J. Moll. Stud. (1998), 64,81-92
© The Malacological Society of London 1998
PHYLOGENETIC ANALYSIS OF MITOCHONDRIAL DNA
AND MORPHOLOGICAL CHARACTERS SUGGEST A NEED
FOR TAXONOMIC RE-EVALUATION WITHIN THE
ALOPIINAE (GASTROPODA: CLAUSILIIDAE)
V. DOURIS, S. GIOKAS1, R. LECANIDOU, M. MYLONAS2 and
G.C. RODAKIS
Department of Biology, Section of Biochemistry and Molecular Biology, University of Athens, 157 01 Athens,
Greece. 'Department of Biology, Section of Ecology and Taxonomy, University of Athens, 15784 Athens,
Greece. 'Department of Biology, University of Crete, P.O. Box 1470, 711 10 Iraclion, Crete, Greece
(Received 10 October 1996; accepted 25 April 1997)
conventional taxonomy. Thus, discrimination of
different species within each genus,.but also of
different genera, is often ambiguous (Nordsieck,
A combined approach of mitochondrial large subunit
ribosomal RNA (lrRNA) sequence analysis and
1984; Gittenberger, 1987; Gittenberger &
cladistic analysis of morphological characters was
Schilthuizen 1996). The suggested polyphyly of
used in an effort to test previously proposed hypotheses
the genus lsabellaria (Nordsieck, 1984; Gittenon the taxonomy and phytogeny of certain taxa within
berger, 1987), recently reinforced by sequence
the land snail family Clausiliidae. Ten populations
analysis of the ITSI rRNA region of some
belonging to three clausiliid subfamilies were exlsabellaria
and Albinaria taxa (Schilthuizen,
amined, focusing mainly on the Alopiinae. Special consideration was given to the Graciliaria-type clausiliar Gittenberger & Gultyaev, 1995) indicates that
taxa currently classified as lsabellaria should be
apparatus (GCA), whose homoplasious character is
reconsidered. Especially in the Peloponnese,
confirmed: the two approaches give congruent results
after its exclusion. The present analysis indicates that
an area where both genera exhibit mosaic
phylogenetic relations, at least within the Alopiinae,
distributions, identification of Albinaria and
have to be reexamined. In view of the above, a new lsabellaria species or subspecies is difficult. As
classification for certain taxa is proposed.
a result certain forms have an uncertain
systematic classification (as Albinaria or lsabellaria) or are considered as hybrids (Nordsieck,
INTRODUCTION
1984; Gittenberger, 1987,1994).
A special case of interest concerns the taxoThe clausiliid subfamily Alopiinae includes the nomic significance of the so called "Graciliaria
genera Albinaria, lsabellaria, Sericata, Agath- type" or "G-type" clausiliar apparatus (GCA)
ylla, Cristataria and Medora. From these, the (Fig. 2), a feature typical within lsabellaria but
taxonomic status of Albinaria, lsabellaria and uncommon in Albinaria (Gittenberger, 1987,
Medora, is uncertain (Nordsieck, 1977; Giusti, 1994). The GCA consists of a set of morphoGrappelli, Manganelli, Fondi & Bullini, 1986). logical characters not found in the majority of
Medora is distributed in Italy and along the the clausiliid taxa. Its presence in several but
Dalmatian coastline, while Albinaria and not closely related clausiliid genera (Idyla,
lsabellaria in the southern Balkan peninsula. Graciliaria, Armenica, Muticaria, Papillifera,
The latter two genera exhibit extreme inter- Neostyriaca, Leucostigma, Ruthenica etc.) has
specific and intraspecific morphological differ- been attributed to independent origins (Nordentiation, although their distribution, whether sieck, 1982; Gittenberger, 1987, 1994; Gittenallopatric or sympatric, is restricted to the rela- berger & Schilthuizen, 1996). It has also been
tively small area shown in Figure 1 (Nordsieck, proposed that the GCA is an effective adapta1974; Zilch, 1977a, 1977b). Clarification of
tion (Nordsieck, 1982), since its construction
evolutionary processes is hindered by the restricts the contact of the animal's soft body
absence of unequivocal criteria governing the with the external environment. In that way it
taxonomic importance of the different concho- could serve as a mechanism of efficient resistlogical and genitalia characters usually used in ance to high temperature and low humidity.
Correspondence lo: G.C. Rodakis
It is widely accepted that the resolution of
ABSTRACT
82
V. DOURIS
classification problems as well as the determination of phylogenetic relations among certain taxa could be facilitated by the parallel use
of molecular and morphological approaches,
although congruence between the two
approaches is not guaranteed (see reviews by
Hillis, 1987; Patterson, Williams & Humphries,
1993). A first attempt at using such combined
methods in land snails, based on the cladistic
analysis of mitochondrial DNA (mtDNA)
restriction site polymorphisms and conchology
(Douris, Rodakis, Giokas, Mylonas & Lecanidou, 1995), has provided congruent results in
Albinaria species, indicating that it is reasonable to employ comparative molecular and
morphological approaches in a broader range
of taxa and taxonomic problems.
In the present paper, we studied populations
from different clausiliid subfamilies, focusing
mainly on Alopiinae genera, as well as populations from the same genus, inspecting them for
an extensive set of morphological characters.
Individuals from the same populations were
also analysed by amplification and sequencing
of mtDNA segments, a procedure that has
proved very informative in revealing phylogenetic histories (Kocher, Thomas, Meyer,
Edwards, Paabo, Villablanca & Wilson, 1989).
Our aim was to evaluate the taxonomic importance of certain conchological characters (like
those concerning the type of clausiliar apparatus) and also to contribute to the resolution of
the systematic status of the Peloponnesian socalled Isabellaria group. The concordance between morphological and molecular approaches
is investigated, a new classification of some
Peloponnesian Isabellaria is proposed, and
possible evolutionary hypotheses are discussed.
MATERIALS AND METHODS
Biological material
Specimens from 10 populations belonging to 3
clausiliid subfamilies (Clausiliinae, Alopiinae, Mentissoideinae) were collected from restricted areas (no
more than 20 m2). The species and collecting sites
(see also map in Fig. 1) are recorded in Table 1.
Morphological data analysis
About ten specimens from each population were
examined for a series of qualitative conchological
characters (see Table 2 and Fig. 2). Genitalia qualitative characters have proved invalid in distinguishing
different genera of the subfamily Alopiinae (Nordsieck, 1977; Kemperman, 1992), and were therefore
excluded from the morphological data set. It should
be noted that exclusion of genitalia characters may
lead to an underestimation of the distance between
taxa belonging to different subfamilies of Clausiliidae. However, the main scope of this work was the
Table 1. List of Clausiliidae populations and collection sites used in the analysis. The collection sites
are numbered as in Figure 1.
Subfamily
Alopiinae
Mentissoideinae
Clausiliinae
Species
Albinaria coerulea
(Rossmassler 1835)
Albinaria discolor
(L. Pfeiffer 1846)
Albinaria grisea
(Deshayes 1833)
Isabellaria haessleini
(Fauer 1978)
Isabellaria campylauchen
(Boettger 1883)
Isabellaria butoti
(Nordsieck 1984)
Isabellaria saxicola
(Pfeiffer 1848)
Sericata sericata
{Boettger 1878)
Idyla bicristata
(Rossmassler 1839)
Clausilia bidentata
(Strom 1765)
Collection Site
Amorgos island, Hora, Cyclades (1)
Monemvassia, Lakonia, Peloponnese (2)
Mount Ymittos, Kessariani, Attica (3)
cross-road to Geraki, on the road from Monemvassia
to Gythio, Lakonia, Peloponnese (4)
Monemvassia, Lakonia, Peloponnese (2)
2 km from Geraki to Kosmas, Lakonia,
Peloponnese (5)
Mount Ymittos, Kessariani, Attica (3)
Mount Dirfy, Steni, Evia (6)
Mount Dirfy, Steni, Evia (6)
Sheffield, England
PHYLOGENETIC RELATIONS WITHIN THE ALOPIINAE
83
Table 2. Conchological characters and their states.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
protoconch colour: 0 = beige, 1 = black;
dorsal keel: 0 = obsolete, 1 = intermediate height, 2 = prominent;
basal keel: 0 = obsolete, 1 = intermediate height, 2 = prominent;
shell development: 0 = gradual, 1 = abrupt;
shell shape: 0 = spindle shape, 1 = club shape;
shell colour: 0 = dark brown, 1 = brown, 2 = beige, 3 = dirty white, 4 = white;
shell transparency: 0 = transparent shell, 1 = not transparent shell;
shell spots; 0 = absent, 1 = dots, 2 = striped;
cervix sculpture: 0 = uniform, 1 = not uniform;
cervix ribbing: 1 = weak, 2 = prominent;
teleoconch ribbing: 0 = imperceptible, 1 = prominent;
protoconch ribbing: 0 = imperceptible, 1 = weak, 2 = prominent;
sutures; 0 = shallow, 1 = deep;
peristome detachment: 0 = present, 1 = absent;
peristome reflection: 0 = present, 1 = absent;
peristome lips: 0 = thin, 1 = medium, 2 = thick;
lip colour: 0 = white, 1 = beige;
apertural shape: 0 = roundish, 1 = pear shape;
clausilial disc end: 0 = pointed, 1 = rounded;
clausilial disc shape: 0 - elongated. 1 = not elongated;
frontal upper palatal fold (FUP): 0 = absent, 1 = present;
distal upper palatal fold (DUP) kyrtosis: 0 = absent, 1 = present;
lunular position: 0 = dorsal, 1 = lateral;
lunula - DUP: 0 = not distinct, 1 = distinct;
lunula: 0 = obsolete, 1 = prominent;
principalis - DUP: 0 = principalis supersedes DUP, 1 = principalis does not supersede DUP;
DUP - principalis contact: 0 = DUP touches principalis, 1 = DUP does not touch principalis;
columellaris position: 0 = basal, 1 = high;
columellaris inclination: 0 = horizontal, 1 = inclined, 2 = vertical;
subcolumellaris: 0 = reaches the apertural lip, 2 = visible from outside. 3 = visible from lateral
view;
columellaris-subcolumellaris end: 0 = subcolumellaris stops before columellaris, 1 =
subcolumellaris does not stop before columellaris;
columellaris - subcolumellaris start: 0: collumellaris starts below subcolumellaris. 1 =
subcollumellaris starts below columellaris;
columellaris shape: 0 = not forked, 1 = forked;
parietalis start: 0 = reaches the lip, 1 = does not reach the lip;
parietalis length: 0 = long, 1 = short;
parietalis height: 0 = intermediate, 1 = prominent;
parallelis; 0 = absent, 1 = present;
spiralis: 0 = absent, 1 = obsolete, 2 = prominent;
fulcrans; 0 = absent, 1 = present;
subclaustralis; 0 = absent, 1 = present;
basalis: 0 = absent, 1 = present;
suturalis; 0 = absent, 1 = present
determination of the intra-subfamily relations. Consequently, only conchological characters that were
not uninformative or difficult to be determined with
accuracy were used. Data were analysed with the
PAUP package (version 3.1.1, Swofford, 1993). Multistate taxa were interpreted as polymorphic. Multistate characters were treated as ordered, because of
the obvious succession of their states, and they were
weighted according to the number of their states,
with base weight 6, so that each character had equal
weight regardless of the number of states (Swofford,
1993). More specifically, we enforced the branchand-bound search option with the furthest addition
sequence. Branches having maximum length zero
collapsed to yield polytomies and topological constraints were not enforced. A second run was enforced
with the same options after excluding characters that
are related with the formation of the GCA; these
characters are: 27 (DUP- principals contact), 37
(parallelis), 38 (spiralis), 39 (fulcrans) and 42 (suturalis).
rtNA
Total DNA was extracted from the foot of each animal, which was incubated in 0.5 ml of lysis buffer (20
mM Tris.Cl pH 7.5, 200 mM NaCI, 20 mM EDTA,
2% SDS.0.5 mg/ml Proteinase K) as 50°C until it was
V. DOURIS
85
PHYLOGENETIC RELATIONS WITHIN THE ALOPIINAE
Figure I.A. Distribution of genus Albinaria (black areas) in the East Mediterranean. B. Distribution of genus
Isabellaria (black areas) in the southern Balkan peninsula. Question marks (?) indicate a very poorly known
northern distribution. C. Map of Greece indicating the collecting sites (black circles) of the populations used in
this study. Each site is numbered as in Table 1. The collecting site of Clausilia (in Britain) is not shown.
D
B
Cs
Cer
Ap
Pr
Su
FUR
PI
Figure 2. Aspects of a Clausiliid shell showing the conchological characters used in the cladistic analysis. A.
frontal view; B. dorsal view; C. schematic frontal view of the aperture; D. schematic dorsal view of the aperture;
E. schematic representation of the normal type clausiliar apparatus; F. schematic representation of Graciliaria
type clausiliar apparatus. Abbreviations: Ap, aperture; Ba, basalis; Bk, basal keel; Cer, cervix; Cl, clausilium
disc; Co, columellaris; Cs, clausilium stalk; Dk, dorsal keel; DUP, distal upper palatalis fold; FUP, frontal upper
palatalis fold; Fu, fulcrans; Lu, lunela; Pa, parietalis; PI, parallelis; Pr, principalis; Pro, protoconch; Sc, subcolumellaris; SI, sulcalis; Sp, spiralis; Su, suturalis; Tel, teleoconch, (modified from Nordsieck 1982).
completely dissolved (usually 30-60 minutes). The
lysate was extracted several times by Phenol/chloroform and the nucleic acids were pelleted with ethanol
precipitation. The pellet was dissolved in 50 JJLI of TE
buffer (10 mM Tris.Cl pH 7.5, 0.1 mM EDTA) and
RNA was removed by RNase treatment (5u.l of 10
mg/ml RNase A were added and the mixture incubated at 37°C for 30 minutes). After the addition of
400 u.1 of TE buffer and 50 u.1 of 4M NaCl, the solution was extracted again with Phenol/chloroform,
and DNA was pelleted with ethanol precipitation
and dissolved in 40 u.1 of TE buffer in order to be used
as template in amplification reactions. DNA concentration was determined spectrophotometrically.
Amplification
Primers for the amplification reaction were designed
after comparison of the published sequences for Albi-
naria turrita (Lecanidou, Douris & Rodakis, 1994)
and A. coerulea (Hatzoglou, Rodakis & Lecanidou,
1995) mitochondrial large ribosomal RNA genes with
the homologous "universal" primers (Palumbi, Martin,
Romano, McMillan, Stice & Grabowski, 1991). The
designed primer pair (16s-l: 5'GGTCTGAACTCAGATCATGT-3', where W stands for A or T) was
used to amplify a region corresponding to bases
12898-13380 of the A coerulea mitochondrial genome
(Hatzoglou et al., 1995). Amplification was carried
out in 100 u.1 of a solution containing 20 mM Tris.Cl
pH 8.3, 50 mM KC1, 0.1% gelatin, 2 mM MgCl2, 200
nM of each deoxynucleotide, 50 pmole of each
primer, 1 u,g template DNA and 2 units of Taq DNA
polymerase (Boeringher Mannheim), in an MJ
Research thermal cycler. Each cycle of the reaction
consisted of denaturation for 1 min at 94°C, hybridisation for 2 min at 48°C, and extension for 3 min
at 72°C. This cycle was repeated 35 times, and after
V.DOURIS
86
the last cycle there as afinalextension step for 20 min
at 72°C.
Sequencing
The amplification reactions were electrophoresed in
2% agarose gels, the gel fragments containing the
amplification products were excised from the gels
and DNA was purified by electroelution (Sambrook,
Fritsch & Maniatis, 1989) and column separation
through a NACS PREPAC minicolumn (BRL, cat.
No 1525NP). The purified DNA was sequenced from
both ends by the dideoxynucleotide method according to the Sequenase ver. 2.0 protocol (United States
Biochemical) as modified for sequencing doublestranded amplification products (McPherson, Jones
& Gurr, 1991). The primers used for the amplification reaction also served as sequencing primers.
Sequence Data Analysis
Sequences were aligned with the aid of the computer
programme CLUSTALW (Thompson, Higgins &
Gibson, 1994). Nucleotide distances were calculated
using the DNADIST programme of PHYLIP package (Felsenstein, 1989) under the assumptions of the
Kimura 2-parameter model (Kimura, 1980). Cladistic
analysis was performed with the PAUP package ver.
3.1.1 (Swofford, 1993) with branch-and-bound search.
Characters were treated as unordered, branches having
maximum length zero collapsed to yield polytomies,
topological constraints were not enforced and trees
were unrooted. Neighbor-joining (Saitou and Nei,
1987) and Maximum-likelihood (Felsenstein, 1981)
trees were constructed using the NEIGHBOR and
DNAML programmes of PHYLIP, respectively. All
trees were submitted to bootstrapping (Felsenstein,
1985), 500 times for parsimony and neighbor-joining
trees, and 100 times for Maximum-likelihood trees.
RESULTS
The conchological character-state matrix is
shown in Table 3. Cladistic analysis of these
data produced a single most parsimonious tree
(length = 594, CI = 0.507, RI = 0.520), shown in
Figure 3A. The following observations can be
made on that tree: a) The genera Clausilia and
Idyla are very well separated from the group
consisting of the genera Albinaria, Sericata and
Isabellaria, even though genitalia characters
were not examined b) Two subgroups are
obvious in the latter group: one containing the
closely related taxa /. saxicola (Ymittos) and S.
sericata (Steni) together with /. campylauchen
(Monemvassia) and A. grisea (Ymittos), and a
second subgroup consisting of the closely related taxa /. butoti (Kosmas) and /. haessleini
(Geraki) together with A. discolor (Monemvassia) and A. coerulea (Amorgos). Thus, it can be
noted that the generally accepted distinction
between the genera Isabellaria and Albinaria is
not supported, since A. grisea clusters with
Isabellaria (I. campylauchen, 1. saxicola), while
/. butoti and /. haessleini are grouped with
Albinaria {A. discolor, A. coerulea). Surprisingly, Sericata sericata and Isabellaria saxicola
comprise a monophyletic group.
The nucleotide sequence alignment of the
Table 3. Conchological character-state matrix. Character numbers are as in Table 2.
Taxon
Clausilia bidentata
Idyla bichstata
Sericata sericata
Isabellaria butoti
Isabellaria campylauchen
Isabellaria haessleini
Isabellaria saxicola
Albinaria coerulea
Albinaria discolor
Albinaria grisea
characters
111111111122222222223333333333444
123456789012345678901234567890123456789012
001000000100100001101000100002100010020010
1
1
1
022010001200101111111000100102111001111001
011001001202111210101000101000001111120111
002003110201001200000100000001001111111001
1
1
021002021101101100001000101001001101100011
2
1 1
1
002003110212001100100111010012011111111000
1
1 2
000001001101110110000000001000001101100111
1
1
122004111201101200100001100102000100020000
1
1 1
002004110101001200100111000012011111020000
1
11
1
001002021101101200000000010001001101020000
1
11 1
1
PHYLOGENETIC RELATIONS WITHIN THE ALOPIINAE
- Clausilia b
Cluiisilia Hih-iuatu
—
Idyla biirisiata
Isabdlaria t tinipytttiuhcn
•
r— Claiisilia bidcnlala
- ld\ la bicrisniltt
• Idyla hicristaiu
- Albinaria griscu
87
- Striciiiu scricaia
^ ^ Isubdlaria saxicola
— Isabdlaria saxia>ta
I— Sericata scricaia
Isabdlaria saxicola
Isabdlaria hacssldni
i—• isabcilariu
hacxacim
Isabdlaria campylauchctt
45
I ... Sericata scricaia
,
'
Albinuriti discolor
ilbinariu cocrulca
Albinaria UK-mica
r Albinaria discolor
•
Isabdlaria bmoli
Isabdlaria biitnti
Isabdlaria cimtpy Imu hen
L_ Isabdlaria hacx.sli.irii
Albinaria grisca
Albinaria grisea
Albinaria cocrtdea
Isabdlaria bmoli
Isabdlaria hacsslcini
Albinaria discolor
B
Figure 3.A. Most parsimonious phylogenetic tree resulting from the cladistic analysis of all conchological characters of Table 2. B. Neighbor-joining tree of the IrRN A sequences, resulting from the distance matrix of Table
4. Numbers indicate bootstrap values. C. Most parsimonious tree from the analysis of the conchological characters, excluding the ones related to the GCA (see Materials and Methods for details).
large rRNA segment analysed is shown in
Figure 4. As can be seen, sequences do not
have the same length; for example Idyla (and
sometimes also Clausilia) has additional
sequence portions, compared to the Albinaria
and Isabellaria lrRNA sequence. Perhaps these
portions form specific secondary structure
domains which are not present in the Alopiinae
lrRNA molecule. Since no valid secondary structure prediction is available for snail lrRNA, we
chose to use only confidently aligned sequence
portions for further analysis. Thus, we excluded regions with large gaps or questionable
alignments (boxed areas in Fig. 4). This resulted in a set of 321 positions on which tree
construction was based.
Nucleotide divergence (number of substitutions per site, according to Kimura's model)
between each pair of sequences was calculated.
The matrix of these distances (Table 4) was
used for construction of the Neighbor-joining
tree shown in Figure 3B. This tree, similarly to
that based on morphological characters, shows
a clear separation of Clausilia and Idyla from
Isabellaria and Albinaria (bootstrap value 100).
Moreover, /. saxicola (Ymittos) and 5. sericata
(Steni) are very well separated (bootstrap
value 100) from all other Isabellaria and
Albinaria. It is worth noting that the three
Isabellaria from the Peloponnese (/. campylauchen (Monemvassia), /. butoti (Kosmas) and
/. haessleini (Gerald) are grouped together
with Albinaria. More specifically, A. grisea
(Ymittos) and /. campylauchen are very close
and clearly form a monophyletic group which is
very well supported by bootstrap values, while
/. haessleini and A. discolor also cluster together. In contrast to the morphology-based
tree, /. saxicola and 5. sericata appear paraphyletic, but close to each other. However,
bootstrap analysis does not favour any particular hypothesis about the branching order,
paraphyly or monophyly for these taxa.
The same observations can be made if the
data set is analysed by maximum likelihood or
parsimony methods. The maximum likelihood
tree topology is almost identical to the topology of the Neighbor-joining tree, with slightly
different branch lengths and bootstrap values.
Parsimony analysis has resulted in two equally
parsimonious trees (length = 201, CI = 0.717,
RI = 0.667). One of these trees has exactly the
same topology with the Neighbor-joining tree,
while the other differs mainly in the branching
order of /. saxicola and 5. sericata. Bootstrap
analysis also indicates this instability.
Comparison of the morphological and
mtDNA trees (Figs 3A and 3B) reveals some
important similarities, a) Clausilia and Idyla in
both trees are distant from each other and from
the rest of the examined taxa, revealing the
extent of evolutionary divergence of the Alopiinae from the other clausiliid subfamilies b)
Isabellaria saxicola and Sericata sericata are
20
30
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
240
250
260
270
280
290
300
310
320
330
360
370
380
390
400
410
420
430
441
(389)
(390)
(389)
(393)
(392)
(390)
(397)
(391)
(403)
(436)
Figure 4. Nucleotide sequences and sequence alignment of the amplified segment of the lrRNA of the ten Clausiliidae analysed. Boxed areas indicate regions with
large gaps or ambiguous alignment that were excluded from the analysis.
A. coerulea
^ T A G A A A A A T T A C C T A A G G G A T A A C A G C A T A A T T T - •TATTAATAAGCTTATGACCTCGATGTTGGACTAGGTACTATTAAGGCTAATCGTTTTAA
A. discolor
T— kTAGAGAAATTACCTAAGGGATAACAGCATAATTT- TAATAATAAGCTTGTGACCTCGATGTTGGACTAGGTACTATTAGGGCTAATCGTTTTAA
A. grisea
T— MAATAAAATTACCTAAGGGATAACAGCATAATTT- TAGTAGTAAGTTTGTGACCTCGATGTTGGACTAGGTACTATTAAGGCTAATCGTTTTAA
I. caopylauchen T — VTAATTAAATTACCTAAGGGATAACAGCATAATTT- TACTAGTAAGTTTGTGACCTCGATGTTGGACTAGGTACTATTAAGGCTAATCGTTTTAA
I. haessleini T — \TAGAGAAATTACCTAAGGGATAACAGCATAATTT- TAATAATAAGCTTGTGACCTCGATGTTGGACTAGGTACTATTAAGGCTAATCGTTTTAA
I. butoti
T— ^CAGTAAAATTACCTAAGGGATAACAGCATAATCT• TAATAATATGTTTGTGACCTCGATGTTGGACTAGGTACTATTAAGGCTAATCGTTTTAA
I. isabellina A S. sericata
A —
- - kAAGTAAAATTACCTAAGGGATAACAGCATAATTTAT—TAATAAGTTTGTGACCTCGATGTTGGACTAGGTACTATTAAAGCTAACCGTTTTAA
C. bidentata
G - VTAAATAAATTACTATAGGGATAACAGCATAATTTTTCATT-TAAGATTGTGACCTCGATGTTGGATTAGGGACTTATTAAATAAACCATTTAAA
I. bicristata cbAAGTATTACTATTAWTAAAAAAATTACTATAGGGATAACAGCATAATTTT-TATTAAAAAGATTGTGACCTCGATGTTGGACTAGGGAATATTTAGGGCAG-CATTCTAA
350
coerulea
-TAATTCTTATTAGAT rTTTTGTTGGGGCAACAATATTTCA \ - A
T A K T A A A T A T A | T T A ATT- -G A A - A G T A A T A A G T C G A T T A - — \ATAAT
discolor
-TCAATCTTGTAAATT rTTTTGTTGGGGCAACAGTATTTCA r - A G C - G
GA M A A A T A T T TCCTT
T-GG—CAGATTAA-TCGATTTT--\ATAAT
grisea
TTAC-TCTAGTACGAT rTTTTGTTGGGGCAACAATATTTCArJ
CATAKTAAATATAkTATT-TA—AAGAATACGTCGATTT—
--TATAAT
canpylauchen
-ATACATCTAGTAAGATtTTTTGTTGGGGCAACAATATTTCA T-AACAAA ^TAAATATT I T A T A — A - - T A T T A T A T G T C G A T T T - — TATAAT
haessleini
CCAATCTTATAAATT j tTTTTGTTGGGGCAACAGTATTTCA:-.
- A A f k T A A A T A T T r A C A T - T A A C C G G A T T A A A T C G A T T T T — \ATAAT
butoti
TCTGTCTGTTAAGAT rTTTTGTTGGGGCAACAATATTTCA
P -TATA-AT—ATGAATATATCGATTT—
--VATAAT
isabellina
-ATGTTAGTTTGATAAlTTTTTGTTGGGGCAACAATATTTCAtr-A-TATAKTAAATATTKACCT-TAA-TAATATACGACGATTT---\ATAAT
ssricata
TAATAGATATAATAT)rTTTTGTTGGGGCAACATTATTTCA(rA
TAT-TATTATGTAACGATTA—
— \ATAAT
- ^TAAATAAT UkA
TTTTAGTCCTTCG CTTTTGTTGGGGCAACAAGTTGACA 3
TTGA iAAAAAATT rAACTCAATAAATTTTAAGGAATTCTTCGAGTTTT
— \CTAAG
c.bidentata
I. bicristata
TTTTAATAGTGGTATTACTAT rTTTTGTTGGGGCAACAACTTACCA TTAGCTAA VTGAATAAT MAATGTAGGTAAAGTATTATAGTTACATATACTTTTTAAT \ A T A A T
A.
A.
A.
I.
I.
I.
I.
S.
230
coerulea
GCAACTTGTCT :AT'-TGATATTACTTTA VATTTA—ATGGTTGAGTGAAAATACTCATAATTT-TAATAATAGACGAGAAGACCCTTAGAATTTTTAAAA SCAATAAG—
discolor
ACAACTTGTCT ^ T'-TAATAATACCTTA W^ATTA—CCTCATGAGTGAAAATACTCTTGAATT-TAATATTAGACGAGAAGACCCTTAGAATTTTAATTA UVTATAAG-grisea
ACAATTTGTCT TAT.'-TCATATTATCTTAKATTTG—CCGTATGAGTGAAAATGCTCATGGTTT-TAAAAATAGACGAGAAGACCCTTAGAATTTTAAAAAakTATTAGTA
canpylauchenACAAATTGTCT ^ T A T G C C A T T A T C T T A K A A T T A.--CCGTATGAGTGAAAATGCTCATGGGTT-TAAAAATAGACGAGAAGACCCTTAGAATTTTAAAAA:ATATTAGCA
haessleini ACAACTTGTCT EAT' - T A A T A A T A C T T T T K A A T T G — C T G C A T G A G T G A A A A T A C T C T T G A A T T - A A A T A A T A G A C G A G A A G A C C C T T A G A A T T T T A A A A A a ^ A A T A A G —
butoti
ATAACTTGTCT CAT
ATAACTTGTCTtTAT-TAATATAAATTTA^AATTG—CCAAATGAGTGAAAATGCTCATGCTTT-GAATAATAGACGAGAAGACCCTTAGAATTTTAAAAArATAATAG-isabellina ATTTCTTGTCT :GCGAATTAATATTTATkAATTT'--TTAAGGAAGTGAAAATACTTCTAATTA-AAATAAAAGACGAGAAGACCCTTAGAATTTTGATTA^CAGACTTsexicata
ATTAATTGTCT TACAGTAAAATAATTTAKAATTAi—CTAAAGAAGTGAAAATGCTTCTGAATATAAATAATAGACGAGAAGACCCTTAGAATTTTTAAGA-TTATATATc. bidentata GGTACATGTCT ^VT
I. bicristata TTTTACTGTClfaTAATTATTTTTATATA\AATTAATCTTAGAAAGTGAAAATACTTTCAGAAA-ATATAATAGACGAGAAGACCCTATAAATTTTAAATAUVGGTAACTT
A.
A.
A.
I.
I.
I.
I.
S.
A.
A.
A.
I.
I.
I.
Z.
S.
TTATCTGCCCAGTGA \A .AAirTTAACGGCCGC-AGTACCTTGACTGTGCAAAGGTAGCATAATAATTTGACTTTTAAATGGAGCCTAGAATGAAAGAAAGAACGTAT
coerulea
discolor
TTATCTGCCCAGTGA TA
ATT IT,AAACGGCCGC-AGTACCTTGACTGTGCAAAGGTAGCATAATCATTTGACTTTTAAATGGAGCCTAGAATGAAAGAAAGAACGTAG
grisea
TTGTCTGCCCAGTGA U3
•TATrTTAACGGCCGC-AGTACCTTGACTGTGCAAAGGTAGCATAATCATTTGACTTTTAATTGGAGCCTAGAATGAAAGAAATAACGTAG
campylauchenTTGTCTGCCCGGTGA
AATrTTAACGGCCGC-AGTACCTTGACTGTGCAAAGGTAGCATAATCATTTGACTTTTAAATGGAGCCTAGAATGAAGGAAGTAACGTAG
haessleini TTATCTGCCCAGTGA TA
ACTrTTAACGGCCGC-AGTACCTTGACTGTGCAAAGGTAGCATAATCATTTGACTTTTAATTGGAGCCTAGAATGAAAGAAAGCACGTAG
butoti
TTATCTGCCCAGTGA !A
AA- rTTAACGGCCGC-AGTACCTTGACTGTGCAAAGGTAGCATAATCATTTGACTTTTAAATGGAGCCTAGTATGAATGAAAGAACGTAG
isabellina TTATCTGCCCAGTGA
A1
'AACGGCCGC-AGTACCCTGACTGTGCAAAGGTAGCATAATCATTTGACTTTTAATTGGAGTCTGGAATGAAAGAAAGCACGTAG
sericata
ATATCTGCCCAGTGA
AATfTTTAACGGCCGC-AGTACCCTGACTGTGCAAAGGTAGCATAATCATTTGGCTTTTAATTGGAGTCTGGAATCAAAGAAGTTACGTAG
'AACGGCCGCTAGTACACTGACTGTGCTAAGGTAGCATAATAAATTGGCTTTTAATTGGAGTCTGGAATAAAAGAGTTCATGGGG
c.bideatata CGCTCTGCCCAGTCA ITTGAAT"I FTTA
I. bicristata ATTTCTGCCCGGTGA M A T -A'TT TTTA
'AACGGCCGC-AGTACACTGACTGTGCAAAGGTAGCATAATAAATTGGCTTTTAATTGAAGTCGGGAATGAAAGAAAATTTGGGA
10
73
a
o
89
PHYLOGENETIC RELATIONS WITHIN THE ALOPIINAE
closely related c) /. campylauchen and A. grisea coincide. Thus, our results indicate that the
are also closely related. In both trees, the tradi- appearance of the GCA can be attributed to
tional distinction between Albinaria and parallelism and that developmental constraints
Isabellaria is not supported. The basic differ- are relatively relaxed in the case of its formaence between the two trees lies in the grouping tion.
of the A. grisea - I. campylauchen pair and of
As far as the proposed "adaptive" function
the /. haessleini -1. butoti pair.
of the GCA is concerned, certain observations
Interestingly, if the morphological characters indicate that a "non-adaptive" hypothesis is
that are related to the type of clausiliar appara- more valid. For the clausiliid taxa distributed in
tus are excluded from the analysis, the result is the east Mediterranean region, summer is the
a unique and more robust tree (length = 496, adverse period. Several mechanisms are emCI = 0.530, RI = 0.571) (Fig. 3C), which is very ployed to increase viability during this period:
similar to the mtDNA tree of Figure 3B as far the snails aestivate (aestivation is "the warm
as the major distinction between Isabellaria weather equivalent of hibernation" - Gould,
and Albinaria is concerned: all the Alopiinae 1985) forming a thick epiphragm, usually in
form a monophyletic group with the Pelopon- clusters on the rock surfaces or in crevices or
nesian Isabellaria forming a monophyletic group under stones (Gittenberger, 1991; Kemperman,
with the Albinaria taxa. Moreover, Isabellaria 1992; Schilthuizen, 1994; Heller & Dolev, 1994,
saxicola remains closely related to Sericata Arad, Goldenberg & Heller, 1995; Giokas,
sericata, while A. discolor and /. haessleini are 1996). Aestivation is triggered by photoperiodism, while awakening by a combination of
now grouped together.
photoperiodism and autumn rainfalls (Heller
& Dolev, 1994; Arad etal, 1995; Giokas, 1996).
Albinaria taxa, which have a more southern
DISCUSSION
distribution than those of Isabellaria (Fig. 1),
One of the major problems in estimating lack the GCA and are generally exposed durphylogenetic relations and testing evolution- ing aestivation, while certain Isabellaria taxa
ary hypotheses is the discrimination between that have the GCA usually aestivate under
homologies and homoplasies. This problem is stones. This may indicate that the GCA is not
especially, obvious in the case of the so-called actually so important for viability of these land
"G-type" clausiliar apparatus (GCA), since it snails as far as resistance to water loss is conis not clear whether it is a result of common cerned. An alternative suggestion is that the
origin or parallelism and, furthermore, whether GCA could offer a selective advantage as a
it is an adaptation or not (Nordsieck, 1982; mechanism of resistance to unknown predators
Gittenberger, 1987, 1994, Gittenberger & (Gittenberger & Schilthuizen, 1996). In any
Schilthuizen, 1996). The GCA has been con- case, its appearance seems to follow a stochastic
mode and thus its exclusion is justified, if a
sidered in the past (Nordsieck, 1979) as a
character set of high taxonomic value, and has more accurate representation of the phylobeen widely used for discrimination between genetic relations is desired.
Albinaria and Isabellaria (Nordsieck, 1982;
An interesting observation on the phyloGittenberger, 1987). On the other hand, the genetic analysis shown in Figure 3 concerns the
"independent origins" argument for the non-monophyletic origin of the genera Albinaappearance of the GCA has already been
ria and Isabellaria. Indications for polyphyly of
established (Nordsieck, 1982; Gittenberger, the genus Isabellaria have been reported pre1987; Gittenberger & Schilthuizen, 1996).
viously; these are based on morphological and
Phylogenetic analysis of the mtDNA data ecogeographical data (Nordsieck, 1984; Gittenamong the taxa used in this study, does not berger, 1987), as well as on some molecular
support the "homology" hypothesis for the data (sequence comparison of the ITS1 nuclear
GCA: taxa with or without it (for example /. rRNA region of some Isabellaria and Albinaria
campylauchen - A. grisea, and /. haessleini - A. taxa; Schilthuizen et al, 1995). A point that
discolor) can be very closely related, while taxa needs to be answered, however, is what this
sharing the GCA feature (such as Idyla and
"polyphyly" actually represents. We propose
Isabellaria can be quite distant (Fig. 3A). that the observed "polyphyly" is an "artifact"
Moreover, if the characters which are related arising from acceptance of the conventional
to the GCA are excluded from the morpho- systematic classification for these taxa. More
logical data set (Fig. 3C), the results of the specifically, the species /. butoti, I. campymorphological and mtDNA analysis almost lauchen and /. haessleini should be considered
90
V. DOURIS
Table 4. Matrix of pairwise Kimura distances (X100).
A.
A.
A.
1.
1.
1.
1.
S.
C.
1.
coerulea
discolor
grisea
campylauchen
haessleini
butoti
saxicola
sericata
bidentata
bicristata
0
7.84
8.13
9.98
6.75
8.88
12.76
14.81
33.41
27.62
0
9.24
8.86
3.61
7.81
12.21
13.62
35.68
27.78
0
3.60
7.39
7.07
14.22
13.55
34.95
29.66
0
8.46
7.79
15.88
13.11
35.05
28.94
as belonging to the genus Albinaria rather than
Isabellaria. Our hypothesis is supported by the
following observations:
(1) The
molecular
and
morphological
approaches are congruent (Fig.3). It should be
noted that, at least as far as the close relation of
/. haessleini and A. discolor is concerned, our
molecular approach, based on mitochondrial
DNA, is in agreement with results obtained
from nuclear DNA sequences (Schilthuizen et
a/., 1995).
(2) The so-called Isabellaria species (/. butoti,
I. campylauchen, I. haessleini) are much closer
to the Albinaria taxa than to Isabellaria saxicola, which is close to Sericata sericata. Moreover, the distances (nucleotide substitutions
per site; x 100 as in Table 4) between /. campylauchen and A. grisea (3.60) and between /.
haessleini and A. discolor (3.61), are much
lower than the distances typically observed
between Albinaria species (7.84 to 9.24 in
Table 4) and falls in the range of distances
observed between "subspecies" or allopatric
populations of the same species (Douris,
Rodakis & Lecanidou, in prep.).
(3) Natural hybridisation phenomena observed
between sympatric populations of /. butoti and
A. grisea (Nordsieck, 1984) and between A.
discolor and /. campylauchen at Monemvassia
(Giokas, 1996) indicate a close relation between
certain Isabellaria and Albinaria taxa.
0
6.69
10.27
11.13
31.54
25.68
0
11.89
12 .03
34 .74
29 .97
0
7 .48
28 .27
22 .18
0
28.47
21.86
0
23.29
(1883), who first described this species as A.
grisea campylauchen, while for /. haessleini (A.
discolor haessleini) one of Gittenberger's classifications (Gittenberger, 1994) is supported.
In the present analysis, two taxa belonging to
other clausiliid subfamilies (Clausilia bidentata:
Clausiliinae and Idyla bicristata: Mentissoideinae) were used as outgroups. Using both
types of data, a very clear separation is
obtained both between Clausilia and Idyla, as
well as between these two and the monophyletic group of the Alopiinae taxa (Fig. 3).
The latter group includes the "Albinaria and
Peloponnesian Isabellaria" cluster mentioned
above, plus Sericata sericata and Isabellaria
saxicola. Our analysis indicates that, unlike the
Peloponnesian Isabellaria taxa, /. saxicola is
very clearly separated from the Albinaria
taxa (Fig. 3). In the morphological analysis, /.
saxicola forms a monophyletic group with S.
sericata (Fig. 3C), while in the mtDNA results
the two taxa appear closely related but paraphyletic, although this branching order is not
well supported by bootstrap analysis (Fig. 3B).
Furthermore, the nucleotide sequence divergence (X100) between /. saxicola and S.
sericata (7.48) is in the same range with the distances typically observed between congeneric
species (Table 4).
Despite the fact that only one Sericata and
one "true" Isabellaria are analysed here, our
observations indicate that phylogenetic relaBased on the above, we propose certain alter- tions within the subfamily Alopiinae are more
ations in the systematic classification of the complicated than those reflected by convenanalysed Peloponnesian Isabellaria taxa. tional classification. Further comparative analNamely, Isabellaria butoti should be considered ysis of morphological and molecular data from
as Albinaria butoti; Isabellaria camplylauchen all Alopiinae genera could provide a more
as Albinaria grisea campylauchen; and Isabel- informative representation of the intra-sublaria haessleini as Albinaria discolor haessleini. family relations and probably lead to the
It must be noted that the proposed classifica- determination of genus-specific morphological
tion for /. campylauchen justifies Boettger characters.
PHYLOGENETIC RELATIONS WITHIN THE ALOPIINAE
ACKNOWLEDGEMENTS
91
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Ecology and Systematics, 18:23-42.
KEMPERMAN, T.C.M. 1992. Systematics and evolutionary history of the Albinaria species from the
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