39°50'N, 71°43'W, Sep- 
tember 18, 1885, 137 fms., cf ML: ap- 
prox. 41.-574638, Bay of Fundy, 
paratype of Octopus Ьа/'гсУ/'/' Verrill, 1873, 
cf ML: 24. 575271, 43° 

38'N, 69°13'W, August 2, 1912, 60 fms., 
9 ML20.-575274, Gulf of Maine, August 
16, 1878, 75 fms., 3 cfcf ML: 21-26.- 
575275, off Cape Cod, August-Septem- 
ber 1 878, 70-94 fms., 6 cf Cf ML: 1 2-26, 
4 99 ML: 10-18.-575276, off Salem, 
41°58'30N, 69°44'W, September 18, 

1879, 4 cfcf ML: 20-27, 9 ML: 39.- 
575277, off Salem, August 1877, 49 fms., 
2 ö'c^ ML: 27, 39, 1 9 ML: 10.-575278, 
42°31'N, 70°20'W, September 2, 1878, 
98 fms., 3 specimens.- 575279, off 
Martha's Vineyard, 39°46'N, 71°05'W, 
February 10, 1880, 487 fms., 9 ML: 39, 
juv. ML: 9.-575280, 40°02'N, 70°23'6W, 
September 4, 1880, 192 fms., cf ML: 14, 
9 ML: 35.-575281, 37°24'N, 74°17'W, 
November 1 6, 1 880, 300 fms., 2 cf cf ML: 
26, 23, 3 99 ML: 17-27.-575282, 
39°49'N, 70°54'W, September 13, 1880, 
225-252 fms., 2 cfcf ML: 20, 38.- 
575285, 37°07'50N, 74°34'20W, Novem- 
ber 18, 1884, 167 fms., cf ML: 37.- 
575288, off Martha's Vineyard, 
39°53'30N, 71°13'30W, August 9, 1881, 
31 9 fms., 2 cfcf ML: 50, one damaged, 9 
ML: 50.-575289, 40°04'N, 69°29' 
30W, September 28, 1884, 58 fms., cf 
ML: 39.-575293, 39°52'20N, 70°58'W, 
October 2, 1880, 372 fms., cf ML: 28.- 
575294, 39°53'N, 70°58'30W, October 2, 

1880, 365 fms., 3 cfcf ML: 38-46.- 
575306, off Gay Head, Martha's Vine- 
yard, 39°57'N, 70°31'30W, August 23, 

1 881 , 225-396 fms., cf ML: 40, 3 9 9 ML: 
21 -49.-575971 , 39°57'N, 70°58'W, Au- 
gust 25, 1 879, 1 75-200 fms., cf ML: 44.- 
RV "Oregon": Stn. 6800, 29°48'N, 
80°09'05W, July 20, 1967, 183 fms., 2 
Cfcf.-Gosnold cruise: Stn. 105, 39° 
51 'N, 70°56'W, August 10, 1972, 875- 
880 m, ö" ML: 51. -Stn. 120, 39°50'N, 
70°32'W, August 16, 1972, 750-775 m, 
cf ML: 65.- RV "Chain": Stn. 243, 
39°30'N, 72°20'W, February 28, 1973, 
474-529 m, cf ML: 21, 9 ML: 27.-Stn. 
244, 39°28'N, 72°18'W, February 28, 
1973, 260-342 m, 9 ML: 44.- Stn. 254, 



220 



MUUS 



39°51'N. 70°47'W, March 2, 1973, 
768-947 m, о' ML: 49. 9 ML: 56.-Stn.?, 
39=31 'N, 78°18'W, February 26, 1973, 
810-900 m, 9 ML: 41.~-Stn.?, 39'31'N, 
75=23W. February 28. 1973. 420-590 m. 
9 ML: 26.- RV "Knorr": Stn. 300, 39 40'N 
72°27'W, November 13, 1973, 110-182 
m, cf ML: 26.-Stn. 301, 39°32'N, 
72=24'W, November 13, 1973, 475-520 
m, 4 ö'o' ML: 20-33, 9 ML: 28.-GI-75- 
08: Stn. 10, 37°53'N, 74°40'08W, Sep- 
tember 9, 1975, 290-340 m, 2 cfO" 99 
(distorted).- Stn. 18, 37=04'07N, 74° 
34'03W, September 10, 1975, 200-215 
m, 3 cfcf, 19. -St. 19. 36"58'04N 
74=37'05W, September 11, 1975, 190- 
250 m, 2 cf Cf , 3 9 9--St. 22, 36°58'06N, 
74=33'08W, September 11, 1975, 880- 
920 m, 3 cTcf ML: 24-42.-Stn. 99, 
36°36'05N 74°39'W, September 20, 

1975. 850-1000 m, 9 ML: 50. 

ZMUC: 

"Albatros IV", cruise 76-02: Stn. 221, 
40°07'N, 69°04'W, March 28, 1976, 465 
m, 9 ML: 30.-Stn. 237, 41°44'N, 
69°46'W, March 30. 1976. 78 m, cf ML: 
27.-Stn. 288, 41 ^53'N, 67"52'W, April 6, 

1976, 52 m, cf ML: 19- Stn. 300, 
42°05'N, 68°15'W, April 7, 1976, 189 m, 
9 ML: 25.-Stn. 305, 42°18'N, 69°12'W, 
April 8, 1 976, 230 m, 9 ML: 38.-Stn. 309, 
42°26'N, 70"06'W, April 8, 1 976, 84 m, cf 
ML: 42.-Stn. 321, 42°45'N, 70°00'W, 
April 14, 1976, 163 m, 9 ML: 41.-Stn. 
322, 42°46'N, 69°35'W, April 14, 1976, 
163 m, cf ML: 25, 9 ML: 43.-Stn. 330, 
42°59'N,68^39'W, April 17, 1976, 187 m, 
2 99 ML: 20, 22. -Stn. 332, 42°45'N, 
67°47'W, April 17, 1976, 200 m, 2 cfcf 
ML: 38, 42.-Stn. 333, 42"34'N, 67°58'W, 
April 17, 1976, 206 m, 9 ML: 20.-Stn. 
339, 42°17'N, 66 49'W, April 18, 1976, 
270 m, (S ML: 26. -Stn. 341, 42^^19'N, 
66'12'W, April 18, 1976, 262 m, 1 spm.- 
Stn. 405, 43 26N, 67'34'W, April 29, 
1976, 218 m, cf ML: 37, 9 ML: 37.-Stn. 
406, 43"06'N, 6748'W, April 29, 1976, 
200 m, 9 ML: 29.-Stn. 409, 43°39'N, 
68^^17'W, April 29, 1976, 190 m, 3 cfcf 
ML: 18-29.- Stn. 426, 43^'29'N, 69° 
01 'W, May 6. 1976. 139 m, 9 ML: 50.- 
Notre Dame Bay, Newfoundland: October 
1975, 3 cfcf ML: 67-74. -USSR "Belo- 
gorsk", cruise 74-11: Stn. 142, 41"16'N, 



68°41'W, October 12. 1974, 80 m, 9 
ML: 32. 

Bathypolypus arcticus (Presch) East Atlantic 

BMNH: 

60°03'N. 05=51 W, August 1 7. 1 880, 540 fms. 
(labelled B. faeroensis) cf ML: 35, 9 ML: 
30. HMS "Triton", Stn. 9, 60°05'N, 
06°2rW, August 23, 1882, 608 fms. 
(syntypes of Benthoctopus sasakii Rob- 
son), cf ML: approx. 32, 9 ML: 27.-Stn. 
40, 57°34'N, 00°01'W, November 27, 
1904, Tow net 100 m, 9 ML: 30.- 
6r27'N, 01°47'W, July 25, 1909, 1240 
m, 9 ML: 43 (labelled Benthoctopus pls- 
catorum) + 9 ML: 20.-61 °42'N, 
02°00'W, July 25, 1909, 1236 m, 9 ML: 
31 (labelled B. faeroensis).-S\n. 77, 
75°16'N, 24°46'E, June 1956, 85 m, 
E. Holt, 2 99 ML: 28, 29.-Cruise IV, 
Stn. 37, 60°25'N, 04°31'W-60°29'N, 04° 
22'W, W of Foula, 1973, 940-910 m, 2 
99 ML: 20, 28. 

IRSNB: 

(v/deAdam, 1939) 66°20'N, 12°28'W, June 
21 , 1 938, 1 80 -220 m, 2 cf cf juv. ML: 1 6, 
10, 9 ML: 13.-66°20'N, 12°28'W, June 
22, 1938, 180-220 m, 9 ML: ca. 18. 

MNHT 

Haul 96, 62°59'N, 09°54'W, July 23, 1979, 
481 m, 9 ML: 46.-BIOFAR project: Stn. 
15, 62°37'68N, 04°40'37W, July 17, 
1987, 683 m, juv. ML: 12.- Stn. 95, 
60°41'51N, 05°18'63W, July 23, 1987, 
803 m, 9 ML: 21. -Stn. 274, 63°00'79N, 
07°49'22W, May 16, 1988, 698 m, 9 ML: 
42.-Stn. 294, 60"26'N, 07°28'W, July 
17, 1988, 1096 m, cf ML: 30.-Stn. 502, 
60^'30'26"N, 08°04'W, July 25, 1 989, 890 
m, cf ML: 30.-Stn. 589, 60°40'N, 
10°00'W, April 9, 1990, 250 m, 9 ML: 
15.- Stn. 726, 60"39'N, 06"54'W, Sep- 
tember 29, 1 990, 400 m, 9 ML: 27. 

IMNH: 

D6-80-50, 67°11'N, 18°30'W, April 20, 1980, 
430 m, cf ML: approx. 41.-B16-80, 
66°50'N, 20"26'W, October 29, 1980, 
415 m, 9 ML: approx. 37.-B3-81-34, 



THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 221 



62°59'N, 20°23'W, March 7, 1 981 , 91 m, 
9 ML: 95.-B4-81-137, 66°37'N, 
12°58'W, April 1, 1981, 320 m, $ ML: 
31. -BIOICE project: Stn. 2080, 
67°22'79N, 1 7°20'77W, July 4, 1 992, 897 
m, 9 ML: 46.-Stn. 2085, 67°15'67N, 
17°26'41W, July 4, 1992, 754 m, 9 ML: 
61. -Stn. 2088, 67°02'35N, 13°25'05W, 
July 4, 1992, 903 m, cf ML: 40.-Stn. 
2134, 6644'46N, 18°54'93W, July 8, 
1992, 504 ГЛ, cf ML: 17.-Stn. 2135, 
66°44'37N, 18°57'32W, July8, 1992,418 
m, cf juv. ML: 26.-Stn. 2320, 64°02'N, 
09°44'W, May 2, 1993, 758 m, cf ML: 
49.-Stn. 2322, 63°55'N, 10°04'W, May 
3, 1993, 627 m, 9 ML: 45.-Stn. 2326, 
63°44'N, 10°09'W, May 3, 1993, 563 m, 
9 ML: 33.-Stn. 2328, 63°20'N, 1 0°57'W, 
МауЗ, 1993,430 m, 9 ML: 54. 



TMDZ: 

M/K "Asterias": Isfjord, Svalbard, July 18, 
1955, 242-228 m, 9 ML: 26.- Isfjord, 
Svalbard, August 14, 1958, 230-215 m, 
C/ juv. ML: 23, 9 ML: 23. 



ZIASP: 

92, Barents Sea, 140-150 m, Kondakov det., 
cf juv. ML: approx. 12.-119, between 
Spitsbergen and Frantz Josef Land, 210 
m, Kondakov det., cf ML: 31.-120, Bar- 
ents Sea, 201 m, cf ML: 19.-Nr. 607, 
Novaya Zemlya, 37 m, 9 ML: 39. 



ZMUB: 

ESE of Vardo (about 70°N, 32°E), August 10, 
1902, 9 ML: approx. 49.-"Solveig I", 
Stn. 52, Kongsfjord, Svalbard, June 22, 
1938, 290-333 m, 9 ML: 42.-42838, 
Stn. 64, Svalbard, 78°25'N, 12°06'E, 
July 7, 1939, 9 ML: 44.-36145, "Sotra", 
Stn. 250, 78°04'N, 14°10'E, September 
9, 1930, cT ML: 38.-"Tovik", Stn. 10, 
77°44'N, 11°45'E, June 30, 1925, 
185-228 m, cf juv. ML: 13.-78°03'N, 
14°10'E, August 30, 1931, 147 m, 9 ML: 
31.-75°00'N 37°20'E, September 1, 
1968, 100-150 m, 9 ML: 57.-18678, 
"Michael Sars", Stn. 37, 62°43'N, 
01°26'E, June 29, 1902, 775 m, cf ML: 
36. 



ZMUC: 

61 °27'N, 01 °27'W, July 25, 1 909, 1 240 m, la- 
belled Polypus faeroensis Russell Type, 
(leg. A. С Stephen). 



Greenland 



ZMUC: 



Lectotype of Octopus arcticus Frosch, 1849: 
cf ML: 42, SW Greenland, August 26, 
1841, K. M. Jorgensen leg.-Paralecto- 
type 9. partly dissected, SW Greenland, 
July 27, 1 840, K. M. Jorgensen leg.- Dis- 
sected male organs pictured by Frosch 
(1849: figs. 1-3).-3 99 in bad shape 
and partly dissected labelled Greenland 
and evidently from the 1840s.- Hol- 
steinsborg, SW Greenland, 1892, Trau- 
stedt leg., 9 ML: 64.-"lngolf": Stn. 124, 
67°40'N, 15°40'W, July28, 1896,932 m, 
2 cf cf ML: 44, 44.-Near Nanortalik, SW 
Greenland, April 15, 1906, 9 ML: 31.- 
Lichtenau fjord, SW Greenland, October 
14, 1910, from stomach of Greenland 
halibut, 9 ML: 45.- Lichtenau fjord, June 
6, 1914, 220 fms., 9 ML: 41. -Lichtenau 
fjord, July 3, 1947, Foul Hansen leg., 9 
ML: 51.-"Godthâb": Stn. 81, 75°35'N, 
65°41 'W, August 1 , 1 928, 490 m, o' ML: 
54, 9 ML: 23.-Stn. 87, 77°05'N, 
71 °1 3' W, August 4, 1 928, 790 m, 1 4 cf Cf 
ML: 32-58, 2 99 ML: 32-46, 1 juv. ML; : 
22.-Stn. 90, 77°17'N, 69°59'W, August 
5, 1928, 930 m, cf ML: 46, 9 ML: 38.- 
Stn. 116, 76°08'N, 80°53'W, August 17, 
1928, 80 m, cf juv. ML: 13.-"Godthâb"s 
summer cruise, Stn. 341 , off Кар Hacker, 
Jameson Land, E Greenland, August 27, 
1933, juv. ML: 15.- Ella Island, Kong 
Oscar Fjord, approx. 73°N, 25°W, Octo- 
ber 10, 1931, 67-68 m, 9 ML: 35.-Ymer 
Island, Frantz Joseph Fjord, E Greenland 
(approx. 73°20'N), August 8, 1932, cf 
ML: 43.- Frantz Joseph Fjord, off Eng- 
dalen, August 7, 1931, 45-36 m, cf ML: 
52.-Lindenow Fjord, 60°30'N, 43°25'W, 
July 17, 1935, 100-150 m, Bertelsen 
leg., cf ML: 36.-Amerdlok Fjord, near 
Holsteinsborg (Sisimiut), July 26, 1938, 
approx. 500 m, 9 ML: 54.-Skovfjord, 
SW Greenland, June 16, 1948, P. 
Hansen leg., cf ML: 58.- Young Sound, 
off Daneborg, approx. 74°15'N, 20°W, 
July 1947, 9 ML: approx. 58.-Bylot 



222 



MUUS 



Sound, 76^3ГМ, 69 09'W, August 20, 
1968, 240 m, Just & Vibe Stn. 45, o' ML: 
54.- Kap Farvel Exp.: Stn. 56, бО'^ТЗ'М, 
44°13'\/V. July 31, 1970. 400-420 m, сГ 
ML: 35. -Stn. 128, 60°0ГМ, 43°59'W, 
August 21. 1970, 530 m, 9 ML: 51. -W 
Greenland, 66'21 N, 54°56'W, August 2, 
1975, 280-290 m. Max Andersen leg., $ 
ML: 12.-Greenland Fish. Invest.: Stn. 
3978, 63°53'N, 51°27'W, May 9, 1968, 
230-250 m, 9 ML: 35.-Stn. 4360, May 
21, 1970, 240-250 m, cf ML: 43, 9 ML: 
34.-Stn. 5101, 66°34'N, 54°15'W, Au- 
gust 10, 1975, 300 m, 2 cfcT ML: 33 + 
juv. 

ZMUO: 

Hoel's Greenland Exp.: Stn. 1101, Kong 
Oscar Fjord, E Greenland (approx. 
72°20'N), August 12, 1930, 55-100 m, cf 
juv. ML: 17.- Stn. 1116, Kong Oscar 
Fjord, August 15, 1930, 250 m, cf ML: 
30.- Stn. 1 1 1 8, Kong Oscar Fjord, August 
16, 1930, 120 m, 1 juv. ML: 26.-Stn. 
1119, Kong Oscar Fjord, Vegasund, Au- 
gust 17, 1930, 190-250 m, 4 cTcT ML: 
37-44. 

American East Coast 

No material. 

Bathypolypus pugnigern. sp. 

IRSNB: 

66°23'N 12°53'W, June 14-15, 1938, 
200-250 m, ö" ML: 22, 9 ML: 18 (vide 
Adam 1939). 

MNHT: 

Haul 25, 63^1 6'N, 09=30'W, July 6, 1979, 500 
m, 2 cfcf ML: 27, 28.-BIOFAR project: 
Stn. 47, 61°02'31"N, 05"54'W, July 19, 
1987, 280 m, 9 ML: 19.- Stn. 80, 60° 



38'89N, 08°27'93W, July 22, 1987, 678 
m, 9 ML: 18.-Stn. 269, 62°49'84N, 08° 
15'55W, May 15, 1988, 510 m, ö' ML 
33. Stn. 503, 60"38'02N, 08°33'54W, 
July 25, 1989, 513 m, o' ML: 18.-Stn 
734, 60'10'06N, 07°57'03W, May 8 
1990, 634 m, cf ML: 31. -Stn. 738 
62°19'N, 10°13'W, October 1, 1990, 749 
m, cf ML: 25.-Stn. 740, 62°29'N, 10° 
02'W, October 1, 1990, 597 m, cf ML: 
18. 

IMNH: 

B5-77-36, 65°40'N, 28°20'W, March 21, 
1977, 1000-970 m, 9 ML: 36.- 85-77- 
40, 65°36'N 29°17'W, March 22, 1977, 
870-910 m, 9 ML: 33.-B5-77-42, 
65°34'N, 29°29'W, March 22, 1977, 
750-760 m, 9 ML: approx. 44.-B5-77- 
46, 65°29'N, 29°33'W, March 23, 1977, 
960 m, 2 cfö' ML: 36, 30, 3 99 ML: 
27-40.- B5-77-50, 65°38'N, 29°27'W, 
March 23, 1977, 450-460 m, 9 ML: ap- 
prox. 32, cf ML: 30.-B5-78-44, 64°58'N, 
27°44'W, March 14, 1978, 860-870 m, 
holotype 19990971. cf ML: 32. 

ZMUO: 

"Dana" Stn. 5840, 62°44'N, 06°06'W, May 14, 
1938, 330 m, cf ML: 19.-Stn. 6001, 
63°33'N, 11°25'W, July24, 1938,322 m, 
5 cfCf (3 juv.) ML: 6-13, 2 99 (1 juv.) 
ML: 15, 23.-Stn. 16437, 64°14'N, 
57°26'W, July 24, 1974, 760 m, cf ML: 
54.- Stn. B5-77-36, 65°40'N, 28°20'W, 
March 21, 1977, 1000-970 m, 9 ML: 
36.-Haul 25, 63°16'N, 09°30'W, July 6, 
1979, 500 m, 2 cfcf ML: 27, 38. BIOFAR 
project: Stn. 503, 60''38'N, 08°33'54W, 
July 25, 1989, 513 m, cf ML: 18.-Stn. 
738, 62°1 9'N, 1 0°1 3' W, October 1 , 1 990, 
749 m, ö' ML: 25.- Stn. 734, 60°10'06N 
07°57'03W, May 8, 1990, 634 m, cf ML: 
31, 



MALACOLOGIA, 2002, 44(2): 223-239 

ULTRASTRUCTURE OF THE SUPPORTING CELLS AND SECRETORY CELLS OF 

THE ALIMENTARY CANAL OF THE SLUGS, LEHMANNIA MARGINATA AND 
BOETTGERILLA FALLENS {PÜLMOHAJA: STYLOMMATOPHORA: LIMACOIDEA)^ 

Ana Maria Leal-Zanchet 

Laboratorio de Histología, Centro de Ciencias da Saude, Universidade do Vale do Rio dos 
Sinos, Av. UNISINOS, 950, 93022-000 Sao Leopoldo- RS, Brasil; ipp@bios.unisinos.br 

ABSTRACT 

The supporting cells and the secretory cells of the alimentary canal of two slugs of the super- 
family Limacoidea, Lehmannia marginata and Boettgerilla pallens, were studied by electronmi- 
croscopy. From the esophagus to the third intestinal region, and in the rectum, ciliated and/or 
nonciliated columnar cells occur In the fourth intestinal region and in the intestinal caecum, the 
latter present only in the intestine of L. marginata, there are nonciliated squamous cells. The ul- 
trastructure of the columnar cells of the crop, stomach and first and second intestinal regions in- 
dicate that these cells are involved in food uptake, digestion and storage of reserve materials. 
Ultrastructural features of ciliated cells of the typhlosoles and the leader folds of the alimentary 
canal indicate the involvement of these cells in the production of currents, thus aiding food and 
faeces transportation. The ultrastructure of the squamous cells of the fourth intestinal regions 
and caecum point to one of the roles of these organs in the absorption of water and ions from the 
faecal pellets. Eight secretory cell types in the alimentary canal of L. marginata and six secretory 
cell types in B. pallens are distinguished by their ultrastructural features, such as diameter and 
contents of secretion granules, development and contents of rough endoplasmatic reticulum, as 
well as diameter of their cisternae, and development of the agranular endoplasmatic reticulum. 

Key words: epithelium, secretion, digestive system, glands cells, intestine, caecum, Limaci- 
dae, Boettgerillidae. 



RESUMO 

As células de revestimento e as células secretoras do tubo digestivo de duas lesmas da su- 
perfamilia Limacoidea, Lehmannia marginata e Boettgerilla pallens, sâo analisadas ultraestru- 
turalmente. Células cilindricas ciliadas e/ou nao ciliadas sao observadas desde o esófago até a 
terceira regiao intestinal bem como no reto. Células pavimentosas nao ciliadas ocorrem na 
quarta regiao intestinal e no ceco intestinal, este último presente apenas em L. marginata. As 
características ultraestruturais das células cilindricas do papo, do estómago e da primeira e se- 
gunda regióes intestinais indicam que essas células auxiliam na absorcáo, na digestáo e no ar- 
mazenamento de material de reserva. Caracteres ultraestruturais das células ciliadas presentes 
ñas tiflossoles e dobras condutoras do tubo digestivo indicam que essas células atuam na pro- 
duçào de correntes, auxiliando no transporte do bolo alimentar e fecal. A ultraestrutura das célu- 
las pavimentosas da quarta regiao intestinal e do ceco sugerem que urna das funçôes desses 
órgaos é a absorçâo de agua e íons do bolo fecal. Oito tipos de células secretoras ocorrem no 
tubo digestivo de L. marginata e seis tipos de células secretoras, em B. pallens, sendo diferen- 
ciadas por características ultraestruturais, tais como diámetro e conteúdo dos granulos secre- 
tores, forma, diámetro e conteúdo das cisternas do retículo endoplasmático granular, bem como 
forma e abundancia relativa das cisternas do retículo endoplasmático agranular, dentro outras 
características. 



Part of a thesis submitted to the Lehrstuhl Spezielle Zoologie of the Eberhard-Karls Univertität Tübingen, Germany, in par- 
tial fulfilment of the requirements for the degree of Doctor of Natural Sciences and supported by a fellowship of the Brazil- 
ian Research Council (CNPq). 

223 



224 



LEAL-ZANCHET 



INTRODUCTION 

Knowledge of the role of the alimentary 
canal of pulmonales in digestion and absorp- 
tion is still incipient, this related to the paucity 
of available information concerning the ultra- 
structure its organs. Babula & Wielinska 
(1 988) described the crop and intestine of De- 
receras reticulatum: Ángulo & Moya (1989) 
studied the intestine of Arion ater: Boer & Kits 
(1990) examined the whole alimentary canal 
of Lymnaea stagnalis: and Franchini & Otta- 
viani (1992) studied intestinal cell types of 
Planorbarius corneus. In addition, Thebskorn 
(1989), Tnebskorn & Künast (1990), and 
Thebskorn & Köhler (1992) analysed ultra- 
structural changes in different cell types of the 
alimentary canal of D. reticulatum and/or 
Arlon lusltanlcus as a background for obser- 
vations on the effects of molluscicides. Dere- 
ceras reticulatum and L. stagnalis. among 
other species of pulmonales, were also the 
object of anatomical and histological investi- 
gations of the alimentary canal (reviewed by 
Runham, 1975, and Luchtel et al., 1997). 

The present work completes a series of 
anatomical and histological (Leal-Zanchet, 
1998), histochemical (Leal-Zanchet, 1999) 
and ultrastructural studies of the alimentary 
canal of limacoid slugs. Leal-Zanchet (1998) 
analysed comparatively the anatomy and his- 
tology of the alimentary canal of six species of 
the superfamily Limacoidea (Dereceras 
laeve, D. rodnae, D. reticulatum, Lehmannia 
marglnata, Malacollmax tenellus, Boettgerllla 
pallens) and the milacid slug Tandonia bu- 
dapestensls. and described the following re- 
gions in this canal: esophagus, crop, stom- 
ach, first, second, third and fourth intestinal 
regions and the rectum, besides an intestinal 
caecum for D. rodnae, D. reticulatum and L. 
marg/nafa (Leal-Zanchet, 1998). 

In the limacoid slugs cited above, as well as 
in T. budapestensis, the epithelium of the ali- 
mentary canal is composed of supporting 
cells and secretory cells, the former being cil- 
iated and/or nonciliated columnar cells from 
the oesophagus to the third intestinal region, 
and in the rectum. The supporting cells of the 
fourth intestinal region and intestinal caecum 
are nonciliated squamous to cuboidal (Leal- 
Zanchet, 1998). 

Eight secretory cell types could be distin- 
guished in the alimentary canal of L. mar- 
glnata (Leal-Zanchet, 1998). Two types of 
mucous cells occur in the proximal and me- 
dian segments of the alimentary tract, type I 



(esophagus, crop, stomach and second in- 
testinal region) and type II (stomach and first 
intestinal region). Two types of secretory 
cells, namely intestinal secreting cells of types 
I and II, occur in the second and third intesti- 
nal regions, respectively. Besides four other 
types of secretory cells, mucous cells of types 
III, IV and V, and cystic cells, occur in the dis- 
tal regions of the tract (third and fourth intesti- 
nal regions, intestinal caecum and rectum). In 
B. pallens, both mucous cells of type V and 
the cystic cells are absent. 

Most of the limacoid slugs are herbivorous 
(Frömming, 1 954), but B. pallens seems to be 
zoophagous (Leal-Zanchet, 1995). Because 
of the apparently diverse feeding habits, as 
well as the differing complexity of the alimen- 
tary canal (length of the alimentary canal, 
presence/ absence of an intestinal caecum 
and number of secretory cell types), Lehman- 
nia marglnata (Limacidae) and Boettgerllla 
pallens (Boettgerillidae) were selected for the 
present investigation. 



MATERIAL AND METHODS 

The animals were collected near the town 
of Tübingen, Baden-Württemberg, Germany, 



Key to Lettering on Figures 



aer: 


agranular endoplasmic reticu 


ar: 


apical region 


bb: 


basal bodies 


bf: 


basal feet 


ci: 


cilia 


er: 


cilia rootlets 


et: 


connective tissue 


f: 


fold 


g: 


Golgi bodies 


gl: 


glycogen 


i: 


interdigitations 


li: 


lipids 


lu: 


lumen 


m: 


mitochondria 


me: 


muscle cells 


mi: 


microvilli 


n: 


nucleus 


ne: 


nerve ending 


nu: 


nucleolus 


r: 


ribosomes 


rb: 


rootlike basal extensions 


rer: 


rough endoplasmic reticulum 


sg: 


secretory granules 


sm: 


secretion mass 


sv: 


synaptical vesicles 


v: 


vesicles 


va: 


vacuoles 


V 1: 


vacuoles of type 1 


vil: 


vacuoles of type 11 


za: 


zonula adhaerens 


zs: 


zonula septata 



ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 



225 



and kept in a cool room at 15°C. They were 
rnaintained humid with decanted water in the 
laboratory for approximately two weeks in 
petri dishes with a certain amount of natural 
soil and litter from the collecting site. Lehman- 
nia marginata was raised on lettuce, carrot 
and cabbage. Boettgerilla pallens consumed 
eggs of limacoid slugs and also accepted 
carrot. Before preparation, the slugs were 
starved for three days. For previous micro- 
anatomical, histological and histochemical 
studies, eight adult specimens of each L. mar- 
ginata and B. pallens were analysed. For 
present ultrastructural studies, two adult 
specimens of each species were examined. 
They were anaesthetised in 5% menthol for 
one hour and killed in the fixative solution (2% 
paraformaldehyde in 0.05 M phosphate buffer 
and 2% glutaraldehyde, pH 7.2) (Plattnert, 
1975), in which they were dissected. The 
whole alimentary tract was transferred to a 
fresh amount of fixative solution, and small 
pieces of the various parts of the alimentary 
canal were separated, labelled and main- 
tained for one hour in a new quantity of fixa- 
tive solution. The material was washed in 
Sörensen's phosphate buffer (Ruthmann, 
1966), post-fixed in 2% osmium tetroxide in 
0.05 M phosphate buffer, washed in 0.025 M 
phosphate buffer, dehydrated in a graded se- 
ries of ethanol, treated with propylene oxide 
and embedded in Epon 812 (Serva). Ultrathin 
sections (60-90 nm), obtained with a LKB-UI- 
trotome I ultramicrotome, were stained with 
Reynolds' (1963) lead citrate and uranyl ac- 
etate. The sections were examined in a 
Siemens Elmiskop 102 transmission electron 
microscope. 



RESULTS 



Columnar Cells 



Both ciliated (Figs. 1, 12) and nonciiiated 
(Fig. 1 3) columnar cells of the alimentary tract 
of L. marginata and B. pa//ens show microvilli. 
The apical zone of the cytoplasm (Fig. 18) is 
devoid of cell organelles, with the exception of 
vesicles and cistemae of the agranular endo- 
plasmic reticulum. In the optical microscope, 
in sections stained by routine histological 
methods, this zone appears as a clear area 
(Leal-Zanchet, 1998). In the ciliated columnar 
cells, the cilia are anchored to the cytoplasm 
by basal bodies from which cross-striated 
rootlets extend into the cytoplasm (Table 2). 



Below the apical zone, there are numerous 
mitochondria (Figs. 17, 18), as well as small 
vesicles and multi-vesicular bodies. The mito- 
chondria present an oval to oblong form. In 
the rest of the cytoplasm, mitochondria are 
only sparsely scattered. The supranuclear cy- 
toplasm of the columnar cells contains nu- 
merous large and slightly electron-dense fat 
droplets, the mean diameter of which is 2.2 ± 
0.4 |jm in the crop of L. marginata and 3.2 ± 
0.4 |дт in the crop of B. pallens, being more 
plentiful in the crop and stomach of both 
species (Figs. 14, 15). Glycogenrosettes were 
often observed in the cytoplasm of the colum- 
nar cells of B. pallens (Fig. 1 7). There are also 
membrane-limited vacuoles in the supranu- 
clear cytoplasm, which can be categorized 
into two types. The vacuoles of type I (Figs. 
12, 14, 21) show a electron-dense peripheral 
zone and a lesser electron-dense central 
core. The mean diameter of the vacuoles is 
1 .3 ± 0.3 and 1 .3 ± 0.1 цт in the crop and 0.7 
± 0.2 and 0.9 ± 0.2 |im in the intestine of L. 
marginata and B. pallens, respectively. In L. 
marginata, the peripheral part has mainly het- 
erogeneous, granular contents (Fig. 14). The 
relative number of vacuoles of type I de- 
creases in the second intestinal region of both 
species. The vacuoles of type II (1 .2 ± 0.3 |im 
in diameter) were observed only in the colum- 
nar cells of the crop, stomach and first and 
second intestinal regions of B. pallens. They 
contain granular, slightly electron-dense ma- 
terial (Fig. 21). In the basal cytoplasm, some 
mitochondria, Golgi bodies, rough endoplas- 
mic reticulum, and fat droplets are present. In 
the optical microscope, in sections stained by 
routine histological methods, type I vacuoles 
show cromophobe contents and type II vac- 
uoles appear acidophillic (Leal-Zanchet, 
1998). 

Neighbouring cells of the alimentary canal 
are interconnected apically by the zonula ad- 
haerens and zonula septata (Fig. 18). The 
zonula adhaerens is located close to the tran- 
sition of the apical to the lateral membranes of 
the cells, followed by the zonula septata, 
which is partly transversed by interdigitations 
(Fig. 1 8). In the middle and basal zones of the 
epithelium, the cells present large intercellular 
spaces, wherein occur connections uniting 
the epithelial cells. 

In the first, second and third intestinal re- 
gions the height of the columnar cells gradu- 
ally decreases (Leal-Zanchet, 1998), the mi- 
crovilli and the cilia rootlets becoming smaller 
as well (Tables 1, 2). 



226 



LEAL-ZANCHET 




FIGS. 1-3. Schematic drawings of a ciliated columnar cell of the first intestinal region (Fig. 1), a ciliated 
columnar ceil of the rectum (Fig. 2) and a squamous cell of the fourth intestinal region (Fig. 3). 



In the ciliated columnar cells of the ty- 
phlosoles and of the leader groove of the 
stomach, the cilia are very numerous and 
densely arranged (Figs. 15-17). Between two 
neighbouring cilia only three microvilli are 
present. The basal bodies of the cilia are in- 
terconnected by well-developed basal feet, 
and the rootlets are thick and very long (Table 
2). In the apical cytoplasm of these cells, the 
mitochondria are more numerous than in the 
ciliated columnar cells of other regions, being 
situated close to the cilia rootlets (Fig. 17). 

In the leader folds of the rectum of B. pal- 
lens, as well as in the ciliated columnar cells 
of the rectum of L. marginata, the microvilli 
and the cilia rootlets are long (Tables 1, 2). 
The apical cytoplasm consists of vesicles, 



channels of the agranular endoplasmic reticu- 
lum (Fig. 19) and ribosomes. In the leader 
folds, the mitochondria zone occupies most of 
the supranuclear cytoplasm, and the mito- 
chondria are arranged parallel to the cilia 
rootlets. In the rectum of Lehmannia mar- 
ginata, where specialized leader folds are ab- 
sent, the mitochondria of the ciliated cells do 
not show such an arrangement, being less 
abundant and scattered in the supranuclear 
cytoplasm (Fig. 2). Numerous poliribosomes 
and some channels of the agranular endo- 
plasmic reticulum are found between the mi- 
tochondria. Poliribosomes and mitochondria 
in a scattered distribution occur in the basal 
cytoplasm. Some vesicles and Golgi bodies 
are found in the zone of the nucleus. 



ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 



227 




FIGS. 4-7. Schematic drawings of mucous cells of types I (Fig. 4) and II (Fig. 5) and intestinal secreting cells 
of types I (Fig. 6) and II (Fig. 7). 



Squamous to Cuboidal Cells 

In the squamous, nonciliated cells of the 
fourth intestinal region and the intestinal cae- 
cum, the microvilli are short (Fig. 3, Table 1). 
An apical mitochondria zone is absent, and 



the supranuclear zone is narrow. Mitochon- 
dria are sparsely scattered in the cytoplasm. 
The apical and middle cell zones show chan- 
nels of the agranular endoplasmic reticulum 
as well as vesicles. Golgi bodies are found in 
the middle zone of the cells. In intranuclear 



228 



LEAL-ZANCHET 





FIGS. 8-11 . Schematic drawings of the cell body of mucous cells of type III (Fig. 8), type IV (Fig. 9), type V 
(Fig. 10) and the cystic cell (Fig. 11). 



TABLE 1 . Length of microvilli in the alimentary canal of Lehmannia mar- 
ginata and Boettgerllla pallens (in |am).-: absent;--: not measured. 





L. marginata 


B. pallens 


Crop 


1.6 ± 0.2 


3.7 ± 0.5 


Stomach (typhlosoles and canal) 


1.9 ± 0.1 


1.9 ± 0.2 


Stomach (other areas) 


1.5 ± 0.1 


1.7 ± 0.3 


First intestinal region 


0.7 ± 0.1 


1.0 ± 0.2 


Second intestinal region 


0.7 ± 0.1 


0.9 ±0.2 


Third intestinal region 


0.4 ± 0.1 


- 


Fourth intestinal region 


0.5 ± 0.1 


0.6 ± 0.08 


Intestinal caecum 


0.5 ± 0.06 


- 


Rectum 


0.7 ■ 0.07 


0.4 • 0.1 



ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 



229 



TABLE 2. Length of cilia rootlets in the alimentary canal of Lehmannia 
marginata and Boettgehlla pallens (in цт).-: absent;--: not measured. 





L. marginata 


B. pallens 


Oesophagus 


0.9 ± 0.2 


- 


Crop 


- 


- 


Stomach (typhlosoles and canal) 


8.7 ± 1.3 


6.0 ± 1.0 


Stomach (other areas) 


2.9 ± 0.4 


2.0 ± 0.5 


First intestinal region 


1.6 ±0.3 


1.7 ±0.4 


Second intestinal region 


0.7 ± 0.1 


1.5 ±0.3 


Third intestinal region 


0.4 ± 0.1 


- 


Rectum 


0.9 ± 0.3 


0.6 ± 0.1 



cytoplasm, there are small vacuoles of type I 
(0.5 ± 0.1 цт in diameter) and ribosomes. 
The granular endoplasmic reticulum is poorly 
developed. The basal membrane shows nu- 
merous folds (107 ± 27 nm wide), which form 
rootlike basal extensions (Fig. 22), containing 
mitochondria, ribosomes and vesicles with 
poor electron-dense contents (83 ± 16.7 nm 
in diameter). These epithelial basal exten- 
sions come close to folds of the muscle cells. 
The muscle folds are wider (0.25 ± 0.06 цт) 
than the epithelial basal extensions and show 
numerous vesicles (83 ± 16.7 nm) with elec- 
tron-dense contents. The connective tissue 
between the folds present collagen fibres, 
which are arranged parallel to the epithelial 
folds. 



Secretory Cells 

The mucous cells of type I, the mucous 
cells of type II, the intestinal secreting cells of 
type I, and the intestinal secreting cells of type 
II are intraepithelial. The mucous cells of type 
III, the mucous cells of type IV, the mucous 
cells of type V, and the cystic cells show a 
subepithelially located cell body and a cell 
neck that extends to the surface of the epithe- 
lium. Cell extensions emerge from the basal 
part of the intraepithelial secretory cells, 
which enter into the connective tissue. Nerve 
endings, which contain electron-dense as well 
as electron-lucent synaptical vesicles, come 
close to these cell extensions (Fig. 23). By the 
subepithelial secretory cells, nerve endings 
make contact with the cell body or with the 
subepithelial part of the cell neck (Fig. 30). In 
the resting phase, the secretory cells show 
microvilli. In the process of secretion, the api- 
cal cytoplasm forms a camber and the mi- 
crovilli are reabsorbed (Figs. 25, 28). 



Mucous Cells of Type I 

The mucous cells of type I (Fig. 4) have a 
widened basal part that contains the nucleus 
and most of the cell organelles (Golgi bodies, 
granular endoplasmic reticulum and mito- 
chondria) (Fig. 24), as well as some secretion 
granules. The middle and apical cell zones 
are performed with secretion granules. Vesi- 
cles containing electron-dense material occur 
between the granules and at the margins of 
the cell. Multi-vesicular bodies and numerous 
ribosomes are found throughout the cyto- 
plasm. Golgi zones are well developed. In L 
marginata, the Golgi lamellae show a width of 
40 ± 1 nm. Vesicles and recently formed se- 
cretion granules are often associated with the 
Golgi bodies. In B. pallens, the Golgi bodies 
usually consist of minimally widened lamellae 
(40 ± 10 nm) with electron-dense contents. 
The rough endoplasmic reticulum (RER) 
shows distended (0.4 ±0.17 цт), branched 
cisternae that contain tubuli (24 ± 4 nm). Usu- 
ally mitochondria occur close to the cisternae 
(Fig. 24). The secretion granules of L mar- 
ginata (2.1 ± 0.3 |дт in diameter) are slightly 
electron-dense, with dense, tube-like scat- 
tered structures, containing at the margins a 
concentration of strong electron-dense mate- 
rial (Fig. 25). In B. pallens, the secretion gran- 
ules (1.7 ± 0.2 (jm in diameter) are electron- 
lucent contents with speckles of granular, 
moderately electron-dense material. The 
granules usually form large secretion masses 
that are membrane-limited (Fig. 26). 



Mucous Cells of Type II 

The mucous cells of type II (Fig. 5) present 
an elongated basal part, which contains very 
numerous RER cisternae as well as numer- 




FIG. 12. Ciliated columnar cell of the oesophagus of Lehmannia marginata. Note the numerous vacuoles of 
type I in supranuclear cytoplasm. Scale bar, 3 цт. FIG. 13. Columnar cell of the crop of Lehmannia mar- 
ginata showing long microvilli. Scale bar, 1 цт. FIG. 14. Supranuclear zone of the cytoplasm of a columnar 
cell of the crop of Boettgerilla pallens. Scale bar, 1 pm. FIG. 15. Apical zone of the cytoplasm of a ciliated 
columnar cell of the typhlosole of the stomach of Lehmannia marginata. The long cilia rootlets are visible. 
Scale bar, 2 pm. FIG. 16. Apical zone of the cytoplasm of a ciliated columnar cell of the typhlosole of the 
stomach of Lehmannia marginata. Note the basal bodies interconnected by the well-developed basal feet. 
Scale bar, 0.3 \.im. FIG. 17. Apical zone of the cytoplasm of a ciliated columnar cell of the typhlosole of the 
stomach of Boettgenlla pallens. Scale bar, 0.7 цт. FIG. 18. Apical zone of the cytoplasm of a ciliated colum- 
nar cell out of the typhlosoles of the stomach of Lehmannia marginata. Scale bar, 1 .3 pm. 



ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 



231 




.»i»^t 



Vii 



/I 





ne 



FIG. 1 9. Apical cytoplasm of a cuboidal cell of the leader fold of the rectum of Boettgerilla pallens. The arrow 
shows channels of the agranular endoplasmic reticulum. Scale bar, 0.3 |.im. FIG. 20. Nonciliated cuboidal cell 
of the fourth intestinal region of Lehmannia marginata. Scale bar, 3 |am. FIG. 21 . Supranuclear cytoplasm of 
a columnar cell and part of a mucous cell of type II of the first intestinal region of Boettgerilla pallens. Scale 
bar, 1 |am. FIG. 22. Rootlike basal extensions of the basal part of a squamous cell of the intestinal caecum 
of Lehmannia marginata and corresponding folds of the muscle cells. Scale bar, 1 цт. FIG. 23. Basal cyto- 
plasm of a mucous cell of type II of the stomach of Lehmannia marginata. The arrow shows tubuli of the rough 
endoplasmic reticulum. Nerve endings come close to the cell. Scale bar, 0.6 [am. 



232 



LEAL-ZANCHET 




FIG. 24. Basal zone of the cytoplasm of a mucous cell of type I of the oesophagus of Lehmannia marginata. 
Scale bar, 1 цт. FIG. 25. Apocrine secretion of a mucous cell of type I of the oesophagus of Lehmannia mar- 
ginata. Scale bar, 1 .3 pm. FIG. 26. Supranuclear cytoplasm of a mucous cell of type I of the crop of Boettge- 
hlla pallens. Scale bar, 2 \im. FIG. 27. Supranuclear cytoplasm of a mucous cell of type II of the typhlosole 
of the stomach of Letimannia marginata. Scale bar, 1 |.im. FIG. 28. Apical part of a mucous cell of type II of 
the stomach of Boettgeriila pallens. The apical cytoplasm forms a camber and the microvilli are reabsorbed 
(arrow). Scale bar, 0.5 цт. 



ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 



233 



ous Golgi bodies and some mitochondria. The 
RER cisternae are dilated (0.4 ± 0.1 |.im) and 
also contain tubuli (28 ± 4 nm in diameter). In 
the basal cytoplasm occur large vacuoles (1 .5 
± 0.1 jim in diameter) containing finely gran- 
ular, electron-dense material (Fig. 27). Occa- 
sionally some secretion granules are present 
in the intranuclear cytoplasm. In the supranu- 
clear cytoplasm, however, the granules are 



very numerous. In L. marginata, the granules 
are electron-lucent with clumps of osmiophillic 
material (Fig. 27). In B. pallens, the granules 
are electron-lucent with flocculent contents 
(Fig. 28). In both species, the granules are 
very small (0.9 ± 0.2 and 0.8 ± 0.2 |.im in L. 
marginata and B. pallens. respectively). Vesi- 
cles and channels of the agranular endoplas- 
mic reticulum as well as ribosomes are pres- 




FIG. 29. Cell body of a mucous cell of type III of the fourth intestinal region of Boettgerilla pallens. Scale bar, 
2.2 |.im. FIG. 30. Subepithelial part of a cell neck of a mucous cell of type III of the fourth intestinal region of 
Lehmannia marginata. Nerve endings make contact with the cell neck. Scale bar, 0.7 |.im. FIG. 31 . Cell body 
of a mucous cell of type IV of the rectum of Lehmannia marginata. Scale bar, 2 цт. FIG. 32. Detail of the cy- 
toplasm of the cell body of a mucous cell of type IV of the rectum of Boettgerilla pallens. Scale bar, 0.2 цт. 
FIG. 33. Detail of the cytoplasm of the cell body of a mucous cell of type V of the third intestinal region of 
Lehmannia marginata. Scale bar, 2 pm. FIG. 34. Detail of the cytoplasm of the cell body of a cystic cell of the 
third intestinal region of Lehmannia marginata. Scale bar, 2.2 |im. 



234 



LEAL-ZANCHET 



ent between the granules. Mitochondria occur 
in the marginal cytoplasm. 

Mucous Cells of Type III 

The cell body of the mucous cells of type III 
(Figs. 8, 29) is occupied by secretion granules 
(1.2 ± 0.1 urn in diameter in L. marginata and 
1 .8 ± 0.4 fim in diameter in B. pallens). These 
granules often fuse to form larger granules. In 
L. marginata as well as in B. pallens. their 
contents show different electron densities, 
slightly to moderately electron-dense (Fig. 
29). The granules are so closely located that 
seldom are other organelles present. The 
RER cisternae occur in the marginal cyto- 
plasm and close to the nucleus. The cisternae 
are distended (0.5 ± 0.2 in L. marginata or 
0.25 ±0.1 ¡am in diameter in B. pallens) and 
contain tubuli (Fig. 30). Golgi bodies and mi- 
tochondria are present at the cell margins. 
Very numerous RER cisternae and some mi- 
tochondria and channels of the agranular en- 
doplasmic reticulum occur in the proximal part 
of the cell neck (Fig. 30). In the apical part of 
the cell, the secretory granules form a large, 
electron-lucent secretory mass. 

Mucous Cells of Type IV 

The secretion granules are smaller (Figs. 9, 
31) than the granules of the mucous cells of 
type III (0.6 ± 0.1 |im in diameter in L. mar- 
ginata and 1 .3 ± 0.3 lam in diameter in B. pal- 
lens). In both species, the secretion granules 
show homogenous, electron-dense contents 
(Fig. 31 ). Usually the granules do not form se- 
cretion masses. Numerous RER cisternae, 
which contain finely granular, electron-dense 
material, occur in the cytoplasm between the 
granules (Fig. 32). The cisternae are usually 
thin (51 ±14 nm in L. marginata and 1 1 5 ± 54 
nm in B. pallens), but in L. marginata some 
cisternae reach 0.17 ± 0.1 цт in diameter. 
Golgi bodies are present at the cell margins 
and close to the nucleus. The apical part of 
the cell neck shows electron-dense secretion 
granules that fuse distal to discharging in the 
lumen. Vesicles and polyribosomes occur in 
the apical cytoplasm. 

Mucous Cells of Type V 

This cell type does not occur in the alimen- 
tary tract of B. pallens. A secretion mass, 
formed by the coalescence of the secretion 



granules, often occupies most of the cell, so 
that the organelles (polyribosomes, mitochon- 
dria and RER cisternae) and the nucleus are 
found in the basal part of the cell body (Fig. 
10). The secretion mass is electron-lucent 
and flocculent (Fig. 33). Channels and vesi- 
cles of the agranular endoplasmic reticulum 
are numerous in the peripheral cytoplasm. 
The RER cisternae are thin (75 ± 20 nm in di- 
ameter) and contain electron-lucent material. 
Golgi complexes are well developed (Fig. 33). 
Occasionally, recently formed secretion gran- 
ules are associated with the Golgi bodies. 



Cystic Cells 

This cell type is absent in the alimentary 
tract of B. pallens. The cell body of the cystic 
cell is occupied by a granular, electron-dense 
secretion mass surrounded by a membrane 
(Figs. 11 , 34). Small vacuoles showing a sim- 
ilar content as well as vacuoles containing 
tubular structures are observed in basal and 
marginal cytoplasm (Figs. 34, 35). Granules 
(0.5 ± 0.4 )jm in diameter) with a homoge- 
neous, stronger electron-dense material also 
occur in the cytoplasm (Fig. 35). The RER is 
often highly developed and shows thin cister- 
nae (113 ± 82 nm). Polyribosomes and chan- 
nels of the agranular endoplasmic reticulum 
are abundant (Fig. 35). Some channels are 
connected to the outer cell membrane. 



Intestinal Secreting Cells of Type I 

The cytoplasm is occupied by the highly de- 
veloped RER and abundant free ribosomes 
(Figs. 6, 38). Some RER cisternae are thin 
(33 ± 5 nm) and show a platelike structure. 
Most of the RER cisternae are, however, di- 
lated (0.22 ± 0.02 цт in diameter) and occur 
scattered throughout the cytoplasm. They 
contain a finely granular, slightly electron- 
dense material. Golgi bodies occur in the in- 
tranuclear cytoplasm as well as around the 
nucleus (Fig. 36). Their lamellae (0.1 ± 0.05 
nm) have an electron-dense material. The cy- 
toplasm also presents channels of the agran- 
ular endoplasmic reticulum, small vesicles 
and vacuoles with heterogeneous electron- 
dense contents (Fig. 38). The round secretion 
granules (2.3 ± 0.2 |jm in diameter in L. mar- 
ginata and 1.5 ± 0.15 (.im in diameter in B. 
pallens) are usually present in the supranu- 
clear cytoplasm. Their content is homoge- 



ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 235 

38 




37 




•^.>7 - 






m 


Ы 


К 


>*,' 


*ч 




1 


P 


v — 






■ 


V 







FIG. 35. Detail of the cell body of a cystic cell of the third intestinal region of Lehmannia margínala. The arrow 
shows a channel of the agranular endoplasmic reticulum. Arrowheads indicate tubular structures in a vac- 
uole. Scale bar, 0.3 цт. FIG. 36. Detail of the cytoplasm of an intestinal secreting cell of type I of the second 
intestinal region of Boettgerilla pallens. Scale bar, 0.7 цт. FIG. 37. Apical cytoplasm of an intestinal secret- 
ing cell of type I of the second intestinal region of Lehmannia margínala. Scale bar, 0.6 |jm. FIG. 38. Supranu- 
clear cytoplasm of an intestinal secreting cell of type I of the second intestinal region of Boetlgerilla pallens. 
Scale bar, 1.0 ¡.im. FIG. 39. Supranuclear cytoplasm of an intestinal secreting cell of type II of the third in- 
testinal region of Boetlgerilla pallens. Scale bar, 1 .0 цт. 



236 



LEAL-ZANCHET 



neous electron-dense (Figs. 36, 37). Numer- 
ous nnitochondria occur in the apical cyto- 
plasm. 

Intestinal Secreting Cells of Type II 

The intestinal secreting cells of type II (Fig. 
7) also possess an extensive RER and abun- 
dant polyribosomes (Fig. 39). The RER cister- 
nae are thinner (119 :r 37 nm) compared to 
those of the intestinal secreting cells of type I. 
They contain finely granular, electron-dense 
material. Golgi bodies are found around the 
nucleus. They show dilated lamellae (67 ± 29 
nm in diameter) with electron-transparent ma- 
terial (Fig. 39). Vesicles that contain electron- 
dense material are often associated with the 
Golgi bodies. The secretion granules that 
show a distinctive limiting membrane are 
larger than those of the intestinal secreting 
cells of type I (3.8 ± 1 .0 [im in diameter in L. 
marginata and 2.5 ± 0.5 цт in diameter in B. 
pallens) and may form secretion masses. 
They contain a flocculent, electron-transpar- 
ent material as well as membrane-like struc- 
tures (Fig. 39). Mitochondria and polyribo- 
somes are often present in the apical 
cytoplasm. Channels of the agranular endo- 
plasmic reticulum and vesicles may also be 
present. 



DISCUSSION 

According to Boer & Kits (1990) the colum- 
nar cells of the alimentary tract of Lymnaea 
stagnalis are involved in the absorption, di- 
gestion and storage of reserve material. Mi- 
crovilli were demonstrated on the surface of 
the columnar and squamous cells of the ali- 
mentary canal of L. marginata and B. pallens. 
However, the absorption of food particles 
should occur mainly through the columnar 
cells of the crop, the stomach, and the first 
and second intestinal regions, where the mi- 
crovilli are long, and numerous mitochondria 
are present in the apical cell zones (Pacheco 
& Scorza, 1971). This would be consistent 
with the presence of alkaline phosphatase in 
the epithelial cells of the crop and intestine of 
Deroceras reticulatum (Babula & Skowron- 
ska-Wendland, 1988) and with the findings of 
Walker (1972), who showed in D. reticulatum 
an uptake of glucose, glycine and palmitic 
acid by the epithelial cells of the crop and in- 
testine. 

A type of vacuole with electron-dense con- 



tents (vacuoles of type I) occurs in the cyto- 
plasm of the columnar cells of the crop, stom- 
ach and first and second intestinal regions of 
B. pallens and L. marginata. Bowen (1970) 
and Babula & Wielinska (1988) also observed 
vacuoles with similar contents by ultrastruc- 
tural studies of epithelial cells of the crop and 
intestine of Arion ater and D. reticulatum, re- 
spectively. According to Bowen (1970) and 
Ángulo et al. (1986), such vacuoles are liso- 
somes, presenting a positive reaction to acid 
phosphatase. The unavailability of histochem- 
ical studies of enzymes in the slugs B. pallens 
and L. marginata make it impossible to point 
out the functions of type I and type II vacuoles. 

The storage of reserve material, such as 
glycogen and lipids, was observed in the 
columnar cells of B. pallens and L. marginata. 
Evidence of the storage of lipids was ob- 
served mainly in the crop, stomach and first 
intestinal region. The storage of glycogen was 
demonstrated by ultrastructural studies, as 
well as histochemically (Leal-Zanchet, 1999). 
Some columnar cells of the esophagus, crop, 
stomach, and intestine of D. reticulatum were 
designated by Thebskorn (1989) as storage 
cells, because of their high lipid and glycogen 
content. 

Ciliated cells occur along the entire alimen- 
tary tract of B. pallens and L. marginata, ex- 
cept for the crop of both species, the fourth in- 
testinal region of B. pallens and the intestinal 
caecum of L. marginata (Leal-Zanchet, 1998). 
In some regions of the stomach and rectum, 
such as the typhlosoles and the transversal 
fold present in the stomach of limacoid slugs 
and the leader folds of the rectum of B. pal- 
lens, we observed that the ciliated cells show 
distinctive features, namely the cilia are 
longer, very numerous and densely arranged 
(Leal-Zanchet, 1998). Furthermore, they pre- 
sent very long cilia rootlets, being intercon- 
nected by well-developed basal feet on the 
basal bodies. According to Zylstra (1972), 
such a contact between the basal bodies 
would be involved in the coordination of ciliary 
movements, and this coordination would be 
crucial in regions involved in the production of 
currents, such as the leader system of the 
stomach. As a matter of fact. Walker (1972) 
studied the ciliary tracts of the stomach of D. 
reticulatum and found three main ciliary path- 
ways: a sorting mechanism in the folds sur- 
rounding the openings of the digestive glands; 
extrusion pathways for material that becomes 
bound up into faecal pellets; and a circulation 
pathway particularly for fine material. After 



ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 



237 



Walker (1 972), the cilia on the typhlosoles and 
accessory fold (= transversal fold) beat mainly 
towards the gastric channels and intestinal 
groove, and cilia in the basis of the gastric 
channels (channels between each tiphlosole 
and the transversal fold) and anterior intesti- 
nal groove (channel between the tiphlosoles) 
transport material posteriorly, whereas cilia of 
the more posterior region of the intestinal 
groove transport material anteriorly. Where 
these two opposite directional movements 
meet, faecal pellets are formed (Walker, 
1972). Physiological studies are not available 
for the rectal leader folds of limacoid slugs, 
but their similar ultrastructural morphology 
may indicate that such folds could be better 
suited to aid faeces transportation than are 
the usual folds (Leal-Zanchet, 1998). 

The squamous cells of the fourth intestinal 
region, the intestinal caecum, and the rectum 
show microvilli on the apical surface, apart 
from numerous folds of the basal membrane, 
which form rootlike basal prolongations be- 
sides well-developed interconnections be- 
tween neighbouring cells. These features are 
characteristic of water- and ion-transporting 
epithelia and would be consistent with the 
findings of Deypup-Olsen & Martin (1987), 
who verified that the distal part of the intestine 
of Ariolimax columbianus plays a significant 
role in osmoregulation. As suggested by Boer 
& Kits (1990) for Lymnaea stagnalis, this 
would indicate that water and ions are ab- 
sorbed from the faecal pellets. In limacoid 
slugs that have an intestinal caecum, espe- 
cially in L. marginata in which this caecum is 
long, this region could provide an additional 
site for water and ions absorption. 

The occurrence of five mucous cell types 
was detected in the alimentary canal of L. 
marginata, as well as in such other limacoid 
slugs as Malacolimax tenellus, Deroceras 
laeve, D. reticulatum, and D. rodnae (Leal- 
Zanchet, 1998). By optical microscopical 
analysis, these cell types were distinguished 
by their distribution along the alimentary tract, 
their position related to the epithelium (intra- 
or subepithelial location), the mean diameter 
of their secretory granules, the morphology of 
the basal part or cell body of the cells, and 
their appearance after histochemical reac- 
tions for protein and mucopolyssaccharides 
(Leal-Zanchet, 1998, 1999). Thus, type I and 
II mucous cells were distinguished on the 
basis of the mean diameter of the secretory 
granules and the morphology of the basal part 
of the cells, characteristics observed by opti- 



cal microscopy and confirmed here. Besides, 
type II secretory cells show a restricted distri- 
bution in the stomach and first intestinal re- 
gion, whereas type I are present in the esoph- 
agus, crop and second intestinal region. Both 
types secret acid mucopolyssaccharides 
(Leal-Zanchet, 1999), but their morphological 
distinctive features and distribution in the ali- 
mentary canal indicate that a more accurate 
histochemical analysis could confirm them as 
different types. Types III, IV and V mucous 
cells, restricted to the distal regions of the ali- 
mentary tract (third and fourth intestinal re- 
gions, intestinal caecum and rectum) were 
distinguished by optical microscopical studies 
due to the different mean diameter of their 
granules and their reaction to trichromical 
stains. Previous histochemical studies 
showed that the three types are involved in 
the secretion of neutral mucopolysaccha- 
rides, but type III is also involved in the secre- 
tion of acid mucopolysaccharides. Besides, in 
B. pallens, where type V mucous cells and 
cystic cells are absent, type IV mucous cells 
could be histochemically distinguished be- 
cause they show positive reaction for protein 
and neutral mucopolysaccharides (Leal- 
Zanchet, 1999). The present investigation 
shows that the granules of types III and IV, in 
both species, have a different electron-den- 
sity and that their rough endoplasmatic reticu- 
lum has a different morphology and distribu- 
tion in the cytoplasm. Type V mucous cell is 
distinguishable by optical studies through the 
elongate morphology of the secretory gran- 
ules. The present ultrastructural studies show 
that they form a eletron-lucent secretion mass 
occupying most of the cell. 

The mucous cells of type I, II and III show 
tubuli in the cisternae of the rough endoplas- 
mic reticulum. The presence of tubuli in the 
rough endoplasmic reticulum has been de- 
scribed by Wondrak (1967), Moya & Rallo 
(1 975), Kessel & Beams (1 984) and Ángulo & 
Moya (1989). According to Kessel & Beams 
(1984), tubular structures within the cisternae 
of the endoplasmatic reticulum appear to be a 
feature of more than one cell type. Further- 
more, according to these authors, their preva- 
lence within secretory cells suggests that 
these tubular structures could be secretory 
products of the cells. 

The secretion of the cystic cells and of the 
intestinal secreting cells of type I and II gave 
positive results for protein (Leal-Zanchet, 
1 999). This would be consistent with the pres- 
ence of a highly developed rough endoplas- 



238 



LEAL-ZANCHET 



mie reticulum, observed in the present inves- 
tigation. The secreting cells of type I. occur- 
ring in the second intestinal region, where the 
columnar cells show ultrastructural features 
that indicate involvement in the absorption of 
food particles, imply that the secretion of this 
cell type is probably of an enzymatic nature 
and may play a role in digestion (Leal- 
Zanchet, 1998). However, additional histo- 
chemical studies, including enzyme histo- 
chemistry, are needed to clarify the role of 
these secretory cells. 

Considering that data obtained in labora- 
tory experiments assume that B. pallens is 
carnivorous, whereas Lehmannia marglnata 
is herbivorous, this living on a specialised diet 
of lichens (Wiktor, 1973), it could be expected 
to find more distinguishing morphological fea- 
tures in the alimentary canal of the two 
species. However, B. pallens shows a mere 
shortening of the intestinal regions (Leal- 
Zanchet, 1998). 



ACKNOWLEDGEMENTS 

Dr. Wolfgang Rähle supervised the doctoral 
thesis. Prof. Dr. Wolfgang Maier is thanked for 
accommodation in his department. Prof. Dr. 
Christian F. Bárdele gave his permission to 
use the Laboratory of Electronmicroscopy. 
Ms. Siegrid Schultheiss is thanked for the EM 
technical assistance. Mr. Fernando Carbayo 
is thanked for the preparation of the drawings. 
Mr. Edward Benya and Mr. Ramon A. Clark 
corrected the English version of the manu- 
script. Thanks are also due to the anonymous 
referees for the valuable suggestions. 



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Revised ms. accepted 6 September 2001 



MALACOLOGIA, 2002, 44(2): 241-257 

SEXUAL MATURATION, SPAWNING, AND DEPOSITION OF THE 

EGG CAPSULES OF THE FEMALE PURPLE SHELL, RAPANA VENOSA 

(GASTROPODA: MURICIDAE) 

Ee-Yung Chung\ Sung-Yeon Kim^, Kwan Ha Park^ & Gab-Man Park^ 

ABSTRACT 

Germ cell development, sexual maturation, and fecundity of female Rapana venosa (Valenci- 
ennes) have been examined by light and electron microscope observations. The Golgi appara- 
tus, vacuoles and mitochondria were involved in the formation of glycogen particles, lipid droplets 
and yolk granules in early vitellogenic oocytes. The modified mitochondria and endoplasmic 
reticulum in late vitellogenic oocytes were involved in the formation of proteid yolk granules near 
the cortical layer. A mature yolk granule was composed of three components: main body (central 
core), superficial layer, and limiting membrane. Monthly changes in the gonadosomatic index 
were closely associated with ovarian developmental phases. Spawning occurred between May 
and early August, when the seawater temperature rose to 18-26°C. The female reproductive 
cycle of this species was divided into five successive stages: early active stage (September to 
February), late active stage (October to April), ripe stage (March to July), partially spawned stage 
(May to August), and recovery stage (June to September). The rate of individuals reaching the 
first sexual maturity was 51.6% in females of 7.1-8.0 cm in shell height, and 100% in those > 
10.1 cm. The total number of egg capsules per individual and the mean number of eggs in an 
egg capsule were 184 to 410 and 976, respectively. Fecundity ranged approximately from 
179,000 to 400,000 eggs/individual with two to four broods (spawning frequencies) during the 
spawning season. The duration of development in egg capsules was 15-17 days at about 20°C. 
Rapana venosa is a species with embryos that hatch as veliger larvae. The sex ratio of female: 
male was not significantly different from 1:1. 

Key word: Rapana venosa, gametogenesis, reproductive cycle, first sexual maturity, fecundity, 
egg capsule, sex ratio. 



INTRODUCTION 

The purple shell, Rapana venosa (Gas- 
tropoda: Muricidae), one of the most Impor- 
tant edible gastropods in Asia (Yoo, 1976; 
Kwon et al., 1993), is abundant along the 
coasts of Korea, China and Japan, especially, 
in silty sand of the intertidal and subtidal 
zones. However, the standing stock of this 
species has gradually been decreasing due to 
extensive loss of habitats from reclamation 
projects and reckless over-harvesting. Thus, 
R. venosa has been identified as a target or- 
ganism that should be carefully managed. 

Prior studies have considered various as- 
pects of the biology of R. venosa, including 
classification (Kuroda & Habe, 1952; Habe, 
1969; Tsi et al., 1983; Wu, 1988; Choe, 1986, 
1992; Higo et al., 1999), morphology (Lee & 
Kim, 1988), biochemical aspects (Yoon, 1986; 
Yoo et al., 1991; Hwang et al., 1991), distribu- 



tion (Gomoiu, 1972; Zolotarev, 1996), habitat 
and age estimation (Chukhchin, 1984; Hard- 
ing & Mann, 1999), reproductive cycle (Chung 
et al., 1 993; Chung & Kim, 1 997), and spawn- 
ing and egg capsule (Hirase, 1928; Habe, 
1960; Amio, 1963; Smagowicz, 1989; Harding 
& Mann, 1999). However, there is still uncer- 
tainty in many aspects of reproductive biology 
of the purple shell. For example, little informa- 
tion is available on ultrastructure of germ cell 
differentiation during oogenesis, the repro- 
ductive cycle, first sexual maturity and fecun- 
dity. Understanding the reproductive cycle 
and the spawning period of R. venosa will pro- 
vide necessary informations needed for the 
determination of age and recruitment period 
of natural populations. Additional information 
on first sexual maturity, fecundity associated 
with egg capsules, sex ratio and reproductive 
strategy are needed to facilitate effective 
management of this important natural re- 



Vaculty of Marine Life Science, Kunsan National University, Kunsan 573-701 , Korea; eychung@kunsan.ac.kr 

^National Fisheries Research and Development Institute, Pusan 619-902, Korea 

^Departrnent of Parasitology, Kwandong University College of Medicine, Kanghung 210-710, Korea 



241 



242 



CHUNG ETAL. 



source. Therefore, the main aim of the pres- 
ent study was to describe vitellogenesis dur- 
ing oogenesis, the reproductive cycle and 
spawning period, first sexual maturity, fecun- 
dity, and sex ratio of R. venosa inhabiting the 
west coast of Korea. 



GSI = Thickness of gonad x IQQ 

Diameter of posterior appendage 
including gonad and digestive gland 



Light and Electron Microscope Observations 



MATERIALSAND METHODS 
Sampling 

Specimens of the purple shell, Я. venosa 
(Valenciennes 1846), were collected monthly 
by subtidal dredging near Biung-do, Choi- 
labuk-do Korea, for one year from June 1994 
to July 1995 (Fig. 1). Purple shells ranging 
from 3.1 cm to 16.9 cm in shell height were 
used for the present study. After the purple 
shells were transported alive to the laboratory, 
shell heights and total body weights were im- 
mediately measured. 

Gonadosomatic Index 

Monthly changes in the mean gonadoso- 
matic index (GSI) (Fig. 2) were calculated by 
the following equation (Chung et al., 1993): 



For light microscopic examination of histo- 
logical preparations, gonad tissues were re- 
moved from shells and preserved in Bouin's 
fixative for 24 h, and then washed with run- 
ning tap water for 24 h. Tissues were then de- 
hydrated in alcohol and embedded in paraffin 
molds. Embedded tissues were sectioned at 
5 "~ 7 jim thickness using a rotary microtome. 
Sections were mounted on glass slides and 
stained with Hansen's hematoxylin-0.5% 
eosin, Mallory's triple stain and PAS stain, and 
examined using a light microscope. 

For electron microscopical observations, 
excised pieces of the gonads were cut into 
small pieces and immediately prefixed in 
2.5% paraformaldehyde-glutaraldehyde in 
0.1 M phosphate buffer (pH 7.4) for 2 h at 4°C. 
After prefixation, the tissues were washed 
several times with the same buffer and then 
postfixed in 1% osmium tetroxide dissolved in 




Biunq-Oo'^"^ 



Sampling area 



'^,CP* 



Gogunsangun-Oo "^ 



D' 




36°0Q 



ЗЗМб' 



I26°I5' 



I2&'45' 



FIG. 1. Map showing the sampling area. 



REPRODUCTION IN RAPANA 



243 




FIG. 2. Anatomy of the female purple shell, Rapana 
venosa, removed from its shell. A, reproductive 
organ of female; B, posterior appendage showing 
the gonad and liver. X, Y and Z denote the sections 
for measurement of GSI, Three sections are spaced 
equally. Abbreviations: A, anus; DO, digestive 
gland; F, foot; MA, mantle; MO, mouth; OP, opercu- 
lum; OV, ovary; PA, posterior appendage; S, stom- 
ach; SC, stomachal caecum. 



0.2 M phosphate buffer solution (pH 7.4) for 
1 h at 4°C. Tissues were then dehydrated in a 
series of increasing concentrations of ethanol, 
cleared in propylene oxide and embedded in 
Epon-Araldite mixture. Uitrathin sections of 
Epon-embedded specimens v\/ere cut with 
glass knives with a Sorvall MT-2 microtome 
and an LKB ultramicrotome at a thickness of 
about 800 ~ 1000 Â. Tissue sections were 
mounted on collodion-coated copper grids, 
doubly stained with uranyl acetate followed by 
lead citrate, and examined with a JEM 100 
CX-2 (80 kv) electron microscope. 

First Sexual Maturity 

A total of 214 female purple shells 
(3.1-17.0 cm in shell height) were used for 



the histological study of first sexual maturity. 
The percentage of sexual mature was deter- 
mined for shell heights that reached maturity 
during the breeding season (from May to late 
August). 

Egg Capsule and Fecundity 

To investigate the number of egg capsules 
per individual and the number of eggs in an 
egg capsule in the laboratory, a total of 25 
adult females of 11.2-14.2 cm in shell height 
were used for observation of spawning be- 
havior in an FRP aquarium (80 cm x 60 cm x 
60 cm) bedded with sand and small gravels. 
An established filtration and aeration appara- 
tus was employed. Purple shells used for this 
experiment were those collected in May to 
June 1994. Length and width of egg capsules 
of the adult purple shells were measured and 
their egg developmental processes were 
checked under a light microscope. 

The purple shells were reared at the spe- 
cific gravity of 1.021 and water temperatures 
of 18.4-23.8°C. Seawater in the rearing FRP 
aquarium was changed every 3 days. Suffi- 
cient amounts of bivalves {Ruditapes philip- 
pinarum, Meretrix lusoria, and Mactra veneri- 
formis) were supplied as food for R. venosa 
during the rearing period. 

Sex Ratio 

Sex ratios in a total of 445 sexually mature 
individuals (>7.1 cm in shell height) were in- 
vestigated from June 1994 to May 1995. Sex 
of individuals was determined by visual ob- 
servation of the presence/absence of the 
penis. Female individuals do not have any 
genital organ (penis) near the tentacles. To 
confirm a clear sexuality for each individual, 
both sexes were confirmed by light micro- 
scopic examination of histological prepara- 
tions of the gonads. A Chi square test on 
goodness-of-fit was used to test the hypothe- 
sis of equal representation of each sex. 



RESULTS 

Morphology and Internal Structures of Re- 
productive Organs 

The purple shell Rapana venosa is dioe- 
cious, and the ovary is located on the surface 
of the digestive gland in the spiral posterior re- 
gion of the shell. The ovary is composed of 



244 



CHUNG ETAL. 



numerous oogenic follicles. With gonad nnatu- 
ration, the external color of the ovary be- 
comes pale yellow. Female individuals do not 
have the genital organ (penis) near the tenta- 
cles (Fig. 2) and can therefore be usually dif- 
ferentiated from males. 

Changes in the Gonadosomatic Index (GSI) 
and Water Temperature 

Monthly GSI changes were measured in fe- 
male samples collected between June 1994 
and May 1995 (Fig. 3). The GSI values slowly 
increased from October through February, 
when seawater temperatures gradually de- 
creased. However, the values rapidly in- 
creased from March, when seawater began to 
increase again and then reached the maxi- 
mum value (mean 1 7.0) in April, when seawa- 
ter temperature rapidly increased. Thereafter, 
the GSI showed relatively lower values in May 
to August, when relatively higher water tem- 
peratures were maintained and spawning oc- 
curred. And the value temporarily reached the 
minimum level in September, when most of 
gonads were completely degenerated or re- 
sorbed after spawning. 



Electron Microscope Observations on Germ 
Cell Development during Oogenesis 

Ultrastructural observations allow the germ 
cell developmental phases during oogenesis 
to be divided into four phases: (1) oogonial 
phase, (2) previtellogenic phase, (3) vitel- 
logenic phase, and (4) mature phase. Char- 
acteristic features in each phase during ooge- 
nesis were as follows: 

Oogonial Phase: Oogonia in the oogonial 
phase, which propagated on the follicular 
wall, were oval in shape and 15 цт in diame- 
ter. They were single or formed a cluster in the 
oogenic follicle. Each oogonium had a large 
nucleus with chromatin, several mitochondria, 
and the endoplasmic reticulum in the cyto- 
plasm (Fig. 4A). At this stage, the granular 
cells and mesenchymal cells were present 
around the oogonia (Fig. 4B). 

Previtellogenic Phase: With cytoplasmic 
growth, several small mitochondria, a well-de- 
veloped endoplasmic reticulum and several 
vacuoles were concentrated around the nu- 
cleus in the cytoplasm of the previtellogenic 
oocyte (Fig. 4C, D). The number of Golgi ap- 



20 



x15 

Ш 
Q 

Z 

О 



о 

(Л 

о 

Q 
< 

о 
о 



10- 



5- 




-25 



-20 



— •- Water temperature 
-S— Gonadosomatic index 



-] — \ \ — 1 — I — 1 — I — \ \ \ 1 r~ 

J JASONDJ FMAM 

1994 1995 

MONTH 



-30 



15 



-10 



LU 

ce 

Cd 

ш 

CL 
UJ 

h- 

Ш 



FIG. 3. Monthly changes in the gonadosomatic index in Rapana venosa and the mean water temperatures. 
Vertical bars represent standard error. 



REPRODUCTION IN RAPANA 



245 



\i|Çr-< 








• < 




.4, 






Щ 


• * 




■ -<^!| 


£?r 






.>'''C . 


тЧКяС^"* ' 


mit 


. F 




fib* 





FIG. 4. Electron micrographs of oogenesis of Rapana venosa (A-F). A, An oogonium, with a large nucleus 
and several mitochondria in the cytoplasm, scale bar - 2 |дт; В, the mesenchymal tissue and the granular 
cells near the oogonia, scale bar = 2 |im; C, a previtellogenic oocyte, with a large nucleus containing chro- 
matin, and several mitochondria, a number of vacuoles and well-developed endoplasmic retícula in the cy- 
toplasm, scale bar = 2 \xm\ D, E, the previtellogenic oocytes, with the Golgi apparatus and glycogen particles 
in the vacuoles, scale bars = 4 |am; F, an early vitellogenic oocyte, with a number of the vacuoles and lipid 
droplets and yolk granules near the nucleus, scale bar - 2 \хт. (Figure continues.) 



246 



CHUNG ETAL 




FIG. 4. (Continued) G, a late vitellogenic oocyte, with yolk granules and proteid yolk granules, scale bar = 2 
urn; H, a late vitellogenic oocyte, with a nunnber of proteid yolk granules and immature yolk granules; I, a ma- 
ture oocyte, with mature yolk granules near the nucleus, scale bar = 2 |лт; J, a mature oocyte, with a mature 
yolk granule being composed of the main body (central core), superficial layer and a limiting membrane, of 
a yolk granule, scale bar = 2 um. Abbreviations: OR, chromatin; ER, endoplasmic reticulum; G, Golgi appa- 
ratus; GC, granular cell; GR, granule; lYG, immature yolk granule; LD, lipid droplet; LM, limiting membrane 
of mature yolk granule; M, mitochondrion; MBy, main body; MC, mesenchymal cell; MYG, mature yolk gran- 
ule; N, nucleus; ОС, oocyte; OG, oogonium; PYG, proteid yolk granule; SL, superficial layer; V, vacuole; YG, 
yolk granule. 



paratus, scattered from the perinuclear region 
to the cortical region of the oocyte in the pre- 
vitellogenic phase, increased (Fig. 4D). At this 
time many vacuoles formed by the Golgi ap- 
paratus appeared near the endoplasmic retic- 
ulum and some mitochondria (Fig. 4E). 

Vitellogenic Phase: In the early vitellogenic 
oocyte, especially with the initiation of yolk 
formation, glycogen particles, lipid droplets 
and a few yolk granules were found in the vac- 
uoles formed by the Golgi apparatus in the 
perinuclear region. Yolk granules diffused to- 
ward the cortical layer, and yolk granules ap- 



peared around the well-developed cortical re- 
gion of early vitellogenic oocytes (Fig. 4F). In 
the late vitellogenic oocyte, accumulation of 
yolk granules occurred in the cortical layer. 
Particularly, yolk granules, proteid yolk gran- 
ules, and glycogen particles were apparent in 
the cytoplasm (Fig. 4G). In addition, a number 
of modified mitochondria and endoplasmic 
reticulum were involved in the formation of 
proteid yolk granules. Glycogen particles, yolk 
granules and proteid yolk granules were ac- 
cumulated in the cytoplasm. Eventually, their 
yolk granules containing several different 
components were intermingled and became 



REPRODUCTION IN RAPANA 



247 



Immature yolk granules in the oocyte in the 
vitellogenic phase (Fig. 4H). 

Mature Phase: In the oocyte in the mature 
phase, small immature yolk granules were 
continuously mixed and became larger ma- 
ture yolk granules in the cytoplasm (Fig. 41). A 
mature yolk granule was composed of three 
components: (1) main body (central core), (2) 
superficial layer; and (3) a limiting membrane 
of a mature yolk granule (Fig. 4J). 

Occurrence of Germ Cells during Oogenesis 
and the Reproductive Cycle 

Based on electron microscopic and histo- 
logical observations of the germ cells and 



other surrounding cells, the gonadal phases 
were classified into five successive stages 
(Fig. 5), which show an annual cycle. Occur- 
rence of germ cells of vahous phases at each 
stage and their characteristics are as follows: 

Early Active Stage: The gonadal volume was 
small, and the follicles occupied approxi- 
mately 25% of the gonad. The follicular walls 
(germinal epithelium) were relatively thick. A 
few oogonia of 15 цт in diameter were pre- 
sent along the follicular walls of the ovary, and 
the previtellogenic oocytes appeared. Previ- 
tellogenic oocytes of 60-70 цт in diameter 
formed an egg-stalk attached to the follicular 
wall (Fig. 6A). The individuals in the early ac- 
tive stage were found from September to 




J JASONDJ FMAM 
1994 1995 

MONTH 



Early active 

Ripe 

Recovery 



Ш Late active 

Ш Partially spawned 



FIG. 5. Frequency of gonadal phases of Rapana venosa from June 1994 to May 1995. 



248 



CHUNG ETAL 




FIG. 6. Photomicrographs of the histological gonadal phases of the female purple shell, Rapana venosa. A, 
Transverse section of oogenic follicles in the early active stage, scale bar = 60 цт; В, С, section of follicles 
in the late active stage, scale bars = 60 цт; D, section of ripe oocytes in the ripe stage, scale bar = 60 |.im; 
E, section of follicles in the partially spawned stage, scale bar = 60 \xm\ F, section of the follicles in the re- 
covery stage, scale bar = 60 \xn\. 



February when seawater terлperatures were 
gradually decreasing. 

Late Active Stage: This stage is character- 
ized by the presence of developing early vitel- 
logenic oocytes. Follicular walls (germinal ep- 
ithelium) of oogenic follicles were thin. A 
number of early vitellogenic oocytes of 
120-140 |im in diameter were attached to the 
follicular walls through each egg-stalk. With 



the initiation of yolk formation, there were nu- 
merous yolk granules in the cytoplasm of late 
vitellogenic oocytes of 150-190 |nm. Some 
mature oocytes were free in the lumen of the 
follicle (Fig. 6B, C). The individuals in the late 
active stage appeared from October to April. 

Ripe Stage: Follicles occupied over 70% of 
the gonad, and follicular walls became very 
thin. Mature oocytes growing up to 230-250 



REPRODUCTION IN RAPANA 



249 



|im diameter became polygonal in shape, and 
contained a number of mature yolk granules 
(Fig. 6D). Mature or ripe ovaries were found in 
March through July, when seawater tempera- 
tures were gradually increasing. 

Partially Spawned Stage: The lumen of the 
oogenic follicle became considerably empty 
because approximately 50-60% of ripe 
oocytes in the lumen were discharged. 
Spawned ovaries were characterized by the 
presence of a few undischarged vitellogenic 
oocytes as well as previtellogenic oocytes in 
the follicle (Fig. 6E). This stage appeared in 
the individuals collected from May to early Au- 
gust, when seawater temperatures were 
18-26°C. 

Recovery Stage: After spawning, each folli- 
cle shrank, and then degeneration or resorp- 
tion of undischarged vitellogenic or mature 
oocytes occurred. Thereafter, the connective 
tissues and a few oogenia and oocytes ap- 
peared on the newly formed follicular walls 
(Fig. 6F). The individuals in the recovery 
stage were found from June to September. 

First Sexual Maturity 

During the breeding season, a total of 214 
individuals (3.1-17.0 cm in shell height) were 
histologically examined to check whether they 
reached maturity and participated in repro- 



duction. The rate of shells of different size that 
reached the first sexual maturity is summa- 
rized in Table 1 . The breeding season of R. 
venosa was found to be from May to August. 
In the case of some individuals with gonad de- 
velopmental stage in the late active stage in 
May through July, it is supposed that they can 
reach maturity except for individuals in the 
early active stage during the breeding sea- 
son. First sexual maturity was 0% in female 
purple shells of 3.1 to 5.0 cm high if they were 
at the early active stage during the breeding 
season. The percentages of first sexual matu- 
rity of female snails of 5.1-6.0 cm and 
6.1-7.0 cm in shell height were 14.3% and 
27.3%, respectively: most of the individuals 
were still in the early active stage. Percentage 
of first maturity in 7.1 to 8.0 cm in shell height 
were over 50%, all of which were at the late 
active, ripe or partially spawned stages. Sex- 
ual maturity was 1 00% for shells over 10.1 cm 
in height. 

Copulatory Behavior and Spawning 

In the experimental aquaria in the labora- 
tory, copulatory interaction between females 
and males could be observed from early April. 
During this time, it was confirmed from histo- 
logical examinations that most of the ovaries 
were filled with a number of late vitellogenic 
oocytes or mature oocytes in the follicle rep- 



TABLE 1. The shell height and first sexual maturity of female purple shell, 
Rapana venosa from early August 1994 to May 1995. 







Number of 


individuals by gonadal stac 


)e* 


Shell height (cm) 


EA 


LA 


Rl 


PS 


RE 


Total 


Mature (%) 


3.1 -4.0 


6 










6 


0.0 


4.1 -5.0 


8 










8 


0.0 


5.1 ~ 6.0 


12 




2 






14 


14.3 


6.1 -7.0 


8 


2 


1 






11 


27.3 


7.1 - 8.0 


15 


2 


11 


3 




31 


51.6 


8.1 ~ 9.0 


5 


2 


9 


4 




20 


75.0 


9.1 - 10.0 


3 




16 


9 




28 


89.3 


10.1 - 11.0 






11 


9 


4 


24 


100.0 


11.1 - 12.0 






9 


9 


2 


20 


100.0 


12.1 - 13.0 






5 


4 


1 


10 


100.0 


13.1 - 14.0 






5 


5 


2 


12 


100.0 


14.1 - 15.0 






5 


6 


2 


13 


100.0 


15.1 - 16.0 






5 


6 


3 


14 


100.0 


16.1 - 17.0 






2 


1 




3 


100.0 


Total 












214 





'Gonadal stage: EA, early active stage; LA, late active stage; Rl, ripe stage; PS, partially 
spawned stage; RE, recovery stage. 



250 



CHUNG ETAL. 



resenting the late active or ripe stages. In the 
peak spawning period (June to July), how- 
ever, spawning in female individuals occurred 
15-30 days later after copulation. This obser- 
vation indicates that copulation occurs one to 
two months earlier than the spawning in fe- 
male individuals. 

Spawning and Number of Spawning 
Frequencies (Broods) in Aquarium 

Egg capsules and spawning intervals of R. 
venosa were counted by visual observation 
(Table 2, Fig. 7). Five out of twenty individuals 
successfully spawned in the experimental 
aquarium kept in the laboratory from May to 
August 1994: a total of 301 egg capsules 
were spawned by No. 1 adult female at inter- 
vals of 1 -4 days (three broods); 410 capsules 
by No. 2 at 1 -2 days (four broods); 306 cap- 
sules by No. 3 at 1 -2 days (three broods); 184 
capsules by No. 4 at 2 days (two broods); and 
No. 5 spawned a total number of 386 egg cap- 
sules at intervals of 1 -3 days (four broods). 

The average time required for a spawning 
of this species was 5.5 h (1 -6 h). According to 
the observation in the laboratory, most 
spawning occurred from night to early morn- 
ing. 




FIG. 7. External feature of deposition of egg cap- 
sules on the shell of Rapana venosa. 



Fecundity, Egg Size, Hatching Shell Length 
and Mode of Development 

The total number of egg capsules per indi- 
vidual and the mean number of eggs in an egg 
capsule of Я. venosa were 184 to 410 and 
976, respectively (Table 2). Thus, fecundity 



TABLE 2. Spawning frequency and the number of egg capsules of Rapana venosa observed from May to 
July 1995. 

Shell Water No. of Spawning Interval 

Ind. height Spawning Spawning temp. egg frequency (day) 

No. (cm) date hours (°C) capsules (No. of broods) 

1 13.5 



14.6 



13.6 



11.2 



14.4 



May 23 1994 


18:34- 


-24:06 


18.4 


113 


May 24 1994 


02:36- 


-08:18 


18.6 


96 


May 28 1994 


01:16- 


-07:08 


20.5 


92 
(301) 


May 24 1994 


23:16- 


-05:16 


19.4 


118 


May 25 1994 


16:31- 


-21:52 


20.1 


107 


May 27 1994 


16:26- 


-22:10 


20.0 


94 


May 28 1994 


04:34- 


-10:08 


20.4 


91 

(410) 


June 15 1994 


22:24- 


-03:50 


21.6 


114 


June 16 1994 


01:04- 


-06:36 


21.8 


98 


June 18 1994 


03:14- 


-08:05 


22.3 


94 
(306) 


June 20 1994 


23:34- 


-04:30 


22.5 


96 


June 22 1994 


05:48- 


-18:18 


22.8 


88 

(184) 


July 24 1994 


22:10- 


-03:09 


22.9 


112 


July 25 1994 


19:18- 


-23:50 


23.0 


95 


July 28 1994 


16:06- 


-20:43 


23.6 


92 


July 29 1994 


05:04- 


-09:43 


23.8 


87 
(386) 



1-4 



1-2 



1-2 



1-3 



REPRODUCTION IN RAPANA 



251 



was estimated to be 179,000 to 400,000 eggs 
per individual. The egg size and hatching 
shell length of R. venosa in Korea were 
230-250 )im and 0.41 x 0.30 mm, respec- 
tively (Table 3). The duration of development 
from fertilized eggs to veliger larvae in egg 
capsules just before hatching of R. venosa, 
were 15 to 17 days at about 20°C under labo- 
ratory conditions (Fig. 8). The mode of devel- 



opment of this species is a planktotrophic 
veliger larval pattern because the embryos 
hatch as planktotrophic veliger larvae. 

Sex Ratio 

Of total 445 purple shells, 228 were fe- 
males and 217 males (Table 4). There was no 
significant difference in the prevalence of 



TABLE 3. Comparison of the sizes of eggs and egg capsules, and larval shells of some species in the 
family Muricidae. 













Shell length 








Number of 


Egg 


Duration of 


of larvae at 




Shell length at 


Egg size 


eggs in an 


capsule 


development 


haching 




hatching (mm) 


(mm) 


egg capsule 


size (mm) 


(days) 


(mm) 


Source 


Hatch as veligers: 














Rapana venosa 


0.26 


790 ~ 1300 


30x25 


13 


0.41 xO.29 


Amio, 1963 


R. venosa 


0.24 


772 1190 


30x26 


15- 17 


0.41 X 0.30 


Present study 


R. bulbosa 


0.28 - 0.32 








0.42 


Thorson, 1940 


Bedevina birileffi 


0.19 


60 90 


3.0 X 0.85 


14 


0.30 - 0.32 


Amio, 1963 


Thais clavigera 


0.19 


160 220 


4.5x1.2 




0.30 - 0.32 


Amio, 1963 


T. bronni 


0.20 


160- 360 


1.0x1.5 




0.32 - 0.34 


Amio, 1963 


T. tissoti 


0.19 


75 




11 


0.32 


Middelfart, 1996 


T. floridana 


0.11 








0.13 


D'Asaro, 1966 


T. carinifera 


0.20 - 0.22 








0.32 0.34 


Thorson, 1940 


Purpura pat и la 


0.24 








0.4 


Lewis, 1960 




FIG. 8. Morphology of veliger larvae before hatching of Rapana venosa. Scale bar = 0.4 mm. 



252 



CHUNG ETAL. 



TABLE 4. Monthly variations in sex ratio of Rapana venosa. 





No. of 


No. of 


Total 


Sex Ratio 


X' (Chi 


Month 


Females 


Males 


Numbers 


(F/(F + M)) 


squared)* 


June 1994 


24 


20 


44 


0.55 


0.37 


July 1994 


20 


26 


46 


0.43 


0.78 


August 1994 


23 


19 


42 


0.55 


0.38 


September 1994 


22 


17 


39 


0.56 


0.64 


October 1994 


18 


24 


42 


0.43 


0.86 


November 1994 


18 


15 


33 


0.55 


0.27 


December 1994 


14 


18 


32 


0.44 


0.50 


January 1995 


14 


19 


33 


0.42 


0.76 


February 1995 


18 


13 


31 


0.58 


0.81 


March 1995 


21 


18 


39 


0.54 


0.23 


April 1995 


19 


15 


34 


0.56 


0.47 


May 1995 


17 


13 


30 


0.57 


0.53 


Total 


228 


217 


445 


0.51 


0.27 



"The critical value for x goodness-of-fit test of equal numbers of fernales and males (1 df) at 95% sig- 
nificance is 3.84. 



each sex (not different from a 1:1 sex ratio, 
X^ = 0.27, p > 0.05), and monthly comparisons 
showed no statistical differences. 

DISCUSSION 
Germ Cell Development and Vitellogenesis 

Our electron microscope observations of 
early vitellogenic oocytes of Rapana venosa 
Indicate that the Golgi apparatus is involved 
in the formation of a number of glycogen 
particles filled vacuoles and small vesicles in 
the perinuclear region of the cytoplasm. Lipid 
droplets and lipid granules are then added to 
the vacuoles and vesicles formed by the Golgi 
apparatus (referred as autosynthetic), as in 
llyanassa obsoleta (Taylor & Anderson, 1 969), 
Biomphalaria glabrata (de Jong-Brink et al., 
1976), Mytilus edulis (Reverberi, 1971), and 
Mactra veneriformis (Chung & Ryou, 2000). 
Therefore, our observation suggests that the 
Golgi apparatus and vacuoles present in the 
perinuclear region are involved in the forma- 
tion of lipid droplets and lipid granules in the 
early vitellogenic oocytes. Some mitochondria 
in late vitellogenic oocytes of Я. venosa ap- 
pear to undergo a process of transformation 
during late vitellogenesis, and some evidence 
affirms that the yolk precursors are derived 
from modified mitochondrial structures found 
in the cytoplasm. The endoplasmic reticulum 
and modified mitochondria are present near 
the Proteid yolk granules. However, we could 
not find pinocytotic tubules, such as those in- 
volved in yolk production in the vitellogenic 
oocytes of Agriolimax reticulatus (Hill & 



Bowen, 1976; Dohmen, 1983), or multivesicu- 
lar bodies as seen in the opisthobranchs 
Hypselodoris tricolor and Godiva banyulensis 
(Medina et al., 1986), and another snail, Physa 
acuta (Terakado, 1974). Accordingly, it Is as- 
sumed that the endoplasmic reticulum and 
modified mitochondria are involved in the for- 
mation of proteid yolk granules (Taylor & An- 
derson, 1969), with R. venosa only synthesiz- 
ing yolk autosynthetically, as seems to occur in 
the majority of gastropods. Exceptions include 
gastropod species {Planorbarlus corneas, 
Lymnaea stagnalis, Hypselodoris tricolor, and 
Gordiva banyulensis) that synthesize yolk by a 
combination of autosynthetic and heterosyn- 
thetic processes (Bottke et al., 1982; Medina 
et al., 1986). 

Occurrence and Resorption of 
Germ Cells during Oogenesis and 
the Reproductive Cycle 

As in most other marine mollusks (Chung et 
al., 1993; Chung & Ryou, 2000), gonad de- 
velopmental phases of R. venosa show an 
annual periodicity (Chung et al., 1993; Chung 
& Kim, 1997), with cyclic change of germ cell 
development during the reproductive cycle. 

According to occurrence of germ cells with 
the gonad developmental stage, the oogenia 
and previtellogenic oocytes appeared in the 
early active stage, and the early and late vitel- 
logenic oocytes occurred in the late active 
stage. Numerous fully mature oocytes in the 
ripe stage were released in the partially 
spawned stage. After spawning, small num- 
bers of undischarged vitellogenic and mature 



REPRODUCTION IN RAPANA 



253 



oocytes were degenerated and resorbed. 
Newly formed oogonia and previtellogenic 
oocytes occurred in the recovery stage. 

Regarding reproductive energy allocated to 
the production of gametes, in particular, the 
continuous production and resorption of germ 
cells may be regard as an adaptation to envi- 
ronmental temperature and food availability 
(Morvan & Ansell, 1988; Paulet, 1990). If the 
reproductive energy allocated to the produc- 
tion of gametes is too large, nutritive reserves 
may not be enough to allow all eggs to reach 
the critical size for spawning and fertilization. 
In this case, the products of gamete atresia 
may be resorbed and the energy reallocated 
to still-developing oocytes or used for other 
metabolic purpose by marine moliusks (Der- 
ange & LePennec, 1989; Motavkine & Varak- 
sine, 1989). Therefore, it is supposed that R. 
venosa should have a reproductive mecha- 
nism to resorb and utilize the high nutritive 
substances rather than releasing hopeless 
gametes. 

Gonadal Development and Maturation 

It is well known that development and mat- 
uration of gametes of prosobranchs are gen- 
erally influenced by several factors, such as 
temperature, food availability, illumination 
(day length), and hormones (Boolootian et al., 
1962; Fretter, 1984). 

We observed that gametogenesis of R. 
venosa initiates at a temperature of about 
3.0°C, with maximum gonadal maturation oc- 
curring in June, when temperatures were high 
and phytoplankton were very abundant. Ac- 
cording to Segal (1956) and Sutherland 
(1970), in temperate prosobranchs, gonadal 
maturation and spawning occur in rapid suc- 
cession during the summer. The importance 
of food is obvious in the limpet Acmaea spp.: 
gametogenic activity of upshore populations 
with less available food is more restricted than 
those of the same species lower on the shore. 
As suggested by Sastry (1963) from a study 
with bay scallops, water temperature seems 
to be the most important parameter for main- 
tenance of gonadal development. In Korean 
coastal waters, growth and production of bi- 
valves that are predated upon by R. venosa is 
relatively high from spring to early summer 
seasons (Kim et al., 1 977; Chung et al., 1 994; 
Lee, 1995) due to the abundance in phyto- 
plankton. Thus, abundant food supply (e.g., 
bivalves) is available to R. venosa during the 
period of gonadal development and matura- 



tion. Therefore, it is suggested that gonadal 
development and maturation of Korean R. 
venosa is closely related to temperature 
change and food availability. Fretter (1984) 
observed that in temperate zones, the sea- 
sonal temperature fluctuation associated with 
changing illumination is a controlling factor in 
gametogenesis. In consequence, gonadal de- 
velopment and maturation of this species may 
be retarded under low illumination, due to the 
decrease in food (bivalves) availability caused 
by diminished primary production of phyto- 
plankton. 

First Sexual Maturity with the 
Gonad Developmental Stage 

All specimens of 3.1-5.0 cm high were in 
the early active stage although collected dur- 
ing breeding seasons, and our gonad histol- 
ogy indicates that none of them could fully de- 
velop: only small numbers of oogonia and 
previtellogenic oocytes were present in the 
oogenic follicle. The size of the oocyte indi- 
cates that they could not have reached matu- 
rity until late August, when spawning was 
ended. Accordingly, the percentage of first 
sexual maturity of female snails ranging from 
3.1 -5.0 cm in shell height is 0%. However, in- 
dividuals 7.1 -8.0 cm in shell height belonged 
to one of the early active, late active, ripe and 
partially spawned stages during the breeding 
season. Sixteen individuals in the late active, 
ripe, and partially spawned stage underwent 
gonadal development, whereas 15 individuals 
in the early active stage did not. It was ob- 
served that among snails of 7.1 -8.0 cm high 
and in the late active stage more than 50% 
reached the first sexual maturity. However all 
snails, in late active, ripe, or partially spawned 
stages, reached it if they were larger 10.1 cm. 
This means that larger individuals can reach 
maturity earlier than smaller individuals. The 
results suggests that because catching purple 
shells < 7.1 cm can potentially cause a dras- 
tic reduction in recruitment, a prohibitory mea- 
sure should be taken for adequate natural re- 
sources management. 

Breeding Pattern 

Our histological observations on the purple 
shell show that spawning of R. venosa on the 
west coast of Korea occurs from late May to 
August. Japanese R. venosa have been re- 
ported to spawn once a year from June to Au- 
gust in the Ariake Sea, Japan (Amio, 1963). 



254 



CHUNG ETAL 



Our results agree well with those described by 
Amio (1963). The slight discrepancy In the 
spawning period between these two studies 
might be related to geographic differences In 
water temperature (Chung et al., 1993). As 
the spawning of R. venosa In Korea occurs 
from late May to August, this species may be 
classified as a summer breeder (Boolootlan et 
al., 1962). 

Morphology of Egg Capsule 

The surface of the exit part of the egg cap- 
sule of R. venosa is flattened and has a chltl- 
nous capsular form. The egg capsule In the 
genus Rapana have a curved sickle shape, 
with a long cylindrical stem, and an albume- 
nous substance being contained in the egg 
capsules. These findings coincide with Amio's 
observation from R. venosa In Japan (Amio, 
1963). Rawlings (1999) described that the 
morphology of egg capsules varies within the 
marine neogastropods, showing differences 
In shape, size, surface texture within families 
(D'Asaro, 1970, 1988, 1991, 1993, 1997; 
Bändel, 1 973). Some species in a genus (Per- 
ron & Corpuz, 1982; Palmer et al., 1990; Mld- 
delfart, 1 996) and sometimes even Individuals 
of the same species population (Rawlings, 
1990, 1994, 1995, 1999) show distinct char- 
acteristics. These morphological differences 
in neogastropods may be associated with en- 
vironmental factors such as physical stresses 
or geographical latitude. 

Number of Eggs In an Egg Capsule 

In the present study, R. venosa in Korea 
spawned 184 - 410 egg capsules each, and 
the mean number of eggs from an egg cap- 
sule was 976. Thus, the fecundity (the total 
number of eggs) from each individual ranged 
from 179,000 to 400,000 eggs. Amio (1963) 
described that the number of eggs In each 
egg capsule of R. venosa in Japan ranged 
from 790 to 1,300. These results indicate a 
close similarity in reproductive output be- 
tween the Korean and the Japanese purple 
shells. 

Egg Size and Hatching Shell Length 

The egg sizes of R. venosa collected In 
Korea (230-250 |im) is slightly smaller than 
those of R. venosa (260 цт) In Japan, and 
shell lengths of veliger larvae at hatching of 



the Korean and Japanese purple shells were 
0.41 X 0.30 mm and 0.41 x 0.29 mm, respec- 
tively. The sizes of eggs and shell length of 
veliger larvae at hatching of R. venosa in 
Korea were similar to R. venosa in Japan, but 
the egg sizes and hatching shell lengths of 
veliger larvae of R. venosa were larger than 
those of Thais clavigera, T. bronni, and Be- 
devina birileffi (Table 3). Spight (1976) and 
Middelfart (1996) described that egg size In 
the egg capsule and shell length of veliger lar- 
vae at hatching are very similar for genera 
within Muricidae (Table 3). The factors affect- 
ing egg size and hatching shell length of R. 
venosa have not been studied yet. 

Duration of Development in the Egg Capsule 

We observed that the duration of develop- 
ment from fertilized eggs to hatching veligers 
ranged from 15-17 days at about 20°C in the 
laboratory. However, Amio (1963) described 
that of R. venosa in Japan took 1 2 days to de- 
velop from trochophore larvae to veliger larvae 
(i.e., about 13 days after fertilization). In gen- 
eral, our results were similar to those of Amio's 
(1963) in the duration of development in egg 
capsules (Table 3). It seems that the duration 
of development in egg capsules, from deposi- 
tion of an egg mass to hatched larvae, varies 
with both internal and external factors, as pro- 
posed by Amio (1963). Middelfart (1996) re- 
ported that duration of development of Thais 
tissoti was 11 days. Accordingly, it is assumed 
that duration of development of this species is 
shorter than R. venosa (Table 3). 

No Evidence of Nurse Eggs as Larval Food 

According to our microscopic observations, 
approximately 80% of eggs in a capsule were 
normally fertilized, and these embryos 
hatched as veliger larvae. The remaining 20% 
failed to develop, but they did not serve as 
nurse eggs for the normally developing lar- 
vae. 

Spight (1976) has classified marine gas- 
tropods into two groups based on hatching 
modes: (1) species with embryos that hatch 
as planktotrophic veliger larvae; and (2) 
species with embryos that hatch as crawling 
young snails (juveniles). Spight (1976) also 
differentiated developing embryos into those 
that consume nurse eggs and those do not 
consume nurse eggs within egg capsules in 
the process of embryonic development. 



REPRODUCTION IN RAPANA 



255 



Of Muricidae species, Siratus senegalensis 
(Knudsen, 1950), Chicoreus torrefactas {Cex- 
nohorsky, 1965), and Thais emarginata (Le 
Boeuf, 1971; Middelfart, 1994) have been re- 
ported to hatch as young snails. According to 
Spight (1976), among Muricidae, 16 species 
hatch as snails consuming nurse cells, and 17 
species hatch as snails without nurse cells. 
Twenty-two species hatch as veligers without 
nurse cells, but four hatch as veliger with 
nurse cells. These species consume 5.9-91 .4 
nurse eggs per embryo during development 
(Spight, 1976), and the size of newly hatched 
young snails is usually large (> 1.15 mm). In 
Pisania maculosa, a species with embryos 
that do not consume nurse eggs, 90% of the 
eggs were not fertilized, 8% became nurse 
eggs, and only 2% developed normally 
(Staiger, 1950). If the encapsulated embryos 
should share a common nutrient of albumen, 
developmentally retarded eggs are likely to be 
fed by surviving embryos (Staiger, 1951). It 
has been suggested that in case of hatching 
size of veliger is < 0.5 mm, R. venosa do not 
use nurse eggs, whereas young snails > 1.0 
mm may use nurse eggs for development. 
Shell size of the veliger larvae of R. venosa 
was relatively small (< 0.41 x 0.30 mm), indi- 
cating that the purple shell hatches as 
veligers without utilization of nurse eggs dur- 
ing development. 

In view of our present findings and those 
observed in other prosobranch gastropods 
(Spight, 1976), an important postulate can be 
made: species that hatch as large (> 1 .0 mm) 
veligers or juvenile snails consume nurse 
eggs during development in the egg capsules, 
whereas those with smaller veligers (< 0.5 
mm) do not. 



ACKNOWLEDGEMENTS 

The authors are grateful to Dr. John B. 
Burch of the University of Michigan and to the 
referees for helpful comments on the manu- 
script. The authors are grateful to the Fish- 
eries Science Institute of Kunsan National 
University for equipment and fund in the pro- 
gram year of 2001. 



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Revised ms. received 10 September 2001 



MALACOLOGIA, 2002, 44(2): 259-272 



IMPACT OF SOIL CHEMISTRY ON THE DISTRIBUTION OF 
ONCOMELANIA HUPENSIS {GASTROPODA: POMATIOPSIDAE) IN CHINA 

Edmund Y. W. Seto\ Weiping Wu^, Dongchuan Qiu^, Hongyun Liu'*, Xueguang Gu^, 
Honggen Chen'', Robert 0. Spear^ & George M. Davis^ 



ABSTRACT 

Oncomelania hupensis subspecies serve as intermediate hosts for the human Schistosoma 
parasite in China. In this study we present a multivariate analysis and comparison soil chemistry 
data associated with the presence or absence of two subspecies, O. hupensis robe rtson i and O. 
hupensis hupensis, that are associated with schistosomiasis disease transmission upstream and 
downstream, respectively, of the soon to be completed Three Gorges Dam on the Yangtze River 
Soil and water samples were collected and analyzed from the Anning River Valley, an 800 km^ 
area in the mountainous region of Sichuan Province and from the Poyang Lake floodplain, a 
4,647 km^ area in Jiangxi Province. A multivariate classification of soil data was able to discrim- 
inate between snail habitat and non-habitat in the Anning River Valley with 85.5% accuracy, and 
revealed that the lack of critical soil conditions potentially excluded 0. hupensis robertsoni Uom 
some sites. The soil classification was also able to correctly identify 72.4% of marginal habitat 
sites that were misclassified by field ecologists. We found that O. hupensis hupensis was less 
dependent upon soil conditions for sites on the Poyang Lake floodplain, and hypothesize that 
changes in water level associated with seasonal flooding may play a larger role than soil in dis- 
criminating between snail habitat and non-habitat in this environment. 

Key words: classification and regression trees, China, Oncomelania hupensis, schistosomia- 
sis. Schistosoma japónica, soil chemistry, snail ecology, Poyang Lake, Anning River, Sichuan, 
Jiangxi. 



INTRODUCTION 

Despite the successful eradication of schis- 
tosomiasis in some regions of China, it still re- 
mains a serious infectious disease that is en- 
demic in 118 counties, infecting nearly one 
million Chinese (Chen & Zheng, 1999). It is 
estimated that over 40 million are at risk of de- 
veloping schistosomiasis in China. Many of 
those at risk live along the Yangtze River, 
which stretches across China, from the moun- 
tains in the west to the coast in the east. Along 
this span exist a multitude of terrains and en- 
vironments, some of which are ideal habitats 
for subspecies of Oncomelania hupensis, 
which senye as the intermediate snail hosts 
for the Schistosoma japónica parasite that 
causes schistosomiasis. 

Current research attempts to better charac- 
terize the different ecologies related to schis- 
tosomiasis transmission (Davis et aL, 1999; 
Zhou et al., 2001). Such work is of critical im- 



portance, as the construction of the new 
Three Gorges Dam on the Yangtze will poten- 
tially result in the emergence of schistosomia- 
sis in entirely new areas (Davis et al., 1999; 
Hotez et al., 1997). The Three Gorges Dam 
will be the world's largest dam. The reservoir 
created by the dam may impact global cli- 
mate, cause dramatic change in the flow pat- 
terns of the Yangtze River that will increase 
snail habitat, and increase the probability of 
snail migration so dramatically that the geo- 
graphic distribution of snail habitats may 
change. A better understanding of which envi- 
ronments can and cannot support snails, and 
how these environments are distributed spa- 
tially now and in the future can potentially lead 
to effective control strategies. This is particu- 
larly true in China, where historically, snail 
control has been an integral part of success- 
ful disease eradication (Chen & Zheng, 1999; 
Sleigh et al., 1998). 
The need for a better ecological under- 



^School of Public Health, University of California, Berkeley, California 94720, U.S.A.; seto@uclink.berkeley.edu. 

^Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, 207 Rui Jin Er Liu, Shanghai 200025, P.R.C. 

^Sichuan Institute of Parasitic Diseases, Chengdu 610041 Sichuan, P.R.C. 

'^Jiangxi Provincial Institute of Parasitic Diseases, Nanchang, P.R.C. 

^School of Public Health, University of California, Berkeley, California 94720, U.S.A. 

^Department of Microbiology and Tropical Medicine, The George Washington University Medical Center, Washington, D.C. 

20037, U.S.A. 

259 



260 



SETO ETAL 



Standing of О. hupensis is most evident in cur- 
rent research in the use of remote sensing 
technologies to identify snail habitats (Spear 
et al., 1999; Zhou et al., 2001). Such studies, 
motivated in large part by the construction of 
the Three Gorges Dam, have been carried out 
both upstream above the dam and down- 
stream below the dam, in current schistoso- 
miasis endemic areas. In such studies it is im- 
portant to realize that different subspecies of 
O. hupensis exist in different regions, and that 
these subspecies have evolved to adapt to 
their particular ecological niches and human- 
parasite interactions. Oncomelania hupensis 
robertsoni exists upstream of the Three 
Gorges Dam site, in the mountains of Sichuan 
Province where the snails live just above the 
waterline in the moist soil and shaded by the 
vegetation of irrigation ditch networks feeding 
agricultural fields. Oncomelania hupensis hu- 
pensis exists downstream of the dam, in the 
lower Yangtze basin, where the snails live in 
the marshes and grasslands of Poyang Lake, 
a low elevation plain, completely enclosed by 
an impressive dyke system, and affected by 
strong seasonal flooding. 

The Anning River Valley in southern Si- 
chuan Province characterizes the mountain- 
ous environment where O. hupensis robert- 
soni live (Figs. 1, 2). The valley, at an 
elevation of 1 ,500 m, is approximately 48 km 
in length, 24 km at its widest point, represent- 



ing approximately 400 km^ of potential snail 
habitat. Here, land is predominantly irrigated 
agriculture, with two growing seasons and 
rice, wheat, corn, tobacco, and various export 
vegetables the major crops grown. Domestic 
animals play a minor role in the agriculture of 
the valley and in schistosomiasis transmis- 
sion. Snails do not live within fields, but rather, 
along the edges of vegetated irrigation 
ditches. Recent prevalence surveys con- 
ducted in the summer of 2000 by collabora- 
tors from the Xichang County Anti-Schistoso- 
miasis Station have shown that the variance 
in human schistosomiasis infection is quite 
large in the valley, ranging from below 10% in 
some areas to above 70% in other areas. On- 
comelania hupensis robertsoni \ha\. live in this 
environment have a smooth shell, which has 
no varix on the outer lip and is relatively short 
(Davis et al., 1995). The subspecies is re- 
stricted to Sichuan and Yunnan provinces 
above the Three Gorges. 

Conversely, the Poyang Lake environment 
where O. hupensis hupensis live, as de- 
scribed by Davis et al. (1999), is vastly differ- 
ent. Poyang Lake of Jiangxi Province is the 
largest lake in China (Figs. 1, 3). During the 
flooding season it regularly fills with water and 
is held back from towns and villages by a se- 
nes of dikes. The time that flooding begins 
depends on the onset of the annual monsoon. 
Typically the flooding is May through October. 




XO km 



Endimic are* 
Provincial boundary 



FIG. 1. Map of schistosomiasis-endemic areas along the Yangtze River. The Anning River Valley, Poyang 
Lake and Three Gorges Dam Area are shown. 



SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 



261 




FIG. 2. Landsat TM satellite image of the Anning 
River Valley. 

During this period only fisherman can venture 
out on the lake when it resembles a vast sea. 
The flooding covers all of the marshland used 
by cattle for grazing. After the rainy season, 
the lake empties, losing as much as 90% of its 
water. All snails are found on the flood plains 
and on the numerous flat marshy islands in- 
side the dikes. All transmission occurs within 
the lake basin, where the cattle graze on the 
marshlands; there are no snails outside the 
dikes. Cattle play a much larger role in trans- 
mission in this environment than in the moun- 
tains of Sichuan Province. The flooding 
drowns adult snails, which cannot withstand 
continuous submersion. During drought, the 
adults move down into the soil and aestivate. 
Oncomelania hupensis hupensis has pri- 
marily ribbed shells in habitats regularly 
flooded and smooth shells in habitats that are 
not flooded (Davis et al., 1995). All popula- 
tions have a varix. The length of shell is, on 



average, greater than that of O. hupensis 
robertsoni. The shell allometry is the same as 
that of O. hupensis robertsoni. The life ex- 
pectancy for snails in this environment is 
about one year (Zhang et al., 1996). On- 
comelania hupensis hupensis is restricted to 
the Yangtze River drainage below the Three 
Gorges; it has spread to Guangxi Province, 
presumably by way of the grand canal from 
Hunan. 

We have studied the characteristic soil con- 
ditions for both environments. We apply mul- 
tivariate statistics to determine which soil con- 
ditions are capable of discriminating between 
areas where O. hupensis exist and where 
they do not. For the Sichuan environment, we 
also look at soil conditions for what we call 
"marginal habitat", which are areas that to the 
field ecologist look suitable as snail habitat, 
but where snails were not found. 



METHODS 

Soil samples from sites in the Anning River 
Valley were collected as part of a remote 
sensing validation study in the summer of 
1998 (Spear et al., 1999). Each site was de- 
fined by randomly generated geographic co- 
ordinates. A global positioning system (GPS) 
receiver was used to navigate to each site. 
Each site consisted of a 30 x 30 m area, 
where a snail survey was conducted and soil 
was collected. A total of 111 sites were visited 
(35 snail, 47 non-snail, and 29 marginal sites). 
Snail status was determined in two manners. 
A snail survey was performed in the sur- 
rounding 30 X 30 m area. The snail survey is 
a systematic search through the ditches for 
the presence of snails (Gu, 1990). The pres- 
ence of a single snail in the survey constitutes 
a positive snail site. In addition to the snail 
survey, a judgment was made by a field ecol- 
ogist as to whether the habitat seemed suit- 
able for snails. There exists sites where no 
snails were found, but where field ecologists 
felt the snails could exist. 

For each site visited, approximately 1 ,000 
cm^ of soil was collected by taking multiple 
samples along ditches within the 30 x 30 m 
area. Soil samples for each site were com- 
bined, dried and well-mixed to constitute a sin- 
gle soil sample for each site. These samples 
were later analyzed using Lamotte Company 
Electronic Soil Lab, Model DCL-12, Code 
1985-01. Measurements were made for pH, 
conductivity, nitrate nitrogen, nitrite nitrogen, 
ammonia nitrogen, phosphorus, potassium, 



262 



SETO ETAL 









<л> 






FIG. 3. Landsat TM satellite image of Poyang Lake at low water, showing TMRC study sites. 




sulfur, copper, iron, manganese, zinc, calcium, 
magnesium, and chlorides. Soil moisture was 
measured using a common garden soil mois- 
ture meter. Soil texture was analyzed using 
Lamotte Company Texture Kit, Code 1067, 
which provided the fractions of sand, silt, and 
clay present in the soil. 

Water samples were also collected at each 
site from the nearest irrigation ditch within the 
30 X 30 m area. Water samples were ana- 
lyzed in the field using Lamotte Company 
GREEN Water Monitonng Kit Code 5848. 
Measurements were made for pH, phospho- 
rus, turbidity, temperature, dissolved oxygen, 
and nitrate. 

The Tropical Medical Research Center 
based in Shanghai conducted snail surveys at 
98 sites in the Poyang Lake environment in 
1 999 and 2000. Sites were chosen based on a 
new randomized snail survey protocol. Ac- 
cording to this protocol, buffalo grazing ranges 
were mapped, and 10,000 m^ "squares" were 
located throughout the grazing ranges to pro- 
vide adequate coverage of the different areas 
of the ranges. Each square is divided into 
numbered grids of 10 m x 10 m, of which 20 
are selected at random each collecting sea- 
son. A frame of 4 m^ is placed in the center of 



each randomly selected grid-square and all 
snails are collected. A GPS record is made of 
each grid-square. The percent area with no 
snails is calculated from the cells with no 
snails. For the area calculated to have snails, 
the mean and standard deviation of snails 
per m^ and the frequency of infections are 
recorded. Usable ecological data and soil con- 
ditions were determined for 87 of the 98 sites. 
Snails were found at 50 of the 87 sites. Eco- 
logical and soil data measured include ambi- 
ent air and soil temperature, humidity, light, 
saturation, pH, nitrate nitrogen, phosphorus, 
potassium, zinc, ammonia nitrogen, calcium, 
chloride, copper, ferric iron, magnesium, man- 
ganese, nitrite nitrogen, sulfate, and soil tex- 
ture. Soil conditions were measured using the 
same Lamotte equipment and methods as that 
used in Sichuan Province for the Anning River 
Valley. 

Univariate statistics were calculated for 
each ecological property measured, allowing 
for comparison between the Anning River Val- 
ley and Poyang Lake environments. To test 
for significant univariate differences in the dis- 
tribution of ecological properties between the 
two environments, Kolmogorov-Smirnov dis- 
tance statistics were calculated (Conover, 



SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 



263 



1980). The Kolmogorov-Smirnov statistic is a 
non-parametric test of the equality of two dis- 
tributions, and was calculated using the Stata 
statistical analysis software (Stata Corpora- 
tion, 2000). 

Multivariate statistics were used to consider 
possible interactions between ecological 
properties in discriminating between snail 
habitat and non-habitat sites. The Classifica- 
tion and Regression Trees (CART) method 
(Breiman, 1984) was used to create a classi- 
fication of habitat versus non-habitat for both 
the Anning River Valley and Poyang Lake en- 
vironments. CART produces optimal binary 
decision trees that describe how different fac- 
tors interact to produce particular classifica- 
tion outcomes. Trees for the Anning River Val- 
ley and Poyang Lake were compared. For 
data from the Anning River Valley, information 
exists on snail habitat, non-habitat, and mar- 
ginal habitat. Only habitat and non-habitat 
sites were used in the CART analysis so that 
the decision tree could subsequently be used 
to predict the outcome for marginal habitat. 
CART analyses were performed using the 
CART for Windows program from Salford 
Systems (Steinberg & Colla, 1997). 



RESULTS 

Univariate statistics for the ecological vari- 
ables related to snail habitats and non-habi- 
tats for the Anning River Valley and Poyang 
Lake environments are presented in Table 1 . 
In general, there is greater overall variation in 
soil conditions for the Anning River Valley 
than in those for Poyang Lake. Snail habitat 
within the Anning River Valley is more variable 
than that in Poyang Lake, as the standard de- 
viations for all but four soil properties (nitrate 
nitrogen, potassium, chloride, and nitrite nitro- 
gen) are larger for snail habitat in the Anning 
River Valley. Similarly, non-habitats within the 
Anning River Valley are more variable than 
those in Poyang Lake, as the standard devia- 
tions for all but four soil properties (potassium, 
ammonia nitrogen, chloride, and nitrite nitro- 
gen) are larger for non-habitats in the Anning 
River Valley. In both regions though, snail 
habitat may be complex, as generally higher 
variances exist in soil conditions associated 
with snail habitat than with non-habitat. 

Table 1 shows that there is overlap in the 
minimum and maximum ranges of soil proper- 
ties for snail habitat and non-habitat, suggest- 
ing that there is no clear univariate method for 



discriminating between habitat and non-habi- 
tat. However, the presence of overlap in the 
ranges does not necessarily indicate that the 
distributions of soil property for snail habitat 
and non-habitat are the same. Kolmogorov- 
Smirnov distance statistics are better suited to 
determining the difference between two distri- 
butions. The distance statistic assesses the 
difference in cumulative distributions between 
snail habitat and non-habitat sites for a partic- 
ular soil property. As an example, the cumula- 
tive distributions for sulfate measurements at 
snail habitat and non-habitat sites in both the 
Anning River Valley and at Poyang Lake are 
shown in Figure 4. It is clear from this figure 
that there is a significant difference in sulfate 
levels for snail habitat versus non-habitat in 
the Anning River Valley. This difference in sul- 
fate is not apparent for Poyang Lake. Corre- 
spondingly, the Kolmogorov-Smirnov statis- 
tics presented in Table 2 reveal a relatively 
large, statistically significant (P < 0.05) D-sta- 
tistic for sulfate in the Anning River Valley ver- 
sus a small, statistically insignificant D-statis- 
tic for sulfate in Poyang Lake. Overally, for the 
Anning River Valley, three soil properties have 
statistically significant (P < 0.05) distances 
(sulfate, ammonia nitrogen, and chloride), 
whereas twice as many distances are statisti- 
cally significant for the Poyang Lake environ- 
ment (zinc, potassium, nitrite nitrogen, silt, 
sand, and calcium). This suggests that a dis- 
crimination of snail habitat from non-habitat 
will depend upon a different set of soil proper- 
ties in the Anning River Valley versus Poyang 
Lake. 

A graphical illustration of the ranges of the 
ecological variables for snail habitat sites in 
the Anning River Valley and Poyang Lake is 
presented in Figure 5. Comparing the ranges 
for snail habitat for the two subspecies, it is 
apparent that for most soil properties there is 
overlap. However, there are distinct differ- 
ences in the range of pH and calcium. More- 
over, although there is some overlap, the lev- 
els of silt, phosphorus, ferric iron, magnesium, 
nitrite nitrogen and sulfate seem different for 
the two subspecies. Again, rather than only 
looking at ranges, Kolmogorov-Smirnov sta- 
tistics were used to compare the distributions 
of snail habitat for the two areas (Table 3). All 
but one of the distance statistics (chloride) 
were statistically significant, reinforcing the 
fact that the two snail subspecies live in very 
different environments. 

To understand how multivariate interactions 
work to discriminate between snail habitat and 



264 



SETO ETAL 



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266 



SETO ETAL 



TABLE 2. Kolmogorov-Smirnov distance between snail habitat and non-habitat for each 
soil property. A comparison of Kolmogorov-Smirnov distances for the Anning River 
Valley and Poyang Lake suggest that the discrimination of snail habitat and non-habitat 
for the two environments depend upon different soil properties. Statistically significant 
results (P < 0.05) are shown in bold type. 





Anning Ri 


iver Valley 


Poyang 
D 


Lake 




D 


P value 


P value 


Soil Moisture 


0.2558 


0.138 






Sand (%) 


0.1318 


0.831 


0.3455 


0.019 


Silt (%) 


0.1792 


0.483 


0.3455 


0.018 


Clay (%) 


0.1045 


0.962 


0.1480 


0.742 


PH 


0.2208 


0.242 


0.2674 


0.119 


Nitrate Nitrogen (lbs/acre) 


0.1695 


0.554 


0.2343 


0.225 


Phosphorus (lbs/acre) 


0.2000 


0.356 


0.1534 


0.714 


Potassium (lbs/acre) 


0.1604 


0.626 


0.4005 


0.005 


Zinc (ppm) 


0.1091 


0.945 


0.4370 


0.001 


Ammonia Nitrogen (lbs/acre) 


0.3623 


0.008 


0.2020 


0.379 


Chloride (lbs/acre) 


0.3266 


0.024 


0.0647 


1.000 


Copper (ppm) 


0.1257 


0.874 


0.2175 


0.298 


Ferric Iron (ppm) 


0.1435 


0.747 


0.2470 


0.174 


Magnesium (lbs/acre) 


0.2916 


0.057 


0.2456 


0.178 


Manganese (ppm) 


0.1864 


0.436 


0.2428 


0.192 


Nitrite Nitrogen (lbs/acre) 


0.1381 


0.777 


0.3695 


0.009 


Sulfate (ppm) 


0.4013 


0.003 


0.2484 


0.170 


Calcium (lbs/acre) 


0.2942 


0.052 


0.3082 


0.048 



non-habitat for each subspecies of snail, 
CART analyses were performed. The CART 
tree forthe Anning River Valley is shown in Fig- 
ure 6. The binary decision tree is read from the 
top down. All 83 data observations are repre- 
sented at the topmost node of the tree, where 
42% of the sites are snail habitat. CART fol- 
lows a brute force algorithm, where every pos- 
sible binary split criterion for every soil prop- 
erty is considered. The criterion that produces 
the best outcome in terms of splitting snail 
habitat from non-habitat is then chosen to split 
the top node into two child nodes. For the An- 
ning River Valley, magnesium is chosen to be 
the best first split. If the level of magnesium at 
a particular site is less than or equal to 1 671 .8, 
that site is passed to the left child. Otherwise, 
the site is passed to the right child. After this 
split, 73 of the original 83 sites are passed to 
the left child node, of which 34.2% are snail 
habitat sites. The remaining ten sites are 
passed to the right child node, where 1 00% of 
the sites are snail habitat. Since all ten sites in 
the right child node are snail habitat, there is 
no need for further splits. However, for the left 
child node with 73 sites, CART determined 
that a chtehon based on sulfur can further dis- 
criminate between snail habitat and non-habi- 
tat sites. This criterion leads to the creation of 
two additional child nodes. This procedure 



continues recursively until child nodes can no 
longer be reliably split by a soil criterion. In the 
Anning River Valley tree magnesium, sulfate, 
phosphorus, and silt are used. Actually, not 
displayed in the tree, but still important are 
contributions from chloride and potassium, the 
values of which were used when values of 
the other four soil properties were missing. 
The six-parameter tree produces a remark- 
ably high 85.5% cross-validated accuracy 
level. To independently test the classification, 
marginal habitat sites -those sites from the 
Anning River Valley where field ecologists felt 
snails could exist, however, where snails were 
not found -were subsequently passed down 
the CART tree. 72.4% of these sites were cor- 
rectly classified as non-habitat. A node of par- 
ticular interest is terminal node T1, where a 
large number of non-habitat sites fall. This 
node represents low magnesium, sulfate, 
phosphorus, and silt conditions. 

For the Poyang Lake environment, a CART 
classification of snail habitat versus non-habi- 
tat revealed that a single soil property, zinc 
was able to correctly classify 63% (cross-vali- 
dated accuracy) of the sites (Fig. 7). The ma- 
jority of the snail sites (39 of 50) fall into ter- 
minal node T2, where zinc levels are between 
2.7 and 6.2 ppm. Recall from the univariate 
analyses that zinc was also identified through 



Sand 
Silt 


ARV 

PL 

ARV 


3 




PL 


12| 



PH 



Zn 



Cu 



Mg 



SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 

63 



267 



11 4l 



24 7 



l36 



Clay ARV 4 

1^^H 



ARV 

PL 4.1 1 



Nitrate ARV 5 

PL lOl 



ARV 0.2 
PL 04| 



ARV 

PL Ol 



|828 
128.8 



647.4 



ARV 

PL ol 



28 



Amm. Nit. ARV 

PL 3.2| 



1 95 
72 

|85.7 
8.5 

|l40 
693 

1 1000 
66 

411 



132 



ARV 20 

PL 30| 



200 



ARV 
PL 













ARV 










PL 0.41 



ARV 1858 
PL 14 4| 



128.: 



124 8 



ARV 

PL ol 



1276 



Nitrite 


ARV 
PL 


0.1 


Sulfate 


ARV 


7.2 




PL 


55| 


Ca 


ARV 






PL 


16| 



1524 



22704 



1 37 

245.5 

14035.2 



1288 



FIG. 5. Illustration of the approximate ranges of soil properties associated with snail habitat in the Anning 
River Valley (ARV) and Poyang Lake (PL) environments. 



Kolmogorov-Smirnov statistics to have a large 
statistically significant distance between snail 
habitat and non-habitat distributions. 

A separate CART classification (Fig. 8) was 
run for the Poyang Lake sites using the same 
six soil parameters as those found to be im- 
portant in discriminating between snail and 
non-snail sites in the Anning River Valley. The 
result of this analysis was a considerably 
more complex eight-terminal node tree. How- 
ever, the accuracy for this complex tree was 



only slightly better (65.6%) than that of the 
simple three-terminal node zinc tree (63%). 
The interactions between the soil parameters 
in Poyang Lake seem to be different from 
those seen in Sichuan. However, the one fac- 
tor that remains important for snails in both re- 
gions is the sufficient availability of silt. 

Soil moisture measurements were col- 
lected for the Anning River Valley. Kol- 
mogorov-Smirnov statistics indicate that the 
difference in the distribution of soil moisture 



268 



SETO ETAL. 



TABLE 3. Kolmogorov-Smirnov distance between 
snail habitat in the Anning River Valley and snail 
habitat in Poyang Lake tor each soil property. All soil 
properties with the exception of chloride are statis- 
tically significant to P < 0.05, which suggests that 
the Anning River Valley and Poyang Lake environ- 
ments are very different. 





D 


P value 


Sand (%) 


1 .0000 


0.000 


Silt (%) 


1.0000 


0.000 


Clay (7o) 


1.0000 


0.000 


PH 


1.0000 


0.000 


Nitrate Nitrogen (lbs/acre) 


0.4327 


0.001 


Phosphorus (lbs/acre) 


0.5306 


0.000 


Potassium (lbs/acre) 


0.3333 


0.016 


Zinc (ppm) 


0.4163 


0.001 


Ammonia Nitrogen (lbs/acre) 


5102 


0.000 


Chloride (lbs/acre) 


0.2286 


0.187 


Copper (ppm) 


0.4748 


0.000 


Ferric Iron (ppm) 


0.6857 


0.000 


Magnesium (lbs/acre) 


1.0000 


0.000 


Manganese (ppm) 


0.7959 


0.000 


Nitrite Nitrogen (lbs/acre) 


0.9714 


0.000 


Sulfate (ppm) 


0.5878 


0.000 


Calcium (lbs/acre) 


1 .0000 


0.000 



between snail habitat and non-habitat sites is 
not significant (Table 2). However, range of 
soil moisture for snail habitat sites is much 
narrower than for non-habitat sites, with snails 
favoring high moisture environments (Table 
1). Corresponding data were not available for 
Poyang Lake. 

Both univariate and multivariate ap- 
proaches were used to analyze water sam- 
ples from the Anning River Valley. Univariate 
statistics for the water samples are presented 
in Table 1. There is considerable overlap in 
the ranges of measured water characteristics. 
However, Kolmogorov-Smirnov statistics indi- 
cate that the distribution of water turbidity for 
snail habitat sites is significantly different (P < 
0.05) from non-habitat sites (Table 4). Multi- 
vahate CART analysis produced a decision 
tree based on water turbidity and temperature 
values. However, there was significant mis- 
classification with this tree, suggesting that 
the variance in water conditions does not sig- 
nificantly affect the existence of snails. Corre- 
sponding water data for Poyang Lake were 
not collected. 



DISCUSSION 

Based on the Kolmogorov-Smirnov statis- 
tics of Table 3, the Anning River Valley is very 



different from Poyang Lake, which is not sur- 
prising given the differences in general ecolo- 
gies for these two regions. Perhaps the most 
important differences between the two envi- 
ronments are pH and calcium levels, for which 
no overlap exists in the ranges for these soil 
factors. Despite these differences, we cannot 
conclude that O. hupensis robertsoni would 
only be able to survive in the mountains of 
Sichuan, and O. hupensis hupensisin Poyang 
Lake. It can be argued that even though high 
pH and high calcium soils do not exist in 
Poyang Lake, O. hupensis hupensis may still 
find such environments suitable, and might be 
able to survive or adapt to the Anning River 
Valley environment. 

The CART classification of snail habitat ver- 
sus non-habitat relies on six soil properties, 
suggesting that the lack of critical levels of 
magnesium, sulfate, phosphorus, and silt may 
prevent the establishment of snails. However, 
one must be careful in interpreting such re- 
sults, as it is difficult to say with certainty that 
there is biological significance to these partic- 
ular soil properties. This is especially true in 
light of the number of different variables con- 
sidered here, many of which are probably cor- 
related. One should also be cautioned that as 
a non-parametric discriminant analysis, 
CART can only create decision rules based 
upon data given. As a consequence, variables 
that do not show up in the tree may not nec- 
essarily be unimportant to snail survival. Such 
variables may indeed be vitally important at 
extremely high or low levels. However, CART 
does not take these extremes into considera- 
tion, as such data do not exist within our 
datasets. 

Nevertheless, the Anning River Valley clas- 
sification tree is remarkably accurate, particu- 
larly in light of its performance in classifying 
marginal habitat sites. Marginal habitat sites 
should be considered as very difficult sites to 
classify correctly since field ecologists essen- 
tially misclassified them as snail habitat. The 
ability of CART to correctly classify 72.4% of 
these sites as truly non-habitat suggests that 
soil conditions are in fact very important, and 
that there is merit to the notion that the lack of 
particular soil conditions can make what 
would look like great snail habitat, marginal at 
best. Such a result is relevant to the changing 
ecologies associated with the Three Gorges 
Dam, where areas suspected of being snail 
habitat may be eliminated as habitat for the O. 
hupensis robertsoni subspecies based on the 
lack of critical soil conditions. Moreover, exist- 



SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 



269 



KEY: 



Percentage 
of snail sites 

Number of sites 
within node 



Terminal Node ID 
Percentage of snail sites 



Number of sites 
ithin terminal node 



42.2 



Mg<= 1671.8 



Мц> 1671.8 



>4.2 



73 



S<=21.5 



S>21.5 




/ 



V 



61.5 



47 \ / 26 \ 

P<= 168.9 P> 168.9 P<= 54.15 P> 54.15 



/ 



1.6 



43 



\ 




; 


T4 




T5 


100 




91.7 



\ 



;5.7 



14 



SILT <= 62.5 SILT > 62.5 4 




36.4 



12 P<= 92.05 P> 92.05 

^ A 



Mg<=681.1 Mg>681.1 

/ Л 







FIG. 6. CART classification of snail habitat versus non-habitat for the Anning River Valley. Units for silt is per- 
cent; magnesium and phosphorus are lbs/acre; and sulfate is ppm. 



Ing regional soil maps may be studied in the 
future for potential correlation with important 
soil factors and snail occurrence, which may 
explain historical patterns of disease-endemic 
areas. 

The comparisons of the variation in soil 
properties between the Anning River Valley 
and Poyang Lake environments suggest that 
the Anning River Valley is a more complex en- 
vironment than Poyang Lake, both in general, 
as well as with respect to snail habitat. Kol- 
mogorov-Smirnov statistics suggest that from 



a univariate standpoint, it is easier to discrim- 
inate between snail habitat and non-habitat in 
the Poyang Lake environment. This is also re- 
flected in the multivariate analyses, in which a 
more complicated multivariate eight-terminal 
node tree was needed to discriminate be- 
tween snail habitat and non-habitat in the An- 
ning River Valley, whereas a more compli- 
cated multivariate eight-terminal node tree 
performed just slightly better than the univari- 
ate three-terminal node classification tree. 
One might expect Sichuan mountain environ- 



270 



SETO ET AL. 



KEY: 




Percentage 
dt" snail sites 

Number of sites 
within node 




Terminal Node ID 
Percentage of snail sites 



Number of sites 
within terminal node 



Zinc <= 2.7 




96 



Zinc > 2.7 



i 



64.1 



64 



Zinc <= 6.2 



Zinc > 6.2 





FIG. 7. CART classification of snail habitat versus non-habitat for Poyang Lake. Zinc split criteria are shown 
in units of ppm. 



TABLE 4. Kolmogorov-Smirnov distance between 
snail habitat and non habitat for each water prop- 
erty in the Anning River Valley. Only the distance for 
turbidity is statistically significant to P < 0.05, which 
suggests that turbidity may play a role in discrimi- 
nating between snail habitat and non-habitat sites 
in the Anning River Valley. 





D 


P value 


PH 


0.1382 


0.826 


Dissolved Oxygen 


0.0794 


1.000 


Nitrate 


0.1563 


0.717 


Phosphorus 


0.2401 


0.221 


Temperature 


0.2451 


0.203 


Turbidity 


0.3240 


0.039 



merits to be more complex because of highly 
variable geology and earth processes associ- 
ated with mountainous terrain. The Poyang 
Lake environment is clearly composed of de- 
positional sediments from flooding, and thus 
might be expected to be more uniform, given 
that these sediments have been accumulating 
from the same sources and mixed over sev- 
eral centuries. 

The Anning River Valley is a much more 
stable environment for snails than Poyang 
Lake, due to the relative lack of seasonal 
flooding. This stability may lead to the estab- 
lishment of different niche habitats for snails in 
the valley. Recent trips to the field reinforce 
this hypothesis. Different snail densities were 
found at different elevations, where different 



land-use and related farming practices soil 
conditions existed at different elevations. In 
the lowlands, more paddy fields and long lin- 
ear irrigation ditches were found, whereas in 
the terraced highlands more complex field 
and ditch structure and considerably higher 
snail density were found. These land-use dif- 
ferences within the valley may correspond to 
the complexity seen in the snail habitat for this 
area. 

As a large flood basin, we hypothesize that 
there is less spatial variation in soil conditions 
for Poyang Lake than for the Anning River Val- 
ley due in part to the deposition, accumula- 
tion, mixing, and dispersal of soil and nutri- 
ents that occurs with flooding over time. It is in 
fact the flooding itself that is most likely the 
largest determinant of snail habitat versus 
non-habitat. Areas that are either too wet or 
too dry for too much of the year will exclude 
snails. Moreover strong evidence is provided 
by Davis et al. (in press) that the severity of 
annual floods can affect whether snails will be 
found in an area one year but not another Soil 
conditions only play a small role in discrimina- 
tion of snail habitat versus non-habitat for this 
area. This is supported by the fact that both 
the simple univariate CART tree based on 
zinc levels, as well as the complex multivari- 
ate CART tree were only able to achieve 
65.6% cross-validated accuracy, in compari- 
son to the 85.5% accuracy of the tree for the 
Anning River Valley. 

There are implications of these findings for 



SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 



271 



KEY: 



Percentage 
of snail sites 



Number of sites 
within node 




Terminal Node ID 
Percentage of snail sites 



Number of sites 
within temiinal node 




K<=85.6 



К > 85.6 




i 



62.3 



P<=2.7 



P>2.7 



Silt <= 20.4 



Silt > 20.4 




i 



¡ 



50.0 



51.1 



47 



13 Mg<=62.4 M2>62.4 S<=''1 7 S>217 

54.5 






FIG. 8. CART classification of snail habitat versus non-habitat for Poyang Lake using same six parameters 
used in the Anning River Valley classification. Units for silt is percent; magnesium, potassium, and phos- 
phorus are lbs/acre; and sulfate is ppm. 



snail control and future research. For O. hu- 
pensis robertsoni'm the mountains, the strong 
relationships between land-use, soil condi- 
tions, and snail habitat suggest that the sur- 
veillance of disease transmission may be very 
much related to the monitoring of changing 
land-use and farming practice. If the level or 
risk associated with particular types of land- 
use is better understood, then land-use 
change, such as those associated with envi- 
ronmental modification and different agricul- 
tural practices, may be effective control 



strategies for O. hupensis robertsoni. Remote 
sensing studies associated with this sub- 
species and environment should focus on 
characterizing differences in land-use and 
crop types, identifying which land cover types 
are most associated with high snail density, 
and determining the spatial relationship be- 
tween these particular land cover types and 
sites of high human exposure. For O. hupen- 
sis hupensis in Poyang Lake, the lack of a 
strong relationship between soil conditions 
and snail habitat suggest that relatively simple 



272 



SETO ETAL. 



remote sensing studies focused on identifying 
grasslands and their relationship to variances 
in water levels and to human exposure sites 
would be most appropriate. Studies of 
Poyang Lake must take into account chang- 
ing water levels associated with the seasonal 
flooding in order to understand the geo- 
graphic distribution of snail habitats overtime. 



ACKNOWLEDGEMENTS 

This study was supported by NASA-Ames 
Joint Research Interchange (NCC2-5102). 
NIEHS Mutagenesis Center at the University 
of California, Berkeley (5 P30 ES 01896-20 
ZESI), NIH-NIAID (1 ROI-AI43961-01A1), 
NSF of China (49825511), the University of 
California Pacific Rim Research Program, 
and N.I.H. grant 1 P50 AI3946, the Tropical 
Medical Research Center, Shanghai, China (I 
P50 AI3946). We acknowledge Ms Zhang 
Jing of the Jiangxi Institute of Parasitic Dis- 
eases for doing the soil chemistry, and the su- 
port of that institute for the intensive GIS field 
work. We thank the Xichang County Anti- 
Schistosomiasis Station for their field support. 



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CHEN, M. & F. ZHENG. 1999, Schistosomiasis 
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CONOVER, W. J., 1980, Statistics of the Kol- 
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DAVIS, G. M.. T WILKE, Y. ZHANG, X. J. XU, С P. 
QiU, С SPOLSKY D. С QIU, Y S. LI. M. Y XIA 
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DAVIS, G. M., W. WU, H. CHEN, H. LIU, J. GUO, D. 
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importance of bovines for human Schistosoma 
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DAVIS, G. M., Z. Yl, G. Y HUA & С. SPOLSKY, 
1995, Population genetics and systematic status 
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133-156. 

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HOTEZ, P J., F ZHENG, X. LONG-QI, С MING- 
GANG, X. SHU-HUA, L. SHUXIAN, D. BLAIR, 
D. R MCMANUS & G. M. DAVIS, 1997, Emerg- 
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SLEIGH. A., X. M. LI, S. JACKSON & K. L. HUANG, 
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SPEAR, R., P GONG, E. SETO, Y ZHOU, В. XU, 
D. MASZLE, S. LIANG, G. DAVIS & X, GU, 1999, 
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STEINBERG, D. & P COLLA, 1997, CART for Win- 
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ZHANG, S. J., Z. D. LIU & L. S. HU, 1996, Studies 
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ZHOU. X. N., I. B. MALONE, T K. KRISTENSEN & 
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Revised ms. accepted 1 December 2001 



MALACOLOGIA, 2002, 44(2): 273-288 

ECOLOGY OF POTENTIAL HOSTS OF SCHISTOSOMIASIS IN 
URBAN ENVIRONMENTS OF CHACO, ARGENTINA 

A. Rumi\ J. A. Bechara^, M. I. Hamann^ & M. Ostrowski de Núñez'* 

ABSTRACT 

Some of the Biomphalaha species living in Chaco, such as B. straminea and B. tenagophila, 
are natural transmitters of schistosomiasis in Brazil, while those of the genus Drepanotrema are 
not intermediate hosts of the disease. The aim of the present work was to analyze the importance 
of a selected set of environmental variables in explaining patterns of distributions and relative 
abundance of planorbid gastropod assemblages. The study sites were located in urban areas of 
Resistencia City, Chaco Province, and the environmental variables measured were substratum 
(macrophytes), water quality (pH, Oj, nutrients, among others), as well as other gastropods (An- 
cylidae, Hydrobiidae and Ampullaridae). Seasonal samplings were carried out in four distinct en- 
vironments. Thirty-one quantitative samples of gastropods and environmental variables were ob- 
tained. In canonical correspondence analysis (CCA), seven environmental variables were 
retained after a stepwise forward selection, from a total of 26, including [N-NH^""], 03%, and the 
macrophytes Eichhornia crassipes, Pistia stratiotes, Panicum elephantipes, Hydrocotyle ranun- 
culoides and Canna glauca. They explained 62% of the variation in planorbid association. Canna 
glauca was the most significant variable, being positively correlated with all of the species of 
Drepanotrema. Axis I separates B. tenagophila from B. straminea, along a gradient related to in- 
creasing 02% and P. eleptiantipes abundance, as well as decreasing [N-NH/] and P. stratiotes. 
Axis И separates D. lucidum, D. anatinum and D. c/mex from the other planorbid species along 
a gradient associated with decreasing abundances of H. ranunculoides and С glauca. Some 
common aquatic macrophytes, and to a lesser extent, dissolved oxygen and ammonium in water, 
may be useful indicators of favorable environmental conditions for potential intermediate hosts 
of schistosomiasis in Chaco Region. 

Key words: Gastropoda, Planorbidae, vector-ecology, schistosomiasis, intermediate-hosts, 
Chaco Region, Paraná River. 



INTRODUCTION 

The southern expansion of mansonic schis- 
tosomiasis in the Neotropical region (Pa- 
raense & Correa, 1987) necessitates devel- 
oping preventive strategies in those areas 
with major risk of disease penetration. In Ar- 
gentina, these zones are related to the 
Guyano-Brazilian subregion, mainly occupied 
by the Del Plata Basin, that includes geo- 
graphic areas such as Chaco, Mesopotamia 
and Pampas (Bonetto, 1994). The most prob- 
able colonization path is along the sub-basin 
of the Paraná River, since the most recent in- 
festation foci of Schistosoma mansoni Sam- 
bon, 1907 (Trematoda: Digenea), discovered 
in southern Brazil, were found in localities 



within that sub-basin in proximity to Argentina. 
One of these foci was located at San Fran- 
cisco do Sul, Santa Catarina State, near the 
headwaters of the Iguazú River (Bernardini & 
Machado, 1981), with Biomphalaria 
tenagophila (d'Orbigny, 1835) as intermediate 
host. The other was located in the Piquiri 
River, a tributary of the Paraná River (Pa- 
raense, 1986), with B. glabrata (Say, 1818) 
the intermediate host. Up to now, the latter 
host has not been detected in Argentina. Bio- 
mphalaria tenagophila was described with 
type locality in "Cantón de las Ensenadas", 
Corrientes Province, Argentina (d'Orbigny, 
1835), and Biomphalaria straminea from Ca- 
racas, Venezuela (Dunker, 1848), was re- 
corded here about 30 years ago. These two 



'CONICET, Facultad de Ciencias Naturales у Museo, División Zoología Invertebrados, Universidad Nacional de La Plata, 
Paseo del Bosque s/n, 1900, La Plata, Buenos Aires, Argentina; alerumi@museo.fcnym.unlp.edu.ar 
^CONICET, Instituto de Ictiología del Nordeste, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste. S. 
Cabra! 2139, 3400, Corrientes Argentina. 

^CONICET, Centro de Ecología Aplicada del Litoral, Ruta 5, km 2,5, Laguna Brava, 3400 Corrientes, Argentina. 
"CONICET, Facultad de Ciencias Exactas y Naturales, Departamento Biología, Universidad Nacional de Buenos Aires, Pa- 
bellón 2, Ciudad Universitaria, 1428, Capital Federal, Buenos Aires, Argentina. 



273 



274 



RUMI ETAL 



latter species are very common in freshwater 
habitats of northeastern Argentina. 

A detailed knowledge of the biology of pos- 
sible hosts of schistosomiasis, as well as the 
host-parasite relationships established in 
both natural and urban environments, is nec- 
essary for any control strategy (Rumi et al., 
1997). Several research studies have been 
conducted in the Paraná and Uruguay river 
basins, including species identification, ecol- 
ogy, demography, population dynamics, and 
spatial distribution of potential intermediate 
hosts of S. mansoni (Bonetto et al., 1982, 
1990: Olazarn, 1978, 1984; Rumi, 1991, 
1993: Rumi & Hamann, 1990, 1992: Rumi & 
Tassara. 1985: Tassara & Bechara, 1983). 
These potential hosts are always species of 
the genus Biomphalaha Preston, 1910 (Mol- 
lusca: Gastropoda: Planorbidae), though the 
studies also treated non-transmitter planor- 
bids of the genus Drepanotrema Preston, 
1910. Other studies consider the host-para- 
site relationships with autochthonous trema- 
todes in natural and urban environments (Os- 
trowski de Núñez et al., 1989. 1991: Rumi & 
Hamann. 1990, 1992). 

Urban environments, like those analyzed in 
the present paper, are particularly interesting 
because of the high probability of schistoso- 
miasis transmission to humans, in case this 
parasitism was found in Argentina. In the 
humid portion of the Chaco Plains, the Paraná 
River and its tributaries frequently generate 
an extensive plain of meanders with hundreds 
of lakes. Many human settlements are well 
established at those places, including small 
towns and medium-sized cities. Therefore, 
the identification of environmental variables 
that would allow a rapid evaluation of the po- 
tential of a habitat to support planorbids might 
be a useful strategy to prevent proliferation of 
the disease. 

As a general rule, the environmental alter- 
ations produced by human activities modify 
the adaptive capacities of host species, as 
well as their abilities to transmit the disease. 
Consequently, it Is necessary to study these 
hosts in natural and urban environments in 
order to contrast their dynamics in different 
conditions. According to ter Braak & Prentice 
(1988), most species occupy a limited area of 
the available habitat, tending to be more 
abundant in their optimal conditions. Conse- 
quently, assemblage composition shifts along 
gradients of environmental variables, and 
species replacements tend to follow the pat- 
tern of spatial and temporal variation of those 



variables. Gradients are not necessarily phys- 
ically continuous in space or time, but it is a 
useful abstraction to explain organism distri- 
butions (Austin, 1985). The concept of spatial 
partition of the life zone also implies the sep- 
aration of species along "resource gradients" 
(Tilman, 1982), "regulator gradients" or "com- 
plex gradients" (Huston, 1994). This scheme 
resulted in two classical models: the environ- 
mental control, in which environmental vari- 
ables regulate the presence and abundance 
of organisms (Whittaker, 1956), and biotic 
control, in which the relationships among or- 
ganisms, such as competition and prédation 
(Connell, 1983), are considered as the main 
factors that structure communities. In an al- 
ternative model, Quinn & Dunham (1983) pro- 
posed that they are not mutually exclusive, 
but both contribute. 

This paper aims to identify and analyze, in 
different planorbid assemblages found in 
urban environments in the city of Resistencia, 
Chaco Province, Argentina. The most impor- 
tant biotic and abiotic variables that explain 
their distribution and patterns of relative abun- 
dance. 



MATERIAL AND METHODS 
Study Area 

The sampling area was located between 
Resistencia and Barranqueras (59°15'S, 
27°44'W), Department of San Fernando, 
Chaco Province (Fig. 1). According to Drago 
(1990), the environments involved in the pres- 
ent study are within in the Paraná River Basin, 
which encompasses the second most impor- 
tant watercourse in South America after the 
Amazon. These environments are on the right 
margin of the alluvial plain of the Middle 
Paraná River. A large plain of meanders, with 
very low slope (10 to 12 cm km"^), runs across 
this sector of the plain. Soils are brown silty- 
clay, with abundant calcareous material in 
some places, and elevated alkalinity. An im- 
portant feature of this alluvial system is the 
great density of subcircular, shallow water- 
bodies (< 5 m depth), organized in a drainage 
arrangement that shows different degrees of 
connection between lotie and lentic waterbod- 
ies. Commonly, they are oxbow lakes and 
other meander-generated lakes and wet- 
lands, locally named "madrejones". Most of 
them are rich in floating, submerged or settled 



POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 



275 




FIG. 1. Geographie location of the study area and sampled biotopes. Scale bar = 1km. 



aquatic vegetation, surrounded by hydrophilic 
gallery forests. In the plain of meanders, lakes 
are found in different states of succession, be- 
ginning with the active meandering river chan- 
nel, followed by recently generated lakes, 
ending in the almost complete coverage of the 
pond basin with palustrine vegetation and 
sediments. Usually these ponds are shallow 
(up to 2 m deep). 



The climate of eastern Chaco is subtropi- 
cal, subhumid mesothermal, with a mean an- 
nual temperature of 23°C, and monthly aver- 
ages between 17°C in July and 28°C in 
January. The mean annual rainfall ranges be- 
tween 950 and 1,100 mm, distributed almost 
uniformly around the year, with peaks in 
spring (September-October) and fall (March- 
April). 



276 



RUMI ETAL 



Sampling Scheme 

Selection of variables, identified a priori as 
presumably important for colonization and de- 
velopment of planorbid populations, was 
based on three criteria: 

(1) Relationships with the substratum- 
mainly composed of aquatic vegetation, 
and described at the scale of their rela- 
tive abundances. The pulmonate snail 
uses macrophytes as sites for reproduc- 
tion, feeding, and shelter. Consequently, 
a decrease in abundance of macrophytes 
generally results in a decrease of gastro- 
pod populations (Thomas & Daldorph, 
1994). 

(2) Relationships with water quality vari- 
ables-including indicators of the trophic 
state of the selected habitats (nutrient 
concentrations), as well as other vari- 
ables related to the snail abundance 
along regulator gradients (pH, oxygen, 
and calcium concentrations). 

(3) Relationships with other members of the 
mollusk assemblages -also referred to a 
scale of relative abundance, represent- 
ing potential competitors or making up a 
part of multispecific associations. 

To include the four climatic seasons, sam- 
plings were carried out quarterly, between 
February 2, 1 988, and October 23, 1 989. This 
area was first widely surveyed to inventory 
habitats. Four sampling sites were selected 
based on their planorbid richness: Negro 
River, at the Municipal Beach of Resistencia 
City; Paikin, a oxbow-lake related to a suburb 
of the same name, used as garbage deposit 
by people living in a group of huts without a 
sewer system; Rural Society, a pond within 
the "Rural Society" of Chaco Province, be- 
longing to the Arazá River corridor, which was 
partially channeled underground; and Golf 
Club, a pond near the Resistencia "Golf Club", 
being probably an old oxbow-lake of the 
Negro River. The Negro and the Arazá rivers 
cross the city of Resistencia NE-SW toward 
the city of Barranqueras, draining into the 
Paraná River (Fig. 1). The present human 
population inhabiting both cities is around 
300,000 people. 

Gastropod sampling was carried out with a 
mean collection time of 41 min (standard devi- 
ation, 18 min) within a range of about 6.5 h 
(from 9:30 to 16:00); snail abundance was de- 
termined as capture per unit effort (CPUE) in 
terms of specimens hour V Collections were 



made with simple meshed, standard samplers 
(Thiengo, 1995), locally named "copos" (1 mm 
mesh size, and 13.5 cm frame diameter). 
Thirty-one samples were obtained from the lit- 
toral zone of the habitats (0.5-1 .0 m deep). 

Collected material was carried alive to the 
laboratory and prepared for identification, dis- 
section of soft parts, counting and measuring. 
The planorbid specimens were relaxed in a 
Nembutal solution 0.01% for approximately 
24 h, according to the method described by 
Paraense (1986). Afterwards, they were sac- 
rificed by immersion in water at 65-70°C for 
45-55 sec, and submerged in fresh water 
equilibrated with air temperature. Finally, the 
prepared material was fixed in a Raillet-Henry 
solution (Paraense, 1976), to facilitate Its 
identification by dissection of the soft parts 
under a stereoscopic microscope. 

At each sampling site, physical and chemi- 
cal variables were monitored immediately be- 
fore the capture of gastropods. Temperature, 
pH and conductivity were measured using 
thermistors, electrode pH-meters, and battery 
conductivity-meters, respectively. In addition, 
water samples were taken with a peristaltic 
pump and transported refrigerated to the lab- 
oratory, where the following variables were 
measured: dissolved oxygen concentration 
(Winkler method), oxygen percentage satura- 
tion, nutrients (nitrites + nitrates, total soluble 
nitrogen, ammonium and total soluble reac- 
tive phosphorus), calcium, hardness, and al- 
kalinity. Chemical analyses where performed 
according to Standard Methods (1980) and 
Golterman et al. (1978). 

Regarding vegetation, the criterion of rela- 
tive dominance was employed, based on vi- 
sual inspection of the sampling area. Plants 
were coded between and 4 for the data ma- 
trix, according to the following scale: = ab- 
sent (0%); 1 = rare (1-25%); 2 = frequent 
(25%-50%); 3 = co-dominance (50-99%); 
and, 4 = complete dominance (100%). The 
surveyed macrophytes belonged to free-float- 
ing covers: Eich horn ia crass ¡pes (Mart.) 
Solms. (Pontederiaceae), Pistia stratlotes L. 
(Araceae), Splrodela intermedia W. Koch. 
(Lemnaceae), Salvinia herzogii de la Sota, 
and S. rotundifolia Willd. (Salvinaceae); set- 
tled with floating leaves: Victoria cruziana 
d'Orbigny (Ninfaceae); settled: Enhydra ana- 
gallis Gardn. (Compositae), Hydrocotyle ra- 
nunculoides L. (Umbeliferae), and Ludwigia 
peploides (И.В.К.) Нага (Onagraceae); emer- 
gent: Panicum elephantipes Nees (Grami- 
neae), Canna glauca L. (Cannaceae); and 



POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 



277 



unidentified cespitóse hydrophilic Gramineae 
(Table 3). 

Statistical Analyses 

Data on gastropod abundance and limno- 
logical variables were transformed to decimal 
logarithm (log(x+1)) to linearize relationships 
among the variables. The multivariate tech- 
nique chosen to explore the relationships be- 
tween environmental variables (independent 
variables) and snails taxocenosis (dependent 
variables) was canonical correspondence 
analysis (CCA) (ter Braak, 1986; ter Braak & 
Prentice, 1988). The program CANOCO 4 (ter 
Braak & Smilauer, 1998) was employed to 
perform the analyses, including adjustment of 
model parameters, goodness of fit tests, and 
plotting the results. This canonical ordination 
technique is a combination of ordination in re- 
duced space and multiple regression. It im- 
plies the representation of samples and 
species constrained by environmental vah- 
ables in the reduced space of orthogonal 
axes, which are in turn linear combinations of 
species relative abundance and environmen- 
tal gradients. This method assume both uni- 
modal (Gaussian) or linear relationships be- 
tween species and environmental variables, 
for long or short environmental gradients, re- 
spectively. It shows the major trends of varia- 
tion of multidimensional data within a reduced 
space of linearly independent canonical axes, 
and it is a useful tool to understand commu- 
nity composition in terms of species distribu- 
tion along different environmental gradients. 
Therefore, given the unimodal proprieties of 
CCA, linear relationships between a given en- 
vironmental variable and species are not re- 
quired, such as in most canonical multivariate 
techniques. 

The unimodal properties of the model allow 
the estimation of optimum and tolerances of 
the species, both indicators of niche attrib- 
utes. Optimum can be approximated by the 
CANOCO 4 output named "spaces-by-envi- 
ronmental table", which are weighted aver- 
ages of species with respect to standardized 
environmental variables. Results are repre- 
sented graphically in a reduced space of or- 
thogonal axes, displaying the distribution of 
species, samples, and/or environmental gra- 
dients in the same bidimentional space (bi- 
plots and triplets). Since triplots with species 
scorers and environmental scorers may be in 
error because they contain the main patterns 
only, this table is useful to verify that infer- 



ences drawn from the plots hold true in the ac- 
tual data on weighted averages (ter Braak & 
Smilauer, 1998). 

Among the 26 environmental variables sur- 
veyed, it was necessary to preselect those of 
higher contribution to the total variability in 
snail relative abundance in order to reduce 
the possibility of an artificial increase in the 
explained variance by environmental factors, 
as well as multicolinearity. Stepwise forward 
selection procedure was employed, using 
Monte Carlo permutation tests, as imple- 
mented in CANOCO 4. A critical rejection 
level of P < 0.10 was applied. The amount of 
variance explained by the whole model and 
the first ordination axes was calculated em- 
ploying Monte Carlo permutation tests, with a 
critical rejection level of P < 0.05. 

RESULTS 
Planorbid Species 

The following planorbid species were found 
(Table 1): Biomphalaria tenagophila (d'Or- 
bigny, 1835), B. straminea (Dunker, 1848), 
Drepanotrema anatinum {оЮгЫдпу, 1835), D. 
/uc/dum (Pfeiffer, 1839), D. kermatoides{ó'Or- 
bigny, 1835), and D. c/mex (Mohcand, 1937). 
In three samples, no planorbids were found; 
these were omitted from the subsequent sta- 
tistical analysis. 

Biomphalaria straminea occurred more fre- 
quently than B. tenagophila (48% and 35%, 
respectively), but the latter showed much 
higher abundances, especially in 1989. Distri- 
butional patterns in space were also different, 
with B. straminea being more common in 
Negro River and Golf Club Pond, whereas B. 
tenagophila was more abundant in Rural So- 
ciety and Paikin ponds. Both species are in- 
termediate hosts of schistosomiasis in Brazil. 

Species of Drepanotrema were much less 
frequent than Biomphalaria (10-16%), but 
abundances were highly variable in time and 
space, with values similar in order of magni- 
tude to those of B. straminea, and never 
reaching the high levels of B. tenagophila. 
Drepanotrema cimex and D. anatinum were 
found almost exclusively in Paikin Pond, a 
biotope where the other species of Drepa- 
notrema also showed the highest frequency. 
In the remaining environments, this genus 
was rarely found, with D. anatinun in Rural 
Society Pond (one presence), D. lucidum in 
Negro River (two presences), and D. kerma- 
toides in Golf Club Pond (two presences). 



278 



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POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 



279 



Environmental Variables 

For all sampling periods and sites, physical 
and chemical variables are shown in Table 2. 
Water temperature varied between 8°C in 
June 1988 and 32°C in February 1988. Aver- 
ages values were similar among sampling 
sites, being only 1-2°C higher in the Rural 
Society Pond and Golf Club Pond, respec- 
tively. These differences may be partly be- 
cause sampling hours were not the same for 
all biotopes. The pH ranged from 6.3 to 8.9 
(October 1989 and February 1988, respec- 
tively), being alkaline in most sampling dates 
in Rural Society Pond, and closer to neutral 
point in the others waterbodies. Specific con- 
ductivity showed marked spatial and temporal 
variations within a range of 90 and 1,600 |iS 
cm"^ (August 1988 and November 1988, re- 
spectively). The highest conductivities were 
measured in Golf Club Pond and the lowest in 
Negro River. Oxygen concentration [O2] and 
oxygen saturation percentage ("/oOg) were 
low or undetectable during the warmest peri- 
ods, increasing during winter and spring, with 
figures as high as 8.1 mg Г^ and 90% satura- 
tion. 

Calcium concentration [Ca""] was also 
highly variable (Table 2), ranging from 9.5 to 
119.4 mg 1"^ in Paikin Pond (July 1989 and 
February 1989, respectively). According to 
Dussart's (1976) classification of mollusk 
habitat regarding calcium concentration, the 
highest mollusk abundance generally corre- 
sponds to the hardest waters ([Ca""] > 40 mg 
r^), while the highest diversity is usually ob- 
served in mid-range waters ([Ca"^] between 
5-40 mg Г^). Sampled environments of 
Chaco generally had waters of medium hard- 
ness, although hard waters were observed on 
some occasions (Table 2). Calcium concen- 
tration was positively correlated with hard- 
ness (r = 0.677, n = 31, P < 0.01), a variable 
that ranged between 36 and 329 mg Г^ (June 
1989 and February 1989, respectively). The 
range of alkalinity due to [HCO"^] was 45-357 
mg r^ (October 1989 and August 1988, re- 
spectively). Rural Society Pond generally 
showed the highest levels of alkalinity, while 
Negro River had the lowest. 

Concerning nutrient concentrations (Table 
2), nitrates and nitrites [N-NO3 + N-NO2], were 
usually below 0.4 mg \~\ reaching exception- 
ally 9.6 mg r^ on November 1988 in Paikin 
Pond. Ammonium concentration [N-NH^"^] 
showed maximum levels in Paikin Pond dur- 



ing 1989, and minimum levels in Rural Soci- 
ety Pond most of the time. This nutrient also 
tended to increase in all waterbodies duhng 
the second sampling year. Total soluble nitro- 
gen (N-SOL) followed the same pattern of 
ammonium. Finally, phosphate concentration 
[P-PO4] was generally below 1.0 mg \~\ 
reaching exceptionally high levels in one sam- 
pling date in Paikin Pond and Rural Society 
Pond. In Negro River, phosphates had the 
lowest concentrations of all waterbodies, 
never being over 0.3 mg \~\ 

Relative abundances of aquatic plants 
are shown in Table 3. Among the most impor- 
tant emergent macrophytes, Panicum ele- 
phantipes was frequent and dominant in Golf 
Club Pond and Negro River, but absent in 
the other waterbodies. On the other hand. 
Canna glauca was always dominant in Paikin 
Pond, and completely absent from the other 
biotopes. Among other settled macrophytes, 
Enhydra anagallis was frequent and important 
in Paikin and Rural Society ponds, while Hy- 
drocotile ranunculoides was present only on 
some dates in Rural Society Pond and Paikin 
Pond. Of free-floating plants (Table 3), Pistia 
stratiotes was dominant in Rural Society Pond 
and during 1989 in Golf Club Pond. Eichhor- 
nia crassipes was completely absent from 
Negro River, and present in variable abun- 
dance and frequency in the remainder of the 
waterbodies. 

Other gastropods that were present with 
planorbids (Table 3), were Heleobias spp. (Hy- 
drobiidae), Stenophysa marmorata (Guilding, 
1828) (Physidae), Gundlachia moricandi 
(d'Orbigny, 1835) (Ancylidae), and species of 
the family Ampullahidae, mainly Pomacea 
canaliculata (Lamark, 1801) and P. scalaris 
(d'Orbigny, 1835). All the gastropods found 
with planorbids were eliminated in the step- 
wise foHA/ard selection of independent vari- 
ables. Pomacea canaliculata was the most 
frequent gastropod considering all samples. 
This species has also a wide distribution in del 
Plata Basin, especially in the Argentinean 
area. 

Canonical Correspondence Analysis 

Seven of the 26 environmental variables 
used in the canonical correspondence analy- 
sis (CCA, Table 4) were retained in the step- 
wise forward selection procedure. For all of the 
selected variables, the variance inflation factor 
(V.I.F.) was below 5, the level suggested for 



280 



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TABLE 4. Summary of major results of the canonical correspondence analysis relating planorbid species to 
the selected environmental variables. 

Canonical Axes I II III 

Eigenvalues 

Cumulative percentage of variance of species data 

Cumulative percentage variance of species-environment relation 

Species-environment correlation 

Correlation of the environmental variables with the axes 

P. elephantipes 

С glauca 

H. ranunculoides 

E. crassipes 

P. stratiotes 

[N-NH41 

%0o 



Total of unconstrained eigenvalues 
Total of canonical eigenvalues 



0.469 


0.401 


0.354 


23.4 


43.4 


61.0 


37.6 


69.8 


98.2 


0.840 


0.837 


0.838 


-0.543 


-0.346 


-0.190 


0.095 


0.684 


0.413 


-0.139 


0.461 


-0.036 


-0.221 


-0.006 


-0.068 


0.385 


-0.274 


-0.587 


0.408 


-0.133 


0.310 


-0.291 


0.268 


-0.237 



2.006 
1 ,247 (62% of explained variance) 



avoiding multicolinearity and over-explanation 
(ter Braak and Smilauer, 1998). Ammonium 
[N-NH^*] and percentage saturation of D.O. 
("oOj) were the water quality variables re- 
tained. The remaining environmental vari- 
ables were all macrophytes, including P. stra- 
tiotes, E. crassipes. H. ranunculoides, P. 
elephantipes. and С glauca. The selected en- 
vironmental variables explain 62% of the total 
vanation in planorbid species relative abun- 
dance. Moreover, the first three canonical 
axes account for 98% of the variance ex- 
plained by these variables. The species-envi- 
ronment relationship was highly significant, 
according to the Monte Carlo permutational 
test (F = 4.46; P < 0.001; 999 permutations), 
as well as the first canonical axis (F = 5.80; P 
< 0.01; 999 permutations). In three samples 
from Negro River and one from Rural Society 
Pond, planorbids were not found, being ex- 
cluded by the CCA program. 

The graphic representation of the species 
positions along the environmental gradients in 
the reduced space of the first three canonical 
axes, yielded the following results (Figs. 2, 3): 

(1) Axis I separates B. straminea and D. lu- 
cidum from B. tenagophila and D. kerma- 
toides. along an increasing gradient of 
[N-NH^"] and P. stratiotes, as well as a 
decreasing gradient of %02 and P. ele- 
phantipes. The remaining species of 
Drepanotrema occupy intermediate posi- 
tions along the first axis gradient. 

(2) Axis II separates D. lucidum, D. anatinum 
and D. cimex from the species of Biom- 



phalaria and D. kermatoides, mainly 
through a decreasing gradient of H. ra- 
nunculoides and С glauca, as well as an 
increasing gradient of P. elephantipes. 
(3) Finally, along axis III, species of Drepa- 
notrema appear more clearly distributed, 
with D. kermatoides on the top, D. anat- 
inum and D. c/mex in the middle, finishing 
with D. lucidum at the opposite end, close 
to B. straminea. Biomphalaria tenago- 
phila is placed on the opposite negative 
side of the planorbid distribution along 
the third axis. This axis is mainly related 
to the independent variables С glauca 
and [N-NH^''] (positively) and P. stratiotes 
(negatively). 

In addition, employing the environment-by- 
species table provided by the program 
CANOCO 4 (Table 5), the position of the opti- 
mum of planorbid species along gradients of 
environmental variables can be better ana- 
lyzed, independently of their correlations with 
canonical axes. With this alternative analytical 
approach, the following results were obtained: 

(1 ) Canna glauca was the most important in- 
dependent variable in the stepwise for- 
ward selection. All species of Drepan- 
otrema appear on the positive side of the 
gradient, with D. anatinum and D. cimex 
placed at the edge. Biomphalaria species 
were on the negative side, with B. 
straminea at the outermost end of the 
gradient. 

(2) Pistia stratiotes, the second most im- 



POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 



283 



CO 
X 

< 

_J 

< 
о 



< 
ü 



Dre lue 




Pan ele 



■1.5 



CANONICAL AXIS I 



+2.0 



FIG. 2. Spatial representation of the samples, species and environnnental variables in the space defined by 
the two first canonical axes. Circles: Paikin pond. Diamonds: Rural Society Pond. Squares: Negro River. Tri- 
angles: Golf Club Pond. Empty symbols: 1 988. Filled symbols: 1 989. Arrows: environmental variables. Stars: 
planorbid species. Bio str: Biomphalaria straminea; Bio ten: B. tenagophila; Dre ana: Drepanotrema ana- 
tinum; Dre cim: D. cimex; D. ker: D. i<ermatoides; Dre lue: D. lucidum; Can gla: Canna glauca: Eic era: Eich- 
hornia crassipes; Hyd ran: Hydrocolyle ranunculoides; Pan ele: Panieum elephantipes and Pis str: Pistia 
stratiotes. 



portant independent variable, had B. 
tenagophila at the positive end of the dis- 
tribution, whereas all species of Dre- 
panotrema were on the negative side. 
Biomphalaria straminea was placed 
close to the center of the distribution. 

(3) Along the gradient of [N-NH/], both 
species of Biomphalaria were clearly 
segregated, with B. tenagophila having 
its optimum at higher concentrations, to- 
gether with D. kermatoides. Moreover, B. 
straminea was placed on the opposite 
end, close to D. lucidum, while D. cimex 
occupied the center of the distribution. 

(4) With respect to "/oOj, planorbid species 
responded inversely as [N-NH/]. 

(5) Dense stands of P. elephantipes sup- 
ported the highest abundances of B. 
straminea, together with D. lucidum. 



(6) Hydrocotile ranunculoides had a ten- 
dency to house populations of D. ana- 
tinum, D. lucidum and D. cimex prefer- 
ably, while Biomphalaha species and D. 
kermatoides were at the negative side of 
the gradient. 

(7) Finally, along the E. crassipes gradient, 
B. straminea and B. anatinum were on 
the positive side, D. cimex, close to the 
center, and the remainder on the nega- 
tive side. 

With respect to sample distribution in the re- 
duced space of the first three axes, four 
groups were observed, each one character- 
ized by a kind of vegetation and a particular 
planorbid assemblage (Figs. 2, 3): 

(1) Samples in Paikin in 1988, characterized 
mainly by C. glauca and H. ranuncu- 



284 



RUMI ETAL 



+ 



Ж 



Dre ker 



Can gla 



N-NH4 




-1.5 



CANONICAL AXIS I 



+2.0 



FIG. 3. Spatial representation of the samples, species and environmental variables in the space defined by 
canonical axes I and III. Circles: Paikin oxbow lake. Diamonds: Rural Society Pond. Squares: Negro River. 
Triangles: Golf Club Pond. Empty symbols: 1988. Filled symbols: 1989. Arrows: environmental variables. 
Stars: planorbid species. Bio str: Biomphalaria straminea: Bio ten: B. tenagophila: Dre ana: Drepanotrema 
anatinum: Dre cim: D. cimex: D. I<er: D. l<ermatoides: Dre lue: D. lucidum: Can gla: Canna glauca: Ele era: 
Eichhornia crassipes: Pan ele: Panicum elephantipes and Pis str: Pistia stratiotes. 



loides. The most common species were 
D. anatinum. D. lucidum and D. cimex. 
The genus Biomphalaria was rare. 

(2) Samples from Rural Society Pond and 
from Paikin Pond in 1989, characterized 
by the presence of P. stratiotes and the 
highest concentrations of ammonium, 
with B. tenagophila and D. kermatoides 
as the most common planorbid species. 

(3) Samples from Rural Society Pond in 
1988, with low-density stands of E. cras- 



sipes, and the presence of B. straminea 
and B. tenagophila also in low densities. 
(4) Samples from Negro River and Golf Club 
Pond, characterized by the presence of 
P. elephantipes, and the highest concen- 
trations of %0^. Biomphalaria straminea 
was the most typical species, while the 
other planorbids were negatively related 
to this kind of vegetation. Within this 
group, two separated clusters of samples 
can be observed, one of them more 



POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 



285 



TABLE 5. Environment-by-species table showing weighted means and standard deviations of environmen- 
tal factors, weighted averages of planorbid species with respect to the seven standardized variables 
("optima"), and back-transformed data to original format as in Tables 2 and 3 (in parentheses). Note that for 
limnological variables, original data were transformed to decimal logarithm. 







Stand 


B. 


B. 


D. 


D. 


D. 


D. 




Mean 


dev. 


tenagophila 


straminea 


anatinum 


lucidum 


kermatoides 


cimex 


C. glauca 


1.460 


1.881 


-0.288 


-0.584 


1.163 


0.536 


0.426 


0.839 








(0.9) 


(0.4) 


(3.6) 


(2.5) 


(2.3) 


(3.0) 


H. ranunculoides 


0.393 


0.930 


-0.196 


-0.158 


0.772 


0.622 


-0.423 


0.3826 








(0.2) 


(0.2) 


(1.1) 


(1.0) 


(0.0) 


(0.7) 


P. elephantipes 


0.884 


1.648 


-0.434 


0.688 


-0.536 


0.393 


0.009 


-0.5361 








(0.2) 


(2.0) 


(0.0) 


(1.5) 


(0.9) 


(0.0) 


E. crassipes 


1.055 


1.558 


-0.185 


0.274 


0.203 


-0.112 


-0.301 


-0.0004 








(0.8) 


(1.5) 


(1.4) 


(0.9) 


(0.6) 


(1.1) 


P. stratiotes 


1.325 


1.775 


0.726 


-0.068 


-0.646 


-0.745 


-0.745 


-0.2038 








(2.6) 


(1.2) 


(0.2) 


(0.0) 


(0.0) 


(1.0) 


[N-NH41 


0.238 


0.276 


0.211 


-0.328 


-0.136 


-0.709 


0.884 


0.0696 








(0.977) 


(0.403) 


(0.586) 


(0.102) 


(2.034) 


(0.807) 


%02 


1.534 


0.493 


-0.193 


0.158 


0.435 


0.499 


-0.789 


0.2062 








(26.5) 


(39.9) 


(55.0) 


(59.2) 


(13.0) 


(42.2) 



clumped, belonging to 1988, and the 
other more dispersed, from 1989. 

These results indicate that there is not a 
clearly defined seasonal variation in the struc- 
ture of planorbid assemblages. The most im- 
portant patterns were spatial or inter-annual, 
as is shown in the triplet of axes I and II (Fig. 
2). There is a defined tendency of most sam- 
ples from all waterbodies to move in a top-left 
to bottom-right direction from 1988 to 1989, 
following a gradient of increasing ammonium 
concentration and decreasing oxygen satura- 
tion. 

DISCUSSION 

The empirical model developed in this paper 
explains a relatively high percentage of ob- 
served variance compared with other commu- 
nity studies (Borcard et al., 1 992), and is in ad- 
dition highly significant. As a general pattern, 
the multivariate model shows that Biom- 
phalaria straminea tends to be relatively more 
important in well-oxygenated environments 
with reophilic vegetation, such as Panicum 
elephantipes. Junk (1973) also found that this 
planorbid species was abundant in the roots of 
"floating meadows" {Paspalum repens Berg, 
and Eciiinociiloa polystachya (H.B.K.)) in 
várzea lakes of the Amazon River. Conversely, 
Biompfialaria tenagopliila, the other potential 
transmitter of schistosomiasis, is more com- 



mon in less oxygenated waters and higher am- 
monium concentrations, conditions that, to- 
gether with pH over 8 and temperatures higher 
than 25°C, may be extremely toxic for aquatic 
animals due to the high proportion of ammonia 
(NH3) (Boyd, 1990). These particular limno- 
logical features are generally associated with 
continuous floating covers of Pistia stratiotes, 
with roots that would function as substratum 
and refuge for B. tenagopliila. Drepanotrema 
kermatoides io\\o\NS a similar pattern to that of 
B. tenagophila, while the association of B. 
straminea with species of Drepanotrema was 
unclear, except for D. lucidum. This latter 
species was also found by Junk (1973) occu- 
pying the same habitat as B. straminea. In this 
sense, the dominance of Canna glauca and 
Hydrocotyle ranunculoides also indicates the 
dominance of Drepanotrema species, as well 
as lower densities of Biomphalaria species, 
particularly B. straminea. 

Among the environmental factors taken into 
account in the CCA, the regulatory effect of 
water temperature on population dynamics 
was already known, as was the importance of 
calcium concentration related to pH, hardness 
and macrophytes, on the distribution and 
abundance of mollusks (Dussart, 1976; Pigott 
& Dussart, 1995; Thomas, 1982; Rumi & 
Hamann, 1992). However, only ammonium 
concentration and percentage of oxygen sat- 
uration were retained as significant limnologi- 
cal variables in the process of forward selec- 



286 



RUMI ETAL 



tion, and they were negatively correlated with 
each other. Biomphalaria tenagophila and D. 
kermatoides were distributed along this lim- 
nological gradient, mainly at the highest am- 
monium concentrations, with optima at 0.98 
and 2.00 mg Г\ respectively, as well as the 
lowest oxygen saturation, with optima at 
26.6% and 13.0°o, respectively. These results 
suggest that the variance explained by the ex- 
cluded variables was either unimportant to be 
selected in the stepwise procedure, or that 
this variation was better explained by oxygen 
and ammonium, or even aquatic plants, which 
were retained first by the statistical method 
employed, due to their larger explained vari- 
ance. Therefore, these results do not neces- 
sarily mean that variables not retained were 
not relevant for planorbids. 

The lack of clear seasonal trends in planor- 
bid communities may be explained because 
the environments in which this study was car- 
ried out are of permanent waters, unlike oth- 
ers of tropical areas, with well-defined sea- 
sonality in rainfall. These observations agree 
with Rumi & Hamann (1992), who compared, 
in another permanent pond of eastern Chaco 
in Corrientes Province, rainfall pattern with 
the relative abundance of Biomphalaria occi- 
dentalis Paraense, 1981, finding also a very 
low correlation between those variables. 
However, in this region of Chaco, inter-annual 
variability in water availability can be very im- 
portant, particularly the surplus after evapora- 
tion, soil infiltration and scouring (Bruniard, 
1997). Since environmental variables as well 
as planorbid population dynamics are related 
to climatic changes, large variations in planor- 
bid composition and abundance in samples of 
the same biotopes from one year to the next 
may be due to different rainy and drought pe- 
riods. For example, different amounts of pre- 
cipitation can affect total biotope area, pro- 
ducing an increase in planorbid density during 
dry periods due to retraction in pond area, or 
an opposite decrease during wet periods due 
to dilution effects. Furthermore, although 
ponds are physically isolated most of the time, 
extensive superficial interconnections among 
wetlands always take place during rainy 
episodes or river floods. Thus, migration 
events in snail populations would be expected 
at those times. These two processes could 
provoke rapid changes in abundance, and 
could partially explain the marked seasonal 
modifications in snail capture observed in the 
present paper. 



Until not long ago, the urban ponds were 
employed for rubbish deposits, which affected 
the water quality and planorbid population 
size, especially during dry climatic events. In 
addition, populations of B. straminea were 
much more abundant during the first year, 
while B. tenagopliila showed an increase in 
population densities during the second year. 

The non-explained variability in planorbid 
relative abundance could be attributed to a 
rather extensive list of possibilities, including 
the colonization patterns in space and time, 
sampling biases, and environmental variables 
not included (i.e., fish and invertebrate preda- 
tors). First, given the limited capacity of 
planorbids to disperse and the time required 
for settled populations to grow, it is possible 
that even with suitable environmental condi- 
tions, populations could not reach maximum 
or stable densities within the sampling period. 
Second, the observed variation range of envi- 
ronmental variables and of planorbid assem- 
blages could be related to the specific envi- 
ronments selected for sampling, as well as 
the sampling technique employed. Thus, in- 
cluding more biotopes and making a greater 
sampling effort could have resulted in a larger 
explained variance or in a somewhat different 
distributional pattern. Finally, the role of 
predators as controls of planorbid populations 
cannot be ignored, given that eastern Chaco 
environments usually hold an abundant and 
diverse fish fauna (Menni et. al., 1992), with 
large proportions of invertebrate-eating, In- 
cluding mollusc-eating, species, such as 
small to medium-sized siluhds, characins, cl- 
chlids and gymnotids. Therefore, given similar 
environmental conditions, the presence or ab- 
sence of predatory fishes could result in dif- 
ferent planorbid densities and species com- 
position. 

From a sanitary standpoint, it is remarkable 
that the species B. tenagopliila and B. 
straminea, living in urban environments in the 
Chaco, are natural transmitters of schistoso- 
miasis in Brazil. Because the presence and 
abundance of these two planorbid species is 
explained chiefly by some species of aquatic 
macrophytes, these variables may be em- 
ployed as indicators for a rapid assessment of 
potential sites of proliferation of B. tenago- 
phila and B. straminea. However, these re- 
sults must be supplemented with new sam- 
plings, as well as experimental field and 
laboratory tests, to confirm the observed 
trends. 



POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 



287 



ACKNOWLEDGEMENTS 

This study was made possible by a grant 
supplied by the Consejo Nacional de Investi- 
gaciones Científicas y Técnicas (CONICET) 
(AGENCIA PICT01 -03453), in Argentina. 
Fieldwork and water analyses were facilitated 
by the logistic support of the Centro de 
Ecología Aplicada del Litoral (CECOAL). Lie. 
Cecilia Longoni de Meabe supervised water 
quality analyses. We also thank Mr. Luis 
Benetti by his valuable collaboration in field- 
work. 

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Revised ms. accepted 22 December 2001 



MALACOLOGIA, 2002, 44(2): 289-305 



EGG MASSES OF CHROMODORID NUDIBRANCHS 
(MOLLUSCA: GASTROPODA: OPISTHOBRANCHIA) 

Nerida G. Wilson 

Centre for Marine Studies, University of Queensland, St Lucia 4072, Queensland, Australia; 

nwilson ©marine. uq.edu.au 

ABSTRACT 

The egg mass characteristics of 20 species of chromodorid nudibranchs are presented, rep- 
resenting the genera Chromodoris. Digidentis, Diversldoris, Glossodoris, Hypselodoris, Noumea 
and Pectenodoris. The egg mass details for 14 of the included species appear previously un- 
recorded. These results combined with observations from the literature (comprising a total of 67 
species from 15 genera) indicate that most genera in the Chromodorididae show only one type 
of egg mass. The exception to this is the genus Ctiromodoris, which includes all three egg mass 
types outlined in this study. Based on anatomical evidence, a group of flat-spawning Chro- 
modoris spec\es has been suggested to belong to a monophyletic clade, which indicates that egg 
mass structure can reflect phylogenetic signal. Genera considered to be more derived among 
the Chromodorididae are more likely to lay an egg mass that is outward sloping or crenulated. 
Cadlinella shows spawning traits (flat egg mass containing extra-capsular yolk) that indicate it 
may be ancestral to the Chromodoris lineage. Developmental data is presented for 11 species 
of Chromodoris. seven of which utilized extra-capsular yolk in their egg masses. The presence 
of extra-capsular yolk appears to correlate with upright egg masses within this genus. However, 
the occurrence of extra-capsular yolk appears to be restricted to Indo-Pacific and Red Sea Chro- 
modoris species, while upright spawns from Caribbean species lack this feature. 

Key words: nudibranch, egg mass, chromodorid, reproduction, extra-capsular yolk 



INTRODUCTION 

All doridinean nudibranch egg masses, in- 
cluding those of chromodorids, take the form 
of a spiral ribbon that is attached to the sub- 
stratum and consists of embryos embedded in 
a gelatinous matrix. The shape of the egg 
mass has been considered to a certain extent 
to characterize particular groupings (Eliot, 
1910; Ostergaard, 1960; Hurst, 1967; Bändel, 
1976; Fernandez-Ovies, 1981; Soliman, 
1987), and some authors have equated the 
taxonomic value of the structure of egg cap- 
sules (and associated coverings) in shelled 
gastropods with shell, radular and opercular 
characters (Andrews, 1935). In a recent 
cladistic analysis, Mikkelsen (1996) confirmed 
that the opisthobranch order Anaspidea had a 
particular egg mass type (string) common to 
all investigated members of the group. 

An extensive literature exists on the 
anatomy and systematics of chromodorids. 
However, comparatively little is known about 
chromodorid reproduction aside from the mor- 
phology of the reproductive system itself 
(Rudman, 1984). In particular, very little is 



known about the structure of the chromodorid 
egg mass in species occurring in Australian 
waters, apart from a few brief descriptions, il- 
lustrations or unpublished theses (Kenny, 
1970; Thompson, 1972; Rose, 1981, 1985; 
Avern, 1986; Marshall & Willan, 1999). Often 
descriptions of chromodorid egg masses are 
brief and include such ambiguous expres- 
sions as "tangled coils" or "loosely coiled" 
(MacGinitie & MacGinitie, 1949; Boucher, 
1983; Johnson & Boucher, 1983). This gives 
no indication as to whether these terms per- 
tain to the distance between the coils, the reg- 
ularity of coiling, or to the actual attachment of 
individual whorls of the spiral mass to the sub- 
stratum. Numerous papers from Japan have 
significantly improved our understanding of 
opisthobranch egg masses (Baba & Hama- 
tani, 1961; Hamatani, 1960a, b, 1961, 1962). 
However, only one account described chro- 
modorid nudibranch egg masses (Baba et al., 
1956). Other studies may inadequately iden- 
tify the parent species or give conflicting data, 
thus rendering the resulting egg mass infor- 
mation less useful (Bändel, 1976; Barash & 
Zanziper, 1980; Rose, 1985). However, many 



289 



290 



WILSON 



photographic nudibranch identification guides 
have helped highlight substantial diversity in 
nudibranch egg mass structure (Gosliner, 
1987; Coleman, 1989: Behrens, 1991; Wells 
& Bryce, 1993; Debelius, 1998). More re- 
cently, websites have also contributed signifi- 
cantly to our knowledge of egg masses (for 
example, www.seaslugforum.net). 

Opisthobranch egg masses were classified 
according to a scheme introduced by Hurst 
(1967). This scheme aimed to describe egg 
masses in a format that would facilitate com- 
panson between taxa. As it was based on 
opisthobranch taxa from the cold temperate 
northwest coast of the USA, chromodorids 
were not included, although nearly all egg 
mass types laid by chromodorids were repre- 
sented in the scheme by other dorids. Hurst's 
scheme consists of four categories, which 
show some taxonomic correlations, for exam- 
ple. Type С (jelly bag attached by string) com- 
mon amongst cephalaspideans and Type D 
(sac-like structure) typical of very small aeolid 
nudibranchs. Doridinean spawns were only 
present in one category that Hurst defined as 
Type A; 

"The egg mass is in the form of a ribbon at- 
tached along the length of one edge, capsules 
occurring throughout most of it. This is com- 
mon amongst dorids, which whilst laying may 
grip the mass between foot and mantle edge 
tending to flatten it, as mentioned by Fretter & 
Graham (1964). This is probably not the sole 
cause of the flattened shape" (Hurst, 1967: 
256). 

Hurst (1967) noted differences in relative 
lengths of the free edge compared to the at- 
tached edge of the masses, but did not incor- 
porate this variation in her classification. Con- 
sequently, any descriptions of doridinean 
spawn masses that employ Hurst's categories 
define the ribbon as simply being upright. This 
does not provide enough information to make 
any comparisons at or below the familial level. 
Bändel (1976) briefly discussed Hurst's 
scheme and proposed 12 of his own group- 
ings. Many of these groupings were based on 
a single opisthobranch species, and all dorid- 
inean egg masses remained in one group, pro- 
viding no improvement over Hurst's original 
scheme. Some subcategories were added to 
Hurst's classification by Fernandez-Ovies 
(1981), who recognised that the scheme did 
not adequately describe variation within each 
'type'. While these subcategories were a sig- 
nificant improvement, they still did not account 
for all the variation caused by differences in the 



length of the free edge of upright egg masses 
and did not account for flat egg masses. In 
fact, Fernandez-Ovies incorrectly listed Chro- 
modoris orientalls {as Glossodoris pallescens) 
laying an upright egg mass, whereas it is actu- 
ally laid flat (Baba et al., 1956). MacGinitie & 
MacGinitie (1949) report that some dorids lay 
flat egg masses, but it is not until much later 
that this shape is formally recognised in a clas- 
sification (Soliman, 1987). 

An unusual feature of egg masses that 
often goes unnoted is the existence of yolk 
reserves external to the capsule, but still con- 
tained within the egg mass. Boucher (1983) 
termed this "extra-capsular yolk" in a paper 
that reported the phenomenon in the saco- 
glossan genera Elysia Risso (Elysiidae) and 
BoseIHa Trínchese (Polybranchiidae) and the 
chromodorid genera Chromodons Alder & 
Hancock and Cadlinella Thiele (Chromodori- 
didae). Risbec (1928) erroneously interpreted 
extra-capsular yolk in the egg masses of Cad- 
linella ornatissima Risbec as crustacean ova. 
A few brief descriptions or illustrations have 
highlighted the presence of extra-capsular de- 
posits in the egg masses of opisthobranchs 
(Gohar & Aboul-Ela, 1957; Marcus & Burch, 
1 965; Gohar & Soliman, 1 967a; Kay & Young, 
1969), but Thompson (1972) appears to be 
the first author to formally recognize these 
bodies as being composed of "yolky material". 
The first and only paper to call attention to the 
widespread occurrence of extra-capsular yolk 
resulted from a taxonomic survey of opistho- 
branchs in the Marshall Islands (Boucher, 
1983). Since that study, no other literature has 
specifically addressed the subject of extra- 
capsular yolk in nudibranch egg masses. The 
actual deposition of extra-capsular yolk varies 
between major taxa, and may take the form of 
granules, caps or blobs in chromodorid nudi- 
branchs (Boucher, 1983) or yolk strings in 
sacoglossans (Clark et al., 1979; Clark & 
Jensen, 1981; Boucher, 1983). Goddard 
(1991) renamed the phenomenon "extrazy- 
gotic" and "extra-embryonic" instead of "extra- 
capsular" in order to include his observation of 
extra yolky polar bodies inside aeolid nudi- 
branch capsules. It is unlikely that all of these 
extra yolk sources are homologous. 

Although most nudibranchs spawn readily 
in captivity, perhaps in response to capture 
and handling stress (Hadfield & Switzer-Dun- 
lap, 1 984), there are few detailed descriptions 
of egg masses. The aims of the present study 
are to increase awareness of structural varia- 
tion in chromodorid nudibranch egg masses, 



CHROMODORID EGG MASSES 



291 



and to highlight the presence of extra-capsu- 
lar yolk in certain taxa. Knowledge of egg 
mass types and extra-capsular yolk may pro- 
vide new data to help evaluate existing phylo- 
genies. 

MATERIALS AND METHODS 

Egg Mass Collection, 
Maintenance and Measurement 

A total of 20 species were collected for this 
study from the east coast of Australia during 
1998-2001. The austral summer is repre- 
sented by the months December, January, 
February; autumn by March, April, May; win- 
ter by June, July, August, and spring by Sep- 
tember, October, and November. The majority 
of specimens were collected using SCUBA, 
although some material was collected in the 
intertidal zone. Animals were kept in holding 
tanks with gentle aeration and flowing seawa- 
ter. There is some evidence to suggest that 
egg masses produced after a prolonged pe- 
riod of stress can be atypical of the species, 
that is, when organ systems begin to deterio- 
rate (J. Havenhand, pers. comm.). To mini- 
mize the likelihood of this effect, only egg 
masses that were produced in the first week 
after capture were used in the study. A portion 
of each egg mass was excised with a scalpel 
and preserved in either glutaraldehyde (3% 
prepared in 0.1 M sodium phosphate buffer 
containing 10% w/v sucrose) or neutral- 
buffered formalin (10%). The remainder of 
each egg mass was maintained in a holding 
tank at a water temperature equivalent to that 
of the collection locality. Portions of the egg 
mass were observed under a compound mi- 
croscope at least every second day, and more 
frequently closer to hatching. The develop- 
mental period was deemed to cease on the 
first day that hatching was observed. Un- 
cleaved ova were measured either from black 
and white photographs taken using an Olym- 
pus BH-2 Nomarski contrast compound mi- 
croscope fitted with an Olympus 0M-2N cam- 
era attachment and Kodak T-max 100 ASA 
film, or from heat images generated from a 
National FIO CCD Video attachment to the 
above microscope, printed on a Sony video 
graphic printer UP-811. To increase the sam- 
ple size for comparisons at generic levels, de- 
scriptions and photographs from the literature 
were included. However, any data that con- 
tained ambiguous terminology or was difficult 
to interpret was excluded. 



Definition of Egg Mass Types 

The egg masses examined in this study 
were classified into three types (Fig. 1) based 
on whether or not they are attached flat on 
the substratum or whether they are attached 
along one edge. Type A egg masses are at- 
tached to the substratum by the broad side 
of the ribbon, and are therefore flat. Type В 
egg masses had a free edge that was either 
shorter than the attached edge, causing the 
ribbon to slope toward the centre of the 
spiral or was equal in length to the attached 
edge, standing upright. Type С egg masses 
had a free edge that was either slightly 
longer than the attached edge, causing the 
ribbon to slope away from the centre of 
the spiral or much longer than the attached 
edge, causing undulations or waves along 
the ribbon in addition to an outward slope. 
In some egg masses, the free edge was so 
long that the ribbon showed tight crenula- 
tions. 

Extra-Capsular Yolk Categories 

Boucher (1983) recognised two categories 
of extra-capsular yolk in nudibranchs and 
these are applied here with some modifica- 
tions. Boucher (1983) defined Type 1 as "cap- 
like bodies associated with individual cap- 
sules". In this study, caps falling into this 
category were further subdivided into (A) 
caps that were distributed equally and (B) 
caps that were distributed unequally (Fig. 2). 
Type 2, as defined by Boucher (1983), was 
"discrete yolk bodies strewn throughout the 
egg mass", and were not observed during this 
study, although are known to occur within the 
genera Cadlinella and Chromodoris (Risbec, 
1928; Gohar & Aboul-Ela, 1957; Boucher, 
1983). 

Developmental Type Definitions 

Where possible, larvae were examined by 
light microscope (Olympus BH-2 Nomarski 
contrast compound microscope) and catego- 
rized into developmental types according to 
the definitions proposed by Thompson (1967) 
and Bonar (1978). A well-developed velum 
and larval retractor muscle, small ova and 
short embryonic period identified plank- 
totrophic larvae, whereas lecithotrophic lar- 
vae typically exhibited a less developed but 
recognisable velum and larval retractor mus- 
cle, prominent propodium, and large ova with 



292 



WILSON 








FIG. 1 . Stylized drawing of the three types of chromodorid egg masses. Type A is laid flat on the substratum, 
Type В is laid upright and may also slope inwards, and Type С is laid upright, slopes outwards and may be 
crenulated. 





A ^^ _ в 

FIG. 2. Extra-capsular yolk. A, equally distributed. B, unequally distributed. Scale bar = 100 pm. 



a longer embryonic period (Thonnpson, 1967). 
Intracapsular development was determined 
to be either metamorphic (undergoing capslar 
metamorphosis) or ametamorphic (not under- 
going a typical veliger stage) (Bonar, 1978). 



RESULTS 

The developmental characteristics of 
twenty chromodorid species are described 
below and summarized in Table 1 . 



CHROMODORID EGG MASSES 



293 






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Chromodorididae 

Chromodohs A\der & Hancock 

Chromodoris collingwoodi Ruäman. 1987 

A single egg mass was observed in March 
from an animal collected at North Stradbroke 
Island, Queensland. The egg mass was up- 
right, consisting of two whorls slightly sloping 
inward to centre of spiral. Ova were yellow, 
and associated with extracapsular yolk of Type 
1A. Each capsule contained a single embryo. 
The embryonic period was six days at 23''C. 
Larvae were planktonic, although the exact 
developmental type was not determined. 

Chromodoris daphne {Angas, 1864) 

A single egg mass was observed in No- 
vember from an animal collected at Welling- 
ton Point, Moretón Bay, Queensland. The egg 
mass was upright, with the broad side of rib- 
bon constricted slightly toward centre of spi- 
ral, giving it a slightly "hour-glass" shape. Two 
whorls were present, containing cream ova 
arranged linearly within the spawn mass. Un- 
cleaved ova were 128 ± 5 цт in diameter 
(n = 8). Extra-capsular yolk of Type 1A was 
present, and each capsule contained a single 
embryo. The embryonic period lasted six days 
at 25-26°C, and the veligers that were re- 
leased were planktotrophic. 

Chromodoris elisabethina Bergh, 1877 

Four egg masses observed from three indi- 
viduals. The adult nudibranchs were all col- 
lected from the Gneehng Shoals, Mooloolaba, 
Queensland, and the egg masses laid in Jan- 
uary and May. All egg masses were laid flat, 
and while all were spiral in shape, they often 
ended askew. The egg masses ranged from 
three to five whorls. Ova were cream and 
there was no extra-capsular yolk observed. 
The rate of deposition of the egg mass for one 
individual was measured at 1 .58 mm/min. Un- 
cleaved ova were 93 ± 5 .um in diameter (n = 
15). Each capsule contained a single embryo. 
The embryonic period lasted 5-6 days at ap- 
proximately 27^0 (n = 1) and 8-10 days at 
20.5-22°C (n = 3). Larvae were planktonic, al- 
though the exact developmental type was not 
determined. 

Chromodoris geométrica Risbec, 1928 

Two incomplete egg masses were ob- 
served in May from two individuals collected 



at the Gneering Shoals, Mooloolaba, Queens- 
land. Egg masses were upnght and consisted 
of only a partial whorl (animals tried to lay their 
spawn on the air-water interface, which could 
not sustain the weight of more than a partial 
egg mass). Ova were cream and extra-cap- 
sular yolk Type IB was present in the egg 
mass. The ova were 82 ± 7 цт (n = 8) in di- 
ameter. Each capsule contained a single em- 
bryo. The embryonic period of one of the 
pieces lasted 10 days at 20.5-22°C. Larvae 
were planktonic, although the exact develop- 
mental type was not determined. 

Chromodons l<uiîeh Rudman, 1 982 

Three egg masses were observed from in- 
dividuals from Cook Island, New South Wales 
(n = 1), and Heron Island, Great Barrier Reef, 
Queensland (n = 2). These were laid in De- 
cember and March respectively. Egg masses 
were laid flat, and consisted of 2-5 whorls. 
The regularity of the coiling varied greatly be- 
tween egg masses laid by different individu- 
als. The ova were pale orange in colour, 
arranged linearly, and no extra-capsular yolk 
was observed. Each capsule contained a sin- 
gle embryo. The embryonic pehod lasted six 
days at 27°C, and also six days at a variable 
temperature range of 23-28°C. Larvae were 
planktonic, although the exact developmental 
type was not determined. 

Chromodoris /can/e/ Pruvot-Fol, 1930 

A single egg mass was observed in August 
from an individual collected from Heron Is- 
land, Great Barrier Reef, Queensland. The 
egg mass was upright, consisting of two 
whorls. The ova were orange and were asso- 
ciated with extra-capsular yolk Type IB. The 
uncleaved ova were 109 ± 2 )jm (n = 7) in di- 
ameter. Each capsule contained two em- 
bryos. The embryonic period was 15 days at 
20-22''C. Larvae were planktonic, although 
the exact developmental type remains unde- 
termined. 

Chromodoris leopardus Rudman, 1987 

A single egg mass was observed in Sep- 
tember from an individual collected in the 
Gneering Shoals, Mooloolaba, Queensland. 
The egg mass was upright, consisting of two 
whorls. The ova were orange and were asso- 
ciated with extra-capsular yolk Type IB. The 
uncleaved ova were 104 ± 3 lam in diameter 



CHROMODORID EGG MASSES 



295 



(n = 10). Each capsule contained one or two 
embryos. The embryonic period lasted 14 
days at 20-22°C. Larvae were planktonic, al- 
though the exact developmental type was not 
determined. 

Chromodoris lochi Rudman, 1 982 

A single egg mass was observed in April 
from a specimen collected at Ribbon Reef 
Number 10, Great Barrier Reef, Queensland. 
The egg mass was laid flat and consisted of 
three whorls. The ova were cream in colour, 
and no extra-capsular yolk was present. Each 
capsule contained a single embryo. The em- 
bryonic period lasted nine days at 25°C, and 
larvae showed a planktotrophic developmen- 
tal pattern. 

Chromodoris robo/ Gosliner & Behrens, 1998 

Two egg masses have been observed from 
two individuals collected from Heron Island, 
and another egg mass was observed from the 
Whitsunday Islands, Great Barrier Reef, 
Queensland. The egg masses from Heron Is- 
land were laid in March and September, and 
were upright and consisted of two whorls. The 
ova were orange and were associated with 
extra-capsular yolk of Type 1 B. The ova were 
101 ± 8 |im in diameter (n = 10). Many cap- 
sules contained multiple embryos; typically 
they contained two, but up to four were ob- 
served. The embryonic period of one egg 
mass lasted 1 1 days at 20-22°C. Larvae were 
planktonic, although the exact developmental 
type remains undetermined. The egg mass 
observed in the Whitsundays was laid in Au- 
gust and consisted of one whorl. This egg 
mass was upright but laid in an irregular spi- 
ral, so that the whorl consisted of short, 
straight sections with a distinct kink that joined 
to another short, straight section. Thus, the 
egg mass appeared to be crenulated, but both 
the free edge and attached edge were equal 
in length. 

Chromodoris strigata Rudman, 1982 

A single egg mass was observed in Qctober 
from an animal collected on Heron Island, 
Great Barrier Reef, Queensland. The egg 
mass was flat, and consisted of five whorls. 
The ova were pale orange and no extra-cap- 
sular yolk was observed. The ova measured 
80 ± 2 |am in diameter (n = 11). Each capsule 
contained a single embryo. The embryonic 



period lasted eight days at 24°C. Larvae were 
planktonic, although the exact developmental 
type remains undetermined. 

Chromodoris tinctoria 
(Rüppell & Leuckart, 1828) 

A single egg mass was observed in March 
from North Stradbroke Island, Queensland. 
The egg mass was upright and consisted of 
three whorls. The orange ova were arranged 
linearly within the egg mass and were associ- 
ated with extra-capsular yolk of Type 1A. 
Each capsule contained two to three em- 
bryos. The embryos died after 11 days at 
22-24°C, and no further details of develop- 
ment could be ascertained. 

Digidentis Rudman 
Digidentisci. arbutus {Burn, 1961) 

A single egg mass was observed in Febru- 
ary from Pt Puer, Tasmania. The animal was 
disturbed while laying, so the egg mass was 
incomplete. However, the spawn was upright 
and quite firm. The ova were orange and no 
extra-capsular yolk was observed. The ova 
measured 491 ± 12 [im (n = 7) in diameter 
and each capsule contained a single embryo. 
The developmental type could not be deter- 
mined, although the large size of the ova po- 
tentially indicates direct development. 

Diversidohs Rudman 

Diversidoris aurantionodulosa 

Rudman, 1987 

Four egg masses were observed in April 
from Pt. Cartwright, Mooloolaba, Queensland. 
The egg masses were upright and consisted of 
one to two whorls each. Two egg masses laid 
in the laboratory sloped inwards very slightly 
while those laid in the field appeared typically 
upright. The ova were cream-white in colour, 
no extra-capsular yolk was observed and each 
capsule contained a single embryo. The em- 
bryonic period lasted 8 days at 25°C, with the 
larvae showing a lecithotrophic developmen- 
tal pattern. 

Glossodoris Ehren berg 
Glossodoris vespa Rudman, 1990 

A single egg mass was observed in May 
from an individual collected in the Gneering 
Shoals, Mooloolaba, Queensland. The egg 
mass was upright, very firm and consisted of 



296 



WILSON 



two whorls. The cream ova measured 300 ± 
1 9 urn (n = 1 0) and no extra-capsular yolk was 
observed. The embryonic period lasted for 56 
days at 17-22^C, and the development pat- 
tern was ametamorphic direct. 



hod took 9-11 days at 20-22°C (n = 2), and 
the resulting veligers were lecithotrophic. 



Hypselodoris zephyra 
Gosliner & Johnson, 1999 



Hypselodoris Stimpson 
Hypselodoris bullocki (Co\\\ng\NOOÓ, 1881) 

Two egg masses were observed from two 
individuals during November on Orpheus Is- 
land, Great Barrier Reef, Queensland. They 
were uphght but with the free edge of the egg 
mass sloping away from the centre of the spi- 
ral. Ova were yellow and no extra-capsular 
yolk was observed. Each capsule contained a 
single embryo, but the developmental type re- 
mains unknown. 



Hypselodoris obscura (Stimpson, 1855) 

Four egg masses were observed in total 
from three individuals. Three egg masses 
were observed from a pair of nudibranchs col- 
lected in April from Amity Point, North Strad- 
broke Island, Queensland, and one egg mass 
was observed in November from an individual 
collected from Wellington Point, Moretón Bay, 
Queensland. The egg masses consisted of 
2-5 whorls. All egg masses were upright but 
ranged from being slightly outward sloping to 
having a crenulated free edge. The ova were 
white, arranged linearly in the egg mass and 
no extra-capsular yolk was observed. The ova 
from one egg mass measured 104 ± 5 цт 
(n = 1 1 ). Each capsule contained a single em- 
bryo. The embryonic period lasted 9-10 days 
at 22 С (n = 2) and 4-5 days at 25-26°C (n = 
1 ). Veligers were planktonic, but the exact de- 
velopment type remains undetermined. 

Hypselodoris sp. Chromodoris geométrica 
Coleman, 1981: 32. Misidentified 

Four egg masses were observed in total 
from three individuals, all from the Gneering 
Shoals, Mooloolaba, Queensland. Three egg 
masses were laid in September and one in 
January. The egg masses ranged from 1 -3 
whorls and were upright with the free edge 
crenulated. Ova were dark orange and no 
extra-capsular yolk was observed. Ova were 
146 ± 4|im in diameter (n = 10). Each capsule 
contained a single embryo. The embryonic pe- 



Two egg masses were observed from a 
single animal in December from Cook Island, 
New South Wales. The egg masses ranged 
from 2-3 whorls and were upright with the 
free edge crenulated. Ova were white and the 
embryonic period took five days at 27°C. 
Veligers were planktonic, although exact de- 
velopmental type was not determined. 



Noumea Risbec 
Noumea norba Marcus & Marcus, 1970 

Four egg masses were observed from two 
individuals in May, from the Gneering Shoals, 
Mooloolaba, Queensland. The egg masses 
ranged from 2-3 whorls and ranged from up- 
right to having the free edge sloping toward 
the centre of the spiral. Ova were cream, 
arranged linearly and measured 83 ± 3 |дт in 
diameter (n = 12). Each capsule contained a 
single embryo. The embryonic period was 
12-14 days at 20.5-22°C (n = 2). Veligers 
were planktonic, although the exact develop- 
mental type was not determined. 



Pectenodoris Rudman 

Pectenodoris trilineata 

(Adams & Reeve, 1850) 

One egg mass was observed in August on 
Heron Island, Great Barrier Reef, Queens- 
land. The egg mass was firm, and consisted 
of one whorl. The ova were pale pink in colour 
and measured 205 ± 11 |am (n = 8). Each 
capsule contained a single embryo. Develop- 
mental details were not recorded. 



DISCUSSION 

Although only a small fraction of the spawn- 
ing details of chromodorid species is known, 
there is some evidence to suggest that egg 
mass morphology is consistent within genera 
and even groups of genera (Table 2). The ob- 
vious exception to this is the presence of mul- 
tiple egg mass types within Chromodoris, the 



CHROMODORID EGG MASSES 



297 



TABLE 2. Egg mass types of chromodorid species 



Species 



Egg Mass Type 
ABC 



Source 



Cadlina luteomarginata 
Cadlina modesta 
Cadlina pellucida 
Cadlinella ornatissima 
Cadlinella sp. 
Tyrinna nobilis 
Chromodoris aspersa 
Chromodoris africana 

Cliromodohs annulata 
Chromodoris aureopurpurea 
Ciiromodoris binza 
Ciiromodoris coi 
Ciiromodoris collingwoodi 
Chromodoris clenchi 
Chromodoris daphne 
Chromodoris elisabethina 
Chromodoris geométrica 
Chromodoris geométrica 
Chromodoris l<uniei 
Chromodoris l<uiteri 
Chromodoris leopardus 
Chromodoris lineolata 
Chromodoris lochi 
Chromodoris magnifica 
Chromodoris orientalis 
Chromodoris perola 
Chromodoris roboi 
Chromodoris strigata 
Chromodoris tinctoria 
Chromodoris willani 
Chromodoris woodwardae 
Glossodoris cincta 

Glossodoris pallida 
Glossodoris plúmbea 
Glossodoris sibogae 
Glossodoris sp. 
Glossodoris sp. 
Glossodoris vespa 
Noumea decussata 
Noumea haliclona 
Noumea norba 
Noumea simplex 
Verconia verconis 
Pectenodoris trilineata 
Digidentis cf arbutus 
Diversidoris aurantionodulosa 
Ceratosoma amoena 
Ceratosoma brevicaudatum 
Ceratosoma magnifica 
Mexichromis cf multituberculata 
Thorunna australis 
Thorunna daniellae 
Thorunna florens 
Thorunna montrouzieri 
Hypselodoris bullocki 
Hypselodoris emma 



Dehne! & Kong, 1979 

Behrens, 1991 

Fernandez-Ovies, 1981 

Boucher, 1983 

Debelius, 1998 

Muniain eta!., 1996 

Gohar & Soliman, 1967b, as C. inornata Pease 

Gohar & Aboul-Ela, 1957, as С quadricolor {RùppeW & 

Leuckart) 
Gohar & Aboul-Ela, 1957 
Baba et al., 1956, as Glossodoris 
Orteaetal., 1994 
Taylor, 2001 
present study 
Orteaetal., 1994 
present study 

Johnson & Boucher, 1983; present study 
Johnson & Boucher, 1983; Chuk, 2001; present study 
Rose, 1981; Fraser, 2001a 
present study; Adams, 2001; Warren, 2001 
present study 
present study 
Kenny, 1970 
present study 

Klussman-Kolb & Wägele, 2001 
Baba et al., 1956, as Glossodoris pallescens Bergh 
Bande!, 1976 
present study 
present study 
present study 
Gill, 2001 
Rudman, 1998a 
Gohar & Soliman, 1967c, as С obsoleta (Rüppell & 

Leuckart) 
Soliman, 1987 

Gohar & Aboul-Ela, 1959, as G. atromarginata Cuvier 
Baba et al., 1956 
Fraser, 2001b 
Ostergaard, 1960 
present study 
Johnson, 2001a 
Avern, 1986; present study 
present study 
Johnson, 2001b 
Debelius, 1998 
present study 
present study 
present study 
Coleman, 2001 
Smith et al., 1989 
Jamieson, 1999, as Miamira 
Miller, 2001a 
S. Johnson, pers. comm. 
Miller, 2001b 
Coleman, 2001 
Rudman, 1998b 
present study 
Marshall & Willan, 1999 

(continues) 



298 

TABLE 2. {Continued) 



WILSON 



Species 



Egg Mass Type 
ABC 



Source 



Hypselodohs festiva 
Hypselodoris longa 
Hypselodohs maculosa 
Hypselodoris obscura 
Hypselodoris sp. 
Hypselodoris whitei 
Hypselodoris zebra 
Hypselodoris zephyra 
Risbecia ghardaqana 
Risbecia pulchella 
Risbecia tryoni 



Baba et aL, 1956, as Glossodoris 

Rudman, 1999 

Johnson, 2000 

present study 

present study 

Johnson & Boucher, 1983, as H. mouaci (R\sbec) 

Geiger, 1999 

present study 

Gohar&Aboul-Ela, 1957 

Gohar & Aboul-Ela, 1957 

Johnson & Boucher, 1983, as Chromodoris 



largest genus in the Chromodorididae. Cur- 
rently, it is estimated that Chromodoris con- 
tains approximately 200 species (Gosliner & 
Draheim, 1996), whereas most other chro- 
modorid genera are considerably less spe- 
ciose and some are monotypic (eg., Dlversi- 
doris. \/ercon/a). Although the total percentage 
of Chromodoris species sampled within the 
present study is very low, all three types of egg 
mass structure were detected (Table 2). 

The nine Chromodoris species that are 
known to exhibit flat egg masses (Type A) 
occur in two colour groups. Rudman (1977, 
1982, 1983) described these groups in order 
to facilitate identification of similarly coloured 
species. The first of these groups, the Chro- 
modoris quadhcolor colour group, contains all 
but two of these flat-spawning species. Based 
on the distribution of mantle glands and on re- 
productive characters, it has been suggested 
that this colour group may represent a dis- 
crete clade within the genus Chromodoris 
(Gosliner & Behrens, 1 998). This provides fur- 
ther evidence that egg masses can potentially 
reflect phylogenetic influence. The remaining 
species that lay a flat egg mass, Chromodoris 
aspersa (Gould) and С orientalis Rudman, 
both belong to the С aspersa colour group 
(Rudman, 1983). These species have long 
been confused, although external colouration 
can be used to reliably separate them (Rud- 
man, 1983). The notai spots in С orientalis 
are black, whereas in С aspersa they are 
deep purple. The precise nature of the rela- 
tionship between the two colour groups re- 
mains to be investigated, but it is interesting to 
note that most recorded Type A spawners in 
Chromodons share a band of orange around 
the mantle. They also typically possess 
translucent orange gills and/or rhinophores, 
and all but С aspersa share the presence of 



black pigment (present as stripes or back- 
ground colour in the С quadhcolor colour 
group and as spots in С orientalis). 

Upright egg masses (Type B) were present 
in at least 1 3 of the 24 species of Chromodoris 
species listed in Table 2. There was some dif- 
ficulty in classifying the egg masses of Chro- 
modoris coi, С. kuniei and C. robot. These 
species all lay ribbons that in most cases are 
upright, but are often attached in short kinks 
that cause them to appear outward sloping. 
These egg masses may also have grooves on 
the broad side of the ribbon running parallel to 
attachment, although the significance of this is 
unclear. The two reports of a clearly crenulated 
egg mass (Type C) occurring in Chromodoris 
warrant further attention. Chromodoris mag- 
nifica falls into the С quadhcolor co\our group 
of Rudman and would thus be predicted to lay 
a flat egg mass similar to all other known mem- 
bers of the group. However, Klussmann-Kolb 
& Wägele (2001 ) report С magnifica laying an 
upright and crenulated egg mass, although 
further observations are desirable to confirm 
the report. Similarly, conflicting reports occur 
regarding the egg mass of Chromodoris geo- 
metries. Boucher (1983) recorded С geomét- 
rica in the Marshall Islands with an upright, or- 
ange egg mass containing extra-capsular 
yolk. This study confirmed that report for spec- 
imens from subtropical eastern Australia, and 
an upright orange ribbon was also reported 
from Papua New Guinea (Chuk, 2001). How- 
ever, Rose (1985) recorded some spawning 
details of С geométrica from temperate east- 
ern Australia and reported an absence of 
extra-capsular yolk. It is only in his unpub- 
lished thesis (1 981 ) that he describes the egg 
mass as being fluted. Although it is quite pos- 
sible that the single specimen that Rose col- 
lected was misidentified, Fraser (2001a) also 



CHROMODORID EGG MASSES 



299 



shows an egg mass of C. geométrica that is 
clearly crenulated. This latter observation fronn 
South Africa also differs from all previous ac- 
counts in that the colour of the egg mass is 
white. It is likely that this egg mass lacked 
extra-capsular yolk as well, as the resulting or- 
ange hue is usually visible to the naked eye. As 
both Rose (1981) and Fraser (2001a) made 
their observations at similar latitudes in the Pa- 
cific and Indian oceans respectively (approxi- 
mately the southermost limits for C. geomét- 
rica), it is possible that the production of 
extra-capsular yolk reserves is related to tem- 
perature. 

An upright egg mass structure (Type B) was 
found in all species of Glossodoris, Noumea, 
Verconia, Pectenodoris, Digidentis and Diver- 



sidoris represented in Table 2. According to 
the first phylogeny proposed for the Chro- 
modorididae (Rudman, 1984), all these gen- 
era are typically considered in the "mid re- 
gion" of evolution within the family, neither 
basal nor highly derived (Fig. 3). Gosliner & 
Johnson's (1999) cladistic analysis found no 
resolution between the lineage containing 
Chromodoris, Ceratosoma and Glossodoris 
and the one containing Noumea, Pecten- 
odoris, Verconia, Thorunna and Digidentis 
(Fig. 4). However, both phytogenies agree 
that the crown group within one lineage con- 
sists of Risbecia + fHypselodoris. This crown 
group (with the exception of IHypselodoris 
zebra) all show egg masses that are either 
outwardly sloping or crenulated. This indi- 




FIG. 3. Egg mass shape mapped onto hypothesized phylogeny of the Chromodorididae (from Rudman, 
1984). 



300 



WILSON 



Type С 




FIG. 4. Egg mass shape mapped onto hypothesized phylogeny of the Chromodorididae (from Gosliner & 
Johnson, 1999). Broken lines indicate that the spawn type illustrated above is also present in the genus. 



cates that the most highly derived forms are 
more likely to lay egg masses that have the 
free edge of the ribbon lengthened, resulting 
in outward sloping or crenulated egg masses. 
As the structure of an egg mass is said to re- 
flect the degree of anatomical complexity of 
the reproductive system (Fretter & Ко Bun, 
1 984), it is likely that more highly derived gen- 
era would lay more complex egg masses. The 
single known exception in Hypselodoris (/-/. 
zebra) shows an upright egg mass. This is the 
only egg mass known for a member of the At- 
lantic/Eastern Pacific clade of the genus. 
Hypselodoris is known to consist of two 
clades, the above-mentioned clade and an 
Indo-Pacific clade (Gosliner & Johnson, 
1999), which is represented by all the other 
Hypselodoris egg masses observed in this 
study. 

The genus Thorunna is considered rela- 
tively derived in both phylogenies of the Chro- 
modorididae, and the egg mass data from 
Table 1 appear to sustain this. Thorunna has 
been proposed as a sister group to both Digi- 
dentis (Rudman, 1984) and Durvilledoris 
(Gosliner & Johnson, 1999). Observations on 
the egg mass of Digidentis offers no firm evi- 
dence in support of this relationship, and the 



egg mass structure of Durvilledoris species 
remains unknown. 

Ceratosoma is closely allied to Chro- 
modoris and Glossodoris, according to the 
phylogeny of Gosliner & Johnson (1 999). This 
contrasts with Rudman (1984), who places it 
in the "hypselodorid" subgroup (also contain- 
ing Digidentis, Thorunna, Durvilledoris, Mexi- 
chromis, Risbecia and Hypselodoris), consid- 
ering Ceratosoma to have a Chromodoris 
ancestor but having diverged early in hypselo- 
dorid evolution. Here, the egg masses of 
three Ceratosoma species are reported to be 
outwardly sloping or crenulated, indicating a 
derived condition. 

Extra-capsular yolk reserves have so far 
been recorded in only two chromodorid gen- 
era, Cadlinella and Chromodoris, with five 
new records in the present study (Table 3). 
While extra-capsular yolk is found in the flat 
egg masses of Cadlinella, the flat egg masses 
of Chromodoris never contain these reserves. 
It is only in the upright egg masses of Indo-Pa- 
cific Chromodoris species that extra-capsular 
yolk is present. Chromodoris binza and Chro- 
modoris clenchi, both found in the Caribbean, 
lay upright egg masses but do not incorporate 
extra-capsular yolk reserves into the egg 



CHROMODORID EGG MASSES 



301 



TABLE 3. Chromodorids that produce extra-capsular yolk. 



Species 


Yolk type 


Original source 


Cadlinella ornatissima 


2, small & pale 


Risbec, 1928 


Chromodons albopunctatus 


2, large & orange 


Boucher, 1983 


Chromodoris albopustulosa 


1A 


Kay & Young, 1969 


Chromodoris annulata 


2, large & orange 


Gohar&Aboul-Ela, 1957 


Chromodoris colling woodi 


1A 


present study 


Chromodoris daphne 


1A 


present study 


Chromodoris decora 


1A 


Kay & Young, 1969 


Chromodoris E-6 


1A 


Boucher, 1983 


Chromodoris fidelis 


1A 


Marcus & Burch, 1965 


Chromodoris galactos 


1A 


Boucher, 1983, as E-57 


Chromodoris geométrica 


1B 


Boucher, 1983 


Chromodoris kuniei 


18 


present study 


Chromodoris leopardus 


1B 


present study 


Chromodoris margínala 


1A 


Boucher, 1983 


Chromodoris preciosa 


not determined 


S. Johnson, pers. comm. 


Chromodoris roboi 


1B 


present study 


Chromodoris rubrocornuta 


not determined 


S. Johnson, pers. comm. 


Chromodoris E-328 


not determined 


S. Johnson, pers. comm. 


Chromodoris E-48 


not determined 


S. Johnson, pers. comm. 


Chromodoris thompsoni 


1A 


Thompson, 1972, as C. loringi 


Chromodoris tinctoria 


1A 


Gohar& Soliman, 1967 


Chromodoris vibrata 


not determined 


S. Johnson, pers. comm. 



mass (Ortea et al., 1994). It will be of great in- 
terest to deternnine whether extra-capsular 
yolk is resthcted solely to Indo-Pacific and 
Red Sea Chromodoris. While Cadlinella sp. 
from the Red Sea does lay flat egg masses, 
they have not yet been examined to deter- 
mine if they also contain extra-capsular yolk 
like Cadlinella ornatissima. 

While Cadlina, Tyrinna and Cadlinella are 
all currently considered basal within the Chro- 
modorididae, there appears to be no indica- 
tion that these three genera are themselves 
closely related (Rudman, 1984). It is therefore 
no surprise that these genera exhibit different 
egg mass types. While varying hypotheses 
regarding the basal chromodorids have been 
proposed or supported (Rudman, 1 984; Muni- 
ain et al., 1996; Gosliner & Johnson, 1999), 
the most recent discussion concludes only 
that the phylogeny of these basal groups re- 
mains unclear (Schrödl & Milien, 2001 ). Given 
that Cadlinella shares a flat egg mass and 
extra-capsular yolk with varying Chromodoris 
species, it is likely that Cadlinella gave rise to 
the Chromodoris lineage. 

There is some concern that egg mass 
structure may reflect environmental rather 
than phylogenetic influences (Wägele & 
Willan, 2000), and is therefore not suitable to 
be used as a character in phylogenetic analy- 
ses. Observations on spawning in the field 
and laboratory have shown some differences, 



which have been incorporated into the egg 
mass classification in this study. Specimens 
of Noumea norba and Diversidoris aurantio- 
nodulosa laid upright ribbons in the field, while 
the same specimens in the laboratory laid egg 
masses that sloped inward. Specimens of 
Hypselodoris obscura lay egg masses that 
range from outward sloping to crenulated. 
Some egg masses, particularly those that are 
thin and flaccid, can appear slightly fluted 
when laid on irregular or uneven substrata. 
However, it is possible to differentiate be- 
tween these and egg masses that are truly 
outward sloping or crenulated by comparing 
the length of both the free and attached 
edges. The regularity of the coiling, that is, the 
space between the whorls of one egg mass, 
differed greatly within a species, suggesting 
this may be affected by environmental condi- 
tions or perhaps the reproductive history of 
the parent. It is not yet possible to make any 
correlation between egg mass type and the 
habitat of the parent nudibranch, since there 
is little information regarding movement within 
the latter. Many species of nudibranch are 
found in both intertidal and subtidal environ- 
ments without showing any obvious change in 
egg mass structure. However, controlled ex- 
periments varying such factors as tempera- 
ture, salinity and water flow are desirable to 
test this idea. 
There are apparently conflicting reports 



302 



WILSON 



where the colour of a egg mass has been re- 
ported to differ between localities, even when 
extra-capsular yolk is absent. Johnson & 
Boucher (1983) reported that Hypselodoris 
maculosa lays a pale pink egg mass, whereas 
Marshall & Willan (1999) recorded it as white. 
While it is possible that a change in prey items 
may trigger a corresponding change in ova 
colour, differences may also reflect subjective 
interpretation of colour. It is also important to 
know whether the animal laying the egg mass 
is identified correctly. Hypselodoris maculosa 
individuals are known to be variable in colour, 
and there is the possibility that a complex of 
species is currently identified as a single 
species. Egg masses may have the potential 
to help separate these complexes but need to 
be used in conjunction with morphological 
data from the parent specimens. Another fac- 
tor that can influence the colour of an egg 
mass is the amount of time that has elapsed 
since it was laid. Gradual colour changes 
occur as the developing embryos use up the 
available yolk. However, while small changes 
in colour may be attributed to such factors, 
real disagreement in colour may reflect differ- 
ences in the identity of the parent. Risbecia 
tryoni has been reported to lay a rose-pink 
egg mass with a crenulated free edge (John- 
son & Boucher, 1983), whereas Marshall & 
Willan (1999) reported the mass as orange 
but do not describe its structure. Marshall & 
Willan (1999) also incorrectly cited Johnson & 
Boucher as attributing extra-capsular yolk to 
this species. 

While the general form of the egg mass is 
usually characteristic of a species or genus, 
Rudman & Avern (1989) found both upright 
and crenulated egg mass types in the rela- 
tively small genus Rostanga (approximately 
13 species). This was also the case for Acan- 
thodorls (Hurst, 1967), in which both upright 
and crenulated egg masses were recorded. 
This indicates that some caution may be nec- 
essary when interpreting phylogenetic signal 
from egg mass structure, as it may be useful 
at different taxonomic levels in different 
groups. The absence of a fossil record means 
there is currently no reliable method of dating 
genera. It may only be in more recent genera 
that egg mass structure remains conservative 
throughout. It is possible that the trend to- 
wards crenulation of egg masses in the more 
derived Chromodorididae is also seen within 
a single "older" genus that has had more time 
to evolve. 

Soliman (1987) recognized the potential 



taxonomic value of egg mass type among 
gastropods, but he recommended that when 
interpreting phylogenetic relationships, pri- 
mary consideration should be given to ana- 
tomical, palaeontological or ecological evi- 
dence. Because no fossil record exists for the 
Nudibranchia, and accurate ecological infor- 
mation is still scarce for most groups, alterna- 
tive characters may be found in reproductive 
data. Egg mass structure may help confirm or 
challenge phylogenetic hypotheses based 
solely on anatomical data, but much work is 
still required to understand the underlying 
causes of observed variation. 



ACKNOWLEDGEMENTS 

Support for this project was obtained from a 
University of Queensland Research Grant, a 
Mollusc Research Grant from the Malacologi- 
cal Society of Australasia, and the Undersea 
Explorer, Port Douglas. I would like to thank 
many people for assistance in collecting ani- 
mals, namely Dan Jackson, Suzie Green, 
David Harris, Shane Litherland and Shireen 
Fahey. Bill Rudman assisted with identifica- 
tions, and Scott Johnson provided valuable 
discussion and observations. Maria del Car- 
men Gómez-Cabrera and Rosa Garcia Novoa 
are thanked for their assistance in translating 
the work of Fernandez-Ovies. This manu- 
script was improved by comments from John 
Healy, Bill Rudman and two anonymous re- 
viewers. I would like to acknowledge the 
Great Barrier Reef Marine Park Authority 
(G98/110), Queensland Parks and Wildlife 
Service (QSE99/489) and the Department of 
Primary Industries, Water and Environment, 
Hobart (P99/00-121) for allowing respective 
collection permits. This manuscript forms con- 
tribution 2002-01 from the Centre of Marine 
Studies, University of Queensland. 



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MALACOLOGIA, 2002, 44(2): 307-324 

ALLOZYMIC TAXONOMY WITHIN THE GENUS MELANOPSIS 

(GASTROPODA: CERITHIACEA) IN ISRAEL: 
A CASE IN WHICH SLIGHT DIFFERENCES ARE CONGRUENT 

Andrzej Falniowski\ Joseph Heller^, Magdalena Szarowska\ & Krystyna Mazan-Mamczarz^ 

ABSTRACT 

Molecular differences between the species of Melanopsis distinguished by Heller were stud- 
ied by means of cellulose acetate gel allozyme electrophoresis on 26 Melanopsis populations 
from Israel: 6 M. buccinoidea Olivier, 1801; 8 M. sau/cy/ Bourguignat, 1853; 1 M. meiostoma 
Heller & Sivan, 2001 ; and 1 1 M. costata Olivier, 1 804, represented by two subspecies: M. costata 
costata Olivier, 1804, and M. costata jordanica Roth, 1839. Fourteen loci (9 polymorphic) were 
scorable: Aat, Alp, Est-1 , Est-2, Gpi, Hbdh, ldh-1 , idh-2, Iddh, Mdh, Mdhp, Mpi, Pgdh, Pgm. Mean 
sample size was 28.983; mean number of alleles per locus: 1 .45 (1 .1 -1 .7); polymorphic loci per 
population: 36.5% (14.3-57.1); mean observed heterozygosity: 0.085 (0.041-0.121); mean ex- 
pected heterozygosity: 0.112 (0.045-0.170). Nei genetic distance (0.000-0.232, mean 0.0761, 
no significant association with geographic distance) and Cavalli-Sforza and Edwards arc genetic 
distance (0.054-0.441 , mean 0.240, significant association with geographic distance) were com- 
puted for pairs of populations. Interpopulation differences were analyzed phenetically (corre- 
spondence analysis, UPGMA), and phylogenetically (neighbor-joining and Fitch-Margoliash ad- 
ditive trees). Despite the slight differentiation of the taxa, the results confirmed the distinctness 
of M. costata costata, M. costata jordanica and M. sau/cy/ although the speciation processes may 
not yet be completed. The molecular vahability of M. buccinoidea was wider than that of all the 
other studied taxa together, and overlapped the variation ranges of each. It may be ancestral 
species to the others, which may have speciated as peripheral isolates. The position of M. meios- 
toma as a distinct species was not confirmed. 

Key words: freshwater snail, species, allozymes, electrophoresis, phenetics, phylogenetics. 



INTRODUCTION 

The freshwater snail genus Melanopsis 
(Férussac, 1807) (Prosobranchia: Melanopsi- 
dae) is one of the most abundant mollusks in 
the lakes, rivers and springs of the Middle 
East, and is widely distributed throughout the 
circum-Mediterranean (Germain, 1921-22; 
Bilgin, 1973; Tchernov, 1973, 1975; Schutt & 
Bilgin, 1974; Banarescu, 1990-95). Its anat- 
omy was described by Bilgin (1973) and Al- 
taba (1991), sperm structure by Afzelius et 
al. (1989), radula vanation by Glaubrecht 
(1993), and allozyme variation in the western 
Mediterranean by Altaba (1991), who found 
considerable genetic differentiation over short 
distances. There are numerous studies on 
mollusk allozymes, devoted to molecular tax- 
onomy (Woodruff et al., 1988; Borsa & Ben- 
zie, 1993; Ponder et al., 1993; Davis, 1994; 
Haase, 1994; Falniowski etal., 1996), includ- 



ing in Melanopsis (Altaba, 1991; Altman & 
Ritte, 1996). 

In Melanopsis, the shell is extremely vari- 
able, with smooth and costate, elongate and 
stout, banded and all-black forms found in a 
relatively small area. Heller et al. (1999) in- 
vestigated the systematics, distribution and 
extent of presumed hybridization of Melanop- 
sis in the central and northern Jordan Valley. 
Basing their study on the shell morphometry 
of 700 snails collected at 35 sites, they recog- 
nized three species: snails with smooth shells 
{M. buccinoidea Olivier, 1801); with straight- 
ribbed shells that almost always extend the 
entire length of the whorl {M. costata Olivier, 
1804); and with tubercle-ribbed shells that do 
not extend the entire length {M. saulcyi Bour- 
guignat, 1853). Melanopsis buccinoidea is 
common in a wide variety of aquatic habitats, 
from small trickles to springs and streams; M. 
costata occurs in the upper Jordan River, 



^Department of Malacology, Institute of Zoology, Jagiellonian University, ul. Ingardena 6, 30-060 Krakow, Poland; 

faln@zuk.iz.uj.edu.pl 

^Department of Evolution, Systematics and Ecology, Hebrew University, Jerusalem, Israel 



307 



308 



FALNIOWSKI ETAL 



Lake Kinneret and lower Jordan River; M. 
saulcyi in streams that border swamps. 

Heller et a!. (1999) also reported the occur- 
rence of presumed hybrids characterized by 
shells with weak ribs, thus defined based on 
shell characters alone. According to those au- 
thors, hybrids were found in narrow zones of 
contact, both between M. buccinoldea and M. 
costata. and between M. buccinoidea and M. 
saulcyi. Outside these zones, the species re- 
tain their distinct conchological identities. The 
narrowness of the hybrid zones suggested to 
Heller et al. (1999) that selection was main- 
taining the parapatric distribution of the 
species involved, with the narrow hybrid 
zones acting as partial barriers to gene flow. 
Between M. buccinoidea and M. costata. the 
hybrid zones were at habitat transition, from 
streams to lake. 

Within M. costata, Heller et al. (1999) dis- 
tinguished three subspecies. Two of them are 
dealt with in the present paper: M. costata 
costata Olivier, 1804 (shell elongate; the 
upper Jordan River, on mud and submerged 
vegetation) and M. costata jordanica Roth, 
1839 (shell usually stout; Lake Kinneret, on 
stones and boulders). The stout shell of M. 
costata jordanica seems correlated with a 
stormy habitat. Recently a new species, M. 
meiostoma. was described from Israel (Heller 
& Sivan, 2000) characterized by a small, 
slender shell with relatively small mouth and 
weak callus. 

The aim of the present study is to test 
whether the morphological differences be- 
tween the species and subspecies distin- 
guished by Heller et al. (1999) are accompa- 
nied by the molecular differences. The 
morphologies are rather finely differentiated; 
in fact, the differences are marked in charac- 
ters that are prone to environmental factors 
(e.g., Gostan, 1966; Palmer, 1985; Falniow- 
ski, 1988). Thus, our null hypothesis is that 
the entire studied group is a single, concho- 
logically variable species, with shell variation 
determined environmentally, not genotypi- 
cally. The latter is not a rare phenomenon 
within the Gastropoda. It is essential in any 
discussion on species distinctness to specify 
the species concept applied. In this study, we 
follow the cohesion species concept, as de- 
fined by Templeton (1989), considering not 
only genetic but also demographic and eco- 
logical factors. In particular, speciation is re- 
garded as the development of cohesion 
mechanisms, not isolation mechanisms. The 
cohesion concept assumes, as cohesion 



mechanisms, not only genetic exchangeabil- 
ity, but also demographic exchangeability. 
The latter means that all the populations of a 
species must share the same fundamental 
niche, as defined by Hutchinson (1 965). Thus, 
to demonstrate that the fundamental niches of 
two taxa are not the same means to prove 
their species distinctness. The cohesion con- 
cept covers the full range of biological reali- 
ties, from completely closed systems of asex- 
ually reproducing taxa to open systems of 
syngameons, with species defined practically 
based solely on demographic exchangeabil- 
ity. It must be stressed that speciation is rather 
a process, not an event (Templeton, 1989). 
Hence, wherever the process is still going on 
and not completed, there obviously will be the 
"bad" species undergoing speciation as op- 
posed to the "good", well-defined ones. The 
cohesion concept is, therefore, what applies 
best to such cases. 

MATERIAL AND METHODS 
Locality Description 

The distribution of the localities is shown in 
Figure 1. 

Melanopsis buccinoidea 

(1) Enot Huga (IG -New Israel Grid-200- 
213). Within a recreation site, a small 
spring that irrigates by way of a recently 
constructed channel into a small pool 
consisting of boulders and cobble; snails 
collected from the channel, at a depth of 
2-3 cm. 

(2) En Hanatziv (IG 197-208). A spring 
within a large natural pool; snails col- 
lected from dead, floating vegetation and 
firm boulders. 

(3) En Raqqat, spring (IG 199-245). Emerg- 
ing 1 5 m from the shore of Lake Kinneret, 
this spring trickles down into the lake 
among boulders and sparse, swampy 
vegetation; snails collected along the 
tnckle, at a depth of 2-3 cm. 

(4) Dan (IG 21 2-295). A lowland stream that 
forms the principal tributary of the Jordan 
River; the physico-chemical aquatic envi- 
ronment extremely stable: annual as well 
as diurnal water temperatures virtually 
constant, at 15.5' С (± 0.5°), chemical 
parameters (NH3, PO^, NO3) vary little, 
dissolved oxygen concentrations uni- 
formly high at 95% of saturation (Heller et 
al., 1997); snails collected from several 



ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 



309 




FIG. 1 . Distribution of studied localities. Black arrow 
in the snnall map at the bottom indicates the area 
presented in the bigger map. Population numbers: 
see text. Symbols of taxa: see Fig. 2. 



small brooks near the spring, from boul- 
ders at a depth of 5-10 cm. 

(6) Banias (IG 21 4-294). A cool spring form- 
ing a major source of the Jordan River; 
snails collected from the artificial pool 
surrounding the spring, at a depth of 
down to 30 cm. 

(7) Nahal Tavor (IG 201-223). A narrow, 
shallow stream covered by dense vege- 
tation; snails collected from within the 
stream, on the muddy substratum, at a 
depth of 2-3 cm. 

Melanopsis meiostoma 

(8) Kefar Haruv (IG 211-240). A small, 
spring that pours into a small cement 
pool; brackishwater (a high, 670 mg/l, 
content of chloride anions); snails col- 
lected from the walls of the pool, at a 
depth of 30 cm. 

Melanopsis costata jordanica 

Lake Kinneret (21 km x 12 km, 170 km^). In 
its natural condition, the water level of Lake 
Kinneret used to fluctuate annually at an av- 
erage of 0.8-1.0 m. In 1964, the lake was 
dammed, its level increased by 1 m, and was 
left at peak levels for much longer periods 
than in its natural condition. Further, in its nat- 
ural condition, the lake's salinity was almost 
400 mg/l (chlohnity). Recent diverting of 
saline springs from the lake has decreased its 
salinity to about 200 mg/l. Daily storms fre- 
quent the lake, the shores of which consist of 
gravel, cobble, stones, mud, sand, and silt. 
There were six localities at Lake Kinneret: 

(9) Ginossar, Kinneret (IG 209-250). A nat- 
ural shore of boulders and stones; snails 
collected along the shore at a depth of 
5-10 cm. 

(10) 208, Kinneret, (IG 209-253). A natural 
shore of boulders and stones; snails col- 
lected at a depth of 5-10 cm. 

(1 1 ) En Raqqat, Kinneret (IG 1 99-245). A nat- 
ural shore of boulders and stones; snails 
collected along the shore at a depth of 
5-10 cm. 

(1 2) En Sheva, Kinneret (IG 201 -252). A nat- 
ural shore of boulders and stones, snails 
collected along the shore at a depth of 
3-10 cm. 

(13) Ha'on, Kinneret (IG 208-237). A shore 
consisting of muddy sand, with a few 
submerged dead reeds; snails collected 
from the reeds, at water level. 

(14) Bet Gavriel, Kinneret (IG 205-234). A re- 
cently constructed retaining wall con- 



310 



FALNIOWSKI ETAL. 



sisted of large basalt boulders; snails col- 
lected at a depth of 5-10 cm. 

Melanopsis costata costata 

(15) Gesher Benot Ya'aqov, upper Jordan 
River (IG 209-269). Muddy substratum, 
willows along the banks; snails collected 
from vegetation, a few cm above the 
water line. 

(16) Gesher Lehavot, upper Jordan River (IG 
209-283). Muddy substratum, willows 
along the bank; snails collected from veg- 
etation a few cm above the water line. 

(17) Eastern Canal of the upper Jordan River, 
(IG 208-278). Muddy substratum, wil- 
lows along the banks; snails collected 
from the muddy substratum. 

(1 8) Bet Hillel (IG 207-289). A tributary of the 
Jordan River, mainly with mud and cob- 
ble along the banks; snails collected from 
the cobble. 

(19) Sede Nehemia (IG 208-288). A tributary 
of the Jordan River; within a recreation 
site; snails collected from the mud and 
from submerged stems, branches and 
tree trunks. 

Melanopsis saulcyi 

(5) Sede Eliyahu (IG 198-204). A small arti- 
ficial pool, with a muddy substratum; 
snails collected from dead vegetation 
floating in the water. 

(20) The same locality as 2. 

(21) Sheluhot (IG 196-209). A cement chan- 
nel in which water from several springs 
flows with a rather strong current; snails 
collected from the walls of the channel, at 
a depth of 30 cm. 

(22) Hamat Gader, 15 m from spring (IG 
213-232). A small artificial pool into 
which the Hamat Gader spring flows; 
snails collected from boulders and stones 
along the shores of the pool. 

(23) The same locality as 7. 

(24) The same locality as 1 . 

(25) Hamat Gader, sphng (IG 213-232). A 
pool in which a spring gushes; snails col- 
lected off logs and various dead 
branches, floating in the water. 

(26) En Malkoah (IG 198-198), a small stag- 
nant water body; on substratum and 
plants. 

Electrophoretic Techniques 

The snails were transported alive to Poland 
and then frozen and stored in a deep freezer, 



at -80°C. Unsexed individuals for electro- 
phoresis were dissected on ice, and their he- 
pato-pancreas tissue was taken for homoge- 
nization. The tissue was homogenized on ice, 
in a glass homogenizer in 50-100 f.il of ho- 
mogenizing solution (100 ml distilled water, 10 
mg NADP, 10 mg NAD, 100 |al ß-mercap- 
toethanol), depending on snail size. The ho- 
mogenates were centhfuged at 11,000 rpm 
for 10 min, then used immediately for cellu- 
lose acetate electrophoresis following the pro- 
tocol of Richardson et al. (1986). The follow- 
ing running buffers were used, depending on 
enzyme system; citrate-phosphate pH 6.4 
(buffer A), phosphate pH 7.0 (buffer B), ortris- 
maleate pH 7.8 (buffer C). The position of ori- 
gin was cathode, the duration of run 1.5 h at 
220 V. Staining protocols followed Richardson 
et al. (1986). The cellogels were from MALTA, 
Italy, all other chemicals from SIGMA, USA. 
Enzyme names and EC numbers are after 
Murphy et al. (1996). 

Numerical Techniques 

Genetic variability parameters and genetic 
distances were computed with BIOSYS-1 
(Swofford & Selander, 1981). One of the two 
distances we had chosen was Nei unbiased 
distance. This is the most commonly used ge- 
netic distance, and so it is the best for com- 
parisons with the literature. The other one was 
Cavalli-Sforza and Edwards arc distance; this 
is not affected by intrapopulation polymor- 
phism and is the most appropriate when a 
group of rather closely related populations is 
concerned. GENEPOP (Raymond & Rousset 
1995) was used to compute exact tests for 
HWE (Guo & Thompson 1992; Rousset & 
Raymond 1995), as well as f (equaling F|g of 
Wright) for each population and for each 
locus. Following the notation and computa- 
tional procedure introduced by Weir & Cock- 
erham (1984) and Weir (1990), F-statistics 
was computed over all populations with 
Weir's FSTAT(Weir, 1990; Goudet, 1995), the 
confidence intervals estimated with jackknife 
and bootstrapping techniques (Weir, 1990). 
The same procedure was applied to each of 
the taxa represented by more than one popu- 
lation (i.e., except M. meiostoma). Mantel 
tests, correspondence analysis, nonlinear 
multidimensional scaling, minimum-spanning 
tree, clustering, and cophenetic distances 
were computed with NTSYSpc v.2.0 (Rohlf, 
1998). Mantel tests were used for testing the 
association between the genetic and geo- 



ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 



311 



graphic distances between the populations, 
as well as for evaluation of the phenograms 
and testing the association between the origi- 
nal and cophenetic distances. Additive neigh- 
bor-joining trees (Saitou & Nei, 1987) and 
Fitch-Margoliash trees (Edwards & Cavalli- 
Sforza, 1964; Fitch & Margoliash, 1967; Swof- 
ford & Olsen, 1990; Swofford et al., 1996) 
were computed with NEIGHBOR and FITCH, 
respectively, of the PHYLIP 3.6 package 
(Felsenstein, 1990). From the same package, 
the program SEOBOOT was applied to gen- 
erate 200 bootstrap pseudosamples. For 
each of them, Cavalli-Sforza and Edwards arc 
distances were computed with GENDIST (the 
source code of Felsenstein modified for arc 
instead of chord version of that distance), and 
the resulted file with 200 pseudosamples of 
distance matrices processed with FITCH with 
the global search option. Finally, the extended 
majority rule consensus tree was computed 
with CONSENSE of PHYLIP 3.6 package. 



RESULTS 

Twelve enzyme systems coded by 14 loci 
gave well resolved and always interpretable 
results: Aat (aspartate aminotransferase, EC 
2.6.1 .1 ., buffer B), Alp (alkaline phosphatase, 
EC 3.1 .3.1 , buffer C), Est-1 (esterase, locus 1 , 
nonspecific, buffer A), Est-2 (esterase, locus 
2, non-specific, buffer A), Gpi (glucose-6- 
phosphate isomerase, EC 5.3.1.9, buffers A, 
C), Hbdh (3-hydroxybutyrate dehydrogenase, 
EC 1 .1 .1 .30, buffer C), ldh-1 (isocitrate dehy- 
drogenase, locus 1, EC 1.1.1.42, buffer C), 
ldh-2 (isocitrate dehydrogenase, locus 2, EC 
1.1.1.42, buffer C), Iddh (L-iditol dehydroge- 
nase, EC 1.1.1.14, buffer C), Mdh (malate 
dehydrogenase, EC 1.1.1.37, buffer C), 
Mdhp [malate dehydrogenase (NADP""), EC 
1.1.1.40, buffer C], Mpi (mannose-6-phos- 
phate isomerase, EC 5.3.1.8, buffers B, C), 
Pgdh (phosphogluconate dehydrogenase, 
EC 1.1.1.44, buffers B, C), and Pgm (phos- 
phoglucomutase, EC 5.4.2.2, buffer C). Nine 
of the loci (64.3%) were polymorphic: Alp, Est- 
1, Est-2, Gpi, Hbdh, ldh-1, ldh-2, Pgdh, and 
Pgm. Allele frequencies in those loci are pre- 
sented in Table 1 . 

Mean sample size was 28.983 (Table 2). 
Mean number of alleles per locus averaged 
1.45, varying from 1.10 in population 8 to 1.7 
in population 12. Mean values for the taxa 
were 1.43 for Melanopsis buccinoidea, 1.10 
for M. meiostoma, 1 .50 for M. costata jorda- 



nica and M. costata costata, and 1 .43 for M. 
saulcyi. On average there was 36.5% of the 
polymorphic loci per population, varying from 
14.3% in population 8 to 57.1% in population 
11 (Table 2). Mean values for the taxa were: 
39.3% for M. buccinoidea, 14.3% for M. 
meiostoma, 40.5% for M. costata jordanica, 
32.9% for M. costata costata, and 36.6% for 
M. saulcyi. Mean observed heterozygosity 
(Table 2) varied from 0.041 in population 1 3 to 
0.121 in population 10, mean for all the popu- 
lations: 0.085. Mean expected heterozygosity 
(Table 2) ranged from 0.045 in population 8 to 
0.170 in population 22, mean for all the popu- 
lations: 0.112. Departures from Hardy-Wein- 
berg equilibrium (HWE), F-statistics, linkage 
disequilibrium, estimates of gene flow, and 
detailed analysis of the genetic structure of 
the studied populations are presented in Fal- 
niowski et a!, (in press). 

The multi-locus (by population) tests per- 
formed with GENEPOP showed statistically 
significant heterozygote deficits in most popu- 
lations. The multi-population (by locus) tests 
indicated a statistically significant deficiency 
of heterozygotes at all loci, except ldh-1 and 
Pgdh. Four populations (8, 17,18, 25) were at 
HWE or had all loci monomorphic. Heterozy- 
gote excess was observed at Est-2 in popula- 
tion 1 0, at Gpi in 21 , and at Pgdh in 1 . All the 
other departures from HWE were heterozy- 
gote deficits. The ratio of the number of popu- 
lations at disequilibrium to the number of pop- 
ulations with a given locus polymorphic varied 
from 9.1% at ldh-1 to 59.1% at Est-1 . Statisti- 
cally significant values of f were found in 13 
populations for Est-1, in 9 for Gpi, in 8 for 
Hbdh, in 7 for Est-2, in 3 for Alp and Pgm, in 2 
for ldh-2 and Pgdh, and in 1 for ldh-1 (Fal- 
niowski et al., in press). The results of FSTAT 
for all populations indicated HWE for Est-2, 
ldh-1, and Pgdh: for all those loci f values 
were not statistically significant and F approx- 
imated 6. The highest values of f were found 
for ldh-2. Alp and Est-1. For all the polymor- 
phic loci, both F and 9 differed significantly 
from 0. The highest 9 was found for Pgm 
(0.770), the lowest for ldh-2. Alp and Gpi. The 
values and confidence intervals for particular 
loci did not overlap. The highest values of F 
were found for Pgm, Est-1 and ldh-2, the low- 
est for Pgdh. F was less than 9 and f varied 
among loci, but the confidence intervals did 
not overlap (Falniowski et al., in press). 

For each pair of populations, genetic dis- 
tances were computed: Cavalli-Sforza and 
Edwards arc distance and unbiased Nei dis- 



312 



FALNIOWSKI ETAL 



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ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 



313 



TABLE 2. Genetic variability at 14 loci in all populations (standard errors in parentheses). 



Population 



Mean 

sample size 

per locus 



Mean no. 

of alleles per 

locus 



Percentage 

of loci 
polymorphic* 



Mean heterozygosity 



Direct count 



HdyWbg 
expected** 



Melanopsis buccinoidea 

1. Enot Huga 

2. En Hanatziv 

3. En Rakkat spring 

4. Dan 

6. Banias 

7. Nahal Tavor 
M. meiostoma 

8. Kefar Haruv 

M. costata jordanica 

9. Ginnossar 
10.208 

11. En Rakkat Kinneret 

12. En Sheva 

13. Ha'on 

14. BetGavriel 
M. costata costata 

15. Gesher Benot Ya'aqov 

16. Gesher Lehavot 

17. Eastern Canal 

18. Bet Hillel 

19. Sede Nehemia 
M. saulcyi 

5. Sede Eliahu 

20. En Hanatziv 

21. Sheluhot 

22. HamatGader 15mf.s. 

23. Nahal Tavor 

24. En Huga 

25. Hamat Gader spring 

26. En Malkoah 



31.9 


±0.1 


1.5 


±0.2) 


8.0 


±0.0 


1.4 


±0.2) 


41.0 


±2.4 


1.4 


±0.1) 


30.9 


±0.9 


1.5 


±0.1) 


29.4 


±0.3 


1.4 


±0.2) 


41.1 


±1.6 


1.4 


±0.1) 


19.0 


±1.9 


1.1 


±0.1) 


36.1 


±1.1 


1.4 


±0.2) 


31.9 


±0.1 


1.4 


±0.2) 


38.1 


±0.9 


1.6 


±0.1) 


31.9 


±0.1 


1.7 


±0.2) 


31.6 


±0.2 


1.4 


±0.2) 


31.1 


±0.6 


1.5 


±0.2) 


32.0 


±0.0 


1.6 


±0.3) 


31.9 


±0.1 


1.5 


±0.2) 


30.0 


±2.5 


1.4 


±0.2) 


32.0 


±0.0 


1.5 


±0.2) 


31.7 


±0.2 


1.5 


±0.2) 


31.4 


±1,7 


1.4 


±0.1) 


31.7 


±0.2 


1.6 


±0.2) 


32.0 


±0.0 


1.4 


±0.2) 


21.0 


±1.3 


1.4 


±0.1) 


24.3 


±1.7 


1.5 


±0.2) 


31.7 


±0.2 


1.5 


±0.2) 


10.0 


±0.6 


1.2 


±0.1) 


41.3 


±0.3 


1.4 


±0.2) 



42.9 
28.6 
42.9 
50.0 
35.7 
35.7 

14.3 

35.7 
35.7 
57.1 
50.0 
28.6 
35.7 

28.6 
35.7 
28.6 
35.7 
35.7 

35.7 
42.9 
35.7 
42.9 
42.9 
42.9 
21.4 
28.6 



0.116 
0.089 
0.092 
0.090 
0.068 
0.053 



0.042 (± 



0.074 
0.121 
0.116 
0.104 
0.041 
0.085 

0.080 
0.055 
0.104 
0.112 
0.106 

0.059 
0.091 
0.063 
0.106 
0.109 
0.054 
0.113 
0.067 



(±0.053) 


0.133 ( 


(±0.062) 


0.140 ( 


(±0.032) 


0.131 ( 


(±0.050) 


0.090 ( 


(±0.033) 


0.117( 


(±0.023) 


0.102 ( 


(±0.029) 


0.045 ( 


(±0.030) 


0.106 ( 


(±0.069) 


0.122 ( 


(±0.041) 


0.165 ( 


(±0.038) 


0.126 ( 


(±0.019) 


0.101 ( 


(±0.036) 


0.101 ( 


(±0.044) 


0.093 ( 


(±0.034) 


0.081 ( 


(±0.049) 


0.099 ( 


(±0.055) 


0.102 ( 


(±0.055) 


0.114( 


(±0.031) 


0.117 ( 


(±0.040) 


0.122 ( 


(±0.040) 


0.078 ( 


(±0.042) 


0.170 ( 


(±0.049) 


0.155 ( 


(±0.022) 


0.117( 


(±0.074) 


0.077 ( 


(±0.033) 


0.111 ( 



t0.053) 
t0.063) 
t0.049) 
t0.039) 

:0.048) 

i0.046) 

10.032) 

10.043) 
10.055) 
tO.055) 
t0.042) 
10.049) 
10.042) 

10.044) 
10.033) 
10.048) 
10.050) 
i0.055) 

:0.048) 
tO.050) 
10.038) 
10.060) 
10.059) 

:0.047) 
tO.044) 

:0.050) 



*A locus is considered polymorphic If more than one allele was detected; ** unbiased estimate (see Nel, 1978) 



tance (Table 3). The mean value of Cavalli- 
Sforza and Edwards arc distance equaled 
0.240, and the Mantel test showed a signifi- 
cant association between the matrices of this 
distance and the geographic distance (r = 
0.52133, t = 8.0717, p = 0.0002). The lowest 
four values were found between populations 

17 and 18 (0.054), 15 and 16 (0.072), 15 and 

18 (0.081), 18 and 19 (0.081), and 15 and 19 
(0.091). The highest values (exceeding 0.4) 
were between population 2 and the following 
populations: 18 (0.441), 19 (0.438), 6 (0.435), 
17 (0.433), 14 (0.429), 4, 16, 15 and 13. The 
mean value of Nel distance equaled 0.076, 
and the Mantel test showed no significant as- 
sociation between Nei distance and geo- 
graphic distance (r = 0.47674, t = 7.6041 , p = 
0.3778). The lowest values were found be- 
tween populations 24 and 25 (0.000); 15 and 
16, 15 and 18, 17 and 18 (0.001); and 18 and 



19 (0.002). The highest values, exceeding 
0.2, were all found for population 2 and the fol- 
lowing: 14 (0.232), 18 (0.229), 16 (0.228), 19 
(0.224), 17 (0.219), 4, 15, 12, 13 and 6. 

The means and ranges of Cavalli-Sforza 
and Edwards arc distance and Nei unbiased 
distance, within and between the studied 
taxa, are listed in Table 4. In general, the val- 
ues are low, and the intraspecific ranges are 
not necessarily narrower than the interspecific 
ones. The mean values of the distances be- 
tween the two subspecies of M. costata are 
similar to the mean intraspecific values for the 
other species. 

The interpopulation allozymic differentiation 
was analyzed both phonetically and phyloge- 
netically. The phenetic analysis included cor- 
respondence analysis of the allele frequen- 
cies and clustering based on the genetic 
distances. The correspondence analysis re- 



314 



FALNIOWSKI ETAL. 



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ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 



315 



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316 



FALNIOWSKI ETAL. 



suited in eigenvalues decreasing slowly: the 
tenth dimension eigenvalue was 0.0087, ex- 
plaining still as much as 2.14% of the total 
variability, and the cumulative variability ex- 
plained by the first ten dimensions explained 
only 93.12°o of the total variability. Thus, we 
decided to present the grouping in only the 
first two dimensions that explained together 
51.81°o of the total vanability. The eigenval- 
ues were 0.1583 and 0.0520, and the per- 
centages of variability explained were 39.00 
and 12.81, respectively. The plot of the col- 
umn factors in the first two-dimension space 
(Fig. 2) shows distinct groups representing M. 
saulcyi (populations 5 and 20-26) and M. 
costata (populations 9-19). Within the latter, 
M. costata jordanica (populations 9-14) are 
not mixed with M. costata costata (popula- 
tions 15-19), although they all form a single, 
compact group. Melanopsis meiostoma (pop- 
ulation 8) lies close to M. saulcyi. On the other 
hand, the populations of M. buccinoidea (1 -4 
and 6-7) are scattered across all the plot, 
mixed with both M. saulcyi and M. costata. 
Nearly the same picture was observed in all 
the ten dimensions analyzed. 
The UPGMA clustehng was computed on 



the genetic distances (Fig. 3). For each 
UPGMA tree, the cophenetic distances were 
calculated, and Mantel tests performed for 
association between the matrices of original 
versus phonetic distances, the so-called 
cophenetic correlation. For Cavalli-Sforza 
and Edwards arc distance r = 0.85446; for Nei 
unbiased distance r = 0.84413. As the as- 
sumptions of the Mantel test were violated 
(the matrices were not independent), only the 
subjective interpretation of the r-values was 
allowed (Rohlf, 1998). The values above 0.8 
and below 0.9 were considered a good fit. 
Both the two phonograms (Fig. 3) show the 
same picture, resembling also the one ob- 
tained with correspondence analysis. Mela- 
nopsis costata costata always forms a cluster, 
and M. costata jordanica forms another clus- 
ter; the two clusters are placed together. Also 
M. saulcyi is grouped in one cluster, which In- 
cludes M. meiostoma. On the other hand, the 
populations that represent M. buccinoidea are 
scattered in the M. saulcyi as well as M. 
costata jordanica clusters. Similar results, al- 
though less clear, and with the stress values 
belonging to the "fair" range, gave nonlinear 
multidimensional scaling, with the minimum- 




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FIG. 2. Correspondence analysis, populations projected on 1st and 2nd dimension. Population numbers: see 
text and Fig. 1. A -Melanopsis buccinoidea Olivier, 1801, B — /W. meiostoma Heller & Sivan, 2001, C — M. 
costata jordanica Roth, 1839, D-/W. costata costata Olivier, 1804, E and F-M sau/cy/ Bourguignat, 1853. 



ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 



317 




FIG. 3. UPGMA clustering trees, based on Cavalli-Sforza and Edwards arc genetic distance (above) and Nei 
unbiased genetic distance (below). Synnbols of taxa: see Fig. 2. 



spanning tree superimposed on the plot, so 
we do not present it here. 

Phylogenetic analysis included distance- 
based additive trees, computed with two tech- 
niques: Neighbor-joining and Fitch-Margo- 
liash (Figs. 4, 5). Surprisingly, the tree 
topologies, which means not the branch 
lengths but the branching patterns, showed 
by neighbor-joining are similar to those re- 
vealed by clustering: the populations repre- 
senting M. saulcyi form one cluster, whereas 
the other includes all the populations of M. 
costata. In the latter, one subspecies is not 
mixed with the other. Melanopsis meiostoma 
falls within the cluster of M. saulcyi, and M. 



buccinoidea is scattered in both clusters. The 
Fitch-Margoliash additive tree technique 
based on Cavalli-Sforza and Edwards arc dis- 
tance resulted in a tree (Fig. 5) with an aver- 
age percent standard deviation of 9.5672 
(6,339 trees analyzed). For unbiased Nei dis- 
tance (Fig. 5), the value was much higher: 
23.2768 (7,083 trees analyzed). Thus, the 
most reliable phylogeny reconstruction is the 
one based on Cavalli-Sforza and Edwards arc 
distance. The phylogenetic trees are drawn In 
the form of a phylogram (Maddison & Maddi- 
son, 1992), with the branch lengths made pro- 
portional to the amount of change, thus en- 
abling one to observe not only the tree 



318 



FALNIOWSKI ETAL 



—О 



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о 



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FIG. 4. Neighbor-joining additive trees, presented in the form of phylograms, based on Cavalli-Sforza and 
Edwards arc genetic distance (above) and Nei unbiased genetic distance (below). Synnbols of taxa: see 
Fig. 2. 



topology, but also the amount of anagenetic 
evolution. Again, there is a distinct group of 
closely related populations that represents M. 
costata jordanica and joins with the somewhat 
less close M. costata costata group. Another 
big cluster includes all the populations of M. 
sauicyi. but also the single population that 
represents M. meiostoma. Also here M. buc- 
cinoidea populations are scattered in both 
clusters, some of them terminating long 
branches. It must be stressed that all the trees 
are unrooted. 
The Fitch-Margoliash tree based on Ca- 



valli-Sforza and Edwards arc distance was 
evaluated using the bootstrap of the original 
frequencies, followed by calculating new dis- 
tances, and Fitch-Margoliash technique ap- 
plied for each one of the new 200 distance 
sets. The extended majority-rule consensus 
tree was similar in topology to the original 
one; it also showed the M. costata costata, M. 
costata jordanica, and M. sauicyi population 
groups as distinct from each, and M. bucci- 
noidea populations scattered all over the tree. 
However, the support of the branches was 
low, about 30-40%. 



ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 



319 



1^ 



О 



r^ 



о 






о 



о 



и 



«А 

^1 



Í— ■ + 



{^'-:1 



гт^"^ 



{? 






&^ 



О 

ж -Э 

О 



О 

о. 






FIG. 5. Fitch-Margoliash additive trees, presented in the form of phylograms, based on Cavalii-Sforza 
and Edwards arc genetic distance (above) and Nei unbiased genetic distance (below). Symbols of taxa: see 
Fig. 2. 



DISCUSSION 

The proportion of polymorphic loci in fresh- 
water gastropods may approach 62% 
(Woodruff et al., 1988); in the freshwater Vi- 
viparus, it ranged from to 33.3% (FalnlowskI 
et al., 1996); In Bythinella, 11 .1 to 55.6% (Fal- 
niowski et al., 1998); from 3.1 to 53.1% in 
Dalhousia; and from to 16% in Fluvidona 
(Ponder et al., 1 996). Hence, the values found 
in Melanopsis, although relatively high for 
prosobranchs, do not exceed the range of 
freshwater gastropods. The value of mean ex- 
pected heterozygosity for all the mollusks 



studied so far is 0.148 (Lazarldou-Dimithadou 
et al., 1994). In fresh- or brackishwater gas- 
tropods, the values are usually lower: 0.002- 
0.136 or 0.021-0.049 in Hydrobia (Haase, 
1993; Davis et al., 1988, respectively) and 
0.04-0.19 on average for various species of 
Oncomelania (Woodruff et al., 1988). On the 
other hand, in Bythinella mean expected het- 
erozygosity ranged from 0.018 to 0.229 (Fal- 
niowski et al., 1998). Thus, the mean ex- 
pected gene diversity in Melanopsis was 
among the highest noted in prosobranchs. 

Despite the criticism concerning Nei ge- 
netic distance (e.g., Swofford & Olsen, 1990; 



320 



FALNIOWSKI ETAL. 



Davis, 1994; Falniowski et al., 1996; Swofford 
et al., 1996), it is the most commonly used 
statistic, thus using it is convenient because 
one can easily make comparisons with the 
rich literature. In this study, contrary to Cavalli- 
Sforza and Edwards arc distance, unbiased 
Nei distance was not correlated with geo- 
graphic distance. This can be explained by 
the fact that Nei distance is intended to reflect 
the number of codon substitutions, thus muta- 
tions, as the main source of variation, and it is 
heavily affected by intrapopulation genotypic 
polymorphism. Thus, it is certainly not appro- 
priate where there is a low level of genetic 
drift, and not necessarily realistic as a quan- 
tificator of the time and/or amount of evolu- 
tionary change after coalescence time. As 
concerns mutations, the highest number 
should be detectable among species, but the 
studied taxa are not geographically distinct: 
their distribution does not show any geo- 
graphic pattern. On the other hand, the high 
level of both genetic drift (shown by the re- 
sults of Ohta's D-statistics and estimates of 
gene flow calculated from 6: Falniowski et al. 
in press) and polymorphism in the studied 
populations has undoubtedly biased the val- 
ues of Nei distance. 

The values of intraspecific Nei distances 
found in Viviparus contectus (Falniowski et al., 
1996) ranged from 0.0014 to 0.0397, mean 
0.0166. In his survey of over 7,000 compar- 
isons of conspecific populations of plants and 
animals, Thorpe (1 983) revealed that only 2% 
of the intraspecific Nei distance estimates ex- 
ceeded 0.10. The highest intraspecific value 
for sexually reproducing gastropod (0.63) was 
reported in Cepaea nemoralis (Johnson et al., 
1 984). Usually the values are lower, for exam- 
ple, 0.001-0.131 in He//x (Lazahdou-Dimitri- 
adouetal., 1994), 0.001 -0.051 in Trochusanó 
rectus (Borsa & Benzie, 1993), 0.000-0.007 
in Haliotis (Brown, 1993), 0.000-0.013 in Hy- 
сУлоЬ/а (Davis et al., 1988), 0.051-0.191 \nOn- 
comelania (Davis et al., 1994). Thus, the val- 
ues found within the presumed species of 
Melanopsis are typical intraspecific distances 
of a rather weakly to quite considerably genet- 
ically differentiated gastropod species. 

The Nei distances between the presumed 
species were also low. They even did not ex- 
ceed in all cases the intraspecific values. In 
M. buccinoidea, indeed, the intraspecific dis- 
tances were often higher than almost any in- 
terspecific distance. The average values were 
about 0.08 between M. buccinoidea and all 



the other species, about 0.05 between M. 
meiostoma and M. saulcyi. about 0.1 between 
M. meiostoma and /W. costata, and about 0.12 
between M. costata and M. saulcyi. The val- 
ues of interspecific distances in the European 
Viviparus (Falniowski et al., 1996) were be- 
tween 0.2306 and 0.9888, mean 0.6871. 
Thorpe (1983) reviewed 900 comparisons of 
interspecific Nei distance for congeners in 
plants and animals and reported a mean dis- 
tance of about 0.40 (from 0.03 to more than 
1); Davis (1994) listed a number of distances 
for mollusks. For gastropods, it may be as 
high as 5.383 in IHaliotis (Brown, 1993), or 
1.726 in Trochus (Borsa & Benzie, 1993), but 
also may not exceed 0.199 for Cristilabrum 
(Woodruff & Solem, 1990), or 0.077 for Stag- 
nicola (Rudolph & Burch, 1989). 

The above figures indicate that there is no 
general rule as to how high the value of Nei 
distance must be to prove species distinct- 
ness. However, the values in Melanopsis are 
rather of conspecific level. The range of in- 
traspecific values, exceeding the ones be- 
tween the taxa, does not confirm the species 
distinctness of the taxa, either. We can con- 
sider them either conspecific, thus not reject- 
ing our null hypothesis, or consider them dis- 
tinct species but the speciation of which 
caused very little change in allozyme pattern. 
A similar case was observed in Cerion (Gould 
& Woodruff, 1986). Secondly, morphology 
may not reflect speciation (Larson, 1989). A 
third possibility that by chance the loci we 
studied are not representative and do not re- 
flect reproductive isolation cannot be ad- 
dressed with our data. Thus, we restrict the 
discussion to the question: are they finely al- 
lozymatically differentiated yet distinct spe- 
cies (or at least distinct biological, units of not 
necessarily a species level) or else, are the 
studied populations conspecific and all their 
shell variation is environmentally induced, as 
postulated by the null hypothesis? 

As it can be seen in Figure 2, the concho- 
logical differences between the studied taxa, 
although rather well marked, are all ex- 
pressed in such characters as the shell di- 
mensions, proportions, and sculpture. There 
is a vast literature describing high, ecologi- 
cally determined variation of shell characters. 
For instance, many prosobranchs that live in 
stormy habitats, where the water movement is 
strong, have ribbed shells (e.g., Wigham, 
1975; Verduin, 1976; Palmer, 1985; Fal- 
niowski, 1988). There were some fine mor- 



ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 



321 



phometric differences in tine radulae of the 
studied taxa (Mazan et al., 2001). On the 
other hand, one can hardly expect well- 
marked differences in radular characters be- 
tween so closely related taxa. Finally, the 
anatomy of Melanopsis is simplified, the 
snails lacking copulatory organs. We did not 
find anatomical differences between the stud- 
ied taxa. Hence, it is the molecular characters 
that are decisive. 

As stated above, the cohesion concept of 
species assumes that the fundamental niches 
of two species cannot be identical. From field 
observations of one of us (J. H.), it seems that 
the fundamental niches of the studied taxa 
are not identical, although the differences are 
rather fine. Thus, the demographic exchange- 
ability of the taxa may be rejected. On the 
other hand, the reasoning may be just re- 
versed: slight differences in ecology may 
create morphology differences between the 
organisms that are basically the same. How- 
ever, in such a case, the morphological and 
ecological differences would not be accompa- 
nied by molecular differences. Although there 
are a few well-documented exceptions (Sza- 
rowska et al., 1998), the neutral theory holds, 
and the vast majority of allozymes seems se- 
lectively neutral; thus, one cannot expect eco- 
logical differentiation in allozyme pattern. 

The results of either phenetic or phyloge- 
netic analyses present a surprisingly congru- 
ent pattern of distinct but close clusters repre- 
senting M. costata costata and M. costata 
jordanica, as opposed to another cluster 
formed by all the populations of M. saulcyi. 
Melanopsis meiostoma invariably adjoins the 
cluster of M. saulcyi. On the other hand, M. 
buccinoidea is scattered in both big clusters. 
Correspondence analysis shows the same 
picture; it also shows that the variability range 
of M. buccinoidea is wider than the variability 
of all the other taxa together. On the other 
hand, the support indices provided by boot- 
strap were low. This may have been due to 
the slight differentiation of the studied popula- 
tions, and thus the high sensitivity of the in- 
ferred trees to any modification of the data. It 
must also be stressed that the number of 
characters, that is the number of alleles 
found, is far too low for the bootstrap to give 
reliable estimates (e.g., Swofford et al., 1 996). 

It is unusual that clustering and phyloge- 
netic techniques of additive trees give such 
congruent results. As a rule, when the dis- 
tances are short and differentiation in general 



is weak, each technique applied will give such 
different results that it does not make sense to 
compute a consensus tree, because too much 
information will then be lost. In Melanopsis, 
however, though the differences are small, the 
different techniques all yield exactly the same 
results. Thus, at least M. costata costata, M. 
costata jordanica, and M. saulcyi, each mor- 
phologically distinct, appear also allozymati- 
cally as distinct biological units, though very 
close to each other. The level of molecular dif- 
ferences between them suggests that they 
speciated not long ago, or even that the speci- 
ation process is still not completed. The latter 
possibility, as stated above, is a good expla- 
nation of those species being not "good". 
Whatever the taxonomic decision, it is evident 
that in the studied case, the allozymic differ- 
entiation accompanies differentiation in shell 
form, thus our null hypothesis of a single, con- 
chologically variable species must be re- 
jected. The two putative subspecies of M. 
costata axe indeed allozymatically, most close 
to each other. As concerns M. meiostoma, its 
species distinctness cannot be confirmed by 
the present data: more specimens and more 
loci must be studied to confirm this. In all trees, 
M. me/ostoma falls within M. saulcyi. 

The most problematic case is M. bucci- 
noidea. Its smooth shell is very characteristic, 
but specimens are found in which the shell 
has weak costae, suggesting hybridization 
with other, ribbed species. Again, it can be in- 
terpreted as a proof of the conspecifity of all 
the Melanopsis populations studied. How- 
ever, conspecifity would hardly result in such 
clearly and univocally distinguishable allozy- 
matic groups of populations as M. saulcyi and 
M. costata. A better explanation of the ob- 
served pattern of distribution of the popula- 
tions of M. buccinoidea in trees and plots is 
that M. buccinoidea is a distinct species, with 
perhaps some level of hybridization with other 
species, and some admixture of hybrids in the 
studied populations (some level of genetic in- 
trogression, which nevertheless cannot be 
proved without genetic markers). However, 
we emphasize again that M. buccinoidea is 
notwithstanding far more variable than all the 
other taxa together. Its wide variability, includ- 
ing all the alleles found in the studied taxa, 
indicates rather that it is not a phenotypic, 
thus polyphyletic group consisting of smooth, 
unribbed forms of both M. costata and M. 
saulcyi. 

Perhaps the most consistent hypothesis 



322 



FALNIOWSKI ETAL. 



with our data is that M. buccinoidea, with its 
high genetic variability, is the ancestral 
species from which the other species in this 
study eventually speciated: M. costata (with 
two subspecies diverging later), and M. 
saulcyi. Genetic variation in each of the pre- 
sumed daughter species is much narrower 
than in M. buccinoidea. which suggests a 
founder effect (Barton. 1989) and the al- 
lopatric speciation of peripheral isolates. On 
the other hand, the founder effect must not 
have been too severe, as the high levels of 
polymorphism are still maintained in the 
daughter species. Later, M. buccinoidea be- 
came sympatric with its daughter species. 
Speciation in Melanopsis. as in Hydrobia 
(Davis. 1993). is perhaps a morphostatic one, 
with little differentiation in morphology, as well 
as in allozymes and ecological niches. Such 
speciation seems not uncommon in gas- 
tropods (Falniowski, 1987, 1992; Giusti & 
Manganelli, 1992). On the other hand, speci- 
ation might have been sympatric, with selec- 
tion restricting the range of genotypic, thus 
also phenotypic variation, which resulted in 
the restricted ranges of environmental vari- 
ability expenenced by the taxa. As concerns 
the potential hybridization, our study has not 
been designed adequately to answer the 
question. However, even if the hybridization 
occurs, it has not resulted in merging the ge- 
netic pools of the studied taxa. Thus, its pos- 
sible occurrence does not reject the species 
distinctness of M. buccinoidea. 



ACKNOWLEDGMENTS 

The molecular work was supported by a 
grant from the Polish Committee of the Scien- 
tific Research BW/IZ/UJ/98 to A. Falniowski, 
and a travel grant from the Hebrew University 
of Israel to J. Heller. 



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FALNIO