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VOL. 39, NO. 1-2 1998
MALACOLOGIA
International Journal of Malacology
Revista Internacional de Malacologia
Journal International de Malacologie
Международный Журнал Малакологии
Internationale Malakologische Zeitschrift
Vol.
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Vol.
Vol.
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Publication dates
Vol. 28, No. 1-2
19 Jan.
28 Jun.
16 Dec
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29 Dec
28 May
30 Nov.
7 Jun.
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1990
1991
6 Sep. 1991
9 Sep
14 Jul.
2 Dec
8 Jan.
13 Nov.
8 Mar.
17 Dec
. 1992
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VOL. 39, NO. 1-2 MALACOLOGIA
CONTENTS
В. А. D. CAMERON & L. M. COOK
Forest and Scrub Snail Faunas from Northern Madeira ........................
KATHERINE COSTIL & STUART E. R. BAILEY
Influence of Water Temperature on the Activity of Planorbarius Corneus (L.)
(BulmonmatasPplanerbidae)e ос
MARIA GABRIELA CUEZZO
Cladistic Analysis of the Xanthonychidae (= Helminthoglyptidae) (Gastropoda:
Stylommatophora:.Helicoidea)" =... ere ees o alo e ae He ee Ros Coe ees Soe
CHRISTOPHE DESBUQUOIS & LUC MADEC
Within-Clutch Egg Cannibalism Variability in Hatchlings of the Land Snail Helix
Aspersa (Pulmonata: Stylommatophora): Influence of Two Proximate Factors ......
GREGORY P. DIETL & RICHARD R. ALEXANDER
Shell Repair Frequencies in Whelks and Moon Snails from Delaware and Southern
Newest nao ati a le GG is SER din mens ehe
ROBERT T. DILLON, JR. & CHARLES LYDEARD
Divergence Among Mobile Basin Populations of the Pleurocerid Snail Genus,
Leptoxis, Estimated by Allozyme Electrophoresis ............................
ALEJANDRA L. ESTEBENET
Allometric Growth and Insight on Sexual Dimorphism in Pomacea Canaliculata
(Gastropoda: Ampullariidae) .. 2... 0... eee eee
BENJAMIN J. GOMEZ, АМА М. ZUBIAGA, М. TERESA SERRANO, & EDUARDO ANGULO
Histochemical and Ultrastructural Identification of Biphasic Granules in the Albumen
Secretory Cells of Arion Subfuscus (Gastropoda, Pulmonata) ..................
SANDRA GORDILLO Y SANDRA N. AMUCHASTEGUI
Estrategias de Depredaciön del Gaströpodo Perforador Trophon Geversianus (Pallas)
(IMürleoldea: lrophOnidae) #2 Fm ote ed ot eee tacit ks aad dsm eas
MARY ELLEN HARTE
Translating Trees into Taxonomy within Veneridae (Bivalvia): A Critique of Two Recent
A ee ee ee ee
J. W. HAWKINS, M. W. LANKESTER, & В. В. A. NELSON
Sampling Terrestrial Gastropods Using Cardboard Sheets .....................
ALEXANDER |. KAFANOV & ANATOLY L. DROZDOV
Comparative Sperm Morphology and Phylogenetic Classification of Recent Mytiloidea
ЕЕ и
MATTY KNIGHT, ANDRE N. MILLER, NEIL S. М. GEOGHAGEN, FRED A. LEWIS,
& ANTHONY R. KERLAVAGE
Expressed Sequence Tags (ESTs) of Biomphalaria Glabrata, an Intermediate Snail
Host of Schistosoma Mansoni: Use in the Identification of RFLP Markers .........
RICHARD M. LEBOVITZ
The Inheritance of an Embryonic Lethal Mutation in a Self-Reproducing Terrestrial
Slug; Déroceras Laeve 4,4: dvr Rene оон оао ое ned оный
CHARLES LYDEARD, JOHN H. YODER, WALLACE E. HOLZNAGEL, FRED G. THOMPSON,
& PAUL HARTFIELD
Phylogenetic Utility of the 5'-Half of Mitochondrial 16S rDNA Gene Sequences for
Inferring Relationships of Elimia (Cerithioidea: Pleuroceridae) ..................
CHARLES S. RICHARDS, CAROLYN PATTERSON, FRED A. LEWIS, & MATTY KNIGHT
Larval Fusion and Development of Conjoined Teratoids in Biomphalaria Glabrata .. .
KEVIN J. ROE & CHARLES LYDEARD
Molecular Systematics of the Freshwater Mussel Genus Potamilus (Bivalvia:
URIONIdAS) EE Re ia es wid eeu Syd oe foes oh nym Siete ye Coste subs Bene dede
PETER D. ROOPNARINE
Translating Trees into Taxonomy within Veneridae (Bivalvia): А Reply to Harte ......
LUIZ RICARDO L. SIMONE
Morphology of the Western Atlantic Haliotidae (Gastropoda, Vetigastropoda) with
Description of a New Species from Brazil ..................................
LAURA R. WHITE
Corrections to White et al., 1996, Molecular Genetic Identification Tools for the
Unionids of French Creek, Pennsylvania Malacologia 38:181-202 ...............
J.B. WOOD, E. KENCHINGTON, & В. К. O'DOR
Reproduction and Embryonic Development Time of Bathypolypus Arcticus, A Deep-
Sea Octopod (Cephalopoda: Octopoda) ...................................
ANA MARIA LEAL-ZANCHET
Comparative Studies on the Anatomy and Histology of the Alimentary Canal of the
Limacoidea and Milacidae (Pulmonata: Stylommatophora) .....................
1998
141
93
173
151
113
207
FT
83
129
175
21
183
123
195
221
59
225
11
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MALACOLOGIA, 1998, 39(1-2): 1-9
SAMPLING TERRESTRIAL GASTROPODS USING CARDBOARD SHEETS
J. W. Hawkins, М. W. Lankester & В. В. A. Nelson
Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario,
P7B 5E1, Canada
ABSTRACT
Cardboard sheets are an efficient way of collecting large numbers of terrestrial gastropods and
are useful for estimating relative densities and determining species composition of snails and
slugs active on the surface of the forest floor, provided sampling is conducted under optimal
weather conditions. Sheets may be less reliable, however, for quantitatively assessing the sub-
terranean component of gastropod communities. Cardboard sheets placed on the forest floor
sampled approximately 1/50 the number of gastropods estimated, using soil cores, to be in the
upper 10 cm of soil beneath the sheets. The numbers collected by the two methods were not cor-
related (p = 0.96), but cardboard sheets produced up to 30 times as many specimens per unit of
sampling time. Gastropod numbers beneath cardboard sheets peaked at near-ground tempera-
tures around 15°C and were augmented by animals moving horizontally over the surface of the
litter from the surrounding area and vertically from the underlying soil, particularly when condi-
tions were wet. Although mean ( + S.E.) gastropod densities determined using sheets allowed
to weather for a year (27.3 + 4.1m“) and new sheets (16.2 + 2.6 п?) were not significantly dif-
ferent (p = 0.09), larger samples might confirm a tendency to prefer weathered sheets. In addi-
tion, 4 of 20 species were collected in greater numbers on weathered sheets, suggesting differ-
ential use of cardboard sheets by those species.
Key words: terrestrial gastropods, sampling, cardboard sheets, horizontal movement, temper-
ature.
INTRODUCTION
Corrugated cardboard sheets have fre-
quently been used in parasitological studies
to collect terrestrial gastropods that serve as
intermediate hosts of metastrongyloid nema-
todes (Lankester & Anderson, 1968; Gleich &
Gilbert, 1976; Kearney & Gilbert, 1978; Up-
shall et al., 1986; Beach, 1992; Lankester &
Peterson, 1996). For this purpose, cardboard
sheets have provided a convenient, time-effi-
cient method of collecting large numbers of
snails and slugs, but whether sample data can
be used to accurately estimate gastropod
population densities and species composition
remains unknown.
Boag (1982) compared the use of card-
board sheets and hard masonite boards for
sampling terrestrial gastropods and con-
cluded that cardboard sheets were effective
for repeatedly sampling the same area over
time but that the technique was subject to po-
tential limitations. He suspected that card-
board sheets may not sample all gastropod
species and life stages equally and that hori-
zontal movement to the sheets may bias den-
sity estimates. In addition, Boag (1990) sus-
pected that sheets may become progressively
more attractive to gastropods the longer they
are exposed to the weather.
Our purpose was to evaluate some con-
cerns raised about using cardboard sheets
to estimate population densities and deter-
mine the species composition of terrestrial
gastropod communities. We tested the hy-
pothesis that the number of gastropods col-
lected using cardboard sheets is correlated
with that deeper within the underlying litter
and soil. In addition, we investigated whether
gastropods migrate horizontally across the
surface and accumulate beneath cardboard
sheets, thereby inflating population estimates.
The relationship between temperature and
the number of gastropods collected was ex-
plored, and collections from new and weath-
ered cardboard sheets were compared.
MATERIALS AND METHODS
Two study sites, in recently restocked
spruce (Picea spp.) plantations, were located
in Fraleigh Township (48°08'N, 89°49'W),
about 60 km southwest of Thunder Bay,
2 HAWKINS, LANKESTER & NELSON
Ontario, within the Great Lakes-St. Lawrence
Forest Region (Rowe, 1972). Site 1 was ona
south facing slope, had shallow, sandy loam
soils and deciduous regrowth consisting pri-
marily of trembling aspen (Populus tremu-
loides) and mountain maple (Acer spicatum).
Site 2 was also on a south facing slope but
had deep, silty soils and regrowth consisting
of trembling aspen, willow (Salix spp.), alder
(Alnus spp.), fireweed (Epilobium angusti-
folium), raspberry (Rubus spp.), and white
birch (Betula papyrifera). Additional informa-
tion on the study sites can be found in Bell et
al. (1996).
Terrestrial gastropods were collected using
unwaxed, corrugated cardboard’ sheets
(80 x 107 cm) placed on the surface of the for-
est floor and weighed down with rocks.
Gastropods located beneath and on top of
each cardboard sheet were identified, with the
aid of Pilsbry (1939-1948), Oughton (1948),
and Burch (1962), counted and removed.
Species too small to be identified in the field
were preserved in glycerin alcohol and identi-
fied later under a dissecting microscope at
16x. Voucher specimens were sent to the
Royal Ontario Museum, Toronto, where iden-
tifications were confirmed.
To determine whether the number of gas-
tropods collected using cardboard sheets was
correlated with that in the soil beneath them,
10 cardboard sheets, in place for five days on
site 1, were examined for gastropods on
September 14, 1993. Immediately thereafter,
three soil cores (8 x 7 x 10 cm) were taken at
random locations from beneath each sheet.
The top and bottom halves (5 cm each) of
each core were placed in separate bags, la-
belled, and stored frozen at -5°C for 2-20
days. Before freezing, an attempt was made
to collect any slugs present in the soil samples
by placing fresh lettuce leaves in the bags and
leaving them overnight in a controlled envi-
ronment chamber at 20°C. The inner surface
of the bag and the lettuce were then inspected
for slugs and snails that moved out of the soil
sample.
Thawed soil samples were placed in an
enamel dissection tray (25 x 45 x 6 cm) half-
filled with water and visually examined for
gastropods. Following this preliminary visual
search, samples were washed through a se-
ries of sieves (Smallest = mesh No. 120, 0.125
mm opening) and the remaining material ex-
amined for the presence of gastropods under
a dissecting microscope (6x). Gastropods
without a visible tissue mass within the shell
were considered dead at the time soil cores
were collected and were not counted in den-
sity estimates. In the case of smaller speci-
mens, this was determined with the aid of a
dissecting microscope.
An experiment was designed to determine
whether cardboard sheets sample only those
gastropods residing in the litter and soil di-
rectly beneath them, or if collections also in-
clude animals moving laterally from the sur-
rounding area. Ten pairs of cardboard sheets
were placed randomly at approximately 10 m
intervals on the ground vegetation at site 2.
Each pair consisted of one sheet enclosed
tightly by a strip of sheet metal (22 gauge, 12
cm high) penetrating 7 cm into the soil and
protruding 5 cm above the soil surface, plus a
control sheet (unenclosed), placed 4 m away.
The above-ground surface of the sheet
metal barrier was coated with automotive
bearing grease and covered with a mixture of
coarse grain, black pepper and cayenne pep-
per (2:1) to discourage gastropods from
crawling over it. All overhanging ground vege-
tation was removed. Pairs of cardboard
sheets were sampled simultaneously for gas-
tropods, between 0800 hrs. and 1000 hrs.,
every 2-3 days from July 22 to August 18,
1994, for a total of 11 sampling days. Gastro-
pods were removed and each sheet was re-
turned to its original position.
The relationship between temperature and
numbers of gastropods collected beneath
cardboard sheets was examined. At each of
30 cardboard sheets on site 2, three type-T
copper-constanten thermocouple leads were
positioned, one 2 cm above the sheet to mea-
sure ambient air temperature, one directly be-
neath, and one 2 cm deep in the litter beneath
the sheet. Thermocouple temperatures were
measured, using a Cole-Parmer digital ther-
mometer (Model 08500-41), five times
throughout the summer of 1994 (0700-1100
hrs.), immediately before collecting all gas-
tropods on the sheet.
An experiment designed to determine if
weathered cardboard sheets sample more
gastropods than new sheets was initiated in
early June 1993. Ten cardboard sheets were
randomly placed on the ground vegetation at
site 2 and sampled five times over the sum-
mer of 1993 as part of a larger gastropod pop-
ulation study (Hawkins et al., 1996). All were
left exposed to the weather over winter. On
May 2, 1994, a new cardboard sheet was po-
sitioned approximately 4 m from each weath-
ered sheet. At the same time, the weathered
SAMPLING TERRESTRIAL GASTROPODS 3
TABLE 1. Gastropods collected on cardboard sheets and from soil cores beneath sheets
Cardboard sheets*
Density (/m?)* Total
Species Total
Zonitoides arboreus ral
Discus cronkhitei 16
Striatura milium 16
Deroceras laeve 48
Vitrina limpida 29
Strobilops labyrinthica 14
Vertigo gouldi 13
Euconulus fulvus 13
Cochlicopa lubrica 0
Columella edentula 13
Zoogenetes harpa 0
Striatura exigua 2
Vertigo modesta 0
Carychium exile canadense 0
Total 235
Soil cores!
Density (/m?)*
8.3 + 92.1 103 613.1 = 1386
1.9 + 0.8 57 339.3 = 81.4
1907 49 291.7 + 70.1
5.6+ 1.0 1 6.0 + 6.0
ЗЕ 1.0 19 11334-23941
16038 9 53.6 + 24.2
1.5 + 0.8 5 29.8 + 23.9
1.5 = 0.5 3 1794127
0 14 83.3 + 33.4
1.5 = 0.5 0 0
0 7 41.7 + 19.9
0:2 + 0/2 1 6.0 + 6.0
0 1 6.0 + 6.0
0 1 6.0 + 6.0
27.5252 270 1607.1 + 272.3
*10 sheets of unwaxed corrugated cardboard (80 x 107 cm)
130 soil cores (8 x 7 x 10 cm)
“mean + S.E. (Im? of surface area)
sheets were moved approximately 1 m from
their over-winter position and any gastropods
adhering to the sheets were removed. Each of
the 10 new sheets was sampled, simultane-
ously with its corresponding weathered sheet,
four times during the 1994 field season (May
20, June 29, July 12, and July 25, 1994).
Simple linear regression (Neter et al., 1989)
was performed to determine whether any re-
lationship existed between density estimates
obtained using cardboard sheets and those
obtained using soil cores. Density estimates
from soil cores were used as the independent
variable. The null hypothesis of no difference
between enclosed cardboard sheets and con-
trol sheets was tested using the Wilcoxon
Rank Sum test (Bradley, 1968). A Kruskal-
Wallis analysis of variance (Bradley, 1968)
was performed to test for differences in gas-
tropod densities between the 11 sample peri-
ods. A repeated measures analysis of vari-
ance (Gumpertz € Brownie, 1993) was
performed to detect differences in near-
ground temperature between the three differ-
ent thermocouple positions. The dependent
variable in this analysis was normalized using
a square root transformation. The Wilcoxon
Rank Sum test was also used to test the null
hypothesis of no difference in the use of new
and weathered cardboard sheets by gas-
tropods. All statistical differences were con-
sidered significant at p < 0.05. Statistical pro-
cedures were performed on SPSS PC-6.1
(Norusis, 1992a, 1992b).
RESULTS
Cardboard Sheets vs. Soil Cores
The mean (+ S.E.) density of gastropods
active on the surface of the forest floor and es-
timated using the cardboard sheet method
was 27.5 + 5.2 т? whereas a mean (+ SE.)
of 1607.1 + 272.3 m” was estimated using
the soil core method to be in a 1 m x 1 m x 10
cm volume of soil (Table 1). Fourteen species,
including 13 snails and one slug (Deroceras
laeve), were collected. Eighty-eight percent of
gastropods collected from the soil cores were
found in the upper 5 cm of the soil and the re-
mainder in the bottom 5 cm. Linear regression
analysis revealed no correlation between the
densities of gastropods estimated using card-
board sheets and soil cores (F = 0.003; p =
0.958). Checking a cardboard sheet required
5-15 minutes with yields ranging from 7-49
gastropods per sheet; 3-4 hours were re-
quired to examine each soil core with a range
of 0-27 gastropods being recovered.
Barrier-Enclosed Sheets
Total mean (+ S.E.) gastropod density was
lower on sheets enclosed with a sheet metal
a HAWKINS, LANKESTER & NELSON
TABLE 2. Mean (+ S.E.) densities (/m?) of terrestrial gastropods collected from cardboard sheets
enclosed with a metal barrier and control sheets (unenclosed)
Barrier enclosed sheets*
Deroceras laeve alkers=l0) 2
Euconulus fulvus 0.23 + 0.060
Columella edentula 0.12 + 0.034
Zonitoides arboreus 0.14 + 0.047
Striatura milium 0.12 + 0.062
Succinea ovalis 0.06 + 0.025
Vitrina limpida 0.03 + 0.018
Vertigo gouldi 0.02 + 0.015
Strobilops labyrinthica 0.04 + 0.021
Cochlicopa lubrica 0.02 + 0.015
Discus cronkhitei 0.03 + 0.018
Pallifera dorsalis 0
Zoogenetes harpa 0.04 + 0.026
Vertigo ovata 0.02 + 0.015
Anguispira alternata 0.01 + 0.011
Gastrocopta tappaniana 0
Total 2.07 + 0.228
Control sheets* p-value!
1.90 + 0.280 p = 0.097
0.20 + 0.058 р = 0.472
0.27 = 0.076 p = 0.328
0.23 + 0.056 p = 0.077
0.04 + 0.026 p = 0.462
0.09 + 0.036 p = 0.974
0.10 + 0.034 p=0.121
0.09 + 0.033 p = 0.088
0.02 + 0.015 p = 0.409
0.04 + 0.021 р = 0.409
0.02 + 0.015 р = 0.652
0.04 + 0.034 р = 0.156
0 р = 0.082
0 р = 0.156
0 p = 0.317
0.01 + 0.011 p = 0.317
3.05 + 0.349 p = 0.038
*sampled with removal 11 times between July 22 and August 18, 1994
p-values based on a Wilcoxon Rank Sum Test
barrier (2.1 + 0.2 m”*) than on control sheets
(3.1 + 0.4 т?) (р = 0.038), although no dif-
ference was detected when individual gastro-
pod species were considered (Table 2).
Cumulative totals of 195 and 287 gastropods
were collected from enclosed and control
cardboard sheets, respectively, over the
course of the experiment. Pallifera dorsalis
and Gastrocopta tappaniana were not col-
lected from the sheets enclosed with a bar-
rier; Zoogenetes harpa, Vertigo ovata, and
Anguispira alternata were not collected from
the control sheets. The mean density of snails
and slugs, from both enclosed and control
cardboard sheets, varied over the 11 sample
periods (p < 0.0001) (Fig. 1). Over the first five
collection days, mean (+ S.E.) gastropod
density was lower under enclosed (2.0 + 0.21
m?) than control sheets (3.9 + 0.26 т?)
(p = 0.0029). However, densities increased
sharply under both, and particularly under
control sheets, following heavy rains on
August 3rd and 7th totalling 35.2 mm and on
August 16 following 3.8 mm of rain (Fig. 1).
Temperature Beneath Sheets
Gastropod collections peaked when the
temperature beneath cardboard sheets was
approximately 15°C and decreased at lower
and higher temperatures. Temperature was
more variable on clear days (4°C to 30°C, x =
13.5 + 0.56) than on overcast days (11°C to
22°C, x= 15.6 = 0.33).
Mean (+ S.E.) temperature beneath the
cardboard sheets (14.4 + 0.4°C) was slightly
cooler than that 2 cm above the forest floor
(15.4 + 0.6°C) throughout July, while both
were similar in August. However, interaction in
temperature among the three thermocouple
positions was observed over the summer (F =
4.99; p < 0.05). Throughout July, the tempera-
tures 2 cm above the forest floor and directly
beneath (0 cm) the cardboard sheets were
higher than those measured 2 cm deep in the
humus layer but, for the first half of August, the
reverse was true.
Weathered Sheets
Total mean (+ S.E.) gastropod densities on
new (16.2 + 2.6 т?) and weathered (27.3 +
4.1 m”) cardboard sheets were not signifi-
cantly different (p = 0.09). However, of the
twenty species of terrestrial gastropod col-
lected (Table 3), Euconulus fulvus, Vertigo
gouldi, Carychium exile canadense, and
Striatura milium were present in greater den-
sities on the weathered sheets than on the
new sheets.
DISCUSSION
Results reported here demonstrate that the
number of terrestrial gastropods present
within the litter and underlying 10 cm of soil is
more than 50 times as great as the number of
SAMPLING TERRESTRIAL GASTROPODS 5
> A. Slugs
Ш Enclosed
® Control
6
5
“E
a 4
E 3
2
1
A E SCS
ES ASS ww
У т т
Sample date
> B. Snails
Ш Enclosed
6 Control
5
E 4
>
5 3
A
2
1
y $. ло + AN
% xv Vv N . o Y
N «У < «У > ys 5 e e yw pe $
Sample date
FIG. 1. Mean (+ S.E.) densities of gastropods removed from cardboard sheets enclosed with a metal barrier
and control sheets (unenclosed), on 11 sample days, July 22 to August 18, 1994. [R = rain greater than 3
mm]. (A) Slugs. (B) Snails.
HAWKINS, LANKESTER & NELSON
TABLE 3. Mean (+ S.E.) densities (/m°) of terrestrial gastropods collected from beneath new
and weathered cardboard sheets
Species New sheets* Weathered sheets* p-value!
Zonitoides arboreus 5.12 + 1.03 6.53 + 1.04 р = 0.187
Strobilops labyrinthica 1.85 + 0.46 4.51 + 1.08 p = 0.072
Euconulus fulvus 1.71 + 0.42 4.51 + 0.85 р = 0.001
Deroceras laeve 2.72 + 0.49 2.23 + 0.35 p=0.712
Vitrina limpida 1.30 + 0.49 1.10 + 0.45 p = 0.889
Discus cronkhitei 0.40 + 0.14 1.30 + 0.38 p=0.110
Vertigo gouldi 0.35 + 0.18 1.10 + 0.36 р = 0.018
Carychium exile canadense 0.09 + 0.09 1.24 + 0.38 p < 0.001
Striatura milium 0.20 + 0.15 1.07 + 0.35 p = 0.008
Succinea ovalis 0.75 + 0.28 0:52 = 0.12 р = 0.665
Columella edentula 0.46 + 0.19 0.78 + 0.32 p = 0.703
Anguispira alternata 0.29 + 0.12 0.69 + 0.29 р = 0.631
Striatura exigua 0.23 + 0.12 0.64 + 0.24 p = 0.278
Cochlicopa lubrica 0.23 + 0.08 0.52 + 0.30 p = 0.863
Gastrocopta tappaniana 0.06 + 0.04 0.20 + 0.07 p = 0.079
Vertigo ovata 0.14 + 0.08 0.12 + 0.09 p = 0.655
Vertigo modesta 0.14 + 0.10 0.06 + 0.04 p = 0.960
Zoogenetes harpa 0.06 + 0.04 0.06 + 0.04 p = 1.000
Punctum minutissimum 0.06 + 0.06 0.06 + 0.06 p = 1.000
Pallifera dorsalis 0.03 + 0.03 0.03 + 0.03 p = 1.000
Total 16.19 + 2.61 27.25 AAS p = 0.086
*sampled with removal four times throughout the summer of 1994 (May 20, June 29, July 12, and July 25)
p-values based on a Wilcoxon Rank Sum Test
snails and slugs active on the surface of the
forest floor and detectable using the card-
board sheet sampling technique. Although the
cardboard sheet and soil core sampling meth-
ods were not correlated, each provides a rel-
ative density estimate of a particular compo-
nent of the gastropod community, one active
on the surface of the forest floor and one pres-
ent within the litter and underlying soil. Kralka
(1986), working in the boreal forest of Alberta,
examined 5 cm deep soil cores and estimated
a total mean gastropod density of 80 m?, with
maximum densities of Discus cronkhitei and
V. gouldi reaching 340 т? and 460 m, re-
spectively. Estimates of gastropod population
densities in boreal forests using cardboard
sheets have ranged from 2-38 m? (Kearney
& Gilbert, 1978; Hawkins, 1995; Lankester &
Peterson, 1996).
Cardboard sheets provided a more time-
efficient method of collecting terrestrial gas-
tropods, yielding up to 30 times as many
specimens as found in soil cores in similar
time periods. In addition, the majority of gas-
tropods found adhering to sheets can be iden-
tified in the field, with the exception of smaller
species, such as Vertigo spp. and Columella
spp., which must be identified beneath a mi-
croscope. Soil cores, however, not only in-
volve a greater effort in the field but also re-
quire considerably more time to extract speci-
mens from the samples in the laboratory.
When using cardboard sheets to sample
terrestrial gastropods, horizontal movement
across the surface of the litter could affect the
number of snails and slugs collected. With re-
peated sampling and removal, the mean num-
ber of gastropods collected beneath both bar-
rier enclosed and control sheets would be
expected to decline if no horizontal movement
of gastropods towards the sheets were occur-
ring. However, if individuals were continually
immigrating from the surrounding area, the
mean density of individuals found beneath the
enclosed sheets should fall to a lower level
than that under control sheets. In fact, this was
observed over the first five collection days.
The mean density recovered beneath en-
closed sheets (2.0 + 0.21 т?) was half that
from unenclosed sheets (3.9 + 0.26 т?) sug-
gesting that horizontal movement does occur.
Numbers increased, however, under both en-
closed and control sheets following separate
rainfalls totalling 35 mm and 4 mm. Although
the increase on both occasions was greatest
beneath the control sheets, indicating that
some increase in horizontal movement had
probably occurred, an increase beneath the
enclosed sheets suggests that individuals
also moved vertically in response to the wet-
SAMPLING TERRESTRIAL GASTROPODS 7
ter conditions (Locasciulli & Boag, 1987). A
more direct demonstration of horizontal move-
ment was provided by Boag (1990), who
marked and released snails beneath ma-
sonite boards and observed that 5% of D.
cronkhitei and 12% of E. fulvus moved from
beneath the boards onto the surrounding lit-
ter.
The extent to which gastropods might actu-
ally be attracted to cardboard sheets has not
been determined, yet there is some empirical
evidence that they do accumulate beneath
more permanent sampling structures (Boag,
1990). Snails and slugs would accumulate be-
neath cardboard sheets if more individuals
move under a sheet than leave, implying that
conditions beneath cardboard sheets are
generally more favourable than those encoun-
tered on the surrounding forest floor. These
animals can be observed moving openly
across the surface of the litter at night and on
overcast and rainy days until moisture, tem-
perature, and/or light conditions become
unfavourable (Boag, 1985; personal observa-
tion). Under natural daily conditions of in-
creasing light intensity and decreasing humid-
ity, their most direct route to refuge would be
downward into the litter and soil. Conditions
that discourage gastropod movement on the
surface may be delayed in onset and be less
severe beneath a cardboard sheet. However,
sheets seldom remain suitable refuge for
long. They dry readily in the sun, and the veg-
etation beneath them dies back if left covered
for more than 2-3 weeks. In some circum-
stances, the litter beneath a cardboard sheet
may be drier than its surroundings, for exam-
ple if a sheet has been put in place before a
light rain or heavy dew occurs. This expected
variability can best be minimized by placing
already dampened sheets in a new location
during, or immediately after, a rainy period,
and by checking the sheets within a few days
and only in the early hours of the morning.
The temperature beneath cardboard sheets
influences the number of gastropods that can
be collected using this method. The mean
temperature immediately beneath the card-
board sheets was slightly lower than the air
temperature 2 cm above them throughout
July, suggesting that the shading effect of the
sheets keeps temperatures cooler than those
on the surrounding forest floor. Collections
were greatest when the temperature beneath
the sheets was approximately 15°C. Boag
(1990) reported the greatest number of gas-
tropods beneath masonite boards at ambient
air temperatures between 7.5°C and 17.5°C
and direct observation of snails in terraria in-
dicated greater activity on the surface of the
litter at temperatures ranging from 6°C to
15°C (Boag, 1985). Snails and slugs most
likely take refuge deeper in the litter and un-
derlying soil when temperatures reach lower
or higher extremes. The time of day and
amount of cloud cover will clearly play a role in
influencing the temperature beneath the
sheets. On sunny, hot days, favourable tem-
peratures beneath the sheets will only prevail
for a limited period of time early in the morn-
ing. On overcast days, however, daytime tem-
peratures will remain cooler and larger num-
bers of gastropods are likely to be found
beneath cardboard sheets later into the day.
The apparent relationship between tempera-
ture beneath the sheets and the number of
gastropods collected should be considered
when using this technique in field studies of
terrestrial gastropods.
Overall, mean densities of gastropods col-
lected from weathered and new cardboard
sheets were not significantly different. How-
ever, the low power of the test (n = 10) anda
reasonably large difference between the mean
(+ S.E.) densities (weathered sheets = 27.3 +
4.1m *;newsheets = 16.2 + 2.6m ©), suggest
that snails and slugs may in fact favour weath-
ered sheets. Four species (E. fulvus, V. gouldi,
C. exile canadense, and S. milium) may pref-
erentially use weathered sheets. Weathered
sheets appeared to absorb more moisture and
retain it longer following a rain. A similar in-
crease in use of weathered masonite boards
was seen by Boag & Wishart (1982) and Boag
(1990). It was suggested that boards exposed
for long periods to the elements may become
more attractive as a result of having dissipated
any possible repellant chemicals, or because
they acquire fungal hyphae and slime trails on
their lower surface. Boag (1990) also sug-
gested that various gastropod species may
use sampling boards differentially, which may
explain, in part, the greater numbers of four
species collected from weathered sheets and
underscores a potential limitation of using
cardboard sheets to estimate relative numbers
and densities of terrestrial gastropods.
We conclude that the cardboard sheet sam-
pling technique is a time-efficient method of
collecting large numbers of terrestrial gas-
tropods that otherwise become difficult to find
in daylight hours. If sampled under optimal
weather conditions, cardboard sheets provide
an acceptable method of quantifying the rela-
tive abundance and species composition of
gastropods active on the surface litter. This
8 HAWKINS, LANKESTER & NELSON
method allows comparison between gastro-
pod communities in different locations and
habitats provided collections are made under
similar weather conditions and in the same
season to account for different reproductive
life histories (Comfort, 1957; Berry, 1966;
Uminski & Focht, 1979; Livshits, 1983). Card-
board sheets are less reliable, however, for
quantitatively assessing the subterranean
component of gastropod communities.
ACKNOWLEDGMENTS
We gratefully acknowledge funding pro-
vided for this work by the VMAP (Vegetation
Management Alternatives Program) under the
Sustainable Forestry Initiative, Ontario Minis-
try of Natural Resources, Sault Ste. Marie,
Ontario. We thank Jackie Hrabok and Cam
Oomen for assisting with gastropod collection
and Karen Watt for helping with the prepara-
tion of the manuscript.
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Hobbes
REPRODUCTION AND EMBRYONIC DEVELOPMENT TIME OF BATHYPOLYPUS
ARCTICUS, A DEEP-SEA OCTOPOD (CEPHALOPODA: OCTOPODA).
J. В. Wood", E. Kenchington'* & В. К. O’Dor'
. Worse, т terms of outright scariness, Are the suckers multifarious ...
ABSTRACT
Mating, brooding, and embryonic development rate of Bathypolypus arcticus, a deep-sea ос-
topod, are described. Live specimens of B. arcticus were collected in the Bay of Fundy, Canada,
and kept in a flow-through system in the lab. Two of the octopods laid and brooded viable eggs.
Brooding and embryological development took over a year at average temperatures of 7.3°C and
7.8°C. Brooding females ate occasionally and left their eggs shortly before dying. Hatchlings
weighed 208 + 17 (SD) mg from the first batch and 283 + 20 (SD) mg from the second batch.
There was no evidence of multiple spawning.
Mating of B. arcticus was also observed. The usually smaller male sits upon the female, en-
veloping much of the female’s mantle in his web, and he inserts his large ligula into her mantle.
One or two large spermatophores are transferred by a combination of mantle pumping and arm
groove peristalsis. A filmed mating sequence lasted 140 seconds.
Key words: cephalopod, Octopoda, Octopodidae, Bathypolypus arcticus, deep-sea, mating,
Bill Watterson, Calvin and
embryonic development, brooding.
INTRODUCTION
Bathypolypus arcticus (Prosch, 1849) is a
small octopodid that rarely exceeds 200 g
(O’Dor & Macalaster, 1983). This species has
been found to depths of 1,543 m (Voss,
1988a,b) and is classified as a deep-sea oc-
topus by Voss (1988b). Bathypolypus arcticus,
most common at depths of 200-600 m, is
widely distributed in the Atlantic Ocean (O’Dor
& Macalaster, 1983). Assuming the three-year
life span estimated by O’Dor & Macalaster, fe-
males brood eggs for a larger percentage of
their life than any octopus studied thus far,
with the possible exception of the iteroparous
Octopus chierchiae (Jata, 1889) (Rodaniche,
1984). Like all deep-sea octopods, female B.
arcticus lay large eggs, from which well-devel-
oped young hatch.
Mature male B. arcticus have the largest
ligula relative to body size of any octopodid.
The ligula is part of the hectocotylus, the mod-
ified third right arm that males use to transfer
their equally large spermatophores while mat-
ing. Mating has not been previously described
for B. arcticus—what males do with their hec-
tocotylus was unknown. Two mating positions
have been observed in the family Octo-
podidae: a distant position in which the male
and female are separated except for the hec-
tocotylus, and one in which the male mounts
the female (Mangold, 1987). Mangold notes
that mating may last from a few minutes to
several hours.
As much as there is to be learned from
cephalopods we can easily obtain, perhaps
even more is yet to be learned from species
that dwell in the deep-sea (Forsythe & Van
Heukelem, 1987). Little laboratory work has
been done with deep-sea cephalopods due to
difficulties in collecting undamaged live spec-
imens and continually providing cold water.
The only previous laboratory information on
brooding and embryonic development of
deep-sea octopods is from O’Dor & Macal-
aster (1983). They reported that a single fe-
male B. arcticus laid eggs in August 1978 and
brooded them until they hatched in July 1979.
They state that the temperature varied be-
tween 3°C and 10°C, but temperatures were
not recorded. The female was essentially ig-
nored until the spring of 1979 when the eggs
‘Biology Department, Dalhousie University, Halifax, Nova Scotia ВЗН 4J1, Canada
“Science Branch, Department of Fisheries and Oceans, P.O. Box 550, Halifax, Nova Scotia, Canada
12 WOOD, KENCHINGTON & O’DOR
were discovered to be developing. She only
had 4 of 40 eggs left in July 1979 and was not
offered food while brooding. This female B.
arcticus died shortly after her eggs hatched.
Very little is known about the life history of
deep-sea octopods. This report describes the
first detailed observations of mating, brood-
ing, and embryonic development time of Ba-
thypolypus arcticus.
METHODS
Eighteen B. arcticus were collected from the
Bay of Fundy off Digby (circa 44.70°N and
65.90°W) on June 8-21 and below Brier
Island (circa 43.80°N and 66.30°W) on
August 22-September 1, 1994. The FRV J. L.
Hart, a 20 m trawler belonging to the
Department of Fisheries and Oceans (DFO),
was used. Animals were collected in scallop
trawls as incidental catch during DFO scallop
stock surveys. The majority of animals came
from tows at depths of 75-100+ m. While on
the boat, specimens were housed in a
portable cooler for as long as a week and kept
at temperatures below 10°C. Additional spec-
imens were collected in the same manner in
the summer of 1995.
The B. arcticus collected in 1994 were kept
together in a flow-through system at DFO's
Halifax laboratory. The animals were housed
in a 91 by 91 cm fibreglass tank (internal di-
mensions). The walls of the tank were 4 cm
thick and contained chilling coils. Water depth
was 31 cm. Water temperature was controlled
by adjusting the amount of ambient and
heated water that entered the system.
Temperature varied with the incoming water
and user demand.
Initially, temperature was recorded with a
mercury thermometer (Fig. 1). In late De-
cember 1994, a min./max. thermometer was
added to the system to record temperature
fluctuations.
The octopoda collected in 1995 were kept
in two flow-through fiberglass tanks at
Dalhousie. Most of the females collected laid
fertile eggs. Temperature was recorded but is
not presented here. The number of eggs laid
in these fertile broods was assessed in May
1996 by removing the females and pho-
tographing their eggs with an underwater
camera. Eggs were much easier to count in
static photographs. (We knocked eight eggs
off when removing the females from their
broods, and these are included in the totals for
the appropriate females.)
During the first months after capture, the
octopodids were hand-fed live crustaceans
and mollusks removed from their shell, and
they were offered amphipods ad libitum. Hand
feeding consisted of bumping the food into
the octopuses arms. Later, sand shrimp
(Crangon) and brittle stars (ophiuroids) were
added т ad libitum quantities to the tank, and
polychaetes, mussels, crabs, amphipods, and
other small invertebrates were added as they
became available.
Octopodids that laid eggs were observed
and occasionally offered food by hand.
However, brooding octopodids were disturbed
as little as possible. Once the eggs started
hatching, brooding octopodids were filmed
with a 24-hour time-lapse VCR under red
light.
To measure mantle length (ML), mantle
width (MW), and interocular width (IOW) of
day-old individuals, they were filmed and mea-
surements were made with an Optimas Video
Analysis System. This system was used to re-
duce stress on the animals. Summers (1985)
used a somewhat similar method, and he
briefly discussed the reliability of using photo-
graphic size determination.
Wet weights +1 mg of 15 hatchlings from
each brood were obtained with a Mettler P163
scale. Members of the first batch of hatchlings
were dried with a tissue to absorb excess
water before being weighed. Many animals
weighed with this method died; several of the
dead hatchlings had tears in their skin.
Because this method damaged the hatchlings
and caused mortality, it was abandoned.
Individuals from the second batch were
weighed in a weighing tray with a micron
screen bottom. This tray was placed on a tis-
sue before being weighed to absorb excess
water. Preserved and recently dead juvenile B.
arcticus were weighed with both methods to
quantify the difference between the two meth-
ods. The second method produced results that
were 10.1% (n = 26) higher. Weights of the first
batch were converted so that they could be
compared to those of the second batch.
Mating and hatching was filmed by Dave
Gaudet (Halifax Cable) with a housed Sony
CCD 3-chip DX3 camera using high-8 format.
The primary autnor induced several eggs to
hatch by handling them and/or adding sugar
to the water.
RESULTS
Adult mortality was highest within the first
few weeks of capture. By September 1994, 12
of the 18 octopodids collected during the pre-
BATHYPOLYPUS ARCTICUS 13
Brooding and Development Temperatures
Temperature
0 60 120 180 240
Time (days)
300 360 420 480 540
FIG. 1. Average and standard deviation of temperatures at which brooding octopuses were kept. The num-
ber next to the average temperature is the sample size for that 60-day period. The horizontal lines show the
brooding period for the two females (1 = first inversion, 2 = second inversion, F = first hatchling, M = median
hatchling, L = last hatchling, D = death).
vious summer were still alive. Of those, eight
were still alive in September 1995. Of the 18
B. arcticus collected in 1994, only one was
male. However, in 1995 the sex ratio was
O0/50 (n= 32).
Average temperature during the brooding
period was 7.3°C for the first female and
7.8°C for the second female (Fig. 1). During
the experiment, the min./max. thermometer
recorded a minimum temperature of —1.8°C
on September 28, 1995, caused by a pump
failure. A similar problem that dropped the
temperature to 0.0°C occurred in fall 1994.
A maximum temperature of 16.7°C was re-
corded by the min./max. thermometer during
July 17-24, 1995. These extreme tempera-
tures probably lasted for only a short time.
On August 16, 1994, a female B. arcticus
was discovered brooding at least three eggs
that were laid the previous night. The female
refused food. There were about ten eggs on
August 17. The female laid more eggs by
August 22 and ate a few amphipods. Due to
our effort to disturb the female as little as pos-
sible while she was laying eggs, eggs were not
counted until several months later. Although
precise observations could not be made, no
noticeable additions of eggs took place after
two weeks. To count the eggs, it was neces-
sary to remove the female for a brief period.
Fifty-five eggs were counted, but only 54 were
accounted for at hatching. This discrepancy is
likely due to difficulties in counting the eggs
while keeping the female away from them.
Eggs were glued individually to the side of the
tank by the female. The glue was initially clear
but it turned green after a few days.
While brooding eggs, the female occasion-
ally ate food offered to her. She took fish pel-
lets, amphipods, Crangon, and crushed mus-
sels. Food was offered by hand-feeding as
described above.
Brooding females would sit on their eggs
and cover most of them with their web. They
often directed their funnel down, which pro-
vided a water current around the eggs. The
video tapes of brooding females revealed that,
although they occasionally moved slightly
away from the eggs, they would always keep a
few arms on the eggs while exploring the
perimeter around the brood with the other
arms. When a probe was used to try to get a
14 WOOD, KENCHINGTON & O’DOR
better look at the eggs, the female would ac-
tively defend them, sometimes by blasting jets
of water atthe probe and/or grabbing it.
An egg was taken on January 20, 1995, 158
days after the first eggs were laid, to deter-
mine if the batch was fertilized. Incirrate em-
bryos, except Argonautidae, flip position in the
egg twice during development (Boletzky,
1987). The egg was viable and the embryo
was in the first inversion. By July 31, 1995
(day 350), most of the embryos had flipped
the second time. At least one egg had not
flipped by August 8, 1995. On August 27,
1995 (day 377), the first octopus hatched. By
October 23, 1995 (day 434), all the octopo-
dids in the first batch had hatched, although
six of the last seven were induced to hatch so
they could be filmed (Figs. 2-5).
Eggs were 11 mm long and 6 mm wide be-
fore hatching. Hatchlings (n = 15) weighed
208 + 17 (SD) mg, had a mantle length of
7.71 + 0.49 (SD) mm, a mantle width of 7.14
+ 0.39 (SD) mm, and an interocular width of
2.30 + 0.20 (SD) mm. The median day of
hatching was October 14, 1995 (day 425),
and assuming the median day of egg laying
was one week after the first egg was laid, the
average octopus in the first batch took 419
days at an average temperature of 7.3°C. This
female died three days after the last egg in her
brood hatched.
On October 30, 1994, a slightly larger fe-
male started laying viable eggs in the lower
left corner of the same tank. Over 50% of
these eggs flipped the second time by
October 23, 1995 (day 359 from first laying of
eggs). The first egg in the second batch
hatched on or a few days before November 8,
1995 (day 375), and the last on December 30,
1995 (day 427) (Fig. 1). The median day of
hatching was December 17, 1995 (day 414).
The average octopus in the second batch took
407 days at 7.8°C to develop. This batch,
counted as they emerged, yielded 36 hatch-
lings with an average weight of 283 + 20 (SD)
mg (n = 15), mantle length of 8.91 + 0.43
(SD) mm, mantle width of 8.60 + 0.84 (SD)
mm, and an interocular width of 2.84 + 0.23
(SD) mm. The second female died two days
before her last egg hatched. Brooding behav-
iour was as noted for the first female, except a
few days before the second female died she
left her eggs, at one point for several hours,
and then returned to them. She was in very
bad condition by this time and appeared to be
having trouble breathing and orienting herself.
Both females moved away from the eggs
shortly before dying. Females were preserved
in formalin after they died. The preserved
specimens weighed 20.82 g and 17.55 g re-
spectively. The two females weighed an esti-
mated 30-40 g prior to laying eggs.
None of the hatchlings from either batch
had any of the outer yolk sac remaining upon
hatching. Video tapes revealed that hatchlings
are able to hang upside down from the water
surface (see Marliave, 1981; Van Heukelem,
1976). A variety of foods were offered to the
first batch of hatchlings. They were initially
hand-fed fresh mussel meat, and bits of gam-
maridean amphipods, mysid shrimp, and
Crangon septemspinosus. Plankton and live
gammaridean amphipods were offered to
some of the hatchlings. Later, they were of-
fered the small burrowing gammaridean am-
phipod Corophium volutator [ad libitum].
Hatchlings from the second batch were rarely
hand-fed and were offered С. volutator ad libi-
tum within a few days of hatching.
Several other females that were collected in
1994 laid eggs on the side of the tank or ina
clay pipe. These eggs disappeared after a few
months. An egg was removed from one of
these females. When this egg was examined
several months later, it was found to be unfer-
tilized. Presumably these batches were not vi-
able and the females ate them. A single male
was kept in the tank with the females.
Nine of the females that survived collection
in 1995 were brooding eggs in May 1996. All
of these broods were fertile, and seven were
able to be counted (the eighth and ninth octo-
pus laid eggs in a plastic pipe). Therefore, the
nine B. arcticus in this study laid 12, 13, 18,
19, 36, 48, 54, 89, and 105 fertile eggs. How-
ever, we estimate that none of these cephalo-
pods weighed 70 or more grams when they
laid their eggs. Macalaster (1976) reports that
70 g is the average size of mature females.
The following observations were made on
the 1995 females that laid eggs. Females
brooding their own eggs can be moved away
from their eggs and then returned to them.
Also, females that are brooding eggs can be
moved to care for another octopuses brood of
fertile eggs. While females don't differentiate
between their own fertile eggs and other
broods of fertile eggs, they seem to be able to
recognize infertile eggs, which we presume
they eat. One female was briefly observed to
be slightly out of arm reach of her eggs but
was rapidly moving back toward them. This
occurred shortly after amphipods were added
to the tank.
Male B. arcticus uses the mounting position
to mate. The smaller male initiated mating by
BATHYPOLYPUS ARCTICUS 15
FIGS. 2-5. Hatching. FIG 2. Swollen egg just prior to hatching. FIG. 3. During hatching the pressure in the
swollen egg pushes out the end of the mantle. FIG. 4. The octopus frees himself from the egg. FIG. 5. The
fully functional hatchling emerges from the egg. Photographs from high-8 video by D. Gaudet.
mounting the female and enveloping her man-
tle in his web (Figs. 7-9). He then inserted his
hectocotylus into the mantle of the female. Its
folded shape suggests that it was actually in-
serted into the oviduct to open it. During mat-
ing, the male contracts his mantle in a dramatic
pumping arch motion (Fig. 9); this may help the
large spermatophore pass to the funnel. We
presume that the spermatophore is then
passed from the funnel to the large grove on
the hectocotylus and that through peristalsis it
is transferred to the ligula which is in or on the
oviduct. The female remained motionless dur-
ing mating, which lasted 140 seconds. Bathyp-
olypus arcticus mates readily—specimens
collected the following year mated in the cooler
that they were kept in while still at sea. These
matings followed the pattern outlined above.
DISCUSSION
This study presents the first detailed data
on the brooding and development time for a
deep-sea octopodid. Bathypolypus arcticus
brood eggs for over 400 days, and if they live
16 WOOD, KENCHINGTON & O’DOR
FIGS. 6-11. Mating Bathypolypus arcticus. FIG. 6. The male octopus (foreground) sees the female octopus
and pounces on her (FIG. 7). FIG 8. He mounts her and inserts his ligula into her mantle cavity. FIG. 9. The
male stretches his mantle dramatically (this happened twice) presumably to help pump the spermatophore
to his ligula. FIG 10. The male removes his ligula and departs (FIG. 11). Illustrations by Rebekah McClean.
BATHYPOLYPUS ARCTICUS 17
for three years, as estimated by O’Dor &
Macalaster (1983), these octopodids spend
over a third of their life brooding. Additionally,
this study describes for the first time the mat-
ing behavior of B. arcticus. Mating in B. arcti-
cus is of interest because these little cephalo-
pods have the largest ligula relative to body
size of any octopod.
In this experiment, brooding behaviour and
developmental time were very consistent be-
tween the two females despite differences in
egg Size, female size, and time of laying. This
suggests that the length of the brooding pe-
riod is relatively fixed at a given temperature.
Brooding B. arcticus in this experiment were
kept at average temperatures of 7.3°C and
7.8°C, which is significantly higher than the
4 + 2°C SD that O’Dor & Macalaster (1983)
reported for wild specimens. Therefore, one
might expect B. arcticus to brood eggs for
even longer in nature, because length of em-
bryonic development has been shown to de-
pend on temperature in cephalopods (Bolet-
zky, 1987, 1994). Ken Drinkwater (personal
communication) reports that average monthly
bottom temperatures in the area where the
octopodids were collected were as warm as
11-12°C in August and September 1994 and
that the average annual temperature was
8.0°C in 1994—these temperatures are ap-
proximately 1.1°C higher than usual. O’Dor &
Macalaster stated that laboratory specimens
experienced mortality with only brief periods
of temperatures from 10°C to 12°C, whereas
Our specimens survived periods of weekly av-
erage temperatures in the 11°C range.
Although O’Dor & Macalaster (1983) found no
evidence of migration, we point out that mi-
gration to warmer water to spawn has not
been ruled out. Villanueva (1992) found evi-
dence of up-slope ontogenic migration in
Bathypolypus sponsalis.
Brooding Bathypolypus arcticus occasion-
ally take food to offset the extremely high en-
ergetic cost of brooding eggs for over a year.
The octopus reported on by O’Dor & Macal-
aster (1983) may have eaten her own eggs to
have sufficient energy to brood the remaining
four through to hatching. If this is true, it shows
that B. arcticus can brood for a year by eating
its own eggs. The cost in such a protracted
brooding period is a sharp decrease in fecun-
dity. An alternative explanation is that this fe-
male only had a few viable sperm from her
mating, which occurred at least five months
earlier. Perhaps she ate the other 36 eggs be-
cause they were not viable.
The long brooding period in B. arcticus must
have substantial costs of time, energy, and risk
of predation. This period may limit B. arcticus
to a semelparous strategy, because the costs
of brooding are not worth the effort unless the
number of eggs and their chance of survival is
sufficiently large. Historicity, or phylogenetic
legacy (Williams, 1992), may also limit many
octopodids to a semelparous strategy.
Parental care is necessary for all incirrate
octopod eggs because they lack a protective
egg case. Without the mother caring for and
defending her eggs, they would be rapidly suf-
focated by fouling organisms (Boletzky, 1994).
However, the length of this brooding period
varies. Bathypolypus arcticus benefit in sev-
eral ways from their large young and long
brooding period. Sibling and non-sibling com-
petition favour smaller broods of larger eggs
(Stearns, 1992). Longer brooding periods
may be selected for in B. arcticus because the
egg stage is likely to have a high survival rate
compared to hatchling. Stearns (1992) noted
that “selection should increase the proportion
of time spent in the safest developmental
stages.” Yampolsky & Scheiner (1996) dis-
cussed demographic reasons that favour
large offspring in cold environments for
exothermic animals.
Because Bathypolypus arcticus are not
found in high densities (O’Dor & Macalaster,
1983) and because they are not very mobile,
chances to mate may be rare. The female’s
ability to store viable sperm for at least five
months (often incorrectly cited as ten months;
O’Dor & Macalaster, 1983) and the male’s
large spermatophores and ligula are likely
adaptations to maximize fitness under such
conditions. It is possible that the large ligula
and spermatophores are selected for in
sperm competition. Cigliano (1995) noted that
octopodids meet the criteria for sperm prece-
dence and that the spoon-shaped ligula could
be used to scoop out competitors sperm.
Another (non-exclusive) possibility is that the
large ligula expands once in the oviduct to an-
chor the hectocotylus during mating. Further
investigation will be needed to confirm or re-
ject these theories.
The successful manipulation of females
between broods suggests that experiments
manipulating brood and egg size may be pos-
sible. An explanation of the experimental
significance of manipulation of offspring size
can be found in Stearns (1992). Such experi-
ments could answer such questions as
whether hatchling behaviour (e.g., planktonic
versus benthic) is a function of hatchling size,
species, or hatchling age. Also, the effect
18 WOOD, KENCHINGTON & O’DOR
of egg size on development time could be
tested within a brood, which would minimize
or eliminate many confounding factors. Work-
ers should test to see if other octopus species
can be manipulated in this manner, because
the long brooding period and cold tempera-
tures required for keeping B. arcticus make it
a poor choice for such experiments.
The fact that a soft-bodied cephalopod can
survive collection in a scallop trawl, days at
sea, and acclimation to laboratory conditions,
indicates the hardiness of B. arcticus. Bolet-
zky & Hanlon (1983) provided a general re-
view of low trauma cephalopod collection
techniques and general culture information.
Less traumatic collection techniques that
specifically target deep-sea cephalopods are
allowing investigators to study more species
of deep-sea octopods in the lab. Researchers
at the Monterey Bay Aquarium and Monterey
Bay Aquarium Research Institute used an
ROV specifically to capture and film cirrate oc-
topuses, and they have been able to keep
these octopuses alive for several months
(Stein-Hunt and Hochberg, personal commu-
nication).
We are just beginning to understand the life
history of deep-sea octopods. Additional stud-
ies involving such modern sampling tech-
niques as video monitoring from submersibles
and remote operated vehicles (Vecchione &
Roper, 1991) will continue to complete the pic-
ture of how deep-sea cephalopods behave in
nature. However, because deep-sea cephalo-
pods cannot always be found, workers must
hope for chance encounters. Telemetry has
provided more continuous records of the be-
haviour of Loligo and Nautilus (Carlson et al.,
1984; O’Dor et al., 1993, 1994) and could also
be employed to study other deep-sea cephalo-
pods. Cost and logistical problems limit the use
of these techniques.
It is widely acknowledged that laboratory
conditions can bias results. However, studies
of cephalopods in their natural environment
are difficult due to their mobility, excellent vi-
sion, and nocturnal habits (Boletzky & Han-
lon, 1983). Laboratory studies are currently
the best, most direct way to look at develop-
ment time, life span, reproductive strategy,
growth rates, and other processes that occur
in individuals over time. Also, the effect of
such variables as temperature, sex, and diet
can be analyzed and separated. Laboratory
conditions allow the experimenter to observe
and manipulate such behaviour as mating or
hatching. Further laboratory studies of hardy
deep-sea species are needed if we want to
understand how these organisms live in their
cold, dark world.
ACKNOWLEDGEMENTS
This project would not have been possible
without the help of the Canadian Department
of Fisheries and Oceans. We acknowledge
the help of Dale Roddick, Mark Lundy, Brenda
Bradford, and the crew of the FRV J. L. Hart.
We thank S. v. Boletzky and J. Voight for
their helpful comments on this manuscript. A
thank you is also due to John Cigliano, who
commented on the mating video. Thanks to
Joyce Chew for helping collect food for the oc-
topodids, to Dave Gaudet for filming them, to
Chris Harvey-Clark for photographing the
eggs, and to Rebekah McClean for illustrating
the mating sequence.
The primary author is supported by a
Killiam Fellowship, and the work by an
NSERC Canada grant to RKO.
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Ta
MALACOLOGIA, 1998, 39(1-2): 21-27
THE INHERITANCE OF AN EMBRYONIC LETHAL MUTATION IN A
SELF-REPRODUCING TERRESTRIAL SLUG, DEROCERAS LAEVE
Richard M. Lebovitz
c/o L.C. Evogen, 3515 Washington Boulevard, #317, Arlington, Virginia 22201, U.S.A.
ABSTRACT
A new lethal developmental mutation (EZL-1, embryonic zygotic lethal-1) in Deroceras laeve
was used as a genetic marker to determine whether self-reproduction is by parthenogenesis or
self-fertilization. An average of 25.7% of the progeny of individuals (N = 38) apparently het-
erozygous for the mutation exhibited the EZL-1 lethal phenotype, a result that can only be ex-
plained if reproduction is meiotic. Histological sections of a fertile slug revealed the presence of
both sperm and eggs in the gonad, consistent with self-fertilization, rather than parthenogene-
sis. In addition to EZL-1, other developmental defects were observed in progeny.
Key words: Deroceras, development, mutation, self-fertilization, slug.
INTRODUCTION
Self-reproduction is widespread, particu-
larly among molluscs (Heller, 1993). Dero-
ceras laeve, the grey garden slug, a pul-
monate, reproduces predominantly, if not
exclusively, by self-reproduction (Foltz et al.,
1982). It is of particular interest to malacolo-
gists because it is unsettled whether they self-
reproduce by self-fertilization or by partheno-
genesis (Heller, 1993; Hoffmann, 1983; Foltz
et al., 1982; Nicklas & Hoffmann, 1981). To
identify the mechanism of reproduction in this
species, the inheritance of a recessive lethal
mutation, embryonic zygotic lethal-1 (EZL-1),
was studied. This is the first report of an em-
bryonic mutation in a terrestrial slug.
EZL-1 mutant embryos complete about
21% of the embryonic stage, arresting after
the acquisition of most of the rudimentary adult
structures, including the tentacles, mantle,
and foot. An average of 25.7% of the progeny
of apparently heterozygous individuals (N =
38) for the mutation exhibited the EZL-1 lethal
phenotype, a result that can only be explained
if reproduction is meiotic (Asher, 1970). Histo-
logical sections of a fertile individual revealed
the presence of both sperm and eggs in
the gonad, consistent with self-fertilization,
rather than parthenogenesis (Hoffmann,
1983; Asher, 1970). п addition to EZL-1, other
developmental defects were observed in prog-
eny.
21
MATERIALS AND METHODS
The founder slugs were isolated from the
wild and maintained on a diet of fresh romaine
lettuce. Subsequent progeny of the founders
were isolated after hatching and cultured indi-
vidually in tupperware dishes containing a
piece of moistened towel. There were no op-
portunities for cross-fertilization. Eggs from
mature, isolated, animals were collected
every one to two days and transferred to a
moistened towel in a petri dish, where they
were stored until hatching. The phenotype of
unhatched eggs was observed under a dis-
secting microscope and scored.
Slugs were fed fresh salad leaves every
one to two days. Environmental conditions
were not controlled, although the slugs were
generally maintained at room or seasonal
temperatures, between about 16°C to about
28°C.
Karotyping was performed on three- to four-
day-old embryos. After removal from the egg
case, embryos were incubated for 30 min in
0.125% colchicine dissolved in 0.45% KCl,
washed in 0.045% KCI for about 30 min, and
then fixed in a cold mixture of 3 parts
methanol to 1 part acetic acid. Fixed embryos
were placed in a drop of 60% acetic acid,
squashed under a siliconized coverslip, and
dried on a hot plate between about 37°C and
47°C. When the slides were completely dried,
the coverslip was removed, and the slides
22 LEBOVITZ
were stained with aceto-orcein for micro-
scopic examination.
For histology, a fertile slug was fixed in 4%
formaldehyde, dehydrated, and embedded in
paraplast. Six um sections were prepared and
stained with Harris hematoxylin-phloxine-
eosin for light microscopic examination.
The values for lifespan, sexual maturity, and
egg number were calculated as the mean with
a standard deviation (+). Statistical analy-
sis was performed by chi-square, using the
test for heterogeneity described in Mather
(1951).
RESULTS
Although five different founder lines were
maintained for a four-year period by self-re-
production alone, only one line, IF, is de-
scribed here. This line was selected because
of its possession of a recessive lethal muta-
tion, providing an opportunity to characterize
the mutation and use it as a genetic marker to
study self-reproduction.
The animals in this study were identified as
Deroceras laeve by H. Lee Fairbanks of
Pennsylvania State University according to
their description by Pilsbry (1948). In all three
animals dissected, an inspection of the dorsal
surface of the internal cavity revealed that the
ovotestis was hidden under the digestive
gland, a characteristic of D. laeve but not D.
reticulatum, a species it closely resembles. In
addition, the rectal caecum and terminal male
genitalia were absent.
Development of the Adult
Animals collected from eight generations
were included in this study. The founder (IF)
was a progeny of an animal captured from the
wild. Ninety animals, predominantly from the
sixth and seventh generations, were selected
for analysis. Only animals that reached matu-
rity and produced viable offspring were in-
cluded. For convenience, the ages discussed
below were calculated from the day of egg de-
position, rather than the actual day of hatch-
ing.
The average lifespan of all 90 animals was
164 + 40.7 days (about 5 1/2 months), rang-
ing from 90 to 250 days. Sexual maturity,
marked by the first deposition of eggs, was
reached at about three months (94.8 + 18.7
days), with a shortest time of 71 days anda
longest of 148. Animals produced eggs for an
average of 57.2 + 35.2 days. Egg production
continued until an average of 12 + 8.8 days
before death.
Very few metaphase figures were observed
in the embryos. Of these, quality of the chro-
mosomal squashes was low and thus pre-
cluded detailed karyotyping. However, of 14
spreads sufficiently clear to count, the chro-
mosome number averaged 50 (data not
shown). Patterson & Burch (1978) reported
that Deroceras are diploid, having a haploid
chromosomal number of 30. The number here
is roughly twice that value, consistent with
diploidy.
Viability and Phenotypes of Deposited Eggs
A mean of 66.5 + 32.5 eggs (N = 90; total
eggs = 5,981), with a range of 6 to 164, were
produced per animal. In addition to producing
eggs with embryos that developed into normal
adults, two other primary egg phenotypes
were observed. First, eggs were frequently
observed that showed very little, if any, em-
bryonic development. Inside such eggs, a
small ball-like structure, varying in size, was
typically observed. These eggs are referred to
as type A. All animals produced this type of
egg. Secondly, a category of eggs that un-
dergo about 20% of normal development and
then arrest were observed. These are embry-
onic/zygotic lethal-1 or EZL-1, the mutation
described here. Not all individuals produced
EZL-1 eggs. It is assumed that those that did
were heterozygous for the gene loci and that
those that did not were homozygous normal
at the EZL-1 locus.
Normal Egg Development
Development of the viable and normal egg
occurred substantially as described in Carrick
(1938) for Agriolimax agrestis. There is no
metamorphosis nor a larval stage. Rather, the
embryo gradually assumes the adult form,
progressing through at least five distinct and
identifiable forms. For the purposes of this
study, the discussion is focused on the pro-
gression of the embryo from stage IV to V, be-
cause this is the stage at which embryonic de-
velopment is arrested in EZL-1.
Embryonic development takes about 14
days. Stage IV, reached at about four days, is
characterized by the appearance of most of
the rudimentary structures of the adult organ-
ism, including the tentacles, mantle, and foot
(Fig. 1A, reproduced from Carrick, 1938). The
LETHAL MUTATION IN DEROCERAS 23
FIG. 1. Development of Deroceras laeve. Stage IV.
A, Right aspect. psr, rudiment of posterior sac; eor,
external opening of right larval nephridia; m, mantle;
a, anterior sac; h, hepatic mss; rrt, rudiment of right
tentacles; f, foot. Stage V. Maximum development of
posterior and anterior sacs. B, Right aspect. C,
Ventral aspect. ps, posterior sac; po, pulmonary ori-
fice; ror, optic rudiment of right anterior tentacle; lor,
optic rudiment of left anterior tentacle; rt1, right an-
terior tentacle; rt2, right posterior tentacle; rt3, third
tentacle of right side; It1, left anterior tentacle; It2,
left posterior tentacle; It3, third tentacle of left side;
st, pedal streaks. Reproduced by permission of the
Royal Society of Edinburgh and R. Carrick from
Carrick (1938-1939).
posterior sac, the respiratory organ of the em-
bryo, and the hepatic mass, another embry-
onic structure, also become visible at this
time. The progression from stage IV to V is
gradual, marked by the enlargement of the
rudimentary stage IV structures into recogniz-
able and significantly differentiated forms.
Stage V is identified by Carrick (1938) when
the hepatic mass reaches its maximal size
(Fig. 1B).
A primary characteristic of the transition
from stage IV to stage V is the differentiation
of the posterior sac from the embryonic foot.
The foot and posterior sac originate from the
same embryonic region. During development
from stage IV to stage V, the two structures
gradually assume characteristic forms, ap-
pearing to comprise different cell types. As the
foot morphologically separates from the pos-
terior sac, it thickens, and two streaks (Fig.
1C, st) appear on its ventral surface, running
from anterior to posterior. After the appear-
ance of the pedal streaks, the mantle and ten-
tacles begin to enlarge, and the embryo grad-
ually becomes more adult. Concomitantly, the
hepatic mass begins to shrink, ending stage
V. The entire sequence takes about seven to
eight days.
After about seven days, midway through de-
velopment, pigmented eye spots appear in
the first optic tentacles. Subsequently, pig-
mentation spreads in a wave across the outer
embryo epithelium. The remainder of develop-
ment until hatching is characterized in Carrick
(1938) and is not discussed here.
Type A Egg Development
An average of 23 + 13.8% (N = 90) eggs
from founder 1F were type A, showing little, in
any, growth past the blastula stage (Stage | ac-
cording to Carrick, 1938). A type A egg was
classified by the appearance in the perivitelline
egg fluid of a small, round mass and a long,
twisted, wispy membranous thread. The latter
was defined by Carrick (1938) as the remains
of the sperm body, but this conclusion has not
been generally accepted (South, 1992). The
type A phenotype cannot be distinguished at
deposition from a viable egg, except that the
former does not develop any further over the
14-day embryonic period. From all appear-
ances, it is likely that this class represents de-
velopmentally inactivated or unfertilized eggs.
A second class of eggs is infrequently iden-
tified in 1F progeny. These eggs, type B, un-
dergo a small amount of growth, arresting one
to two days after egg deposition. B exhibits the
same characteristic ball-like morphology as
A, but it is about two to three times larger. It is
uncertain at what development type B arrests,
but the absence of visible structures suggests
24 LEBOVITZ
TABLE 1. Percent frequency of egg phenotypes produced by homozygous and heterozygous
EZL-1 Deroceras laeve.
TOTAL EGGS
Group 1, 1141
homozygotes (N = 22)
Group 2, 2181
homozygotes (N = 30)
Group 3, 2659
heterozygotes (N = 38)
that it has not, or has just, entered stage Ill
(Carrick, 1938) when visible morphological
differentiation begins. The tissue mass inside
the perivitelline sac of type B eggs often is
“fuzzy, rather than a well-defined structure as
in type A, possibly indicating tissue necrosis.
It is possible that B defines an early embry-
onic (or maternal effect) mutation, but this has
yet to be confirmed. B represents less than
3% of the total eggs and was included in the
type A class for all calculations, because it is
only qualitatively different and therefore diffi-
cult to score separately.
EZL-1 Egg Development
In the line from founder 1F, an embry-
onic/zygotic lethal mutation (EZL-1) was first
observed in the fourth generation, when the
phenotypes of unhatched embryos were first
inspected for defects. It was observed in all
subsequent generations.
The lethal mutation was first distinguished
on day 4 of the 14-day development period,
about 21% of the way through its develop-
ment. Developmental arrest occurs during
stage V when the embryonic foot and poste-
rior sac start to become distinguishable struc-
tures in the rudimentary foot of the zygote
(Fig. 1C). In the majority of these embryonic
lethals, although the foot and posterior sac
became distinguishable from one another, the
foot is enlarged but arrests before or when the
pedal streaks would become visible in a nor-
mal embryo. The posterior sac, on the other
hand, looks perfectly normal in EZL-1. Its
characteristic movement throughout the nor-
mal 14-day period when egg development
was examined suggests that the EZL-1 zy-
gote is alive, but that its development halted.
All development appears to cease in EZL-1.
For example, during normal development, the
optic tentacles begin to enlarge after the ap-
pearance of the pedal streaks, when the he-
% Type A (not fertilized or
% EZL-1 not activated)
1.3251 20.8 + 16.4
range: 0-7.4
OE 21.4 + 13.7
range: 0-7
207/1916 25.8 + 11.8
range: 8.6-61.5
patic mass reaches its maximum (i.e., stage
V). In EZL-1, the zygote acquires the rudi-
ments of the optic tentacles, but they do not
appear to expand or acquire any visible struc-
ture, such as the pigmented eyespots ob-
served in normal embryos.
Frequency of EZL-1 Phenotype
Because animals carrying the EZL-1 muta-
tion could not be distinguished phenotypically
from non-carriers, genotype was deduced
from the frequency of the mutation appearing
in their progeny. To calculate the percentage
of eggs exhibiting the EZL-1 lethal phenotype,
the number of type A eggs was subtracted
from the total eggs and the result was divided
into the number of eggs scored as EZL-1. This
adjustment was done to eliminate type A eggs
from the analysis, on the assumption that they
represent a class of eggs that are either un-
fertilized or unactivated. The results are sum-
marized in Table 1.
Group 1 comprises three generations de-
scended from (and including) a fifth genera-
tion animal that apparently did not possess
the EZL-1 gene mutation. An average of 1.3%
of the eggs were scored as EZL-1. The values
ranged from 0% to 7.4%. It is assumed that
the eggs scored as EZL-1 (since all data were
collected blindly) died for other reasons, but at
a stage similar to the one at which EZL-1 ar-
rests. In some of these, it had been noted that
the phenotype was not characteristic of EZL-
1, that is, later developmental arrest or differ-
ent-sized embryo.
Groups 2 and 3 were collected from de-
scendants of three fourth-generation animals.
These animals were from the same founder
as Group 1, but from a lineage that diverged
at the third generation. The animals were clas-
sified into two groups, using 7% EZL-1 as a
cut-off, because that was the highest value
observed in Group 1. Based on this value,
LETHAL MUTATION IN DEROCERAS 25
Group 2 possessed an average of 1% EZL-1
eggs, similar to Group 1, the animals that do
not have the EZL-1 mutation. The average
number of eggs with the EZL-1 phenotype in
Group 3, however, was 25.7%, with only four
of the 38 animals being lower than 17% (that
is, 8.6, 12.7, 14.3, and 15). This result for the
pooled data (normal = 1,450 eggs; EZL-1 =
481 eggs) is consistent with the expected
number for self-fertilization by heterozygotes
(x? = 0.027, df = 1, 0.95 > > 0.90). When
Group 3 was analyzed as a class, using chi-
square to test heterogeneity (Mather, 1951),
the y? value was high (P < 0.05), suggesting
that the class was heterogeneous. Homo-
geneity was established (y = 48.2, df = 35,
0.30 > P > 0.20) by eliminating from Group 3
the two animals with the highest EZL-1 fre-
quency (43% and 62%). As discussed below,
other lethal developmental defects have been
noticed in slug progeny, albeit at a much lower
occurrence than EZL-1. Thus, the appearance
of EZI-1 in these two slugs (as well as others)
could have been inflated by scoring other de-
velopmental defects that resemble but are not
EZL-1. Figure 2 is a frequency histogram of
the % EZL-1 per animal.
Other Developmental Defects
The appearance of an EZL-1 like pheno-
type was observed an average of 1.1% (+
1.9%, N = 52) in lines apparently homozygous
wild-type for the EZL-1 gene locus (Groups 1
and 2). Other lethal developmental defects
were noted, as well. These phenotypes in-
cluded: developmental arrest at earlier stages
than EZL-1, either with a distinct morphology
or comprising a bulbous, undifferentiated
mass; incomplete to fully pigmented embryos;
and fully developed embryos that did not
emerge from the egg shell. When added to the
EZL-1-like phenotype, the mean value of de-
velopmental defects for the apparently ho-
mozygous wild-type animals was 3.4% (+
319%; М= 52).
Histology of the Gonad
The simultaneous presence of both sperm
and egg in slug gonads is well documented
(South, 1992). In D. reticulatum, for example,
all individuals are hermaphroditic, and the
gonad can contain both eggs and sperm at the
same time (Runham & Laryea, 1968). Both
sperm and eggs were seen in the gonad of one
fertile D. laeve. The spermatids are arranged in
Frequency
% EZL-1
FIG. 2. Frequency histogram displaying percent
eggs exhibiting the EZL-1 phenotype from individ-
ual slugs. Data collected from 38 slugs character-
ized as heterozygous for the EZL-1 locus.
clusters, suggesting their origin from individual
spermatogonia (Fig. 3A). Mature spermatozoa
were abundant in the hermaphroditic duct (Fig.
3B). Because aphallic slugs, lacking most of
the terminal structures of the male reproduc-
tive system, were fertile, it is suggested that
sperm production is dissociated from the de-
velopment of the reproductive tract required
for transporting sperm.
DISCUSSION
The EZL-1 mutation in D. laeve is clearly a
recessive mutation at a single gene locus.
First, it is transmitted through successive gen-
erations, segregating between carrying and
non-carrying slugs. Secondly, an apparently
heterozygous individual produces an average
of 25.7% eggs exhibiting the EZL-1 pheno-
type, consistent with either simple Mendelian
inheritance by self-fertilization or meiotic par-
thenogenesis with a high frequency of recom-
bination (Asher, 1970; Hoffmann, 1983). The
detection of both sperm and egg in the gonad
favors self-fertilization, in agreement with Foltz
et al. (1984), and all other pulmonates (Heller,
1993), but contrary to the conclusions of
Nicklas & Hoffmann (1981) and Hoffmann
(1983).
EZL-1 is an embryonic lethal mutation that
arrests during stage V of development
(Carrick, 1938). Its progression from the blas-
tula to stage IV is visibly normal, acquiring
most, if not all, of the rudimentary adult struc-
tures. However, the progression from stage IV
to the adult in EZL-1 is arrested. Differen-
tiation, for example, of the foot rudiment into
the posterior sac and foot is abnormal in these
26 LEBOVITZ
FIG. 3. Light micrograph showing the reproductive tract of Deroceras laeve. A, Section through gonad re-
vealing a cluster of spermatids (arrow head) between egg cells (e). B, Hermaphroditic duct filled with mature
spermatozoa. Sperm nuclei are darkly stained (box).
mutants, and the optic tentacles do not signif-
icantly differentiate from their rudimentary el-
ements.
It is likely that the EZL-1 locus is a zygoti-
cally active gene, rather than a maternally ac-
tive one. Maternally active genes are con-
tributed by the maternal chromosome of the
oocyte, responsible for setting up the spatial
patterns in the embryo. Once the spatial orga-
nization is set out, differential expression of
the zygotic genes is triggered, and embryonic
development can be completed. In EZL-1 mu-
tations, development is visibly normal until
stage IV, when the acquisition of all of the
adult rudimentary structures is complete.
Further development, however, is aberrant.
Thus, the initial spatial patterning of the em-
bryo is normal, suggesting that the perfor-
mance of the maternal genes is normal. On
the other hand, differentiation of the embryo is
abnormal, arresting about one-quarter into
development, making it likely that the defect is
zygotic, a consequence of the malfunction of
a zygotically active gene.
The number of eggs in self-fertilizing slugs
that failed to hatch was high. For example, in
apparently homozygous individuals, about
23% of the eggs did not develop at all, and
3.4% of the developing eggs exhibited a range
of lethal phenotypes. The failure of eggs to
hatch into viable offspring has been observed
with cross-fertilizing snails as well (Doums et
al., 1994; Jarne & Delay, 1990; Rollinson et
al., 1989). In one of these studies (Jarne &
Delay, 1990), a significant difference in egg
“hatchability” was observed between self- and
cross-fertilizing Lymnaea peregra snails. It
was Stated that the disparity was due to well-
formed snails that did not hatch, rather than
eggs arresting at an early developmental
stage. This result was interpreted as a de-
crease in fitness produced by self-fertilization,
which these authors called self-fertilization
depression. The studies presented here sug-
gest the possibility that self-fertilization de-
pression can be caused by a recessive lethal
mutation. For example, the number of un-
hatched eggs produced by EZL-1 heterozy-
gotes (EZL-1 eggs plus type A) is about two-
fold greater than for homozygous wild-type
slugs (type A eggs only) at the EZL-1 gene
locus. Thus, the presence of a lethal mutation
decreases egg “hatchability” in self-fertiliza-
tion, a result that could be avoided by cross-
fertilization with non-carrying animals. It is in-
teresting that in the studies reported here with
self-fertilizing slugs, the largest class of un-
hatched eggs exhibit no, or little, embryonic
development. Further studies are needed to
explain this observation.
ACKNOWLEDGEMENTS
Many thanks to Beth Fricano of the Natural
History Museum of the Smithsonian Insti-
tution, Washington, D.C., for performing the
histology; to Dr. Lee Fairbanks of Pennsylvania
State University, Beaver Campus, for identify-
ing the slug species; to Dr. Teresa Tansey for
LETHAL MUTATION IN DEROCERAS 27.
scientific advice and encouragement; to Hien
Truong for technical assistance; and to Dr.
Anthony Zelano for encouragement.
LITERATURE CITED
ASHER, J. H., 1970, Parthenogenesis and genetic
variability. II. One-locus models for various diploid
populations. Genetics, 66: 369-391.
CARRICK, R., 1938-1939, The life-history and de-
velopment of Agriolimax agrestis L., the grey field
slug. Transactions of the Royal Society of
Edinburgh, 59: 563-597.
DOUMS, C., B. DELAY & P. JARNE, 1994, A prob-
lem with the estimate of self-fertilization depres-
sion in the hermaphrodite freshwater snail Buli-
nus truncatus: the effect of grouping. Evolution,
48: 498-504.
FOLTZ, D. W., H. OCHMAN & R. K. SELANDER,
1984, Genetic diversity and breeding systems in
terrestrial slugs of the families Limacidae and
Arionidae. Malacologia, 25: 593-605.
FOLTZ, D., W., B. M. SCHAITKIN & R. K. SE-
LANDER, 1982, Gametic disequilibrium in the
self-fertilizing slug Deroceras laeve. Evolution,
36: 80-85.
HELLER, J., 1993, Hermaphroditism in molluscs.
Biological Journal of the Linnean Society, 48:
19-42.
HOFFMANN, В. J., 1983, The mating system of the
terrestrial slug Deroceras laeve. Evolution, 37:
423-425.
JARNE, P. & B. DELAY, 1990, Inbreeding depres-
sion and self-fertilization in Lymnaea peregra
(Gastropoda: Pulmonata). Heredity, 64: 169-175.
MATHER, K., 1951, The measurement of linkage in
heredity. New York: John Wiley & Sons, Inc.
NICKLAS, N. L. & В. J. HOFFMANN, 1981, Apo-
mictic parthenogenesis in a hermaphroditic ter-
restrial slug, Deroceras laeve (Muller). Biological
Bulletin, 160: 123-135.
PATTERSON, С. М. & J. B. BURCH, 1978, Chromo-
somes of pulmonate molluscs. Pp. 171-217, in
Pulmonates, Vol. 2A, Systematics, evolution, and
ecology, V. FRETTER & J. PEAKE, eds., Academic
Press, London.
PILSBRY, H. A., 1948, Land Mollusca of North
America. Monograph of the Academy of Natural
Sciences of Philadelphia, 2(2).
ROLLINSON, D., R. A. KANE & J. R. L. LINES,
1989, An analysis of fertilization in Bulinus cerni-
cus (Gastropoda: Planorbidae). Journal of
Zoology (London), 217: 295-310.
RUNHAM, N. W. & А. A. LARYEA, 1968, Studies on
the maturation of the reproduction of reproductive
system of Agriolimax reticulatus (Pulmonata:
Limacidae). Malacologia, 7: 93-108.
SOUTH, A., 1992, Terrestrial slugs: biology, ecology
and control. Chapman and Hall, London.
Revised ms. accepted 20 September 1996
MALACOLOGIA, 1998, 39(1-2): 29-38
FOREST AND SCRUB SNAIL FAUNAS FROM NORTHERN MADEIRA
R. A. D. Cameron! & L. M. Cook?
ABSTRACT
The island of Madeira has two major natural vegetation types, a damp forest association at
higher altitudes, with below it a dry association of grasses, herbs and low scrub. The open scrub
is predominantly on the south coast and the eastern peninsula. The land mollusc fauna of the
high forest and the north coast has been surveyed, and compared with that of other regions.
Presence or absence of 84 species, 56 of them endemic, has been recorded in 51 samples. The
forest samples are very clearly separated from those of the other regions; species in the families
Pupillidae and Vitrinidae have radiated there, whereas radiation of Helicidae is characteristic of
the other areas. Where non-endemics are present, they increase the species richness and do not
displace endemics. There is no evidence of subdivisions within the forest fauna. Previous work
has shown that at lower and drier locations an eastern peninsula fauna is differentiated from that
of the south coast. The existence of the forest accounts for some of the species richness of the
Madeiran archipelago, but much of it is attributable to species proliferation, especially in the
Helicidae, between similar scrub habitats on different islands and on different parts of the same
island.
Keywords: land snail, Mollusca, Madeira, competition, endemism.
INTRODUCTION
The Madeiran group is in the Atlantic
Ocean, 900 km southwest of Portugal and
800 km west of Morocco at 33°N 17°W. The is-
land of Madeira is 58 km at its largest dimen-
sion and rises to an altitude of 1860 m. There
are two other clusters of islands, Porto Santo
with its offshore islets and the three Deserta
islands, all considerably lower than Madeira it-
self. The archipelago is exceptionally rich in
land molluscs, with a high frequency of en-
demic species. In a count by Waldén (1984)
there are 261 + 3 taxa, of which 193, or
73.9%, are endemic. Waldén's (1983) list con-
tains 216 species.
The distribution of the land snail fauna has
been examined in detail in the southeast and
eastern part of Madeira (Cameron & Cook,
1992; Cook et al., 1990). These areas have a
predominantly dry-zone fauna, but with some
species found also in the montane forest re-
gion. Some montane species occur, along
with those characteristic of the modern east-
ern fauna, in the fossil deposit on the eastern
peninsula (Cook et al., 1993; Goodfriend et
al., 1994, 1996). The distribution of species in
Porto Santo has also been surveyed (Came-
ron et al., 1996).
The territory from the peninsula to the west-
ern extremity of the island in the mountains,
mostly on the north side of the main dividing
ridge, has now been investigated. This area
consists of steeply sloping valleys and moun-
tainsides, covered in many places by indige-
nous humid laurel forest (Sjogren, 1972). The
collections allow comparison with the lower-
lying and dryer south coast and the eastern
peninsula of Madeira, and also with Porto
Santo and the Desertas. In order to under-
stand how the fauna evolved, it is necessary
to Know the extent to which species are lo-
calised within islands and whether there is
any evidence of competition, which might limit
the diversity at a given locality or lead to se-
lection for divergence between taxa.
MATERIALS AND METHODS
Fifty-one samples were examined, stretch-
ing from Porto Moniz in the west to the Boca
do Risco to the east of the north coast, and
from there along the peninsula and the south-
east coast, a linear distance of about 70 km.
Site locations are shown in Figure 1. Each
site was examined by two people for about
half an hour, searches being made on rock
faces, litter and living vegetation. About 5 | of
soil and litter were collected at each site, tak-
‘Division of Adult Continuing Education, University of Sheffield, Sheffield $1 4ET, United Kingdom
“The Manchester Museum, University of Manchester, Manchester M13 9PL, United Kingdom
30 CAMERON & COOK
ing small amounts from favourable patches
within the sample area. Material so collected
was air-dried and searched in the laboratory
after sieving. Material passing through a 0.5
mm mesh aperture was discarded. Searching
strategies of this kind give reasonably robust
and reliable indications of presence and ab-
sence. Different and more time-consuming
techniques would be needed to estimate den-
sities (Cameron, 1973, 1992). For the pur-
pose of this study, presence and absence
data are sufficient. A set of specimens of crit-
ical endemic species has been deposited with
the Manchester Museum. Nomenclature and
classification follow Waldén (1983), Cook et
al. (1990), Groh & Hemmen (1986) and
Holyoak & Seddon (1986). There were 84
species recorded, of which 56 are endemics.
The mean number of species in the 51 sam-
ples was 14.0, with a standard deviation of
4.7. The richest sample contained 31 species,
the poorest 6.
Salient features of the habitat, including
dominant vegetation structure, the balance of
native and alien plant species, and altitude,
were noted and used to classify the sites into
the following categories.
A (Sites 1-5): southern coastal scrub, firmly
within the Aeonio-Lyntathion alliance (Sjö-
gren, 1972), usually with introduced Opuntia
tuna present. All sites subject to some degree
of past agricultural disturbance, including oc-
casional burning.
B (Sites 6-8): sites on the Ponta de Sao
Lourenco, all open with field layer only present
and with calcareous sand in the substrate.
Floristically, they fall into the Biserrulae-
Scorpiurietum association within the Aenio-
Lytanthion alliance (Sjogren, 1972; Натр-
shire, 1984). These are the richest and least
disturbed sites on the Ponta as reported by
Cook et al. (1990).
C (Sites 9-15): coastal cliff and slope sam-
ples from the north coast. These also fall in
the Aeonio-Lytanthion alliance of Sjogren
(1972), but with more native scrub elements
and higher moisture levels than on the south
coast.
D (Sites 16-21): coastal cliff and slope
samples from the north coast, as for C above
but in which there are significant elements of
the Clethro-Laurion alliance, the vegetation of
typical native laurel forest (Sjogren, 1972),
which descends to sea level in places along
the north coast.
E (Sites 22-33): inland sites (at least 1 km
from the sea) below 600 m, clearly within the
Clethro-Laurion alliance, but generally dis-
turbed and part cleared, with non-native trees
often dominating the vegetation.
Е (Sites 34-41): native /aurisilva above 600
m but below 900 m above sea level in which
native species typical of the Clethro-Laurion
predominate.
G (Sites 42-51): native laurisilva above 900
m but below 1,400 m, as for F, but with Erica
species often more important. At and above
1,300-1,400 m Jaurisilva is frequently re-
placed by Erica scrub or by overgrazed mon-
tane grassland (Sjögren, 1972).
The topography and land-use pattern of the
island is such that there is some geographical
clustering of sites placed in the same cate-
gory. Categories C, D and E are more hetero-
geneous than the others, and note should be
taken of the following sites.
Site 12 (Group C): extremely arid, very
heavily grazed and uniquely on Pleistocene
limestone rather than volcanic rock.
Site 14 (Group C): very open, with no sur-
viving laurisilva elements, but with springs
and permanently moist soil in places.
Site 20 & 21 (Group D): both with more or
less complete native forest cover or shaded,
wet rocks with native Clethro-Laurion vegeta-
tion, although very close to the sea.
Sites 22 & 23 (Group E): both clearly within
the Clethro-Laurion zone altitudinally, but in
an area of long-term forest clearance with
much open ground and non-native vegetation
typical of grassland and scrub.
RESULTS
General Patterns
Table 1 shows the percentage occurrence
of each species in the samples within each
sample group as defined above. A full matrix
of species and sites is available from the au-
thors, and a copy has been deposited with the
collections in the Manchester Museum.
There is evidence of both geographical and
ecological patterns in the distribution of
species. Table 2 lists the species found only in
predominantly Aeonio-Lytanthion habitats
and their derivatives (groups A, B and C), only
in predominantly Clethro-Laurion habitats
(groups D, E, F and G) and also those found
in combinations of C, D and E, which are to
some extent intermediate between the ex-
tremes of + В and F + G. Thirty-five species
out of 84 show restriction to one or other of the
MADEIRAN FOREST SNAILS 31
Porto Moniz
Säo Jorge
Funchal
FIG. 1. The island of Madeira showing location of sampling sites. Open circles: sites in groups A and В (south
coast and peninsula), closed circles: sites in groups C and D (dry and wetter north coastal samples), open
triangles: group E (inland, wooded or disturbed, below 600 m), closed triangles: groups F and G (woodland,
6-1,400 m). Approximate position of 600 m contour is shown. Maximum distance from west to east is 58 km.
major vegetation alliances, and a further 12
are restricted to intermediate habitat types,
which may represent a third, extensively dis-
turbed faunal grouping. Helicidae, generally
the largest species with the thickest shells,
constitute a far higher proportion of restricted
species in the open and intermediate habitats
than they do in the forests (13 out of 30 in the
former, 2 out of 17 in the latter). Relatively few
non-endemics are restricted to forest sites.
To investigate the distinctions between cat-
egories further, we carried out a cluster analy-
sis on individual samples. The basis for such
analyses is a matrix of values of an index of
similarity in species composition between all
pairs of samples. The Jaccard Index is one
such index, calculated by dividing the number
of species in common between two sites by
the total number of species present in both.
When there are different numbers of species
in the samples being compared, this index
measures both the taxonomic similarity and
the difference in number of species, because
the maximum possible value is number of
species in the sample with the fewer species
as a fraction of the total in that with the larger
number. Cook et al. (1972) normalized the
Jaccard Index by multiplying it by the ratio of
the larger species number to the smaller, call-
ing the result maximum similarity. This would
be appropriate if all the variation in species
number was regarded as sampling error, and
we were concerned exclusively with taxo-
nomic similarity. However, a measure that is
affected to some extent by difference in spe-
cies richness as well as species composition
would be preferable to either of these indexes,
because it gives a better idea of the ecologi-
cal similarity of the sites. When applied to
presence/absence data, the Nei Index (Nei,
1987) is the number of species in common di-
vided by the geometric mean of the number of
species in each sample of the pair, providing
the right kind of balance. It is therefore an ap-
propriate measure, and has been used to ex-
amine pairwise similarity. Figure 2 shows the
dendrogram of faunal affinities for all species
using the Nei Index and UPGMA clustering. !t
confirms the habitat distinctions noted above.
It is clear that the data fall into two major clus-
ters (Table 3). All samples of groups A, B and
C fall into one of these, and all those of groups
F and G into the other. Samples in groups D
and E are distributed between the two. There
are clear separations between groups A and
В and between these and groups Е, F and С.
Groups C, D and E are more scattered; much
of the variation being explicable in terms of
the specific site characteristics noted above.
We therefore conclude that the groups recog-
nise differences in ecology and/or geography
that genuinely influence the faunal composi-
tion. Intermediate habitats have mixtures of
species from the two major faunas.
The Nei index is usually applied to fre-
quency data, and may be used to examine the
fraction of sites occupied by each species in
each group as shown in Table 1. The result is
shown in Figure 3. Again, all species were in-
cluded; when non-endemics are excluded, the
32 CAMERON & COOK
TABLE 1. Samples grouped into seven geographical and habitat categories, showing fraction of sites
occupied in each group (to nearest per cent). Endemic species (1 to 56) are listed before non-endemics
(57 to 84), otherwise species are listed in the systematic order given by Walden (1983). Authorities for the
names are in Walden (1983), Groh & Hemmen (1986), and Holyoak & Seddon (1986).
A B С D E F G Group
ENBEMIESFTFF IF мя SS Sn Tg © = ne
0 66 42 83 91 100 100 1. Craspedopoma mucronatum
0 0 14 50 33 50 20 2. C. neritoides
20 0 0 0 0 0 0 3. С. monizianum
0 0 14 50 16 37 10 4. С. trochoideum
0 0 14 33 33 62 80 5. Columella microspora
40 33 57 16 0 0 0 6. Staurodon saxicola
0 0 0 0 0 37 50 7. Leiostyla cheilogona
0 0 0 0 0 0 10 8. L. filicum
0 0 0 16 0 0 0 9. [. vincta
0 0 14 16 16 0 O 10. [. irrigua
0 0 14 33 8 50 30 — 11.1. ошеапа
0 0 0 0 0 0 20 12. L. concinna
0 0 0 0 0 25 10 13. L. laurinea
0 0 14 16 8 12 O 14.1. sphinctostoma
0 0 14 0 0 0 10 15.L. arborea
0 0 14 16 8 0 O 16.L. fusca
20 0 57, 66 0 0 O 17.L. recta
80 33 14 0 0 0 O 18.L. millegrana
0 0 0 0 0 25 10 19. Lauria fanalensis
0 0 0 0 0 37 50 20. Hemilauria limneana
0 0 0 0 0 12 O 21. Phenacolimax nitidus
0 0 0 33 15 100 89 22.Р marcidus
0 0 14 33 58 62 80 23. P ruivensis
0 0 0 16 16 12 O 24.Р behnii
0 0 0 16 8 12 30 25. P albopalliatus
0 0 14 0 0 0 O 26. Janulus stephanophora
80 33 42 50 0 0 O 27. J. bifrons
0 0 14 33 58 12 20 28. Amphorella tornatellina
0 100 0 16 0 0 O 29. А. cf. minor
40 0 0 0 0 0 O 30. А. mitriformis
0 33 0 0 0 0 O 31. А. cf. iridescens
80 33 28 33 0 0 O 32. Pyrgella leacockiana
40 0 0 16 0 0 O 33. Boettgeria deltostoma
60 66 14 0 0 0 O 34. В. depauperata
0 66 71 33 0 0 O 35. В. exigua
0 0 0 16 0 62 50 — 36. В. crispa
40 100 28 16 0 0 O 37. Heterostoma paupercula
0 0 14 0 0 0 10 38. Spirorbula latens
0 0 14 0 0 0 O 39. $. squalida
40 100 0 0 0 0 0 40. Caseolus compactus
60 0 0 0 0 0 0 41. С. leptostictus
20 0 0 0 0 0 O 42. Disculella maderensis
0 0 57 16 8 0 0 43. Actinella lentiginosa
0 0 14 0 0 0 O 44. А. actinophora
100 0 0 0 0 0 O 45. A. arcta
0 0 14 16 16 0 10 46. А. fausta
0 0 0 16 16 0 O 47. А. carinofausta
0 0 0 0 0 0 10 48. А. obserata
100 100 100 100 16 0 O 49. À. nitidiuscula
0 0 0 0 16 0 O 50. А. giramica
20 0 0 0 0 0 O 51. Discula tabellata
100 100 85 50 0 0 O 52.0. polymorpha
60 0 71 66 66 50 10 53. Leptaxis erubescens
0 0 0 16 0 0 20 54.L. furva
0 0 14 33 58 87 100 55. L. membranacea
100 100 28 0 8 0 O 56.L. undata
MADEIRAN FOREST SNAILS 33
TABLE 1. (Continued)
A B С D E
NON-ENDEMIES= 22-2222 Ce
0 0 14 0 0
0 0 14 33 58
0 0 57 0 16
80 0 57 100 66
20 0 14 33 8
0 0 14 0 0
60 33 42 16 0
100 0 100 66 50
80 0 14 0 0
40 33 14 0 0
0 0 0 0 0
0 0 0 0 16
0 0 0 50 50
100 33 85 66 13
0 0 0 33 25
20 0 0 0 0
60 33 85 83 91
0 0 14 0 16
80 0 100 83 8
0 0 0 16 16
0 0 0 0 8
0 0 14 83 58
60 33 0 0 0
20 0 0 0 0
60 33 0 0 0
0 33 0 0 0
0 33 0 0 0
0 0 28 0 8
5 3 7 6 12
19 14 30 31 21
13 i 16 12 16
32 21 46 43 37
pattern is almost identical. The impression
gained from presence/absence data is con-
firmed. Groups A and B, representing the fau-
nal composition of the Aeonio-Lytanthion al-
liance on the southern and eastern parts of
the island, are similar to each other and form
a pair distinct from the rest. At a higher level of
similarity, categories F and G, the higher alti-
tude Clethro-Laurion, separate off from C, D
and E, which contain species characteristic of
the laurel forest plus others introduced by dis-
turbance and by natural spreading to those
coastal areas resembling the eastern sites.
They include sites 22 and 23 (exceptionally
dry and open for group E), which resemble the
open habitat section, and sites 14, 20 and 21
(in groups C and D), which have above aver-
age moisture or Clethro-Laurion characteris-
tics. The faunal separation of the south coast
and the high laurel forest is due not only to the
species restricted to them but also to marked
E G Group
12 10 57. Carychium minimum
75 20 58. C. tridentatum
0 O 59. Cochlicopa lubrica
75 O 60. С. lubricella
12 O 61. Columella aspera
0 О 62. Truncatellina callicratis
0 O 63. Vertigo pygmaea
0 O 64. Lauria cylindracea
0 O 65. Vallonia costata
0 O 66. V. pulchella
0 10 67. Acanthinula aculeata
87 69 68. Plagyrona placida
37 10 69. Punctum pygmaeum
87 69 70. P pusillum
12 10 71. Helicodiscus singleyanus
0 O 72. Намайа miniscula
87 30 73. Vitrea contracta
12 O 74. Nesovitrea hammonis
0 0 75. Oxychilus cellarius
0 0 76. Zonitoides arboreus
0 0 77. Oxychilus alliarius
87 40 78. Euconulus fulvus
0 O 79. Cecilioides acicula
0 O 80. Ferrusacia folliculus
0 O 81. Caracollina lenticula
0 O 82. Cochlicella barbara
0 O 83. Theba pisana
0 O 84. Helix aspersa
8 10 Total sites in group
19 23 Endemic species
11 9 Non-endemic species
30 32 Total species in group
differences in proportion in several of the
other, non-restricted species.
There is also a less obvious correlation be-
tween similarity and geographical position.
Sites within groups A and B are close both ge-
ographically and in terms of affinity, and there
is a collection of group C and D sites between
Boca do Risco and Sáo Jorge, which also
show high affinity levels. No geographical pat-
tern is visible in the forest sites.
Ratio of Non-endemics to Endemics
When non-endemics are introduced to an
endemic fauna, one possible outcome is a
negative relation between numbers of en-
demic and non-endemic species in samples.
This may occur because non-endemics thrive
in habitats to which endemics are not adapt-
ed, and vice versa, or because there is direct
competition between the species. Such a
CAMERON & COOK
TABLE 2. Species in the survey which come from (a) Clethro-Laurion forest or from lower
altitude habitats of similar type (DEFG), (b) intermediate and disturbed habitats (CDE) or
(c) from Aeonio-Lytanthion scrub or the damper sites of group C (ABC).
Groups DEFG CDE ABC
ENDEMICS
Cyclophoridae ------------ -
Pupilidae == 2282 S222 22 se nat nn de Da Еее eee
Leiostyla cheilogona L. vincta L. millegrana
L. filicum L. arborea
L. concinna L. fusca
L. laurinea
Lauria fanalensis
Hemilauria limneana
NV ae a ee a
Phenacolimax nitidus
P marcidus
P behnii
P albopalliatus
Zonitidaer-r-2 25222! ose e e se oe eee eee Pa
Ferussacidac ee a a ee à
Amphorella mitriformis
A. cf. iridescens
Clausiliidae ------------------------------------------------------
Boettgeria crispa B. depauperata
Helicidae --------------------------------------------------------
Spirorbula squalida
Caseolus compactus
C. leptostictus
Disculella maderensis
Actinella arcta
A. obserata A. lentiginosa
A. carinofausta
A. giramica
Discula tabellata
Leptaxis furva
NON-ENDEMICS
Cochlicopidae ----------------------------------------------------
WW EY oy at I ae ee ee Ir SS ae
Vallonia costata
V. pulchella
Acanthinula aculeata
Plagyrona placida
EndodOn idees e A ae ae rae eee ee
Punctum pygmaeum
Helicodiscus singleyanus
Zonitidaet a a a Se ees Se ARE A A E
Oxychilus alliarius
Zonitoides arboreus
RenusSaclidae’ ze ne 2225225224 e ee ве
Cecilioides acicula
Ferrusacia folliculus
Helicidac==-== Вы. A A A
Caracollina lenticula
Cochlicella barbara
Theba pisana
Helix aspersa
MADEIRAN FOREST SNAILS 35
Sample Distance
0 20 40 60 80 100
FIG. 2. Similarity of the 51 samples in the survey. Scale shows distance as a fraction of the maximum value
for the analysis.
36 CAMERON & COOK
TABLE 3. The seven defined habitat groups divided between the two main clusters
of Figure 2. Groups, based on habitat type and reflecting to some extent geo-
graphical location, are defined in the text.
Defined groups
Derived cluster A B С
1 5 3 74
2 0 0 0
Total 5 3) if
Category Distance
0 20
arm mo num LS
D E F G Total
4 4 0 0 23
2 8 8 10 28
6 12 8 10 51
40 60 80 100
FIG. 3. Similarity of the seven categories of samples derived from the frequency of species in the samples
within categories (Table 1). Scale shows distance as a fraction of the maximum value for the analysis.
relation occurs, for example, in our study of
the snails of Porto Santo (Cameron et al.,
1996), where there is a negative relation of
endemics to non-endemics on a rocky/undis-
turbed to sandy/disturbed axis. To test for a
similar effect on Madeira, the association of
the number of non-endemic species with the
number of endemic species has been exam-
ined. The correlation coefficient r is 0.263, for
which t = 1.906 (P < 0.06). Thus, there is no
evidence of a negative association; the rela-
tion is positive, and nearly significant, the
slope of the reduced major axis being 0.757.
It could result from the fact that sites vary in
their suitability for molluscs, good sites being
favourable to non-endemics and endemics
alike. The feature most likely to lead to high
species number may be microhabitat hetero-
geneity. At any rate, presence or absence of a
species appears to depend on habitat suit-
ability, with no evidence of negative associa-
tion between the two categories.
DISCUSSION
Cook et al. (1990) demonstrated differ-
ences between snail faunas from open habi-
tats in the south and east of Madeira, which
suggested a pattern of geographical differen-
tiation independent of present habitats.
Similar, indeed more striking differentiation of
this kind is found in the neighbouring island of
Porto Santo (Cameron et al., 1996). This
study extends the survey to the high altitude
forest and open and intermediate habitats
along the north coast of Madeira.
There is a radical difference between the
fauna of the Clethro-Laurion forest and of the
open habitats and scrubby areas at lower alti-
tudes. In the forest, the climate is cooler, rain-
fall is higher, and soils are less rich in calcium
(Sjogren, 1972). Table 4 illustrates the differ-
ent balance of families in Clethro-Laurion
sites from those of open habitats on Madeira
and on the other islands of the archipelago,
which are even more arid. The faunas have
substantial representation of small, thin-
shelled species (especially Pupillidae) and
semi-slugs (Vitrinidae), and are less domi-
nated by Helicidae species, only six of which
were found above 600 m altitude. In the open
sites, there is again evidence of geographical
differentiation, albeit complicated by distur-
bance and small-scale mosaics of forest and
open habitats. Faunas from mid- to high alti-
MADEIRAN FOREST SNAILS 37
TABLE 4. Species distribution between families in samples collected in different parts of the archi-
pelago. Madeira north side (Groups D to G): data from this paper. Madeira fossil: samples 45 thou-
sand years old or older from the sand bed on the eastern peninsula from Cook et al. (1993). Madeira,
eastern peninsula and Desertas: contemporary samples from Cook et al. (1990). Porto Santo: con-
temporary samples from Cameron et al. (1996).
Madeira Madeira Madeira Porto
Family N side fossil peninsula Desertas Santo
Endemics
Pupillidae 13 TÉ 4 0 5
Ferussaciidae 3 3 5 1 8
Clausiliidae 3 1 4 1 1
Vitrinidae 5 2 2 0 1
Helicidae 12 18 12 10 32
Others 6 6 5 1 0
Non-endemics (all families) 19 2 21 0 9
Total 61 39 53 13 56
tude forests, free from gross disturbance, do
not show such effects, although distances be-
tween sites are the same, or greater than
those involved in open habitat comparisons.
In drawing the distinction between the rela-
tively open Aeonio-Lytanthion and Clethro-
Laurion alliances, Sjögren (1972) noted that
on the drier south side of the island the
Aeonio-Lytanthion gives way to forest be-
tween 300 and 700 m above sea level,
whereas on the wetter north it is rare for it to
ascend above 300 m; Clethro-Laurion or de-
rivatives frequently approach sea-level. While
modest changes in altitudinal range of the al-
liances (for example, in response to Pleisto-
cene climatic changes) could isolate sections
of open habitat on the south coast, and extin-
guish it in the north, the forest area would fluc-
tuate is size but remain as a largely continu-
ous block. Extension of forest faunas in the
past is suggested by the subfossil data in
Table 4, and confirmed by detailed analysis
(Cook et al., 1993).
Although forest faunas are more uniform,
there is some altitudinal differentiation, with
species characteristic of low or high altitudes.
There are some species-rich genera. Of
these, Leiostyla species are small, rare, cryp-
tic and apparently patchily distributed. Our
knowledge of them is as yet too limited to say
whether they show geographical differentia-
tion. On the present evidence, however, the
extensive proliferation of distinct species of
limited distribution but similar habitat, which
contributes so much to the species richness
of the archipelago, is largely a dry-habitat
phenomenon. Forests, especially at higher al-
titudes, have few non-endemic species, and
some of those that do occur may be natives
rather than introductions. We have no evi-
dence pointing to adverse effects of non-
endemics on endemics or to the presence of
habitats which favour non-endemics over en-
demics. The greater number and higher den-
sity of non-endemics in open habitats proba-
bly reflects the richer conditions there,
created to some extent by human activity, in
which both categories can flourish.
ACKNOWLEDGEMENTS
We thank Dr. Mary Seddon and the
National Museum of Wales for essential as-
sistance in species identification.
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MALACOLOGIA, 1998, 39(1-2): 39-57
COMPARATIVE STUDIES ON THE ANATOMY AND HISTOLOGY
OF THE ALIMENTARY CANAL OF THE LIMACOIDEA AND MILACIDAE
(PULMONATA: STYLOMMATOPHORA)'
Ana Maria Leal-Zanchet
Laboratorio de Histologia, Centro de Ciéncias da Saude, Universidade do Vale do Rio dos
Sinos, Caixa Postal 275, 93022-000 Sao Leopoldo-RS, Brasil
ABSTRACT
The anatomy and histology of the digestive tract of Deroceras laeve, D. reticulatum, D. rodnae,
Lehmannia marginata, Malacolimax tennelus, Boettgerilla pallens and Tandonia budapestensis
are described comparatively. The alimentary canal is composed of oesophagus, crop, stomach,
intestine and rectum. An intestinal caecum is present in D. reticulatum, D. rodnae and L. mar-
ginata. The intestine is subdivided into four histologically distinct regions. From the oesophagus
to the third intestinal region, and in the rectum, the alimentary canal is lined by a simple colum-
nar to cuboidal epithelium. In the fourth intestinal region and in the intestinal caecum, the ep-
ithelium is simple squamous to cuboidal. Supporting ciliated and nonciliated cells are found in the
epithelium. Both types carry microvilli. Eight gland cell types can be distinguished: mucous cells
of types |-V, cystic cells, and intestinal secreting cells of types | and II. The epithelium of the ali-
mentary canal is surrounded by a layer of connective tissue and two muscle layers, an inner lon-
gitudinal muscle layer and an outer circular muscle layer.
Key-words: anatomy, histology, alimentary canal, secretory cells, Limacidae, Agriolimacidae,
Boettgerillidae, Milacidae.
INTRODUCTION
According to Likharev & Wiktor (1980), the
superfamily Limacoidea is formed by the fam-
ilies Limacidae, Agriolimacidae and Boett-
gerillidae, whereas the family Milacidae be-
longs to the superfamily Zonitoidea. Little
information exists on the anatomy and histol-
ogy of the digestive system of the limacids
and agriolimacids. Simroth (1885). Quick
(1960) and Wiktor (1973), in their systematic
studies on the group, only described the
course of the alimentary canal. Studies on the
anatomy and histology of the alimentary canal
of Limax, Arion and Helix have been carried
out by Gartenauer (1875) and Baecker
(1932). Walker (1972) examined the digestive
system of Deroceras reticulatum, emphasiz-
ing the physiology of the crop, stomach and
intestine. More recently, only a few observa-
tions have been made on the histology of the
alimentary canal of a limacoid species (Ba-
bula & Skowronska-Wendland, 1988). Мо
data are found in the literature on the anatomy
and histology of the digestive system of the
families Boettgerillidae and Milacidae. The
purpose of this paper is to report on a com-
parative anatomical and histological study of
the alimentary canal of six limacoid species
and a milacid species.
MATERIALS AND METHODS
The following species were studied:
Malacolimax tenellus (Muller, 1774) and Leh-
mannia marginata (Muller, 1774) (Limacidae),
Deroceras laeve (Muller, 1774), D. reticulatum
(Muller, 1774) and D. rodnae (Grossu & Lupu,
1965) (Agriolimacidae), Boettgerilla pallens
(Simroth, 1912) (Boettgerillidae), and Tan-
donia budapestensis (Hazay, 1881) (Milaci-
dae). The animals were collected next to the
city of Tubingen, Baden-Wurttemberg, Ger-
many, and kept in a cool room at 15°C (Leal-
Zanchet, 1995). The species are herbivorous,
except for B. pallens (Leal-Zanchet, in press).
In the laboratory, the animals were fed cab-
bage, lettuce and carrots. For the anatomical
studies, the slugs were anaesthetized in a 5%
‘Part of a thesis submitted to the Lehrstuhl Spezielle Zoologie of the University of Tübingen, Germany, in partial fulfillment
of the requirements for the degree of Doctor of Natural Sciences.
40 LEAL-ZANCHET
solution of menthol for three to four h, fixed in
4% formaldehyde for 24 to 48 h and trans-
ferred to 70% ethanol. The slugs were pro-
gressively dissected with the aid of a binocu-
lar microscope. The digestive tract was
uncovered and drawn under a camera lucida.
Finally, the alimentary canal was opened lon-
gitudinally to observe its internal morphology.
For the light microscopy studies, the slugs
were anaesthetized in 5% menthol for two and
fixed in Susa or 4% paraformaldehyde/glu-
taraldehyde. Tissues fixed with Susa were de-
hydrated in ethanol, embedded in Paraplast,
and serially sectioned at 6 um. The sections
were stained with haematoxylin/eosin, or with
the triple stain methods of Masson-Goldner
(MG) and Azan-Heidenhain (AZ) (Romeis,
1989). This method was used to delimit histo-
logically the organs and regions of the diges-
tive system, and also for a first characteriza-
tion. Small fragments of the various parts of
the alimentary canal were fixed in 4%
paraformaldehyde/glutaraldehyde, washed in
phosphate buffer, dehydrated in ethanol and
embedded in historesin. The 2 um thin sec-
tions were stained with methylene blue and
basic fuchsin (МЕ) (Bennett et al., 1976). This
second method gives the best results for de-
tailed histological studies.
For measurements of the epithelium and
the gland cells we used a micrometric glass.
Four animals of each species were measured.
All measurements were made on the material
fixed in paraformaldehyde/glutaraldehyde and
embedded in historesin. They were made on
the apices of the folds and between the folds.
The mean values were given in the tables.
RESULTS
Anatomical Features
The narrow oesophagus (oe) rises from the
buccal mass (Fig. 2, bcm) and widens poste-
riorly to form the crop (cr). From this emerges
the small stomach (s), which is followed by the
long intestine (Fig. 2, i). The ducts of the di-
gestive gland open into the stomach (Figs. 3,
4). The intestines of Lehmannia marginata,
and Malacolimax tenellus have two forward-
directed loops, whereas the intestines of
Deroceras laeve, D. reticulatum, D. rodnae,
Tandonia budapestensis and Boettgerilla pal-
lens have only one forward directed loop
(sensu Quick, 1960). From the second intesti-
nal loop of Lehmannia and Malacolimax, or
the single loop of the other species, arises the
terminal branch of the intestine, which goes
forward and enters into the body wall and is
then called rectum (Fig. 1).The rectum goes
through the pallial complex for a short way
and unites with the ureter into the anus, which
is situated in the right anterior part of the body.
A blind tube, the intestinal caecum (Fig. 2, c),
is associated with the terminal part of the in-
testine of L. marginata, D. reticulatum and D.
rodnae (Fig. 1).
The oesophagus has longitudinal folds (Fig.
5, If), and the crop has a smooth wall without
folds, containing only narrow elevations (Fig.
KEY TO LETTERING ON FIGURES
as: apocrine secretion
bem: buccal mass
c: intestinal caecum
ca: clear area
cc: ciliated columnar cells
cct: cells of the connective tissue
ce: cuboidal epithelium
ci: cilia
cia: ciliated area
cm: circular muscle layer
cn: cell neck
cr: crop
ct: connective tissue
cy: cystic cells
dg: digestive gland
dgd: duct of the digestive gland
dgo: opening of the digestive gland
e: elevation
i: intestine
ic: intestinal secreting cells
ic |: intestinal secreting cells of type |
ic Il: intestinal secreting cells of type II
lef: leader fold
If: longitudinal fold
Ig: leader groove
Im: longitudinal muscle layer
lu: lumen
mc: mucous cells
me |: mucous cells of type |
me Il: mucous cells of type Il
me Ill: mucous cells of type III
me IV: mucous cells of type IV
me V: mucous cells of type V
mi: microvilli
n: nucleous
nc: nonciliated columnar cells
nu: nucleolus
oe: oesophagus
s: stomach
sac: strong acidophilic cells
se: squamous epithelium
sg: salivary glands
sgd: duct of the salivary gland
sgr: secretory granules
sl: subepithelial layers
sm: secretion mass
sv: supranuclear vacuoles
t,, t,: typhlosoles
tf: transversal fold
va: vacuole containing amorphous material
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 41
FIG. 1. Schematic diagrams comparing the digestive system of (1) Deroceras laeve, (2) Deroceras reticula-
tum, (3) Deroceras rodnae, (4) Malacolimax tenellus, (5) Lehmannia marginata, (6) Boettgerilla pallens and
(7) Tandonia budapestensis. A: first intestinal region, B: second intestinal region, C: third intestinal region, D:
fourth intestinal region, E: rectum, F: intestinal caecum.
First intestinal region Ha
> —
2 Second intestinal region We
6, e) due to the irregular height of the epithe-
lium. The crop of Tandonia budapestensis is
an exception, with the wall having transverse
folds (Fig. 28). The stomach contains three
conspicuous folds, two longitudinal typhlo-
soles and a transverse fold (Figs. 4, 7). Each
typhlosole originates close to an opening of
the duct of the digestive gland and runs pos-
teriorly as far as the posterior limit of the stom-
ach (Fig. 4, t,, t,). The transverse fold is
closely triangular and extends between the
two openings of the ducts of the digestive
gland (Fig. 4, f). A leader groove occurs be-
tween the typhlosoles, as well as between the
transverse fold and the typhlosoles (Fig. 4, Ig).
The intestine can be subdivided into four re-
gions. Figure 1 shows the arrangement of the
intestinal regions in relation to the intestinal
loops. The first and second intestinal regions
have smooth walls without folds, being differ-
entiated only by their histological features.
However, in L. marginata and M. tenellus, only
small longitudinal folds are present in the first
intestinal region (Fig. 8, If). The third intestinal
region has numerous longitudinal folds (Fig.
10, If), and the fourth region has a smooth
wall, except for a ciliated area (Fig. 11, cia).
This occurs in limacids and agriolimacids, but
not in В. pallens and T. budapestensis. The in-
Third intestinal region
Rectum
Fourth intestinal region | Intestinal caecum.
testinal caecum has no folds. The rectum has
a smooth wall in its proximal third and abun-
dant longitudinal folds in its distal two thirds
(Figs. 12-16, If). Among these, three to five
differentiated folds occur in Agriolimacidae,
Boettgerillidae and Milacidae (Figs. 13-16,
lef). There are five of these folds in B. pallens
and 7. budapestensis, where they are most
developed (Figs. 13, 14, 43). In Deroceras,
there are two to three leader folds (Figs. 15,
16, 44).
Histological Features
Epithelium: Oesophagus—The oesophagus
is lined by a columnar simple epithelium. In
the proximal third of the oesophagus, its sur-
face is cuticularized, but in most of the oe-
sophagus the epithelium carries cilia and mi-
crovilli (Fig. 17). The cilia are especially
numerous on the crests of folds (Figs. 5, 26).
The epithelium of the oesophagus consists
of ciliated columnar cells (cc), nonciliated
columnar cells (nc), and mucous cells (Fig. 17,
mc |). Both ciliated and nonciliated columnar
cells bear microvilli. The columnar cells show
an acidophilic cytoplasm, in which supra-
nuclear vacuoles (Fig. 17, sv) with strong aci-
dophilic or cromophobe contents can be seen.
42 LEAL-ZANCHET
N JA
RS, o
ET
ER
FIG. 2. Dorsal view of the digestive system of
Lehmannia marginata. Scale bar 1 mm.
The most apical part of the cells is a clear area
(Fig. 17, ca). The oval nucleus lies in the mid-
dle or basal third of the cells (Fig. 17, n).
The columnar cells of the alimentary canal
show an apocrine secretion (Figs. 17, 18, 22,
as), and eventually a holocrine secretion in
which strong acidophilic columnar cells are
discharged into the lumen (Figs. 30, 31, sac).
The mucous cells of the oesophagus are
termed type | (Fig. 17, mcl). They are intraep-
ithelial and flask shaped, with a broadened
base and a long neck, and they contain nu-
merous weakly or strongly basophilic granules
(MF). The oval or elongated nucleus is basally
located and surrounded by a strong acidophilic
FIG. 3. Ventral view of the crop and the stomach of
Deroceras reticulatum.
FIG. 4. Internal morphology of the stomach of
Lehmannia marginata. Scale bar 1 mm.
cytoplasm. In the paraffin sections, the gran-
ules and the remaining cytoplasm of the mu-
cous cells cannot be distinguished; the mu-
cous cells show a foamy content that stains
light green with MG and light blue with AZ.
Crop—The columnar epithelium of the crop
is higher than that of the oesophagus (Table
1). In the crop, only nonciliated columnar cells
(nc) and mucous cells of type | (Figs. 18, 27,
mcl) are present. The columnar cells of the
crop are clearly distinguishable because of
the presence of very abundant large vacuoles
containing amorphous material (Figs. 18, 27,
va). The contents of the vacuoles can be re-
moved by histological methods. Supranuclear
vacuoles (Fig. 18, sv) with strong acidophilic
or chromophobe contents, similar to those of
the oesophagus, are also present. The oval or
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 43
FIG. 5. Cross-section of the oesophagus of
Lehmannia marginata.
FIG. 6. Cross-section о the crop of Lehmannia mar-
ginata. Scale bar 0.1 mm.
elongated nucleus lies in the middle or basal
third of the cells (Fig. 18, n). Type | mucous
cells are similar to those of the oesophagus
(Figs. 18, 27, mel).
Stomach— The ciliated columnar epithe-
lium of the stomach usually is lower than that
of the crop (Table 1). In the stomach, the cili-
ated columnar cells far outnumber the noncil-
iated cells. The latter occur only in small areas
adjoining the typhlosoles (Fig. 7). The cyto-
plasm of the columnar cells is similar to that of
the corresponding cell types of the crop, but
the number of vacuoles containing amor-
phous material is lower (Fig. 19, va). The
columnar cells of the leader groove (Fig. 20),
located between the two typhlosoles, differ
from the other columnar cells of the stomach
by their strong acidophilic cytoplasm contain-
ing rare vacuoles. In addition, the epithelium
of the leader groove and of the typhlosoles is
om
FIG. 7. Cross-section of the stomach of Lehmannia
marginata.
FIG. 8. Cross-section of the first intestinal region of
Lehmannia marginata.
taller than in the rest of the stomach and has
long cilia (Fig. 20).
In the stomach, the mucous celis are more
abundant than in the crop (Table 3). Two types
of mucous cells can be distinguished. The
type | mucous cells are found in most of the
stomach and are similar to those of the crop in
shape and morphology (Fig. 19, mcl). The
type ll mucous cells occur only in the leader
groove (Figs. 20, 31, mcll). However, in В. pal-
lens and T. budapestensis, type |! mucous
cells are found in the entire stomach. They are
intraepithelial and have a long base with an
acidophilic cytoplasm containing many vac-
uoles. The nucleus lies distal at the base. The
long neck shows numerous small and strong
basophilic secretory granules (Table 2, Fig.
20).
First Intestinal Region—This region has a
high columnar epithelium (Table 1) with a
pseudostratified appearance because the nu-
44 LEAL-ZANCHET
FIG. 9. Cross-section of the second intestinal region
of Lehmannia marginata.
FIG. 10. Cross-section of the third intestinal region
of Lehmannia marginata. Scale bar 0.1 mm.
clei tend to be arranged in two rows: the round
nuclei of the abundant mucous cells form a
row at the base of the epithelium, and the
elongated nuclei of the columnar cells lie in
one or two rows halfway up in the epithelium
(Figs. 21, 33, n).
Only ciliated columnar cells occur in the first
intestinal region (Fig. 8). Their cytoplasm pos-
sess a small number of vacuoles with amor-
phous contents (Fig. 21, va), and is otherwise
similar to that of the columnar cells of the
crop. Due to the pressure of the numerous
mucous cells, the base of the columnar cells
becomes thinner and their nuclei lie in the
middle or apical third (Figs. 21, 33).
The abundance of mucous cells is the more
conspicuous feature of this intestinal region
(Table 3, Fig. 33). The mucous cells are
mainly of type Il (Fig. 21, mc Il), but some mu-
cous cells of type | may also be present (Table
4).
Second Intestinal Region—The second in-
testinal region is characterized by the pres-
ence of type | intestinal secreting cells (Fig.
FIG. 11. Cross-section of the fourth intestinal region
of Lehmannia marginata.
FIG. 12. Cross-section of the rectum of Lehmannia
marginata. Scale bar 0.1 mm.
22, ic |). The columnar epithelium is usually
lower than in the first intestinal region (Table
1). Ciliated and nonciliated columnar cells
occur in the second intestinal region. Both cell
types contain rare vacuoles with amorphous
contents. The nucleus of the columnar cells is
located in the basal or middle third (Fig. 22).
The number of mucous cells is relatively
small in the second intestinal region (Table 3).
Morphologically, the cells are similar to the
type | mucous cells of the oesophagus and
crop (Fig. 22, mc |).
The intestinal secreting cells of type | (Figs.
22, 34, ic |) have a claviform shape. The large,
oval nucleous is rich in chromatin and has a
conspicous nucleolus (Fig. 34, nu). The basal
cytoplasm stains violet (MF). The numerous
secretory granules are found in the supranu-
clear cytoplasm (Fig. 22, sgr). The granules
stain red with MG and MF.
Third intestinal region—In the third intesti-
nal region, type Il intestinal secreting cells are
found (Figs. 23, 35). Ciliated cells occur only
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 45
FIG. 13. Cross-section of the rectum of Tandonia
budapestensis.
FIG. 14. Cross-section of the rectum of Boettgerilla
pallens. Scale bar 0.1 mm.
at the crests of the folds (Fig. 10). The re-
maining cells are nonciliated. The cytoplasm
of the columnar cells has a small number of
supranuclear vacuoles (Fig. 23, sv).
The intestinal secreting cells of type II (Figs.
23, 35, ic Il) have granules, the contents of
which were not stained by any of the methods
used. The granules are larger than those of
the intestinal secreting cells of type 1. Other
features of type Il intestinal secreting cells are
similar to those of type 1.
The gland cells become more abundant
than in the second intestinal region (Table 3).
In L. marginata and M. tenellus, four types of
gland cells (mucous cells of type III, IV and V,
and cystic cells) occur in the third and fourth
intestinal regions, in the intestinal caecum,
and in the rectum. In D. rodnae and D. re-
ticulatum, all four types are also present, but
their occurrence is different (Table 4). Only
three of these gland cell types can be seen in
FIG. 15. Cross-section of the rectum of Deroceras
rodnae.
FIG. 16. Cross-section of the rectum of Deroceras
laeve. Scale bar 0.1 mm.
D. laeve, and only two of them in B. pallens
and T. budapestensis (Table 4). These cell
types were identified and described with the
aid of historesin sections. With the exception
of the cystic cells, the gland cells cannot be
distinguished on paraffin sections, remaining
unstained or staining light green with MG and
light blue with AZ.
The mucous cells of type Ill (Figs. 23, 25,
36, 38, 42, 45, Table 2, mc Ill) have an aci-
dophilic cytoplasm and granules staining light
or dark blue (MF). The cells are usually subep-
ithelial. Their cell body is located in the subep-
ithelial connective tissue or external to the
ring muscle layer (Figs. 25, 38).
The mucous cells of type IV (Figs. 25, 38,
39, mc IV) are also usually subepithelial. Their
cell body has a smaller diameter than that of
type Ш mucous cells (Table 2). The nucleus,
however, is larger than that of type III mucous
cells. The acidophilic cytoplasm has numer-
ous small secretory granules that stain red
(MF).
46 LEAL-ZANCHET
FIG. 17. Semi-schematic drawing of part of a trans-
verse section of the oesophagus of Lehmannia
marginata.
FIG. 18. Semi-schematic drawing of part of a trans-
verse section of the crop of Lehmannia marginata.
Scale bar 10 um.
The mucous cells of type V (Figs. 23, 25,
47, Table 2, mc V) are always subepithelial
and are easily distinguishable from the other
mucous cells by their shape and by their con-
tents. The cell body has a sacculiform shape,
so that its transition to the cell neck is very
gradual, and the cell body is small and located
close to the epithelium. Most of the cell is filled
with elongated secretory granules that stain
blue with MF. The granules often coalescens,
forming a secretion mass. The nucleus is pe-
ripheral and oval to elongated in shape and is
surrounded by acidophilic cytoplasm.
The cystic cells (Figs. 23-25, 46, cy), like
the mucous cells of type V, are always subep-
ithelial, but have a very large sacculiform cell
body (Table 2). The cystic cells are filled with
an amorphous secretion that stains pink with
MF and red with MG. Basophilic cytoplasm
can be seen in a peripheral zone of the cell
body. The nucleus is basally located.
Fourth Intestinal Region—The epithelium
of the fourth intestinal region is squamous to
cuboidal, and two to three times lower than in
the third intestinal region (Table 1, Figs. 25,
37-39). The cells are nonciliated, except for
the longitudinal ciliated area (Figs. 11, 25, 37).
Both nonciliated and ciliated cells have mi-
crovilli. The cytoplasm of the cells of the fourth
region seldom has supranuclear vacuoles
(Fig. 25). The basal part of the cells is highly
folded. In the fourth intestinal region of B. pal-
lens and T. budapestensis, the ciliated area is
absent.
The epithelium of the ciliated area is higher
and has long cilia (Figs. 25, 37). Apical cells
are more acidophilic than the other cells of the
region.
The gland cells (Table 4) are usually more
abundant than in the third intestinal region
(Table 3). In the fourth intestinal region, the
gland cells are always subepithelial (Fig. 25).
The intestinal secreting cells are absent. The
cell body of the mucous cells of type Ш and IV,
as well as that of the cystic cells, is located ex-
ternal to the muscle layers.
Intestinal Caecum— The epithelium of the
intestinal caecum is squamous and lower
than that of the fourth intestinal region (Table
1, Figs. 24, 40, 41). The cytoplasm of the
squamous cells is similar to that of the fourth
intestinal region. The number of gland cells is
small (Tables 3, 4).
Rectum—The epithelium of the rectum is
cuboidal to columnar. The epithelial cells of
the leader folds (Figs. 43, 44, 46) are higher
and show features similar to those of the
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 47
TABLE 1. Epithelial height of the digestive tube of Limacoidea and Milacidae (um).
B. lee D.
pallens marginata laeve
Oesophagus 49.7 47.0 315
Crop 79.8 65.0 55.0
Stomach 52.5 41.5 53.8
1. intestinal region 56.0 61.0 58.3
2. intestinal region 53.8 54.0 42.8
3. intestinal region 34.0 37.8 33.3
4. intestinal region 10.5 15:5 15.0
Caecum — 13.8 —
Rectum Teil 10.0 ES
leader fold of the fourth intestinal region. The
rectum of L. marginata and M. tenellus, with-
out leader folds, has ciliated cells in the crests
of the folds (Figs. 12, 42, 47). In the rectum of
B. pallens, T. budapestensis and D. laeve
(Figs. 13, 14, 16), ciliated cells occur only in
the leader folds, and all other cells are noncil-
iated. In D. rodnae and D. reticulatum, ciliated
cells occur in the leader folds, as well as in the
crest of other folds (Fig. 15).
In the rectum, the gland cells (Table 2) are
again fewer than in the fourth intestinal region
(Table 3). The gland cells are still fewer in the
leader folds (Figs. 13-16, 43, 44). The cell
body is located in the subepithelial connective
tissue or external to the muscle layers (Fig.
45).
Subepithelial Layers: The epithelium of the
alimentary canal is surrounded by a thin layer
of connective tissue and two muscle layers,
an inner longitudinal layer, and an outer
circular layer. The longitudinal muscle layer is
rather irregular; some longitudinal muscle
bundles were observed within or externally to
the circular layer.
The muscle layers are thicker in the oe-
sophagus (Fig. 26), in the third intestinal re-
gion (Fig. 36) and in the rectum (Figs. 42, 44).
The longitudinal layer is thicker in the oesoph-
agus and in the third intestinal region,
whereas in the rectum both muscle layers are
well developed.
In the other organs and regions, the muscle
layers are thin. In the crop (Fig. 27), in the
stomach (Fig. 32), and in the first and second
intestinal regions (Figs. 33, 34), the longitudi-
nal layer is the less developed; in the fourth in-
testinal region (Figs. 37, 38), the circular layer
is the least developed. In the intestinal cae-
D. D. M. T. buda-
reticulatum rodnae tenellus pestensis
40.3 46.5 ARS) 29.0
63.0 58.5 72.3 3748
49.0 45.1 58.5 56.3
62.5 57.3 61.3 32.5
42.0 ES TES) 41.8
21.3 32.8 30.3 17.5
10.1 6.8 9.8 10.0
6.3 6.0 — —
13.4 11.4 14.0 12.1
cum (Figs. 24, 40, 41), the muscle layers are
poorly developed and not well demarcated.
DISCUSSION
The light microscope observations demon-
strated that the alimentary canal of the
Limacoidea and Milacidae is divisible into five
morphologically distinct organs: vesophagus,
crop, stomach, intestine and rectum. In the in-
testine of the species studied here, | distin-
guish four histologically different regions,
whereas other pulmonates have only three in-
testinal regions (Carriker & Bilstad, 1946;
Moussa et al., 1983; Roldan & Garcia-
Corrales, 1988; Boer 4 Kits, 1990). Walker, in
Runham (1975) subdivided the intestine of
Deroceras reticulatum into only three intesti-
nal regions. He termed our fourth intestinal re-
gion as rectum without mentioning the actual
rectum, which is located in the pallial cavity.
Another peculiar feature of the intestine of
some Limacidae and Agriolimacidae is the
presence of an intestinal caecum. This can be
long, as in Lehmannia marginata, or short, as
in Deroceras reticulatum and D. rodnae. The
histological and ultrastructural features of the
caecum imply the probably function of this
organ. A simple columnar epithelium is pres-
ent from the oesophagus to the third intestinal
region and in the rectum. In the fourth intesti-
nal region and in the intestinal caecum, how-
ever, the epithelium is simple squamous, with
cells showing distinct ultrastructural features
that are characteristic of water- and ¡on-trans-
porting epithelia (Leal-Zanchet, in preparation
b). This would indicate that water and ¡ons are
absorbed from the faecal pellets. This was
also suggested by Boer & Kits (1990) for
48 LEAL-ZANCHET
FIG. 19. Semi-schematic drawing of part of a trans-
verse section of the stomach (out of the ty-
phlosoles) of Lehmannia marginata.
FIG. 20. Semi-schematic drawing of part of a trans-
verse section of the stomach of Lehmannia mar-
ginata showing the leader groove. Scale bar 10 um
Lymnaea stagnalis and is consistent with the
findings of Deyrup-Olsen (1987), who verified
that the distal part of the intestine of Ariolimax
columbianus plays a significant role in os-
moregulation.
Most of the epithelium of the alimentary
canal shows ciliated supporting cells. The cilia
play a role in the transport of the food bolus
and the faecal pellets (Roldan & Garcia-
Corrales, 1988; Boer & Kits, 1990). In the
== Ss. S u se
А Le
=X=== <E— cct
SS =
SSS = =3=3
cm EE mm
FIG. 21. Semi-schematic drawing of part of a trans-
verse section of the first intestinal region of Leh-
mannia marginata.
FIG. 22. Semi-schematic drawing of part of a trans-
verse section of the second intestinal region of
Lehmannia marginata. Scale bar 10 um.
stomach and in the rectum, however, there
are distinct folds with cilia that are longer and
very numerous. In addition, ultrastructural
data show that these cilia have very long roots
and are interconnected by well-developed
basal feet on the basal bodies (Leal-Zanchet,
in preparation b). In the stomach, such folds
are the typhlosoles and the transversal fold,
and in the rectum they are termed leader
folds. The cilia of the typhlosoles, the trans-
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA
49
TABLE 2. Length and width of the cell body (CB), of the nucleus (N) and diameter of the secretory cell gran-
ules (SGR) of gland cells of the digestive tube of Limacoidea and Milacidae (um).
L. D. T. buda-
В. pallens marginata О. laeve reticulatum D.rodnae M.tenellus pestensis
MCI N TD 5:3 8:2 Xx 5:8 8.5 x 5.7 6.8 x 5.1 8.8 x 5.3 00х70 86х55
SGR 2.0 23 2.0 1.8 12% 2.1 ПИ
MCI N 8.0 x 6.4 125% 50 9.5 x 5.0 8.0 x 5.5 11.0:6:5 SOX 723 95x55
SGR 0.5 0.5 0.5 0.5 0.5 0.5/2.0 0:5
Ме" СВ” 20'9'х 15:9 119.3%°15.1 20.6 х 13.2 18.0х 10:9’ 21.0315 20,3 x158 265 x157
М Tees) 10.4 x 6.8 7.3 x 4.9 8.5 x 4.6 8.0 x 5.0 84x63 84x64
SGR 1.5 1.4 1.4 17. 2 2.3 1.9
MC IV СВ 204x135 16.5 х 12.0 19.8 x 12.8 16.9 x 10.4 20.2 x 14.9 17.6 x 12.0 19.2 x 14.7
N 9.3 x 7.6 9.4 x 6.4 10.1 x 6.8 7.6 x 5.6 7.0 x 6.0 89x73 9.4х7.6
SGR 122 0.9 0.9 0.9 1.2 0.9 182
MCV CB — 11.4 x 11.9 — 11.8 х 9:5" 14:0х6.5 “14:0 x'6:5 —
N — 6.8x 3.7 5.0 x 3.2 6.5 x 3.8 6.5 x 3.8 —
SGR — — — — — — —
CY CB — 27.2 < 17.5 — 21 18.8 2310 x 17.5 17.7 х 13:5 —
N = 12.4 x 6.8 — 14.3х 6.7 10.2х53 13х48 —
SGR — — — — — — —
TABLE 3. Frequency of mucous cells in the digestive tube of Limacoidea and Milacidae.
В. E: D. D. M. T. buda-
pallens marginata D.laeve reticulatum rodnae tenellus pestensis
Oesophagus 15% 9% 5% 3% 3% 4% 10%
Crop 8% 9% 5% 9% 10% 8% 8%
Stomach 18% 10% 11% 13% 13% 10% 18%
1. intestinal region 54% 41% 35% 44% 46% 35% 55%
2. intestinal region 15% 8% 15% 4% 6% 13% 4%
3. intestinal region 22% 18% 19% 16% 21% 11% 27%
4. intestinal region 34% 17% 26% 16% 21% 18% 36%
Caecum — 4% — 8% 7% — —
Rectum 27% 21% 20% 25% 22% 19% 44%
versal fold and the rectal leader folds have a
distinct function. This would be consistent with
the studies of Walker (1972) on the physiology
of the stomach of Deroceras reticulatum; he
showed that the typhlosoles, the transversal
fold, and the leader groove play an important
role in the transport of fine material. No data
were found about the physiology of the rec-
tum. The rectal leader folds seem to be better
suited to aid faeces transport than the usual
folds. In the rectum of other pulmonates, the
presence of leader folds has not yet been de-
scribed.
In some regions of the alimentary tract of
the Limacoidea and Milacidae, the epithelium
is unciliated, namely in the crop, in the fourth
intestinal region, and in the intestinal caecum.
The absence of cilia in the crop was also ob-
served in other Stylommatophora (Ghose,
1963; Rigby, 1963, 1965; Roldan & Garcia-
Corrales, 1988). In the fourth intestinal region
of Boettgerillaand Tandonia, the epithelium is
completely unciliated, but in Malacolimax,
Lehmannia and Deroceras a reduced ciliated
area is present. According to Runham (1975),
the variation in the presence or absence of
cilia in the organs of the alimentary canal may
reflect the relative importance of cilia and
muscles for transport and mixing of food ma-
terial. In the crop, the muscle layers alone are
responsible for mixing food material with
the crop juice (Runham, 1975) and also for
transport of the food material towards the
stomach. In the fourth intestinal region, the cil-
lated area present in Malacolimax, Lehman-
nia and Deroceras seems to aid faeces trans-
port towards the rectum together with the
muscle layers.
50
LEAL-ZANCHET
TABLE 4. Distribution of gland cells in the intestine, caecum and rectum of Limacoidea and Milacidae.
B. [E D. reticula- D. M. T. buda-
pallens marginata D.laeve tum rodnae tenellus pestensis
1. intestinal region MC Il MC II MC II MC II MC Il MC Il MC II
MC I MC | MC | MC | MC |
2. intestinal region IC | IC | IC | IC | IC | IC | IC |
MC | MC | MC | MC | MC | MC | MC |
3. intestinal region IC Il IC Il IC Il IC Il IC Il IC Il IC Il
MC III MC Ill MC III MC Ill MC Ill MC III MC Ill
MC IV MC IV MC IV MC IV MC IV MC IV MC IV
MC V MC V
CY CY
4. intestinal region MC Ill MC Ill MC Ill MC Ill MC Ill MC Ill MC III
MC IV MC IV MC IV MC IV MC IV MC IV MC IV
МСУ MC V CY МСУ
CY CY CY
Caecum MC III MC III MC III
— MC IV MC IV MC IV — —
MC V MC V CY
CY CY
Rectum ME Il MC III MC III MC Ill MC III MC III MC Ill
MC IV MC IV MC IV MC IV MC IV MC IV MC IV
МСУ МСУ МСУ MC V МСУ
CY CY CY CY
The presence of five mucous cell types is
now reported for the alimentary canal of li-
macids and agriolimacids. Type | mucous cells
are present in the proximal regions of the ali-
mentary canal, such as the eosophagus, the
crop, in parts of the stomach, and also in the
second intestinal region. Type ll mucous cells
are found in the stomach and in the first in-
testinal region. The mucous cells of type Ill, IV
and V occur in distal regions of the canal, such
as the third and fourth intestinal regions, the
intestinal caecum, and the rectum. The mu-
cous Cells of type | and II are intraepithelial,
whereas the mucous cells of type Ill, IV and V
are subepithelial. The mucous cells of type V
are absent in Boettgerilla pallens and Tan-
donia budapestensis.
The functional role of the mucus would be
the lubrification of the lumen, helping in the
transport of food and faeces, the clumping of
food particles for the formation of the food
bolus, the formation of the faecal string, and
the compaction of the faecas (Carriker &
Bilstad, 1946; Pereira & Breckenridge, 1981).
The mucous cells of type | of the Limacoidea
and Milacidae, the occurrence of which is lim-
ited to the proximal parts of the tract, must be
related to the formation of the food bolus. The
formation of the faecal pellets that takes place
in the distal part of the stomach (Walker,
1972) should involve the mucous cells of type
|. The mucous cells of the distal regions of the
alimentary canal—types III, IV and V—should
be concerned with the compaction of the fae-
ces.
Gland cells that are termed intestinal se-
creting cells have been described for the ali-
mentary canal of various pulmonates (Haff-
ner, 1924; Baecker, 1932; Walker, in Runham,
1975; Roldan & Garcia-Corrales, 1988; Leal-
Zanchet et al., 1990; Franchini & Ottaviani,
1992). The intestinal secreting cells described
by these authors are similar to the type | in-
testinal secreting cells of the limacoids. We
observed also another type of gland cells (i.e.,
intestinal secreting cells of type Il) that are
clearly distinguishable from intestinal secret-
ing cells of type 1. The secreting cells of type |
and II occur in all the species studied in the
present investigation. The occurrence of the
intestinal secreting cells of type | and II in
the second and third intestinal regions, re-
spectively, and their positive reaction to pro-
tein (Leal-Zanchet, in preparation a), indicate
that the secretion of these cells is probably of
an enzymatic nature and may play a role in di-
gestion.
The occurrence of cystic cells in the ali-
mentary canal of gastropods has not yet been
described, but similar cells are known in the
salivary glands of pulmonates (Blain, 1957;
Bani, 1964, Boer et al., 1967). In Limacidae
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 51
icll
==>
FIG. 23. Semi-schematic drawing of part of a trans-
verse section of the third intestinal region of
Lehmannia marginata.
FIG. 24. Semi-schematic drawing of part of a trans-
verse section of the intestinal caecum of Lehmannia
marginata. Scale bar 10 um.
and Agriolimacidae, the secretion of the cystic
cells is positive to histochemical reactions for
protein (Leal-Zanchet, in preparation a), but
the exact role of the cystic cells is still unclear.
The cystic cells occur in the distal regions of
the alimentary canal of the limacids and agri-
olimacids, except in Deroceras laeve, but are
absent in Boettgerilla and Tandonia.
The alimentary diet of the species studied
in the present investigation differs widely.
Deroceras and Tandonia are herbivorous.
ct
cy
FIG. 25. Semi-schematic drawing of part of a trans-
verse section of the fourth intestinal region of
Lehmannia marginata. Scale bar 10 um.
Lehmannia lives on a specialized diet of
lichens, whereas Malacolimax feed prefer-
ently on fungus (Wiktor, 1973). Little is known
about the diet of Boettgerilla pallens, but
some data obtained in laboratory experiments
suggest that this species is carnivorous (Leal-
Zanchet, in press). Although the animals have
a distinct diet, only few distinguishing anatom-
ical and histological features were observed.
Boettgerilla presents a mere shortening of the
intestinal regions, a characteristic observed in
others carnivorous slugs, such as Daude-
bardia (Zonitidae) and Diplompharus (Rhyti-
didae) (Wiktor, 1983; Tillier, 1989). If Boett-
gerilla is carnivorous, the occurrence of a
powerful protease in its digestive system
would be expected. At present, having com-
pleted anatomical, histological, histochemical
(Leal-Zanchet, in preparation a) and ultra-
structural (Leal-Zanchet, in preparation b)
studies, we still cannot relate the different
diets of the Limacoidea and Milacidae to their
distinct histological features. An investigation
of the enzymes of Limacoidea and Milacidae
would clarify many questions.
52 LEAL-ZANCHET
FIG. 26. Cross-section of the oesophagus of Malacolimax tenellus showing a fold. Scale bar 50 um.
FIG. 27. Cross-section of the crop of Malacolimax tenellus. Note the abundant vacuoles with amorphous con-
tents. Scale bar 25 um.
FIG. 28. Cross-section of the crop of Tandonia budapestensis showing a fold. Scale bar 25 um.
FIG. 29. Cross-section of the distal part of the stomach of Lehmannia marginata demonstrating the ty-
phlosoles and the leader groove. Scale bar 80 um.
FIG. 30. Cross-section of the stomach of Lehmannia marginata demonstrating the lining epithelium of a ty-
phlosole. Note the strong acidophilic columnar cells and the mucous cells of type Il. Scale bar 25 um.
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 53
FIG. 31. Cross-section of the stomach of Lehmannia marginata showing the lining epithelium of the leader
groove. The holocrine secretion of a strong acidophilic columnar cell can be seen. Scale bar 25 um.
FIG. 32. Cross-section of the stomach wall (out of the typhlosoles) of Lehmannia marginata. Note the apoc-
rine secretion of the columnar cells. Scale bar 25 um.
FIG. 33. Cross-section of the first intestinal region of Lehmannia marginata showing the numerous mucous
cells of type Il. Scale bar 25 um.
FIG. 34. Cross-section of the second intestinal region of Malacolimax tenellus demonstrating the intestinal
secreting cells of type I. Scale bar 25 um.
FIG. 35. Cross-section of the third intestinal region of Malacolimax tenellus. The cell body of a mucous cell
oftype V can be seen. Scale bar 25 um.
FIG. 36. Cross-section of the third intestinal region of Boettgerilla pallens. Scale bar 25 um.
54 LEAL-ZANCHET
FIG. 37. Cross-section of the fourth intestinal region of Lehmannia marginata. Exceptionally, the circular layer
lays directly below the epithelium. Note the ciliated area. Scale bar 25 um.
FIG. 38. Cross-section of the fourth intestinal region of Boettgerilla pallens. A ciliated area is absent. Scale
bar 25 um.
FIG. 39. Cross-section of the fourth intestinal region of Deroceras laeve. A ciliated area can be seen. Scale
bar 25 um.
FIG. 40. Cross-section of the intestinal caecum of Lehmannia marginata. Scale bar 25 um.
FIG. 41. Cross-section of the intestinal caecum of Deroceras reticulatum. Scale bar 25 um.
FIG. 42. Cross-section of the rectum of Lehmannia marginata showing a fold. Scale bar 25 um.
HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 55
т E >
a
ncV
a = e
=
wa.
Br
< = tas
ex
%
#
Ss
+ cm
FIG. 43. Cross-section of the rectum of Boettgerilla pallens showing the well developed leader folds. Cilia are
absent outside the leader folds. Scale bar 25 um.
FIG. 44. Cross-section of the rectum of Deroceras laeve. Small leader folds and well-developed muscle lay-
ers can be seen. Scale bar 25 um.
FIG. 45. Cross-section of the rectum of Deroceras reticulatum. Note the numerous mucous cells of type III
and two cell necks of mucous cells of type V. Scale bar 25 um.[
FIG. 46. Cross-section of the rectum of Deroceras reticulatum demonstrating the large cell body of a cystic
cell. Scale bar 25 um.
FIG. 47. Cross-section of the rectum of Malacolimax tenellus. Observe the small cell body of a mucous cells
of type V. Scale bar 25 um.
56 LEAL-ZANCHET
ACKNOWLEDGEMENTS
The author wishes to thank Dr. Wolfgang
Rähle for supervising her doctoral thesis and
Prof. Dr. Wolfgang Maier for providing space in
his department. Thanks are also due to Dr.
Dieter Bunke for discussion about the histol-
ogy and for help with the historesin technique,
and Dr. Klaus Eisler and Miss Martina
Hohloch for assistance with the photography.
The help of Mr. Neuri Zanchet and Miss
Irmlind Heinze in collecting specimens is also
gratefully acknowledged.
RESUMO
Estudo comparativo da anatomia e histolo-
gia do tubo digestivo dos Limacoidea e
Milacidae (Pulmonata: Stylommatophora)
Descreve-se comparativamente a anato-
mia e histologia do tubo digestivo de Deroc-
eras laeve, D. reticulatum, D. rodnae, Leh-
mannia marginata, Malacolimax tennelus,
Boettgerilla pallens e Tandonia budapesten-
sis. О tubo digestivo destes animais é com-
posto pelo esöfago, papo, estömago, intestino
e reto. Um ceco intestinal estä presente em
О. reticulatum, D. rodnae e L. marginata. O in-
testino pode ser subdividido em quatro re-
giöes histologicamente distintas. Do esöfago
a terceira regiáo intestinal, e no reto, o tubo di-
gestivo é revestido em sua maior parte por
um epitélio cilíndrico a cúbico simples. Na
quarta regiao intestinal e no ceco o epitélio
apresenta-se pavimentoso a cúbico simples.
As células epiteliais de suporte podem ser ci-
liadas ou nao, mas apresentam sempre mi-
crovilos. Distinguem-se também oito tipos
celulares secretores: células mucosas do tipo
|, células mucosas do tipo Il, células mucosas
do tipo Ш, células mucosas do tipo IV, células
mucosas do tipo V, células císticas, células
secretoras intestinais do tipo | e células se-
cretoras intestinais do tipo II. Subepitelial-
mente, encontram-se uma camada de tecido
conjuntivo frouxo e duas camadas muscu-
lares, uma longitudinal interna e outra circular
externa.
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MALACOLOGIA, 1998, 39(1-2): 59-75
MORPHOLOGY OF THE WESTERN ATLANTIC HALIOTIDAE (GASTROPODA,
VETIGASTROPODA) WITH DESCRIPTION OF A NEW SPECIES FROM BRAZIL
Luiz Ricardo L. Simone
Secáo de Moluscos, Museu de Zoologia da Universidade de Sao Paulo, Caixa Postal 7172,
CEP 01064-970, Sao Paulo, SP. Brazil
ABSTRACT
Haliotis aurantium, new species, is described from the southeastern coast of Brazil and com-
pared morphologically with Haliotis pourtalesii, which inhabits the Gulf of Mexico and the
Caribbean Sea. These species differ mainly in characters of the head, epipodium, metapodium
and digestive system. The species are also compared anatomically with other species of Haliotis
based on published descriptions and on comparative dissections of H. lamellosa and H. tuber-
culata.
INTRODUCTION
The finding of a haliotid in the western
Atlantic excited considerable interest (Hender-
son, 1915; Harry, 1966; Klappenbach, 1968;
Sarasua, 1968; Merrill & Petit, 1969; Guince,
1969; Nijssen-Meyer, 1969; Silva & Guerra,
1981; Martinez & Ruiz, 1994), although until
now few specimens with soft parts have been
found (Titgen & Bright, 1985).
All western Atlantic haliotid specimens
have been identified as Haliotis pourtalesii
Dall, 1881, with apparently in two disjunct
populations, one from North Carolina to Cuba
and other in Brazil (Rios, 1985; Titgen &
Bright, 1985). These identifications were
based only on shell characters, because no
anatomical information has appeared to date.
Photos and a brief description of the head-
foot and color pattern of a specimen from the
Gulf of Mexico were given by Titgen & Bright
(1985).
Specimens from the two regions were care-
fully compared in their morphology, showing
that, despite the similarity in their shells, the
specimens from these regions are sufficiently
distinct to be regarded as separated species.
Few papers provide anatomical information
on haliotids (e.g., Freure, 1905; Crofts, 1929,
1937, 1955; Campbell, 1965; Russell & Evans,
1989). The present paper provides an anatom-
ical description, as a basis for comparison be-
tween the two western Atlantic species, as well
as for use in a future systematic rearrange-
ment of the family, which has about 70 species
in the single genus Haliotis Linné, 1758
(Abbott & Dance, 1983).
59
The complex history of the discovery of H.
pourtalesii in waters near Florida and the loss
of its type specimen have been discussed
elsewhere (e.g., Foster, 1946; Titgen & Bright,
1985). Also there are misidentifications based
on young specimens of other species (Abbott,
1974) and based on similar species occurring
in Pacific waters (H. dalli Henderson, 1915; H.
roberti McLean, 1969), demanding care in lit-
erature analysis.
MATERIAL AND METHODS
The specimens from Brazil are in the col-
lection of the Museu de Zoologia da Univer-
sidade de Sao Paulo (MZSP), some of them
collected by Instituto Oceanografico da Uni-
versidade de Sao Paulo (IOUSP) in the pro-
ject “Monitoramento Ambiental Oceanico da
Bacia de Campos.” The northern specimens
are in the collection of the National Museum
of Natural History (USNM) and Marine Invert-
ebrate Museum, Rosenstiel School of Marine
and Atmospheric Science, University of Miami
(UMML). Those with soft parts are preserved
in 70% ethanol.
Two Brazilian and five northern specimens
were available with soft parts for dissections,
which were made using standard techniques.
Some organs, such as the buccal mass and
pallial organs, were dehydrated in ethanol se-
ries, stained with carmine, fixed and cleared in
creosote. All drawings were made with the aid
of a camera lucida. Shells, radulae and jaws
were also examined using SEM in the Lab-
oratorio de Microscopia Electrönica do Insti-
60 SIMONE
tuto de Bioci@ncias da Universidade de Säo
Paulo. The shells were not coated with gold.
Odontophoral muscles were examined by di-
rect dissection, although the jugal muscles
were not seen in detail. The nomenclature of
buccal musculature follows Fretter & Graham
(1962).
Anatomical comparison with other Halio-
tidae is based on the literature (Fleure, 1905;
Crofts, 1929, 1937, 1955; Fretter & Graham,
1962; Campbell, 1965, digestive system; Rus-
sell & Evans, 1989, circulatory system) and
on comparative examination of two lots of the
MZSP collection: MZSP 13340, Haliotis tuber-
culata Linne, 1758, 5 specimens in 70%
ETOH from Trieste, Italy; MZSP 28202, Halio-
tis lamellosa Lamarck, 1822, 1 specimen in
70% ETOH from Trieste, Italy.
In the figures the following abbreviations
are used: ac: anterior cartilages; af: accessory
oesophageal fold; al: aperture of left oe-
sophagea! pouch; an: anus; ar: aperture of
right oesophageal pouch; cm: main (right) col-
umellar muscle; da: direct anterior radular
tensors; dg: digestive gland; dr: direct radular
tensor muscle; ef: efferent gill vessel; ep: epi-
podium; ff: dorsal epipodial flap; gc: gastric
caecum; go: gonad; hz: horizontal muscle; if:
intermediary epipodial flap; im: intertentacular
mernbrane; in: intestine; ja: jaws; la: left auri-
cle; Ic: left columellar muscle; lg: left gill; Ih: left
hypobranchial gland; Ik: left kidney; Im: lateral
protractor muscle; Ip: left oesophageal pouch;
ma: main epipodial tentacle; mb: mantle bor-
der; mt: metapodium; nr: nerve ring; oa: outer
approximator muscle of cartilages; od: odon-
tophore; oe: oesophagus; om: ommatho-
phore; os: osphradium; pc: posterior cartilage;
pr: pigmented region of dorsal epipodial flap;
pv: posterior ventral radular tensor muscle; ra:
right auricle; rd: radula; rg: right gill; rh: right
hypobranchial gland; rk: right kidney; rp: right
oesophageal pouch; rs: radular sac; rt: rec-
tum; sa: sorting area; sf: pigmented multipapil-
late tentacles surrounding ma; sl: pallial slit;
sn: snout; sr: subradular membrane; st: stom-
ach; tc: metapodial tentacle covered with long
cilia; te: cephalic tentacle; tm: metapodial ten-
tacle; ts: slit pallial tentacle; ty: gastric ty-
phlosole; vp: ventral buccal protractor muscle;
ve: ventricle; vf: ventral epipodial flap.
Abbreviations of institutions: MNRJ: Museu
Nacional do Rio de Janeiro; MORG: Museu
Oceanografico da Fundacäo Universidade de
Rio Grande; MZSP: Museu de Zoologia da
Universidade de Sao Paulo; UMML: Marine
Invertebrate Museum, Rosenstiel School of
Marine and Atmospheric Science, University
of Miami; USNM: National Museum of Natural
History, Smithsonian Institution.
SYSTEMATICS
Haliotis aurantium, new species (Figs. 3-9,
11-13, 18-35)
Haliotis pourtalesii: Klappenbach, 1968: 1-2;
Rios, 1970: 16, pl. 1; Silva & Guerra,
1971: 49-50, figs. 1-4; Rios, 1975: 11, pl.
1, fig. 4; Rios, 1985: 10, pl. 5, fig. 35; Rios,
1994: 22, pl. 5, fig. 39 (non Dall, 1881).
Types: Holotype, MZSP 28201, from type lo-
cality; paratypes: MZSP 18482, 1 shell,
off Ubatuba, Sao Paulo, 24°07'S
44°06'W, 150 m depth; MZSP 19569, 2
shells, 22°27'6"S 40°30'W, off Cabo de
Sao Tome, Rio de Janeiro, Brazil, 95 m
depth (11/11/1969); MZSP 28391, 1 spec-
imen, 21°05'S 41°19'W, east of Ponta do
Ubu, Espirito Santo, Brazil, 48 m depth
(E. C. Oliveira Fo. col., 1986).
Type Locality: Brazil, Rio de Janeiro, off
Campos Bay (sta. 21), 22%06'06"S
40°08'38"W, 95 m depth (R. V. Astro-
garoupa, 22/vii/1991).
Diagnosis
Minute southwest Atlantic species with un-
pigmented head-foot and mantle; two tenta-
cles in mantle slit; epipodial tentacles ran-
domly arranged; pair of large epipodial
tentacles posteriorly; pair of metapodial tenta-
cles sometimes present; lobed snout border;
left pouch of buccal mass covering ventral
surface of odontophore; several pairs of lat-
eral radular protractor muscle.
Description
Shell (Figs. 3-7). Auriform, fairly thin, subel-
liptical, up to 15 mm in length, few more than
three whorls (Fig. 4). Color of exposed areas
from homogeneous vivid reddish orange in
living specimens to pale yellow in eroded
specimens. Protoconch (Fig. 5) of two whorls,
low, sculptured by several minute, uniform,
spiral threads. Spire small, low, submarginal,
situated on posterior fourth of shell (Figs. 4,
7). Aperture subelliptical, nacreous. Base of
shell concave with some lateral torsion.
Columella with a sulcus inside raised parietal
margin of aperture (Fig. 6). Three to four oval
WESTERN ATLANTIC HALIOTIDAE 61
FIG. 1-7; shells in SEM. (1) and (2) left and dorsal view of Haliotis pourtalesii USNM 833627, scales =2 mm;
(3
(4
(6
shell pores (tremata) open, preceded by sev-
eral closed, all of them oval, with elevated
margins (Figs. 4, 6, 7). Spiral sculpture of
sharp, rather widely shaped cords, between
which finer threads are occasionally interca-
lated (Fig. 3). About 30 cords and threads in
area between suture and outer margin of
) detail of middle-outer region of body whorl of Haliotis aurantium, MZSP 18482, paratype, scale = 0.5 mm;
) to (7) Haliotis aurantium holotype: (4) dorsal view, scale =2 mm; (5) detail of protoconch, scale = 0.5 mm;
) ventral view, scale = 2 mm; (7) left view, scale = 2 mm.
body whorl. Lateral portion of body whorl with
three cords followed by strong, angular, pe-
ripheral ridge or carina (Fig. 7). Immediately
below this, three or four more cords present
(Figs. 6, 7). Axial sculpture consisting of radi-
ating lamellae, which roughly correspond to
pores in their position, considerably variable,
62 SIMONE
sometimes missing. Minute uniform axial
cords between spiral cords occasionally pre-
sent. Surface with very fine growth lines. No
periostracum apparent.
Head-Foot. Head somewhat protruding
(Fig. 21). Tentacles stubby, short, broad, cov-
ered with long cilia, pigmented by regular pale
brown, successive transverse bands (Fig. 21).
All other structures without pigment.
Ommatophore well developed, in outer basal
region of tentacles (Figs. 18, 19, 21), with
dark, vesicular, opened eyes. Intertentacular
membrane a semi-transparent, thin flap (Figs.
21, 26, 27) between the two cephalic tenta-
cles, covering anterior region of snout and
inner region of tentacles (Fig. 21). Snout well
developed (Figs. 21, 25), cylindrical, broad,
with irregular ventral margin. Foot large, about
same size as shell aperture (Fig. 19), without
pigment. Epipodium with many lobed tenta-
cles (Figs. 19, 22, 23), without pigment, uni-
form in size, some of them covered with long
cilia, apparently without special organization
(Fig. 22); in posterior extremity of epipodium
two epipodial tentacles larger and longer (Fig.
23), and a median area without tentacles (Fig.
23). In the holotype, a pair of long metapodial
tentacles present on posterior border; dis-
sected paratype without this structure. Main
(right) columellar muscle very large, circular in
section (Fig. 18). Secondary (left) columellar
muscle very small (Fig. 25: Ic).
Mantle border. Trifolded and simple, without
pigment. Slit deep (Figs. 18, 20), with two ten-
tacles covered with long cilia, one on the left-
anterior border and other on the right-poste-
rior border of slit (Figs. 18, 20).
Pallial cavity. Short, about half of body whorl
(Fig. 20). Gills short, bipectinate, right gill
shorter than left (Fig. 20). Afferent gill vessel in
base of gill’s insertion. Efferent vessel be-
tween two flaps of each gill leaflet, inserting in
gill sub-terminally, anterior to posterior ex-
tremity of gill (Fig. 20). Hypobranchial glands
present, left larger, with several transverse,
uniform furrows; right much smaller, with three
oblique furrows. Both hypobranchial glands
situated at left of slit (Figs. 18, 20, 24). Rectum
between both hypobranchial glands, slightly
free in posterior half of pallial cavity; anus
papillated near posterior extremity of slit (Figs.
20, 24).
Circulatory and excretory systems. Kidneys
and pericardium situated ventrally, in mid-left
side of animal just behind pallial cavity (Fig.
24). Left kidney short, broad, with a short
8
FIG. 8-13. Jaws and radula in SEM: (8) jaws of
Haliotis aurantium, scale = 200 um; (9) detail of
same, scale = 20 um (10) detail of central region
of jaws of Haliotis pourtalesii, scale = 50 um;
(11) radula of Haliotis aurantium, scale = 100 um;
(12) detail of same, central region, scale = 50 um;
(13) detail of Fig. 11, marginal region, scale =
50 um.
WESTERN ATLANTIC HALIOTIDAE 63
FIGS 14-17. Radulae of Haliotis pourtalesii in SEM: (14) USNM 833627, scale = 100 um; (15) UMML
30-8376, scale = 100 um; (16) detail of Fig. 14, marginal region, scale = 100 um; (17) the same, central re-
gion, scale = 100 um.
papillated nephrostome in ventral base of rec-
tum. Right kidney long, thin, Iying right margin
of pallial cavity (Fig. 24), its nephrostome a
longitudinal slit in its anterior extremity.
Ventricle large, surrounding intestine; left auri-
cle anterior to ventricle and right auricle ven-
tral to it.
Digestive system. Mouth in snout, covered
internally by pavement-shaped papillae (Fig.
28). Jaws two small plates (Figs. 8, 28), with
rounded borders, situated in mid-dorsal region
of mouth; median and anterior region of each
plate with a small, sharp pointed projection
(Figs. 8, 9). Buccal mass very large, complex;
odontophore surrounded by two glandular oe-
sophageal pouches (Figs. 25-27), both with
inner surface covered by many tall villiform
papillae (Fig. 28). Left pouch broad, short, cov-
ering ventral and lateral-left surfaces of odon-
tophore. Right pouch narrow, long, beginning
at right and running obliquely (Figs. 26, 27).
Both pouches open in ventral-anterior region
of oesophagus in two separated, tall, ring-like
folds; a third short accessory fold also at this
position (Fig. 28: af). Odontophore short, with
very long radular sac, extending behind buccal
mass and terminating near stomach (Fig. 25).
Radula (Figs. 11-13): rachidian teeth broad,
short, each with a large curved terminal cusp
and two lateral bolsters (Fig. 12); first lateral
tooth with triangular base and small rounded
cusp; second lateral tooth long, with a some-
what rectangular base and a lateral-terminal,
hook-like cusp; third lateral tooth the largest,
with along, irregular, curved base, and a large,
long, sharp cusp, of almost the same length as
base; fourth and fifth lateral teeth similar to
third, but narrow; fifth narrowest, sharply
pointed. About 32 pairs of marginal teeth per
row (Fig. 13), with a long stalk; main cusp
rounded, curved, spoon-like, flanged on each
side by two small, sharp secondary cusps;
marginal teeth gradually decrease in size lat-
erally. Odontophore muscles (Figs. 31-35)
consisting of: pair of ventral buccal protractor
muscles, with their origin in ventral-lateral
inner surface of peribuccal wall, and their in-
sertion in ventral lateral region of posterior car-
tilages (Figs. 31, 32: vb); pair of direct radular
tensor muscles, their origin in mid-ventral re-
gion of posterior cartilages and insertion on
lateral angles of ventral edge of radula (Figs.
31, 33, 34: dr); pair of muscles as outer ap-
proximator of cartilages, their origin in anterior
surface of posterior cartilages and insertion in
outer lateral surface of anterior cartilages
64 SIMONE
FIGS. 18 to 20.Haliotis aurantium n. sp. anatomy: (18) topography of the holotype specimen in dorsal view;
(19) the same in ventral view, scale = 2 mm; (20) pallial organs, mantle deflected, inner-ventral view, scale =
1mm.
(Figs. 33-35: oa); pair of small posterior ven-
tral radular tensor muscles, their origin in ven-
tral inner surface of peribuccal wall and inser-
tion in mid-ventral region of radular sac (Figs.
31, 32: pv); several pairs of small lateral pro-
tractor muscles, their origin in dorsal inner sur-
face of peribuccal wall and insertion in dorsal-
mid surface of radula (Fig. 32; Im); pair of direct
anterior radular tensors, their origin in ventral-
dorsal surface of posterior cartilages and in-
sertion in lateral borders of sub-radular mem-
brane and ventral surface of radula up to mid
line, in a “М” shape (Fig. 33: da); and horizon-
tal muscle, uniting ventral edge of both anterior
cartilages (Figs. 31, 35: hz). Anterior odon-
tophoral cartilages long, flattened, anteriorly
sharp, posteriorly broad, with rounded borders
(Fig. 35). Posterior odontophoral cartilages
very short, elliptic, situated in outer posterior
extremity of anterior catilages (Fig. 35).
Oesophagus short, flattened tube (Figs. 25,
26), with about eight internal longitudinal folds
(Fig. 28). Stomach very large, U-shaped, near
mid line in posterior region of animal (Fig. 18).
WESTERN ATLANTIC HALIOTIDAE 65
tm 23
FIG. 21 to 24.Haliotis aurantium n. sp. anatomy: (21) detail of head, frontal view, mantle removed, scale = 1
mm; (22) detail of left-posterior fourth of epipodium, scale = 1 mm; (23) detail of posterior extremity of foot,
ventral view, scale = 1 mm; (24) semi-diagrammatic drawn of cleared pericardial and nearby structures, ven-
tral-right view, scale = 1 mm.
Oesophageal branch of stomach ventral, con-
ical, with a very small caecum (Fig. 26).
Internally oesophagial branch of stomach with
mosaic of low, irregular folds near oesophagus
opening (Fig. 30), where three longitudinal
folds begin, two of them contouring posterior
extremity of stomach, becoming weaker pos-
teriorly; the third fold becoming larger and run-
ning to intestinal branch of stomach where it
becomes weaker (Fig. 30: ty). Some radial
muscle fibers in stomach wall originating be-
tween oesophageal and intestinal branches of
stomach (Fig. 30: Ic) and inserting in small left
columellar muscle (Fig. 25: Ic). Intestinal
branch of stomach dorsal, conical, larger than
oesophageal branch (Fig. 26); two typhlosoles
running alongside gastric intestinal branch
from caecum into intestine, one of them pre-
senting in its mid region a series of oblique
folds, differentiating a small sorting area (Fig.
29:sa). Other regions of stomach inner surface
smooth, covered by thin greenish cuticle (Figs.
29, 30). Intestine long, with thin transparent
walls, running near right side of head, when it
twists and returns to posterior region near
stomach (Figs. 25, 27); in this posterior region,
itis sigmoid, running through pericardium (Fig.
24) and exiting into pallial cavity (Figs. 20, 25,
26). Intestine and stomach full of gravel.
Digestive glands large, green, with mosaic
of irregular brown spots on its surface (Figs.
18, 25) and occupying visceral mass ventral to
gonad, surrounding stomach (Fig. 25).
Genital system. Very large ovary occupying
all of spire and part of body whorl (Figs. 18,
25: go), pale cream in color. Ovary with three
lobes (Fig. 18), one within spire, one posterior
to main columellar muscle, the third in left side
of this muscle (Fig. 18). Oviduct, which proba-
bly runs within right kidney, not seen. Ventral
limit of gonad at the digestive gland and stom-
ach (Fig. 25). Male not examined.
66 SIMONE
FIG. 25 to 29. Haliotis aurantium n. sp. anatomy: (25) cephalic organs and visceral mass, ventral view, foot
and mantle removed; (26) extracted head and digestive ducts, right-dorsal view; (27) detail of anterior region
of digestive system, dorsal view, head tegument partially removed; (28) snout, buccal mass and esophagus
opened longitudinally, ventral view; (29) stomach opened longitudinally in intestinal branch. Scales = 1 mm.
WESTERN ATLANTIC HALIOTIDAE 67
Nervous system. Only circum-oesophageal
region examined, agreeing closely with that
described by Fleure (1905) and Crofts (1929)
for Haliotis tuberculata.
Measurements (respectively length in mm,
width in mm, number of whorls, of opened
pores, of closed pores). Holotype, MZSP
28201: 12.9 by 8.7, 3.2, 4, 14; Paratypes:
MZSP 18482: 15.0 by 10.5, 3.2, 3, 16; MZSP
19569: 13.2 by 9.0, 3.0, 4, 13 and 7.2 by 5.2,
2.1, 4, 8.
Habitat. From 77 to 150 m depth, on gravel,
generally associated with Laminaria sp.
Etymology. The specific name refers to the
orange color of the external shell surface
(Latin, aurantium).
Material Examined. Types.
Material Available. BRAZIL; Amapa; MORG
15000, 1 shell, off Cabo Orange, 100 m (R. V.
Almirante Saldanha, 30/xi/1988). Espirito
Santo; MNRJ-HSL 6603, 2 specimens, off
Vitoria (lost); MORG 15930, 1 shell, off
Vitoria, 87 m (R. V. A. Saldanha, 24/iv/1969);
ММА] 3577, 20 shells, 20°37'05”S
39°59'00"W, 87 m (R. V. A. Saldanha, sta.
DHN 2027, 24/ix/1971). Rio de Janeiro;
MNRJ 3554, 14 shells, 21°56'05"$
40°07'00"W, 77 m (В. V. A. Saldanha, sta
DHN 2012, 11/ix/1969); MNRJ 3578, 4 shells,
off Cabo Frio, 23°05'00"S 40%05'00"W, 111 т
(R. V. A. Saldanha, sta. DHM 2168); MORG
15931, 1 shell, off Cabo Frio, 90 m (R. V. A.
Saldanha, 9/ix/1969); MORG 26226, 1 shell,
off Cabo Frio (R. V. A. Saldanha, x/1986, on
Laminaria); MORG 15929, 1 shell, off Cabo
de Sao Tomé, 77 m (R. V. A. Saldanha,
11/iv/1969). Rio Grande do Sul; MORG
17467, 1 shell, off Conceicao, 126 т (В. V.
Mestre Jerönimo, 2/x/1972).
Haliotis pourtalesii Dall, 1881 (Figs. 1, 2, 10,
14-17, 36-45)
Haliotis (Padollus) Pourtalesii Dall, 1881: 79
[Gulf Stream near Florida Reefs, 180 m
(31/ili/1869)] (described from memory).
Haliotis pourtalesii: Dall, 1889: 168;
Henderson, 1911: 81 [neotype]; Cooke,
1914: 103; Henderson, 1915: 660, pls.
45, 46; Smith, 1937: 78, pl. 29, fig. 3;
Foster, 1946: 38-40, pl. 22, figs. 1, 2;
Parker, 1960; Harry, 1966: 207-208, pl.
30; Jung, 1968; Sarasua, 1968: 1-8, figs.
1, 2; Merrill & Petit, 1969: 117; McLean,
1969: 115; Guice, 1969: 140; Abbott,
1974: 18, fig. 30; Titgen & Bright, 1985:
147-152 figs. 1, 2; Abbott & Dance, 1983:
19, fig.; Ode, 1986: 69-73; Martinez &
Ruiz, 1994: 63-64, figs. 1-2.
Type: Neotype USNM 271601 [3 miles off
Sand Key, Florida, 49 m, 1913]
Diagnosis
Minute northwest Atlantic species with pig-
mented epipodium and metapodium; three
tentacles in mantle slit; epipodial tentacles
arranged in three layers around well-devel-
oped (main) tentacles; without large epipodial
tentacles posteriorly; without metapodial ten-
tacles; snout border papillated; ventral surface
of odontophore free of pouches; only one pair
of lateral radular protractor muscles.
Description
Shell. Figs. 1, 2.
Head-foot. Head somewhat protruding (Fig.
39). Tentacles long, narrow, covered with long
cilia; pigmented by regular dark brown, suc-
cessive, well-spaced transverse bands and a
mid longitudinal band (Fig. 39). Dark brown
spots abundant in dorsal and ventral epi-
podium faces and dorsal face of metapodium,
scanty in metapodial sole. Ommatophore well
developed, on outer basal region of tentacles,
with dark, vesicular, open eyes (Figs. 36, 37,
39). Intertentacular membrane semitranspar-
ent, thin, covering anterior region of snout and
inner region of cephalic tentacles (Fig. 39).
Snout well developed, cylindrical, broad, with
regular small, abundant papillae on its ventral
border (Fig. 39). Foot large, about same size
as shell aperture (Fig. 37). Epipodium with
many tentacles (Fig. 37) arranged as follows
(Fig. 41):(1) a dorsal flap (ff) fringed by flat-
tened, polytomic tentacles in a uniform zigzag
pattern; regions nearest foot with dark pig-
ment in dorsal and ventral faces (pr), other re-
gions white; (2) intermediary flap (if) with con-
spicuous, large, white, sharp tentacles (called
“main” tentacles), covered with long cilia (ma);
bases of these main tentacles, which are ven-
tral to pigmented region of superior flap (pr),
surrounded by two (one on each side) large,
multipapillated, dark-brown colored tentacles
(sf); between these structures, many other
short tentacles, with rounded tips, without pig-
ment nor evident cilia; (3) ventral flap (vf) ex-
tremely rich in tentacles, some of them longer
68 SIMONE
x
SS
SECS ASIAN
FIG. 30 to 35. Haliotis aurantium n. sp. anatomy: (30) oesophageal branch of stomach opened longitudinally;
(31) ventral view of odontophore; (32) dorsal view of same; (33) ventral view of radular ribbon and subradu-
lar membrane showing the “M” in shape insertion of “da”; (34) lateral-right view of odontophore, direct ante-
rior radular tensor muscle (da) partially sectioned; (35) dorsal view of odontophore with part of its muscles
removed, right anterior cartilage deflected. Scales = 1 mm.
(tc), sharp, covered with long cilia, similar to
but smaller than main tentacles; other tenta-
cles short, without pigment, with rounded tips
without evident cilia. Epipodium on each side
begining near snout and ending at posterior
extremity of foot, where it unites with metapo-
dial sole (Fig. 40); practically no region without
tentacles. Number of main epipodium tenta-
cles in each side from 10 to 12. Main columel-
lar muscle very large, circular in section (Fig.
36). Secondary (left) columellar muscle very
small, with some fibers attached to mid wall of
stomach (Fig. 42: Ic).
Mantle border. Trifolded, simple, depig-
mented. Slit deep, with three sharp tentacles
covered with long cilia, two of them about mid
region of the slit (one in each side), and the
third in posterior extremity of slit (Figs. 36, 38).
Pallial cavity. Short, about half of body whorl
(Figs. 36, 38). Gills somewhat long, bipecti-
WESTERN ATLANTIC HALIOTIDAE 69
FIG. 36 to 38. Haliotis pourtalesii anatomy: (36) topography of the specimen USNM 833627 in dorsal view,
scale = 2 mm; (37) same in ventral view, scale = 2 mm; (38) pallial organs, mantle deflected, inner-ventral
view, scale = 1 mm.
70 SIMONE
FIG. 39 to 42. Haliotis pourtalesii anatomy: (39) detail of head, frontal view, mantle removed, scale = 1 mm:
(40) detail of posterior extremity of foot, ventral view, scale = 1 mm; (41) detail of left-posterior fourth of
epipodium, scale = 1 mm; (42) cephalic organs and visceral mass, ventral view, snout opened longitudinally,
scale = 1 mm.
nate; right gill somewhat shorter than left (Fig.
38). Afferent gill vessel in base of gill’s inser-
tion. Efferent vessel between two flaps of each
gill leaflet, inserting in gill sub-terminally, an-
terior to posterior extremity of gill. Hypo-
branchial glands present, left very large (Fig.
38), with about 18 transverse, successive,
uniform folds; right smaller, with two oblique,
somewhat curved folds (Figs. 36, 38). Both hy-
pobranchial glands at left of slit (Fig. 38).
Rectum lying between hypobranchial glands,
slightly free in posterior half of pallial cavity;
anus papillated, opening in posterior third of
slit (Fig. 38).
Circulatory-excretory systems. Kidneys and
pericardium in posterior left side of animal,
just behind pallial cavity. These structures are
very similar to those of the preceding species
(Figs. 24, 38).
Digestive system. Mouth in snout, covered
WESTERN ATLANTIC HALIOTIDAE 71
internally by smooth walls (Fig. 42). Jaws two
somewhat large plates (Fig. 42), brown, in
mid-dorsal region of buccal cavity, lateral and
anterior border with a sharp edge, median-an-
terior region with a sharp projection (Fig. 10).
Buccal mass large, complex; odontophore
(Fig. 42) surrounded, except in its ventral re-
gion, by two glandular pouches, both with
inner surface covered by many tall, villiform
papillae (Fig. 43). Left pouch broad, short,
covering left side of odontophore. Right pouch
narrow, long, running obliquely from right side
of odontophore. Both pouches opening in ven-
tral-anterior region of oesophagus in sepa-
rate, tall, ring-like folds, that of left chamber
more anteriorly (Fig. 43). Radular sac very
long, running close to and attached to dorsal
region of buccal mass and oesophagus (Fig.
42). Radula (Figs. 14-17): similar to that of
preceding species, except for the marginal
teeth, which have a much longer, sharp cen-
tral cusp (Fig. 16); and in being more abun-
dant, with about 40 pairs per row (Figs. 14,
15). Odontophore similar to that of preceding
species (Figs. 44, 45), including cartilages
and intrinsic muscles; except lateral protractor
muscle of radula (Fig. 45: Ip), which in H. pour-
talesii is a single, larger pair.
Oesophagus short, flat (Fig. 42), with about
ten internal longitudinal folds (Fig. 43).
Stomach very large, U-shaped; walls irregu-
lar, with two ventral (oesophageal) and three
dorsal (intestinal) chambers and a well-devel-
oped, narrow spiral caecum in right dorsal re-
gion of stomach, with about one whorl (Fig.
36: gc). Stomach and intestine of studied
specimen with a large quantity of clear gravel
and organic materials including foraminiferan
shells and unidentified bristles.
Intestine, rectum and digestive gland char-
acters similar to those of preceding species
(Fig. 42).
Genital system. Gonad with only two lobes:
a lobe within spire and another just posterior
to main columellar muscle (Fig. 36). Ventrally,
this gland terminates at the digestive gland
and stomach (Fig. 42).
Measurements (respectively length in mm,
width in mm, number of whorls, of opened
pores, of closed pores). USNM 833627: 17.8
by 12.5, 3.2, 6, 18. UMML 30-8376: a) 20.1 by
15.0, 3.1, 5,18; 6) 17.6 by-11..3, 3.0,.5, 17;.0)
29570, 2,5,.5;.12:9).21.65у.150,;3.2:5,
1858).19.6: by 13.0, 3:0,-5,.17; 1) 13.7 by 9.9,
Э.0, 6, 116;.9) 12.9 by 9.1, 2:3,5, 14.
Habitat. Depth from 50 to 160 m. In the lit-
erature, the habitat is referred to as bottoms
with rocks, stones sand and shell debris, or
reef (Nijssen-Meyer, 1969).
Material examined: USNM 833627, 1 spec-
imen, U.S.A., Gulf of Mexico, off Florida,
25°16'55"N, 83°37 47'°W, «74m depth
(15/viii/1984). UMML 30-8376, 4 specimens
+ 5 shells, off VENEZUELA, 11%03'N
65°59'W, 69-155 т (R. V. Pillsbury sta. P-736,
22/vii/1968).
DISCUSSION
Haliotis pourtalesi and H. aurantium are
atypical Haliotidae. Generally, haliotids are
large gastropods, over 150 mm in length,
whereas both these species are less than 25
mm. Haliotids generally are common and
occur in shallow waters, whereas these
species are rare and found in deep water
(slope). However, both have the same general
anatomical characters of the family, modified
due to miniaturization.
Haliotis aurantium differs from H. pourtale-
sii in having: (1) a smaller size; (2) only two
tentacles on the mantle slit (not three); (3)
only transverse bands in the cephalic tenta-
cles (without a longitudinal band); (4) foot
without pigment; (5) epipodial tentacles of a
simpler structure (see below); (6) a pair of
larger tentacles on the posterior extremity of
the epipodium and a proportionally large area
with out tentacles posteriorly; (7) a pair of
large tentacles on the posterior extremity of
the metapodium sometimes present (absent
in paratype) (Fig. 23: tm); (8) right gill much
shorter than the left (in H. pourtalesii both gills
are about the same size and are proportion-
ally longer than in H. aurantium); (9) left hypo-
branchial gland with three folds (not two);
(10) right hypobranchial gland proportionally
smaller and with weaker transverse folds; (11)
pericardial structures situated more anteri-
orly; (12) snout bordered by lobes (without
small papillae); (13) marginal teeth with
rounded main cusp (those of H. pourtalesii are
sharp); (14) left pouch of the buccal mass cov-
ering the ventral surface of the odontophore
(H. pourtalesii has this region free); (15) sev-
eral pairs of small lateral protractor muscles of
the radula (not one large pair); and (16) kid-
neys and pericardium more ventrally placed.
Both species show considerable shell vari-
ation, from specimens with well-developed
axial ridges to specimens lacking them (e.g.,
the figures of Sarasua, 1968). There is also
variation in the number of spiral ridges, which
72 SIMONE
FIG. 43 to 45. Haliotis pourtalesii anatomy: (43) anterior region of digestive system, odontophore removed,
left pouch (Ip) and oesophagus opened longitudinally; (44) ventral view of odontophore; (45) same in dorsal
view. Scales = 1 mm.
apparently increases with shell growth. Be-
tween the spiral ridges, shells sometimes
have delicate axial and uniform threads (Fig.
3). Shared shell characters of these species
are small size (up to 25 mm), the reddish-or-
ange color of the exposed areas, and the
prominent borders of the tremata. A possible
difference between the species is the number
of open pores; in specimens of H. aurantium,
they varied from 3 to 4, whereas H. pourtale-
sii had from 5 to 6 open pores. Analysis of
more specimens is necessary to establish
whether this is consistent. Comparative exam-
ination of available shells, as well as those fig-
ured in the literature, no shell character exclu-
sive of a species was found. Thus, it is difficult
to separate them using only the shell, al-
though, if a specimen is found from North
Carolina to the Caribbean Sea it is probably
H. pourtalesii, whereas if collected in Brazil
(from Espirito Santo to Rio Grande do Sul), it
is probably H. aurantium. This criterion was
used in the synonymic list, but the anatomical
study of specimens from all localities was not
undertaken and perhaps examination of addi-
tional specimens could modify this concept. In
both areas, a considerable number of speci-
mens were found. Between these areas shells
have been recorded from three localities: off
Surinam (Nijssen-Meyer, 1969), off Para
WESTERN ATLANTIC HALIOTIDAE 73
River (Rios, 1975) and off Maranhäo (Kempf
& Matthews, 1968). Although this could be
due to transportation, further sampling in
these areas should clarify the distribution of
these taxa. Dr. Mello (Museu de Malacologia
of the University of Pernambuco), has not ob-
tained haliotids from dredge samples in the
northeastern coast of Brazil (pers. comm.).
The epipodial tentacles of H. aurantium
(Fig. 22) differ from those of H. pourtalesii
(Fig. 41) in being entirety unpigmented and in
having no specific arrangement around the
main tentacles. The characters of the epipo-
dial tentacles of H. pourtalesii have some sim-
ilarity to those of the Mediterranean H. lamel-
losa and H. tuberculata, (pers. obs), but these
differ from H. pourtalesii in having only two
epipodial tentaculated flaps, the main tenta-
cles inserting dorsally in the dorsal flap; the
ventral flap has a similar organization to the
dorsal one, but its main tentacles are smaller,
ventrally inserted, and situated between the
main tentacles of the dorsal flap. Haliotis tu-
berculata has a practically straight epipodium,
with three or four small undulations between
the main tentacles (Crofts, 1929: pl. 1); H.
lamellosa, in contrast, has two strong undula-
tions between the main tentacles. Epipodial
tentacle characters have been used in hali-
otids by Owen, et al. (1971) for separating
seven eastern Pacific abalone species, and
even their hybrids. Using the good figures of
that paper, it is clear that the species studied
herein differ considerably from those taxa.
Haliotis aurantium and H. pourtalesii differ
anatomically from H. tuberculata (Fleure,
1905; Crofts, 1927, 1937, 1955; person. obs.)
and from H. lamellosa (pers. obs.) in several
characters: the cephalic tentacles possess
spots (not of uniform color); the intertentacu-
lar membrane simple and free in its lateral
margins (H. lamellosa has minute lobes in lat-
eral margins, and in H. tuberculata the lateral
regions are attached to omatophores); as well
as the other characters of epipodial tentacles
(noted above); the epipodium of Atlantic
species begins abruptly near the snout (in the
Mediterranean species, there is a coiled ex-
pansion in each side, which partially covers
the snout); gill proportionally shorter and with
fewer leaflets; hypobranchial glands propor-
tionally smaller and simpler (those species
have strong and tall folds, H. pourtalesii has
low folds and H. aurantium only furrows); rec-
tum only covered by tegument (both Medi-
terranean species have the posterior region of
the rectum covered on both sides by the hy-
pobranchial glands); anus long and papilliform
(H. tuberculata and H. lamellosa have a short,
broad anus); stomach shorter with clear de-
limitation; and gastric spiral caecum much
shorter (which also differentiates it from H.
cracherodii, see Campbell, 1965). With regard
to the tentacles of the mantle slit, H. pourtale-
sii, H. tuberculata and H. lamellosa are similar
in having three tentacles in somewhat the
same disposition; H. aurantium has only two
(there is no tentacle situated just in angulated
posterior extremity of slit).
The auriform shell with tremata, the com-
plex tentaculate epipodium and the lack of an
operculum, are known synapomorphic char-
acters of the Haliotidae within the Vetigas-
tropoda. At least three additional synapomor-
phies are offered here: (1) the insertion in the
stomach of some fibers of the right retractor
muscle, (2) the insertion in a “М” shape of the
direct radular anterior muscle, and (3) the in-
tertentacular membrane of the head (“head
pleat” of Crofts, 1927).
Schremp (1981: 1125, pl., fig. 1) called a
Pliocene haliotid found in the Imperial
Formation of California Haliotis pourtalesii.
Because this identification is based only on
the shell, this specimen might instead be the
Pacific Haliotis roberti McLean, 1969, consid-
ered a synonym by that author.
ACKNOWLEDGMENTS
| thank Dr. Airton S. Pararam and Cyntia
Miyaji, IOUSP, and Dr. Alvaro Migotto, Centro
de Biologia Marinha, USP, for the specimen of
Haliotis aurantium with soft parts; Tyjuana
Nickens and Mike Sweeney, USNM, and Dr.
José H. Leal, Rosenstiel School of Marine and
Atmospheric Science, University of Miami, for
the loan of H. pourtalesii specimens. For
search of haliotids in the collection, | am grate-
ful to Dr. L. C. F. Alvarenga, Museu Nacional da
Universidade Federal do Rio de Janeiro; Dr.
Rosa L. S. Mello, Museu de Malacologia,
Universidade Federal Rural de Pernambuco;
Dr. Eliezer C. Rios, Museu Oceanograafico da
Fundacao Universidade do Rio Grande; Yae В.
Kim, American Museum of Natural History,
New York; Dr. Kenneth J. Boss, Museum of
Comparative Zoology, Cambridge; Edward
Gilmore, Academy of Natural Sciences, Phila-
delphia; and John Slapcinsky, Field Museum
of Natural History, Chicago. For helping SEM
exams | thank Marcio V. Cruz and Enio Mattos,
Instituto de Biociéncias, USP. | specially thank
74 SIMONE
also Dr. Winston F. Ponder, Australia, for valu-
able revision on manuscript.
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Received ms. accepted 3 March 1997
MALACOLOGIA, 1998, 39(1-2): 77-81
HISTOCHEMICAL AND ULTRASTRUCTURAL IDENTIFICATION OF BIPHASIC
GRANULES IN THE ALBUMEN SECRETORY CELLS OF ARION SUBFUSCUS
(GASTROPODA, PULMONATA)
BENJAMIN J. GOMEZ’, АМА М. ZUBIAGA?, M. TERESA SERRANO? & EDUARDO ANGULO!
ABSTRACT
Two types of globules, differing in size, structure, and nature, are demonstrated within each se-
cretory cell of the albumen gland of Arion subfuscus. Large secretory globules, with a homoge-
neous granular content, are rich in proteins and carbohydrates, with the protein-reacting groups
probably partially masked by intimate junction with the polysaccharides. Smaller secretory glob-
ules lie inside the large ones and are more electron-lucent and foam-like in substructure, con-
taining mostly glycosaminoglycans but also proteins. Lectin-sugar specificities show that the car-
bohydrates synthesized by the gland are mainly galactogen, but other neutral polysaccharides,
such as glycogen, are also present in the secretory vesicles. The reactions of each type of glob-
ule to the lectins are also very different.
Key words: albumen gland, lectin histochemistry, ultrastructure, Arion subfuscus.
INTRODUCTION
The albumen gland is a large female ac-
cessory gland that surrounds the distal part of
the ovotestis duct, enclosing the carrefour
system and the most proximal portion of the
spermoviduct in stylommatophorans. It is
composed of a large number of secretory
branched tubuies, which all open into the slit-
like central lumen of the gland (Tompa, 1984).
This gland synthesizes the perivitelline fluids
of the molluscan egg, which is composed
mainly of galactogen (Bayne, 1967; Meenak-
shi & Scheer, 1968), a polysaccharide of
galactose (Duncan, 1975). Besides galacto-
gen, some proteins mineral salts, and mono-
saccharides are present in the albumen gland
secretions (Runham, 1988).
The tubules of the albumen gland in gas-
tropods contain two cell types — the large
glandular cells and the small centrotubular
cells (Jong-Brink, 1969; Breckenridge & Fallil,
1973; Els, 1978).
It is the aim of this study to investigate in
detail the nature of the albumen gland secre-
tions of the arionid species Arion subfuscus
(Draparnaud, 1805), by means of general
and lectin histochemical methods. The ultra-
structure of secretory granules is also de-
scribed.
MATERIALS AND METHODS
For general and lectin histochemical stud-
ies the albumen glands were fixed in Carnoy’s
fluid (Culling, 1974) for six h, dehydrated with
alcohol, and embedded in paraffin wax. Sec-
tions, 6-8 um thick, were obtained.
Neutral carbohydrates were detected by the
periodic acid Schiff technique (PAS). Acetyl-
ation followed by saponification, and diastase
treatment were used as controls and also for
glycogen demonstration (Martoja & Martoja-
Pierson, 1970). Alcian Blue staining (AB) was
used at pH = 0.5 to stain strongly sulphated
mucosubstances and at pH = 2.5 for carboxy-
lated and weakly sulphated mucosubstances
(Bancroft & Stevens, 1982). The combined
High Iron Diamine with Alcian Blue (HID-AB)
was used to stain sulphated mucosubstances
black and non-sulphated acidic mucosub-
stances blue (Culling, 1974). The Mercuric
Bromophenol Blue (BB) with deamination
(Chapman, 1975), as well as Chloramine T-
Schiff techniques (Martoja & Martoja-Pierson,
1970), were used for detection of proteins.
Bock’s technique (Bock et al., 1976) was used
for demonstrating protein disulphide groups.
In order to detect lipid material, glands were
fixed in Baker’s formol-calcium and sections
were stained with Sudan Black B.
Departamento de Biologia Celular y Ciencias Morfolögicas. Facultad de Ciencias, Universidad del País Vasco, Apdo. 644-
48080, Bilbao, Spain
“Departamento de Biologia Animal y Genética. Facultad de Ciencias, Universidad del País Vasco, Apdo. 644-48080, Bilbao,
Spain
78 GOMEZET AL.
TABLE 1. Lectins employed and their major binding specificities: Fuc: fucose; Gal: galactose;
GalNac: N-acetylgalactosamine; Сс: glucose; GlcNac: N-acetylglucosamine; Man: mannose;
NeuAc: neuraminic acid or sialic acid.
Taxonomic name of the source Acronym
Ulex europaeus UEA-I
Canavalia ensiformis Con A
Limax flavus LFA
Arachis hypogaea PNA
Ricinus comunis RCA:
Glycine max SBA
Carbohydrate binding specificity
L-Fuc; a-1,2 Gal B-1,4 GIcNAc, B-1,6
a-D-Man > a-D-Glc >> a-D-GIcNAc
NeuAc a-2,3/6Gal; NeuAc a-2,3/6GalNAc
Gal В-1,3 GalNAc > Gal
Gal В-1,4 GIcNAc; Gal
D-GalNac > D-Gal
Lectin histochemistry was used according
to Welsch & Schumacher (1984). Deparaf-
fined sections were incubated for 30 min with
lectins coupled to fluorescein isothiocyanate
(FITC) : PNA (Peanut agglutinin); SBA (Soy-
bean agglutinin); LFA (Limax flavus aggl.);
UEA | (Ulex europeus | aggl.); RCA | (Ricinus
communis aggl.); Con A (Concanavalin A)(for
lectin specificities, see Table 1). After incuba-
tion, the sections were rinsed in phosphate
buffered saline (pH = 7.4) for 2 h. Preparations
were studied by fluorescence microscopy.
Sections not treated with FITC-lectins were
used as controls to observe the autofluores-
cence of the tissue. The specificity of the
staining (Goldstein & Hayes, 1978) was
tested by preincubating the lectin in a solution
of the appropriate reactive sugar (Zubiaga et
al., 1990).
For transmission electron microscopy, tis-
sues were fixed for 2 h in 25% Karnovsky in
0.1M cacodylate buffer at pH 7.3 (Glauert,
1981), postfixed for 1 h in 1% osmium tetrox-
ide in the same buffer and embedded in
Araldite. Ultrathin sections were stained with
uranyl acetate and lead citrate. The grids were
observed in a Zeiss EM 9 and Philips EM 300
electron microscopes.
RESULTS
Ultrastructure
In the secretory cells of the albumen gland
of Arion subfuscus, the organelles involved in
the synthesis of secretory materials are abun-
dant and include flattened and parallely
arranged rough endoplasmic reticulum cister-
nae and Golgi stacks with many vesicles bud-
ding from them (Fig. 1). The middle and apical
portions of the cells are filled by secretory
granules (large secretory globules, 2-3 um in
FIG. 1. TEM micrograph showing the matrix of large
secretory globules (sg) containing small globules
(*). Rough endoplasmic reticulum cisternae are flat-
tened and abundant (rer). Arrows indicate Golgi
system. x 15000.
diameter) with a matrix of finely granulated
material, homogeneously distributed (Fig. 1).
Inside these large globules, there are small
aggregates of irregular lumps (small secre-
tory globules, 0.3-0.4 um in diameter), which
contain a fine granulation (Fig. 1).
Histochemistry
The general histochemical tests show that
the composition of the albumen gland secre-
tion is complex. The small globules show a re-
action distinct from that of the general matrix
of the large globules in which they are in-
mersed. Within each large globule there are
one or two small ones. The results of the his-
tochemical tests carried out are shown in
Tables 2 and 3.
The small globules react strongly to the AB
technique at both pH levels, and they stain
black with the HID-AB technique indicating
that they are composed mostly of sulfo-
mucins. Moreover, these small globules are
IDENTIFICATION OF BIPHASIC GRANULES
19
TABLE 2. Results of the histochemical tests of the albumen gland tubules of Arion subfuscus. Three portions
of the secretory cells have been distinguished: 1, cytoplasm; 2, large secretory globules; 3, small secretory
globules; —, negative reaction; +, weak reaction; ++, moderate reaction; +++, strong reaction.
Secretory cells
Staining technique 1
AB pH = 0.5 -
AB pH = 2.5 +
HID-AB +
blue
PAS ++
PAS-diastase +
PAS-acet -
PAS-acet.-sap ++
Deamination-PAS ++
Bromophenol blue ++
Chloramine T-Schiff ++
Bock -
Sudan Black -
2
3 Centrotubular cells
+++ =
+++ -
+++ -
black =
= ++
- +
= +
- ++
- +
+ +
+ +
++ -
TABLE 3. Lectin specificities of the different portions of the secretory cells of the Arion subfuscus albu-
men gland. —, negative reaction; +, weak reaction; ++, moderate reaction; +++, strong reaction.
Lectins PNA SBA
Cytoplasm - Ea
Large globules ++ +++
Small globules +++ =
PAS-negative, whereas exhibiting a strong re-
action to the Bock technique for protein SH
groups.
The large globules show a strong reaction
to the PAS technique, even after treatment
with diastase or after acetylation followed by
saponification. They also show moderate re-
actions to tests for protein.
The reactions of each type of globule to the
lectins are different. The large granules bind
specifically to PNA, SBA, RCA 1, and Con A
(Fig. 2), whereas the small granules bind to
PNA, LFA, and UEA | (Fig. 3).
DISCUSSION
Previous histochemical and biochemical
studies have shown that the albumen gland of
gastropods produces a galactogen and pro-
tein-rich nutritive fluid for the developing em-
bryos (Grainger & Shilliote, 1952; Bayne,
1967; Meenakshi & Scheer, 1968; Okotore et
al., 1981; Dictus & Jong-Brink, 1987). The pro-
tein percentage varies from one species to an-
other. Thus, in the pulmonates Biomphalaria
glabrata (see Jong-Brink, 1969) and Achatina
LFA UEAI RCAI ConA
- _ + 4
- - +++ +
+++ +++ -
fulica (see Ramasubramaniam, 1979) the pro-
tein content is high, whereas in Deroceras
laeve only a small amount of protein has been
demonstrated (Els, 1978). We have found that
protein is present in the secretory globules of
the albumen gland of Arion subfuscus, as well
as in the cytoplasm of the secretory cells, but
the reaction to protein tests is only moderate.
Nevertheless, the rough endoplasmic reticu-
lum is very abundant. The protein-reacting
groups could be intimately joined with poly-
saccharides and thus masked. In this sense,
Bayne (1967) could not separate the proteins
from sugars by electrophoretic studies, but he
found great amounts of free amino-acids in al-
bumen gland homogenates. This fact, together
with the near absence of digestion of secretory
granules by pronase (Kress & Schmekel,
1992), indicates that there is a very close
union between both components.
The presence of galactogen in the albumen
gland has been shown using different metho-
dologies (Bolognani-Fantin & Vigo, 1968;
Varute & Nanaware, 1972). In this work, we
have used lectin histochemical methods to
detect galactogen. The secretory granules of
Arion subfuscus show a strong reaction with
80 GOMEZET AL.
FIG. 2. Lectin histochemistry. RCA | binding to large
secretory globules (arrows). Arrow heads indicate
the nuclei of cells. x40.
FIG. 3. Lectin histochemistry. LFA binding to small
secretory globules (small arrows) which are present
inside the large secretory globules (arrows). Arrow
heads indicate the nuclei of cells that show a weak
reaction to LFA. x65.
PNA, SBA, and RCA 1. These three lectins
bind specifically to galactose residues (Gold-
stein & Hayes, 1978; Zubiaga et al., 1990).
According to Okotore et al. (1981) and
Okotore & Uhlenbruck (1982), these residues
are probably D-Gal $1, 3D-Gal.
Moreover, the secretory granules of A. sub-
fuscus as well as the cytoplasm of secretory
cells also react with Con A. This indicates the
additional presence of glucose or mannose
sugar residues (Goldstein & Hayes, 1978)
free in the cytoplasm as well as inside secre-
tory vesicles. The strong positive reaction of
the cytoplasm and secretory granules of the
albumen gland of this species with the PAS
technique decreases after acetylation and di-
astase treatment. Thus, the general histo-
chemical tests also indicate the presence of
glycogen and other neutral carbohydrates
(Culling, 1974; Bancroft & Stevens, 1982) dif-
ferent from galactogen (Rangarao, 1963;
Bolognani-Fantin 8 Vigo, 1968) inside secre-
tory globules.
On the other hand, the strong reaction with
AB at pH 0.5 together with the black-staining
reaction with HID-AB, indicates the presence
of sulphated acidic mucosubstances in the
small granules contained in the large ones
of the albumen gland secretion of Arion sub-
fuscus. We conclude that the small and
foamy-looking globules contain mainly gly-
cosaminoglycans. Acidic groups have also
been reported in the albumen gland of other
gastropods (Rangarao, 1963; Bayne, 1967),
but the secretory granules have always been
described as ultrastructurally homogeneous
without smaller clear granules inside (Nieland
8 Goudsmit, 1969).
As with other stylommatophorans (Bayne,
1967; Ramasubramaniam, 1979), no lipid has
been detected in albumen secretions of Arion
subfuscus.
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OKOTORE, R. & G. UHLENBRUCK, 1982, Addi-
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GULO, 1990, Identification and carbohydrate
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Revised ms. accepted 25 February 1997
Fr
MALACOLOGIA, 1998, 39(1-2): 83-91
ESTRATEGIAS DE DEPREDACION DEL GASTROPODO PERFORADOR
TROPHON GEVERSIANUS (PALLAS) (MURICOIDEA: TROPHONIDAE)
Sandra Gordillo у Sandra N. Amuchástegui
Centro Austral de Investigaciones Cientificas (CADIC, CONICET), C.C. 92 (9410) Ushuaia,
Tierra del Fuego, Argentina
RESUMEN
Se analizaron las diferentes estrategias de depredacion utilizadas por Trophon geversianus
(Trophonidae) sobre cuatro especies de bivalvos típicos de la región fueguina: Mytilus chilensis,
Aulacomya atra, Hiatella solida y Tawera gayi.
Los datos fueron obtenidos utilizando tres metodologías: análisis de valvas perforadas trans-
portadas sobre la playa actual, estudio de la depredación bajo condiciones de laboratorio, y ob-
servación de la depredación en poblaciones naturales durante la bajamar.
Se concluye que (1) no todas las perforaciones producidas por los murícidos sensu lato son
cilíndricas; (2) en las distintas presas hay variación en la forma y sitio de las perforaciones; y (3)
la intensidad de depredación es baja a moderada.
Palabras clave: depredación, gastrópodos, perforaciones, Tierra del Fuego.
ABSTRACT
Predation strategies of Trophon geversianus (Trophonidae) on four bivalve species: Mytilus
chilensis, Aulacomya atra, Hiatella solida and Tawera gayi, from the fuegian region were ana-
lyzed.
Data were obtained by three methods: analysis of bored shells transported on modern beachs,
study of predation under laboratory conditions, and observation of predation on populations in
the field.
We conclude that (1) not all muricid holes are cylindrical; (2) there is variation among prey
species in the shapes of holes and in the sites of drilling; and (3) predation intensity is low to mod-
erate.
Key words: predation, gastropods, drillholes, Tierra del Fuego.
INTRODUCCIÓN
Una característica notable de la mayoría de
los moluscos es su capacidad para secretar
un exoesqueleto calcáreo de protección.
Otra, irónicamente, es la habilidad de algunos
de estos moluscos de excavar o perforar di-
chos exoesqueletos mediante un proceso
mecánico-químico, descripto por Carriker &
Williams (1978) y Carriker (1981), entre otros.
Es ampliamente conocido que los princi-
pales responsables de dichas perforaciones
son distintas especies que pertenecen a dos
grupos dentro de Gastropoda. Por un lado, y
entre los mesogastrópodos, figuran los natici-
dos (Naticidae). Por otro lado, y entre los
neogastrópodos, la superfamilia Muricoidea
comprende los murícidos sensu stricto (Mu-
ricidae), táididos (Thaididae) y trofónidos
(Trophonidae) con especies perforadoras de
valvas.
83
Los bivalvos epifaunales son atacados par-
ticularmente por los murícidos sensu lato, es
decir por representantes de las distintas fa-
milias que integran el grupo Muricoidea
(Kabat, 1990). Los bivalvos infaunales, en
cambio, son comúnmente víctimas de los
natícidos (Kabat, 1990; Anderson et al.,
1991), aunque también pueden ser presas de
algunos Muricoidea como lo mencionan
Vermeij (1980) y Gordillo (1994) para distintas
especies.
La morfología de la perforación ha sido uti-
lizada por distintos autores para diferenciar
familias. La perforación de los murícidos
sensu lato es cilíndrica con bordes casi rec-
tos; mientras que la perforación de los natici-
dos, en contraste, tiene una forma más pa-
raboloide y bordes biselados (Carriker, 1981;
Aitken 4 Risk, 1988; Kabat, 1990; Anderson
et al., 1991). Un caso atípico dentro de
Muricoidea lo constituyen los trofónidos, ca-
84 GORDILLO & AMUCHÄSTEGUI
paces de realizar perforaciones que varian
entre cilindrica y cono-truncada, segun la
presa (Gordillo, 1994, 1995a).
Para Tierra del Fuego, trabajos anteriores
realizados por Gordillo (1994, 1995a) y Amu-
chästegui (1995) describen la depredaciön de
Trophon geversianus en distintas especies
que viven en el litoral fueguino. El objectivo de
este trabajo ha sido reunir y sintetizar toda la
información recabada sobre la depredación
de los trofönidos que caracterizan esta región.
UBICACIÓN Y CARACTERIZACIÓN DEL
ÁREA DE ESTUDIO
El Canal Beagle se ubica entre la Isla
Grande de Tierra del Fuego, la Isla Navarino
y la Isla Hoste a los 54°LS y 68°LO. Este
canal es de aguas tranquilas, con una profun-
didad media de aproximadamente 150-200
m y un litoral rocoso con playas de rodados.
El canal posee un régimen de mareas de de-
sigualdades diurnas con una amplitud media
de 1 metro.
La temperatura media anual es de 5.5°C
con una amplitud térmica moderada. La sali-
nidad superficial del agua varía entre 27 y 31
gramos/litro (Iturraspe et al., 1989). La tem-
peratura estival del agua oscila en 11-12°C,
mientras que en invierno es de 3-4°C
(Iturraspe et al., 1989).
MATERIAL Y MÉTODO
El principal depredador considerado fue el
trofónido Trophon geversianus y, en segunda
instancia, Xymenopsis muriciformis. Las pre-
sas estudiadas fueron cuatro especies de bi-
valvos típicos de la región: Mytilus chilensis,
Aulacomya atra, Hiatella solida y Tawera gayi.
El Apéndice presenta la posición taxonómica
de las especies tratadas según Gordillo
(1995b). El material estudiado procede de
distintos sitios de las zonas mesolitoral e
infralitoral del Canal Beagle. Los es-
pecimenes se obtuvieron por recolección
manual durante la bajamar o con buceo
autónomo a profundidades de hasta 3 me-
tros.
Los datos sobre los que posteriormente se
aplicaron los distintos parámetros fueron
obtenidos utilizando tres metodologías:
Análisis de valvas perforadas transporta-
das sobre la playa actual: el material procede
de distintas playas indicadas en la Figura 1.
En cada sitio, y dentro del supralitoral, se
tomó como unidad de muestreo un cuadrante
de 0.5 x 0.5 m con un número de réplicas pre-
determinado. En cada cuadrante se contabi-
lizaron todas las valvas de las cuatro es-
pecies que aparecieron en sus respectivas
superficies.
Estudio de la depredación bajo condiciones
de laboratorio: se utilizaron acuarios de vidrio
de distinta capacidad, según la experiencia,
variando la cantidad de depredadores y pre-
sas (Tabla 1). Los ejemplares fueron recolec-
tados del mesolitoral e infralitoral de distintas
localidades del Canal Beagle (Fig. 1). Los
acuarios se mantuvieron en la cámara fría del
CADIC a una temperatura de 2 a 9°C. Dia-
riamente se realizaron observaciones sobre
el comportamiento de los depredadores y se
retiraron las valvas vacías.
Observación de la depredación en condi-
ciones naturales durante la bajamar: se ana-
lizó la presencia de Trophon geversianus en
una superficie que varió, según las carac-
teristicas del sitio elegido, entre 2.5 y 50 mf.
Estos muestreos se realizaron mensualmente
durante la bajamar (con un nivel menor a 0.45
m) en las localidades indicadas en la Figura 1.
Se midió la longitud de los ejemplares halla-
dos, consignando si depredaban o no, parti-
cularmente sobre Mytilus chilensis por ser la
presa más abundante del mesolitoral.
Los parámetros analizados fueron los si-
guientes:
Morfología de la perforación: para analizar
este parámetro se midieron los diámetros ex-
ternos e internos (mm) de la perforación, uti-
lizando una lupa binocular con retículo.
Selectividad por el sitio a perforar: para de-
terminar la preferencia con respecto al sitio de
perforación en las distintas especies, la su-
perficie de la valva fue dividida en regiones
según muestra la Figura 2. Para analizar este
parámetro se sometieron los datos a la prueba
estadística de Chi-cuadrado (X?). Para este y
otros parámetros de selectividad se hace re-
ferencia a “selectivo” para expresar la prefer-
encia o comportamiento estereotípico y “no
selectivo” para denotar un comportamiento
aleatorio.
Selectividad por la valva derecha о iz-
quierda: para analizar la preferencia por al-
guna de las dos valvas opuestas, los valores
obtenidos también se sometieron a una
prueba de Chi-cuadrado (X?).
Selectividad por un tamaño de la presa: en
valvas transportadas, para determinar la exis-
tencia de selectividad por un tamaño de valva
DEPREDACION DE TROPHONIDAE 85
TABLA 1. Experiencias de laboratorio. Se incluye el tamano y el numero o peso de las presas y de
los depredadores y la capacidad de los acuarios.
ACUARIOS PRESAS
Acuario A Mytilus chilensis
100 gr. (x < 30 mm)
100 gr. (30 mm < x < 50 mm)
100 gr. (50 mm < x < 70 mm)
Acuario B igual al Acuario A
Acuario C Aulacomya atra
100 gr. (x < 30 mm)
100 gr. (30 mm < x < 50 mm)
100 gr. (50 mm < x < 70 mm)
Acuario D igual al Acuario C
Acuario E Mytilus chilensis
п = 25 (24 тт <x <65 mm)
Тамега дау!
п =3 (31 mm; 31.7 тт у 33 mm)
Acuario Е Тамега дау!
п = 20 (20 тт <x = 36 mm)
Hiatella solida
n=5 (20 mm <x < 45.4 mm)
en particular, se relacionö la distribuciön de
frecuencias por tamano (longitud), consi-
derando separadamente las valvas no per-
foradas, y las valvas perforadas. Con los datos
obtenidos en laboratorio se calculó la relación
entre la cantidad de presas ofrecidas y las pre-
sas consumidas según su tamaño. Para
analizar este parámetro, y en ambos casos, se
aplicó la prueba de Chi-cuadrado (Х?).
Correlación entre el tamaño del depredador
y el tamaño de la presa: se calculó en forma
directa mediante la medición de los espe-
címenes que fueron observados depredando
y de sus presas. Se realizó bajo condiciones
de laboratorio y en poblaciones naturales en
el caso de Mytilus chilensis. Para expresar la
correlación se calculó el coeficiente de cor-
relación (r).
Exito-fracaso de la depredación (drilling
success; Tull & Bohning-Gaese, 1993): se
partió del supuesto de que una perforación in-
completa constituye un evento fallido de
depredación. Este parámetro expresa el por-
centaje de éxito y resulta de dividir el número
de perforaciones completas por el número
total de intentos, dado por la suma de las per-
foraciones incompletas y completas. Los por-
centajes de depredación obtenidos se expre-
DEPREDADORES CAPACIDAD
6 Trophon geversianus 19 litros
(23 mm < x < 45.6 mm)
6 Trophon geversianus 19 litros
(22.7 mm < x < 43.3 mm)
6 Trophon geversianus 19 litros
(18.5 mm < x < 52.3 mm)
6 Trophon geversianus 19 litros
(22.7 mm s x < 45.3 mm)
3 Trophon geversianus 4 Vo litros
(30 mm; 48 mm y 54 mm)
6 Trophon geversianus 4 Vo litros
(33.0 mm < x < 51.5 mm)
saron según la siguiente escala: 0-20%: muy
poco exitosa; 20-40%: poco exitosa; 40-60%:
moderadamente exitosa; 60-80%: exitosa y
80-100%: muy exitosa.
Indice de depredación (predation rate,
Vermeij, 1980): la proporción de depredación
se calculó tomando la proporción de valvas
perforadas en relación al número total de val-
vas. Los valores obtenidos se expresaron
según la siguiente escala: 0.0-0.25: bajo;
0.25-0.50: moderado; 0.50-0.75: elevado y
0.75-1.0: muy elevado.
Tiempo de depredación: un ciclo de depre-
dación está dado por el período de alimen-
tación y el de descanso hasta que se inicia un
nuevo período de alimentación. Para determi-
nar el tiempo de depredación, y siempre bajo
condiciones de laboratorio, se calculó el tiem-
po transcurrido (en días) desde que el depre-
dador se apoyó sobre la presa hasta que la
abandonó. Luego se tomó, también en días, el
período de descanso transcurrido hasta que
un mismo depredador atacó a otra presa. El
tiempo de depredación se expresó arbitraria-
mente en: “variable” cuando el desvío resultó
mayor o igual a la media aritmética dividida 2
y “estable” cuando el desvío fue menor a dicho
cociente.
86 GORDILLO & AMUCHASTEGUI
|
7 ARGENTINA” 707"
Dean id
Ensenada al) Ushuaia
|. Navarino
О Depredacién in situ @ Especimenes para acuario
eee CHILE
ARGENTINA
A Valvas transportados
FIG. 1. Mapa de Ubicación. Los símbolos señalan los sitios de procedencia de las muestras.
B
IA
ВВ
FIG. 2. Sectores en que se subdividieron las valvas
de las presas para analizar la selectividad por el
sitio a perforar. A, Mytilus chilensis. В, Тамега gayi.
C, Hiatella solida.
RESULTADOS
Morfologia de la Perforaciön
Las perforaciones producidas por Trophon
geversianus mostraron algunas diferencias
morfolögicas en las distintas presas (Tabla 2,
A; Fig. 3).
En Hiatella solida, Gordillo (1994) observö
que Trophon geversianus realiza perfora-
ciones cono-truncadas, tal como se esque-
matiza en la Figura 3, A.
Las perforaciones excavadas por este
mismo depredador en valvas de Mytilus chi-
lensis también son de tipo conotruncado, si-
milares a las descriptas previamente por Gor-
dillo (1994, 1995a) para Тамега gayi (Fig. 3,B).
Sobre Aulacomya atra, las perforaciones
producidas por Trophon geversianus se difer-
encian de las anteriores en que se asemejan
mas a un cilindro simple (Fig. 3, С).
En relacion al depredador Xymenopsis mu-
riciformis se constató que bajo condiciones
de laboratorio, este produce en Mytilus chilen-
sis perforaciones que se asemejan mas a la
morfologia cilindrica, por las menores diferen-
cias entre los diámetros externo e interno; i.e.
sobre un total de 19 ejemplares de esta es-
pecie, los diámetros externo e interno fueron
respectivamente 1.79 + 0.2 mm y 1.22 + 0.16
mm. En Tawera gayi y Hiatella solida, bajo
condiciones de laboratorio, solamente fueron
observadas perforaciones de este tipo en un
caso para cada especie. Algunas valvas
transportadas de Tawera gayi (n = 14) pre-
sentaron una morfología similar, razón por la
cual fueron atribuidas a este depredador. En
estas perforaciones se obtuvo un diámetro
único de 1.81 + 0.30 mm, ya que no se notó
diferencias entre los dos diámetros.
Finalmente, se observó que las perfora-
ciones incompletas dejadas por Trophon ge-
versianus y Xymenopsis muriciformis en las
distintas presas coincidían en tener el fondo
plano.
Sitio de Perforación
Las zonas elegidas por los depredadores
variaron según las distintas presas conside-
radas (Tabla 2, B).
En las valvas de Mytilus chilensis se ob-
servó que alrededor del 50% de las valvas
(53% en el primer caso y 59% en el segundo)
estaban perforadas en los sectores Пу Ill, que
corresponden a la zona media ventral y dorsal
respectivamente, por lo que se consideró que
hay preferencia por la zona media.
DEPREDACION DE TROPHONIDAE 87
=
B
И
VISTA DORSAL:
M ext
O
2 тт
==
oO
FIG. 3. Morfologia de la perforaciones producidas por Trophon geversianus en A, Hiatella solida. B, Mytilus
chilensis у Тамега gayi. С, Aulacomya atra Referencias: дех, diámetro externo. 0, , diámetro interno [Tomado
de Gordillo (1994)].
Con respecto al mitilido Aulacomya atra, y
sólo bajo condiciones de laboratorio, se pudo
constatar que las perforaciones se encon-
traron en el 100% de los casos en proximi-
dades del borde de las valvas, lo que marcó
una gran diferencia con el sitio que Trophon
geversianus elige para perforar las valvas de
Mytilus chilensis.
En las valvas de Tawera gayi hubo prefe-
rencia por el sector V o medio central donde
se concentraron el 50% de las perforaciones
(Gordillo, 1994).
En Hiatella solida el 77% de las perfora-
ciones se encontraron en el sector Il o poste-
rior en torno a una cóstula radial caracterís-
tica de la especie (línea punteada de la Fig. 2,
C), descripto con anterioridad por Gordillo
(1994). Una proporción de las mismas (61%)
se halló dentro de la zona de inserción del
músculo posterior.
Respecto a este parámetro, también se ob-
tuvo que el segundo depredador considerado,
Xymenopsis muriciformis, eligió el sector um-
bonal en valvas de Tawera gayien el 100% de
los casos (n = 14). Sobre Mytilus chilensis (n
= 19) no se halló selectividad por un sitio par-
ticular, estando las perforaciones distribuidas
en toda la superficie de la valva. En Aula-
comya atra se produjo un único caso de per-
foración por Xymenopsis muriciformis en que
la misma se ubicó en el borde de la valva,
similar a Trophon geversianus.
Preferencia por la Valva Derecha o Izquierda
Para las distintas presas se observó que no
hubo preferencia por una de las valvas, ya
sea derecha o izquierda (Tabla 2, C). Este
parámetro no pudo calcularse para Aulaco-
mya atra ya que las perforaciones ubicadas
en el borde abarcaron en la mayoría de los
casos las dos valvas opuestas.
Selectividad por un Tamaño
Hubo variaciones en los resultados obteni-
dos en relación a este parámetro (Tabla 2, D).
Con los datos de laboratorio se obtuvo la
88 GORDILLO & AMUCHÄSTEGUI
proporciön de presas ofrecidas y consumidas
por Mytilus chilensis y Aulacomya atra.
Para el caso de Mytilus chilensis se ob-
servö una marcada preferencia de Trophon
geversianus por las presas mas grandes
(50-70 mm). En Aulacomya atra los ejem-
plares seleccionados comprendieron un ran-
go mas amplio, ya que el depredador prefirió
los ejemplares medianos (30-50 mm) y los
grandes (50-70 mm).
En valvas transportadas de estas dos es-
pecies, y en base a la distribuciön de frecuen-
cias de valvas perforadas y no perforadas
también se observó preferencia por las valvas
más grandes.
En cambio, con la misma metodología, en
Tawera gayi y Hiatella solida, no se observó
selectividad por un tamaño particular. En
estas dos especies, la mayor proporción de
perforaciones sobre valvas con un largo
promedio de 31 mm se relaciona directa-
mente con la mayor cantidad de valvas de
dicho tamaño y no con una selección del
mismo.
Relación Entre los Tamaños de la Presa y
del Depredador
Según la especie y la metodología em-
pleada, los resultados variaron entre sin cor-
relación hasta una ligera correlación positiva
(Tabla 2, E).
En Mytilus chilensis, y bajo condiciones
naturales, este parámetro mostró una ligera
correlación positiva. Sin embargo, los resulta-
dos de laboratorio indicaron ausencia de cor-
relación.
En Aulacomya atra con los datos de labora-
torio se obtuvo una correlación positiva entre
los tamaños del depredador y de sus presas.
Bajo condiciones naturales, solamente en
cuatro oportunidades se observó a Trophon
geversianus depredando sobre ejemplares
chicos (menores a 30 mm) de Aulacomya atra.
Finalmente, con los datos de laboratorio de
Tawera gayitampoco se encontró correlación
entre estas variables.
Perforaciones Incompletas: Éxito-Fracaso de
la Depredación
Los resultados mostraron (Tabla 2, F) que
la depredación de Trophon geversianus varió
entre muy exitosa en Aulacomya atra (98.7-
100%); muy exitosa a exitosa en Hiatella so-
lida (63.7-100%); y exitosa en Tawera gayi
(64.4-74.1%) y en Mytilus chilensis (79-
93.3%).
Para estas especies las valvas presentaron
una sola perforación, ya sea completa o in-
completa. Una excepción la constituye la
depredación en Tawera gayi bajo condiciones
de laboratorio, en que algunas presas fueron
atacadas simultáneamente por dos depre-
dadores distintos, resultando perforadas las
dos valvas en un 25% de los casos.
Proporción de Valvas Perforadas:
Indice de Depredación
El índice de depredación resultó bajo para
Mytilus chilensis, Aulacomya atra y Tawera
gayi, mientras que fue moderado en el caso
de Hiatella solida (Tabla 2, G).
Tiempo de Depredación
Se desconoce aún el tiempo que emplea el
depredador en excavar la perforación; sin em-
bargo, los resultados (Tabla 2, H) hacen refe-
rencia al tiempo promedio transcurrido desde
que el depredador se apoyó sobre la presa
hasta que la abandonó.
Los tiempos de depredación y de descanso
fueron muy variables en Mytilus chilensis y
Aulacomya atra, y relativamente más estables
en Tawera gayi y Hiatella solida, aunque el
número de observaciones en estas dos es-
pecies fue menor.
DISCUSIÓN
En relación a la morfología de la per-
foración es importante destacar que la per-
foración cono-truncada que Trophon gever-
sianus excava en valvas de Mytilus chilensis,
Tawera gayi y Hiatella solida se asemeja más
a la perforación típica de los natícidos que a
la perforación producida por los murícidos
sensu lato.
Sin embargo, una forma de diferenciar
estas perforaciones de aquellas producidas
por los natícidos es a través de las perfora-
ciones incompletas; ya que en los natícidos
éstas presentan un fondo deprimido con una
prominencia o giba central (Aitken 8 Risk,
1988; Kabat, 1990), mientras que la per-
foración incompleta realizada por los dos
trofónidos Trophon geversianus y Xymenop-
sis muriciformis se caracteriza por tener el
89
2
DEPREDACION DE TROPHONIDAE
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90 GORDILLO & AMUCHASTEGUI
fondo plano, y sin ninguna prominencia cen-
tral.
En Aulacomya atra la perforaciön dejada
por Trophon geversianus es relativamente
mas cilindrica, es decir con menor diferencia
entre diametros externo e interno. Este tipo de
perforacion se asemeja mas a la perforacion
producida por el otro depredador Xymenopsis
muriciformis sobre valvas de Mytilus chilen-
sis, Tawera gayi y Hiatella solida.
Sólo en una oportunidad se observó una
perforación producida por Xymenopsis murici-
formis en una valva de Aulacomya atra, y si se
compara su morfología con la perforación de
Trophon geversianus sobre la misma presa,
no se detectan diferencias morfológicas, in-
cluso tampoco por su ubicación ya que ambos
depredadores dejan sus marcas en el borde.
También cabe mencionar en relación a
Mytilus chilensis y Aulacomya atra que ejem-
plares previamente perforados por Trophon
geversianus fueron capaces de reparar su
valva en la zona interna exactamente opuesta
al sitio de la perforación. Este mecanismo de
reparación también fue observado por Grif-
fiths & Blaine (1994) en Mytilus galloprovin-
cialis del hemisferio norte.
Otro aspecto que merece ser destacado es
la selectividad por un sitio particular de la
valva. El registro fósil muestra que en los
murícidos sensu lato las perforaciones
pueden estar distribuidas al azar (Aitken &
Risk, 1988; Tull 8 Bohning-Gaese, 1993) o ser
selectivas (Vermeij et al., 1989; Guerrero &
Reyment, 1988). Las observaciones de este
trabajo, y las obtenidas previamente por
Gordillo (1994) y Amuchástegui (1995), tam-
bién indican selectividad en relación a este
parámetro, en coincidencia con lo señalado
por Vermeij et al., (1989) y Guerrero &
Reyment (1988) para otras especies de este
grupo. El sitio elegido por Trophon gever-
sianus para realizar su perforación varía en
las distintas presas, y estaría condicionado
por la morfología de la presa en relación a su
hábitat.
En relación a la selectividad por una valva
en particular, los resultados muestran que no
existe selectividad o preferencia por una de
las valvas ya sea derecha o izquierda, lo que
se atribuye a que las presas exponen indistin-
tamente las dos valvas al depredador.
Las diferencias registradas al analizar la
selectividad por un tamaño particular de las
presas quizás se relacione con otros aspec-
tos no considerados en este trabajo como la
existencia de otros depredadores, o con los
tamaños relativos que alcanzan las presas.
La selectividad observada en Mytilus chilen-
sis coincide con la selectividad registrada por
Zaixso & Bala (1995) en poblaciones natu-
rales de esta especie a una menor latitud, en
Santa Cruz (Argentina). La ausencia de se-
lectividad por tamaño en Tawera gayi y
Hiatella solida concuerda con los resultados
obtenidos por Aitken & Risk (1988) para la es-
pecie antes mencionada del hemisferio norte.
El éxito de depredación con un rango varia-
ble entre un 63.7-100% entre las distintas
presas permiten considerar a Trophon gever-
sianus como un depredador eficiente. Esta
característica podría constituir una ventaja
evolutiva si se considera que una perforación
incompleta representa un gasto energético
inútil, tal como lo considera Kabat (1990) para
los natícidos.
Los índices de depredación que variaron
entre bajos a moderados indicarían que Tro-
phon geversianus es responsable de la mor-
talidad de una proporción baja a moderada de
las poblaciones de las distintas presas.
Finalmente, para interpretar las variaciones
de los tiempos de depredación de Trophon
geversianus sobre las distintas especies,
además del tamaño de la presa habría que
analizar otros aspectos no considerados en
este trabajo, como el grosor de la valva y/o su
naturaleza mineralógica.
AGRADECIMIENTOS
Las autoras agradecen al Prof. Geerat J.
Vermeij y a un árbitro anónimo por sus valio-
sos comentarios. También al Dr. Nermesio
San Román por el apoyo brindado como en-
cargado de la sección de Biología Marina del
CADIC.
Este trabajo ha sido subsidiado con fondos
propios y realizado como parte del trabajo de
Investigador Asistente del Consejo Nacional
de Investigaciones Científicas y Técnicas
(CONICET) de S.G. y de una beca de la
Provincia de Tierra del Fuego otorgada a S.A.
LITERATURA CITADA
AMUCHASTEGUI, S., 1995, Depredación del mejil-
lón y cholga del Canal Beagle por gastrópodos
perforadores de valvas. Informe técnico no publi-
cado de la Secretaría de Planeamiento de la
Provincia. 58 pp.
AITKEN, A. E. & M. J. RISK, 1988, Biotic interac-
tions revealed by macroborings in arctic bivalve
molluscs. Lethaia, 21: 339-350.
DEPREDACION DE TROPHONIDAE 91
CARRIKER, M. R., 1981, Shell penetration and
feeding by naticacean and muricacean predatory
gastropods: a synthesis. Malacologia, 20 (2):
403-422.
CARRIKER, M. R. & L. G. WILLIAMS, 1978, The
chemical mecanism of shell dissolution by preda-
tory boring gastropods: a review and an hypothe-
sis. Malacologia, 17 (1): 143-156.
GORDILLO, S., 1994, Perforaciones en bivalvos
subfösiles y actuales del Canal Beagle, Tierra del
Fuego. Ameghiniana (Rev. Asoc. Paleont. Ar-
gent.), 21 (2): 177-185. Buenos Aires.
GORDILLO, S., 1995a, Muricid gastropod preda-
tion on Holocene bivalves from Tierra del Fuego,
Subantarctic Region. En: International Sympo-
sium on the Paleobiology and Evolution of the
Bivalvia, Abstracts, 10. Drumheller, Canada.
Gordillo, S., 1995b, Moluscos australes. Bivalvos y
caracoles de las costas del extremo sur de
America. Zagier & Urruty Publ., 115 pp. Buenos
Aires.
GRIFFITHS, С. L. & М. J. BLAINE, 1994, Non-fatal
cropping of large mussels by drilling whelks,
Nucella cingulata (Linnaeus, 1771). Journal of
Molluscan Studies, 60: 346-348.
GUERRERO, S. & A. REYMENT, 1988, Differen-
tiation between the traces of predation of muri-
cids and naticids in Spanish Pliocene Chlamys.
Estudios Geologicos, 44: 317-328.
ITURRASPE, R., В. SOTTINI, С. SCHROEDER & J.
ESCOBAR, 1989, Hidrologia y variables climati-
cas del Territorio de Tierra del Fuego. Informacion
basica. Contribucion CADIC, 7. Ushuaia.
KABAT, A. R., 1990, Predatory ecology of naticid
gastropods with a review of shell boring preda-
tion. Malacologia, 32 (1): 155-193.
TULL, D.S.& K. BOHNING-GAESE, 1993, Patterns
of drilling predation on gastropods of the family
Turritellidae in the Gulf of California. Paleobiology,
19: 476-486.
VERMEIL, С. J., 1980, Drilling predation of bivalves
in Guam: some paleoecological implications.
Malacologia, 19: 329-334.
VERMEIL, G.J., Е. С. DUDLEY 4 Е. ZIPSER, 1989,
Successful and unsuccessful drilling predation in
Recent pelecypods. The Veliger, 32: 266-273.
ZAIXSO, Н. Е. & L. O. BALA, 1995, Relaciones de
tamano en la predaciön de Trophon geversianus
sobre mitilidos. Resumenes del VI Congreso
Latinoamericano de Ciencias del Mar, 206. Mar
del Plata, Argentina.
Revised ms. accepted 27 May 1997
APENDICE
Ubicaciön sistematica de las especies
consideradas
Clase Gastropoda
Subclase Prosobranchia
Orden Neogastropoda
Superfamilia Muricoidea
Familia Trophonidae
Trophon geversianus (Pallas)
Xymenopsis muriciformis (King)
Clase Bivalvia
Subclase Pteriomorpha
Orden Mytiloida
Familia Mytilidae
Mytilus chilensis (Hupe)
Aulacomya atra (Molina)
Subclase Heterodonta
Orden Veneroida
Familia Veneridae
Tawera gayi (Hupe)
Orden Myoida
Familia Hiatellidae
Hiatella solida (Sowerby)
MALACOLOGIA, 1998, 39(1-2): 93-111
CLADISTIC ANALYSIS OF THE XANTHONYCHIDAE (= HELMINTHOGLYPTIDAE)
(GASTROPODA: STYLOMMATOPHORA: HELICOIDEA)
Maria Gabriela Cuezzo
Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucuman-
CONICET, Miguel Lillo 205, 4000 Tucuman, Argentina
ABSTRACT
A cladistic analysis of the family Xanthonychidae (= Helminthoglyptidae) was carried out. The
data set consisted of 34 characters and 25 terminal taxa (including the four outgroups: Oreohelix,
Neohelix, Helix, and Bradybaena). Two different analyses were performed using the program
Hennig86. The preferred hypothesis is represented by one of the two trees obtained after suc-
cessive weighting in the first analysis, which has the same topology of one of the original set of
50. The conclusions of this study suggest that: (1) the family Xanthonychidae as defined by
Pilsbry (1939) and Zilch (1959-1960) is a paraphyletic group, (2) Bradybaenidae, Helicidae and
Xanthonychidae form a monophyletic group and therefore should be analyzed together as a unit,
(3) based upon the preferred hypothesis, three monophyletic groups can be defined, although
taxonomic changes will have to wait until a new analysis of the Bradybaenidae-Xanthonychidae-
Helicidae group is performed.
Key Words: Stylommatophora, Helicoidea, Xanthonychidae, cladistics, phylogeny, characters.
INTRODUCTION
The Xanthonychidae (= Helminthogly-
ptidae) are a large group of land snail and
semislug genera distributed along the Am-
ericas. Pilsbry (1939), while describing the
land snail fauna of North America, established
the family Helminthoglyptidae, composed of
all the “American dart-bearing helices.” In this
work, he named only five (Helminthoglyp-
tinae, Sonorellinae, Humboldtianinae, Cepo-
linae and Epiphragmophorinae) out of eight
subfamilies he included in the Helmintho-
glyptidae. The remaining three subfamilies
are probably the Central American groups
(Pilsbry, 1900, 1927): Lysinoinae, Xanthony-
chinae, and Metostracinae. Zilch (1959-1960)
gave a complete list of the subfamilies and
genera included in the Helminthoglyptidae.
Richardson (1982) catalogued the Helmintho-
glyptidae at species level.
Baker (1943, 1959) pointed out that the
name Xanthonychidae Strebel & Pfeiffer,
1879, has priority over Helminthoglyptidae
Pilsbry, 1939. Xanthonyx was also the first
genus of an American helicoid described
(Fischer & Crosse, 1872). However, only a few
authors adopted Xanthonychidae as the cor-
rect name for the family (Nordsieck, 1987).
Helminthoglyptidae continued to be used to
93
refer to the North American helicoids (Roth,
1996), while Xanthonychidae was employed
for the Central and South American helicoids
(Emberton, 1991; Miller & Naranjo-Garcia,
1991; Schileyko, 1991). Other authors, how-
ever, continued using the name Helmintho-
glyptidae for all the American helicoids (North,
Central and South) (Zilch, 1959-1960; Solem,
1978; Richardson, 1982). For a complete
chronologic review of previous studies and
tendencies, see Roth (1996). As a result,
whenever the names Helminthoglyptidae or
Xanthonychidae are used, it is not clear to
which genera the authors are referring, unless
they state which convention they are follow-
ing.
For years many authors (Pilsbry, 1939;
Nordsieck, 1987; Miller & Naranjo-Garcia,
1991; Schileyko, 1991) have searched for a
coherent definition of the family. Pilsbry’s
(1939) was the concept that has lasted
longest. His definition of the Xanthonychidae
(= Helminthoglyptidae) is a combination of
character states, none of which represents
true synapomorphies for the group. The geo-
graphic distribution became important in the
identification of the genera. Similarly, the ab-
sence of clearly defined synapomorphies lead
to poor definitions of the other families in-
cluded in Helicoidea (Scott, 1996). Recent
94 CUEZZO
studies have appeared reviewing the Heli-
coidea (Nordsieck, 1987; Schileyko, 1991),
but they failed to propose their hypotheses in
a testable way. The only two studies using
cladistic analysis to formulate phylogenetic
hypotheses within Helminthoglyptidae are
Pearce (1990) and Roth (1996).
The anatomy of several components of the
Xanthonychidae is still poorly known. Although
several papers have been published with
anatomical data on the Central American
(Binney, 1879; Pilsbry, 1894, 1900; Baker,
1942; Thompson, 1959; Miller, 1987; Tillier,
1989; Cuezzo, 1996) and South American
taxa (Hesse, 1930; Fernandez & Rumi, 1984;
Tillier, 1989; Cuezzo, 1997), they are still the
least known components of the Xanthony-
chidae.
Three different problems occurred in the
published literature of the Xanthonychidae:
(1) Monographic revisions are scarce, and
most of the existing studies focus on the de-
scription of single species upon which phylo-
genetic hypotheses are built (Pilsbry, 1900;
Pilsbry & Cockerell, 1937; Hylton Scott, 1951,
1962; Parodiz, 1955; Hass, 1962; Miller, 1970,
1971, 1976a, b, 1981, 1985). (2) The taxo-
nomic differences among the genera included
in the Xanthonychidae are mainly based on
two organs: mucous glands and dart sac (=
dart complex), with different combinations of
their character states (generally shape and
number). In this way, phylogenetic assump-
tions and classifications (Miller & Naranjo-
Garcia, 1991; Schileyko, 1991) had been car-
ried out on single characters or at least
single-organ systems (generally the genital
system) (Bieler, 1992). (3) Phylogenetic rela-
tionships of the genera are established on ar-
bitrarily narrative character transformations
(Nordsieck, 1987; Miller & Naranjo-Garcia,
1991; Schileyko, 1991). As stated by Nixon &
Carpenter (1993), there is no clear way to es-
tablish whether a character is “primitive” or
“derived” prior to cladistic analysis, and actu-
ally there is no need for the creation of “evolu-
tive scenarios” to explain the possible direc-
tion of character transformation.
This study was undertaken (1) to test the
monophyly of the Xanthonychidae (= Hel-
mintoglyptidae) (2) to carry out a cladistic
analysis in order to hypothesize the relation-
ships among the components of the family in
a testable way, and (3) to review the current
classification of the Xanthonychidae based on
the cladograms obtained.
MATERIALS AND METHODS
The adult alcohol preserved snails used for
this study belong to the following Institutions:
ANSP = Academy of Natural Sciences of
Philadelphia, Pennsylvania, USA
INBIO = Instituto de Biodiversidad, Costa
Rica.
UF = Florida Museum of Natural Sciences,
Gainesville, Florida, USA
FMNH = Field Museum of Natural History,
Chicago, Illinois, USA
FML = Fundacion Miguel Lillo, Тиситап,
Argentina
In the case of the material from ANSP, the
first number cited after the institution corre-
sponds to dry lots consulted every time that a
verification of the species determination was
necessary. A number preceded by the letter
“A” (in the case of material from ANSP or
FML) corresponds to alcohoi-preserved ma-
terial used in this study. All catalogue numbers
from other institutions correspond to alcohol-
preserved material. A complete list ofthe taxa
studied is documented in Appendix 1.
The characters used in this study (except
characters 30 and 31, which are based on
published literature) are based on a selection
made after a study of the anatomy of the pal-
lial, digestive, reproductive and nervous sys-
tems, and external morphology of the type
species of each genus (Appendix 1). Speci-
mens were dissected under a Wild M3C mi-
croscope. Illustrations were made with the aid
of a camera lucida. Illustrations and a brief de-
scription of the characters are included only
when there is no agreement with the literature
or a Clarification of a specific character is
needed. Shell characters have not been con-
sidered for this analysis under the assumption
that they are inadequate for reconstructing
phylogenetic history (Nordsieck, 1986; Em-
berton, 1995). However, the study of the
sculpture, apertural barriers and composition
of the shell could lead in the future to informa-
tive characters (Solem, 1978; Emberton,
1995).
Cladistic analyses were carried out using
the computer program Henning86 (Farris,
1988). The program DADA (Nixon, 1992) was
used for the construction of the data matrix
and CLADOS (Nixon, 1992) for the analysis of
the character distribution on the trees. Jack-
knife, a statistical test for homoplasy, was per-
formed with the program NONA (Goloboff,
1993). FQ, a program provided by P. Goloboff,
PHYLOGENETICS OF XANTHONYCHIDAE 95
integrated jackknife results from 50 replicates.
FQ reads a “tread” statement (resulting trees
in parenthetical notation) and calculates the
majority rule consensus tree showing the fre-
quencies above 50% (frequencies of 100%
are not indicated).
The frequency index is:
> fi
Wa)
where fi is frequency of group i of consensus
tree, and T is the number of terminals, note
that these depend on the cutoff value.
The data set includes 34 characters and
25 taxa (including the outgroups) (Table 1).
Thirteen characters are multistate and were
coded as non-additive so that any state could
transform into any alternative state at an equal
cost. Character polarity is derived from the
analysis rather than being an a priori assump-
tion (Nixon & Carpenter, 1993). The assign-
ment of “missing character” (“?”) to some taxa
is not mentioned in the character description
but indicated in the data matrix. Some apo-
morphies, although not informative for con-
structing phylogenies, were included among
the characters because they are usfull for the
characterization of certain terminals. Cladistic
analysis was initially carried out with Hennig86
using the commands “mh*;bb*;”. Successive
weighting (Farris, 1969; Carpenter, 1988) was
used after the initial runs. From the cladograms
obtained after successive weighting, one
cladogram was selected. When more than one
tree was obtained, they were sumarized in a
Nelson consensus tree.
RESULTS
Selection of the Ingroup and Outgroup Taxa
The xanthonychid (= helminthoglyptid) in-
group taxa considered in this analysis are
those included in the family by Zilch (1959-
TABLE 1. Data set containing thirty four characters and twenty five taxa used
in analysis #1. Two additional characters (#35-36) were used in analysis #2.
10 20 30
Outgroups 123456789 123456789 123456789 1234 56
Oreohelix -000000100 3011101000 0000000001 01100 00
Neohelix -000001002 1011101000 0000000001 70010 00
Helix -000000001 0011101120 1000000000 10010 11
Bradybaena -000000011 0000101021 0000000100 10011 24
Xanthonychidae
Cepolis -000000011 0021131111 0100001200 10011 23
Polymita -000002011 1011131111 0100001200 10011 23
Dialeuca -00000200? 0000130011 0100001200 10021 23
Helminthoglypta -000000011 0000111111 0010002100 10021 22
Epiphragmophora -000000011 0100101111 0000002200 10011 24
Monadenia -000002012 1021111111 0000003000 10021 21
Sonorella -000002012 0022101100 0000000000 10021 00
Eremarionta -000000010 1021101021 0021000100 10021 22
Micrarionta —000000010 7021101121 0001000700 10021 22
Humboldtiana -000002012 1021101120 1000000000 10021 13
Charodotes -000000011 0000121111 0010002100 10021 22
Plesarionta -000002011 1021101121 0001000000 10011 22
Bunnya -010010011 2012101120 1000113010 70011 13
Tryonigens -100000010 0021001000 0000000000 10020 00
Trichodiscina -0000000?0 0022101121 0000000000 10021 24
Cryptostrakon -001110010 0011201121 0000000000 ?0012 21
Lysinoe —100001010 1021101120 1000000100 10010 13
Leptarionta -10000?01? 2072101121 0000000000 10021 24
Metostracon —001110010 0011101121 0000023000 20010 24
Xanthonyx -010010010 2021101120 100001?000 ?0010 14
Xerarionta -000002010 1000101121 1001000000 10011 32
96 CUEZZO
1960), with the exception of Averellia Ancey,
Dinotropis Pilsbry & Cockerell, Sonorelix
Berry, and Setipelis Pilsbry, for which no alco-
hol-preserved material was available. For the
purpose of the analysis, subgenera as well as
genera (sensu Zilch, 1959-1960) have been
used as taxonomic units when material was
available. This is the case for Plesarionta
Pilsbry, Xerarionta Pilsbry, and Eremarionta
Pilsbry, which were considered by Zilch to be
subgenera of Micrarionta Ancey. Charodotes
Pilsbry, considered a subgenus of Helmintho-
glypta Ancey by Zilch, and Trichodiscina
Martens, considered a subgenus of Averellia,
are also treated as separate terminals.
For each genus, the type species, when
available, was used as representative of the
group. The only exceptions are Xanthonyx
Crosse & Fischer, Eremarionta Pilsbry, Lepta-
rionta Fischer & Crosse, and Xerarionta
Forbes. Consequently, other species were se-
lected as representative of those genera.
For the selection of outgroups, a previous
cladistic analysis was used (Nixon & Car-
penter, 1993). Based on Emberton (1991), the
Bradybaenidae, Helicidae, Oreohelicidae, and
Polygyridae were chosen as outgroups. Al-
though the use of more than one outgroup is
not necessary, this option was prefered, with
the idea that the cladistic inferences might be
better founded. Outgroups were treated as all
the other terminals in order to test the mono-
phyly of the ingroup. The Bradybaenidae were
traditionally assumed to be the sister group of
the Helminthoglyptidae (Schileyko, 1978;
Roth, 1996). In each case, the nominal genus
was used as representative of its family, except
in the case of Polygyridae.
My results are presented in Figures 1-3
and are discussed below.
Character Descriptions
External Morphology:
Character 1: Tail keeled (Figs. 4, 6):
Longitudinal row of plaques in mid-dorsal
tail. Character states: (“0”) absent; (“1”) pre-
sent.
Character 2: Tail horn (Figs. 5, 6):
Projection on the end of the tail with the ap-
pearance of a horn. Character states: (“0”) ab-
sent; (“1”) present.
Pallial System:
Character 3: Lobes of kidney:
This character is generally associated with
the reduction in space due to the limacization
process (Tillier, 1984). Character states: (“0”)
absent; (“1”) present.
Character 4: Position of the heart relative to
the kidney (Fig. 7):
The common position of the heart in
Stylommatophora is to the left of the kidney
with the roof of the lung observed ventrally.
However, the heart is partially surrounded by
the kidney in Cryptostrakon and Metostracon.
Although this character has usually been as-
sociated with the morphology of a semislug, in
the other two зет ид genera of the
Xanthonychidae, the position of the peri-
cardium is to the left of the kidney, as in most
of snails. Character states: (“0”) to the left of
the kidney; (“1”) partially surrounded by the
kidney.
Character 5: Relation of the mantle to the
shell:
Character states: (“0”) the mantle does not
enclose the shell; (“1”) mantle entirely en-
closes the shell.
Character 6: Diaphragm:
The diaphragm or lung floor forms the base
of the pulmonary cavity. Character states: (“0”)
diaphragm thin, transparent, and membra-
nous; (“1”) diaphragm thick, muscular, not
transparent; (“2”) diaphragm thin but not
transparent showing some muscular strands.
Character 7: Anus position (figure and
description in Emberton, 1991). Character
states: (“0”) near mantle collar; (“1”) recessed
from collar.
Reproductive System:
Character 8: Fertilization Pouch-Sperma-
thecal complex (FPSC):
Character states: (“0”) the FPSC is com-
pletely free of the albumen gland; (“1”) FPSC
totally or partially embedded in the base of the
albumen gland.
Character 9: Penial sheath:
Character states: (“0”) penial sheath ab-
sent; (“1”) sheath thin, membranous; (“2”)
sheath thick, muscular.
Character 10: Internal penial structure:
Character states: (“0”) with longitudinal
ridges; (“1”) with one or two pilasters; (“2”)
smooth or with wrinkles; (“3”) basal portions
with several pilasters and upper portion with
pustules.
Character 11: Penial muscular band: (de-
scribed and illustrated in Cuezzo, 1997).
Character states: (“0”) absent; (“1”) present.
Character 12: Verge (Figs. 8, 9):
Character states: (“0”) verge absent; (“1”)
verge present, with thin projections or termi-
PHYLOGENETICS OF XANTHONYCHIDAE 97
Oreohelix
Neohelix
Helix
Monadenia
Sonorella
Eremarionta
Micrarionta
Humboldtiana
Plesarionta
Tryonigens
Trichodiscina
Lysinoe
Leptarionta
Xerarionta
80 Bunnya
Xanthonyx
86 Cryptostrakon
Metostracon
Bradybaena
Epiphragmophora
78 84 Helminthoglypta
Charodotes
Cepolis
us Polymita
Dialeuca
FIG. 1. Consensus tree generated from 50 trees. Numbers in the nodes are the frequencies above 50% ob-
tained with FQ after Jackknife (frequencies of 100% are not indicated).
701003132533
Oreohelix
9 13 24 28 34
ES 8 Bunnya
E
e TE al
& 12
DIANA E =
Es Aanthonyx
170918729730. h 14 34
a EE > 4 19 y № № Cryptostrakon
1] . 25 26
&@-Metostracon
2 3
14
Tryonigens
8 arme
о
& Lysinoe
1
17
É—Bradybaena
0
15
» 33 Mé Helminthoglypta
ES Charodotes
2 18 26 2
E и 19
1 2 #-Еррйгаеторйога
= 10
2
15721026
a ares Polymita
33
HEH Dialeuca
00720722
„ fTrichodiscina
ES 1 10
St Leptarionta
72
15 18 26
НЕ Я Мопааеша
33 о =
E 10 13 18
| 19 TÉ #-Sonorella
Humboldtiana
10 1
= 9
; sur Plesarionta
=
12 1320
35 не Aerarionta
= 0
| 17222027
Eremarionta
D) 2 Pen
Micrarionta
FIG. 1a. Prefered phylogenetic hypothesis for Xanthonychidae, generated by Henig86 and constructed using
CLADOS, from data in Table 1. Filled dash marks represents synapomorphies, gray dash marks represents
homoplasies. There are no reversals.
PHYLOGENETICS OF XANTHONYCHIDAE 99
72107319032
Oreohelix
E-Neohelix
1 14
5
8 12
EZ.
Bradybaena
6 9 2107512513726
2 Е 1-Monadenia
DE; IS
» я x [Helminthoglypta
1 7 2 ь
а Charodotes
Е 11 5
Epiphragmophora
1
a 16 17 33
18 35 2 a Dialeuca
Fa 15 21 26 36
a or E 12
LS TENTE x ГВ Серой$
т
610% 12
E =-Polymita
1
9
&-Plesarionta
1
12 13) 35
: Xerarionta
23 al
1722 27
E —Eremarionta
1
Micrarionta
HHumboldtiana
1 6 27
3 Lysinoe
9 13 24 28 34 36
36
ES 2 10 25 26 an
i 2 173 =
Mae ® Xanthonyx
re 7
a 14 34 36
3 &@-Cryptostrakon
3 35 =
Metostracon
FIG. 2. Cladogram #2 for Xanthonychidae, generated by Henig86 and constructed using CLADOS.
Alternative hypothesis adding characters 35 and 36. Filled dash marks are synapomorphies, gray dash
marks are homoplasies.
100 CUEZZO
Oreohelix
Neohelix
Tryonigens
Sonorella
Trichodiscina
Leptarionta
Bradybaena
Monadenia
Helminthoglypta
Charodotes
Epiphragmophora
Dialeuca
Cepolis
Polymita
Eremarionta
Micrarionta
Plesarionta
Xerarionta
Humboldtiana
Lysinoe
Helix
Bunnya
Xanthonyx
Cryptostrakon
Metostracon
FIG. 3. Consensus of 22 trees, second analysis, generated by Henig86 and constructed by CLADOS.
PHYLOGENETICS OF XANTHONYCHIDAE 101
NY
pel
FIG. 4. Tryonigens: Tail keeled (Tk), character 1(1). Bar = 0.1 cm.
FIG. 5. Bunnya: Tail horn (Th), character 2(1). Bar = 3 mm.
FIG. 6. Epiphragmophora: Tail morphology, character 1(0), 2(0). Td = tail middorsal groove. Bar = 1 cm.
FIG. 7. Bradybaena: Position of heart (H) respect to the kidney (K), character 4(0). Bar = 3 mm.
FIG. 8. Polymita: Portion of the penial complex showing small verge (Ve), character 13(1), with terminal thin
projections (P), character 12(1). Bar = 3 mm.
FIG. 9. Sonorella: Penial complex showing verge (Ve) without terminal projections, character 12(2), occupy-
ing half of the penial sac, character 13(2). Bar = 3 mm.
nal papillae; (“2”) verge present, but without
terminal papillae in which case it is stout.
Character 13: Relative size of the verge with
respect to penial sac (Figs. 8, 9):
Character states: (“0”) absent; (“1”) present,
small to medium sized verge, generally lo-
cated in the upper portion of the penial sac;
(“2”) present, large verge, occupying half or
more of the penial sac.
Character 14: Atrium:
Character states: (“0”) absent; (“1”) present,
with longitudinal folds; (“2”) present, with
transversal thin folds.
Character 15: Atrial sac (Figs. 10, 11):
102 CUEZZO
The atrial sac is a projection or prolongation
of the atrium. In general, both the atrium and
the atrial sac presents the same internal
sculpture. Character states: (“0”) absent; (“1”)
present, with internal sculpture consisting of
thin folds; (“2”) present, with a wide pilaster in
the internal wall; (“3”) present, with small ir-
regular pustules in internal wall.
Character 16: Epiphallus:
The epiphallus is the portion of the penial
complex between the penis and the insertion
of the vas deferens. Its delimitation is easy
when the penis bears a verge that clearly
marks the limit between penis and epiphallus.
When the verge is absent, the internal struc-
ture of the epiphallus is an important element
in determining its limits. The portion of the pe-
nial complex termed “penis or preputial cham-
ber” (Gregg & Miller, 1976; Miller, 1981), and
“double tube of the upper part of penis” (Miller,
1985) are considered here to be homologous
to the lower portion of the epiphallus:
Character states: (“0”) absent; (“1”) present.
Character 17: Flagellum:
In the penial complex, the epiphallus con-
tinues as a blind duct that can have different
lengths and that decreases in diameter to-
ward the tip. In the type species of the genus
Sonorella, a reduced flagellum is present,
known in the literature as “epiphallic caecum.”
Because of its position this structure is con-
sidered here to be homologous to the flagel-
lum present in the other Xanthonychidae.
Character states: (“0”) absent; (“1”) present.
Character 18: Dart Sac insertion (Figs. 12,
13):
The dart sac is a muscular blind sac usually
containing a calcareous dart, which functions
in stimulation during copulation. Character
states: (“0”) absent; (“1”) present, one dart sac
inserted in the atrium or in the atrial sac, cylin-
drical to round; (“2”) present, one to four dart
sacs seated on the vagina.
Character 19: Mucous glands inserted in dart
sac (Fig. 14):
The mucous glands that insert in the dart
Sac are considered homologous because
they share the same position and probable
function. Character states: (“0”) absent; (“1”)
present, generally bearing one or more ducts.
Character 20: Mucous glands inserted in
vagina (Fig. 15):
The mucous glands inserted in the vagina
are considered to be homologous because
they share the same position and probably the
same function. Character states: (“0”) absent;
(“1”) present.
Character 21: Mucous glands inserted in
atrial sheath (Fig. 12):
The ducts of these glands are inserted be-
tween the folds of the atrial sheath. These
glands are not homologous to others in the re-
productive system. Character states: (“0”) ab-
sent; (“1”) present.
Character 22: Bulbous reservoirs on mucus
gland ducts (Figs. 16, 17):
The reservoirs are swellings in the ducts.
Character states: (“0”) absent; (“1”) present,
with glands ending in a common duct and
(“2”) present, with glands ending in separate
ducts.
Character 23: Distal portion of mucous glands
(Figs 17):
Character states: (“0”) glands not expanded
in their distal portion; (“1”) distal portions of
mucous glands expanded, flattened and
spread upon vagina, dart or base of penis.
Character 24: Vaginal diverticulum:
The vaginal diverticulum is a round, cecil
evagination in the ventral side of the vagina
under the row of dart sacs. There is no possi-
bility that it is another dart sac, because the
internal structure is completely different. Also,
no dart was found in the interior. Whereas the
dart sacs have two thin dart papillae, the vagi-
nal diverticulum has only longitudinal folds in
its interior. Character states: (“0”) absent; (“1”)
present.
Character 25: Albumen gland:
Character states: (“0”) albumen gland bean
shaped, located in the visceral mass; (“1”) al-
bumen gland bilobate, located in the visceral
mass; (“2”) albumen gland straight, located in
the pedal cavity.
Character 26: Vas deferens:
Character states: (“0”) vas deferens does
not loop around any structure; (“1”) vas defer-
ens looped around the penial retractor muscle
close to its insertion in the epiphallus; (“2”)
vas deferens looped around the dart sac;
(“3”) vas deferens looped around penis-
epiphallus.
Character 27: Basal genital sheath:
The observation of this character has been
affected in the past by the tendency to clean
the genitalia from connective tissue or other
membranous tissue before any observations
are made. However, many genera of the
Xanthonychidae show a conspicuous basal
genital sheath that overlaps the basal female
and male terminal genitalia. Character states:
(“0”) absent; (“1”) present, formed by mem-
branous tissue; (“2”) present, composed of
thin muscular tissue.
Ash
FIG. 10. Charodotes: Lower genitalia showing atrial sac (As) with internal wide pilaster, character 15(2). Bar
=3mm.
FIG. 11. Dialeuca: Lower genitalia showing atrium (A) and atrial sac (As) with irregularly distributed pustules
in the internal wall, character 15(3). Bar = 3 mm.
FIG. 12. Cepolis: Terminal genitalia. Dart sac (Ds) cilindrical inserting in the atrial sac (As), character 18(1).
There are another pair of glands (Mg) inserted in the atrial sheath (Ash), character 21(1). Bar = 0.5 cm.
FIG. 13. Bunnya: Terminal genitalia. Dart sacs (Ds) seated on the vagina, character 18(2). Ba = bursa copu-
latrix appendix; Bc = bursa copulatrix. Bar = 2 mm.
104 CUEZZO
Character 28: Bursa copulatrix sac appendix
(Fig. 13):
Thick and short appendix in the bursa cop-
ulatrix sac. Character states: (“0”) absent; (“1”)
present.
Character 29: Bursa copulatrix duct swollen at
the base:
Character states: (“0”) absent; (“1”) present.
Character 30: Copulation modality:
Character states were selected from the
published data (Webb, 1947, 1948, 1959,
1972; Emberton, 1985): (“0”) copulation is one
sided; (“1”) copulation is reciprocal.
Character 31: Reproductive modality:
Character states were selected from pub-
lished information (Solem, 1978; Tompa,
1984): (“0”) oviparous; (“1”) ovoviviparous.
Character 32: End of penial retractor muscle:
Character states: (“0”) penial retractor in-
serts without divisions; (“1”) penial retractor
splits in branches.
Digestive System:
Character 33: Internal structure of the oe-
sophageal crop:
The wall of the oesophageal crop presents
different kinds of interior sculpture indepen-
dent of the thickness of the wall. Character
states: (“0”) wall with longitudinal ridges that
can extend along all the crop length or only
portions of it length; (“1”) wall with pustules or-
dered as longitudinal cords or irregularly dis-
tributed.
Nervous System:
Character 34: Fusion of the visceral ganglion
(illustrated in Emberton & Tillier, 1995):
The visceral ganglion is located in the ven-
tral chain of the nervous system. Although the
fusion of the ganglia is traditionally associated
with the limacization process, surprisingly non
fusion of the visceral ganglion was observed
in the case of two semislug genera. This char-
acter is discussed by Emberton & Tillier
(1995). Character states: (“0”) absent; (“1”)
present, fused with left parietal ganglion; (“2”)
present, fused with both parietal ganglia.
Characters Added in Analysis #2
Character 35: Position of mucous glands
Character states: (“0”) absent; (“1”) inserted
in vagina; (“2”) inserted in dart sac or close to
its base; (“3”) inserted in vagina and dart sac.
Character 36: Type and shape of mucous
glands (according to Miller & Naranjo Garcia,
1991)
Character states: (“0”) absent; (“1”) tubular;
(“2”) membranous; (“3”) round compact; (“4”)
vesicular club-shaped.
Cladistic Analysis
Using the program Hennig86, two different
analyses were carried out: (1) The initial analy-
sis using the command “mh*; bb*;” produced
50 trees of 99 steps in length, Cl = 52, RI = 62.
A consensus tree summarizing the 50 original
trees obtained is presented in Figure 1.
Caution should be used to interpret it, because
consensus trees contain less information,
being generated from fundamental clado-
grams instead of original information. After
performing succesive weighting, two trees
were retained, each with length 372, Cl = 79
and RI = 78. One of them (Fig. 1a) is identical
to one of the trees of the original set. The other
differs only in the position of Micrarionta. In
one of the trees, its position is resolved, but
there are no characters supporting this resolu-
tion, whereas in the other, there is no resolu-
tion on the position of Micrarionta. Conse-
quently, the Nelsen consensus tree (after
succesive weighting) has the same topology
as the one showing the unresolved trichotomy
for (Plesarionta, Xerarionta) + Eremarionta +
Micrarionta. There are four synapomorphies
(characters 17, 18, 29, 30) supporting the
monophyly of the ingroup (Bradybaenidae-
Xanthonychidae-Helicidae). Helix is the sister
group of the Bradybaenidae-Xanthonychidae
complex, supported by character 8. The out-
groups Oreohelix and Neohelix are clearly
separated from the other genera. Within
the ingroup, three monophyletic groups are
clearly defined: (a) first, the genera Bunnya,
Xanthonyx, Cryptostrakon and Metostracon
with character 5 as synapomorphy; (b) sec-
ond, Helminthoglypta, Charodotes, Epiphra-
gmophora, Cepolis, Polymita and Dialeuca,
supported by character 26; (c) and third, Ere-
marionta, Micrarionta, Plesarionta and Xerari-
onta, supported by character 23.
(2) A second analysis for comparative pur-
poses has been made by adding to the same
matrix two characters: (a) type and shape of
mucous glands (character states as defined
by Miller & Naranjo-Garcia, 1991; #35 in Table
|) and (b) mucous glands insertion (assuming
that all the mucous glands are homologous,
as it is traditionally considered, with the ex-
ception of the glands inserted in the atrial
sheath in the Cepoliinae, #36 in Table |).
Concurrently, characters 19 and 20 (mucous
PHYLOGENETICS OF XANTHONYCHIDAE 105
FIG. 14. Metostracon: Terminal genitalia. Mucous glands (Mg) inserted in dart sac (Ds), character 19(1). Bar
= 0.5 ст»
FIG. 15. Humboldtiana: Terminal genitalia. Mucous glands (Ма) inserted т vagina (V), character 20(1). Ds =
dart sac. Bar = 0.5 cm.
FIG. 16. Helminthoglypta: Terminal genitalia. Bulbous reservoirs (Br) on mucous glands ducts ending in a
common duct (Мда), character 22(1). Bar = 0.5 cm.
FIG. 17. Eremarionta: Terminal genitalia. Bulbous reservoirs (Br) on separate mucous glands ducts (Мда),
character 22(2). Distal portion of mucous glands (Mg) expanded and spreaded on vagina and base of penis,
character 23(1). Bar = 1 mm.
106 CUEZZO
gland insertion in vagina and in dart sac) were
deactivated. This analysis produced initially
40 trees, L = 106, Cl = 52 and RI = 62, and
after succesive weighting the number was re-
duced to 22 trees, L = 391, Cl = 82, RI = 84.
One of them is illustrated in Figure 2. This tree
has the same topology as one of the original
set and is very similar to the consensus tree
(Fig. 3). The differences with the first analysis
are: (a) Sonorella and Tryonigens, the genera
that lack a dart complex, are located basally in
the cladogram, but they do not form a mono-
phyletic group; (b) excluding Tryonigens, the
synapomorphy for the rest of the genera is the
presence of flagellum (character 17); (c)
Monadenia is the sister group of Helmin-
thoglypta-Charodotes, this relationship sup-
ported by character 15 (atrial sac with internal
thin folds). The other two monophyletic groups
described in analysis #1: Bunnya + Xanthonyx
+ Cryptostrakon + Metostracon (supported by
character 5) and Plesarionta + Xerarionta +
Micrarionta + Eremarionta (Supported by
character 23), are also clearly delimited in the
second analysis; (d) the genus Helix repre-
senting the Helicidae is internested in the in-
group.
DISCUSSION
Both cladistic analyses suggest that the
family Xanthonychidae, or Helminthogly-
ptidae as it was defined by Pilsbry (1939) and
subsequently treated by various authors
(Baker, 1959; Zilch, 1959-1960; Schileyko,
1991), is a paraphyletic group. The results ob-
tained (Figs. 1, 1a, 2, 3) support the idea that
the families Bradybaenidae and Helicidae
should be jointed with Xanthonychidae, be-
cause they form a monophyletic group.
Baker's (1959) concept: “Since the sizes of
families are matters of convenience or custom,
we Americans can leave to the wisdom of our
Old World colleagues the advisability of a sep-
arate family for the genera of their home lands”
had prevailed since 1959, and the Xantho-
nychidae (American Helicoids) had been
maintained as a separate family from the Bra-
dybaenidae (mostly Asian Helicoids) and
Helicidae (mainly European) without much
justification. Consequently, geographic data
become important in the identification of cer-
tain genera of the Bradybaenidae-Xanthony-
chidae-Helicidae group.
The cladogram illustrated in Figure 1a is
chosen as the preferred hypothesis, because
is the one that best explained the data: (1) the
mucous glands are not homologous structures
as they are traditionally considered in pub-
lished literature, because they have different
positions and, therefore, should be treated as
different characters; (2) this hypothesis (clado-
gram #1a) shows closest relationship between
xanthonychid and bradybaenid snails, with
Helix, representing the Helicidae, their sister
group.
The position of the Helicidae, however,
should be carefully reconsidered when more
genera of Helicidae and Bradybaenidae can
be studied along with the Xanthonychidae.
Four synapomorphies (Fig. 1a) support the
Bradybaenidae-Xanthonychidae-Helicidae
group (presence of flagellum, dart sac in-
serted in vagina, bursa copulatrix duct not
swollen at the base, and copulation recipro-
cal). One synapomorphy supports the mono-
phyly of the Bradybaenidae-Xanthonychidae
group (fertilization pouch-spermathecal com-
plex embedded in the base of the albumen
glana).
Although it is clear that it would be prema-
ture to translate this phylogenetic hypothesis
(cladogram #1a) into a new classification (too
many branches of the cladogram are not
strongly supported, and the relationships of
the genera could also change when more taxa
of the Helicidae and Bradybaenidae are stud-
ied), three monophyletic groups are well de-
fined and delimited based upon the analyses
performed (cladogram #1a). The first is the
semislug group composed of Cryptostrakon +
Metostracon + Bunnya and Xanthonyx. The
monophyly of this group is supported by char-
acter 5 (mantle entirely concealing the shell).
Other characters, such as shell reduction, kid-
ney size reduction and compactation, rotation
of the longitudinal axis of kidney, pulmonary
cavity short and reduced, presence of sec-
ondary respiratory structures (such as alveoli),
detorsion of digestive tract, position of the
stomach, oesophageal crop contained in foot
cavity, digestive gland invading the pulmonary
cavity, presence of air sacs, have been hy-
pothesized by Solem (1978) and Tillier (1983,
1984, 1989) as being correlated with the lima-
cization process. Forthis reason, none ofthem
were used in the final analysis (#1a) in order to
avoid redundant characters (in case Solem
and Tillier are correct). However, when these
characters were included in preliminary analy-
ses, they appeared as synapomorphies of the
semislug group. At this point, it is difficult to test
whether Solem and Tilliers hypotheses are
PHYLOGENETICS OF XANTHONYCHIDAE 107
simply evolutionary scenarios because, as
proposed, they appear to be based on circular
reasoning. Characters 3 and 4, although de-
scribed also as correlated with the limaciza-
tion process, were maintained in the analysis,
because the lobes of the kidney (character
3) and the position of the heart internested in
the kidney (character 4) are present only in
Cryptostrakon and Metostracon but not in the
other semislug genera. Similarly, character 5
(mantle entirely enclosing the shell) has been
used, because it is not possible to correlate
it with a “semislug state.” The reason for this
is that there are some semislugs that have
the shell exposed — Vitrina: Vitrinidae;
Binneya Cooper (Pilsbry, 1939); Pellicula de-
pressa: Bulimulidae (Van Mol, 1968, 1971) —
others with the shell partially enclosed by
the mantle — Austenia, Parmarion: Heli-
carionidae (Solem, 1966) — and still in others
the shell is completely concealed by the man-
tle — Peltella ¡heringi: Bulimulidae (Van Mol,
1968), Malagarion paenelimax: Helicarioni-
dae (Tillier, 1979) and the xanthonychid
genera studied here. In other words, it is not
possible to infer that all semislugs have
the shell concealed by the mantle due to
the changes in shape (mainly reduction of
visceral mass). Also, if the trend towards a
“slug stage” is a real phenomenon, all slugs
should have the shell completely concealed
by the mantle, which is true only in some
cases (some Arionidae) but not in others, for
example in Testacella (Testacellidae) and
Daudebardia (Zonitidae). Solem (1978) and
Tilliers (1983, 1984, 1989) argumentation
that all the characters mentioned above
represent compensations for space alter-
ations in the process of slug evolution must be
reconsidered with a historical perspective.
Within the Xanthonychidae, the semislug
stage has occured only once, and thus the
monophyly of the semislug genera is sup-
ported in this study.
The second monophyletic group delimited
in the two analyses and also well supported
by the statistical test of homoplasy (Fig. 1) is
composed of Helminthoglypta + Charodotes +
Epiphragmophora + Cepolis + Dialeuca and
Polymita. This group occurs in all most parsi-
monious cladograms and thus is “unequivo-
cally supported” by the data. The monophyly
of this group is supported by “vas deferens
looped around the dart sac,” a character that
later changes to “looped around the penial re-
tractor muscle” in the clade Cepolis + Polymita
+ Dialeuca. The muscular basal genital
sheath is the synapomorphy supporting the
relationship between the Cepoliinae and
Epiphragmophora. Baker (1943) had pointed
out that Helminthoglypta and Cepolis, placed
by Pilsbry (1939) in different subfamilies,
could be related based on the common pres-
ence of the “dart sheath” (considered here as
the basal genital sheath, character 27). Later,
Baker (1961) proposed that the Cepolinae
(Cepolis, Polymita, and Dialeuca) should be
included with Helminthoglypta and Micra-
rionta in the Helminthoglyptinae as defined by
Pilsbry (1939). However, both Nordsieck
(1987) and Roth (1996) denied any relation
between the two genera because of the “com-
pletely differently constructed glands” that
they possess. In this study, the clade com-
posed by Helminthoglypta + Charodotes +
Epiphragmophora + Cepolis + Polymita and
Dialeuca appeared as a monophyletic group
with the addition of Monadenia in the second
analysis (cladogram +2). The genus Brady-
baena (representing the Bradybaenidae) is
the sister group of this clade in both analyses,
although its position is not strongly supported
by any synapomorphy.
The third monophyletic group is composed
of Eremarionta + Micrarionta + Plesarionta
and Xerarionta. The monophyly of this group
is sustained by character 23 (expansioned
and spread of distal portion of mucous
glands). Eremarionta, Plesarionta and Xera-
rionta were originally proposed as “sections”
or subgenera of Micrarionta. Later, they were
elevated to genera based on “major differ-
ences” that were unfortunately not well de-
tailed (Bequaert 4 Miller, 1973; Miller, 1981).
My results are consistent with Pearce's (1990)
hypothesis of the relationships of these gen-
era (Fig. 1a).
Because the type and shape of the mucous
glands has been traditionally important in the
various definitions of Xanthonychidae-Hel-
minthoglyptidae-Helicidae, and often used as
the only justification for splitting the families
(Pilsbry, 1939; Miller 8 Naranjo-Garcia, 1991),
the second analysis was performed for com-
parative purposes. In this analysis, there are
three synapomorphies supporting the in-
group: bursa copulatrix duct not swollen at
base, copulation reciprocal, and fertilization
pouch-spermathecal complex buried in the al-
bumen gland. Helix is internested in the in-
group. The hypothesis proposed in Figure 2,
treating the mucous glands as homologous
structures, suggests that the dart sac and mu-
cous glands were originally absent in the
108 CUEZZO
basal clades Tryonigens and Sonorella, and
secondarily acquired (Figs. 2, 3) in the rest of
Xanthonychidae. The hypothesis proposed in
Figure 1, 1a, treating the mucous glands as
non-homologous structures, suggests, how-
ever, the opposite situation: the dart sac and
mucous glands inserted in the vagina were
originally present and secondarily lost in sep-
arate groups. Comparisons among the clado-
grams presented (Figs. 1a, 2) are difficult to
make because a different matrix was used for
the analysis. Even if we compare the behavior
of characters 19 and 20 of the first analysis
and 35 and 36 of the second, which refer to
the same structure coded differently, the mu-
cous glands are highly homoplastic and there-
fore appear to be unreliable as characters for
phylogenetic reconstruction. In any case, the
mucous glands and dart sac have been over-
valued in past studies, and many other non-
genital characters have not been considered
in previous classifications of the traditional
Xanthonychidae.
The question of whether the genera that
lack the dart complex form a monophyletic
unit or whether the absence of these struc-
tures are products of parallel evolution has
been discussed several times (Nordsieck,
1987; Schileyko, 1991). Based on the clado-
grams obtained in this study, neither analysis
supports the idea of monophylly of taxa that
lack dart complex, represented here by Tryo-
nigens and Sonorella. The taxonomic position
of Monadenia, originally placed in the
Helminthoglyptinae by Pilsbry (1939) but later
moved to Bradybaenidae (Miller & Naranjo-
Garcia, 1991), remains controversial. In this
study, the “swollen” in the terminal genitalia
present in Monadenia was interpreted as an
atrial sac. When the shape of the mucous
glands are taken into consideration and the
mucous glands are considered homologous
structures (Fig. 2), the position of Monadenia
is similar to the one traditionally accepted
and proposed by Pilsbry (1939). However, it
could be concluded that the relationships of
Monadenia will remain obscure until a phy-
logeny of the Bradybaenidae-Xanthonychi-
dae-Helicidae is proposed.
(a) The family Xanthonychidae (= Helmin-
thoglyptidae) as defined and used by Pilsbry
(1939) and Zilch (1959-1960) is paraphyletic.
(6) Xanthonychidae-Bradybaenidae-Heiici-
dae conform a monophyletic group.
(c) The preferred hypothesis (Fig. 1a) sup-
ports the delimitation of three monophyletic
groups within the traditionally named Xan-
thonychidae.
(d) Some characters used in previous studies,
such as the shape and number of mucous
glands, are of poor value for the reconstruc-
tion of the phylogeny of the Xanthonychidae.
Considering the monophyly of the Brady-
baenidae-Helicidae-Xanthonychidae well es-
tablished, further cladistic studies are needed
as a basis for a revised, testable and informa-
tive classification of its components.
ACKNOWLEDGMENT
This study was partially done while | was
beneficiated with a Jessup Fellowship award-
ed by The Academy of Natural Science of
Philadelphia, without which this work would
not have been possible. | am deeply indebted
to George Davis for his consistent support
and encouragement. | am grateful to Quentin
Wheeler, not only for providing laboratory fa-
cilities at Cornell University but also for his
contagious enthusiasm for taxonomy, “the big
science” and for his unconditional support.
Thanks are extended to Eduardo Dominguez,
Pablo Goloboff, Diana Silva, and Quentin
Wheeler for reviewing the manuscript and to
P. Goloboff for providing the computer pro-
gram NONA and FQ. David Robinson facili-
tated the access to the collection in the
Academy of Natural Sciences of Philadelphia.
| also thank the following persons for the loan
of material of their institutions: George Davis
(ANSP), Fred Thompson (UF), Zaidett Bar-
rientos (INBIO), and Rüdiger Bieler and John
Slapcinsky (FMNH).
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APPENDIX |
Taxa studied:
Bradybaena similaris (Férussac):
ANSP 434, A8774: West Java, Bogor, Java.
Feb. 1973.Bunnya bernardinae Baker
ANSP A16728: Ruins of Monastery 20 km
southwest of Mexico City, El Desierto de
Los Leones to La Venta, Distrito Federal,
Mexico. July 1926. H.B. Baker!
Cepolis cepa (Muller):
UF 46191: Dept. du Sud, SE slope of Morne
Formon, Haiti. 1500 m. Thompson! Jan.
1984.
UF 235002: Dept. Quest, 3 km S. Kenscoff,
Haiti. 1470 m. Thompson & Auffenberg!
UF 48281: Dept. du Sud, Boia Dirant, SW of
Morne Formon, 1250 m. June 1984, K.A.
& R.W.P!
Charodotes traski (Newcomb):
ANSP A14976: 12 miles E. of Las Cruces,
Baja California, Mexico. 1918. H. N.
Lowe!
Cryptostrakon gabbi Binney:
ANSP 246310, A9639: Costa Rica. Lec-
totypes selected by Baker (1963).
Cryptostrakon corcovadensis Cuezzo (т
press):
INBIO 468080: P. N. Corcovado, Estacion
Sirena, Sendero a Rio Los Patos, 10 mt.,
Madrigal, Puntarenas Province, Costa
Rica. 18 Aug. 1994. Marianella Segura.
INBIO 468087: Madrigal, Puntarenas Prov-
ince, Р. М. Corcovado, Estacion Sirena,
Sendero a Los Patos, Costa Rica, 10 m.
14 Aug. 1994. Marianella Segura!
INBIO 408059: Madrigal, Puntarenas Prov-
ince, P. N. Corcovado, Estacion Sirena,
Sendero Los Espaveles, 20 m. 12 Aug.
1994. Marianella Segura!
Dialeuca nemoraloides (Adams):
ANSP A12685: Mandeville, Manchester
Parish, Jamaica. June 1933. H. Baker!
Epiphragmophora hieronymi Doering:
FML A100: Quebrada del Tala, Catamarca,
Argentina. Jan. 1993. Dominguez!
PHYLOGENETICS OF XANTHONYCHIDAE A
Eremarionta rowelli (Newcomb):
ANSP A11327B: Needles Peaks, Topock, Mo-
have County, Arizona, USA. Ex Ferriss.
ANSP 164956, A11327G: Rocky Hills, Punta
Libertad. Feb. 1935. H. N. Lowe!
Helminthoglypta arrosa (Gould, in Binney):
ANSP A11345: 12 miles from end of Point
Reyes, Marin County, California, USA. H.
N. Lowe!
Helminthoglypta tudiculata (Binney):
ANSP 94237 A11344F: Oceanside, San Di-
ego County, California, USA. Sept. 1907.
Ex H. G. Eaton.
Humboldtiana humboldtiana (Valeciennes, in
Pfeiffer):
ANSP A13281: El Desierto de Los Leones,
Mexico.
Leptarionta quillarmodi (Shuttleworth):
ANSP A16727: 4 km North and slightly east of
Cordova, foothills east and north of
Toxpam (Hacienda de San Francisco),
Cerro de Las Palmas, Vera Cruz, Mexico.
June 1926. H. B. Baker!
Lysinoe ghiesbreghti (Nyst):
FMNH 206294/1: 2400 m, on trail, 1 km SW of
Esquipulas Palo Gordo, San Marcos,
Guatemala. 28 July 1980. Ken Young!
UF 190195: Alta Verapaz Prov., Guatemala.
10.5 Km. SE of Tactic. Feb. 1991. Е. Т. &
S. P. Christman!
Metostracon mima Pilsbry:
ANSP 77245, A9636: Morelia, Michoacan,
Mexico. S. N. Rhoads! Holotype.
ANSP A9635F: Uruapam del Progresso, Mi-
choacan, Mexico. S. N. Rhoads! Para-
type.
Metostracon mima Pilsbry:
ANSP A9410D: Near Alvarez at km 53, San
Luis de Potosi, Mexico. July 1934. H. A.
Pilsbry!
ANSP A9411A: Km 42, Potosi and Rio Verde
Railroad, San Luis de Potosi, Mexico.
Aug. 1934. H. A. Pilsbry!
Micrarionta facta (Newcomb):
ANSP 10789, A11342: Santa Barbara Island,
Santa Barbara County, California, USA.
Newcomb!
Micrarionta sp.:
ANSP 130897, A113321: 5 mi W of Leach
Spring, Granite Mountains, California,
USA. 1922. Ferris!
Monadenia fidelis (Gray):
ANSP 158283, A16079: Riverdale, Multno-
mah County, Oregon, USA. August 1929.
H. B. Baker!
ANSP 158278 A16078: About 13 mi N of
Klamath Falls, E side of upper Klamath
Lake, Ouxy siding, Klamath County,
Oregon, USA. July 1929. H. B. Baker!
Plesarionta stearsiana (Gabb):
ANSP 66091 A11336: Coronado Island, San
Diego County, California, USA. 1895.
A. W. Anthony!
ANSP 146098 A11332E: Near San Vicente
Mission, Baja California Norte, Mexico.
Dec. 1927. L. G. Ingles!
Polymita picta Born:
ANSP A13209: Cuba,
ANSP 154067, A9341: Mandinga de Yumuri,
Oriente Province, Cuba. Welch!
Sonorella hachitana (Dall)
ANSP A10367: Florida Mountains, Luna
County, New Mexico. 1906. H. A. Pilsbry!
Trichodiscina cordovana (Pfeiffer):
ANSP A16732: Steep Valley down from sad-
die, Twin Peak Valley, Estado Puebla,
Mexico. July 1926. H. B. Baker!
ANSP A16734: Steep valley down from sad-
die, Twin Peak Valley, Estado Puebla,
Mexico. July 1926. Н. В. Baker!
Tryonigens remondi (Tryon):
ANSP 166233, A9415A: Hills around Panuco,
Sinaloa, Mexico. Aug. 1935. H. A. Pilsbry!
Xanthonyx sp.:
ANSP A16735: 14 km from Cordoba towards
Orizaba on Mexican railroad, hills south-
east of town, on opposite side of canyon
Sumidero, Vera Cruz State, Mexico. June
1926. H. B. Baker!
Xerarionta kelletti (Forbes):
ANSP 138972, A11332A: West of North Bay
isthmus, hillside west of Isthmus Cove,
Santa Catalina Island, Los Angeles
County, California, USA. 1925. H. A. Pils-
bry! ANSP A138973, A11332B: Avalon,
Santa Catalina Island, Los Angeles
County, California, USA. 1925. H. A.
Pilsbry!
MALACOLOGIA, 1998, 39(1-2): 113-121
DIVERGENCE AMONG MOBILE BASIN POPULATIONS OF THE PLEUROCERID
SNAIL GENUS, LEPTOXIS, ESTIMATED BY ALLOZYME ELECTROPHORESIS
Robert T. Dillon, Jr.' and Charles Lydeard*
ABSTRACT
Although the Mobile River Basin of Alabama was historically a center of great pleurocerid di-
versity, populations today are small and scattered. We obtained samples of all four nominal
species of Mobile Basin Leptoxis currently extant: L. ampla (3 populations), L. picta (1 popula-
tion), L. plicata (2 populations), and L. taeniata (2 populations). Gene frequencies at nine variable
enzyme loci were determined for about 30 individuals from each population using horizontal
starch gel electrophoresis. Samples of about 30 individuals from three populations of the wide-
spread Leptoxis praerosa were analyzed as controls. Within populations, 18 of 99 loci were poly-
morphic, none showing genotype frequencies significantly different from Hardy-Weinberg ex-
pectation. Between populations within species, statistically significant divergence was apparent
at most loci. Comparisons among the nominal species showed L. praerosa and L. plicata to be
quite distinct from each other, and from all other populations. Much lower levels of divergence
among populations nominally L. picta, L. ampla, and L. taeniata seem more consistent with a hy-
pothesis of geographic isolation than reproductive isolation. We refer to these three taxa as the
“Leptoxis picta group.” Our results compare favorably in most respects with previously published
data on mitochondrial 16S rRNA gene sequence divergence among these taxa, especially in the
genetic distinction of L. plicata. The relationships within the L. picta group warrant further study.
Key words: genetics, isozymes, speciation, freshwater, gastropods, Alabama, endangered
species.
INTRODUCTION
The rivers and streams of Alabama’s
Mobile River Basin have recently attracted at-
tention as a center of endemism for a variety
of aquatic life, including turtles, fish, bivalves
and prosobranch snails (Lydeard & Mayden,
1995). Based primarily on the revisions of
Goodrich (1922, 1924, 1936, 1941), Burch
(1989) recognized 77 species of pleurocerid
snails from the region, 95% of which were un-
known outside the Mobile Basin. Burch’s list
included 6 species of Gyrotoma, 5 species of
Pleurocera, 52 species of Elimia (synonymiz-
ing Goniobasis as used by Goodrich), and 14
Leptoxis (lowering Anculosa, as used by
Goodrich, to subgeneric level). During the
present century, however, most of the larger
rivers of the Mobile Basin have been im-
pounded for hydroelectric power, channel-
ized, or otherwise modified for navigation. The
Mobile Basin pleurocerid fauna has also been
adversely impacted by changing patterns of
land use, first from siltation due to intensive
agriculture, and more recently from pollution.
Lydeard & Mayden (1995) presumed extinct
29 species of Mobile Basin pleurocerids, in-
cluding all six species of the endemic genus
Gyrotoma.
The Leptoxis species of the basin have
been the object of special concern. Of the 11
species of Leptoxis known historically from
the Coosa River, Bogan & Pierson (1993a)
found only L. taeniata (Conrad, 1834), appar-
ently restricted now to but a few small tribu-
taries. Of the four Leptoxis species docu-
mented from the Cahaba River, only L. ampla
(Anthony, 1855) apparently survives, inhabit-
ing a 30 km reach of the main river and sev-
eral smaller Cahaba tributaries (Bogan &
Pierson, 1993b). The only Leptoxis population
remaining in the Black Warrior drainage is L.
plicata (Conrad, 1834), restricted to a short
reach of Locust Fork. Based on these data, as
well as extensive U. S. Fish & Wildlife Service
field records, Hartfield (1997) identified L. tae-
niata, L. ampla, and L. plicata as candidates
for addition to the U. S. list of endangered and
threatened wildlife and plants. The status of
the only other nominal Leptoxis species
"Department of Biology, College of Charleston, Charleston, South Carolina, 29424, U.S.A.
“Aquatic Biology Program, Department of Biological Sciences, University of Alabama, Р.О. Box 870344, Tuscaloosa,
Alabama 35487, U.S.A.
114 DILLON AND LYDEARD
known with certainty to have survived in the
Mobile Basin, L. picta (Conrad, 1834) of the
main Alabama River, continues to be moni-
tored.
But Hartfield noted that the genetic relation-
ships among these four nominal species of
Leptoxis are poorly understood. They are dis-
tinguished primarily by minor attributes of
shell shape and size, traits long known for cli-
nal variability (Goodrich, 1934, 1935). The
non-genetic component of some aspects of
pleurocerid shell morphology is well-docu-
mented (Chambers, 1982; Dillon, 1984a).
In light of these concerns, Lydeard et al.
(1997) surveyed 15 pleurocerid populations
from the Mobile Basin: seven Elimia and four
Pleurocera, in addition to the four nominal
Leptoxis species. A molecular phylogeny con-
structed from mitochondrial 16S rRNA gene
sequences suggested that Alabama Elimia
and Pleurocera are sister taxa. The four
Leptoxis species were quite different from the
Elimia/Pleurocera group, and depicted as pa-
raphyletic. Levels of sequence divergence
were low between L. taeniata and L. ampla,
with L. picta and L. plicata appearing more
distinct.
Allozyme electrophoresis is an older and
more established technique for evaluating the
specific status of pleurocerid populations, es-
pecially the large genus Goniobasis.
Extensive surveys of variation at allozyme-en-
coding loci, involving at least 11 species and
58 populations, have established that
Goniobasis shows unusually low levels of het-
erozygosity, high levels of divergence
between populations within species, and
very few shared alleles at any locus when
compared among species (Chambers,
1978, 1980; Dillon, 1984b, 1988a; Dillon &
Davis, 1980; Bianchi et al., 1994; Stiven &
Kreiser, 1994). Recent evidence suggests
similar trends in Leptoxis, although intrapopu-
lation variation may be somewhat greater, and
interpopulation variation less (Dillon &
Ahlstedt, 1997).
The purposes of the present work are
twofold. We survey the allozyme divergence
displayed by populations representing the
four nominal species of Mobile River basin
Leptoxis to gather further evidence regarding
their genetic distinction. We also compare the
levels of allozyme divergence estimated here
to the DNA sequence divergence estimates of
Lydeard et al. (1997), as a possible guide to
the future application of the newer technology.
METHODS
We analyzed eight populations of Alabama
Leptoxis assigned to four species (Appendix).
Our L. taeniata populations were sampled
from Buxahatchee and Choccolocco creeks,
two tributaries of the Coosa River. As no
Leptoxis inhabit the 50 km reach of the Coosa
River separating these two creeks, gene flow
between the populations we designated
Taebux and Taechc, respectively, would seem
to be negligible at present. We obtained sam-
ples of L. ampla from three shoals of the
Cahaba River separated about 20 river km
from each other, labeled Ampcah1, Ampcah2,
and Ampcah3 from upstream down. Our two
samples of L. plicata are from Locust Fork,
РШос1 about 15 river km upstream from
Pliloc2. Our single sample of L. picta (Picala)
was collected by boat from limestone walls
and outcrops in the lower Alabama River.
We selected three populations of the well-
characterized Leptoxis praerosa (Say, 1821)
to provide calibration for our analysis.
Populations of this species are common and
widespread throughout the Ohio, Cumber-
land, and Tennessee river drainages. Our L.
praerosa came from three tributaries of the
Tennessee River, the Elk River (Praelk), the
Duck River (Praduk), and the Sequatchie
River (Praseq). Leptoxis from the Sequatchie
and Duck rivers have been previously ana-
lyzed by Dillon & Ahlstedt (1997). Analyzing
all 11 populations together, we were able to
evaluate observed levels of genetic diver-
gence among nominal Alabama species by
comparison to divergence among Leptoxis
populations known to be conspecific, isolated
at approximately equivalent distances.
The geographic relationships among the 11
populations analyzed in this work are mapped
in Figure 1, and locality data and sample sizes
are given in the Appendix. Although our sam-
ple sizes were in most cases greater than 30,
only 21 individual L. picta were available. The
Appendix also provides catalog numbers for
voucher specimens deposited in the Aca-
demy of Natural Sciences of Philadelphia.
Our equipment and techniques for hori-
zontal starch gel electrophoresis of whole an-
imal homogenates have been previously de-
scribed (Dillon, 1985, 1992). Samples were
initially run on gels of five different buffer sys-
tems and stained to visualize 13 different en-
zymes. We simultaneously screened these
gels and stains by requiring that clearly inter-
LEPTOXIS POPULATION GENETICS 115
Pliloc
Taechc
Picala
100 km
@
FIG. 1. The state of Alabama, showing major
drainages and sample sites. (1) Tombigbee River,
(2) Black Warrior River, (3) Cahaba River, (4) Coosa
River, (5) Tallapoosa River, (6) Alabama River, (7)
Mobile Bay, (8) Tennessee River.
pretable polymorphism be present in an initial
comparison of Praduk and Ampcahl, se-
lected as the most different pair of populations
in our study. Ultimately we identified the prod-
ucts of nine putative gene loci for detailed
analysis over all 11 populations.
The Poulik buffer (Poulik, 1957) was used to
resolve glucose phosphate isomerase (GPI,
EC 5.3.1.9) and octopine dehydrogenase
(ODH, EC 1.5.1.11). The AP6 buffer (Clayton
& Tretiak, 1972) was used to resolve mannose
phosphate isomerase (MPI, EC 5.3.1.8), 6-
phosphogluconate dehydrogenase (6PGD,
EC 1.1.1.44), and isocitrate dehydrogenase
(IDH, EC 1.1.1.42). The products of two puta-
tive loci were apparent on the IDH gel, one mi-
grating cathodally (“IDHF”) and the other an-
odally (“IDHS”). The TEB8 buffer (buffer Ш of
Shaw & Prasad, 1970) was also employed for
IDHF, xanthine dehydrogenase (XDH, EC
1.2.1.37), and esterase (EST1, EC 3.1.1.2).
Superoxide dismutase (SOD, EC 1.15.1.1)
activity was visualized as light bands on ТЕВ8
gels darkly stained for XDH or IDH.
Allozyme phenotype has been shown to re-
sult from simple Mendelian inheritance of
codominant alleles at the 6PGD locus by
Chambers (1980), working with Goniobasis
floridana. Dillon (1986) reported similar find-
ings for GPI, ODH, and EST1, based on a
mother-offspring analysis in Goniobasis prox-
ima. Although the esterase stain employed
here (a-napthyl acetate as substrate) yields a
complex, multi-banded phenotype for each in-
dividual, only the slowly-migrating, strongly
staining products of the single locus desig-
nated EST1 by Dillon (1986) were accorded a
genetic interpretation in the present work.
Population Praduk served as the standard
for allelic designations. We adopted here the
same designations used for this population by
Dillon & Ahlstedt (1997) for the four shared
loci (EST1, GPI, MPI, and ODH), and labeled
all new alleles accordingly. For the five loci not
reported by Dillon & Ahlstedt (IDHS, IDHF,
XDH, 6PGD, and SOD), the most common al-
lele in Praduk was considered to migrate 100
mm and all other alleles labeled by their rela-
tive electrophoretic mobilities in millimeters
faster or slower.
Gene frequencies, tests to Hardy-Weinberg
expectation (by chi-square, with pooling for
rare genotypes), and Nei’s (1978) unbiased
genetic identities and distances were calcu-
lated using BIOSYS version 1.7 (Swofford &
Selander, 1981). Tests for homogeneity be-
tween populations within nominal species
were by Fisher’s exact method in 2 x 2 cases,
otherwise by chi-square contingency tests,
pooling the rarest rows or columns as neces-
sary. We analyzed the matrix of genetic dis-
tances using the multidimensional scaling
module of STATISTICA (Release 5.0, StatSoft,
Inc.), with a standard Guttman-Lingoes (prin-
cipal component) starting configuration. The
distances between any pair of populations
sharing no alleles at any locus (i.e., similarity =
0.0) were set to 5.0, a figure greater than any
value actually observed.
116 DILLON AND LYDEARD
RESULTS
Example shells from each of the four nomi-
nal Alabama Leptoxis species are shown in
Figure 2. Their differences were not striking.
The shells of L. picta tended to be heavier,
with a higher spire, while those of L. ampla
were lower and more rounded, and L. taeniata
intermediate. Apical erosion made spire
height difficult to evaluate, however, espe-
cially in the L. ampla population. The shells of
L. plicata were less eroded, with more shoul-
dered whorls. They were characterized by low
folding (or plication) on the whorl periphery,
barely visible in Figure 2. Although such plica-
tions have been reported to occur in L. ampla,
we saw no evidence of them in our samples.
Gene frequencies are given in Table 1.
Levels of intrapopulation variation were low,
although perhaps not quite as low as in the
better-studied Goniobasis. Over all9 x 11 =
99 loci, we found 18 polymorphic as judged by
the 95% criterion. Genotype frequencies at
none of these 18 loci differed significantly
from Hardy-Weinberg expectation.
All nominal species for which more than one
population was sampled are listed in Table 2,
along with the loci at which any intraspecific
polymorphism was observed. Every nominal
species showed significant interpopulation al-
lelic frequency difference in at least one locus.
This was especially striking at the ODH locus
in L. ampla , and at both the ODH and EST1
loci in L. plicata, where the most common al-
lele changed over distances as short as 15
river km. Not only did the three L. praerosa
populations differ significantly from each other,
the present Praseq and Praduk populations
differed from the Sequatchie and Duck sam-
ples of Dillon & Ahlstedt (1997) located 20-30
km downstream.
Figure 3 shows Nei’s unbiased genetic
identities among all pairs of Leptoxis popula-
tions. The three L. praerosa populations were
strikingly different from all others, as were the
two L. plicata populations. The levels of ge-
netic identity among L. ampla, L. picta, and L.
taeniata populations were much higher.
Figure 3 also depicts the Nei’s genetic dis-
tances in two dimensions, from multidimen-
sional scaling. After 100 iterations, the stress
for this solution was 0.0015. The six popula-
tions comprising L. ampla, L. picta, and L. tae-
niata occupy one end of the long axis of the
scale, the three L. praerosa the other end, and
L. plicata appears intermediate.
DISCUSSION
The species concept under which the pleu-
rocerid fauna of the Mobile Basin has been
described and revised differs substantially
from the biological concept in currency today.
In his (1922) monograph on the “Anculosae”
(Leptoxis) of Alabama, Goodrich wrote, “That
collection of individuals in the Pleuroceridae
may be called a species whose predominant
characters are not the predominant charac-
ters of another collection of individuals. If we
see only a few specimens of a single species
its own peculiar characters may often seem to
be submerged by characters linking it with an-
other species. But in a long series the individ-
ual characters stand out, and we are com-
pelled then to recognize the existence of
definable differences and to proceed to de-
scribe them and provide the label of a name.”
5mm
FIG. 2. Example shells of four Mobile Basin Leptoxis species. From left, L. picta (Picala), L. taeniata (Taechc),
L. ampla (Ampcah1), and L. plicata (Pliloc2).
LEPTOXIS POPULATION GENETICS 1417
TABLE 1. Gene frequencies at nine allozyme loci for 11 populations of Leptoxis
Атр- Amp- Атр-
Locus Allele cah1 cah2 cah3 Picala Taechc Taebux Pliloci Pliloc2 Praduk Praelk Praseq
GPI 108 0.024 0.726 0.726
104 1.000 0.855 0.625 0.976 1.000 1.000
97 0.274 0.274 1.000 1.000 0.838
94 0.145 0.375
90 0.162
MPI 100 1.000 1.000 0.016
98 0.929 1.000 1.000
95 1.000 1.000 1.000 0.071 1.000 0.984 1.000
EST 106 0.613 0.387
105 1.000 1.000 1.000
104 1.000 1.000 1.000 1.000 1.000 1.000
99 0.387 0.613
6PGD 106 1.000 1.000 1.000 1.000 1.000 1.000 0.767 0.900
100 0.233 0.100 1.000 1.000 0.952
94 0.048
ООН 121 0.355 0.242
118 0.065 0.048
1115 0.323 0.532 0.855 0.177 0.054
113 0.258 0.177 0.145 0.823 0.203
110 0.057 0.682
107 1.000 0.943 0.318 1.000 1.000 1.000 0.743
IDHF 103 1.000 1.000
100 1.000 1.000 1.000
98 1.000 1.000 1.000 1.000 0.875 1.000
95 0.125
IDHS 103 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
102 0.177
100 1.000 1.000 0.823
XDH 103 1.000 1.000 1.000 1.000 1.000 1.000
100 1.000 1.000 1.000 1.000 1.000
SOD 110 1.000 1.000 1.000 1.000 1.000 1.000
100 1.000 1.000 1.000 1.000 1.000
Goodrich often reported that Mobile Basin
species overlapped not just in character, but
in geographic range as well. Populations iden-
tified by Goodrich (1922) as L. picta histori-
cally inhabited the lower Coosa River, the
lower Cahaba River, and the Alabama River
downstream to Claiborne, Monroe County.
Goodrich reported the range of L. taeniata as
substantially identical to that of L. picta, ex-
cept that L. taeniata extended further up the
Coosa River and its tributaries. Goodrich
listed L. ampla from both the Coosa and
Cahaba rivers and their tributaries, although
not from the main stem of the Alabama River.
Goodrich did not consider that the geographic
range of L. plicata overlapped with those of L.
picta, L. taeniata, or L. ampla. He recorded L.
plicata as occurring in the Black Warrior River,
the Tombigbee River, and their tributaries
only.
The concept of the species differs today, as
does the distribution of Leptoxis in the Mobile
Basin. Under the biological species concept,
local variation in gene frequencies (and by ex-
tension, external appearances) is a not-unex-
pected consequence of limited gene flow in
DILLON AND LYDEARD
118
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LEPTOXIS POPULATION GENETICS M9
TABLE 2. The probability of homogeneity among nominally conspecific popula-
tions of Leptoxis (p, from Y tests, р, from Fisher's exact tests). A subscript “c” indi-
cates that rows or columns were combined for the test. The table is blank for loci
where conspecific populations were not polymorphic.
L. ampla L. taeniata L. plicata L. praerosa
GPI Py. < 0.001 р, = 1.0 р, < 0.001
EST1 p, = 0.019
6PGD p, = 0.085
ODH p, < 0.001 Py. = 0.062 p, < 0.001
IDHF p, = 0.006
IDHS Py, < 0.001
populations of organisms with dispersal capa-
bilities as low as freshwater snails. For exam-
ple, a culvert placed in a small North Carolina
stream in the 1950s caused significant diver-
gence at the ODH locus between upstream
and downstream populations of G. proxima
over a distance of just 10 meters (Dillon,
1988b). That this did not comprise a speciation
event became clear when the barrier was re-
moved, and the genetic difference disap-
peared. Indeed, geographically isolated popu-
lations of G. proxima sharing no alleles at as
many as six allozyme loci have nevertheless
demonstrated no evidence of reproductive iso-
lation when transplanted (Dillon, 1986,
1988a). Evidence of similar interpopulation di-
vergence is clear in our three samples of L.
ampla, our two samples of L. taeniata, and our
two samples of L. plicata. Whether the signifi-
cant differences highlighted in Table 2 are due
to some unrecognized barriers to dispersal, or
whether they may be due to isolation by dis-
tance alone, cannot be told at present. But it is
clear that our three populations of L. ampla, for
example, do not constitute different species. It
is also clear that, extending the levels of diver-
gence illustrated within L. ampla down the 120
km length of the Cahaba River as was the sit-
uation earlier in this century, the Leptoxis of the
Alabama River would be expected to show
striking genetic differences with the Leptoxis of
the headwaters, through isolation by distance.
There is little expectation, however, that repro-
ductive isolation will evolve in such a circum-
stance, or that headwaters populations and
populations from the main river will speciate
parapatrically.
The divergence among L. taeniata, L. picta,
and L. ampla is negligible, given their geo-
graphic distance. Leptoxis picta has uncom-
mon alleles at the СР! and MPI loci not de-
tected in L. taeniata, and one L. taeniata pop-
ulation has an allele at IDHF not seen in L.
picta. The levels of divergence appeared
somewhat greater between L. ampla and L.
picta/taeniata, due to the results at the MPI
locus. But although L. ampla is fixed for an al-
lele not seen in L. taeniata, Table 1 shows that
both MPI alleles are found in the L. picta pop-
ulation that may have connected them in the
main Alabama River. Such small and clinal dif-
ferences are not comparable to those nor-
mally displayed by species of pleurocerid
snails presumed distinct, as illustrated by L.
praerosa and L. plicata. We therefore refer to
all three of these taxa, L. picta, L. taeniata,
and L. ampla, as the “Leptoxis picta group.”
In most respects, our findings coincide with
those based on 16S rRNA gene sequence di-
vergence. Lydeard et al. (1997) also found L.
plicata to be quite distinct from all other Mobile
Basin Leptoxis, unique at about 20% of its nu-
cleotide bases. Lydeard’s mtDNA phylogeny
depicted the three members of the L. picta
group as a single clade when transversions
were weighted more than transitions. But while
very little sequence divergence was apparent
between L. taeniata and L. ampla (only about
2%), Lydeard reported about 20% sequence
divergence between L. taeniata/ampla and L.
picta. So our finding that L. picta and L. taeni-
ata are indistinguishable in their allozyme fre-
quencies at nine loci was quite unexpected.
A similar discrepancy between allozyme
and mtDNA divergence in oysters was attri-
buted to balancing selection at multiple en-
zyme loci by Karl & Avise (1992), although
much more data would be required before
such a suggestion could be made in our case.
Lydeard et al. (1997) only analyzed a single
individual for each nominal Leptoxis species.
There is a clear need for additional surveys of
120 DILLON AND LYDEARD
16S rRNA sequence divergence focused
below the species level.
A complete understanding of the genetic re-
lationships among L. picta, L. taeniata, and L.
ampla would have required samples from
populations inhabiting the lower regions of the
Cahaba and Coosa rivers, where the three
nominal species were once reported to co-
occur. All such populations are long extinct.
Regardless of their specific status, the levels
of genetic diversity displayed by the small
populations of the L. picta group that remain
today, as evidenced by both mtDNA and al-
lozyme studies, argue strongly for conserva-
tion measures. The L. plicata population re-
stricted now to just 20 km of Locust Fork
(Hartfield, 1997) is clearly a unique species by
all measures, and deserves immediate pro-
tection.
ACKNOWLEDGMENTS
We thank Wallace Holznagel, John Yoder
and J. Malcolm Pierson for assistance in the
field, Gary Rosenberg for helpful discussions,
and Paul Hartfield for reading the manuscript.
This research was supported by a Research
Grants Committee Award (2-67767) from the
University of Alabama, a contract with the U.
S. Department of the Interior (1448-0004-04-
929), and the National Science Foundation
(DEB-9527758).
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APPENDIX
Locality data, sample sizes, and (where ap-
plicable) catalog numbers for voucher speci-
mens deposited in the Academy of Natural
Sciences of Philadelphia
Ampcah1—Cahaba River at Co. 52, 1.5 km W
of Helen, Shelby County, Alabama. Same
station as CA-61 of Bogan & Pierson
(1993b). N = 25 Leptoxis ampla.
Ampcah2—Cahaba River at River Road, 3 km
S of intersection of Co. 1 and Co. 13.
Shelby County, Alabama. N = 36 Leptoxis
ampla.
Ampcah3—Cahaba River at Co. 24, Bibb
County, Alabama. Just downstream from
station CA-64 of Bogan & Pierson
(1993b), and 10 km downstream from the
L. ampla site of Lydeard et al. (1997). N =
24 Leptoxis ampla. ANSP 400111.
Picala—Alabama River about 2 km south of
U.S. 84 crossing, Monroe County, Ala-
bama. Same site as the L. picta site of
Lydeard et al. (1997). N = 21 Leptoxis
picta. ANSP 400112.
Pliloc1—Locust Fork, 0.4 km upstream from
the Mount Olive Road boat ramp,
Jefferson County, Alabama. About 20 km
south of L. plicata site of Lydeard et al.
(1997). N = 31 Leptoxis plicata. ANSP
400113.
Pliloc2—Locust Fork, at shoal 10 km down-
stream from the Mount Olive Road boat
ramp, Jefferson County, Alabama. N = 31
Leptoxis plicata.
Taebux—Buxahatchee Creek, Shelby County,
Alabama. N = 34 Leptoxis taeniata.
Taechc—Choccolocco Creek, Talladega
County, Alabama. Same site as the L.
taeniata site of Lydeard et al. (1997). М =
32 Leptoxis taeniata. ANSP 400115.
Praduk—Duck River at Shelbyville, Bedford
County, Tennessee. About 30 km up-
stream from Duck River site of Dillon &
Ahlstedt (1997). N = 31 Leptoxis prae-
rosa.
Praelk—Elk River at Stump Shoals Public
Access near US 64 bridge, 8 km E of
Fayetteville, Lincoln County, Tennessee.
М = 31 Leptoxis praerosa. ANSP 400114.
Praseg—Sequatchie River at Tn 28 bridge,
Whitwell, Marion County, Tennessee.
About 20 km upstream from Sequatchie
site of Dillon & Ahlstedt (1997). N = 37
Leptoxis praerosa.
e
MALACOLOGIA, 1998, 39(1-2): 123-128
LARVAL FUSION AND DEVELOPMENT OF CONJOINED TERATOIDS IN
BIOMPHALARIA GLABRATA
Charles S. Richards, Carolyn Patterson, Fred A. Lewis & Matty Knight
Biomedical Research Institute, 12111 Parklawn Drive, Rockville, Maryland 20852
ABSTRACT
Snails of a genetically isolated laboratory stock of Biomphalaria glabrata, if mated to snails of
certain other stocks, produce polyzygotic egg capsules. When two or more embryos occupy a
single capsule, some embryos spontaneously fuse during the trochophore larval stage into ter-
atoids of usually two, but up to seven, conjoined snails. We have observed more than 950 of
these fused teratoids, with some dizygotic conjoined twins surviving to adulthood. Allophenic
cell-to-cell adhesions lead to unusual patterns of development, apparently determined by the
areas of the embryos that are fused.
Key words: Biomphalaria, Pulmonata, development.
INTRODUCTION
Observations of conjoined twinning in mol-
lusks have been previously reported [re-
viewed by Bigus (1981) and Mason &
Copeland (1988)], most often as rare anom-
alies in otherwise normal populations. The un-
expected occurrence of large numbers of
spontaneously conjoining embryos in one of
our snail stocks and the consistency with
which we obtain these teratoids have pro-
vided an abundance of material for study.
Allophenic cell membrane junctions, the de-
velopment of conjoined embryos, and the un-
usual morphological patterns that result are
areas of interest that lead us to continue the
culture of these teratoids.
The pulmonate gastropod Biomphalaria
glabrata is a simultaneous hermaphrodite and
is an important intermediate host of the para-
sitic trematode Schistosoma mansoni. We
maintain several named stocks of this snail in
genetic isolation to conserve phenotypic vari-
ations in pigment and in susceptibility to
S. mansoni (Richards & Shade, 1987). Pig-
mentation markers (Newton, 1954; Paraense,
1955) are useful in elucidating inheritance
patterns of the susceptibility-resistant pheno-
types (Richards, 1985; Richards et al., 1992).
MATERIALS 8 METHODS
Snails of one genetic stock, M3 636, pro-
duced a large number of polyzygotic eggs
after out-crossing with a different stock (10R2
123
was used most often). M3 636 stock snails
were isolated as juveniles and reared individ-
ually in 400 ml beakers until the onset of egg
production by self-fertilization. Each snail was
then put in a new 400 ml beaker with a part-
ner of a different snail stock. After 2-7 days,
during which each snail fertilized the other,
the snails were re-isolated. A piece of clear
plastic sheeting was floated in the beaker of
each isolated snail. Most snails preferentially
deposit clutches onto this plastic, which can
then be removed from the beaker for exami-
nation under a low-power dissecting micro-
scope. Hybrid pigmentation of offspring from
the clutches of both parents indicated the two
snails had each functioned as a male in the
cross.
Many (> 200) egg clutches that contained
polyzygotic egg capsules were sketched at in-
tervals of their development to record the
number of zygotes originally deposited in
each capsule and the timing and pattern of
embryo fusion.
Some conjoined teratoids apparently had
difficulty hatching on their own, in which
cases we used dissecting needles to break
the egg capsule. Survival of teratoids after
hatching was very limited until we began
feeding the neonate teratoids with cultured
Nostoc sp. (Liang et al., 1987). This filamen-
tous cyanobacterium, in addition to being nu-
tritionally sufficient, reduced the need for
active foraging and allowed simultaneous
feeding by component snails fused in a con-
figuration that made motility and feeding
problematic.
124 RICHARDSET AL.
RESULTS
Polyzygotic Egg Capsules
Because we had not previousiy observed
significant polyzygocy in self-fertilizing snails
or in isogenic interbreeding populations of
laboratory snail stocks, the production of large
numbers of polyzygotic egg capsules by an
out-crossed snail suggested that crossing ge-
netically distant snails may trigger polyzygocy.
Most M3 636 snails exhibit a degree of self-
sterility (Paraense, 1993) and produce, by
self-fertilization, very few clutches, each of
which contains few or no viable eggs. After
mating with a snail from a different stock, M3
636 snails would generally produce normal
clutches for several days before producing
polyzygotic egg capsules. Polyzygocy in-
creased during the next 4-6 weeks and then
declined until only a few small clutches were
produced. A subsequent out-crossing would
often induce another cycle of polyzygocy.
Egg capsules that contain multiple zygotes
are not larger than monozygotic capsules.
Multiple embryos share the single portion of
nutritive capsular fluid and are smaller at
hatching than snails from normal monozy-
gotic egg capsules. In clutches that contain
both single embryo and polyzygotic egg cap-
sules, it is sometimes evident that the egg
capsules deposited first are those that are
monozygotic. The egg capsules deposited
last in the clutch contain increasing numbers
of zygotes, as if the snail were unable to pro-
vide enough capsular fluid or other material to
accommodate available zygotes. Zygotes are
often observed at one end of the clutch, not
contained in egg capsules, but loose in the
fluid that surrounds the egg capsules.
Fusion of Larval Snails
Early observations of conjoined twins indi-
cated that it was unlikely that they resulted
from incomplete division of a single embryo.
Conjoined snails of different pigment pheno-
types (Fig. 1) confirmed that fusion of individ-
uals occurred. In our many observations of
new clutches, which were sketched and
recorded at intervals in their development, we
saw that the polyzygotic egg capsules con-
tained excess fertilized oocytes and that divi-
sion of a single zygote into multiple germs did
not occur.
For embryos to fuse they must make con-
tact. When the clutch is first deposited, the zy-
FIG. 1. Conjoined Biomphalaria glabrata twins with
one albino component and one black-eye (arrow)
component. (length about 1 mm)
gotes are non-motile and are spaced through-
out the viscous capsular fluid. Cleavage con-
tinues with little change in the size of the em-
bryo through the blastula stage. During this
interval, up to about 24 hrs after the clutch is
deposited, the embryos become more dense,
and gravity acts to bring the embryos, now
blastulae, together at the lowest point in the
spherical egg capsule. Though the embryos
seem to be in close contact, we have not seen
fusion at this stage. With gastrulation, the
vitelline membrane that had surrounded and
isolated the embryo is lost (Kawano et al.,
1992; Arambasic et al., 1989), the embryo in-
creases in size, and the prototroch forms. The
first larval motility is seen in this trochophore
larva. We see fused embryos only after the
multiple embryos in a polyzygotic capsule
begin to move by means of the ciliated pro-
totroch cells (Fig. 2). However, there may be
some asynchrony in the development of em-
bryos within the same egg mass, and the
growing embryos may crowd each other in a
polyzygotic egg capsule to the extent that fu-
sion or the absence of fusion is not discern-
able. Also, because of the large number of
polyzygotic egg capsules produced in our lab-
oratory, we do not follow every clutch through-
out larval development. Some conjoined twins
are first observed when the component snails
are long past the early trochophore stage.
Fusion of separate embryos may sometimes
occur in late larval stages.
DEVELOPMENT OF CONJOINED TERATOIDS 125
FIG. 2. Four egg capsules from a clutch laid by a M3 636 snail after crossing with a 10R2 snail: motile tro-
chophore larvae of which three pairs (arrows) have fused.
Development of Conjoined Embryos
The differentiation and development of ter-
atoids subsequent to larval fusion is, of
course, affected by the regions of the em-
bryos that are fused. Larval snails fuse in
many configurations. Most teratoids with more
than two components do not develop long
past the trochophore stage (Michelson &
Schork, 1958; Bigus, 1981). Therefore, the fol-
lowing generalizations will be discussed only
in relation to the development of conjoined
twins.
Fusion occurs between homologous struc-
tures. For example, two heads may be fused
dorsally (Fig. 3) or laterally, occasionally two
individuals may be joined at the edge of the
foot, or the shell fields of two embryos may
fuse and result in a two-headed individual
under one shell (Fig. 4). Development of a twin
that would have resulted from fusion of a
pretrochal area of one component embryo
with a posttrochal area of the other compo-
nent embryo (Kawano et al., 1992) has not
been seen.
Every conjoined twin that developed and
survived to the veliger stage had two heads
(Fig. 5). Mouth parts, including the radula and
anterior esophagus, were not fused in surviv-
ing conjoined twins. Occasionally tentacles
and eyes were partially fused, displaced, or
distorted, but there was individual develop-
ment of these structures in both component
snails.
For twins that had fused in a configuration
allowing normal development of two shells—
for example, fusion of dorsal areas of the
heads—each component developed a com-
plete anatomy of basically normal morphol-
оду.
The fusion of the shell fields of two embryos
often led to the formation of an aberrant shell
shared by the two components. Often these
teratoids would initially have two hearts, two
kidneys, and other separate viscera. As de-
velopment progressed with the single shell
sheltering the two-headed teratoid, usually
only one heart would persist and other inter-
nal organs would seemingly coalesce into an
internal anatomy grossly similar to that of a
normal individual. These individuals, however,
did not produce eggs, although one lived sev-
eral weeks (Fig.4).
Three sets of conjoined twins fused in con-
figurations that allowed one component to
develop fairly normally while the other compo-
nent, after some early development, degener-
ated and became a vestigial tumor-like mass
on the larger component. In these cases,
head and mouth parts of the smaller compo-
nent, although initially present, were lost. The
larger component snails continued to develop
and two produced offspring.
Conjoined snails, often with heads oriented
126 RICHARDSET AL.
FIG. 3. (a) Teratoid that survived several weeks and produced offspring (each shell ~4 mm diameter) and
(b) a stained section through the separate brains and shared cephalic sinus (arrow) of this teratoid.
FIG. 4. Conjoined pair sharing one aberrant shell
(grid = 2 mm). This teratoid initially had two hearts,
only one of which persisted.
FIG. 5. A teratoid with one component that initially
had an everted mouth and was unable to feed. The
other component fed normally, developed a com-
plete anatomy, and produced offspring. The mouth
of the non-feeding component reverted to the nor-
mal configuration concurrent with the eversion of its
preputium (shell diameter approximately 5 mm).
DEVELOPMENT OF CONJOINED TERATOIDS 127
in different planes or facing opposite direc-
tions, are sometimes unable to hatch without
assistance and do not forage and feed effi-
ciently. Nearly all teratoids are considerably
smaller than individual snails ofthe same age,
and most do not approach normal adult size.
DISCUSSION
Polyzygocy is a necessary condition for the
formation of the conjoined teratoids described
in this report. The highly determinate spiral
cleavage in B. glabrata and other mollusks
would all but preclude monozygotic twinning
(Bigus, 1981; Crabb, 1931). One previous re-
port (Eyster, 1995) of conjoined teratoids in-
volves one of the many mollusks for which
polyzygocy is normal. Mason & Copeland
(1988) report some increase in the frequency
of double embryo egg capsules in one gener-
ation of selected breeding of the normally
mono-embryonic pulmonate slug Lehmannia
valentiana. Studies of other conjoined mol-
lusks for which polyzygocy is unusual have
not demonstrated any heritability of the trait
(Bigus, 1981; Crabb, 1931; Hall, 1925). In the
past, we have seen occasional polyzygotic
egg capsules in most of our laboratory stocks
of B. glabrata, but attempts in these cases to
increase its frequency by selection always
failed. The fact that snails of one of our genet-
ically isolated stocks of B. glabrata, when
mated with snails of certain other stocks, con-
sistently produce polyzygotic egg capsules in-
dicates that there is indeed a genetic factor in
this case of polyzygocy.
Bigus (1981) reports that polyzygocy in
Physa acuta increases as reproductive sen-
escence approaches, concurrent with a re-
duction in the thickness of the capsule and in
the amount of capsular fluid. Although we
have found no age dependence in our study,
the very small clutch size of most self-fertiliz-
ing M3 636 snails suggests that polyzygocy
may serve as a reproductive strategy that
maximizes number of offspring when re-
sources for egg capsule or clutch production
are for some reason limited or declining while
production of zygotes continues or increases.
Larval motility does not limit fusion and may
in some instances be necessary to bring the
trochophore larvae into contact with each
other. The embryos of B. glabrata do not move
until the vitelline membrane is lost. Leh-
mannia embryos lack a vitelline membrane
and are reportedly motile as zygotes when
the polar bodies are visible (Mason & Cope-
land, 1988). Although these authors observed
early paired zygotes of Lehmannia in appar-
ent contact, they do not state when in embry-
onic development fusion may have occurred,
only that teratoids survived to hatch. Eyster
(1995) increased the number of conjoined ter-
atoids, first observed as veliger larvae, by
subjecting the polyzygotic egg capsules of
Crepidula to acidified seawater, possibly dis-
rupting or destroying the vitelline membrane.
We have not been able to demonstrably re-
move this membrane from B. glabrata em-
bryos and have not seen larval motility, indi-
cating loss of the membrane, before the early
trochophore stage.
By the trochophore larva stage, the mor-
phogenetic fields of B. glabrata have been es-
tablished (Camey & Verdonk, 1970; Kawano
et al., 1992), and cell membranes on the ex-
terior of the embryo are evidently primed to
form cell-to-cell junctions (Serras et al., 1990).
Further study of live and fixed, whole-mount
and sectioned, fused embryos will help deter-
mine which cells form these junctions, the na-
ture of the junctions, whether non-homolo-
gous areas of separate embryos can fuse,
and the effect on development of communica-
tion between allophenic cells.
The morphologies of conjoined teratoids in
our laboratory are analogous to morphologies
reported for teratoids of other molluscan
species, and most closely resemble those re-
ported by Bigus (1981) for Physa acuta, an-
other pulmonate snail. The survival to maturity
of bizarrely configured teratoids of Lehmannia
(Mason & Copeland, 1988) may be due to the
lack of shell to interfere with mobility or to limit
the spatial arrangement of multiple organs.
From the morphologies we have observed,
itis apparent that the development of head re-
gions is not as greatly modified or suppressed
as the development of other regions often is.
We cannot readily determine the extent to
which internal organs are mosaic in a devel-
oped teratoid, but the head and mouth parts
are clearly distinct. When separate hearts are
initially present, in Some cases, one will per-
sist as the other seems to be resorbed. Other
organ systems, including the kidney, repro-
ductive tract, and the digestive tract posterior
to the mouth, develop later than the heart and
are not as readily visible under the shell as is
the beating heart. To what degree these and
other systems are histologically a mosaic of
genetically different cells or are the result of
suppression or degeneration of one genetic
128 RICHARDSET AL.
line of cells while the other cell line develops
is not known.
ACKNOWLEDGEMENTS
We are grateful to Dr. J. A. M. van den
Biggelaar for comments on an early draft of
this manuscript. This work was supported by
grant Al-27777 from the National Institute of
Allergy and Infectious Diseases.
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Revised ms. accepted 27 August 1997
MALACOLOGIA, 1998, 39(1-2): 129-139
COMPARATIVE SPERM MORPHOLOGY AND PHYLOGENETIC CLASSIFICATION
OF RECENT MYTILOIDEA (BIVALVIA)
Alexander |. Kafanov & Anatoly L. Drozdov
Institute of Marine Biology, Far East Branch, Russian Academy of Sciences, Vladivostok
690041, Russia
ABSTRACT
In sperm morphology, the genera of Recent Mytiloidea that have been studied thus far —
Adula, Arcuatula, Aulacomya, Brachidontes, Choromytilus, Crenomytilus, Modiolus, Musculista,
Musculus, Mytilus, Perna, Semimytilus and Septifer — may be classified into several groups
based on size of the tip, mode of chromatin packing, number of mitochondria, and presence or
absence of an axial rod in the acrosomes. Spermatozoa of Modiolus might be regarded as a
basal or plesiomorphic type within the Mytiloidea. The available sperm morphology data, to-
gether with conchological characters, is adequate for suggesting a modified classification of
Recent Mytiloidea, representing a sort of trade-off between the arrangements of Soot-Ryen
(1969) and Scarlato & Starobogatov (1979, 1984). Only one family (Mytilidae) is conventionally
acknowledged among Recent Mytiloidea. The family Septiferidae Scarlato & Starobogatov, 1979,
has been ranked as a tribe in the subfamily Modiolinae. The subfamily Perninae Scarlato &
Starobogatov, 1979, non Zittel, 1895, has been abolished.
Key works: Mytilidae, sperm morphology, classification.
INTRODUCTION
It is commonly accepted that, besides the
Triassic Mysidiellidae Cox, 1964, the super-
family Mytiloidea includes the large family
Mytilidae Rafinesque, 1815, incorporating at
least 57 Recent and fossil taxa of the genus
group. Numerous Mytilidae are of commercial
importance in fisheries and aquaculture and
are of biostratigraphical use. For these rea-
sons, the systematics of the family is of long-
standing interest to researchers.
After the revision by Soot-Ryen (1969) for
the Treatise on Invertebrate Paleontology, it
became the usual practice to subdivide
Mytilidae into four or five separate subfamilies:
Mytilinae, Crenellinae Gray, 1840, Musculinae
Iredale, 1939, Lithophaginae H. Adams & А.
Adams, 1857, and Modiolinae Keen, 1958
(Kafanov, 1987). Subsequently, these subfam-
ilies were supplemented by the monotypic
Dacrydiinae (Ockelmann, 1983: 112).
A very different scheme was suggested by
Scarlato & Starobogatov (1979, 1984; Staro-
bogatov, 1992), who subdivided the family My-
tilidae into four separate families — Mytilidae,
Septiferidae Scarlato & Starobogatov, 1979,
Crenellidae, and Lithophagidae — and they
established an additional 13 subfamilies. Un-
fortunately, their diagnoses of the taxa are ex-
129
tremely brief and they did not give arguments
to support their classification. At the same
time, a need for new morphological criteria for
the classification of the Mytiloidea and the tax-
onomic importance of sperm morphology in
other groups made us pay attention to specific
features of spermatozoon morphology.
TAXONOMIC IMPORTANCE OF
SPERMATOZOON MORPHOLOGY
In Recent years, gamete ultrastructure has
been used extensively for solving various sys-
tematic and phylogenetic problems in the
Metazoa (Afzelius, 1979; Jamieson & Rouse,
1989; Ferraguti & Gelder, 1991; Justine, 1991;
Jamieson et al., 1995). Species specificity for
spermatozoa ultrastructure was initially estab-
lished for Mammalia (Bishop & Austin, 1957)
and subsequently confirmed for other animal
groups (Baccetti & Afzelius, 1976), including
bivalve molluscs (Drozdov & Reunov, 1986b).
Structural features of spermatozoa have been
successfully used for identification of sibling
species (Meier et al., 1972; Aksenova, 1978).
Species specificity of spermatozoon morphol-
ogy, acrosome structure in particular, is
thought (Popham, 1979) to contribute to re-
productive isolation of closely related species
130
on a cytological level. In the opinion of a num-
ber of researchers (Ockelman, 1964, 1965;
Popham, 1979; Franzen, 1970, 1983; Drozdov
& Kasyanov, 1985; Pashchenko & Drozdov,
1991), the spermatozoon structure of bivalve
molluscs is dependent on the structure of egg
membrane and specific insemination features.
A significant amount of data is presently
available concerning gamete-specific mor-
phological features for families of various ani-
mal groups: scleractinias (Steiner, 1991),
archiannelids (Franzen, 1982; Franzen &
Sensenbaugh, 1984), and chitons (Hodgson
et al., 1988; Pashchenko & Drozdov, 1994,
1997). Such features also are known in the
Bivalvia (Karpevich, 1961, 1964; Gharago-
zlou-Van Ginneken & Pochon-Masson, 1971;
Thompson, 1973; Popham, 1974, 1979; Max-
well, 1983; Drozdov & Kasyanov, 1985;
Eckelbarger et al., 1990; Hodgson et al.,
1987, 1990; Healy, 1995, 1996). In particular,
acrosomal morphology displays characteristic
features that provide information on phyloge-
netic relationships (Baccetti, 1970).
Spermatozoon morphology of Mytiloidea
KAFANOV AND DROZDOV
has been dealt with in several papers (Table
1), and sufficient data now exists for a review
of previous classifications.
GENERIC FEATURES OF
SPERMATOZOON MORPHOLOGY
IN MYTILOIDEA
All mytiloidean sperm show essentially the
same organization of the midpiece, that is,
spherical mitochondria are grouped in a ring
around the proximal and distal centrioles
(centrioles arranged at approximately 90° to
each other), a small rootlet connects the prox-
imal centriole to the nucleus, and a satellite
fibre complex of nine terminally forked fibres
anchors the distal centriole to the plasma
membrane. Some generic differences in sper-
matozoon morphology are given below and in
Table 2.
Modiolus Lamarck, 1799 (Fig. 1a). Large-
sized spermatozoa with flask-shaped head
and pointed acrosome. Barrel-shaped nu-
cleus 2.0 um in length and 2.7 um in diameter.
TABLE 1. References to the morphology of spermatozoa in Recent Mytiloidea
Species
Adula falcatoides Habe, 1955
Arcuatula capensis (Krauss, 1848)
Aulacomya ater (Molina, 1782)
Brachidontes semistriatus (Krauss, 1848)
Choromytilus chorus (Molina, 1782)
Choromytilus meridionalis (Krauss, 1848)
Crenomytilus grayanus (Dunker, 1853)
Modiolus americanus (Leach, 1815)
M. kurilensis Bernard, 1983
M. modiolus (Linnaeus, 1758)
Musculista senhousia (Benson, in Cantor, 1842)
Musculus discors (Linnaeus, 1767), including
M. laevigatus (Gray, 1824)
Mytilus chilensis Hupe, 1854
M. coruscus Gould, 1861
Mytilus of the group of M. edulis [M. edulis Linnaeus,
1758 + M. trossulus Gould, 1850]
M. galloprovincialis Lamarck, 1819
Perna perna (Linnaeus, 1758)
P viridis (Linnaeus, 1758)
Semimytilus algosus (Gould, 1850)
Septifer keenae Nomura, 1936
Authors
Reunov & Drozdov, 1986
Reunov & Hodgson, 1994
Hodgson & Bernard, 1986a; Garrido & Gallardo,
1996
Reunov & Hodgson, 1994
Garrido & Gallardo, 1996
Hodgson & Bernard, 1986a
Drozdov, 1979, 1983; Drozdov & Mashansky, 1979;
Drozdov et al., 1981; Drozdov & Kasyanov, 1985
Hylander & Summers, 1977
Drozdov & Kasyanov, 1985; Drozdov & Reunov,
1986a
Franzen, 1955
Drozdov, 1992
Kaufman, 1977; Franzen, 1983; Drozdov &
Kasyanov, 1985
Garrido & Gallardo, 1996
Reunov & Drozdov, 1987
Niijima & Dan, 1965; Longo & Dornfeld, 1967;
Endo, 1976; Drozdov & Reunov, 1986b; Hodgson
& Bernard, 1986b
Hodgson & Bernard, 1986b; Crespo et al., 1990;
Drozdov, 1992
Boucart et al., 1965; Bernard & Hodgson, 1985;
Hodgson & Bernard, 1986a
Drozdov, 1992
Garrido & Gallardo, 1996
Reunov & Drozdov, 1986
131
MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA
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132 KAFANOV AND DROZDOV
FIG. 1. Structural pattern of spermatozoa of the subfamily Modiolinae: a—Modiolus kurilensis, b—Aulacomya
ater, c—Adula falcatoides, d—Brachidontes semistriatus, e—Choromytilus meridionalis, f—Musculista sen-
housia, g—Arcuatula capensis, h—Septifer keenae. A: acrosome; N: nucleus; AR: axial rod; m: mitochondria;
de: distal centriole; pc: proximal centriole; AV: acrosomal vesicle; PM: periacrosomal material. Bar = 1 um.
No axial rod typically present. Acrosomal
complex of two parts: cone-shaped acrosomal
vesicle and periacrosomal granular material
arranged between the vesicle and nucleus.
On side of nucleus, acrosomal vesicle with a
contraction filled by periacrosomal material.
Midpiece of spermatozoon formed by two mu-
tually perpendicular centrioles, surrounded by
13-14 mitochondria.
Aulacomya Morch, 1853 (Fig. 2b). Sperms
with flask-shaped head, containing a barrel-
shaped nucleus of about 2.2 um length anda
large (3.7 um long) cone-shaped acrosomal
vesicle with a large cavity filled by granular pe-
riacrosomal material. The midpiece of sper-
matozoon with five, sometimes six, spherical
mitochondria.
Adula H. & A. Adams, 1857 (Fig. 1c).
Acrosomal complex (1.4 um long) with a con-
ical electron-dense acrosomal vesicle with a
contraction in a distal acrosomal part, filled
by electron-lucent periacrosomal material.
Middle part of spermatozoon formed by five
mitochondria encircling two centrioles.
Brachidontes Swainson, 1840 (Fig. 1d),
and Arcuatula Lamy, 1919, ex Jousseaume,
MS (Fig. 1g). Sperm head of B. semistriatus
4.5 um long, containing a barrel-shaped nu-
cleus (2.0 x 1.9 um) with minute anterior hole.
Nucleus adjoining flask-shaped acrosome
of 2.5 um length, with a long anterior projec-
tion.
Sperm of A. capensis with a relatively long
head, a barrel-shaped nucleus (length, 1.2
um, average diameter 1.5 um) and a small
conical acrosome, 0.8 um long).
Choromytilus Soot-Ryen, 1952 (Fig. 1e).
Sperms of C. meridionalis with a compara-
tively rounded nucleus of 1.6 um diameter
and a 2 um-long acrosome crowning the
head. Acrosome with a large-sized conic cav-
ity filled by granular electronically compact
material. The middle part of spermatozoon in-
cludes four (sometimes five) spheric mito-
chondriae of 6 um diameter.
Musculista Yamamoto & Habe, 1958 (Fig.
1f). Unlike Modiolus, spermatozoa compara-
tively small-sized (bullet-shaped head of
sperm about 2.5 um, middle piece of sperma-
tozoon 0.4 um, top head width 1.2 um). Flask-
shaped acrosome with two components: acro-
somal vesicle widened distally and narrowed
towards the top, possessing a contraction in
the distal part filled by globular periacrosomal
material. Proximal and distal centrioles per-
pendicular.
MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA 133
Septifer (Mytilisepta) Habe, 1951 (Fig. 1h).
Sperms with a bullet-shaped head of 1.2
length and 1.4 um width. Conical acrosome
0.7 um-long with cupola-shaped acrosomal
vesicle filled with electron-lucent periacroso-
mal material. Two centrioles surrounded by
five mitochondriae of 0.4 um diameter.
Mytilus Linnaeus, 1758 (Figs. 2a, b, d, e),
Crenomytilus Soot-Ryen, 1955 (Fig. 2c), and
Perna Retzius, 1788 (Figs. 2f, g). Sperma-
tozoa of several species of these genera with
flask-shaped head between 3.6 um (Perna
viridis) and 7.0 um (Mytilus coruscus) in
length. Barrel-shaped nucleus with pointed
acrosome anteriorly. Posteriorly, nucleus ad-
jacent to middle part of spermatozoon con-
taining five spheric mitochondria and two cen-
trioles.
Musculus Rodding, 1798 (Fig. За, b).
Sperms with extended cone-shaped head of
about 8.0 um length and 0.8 um base diame-
ter. Acrosomal complex of two components:
apical part with an acrosomal vesicle of ap-
proximately 0.8 um diameter, surrounded by a
membrane. Periacrosomal material branching
off from vesicle and consisting of a bundle of
actine threads entering nucleus channel and
a small amount of globular material. In some
specimens, the axial rod reaching midpart of
spermatozoon (M. discors from North Pacific),
whereas in other specimens only reaching
middle of nucleus (Same species from North
Atlantic and Arctic Ocean). Midportion of
sperm with four spheric mitochondriae of 0.4
um diameter encircling two perpendicular
centrioles.
TAXONOMIC ANALYSIS
Although a thorough taxonomic analysis
would require the availability of sperm mor-
phology data for a larger number of genera,
the available data (Table 1) is adequate for
some conclusions on alternatives for the sys-
tematics of the Mytiloidea (Table 3). The more
so because the taxonomic value of a charac-
ter depends on its adaptive significance
FIG. 2. Structural pattern of spermatozoa of the subfamily Mytilinae: a—Mytilus coruscus, b—Mytilus trossu-
lus, c—Crenomytilus grayanus, d—Mytilus galloprovincialis, e—Mytilus edulis, f—Perna perna, g—Perna
viridis. Bar = 1 um.
134 KAFANOV AND DROZDOV
FIG. 3. Structural pattern of spermatozoa of the
subfamily Musculinae: aa b—Musculus discors.
Note that the length of the axial rod varies among
specimens of one species. Bar = 1 um.
(Mayr, 1969), there is no reason to treat sper-
matozoon morphology as less taxonomically
important than the conventional conchologi-
cal characters.
In terms of the presence or absence of an
axial rod in acrosomes of intact spermatozoa,
all the analysed representatives of Mytiloidea
may be clearly classed into two major groups:
those possessing no axial rod (Modiolus, Mu-
sculista, Septifer, Adula, Choromytilus, Aula-
comya, Brachidontes, Semimytilus, and Ar-
cuatula) and those possessing an axial rod
(Mytilus, Crenomytilus, Perna, and Muscu-
lus). The axial rod of the acrosome consists of
a bundle of actine filaments which, in the
course of acrosomal reaction, form the basis
for the formation of acrosomal filament (Droz-
dov & Mashansky, 1979; Drozdov et al., 1981;
Drozdov & Podgornaja, 1982; Drozdov, 1992).
Because the acrosomal reaction has a role in
fertilization and the onset of ontogenesis, it
should be assumed that the presence or ab-
sence of acrosomal the axial rod is of great
taxonomic importance. The absence of an
acrosomal axial rod provides evidence con-
cerning the more primitive spermatozoon
structure in animals with external fertilization
(Popham, 1979).
Spermatozoons of Modiolus are distin-
guished by an unusually large head with a
large amount (3.99 pkg) of loosely packed
(0.42 pkg/um?) DNA (Tuturova, 1989), large,
variable number (10-14) of mitochondria in
the middle part of the spermatozoon and, as
previously mentioned, the absence of an axial
rod in the acrosome. These characters pre-
sent strong evidence concerning the apomor-
phic structure of the spermatozoa of Modio-
lus, which is confirmed by its long (Devon-
ian through Recent) geological history com-
pared to the remaining Mytilidae (Soot-Ryen,
1969).
In terms of acrosomic rod presence/ab-
sence, Recent Mytiloidea form two major
groups around Modiolus and Mytilus. The
rank of these taxa is open to debate, because
there are no objective criteria for establish-
ment of taxa above the species rank (Mayr,
1969). Analoguous information about other
superfamilies provides a perspective on how
many families should comprise the superfam-
ily Mytiloidea. In this connection, Cardioidea is
notable. For instance, the spermatozoa mor-
phology of Keenocardium californiense (De-
shayes, 1839) (Clinocardiinae) and various
representatives of Lymnocardiinae are very
different from each other (Fig. 4). Never-
theless, in the most modern classification
(Schneider, 1992, 1995), the Cardioidea in-
cludes only one family Cardiidae. Among
Recent Mytiloidea, the degree of morphologi-
cal variability of spermatozoa is much less.
Because all the spermatozoon types of
Mytiloidea may be derived from that of
Modiolus, as well as the fact that even the clas-
sification of Scarlato & Starobogatov (1979,
1984) imparts only subfamily status to Modio-
MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA 135
TABLE 3. Comparison of the classifications of Recent Mytiloidea by Soot-Ryen (1969), Scarlato &
Starobogatov (1979, 1984) and proposed system
Soot-Ryen, 1969
fam. Mytilidae
Rafinesque, 1815
subfam. Mytilinae
Rafinesque, 1815
subfam. Modiolinae
Keen, 1958
subfam. Crenellinae
Gray, 1840
subfam. Lithophaginae
H. Adams & A. Adams, 1857
Scarlato & Starobogatov,
1979, 1984
fam. Mytilidae
Rafinesque, 1815
subfam. Mytilinae
Rafinesque, 1815
subfam. Arcuatulinae
Scarlato & Starobogatov, 1979'
subfam. Musculinae
Iredale, 1939
subfam. Modiolinae
Keen, 1958
subfam. Brachidontinae
Scarlato & Starobogatov, 1979?
subfam. Perninae
Scarlato & Starobogatov, 1979°
subfam. Trichomyinae
Scarlato & Starobogatov, 1979
fam. Septiferidae
Scarlato & Starobogatov, 1979
subfam. Septiferinae
Scarlato & Starobogatov, 1979
subfam. Limnoperninae
Scarlato & Starobogatov, 1979
fam. Crenellidae
Gray, 1840
subfam. Crenellinae
Gray, 1840
subfam. Botulinae
Scarlato & Starobogatov, 1979*
fam. Lithophagidae
H. Adams 4 A. Adams, 1857
subfam. Lithophaginae
H. Adams & A. Adams, 1857
subfam. Adulinae
Scarlato & Starobogatov, 1979
Proposed herein
fam. Mytilidae
Rafinesque, 1815
subfam. Mytilinae
Rafinesque, 1815
subfam. Musculinae -
Iredale, 1939
subfam. Modiolinae
Keen, 1958
tribe Modiolini
Keen, 1958
tribe Brachidontini
Scarlato & Starobogatov,
1979
tribe Septiferini
Scarlato & Starobogatov,
1979
subfam. Crenellinae
Gray, 1840
subfam. Lithophaginae
H. Adams & A. Adams, 1857
Notes. 'Туре-депиз not given т the original publication. Established (according to Sysoev & Kantor, 1992), by name forma-
tion, on Arcuatula Lamy, 1919, ex Jousseaume MS, non Gugenberger, 1934, nec Soot-Ryen, 1955. “Nom. correct. (Kafanov,
1987) pro Brachiodontinae Scarlato & Starobogatov, 1979. “Invalid because preoccupied repeatedly as family-group name
based on Perna Bruguiere, 1789, non Retzius, 1788 [Pernaridia Rafinesque, 1815; Pernadae Fleming, 1828, also Guilding,
1828; Pernidae Zittel, 1895]. “Dacrydiinae Ockelmann, 1983, may be a junior synonym.
linae, there is no sound basis for identifying
any other separate families among Recent
Mytiloidea.
The genera grouped around Modiolus differ
in levels of synplesio- and synapomorphy (in
the sense of Hennig, 1950, 1966). Morpho-
logical evolutionary transformations of sper-
matozoons of the Modiolus-type were then
accompanied by compactification of DNA,
diminution of spermatozoon head size, and by
the development of an acrosomal axial rod.
The most advanced spermatozoons appear
to be those of Septifer, possessing a small-
sized bullet-shaped head with a negligible
(1.29 pkg) but very tightly packed (0.72
pkg/u°) amount of DNA (Tuturova, 1989) and
a minute acrosome. Spermatozoons of other
genera characterized by the absence of an
axial rod in the acrosome (Musculista, Adula,
Choromytilus, Semimytilus, Arcuatula), being
variable in details, occupy an intermediary po-
sition between Modiolus and Septifer. In this
136 KAFANOV AND DROZDOV
a b ©
d
e Е g
FIG. 4. Structural pattern (light microscope) of spermatozoa in cardiid subfamilies Lymnocardiinae Stoliczka,
1870 (a-f), and Clinocardiinae Kafanov, 1975 (g). Species: a—Hypanis (Monodacna) colorata (Eichwald,
1829), b—Hypanis (Monodacna) sp. 1, c—Hypanis (Monodacna) sp. 2, d—Hypanis (Adacna) laeviuscula
(Eichwald, 1829), e—Hypanis (Adacna) vitrea (Eichwald, 1829), —Hypanis (Adacna) minima (Ostroumoff,
1907) (Figs. a-f from: Karpevich, 1964), g—Keenocardium californiense (Deshayes, 1839) (from Drozdov,
1992). Ваг = 8 um.
case, the spermatozoons of Modiolus and
Musculista are most similar, closely corre-
sponding to their allocation to the same sub-
family Modiolinae by Scarlato & Starobogatov
(1979, 1984) and Scarlato (1981).
Among spermatozoa of Mytilus-type, pos-
sessing an axial rod, the most primitive are
those of Musculus, which possess a long (ap-
proximately 8 um), narrow head crowned with
a minute acrosomal vesicle, from which an
axial rod almost reaches the middle part of
spermatozoon and which also possess five mi-
tochondriae. These features may indicate
some specialization, because of the large
(about 600 um) eggs of Musculus compared to
those of Mytilus (about 70 um) and to features
of their insemination (Drozdov & Kasyanov,
1985).
In spermatozoon structure, as well as con-
chological features (Siddall, 1980), the sub-
family Perninae Scarlato & Starobogatov,
1979, appears to be paraphyletic. Although a
close taxonomic relation among Perna,
Mytilus, Choromytilus, and Aulacomya, was
proposed by Soot-Ryen (1952), spermatozoa
of Perna show no morphological differences
from those of Mytilus and Crenomytilus,
whereas spermatozoa of Chromytilus and
Aulacomya prove to be similar to the sperma-
tozoa of Modiolus. This makes it impossible to
consider Choromytilus Soot-Ryen, 1952, to be
a subgenus of Perna Retzius, 1788, as was
suggested by Soot-Ryen (1969). In any case,
however, Perninae Scarlato & Starobogatov,
1979, is invalid for nomenclatural reasons
(Table 2).
The available spermatozoan data, together
with conchological characters, is adequate for
suggesting an slightly modified systematics of
Recent Mytiloidea, representing a trade-off
between the classifications of Soot-Ryen
(1969) and Scarlato & Starobogatov (1979,
1984) (Table 3). The subfamily status of Litho-
phaginae in this system is the result of its bor-
ing habitat, generally uncommon for mytilids,
resulting in major transformations in shell
morphology. In any case, the Lithophaginae is
more closely related to Modiolinae than to the
Mytilinae.
ACKNOWLEDGEMENTS
The manuscript of the present article was
critically reviewed by Dr. Eugene V. Coan
MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA 137
(California Academy of Sciences, San Fran-
cisco, USA), by three anonimous reviewers,
and by Dr. Yaroslav I. Starobogatov (Zoo-
logical Institute, Russian Academy of Sci-
ences, St. Petersburg, Russia). These per-
sons made a number of important comments.
Dr. Klara F. Tuturova (Institute of Marine
Biology, Far East Branch, Russian Academy
of Sciences, Vladivostok, Russia) kindly per-
mitted to make use of the material from her
unpublished Ph.D. dissertation. Mrs. Elena S.
Kornienko (Institute of Marine Biology, Vladi-
vostok, Russia) made the drawings for the
paper and rendered constant technical assis-
tance. Initial translation of the Russian text
into English was made by Mr. Sergei V.
Solovyev (Research Institute for Nature
Conservation of the Arctic and North, St.
Petersburg, Russia). Assistance of all the
above persons is deeply appreciated.
The research described in this publication
was made possible in part by grants #95-04-
11134 and #96-04-49702 from the Russian
Foundation for Basic Research and by Grant
of INTAS #93-2176.
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MALACOLOGIA, 1998, 39(1-2): 141-150
INFLUENCE OF WATER TEMPERATURE ON THE ACTIVITY OF PLANORBARIUS
CORNEUS (L.) (PULMONATA, PLANORBIDAE)
Katherine Costil' & Stuart Е. В. Bailey?
ABSTRACT
This study complements our previous study of the influence of temperature on the life-history
traits of the freshwater snail Planorbarius corneus by examining the effect of temperature on ac-
tivity. The activity of 24 groups of three individuals was recorded over 24 h та 12 h light:12 h
dark photoperiod with six groups at each of four temperatures — 10, 15, 20 and 25°C.
Despite considerable variations between individuals, activity increased significantly with tem-
perature, principally due to an increase in time spent feeding. Time spent on non-displacement
movements declined above 10°C, but time spent on locomotor activity did not alter with temper-
ature. However, the speed and total distance moved increased significantly with temperature, as
did the number of contacts made with the water surface and with other individuals. Locomotion,
feeding and non-displacement movements showed no diurnal rhythms at any temperature. The
present study shows that a behavioural component contributes to the influence of temperature
on life-history traits.
Key words: freshwater snail, behavioural ecology, temperature, light, locomotor and feeding
activities.
INTRODUCTION
Planorbarius corneus is a large basom-
matophoran snail, common in eutrophic
ponds of northwest France (Costil & Clement,
1996). It is often collected in association with
Lymnaea stagnalis, and both species con-
tribute to communities showing high species
richness and diversity (Costil, 1994b). Pla-
norbarius corneus plays an important part in
the invertebrate communities of eutrophic
ponds in this region, where it is eaten by both
fish and birds.
Natural populations of freshwater snails
have to adapt to various abiotic constraints,
for example, climate, desiccation and water
chemistry. Temperature is particularly impor-
tant for freshwater pulmonates, because they
inhabit shallower waters than most proso-
branch molluscs, and thus experience a sea-
sonal range of temperatures almost as great
as that on land (MacMahon, 1983). We have
previously studied the life-history traits of
P corneus and demonstrated the importance
of water temperature in the control of these
snail populations (Costil, 1994a; Costil &
Daguzan, 1995a, b). To complete this study,
we have now examined the impact of temper-
ature on the activity of P corneus.
Activity level is an important component of
adaptive strategy. For example, locomotion in
most animal species is an essential compo-
nent of fitness, being necessary for both re-
production (finding mates and oviposition
sites) and survival (finding food and avoiding
predators) (Calow, 1974). Most studies of mol-
luscan activity have concerned land pul-
monates, and concentrated on their diurnal
activity rhythms (e.g., Rollo, 1982; Dainton &
Wright, 1985; Ford & Cook, 1987; and Wareing
& Bailey, 1985, on slugs; and Cameron,
1970a, b; Bailey & Lazaridou-Dimitriadou,
1986; and Lorvelec, 1988, on helicid snails).
The fewer studies on the activity of freshwater
snails have focused on locomotion of tropical
species (Beeston & Morgan, 1977; Chaudry &
Morgan, 1983; Pimentel-Souza et al., 1984),
the North American planorbid Helisoma trivol-
vis (Kavaliers, 1981) and the European spe-
cies Lymnaea stagnalis (MacDonald, 1973),
Ancylus fluviatilis and Planorbis contortus
(Calow, 1974). In P corneus, locomotion in-
volves mucus, muscles and cilia covering the
sole of the foot. Deliagina & Orlovsky (1990)
established the locomotory repertoire of this
species and the nervous control of locomotion.
Little attention has been paid to the time
budget of different activities, and the effect of
‘Laboratoire de Zoologie et d’Ecophysiologie, Campus de Beaulieu, Universite de Rennes |, 35042 Rennes Cedex, France.
2School of Biological Sciences, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom
142 COSTIL AND BAILEY
temperature on this budget, or on the conse-
quences of increased speed of movement in-
duced by elevated temperatures on distances
travelled and encounters made, for example,
with other individuals and the water surface.
This study addresses those aspects.
METHODS
The snails were collected from a pond near
Rennes, northwest France, where the life
cycle of the population had been studied
(Costil & Daguzan, 1995b). The snails were
immature when collected and belonged to the
same cohort. Brought into the laboratory at
18°C, they grew to maturity and after nine
months were grouped into four experimental
groups of 18 snails, each group having a sim-
ilar mean diameter (close to 18.8 mm).
The water temperature of each group was
gradually adjusted to one of the four experi-
mental temperatures of 10, 15, 20 or 25°C.
The experimental temperatures are within the
range generally recorded in the region. After
an acclimatization period of at least three
weeks at the target temperature, the activity of
the snails was studied by filming them with a
video-camera for 24 h. The advantages and
disadvantages of this technique were empha-
sised by Bailey (1994). The recording appara-
tus consisted of a black-and-white camera
sensitive to dim red light (6300 to 7500 A), a
monitor, and a time-lapse video-recorder.
Recordings were made at one frame per sec,
so that 24 h of recording could be played back
in one hour at normal speed to analyse activ-
ity.
A total of 72 snails were tested, 18 at each
temperature. The snails were filmed in six
groups of three. Individuals were identified by
painting spots on one snail, stripes on a sec-
ond, and leaving the third snail unpainted. The
snails were fed with rectangular pieces of let-
tuce (5 x 7 cm) which were weighted down to
the bottom of the tank. Each tank (23 x 17 x 9
cm) was filled with 1800 ml of pond water. To
aid the recording of distances moved by the
snails, a grid of lines was drawn on the bottom
and sides of the tank. There was no artificial
aeration of the water in the tanks. Recordings
were made in a photoperiod of 12 h light (from
08h00 to 20h00) and 12 h dark. As in
Lorvelec’s study (1988), a red light was used
for filming during the dark phase, since pul-
monates respond only weakly to long wave-
lengths (Kerkut & Walker, 1975). When the
lights went on or off, the snails either did not
react, or immediately stopped crawling for a
short period, usually a few seconds.
Activity was analysed for each individual
snail, recording its behaviour in each of the
288 5-min units over the 24 h. Snail activity
was classified into five categories:
—inactive (I);
—locomotion (L); a displacement of 2 cm or
more in the 5 min;
—moving without displacement (M), often
consisting of rocking movements of the shell;
—feeding (F), with obvious scraping move-
ments with the head on the lettuce;
—copulating (C).
If a snail showed two activity categories
(e.g., locomotion and feeding) within a five-
min time unit, both categories were scored as
a half unit. More than two categories never oc-
curred within one time unit.
The following additional variables were also
calculated for each individual:
—longest period of inactivity in 24 h (LPI), in
min;
—total distance covered in 24 h (TD), in m;
—mean speed (MNS), the total distance
moved divided by five times the number of
five-min units spent locomoting, in cm min”?;
—maximum speed (MXS), recorded over any
5-min period;
—number of contacts with the water surface
in 24 h (CWS), including contacts with head or
foot of a snail near the walls of the tank as well
as contacts when the snail crawls with foot ex-
tended on the surface;
—number of contacts with fellow snails in 24
h (CFS).
The data on inactivity (| and LPI) were sub-
jected to non-parametric tests (Mann Whitney
test and Kruskal Wallis test), because the
conditions of normality and equivariance re-
quired for an analysis of variance were not
met, even after various transformations.
The data on activity were subjected to mul-
tivariate analyses of variance (MANOVA), pre-
ceeded by univariate analyses and Newman
Keuls tests (Morrison, 1967; Dagnelie, 1977).
The effects of temperature and light on the dif-
ferent types of activity were tested using a two-
way MANOVA with two factors (temperature
and light), 18 replicates, and three variables
(times spent locomoting, feeding, and in non-
displacement movements). The effect of tem-
perature on activity was tested with a one-way
ACTIVITY OF PLANORBARIUS CORNEUS 143
MANOVA applied to eight variables (L, F, M,
MNS, MXS, TD, CWS and CFS); M and TD
were transformed to Log (x + 1) to meet the re-
quirements of the analysis of variance. After
MANOVA, we used multiple comparisons
which were in agreement, and thus the results
of WILKS test and ROY test alone are pre-
sented here. Calculations were performed with
STATVIEW (1988) and STAT-ITCF (1988) pro-
grams.
RESULTS
Egg masses were laid at 20 and 25°C, but
the egg masses of Planorbarius corneus are
almost colourless, and not sufficiently visible
to enable us to reliably distinguish ovipositing
during recording. Mating activity was ob-
served only at 20°C. Two matings occurred:
one began at 18h30 and lasted for 5.33 h, the
other began at 5h40 and lasted 6.50 h. To
compare the results at 20°C with the others,
we have excluded those 5.6% of time units
which were occupied by copulatory activity,
and brought the percentage occupied by
other activities back to 100.
As the high standard deviations given in
Table 1 indicate, there are considerable differ-
ences between individuals, and at all temper-
atures, some snails were continuously active.
For example, at 10°C, three snails were con-
tinuously active while four other snails were
inactive for three quarters of the time. At 15°C,
the percentage of time that individuals spent
feeding varied from 1 to 75%. At 20°C, the dis-
tance covered by individual snails varied from
less than three to more than 32 m. At 25°C,
some snails made ten times more contacts
with the surface than other snails.
Temperature significantly affects both the
total time spent inactive (H = 28.01, p =
0.0001, Kruskal Wallis test), and the longest
period of inactivity (H = 26.89, p = 0.0001,
Kruskal Wallis test). The time spent inactive
decreases at higher temperatures, and this
decrease was most noticeable between 15
and 20°C (Fig. 1). The mean length of the
longest period of inactivity (ЕР!) was nega-
tively related to temperature (Fig. 2); LPI at
25°C was 35 min, but at 10°C it was > 21 h.
The proportion of time spent feeding in-
creases with temperature, from 17.1% at
10°C to 56.2% at 25°C. The increase in time
devoted to feeding accounts for most of the
decline in inactivity seen with increasing tem-
perature. Non-displacement movements oc-
cupy 10.2% of the time at 10°C, but only 3.3%
to 5.4% of the time at higher temperatures.
Time spent in locomotion was independent of
temperature. Figure 3 shows no consistent or
10°C 15°C
INACTIVITY
MOVEMENT
FEEDING
LOCOMOTION 25°C
MATING 2
Corrected
20°C
20824
FIG. 1. Mean percentages of inactivity and different
types of activity in Planorbarius corneus individuals
at four temperatures (10, 15, 20 and 25°C) over 24
h corresponding to 288 activity units of 5 min. The
results obtained at 20°C were corrected by exclud-
ing the activity units corresponding to mating.
TABLE 1. Mean numbers (+ S.D.) of activity units spent by the individuals of Planorbarius corneus in inactiv-
ity and different types of activity (locomotion, feeding, movement without displacement and mating) at 10, 15,
20 and 25°C (n = 18). The results obtained at 20°C were corrected by excluding the activity units corre-
sponding to mating, thus bringing back the total number of activity units to 288.
TEMPERATURE INACTIVITY LOCOMOTION FEEDING MOVEMENT MATING
10°C 97.28 + 103.89 112.00 + 71.90 49.28 + 45.96 29.4 + 22.11 0
15°C 79.06 + 65.63 87.61 = 57.32 107.72 = 64.58 13.61 + 9.78 0
20°С 9.39 + 10.36 114.08 = 43.08 138.81 + 34.11 9611727 16.11 + 31.29
Corrected 20°C 10.00 + 12.02 118.92 + 41.29 148.86 = 43.27 10.22 + 12.02 0
2556 5.28 + 4.87 105.08 + 48.68 161.97+47.30 15.67 + 10.13 0
144 COSTIL AND BAILEY
LPI (min)
1400
© Mean
М Maximum
Temperature
FIG. 2. Mean (+ S.D.) and maximum values of the
longest period of inactivity (LPI) in Planobarius
corneus put at 10, 15, 20 and 25°C.
(%) Inactivity
cor 20°C 25°C
Temperature
cor 20°C 25°C
Temperature
10°C 152€
(%) Feeding
cor 20°C 25°C
Temperature
10°C 15°C
(%) Movement
10°C 15°C
cor 20°C 25°C
Temperature
Е] Light period Ш Dark period
FIG. 3. Mean percentages of inactivity and different
types of activity occurring during a 12h light and 12h
dark period in Planorbarius corneus at 10, 15, 20
and 25°C.
sizeable differences between the light and
dark phases in the proportion of time spent in
any activity category, and applying a two-way
MANOVA using both Wilks test and Roy test,
no significant differences were found for either
light or the light x temperature interaction
(Table 2). Temperature emerges as the only
factor influencing activity. As Table 3 shows,
the time spent feeding changes significantly
throughout the temperature range, while non-
displacement movements only alter signifi-
cantly over the lower part of the range, and
there are no significant differences in the time
spent in locomotor activity over the range of
temperatures employed.
In general, the higher the temperature, the
faster the snails moved (Fig. 4). At 25°C, the
mean speed was on average 3.5 cm min !,
with a maximum speed of 7.5 cm min !. The
mean distances covered at 25°C were twice
and four times those recorded at 15 and 10°C
respectively, and one snail at 25°C covered 42
m in 24 h. The number of contacts between
snails increased with temperature to reach a
mean of 45.3 encounters in 24 h at 25°C,
while at 10°C four snails never encountered a
fellow snail. When two P corneus snails met,
one snail generally pushed the other and con-
tinued on its way. At the three highest temper-
atures, phoretic behaviour (carrying other in-
dividuals) was often observed. Individuals at
25°C has significantly more contacts with fel-
lows than individuals at 10°C, probably be-
cause, although they do not move for any
longer period of time, they move faster, and
cover more distance.
The number of contacts with the water sur-
face in general increased with temperature;
the mean values are lowest at 15°C and high-
est at 25°C. However, the differences were not
significant Although these contacts with sur-
face included breathing visits, it is very difficult
to be see breathing from a videorecording,
and only in a few instances were lung ventila-
tions with the pneumostome obviously open
observed. The results of a one-way MANOVA
applied to eight activity variables (L, F, M, TD,
MNS, MXS, CWS and CFS) show a signifi-
cant effect of temperature (Wilks Test, F =
9.327, p < 0.0001; ROY Test, eigen value =
5.179, critical value = 0.414, p < 0.001).
Among these variables, temperature affects
time spent feeding, total distance, mean and
maximum speeds, and number of encounters
with fellow snails (Table 4).
DISCUSSION
The large differences in the level of activity
of individual snails under identical conditions
that we observed were also noted by
ACTIVITY OF PLANORBARIUS CORNEUS 145
TABLE 2. Results of a two-way multivariate analysis of variance using two different tests (Wilks
test and Roy test) and applied to three variables (locomotion, feeding, movement without dis-
placement) of Planorbarius corneus activity.
WILKS TEST
FACTORS в р
TEMPERATURE 10.807 0.0000
LIGHT 0.330 0.8062
INTERACTION
Temp. x Light 0.451 0.9062
ROY TEST
Eigen Value Critical Value p
0.723 0.102 0.0000
0.007 0.060 0.8065
0.021 0.102 0.9202
TABLE 3. For each activity, temperatures for which some significant differences were calculated
with Roy multiple comparison method used after the two-way multivariate analysis of variance.
LOCOMOTION
Temperatures (°C) NO
for which some
differences were
calculated
MacDonald (1973), who found that the se-
quences and rates of activity patterns of indi-
vidual L. stagnalis were so different as to pre-
clude summing results. Such heterogeneity
necessitates the use of sufficient numbers of
individuals, and cautious interpretation of re-
sults. Video recordings often fail to show
whether snails and slugs feed all the time that
they are in contact with food (Bailey, 1994),
but the use of visible scraping movements
proves to be an efficient definition of feeding,
and allows a firm relationship to be estab-
lished between increasing temperature and
increase in time spent feeding.
Despite the inter-individual differences, this
study has demonstrated that Р corneus
spends more time active, or, conversely, less
time inactive, at higher temperatures. This
may seem an unremarkable conclusion, but it
does, in fact, contrast with Cameron’s (1970b)
observations on helicid snails, and Dainton &
Wright's (1985) data on a terrestrial slug. Ford
& Cook (1987) also showed that Limax pseu-
doflavus is initially stimulated when the tem-
perature is lowered from 17°C to 4°C.
Furthermore, the present study shows that
the component activities (locomotion, non-
displacement movement, feeding, mating)
show different and non-linear relationships to
temperature. The time spent in locomotion is
not affected by temperature. The principal
component of activity which increases with
temperature is the time spent feeding. This ac-
tivity levels off between 20 and 25°C, and it is
possible that at higher temperatures, feeding
FEEDING MOVEMENT
10-15 10-20
10-20
20-25
15-25
would be depressed, as shown in L. stagnalis
at 30°C (MacDonald, 1973). Rollo (1982) also
found a curvilinear relationship between tem-
perature and activity in the slug Limax max-
imus, and no activity was found above 19.5°C.
Non-displacement movement of P corneus is
not linearly related to temperature, the mean
value being minimum at 20°C and maximum
at 10°C (the sole significant difference): more-
over, this activity showed no significant rela-
tionship to temperature when the MANOVA
was applied to a higher number of variables.
The observations of egg-laying and mating
at higher temperatures are consistent with our
previous demonstration that the population of
P corneus from which these animals were
taken reproduces at and above 15°C and is
most fecund at 20°C, all reproduction para-
meters being affected by temperature (Costil
& Daguzan, 1995a). No complex courtship
was seen, the first contact between partners
occurring about 10 min before the start of cop-
ulation. Durations of mating varying from 3 to
5 h are generally reported for planorbid
species (Madsen et al., 1983), slightly shorter
than the times noted here. However, the low
number of observed matings do not allow us
to draw any firm conclusions.
A snail’s activity pattern is an energetic com-
promise between different activities, and pedal
mucus production accounts for 13-32% of the
energy assimilated (Calow, 1974). According
to Denny (1980), “it seems likely that the high
cost of movement has affected the lifestyle of
these animals, for example by limiting the dis-
146 COSTIL AND BAILEY
Temperature
50- тр
10 15 20
Temperature
Temperature
O Mean Minimum [sa] Maximum
FIG. 4. Mean (= S.D.), minimum and maximum val-
ues of activity variables calculated in Planorbarius
corneus put at 10, 15, 20 and 25°C. MNS = mean
speed for 24 h filmed; MXS = maximum speed; TD
= total distance covered by snails; CFS = number of
meetings with the fellow snails; CWS = number of
contacts with the water surface.
tance over which it is profitable to crawl in
search of mates or food.” Mouritsen & Jensen
(1994) found that infection by larval trema-
todes reduced locomotor activity of Hydrobia
ulvae, and suggested that the energy for loco-
motion is probably re-allocated to parasite me-
tabolism and excessive growth of the host.
The persistence of locomotion when tem-
perature decreases is important for species
that make regular seasonal migrations or bur-
row into the substratum in winter. Cheatum
(1934) reported that during autumn, when
temperatures are declining, some species mi-
grate from shallow littoral waters to overwinter
in deeper waters. Several authors have con-
firmed such migrations, but there is no definite
information on Р corneus. Caution is required
when attempting to extend laboratory obser-
vations on the effect of temperature on speed
of movement to field conditions: movement
over a substratum of mud, plants and stones
could be expected to be slower than on the
glass of an aquarium tank. Furthermore,
Calow (1974) showed that the speed of
Planorbis contortus is affected by starvation,
food availability and water movement,
whereas Dimock (1985) found that after three
weeks in an aquarium, there was a 50% re-
duction in speed and a significant decrease in
overall activity in the marine mud _ snail
llyanassa obsoleta. Nonetheless, DeWit
(1955) reported a maximum speed of 7 cm
min”! in P corneus, close to our highest
recorded speed, and speeds of 8.5 cm min !
for the slightly larger L. stagnalis and 6.5 cm
min”? for the smaller Physa fontinalis. In our
experiments, the mean distance travelled in
24 h ranged from 4.38 m at 10°C to 19.29 m
at 25°C, with a maximum value of 42 m. Boss
et al. (1984) recorded much smaller displace-
ments in 24 h by marked individuals of three
aquatic species of snail — less than 40%
moved one metre or more from the release
point, and the maximum distance moved in
the speediest species, Physa integra, was
less than seven m. However, these authors
observed snail positions only at intervals and
did not follow the complete tracks of the
snails. The direction of movement is also im-
portant, and Deliagina & Orlovsky (1990) re-
ported that when searching for food, P
corneus exhibited very sinuous tracks, turning
at irregular intervals. In random movement,
the mean displacement is close to the square
root of tracklength (Bailey, 1989).
Freshwater pulmonates renew the air in the
mantle cavity lung by periodic visits to the sur-
ACTIVITY OF PLANORBARIUS CORNEUS 147
TABLE 4. Temperatures for which some significant differences were calculated with Roy multiple comparison
method used after the one-way multivariate analysis of variance. L = locomotion, Е = feeding, М = movement,
TD = total distance covered by the snails, MNS = mean speed, MXS = maximum speed, CWS = number of
contacts with water surface, CFS = number of meetings with fellow snails.
В Е М
Temperatures for which NO 10-20 NO
some differences were 10-25
calculated
face. The interval between lung ventilations
varies considerably between species: in nine
species studied by Cheatum (1934) the range
was from 52 min in Lymnaea palustristo 1493
min in Physa sayii crassa. The pseudobranch
of P corneus enables this species to obtain
over 50% of its total oxygen uptake by cuta-
neous exchange at much lower levels than the
similarly sized L. stagnalis, and the high affin-
ity of planorbid haemoglobin makes better
use of pulmonary oxygen, allowing it to re-
main submerged for extended periods, even
burrowing into substrate (Jones, 1961, 1964).
Water contains less dissolved oxygen at
higher temperatures, and at 11°C, the aver-
age interval between breathing periods for
Cheatum’s nine species was nearly 22 times
that in water at 21°C. In L. stagnalis, the per-
centage of time breathing increased with tem-
perature (MacDonald, 1973). Although snails
would be expected to visit the surface more
frequently merely because of the increased
distances they moved at higher temperatures,
surface visits increase in response to lowered
oxygen tensions even at the same tempera-
ture (Jones, 1961).
Our conclusion that snail activity was unaf-
fected by the presence or absence of light,
might appear surprising, given the well-known
nocturnality of terrestrial gastropods (e.g.,
Limax pseudoflavus, Ford & Cook, 1987;
Helix aspersa, Lorvelec, 1988; and Helix luco-
rum, Bailey & Lazaridou-Dimitriadou, 1986).
Unlike terrestrial snails and slugs, however,
freshwater gastropods are not subjected to
the low humidity that reduces the activity of
helicids (Cameron, 1970a). Thus, they are not
constrained to place most of their activities
during the night. Several studies on the influ-
ence of light, reviewed by MacMahon (1983),
suggest that the activity of freshwater gas-
tropods increases during darkness, or at dusk
and dawn. In Bulinus tropicus, locomotion,
feeding, excretion, ovipositing and hatching
all follow a diurnal pattern (Chaudry &
TD MNS MXS CWS CFS
10-20 10-20 10-20 10-25 NO
10-25 20-25 20-25
15-25
Morgan, 1983). Endogenous dawn and dusk
activity peaks are reported in the prosobranch
Melanoides tuberculata by Beeston & Morgan
(1979). Infected individuals of L. stagnalis are
most active in the first few hours of illumina-
tion (Anderson et al., 1976). In the absence of
food, P corneus exhibited maximum locomo-
tor activity during the day (Deliagina &
Orlovsky, 1990). Truscott et al. (1995) found
maximum activity in Lymnaea stagnalis in the
morning, and minimum activity around mid-
night, but the differences were small. Intertidal
gastropods could constitute an intermediate
case: the pulmonate limpets Siphonaria
capensis are active at low tide both by day
and night in intertidal pools, whereas limpets
exposed to air at low tide are active only dur-
ing nocturnal or late evening low tides (Branch
& Cherry, 1985). Nevertheless, the siphonar-
ian species S. sirius, inhabiting the low shore,
is active only during daytime and when awash
and submerged both at ebb and flood tides
(lwasaki, 1995). Barnes (1986) suggested
that the activity variation in Hydrobia ulvae
was most likely a direct response to changes
in light intensity and water cover.
The tropical planorbid Biomphalaria gla-
brata exemplifies the great variation in activity
that can be encountered in a single species
under the influence of several factors, includ-
ing parasitization. Moreover, activity, in com-
mon with many life-history traits, probably
differs between different populations of a spe-
cies. Pimentel-Souza et al. (1984) reported a
dusk and a dawn peak of activity in B.
glabrata. The levels of locomotion are similar
in constant darkness and constant light, al-
though significantly lower than levels shown
under a natural cycle of illumination. Biom-
phalaria glabrata shows more locomotor ac-
tivity by day than by night, with a distinct max-
imum in the second hour of light, in contrast to
feeding and egg-laying activities (Hien &
Disko, 1981). Gerard (1996) also found that
uninfected B. glabrata move less during the
148 COSTIL AND BAILEY
night than during the day, but the locomotor
activity of infected individuals is not influenced
by the time of day or night.
The importance of light in directly control-
ling activity or synchronising the endogenous
rhythm of activity of terrestrial gastropods has
diverted attention away from the subtle effects
of temperature in terrestrial species. However,
Cameron (1970b) found significant effects of
temperature and time on the activity of three
species of land snails. All species had their
maximum daytime activity at 8°C but became
increasingly nocturnal as temperature in-
creased. Arianta arbustorum, the least noctur-
nal species, reached maximum activity at 8°C,
but Cepaea nemoralis and C. hortensis were
most active at 22°C. In aquatic snails, also,
temperature interacts with endogenous activ-
ity cycles in subtle ways: Kavaliers (1981)
showed that the planorbid Helisoma trivolvis
has a circadian rhythm of behavioural ther-
moregulation, selecting maximum tempera-
tures of 21-22°C in a thermal gradient during
the dark phase, and minimum temperatures
(17-18°C) during the light phase (Kavaliers,
1981). This rhythm had an endogenous basis,
and temperature selection continued in con-
stant darkness.
Temperature and light influence molluscan
behaviour in different ways. From the present
results and those already discussed, we may
conclude that temperature, light and endo-
genous circadian rhythm act together to con-
trol the behaviour of terrestrial gastropods,
whereas temperature is the most important
factor controlling the behaviour of freshwater
species. Interspecific differences probably re-
flect different environmental constraints to
which different species have adapted. Basom-
matophoran snails, including the planorbids,
are pulmonates that have secondarily re-
adapted to aquatic life, and different species
show varying degrees of re-adaptation (Rus-
sell-Hunter, 1978). It would be of greatest in-
terest to compare the influence of tempera-
ture and light on the behaviour of a less
aquatic species, such as L. truncatula, with
that ofthe more aquatic P corneus.
In northwest France, P corneus has a
spring generation each year, and sometimes
an autumn one as well (Costil & Daguzan,
1995b). Compared to Planorbis planorbis, Р
corneus appears to be more influenced by cli-
mate, and there are strong differences in the
growth patterns of the spring and autumn
generations. Shell growth was fastest in
Spring, and very slow or nil in Winter. In the
laboratory, growth increases with temperature
(Costil, 1994a): this is connected to the en-
hancement of metabolic processes with a
temperature coefficient of about two (Ricklefs,
1990). However, the present study demon-
strates an additional behavioural explanation
for enhanced growth. The increased feeding
activity at higher temperatures is important in
meeting the increased requirements for
growth and reproduction.
This study also provides a behavioural ex-
planation for the precocious senility at higher
temperatures. At 10°C, growth is slow, and
there are many mortalities (Costil, 1994a);
this is accompanied by inactivity, slow loco-
motion, and little feeding activity. However, the
snails reared at the two highest temperatures
expended energy on fast somatic growth and
then became senile earlier. Life expectancy
from hatching falls from 2.64 years at 15°C to
1.96 y at 20°C and 1.26 y at 25°C (Costil,
1994a). Snails at the two highest tempera-
tures were rarely inactive and moved quickly.
Our wider study demonstrates the impor-
tance of temperature on the life history of P
corneus, and the present study emphasises
the induction of life history traits by behavioural
components, as well as physiological ones.
ACKNOWLEDGEMENTS
Thanks are due to J. L. Foulon for technical
help.
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MALACOLOGIA, 1998, 39(1-2): 151-165
SHELL REPAIR FREQUENCIES IN WHELKS AND MOON SNAILS
FROM DELAWARE AND SOUTHERN NEW JERSEY
Gregory P. Dietl! & Richard В. Alexander?
ABSTRACT
More than 1,500 specimens combined from the extant moon snails Euspira heros (Say) and
Neverita duplicata (Say), and the whelks Busycon carica (Gmelin) and Busycotypus canalicula-
tus (Linnaeus) from southern New Jersey tidal flats were examined for breakage-induced shell
repair. Additionally, 500 specimens of E. heros and N. duplicata and Busycon scalarispira
(Conrad) from the Miocene Kirkwood Formation collected from northern Delaware were exam-
ined. On each specimen, body whorl diameter (WD) and apertural lip thickness (ALT) atthree po-
sitions, namely, anterior-most, mid-length, and posterior-most location on the lip, were measured
and number of scars per final whorl counted. Although mean number of repairs per specimen dif-
fered among the five species, averages were comparable for Recent moon snails (1.1 for N. du-
plicata and 1.0 for E. heros) and whelks (5.3 for B. carica and 5.2 for B. canaliculatus). Mean re-
pairs/specimen were appreciably lower for Miocene naticids (0.4) and the melongenid (0.7).
Repair frequencies/shell ranged from zero to 12 for Recent whelk species and Recent N. dupli-
cata, zero to seven for the Recent E. heros, and zero to four for Miocene moon snails and whelks.
Only four and three percent of shells of B. canaliculatus and B. carica lack repairs, whereas 48%
and 57% of Recent E. heros and N. duplicata, respectively, lack repairs. The majority of shells in
every size class of Miocene whelks and moon snails lacked repairs, save for the largest size class
of B. scalarispira. Repair frequencies accumulated at a greater rate (regression line slope) and
are more strongly correlated (higher r value) with WD and ALT for thicker lipped Recent whelks
than either thinner lipped moon snails or Miocene whelks. Presence of an umbilical plug in N. du-
plicata renders posteriorly located breaks on the apertural lip repairable, unlike the plug-lacking
E. heros. Greater rate of scar accumulation with increasing shell size among Recent naticids re-
flects increase in breakage-localizing shell thickness since the Miocene. Greater mean repair fre-
quencies in Recent whelks relative to Recent moon snails is attributed to the additive effect of
sublethal predation on whelks plus prey-induced apertural lip fracture during valve-wedging by
feeding whelks. The substantially lower frequency of repair/shell in B. scalarispira versus B. car-
ica and B. canaliculatus suggests that the habit of shell-wedging of prey had not yet evolved, or
was ineffectually practiced by Miocene whelks.
Key words: shell repair, moon snails, whelks, durophages, shell-wedging.
INTRODUCTION
Shell breakage by durophagous predators
is potentially a more important agent of an-
tipredatory adaptive selection with respect to
gastropod armor today than in pre-Cre-
taceous time. The most effective shell-break-
ing predators in Recent gastropod communi-
ties, brachyuran crabs, elasmobranchs and
teleost fishes, were rare, absent or had not yet
achieved durophagy in the Paleozoic and
early Mesozoic eras. Vermeij et al. (1981) pos-
tulated that “if all breakage were lethal, there
would be no selection between weak and
strong shell variants, and no shells would
show the scars that record nonlethal injury.”
Therefore, breakage-induced shell repair, al-
though not a measure of the magnitude of se-
lection, is a conservative estimate for the im-
portance of selection in favoring the evolution
of durophage-resistant gastropod armor. Low
frequencies of repair implies either that shell-
breaking predators were rare or that most
shells were successfully lethally broken, re-
gardless of predator abundance. High fre-
quencies of breakage-induced shell repair
(among other defenses) reveals the effective-
ness of shell characteristics in protecting the
snail against contemporaneous durophages.
Repair frequencies are expected to vary
within and between snail species both spa-
tially and temporally.
Department of Ecology and Evolution, State University of New York, Stony Brook, New York 11794-5245, U.S.A.
Department of Geological and Marine Sciences, Rider University, Lawrenceville, New Jersey 08648-3099, U.S.A.
151
152 DIETL & ALEXANDER
Several factors influence differences in
accumulation of repairs of sublethal shell
breakage on different gastropod species with
unornamented, oval to tear dropped-shaped
apertures, among taxonomically closely and
distantly related species. Shell size, thickness,
strength, and shape о the gastropod prey is of
primary importance (Vermeij, 1976, 1977;
Curry & Kohn, 1976; Palmer, 1979; Vermeij &
Curry, 1980; Kitchell et al., 1966; Bertness &
Cunningham, 1981), but other factors, includ-
ing differences in diversity and abundance of
shell-breaking predators (Vermeij, 1987), size
refugia from predators, within-habitat refugia
(e.g., vegetated vs. barren substrata or dura-
tion of intertidal exposure) from predators,
age-structure of different prey species popula-
tions, difference in locomotion or escape habit
of prey species, tissue toxicity and palatability,
as well as differences in mode of subjugation
and invasion of prey of compared predatory
snail species influence repair frequencies. In
order to determine the importance of any one,
or set of factors, it is desirable to control or
eliminate many other variables. Studies of co-
habitating gastropod species afford the oppor-
tunity to eliminate differences in frequencies of
shell repair due to disparities in predator
abundances and within-habitat refugia, and
focus on the architecture and size of the prey
species and differences in their escape and/or
feeding habits. Accordingly, the low intertidal to
shallow subtidal, vegetation-barren, shifting,
rippled, fine sandy bottomed community of the
New Jersey coast offers an opportunity to
study repair frequencies on shells of four large,
burrowing, predatory gastropods that are
prone to durophagous predation.
Durophagous predators on snails crush or
peel their prey. Crushing involves compression
between two rigid surfaces, such as the claws
of many crabs (Vermeij, 1976, 1977, 1978;
Zipser & Vermeij, 1978; Brown et al., 1979).
Peeling involves breakage of the outer shell lip
in a spiral direction (Muntz et al., 1965; Shoup,
1968; Vermeij, 1978; Vermeij et al., 1980).
Shell characteristics that deter durophagy in-
clude a low spire, small aperture, thick outer
lip, tight coiling, external sculpture, and retrac-
tility (Vermeij, 1982). In the case of moon snails
and whelks, deeply embayed fractures (Fig. 2-
1, 2-8) and scalloped fractures (Figs. 2-3, 5) in
the body whorl suggest that peeling and crush-
ing are employed by crabs preying on whelks
and moon snails. The four large gastropods
common to the southern New Jersey coast are
the globose naticids Euspira heros (Say),
Neverita duplicata (Say) and the subpyriform
melongenids Busycon carica (Gmelin) and
Busycotypus canaliculatus (Linnaeus) (Figs.
1, 2). Their common shell-crushing and shell-
peeling predator is the blue crab Callinectes
sapidus. Sampled gastropod species from
New Jersey are compared to the fossil gastro-
pod assemblage from the Miocene Kirkwood
Formation of Delaware (Ward, 1992), which in-
cludes both E. heros and N. duplicata, plus the
whelk Busycon scalarispira (Conrad).
Objectives of this investigation were deter-
mination of significant differences, if any, in: (1)
size-dependent accumulation of shell repair
among the co-existing species; and (2), rela-
tion between apertural lip thickness at se-
lected anteroposterior positions and number
of repairs per shell among the co-existing
modern and fossil species. Furthermore, this
study utilizes several perspectives. The evolu-
tionary perspective deals with whether or not
repair frequencies for the temperate E. heros
and WN. duplicata are similar to congeneric and
conspecific fossil populations from the Late
Cretaceous to Miocene. The latitudinal per-
spective bears on the issue of different abun-
dance and diversity of predators on whelks
and moon snails from different communities.
Are repair frequencies in these temperate lati-
tude gastropods higher or lower than those re-
ported on tropical congeneric Recent assem-
blages (Vermeij, 1982)? The architectural-size
perspective focuses on whether or not co-
habitating, confamilial and congeneric species
with differing shell thicknesses differ in their
frequency of breakage-induced repair (i.e., E.
heros vs. N. duplicata and B. carica vs. B.
canaliculatus). The trophic habit perspective
focuses on possible differences in repair fre-
quencies among co-habitating melongenids
versus naticids given that whelks risk, and
moon Snails do not risk, fracture of their aper-
tural lip during invasion of their molluscan prey.
MATERIALS AND METHODS
Empty and hermit crab-occupied shells of
the naticid snails N. duplicata and E. heros
and the melongenid whelks B. carica and
B. canaliculatus were collected from Here-
ford Inlet (HI), Cape May County, and Longs-
port Beach in Great Egg Harbor (GEH),
Atlantic County, New Jersey. Collections of E.
heros, N. duplicata, and B. scalarispira from
the Miocene Kirkwood Formation of Delaware
were loaned by Lauck Ward, Virginia Museum
SHELL REPAIRS IN MOON SNAILS AND WHELKS 153
FIG. 1. Apertural view of Busycotypus canaliculatus and Euspira heros showing position of maximum whorl
diameter (WD) measured for body whorl and apertural lip thickness (ALT) measured (A) anteriorly near junc-
tion with penultimate whorl, (B) at lip mid-length, and (C) posteriorly at siphonal canal in whelks and nearest
umbilicus in moon snails.
of Natural History. The fauna of the Kirkwood
Formation correlates with the Calvert For-
mation of Maryland, which is the lowest of the
Chesapeake Group (Richards & Harbison,
1942). Whorl diameter (WD) and apertural lip
thickness (ALT) at three positions, namely, the
anterior-most, mid-length, and posterior-most
location on the lip (Fig. 1) were measured with
Vernier calipers to the nearest 0.05 mm on
more than 1,500 specimens combined from
the extant moon snails N. duplicata and E.
heros, and the whelks B. carica and B.
canaliculatus. These same measurements
were made on more than 500 fossil speci-
mens of E. heros, N. duplicata and B. scalar-
ispira. Traces of sublethal breakage may be
seen as scars that cut across the growth lines
or axial sculpture of the shell (Fig. 2). Number
of sublethal scars per final whorl were
counted for each specimen and the repair fre-
quency determined. Repair frequency was
calculated as the number of repairs per shell
divided by the total sample size, following the
definition by Vermeij et al. (1982), Vermeij &
Dudley (1982), and Schindel et al. (1982).
Accordingly, the number of repair scars per
shell, an index of repair frequency, was re-
gressed on WD, the index of size, for each
species in the Recent and Miocene samples
to determine if repairs were correlated with
size (age) for either moon snail and/or whelk
species. Both linear and second-order polyno-
mial regressions were executed for number of
repairs regressed on WD. The greater correla-
tion coefficient generated by these two meth-
ods was used in statistical comparisons
(Tukey-HSD test) of r values within and be-
tween species. Slope of the regression equa-
tions, beta, were also statistically compared
(F-test) between and within species in order
to evaluate if the rate of accumulation of re-
pairs with increasing size differ within and be-
tween species.
Shells of all species were sorted according
to WD into six size-class divisions, namely,
class 1, < 19 mm; class 2, 20-39 mm; class 3,
40-59 mm; class 4, 60-79 mm; class 5,
80-99 mm; and class 6, 100-119 mm. Repair
frequencies were calculated for specimens in
each size class by dividing the total number of
repairs in the sample by the number of shells
examined in the size class (Table 1). The pro-
portion of shells with a given number of scars
per final whorl, which ranged from zero to 12,
154 DIETL & ALEXANDER
FIG. 2. Specimens of Busycotypus canaliculatus (1-2), Busycon carica (3-5), Neverita duplicata (6, 8), and
Euspira heros (7) with shell repairs. Multiple repairs in body whorl illustrated in 3, 7. Anteriorly concentrated
repair in apertural lip shown in 2, mid-length lip repair in 4, 8, and posteriorly concentrated repair near um-
bilicus of moon snail shown in 6. Repair that extends anteriorly across whorl shoulder to penultimate whorl
of juvenile whelk shown in 5. Width of bar equals one cm.
SHELL REPAIRS IN MOON SNAILS AND WHELKS 155
TABLE 1. Frequency of shell repair in relation to size class (based on whorl diameter).
N = number of specimens; f = shell repair frequency. Repair frequencies are calculated
as total repairs in a sample divided by number of shells examined in that sample.
Taxon Location
E. heros GEH
Recent
HI
E. heros Kirkwood
Miocene Fm; Delaware
N. duplicata GEH
Recent
HI
N. duplicata Kirkwood
Miocene Fm; Delaware
B. carica HI
Recent
B. canaliculatus HI
Recent
B. scalarispira Kirkwood
Fm; Delaware
Size Class (mm) N f
<19 0 0.00
20-39 23 0.69
40-59 29 1.00
60-79 10 2.10
<19 12 0.58
20-39 79 0.72
40—59 86 0.96
60-79 33 1.51
80-99 13 1.54
<19 71 0.48
20-39 28 0.18
40-59 7 0.57
<19 0 0.00
20-39 64 0.61
40-59 324 1.05
60-79 53 2.13
<19 17 0.35
20-39 193 0.73
40-59 207 137
60-79 132 2.04
<19 69 0.26
20-39 82 0.73
40-59 5 0.80
<19 12 1.67
20-39 49 3.80
40-59 20 4.55
60-79 28 6.10
80-99 40 7.00
<19 3 0.33
20-39 41 3.00
40-59 21 3.42
60-79 28 5.03
80-99 42 7.78
100-119 10 7.90
<19 24 0.13
20-39 28 0.43
40-59 58 0.59
60-79 66 0.83
80-99 25 1.32
100-119 3 1.67
Locations: GEH = Great Egg Harbor, Atlantic County, NJ; HI = Hereford Inlet, Cape May County, New
Jersey. Kirkwood Fm = Pollac site, Kent County, Delaware.
was determined for each size class in each
species. Hereafter this calculation is referred
to as “proportion of repair” (Table 2). One-
way ANOVA was used to determine if mean
number of repairs per specimen was signifi-
cantly different between the five species. A
Kolmogorov-Smirnov test was used to deter-
mine if there were differences in the percent-
frequency of shells with increasing number of
repair scars per shell between contemporane-
ous and noncontemporaneous whelk and
moon snail species (Table 3).
156
DIETL & ALEXANDER
TABLE 2. Proportion of shells of Euspira heros, Neverita duplicata, Busycon carica, B. scalarispira, and
Busycotypus canaliculatus, with increasing number of repairs per shell (0-12) in relation to size class defined
by whorl diameter (mm).
Taxon &
Geol. Age LOC
Eh GEH
Recent
HI
Nd GEH
Recent
HI
Ben HI
Recent
Bcr HI
Recent
Nd
Miocene
Kirkwood
Eh
Miocene
Kirkwood
Bs
Miocene
Kirkwood
Species: Eh - Euspira heros; Nd -
Size Class
(mm)
<19
20-39
40-59
60-79
80-99
<19
20-39
40-59
60-79
80-99
<19
20-39
40-59
60-79
<19
20-39
40-59
60-79
<19
20-39
40-59
60-79
80-99
100-119
<19
20-39
40-59
60-79
80-99
100-119
<19
20-39
40-59
<19
20-39
40-59
<19
20-39
40-59
60-79
80-99
100-119
0 1
0.16 0.12
0.67 0.33
0.17 0.08
0.08 0.06
0.07
0.76 0.21
0.70 0.21
0.50 0.50
0.66 0.24
0.82 0.18
0.88 0.12
0.71 0.18
0.65 0.21
0.50 0.30
0.24 0.32
0.50
0.12
0.07
0.03
0.03
0.06
0.06
0.07
0.09
0.12
0.36
0.33
0.25
0.16
0.05
0.04
0.02
0.04
0.04
0.02
0.02
0.04
0.33
Number of repairs per shell
4
0.10
0.05
0.01
0.09
0.02
0.02
0.06
0.01
0.06
0.08
0.10
0.11
0.25
0.07
0.16
0.11
0.17
0.05
0.08
0.14
0.04
0.06
0.04
5
0.04
0.01
0.03
0.01
0.02
0.02
0.12
0.13
0.21
0.14
0.12
0.10
0.37
0.10
0.10
0.25
6
0.10
0.03
0.08
0.01
0.08
0.02
0.04
0.03
0.11
0.07
0.10
0.14
0.11
0.10
0.18
0.17
7
0.01
0.03
0.04
0.03
0.05
0.11
0.17
0.20
0.06
0.05
0.17
0.10
0.08
8
0.02
0.08
0.04
0.14
0.04
0.14
0.10
0.17
0.05
0.04
0.17
0.11
0.10
0.13
0.13
0.03
0.04
0.12
0.40
0.02
0.07
0.13
0.04
0.07
0.10
0.05
0.04
0.05
0.02
0.03
0.04
0.05
0.08
Neverita duplicata; Всп - Busycotypus canaliculatus; Всг - Busycon carica.; Bs - Busycon
scalarispira; LOC = Locations; GEH - Great Egg Harbor, Atlantic County, New Jersey; HI - Hereford Inlet, Cape May County,
New Jersey; Miocene Kirkwood Fm - Pollac site, Kent County, Delaware.
Vermeij (1982) reported that species with
thickened lips have significantly lower fre-
quencies of repair than do thin-lipped species,
because thick-lipped species experience
many unsuccessful attacks that leave the lip
unscathed and, therefore, unrepaired. Ac-
cordingly, a two-way ANOVA was used for
both Recent and Miocene moon snails and
SHELL REPAIRS IN MOON SNAILS AND WHELKS 157
TABLE 3. Multiple comparison Fisher PLSD test for equality among mean number of repairs and
Kolmogorov-Smirnov test for differences in percent-frequency of shells with increasing number of repair
scars per shell.
Comparison
Within naticids
Neverita duplicata (Recent) vs. Euspira heros (Recent)
vs. E. heros (Miocene)
vs. N. duplicata (Miocene)
E. heros (Recent) vs. E. heros (Miocene)
vs. N. duplicata (Miocene)
E. heros (Miocene) vs. N. duplicata (Miocene)
Within melongenids
Busycotypus canaliculatus (Recent) vs. Busycon carica (Recent)
vs. B. scalarispira (Miocene)
B. carica (Recent) vs. B. scalarispira (Miocene)
P-values: ns = nonsignificant
*=</0105
ea <0: 0)
*^* = < 0.001
whelks treated separately to determine if
mean thickness of the apertural lip for each
species varies significantly anteriorly to pos-
teriorly, that is, at lip positions A, B, or C (Fig.
1). Because repairs are often localized along
a portion of the apertural margin (Figs. 2-4),
the number of repair scars per shell was re-
gressed on thickness of the apertural lip sep-
arately at positions A, B, or C for each species
to determine the susceptibility of differing
parts of apertural lip to sustaining repeated,
repairable, breakage. As with regressions in-
volving size (WD), both linear and second
order polynomial regressions of the number of
repairs versus apertural lip thickness were
generated for each sample of each species.
The greater r value generated by either
method was used in statistical comparisons of
correlation coefficients (Tukey-HSD test).
Similarly, the slopes of the regression lines,
beta, were statistically compared (F-test) to
evaluate possible significant differences in the
rate of accumulation of repairs with increasing
lip thickness between and within species.
RESULTS
Frequency of shell repair is greater in all
size classes of Recent whelks relative to the
same size class among Recent moon snails
(Table 1). However, frequencies of shell repair
are similar among same size classes of com-
pared Miocene whelks versus Miocene moon
snails (Table 1). Number of repair scars per
specimen ranged form zero to 12 for both
Fisher PLSD K-S Chi-square
0.225ns 1.5ns
0.334* 355
0.249* Gr
0.379* 2.1.8:
0.296* INE
0.394ns 0.173ns
0.378ns 2.1ns
0.365* ИЗ
0.346* 220.77
Recent whelk species, zero to 12 for Recent
N. duplicata, and zero to seven for Recent E.
heros (Table 2). Only four and three percent of
B. carica and B. canaliculatus, respectively,
lack repairs, whereas 48% and 57% of Recent
E. heros and N. duplicata, respectively, lack
repairs. Recent whelks display significantly
higher proportions of shells with more than six
repairs relative to moon snails (Table 2). There
is a significant difference (p < 0.001) between
noncontemporaneous congeneric melon-
genids in percent-frequency distributions of
shells with increasing number of repair scars
per shell, as well as noncontemporaneous
confamilial and conspecific Miocene and
Recent naticids (Table 3). Furthermore, the
thinner Recent moon snail N. duplicata and
whelk B. canaliculatus (Table 4) do not have
percent-frequency distributions of shells with
increasing number of repair scars per shell
different from their thicker-lipped Recent rela-
tives E. heros and B. carica, respectively
(Table 3). Among Miocene naticid shells, all
size classes are dominated by shells without
repairs. The maximum number of repairs per
shell for any size class is four (Table 2).
Among Miocene whelks, no shell had more
than four repairs, and the majority of the spec-
imens (72%) in the 19-79 mm size classes
lacked repairs (Table 2).
The apertural lip of E. heros is thicker on av-
erage at all three positions (Fig. 1) relative to
N. duplicata (Table 4), although the shell of the
latter species is more massive due to an um-
bilical plug. Average thickness of the apertural
lip increases by a factor of three at positions A
158 DIETL & ALEXANDER
0.7
BcnO
0.6
BcrO
Correlation Coeff. (Repairs on WD)
Miocene Recent
FIG. 3. Distribution of correlation coefficient, r, for
number of repairs on shell regressed on size (in-
dexed by whorl diameter, WD) for Miocene versus
Recent samples of Euspira heros (Eh), Neverita du-
plicata (Nd), Busycon carica (Bcr), Busycotypus
canaliculatus (Ben), and Busycon scalarispira (Bs).
Multiple comparison Tukey-HSD test reveal correla-
tion coefficients are significantly different (p < 0.01)
among naticids only in comparisons between Nd
(Recent vs. Miocene). Among melongenids, r val-
ues are significantly different in comparisons of Bs
vs. Ber and Bs vs. Всп (р < 0.05). Greater r value be-
tween linear or second order polynomial regression
for asample used in all comparisons.
and B, and a factor of four at position C (Fig.
1) from Miocene to Recent samples of E.
heros (Table 4). Average thickness values at
the same three positions on the apertural lip
more than doubled from Miocene to Recent
samples of N. duplicata (Table 4). Among
whelks, shells of B. carica are on average
twice as thick as those of B. canaliculatus at
position A, but is no thicker at position C than
B. canaliculatus or B. scalarispira (Table 4).
Mean number of repairs per specimen was
different among the five species (ANOVA, p =
0.0001), although the average was compara-
ble for the two moon snails, namely 1.1 and
1.0 for Recent confamilial N. duplicata and E.
heros, respectively (Table 3). Miocene nati-
cids N. duplicata (0.36) and E. heros (0.41), as
well as the Recent whelks B. carica (5.3) and
Regression Slope (Repairs on WD)
Recent
Miocene
FIG. 4. Distribution of slope of regression lines,
beta, for number of repairs on shell regressed on
size (indexed by whorl diameter, WD) for Miocene
versus Recent samples of Euspira heros (Eh),
Neverita duplicata (Nd), Busycon carica (Всп,
Busycotypus canaliculatus (Всп), and Busycon
scalarispira (Bs). Multiple comparison F-tests re-
veal slopes of regression lines are significantly dif-
ferent (p < 0.001) in comparisons between Nd
(Recent) and all other naticid samples. Among me-
longenids, regressions lines for Recent whelks (Всп
and Ber) have significantly greater slopes (р <
0.001) than that for the Miocene whelk (Bs). Slope
of regression line of Bcn is significantly greater (p <
0.001) than that for Bcr. Slopes for regression line of
Recent whelks are significantly greater (p < 0.001)
than all naticids.
B. canaliculatus (5.2), also show comparable
averages (Table 3). Mean number of repairs
for Miocene B. scalarispira (0.7) and Miocene
naticids are significantly different (p < 0.05)
than Recent congeneric whelks and Recent
naticids (Table 3). Among naticids, mean val-
ues are substantially greater for Recent tem-
perate latitude N. duplicata and E. heros, ver-
sus Recent tropical naticids and Upper
Triassic to Miocene naticids (Table 5). Among
the melongenids, Recent species have a
seven times greater repair frequency than the
Miocene species (Table 6).
Repair frequency is more positively corre-
lated with size and apertural lip thickness for all
SHELL REPAIRS IN MOON SNAILS AND WHELKS 159
TABLE 4. Two-way ANOVA for moon snails indicates that species (р = 0.0001) and position (p =
0.0001) are significant factors in mean differences in apertural lip thickness (ALT). See Fig. 1 for
location of position A, B, and C on apertural lip. Factor interaction (species in combination with
ALT position) is also significant (p = 0.0001). Two-way ANOVA for whelks indicates that species
(p = 0.0001) and position (p = 0.0001) are significant factors in mean differences in ALT. Factor
interaction (species in combination with position) is also significant (p = 0.0001); n = sample size.
E +4 KA
Mean ALT (mm) at
Taxon position A
E. heros 0.89
Recent n = 241
E. heros 0.29
Miocene п = 81
N. duplicata 0.75
Recent n = 779
N. duplicata 0.37
Miocene п = 216
В. canaliculatus 1.26
Recent n = 143
B. carica 2.11
Recent r= 173
B. scalarispira 1.99
Miocene п = 194
Mean ALT (mm) at Mean ALT (mm) at
position B position C
0.76 2.08
п = 241 п = 240
0.22 0.50
п = 80 п = 80
0.68 1.62
п = 780 п = 780
0.26 0.66
п = 222 п = 198
1.36 1.30
п = 143 п = 143
1.60 1.28
п = 173 п = 173
1:73 1.29
п = 195 п = 195
TABLE 5. Сотрайзоп of repair frequencies, f (the number of scars рег shell), in umbilicate species from the
present study with extant and fossil populations.
—————"—"—"—"—"—"—"—"—…—…—…—…"…"…"…"…—…—…—…—————
Geological
Taxon Age/Formation Location f Reference
Euspira heros Recent Southern New Jersey 1.0 Present study
Neverita duplicata Recent Southern New Jersey 1.1 Present study
Euspira Sp. Recent Tropical 0-0.05 Vermeij, 1982
Polinices tumidus Recent Aru Islands 0.10 Vermeij, 1982
P uber Recent Panama 0.13 Vermeij, 1982
Natica chemnitzii Recent Panama 0.50 Vermeij, 1982
E. heros Miocene (Kirkwood Delaware 0.41 Present study
Fm.)
N. duplicata Miocene (Kirkwood Delaware 0.36 Present study
Fm.)
E. rectilabrum Late Cretaceous Mississippi 0.71 Vermeij & Dudley,
(Ripley Formation) 1982
Euspira sp. Late Cretaceous Alabama 0.64 Vermeij & Dudley,
(Ripley Formation) 1982
Amauropsis Upper Triassic St. Costalaresc, Italy 0.051 Vermeij et al.,
paludinaris Cassian Gr. 1982
three positions (Fig. 1) for Recent melon-
genids versus Recent naticids (Figs. 3, 5, 7, 9).
Furthermore, rate of accumulation of repairs is
significantly greater for Recent whelks versus
moon snails based on slopes of the regression
lines for repairs regressed on WD and ALT
(Figs. 4, 6, 8, 10). Repair frequency is more
positively correlated with size and apertural lip
thickness in Recent whelks relative to Miocene
whelks (Figs. 3, 5, 7, 9). In contrast, the only
significant difference occurs in the r values of
repair frequency regressed on size for
Miocene versus Recent samples of N. dupli-
cata (Fig. 3). No significant differences occur in
160 DIETL & ALEXANDER
TABLE 6. Comparison of repair frequencies, f (the number of scars per shell), in extant and fossil
whelk populations.
Geological
Taxon Age/Formation Location f Reference
Busycon carica Recent Southern New Jersey 5.3 Present study
Busycotypus
canaliculatus Recent Southern New Jersey 5.2 Present study
Busycon Miocene (Kirkwood Delaware 0.7 Present study
scalarispira Fm.)
0.5
0.4
0.3
0.2
0.1
Correlation Coeff. (Repairs on ALT-A)
>
Recent
Miocene
FIG. 5. Distribution of correlation coefficient, r, for
number of repairs on shell regressed on apertural
lip thickness at position A (see Fig. 1) for Miocene
versus Recent samples of Euspira heros (Eh),
Neverita duplicata (Nd), Busycon carica (Bcr),
Busycotypus canaliculatus (Ben), and Busycon
scalarispira (Bs). Multiple comparison Tukey-HSD
test reveal correlation coefficients are significantly
different (p < 0.05) only in comparison between B.
carica and B. scalarispira. Greater r value between
linear or second order polynomial regression for a
sample used in all comparisons of correlation coef-
ficients.
comparison of r values involving shell thick-
ness (Figs. 5-10). Furthermore, no significant
difference occurs between compared correla-
tion coefficients or slopes of regression lines
for Miocene and Recent samples of E. heros
for either size or lip thickness (Figs. 3-10).
pod ud judo fdo pd od
Sem Dana
>
Regression Slope (Repairs on ALT-A)
>
SI
Recent
Miocene
FIG. 6. Distribution of slope of regression lines,
beta, for number of repairs on shell regressed on
apertural lip thickness at position A (see Fig. 1) for
Miocene versus Recent samples of Euspira heros
(Eh), Neverita duplicata (Nd), Busycon carica (Bcr),
Busycotypus canaliculatus (Bcn), and Busycon
scalarispira (Bs). Multiple comparison F-tests re-
veal slopes of regression lines are significantly dif-
ferent only in comparisons involving melongenids.
Regression lines for Recent whelks (Ben and Ber)
have significantly greater slopes (p < 0.001) than
that for the Miocene whelk (Bs) and Recent naticids
(Eh and Nd). Slope of regression line of Всп signifi-
cantly greater (p < 0.001) than that for Ber.
DISCUSSION
The significant correlation (p < 0.05) be-
tween the number of repairs per shell and size
(WD) for Recent whelk and moon snail species
(Fig. 3) indicates that age-dependent accumu-
SHELL REPAIRS IN MOON SNAILS AND WHELKS 161
Correlation Coeff. (Repairs on ALT-B)
Recent
Miocene
FIG. 7. Distribution of correlation coefficient, r, for
number of repairs on shell regressed on apertural
lip thickness at position B (see Fig. 1) for Miocene
versus Recent samples of Euspira heros (Eh),
Neverita duplicata (Nd), Busycon carica (Bcr),
Busycotypus canaliculatus (Ben), and Busycon
scalarispira (Bs). Multiple comparison Tukey-HSD
test reveal correlation coefficients are significantly
different (P < 0.05) only in comparison between B.
carica and B. scalarispira. Greater r value between
linear or second order polynomial regression for a
sample used in all comparisons of correlation coef-
ficients.
lation of scars increased as exposure time to
potential predators increased over the life
span of the prey (Fig. 3). In most Recent gas-
tropods, incidence of sublethal shell breakage
increases as shell length increases (Vermeij et
al., 1980; Zipser & Vermeij, 1980; Vermeij,
1982; Vermeij & Dudley, 1982; Vermeij et al.,
1982; Raffaelli, 1978; Dudley, 1980). Similarly,
Alexander (1989) showed that the frequency
of repaired valves correlates significantly with
valve surface area (index of size) for Late
Ordovician brachiopods. Large size also de-
creases the probability that a shell-breaking
predator will lethally fracture the shell (Hughes
& Elner, 1979; Seed, 1978; Elner & Raffaelli,
1980; Preston et al., 1996), which may also ex-
plain why successively larger size classes
have greater frequencies of shell repair for all
five gastropod species in this investigation
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
-0.2
Regression Slope (Repairs on ALT-B)
Recent
Miocene
FIG. 8. Distribution of slope of regression lines,
beta, for number of repairs on shell regressed on
apertural lip thickness at position B (see Fig. 1) for
Miocene versus Recent samples of Euspira heros
(Eh), Neverita duplicata (Nd), Busycon carica (Bcr),
Busycotypus canaliculatus (Ben), and Busycon
scalarispira (Bs). Multiple comparison F-tests re-
veal slopes of regression lines are significantly dif-
ferent only in comparisons involving melongenids.
Regression lines for Recent whelks (Всп and Ber)
have significantly greater slopes (p < 0.001) than
that for the Miocene whelk (Bs) and Recent naticids
(Eh and Nd).
(Table 1). However, repaired fractures do not
continue to accumulate on shells of E. heros
(maximum = 6) from the penultimate to the
largest size classes (60-79 vs. 80-99) as they
do between the two largest size classes of N.
duplicata (Table 2), suggesting a possible size
refugia from predation for E. heros. In contrast,
the largest individuals of N. duplicata had a
WD in the 60-79 mm size class (Table 2) and
had scars near the periphery of the apertural
lip.
Failure to continue to accumulate repairs on
shells of E. heros with increasing size beyond
the 80 mm WD threshold in the population at
Hereford Inlet (Table 2) may account for the
lower correlation coefficient and slope value
between repair frequency and WD for E. heros
relative to the slightly smaller N. duplicata
(Figs. 3-4). Preston et al. (1996) commented
162 DIETL & ALEXANDER
ALT-C)
Correlation Coeff. (Repairs on
Recent
Miocene
FIG. 9. Distribution of correlation coefficient, r, for
number of repairs on shell regressed on apertural
lip thickness at position C (see Fig. 1) for Miocene
versus Recent samples of Euspira heros (Eh),
Neverita duplicata (Nd), Busycon carica (Bcr),
Busycotypus canaliculatus (Bcn), and Busycon
scalarispira (Bs). Multiple comparison Tukey-HSD
test reveal correlation coefficients are significantly
different (p < 0.01) only in comparison of B. carica
vs. B. scalarispira. Greater r value between linear or
second order polynomial regression for a sample
used in all comparisons.
that with increasing shell size, more attacks
from predators will fail to break the thicker lip.
Rate of accumulation of repairs is thus ex-
pected to decline when the predator can no
longer readily inflict damage to the thicker
shelled gastropods, a contrast to durophagy
on skeletonized invertebrates with delicate ap-
pendages that can be sheared off at any body
size, such as with crinoid arms (Oji, 1996).
The significantly greater correlation coeffi-
cient (p < 0.05) between repair frequency and
lip thickness at position C (r = 0.16) vs. posi-
tion A (r = 0.06) or B (r = 0.06) (Fig. 9 vs. Figs.
5 and 7) for both Recent and Miocene N. du-
plicata may indicate where the crushing ele-
ments of the durophage more often exerted
their peeling force, namely posteriorly near
the umbilicus (Fig. 1, position C). Peeled
shells of N. duplicata more often have shell
material of the body whorl closest to the um-
1.5
Regression Slope (Repairs on ALT-C)
>
SQ
Recent
Miocene
FIG. 10. Distribution of slope of regression lines,
beta, for number of repairs on shell regressed on
apertural lip thickness at position C (see Fig. 1) for
Miocene versus Recent samples of Euspira heros
(Eh), Neverita duplicata (Nd), Busycon carica (Ber),
Busycotypus canaliculatus (Всп), and Busycon
scalarispira (Bs). Multiple comparison F-tests re-
veal slopes of regression lines are significantly dif-
ferent (p < 0.001) among naticids only in compar-
isons between Recent N. duplicata and Recent E.
heros. Regression lines for Recent whelks (Всп and
Bcr) have significantly greater slopes (p < 0.001)
than that for the Miocene whelk (Bs) and Recent
naticids (Eh and Ма).
bilical plug removed, but retain a wrapped
around “awning” of shell material anteriorly,
nearest the suture with the penultimate whorl.
The aperture is widest posteriorly at position
C, in N. duplicata, allowing the claw of a crab
to secure the deepest purchase onto the lip
and into the body whorl. Hence most sublethal
shell breakage originates posteriorly on the
lip. Thus, the thickness of the lip posteriorly
(position C), where peeling forces are exerted
on the shell, may be more important than the
thickness of the lip anteriorly (position A) in
the determination if a fracture initiated by a
durophage will be localized by the prey. The
significantly greater correlation of repair fre-
quency with lip thickness at position C rather
than position A is harmonious with that rea-
soning.
Among repaired shells the umbilical plug
SHELL REPAIRS IN MOON SNAILS AND WHELKS 163
functioned to stop propagation of fractures
through this posterior region into the basal
cavity and thereby render repairable any pos-
teriorly localized breakage. Vermeij (1987)
stated that the structural weakness of the um-
bilicus can be strengthened by the formation
of the umbilical plug closing the basal cavity.
Conversely, lack of correlation between ALT at
any position and number of shell repairs for
the other Recent moon snail, E. heros (Figs. 5,
7, 9) may indicate an architectural weakness
of this unplugged umbilicate design to local-
ized fractures initiated in the apertural lip. As
the probability of lethal fractures increased in
this unplugged moon snail, frequency of sub-
lethal fractures decrease. The statistical con-
sequence is diminished correlation between
ALT and repair frequency in shells of E. heros
(Vermeij, 1982).
The better correlations between ALT at any
lip position for whelks versus moon snails
(Figs. 5-10) may reflect the fact the whelks
have a higher probability of accumulating sub-
lethal fractures on their shell as a conse-
quence of their feeding habit. They wedge
apart their bivalve prey (Colton, 1908; Warren,
1916; Carriker, 1951), an activity that is not
likely to induce an unrepairable fracture.
Indeed, most repairs are scallop-like, shallow
indentations from the normal axial growth line
(Figs. 2, 3), a pattern suggestive of breakage
during valve-wedging of bivalve prey. Only
deep, irregular embayments from the normal
axial growth line (Fig. 2-1) indicate repairs of
shell-breakage more likely to have been in-
flicted by blue crabs. Thus, the feeding habit of
the whelk is probably more important than its
victimization by crabs in accumulation of shell
repairs with increasing size and apertural
thickness (Tables 1, 2).
Vermeij (1997) speculated that predator-
prey escalation between armored gastropods
and their shell-breaking enemies can be used
as a prediction that shell-breakage became
increasingly important as a selective factor for
gastropods through Mesozoic and Cenozoic
time. Interpretations of repair frequencies
cannot estimate either predator intensity or
predator efficiency but can reflect the effec-
tiveness of the predator’s crushing ability and
the prey’s resistance to crushing (Schindel &
Vermeij, 1982). Based on these assumptions,
Vermeij, after several investigations, con-
cluded that repair frequencies remained con-
stant from the Late Carboniferous to the Late
Triassic (Schindel et al., 1982; Vermeij et al.,
1982), increased from the Late Triassic to the
Late Cretaceous (Vermeij & Dudley, 1982),
and then again became relatively constant
approaching Recent levels (Vermeij, 1982).
These trends, coupled with an increase in
power and diversification of shell-breaking
predators from the Mesozoic to Cenozoic,
formed the basis of Vermeij’s conclusions.
Vermeij & Dudley (1982) reported that
Euspira sp. from the Ripley Formation (Late
Cretaceous) had higher frequencies of repair
(0.71, Table 5) than do Recent tropical popula-
tions of Euspira sp. (0-0.5; Vermeij, 1982).
Data in this investigation show a frequency of
repair which is even higher (1.0) than those of
Late Cretaceous members (0.71), suggesting
higher incidence and stronger expression of
breakage-resistant armor in temperate mem-
bers of this family. However, repair frequency
in the Miocene E. heros is lower (0.4) than that
reported for the Late Cretaceous (Table 5), ev-
idence in support of Vermeij’s contention that
repair frequencies did not continue to increase
after the Cretaceous. Disparity in repair fre-
quencies between Recent tropical (0.05) and
temperate Euspira sp. (1.0) may reflect differ-
ences in both the strength and the abundance
of predators relative to the strength and abun-
dance of the prey. Similarly, repair frequencies
for N. duplicata (1.1) are higher than ecologi-
cally similar Recent tropical populations of
Polinices tumidus (0.1) and Polinices uber
(0.13)(Vermeij, 1982) (Table 5). Vermeij (1983)
reported that an increase in relative abun-
dance of the predator would increase the fre-
quency of repair.
Size of individual shells is important in com-
parison of repair frequencies. Vermeij (1982)
sorted 21 specimens of Natica chemnitzii
among four size classes, that is, 5-9 mm;
10-19 mm; 20-29 mm; and 30-39 mm. The
repair frequency of 0.67 in the 20-29 mm size
class (n = 9; Vermeij, 1982), is comparable to
the repair frequency values in the 20-39 mm
size class in this study, namely, 0.61 (GEH)
and 0.73 (HI) for N. duplicata and 0.69 (GEH)
and 0.72 (HI) for E. heros (Table 1). However,
for all size classes, repair frequency for shells
of N. chemnitzii was 0.50 compared with 1.1
and 1.0 for N. duplicata and E. heros, respec-
tively (Table 5). This difference may be the re-
sult of the inclusion of large numbers of shells
in the 40-99 mm size classes in this study.
The higher frequency of repair for both B.
canaliculatus (5.2) and B. carica (5.3) is attri-
buted to a combination of shell breakage from
crabs preying on whelks combined with aper-
tural lip fracture (Fig. 2) during attempts by
whelks to employ the outer shell lip to wedge
open the valves of tightly closed bivalve prey.
164 DIETL & ALEXANDER
The rate of accumulation of repairs, reflected
in the slopes of repair frequencies regressed
on size and lip thickness (Figs. 4, 6, 8, 10), is
greater for whelks in comparison to moon
snails. Because the shell lip of predatory
whelks is often slightly damaged during at-
tacks on prey, valve-wedging species fre-
quently have a high incidence of repaired
breaks than non-valve-wedging species. The
frequencies of repair for Recent whelk species
in this study are higher than that reported for
any other gastropod in the literature. The fre-
quency is more than seven times greater than
that for B. scalarispira from the Miocene.
Vermeij (1987: 182) suggested that the lip-
wedging technique may not have evolved until
the Middle Pliocene on the Atlantic Coastal
Plain of North America, although no evidence
was presented. The majority of repaired frac-
tures in B. scalarispira extend across the
varices onto the abaxial edge of the sutural
shelf of the final whorl (Fig. 2-5). In contrast,
most repairs in Recent whelks were concen-
trated at the lip mid-length (Fig. 2-4, 2-8).
Thus, varices appear to be effective in limiting
the extent of unrepairable damage from lip-
wedging but not the direct attention to the lip by
the durophage. This discrepancy suggests
that Miocene melongenids had not evolved, or
perfected, valve-wedging, and as a conse-
quence the frequency of repairs probably re-
flects the contribution of predators sublethally
peeling the whelks.
ACKNOWLEDGMENTS
We are grateful to Joanne Dietl, Walt Bien,
and Richard Trub for assistance in collection
and measuring of specimens, and editing the
manuscript. We also appreciate the sugges-
tions of Mel Carriker and Charles Jansen to
improve the manuscript. We are especially
grateful to Lauck Ward for the loan of the gas-
tropod collections from the Miocene Kirkwood
Fm of Delaware. Critical review of the manu-
script by Geerat Vermeij and an anonymous
reviewer is also appreciated.
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Revised ms. accepted 12 May 1997
MALACOLOGIA, 1998, 39(1-2): 167-173
WITHIN-CLUTCH EGG CANNIBALISM VARIABILITY IN HATCHLINGS OF THE
LAND SNAIL HELIX ASPERSA (PULMONATA: STYLOMMATOPHORA):
INFLUENCE OF TWO PROXIMATE FACTORS
Christophe Desbuquois & Luc Madec
U.M.R. CNRS 6553, Laboratoire de Zoologie et d’Ecophysiologie (U.A. INRA), Faculte des
Sciences, Université de Rennes 1, Avenue du General Leclerc, 35042 Rennes CEDEX, France
ABSTRACT
This study investigated, under laboratory conditions, the influence of two proximate factors,
temperature and humidity, on the rate of egg cannibalism in hatchlings of Helix aspersa. Nine
combinations were tested involving three different temperatures (15, 20, 25°C) and relative hu-
midities (40, 70, 100% R.H.). Each new-born snail was maintained with a conspecific egg of its
laying, previously incubated at 15°C instead of 20°C for the tested snails, so that its incubation
duration was enhanced. Three measurements were carried out, namely the percentage of can-
nibalism, the snail mortality and the egg desiccation. Temperature, humidity and their interaction
had a significant influence on egg cannibalism. For every combination of these two factors, the
percentage of hatchlings having consumed the available egg increased with time. Those snails
not fed could survive at least four days at high humidity. However, four days after hatching, non-
cannibalistic hatchlings exhibited a higher mortality than cannibalistic ones, essentially due to
humidity constraint. Hence, egg cannibalism may represent an adaptive mechanism to resist to
adverse climatic conditions, such as high temperature and low humidity. In natural conditions, a
higher rate of egg cannibalism might be expected at low temperature and low humidity in com-
parison with the one observed in this experiment. Egg cannibalism by juveniles might improve
survival during adverse conditions (via water ingestion) and would be able to affect other life his-
tory traits, such as the subsequent growth rate of juveniles having consumed one egg immedi-
ately after the hatching. It might represent a mean to avoid juvenile size-dependent mortality due
to the seasonal variations of abiotic conditions.
Key words: nutrition, intraspecific oophagy, egg cannibalism, proximate factors, Helix aspersa.
INTRODUCTION
Egg cannibalism in hatchlings of different
land snail species has been reviewed by Baur
(1992). In pulmonates, two species have been
especially investigated, namely Helix pomatia
Linnaeus (Baur, 1988a,d) and Arianta arbus-
torum Linnaeus (Baur 1988a-c, 1993; Baur 8
Baur, 1986). However, few studies deal with
this aspect of juvenile nutrition in Helix as-
persa Múller, although Elmslie (1988) demon-
strated its existence in hatchlings of this
species and Fearnley (1993) gave hypothe-
ses on its consequences in life history. As in
H. pomatia (Baur, 1988d), oophagy is re-
stricted to new-born snails during the hatch-
ing period.
Different life history traits may be affected
by oophagy; for example, subsequent sur-
vivorship and growth enhancements were ob-
served in the snail H. aspersa having con-
sumed one egg immediately after hatching
(Desbuquois, 1997). Adaptation for terrestrial
167
life involves strategies of survival and devel-
opment under various proximate factors.
Special forms of behaviour, namely aestiva-
tion and hibernation, may have evolved in
land snails to offer some resistance to ad-
verse abiotic conditions (Riddle, 1983). Such
dormancies have a diapause value in H. as-
persa (Bailey, 1981, 1983; Lorvelec 1988)
and, in the context of life history theory, are
often related with an adversity selection
(Greenslade, 1983). In the same way, egg
cannibalism might be one of these behav-
ioural tactics with important repercussions on
survival during winter via the size reached by
juvenile snails before diapause (Biannic,
1995).
Within-clutch egg cannibalism variation can
be related to microhabitat differences, espe-
cially in temperature and moisture (Baur,
1994; Baur & Baur, 1986), which were also
subject, in Western Europe, to great variation
during daily and annual cycles. Thus, the aim
of our study is to test the influence of this vari-
168 DESBUQUOIS & MADEC
ation during hatching, on the extent of egg
cannibalism in hatchlings of H. aspersa.
MATERIAL AND METHODS
Relevant Natural History
The hermaphroditic land snail H. aspersa
lays its eggs in batches in a nest excavated in
the soil. One to three clutches are deposited
per breeding season, with a mean clutch size
of around 100 eggs. Newly hatched snails
might remain in their nest from 3 to 16 days
after hatching (6 days on average) before
emergence (Herzberg & Herzberg, 1962); this
may allow first born hatchlings to feed on un-
hatched eggs. Juvenile and adult snails are
herbivorous.
In the present experiment, adults of H. as-
persa were collected in August 1994 in a pop-
ulation living in the salt-pans at Guerande
(South Brittany, France). They were main-
tained in hibernation in a room at 4°C for three
months before breeding.
Breeding
The study was carried out on egg-layings
obtained from 20 January to 23 March 1995
from adults reared in polythene boxes under
constant artificial conditions (temperature:
20 + 1°C, relative humidity: 80 + 5%, 18/6
light/dark cycle) promoting reproduction
(Daguzan, 1981). They were fed with a cereal
composed snail food (produced by the com-
pany Arrivé) supplied ad /ibitum and renewed
twice a week. Four laying jars containing moist
soil were placed in each cage allowing snails
to deposit their clutches.
Experimental Conditions
After each clutch was washed, it was di-
vided into two equal groups of eggs, which
were incubated in small Petri dishes (diame-
ter: 55 mm) lined with moistened filter paper to
obtain air moisture saturation. Half of the eggs
were placed at 15°C to slow down the embry-
onic development, the other at 20°C to obtain
the snails that will be fed with the 15°C-incu-
bated eggs. This method, used by Baur
(1988a, 1993) and Baur & Baur (1986), al-
lowed to reproduce the natural hatching asyn-
chronism, that is, a delay between the first
and the last hatching. According to Le Calve
(1987), these conditions lead to a hatching
delay of about nine days for the 15°C-incu-
bated eggs.
Each new-born snail (one day old) was
placed on a moistened filter paper disc in a
small aluminium container (diameter: 20 mm;
height: 6 mm), open at the top, and received a
conspecific egg incubated at 15°C.
Animals tested were maintained in the
shade at different temperatures in hermeti-
cally closed plexiglass boxes (24 x 18 x 10
cm). Constant humidities were obtained within
the boxes by means of NaOH/water mixtures
(Madge, 1961), which were introduced more
than three days before starting the experi-
ment. Three temperatures (15, 20, 25°C) and
relative humidities (40, 70, 100% R.H.) and
their eventual interactions were tested. The
ranges of air temperature and relative humid-
ity chosen were commonly encountered in
South Brittany during the breeding season of
this species. Containers were maintained
above the NaOH solution with a wired prop
placed 2 cm above the bottom of the boxes
and were covered with a polythene net. The
boxes were opened daily in order to allow ob-
servations and air renewal; at the same time,
a drop of water was deposited on each disc of
paper.
Measurements Used
Egg predation was observed under 12.5x
magnification; the rate of egg cannibalism
(percent) was defined as the ratio of hatch-
lings having consumed the egg divided by the
number of snails tested per batch. Snail mor-
tality was equal to the number of hatchlings
dead divided by the number of snails tested
(individuals of all clutches were considered to-
gether). When an egg was dehydrated, his
weight became very low and his colour white
and opaque. Egg desiccation was the delay
(in days) before each egg not consumed in a
batch was dehydrated. Three or four clutches
were used for each combination of tempera-
ture and humidity. A total of 1,555 hatchlings
was tested for egg cannibalism in the different
thermohygrometric associations (i.e., from
130 to 260 hatchlings per combination).
Statistical Analyses
First, we calculated regressions of the per-
centage of egg cannibalism on the age in the
different thermohygrometric conditions. Then,
the effects of temperature, relative humidity
and their eventual interactions on egg canni-
ENVIRONMENTALLY-INDUCED VARIATION OF EGG CANNIBALISM 169
balism were tested using ANOVA on residuals
of regressions, that is, log(age) was intro-
duced as a covariable in the analysis. SNK
multiple comparison tests were carried out
when means were heterogeneous. The per-
centages of mortality were compared using
x2 tests of association. The assumption of
normality of the residuals was checked using
Lilliefors test on BIOMECO (1988). Other
analyses were run with MINITAB (1991).
RESULTS
Egg Cannibalism
For every combination of temperature and
humidity, the percentage of hatchlings having
consumed the available egg increased with
the logarithm of time (Fig. 1). Temperature
significantly affected egg cannibalism by
hatchlings (Table 1; ANOVA, P < 0.001):
oophagy was highest at 20°C and 25°C and
significantly lower at 15°C (SNK tests, P <
0.001). The percentage of egg cannibalism
was also different according to relative humid-
ity (Table 1; P < 0.001) and was higher at
100% R.H. than at the two other humidities
(SNK tests, P < 0.005). Moreover, a significant
interaction between the two factors studied
was found (Table 1; P < 0.001) especially in-
volving the 20°C-100% R.H. and 25°C-100%
R.H. modalities (SNK tests, P < 0.05).
Other Mortality Factors
Snail survival and egg desiccation were
highly affected by humidity and, to a lesser ex-
tent, by temperature.
Thus, hatchling mortality was higher and
earlier at 40% R.H., much lower and later at
100% R.H. After 4 days, mortality was signifi-
cantly higher in non-cannibalistic hatchlings,
that is, snails having not consumed the avail-
able egg, except for 15°C-100% and 20°C-
100% В.Н. (y2 test, P < 0.001), where no mor-
tality occurred (Table 2A). In low relative
humidity (40% R.H.) or high temperature
(25°C) conditions, lethal water loss was espe-
cially important in non-cannibalistic hatch-
lings. After 6 days, results were not different
but several combinations could not be tested
because of egg desiccation which prevented
snails from cannibalism (Table 2B). Obviously,
humidity drastically affected the time of dehy-
dration of the eggs (Table 3). At 100% relative
humidity, eggs did not suffer from desiccation
with time so they could be consumed more
than ten days after the beginning of the ex-
periment. At 40% R.H., all eggs were dehy-
drated after 2 to 4 days. At 70% R.H., egg des-
iccation increased with temperature. Hence,
egg availability duration for potentially canni-
balistic hatchlings was different according to
thermohygrometric conditions.
DISCUSSION AND CONCLUSIONS
The present experiment shows that egg
cannibalism is influenced by the two environ-
mental variables studied which might operate
at two levels: (i) hatchling survival, and proba-
bly activity and time spent by hatchlings in the
nest, that is, egg cannibalism opportunity, and
(ii) egg desiccation and hatching asynchro-
nism of the clutch, that is, egg availability.
Obviously, the longer a hatchling is in pres-
ence of non-dehydrated eggs, the more likely
it is to eat them.
As was recorded in other mollusc species
(Machin, 1975), juveniles of H. aspersa are
particularly sensitive to dehydration: at 15°C
and 20°C, water loss occurs under 90% rela-
tive humidity; at a temperature of 25°C, three-
month-old snails lose water even if they are
maintained at 100% relative humidity (Cha-
rrier, 1980). Klein-Rollais (1993) showed that
the rate of water ingestion of juvenile snails
during the first weeks of life was highest
above 20°C, and decreased when the relative
humidity increased from 60% to 100%. Under
mild climatic conditions, individuals may sur-
vive more than four days without feeding,
whereas under harsh conditions of tempera-
ture and relative humidity, mortality is very
high in non-cannibalistic hatchlings. Thus,
snail mortality is greatly influenced by temper-
ature-humidity interaction through dehydra-
tion and impossibility of water intake. There-
fore, egg cannibalism, through water intake,
might be considered as an adaptive mecha-
nism which allow some resistance to condi-
tions which promote water loss.
In H. aspersa, locomotor activity is influ-
enced by snail water content (Charrier, 1980;
Biannic, 1995), which is itself dependent on
temperature and humidity (Klein-Rollais,
1993). Thus, as in other slug and snail species
(Prior, 1985), activity of H. aspersa is also
closely related to these two factors (Herzberg
& Herzberg, 1962; Dan, 1978). Moreover, ju-
veniles are active in a higher range of temper-
atures and humidities than adults (Biannic,
170 DESBUQUOIS & MADEC
40% RH y=5.42log(x) + 3.05 r=0.27 p=0.554
70% RH y = 19.58log(x) - 1.96 r=0.52 p= 0.039
100% ВН y = 38.43log(x) - 10.06 г=0.67 p= 0.001
egg cannibalism (%)
age (days)
40% RH y = 12.33log(x) - 1.16 r=0.39 p= 0.268
70% RH y=50.77log(x) + 4.97 r=0.59 p= 0.050
e
Е 100% RH y = 105.71log(x) - 8.33 r=0.91 р= 0.001
3
2
;
58
o
age (days)
40% RH y=41.34log(x) - 5.13 r=0.83 p=0.010
> 70% ВН y = 12.40log(x) + 6.20 r=0.49 p=0.216
Е 100% RH y = 84.66log(x) + 6.18 г= 0.92 p = 0.001
3
ES
-
50
>
age (days)
FIG. 1. Regressions between the rate of egg cannibalism and the age of hatchlings of Helix aspersa in rela-
tion to ambient temperature (T) and relative humidity (RH).
1995). Thus, the two factors tested are indi- desiccation which influences egg availability.
rectly able to affect the extent of egg canni- Eggs are particularly sensitive to dehydration
balism. (Machin, 1975; Riddle, 1983). At 25°C, the rate
In addition, egg cannibalism was also de- of desiccation of isolated eggs of H. aspersa
pendent on these two abiotic factors via egg placed at 25% R.H. is 6.4 times higher than for
ENVIRONMENTALLY-INDUCED VARIATION OF EGG CANNIBALISM
171
TABLE 1. Analysis of variance for egg cannibalism by hatchlings of Helix aspersa according to
ambient temperature and relative humidity
Source of variation Degrees of freedom
Temperature (T) 2
Relative humidity (RH) 2
T x RH 4
Error 102
Sum of squares F-values P
33.09 20.87 <0.001
18.03 24.61 <0.001
18.67 11.59 <0.001
41.07
TABLE 2. Mean values of natchling mortality (%) of Helix aspersa according to ambient tem-
perature and relative humidity (left value: cannibalistic hatchlings; right value: non-cannibalistic
hatchlings) (number of clutches in parentheses)
A. After four days of life. B. After six days of life (When no value appears, the experiment was
stopped before day six because all the eggs were dehydrated)
Temperature (°C)
A Relative humidity (%) 15 20 25
40 28.6/85.2 (3) 16.7/53.5 (3) 0.0/43.8 (3)
70 0.0/6.9 (3) 0.0/3.3 (3) 0.0/19.4 (3)
100 0.0/0.0 (4) 0.0/0.0 (3) 0.0/4.8 (3)
Temperature (°C)
B Relative humidity (%) 15 20 25
40 — — —
70 0.0/8.3 0.0/3.8 —
100 0.0/0.0 0.0/20.0 0.0/4.8
eggs maintained at 90% R.H. (Bayne, 1968).
Thus, egg cannibalism decreased when tem-
perature increased and/or humidity decreased
because eggs lost water and became uneat-
able.
There is also a negative correlation between
the humidity and the durations of incubation
and hatching in H. aspersa (Guéméné 8
Daguzan, 1983) and a positive one between
temperature of incubation and hatching syn-
chronism (Le Calvé & Daguzan, 1989). Ther-
mohygrometric conditions can increase hatch-
ing asynchronism of clutches incubated in
natural conditions and lead to egg cannibal-
ism. The occurence of oophagy is also related
to the time spent by hatchlings in the nest.
Temperature enhances this period from four
days at 25°C to 10 days at 15°C (Le Calvé,
1987). A low soil humidity may also increase
this delay, because dry soil might prevent
snails from emerging.
In natural clutches, as eggs are arranged in
groups, egg dehydration is significantly lower
and outer eggs may dry more rapidly (Bayne,
1969). Therefore, in a dry soil, outer eggs of
the clutch dehydrate more rapidly and hatch
later than the inner ones, so that they may be
TABLE 3. Range of egg dehydration time (in days)
of all eggs available of several batches of Helix
aspersa according to ambient temperature and rel-
ative humidity (number of clutches in parentheses)
Temperature (°C)
Relative
humidity (%) 15 20 25
40 2-4 (3) 2-4 (3) 2-4 (3)
70 >10 (3) 2-5 (3) 2-4 (3)
100 >10 (4) >10 (3) >10 (3)
consumed by newly hatched snails from the
internal eggs, as hypothetised by Baur 8 Baur
(1986) for A. arbustorum. In A. arbustorum,
Baur (1988b) noted a preference for wet ovi-
position sites and assumed that this choice in-
duced a higher hatching success. Egg canni-
balism might therefore be influenced by the
choice of the oviposition site (parental manip-
ulation), which produces an alteration of the
hatching asynchronism (Baur, 1992).
In snail farms, the rates of hatching are
often around 70% to 90% (Daguzan, 1981);
although no value on egg cannibalism are
available in the literature, it seems to be rare.
172 DESBUQUOIS & MADEC
In the wild, numerous causes of egg mortality
exist in the clutches, but the extent ofegg can-
nibalism is unknown.
High rates of egg cannibalism observed in
this experiment compared with farm studies
and probably wild conditions might be ex-
plained by two reasons: (i) the incubation time
was artificially prolonged for half of the hatch;
natural hatching asynchronism is probably
lower in field conditions for the reason that the
eggs of a clutch never undergoes such differ-
ent environmental conditions, that is a range
of 5°C in a egg laying site. In the case of high
temperature and humidity in natural condi-
tions or in snail farms where incubation condi-
tions were nearly constant, the hatching syn-
chronism of the eggs might have prevented
egg cannibalism, (ii) in snail farms and when
the conditions were favourable in the wild, the
time spent by hatchlings in the nest is low.
Thus, the length of time during which hatch-
lings may consume eggs is lower than in this
experiment. On the other hand, egg cannibal-
ism in natural conditions may increase at low
temperature and low humidity in comparison
with our results because hatchings were not
synchronized and the length of time spent in
the nest was high.
The low availability of an alternative food
tends to favour cannibalism in different spe-
cies (Elgar & Crespi, 1992). However, prelimi-
nary experiments demonstrated that new-
born hatchlings of H. aspersa exhibited similar
rate of cannibalism in presence and absence
of humus (unpublished data). Thus, the lack of
an alternative food can not explain the high
rates of egg cannibalism observed in this ex-
periment.
According to Baur & Baur (1986), egg can-
nibalism during dry weather might be a sur-
vival mechanism for the reason that nutritional
and energetic benefits of egg consumption
allow an increase in growth and survival of
hatchlings. In addition, water intake by egg
feeding leads, in those dry conditions, to a re-
hydration of hatchlings, which gives them the
opportunity to wait for humidity and therefore
increases their survival. Because of a juvenile
size-dependent mortality based on harsh con-
ditions encountered in winter for European
populations of Helix aspersa, a high juvenile
growth rate might represent an interesting ele-
ment to avoid mortality due to low tempera-
tures during winter, because juveniles with
shell breadth below 19 mm are not able to re-
ally hibernate and thus, exhibit high mortality
(Biannic, 1995). The plasticity of egg size and
the correlation between egg size and hatchling
size may lead to a seasonal enhancement of
the juvenile growth rate, for autumnal clutches
(Madec, 1989; Madec et al., in press). Egg
cannibalism may be an alternative solution
favouring higher growth rates. On that ac-
count, egg cannibalism and its plasticity, via
their action on juvenile survival and on size at
maturity (in preparation), may become an im-
portant component of the fitness in popula-
tions subject to periods of dryness.
ACKNOWLEDGEMENTS
We would like to express our grateful
thanks to L. Chevalier and R. Spittal for cor-
recting the English text, and to anonymous
referees for helpful advices.
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Revised ms. accepted 30 September 1997
MALACOLOGIA, 1998, 39(1-2): 175-182
EXPRESSED SEQUENCE TAGS (ESTs) OF BIOMPHALARIA GLABRATA,
AN INTERMEDIATE SNAIL HOST OF SCHISTOSOMA MANSONI:
USE IN THE IDENTIFICATION OF RFLP MARKERS
Matty Knight', Andre N. Miller', Мей $. М. Geoghagen”, Fred A. Lewis’,
& Anthony В. Kerlavage”
ABSTRACT
To identify some of the genes expressed in the snail Biomphalaria glabrata, a major interme-
diate host for the trematode parasite Schistosoma mansoni, we sequenced random cDNA clones
from either a whole body or a cerebral ganglia cDNA library to generate 111 expressed sequence
tags (ESTs). Searches of existing public databases showed that the majority of the snail se-
quences (54%) showed no significant homology to genes listed in either DNA or protein data-
bases. Thirty one EST sequences showed significant matches with other genes in the databases.
These included genes involved in gene expression, such as ribosomal proteins and translation
factors, and those involved in cell communication, such as acetylcholine receptor and ATP-de-
pendent transporter. Some ESTs used as probes demonstrated the occurrence of restriction
fragment length polymorphisms (RFLPs) between parasite-resistant and parasite-susceptible
snail stocks. Southern hybridization of parasite DNA with the snail EST encoding the acetyl-
choline receptor as probe showed the presence of a related sequence in the parasite genome,
with the heterologous probe, indicating that this may be a useful method to identify closely re-
lated genes between the host and parasite.
Key words: Biomphalaria glabrata, cDNA libraries, Expressed Sequence Tags (ESTs), RFLP,
Schistosoma mansoni resistant/susceptible.
INTRODUCTION
Considerable progress has been made in
the analysis of genes of complex organisms
by partial sequencing of random cDNAs. This
method of generating expressed sequence
tags (ESTs), first developed by Adams et al.
(1991) for the human genome project, has
rapidly expanded our knowledge of the iden-
tity and diversity of transcripts in organisms
for which relatively little genetic information
previously existed. For the parasitic helminths,
Schistosoma mansoni and Brugia malayi for
example, ESTs compiled in recent years have
greatly expanded the number of cloned tran-
scripts listed in DNA and protein databases
from these organisms (Franco et al., 1995;
Blaxter et al., 1996; Johnston, 1997). The
identification of previously unknown genes
from these invertebrates, and homologies
with those from other organisms, may lead to
a better understanding of their biology and to
more effective treatment of the diseases they
cause.
Much less is known about the genes of the
vectors or intermediate hosts that transmit
these parasites. For the mollusc Biomphalaria
glabrata, an important intermediate host of
Schistosoma mansoni, infection by the para-
site is influenced by both snail and parasite
genes (Richards, 1973, 1975). In adult snails,
resistance to parasite infection is controlled by
a single gene, which is inherited in Mendelian
fashion, with resistance dominant. In juvenile
snails, resistance is believed to be a polygenic
trait, which is based on the complex interac-
tion of other yet unknown genetic factors
(Richards & Merrit, 1972). A more detailed un-
derstanding of both parasite and snail genes
involved in this parasite-host interaction may
lead to new methods for schistosomiasis con-
trol.
Compared to some other invertebrates that
serve as intermediate hosts or vectors for
major human diseases, relatively little is
known about the molecular make-up of B.
glabrata; no genetic or physical maps exist,
and very few genes have been analyzed. A
‘Biomedical Research Institute, Rockville, Maryland 20852, USA
The Institute for Genomic Research, Gaithersburg, Maryland 20850, USA
175
176 KNIGHT ET AL.
search of sequences in GenBank showed only
five B. glabrata sequences listed. Relatively
few molluscs have been karyotyped (Burch,
1967; Patterson & Burch, 1978), and few stud-
ies have been conducted on the cytological
analyses of chromosomes from different
strains of B. glabrata chromosomes (Goldman
et al., 1984). Based on the genome size of an-
other closely related freshwater pulmonate
gastropod, Lymnaea stagnalis, the B. glabrata
genome is expected to be about 5.0 x 10° bp
(Boer et al., 1977).
Using snails of different parasite suscepti-
bility phenotypes, we previously described the
identification of restriction fragment length
polymorphisms (RFLP) and random amplified
polymorphic DNA (RAPD) markers that can
be used to distinguish some of our laboratory
maintained, genetically selected resistant and
susceptible snails (Knight et al., 1991; Larson
et al., 1996). In the present study, we have
used the isolation of ESTs to identify B.
glabrata genes and to search for polymor-
phisms within some of these genes. This may
be useful for conducting genetic linkage stud-
ies to identify genes associated with either re-
sistance or susceptibility in B. glabrata. In this
paper, we describe the identification of new B.
glabrata ESTs from a survey of whole body
and cerebral ganglia directional cDNA 1-
braries from a S. mansoni-resistant snail (BS-
90), and report the occurrence of RFLPs with
some of these genes.
MATERIALS AND METHODS
Snails
The BS-90 snail line of В. glabrata is resis-
tant at any age to S. mansoni infection
(Paraense & Correa, 1963) and was made
available to us by Dr. E. S. Loker (University of
New Mexico). The M-line snail was selected
for high susceptibility to S. mansoni infection
by Newton (1955). Both snail lines were main-
tained as previously described (Miller et al.,
1996).
RNA and DNA Extraction
Snails used for nucleic acid extraction were
cleaned and kept overnight in sterile water
containing 0.1 mg/ml ampicillin. RNA was ex-
tracted from either the whole body or cerebral
ganglia. For whole body extraction, snails
were crushed with a pestle into a fine powder
under liquid nitrogen on dry ice in a chilled
mortar. RNA was extracted with Rnazol as de-
scribed by the manufacturer (Sinna Biotech).
Cerebral ganglia were dissected from 10 indi-
vidual adult snails (12-14 mm), plunged di-
rectly into Rnazol on ice, and extracted imme-
diately. DNA was extracted from adult snails
(10 mm) by a combination of the methods de-
scribed by Knight et al. (1991) and Winnepen-
ninckx et al. (1993). Briefly, snails were
crushed into a fine powder as described
above, and the powder was mixed, by inver-
sion, into 10 ml pre-warmed (60°C) extraction
buffer containing 2% cetyltrimethylammonium
bromide (CTAB) (w/v), 1.4M NaCl, 0.2% (v/v)
B-mercaptoethanol, 20 mM EDTA pH 8.0, 100
mM Tris-HCI pH 8.0 and 100 ug/ml proteinase
K (Boehringer Mannheim) and incubated for 1
h at 60°C. Samples were extracted sequen-
tially with an equal volume of phenol, phe-
nol/chloroform (1:1) and chloroform. The
aqueous phase was digested with RNase A
(20 ug/ml) for 1 h at 37°C, and extractions
were repeated as described above. DNA was
recovered by spooling after the addition of 2.5
volumes of ethanol (—20°C). Spooled DNA
was washed in 70% ethanol (—20°C), air
dried and resuspended in sterile dH,O at a
concentration of 0.1 mg/ml. Restriction en-
zyme digestions of DNA samples were per-
formed as described by Knight et al. (1991),
except that DNA was heated for 5 min at 65°C
before enzymatic digestion. Digestions were
done overnight at 37°C with buffer supplied by
the manufacturer (New England Biolabs,
Massachusetts).
Construction of cDNA Libraries
The whole body snail directional cDNA li-
brary was prepared from 5 ug of poly A+ se-
lected MRNA in the phage vector AZAP using
the AZAP-cDNA synthesis kit according to
manufacturer's instructions (Stratagene, Cal-
ifornia). Briefly, first strand synthesis with re-
verse transcriptase was prepared by priming
with Xho l-oligo-dT primer, followed by second
strand synthesis with RNase H and DNA poly-
merase |. The final cDNA product was blunt-
ended and, after ligation of EcoR | linkers and
kinase treatment, was size selected on a
Sephacryl S-400 column. The cDNA recov-
ered was digested with EcoR | and Xho | and
cloned directionally (EcoR | at the 5’ end
and Xho | at the 3’ end) into the AZap vector
EcoR \/Xho | phosphatase treated arms.
Packaging was performed using packaging
EXPRESSED SEQUENCE TAGS OF BIOMPHALARIA 1777
extract (Gigapack gold) from Stratagene and
plated out on E. colistrain XL1-blue MRF’. The
library consisted of 1.1 x 10° independent re-
combinants with average insert size of 1000
bp.
The cDNA library of cerebral ganglia was
constructed as described above, with the ex-
ception that first strand cDNA was prepared
from total RNA (13.3 ug) extracted from cere-
bral ganglia from snails exposed for 5 h to 25
S. mansoni miracidia. The AZap cerebral gan-
glia directional cDNA library has 1.0 x 10° re-
combinants. Phagemids were prepared by
mass excision of the libraries by co-infection
with helper phage R408 and plating on E. coli
(Sure strain) according to the manufacturer’s
instructions (Stratagene). Individual random
colonies, selected by plating on IPTG/Xgal
plates, were transferred in ordered array into
Super broth (100 ul) in a 96-well microtitre
plate. Phagemid DNA was prepared from
each well after 37°C overnight incubation,
with the mini-prep DNA isolation kit (Wizard,
Promega, Wisconsin). Partial sequencing was
on double stranded templates in the forward
and reverse directions with fluorescent M13
universal primers and automated sequencers
(Applied Biosystems 373A) (Adams et al.,
1995). Nucleotide and protein sequence
searches were conducted as described by
using the algorithms BLAST (Altschul et al.,
1990) and BLAZE (Brutlag et al., 1993), re-
spectively.
Southern Hybridization
Restriction enzyme digested DNA was
loaded (10 ug/lane) onto 0.8% TBE agarose
gels and resolved by horizontal flat bed elec-
trophoresis. Southern transfer onto nylon
membranes (Nytran, Schleicher & Schuell)
was performed using 10x SSC according to
the standard method (Southern, 1975). DNA
was immobilized by UV cross-linking and bak-
ing for 2 h at 80°C.
Hybridizations were performed overnight at
42°C in the presence of 50% formamide and
10% Dextran sulfate in 2x SSPE, 5x
Denharts, 1% SDS and 100 ug/ml of soni-
cated salmon testes DNA. Probes were made
from individual phagemid DNA by labeling
with ®P-dCTP (6000 Ci/mmole, Amersham)
by the random priming method as described
by Feinberg & Volgelstein (1983). Blots were
washed at 55°C in 0.2x SSC and 0.1% SDS
and set up for autoradiography at —70°C, for
2-5 days, with intensifying screens.
RESULTS & DISCUSSION
Sequence Analysis and Identification of
B. glabrata ESTs
Of the 190 clones sequenced (95 from each
library), 111 provided usable sequences. An
average of 322 bases was obtained from ei-
ther the 5’ or 3’ ends. The standard for elimi-
nation of unwanted and ambigous sequences
(vector and poly A tail) was performed as de-
scribed by Adams et al. (1995). Searches of
peptide sequences were performed from all
six possible reading frames. Table 1 shows the
number of ESTs divided into categories which
represent (1) sequences that show significant
homology to database sequences, (2) se-
quences that show no significant matches,
and (3) sequences of mitochondrial DNA. As
indicated, significant matches were detected
with 28% of the snail sequences, 54%
showed no homology to existing database se-
quences, and 18% of the sequences corre-
sponded to mitochondrial DNA. Because the
RNA utilized to construct the cerebral ganglia
library was not poly-A selected, we expected
to identify large numbers of ribosomal RNA
sequences, but none were detected among
the clones we sequenced. On the other hand,
the higher number of EST sequences corre-
sponding to mitochondrial-related sequences
in the cerebral ganglia library (14), compared
to the whole adult library (5), may be the result
of not using poly-A RNA as starting material in
the construction of the former library.
The 31 EST sequences that showed signifi-
cant matches with other genes in the data-
bases are listed in Table 2. Several sequences
identified corresponded to genes involved in
protein/gene expression (ribosomal protein,
translation elongation factor 1) or to genes in-
volved in cell communication (acetylcholine re-
ceptor, ATP-dependent transporter). From
the neural tissue library, we identified se-
quences with significant homologies (57%-
TABLE 1. Summary of В. glabrata ESTs
Cerebral
Libraries Whole Snail Ganglia Total
Database Match 15 16 31
Unknown 31 29 60
Mitochondrial 5 15 20
Total 51 60 111
178 KNIGHT ET AL.
TABLE 2. Database similarities of B. glabrata ESTs. ESTs with database matches are listed with their puta-
tive identification, the length, percent identity, and percent similarity ofthe match, and the accession number
of the sequence matched. Match lengths are in nucleotides. In addition to those listed above, EST188741
had 83% nucleotide identity with GB:M69023, which is misidentified as a human globin gene. We counted
this EST as unknown.
est# putative ID
Adult library
EST188651 acetylcholine receptor
EST188652 possible glycoprotein
EST188653 possible glycoprotein
EST188654 cystatin
EST188655 cystatin
EST188656 endo-1,3-beta-glucanase
EST188657 endo-1,3-beta-glucanase
EST188658 major secreted protein MPB70
EST188659 major secreted protein MPB70
EST188660 major secreted protein MPB70
EST188661 moesin
EST188662 ribosomal protein L13
EST188663 ribosomal protein L17
EST188664 ribosomal protein L17
EST188665 ribosomal protein S20
Cerebral ganglia library
EST188671 antigen HuD, neuronal
EST188672 ATP-dependent transporter
EST188674 DNA topoisomerase II
EST188675 DNA topoisomerase II
EST188676 FMRFamide precursor
EST188677 FMRFamide precursor
EST188678 FMRFamide precursor
EST188679 globin
EST188680 heat shock protein 90
EST188681 heat shock protein 90
EST188682 proclotting enzyme precursor
EST188683 ribosomal protein L41
EST188684 ribosomal protein S17
EST188685 ribosomal protein S17
EST188686 serine protease
EST 188687 translation elongation factor 1, alpha
61% similarity) to the neuropeptide FMRF-
amide precursor. Anumber of sequences from
both libraries were highly redundant. For ex-
ample, the EST sequences for ribosomal pro-
teins occurred frequently as did sequences for
housekeeping genes, hsp90 and DNA topo-
isomerase. The high frequency of certain
clones may either reflect the metabolic state of
the tissue samples when RNA was isolated
(abundant transcripts) or a bias created during
the manipulation (amplification and mass ex-
cision) of the libraries. Using the partial EST
sequence for globin, the corresponding full-
length cDNA has been isolated and se-
quenced from the cerebral ganglia library
(Dewilde et al., manuscript submitted). The
len %id %sim acc#
182 30.6 51.6 SP:P22770
233 34.2 51.9 GP:912490
233 34.2 51.9 GP:912490
155 36.5 SIT. PIR:S12913
239 30.9 48.2 PIR:A29632
296 45.5 58.6 GP:144808
242 47.6 59.8 GP:144808
119 42.5 60.0 PIR:A37195
119 45.0 62.5 PIR:A37195
119 45.0 62.5 PIR:A37195
182 59.7 88.7 GP:623040
119 50.0 65.0 SP:P26373
344 93.0 95.6 PIR:JC1253
242 91.4 95.1 PIR:JC1253
218 93.2 95.9 GP:292443
281 41.5 54.3 GP:179537
296 53.0 70.0 SP:P40024
104 42.9 60.0 SP:Q01320
104 42.9 60.0 SP:Q01320
233 43.8 61.2 SP:P42565
262 538 60.0 SP:P42565
143 3723 56.9 SP:P42565
389 30.8 49.2 SP:P02215
374 88.8 97.6 GP:256089
338 81.6 90.4 GP:256089
242 357 54.8 SP:P21902
74 92.0 92.0 GP:36136
164 78.2 89.1 GP:337501
101 82.4 82.4 GP:337501
500 29.2 47.0 GP:868212
293 79.6 85.7 GP:214111
111 B. glabrata EST sequences discussed in
this manuscript have been deposited in the
dbEST database of the National Center for
Biotechnology Information with the follow-
ing accession number: dbEST:1193734 to
1193844; GenBank: AA547685 to AA547795.
Identification of Polymorphic ESTs
Hybridization patterns were compared be-
tween genomic DNA from parasite-resistant
(BS-90) and -susceptible (M-line) snails,
using the ESTs as probes. As shown in Table
3, RFLPs were detected with some ESTs.
Most polymorphisms were observed with re-
striction enzymes Hind Ш and Hinf Ш. RFLP
179
EXPRESSED SEQUENCE TAGS OF BIOMPHALARIA
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180 KNIGHT ET AL.
analysis was conducted on either individual or
pooled snail DNA samples. Linkage studies,
using these EST RFLPs as probes, will be
performed on progeny snail DNA derived from
a cross between the resistant and susceptible
snails as part of an ongoing study to identify
sequences associated with either the resis-
tant or susceptibility loci in B. glabrata.
Hybridization of parasite DNA using heterol-
ogous snail ESTs as probe, in some cases,
demonstrated the occurrence of related se-
quences in the parasite genome. For example,
Figure 1 shows the hybridization of the
snail EST encoding acetylcholine receptor
(EST188651) to Hae Ш digested DNA from the
resistant snail (Lane 1), susceptible snail
(Lane 2) and S. mansoni (Lane 3). As indi-
cated, the snail probe detects a major frag-
ment (590bp) in both the snail and parasite
genomes. Considerable sequence homolo-
gies have previously been reported between
parasite and snail genes (Dissous et al., 1990;
Weston et al., 1994). Cross hybridization stud-
ies, using these snail probes, may be a useful
strategy to identify and clone corresponding S.
mansoni genes for which no sequence infor-
mation currently exists. Conversely, ESTs that
are currently being generated from the para-
site (Franco et al., 1995; Neto et al., 1997),
may serve as useful heterologous probes for
genome studies of the intermediate host.
Unlike the parasite, which shows an amino
acid codon preference for A/T in the third base
position (Meadows & Simpson, 1989), analy-
sis of the partial amino acid sequences gener-
ated from the B. glabrata ESTs does not reflect
a similar bias (data not shown). Full-length se-
quences will, however, be required in order to
assess relative structural similarities and di-
vergences between snail ESTs and related
parasite sequences.
This study shows that the generation of B.
glabrata ESTs is a useful approach that
should quickly expand our knowledge on the
molecular biology of this organism. Although
most of the sequences identified in this study
showed no homology to sequences listed in
existing public data bases, indicating they rep-
resent novel snail-related sequences, the ac-
cumulation of such sequences will help in our
collective efforts in this field towards the iden-
tification of genes involved in the genetic con-
trol of parasite infection in this snail host. The
ability to identify RFLP EST sequences pro-
vides a useful method to limit the search of
probes to known genes for which a biological
function can be ascribed. These polymorphic
Kbp 1.2.8
1.4 —
1.1 —
0.87 — Y +
¥
0.60 —
+ a
0.31 —
0.28 —
De. «&
FIG. 1 Southern blot of Hae Ill digested DNA from
resistant (1) and susceptible (2) B. glabrata snail
lines and S. mansoni (3) hybridized with EST probe
EST188651 (acetylcholine receptor).
EST markers can form the basis to begin to
create a physical map of the B. glabrata
genome.
The comparative analysis of the profile of
transcripts obtained from the two libraries
screened in this study suggests that for B.
glabrata, the generation of ESTs from various
tissues, rather than the whole snail, may be a
more meaningful strategy to adopt in the iden-
tification of transcripts that may be relevant to
a particular biological phenomenon. In this
context, work being conducted in our labora-
tory is employing an EST strategy to compare
the profile of transcripts, as described by Lee
et al. (1995), between hemocytes of resistant
B. glabrata snails, with and without parasite
exposure.
In summary, ESTs have been generated by
partial sequencing of clones from B. glabrata
cDNA libraries. Most sequences showed no
homology to sequences in existing data
bases. Some snail ESTs may serve as useful
probes to identify homologous genes in the
parasite. RFLPs identified with ESTs provides
useful markers to conduct genetic linkage
EXPRESSED SEQUENCE TAGS OF BIOMPHALARIA 181
studies for the identification of the parasite re-
sistance/susceptibility loci in this snail host.
ACKNOWLEDGMENT
This work was supported by NIH grant Al
Zeit.
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MALACOLOGIA, 1998, 39(1-2): 183-193
PHYLOGENETIC UTILITY OF THE 5'-HALF OF MITOCHONDRIAL 16$ rDNA
GENE SEQUENCES FOR INFERRING RELATIONSHIPS OF ELIMIA
(CERITHIOIDEA: PLEUROCERIDAE)
Charles Lydeard', John H. Yoder', Wallace E. Holznagel', Fred G. Thompson” & Paul Hartfield®
ABSTRACT
Mitochondrial 16S rDNA sequences have proven useful for mesolevel phylogenetic questions.
To date, most published studies have used primers that amplify the conservative 3’-half of the
gene. We recently developed primers that amplify an approximately 550 bp portion of the more
variable 5'-half of the gene. The primers work well for a wide range of gastropods tested. Because
the 5'-half of the gene exhibits greater variation than the 3'-half, we wanted to determine whether
there is sufficient phylogenetic signal for resolving relationships among closely related taxa. We
examined the utility of the 5’ portion by assessing relationships within the pleurocerid genus
Elimia of the Mobile River basin. Although the 433 bp data matrix possessed significant phylo-
genetic signal, and the resultant 320 most parsimonious trees had some aspects that were well
resolved, most of the phylogenetic signal seemed to be partitioned among genera. Only 36 phy-
logenetically informative sites were found within Elimia, which is too few to resolve all nodes for
such a diverse assemblage. We recommend the continued use of the 16S rDNA gene for stud-
ies examining intergeneric relationships of molluscs, and suggest researchers employ mito-
chondrial protein coding genes for interspecific studies.
Key words: mollusks, Cerithioidea, Pleuroceridae, Elimia, mitochondrial DNA, 16s rDNA.
INTRODUCTION
The introduction of conserved “universal”
primers (Kocher et al., 1989), which permit
amplification of specific regions of homolo-
gous DNA via the polymerase chain reaction
(PCR) (Saiki et al., 1985), has offered tremen-
dous opportunities for systematic studies.
Many of the earlier molecular phylogenetic
studies of molluscs have employed 18S or
28S nuclear ribosomal sequence data in an
attempt to resolve relationships among dis-
tantly related taxa. For example, the relation-
ships of molluscs to other metazoans (e.g.,
Ghiselin, 1988; Winnepenninckx et al., 1994),
gastropod relationships (e.g., Emberton et al.,
1990; Tillier et al., 1992; Rosenberg et al.,
1994), and unionoidean bivalve relationships
(e.g., Rosenberg et al., 1994). Although these
genes appear useful for resolving some as-
pects of higher-level relationships among mol-
luscs, they are too conservative to be of much
use for studying relationships among more
closely related genera or species.
Mitochondrial DNA has proven to be quite
useful for studying evolutionary relationships
of animals (Brown, 1985; Avise et al., 1987;
Moritz et al., 1987). Sequencing the complete
genome, although relatively labor intensive,
has provided data for studying molluscan rela-
tionships based both on nucleotide homology
and on mitochondrial gene order (Boore &
Brown, 1994). In contrast, restriction fragment
or site analysis of the mtDNA genome pro-
vides information for intraspecific population
structure (e.g., Reeb & Avise, 1990; Liu et al.,
1996). On the other hand, molecular phyloge-
netic studies employing mitochondrial DNA
sequence data are only recently becoming
more commonly conducted in malacology. The
¡Aquatic Biology Program, Department of Biological Sciences, University of Alabama, Box 870345, Tuscaloosa, Alabama,
35487 USA
2Florida Museum of Natural History, Museum Road, University of Florida, Gainesville, Florida, 32611 USA
3U.S. Fish and Wildlife Service Endangered Species Office, Dogwood View Parkway, Suite A, Jackson, Mississippi, 39213
USA
184 LYDEARDET AL.
delay is most likely due to the fact that many of
the first universal primers reported in the liter-
ature do not work well if at all for molluscs
(Spolsky et al., 1996). Thus, more effort is re-
quired on the part of the investigator to design
primers that will work efficiently and yield suf-
ficient variation for the question in hand.
Folmer et al. (1994) developed primers that
amplify a portion of the protein coding gene,
cytochrome c oxidase subunit | from a wide
range of invertebrate taxa, including molluscs.
These primers have since been used to exam-
ine the evolution of gender-associated mito-
chondrial lineages in bivalves (Hoeh et al.,
1996), and are now being used in a variety of
other systematic studies of freshwater unionid
mussels (Roe & Lydeard, 1998; Hoeh, pers.
comm.; Liu & Mulvey, pers. comm.) and gas-
tropods (Harasewych, pers. comm.). Primers
for another protein coding gene, cytochrome b,
have also been developed, which have proven
useful for studying relationships within the
gastropod genus Nucella (Collins et al., 1996)
and for intraspecific relationships within the
gastropod genus Oncomelania (Spolsky et al.,
1996). Mitochondrial protein coding genes ap-
pear to be useful for a wide range of system-
atic questions. This can be attributed to rate
variation among codon positions. For exam-
ple, third codon positions are under fewer se-
lective constraints and hence evolve faster
than first and second codon positions. Thus,
sufficient variation may be found to estimate
relationships even among closely related taxa.
In contrast, for deeper phylogenetic questions,
substitutions in the third codon position can be
downweighted or dropped (e.g., Lydeard &
Roe, 1997).
Mitochondrial ribosomal genes have been
of considerable use for systematics studies
(Hillis & Dixon, 1991). The genes are thought
to be more conservative than protein coding
genes and are therefore more useful for
meso-level phylogenetic questions. Recently,
the mitochondrial 16S rDNA gene has been
used successfully for estimating relationships
among genera of North American freshwater
unionids (Lydeard et al., 1996). The primers
used by Lydeard et al. (1996) were designed
by modifying the universal primers of Palumbi
et al. (1991), which amplify a 550 bp fragment
of the 3’ half of the gene. In another study,
Lydeard et al. (1997) developed primers that
extended into the 5’ half of the gene, which is
rarely sequenced in phylogenetic studies. The
combined data matrix of nearly 900 bp was
used to study relationships among three gen-
era of pleurocerid gastropods of the Mobile
River basin. Interestingly, the 5’ half of the
gene exhibits more variation than the 3’ half
(Gutell et al., 1992; Lydeard et al., unpubl.),
which lead us to believe the gene might be
useful for assessing relationships among
more closely related taxa. Here, we present
our analysis of the utility of the 5’ half of the
16S rDNA gene for examining relationships
within the gastropod genus Elimia. The pri-
mers used to amplify this region work well on
a wide range of gastropod species, so our
findings will be of interest to other malacolo-
gists interested in examining systematic rela-
tionships among similarly divergent taxa.
MATERIALS AND METHODS
Specimens Studied
Elimia were collected from various localities
throughout the Mobile River basin, but with a
particular emphasis on Coosa River species
(Appendix). Twenty-three Elimia specimens
representing eleven nominal species were in-
cluded in the analysis, along with three
Pleurocera prasinatum and one Leptoxis tae-
niata as outgroup taxa (Table 1).
Sequence Procurement, Alignment,
and Analysis
Genomic DNA was isolated from frozen or
80% ethanol-preserved specimens (typically
the proboscis or entire head of the snail; less
tissue seems to yield better quality DNA) by
standard phenol-chloroform extractions. Mito-
chondrial DNA sequences were obtained for
an amplified segment of the mitochondrial
16S rDNA gene using primers SNLOO2 (5’-
aaatgattatgctacctttgc-3" and SNL-448 (5'-
gaaatttcattcgcactag-3’). These primers were
designed by Lydeard et al. (1997) specifically
for pleurocerids and related gastropods, and
amplify an approximately 550 bp fragment at
the 5’ end.
Approximately 50 - 500 ng of genomic DNA
provided template for double-stranded ampli-
fications via PCR in 25 ul of Tris (67 mM, pH
8.8) containing 2 mM MgCl2, 1 mM of each
dNTP, 1 uM of each primer, and Tag poly-
merase (1.25 units, Perkin-Elmer-Cetus). The
amplification regime consisted of 30 cycles of
denaturation at 92°C for 40 s, annealing at
52°C for 60 s, and extension at 72°C for 90 s.
Single-stranded DNA was produced for se-
16S rDNA GENE SEQUENCES IN ELIMIA 185
TABLE 1. Taxa and number of specimens included
in the present study.
Pleuroceridae
Elimia catenaria group
E. crenatella (2)
Elimia carinocostata group
E. carinocostata (9)
Elimia gerhardtii group
Е. gerhardtii (3)
Elimia haysiana group
E. alabamensis (1)
E. haysiana (1)
Elimia hydei group
E. hydei (1)
Elimia olivula group
E. cylindracea (1)
E. olivula (1)
E. showalteri (1)
Elimia vanuxemiana group
E. fascinans (2)
E. caelatura infuscata (2)
Pleurocera prasinatum (3)
Leptoxis taeniata (2)
quencing via asymmetric amplification
(Gyllensten & Erlich, 1988) using low-melt
agarose (FMC BioProducts) purified double-
stranded PCR products as templates.
Reaction conditions for asymmetric PCR
were as above with the exceptions that one
primer was held limiting, and the final volume
of the reaction cocktail was increased to 50
uL. Asymmetric reactions were conducted for
each specimen using both amplification
primers in limited quantity in separate reac-
tions. Thermal cycling was performed in a pro-
grammable heating block (Perkin-Elmer-
Cetus) with negative (-DNA) controls included
with each reaction set.
Following purification by centrifugal filtra-
tion (Millipore Ultra-free-MC 30,000), single-
stranded DNA was sequenced by dideoxy
chain termination using Sequenase Version
2.0 (United States Biochemical) and instruc-
tions supplied by the manufacturer. Both
strands were sequenced using the appropri-
ate amplification primer as a sequencing
primer. The sequencing reaction products,
which included 32$ to permit autoradiographic
visualization, were run on 6% polyacrylamide
gels (Long Ranger, FMC BioProducts) from 2
to 4.5 h. Following electrophoresis, all gels
were vacuum-dried and exposed to X-ray film
for 48 - 120 h.
Sequences were initially entered in the soft-
ware program XESEE version 3.0 (Cabot &
Beckenbach, 1989) and subsequently aligned
using CLUSTALW version 1.5 (Thompson et
al., 1994) and visual inspection. In addition, a
consensus sequence of the ingroup taxa was
superimposed over the hypothesized sec-
ondary structure of the fruitflies Drosophila
yakuba and D. melanogaster (Gutell & Fox,
1988; Gutell et al., 1992) in an attempt to fur-
ther refine the alignment and identify regions
corresponding to hypothesized loops and
stems. Pairwise percent sequence differences
were calculated using Kimura’s two-parame-
ter model (Kimura, 1980), which corrects for
multiple hits using the software package
MEGA (Kumar et al., 1993).
The phylogenetic analyses were conducted
using maximum parsimony of the orthologous
sequences using the heuristic search option
(10 replicates) of PAUP (version 3.1.1;
Swofford, 1993). We employed the following
options in PAUP: uninformative characters
were ignored, only minimal trees were kept,
gaps were treated as missing data, and zero-
length branches were collapsed. A bootstrap
analysis (Felsenstein, 1985) with 100 itera-
tions was conducted to estimate the internal
stability of the data matrix. Although we usu-
ally prefer to run more iterations, this particu-
lar data set tested the limits of memory for
more than 100 replicates. A skewness test
statistic (g1) was calculated based on the dis-
tribution of tree lengths of a random sample of
10,000 topologies. Data matrices with a
strong phylogenetic signal are significantly
more structured than random data (Hillis &
Huelsenbeck, 1992).
RESULTS
Multiple sequence alignment of the ampli-
fied region from our selected taxa resulted in
a data matrix consisting of 433 nucleotide po-
sitions (Fig. 1) including insertions and dele-
tions. Several DNA sequences from conspe-
cific specimens were identical, and therefore
combined in subsequent analyses: Elimia cre-
natella (cren18 = 41A-2), E. carinocostata
(5A-2 = 11A-1), E. caelatura infuscata (16-1 =
16-2), and Leptoxis taeniata. (2 individuals, 1
locality). Thus, 25 unique sequences from 28
individuals. Of the 433 nucleotide positions
examined, 135 (31%) are variable among all
taxa, including the outgroups, and 71 (16%)
are potentially phylogenetically informative.
Within the genus Elimia, 75 (17%) are vari-
able and 36 (8%) are potentially phylogeneti-
cally informative.
1 . o o > a 60
E. carino 11A-1,5A-2 GACGAGAAAATAATTATAAAATATTAATTA-TTTCATAAAATATTTCTCGATTAATTTTT
E. carino 11A-2 ec... :..... Oleletelatatefalefatetotefoleteletetelermieiefeleteierereieinieisteie cesser С
E. carino 11B-1 noodoon0000d00000000e соо Erecrceeececcec-cr-cc-cc-c-cece od
E. carino 11B-2 elev Lioieleicicheleychotetelotsicisleleievoisteteictetehels 900800 slofeteletetafeVeketeferoletotenetelntefeieke
E. carino 47A-2 59566096 OVINA =felefatelsie.eleieie siofederereteietererctetorerereiatete
E. gerhard 10-1 NNN...... ооовообъововововаоа ао SIDAD DUDAS OSOS
E. gerhard 10-2 oeDeccccccce ооо совосео о 00 ODO OOO eletololalataletelerotekefefeke
E. gerhard 42-1 NNN....... HO DA 00000 0 0010 ala messes. soso...
E. alabamensis combo dove 000000000000 SHOT RAR DOOR OOOO
E. haysiana ооо ба о ADOC OD CODCOD AGCCOGO one secs ses VOS ere
E. olivula ооо дою6а овоозоововасывововов ее ее
Е. су11пагасеа Теа Soagecoono0000 000000 Coco adasaa JTONO DONOSO
E. hydei Doro с ооо ссеваеосо С-.----- ss Lele risas Ааа е
Е. carino 46А-2 МЫМЫМ еее еее ос OOO ODIO асов oO
E. carino 49A-2 ste Tleroleletejatelefererststefekelstersterere steleletere se CTes- SOON ..
E. carino 49B-2 AMOO OOOO OO ROO == CT... [Gauss А-а-а
E. fascin 7-1 INEA a Geteteiele/nleleiee ee less ter lito
E. fascin 7-2 ale DierorefokeleteVoraraze ее Пес is
E. cael inf (2) ale Veteforeteteferoreteisiala,e эро NN o sao Gio tatoo atea Seesen
E. crenatella (2) Less. Clefeteejeleletatereisterslefe/eiereie lave} oil OSO OOOO eee
E. showalteri saone SOS aan ее бое ec
P. pras 12A-1 TA (dados baba ooo > Lis cic cle OOOO Termes.
P. pras 12B-1 Se DA еее еее ет. po bo see s-css-sccee
P. pras 12B-2 OOOO OO OOOO oo... 00.0 Lies sise oies ce ste = lle - ec ооо
L. taen (2) CGGCT:G-S CE Create AOS А... ТСС. СТО:
61 o 5 5 = 5 120
Е. carino 11А-1,5А-2 TTGAGGATAAGCTCGAAAAAAAGTTAAGAAATTTTACTAATTTAGGTT---ATTATGTGG
Е. carino 11A-2 FOOD SOS IDOLO csscese === ore lelado
E. carino 11B-1 ° eeiseemeecene cles ces siecle eos es serce ее ЕЕ ее 6600
E. carino 11B-2 FONDOS OSONA SOUUDOOOCOOUDOOOCOCODOG see ——— eee es siele
Е. carino 47A=2 зе arelatereYe/erefolerelerefererefekarere aferekorerefeieteferersterete === OOOO о
E. gerhard 10-1 atatetalelarereye ors olelsiereretalateferateleratotereierotevetonelcveters) etarare) OO SS O LO OOOO
E. gerhard 10-2 = ..oooooo... еее sieste еее соо еее === its
E. gerhard 42-1 съ oooooo... tales oa alot soso see ss.
E. alabamensis ее еее ce Sec eseeses eee secte eee secs.
E. haysiana as ss S0ODÍNE ---...... Stele
E. olivula о OOD (dosgooaanoacoboo daba 000 0550000 с
Е. cylindracea 00 DO UOD IE Creole siete aaa OO IO OOO SON
E. hydei concrrocscsananasa ss... С.А. ..-Т.....С.....---б.С.....-
Е. carino 46A-2 Мое = BOOOUCOD DUD OH ec sens see === OO
E. carino 49A-2 бабоовеьаосзьсаоносоасасоюо босзос OOOO DO es === еее
Bier Carino) 49B=2) еее еее еее Siafeislateistera/siaslele/sie.e.e os...
El tascin 7"... otras еее еее еее ее ses еее Coco...
E. fascin 7-2 ec cLecr-crecr 000000000 Vo dona aaa == Cilia
E. cael inf (2) ooneone.e ela GA - 2e ce eee secs cts cos...
E. crenatella (2) tala see === ecelseie ss see ее А. ть. A
Е. showalteri se... SOC ODO MOO nOOCOOO SOTO еее OOO ss...
Р. pras 12A-1 съ Се Ты СААСье,- = ....---б........
Р. pras 12B-1 = ..... OSCAR Gaelle: Gle Gestes ....---С6........
Р. pras 12B-2 ..... ее отеле OC Gras lalalala СТ.С.........---6........
Г. taen (2) ооо caia == С. А.АА. TGTG ¡GC Ets
121 . 5 > с E 180
E. carino 11A-1,5A-2 GCTTAAAATTGGCCATCATAAGAGTTTGTTATAAAACAATAATCTTAATATTTAAGATAA
ESP carıno ПА еее micieleretsreiel clotelate(oletete\elatataletata
Es carıino ВЕ И ое ее sis so oies els ass sis a еее sie ss e SOO DOUO SONO
Е. (CALINO A 1B=2 le ET = blesse cesser 40090000 ODO ODDOCOOORUGOOODOD :
Е. Carino: ATA-2 сене еее сес else eine.“ FOSO OSA COLO O a Tasse see sieste
Е. gerhard 10-1 sisas els aie ss ee 0 ele ee ee еле еде ADO OO OOO ODO DO CONO Bac
Е. gerhard 10-2 .ecccccccce SOO GUD ее ее sjoie sie еее ee OOOO esse sosie ee eeleie ее
Е. gerhard 42-1 — —=—§ wcccccccccccccce olalekeLehefefeleiseisteie.oleleieteterelafereye ee ses se sie ess esse T.
Bi, alabamensis eses еее аа ое еее OOOO >
Е. haysiana ое oa еее о соевое оао OOOO 500
Е. Olivula === ооо еее ее а еее oies < еее ее evo ое осел esse
Е. cylindracea == weececcccccces A elotese\cvelole/eeleisieieteleveleleleiels efeleielCleteralelore 00 ORO Sa
Е пуае еее. еее ее бе CATA et
E. carino 46А-2 i sacsccscsoe ео осо steinLele GOGO COSC CHOCO OOOO 5
Es CArino ЧА ое ss sets ess Trae lata levels
Es ‘Carino 49B=2 = се еее SSA E ооо ess.
Esktascın 1-1 ен e sons aa ssesanislals sa o se sise se esiols ее асе аа
Е. fascin 71-2 soso. OOOO Noe oc s os ses OSO Мое еее ее
Е. cael Inf (2) a Tiana. sis: еее вооон осоо Тео Ases
E. crenatella (2) ....... cs А...... eXelehofefeletelexoieterefelererere (A OO ein/eteie/e:e
Е. showalteri еее > So odo Nba OS O То Ge ee cie 006
Р. pras 12A-1 oo... ses ее pes ces» ODIA O OO ASAS SO OOO ONO E
Р. pras 12B=1. mese conrro.naso.. OOOO IO OOOO Ce Сб -- ....А....
Р. pras 128-2 “000.0 TOO DADO еее еее Се бе. Ас
в. taen (2) ее еее sie = Les «Geo нео сое песо Ана еее mie OIDO
FIG. 1. Ап aligned data matrix of 433 nucleotide positions of mitochondrial 16S rDNA sequences for 25 pleu-
rocerid specimens. Dashes correspond to gaps and N's are missing data. E. carino = Elimia carinocostata;
E. gerhard = E. gerhardtii; E. fascin = E. fascinans; E. cael inf = Elimia caelatura infuscata; Р. pras =
Pleurocera prasinatum; L. taen = Leptoxis taeniata. Numbers following the name of the species is the spec-
imen number. Locality information of the specimens can be found in the Appendix.
carino 11A-1,5A-2
carino 11A-2
carino 11B-1
carino 11B-2
carino 47A-2
gerhard 10-1
gerhard 10-2
gerhard 42-1
alabamensis
haysiana
olivula
cylindracea
hydei
carino 46A-2
carino 49A-2
carino 49B-2
fascin 7-1
fascin 7-2
cael inf (2)
crenatella (2)
showalteri
pras 12A-1
pras 12B-1
pras 12B-2
taen (2)
carino 11A-1,5A-2
carino 11A-2
carino 11B-1
carino 11B-2
carino 47A-2
gerhard 10-1
gerhard 10-2
gerhard 42-1
alabamensis
haysiana
olivula
cylindracea
hydei
carino 46A-2
carino 49A-2
carino 49B-2
fascin 7-1
fascin 7-2
cael inf (2)
crenatella (2)
showalteri
pras 12A-1
pras 12B-1
pras 12B-2
taen (2)
carino 11A-1,5A-2
carino 11A-2
carino 11B-1
carino 11B-2
carino 47A-2
gerhard 10-1
gerhard 10-2
gerhard 42-1
alabamensis
haysiana
olivula
cylindracea
hydei
carino 46A-2
carino 49A-2
carino 49B-2
fascin 7-1
fascin 7-2
cael inf (2)
crenatella (2)
showalteri
pras 12A-1
pras 12B-1
pras 12B-2
taen (2)
FIG. 1. (Continued)
181 . . . . . 240
ATATATTTTATTCTAATTTTTT-ACAGAAATAAAGACCTCAATTAATAAATGCCTTATAC
css. soso. sons. so...
ss. See een nennen ne cs...
хо eee ou. ss oe
.e..o oso... eee eee ...o.o.o Ts зоо... о
cocos -..... Toccoooooomoooooocooo.o».. T.......
coros о осо оное осно sons. T....... ce. 0... Corse
хе». о ооо ооо nn... ....—— ооо ооо ооо ооо ооо ооо ооо
..oooo.o...so ооо ооо То ое ооо ооо ооо ооо ооо ооо
css... ES een nennen о «Азове ee
css. See оо ооо ооо оо ооо ооо оное оное ae? Ce a о
.......... вое ооо о о То ооо о ооо о ооо о ооо ооо nn.
......... T.oooooo..«AC.T.ooooooooo..«GTGCTG..ooooooooAcorno....
css. cee c cece cc оо То ооо ооо ооо cccccccccceGecccccccccccccs
astas „оса ооо ооо о ооо ооо сено е о ТС. « „С. сосоеооооовове
беоне ооо ооо оо то ооо ооо ооо ооо Ge... GG...
soso. ecco cece eNe wee cccees с........ ...ТТ......
........ coosorncrrrrrnorrsosnronrornononil......
ss... coo oo о ооо ово nennen. Сео соо е о .
241 . . . . В 300
СТАТССТАССАТСАСТАТТААААСТТТТТАТАТТСТААСАААСТТТТАТСТАТТТТТСТТ
ss... Cece emer cere ccc оо ооо ооо ось soso... N..
ss... зоо ооо оо ооо ооо оо ооо о ооо о ооо ооо ооо ооо ово ооо ооо
nsc nsc.
soso. nee“
cece rere rec ernennen nenne Cece ccc оо ооо ооо sere nennen nee
coo oosonnrnoosssss.o вое ео оо оо ооо оо о ооо ооо ооо ооо ооо ооо ее
зоо соо оо ооо ооо о о во ооо в ооо ооо ооо ооо воно Adoooooooooooooooo
sonne ооо а о ооо ооо ооо ово осень
ооо соо ооо ооо ооо ооо ооо са en oo ео оо оса ово ооо ооо вовооо о
commons». зоо оо соо ооо о о ое о ооо ооо ооо ооо ооо сосен
conose... кое ооо ооо ооо оо о = То еее ео вооон
еее ооо ооо ооо ооо оо в «С. Ань о AT..o.oooooo.. ....А. . «Ань.
зоо ооо оо ео в ово nenne сес AAA о Ме сео ооо ново сонае
ооо ео оо ооо ооо оо еь ное conso То ооо оо о ооо ооо ооо оное о
сот ee оо оо по ово носов оное ооо Т.е ооо о
concerns.» coosorrroTrccnonorooroccccon on».
соо ооо ооо вое ssso с ъзеофосовое Тео о А еее
soso. „ооо ооо ооо ооо во То синь ео во ооо со ооо ооо вое
иене ооо о о ооо оо ооо еее в А....С.Т. зоне ьное os...
ооо ое ооо ооо ооо оное о о оАооеоое Тосс оо сооьоооое воно
соо ооо ово о со ооооое ‚Тен. Со. CGT .. оьсьоньвое A.ooooooo»..
ss... os. ssssseTasseoCeseCGTssssscscsscosse Boo... и...
weer eee «Азове о + + = « ССА. ..
301 . . . . . 360
СААААААААТАТТСАТТСААТТАААТТАСТТААААССААСТССССААААТТААТССТТСС
sous. AAA AAA
еее ооо ооо еее ооо sssssms ressens
nn CR rennen. sms...
ss... ccc emer crc ооо со оо о eGeeccccccccceseesssccssesses
sons. CR ary
...... Terre
..... CR T Terre nennen.
Deere nee
Deren ee een ne
sos... i i ее i i ie воно в
«Too... ss... А. еее ооо оо оооооое о sos.
„Т.о оо А ооо ооо ово ооо оо ооо а оо оз ооо зов ово соевое
188 LYDEARDET AL.
361 с = 5 420
E. carino 11А-1,5А-2 CCTGTTT-ATCAAAAACATGGCTCTCTGAATTCATTTTTATAGAGAGTCAGGCCTGCCCA
ENCarinON TA A NT ee cecile = со оово во осабосоооо росс оборо восовоебоев
Е. carino 11B-1 ....... особо рососо во овобосовососо ово (Чебооо<овьвобовисоессва
E. carino 11B-2 ....... 506000002000 Stel S1e1sTeleretsfeletetererejele,e argo onoooo Dona
в. CAarinO Аааа ее ее sole еее еее еее < еее а а еее
Е. gerhard 10-1 „ооо ооо оо оо вое оо ооо ооо ооо ооо о Gosse ose
Е. gerhard 10-2 ....... —ieje/efalejejeinlelnlele/eialeje/ejeleie/nie/e,e,eleie/e eee e sise os eee + == eee = sie
Е. gerhard 42-1 ....... feia еее еле ее едете еле sielciejeyeieielcieicie С. еее
Е. alabamensis = e«eeeee —обооссвообовааоворсоссововособовоовобооооооове сос ово
Е. haysiana ....... materiels scies eceele sise cc -cccccole- ce esc: li.)
ECOLIVULA Te masse eos see selela=sleis see elec siecle 1e eee - ec ---i- ci
Е. cylindracea === ....... Se HOODEO DOOD DDO UD OUND ооо орооворооносовавоовосоово вос
E.chyder о аа Toe sels ee aleja Mo etais sieste ces pie cisleists
E. carino 46А-2 ....... —Ieheleferaj alts NNicteislerelere SOS т TUDO coo So 00 oO co Ono
E. carino 49А-2 11111. moe еее © т... Mise c—serceceesscesc close
Е. carino 49B=2 „еее eos Е О SES NOOO OOOO OOOO
Е. Расти II ce mere cecile Mes sais eee ses es OSADO
РЕВ 2 ee ee eee sciences oise sise oc sioiele Tee cesse Guaca lla
E. cael inf (2) === „ооо ооо ооо ооовоноооооо о Tia letal lalala
Е. crenatella (2) ....... ala ala sie oiete GI Ace Gon ес ее а
ES showalterin ее ое ее еее IDO SO OS COSO ooo uconon OC
Р. pras 12A-1 ....... cece ccc rororas. T..C...T..... O OOOO ODO
Р. pras 12B-1 ....... SAO IIS OR To. С.Т... A ROO
P. pras 12B-2 ....... nas +00) Т..С...Т..... А cles sie ее
в. taen (2) 5 mess ec sise clos eee т...... SIS OOOO SONO COSO OOOO OS
421 .433
carino 11A-1,5A-2 GTGAATAATATTT
Carino 11А-2 i cwrrsicccecscce
Carino 11B-1 ое
carino 11B-2 ....000..000°
carino ATA-2 ceseseeenece.e
gerhard 10-1 ......... NNNN
gerhard 10-2
gerhard 42-1
alabamensis
haysiana
olivula
cylindracea
hydei
carino 46A-2
carino 49A-2
carino 49B-2
fascin 7-1
fascin 7-2
cael inf (2)
crenatella (2)
showalteri
pras 12A-1
pras 12B-1
pras 12B-2
ss...
ss...
ss...
ss...
ss...
ss...
ss...
CRE
ss...
ss...
Pom ba ba u мы da o bu du do bu du Dn it bo
taen (2) ee... ATT.NNNN
FIG. 1. (Continued)
Pairwise percent sequence differences cor-
rected for multiple hits by the two-parameter
method of Kimura (1980) were from 0% to
3.93% within Elimia species, and 0.3% to
11.08% among Elimia species. Intergeneric
values ranged from 10.64 to 15.33% for com-
parisons of Elimia and Pleurocera and
17.75% to 23.29% for comparisons of Elimia
and Leptoxis. All pairwise genetic distances
are shown in Table 2.
Maximum parsimony analysis was con-
ducted treating all base substitutions as
equally weighted based on a previous analy-
sis of nucleotide substitutions within pleuro-
cerids (Lydeard et al., 1997). Maximum parsi-
mony analysis resulted in 320 equally most
parsimonious trees (tree length = 207; consis-
tency index = 0.619, excluding uninformative
characters). The gi statistical analyses
showed the data were significantly skewed (p
= 0.01), suggesting the data contain signifi-
cant phylogenetic signal. Phylogenetic signal
was stronger when outgroup taxa were in-
cluded in the 91 statistical analysis (91 =
—1.84) than when they were excluded (g1 =
—0.583). A strict consensus tree of the 320
equally most parsimonious trees and a phylo-
gram of one of the 320 equally most parsimo-
16S rDNA GENE SEQUENCES IN ELIMIA 189
TABLE 2. Estimated percentage nucleotide sequence difference (Kimura’s 2-parameter) among pairwise
comparisons of taxa based on mitochondrial 16S rDNA sequences
cren car11A1 car11A2 car11B1 car11B2 car46A2 car47A2 car49A2 car49B2
E.cren — 6.99 TT 6.99 6.72 6.67 6.74 TES 7.02
E.car11A1 = 0.24 0.23 0.47 1.98 0.71 2.93 3.67
E.car11A2 — 0.47 0.71 2.23 0.95 3.19 3.93
Е. car11B1 = 0.23 1.98 0.95 2.93 3.67
E.car11B2 — 1.98 0.71 2.68 3.42
Е. car46A2 — 1.98 1:23 1.98
E. car47A2 — 2.68 3.42
E. car49A2 — 1.20
E. car49B2 =
E. ger10-1
E. ger10-2
E. ger42-1
E. ala
Е. hay
E. hydei
E. cyl
E. oli
E.sho
E. fas7-1
E. fas7-2
Е. cael
Р. pra12A-1
P. pra12B-1
P. pra12B-2
ger10-1 gert0-2 ger42-1 ala hay hyd cyl oli sho fas7-1 fas7-2 cael
cren 7.10 7.26 7.06 6.99 7.26 11.08 6.99 7.52 4.16 7.29 7.02 7.04
.cart1A1 0.72 0.71 0.71 0.71 0.71 10.20 1.43 0.95 4.63 3.64 3.90 3.15
. car11A2 0.96 0.95 0.72 0.95 0.95 10.50 1.67 1.19 4.90 3.90 4.16 3.41
. car11B1 0.96 0.95 0.96 0.95 0.95 10.48 1.67 1.18 4.63 3.90 4.15 3.40
. car11B2 0.96 0.71 0.96 0.71 0.71 10.20 1.43 0.95 4.38 3.64 3.90 3.15
. car46A2 2.74 2.48 2.74 2.48 2.48 9.99 2.23 2.74 4.78 3.51 3.77 3.00
.car47A2 1.21 0.95 1.20 0.95 0.95 10.22 1.67 1.19 4.39 3.40 3.65 2.91
. car49A2 3.45 3.18 3.47 3.18 3.18 9.20 2.93 3.43 4.42 3.18 3.43 2.68
car49B2 3.71 3.92 3.71 3.92 3.92 9.99 3.67 4.18 4.15 3.42 3.16 2.92
mmmmmmmmmmmmmmmmmmmmm
ger10-1 — 1.20 0.96 1.20 1.20 10.94 1.94 1.45 4.71 3.70 3.44 3:71
ger10-2 = 1.20 0.95 0.95 10.20 1.67 1.18 4.89 3.90 4.15 3.40
ger42-1 = 1.20 1.20 10.88 1.93 1.44 4.68 4.19 3.93 3.69
ala — 0.95 9.91 1.67 1.18 4.63 3.39 3.64 3.40
hay — 9.33 1.66 0.23 4.37 3.90 4.15 3.40
hydei — 10.17 9.64 8.81 8.30 8.57 8.87
cyl — 1.43 4.62 3.64 3.90 3.15
oli — 4.63 4.15 4.40 3.65
sho — 3.89 3.64 3.65
fas7-1 — 0.24 1.92
fas7-2 — 2417
cael =
P. pra12A-1 P. pra12B-1 P. pra12B-2 L. tae
Е. сгеп 15.33 14.96 14.93 23.29
Е. car11A1 12.03 11.70 11.72 19.72
Е. car11A2 12.36 12.02 12.05 20.02
Е. car11B1 12.03 11.70 1172 20.06
Е. car11B2 11.74 11.40 11.43 20.06
E. car46A2 11.78 11.50 11:53 17.75
Е. car47A2 11.77. 11.43 11.46 19.78
E. car49A2 12110 11.82 11.85 19.44
E. car49B2 12:33 12.05 12.08 21.14
E. ger10-1 1247 11.89 11.91 19.25
E. ger10-2 11.74 11.40 11.43 20.40
E. ger42-1 12.77 12.42 12.45 19.02
Е. ala 12.33 11.99 12.02 19.06
E. hay 12.33 11.99 12.02 19.72
E. hydei 15.27 14.59 14.66 23.09
E. cyl 10.87 10.64 10.66 19.39
E. oli 12.03 11:70 11:22 20.06
E. sho 13.47 13.12 13.09 21.11
E. fas7-1 11.50 TZ 11.14 19.47
E. fas7-2 11.79 11.46 11.43 19.81
E. cael 12.12 11.78 11:76 20.21
P. pra12A-1 — 0.47 0.71 24.48
P. pra12B-1 — 0.24 24.09
P. pra12B-1 — 24.04
L. tae =
190 LYDEARDET AL.
. crenatella (2)
. showalteri
. carino11A-1,5A-2
. carino11A-2
. carino11B-1
. carino11B-2
. carino47A-2
. gerhard10-1
. gerhard42-1
. gerhard10-2
alabamensis
haysiana
olivula
. cylindracea
. carino46A-2
. carino49A-2
. carino49B-2
. fascin7-1
. fascin7-2
. cael inf (2)
hydei
. pras12A-1
. pras12B-1
. pras12B-2
. taen (2)
88
63
58 80
59
94
100
DU UMMMMMMMMMMMMMMMMMMMMM
r
FIG. 2. A strict consensus tree of 320 most parsi-
monious trees obtained in the maximum parsimony
analysis of the mtDNA sequence data using equal
weighting for all substitutions. Bootstrap values are
noted to the left of the corresponding node. Tree
length = 207; consistency index = 0.619, excluding
uninformative characters.
nious trees is shown in Figures 2 and 3, re-
spectively. Bootstrap values are reported for
each node of the strict consensus tree. Nodes
without numbers have bootstrap scores of
50% or less.
DISCUSSION
The molecular phylogeny obtained here
shows strong support for the monophyly of
Elimia of the Mobile River basin. The basal-
most species is Elimia hydei, which is sister to
a large unresolved clade of remaining Elimia
species. The unresolved polytomy is com-
prised of four clades: (1) E. caelatura infus-
cata; (2) E. fascinans; (3) E. crenatella + E.
showalteri; and (4) E. carinocostata + E. ger-
hardtii + E. alabamensis + Е. haysiana + E.
olivula + Е. cylindracea. There appears to be
little congruence between the molecular phy-
logeny and the current classification scheme
of pleurocerids (Burch, 1980). For example,
E. crenatella (2)
E. showalteri
E. carino11A-1,5A-2
E. carino11A-2
E. carino11B-1
E. carino11B-2
E. carino47A-2
E. gerhard10-1
E. gerhard42-1
E. gerhard10-2
E. alabamensis
E. haysiana
E. olivula
E. cylindracea
E. carino46A-2
E. carino49A-2
E. carino49B-2
E. cael inf (2)
E. fascin7-1
E. fascin7-2
E. hydei
P. pras12A-1
P. pras12B-1
P. pras12B-2
L. taen (2)
FIG. 3. A phylogram representing one of the 320
most parsimonious trees obtained in the maximum
parsimonious analysis of the mtDNA sequence
data using equal weighting for all substitutions. Tree
length = 207; consistency index = 0.619, excluding
uninformative characters. Branch lengths reflect
total number of substitutions.
the E. olivula group (E. cylindracea, E. olivula,
and E. showalteri) and the E. haysiana group
(E. alabamensis and E. haysiana) are para-
phyletic. However, some of the most parsimo-
nious trees depict E. fascinans + E. caelatura
infuscata of the Е. vanuxemiana group as sis-
ter taxa. Of course, it is worth noting that the
classification scheme of pleurocerids is pre-
Hennigian, and many of the groups are no
doubt recognized by shared plesiomorphic
characters. Within the largest of the four afore-
mentioned clades, E. carinocostata (speci-
mens 46A-2, 49A-2, and 49B-2) is depicted
as being sister to a clade that contains
E. cylindracea + E. olivula + E. haysiana +
E. alabamensis + E. gerhardtii + E. cari-
nocostata (specimens 5A-2, 11A-1, 11A-2,
11B-1, 11B-2 and 47A-1), rendering E. ca-
rinocostata paraphyletic. Elimia cylindracea is
the next most-basal member of the largest of
the four clades, and it is sister to the remain-
ing Elimia species. The only resolved relation-
16S rDNA GENE SEQUENCES IN ELIMIA 191
ships within this large clade are E. haysiana
+ Е. olivula and E. gerhardtii (10-1 + 42-1).
The relationship of the remaining Е. gerhardtii
(10-2) remains uncertain. The phylogeny ob-
tained in the present study is consistent with
the findings presented by Lydeard et al.
(1997).
Of the five Elimia species that had more
than one specimen sequenced, E. crenatella
(identical sequences), E. caelatura infuscata
(identical sequences), and E. fascinans are
depicted as monophyletic. However, the
monophyly of the aforementioned species
should be tested with additional specimens
and sequence data. Three Elimia gerhardtii
specimens were sequenced from two sepa-
rate locales. Interestingly, E. gerhardtii speci-
mens from different locales are sister taxa, but
this clade does not include the third E. ger-
hardtii specimen. Eight E. carinocostata spec-
imens were sequenced. The three most-basal
E. carinocostata specimens (46A-2, 49A-2,
49B-2) were collected in headwater streams
of the Coosa River, whereas the remaining E.
carinocostata specimens were obtained in
sites located further downstream.
Genetic differentiation among Elimia
species was generally low. This was under-
scored particularly in the E. carinocostata (in
part) + E. gerhardtii + E. alabamensis + E.
haysiana + E. olivula + E. cylindracea clade.
Most species of this large clade differed by no
more than 1.8%. Confounding this problem, is
the presence of intraspecific variation that is
equal to or exceeds the amount of variation
present among several of the species. The low
genetic variation detected among the afore-
mentioned species can be interpreted in two
ways. First, they represent a single evolution-
ary entity, and therefore should be syn-
onymized or second, they represent valid
species, but the gene is simply too conserva-
tive to detect any significant differences. We
recommend a more detailed investigation of
the genus using other more potentially useful
genetic markers before any formal taxonomic
decisions be made.
Elimia is the second most diverse genus of
freshwater gastropods in North America.
Burch (1988) lists 83 species within the
genus, but this number is likely to change fol-
lowing more detailed studies. Support for this
claim comes from Hershler's (1994, 1998) re-
view of the hydrobiid genus Pyrgulopsis,
which is now considered the most diverse
genus of North American gastropods. Prior to
Hershler's reviews, Elimia would have ranked
first, and the hydrobiid genus Somatogyrus
would have ranked second, with 35 species
(Burch, 1988). This dramatic increase in the
number of hydrobiid species underscores the
need for detailed monographic studies of all
freshwater gastropods (Hershler, 1996).
The 5'-half of the mitochondrial 16S rDNA
gene seems to be of limited utility for assess-
ing relationships among closely related Elimia
species. Although it is evident that there is sig-
nificant variation among more distantly re-
lated Elimia, there were only 36 phylogeneti-
cally informative sites, which is not very many
when examining relationships among 20+
taxa. Despite exhibiting more variation than
the 3’-half of the gene, most phylogenetic sig-
nal seems to be partitioned among genera.
Although we recognize that evolutionary rate
differences exist among taxa, and that pilot
studies should be carried out before under-
taking any major sequencing project, the 5'-
half of the ribosomal gene is likely to be of use
to investigators interested in resolving rela-
tionships among molluscan genera.
ACKNOWLEDGMENTS
We thank Rob Dillon, Jr., К. Roe, L.
Thompson, P. J. West, and an anonymous re-
viewer for helping to improve the quality of the
manuscript. This research was supported by a
Research Grants Committee Award (2-67767)
from the University of Alabama, a contract with
the U.S. Department of the Interior (#1448-
0004-95-938), and the National Science
Foundation (DEB-9527758, DEB-9707623)
to CL. John Yoder was a participant of a
NSF Undergraduate Research Supplement
Award. GenBank accession numbers for se-
quences are U73761 to U73767, U73771, and
AF050037 to AF050053. Vouchers of speci-
mens have been deposited at the Florida
Museum of Natural History.
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APPENDIX
Elimia alabamensis —
Coosa River just below Mitchell Dam, down-
stream of fishing platform (east bank),
Coosa Co., Alabama.
Elimia caelatura infuscata— Kahatchee Creek,
on Co. Rd. 008 (Childersburg Parkway),
Talladega Co., Alabama.
Elimia carinocostata —
(5A-2) Camp Branch Creek, 3 miles W of
Shelby on Co. Rd. 42, Shelby Co.,
Alabama.
(11A-1, 11A-2, 11B-1, 11B-2) Waxahatchee
Creek, 2.7 miles W of Shelby, Shelby Co.,
Alabama.
(46A-2) Beaver Creek, Co. Rd. 26 bridge, St.
Clair Co., Alabama.
(47A-2) Shoal Creek, at Co. Rd. 21 bridge, 4.7
miles NW of Ragland, St. Clair Co.,
Alabama.
(49A-2, 49B-2) Little Canoe Creek, Etowah
Co. line on St. Clair Co. Rd. 44, St. Clair
Co., Alabama.
Elimia crenatella —
(cre 18) Cheaha Creek at Co. Hwy 005, 5.1
mile SSW of Eastaboga, Talladega Co.,
Alabama.
(4142) Yellow Leaf Creek, 2 mile $ of
Westover on Co. rd. 51, Shelby Co.,
Alabama
Elimia cylindracea —
Noxubee River, 6 river miles above Alabama
state line, Noxubee Co., Mississippi.
Elimia fascinans —
Shoal Creek, Pine Glen Recreation Area,
Cleburne Co., Alabama.
Elimia gerhardtii —
(10-1, 10-2) Weogufka Creek, 0.8 miles NW
of Moriah on Co., Rd. 15, Coosa Co.,
Alabama.
(42-1) Cheaha Creek, Co. Hwy 005, Talladega
Co., Alabama.
Elimia haysiana —
Coosa River, main channel about 2.6 miles
downstream of Jordan Dam, 4.0 miles
NW of Wetumpka, Elmore Co., Alabama.
Elimia hydei —
Locust Fork of Black Warrior River at Warrior,
0.3 miles E of U.S. Hwy 31 on unnum-
bered Co. rd., Jefferson Co., Alabama.
Elimia olivula —
Alabama River ca. 1.5 miles downstream of
US Hwy 84, ca. 300 m upstream from
grain elevator, Monroe Co., Alabama.
Elimia showalteri —
Cahaba River at Booth’s Ford, 4.7 mile NW of
Pea Ridge, Shelby Co., Alabama.
Pleurocera prasinatum —
Coosa River, public boat ramp, just S of hwy
22 intersection with Coosa River S of
Mitchell Dam, Chilton Co., Alabama.
Leptoxis taeniata —
Buxahatchee Creek, 5 miles ESE of Calera,
Hiawatha Rd. off Co. Rd. 86, Shelby Co.,
Alabama.
MALACOLOGIA, 1998, 39(1-2): 195-205
MOLECULAR SYSTEMATICS OF THE FRESHWATER MUSSEL GENUS
POTAMILUS (BIVALVIA: UNIONIDAE)
Kevin J. Roe & Charles Lydeard
Aquatic Biology Program, Department of Biological Sciences University of Alabama, Box
870345, Tuscaloosa, AL 35487-0345, USA
ABSTRACT
Few explicit hypotheses for the relationships of unionid mussels exist. The absence of explicit
phylogenetic hypotheses is problematic and is in part responsible for the lack of taxonomic sta-
bility seen in this group. In this paper we examine the relationships of mussels in the genus
Potamilus, based upon the DNA sequences of a 600 base pair portion of the first subunit of the
mitochondrial cytochrome с oxidase (COI) gene. We also examine the genetic distinctiveness of
populations of the inflated heelsplitter P inflatus. The molecular phylogeny indicates that
Potamilus is paraphyletic with Leptodea fragilis and Lampsilis ornata nested between P capax
and the remaining Potamilus species. With the exception of P capax, the remaining Potamilus
species are depicted as monophyletic and form three distinct clades: (1) a reciprocally mono-
phyletic P inflatus clade; (2) a P ohiensis/P amphichaenus clade; and (3) a Р purpuratus/P р. col-
oradoensis/P alatus clade. While bootstrap values indicate a high degree of support for these
three clades, relationships among these three clades are not as strongly supported.
The genetic distinctiveness of two populations of the inflated heelsplitter exceeds that seen be-
tween some other species in the genus. These populations represent geographically isolated,
genetically distinct entities, and we therefore recommend the recognition of both the Amite and
the Black Warrior populations of P inflatus as separate species.
Key words: Unionidae, Potamilus, cytochrome с oxidase subunit |.
INTRODUCTION
The freshwater mussel genus Potamilus
Rafinesque, 1818 (Bivalvia: Unionidae), cur-
rently contains six species: Р alatus (Say,
1817), Р amphichaenus (Frierson, 1898), Р
capax (Green, 1832), P inflatus (|. Lea, 1831),
Р ohiensis (Rafinesque, 1820), and P purpu-
ratus (Lamarck, 1819) (Turgeon et al., 1988;
Williams et al., 1992). In addition to these
taxa, Simpson (1914) included P (Lampsilis)
coloradoensis (l. Lea, 1856), which is now
generally considered a western form of P pur-
puratus. Potamilus is distributed in the St.
Lawrence and Mississippi drainages and in
Gulf drainages from Alabama to Texas
(Valentine & Stansbery, 1971; Burch, 1975;
Clarke, 1981). The type species for the genus
was designated as Unio alatus Say, 1817, by
Morrison (1969).
The genus Potamilus in its current form was
first recognized as a natural assemblage of
species by Frierson (1927) in the synonymous
genus Proptera Rafinesque, 1819. Several re-
searchers have proposed classifications that
render the genus paraphyletic (Simpson,
195
1914; Hoggarth, 1988; Burch, 1975) and have
placed mussels currently assigned to Pot-
amilus in the genus Lampsilis (Р capax)
(Simpson, 1914), the genus Leptodea (Р lae-
vissima [= ohiensis, Р amphichaenus)
(Burch, 1975) or the resurrected genus Las-
tena (Hoggarth, 1988). Whereas Potamilus is
generally perceived as a natural group by
freshwater malacologists, it has not yet
achieved taxonomic and nomenclatural stabil-
ity, as evidenced by the continual change in
generic assignments over the last 170 years.
Even after successful petitioning by Bogan et
al. (1990) of the International Commission on
Zoological Nomenclature for the retention of
Potamilus (BZN, 1992), Proptera, a junior
synonym of Potamilus, appears in publica-
tions as late as 1993 (e.g., McMahon, 1993).
While many descriptions of the genus include
the presence of a posterior wing as diagnos-
tic, this character alone does not discriminate
members of Potamilus from their putative sis-
ter genus Leptodea (Ortmann, 1912; Valen-
tine & Stansbery, 1971). Ortmann’s (1912)
statement that “this genus (Potamilus) stands
in all characters except the glochidia, by that
196 ROE & LYDEARD
of Paraptera [= Leptodea] supports the simi-
larity of these two genera. Valentine &
Stansbery (1971) stated that the only unique
feature that defines Potamilus is the posses-
sion of axe-head shaped or ligulate glochid-
ium (Fig. 1), and Utterback (1915) noted that
with the exception of the unique glochidia and
the more developed hinge, “this genus
(Potamilus) stands with Lasmonos [= Lep-
todea]” A phenetic analysis by Hoggarth
(1988) of the utility of glochidia morphology
for deducing the relationships among North
American freshwater mussels indicated that
Potamilus is not a monophyletic group and
that Р ohiensis and Р amphichaenus are
more closely related to mussels in the genus
Leptodea than to other members of Po-
tamilus. Hoggarth’s analysis indicated two
distinct groups of mussels within Potamilus:
those with lateral hooks on the ventral valve
edges (alatus, capax, purpuratus) and those
without such hooks (ohiensis, amphichaenus,
inflatus). He concluded that the glochidia bore
only a superficial resemblance to each other,
and implied that the axe-head shaped
glochidia were not homologs.
The historic lack of taxonomic stability of
Potamilus reflects the fact that no detailed or
comprehensive cladistically based study has
been conducted on this genus. Despite in-
creasing interest in freshwater mussels, only
a few cladistically based analyses have been
published to date (Hoeh, 1990; Hoeh et al.,
1996; Lydeard et al., 1996; Mulvey et al.,
1997). The primary objective of this study is to
test the monophyly of Potamilus using a mol-
ecular data set composed of the DNA se-
quences of a portion of the first subunit of the
mitochondrial cytochrome с oxidase (COI)
gene, and develop hypotheses for relation-
ships within the genus.
Additionally, we wish to examine the level of
intraspecific genetic variation in the inflated
heelsplitter, P inflatus. Potamilus inflatus was
known from the Amite and Tangipahoa rivers
in Louisiana, the Pearl River in Mississippi,
and the Black Warrior, Coosa, and Tombigbee
rivers in Alabama. Presently, it is limited to the
lower and middle reaches of the Amite River,
and a portion of the Black Warrior River. In
1990, the U.S. Fish and Wildlife Service listed
P inflatus as a threatened species, because of
its diminished range and potential threats to
its continued survival in those rivers where it
still occurs (USFWS, 1992). Knowledge of
how genetic variation is partitioned in P infla-
tus will aid in making management decisions
concerning this species.
MATERIALS AND METHODS
Twenty-four specimens representing ten
species and five genera were included in the
analysis (Table 1). Genomic DNA was isolated
from fresh frozen or ethanol preserved tissues
using the QlAamp Tissue Kit (QIAGEN
#29304) following manufacturers instructions.
Care was taken to use only somatic tissues as
unionid mussels exhibit bi-parental inheri-
tance of mitochondria (Hoeh et al., 1996; Liu et
al., 1996b). Double-stranded and single-
stranded DNA was generated via the poly-
merase chain reaction (PCR) using the
primers LCO1490 and HCO2198 (Folmer
et al., 1994). Approximately 100 ng of geno-
mic DNA provided the template for double
stranded reactions performed in a 25 ul solu-
tion containing each dNTP at 0.1 mM, each
primer at 1.0 uM, 40 mM MgCl,, 2.5ul 10X Taq
buffer, and 0.6 units of AmpliTaq polymerase.
Reactions were amplified for 32 cycles at 94°
for 40 sec, 55° for 60 sec, and 72° for 90 sec.
The amplified DNA was gel purified and then
used as template for single-stranded amplifi-
cation (Gyllensten & Erlich, 1988) using the
same conditions and primer pair, with the H-
primer used in limited quantity. Single stranded
DNA was concentrated on Millipore Ultrafree
MC filters, and sequenced using the Se-
quenase version 2.0 kit (U.S. Biochemical) and
°°S-labeled dATP following the manufacturers
instructions. The heavy strand was sequenced
using overlapping primers: HCO2198 (5'-
taaacttcagggtgaccaaaaaatca-3), UNICOIH
(5'-tcagcaaccaacccaggag-3’), and HUNI-
COIC (5’-aacaacactctctaccaaag-3').
DNA sequences were visualized via autora-
diography, and aligned by eye using the soft-
ware package XESEE (Cabot & Beckenbach,
1989). P-distances (uncorrected for multiple
hits) and Kimura’s “two parameter” distances
(Kimura, 1980) were calculated using the soft-
ware package MEGA (Kumar et al., 1993).
Prior to phylogenetic analysis, the DNA se-
quences were examined for evidence of satu-
ration by plotting the number of transversions
and transitions at each codon position vs. p-
distance. Trees were generated under maxi-
mum parsimony using PAUP version 3.1.1
(Swofford, 1993). Trees were rooted using
Fusconaia cerina (Conrad, 1838) and
MOLECULAR SYSTEMATICS OF POTAMILUS 197
A B
FIG. 1. (A) Glochidia of Potamilus purpuratus,
showing the axe-head shape and lateral hooks.
Redrawn from Surber (1915). (B) Glochidia of
Lampsilis cardium for comparison. Redrawn from
Surber (1912). Bar = 100 um.
Obliquaria reflexa (Rafinesque, 1820).
Bootstrapping (Felsentein, 1985) was em-
ployed to measure the internal stability of the
topologies generated using 200 iterations.
Skewness of tree-length distributions as a
measure of phylogenetic signal (Hillis &
Huelsenbeck, 1992) was estimated by gener-
ating 10,000 random trees.
RESULTS
Sequence Variation
DNA sequencing procedures yielded ~600
base pairs of COI sequence for 24 taxa for a
total of 14,400 nucleotides (Genbank acces-
sion numbers AFO 49499-AFO 49522). Pre-
liminary analysis of the sequence data re-
vealed 182 variable sites, 151 of which were
phylogenetically informative. Of those sites
that were phylogenetically informative 16
were at the first position, 10 were at the sec-
ond position, and 125 were at the third.
Translation of codons into amino acids indi-
cates 23 variable residues. Pairwise percent
sequence differences corrected for multiple
hits using the “two parameter” model (Kimura,
1980) ranged from 0 to 2.6% for intraspecific
comparisons. Values for interspecific compar-
isons within Potamilus were between 1.2%
and 14.5%. Pairwise comparisons for all taxa
are presented in Table 2.
Scatterplots of pairwise genetic sequence
differences versus the absolute number of
transitions and transversions are presented
for each codon position in Figure 2. Trends re-
vealed by the scatterplots are typical for those
seen in other protein coding genes (Roe et al.,
1997a; Lydeard & Roe, 1997), transversions
were relatively rare at first and second posi-
tions, not exceeding four and two substitu-
tions respectively for any comparison. Trans-
versions were considerably more common at
the third codon position. A slight decrease in
the number of transitions relative to the num-
ber of transversions at the third position pro-
vides evidence that some saturation is pre-
sent. Saturation has the potential to affect
phylogenetic analyses, therefore differential
weighting of substitutions in the third codon
position was employed.
Phylogenetic Analyses
Based on the analysis of nucleotide substi-
tution patterns, phylogenetic analyses were
performed under maximum parsimony using
equal weighting and weighting transversions
2x transitions at the third codon position.
The g' values (—0.362894, —0.625367) for
weighted and equal weight analyses indicate
the presence of significant phylogenetic signal
(p = 0.01). Parsimony analysis of the data
using equal weighting of transitions and trans-
versions resulted in five equally parsimonious
trees (Cl = 0.636, RC = 0.517, 352 steps), the
strict consensus of which is presented in
Figure 3. Analysis of the data weighting trans-
versions 2x transitions resulted in two equally
parsimonious trees, which are presented in
Figure 4. With the exception of the equivocal
placement of Р р. coloradoensis, the two trees
from the weighted analysis represent a single
topology, identical to two of the five trees from
the equal weight analysis. Whereas differ-
ences exist between the trees generated using
transversion weighted and equal weighted
parsimony analysis, all topologies depict
Potamilus as paraphyletic. In addition, all
topologies support the monophyly of all
species with the exception of the P purpuratus
clade. All topologies also support the sister
relationships of P ohiensis and PR am-
phichaenus, and the reciprocal monophyly of
the Amite and Black Warrior populations of P
198 ROE & LYDEARD
TABLE 1. Localities and number of specimens included in this study.
SPECIES # INDIVIDUALS LOCALITY
Potamilus alatus' 1 Elk River, Limestone Co., AL., 29 September 1994.
P alatus? 1 Clinch River, Hancock Co., TN., 12 August 1994
P amphichaenus' 1 B.A. Steinhagen Resevoir, Neches River Dr., Tyler
Co., TX., 28 January 1996.
P amphichaenus? 1 Sabine River, at US Highway 59, Panola Co., TX., 5
July 1995.
Р capax 2 Iron Mines Ck., ~1.25 mi. W. of AR. Highway. 140
and Red Oak Baptist Church, Poinsett Co., AR.,
26 October 1994.
P ohiensis' 1 St. Francis floodway, near Wittsburg, Cross Co., AR.,
16 July 1995.
P ohiensis? 1 Lake Arrowhead, Little Wichita River, Red River Dr.,
Clay Co., TX., 12 July 1994.
P purpuratus' 2 Cahaba River, below Cooper Island, Bibb Co., AL.,
15 September 1994.
P purpuratus” 1 Cahaba River, ~1 mi. downstream of Hwy. 24, Bibb
Co., AL., 30 June 1993.
Р р. coloradoensis 1 Twin Buttes Resevoir, Concho River Dr., Тот Green
Co., TX., 30 August 1993.
P inflatus 4 Amite River, above Port Vincent, Baton Rouge Pa.,
LA., 3-4 August 1994.
P inflatus 4 Black Warrior River, (river mile 327.3), Tuscaloosa
Co., AL., 15 October 1994.
Leptodea fragilis' 1 Cahaba, River, above AL. Highway 58, Centreville,
Bibb Co., AL., 14 November 1994.
L. fragilis? 1 Elk River, upstream of AL Highway 127, Limestone
Co., AL., 14 October 1996.
Lampsilis ornata 1 Cahaba, River, above AL. Highway 58, Centreville,
Bibb Co., AL., 14 November 1994.
Obliquaria reflexa 1 Cahaba, River, above AL. Highway 58, Centreville,
Bibb Co., AL., 14 November 1994.
Fusconaia cerina 1 Cahaba River, ~1 mi. downstream of Hwy. 24, Bibb
Co., AL., 30, June 1993.
inflatus. Weaker support was found for some
deeper nodes as evidenced by the low boot-
strap values.
DISCUSSION
Phylogenetic Analysis
The СО! data do not support the recogni-
tion of Potamilus as a monophyletic group.
Whereas the majority of the species of
Potamilus form a natural assemblage, the
placement of Lampsilis ornata and Leptodea
fragilis nested between P capax and the re-
maining members of Potamilus renders the
genus paraphyletic. The single morphological
character that serves to unite members of
Potamilus is the possession of axe-head
shaped glochidia. Hoggarth (1988) suggested
only a “superficial resemblance” between the
glochidia of P amphichaenus, P. ohiensis and
those of P alatus, P purpuratus and Р capax,
and recommended that mussels with axe-
head shaped glochidia possessing hooks
(alatus, capax and purpuratus) should remain
in Potamilus, while those that lacked hooks
(amphichaenus, inflatus and ohiensis) should
be placed in the resurrected genus Lastena
Rafinesque, 1820. Hoggarth had not exam-
ined the glochidia of P inflatus and placed it in
Lastena on the basis of the morphology of
adult shells. His phenetic analysis indicated
that Lastena was more closely allied to
Leptodea than to Potamilus. Within Lastena,
Hoggarth placed Р ohiensis and Р am-
phichaenus as sister to Р inflatus. However,
recent examination of the glochidia of P infla-
tus revealed the presence of large supernu-
merary hooks (Roe et al., 1997b). Based on
Hoggarth’s criteria, Р inflatus should have
been placed in a group containing P alatus, Р
purpuratus and P capax, all of which have
glochidia that possess hooks. The molecular
phylogeny (Fig. 4) agrees with the classifica-
tion of Hoggarth (1988) in the recognition of P
MOLECULAR SYSTEMATICS OF POTAMILUS 199
TABLE 2. Pairwise genetic distances based on Kimura’s “two parameter” model. Values are percentages.
Р. pl R P. R P. P. R P. P. Р Р.
inf.w1 inf.w2 inf.w3 inf.w4 inf.at inf.a2 inf.a3 inf.a4 purp1 purp.2 purp.c. alatusi
P. inf.w1 0.00 0.00 0.34 2.46 2.44 2.62 2.08 9.68 10.16 10.55 10.16
P. inf.w2 0.00 0.34 2.12 2.29 2.47 1.93 9.55 9.51 9.70 9.88
P. inf.w3 0.34 2.29 2.45 2.62 2.09 9.49 9.82 10.39 10.00
P. inf.w4 2.46 2.08 2.26 2.07 9.26 9.72 10.10 9.72
P. inf.a1 0.35 0.35 0.17 9.36 9.32 9.49 9.10
P. inf.a2 0:17 0.17 9.13 9.58 107 9.18
P. inf.a3 0.34 9.49 9.82 10.19 9.39
P. inf.a4 9.09 9.53 10.32 9.34
P. purp.1 0.00 1.40 1:22
P. purp.2 1.55 1.38
P. purp.col. 1.20
P. alatus1
P. alatus2
Р. capax1
Р. capax2
P. ohien.1
P. ohien.2
P.amph.1
Р. amph.2
L. frag.1
L. frag.2
L. ornata
O. reflexa
F. cerina
Р. Р. PR: P. Р. PR P. IL: IE IS O. Е
alatus2 capaxi capax2 ohient ohien.2 amph.1 amph2 frag.1 frag.2 ornata reflexa cerina
P. inf.w1 10.11 14.40 14.48 12.40 13.02
P inf.w2 9.83 1442 1448 12.39 13.00
P inf.w3 9.92 1428 1431 12.47 13.09
P inf.w4 9.66 13.91 14.02 12.16 12.76
P.inf.at 9.02 1424 1388 11.13 11.75
Р inf.a2 911 1425 i444 1120 11.82
P inf.a3 931 1408 1389 11.02 11.63
P inf.a4 9.27 1399 1388 10.98 11.56
P. purp.1 1.23 13.79 1362 1018 10.56
P. purp.2 1.40 1419 1388 10.82 11.20
P. purp. c. 1.22 13.52 13.18 10.19 10.56
P. alatus1 0.00 13.12 12.99 9.98 10.37
P. alatus2 13.33 13.16 9.92 10.32
P. capax1 0.00 13.54 14.15
P. capax2 13.40 14.04
P. ohien.1 0.34
P. ohien.2
P. amph.1
Р. amph.2
L. frag. 1
L. frag. 2
L. ornata
O. reflexa
F. cerina
12.88 12.80 9.95 9.61 14.48 16.43 14.92
12.89 12.81 9.90 9.55 14.70 16.69 14.93
12.96 12.88 9.81 9.46 14.34 16.53 14.79
12.64 12.57 9.51 9.18 13.98 16.15 14.65
11.59 11.54 9.09 8.74 12.95 16.26 14.95
11.66 11.61 8.98 8.64 12.98 16.02 15.17
11.47 11.42 9.40 9.06 12.82 15.86 15.45
11.42 11.38 9.13 8.79 12.94 15:55 15:11
11.64 11.61 7.16 1.2 11.74 14.51 14.37
12.28 12.24 7.62 7.67 12.16 14.91 14.55
11.66 11.61 8.20 8.25 12.35 16.84 16.27
11.27 11.22 7.24 7.29 11.33 15.75 14.79
11.22 11.18 7.36 7.41 11.32 16.03 14.83
14.28 14.17 11.42 11.29 13.87 16.77 17.44
14.14 14.04 11.30 11.16 13.98 16.89 17.57
4.68 4.39 9.76 9.42 14.49 17.10 16.96
5.24 4.94 10.34 10.00 14.66 17,52 17.59
0.17 10.78 10.44 16.65 17.84 17.37
10.77 10.43 16.26 17.63 17.19
1.03 9.87 13.41 13.64
9.33 13.49 12.67
15.02 16.62
13.44
Note Taxon abbreviations: P. inf.w1-4, Potamilus inflatus-Black Warrior River; P. inf.a1-4, Potamilus inflatus-Amite River; P.
purp.1-2, Potamilus purpuratus; P. purp. col., Potamilus purpuratus coloradoensis; P. alatus1-2, Potamilus alatus; P.
capax1-2, Potamilus capax; P. ohien.1-2, Potamilus ohiensis; P.amph.1-2, Potamilus amphichaenus; L. frag.1-2, Leptodea
fragilis; L.ornata, Lampsilis ornata; O.reflexa, Obliquaria reflexa; Е. cerina, Fusconaia cerina.
amphichaenus, P inflatus, and Р ohiensis as
a natural group; however, it is not due to the
shared absence of hooks. Clearly, given the
homoplastic nature of hook development this
character appears to be of limited phylogenet-
ically utility.
The phylogenetic position of P capax is
problematic. In analyses of the molecular
data, P capax is depicted as the most basal
member of the in-group in the weighted analy-
sis, and is the most basal or second most
basal member in the equal weight analysis.
# of TS & TV
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
P-distance
# of Ts & TV
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04
P-distance
# of TS & TV
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
P-distance
FIG. 2. Scatter plots of number of nucleotide substitutions (transitions (TS) = open circles, transversions (TV)
= filled circles) versus genetic difference (p-distance) at (A) first, (B) second and (C) third codon positions.
MOLECULAR SYSTEMATICS OF POTAMILUS 201
. reflexa
. inf.w1
. inf.w2
. inf.w3
84
100
1 . inf.w4
. inf.al
. inf.a4
inf.a2
inf.a3
. purp.1
. purp.2
O
P
P
P
P
59 Р
Р
2
Р.
Р
Р
P. риф. col.
P
P
P
P
P
P
L
L
P
P
L
F
94
97
99
. alatus1
. alatus2
. ohien.1
88
100
. ohien.2
100 . amph.1
. amph.2
98 97 . frag.1
. frag.2
100 . сарах1
. capax2
. ornata
. сеппа
FIG. 3. Strict consensus tree for five equally parsi-
monious cladograms based on maximum parsi-
mony analysis using equal weighting of all substitu-
tions. Numbers correspond to the percentage of
bootstrap replicates where the clade was found
(200 total replications). Only values greater than
50% are shown. Taxon labels follow Table 2.
The placement of P capax outside the re-
maining members of Potamilus indicates pos-
sible affinities with other genera. Potamilus
capax had been placed in Lampsilis by
Simpson (1914) based on similarities of the
adult shells, particularly L. satura (l. Lea,
1852) (Valentine & Stansbery, 1971). Based
on glochidia morphology, Coker & Surber
(1911) indicated that capax was not a
Lampsilis but a Potamilus. The molecular evi-
dence presented here indicate no support for
the placement of P capax in Potamilus; for the
present, we withhold a formal recommenda-
tion concerning the generic affinity of P capax
until a more inclusive analysis can be per-
formed, including the type species of both
Leptodea and Lampsilis.
Our analyses suggest that Р р. coloradoen-
sis may represent a species distinct from P
purpuratus (Fig. 4B). Simpson (1914) also rec-
ognized P coloradoensis (|. Lea, 1856) as a
distinct species, although he admitted he was
doubtful of its validity. The placement of the
specimen referable to Р coloradoensis in our
analysis is equivocal, either being sister to P
purpuratus or P alatus. Examination of adult
shells reveals differences in periostracum and
nacre color between Р р. coloradoensis and Р
purpuratus shells from east of the Mississippi
River. Specimens of P alatus are generally dis-
tinguishable from those of P purpuratus, but
examination of the glochidia of representa-
tives of these taxa reveals no detectable dif-
ferences. Based upon genetic distances Р р.
coloradoensis is phenetically more similar to P
alatus (1.2%) than to PR purpuratus (1.5%).
Genetic distances between these taxa exceed
the intraspecific variation observed in all other
species included in the study, with the excep-
tion of P inflatus. Further research involving
representatives of P purpuratus and P alatus
from throughout their respective ranges is nec-
essary to resolve the relationships of this
clade. For the present, we recommend caution
in treating Р р. coloradoensis and Р purpura-
tus as the same evolutionary entity.
Both P ohiensis and Р amphichaenus were
placed in the genus Leptodea by Burch
(1975), however no support for the sister rela-
tionships of Leptodea and these taxa is found
in this analysis. The molecular data do provide
strong support for the sister relationships of Р
ohiensis and Р amphichaenus, and indicate
they represent distinct evolutionary entities,
more closely related to other members of
Potamilus than to L. fragilis.
The paraphyletic nature of Potamilus raises
questions about the monophyly of other
closely related unionid genera, such as
Leptodea. Leptodea contains three species:
L. fragilis, L. ochracea and L. leptodon. Of
these, L. ochracea was assigned to Lampsilis
by several authors (Simpson, 1914; Johnson,
1970; Burch, 1975) because of similarities in
appearance of adult shells, particularly to
Lampsilis cariosa. Morrison (1975) placed it in
Leptodea because it lacked the mantle flaps
often seen in species of Lampsilis. Hoggarth
(1988) found the glochidia of L. ochracea to
be more similar to L. fragilis and recom-
mended retaining it in Leptodea. The type
species, Leptodea leptodon, was originally
assigned to Leptodea by Rafinesque (1820).
It was also placed in Lampsilis by Simpson
(1914). This species has always been consid-
ered rare (Oesch, 1984) and has become
very difficult to find recently. Ultimately, any
taxonomic revision of these taxa must include
type species. Future phylogenetic analyses
including these and other allied taxa are
needed in order to more fully resolve relation-
ships among these genera.
202 ROE & LYDEARD
O. reflexa
inf.w1
inf.w2
inf.w3
83
inf.w4
inf.al
inf.a4
inf.a2
25 59
inf.a3
ohien.1
10
ohien.2
amph.1
uke 14 amph.2
purp.1
purp.2
99 1
Vat 14 4
alatus1
alatus2
frag.1
frag.2
100
100 10
5
o
A Ud al UI SUR RU qu) ae) qu
3% ornata
100 P. capax1
35 P. capax2
F. cerina
purp. col.
O. reflexa
inf.w1
= inf.w2
inf.w3
inf.w4
inf.ai
inf.a4
inf.a2
25 59
inf.a3
ohien.1
ohien.2
amph.1
amph.2
purp.1
purp.2
purp. col.
alatus1
alatus2
frag.1
frag.2
14 14
99
15
13
27 ornata
capax1
capax2
пе TO Geile ооо Ue OU UU 0-00
cerina
FIG. 4. (A, B). Two equally parsimonious cladograms based on maximum parsimony analysis weighting trans-
versions 2x transitions at the third codon position. Numbers above the branches correspond to the percent-
age of bootstrap replicates where the clade was found (200 total replications). Only values greater than 50%
are shown. Boldface numbers below the branches correspond to the number of nucleotide substitutions at
those nodes. Taxon labels follow Table 2.
Conservation Genetics of Potamilus inflatus
DNA sequence data have been used to
clarify relationships both between and within
species for a large variety of organisms from
whales (Milinkovitch et al., 1993) to hermit
crabs (Cunningham et al., 1992). However,
very few intraspecific comparisons of DNA se-
quences exist for studies involving unionids
(Liu et al., 1996a; Mulvey et al., 1997).
Intraspecific studies are necessary for wise
management decisions concerning endan-
gered and threatened species. Phylogenetic
analysis of sequence data of the COI gene in-
dicates that populations of P inflatus from the
Amite River, Louisiana, and the Black Warrior
River, Alabama, are reciprocally monophyletic
(Figs. 3, 4) and represent distinct evolutionary
entities (Moritz, 1994; Mayden & Wood,
1995). Genetic distances and the number of
nucleotide substitutions that separate these
two populations were compared with the num-
ber of substitutions that separate well-estab-
lished species. Examination of genetic dis-
tances reveals that the two populations of P
inflatus are more distinct genetically than Р
purpuratus is from P alatus (Table 2).
Examination of nucleotide substitution pat-
terns reveals that a total of 12 diagnostic sub-
stitutions separate the two populations of P in-
flatus, whereas P alatus and Р purpuratus are
separated from each other by eight substitu-
tions. In another comparison of congenerics,
P ohiensis and P amphichaenus are sepa-
rated by 26 substitutions.
Nucleotide substitutions are considered by
some researchers to accumulate at a similar
rate for closely related taxa (Wilson et al.,
1987; Vigilant et al., 1991; Wayne et al., 1991;
Li, 1993). If this is true for Potamilus, it would
indicate a more distant divergence time for the
two populations of Р inflatus than that for
some conspecifics. Alternatively, the differ-
ences observed could indicate an increased
rate in nucleotide substitutions for the inflatus
clade. In either case, based on these data, a
strong argument can be made for the recogni-
tion of the Black Warrior and Amite popula-
MOLECULAR SYSTEMATICS OF POTAMILUS 203
tions of P inflatus as distinct species. To date
no conchological characters have been found
that support the molecular data, and discrimi-
nation between these two species is based
solely upon DNA sequence data. The recog-
nition of cryptic unionid species is not without
precedent. Davis (1983) identified allozymic
differences for two phenotypically similar
species of Uniomerus. The degree of genetic
differentiation observed between populations
of P inflatus was greater than that seen in a
comparison of two other morphologically dis-
tinct species of Potamilus and exceeded in-
traspecific values for all other species. The
current geographic isolation of these two pop-
ulations can only lead to further genetic differ-
entiation of these entities and has serious im-
plications for any plans to reintroduce P
inflatus in areas where it once occurred. Other
studies involving mitochondrial DNA variation
in unionids have come to similar conclusions
regarding the protection of genetically distinct
forms. For example, in a study of the conser-
vation genetics of two unionid genera, Mulvey
et al. (1997) confirmed the distinctiveness of
Amblema neislerii (|. Lea, 1858) and A. plicata
(Say, 1817) using allozyme and DNA se-
quence data. Mulvey et al. (1997) recom-
mended additional protection for A. neislerii
because of its restricted range and particular
habitat requirements. In another study, Liu et
al. (1996b) urged caution regarding any ef-
forts aimed at re-establishing populations of
the giant floater, Pyganodon grandis, in
Colorado, because of observed mitochondrial
DNA differentiation between different river
drainages. Given the unique genetic status of
the Amite and Black Warrior forms of P infla-
tus, we recommend that each should be man-
aged as a distinct evolutionary entity.
The utility of the СО! gene for elucidating
relationships at the species level in our study
is based primarily on the relatively high num-
ber of substitutions at the third codon position.
The relative lack of support, as measured by
bootstrapping, for deeper nodes in the phy-
logeny is due in part to the smaller number of
variable sites at the first and second positions.
It is possible that sequencing a larger portion
of the COI gene would result in higher support
for these internal nodes. Lydeard & Roe
(1997) found that the complete cytochrome b
gene proved useful for diagnosing relation-
ships of representative actinopterygian fishes,
contrary to previous studies based on only a
portion of the gene. These studies questioned
the usefulness of this particular gene for re-
solving deeper phylogenetic relationships
(Hillis & Huelsenbeck, 1992; Graybeal, 1993),
but merely lacked sufficient data to address
the question at hand.
Historically, much of the uncertainty sur-
rounding the placement of particular unionid
species in one genus or another can be at-
tributed to the use of characters of unknown
phylogenetic utility and the absence of any
objective analysis. In the case of Potamilus,
the phylogenetic analysis of an independent
molecular data set indicates that such charac-
ters as glochidia shape and spines on
glochidia may be homoplastic and thus not
useful in diagnosing natural groups of mus-
sels. Further investigations involving Po-
tamilus and other genera are warranted and
should include morphological as well as mol-
ecular characters. Davis (1983) recom-
mended the use of multiple data sets for re-
solving relationships between unionid taxa.
The use of multiple data sets, such as mor-
phological and molecular characters, both in-
dependently and in a total evidence approach
(Kluge, 1989) would provide a more accurate
test of the phylogenetic utility of molecular and
traditional morphological characters in an
evolutionary context and provide much
needed insight into the evolution of these
traits.
ACKNOWLEDGMENTS
We wish to thank the following individuals
for their assistance in procuring specimens:
R. Howells, Texas Parks and Wildlife; J. Harris
and R. Doster, Arkansas State Highway and
Transportation Department, and P. Hartfield,
USFWS. A special thanks to P. Hartfield for
bringing the P inflatus question to our atten-
tion. This research was supported by the
Conchologists of America, the Hawaiian
Malacological Society (to K. J. В.) and USFW
Contract #43910-5-0098, National Science
Foundation DEB-9527758 and 9707623 (to
C. L.). We also thank David Neely for the
glochidium illustration and A. M. Simons, A.
Bogan, P. Harris and two anonymous review-
ers for their comments on drafts of this manu-
script.
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Revised ms. accepted 21 October 1997
Zur |
| e ot à y
FUNDE à PESA
MALACOLOGIA, 1998, 39(1-2): 207-213
ALLOMETRIC GROWTH AND INSIGHT ON SEXUAL DIMORPHISM IN POMACEA
CANALICULATA (GASTROPODA: AMPULLARIIDAE)
Alejandra L. Estebenet
Departamento de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, San Juan
670 - 8000, Bahia Blanca, Argentina
ABSTRACT
The shape changes associated with increase in size and sexual dimorphism in Pomacea
canaliculata are described using bivariate and multivariate statistical analysis. Allometric growth
was found in the studied population, the shell becoming relatively more globose and both aper-
ture and operculum becoming rounder as shell height increases. Related to this ontogenetic
change in shell shape is a relative increase in body dry weight. Adult snails show sexual dimor-
phism, males having both aperture and operculum rounder than females. Because juvenile snails
do not exhibit these differences in aperture form, shell dimorphism seems to be associated with
sexual maturity; that is, it is possibly related to the development of the penial complex. In sum-
mer, adult females of any given shell height weigh significantly more than males of equivalent
size; this being possibly due to the remarkable development of the albumen gland in this period.
Shell dry weight shows great variability in similarly sized snails, this fact being partially ascrib-
able to the seasonal growth pattern of P canaliculata in waters showing thermal seasonality.
Key words: Sexual dimorphism, Pomacea, growth.
INTRODUCTION
Freshwater snails in the family Ampu-
llariidae have a tropical and subtropical distri-
bution. The literature describing shell shape in
adult snails is extensive. Many species have
been described according to external shell
characters. However, shell shape changes as-
sociated with increase in size have not been
explored. Allometric relationships between
some linear shell measurements and snail
weight have been investigated for adult
Pomacea canaliculata (Lamarck 1822) (Gue-
des et al., 1981; Cazzaniga, 1990), but the
values of proportionality constants cannot be
related to specific or relative growth unless we
are dealing with ontogenetic data (Shea,
1985).
Because young P canaliculata have no de-
veloped sexual structures (penial complex
and albumen gland) (Hylton Scott, 1957),
their sex can be determined only by the mi-
croscopical analysis of the gonad. Cazzaniga
(1990) reported external differences between
sexes in adult PR canaliculata; however, shell
207
shape in undifferentiated juvenile snails was
not analyzed.
In this paper, ontogenetic changes in shell
form and sexual dimorphism in P canaliculata
are analyzed using bivariate and multivariate
Statistics.
MATERIALS AND METHODS
Live P canaliculata were collected from
standing waters in an artificial pond at Paseo
del Bosque, La Plata city, Argentina, in
January and February 1989. Their shells were
cleaned of adhering matter (mostly algae),
and they were then starved in aquaria with tap
water for 18 h to empty their guts. The snails
were killed by immersion into warm water (75
°C) for 3-5 min.
The sex of each snail was determined by
the presence of the penial complex or the al-
bumen gland; the snails were classed as un-
differentiated (or juveniles) when these sexual
structures could not be clearly identified.
The shells, soft parts and opercula were
208 ESTEBENET
dried separately at 80 °C for 48 h and weighed
оп a scale to the nearest 0.1 mg.
The following seven measurements were
taken on each specimen: shell height (SH),
spire height (SpH), operculum height (OH),
aperture height (AH), shell width (SW), aper-
ture width (AW) and operculum width (OW).
All measurements were made along straight
lines either parallel or perpendicular to the
imaginary axis shown on Figure 1. Shells <25
mm high were measured using a stereoscopic
microscope fitted with a camera lucida. Some
shell reference points were projected on a
graph paper ruled in squared millimeters.
From lines that joined these points, | obtained
the linear measurements for each snail to the
nearest 0.5 mm. Shells >25 mm high were
measured also from a plane projection but ob-
tained from X-ray images.
A total of 363 shells were measured (171 ju-
veniles, 89 females, 103 males). Because
some parts were spoiled while separating tis-
sue from shell and operculum, only 226 snails
were used to weight operculum and body (57
juveniles, 80 females, 89 males) and 233
snails to weigh shell (64 juveniles, 80 females,
89 males).
The power function у = ах? was used to de-
scribe the relationships among the linear and
weight measurements. Least squares regres-
sion analyses were carried out for the whole
population, and for males and females sepa-
rately. The logarithm of SH was used as inde-
pendent variable in all regressions. All the
slopes of the regressions involving whole pop-
ulation were compared with isometry (b = 1 for
linear variables or b = 3 for weight variables)
using t statistics (Sokal & Rohlf, 1979).
Analysis of covariance was used to test the
equality of regression coefficients and inter-
cepts between sexes (BMDP1V, Statistical
Software, UCLA, 1982). Residual analysis
was performed in all cases to test the basic
assumptions of the linear regression analysis
and to assess the adequacy of the linear
model.
Stepwise discriminant function and cano-
nical variate analysis were performed
(BMDP7M, Statistical Software, UCLA, 1982)
in order to reveal which variables contributed
to discriminating the sexes. Only the linear,
not weight, variables were used. Allometric
growth exists in all characters measured (see
results); all variables were therefore log-trans-
formed before the analyses were carried out.
Twenty eight newly hatched snails from a
single egg-mass were reared in the labora-
tory. They were placed individually in cylindri-
cal plastic tubes submerged in an aquarium
with aerated, warm (25 + 1°C), tap water
(hardness 90 to 96 ppm CaCO,). The snails
were fed with lettuce, and the water was
changed periodically at the time the linear
shell dimensions were measured. When the
snails were approximately 25 mm high, their
sexes were determined (14 males and 14 fe-
males).
RESULTS
Table 1 is a statistical summary of the 10
measured variables.
Analyses of residual plots showed no de-
partures from the assumptions of the regres-
sion model; the linear log-log regression ac-
curately describes the relationships between
the pairs of selected variables, the only ex-
ception being log shell weight (SWt) - log shell
height (SH) regression.
Table 2 shows the different allometric rela-
tionships calculated for the whole population.
The null hypothesis that b = 1 was rejected in
all regressions involving linear variables. The
ratios of shell width, aperture width, opercu-
lum width and spire height to shell height
(SW/SH, AW/SH, OW/SH and SpH/SH re-
spectively) increase as the shell increases in
size. The dry weight of the soft parts (BWt)
and operculum (OW?) also increase relative to
SH (the null hypothesis that b = 3 was re-
jected).
Table 3 shows the results of the ANCOVA
on the two sexes of Р canaliculata. Except for
log SW and log SpH, the slopes or intercepts
of the regression lines were significantly dif-
ferent between the sexes. The values of b for
aperture and operculum variables were signif-
icantly larger for males. The slopes of the log
BWt-log SH and log OWt-log SH regressions
did not differ between sexes, but the inter-
cepts were larger for the females.
The linear log SWt - log SH regression was
not appropriate to describe the relationship
between both variables, because the ratio of
specific growth rates changes during the on-
togeny. Residual plot showed that residual
variance increases along the independent
variable.
In this study, the best discriminating model
for separating sexes uses only three of the
seven available measurements (SW, OH,
SEXUAL DIMORPHISM IN POMACEA 209
OW
FIG. 1. Variables measured on the shell and the op-
erculum of Pomacea canaliculata (SH: shell height,
SpH: spire height, AH: aperture height, SW: shell
width, AW: aperture width, OH: operculum height
and OW: operculum width).
OW). The other four variables were not in-
cluded, because they contribute no additional
discriminating information. A discriminant
score was computed for each snail by multi-
plying each of three characters by the corre-
sponding coefficient and adding together
these products (Fig. 2). There was a signifi-
cant difference between the mean discrimi-
nant scores of the two sexes; that is, the func-
tion will discriminate, significantly, between
the two groups (Wilk’s lambda = 0.534 or Y =
118, d.f = 3, p < 0.0001). The percentage of
correctly allocated individuals by sex using
this function was greater than 78% for the
males and 85% for the females. The results of
canonical variate analysis showed that OW
had the greatest importance in separating
sexes (Table 4).
The growth rate (measured as increase in
SH) was similar in males and females reared
in the laboratory, at least until sexual maturity
was reached (Fig. 3). The form of the aperture
(AW/AH) was compared between sexes twice
during growth. The first comparison was made
when the snails were 7.42 + 0.707 mm high
(mean + SD); there was no significant differ-
ence (male mean ratio: 0.7605 + 0.0255 SD,
female mean ratio: 0.7636 + 0.0269 SD; t =
0.313, p > 0.10, d.f. = 26). However, when the
snails were 23.51 + 2.996 mm high (mean +
SD), the aperture was significantly rounder in
males than in females (male mean ratio:
0.8042 + 0.0302 SD; female mean ratio:
0.7698 + 0.0427 SD; t = 2.461, р < 0.05, d.f. =
26).
DISCUSSION
Allometric growth occurs in Pomacea
canaliculata, the shell becoming relatively
more globose, and both aperture and opercu-
lum becoming rounder as the shell increases
in size. Related to this ontogenetic change in
shell shape, there is a relative increase in the
dry weight of the soft parts. The positive al-
lometry of AW and SW relative to SH may be
related to a positive allometric growth of the
foot.
Though SpH shows positive allometric
growth with respect to SH, it is a widely vari-
able character within populations. The shell of
P canaliculata usually has a short spire.
However, in our material, snails range from
those whose shell apex does not exceed the
plane that delimits the superior edge of the
last whorl, to snails with a much higher spire.
This variation has been noted by earlier au-
thors (d’Orbigny, 1847; Barattini, 1939; Hylton
Scott, 1957) and prevents SpH be used as a
diagnostic character.
Adults of Р canaliculata from Paseo del
Bosque show sexual dimorphism, as shown
by Cazzaniga (1990) for another population of
the same species. The external differences
between males and females are manifested
by both the form of the aperture and the oper-
culum. The AW/SH ratio increases with in-
crease in size. However, in females AW in-
creases less rapidly than in males. Another
ampullariid, Marisa cornuarietis (L.), also ex-
hibits positive allometric growth of AW
(Demian & Ibrahim, 1972), and the differ-
210 ESTEBENET
TABLE 1. Statistic summary of the variables measured on Pomacea canaliculata. Linear measurements are
in mm; weights are in mg.
Variable Mean SD Minimum Maximum N
Shell height 35.21 15.61 8.84 80.00 363
Shell width 29.38 13.65 F2 70.50 363
Spire height 4.10 2.10 0.88 10.00 363
Aperture height 24.69 10.45 6.28 54.00 363
Aperture width 19.01 8.79 4.80 42.50 363
Operculum height 21.88 9.53 5.64 49.00 363
Operculum width 15.03 6.95 3.64 33.50 363
Body weight 1480 1250 110 6130 226
Shell weight 3865 2795 253.8 14946 233
Operculum weight 90 70 6 370 226
TABLE 2. Values for different linear regressions for a population of Pomacea canaliculata, with tests of sig-
nificance of deviation from the values of isometry (values of test t)
Isometry t
Regression N b + SE b= Р а В?
log SW/log SH 363 1.042 = 0.005 8.13 <0.0001 -0:147. 0.99
log SpH/log SH 363 1.070 + 0.020 3.47. <0.001 —1.053 0.88
log AH/log SH 363 0.945 + 0.004 12.08 <0.0001 —0.067 0.99
log AW/log SH 363 1.025 + 0.006 AS <0.0001 -0.308 0.99
log OH/log SH 363 0.967 = 0.005 5.61 <0.0001 -0.156 0.98
log OW/log SH 363 1.015 + 0.006 2.5 <0.02 -0.395 0.97
DIS
log BWt/log SH 226 3.456 + 0.062 7.36 <0.0001 -2.590 0.93
log OWt/log SH 226 3.253 + 0.068 3.68 <0.001 -3.470 0.91
TABLE 3. Values for different linear regressions in males and females Pomacea canaliculata.
Regression Sex N b+SE a В? F*
log SW/log SH females 89 1.020 + 0.021 -0.104 0.96 Рь NS
males 103 0.984 + 0.022 -0.051 0.95 F,NS
log SpH/log SH females 89 1.084 + 0.103 -1.083 0.56 Рь NS
males 103 0.987 + 0.103 -0.912 0.48 F,NS
log AH/log SH females 89 0.886 + 0.019 -0.032 0.96 Fy 13.707
males 103 0.983 + 0.018 -0.127 0.97
log AW/log SH females 89 1.048 = 0.021 -0.353 0.96 F, 3.697
males 103 1.114 + 0.027 -0.445 0.94
log OH/log SH females 89 0.924 + 0.023 -0.083 0.95 Рь 5.44*
males 103 1.003 + 0.024 -0.209 0.94
log OW/log SH females 89 0.975 + 0.030 -0.342 0.92 Ep Soils:
males 103 1.123 + 0.040 -0.545 0.88
log BWt/log SH females 80 3.229 + 0.154 -2.145 0.86 F, NS
males 89 3.081 + 0.112 -2.015 0.90
females common slope -2.034 Е. 59:63
males 3.164 + 0.086 -2.151
log OWt/log SH females 80 3.076 + 0.149 -3.230 0.85 Рь NS
males 89 3.365 + 0.172 -3.590 0.82
females common slope -3.326 F 34.7777
males 3.204 + 0.113 -3.443
#F test for null hypothesis that Dmales = Diemales Fo OF Amales = Afemales Fa
NS: not significant (р > 0.05) *p<0.05 **p<0.01
SEXUAL DIMORPHISM IN POMACEA All
-3.562
-2.149
-0.736
0.676
2.089
3.620
15 10 5
me Males
a Females
F=89
O
ayi
5 10 19
RELATIVE FREQUENCY (%)
FIG. 2. Frequency histograms of discriminant scores (DS) for male and female Pomacea canaliculata. DS =
-35.42(logOW) + 22.91(logOH) + 19.31(logSW) - 18.07
ences between the sexes in this species are
greater than in P canaliculata.
Multivariate analysis showed OW is a better
discriminant between sexes than AW, be-
cause OW grows with positive allometry re-
spective to SH in males, whereas in the fe-
males the growth is isometric.
In P canaliculata, the penial complex, which
arises as a ventral outgrowth from the mantle
edge, appears to develop at the same rate in
both sexes until the gonad becomes active,
when its growth is arrested in females. This
occurs when the females are 8 mm in diame-
ter (Andrews, 1964). Around this point, the
aperture form is still similar between sexes.
The sexual dimorphism in the aperture form
seem therefore to be associated with the sex-
ual maturity, possibly with the posterior devel-
opment of the penial sheath in the males.
About 50% of the males of P canaliculata
greater than 40 mm in SH show a slight re-
TABLE 4. Results of canonical variate analysis for
shape differences between sexes.
Standardized coefficients
log SW 1.487
log OW -3.655
log OH 2.188
Eigenvalue 0.871
Canonical correlation 0.682
flection of the free edge of the peristomal lip.
This feature was only present in three of the
89 females analyzed. This other differential
shell feature was present in some males of M.
cornuarietis (Demian & Ibrahim, 1972).
Sexually dimorphic growth and/or survivor-
ship patterns resulting in sexually dimorphic
sizes are known for many freshwater proso-
branchs (Browne, 1978; Aldrige, 1982;
212 ESTEBENET
SHELL HEIGHT (mm)
<+ Females
# Males
5 =I] T T T T
0 20 40 60 80 100 120
DAYS
FIG. 3. Pattern of growth of Pomacea canaliculata
reared under laboratory conditions (mean + 95%
confidence interval).
Jokinen, 1982; Jokinen et al., 1982; Brown &
Richardson, 1992). Females reach sizes
greater than males in several species of
Ampullariidae: Pila spp. (Keawjam, 1987),
Pomacea urceus (Muller) (Lum Kong & Kenny,
1989) and Marisa cornuarietis (Demian &
Ibrahim, 1972). Burky (1974) reported that P
urceus males can attain a maximal size simi-
lar to that of females, but usually the proportion
of males in the largest size classes is less than
those of females. In my material, the maximal
size attained by one male was 62.5 mm (SH)
whereas around 12% of the females were
larger, up to 80 mm. This size difference could
be attributed to a sexually dimorphic growth
pattern (Estebenet & Cazzaniga, 1997).
Females of any given size weigh signifi-
cantly more than males. Because there was
not marked shell form differences between
sexes, this could be attributed to the sampling
date (Summer) that coincided with reproduc-
tive season (from late spring to late summer).
The albumen gland grows up to represent
68% of the dry body mass in a reproductive
active female, this being why Guedes et al.
(1981) discarded reproductive active females
to obtain reliable regressions for biomass es-
timation in Р canaliculata. Bourne & Berlin
(1982) determined a similar weight difference
between sexes of Pomacea dolioides
(Reeve). It is therefore probable that the re-
gressions involving soft parts weight are sea-
sonally variable. This fact could be extended
to other temperate ampullariid populations
with seasonal reproductive patterns.
A linear model did not accurately describe
the SWt-SH relationship. Great variability in
SWt exists among similar sized snails that can
be partly ascribed to seasonal pattern of
growth showed by Р canaliculata in waters
with marked thermal seasonality (Estebenet &
Cazzaniga, 1992). Snails hatching at the be-
ginning of the breeding season grow rapidly;
they generally have a thin shell and are often
similar sized as snails hatched at the end of the
previous breeding season, the later generally
having a heavier shell. Another source of vari-
ation in the SWt could be the sex of the snails,
because Cazzaniga (1990) determined that
shells of P canaliculata males are significantly
heavier than the shells of females.
ACKNOWLEDGMENTS
This work was funded with grants by CON-
ICET (Consejo Nacional de Investigaciones
Científicas y Técnicas, Argentina: PI.D. +
3368-80092), CIC (Comisión de Investiga-
ciones Cientificas de la Provincia de Buenos
Aires, Argentina), and UNS (Universidad
Nacional del Sur).
| am grateful to Dr. Néstor J. Cazzaniga for
his critical reading of the manuscript and en-
couragement and to Lic. Pablo R. Martín for
his valuable comments along the course of
this work.
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Revised ms. accepted 1 April 1997
MALACOLOGIA, 1998, 39(1-2): 215-219
LETTERS TO THE EDITOR
TRANSLATING TREES INTO TAXONOMY WITHIN VENERIDAE (BIVALVIA):
A CRITIQUE OF TWO RECENT PAPERS
Mary Ellen Harte
1180 Cragmont Ave., Berkeley, California 94708, USA
Cladistic analysis has been developed over
the past two decades into a scientifically rig-
orous method for determining probable evo-
lutionary relationships among taxa, and it is
increasingly used throughout all biological dis-
ciplines concerned with evolution. In each
case, the end result is a rooted or unrooted
tree of taxa—commonly referred to as a clado-
gram—from which phylogenetic inferences
are made. At the same time, various phenetic
algorithms have been developed to create
rooted or unrooted trees of taxa based on bio-
molecular data (e.g., Neighbor-Joining; Saitou
& Nei, 1987), and these have been used to val-
idate or refute existing taxonomies of organ-
isms (e.g., Sibley & Ahlquist, 1990; Albert et
al., 1992; Simon et al., 1993; Wainright et al.,
1993; Halnych et al., 1995).
Two recent papers, Roopnarine (1996) in
Malacologia and Canapa et al. (1996) pub-
lished elsewhere, utilize such algorithms to
study the bivalve family Veneridae and attempt
to apply the resulting inferences to current tax-
onomy. An examination of these papers indi-
cates some taxonomic and methodological
misapplications to an already taxonomically
controversial group.
A STUDY OF THE CHIONINAE
Roopnarine (1996) focuses on the venerid
subfamily Chioninae, which represents a pro-
lific evolutionary radiation centered in North
America, judging by the number of current
and extinct taxa. He collected data on 25 con-
chological characters from six extinct and ten
Recent American chionine species, most re-
cently classified into four genera and 11 sub-
genera (Keen, 1969; Fischer-Piette 8 Vuka-
dinovic, 1977). Two of the four genera are
215
represented by their type species; nine of the
11 subgenera are represented by their type
species.
Character Definitions
Of the 25 characters, inadequately defined
character states are present among the fol-
lowing seven: pallial sinus depth, spacing of
commarginal sculptural elements, anterior de-
velopment of commarginal elements, ventral
margin crenulation, width of anterior cardinal
tooth, posterior cardinal tooth shape, and
width of central cardinal tooth. For example,
the character of pallial sinus depth is defined
as greatly reduced to absent for one state,
and present but short for another. It is not
clear where the demarcation is between these
two states. Similarly, | disagree with the as-
signment of a character state in one of the
taxa for spacing of commarginal elements, but
the ambiguous definition of states (“close,
widely spaced, widely spaced but narrowing
later’) allows independent workers to reach
different conclusions. Such ambiguities exist
in the other six characters, allowing for a sig-
nificant amount of subjectivity in the analysis,
and preventing other workers from replicating
the results.
A second source of ambiguity arises be-
cause Roopnarine fails to define what per-
centage of examined specimens must have a
character state in order for it to be considered
present in the species. For example, in defin-
ing types of sculpture present, he asserts that
“radials are absent in Mercenaria” (p. 140;
presumably M. mercenaria, the only species
included in his study), countering the obser-
vations of previous workers (Jones, 1979;
Harte, 1992), who note the characteristic
presence of radial sculpture in that species.
216 HARTE
Representation of Genera
Inadequate representation of some of the
genera weakens his analysis. The taxonomic
placement of Chione (Puberella) based on the
consensus tree is problematic, but its very
presence is weakened because it was not
represented by its type species. Two other
taxa in the analysis, Protothaca (Leukoma)
and Timoclea (Glycydonta), are parts of much
larger groups that would require more analy-
sis of related taxa to adequately assess their
taxonomic status. If Roopnarine represented
all of the Chione subgenera solely by their
subgeneric names on his cladograms, how-
ever, he should have treated Leukoma and
Glycydonta in the same manner. Using their
generic classifications implies relationships
that might not be taxonomically valid to those
much larger groups. To justify inclusion of
Protothaca and Timoclea in the cladograms,
type species of the taxa should have been
used.
Not using type species of taxa could possi-
bly influence the results. For example, in his
resulting consensus tree, Roopnarine (1996:
fig. 20) shows Timoclea grouped with Proto-
thaca in a clade separate from that of Chione
s.s. In the study, the species representing
Protothaca and Timoclea were Р (Leukoma)
asperrima (Sowerby) of the east Pacific, and
T. (Glycydonta) marica (Linnaeus), ofthe west
Pacific, respectively. The type species of
Timoclea s.s., Timoclea ovata (Pennant), an
eastern Atlantic species, has different charac-
ter states for at least some of the characters.
Whether these different character states
might make it more closely related to the type
species of Chione s.s., Chione cancellata
(Linnaeus), a western Atlantic species, than
to P asperrima, remains untested.
Roopnarine said that specimens of some of
the type species were not available to him, but
these species are not rare. For example,
Timoclea ovata is present in the UCMP col-
lection, one of several he used in his study,
and availability of specimens is adequate
through the current museum loan system of
the major collections in North America.
Classification
Assuming these two sources of ambiguity
do not seriously interfere with the cladistic re-
sults, can one justify his proposed taxonomic
revision based on his analysis and its minimal
translation into the taxonomy? Here, | believe
he is only partly successful.
Reducing taxonomic ambiguity is a major
consideration for creating hierarchical, supra-
specific taxa. Thus, the introduction of sub-
genera into a classification clearly aligns
some generic groups with one genus and not
another, reducing ambiguity in intergeneric
relationships. While taxonomic hierarchy does
not have the flexibility for incorporating pre-
cisely the hierarchical information offered in a
cladistic network, major cladistic hierarchical
elements can be incorporated into the taxo-
nomic framework, which can significantly im-
prove its information content and reduce its
taxonomic ambiguity. By demonstrating that
Chione, as previously defined, was para-
phyletic, Roopnarine justifies its breakup, but
not, on the basis of his results and the stan-
dard of minimizing taxonomic ambiguity, his
creation of genera from almost all former sub-
genera of Chione. For example, Roopnarine
(1996) (Fig. 1) illustrates that the clade con-
taining Chione subdivides into two clades. If
he had incorporated this information into
current chionine taxonomy following the taxo-
nomic seniority rule, he could improve chion-
ine taxonomy by proposing that Anomalo-
cardia, the senior taxon of one clade, remain
a genus under which the other members of
that clade—Jliochione, Lirophora, and Panchi-
one—are subsumed as subgenera. He did
propose that the senior taxon of the second
clade, Chione, remain a genus, under which
the other supraspecific taxon of that clade,
Chionista, was subsumed as a subgenus. He
also proposed, justifiably, that Chionopsis
stand as a genus based on its solitary loca-
tion in his cladograms. Taken in combination,
these steps reduce ambiguity in chionine
taxonomy, thereby justifying the revision, and
they incorporate much, but admittedly not all,
plausible evolutionary information derived
from the analysis. But elevation of all subgen-
era except Chionista to generic rank indicates
an underutilization of cladistic hierarchical in-
formation, creating unnecessary taxonomic
ambiguity by decreasing the hierarchical infor-
mation within the existing taxonomy.
A STUDY OF VENERID PHYLOGENY
The second paper, Canapa et al. (1996), uti-
lized biomolecular data from a length of 16s
rRNA to construct two Neighbor Joining trees
for nine Mediterranean venerid species from
TRANSLATING TREES INTO TAXONOMY 217
Mercenaria
Puberella
Chionopsis
Protothaca
Timoclea
Chionista
à. Chione
Anomalocardia
Iliochione
Lirophora
Panchione
FIG. 1. Consensus tree after Roopnarine (1996: fig.
20, lower figure).
the five largest presently recognized subfami-
lies: Chamelea gallina (Linnaeus) [Chioninae],
Dosinia lupinus (Linnaeus) [Dosiniinae],
Callista стопе (Linnaeus) and Pitar rudis
(Poli) [Pitarinae], Tapes decussatus (Lin-
naeus), Tapes philippinarum (Adams 4
Reeve), and Venerupis aurea (Gmelin) [Tape-
tinae], and Venus verrucosa Linnaeus [Ve-
nerinae]. Three of the species, Chamelea gal-
lina, Callista chione and Venus verrucosa are
the type species for these genera, and the last
is the nominal genus of the subfamily
Venerinae and the Veneridae. The authors
concluded that the resulting trees (Canapa et
al., 1997) (Figs. 2, 3) support the results of tra-
ditional classification at the subfamily level but
do not support the concept of a genus Tapes.
Indeed, Chioninae and Venerinae, long con-
sidered to be closely related, fall within the
same clade on both trees, and each of the
other subfamilies forms a distinct clade, al-
though the relationships among those clades
differ substantially between their two trees.
Contrary to the papers assertions, how-
ever, the data do not disprove the concept of
the genus Tapes. The tapetine species used
in this study do not represent true Tapes, but
rather belong to the subgenus Ruditapes
(Fischer-Piette & Metivier, 1971). In order to
prove or disprove the concept of Tapes, both
the species representing the genus Tapes
s.s.—T. literatus (Linnaeus), an Asian
species—and at least one of the 3-4 addi-
tional Asian species considered to be mem-
bers of Tapes s.s., should have been included
in the study. If the tapetine species included in
the study had fallen among these Asian
species in the resulting trees, then this would
indeed disprove the concept of Tapes. While
the choice of species in this study reflects the
accessability of material (local Mediterranean
venerid species), this also limits what one can
interpret, taxonomically, from the analyses.
As it is, a more accurate taxonomic inter-
pretation of the analyses indicates some
equally interesting insights and illustrates
some of the limitations presented by the
species used. For example, Ruditapes decus-
satus is the type species of Ruditapes. How-
ever, what malacologists identify as Rudi-
tapes decussatus in the Mediterranean might
not be the same species as from the type lo-
cality in the Indian Ocean, according to
Fischer-Piette & Metivier (1971: 28).
Venerupis aurea is not the type species of
Venerupis; that honor belongs to the British
Venerupis perforans Montagu, 1803, which in
turn is a synonym of Venerupis corrugata
Gmelin, 1791 (Fischer-Piette & Métivier,
1971). Fischer-Piette & Métivier (1971)
lumped several nominal taxa under V. corru-
gata, resulting in a geographic distribution
from Norway to Natal, Mozambique, and be-
yond, and thus presenting the possibility that
this is not one wide-spread species but a se-
ries of similar taxa. This, then, limits what can
be inferred about the integrity of Venerupis.
What can be inferred about these taxa?
Since Venerupis aurea falls among the
Ruditapes, it calls into question the placement
of М aurea within Venerupis, the correct
placement of the Mediterranean Ruditapes
decussatus within Ruditapes, OR, if one as-
sumes that the Mediterranean Ruditapes de-
cussatus is indeed a valid representative of
that taxon, the integrity of Ruditapes as a
taxon. With respect to the last possibility,
Venerupis Montagu, 1803, was established
long before Ruditapes Chiamenti, 1900. Thus,
if species of Venerupis are found to fall within
Ruditapes, then Ruditapes would be a junior
synonym of Venerupis. The data presented do
not indicate which of the three possibilities
might be correct, so no such inference can be
made.
CONCLUSION
Careful study of these papers illustrates
that the species chosen for analyses will limit
218
Crassostrea gigas
Crassostrea gigas
58
HARTE
Ruditapes decussatus
Venerupis aurea
Ruditapes philippinarum
Dosinia lupinus
Chamelea gallina
Venus verrucosa
Pitar rudis
Callista chione
Venus verrucosa
Chamelea gallina
Dosinia lupinus
Pitar rudis
Callista chione
Ruditapes philippinarum
Venerupis aurea
Ruditapes decussatus
FIG. 2. Neighbor-Joining trees constructed with pairwise distances calculated following an application of two-
parameter correction for multiple substitutions (upper), and calculated on the basis of the sole transversions
(lower), modified after Canapa et al. (1996: figs. 2, 3). The numbers represent the percentage of 100 boot-
strap replications in which a given node appeared.
what can be interpreted about higher taxa. In
order to make significant inferences about
generic and subgeneric taxonomy from the
trees of cladistic or other types of analyses,
studies must include at least the type species
of those taxa, and preferably other species
belonging to the same taxon or, in the case of
larger taxa, that represent adequately the di-
versity of the taxon. In morphological cladis-
tics, character states must be carefully de-
fined. An initial review of the taxonomy of the
group to be analyzed should be conducted, so
as to optimize choice of species for insights
into the taxonomy. To ensure that the material
TRANSLATING TREES INTO TAXONOMY 219
is indeed the originally described species, the
specimens should come from the same geo-
graphic locale as the originally described type
specimens for the species. Conversely, if
availability limits choice, this must be reflected
in the limits of the resulting discussion of the
taxa and presentation of cladograms.
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1992, Carnivorous plants: phylogeny and struc-
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revisions des Veneridae (Moll. Lamellib.) Chion-
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LAKE, 1995, Evidence from 18s ribosomal DNA
that the lophphorate are protostome animals.
Science, 267: 1641-1643.
HARTE, М. Е, 1992, An eastern Pacific Mercenaria
and notes on other chionine genera (Bivalvia:
Veneridae). The Veliger, 35: 137-140.
JONES, C., 1979, Anatomy of Chione cancellata
and some other chionines (Bivalvia: Veneridae).
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KEEN, A. M., 1969, Veneracea. Pp. 670-690, in
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Mollusca 6. Bivalvia, vol. 2. В. С. MOORE, ed. The
Geological Society of America and The University
of Kansas.
ROOPNARINE, P.D, 1996, Systematics, biogeogra-
phy and extinction of chionine bivalves (Bivalvia:
Veneridae) in tropical America: Early Oligocene—
Recent. Malacologia, 38: 103-142.
SAITOU, N. & M. NEI, 1987, The Neighbor-Joining
method: a new method for reconstructing phylo-
genetic trees. Molecular Biological Evolution, 4:
406-425.
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and classification of birds. Yale University Press,
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SIMON, L., J. BOUSQUET, В. С. LEVESUE 8 MAU-
RICE LALONDE, 1993, Origin and diversification
of endomycorrhizal fungi and coincidence with
vascular land plants. Nature, 363: 67-69.
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Revised ms. accepted 22 April 1997
MALACOLOGIA, 1998, 39(1-2): 221-224
TRANSLATING TREES INTO TAXONOMY WITHIN VENERIDAE (BIVALVIA):
A REPLY TO HARTE
Peter D. Roopnarine
Department of Biology, Southeast Missouri State University, Cape Girardeau,
Missouri 63701, U.S.A.
Harte (this volume) has initiated, in com-
ments on two papers (Capana et al., 1996;
Roopnarine, 1996), an interesting and much
needed discussion of the status of venerid
systematics. The major focus of her commen-
tary on Roopnarine (1996) is the selection
and utilization of morphological characters for
a phylogenetic analysis of the Chioninae, and
the subsequent revision of the taxonomy of
that subfamily on the basis of the phylogenetic
analysis. In recognition of the fact that modern
phylogenetic analysis is a dynamic process of
hypothesis construction, testing and restruc-
turing, | welcome her comments on my paper
and below will admit shortcomings of my
study but will also defend what | believe are
valid and valuable contributions.
Character Definitions
Harte’s first source of objection is the de-
scriptive nature of several conchological char-
acters employed in the phylogenetic analysis,
for example pallial sinus depth. It should be
made clear that, while the explanations of
character states (for example, “greatly re-
duced to absent” versus “present but short”)
may be verbally ambiguous, morphologically
they are not. Close examination of taxa in-
volved, such as Chione (von Mühlfeld) and
Lirophora Conrad, reveals that these charac-
ter states are consistently identifiable and
separable. Certainly a more robust method of
presentation that would permit precise repli-
cation by other workers, besides productive
interworker Communication, would be the
quantitative description of such characters.
Attempts have been made to incorporate
quantitative description of continuously vary-
ing characters into phylogenetic analyses,
such as gap coding (Archie, 1985) and more
recently thin plate spline decomposition of
landmark data (Naylor, 1996; Fink & Zelditch,
1995; Zelditch et al., 1995), but much remains
to be done in this area. In fact, salient argu-
ments have been presented against the feasi-
221
bility of such approaches (e.g., Bookstein,
1994).
One other reason for apparent character
ambiguity is a less fundamental one. While
many characters can be coded as dichoto-
mously discrete states, several have addi-
tional states that represent single taxa. An ex-
ample is ventral margin crenulation, which is
normally either very regular and fine, or alter-
natively consists of significantly larger,
coarser and more variable subunits (for ex-
ample, Lirophora versus Chione). All taxa in-
cluded in my analysis could be coded as one
state or the other with the single exception of
Chionista Keen, which has marginal crenula-
tions intermediate in relative size and transi-
tions posteriorly from regular to coarse and ir-
regular. The trichotomous nature of this
character then is based on one taxon out of
sixteen. However, the potential ecomorpho-
logical importance of this character (Vermeij,
1987, 1993) justifies its inclusion in the analy-
sis.
Another cited source of ambiguity is the fail-
ure to report the relative occurrence of a char-
acter state in a species. Only character states
that were recognized as invariable with re-
spect to relevant taxa were analyzed. Char-
acter conditions resulting from ontogenetic or
environmental variation were purposefully ex-
cluded. Such an example would be the radials
of Mercenaria mercenaria (L.) cited by Harte.
These are typically obvious on the smoothed
central portions of adult valves, and have even
been used in the past as support of subspe-
cific recognition (for example, M. mercenaria
subradiata Palmer). The appearance of radial
lines on the surfaces of smoothed, worn or
eroded venerid valves that normally possess
only concentric or commarginal sculpture is
very common, but these radials should not
necessarily be recognized as primary sculp-
ture. They are perhaps indeed of phylogenetic
importance, an issue not explored in my
paper, but until they are examined in that con-
text should not be considered homologous
222 ROOPNARINE
with the prominent and primary radial sculp-
ture of taxa such as Chione, Chionopsis
Olsson and Puberella Fischer-Piette. Harte
(1992) makes such an assumption (in addi-
tion to the possession of a rugose nymph)
when arguing for the assignment of the
species Lirophora kellettii (Hinds) to genus
Mercenaria. As discussed in Roopnarine
(1996), this species is more properly assigned
to Panchione Olsson (Early Miocene-Recent)
because of its obvious and overwhelming sim-
ilarity to the numerous described extinct
species also assigned to the genus, for exam-
ple P mactropsis (Conrad) and Р шосута
(Dall). Panchione kellettii bears faint radial
lines on its surface, but this is characteristic of
all species of Panchione, but certainly not
Mercenaria.
Representation of Genera
The absence of type species for some of
the genera used in the systematic analysis
does introduce a certain amount of ambiguity
in the results if they are viewed at a level of
phylogenetic universality beyond mere con-
sideration of former genus Chione. The extent
and effect of the ambiguity of course remains
itself ambiguous until the data are reanalyzed
with the inclusion of the type species. | do not
believe that inclusion of the type or additional
species of Puberella will alter the resulting
cladograms in any way, because the species
are morphologically very similar, being sepa-
rated primarily by time, geography, and such
labile characteristics as shell size (e.g.,
Stanley & Yang, 1987). Increasing the cover-
age of Protothaca Dall and Timoclea Brown in
the analysis was unnecessary, because the
major focus was to analyze the evolutionary
histories of the tropical American Chione sub-
genera, and to place their histories in a
testable phylogenetic framework. The phylo-
genetic relationships of these two genera,
which range far beyond tropical American wa-
ters, to the former Chione subgenera is un-
doubtedly of great interest. That analysis
would definitely require the examination of
generic type species, as well as subgeneric
types, a process that | am currently undertak-
ing as an analysis of subfamily Chioninae.
Moreover, varying degrees of relationship of
say Timoclea (Timoclea) and T. (Glycydonta)
to Chione would lead to much more than a
simple change of Timoclea's position on the
current consensus tree. It would instead imply
paraphyly of that genus and would require its
entire taxonomic reconstruction.
Classification
In converting the results of my phylogenetic
analyses to a revised hierarchical taxonomy
of Chione, | relied upon two criteria: (1)
changes to the existing taxonomy should be
minimized, and (2) the revised taxonomy
should be logically consistent with the under-
lying hypothesis of phylogeny (Wiley et al.,
1991; deQueiroz & Gauthier, 1992). As Harte
points out, the revised taxonomy is consistent
with the phylogenetic results, but alternative
revisions are available. Her major emphasis
and point of contention seems to be the ele-
vation of subgenera in the Lirophora clade
(Anomalocardia Schumacher, /liochione
Olsson, Lirophora and Panchione) to generic
status. She suggests instead that the hierar-
chical information implied by the topology of
this subclade could be retained in a revised
taxonomy by subsuming all subgenera under
the historically senior taxon, in this case
Anomalocardia. This alternative, while simpler
and almost as informative, would unfortu-
nately be inconsistent with the cladistic re-
sults. Anomalocardia and lliochione spring
from an unresolved polytomy, along with a
branch bearing Lirophora and Panchione. The
placement of lliochione with respect to the
other two branches is therefore unknown, and
additional information may well place it out-
side of Harte’s Anomalocardia (Fig. 1). The re-
sulting Anomalocardia, while monophyletic,
would conflict with the hierarchical structure
implied by the phylogeny. Interestingly, the re-
cent description of an extinct chionine genus
(Roopnarine, in press) (Fig. 2) also from trop-
са! America partially supports Harte's sug-
gestion. It may now be reasonable to consider
lliochione a subgenus of Anomalocardia.
| would like to reiterate that the taxonomic
revision of Chione suggested in Roopnarine
(1996) merely gives phylogenetic and paleon-
tological support to revisions implemented by
previous workers. For example, Olsson (1961)
regarded Chionopsis as a genus distinct from
Chione; Keen (1969) treats Panchione as dis-
tinct from Lirophora; and Woodring (1982)
and Ward (1992) treat Lirophora as a generic
rank taxon. Finally, at the risk of portraying
myself as an unrepentant cladist, | must point
out that the maintenance of an artificial sys-
tem of hierarchical categorization and classifi-
cation is one of convenience (albeit a very in-
A REPLY TO HARTE 223
Mercenaria
Puberella
Chionopsis
Protothaca
Timoclea
Chionista
Chione
Anomalocardia
Iliochione
Lirophora
Panchione
Mercenaria
Puberella
Chionopsis
Protothaca
Timoclea
Chionista
Chione
Anomalocardia
Iliochione
Lirophora
Panchione
Mercenaria
Puberella
Chionopsis
Protothaca
Timoclea
Chionista
Chione
Iliochione
Anomalocardia
Lirophora
Panchione
FIG. 1. Three possible solutions to the polytomy pre-
sented in Roopnarine (1996: figs. 4, 20). The upper
two cladograms would support the inclusion of
lliochione within a new genus Anomalocardia, but
the lowest cladogram would be incompatible with
the hierarchical structure implied by such an inclu-
sion (that is, genus Anomalocardia could then also
include genera Chione, Protothaca, Timoclea, etc.).
Mercenaria
Puberella
Chionopsis
Chione
Protothaca
Timoclea
Liromissus
Lirophora
Panchione
Iliochione
Anomalocardia
FIG. 2. Strict consensus tree of four equally most
parsimonious cladograms illustrating the relation-
ship of Liromissus Roopnarine (a recently described
genus endemic to the late Neogene of Venezuela) to
other chionine genera (consistency index = 0.426)
(Roopnarine, in press).This solution supports the in-
clusion of /liochione within Anomalocardia as a sub-
genus. Note the loss of resolution, with respect to
Chione, Chionopsis, Protothaca, Puberella and
Timoclea in the other portion of the tree. The corre-
spondence between addition of a taxon and loss of
resolution suggests the need for more characters,
possibly non-conchological (although this would be
difficult for the extinct Liromissus).
valuable one). The Linnean system of taxo-
nomic classification does not necessarily lend
itself to descriptions of history, and it was
never intended to, nor will it ever be capable of
capturing fully the depth of phylogenetic rela-
tionships implied by cladistic hypotheses.
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Revised ms. accepted 22 April 1997
MALACOLOGIA, 1998, 39(1-2): 225
CORRECTIONS TO WHITE ET AL., 1996, MOLECULAR GENETIC
IDENTIFICATION TOOLS FOR THE UNIONIDS OF FRENCH CREEK,
PENNSYLVANIA MALACOLOGIA 38:181-202
Laura R. White
School of Forest Resources, The Pennsylvania State University, University Park,
Pennsylvania 16802, U.S.A.
It has recently come to my attention that several errors appeared in the “Laboratory
Techniques” section of the article “Molecular genetic identification tools for the unionids of French
Creek, Pennsylvania” [White et al., Malacologia, 1996, 38(1-2): 181-202]. Specifically, the con-
centration of proteinase K in the standard phenol-chloroform nucleic acid extraction protocol
should be 0.05 ug/ul (instead of 5 ug/ul); the concentration of MgCl, in the manufacturer-supplied
amplification buffer at 1x final concentration should be 1.5 mM (instead of 15 mM); and the con-
centration of gelatin should be 0.001% (instead of 0.01%). | regret any inconvenience or confu-
sion that these errors might have caused.
7 May 1997
The editor-in-chief of Malacologia welcomes let-
ters that comment on vital issues of general im-
portance to the field of Malacology, or that com-
ment on the content of the journal. Publication is
dependent on discretion, space available and, in
some cases, review. Address letters to: Letter to
the Editor, Malacologia, care of the Department of
Malacology, Academy of Natural Sciences, 1900
Benjamin Franklin Parkway, Philadelphia, PA
19103-1195, U.S.A.
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MALACOLOGIA, 1998, 39(1-2): 227-234
INDEX
Page numbers in italics indicate figures of Anomalocardia 216, 217, 222, 223
taxa. No new taxa appear in this number of arborea, Leiostyla 32, 34
Malacologia. arboreus, Zonitoides 3, 4, 6, 34
arcta, Actinella 32, 34
Achatina fulica 79 arcticus, Bathypolypus 11-19
Acer spicatum 2 Arcuatula 129, 131, 132-135
acicula, Cecilioides 33, 34 capensis 130, 132, 132
Actinella actinophora 32 Arcuatulinae 135
arcta 32, 34 Argonautidae 14
carinofausta 32, 34 Arianta arbustorum 148, 167, 171
fausta 32 Ariolimax columbianus 48
giramica 32, 34 Arion 39
lentiginosa 32, 34 subfuscus 77-81
nitidiuscula 32 Arionidae 107
obserata 32, 34 aspera, Columella 33
actinophora, Actinella 32 aspera, Helix 33, 34, 145, 167-173
aculeata, Acanthinula 33, 34 asperrima, Protothaca 216
acuta, Physa 127 ater, Aulacomya 130, 132
Adacna 136 atra, Aulacomya 83-91, 87
Adula 129, 131, 132, 134, 135 Aulacomya 129, 131, 132, 134, 136
falcatoides 130, 132 ater 130, 132
Adulinae 135 atra 83-91, 87
Aenio-Lytanthion 30 aurantium, Haliotis 59-75, 61, 62, 64-66, 68
Aeonio-Lyntathion 30, 33, 34, 37 aurea, Venerupis 217, 218
agrestis, Agriolimax 22 Austenia 107
Agriolimacidae 39, 41, 47, 51 Averellia 96
Agriolimax, agrestis 22 barbara, Cochlicella 33, 34
alabamensis, Elimia 185-191 Bathypolypus arcticus 11-19
alatus, Potamilus 195, 196, 198-202 sponsalis 17
alatus, Unio 195 behnii, Phenacolimax 32, 34
albopalliatus, Phenacolimax 32, 34 Betula papyrifera 2
algosus, Semimytilus 130 bifrons, Janulus 32
alliarius, Oxychilus 33, 34 Binneya 107
Alnus 2 Biomphalaria glabrata 79, 123, 124, 127, 147,
minor 32 175-182, 180
mitriformis 32, 34 Biserrulae-Scorpiurietum 30
tornatellina 32 Boettgeria crispa 32, 34
alternata, Anguispira 4, 6 deltostoma 32
Amauropsis paludinaris 159 depauperata 32, 34
Amblema neislerii 203 exigua 32
plicata 203 Boettgerilla pallens 39-57, 41, 45, 53-55
americanus, Modiolus 130 Boettgerillidae 39
amphichaenus, Potamilus 195-202 Botulinae 135
Amphorella iridescens 32, 34 Brachidontes 129, 131, 132, 134
ampla, Leptoxis 113-121, 116 semistriatus 130, 132, 132
Ampullariidae 207, 212 Brachidontinae 135
Anculosa 113 Bradybaena 93, 95, 97-100, 107, 107
Anculosae 116 Bradybaenidae 93, 96, 104, 106-108
Ancylus fluviatilis 141 Brugia malayi 175
Anguispira alternata 4, 6 budapestensis, Tandonia 39-57, 41, 45, 52
angustifolium, Epilobium 2 Bulimulidae 107
zer
228 INDEX
Bulinus tropicus 147
Bunnya 95, 97-100, 101, 103, 104, 106
Busycon carica 151-165, 154
scalarispira 151-165
Busycotypus canaliculatus 151-165, 153, 154
caelatura infuscata, Elimia 185-191
californiense, Keenocardium 134, 136
callicratis, Truncatellina 33
Callinectes sapidus 152
Callista chione 217, 218
canadense, Carychium exile 3, 6, 7
canaliculata, Pomacea 207-213
canaliculatus, Busycotypus 151-165, 153, 154
cancellata, Chione 216
capax, Potamilus 195, 196, 198-202
capensis, Arcuatula 130, 132, 132
capensis, Siphonaria 147
Caracollina lenticula 33, 34
Cardiidae 134
Cardioidea 134
carica, Busycon 151-165, 154
carinocostata, Elimia 185-191
carinofausta, Actinella 32, 34
cariosa, Lampsilis 201
Carychium canadense exile 3, 6, 7
minimum 33
tridentatum 33
Caseolus compactus 32, 34
leptostictus 32, 34
catenaria, Elimia 185
Cecilioides acicula 33, 34
Cepaea hortensis 148
nemoralis 148
Cepoliinae 93, 104, 107
Cepolis 95, 97-100, 103, 104, 107
cerina, Fusconaia 196, 198-202
Cerithioidea 183
Chamelea gallina 217, 218
Charodotes 95-100, 103, 104, 106, 107
cheilogona, Leiostyla 32, 34
chemnitzii, Natica 159, 163
chierchiae, Octopus 11
chilensis, Mytilus 83-91, 86, 87, 130
chione, Callista 217, 218
Chione 216, 217, 221-223
cancellata 216
Chioninae 215, 217, 221
Chionista 216, 217, 221, 223
Chionopsis 216, 217, 222, 223
Choromytilus 129, 131, 132, 134-136
chorus 130
grayanus 130
meridionalis 130, 132, 132
chorus, Choromytilus 130
Clausiliidae 34, 37
Clethro-Laurion 30, 33, 34, 36, 37
Clinocardiinae 134, 136
Cochlicella barbara 33, 34
Cochlicopidae 34
Cochlicopa lubrica 3, 4, 6, 33, 34
lubricella 33
colorata, Hypanis 136
columbianus, Ariolimax 48
Columella aspera 33
edentula 3, 4, 6
microspora 32
compactus, Caseolus 32, 34
concinna, Leiostyla 32, 34
contortus, Planorbis 141, 146
contracta, Vitrea 33
corneus, Planorbarius 141-150
cornuarietis, Marisa 209, 211, 212
Corophium volutator 14
corrugata, Venerupis 217
coruscus, Mytilus 130, 133, 133
costata, Vallonia 33, 34
cracherodii, Haliotis 73
Crangon 12, 13
mucronatum 32
neritoides 32
septemspiosus 14
trochoideum 32
Craspedopoma monizianum 32, 34
crenatella, Elimia 185-191
Crenellinae 129, 135
Crenomytilus 131, 133, 134, 136
grayanus 133
Crepidula 127
crispa, Boettgeria 32, 34
cronkhitei, Discus 3, 4, 6, 7
Cryptostrakon 95-100, 104, 106, 107
Cyclophoridae 34
cylindracea, Elimia 185-191
cylindracea, Lauria 33
Dacrydiinae 129, 135
dalli, Haliotis 59
Daudebardia 51, 107
decussatus, Ruditapes 217, 218
decussatus, Tapes 217
deltostoma, Boettgeria 32
depauperata, Boettgeria 32, 34
depressa, Pellicula 107
Deroceras 21-27
laeve 3, 4, 6, 21-27, 39-57, 41, 45, 54, 55,
79
reticulatum 22, 25, 39-57, 41, 42, 54, 55
rodnae 39-57, 41, 45
Dialeuca 93, 95-98, 103, 104, 107
Dinotropis 96
Diplompharus 51
discors, Musculus 130, 133, 134
Discula polymorpha 32
tabellata 32, 34
Disculella maderensis 32, 34
Discus cronkhitei 3, 4, 6, 7
dolioides, Pomacea 212
dorsalis, Pallifera 4, 6
Dosinia lupinus 217, 218
Dosiniinae 217
duplicata, Neverita 151-165, 154
edentula, Columella 3, 4, 6
edulis, Mytilus 130, 133
Elimia 113, 114, 183-193
alabamensis 185-191
caelatura infuscata 185-191
carinocostata 185-191
catenaria 185
crenatella 185-191
cylindracea 185-191
fascinans 185-191
gerhardtii 185-191
haysiana 185-191
hydei 185-191
olivula 185-191
showalteri 185-191
vanuxemiana 185
Endodontidae 34
Epiphragmophora 95, 97-100, 101,104, 107
Epiphragmophorinae 93
Epilobium angustifolium 2
Eremarionta 95-100, 104, 105, 106, 107
Erica 30
erubescens, Leptaxis 32
Euconulus fulvus 3, 4, 6, 7, 33
Euspira 159, 163
heros 151-165, 153, 154
rectilabrum 159
exigua, Boettgeria 32
exigua, Striatura 3, 6
falcatoides, Adula 130, 132
fanalensis, Lauria 32, 34
fascinans, Elimia 185-191
fausta, Actinella 32
Ferrusacia folliculus 33, 34
Ferussaciidae 34, 37
filicum, Leiostyla 32, 34
floridana, Goniobasis 115
fluviatilis, Ancylus 141
folliculus, Ferrusacia 33, 34
INDEX
229
fragilis, Leptodea 195, 198-202
fulica, Achatina 79
fulvus, Euconulus 3, 4, 6, 7, 33
furva, Leptaxis 32, 34
fusca, Leiostyla 32, 34
Fusconaia cerina 196, 198-202
gallina, Chamelea 217, 218
galloprovincialis, Mytilus 90, 130, 133
Gastrocopta tappaniana 4, 6
gayi, Tawera 83-91, 86, 87
gerhardtii, Elimia 185-191
geversianus, Trophon 83-91, 87
giramica, Actinella 32, 34
glabrata, Biomphalaria 79, 123, 124, 127,
147, 175-182, 180
Glycydonta 216, 222
Goniobasis 113-116
floridana 115
proxima 115, 119
gouldi, Vertigo 3, 6, 7
grandis, Pyganodon 203
grayanus, Choromytilus 130
grayanus, Crenomytilus 133
Gyrotoma 113
Haliotidae 59-75
Haliotis 59, 67
aurantium 59-75, 61, 62, 64-66, 68
cracherodii 73
dalli 59
pourtalesii 59-75, 61-63, 69, 70, 72
lamellosa 59, 60, 73
roberti 59, 73
tuberculata 59, 60, 67, 73
hammonis, Nesovitrea 33
harpa, Zoogenetes 3, 4, 6
Hawaiia miniscula 33, 34
haysiana, Elimia 185-191
Helicarionidae 105
Helicidae 34, 36, 37, 93, 96, 106, 107
Helicodiscus singleyanus 33, 34
Helicoidea 91, 106
Helisoma trivolvis 141, 148
Helix 39, 93, 95, 97-100, 104, 106, 107
aspersa 33, 34, 147, 167-173
lucorum 147
pomatia 167
Helminthoglypta 95-100, 104, 105, 106, 107
Helminthoglyptidae 93-111
Hemilauria limneana 32, 34
heros, Euspira 151-165, 153, 154
Heterodonta 91
Heterostoma paupercula 32
Hiatella solida 83-91, 86, 87
230 INDEX
Hiatellidae 91
hortensis, Cepaea 148
Humboldtiana 95, 97-100, 105
Humboldtianae 93
Hydrobia ulvae 146, 147
hydei, Elimia 185-191
Hypanis colorata 136
laeviuscula 136
minima 136
vitrea 136
iheringi, Peltella 107
lliochione 216, 217, 222, 223
llyanassa obsoleta 146
inflatus, Potamilus 195-203
integra, Physa 146
iridescens, Amphorella 32, 34
irrigua, Leiostyla 32
Janulus bifrons 32
stephanophora 32, 34
keenae, Septifer 130, 132
Keenocardium californiense 134, 136
kellettii, Lirophora 222
kellettii, Panchione 222
kurilensis, Modiolus 130, 132
labyrinthica, Strobilops 3, 4, 6
laeve, Deroceras 3, 4, 6, 21-27, 39-57, 41, 45,
54.5519
laevigatus, Musculus 130
laevissima [= ohiensis], Potamilus 195
laeviuscula, Hypanis 136
lamellosa, Haliotis 59, 60, 73
Laminaria 67
Lampsilis 195, 201
cariosa 201
ornata 195, 198-202
satura 201
ventricosa 197
Lasmonos 196
Lastena 195, 198
latens, Spirorbula 32
Lauria cylindracea 33
fanalensis 32, 34
laurinea, Leiostyla 32, 34
leacockiana, Pyrgella 32
Lehmannia 127
marginata 39-57, 41-44, 46, 48, 50-54
valentiana 127
Leiostyla 37
arborea 32, 34
cheilogona 32, 34
concinna 32, 34
filicum 32, 34
fusca 32, 34
irrigua 32
laurinea 32, 34
loweana 32
millegrana 32, 34
recta 32
sphinctostoma 32
vincta 32, 34
lentiginosa, Actinella 32, 34
lenticula, Caracollina 33, 34
Leptarionta 95-100
Leptaxis erubescens 32
furva 32, 34
membranacea 32
undata 32
Leptodea 195, 196, 198, 201
fragilis 195, 198-202
leptodon 201
ochracea 201
leptodon, Leptodea 201
leptostictus, Caseolus 32, 34
Leptoxis 113-121, 116
ampla 113-121, 116
picta 113-121, 116
plicata 113-121, 116
praerosa 113-121
taeniata 113-121, 116, 184-191
Leukoma 216
Limacidae 39, 47
Limacoidea 39-57
Limax 39
maximus 145
pseudoflavus 145, 147
limneana, Hemilauria 32, 34
Limnoperninae 135
limpida, Vitrina 3, 4, 6
Liromissus 223
Lirophora 216, 217, 221-223
kellettii 222
literatus, Tapes 217
Lithophagidae 135
Lithophaginae 129, 135, 136
Loligo 18
loweana, Leiostyla 32
lubrica, Cochlicopa 3, 4, 6, 33, 34
lubricella, Cochlicopa 33
lucorum, Helix 147
lupinus, Dosinia 217, 218
Lymnaea palustris 147
peregra 26
stagnalis 141, 145, 146, 175
truncatula 148
Lymnocardiinae 134, 136
Lysinoe 95, 97-100
INDEX 231
Lysinoinae 93 laevigatus 130
mactropsis, Panchione 222 Myoida 91
maderensis, Disculella 32, 34 Mysidiellidae 129
Malacolimax tennelus 39-57, 41, 52, 53, 55 Mytilidae 91, 129, 131, 134, 135
Malagarion paenelimax 107 Mytilinae 129, 133, 135, 136
malayi, Brugia 175 Mytilisepta 133
mansoni, Schistosoma 123, 175-177, 180- Mytiloida 91
182, 180 Mytilus 129, 131, 133, 134, 136
marcidus, Phenacolimax 32, 34 chilensis 83-91, 86, 87, 130
marginata, Lehmannia 39-57, 41-44, 46, 48, coruscus 130, 133, 133
50-54 edulis 130, 133
marica, Timoclea 216 galloprovincialis 90, 130, 133
Marisa cornuarietis 209, 211, 212 trossulus 130, 133
maximus, Limax 145 Mytiloidea 129-139
Melanoides tuberculata 147 Natica chemnitzii 159, 163
membranacea, Leptaxis 32 Naticidae 84
Mercenaria 215, 217, 222, 223 Nautilus 18
mercenaria 215, 221 neislerii, Amblema 203
mercenaria subradiata 221 nemoralis, Cepaea 148
meridionalis, Choromytilus 130, 132, 132 Neogastropoda 91
Metostracinae 93 Neohelix 93, 95, 97-100, 104
Metostracon 95-100, 104, 105, 106, 107 neritoides, Crangon 32
Micrarionta 95-100, 104, 106, 107 Nesovitrea hammonis 33
microspora, Columella 32 Neverita duplicata 151-165, 154
Milacidae 39, 41, 47, 49-51 nitidiuscula, Actinella 32
milium, Striatura 3, 4, 6, 7 nitidus, Phenacolimax 32, 34
millegrana, Leiostyla 32, 34 Nostoc 123
minima, Hypanis 136 Nucella 184
minimum, Carychium 33 Obliquaria reflexa 197-202
miniscula, Hawaiia 33, 34 obserata, Actinella 32, 34
minor, Alnus 32 obsoleta, llyanassa 146
minutissimum, Punctum 6 ochracea, Leptodea 201
mitriformis, Alnus 32, 34 Octopoda 11
modesta, Vertigo 3, 4, 6 Octopodidae 11, 12
Modiolinae 129, 132, 135, 136 Octopus 11-18, 15, 16
modiolus, Modiolus 130 chierchiae 11
Modiolus 129-132, 134, 135 ohiensis, Potamilus 195-202
americanus 130 olivula, Elimia 185-191
kurilensis 130, 132 Oncomelania 184
modiolus 130 Opuntia tuna 30
Monadenia 95, 97-100, 106-108 Oreohelicidae 96
monizianum, Craspedopoma 32, 34 Oreohelix 93, 95, 97-100, 103
Monodacna 136 ornata, Lampsilis 195, 198-202
mucronatum, Crangon 32 ovalis, Succinea 4, 6
Muricidae 84 ovata, Vertigo 4, 6
muriciformis, Xymenopsis 84, 86-91 ovata, Timoclea 216
Muricoidea 83, 84, 91 Oxychilus alliarius 33, 34
Musculinae 129, 134 Padollus 67
Musculinae 135 paenelimax, Malagarion 107
Musculista 129, 131, 132, 134-136 pallens, Boettgerilla 39-57, 41, 45, 53-55
senhousia 130, 132 Pallifera dorsalis 4, 6
Musculus 129, 131, 133, 134, 136 paludinaris, Amauropsis 159
discors 130, 133, 134 palustris, Lymnaea 147
232
Panchione 216, 217, 222, 223
kellettii 222
mactropsis 222
ulocyma 222
Paraptera 196
Parmarion 107
papyrifera, Betula 2
paupercula, Heterostoma 32
Pellicula depressa 107
Peltella iheringi 107
peregra, Lymnaea 26
perforans, Venerupis 217
perna, Perna 130, 133
Perna 129, 131, 133-136
perna 130, 133
viridis 130, 133, 133
Pernadae 135
Pernidae 135
Perninae 129, 135, 136
Phenacolima ruivensis 32
Phenacolimax albopalliatus 32, 34
behnii 32, 34
nitidus 32, 34
marcidus 32, 34
ruivensis 32
philippinarum, Ruditapes 218
philippinarum, Tapes 217
Physa acuta 127
integra 146
sayii 147
Picea 1
picta, Leptoxis 113-121, 116
Pila 212
pisana, Theba 33, 34
Pitar rudis 217, 218
Pitarinae 217
placida, Plagyrona 33, 34
Plagyrona placida 33, 34
Planorbarius corneus 141-150
planorbis, Planorbis 148
Planorbis contortus 141, 146
planorbis 148
Planorbidae 141
Plesarionta 95-100, 104, 106, 107
Pleurocera 114
prasinatum 184-191
Pleuroceridae 116, 183
plicata, Amblema 203
plicata, Leptoxis 113-121, 116
Polinices tumidus 159, 163
uber 159, 163
Polygyridae 96
Polymita 95, 97-100, 101, 104, 107
INDEX
polymorpha, Discula 32
Pomacea 207
canaliculata 207-213, 209
dolioides 212
urceus 212
pomatia, Helix 167
Populus tremuloides 2
Potamilus 195-205
alatus 195, 196, 198-202
amphichaenus 195-202
capax 195, 196, 198-202
inflatus 195-203
laevissima 195
ohiensis 195-202
purpuratus 195-202, 197
purpuratus coloradoensis 195, 197-202
pourtalesii, Haliotis 59-75, 61-63, 69, 70, 72
praerosa, Leptoxis 113-121
prasinatum, Pleurocera 184-191
Proptera 195
Protothaca 216, 217, 222, 223
asperrima 216
proxima, Goniobasis 115, 119
pseudoflavus, Limax 145, 147
Pteriomorpha 91
Puberella 216, 217, 222, 223
pulchella, Vallonia 33, 34
Pulmonata 77, 123, 141, 167
Punctum minutissimum 6
pusillum 33
pygmaeum 33, 34
Pupillidae 34, 36, 37
purpuratus, Potamilus 195-202, 197
purpuratus coloradoensis, Potamilus 195,
197-202
pusillum, Punctum 33
Pyganodon grandis 203
pygmaea, Vertig 33
pygmaeum, Punctum 33, 34
Pyrgella leacockiana 32
Pyrgulopsis 191
recta, Leiostyla 32
rectilabrum, Euspira 159
reflexa, Obliquaria 197-202
reticulatum, Deroceras 22, 25, 39-57, 41, 42,
54, 55
Rhytididae 51
roberti, Haliotis 59, 73
rodnae, Deroceras 39-57, 41, 45
Rubus 2
rudis, Pitar 217, 218
Ruditapes 217
decussatus 217, 218
INDEX
philippinarum 218 Tapetinae 217
ruivensis, Phenacolima 32 tappaniana, Gastrocopta 4, 6
Salix 2 Tawera gayi 83-91, 86, 87
sapidus, Callinectes 152
satura, Lampsilis 201
Saxicavidae 90
saxicola, Staurodon 32
sayli, Physa 147
scalarispira, Busycon 151-165
Schistosoma mansoni 123, 175-177, 180-182,
180
Semimytilus 129, 131, 133, 135
algosus 130
semistriatus, Brachidontes 130, 132, 132
senhousia, Musculista 130, 132
septemspiosus, Crangon 14
Septiferidae 129, 135
Septiferinae 135
Septifer 129, 131, 133-135
keenae 130, 132
Setipelis 96
singleyanus, Helicodiscus 33, 34
sirius, Siphonaria 147
Siphonaria capensis 147
sirius 147
solida, Hiatella 83-91, 86, 87
Somatogyrus 191
Sonorella 95, 97-100, 101, 102, 106, 108
Sonorelix 96
Sonorellinae 93
sphinctostoma, Leiostyla 32
spicatum, Acer 2
Spirorbula latens 32
squalida 32, 34
sponsalis, Bathypolypus 17
squalida, Spirorbula 32, 34
stagnalis, Lymnaea 141, 145, 146, 175
Staurodon saxicola 32
stephanophora, Janulus 32, 34
Striatura exigua 3, 6
milium 3, 4, 6, 7
Strobilops labyrinthica 3, 4, 6
Stylommatophora 49, 93, 96, 167
subfuscus, Arion 77-81
subradiata, Mercenaria mercenaria 221
Succinea ovalis 4, 6
tabellata, Discula 32, 34
taeniata, Leptoxis 113-121, 116, 184-191
Tandonia budapestensis 39-57, 41, 45, 52
Tapes 217
decussatus 217
literatus 217
philippinarum 217
233
tennelus, Malacolimax 39-57, 41, 52, 53, 55
Thaididae 84
Theba pisana 33, 34
Teratiodae 123-128
Testacella 107
Testacellidae 107
Timoclea 216, 217, 222, 223
marica 216
ovata 216
tornatellina, Alnus 32
tremuloides, Populus 2
Trichodiscina 95-100
Trichomyinae 135
tridentatum, Carychium 33
trivolvis, Helisoma 141, 148
trochoideum, Crangon 32
Trophon geversianus 83-91, 87
Trophonidae 83-91
tropicus, Bulinus 147
trossulus, Mytilus 130, 133
Truncatellina callicratis 33
truncatula, Lymnaea 148
Tryonigens 95, 97-100, 107, 106, 108
tuberculata, Haliotis 59, 60, 67, 73
tuberculata, Melanoides 147
tumidus, Polinices 159, 163
tuna, Opuntia 30
uber, Polinices 159, 163
ulocyma, Panchione 222
ulvae, Hydrobia 146, 147
undata, Leptaxis 32
Unio alatus 195
Uniomerus 203
Unionidae 195, 225
urceus, Pomacea 212
valentiana, Lehmannia 127
Valloniidae 34
Vallonia costata 33, 34
pulchella 33, 34
vanuxemiana, Elimia 185
Veneridae 91, 215, 217, 221
Venerinae 217
Veneroida 91
Venerupis 217
aurea 217, 218
corrugata 217
perforans 217
ventricosa, Lampsilis 197
Venus verrucosa 217, 218
verrucosa, Venus 217, 218
234 INDEX
Vertigo gouldi 3, 6, 7 Vitrinidae 34, 36, 37, 107
modesta 3, 4, 6 volutator, Corophium 14
ovata 4,6 Xanthonychidae 93-111
pygmaea 33 Xanthonyx 93, 95-100, 104, 106
Vetigastropoda 59, 73 Xerarionta 95-100, 104, 106, 107
vincta, Leiostyla 32, 34 Xymenopsis muriciformis 84, 86-91
viridis, Perna 130, 133, 133 Zonitidae 34, 51, 107
Vitrea contracta 33 Zonitoidea 39
vitrea, Hypanis 136 Zonitoides arboreus 3, 4, 6, 34
Vitrina limpida 3, 4, 6 Zoogenetes harpa 3, 4, 6
Ww <
17. Tables are to be used sparingly, and
vertical lines not atall. ^
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ANA MARIA LEAL-ZANCHET — |
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No TOM OS O e CONTENTS UN ee, |
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J. M. HAWKINS, M.W. LANKESTER, &R. R. ANE NELSON. +, y A 6
| Sampling Terrestrial Gastropods Using Cardboard Sheets rt ae EA К
| J. B. WOOD, E. KENCHINGTON, & R. K.O’DOR м PR LEA 53
a Reproduction and. Embryonic Development Time of Bathypolypus. Arcticus, A Deep-
7 a Sea Octopod (Cephalopoda: Octopoda) 0h. CL SE ое ВВ ont
' RICHARD M. LEBOVITZ | > :
\ l . The Inheritance of an Е Lethal Mutation in a Solf-Reproducing Terrestrial _ Es;
Slug, Deroceras Laeve EN Oe Be Sit ain pl shen the -- e
| В. A.D. CAMERON & L. М. COOK. SAN \ „u. EN
ot
Forest and Scrub Snail Faunas from Northern Madeira ...... RAR с CEN
f <
к oe кв
Comparative Studies on the Anatomy and Histology of the Alimentary Canal of т. к
} Limacoidea and Milacidae (Pulmonata: Stylommatophora) E . lo A AA fat
LUIZ RICARDO L.SIMONE — | 00 N,
Morphology of the Western Atlantic Haliotidae (Gastropoda A with Jar
| Description ofa New Species from A A Sais PATES à Ye 5
BENJAMIN J. GOMEZ, ANA M. ZUBIAGA, M. TERESA SERRANO, & EDUARDO ANGULO -
Histochemical and Ultrastructural Identification of Biphasic Granules in the Albumen
| ces | Secretory Cells of Arion Subfuscus (Gastropoda, Bulmenata} A A ER tu
SANDRA GORDILLO Y SANDRA N. AMUCHÄSTEGUI
- Estrategias de Depredaciön del Gastrópodo Perforador Trophon Geversianus (Pallas) |
- (Muricoidea: Traphanidae)ir set la a ER See NG Josie o: ea
MARIA GABRIELA CUEZZO › a E \
Cladistic Analysis of the Xanthonychidae (= Halmininoalyalidae) (Gastropoda:
Stylommatophora: Helicoidea) ......... gE Gy Tee aaa N, est AN |
ROBERT T. DILLON, JR. & CHARLES LYDEARD > | |
Divergence Among Mobile Basin Populations of the Pleurocerid Snail Genus,
Leptoxis, Estimated by Allozyme Electrophoresis ............................ à
CHARLES $. RICHARDS, CAROLYN PATTERSON, FRED A. LEWIS, & MATTY KNIGHT =
Larval Fusion and Development of Conjoined Teratoids in Biomphalaria Glabrata .
ALEXANDER |. KAFANOV & ANATOLY L. DROZDOV
Comparative Sperm Morphology and Phylogenetic Classification of Recent Modes
(Bala) LR do PU SR EE ло а В
KATHERINE COSTIL 8 STUART Е. В. BAILEY ) Ae
Influence of Water Temperature on the Activity of Planorbarius Corneus (L.)
(Pulmonata, Planorbidae)*.............» sce hess wee eee Pie В
GREGORY P. DIETL & RICHARD R. ALEXANDER |
Shell Repair Frequencies in Whelks and Moon Зпай$ from Delaware and Southern
New Jersey us en en abit SE ot, TE aaa AE he ate
CHRISTOPHE DESBUQUOIS & LUC MADEC | (
Within-Clutch Egg Cannibalism Variability in Hatchlihgs of the Land Snail Hele a
Aspersa (Pulmonata: Stylommatophora): Influence of Two Proximate Factors ...... |
MATTY KNIGHT, ANDRE N. MILLER, NEIL S. М. СЕОСНАСЕМ, FRED A.LEWIS, | > 1 ae
& ANTHONY R. KERLAVAGE 0
Expressed Sequence Tags (ESTs) of Biomphalaria Glabrata, an Intermediate Snail
Host of Schistosoma Mansoni: Use in the Identification of RFLP Markers ......... 34 175
CHARLES LYDEARD, JOHN H. YODER, WALLACE E. HOLZNAGEL, FRED G. THOMPSON, ¿Y 7
& PAUL HARTFIELD 7a
Phylogenetic Utility of the 5'-Half of Mitochondrial 16S rDNA Gene Sequences for. j=
Inferring Relationships of Elimia (Cerithioidea: Pleuroceridae) ..... со 183 |
KEVIN J. ROE & CHARLES LYDEARD oe
Molecular Systematics of the Freshwater Mussel Genus Potamilus (Bivalvia: a
уповает (PRET RS VAN SEE Sn ЛЬ à 195
ALEJANDRA L. ESTEBENET | 4
Allometric Growth and Insight on Sexual Dimorphism in Pomacea Canaliculata
(Gastropoda:‘Ampullaniidae).. бе A - 207.
LETTERS TO THE EDITOR & Е
MARY ELLEN HARTE 4 an
Translating Trees into Taxonomy within Veneridae (Bivalvia): A Critique of Two Recent \
Papers‘... u na ое Ale iq le RE АЕ EC EEE EEE 215
PETER D. ROOPNARINE i
Translating Trees into Taxonomy AN Veneridae (EVANS A Reply to Harte ....-. 221
LAURA В. WHITE
Corrections to White et al., 1996, Molecular Genetic Identification Tools for the
Unionids of French Creek, Pennsylvania Malacologia 38:181-202 .....:......... 2237)
A ES! | Pa a u $ ‘0 A к
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