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Molecular phylogeny of the land snail genus Alopia(Gastropoda: Clausiliidae) reveals multiple inversionsof chirality
ZOLTÁN FEHÉR1, LÁSZLÓ NÉMETH2, ALEXANDRU NICOARA3 andMIKLÓS SZEKERES4*
1Department of Zoology, Hungarian Natural History Museum, Baross u. 13, H-1088 Budapest,Hungary2Rekettye u. 24, H-1155 Budapest, Hungary3Department of Ecology and Environmental Protection, Lucian Blaga University, Str. Oituz 31,R-550337 Sibiu, Romania4Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences,Temesvári krt. 62, H-6726 Szeged, Hungary
Received 31 July 2012; revised 4 November 2012; accepted for publication 15 November 2012
Whereas the vast majority of gastropods possess dextral shell and body organization, members of the Clausiliidaefamily are almost exclusively sinistral. Within this group a unique feature of the alpine genus Alopia is thecomparable representation of sinistral and dextral taxa, and the existence of enantiomorph taxon pairs that appearto differ only in their chirality. We carried out a molecular phylogenetic study, using mitochondrial cytochrome coxidase subunit I (COI) gene sequences, in order to find out whether chiral inversions are more frequent in thisgenus than in other genera of land snails. Our results revealed multiple independent inversions in the evolutionaryhistory of Alopia and a close genetic relationship between members of the enantiomorph pairs. The inferred COIphylogeny also provided valuable clues for the taxonomic division and zoogeographical evaluation of Alopia species.The high number of inverse forms indicates unstable fixation of the coiling direction. This deficiency and theavailability of enantiomorph pairs may make Alopia species attractive experimental models for genetic studiesaimed at elucidating the molecular basis of chiral stability.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 167, 259–272.doi: 10.1111/zoj.12002
ADDITIONAL KEYWORDS: Alopia – chirality – Clausiliidae – enantiomorph – Mollusca – phylogeny –speciation.
INTRODUCTION
In contrast to the predominantly bilateral symmetryof most animals, snails develop in an asymmetricfashion, which is easily recognizable by the helicalorganization of their body. The vast majority of taxadisplay dextral chirality, characterized by the clock-wise growth of the shell when viewed from its apex.Rare mutant populations of opposite, sinistral coiling
have proven instrumental for investigating themolecular genetic background of chiral determina-tion. These studies revealed that coil directiondepends on the polar orientation of cells at the earlyembryonic stage, which is established by the correctperformance of polarity-setting cytoskeletal elements(Crampton, 1894; Shibazaki, Shimizu & Kuroda,2004; Kuroda et al., 2009). Although the key functiondetermining chirality has not yet been identified,genetic studies in species of four gastropod super-families indicated that it is a maternally inheriteddominant cytoplasmic factor encoded by a single gene*Corresponding author. E-mail: szekeres@brc.hu
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Zoological Journal of the Linnean Society, 2013, 167, 259–272. With 4 figures
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 167, 259–272 259
(Sturtevant, 1923; Degner, 1952; Asami, Gittenberger& Falkner, 2008). Mutations in this gene can haveconsiderable evolutionary consequences. For instance,altered body structure can make inverse individualsless likely targets for predation (Hoso, Asami & Hori,2007), and severely reduced mating success with non-inverted members of the population can lead to repro-ductive isolation (Gittenberger, 1988). These effectscan enhance diversification and lead to the appear-ance of new subspecies and species.
Despite the ancestral dominance of dextrality ingastropods, sinistral coiling can be found at varioustaxonomic levels. In dextral species there are severalreports of rare inverted individuals, as well as ofsmall populations with frequent or uniform sinistral-ity. Also, there are entire species, genera, and evenfamilies of gastropods with dominant sinistrality(for examples, see Davison et al., 2005). One of thesinistral families is that of the clausiliids (Clausili-
idae, door snails), characterized by a spindle-shapedshell that is equipped with intricate closing (clausil-iar) apparatus. Situated inside the last whorl, thisstructure efficiently seals the entrance when the snailretreats, and consists of multiple lamellae and plicaethat are useful morphological markers for taxono-mists. Sinistrality is very stringently determined inmost clausiliid subfamilies, but not in the relativelyyoung and species-rich Alopiinae that dominate theeastern Mediterranean basin. In this subfamily anumber of genera also include dextral species (Git-tenberger & Uit de Weerd, 2006; Nordsieck, 2007).
Among the Alopiinae genera that include dextralspecies, Alopia H. & A. Adams, 1855, a south-eastEuropean genus endemic to the Carpathian Moun-tains (Fig. 1), is of particular interest. In contrast tothe other genera composed of predominantly sinistralspecies, in this genus sinistral and dextral formsare represented comparably, with 50 and 23 taxa,
Figure 1. Sampling sites of the Alopia material studied from the Romanian and Slovakian Carpathians. The taxa andlocalities corresponding to the numbered dots are identified in Table 1. The insets show the position of the Carpathians(black shaded) in Europe and an enlarged map of the south-eastern Carpathian ranges (upper right and lower left corners,respectively).
260 Z. FEHÉR ET AL.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 167, 259–272
respectively. Furthermore, Alopia includes enantio-morph (oppositely coiled, but otherwise seeminglyidentical) taxon pairs that always occur in contiguousranges (Wagner, 1914; Soós, 1943). Considering thatin other clausiliid genera chiral inversions are veryrare, the null hypothesis of our current study wasthat Alopia is not an exception from this rule. In thiscase the unusually high number of dextral formsmight be explained by a reversion that happenedearly during Alopia diversification, and then servedas a basis for a monophyletic lineage of dextralspecies. The alternative hypothesis was that thedextral taxa of Alopia could have resulted from mul-tiple independent reversion events. Clarifying whichof these hypotheses was correct promised intriguinginformation in broader contexts. If dextral and sinis-tral lineages evolved in parallel, enantiomorph pairswould exemplify extreme morphological convergence.If, however, the phylogenetic history of the genusincluded multiple inversions, then the genetic deter-mination of chirality is less stable than in othergenera, offering Alopia species as uniquely suitableobjects for studying the genetic background of chiralstability. Either way, ascertaining the phylogenies ofsinistral and dextral forms could also provide impor-tant phylogenetic background for Alopia taxonomy.
As are most genera of the Alopiinae, Alopia is anobligate rock-dwelling genus with numerous, mostlypolytypic, species (Nordsieck, 2007). Because of therestricted vagility of the animals and the scattereddistribution of their preferred habitat type (barelimestone outcrops), Alopia populations occur in well-defined, isolated patches. Populations of shared mor-phological characters and geographical ranges havetraditionally been considered as subspecies, and cur-rently there is wide consensus regarding their delimi-tations (Szekeres, 1976; Grossu, 1981; Nordsieck,2008). However, the evaluation of taxonomic relation-ships between these subspecies, and particularlythe enantiomorph pairs, has long been controversialbecause of a disagreement over the homologousor homoplastic origin of the dextral subspecies. Oneattempt at classification was based on the notion thatthe far-reaching similarity of enantiomorphs, regard-less of chirality, indicated close evolutionary relation-ships, and that classification should rely primarilyon other morphological characters of the shells (e.g.sculpture or closing apparatus) and soft organs (e.g.genitalia), rather than chiral differences (Bielz, 1861;Wagner, 1914). Accordingly, these authors regardedenantiomorph pairs as subspecies of the samespecies. The other, contrasting approach assumedparallel evolution of sinistral and dextral lineagesfrom the onset of Alopia diversification, concludingthat taxa of opposite chirality cannot be classifiedwithin the same species (Kimakowicz, 1894). Later
systematic studies merely followed up these two clas-sification concepts, favouring either the former (Soós,1943; Szekeres, 2007) or latter (Soós, 1928; Grossu,1981; Nordsieck, 2008) approach. Therefore, findingout which of the two concurring approaches faith-fully reflects the evolutionary relationships withinthe genus necessitated ascertaining the phylogeneticorigin of dextrality.
The aim of this study was to test our hypotheses onthe incidence of chiral inversions by elucidating theevolutionary relationships within Alopia, especiallythose of the enantiomorph taxon pairs, based on amolecular phylogenetic analysis. The gene sequencebest suited for this purpose is mitochondrial cyto-chrome c oxidase subunit I (COI), because its highdivergence rate (Pons et al., 2010) offers good resolu-tion, even at the infraspecific level, it has beensuccessfully used for inferring close phylogenetic rela-tionships in other genera of the Alopiinae (Uit deWeerd, Schneider & Gittenberger, 2005 and Uit deWeerd, Schneider & Gittenberger, 2009), and hasbeen proposed to serve as the central barcodingmarker for the identification of animal taxa (Hebertet al., 2003). Considering COI data as indicators ofgenetic divergence, we propose a taxonomic divisionof the genus that is compatible with both the molecu-lar and morphological characters. Relying on ourphylogenetic results we discuss the zoogeographicalbackground of Alopia diversification, and make anattempt at estimating the start date of this process.
MATERIAL AND METHODSSNAIL SAMPLES
The Alopia samples used in this study were collectedfrom the entire geographical range of the genus (Fig. 1;Table 1). Sampling was designed to include most (64)of the 73 recognized taxa, and all those of disputedspecies affiliations. In order to avoid ambiguous iden-tification, each taxon was sampled at its type localityor one of its localities well known in the literature.Further clausiliid samples of the subfamilies Alopiinae[Herilla ziegleri dacica (Pfeiffer, 1848)] and Clausili-inae [Vestia elata (Rossmässler, 1836)] were used asout-groups. The list of samples, with full names andlocality information, are given in Table 1. All material,preserved in 99% ethanol, has been deposited at theMollusca Collection of the Hungarian Natural HistoryMuseum, Budapest (for collection numbers see theGenBank records listed in Table 1).
DNA ISOLATION AND SEQUENCING
DNA was prepared from foot tissue of ethanol-preserved specimens using QIAamp DNA Mini Kit(Qiagen, Valencia, CA, USA). A 655-bp segment of the
MULTIPLE CHIRAL INVERSIONS IN ALOPIA 261
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 167, 259–272
Tab
le1.
Dat
aof
the
Alo
pia
and
out-
grou
psa
mpl
esu
sed
inth
isst
udy
Sam
ples
Loc
alit
yG
eogr
aph
icpo
siti
onG
enB
ank
1A
.alp
ina
Kim
akow
icz,
1933
Bu
cegi
Mou
nta
ins,
Bat
rân
a45
°24′
16″N
,25
°25′
51″E
;18
90m
JQ91
1783
2A
.bie
lzii
biel
zii
(Pfe
iffe
r,18
49)
Hu
ned
oara
45°4
4′59
″N,
22°5
3′16
″E;
250
mJQ
9117
843
A.b
ielz
iicl
ath
rata
(Bie
lz,
1856
)S
lova
kK
arst
,Z
adie
l48
°37′
21″N
,20
°49′
52″E
;36
0m
JQ91
1785
4A
.bie
lzii
mad
ensi
s(F
uss
,18
55)
Ard
eun
ear
Bal
sa45
°02′
07″N
,23
°08′
46″E
;50
0m
JQ91
1786
5A
.bie
lzii
ten
uis
(Bie
lz,
1861
)G
ilau
Mou
nta
ins,
Bel
ioar
a46
°29′
31″N
,23
°22′
58″E
;11
20m
JQ91
1787
6A
.bog
aten
sis
angu
stat
a(B
ielz
,18
59)
Per
san
iM
oun
tain
s,C
oman
a45
°54′
59″N
,25
°20′
00″E
;58
0m
JQ91
1788
7A
.bog
aten
sis
boga
ten
sis
(Bie
lz,
1856
)A
Per
san
iM
oun
tain
s,B
ogat
a45
°56′
49″N
,25
°22′
45″E
;56
0m
JQ91
1789
8A
.bog
aten
sis
boga
ten
sis
(Bie
lz,
1856
)B
Per
san
iM
oun
tain
s,V
ârgh
is46
°13′
09″N
,25
°32′
42″E
;64
0m
JQ91
1790
9A
.can
esce
ns
can
esce
ns
(Ch
arpe
nti
er,
1852
)C
iuca
sM
oun
tain
s,C
iuca
s45
°31 ′
16″N
,25
°55′
20″E
;18
50m
JQ91
1791
10A
.can
esce
ns
hau
eri
(Bie
lz,
1859
)C
iuca
sM
oun
tain
s,D
un
gu45
°33′
13″N
,25
°54′
00″E
;12
40m
JQ91
1792
11A
.can
esce
ns
stri
atic
olli
s(K
imak
owic
z,18
94)
Ciu
cas
Mou
nta
ins,
Tesl
a45
°29′
26″N
,25
°53′
22″E
;13
40m
JQ91
1793
12A
.gla
uca
(Bie
lz,
1853
)A
Has
mas
Mou
nta
ins,
Bic
az46
°48′
45″N
,25
°49′
06″E
;80
0m
JQ91
1794
13A
.gla
uca
(Bie
lz,
1853
)B
Rar
auM
oun
tain
s,R
arau
47°2
7′19
″N,
25°3
2′56
″E;
1410
mJQ
9117
9514
A.g
lori
fica
dec
epta
ns
Del
i&
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kere
s,20
11P
iatr
aC
raiu
lui
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nta
ins,
Sea
caP
ietr
elor
45°2
8′19
″N,
25°1
3′30
″E;
980
mJQ
9117
9615
A.g
lori
fica
eleg
anti
ssim
aN
ords
ieck
,19
77P
iatr
aC
raiu
lui
Mou
nta
ins,
Dâm
bovi
cioa
ra45
°26′
33″N
,25
°13′
20″E
;91
0m
JQ91
1797
16A
.glo
rifi
cagl
orifi
ca(C
har
pen
tier
,18
52)
Pia
tra
Cra
iulu
iM
oun
tain
s,C
rapa
tura
45°3
3′21
″N,
25°1
5′27
″E;
1550
mJQ
9117
9817
A.g
lori
fica
inte
rced
ens
(Sch
mid
t,18
57)
AP
iatr
aC
raiu
lui
Mou
nta
ins,
Dâm
bovi
cioa
ra45
°26′
35″N
,25
°23′
20″E
;92
0m
JQ91
1799
18A
.glo
rifi
cain
terc
eden
s(S
chm
idt,
1857
)B
Pia
tra
Cra
iulu
iM
oun
tain
s,S
eaca
Pie
trel
or45
°28′
38″N
,25
°13′
35″E
;11
00m
JQ91
1800
19A
.glo
rifi
cam
agn
ifica
Kim
akow
icz,
1962
Pia
tra
Cra
iulu
iM
oun
tain
s,P
odu
Dâm
bovt
ei45
°25′
31″N
,25
°11′
28″E
;81
0m
JQ91
1801
20A
.glo
rifi
casu
bita
(Kim
akow
icz,
1894
)P
iatr
aC
raiu
lui
Mou
nta
ins,
Col
tuC
hil
iilo
r45
°34′
07″N
,25
°14′
49″E
;10
80m
JQ91
1802
21A
.glo
rifi
cavr
ance
ana
Gro
ssu
,19
67V
ran
cea
Mou
nta
ins,
Tis
aru
45°5
6′26
″N,
26°3
5′06
″E;
600
mJQ
9118
0322
A.g
ross
uan
agr
ossu
ana
Nor
dsie
ck,
1977
Vâl
can
Mou
nta
ins,
Soh
odol
45°0
8′11
″N,
23°0
8′22
″E;
390
mJQ
9118
0423
A.g
ross
uan
an
emet
hi
Del
i&
Sze
kere
s,20
11V
âlca
nM
oun
tain
s,S
ohod
ol45
°10′
47″N
,23
°07′
58″E
;45
0m
JQ91
1805
24A
.lis
chke
ana
boet
tger
i(K
imak
owic
z,18
83)
Pia
tra
Cra
iulu
iM
oun
tain
s,R
âuM
are
45°3
2′30
″N,
25°1
7′45
″E;
820
mJQ
9118
0625
A.l
isch
kean
acy
baea
(Kim
akow
icz,
1894
)P
iatr
aC
raiu
lui
Mou
nta
ins,
Râu
Mar
e45
°31′
53″N
,25
°17′
15″E
;85
0m
JQ91
1807
26A
.lis
chke
ana
galb
ina
Kim
akow
icz,
1943
Pia
tra
Cra
iulu
iM
oun
tain
s,M
agu
raS
at45
°31′
36″N
,25
°17 ′
58″E
;10
90m
JQ91
1808
27A
.lis
chke
ana
lisc
hke
ana
(Ch
arpe
nti
er,
1852
)P
iatr
aC
raiu
lui
Mou
nta
ins,
Râu
Mar
e45
°31′
36″N
,25
°16′
08″E
;94
0m
JQ91
1809
28A
.lis
chke
ana
live
ns
(Bie
lz,
1853
)P
iatr
aC
raiu
lui
Mou
nta
ins,
Pes
tera
45°3
0′43
″N,
25°1
7′37
″E;
970
mJQ
9118
1029
A.l
isch
kean
asa
rkan
yiS
zeke
res,
2007
Pia
tra
Cra
iulu
iM
oun
tain
s,M
atei
as45
°18′
17″N
,25
°08′
52″E
;10
80m
JQ91
1811
30A
.liv
ida
bipa
lata
lis
(Kim
akow
icz,
1883
)B
uce
giM
oun
tain
s,G
aura
45°2
6′25
″N,
25°2
5′27
″E;
1900
mJQ
9118
1231
A.l
ivid
ad
ean
ian
a(C
ooke
,19
22)
Pia
tra
Cra
iulu
iM
oun
tain
s,M
agu
raM
ica
45°3
1′41
″N,
25°1
8′47
″E;
1290
mJQ
9118
1332
A.l
ivid
aju
lii
(Wag
ner
,19
14)
Bih
orM
oun
tain
s,O
rdân
cusa
46°2
7′55
″N,
22°5
0′14
″E;
820
mJQ
9118
1433
A.l
ivid
ali
vid
a(M
enke
,18
28)
AB
uce
giM
oun
tain
s,Ia
lom
ita
45°2
1′44
″N,
25°2
5′44
″E;
1490
mJQ
9118
1534
A.l
ivid
ali
vid
a(M
enke
,18
28)
BB
uce
giM
oun
tain
s,P
eles
45°2
1′21
″N,
25°3
2′02
″E;
1020
mJQ
9118
1635
A.m
acia
na
Bad
arau
&S
zeke
res,
2001
Gil
auM
oun
tain
s,B
elio
ara
46°2
9′46
″N,
23°2
1′58
″E;
1300
mJQ
9118
1736
A.m
afte
ian
am
afte
ian
aG
ross
u,
1967
Fag
aras
Mou
nta
ins,
Vâl
san
45°2
0′04
″N,
24°4
4′21
″E;
680
mJQ
9118
1837
A.m
afte
ian
ava
leri
aeS
zeke
res,
2007
Cet
aten
i45
°12′
35″N
,25
°12′
36″E
;55
0m
JQ91
1819
262 Z. FEHÉR ET AL.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 167, 259–272
38A
.mar
iae
coro
nat
aK
imak
owic
z,19
43L
otru
Mou
nta
ins,
Pia
tra
Fet
ii45
°23′
48″N
,23
°54′
17″E
;13
70m
JQ91
1820
39A
.mar
iae
hil
deg
ard
aeK
imak
owic
z,19
31C
apat
âna
Mou
nta
ins,
Bu
ila
45°1
4′19
″N,
24°0
6′27
″E;
1350
mJQ
9118
2140
A.m
aria
em
aria
eK
imak
owic
z,19
31C
apat
âna
Mou
nta
ins,
Bu
ila
45°1
4′07
″N,
24°0
6′11
″E;
1300
mJQ
9118
2241
A.m
aria
eso
osi
Bra
ndt
,19
61C
apat
âna
Mou
nta
ins,
Olt
et45
°12′
03″N
,23
°47′
00″E
;67
0m
JQ91
1823
42A
.mes
chen
dor
feri
(Bie
lz,
1858
)P
ersa
ni
Mou
nta
ins,
Mag
ura
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lei
45°4
2′26
″N,
25°2
4′33
″E;
1040
mJQ
9118
2443
A.m
onac
ha
(Kim
akow
icz,
1894
)A
Bu
cegi
Mou
nta
ins,
Ialo
mit
a45
°23′
38″N
,25
°26′
15″E
;16
00m
JQ91
1825
44A
.mon
ach
a(K
imak
owic
z,18
94)
BL
otru
Mou
nta
ins,
Târ
nav
a*45
°21′
15″N
,23
°52′
34″E
;17
20m
JQ91
1826
45A
.nef
asta
hel
enae
Kim
akow
icz,
1928
Ciu
cas
Mou
nta
ins,
Gro
psoa
ra45
°28′
56″N
,25
°57′
50″E
;12
80m
JQ91
1827
46A
.nef
asta
mau
riti
iK
imak
owic
z,19
28C
iuca
sM
oun
tain
s,G
rops
oara
45°2
9′43
″N,
25°5
7′34
″E;
1550
mJQ
9118
2847
A.n
efas
tan
efas
ta(K
imak
owic
z,18
94)
Ciu
cas
Mou
nta
ins,
Bra
toce
a45
°29′
46″N
,25
°53′
52″E
;16
60m
JQ91
1829
48A
.nef
asta
zaga
ni
Sze
kere
s,19
69C
iuca
sM
oun
tain
s,G
rops
oara
45°2
8′57
″N,
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MULTIPLE CHIRAL INVERSIONS IN ALOPIA 263
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COI gene, between nucleotides 39 and 693 relative tothe translational start codon (Hatzoglou, Rodakis &Lecanidou, 1995), was amplified by polymerase chainreaction (PCR) as described in Fehér et al. (2009).Primers PF372 5′-TCAACGAATCATAAAGATATTGG-3′ and PR373 5′-TATACTTCAGGATGACCAAAGAATCA-3′ were designed by modifying the Albinariaprimers L1490-Alb and H2198-Alb (Gittenberger, Piel& Groenenberg, 2004), respectively. Isolated and puri-fied PCR products were sequenced on both DNAstrands using an ABI Prism 3100 Genetic Analyzer(Applied Biosystems, Carlsbad, CA, USA).
PHYLOGENETIC ANALYSIS
DNA sequences were of equal length (655 bp) andshowed proper open reading frames (ORFs), allowingunambiguous alignment. Identical sequences werecollapsed into haplotypes. All sequence data havebeen deposited in GenBank (accession numbersJQ911783–JQ911855; Table 1).
The appropriate model for nucleotide substitu-tion (HKY + I + G) was selected by jModelTest 0.1.1(Guindon & Gascuel, 2003; Posada, 2008) using theBayesian Information Criterion (BIC). The inverte-brate mitochondrial code table, as implemented inMEGA 5.0 (Tamura et al., 2011), was used to deduceencoded amino acid sequences.
The molecular clock hypothesis was tested bylikelihood ratio test in MEGA 5.0, using the topologyshown in Figure 2, and the null hypothesis ofequal evolutionary rate throughout the treewas rejected (log L0 = -4582.32, log L1 = 4665.43,D = 166.22, d.f. = 67, P < 0.000).
Haplotypes were analysed by various methodsand settings in order to test the method dependenceof phylogenetic tree topology and root position.An unconstrained Bayesian tree was inferred byMrBayes 3.2.1 (Ronquist et al., 2012) using thefollowing parameters: HKY + I + G model of sequenceevolution; a four-chain (one cold, three heated;T = 0.2) Metropolis-coupled Markov chain Monte
Figure 2. Unrooted phylogenetic tree inferred from Alopia COI sequences by unconstrained Bayesian analysis.Sequences are identified with the subspecific names of the sinistral (normal print) or dextral (inverted print) taxa of theirorigin (see Table 1). Major clades are marked with upper case letters and numbering. The sequences labelled proclivisand wagneri (clade C1), as well as intercedens B and deceptans (clade D3), belong to identical haplotypes. Scale bar:0.1 substitutions per site.
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Carlo (MCMC) analysis run for 106 generations; treessampled every 100 generations, starting after aburn-in of 105 generations. Neighbor-joining (NJ),maximum likelihood (ML) and maximum parsimony(MP) analyses were performed with MEGA 5.0. ForML analysis we used the HKY + I + G model ofsequence evolution with five gamma rate categoriesand the nearest-neighbor interchange heuristicsearch strategy. The MP tree analysis was performedusing the close-neighbor interchange heuristicsearch strategy with random additions to ten initialsequences. The NJ analysis was performed under theKimura two-parameter (K2P) model of substitution.Bootstrap values of the ML, MP, and NJ analyseswere calculated with 1000 bootstrap replicates.
To define the most likely rooting site, wealso carried out likelihood mapping (Strimmer &von Haeseler, 1997) as implemented in TREE-PUZZLE 5.2 (Schmidt et al., 2002). This method canmanage a maximum of four clusters, and thereforethe unconstrained Bayesian tree (Fig. 2) was dividedinto three trifurcation-separated subgroups in sixcombinations, and these were analysed against thecluster of the out-groups (Herilla and Vestia). Thefollowing combinations were tested: (1) clades C/B/A + D + E + F/out-groups; (2) clades C + B/A/D + E +F/out-groups; (3) clades A + B + C/F/E + D/out-groups;(4) clades E/D/A + B + C + F/outgroups; (5) cladesC1/C2/A + B + D + E + F/out-groups; and (6) A1/A2/B + C + D + E + F/out-groups.
To estimate divergence times, we used a data setthat included representatives of all Alopia clades and20 additional sequences of Carinigera Möllendorff,1873 downloaded from the GenBank database. Baye-sian analyses were performed using BEAST 1.4.6(Drummond & Rambaut, 2007), with the followingsettings: HKY + I + G model of sequence evolutionwith five gamma rate categories, Yule tree prior, anda relaxed (uncorrelated lognormal) clock assumption.Two analyses were carried out using clock rates of 1or 8.6% (‘ucld.mean’ parameters). Following a burn-inof 106 cycles, every 1000th tree was sampled from 107
MCMC steps. Convergence of the chains to the sta-tionary distribution was checked by visual inspectionof plotted posterior estimates using the TRACER 1.3(Rambaut & Drummond, 2007). The effective samplesize for each parameter sampled from the MCMCanalysis was always found to exceed 100. Sampledtrees were annotated to a maximum clade credibilitytree.
USE OF TAXONOMIC NAMES
Recent concepts of Alopia classification (Grossu, 1981;Szekeres, 2007; Nordsieck, 2008) have been basedon conflicting principles, and none of them is in full
agreement with our results. A comprehensive system-atic revision of the genus is beyond the scope of thepresent study, and will be provided in a follow-uptaxonomic publication. Nevertheless, based on ourresults we make taxonomic statements when thisis essential for the consistency of the classificationthat we use. For easy comparison, our names and anassessment of earlier taxonomic nomenclature areprovided in Appendix S1.
RESULTSCOI PHYLOGENY IN THE GENUS ALOPIA
The COI gene sequences were determined from 71Alopia samples (Table 1) belonging to 69 haplotypes.Within the amplified 655-bp region we identified206 variable positions, corresponding to 31.4% of thenucleotides. The highest observed intrageneric valueof pairwise sequence divergence was 13.0%. Most ofthe detected variability had no effect on the deducedamino acid sequence, except those at nucleotide posi-tion 470, causing the replacement of proline byalanine or serine in 11 haplotypes, and unique muta-tions at positions 167, 315, 358, 360, 470, 480, and525, leading to amino acid substitutions in sevendistinct haplotypes.
An unconstrained Bayesian tree generated from theCOI sequences (Fig. 2) featured six basic evolutionarylineages (clades A–F). Supporting posterior probabil-ity values of the node positions are given in Appen-dix S2. All inference methods used (also includingML, MP, and NJ) yielded congruent topologies withinthe basic clades (data not shown) and nearly identicalrelative positions for the major clades (Appendix S2).By contrast, these methods failed to identify a con-sistent root position relative to the out-groups. Fur-thermore, likelihood mapping determined two, almostequally likely root positions that did not match anyof those assigned by the above inference analyses(Appendix S3).
A recent analysis of the molecular evolution ofmitochondrial genes in beetles (Pons et al., 2010)revealed an overall 8.6% per million years (Myr)divergence rate for the COI sequences. Based on thisclock rate, which is similar to that determined forsalamander species (Mueller, 2006), our Bayesiananalysis using an uncorrelated lognormal relaxedclock model estimated the divergence of the majorAlopia clades at 1.2 Mya, with a 95% highest poste-rior density (HPD) interval of 1.6–0.8 Myr (for details,see Appendix S4).
SPECIATION IN ALOPIA INVOLVED MULTIPLE
INVERSIONS OF CHIRALITY
The COI-based phylogram shows differential repre-sentation of the sinistral and dextral taxa in the
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major evolutionary lineages (Fig. 2). Clades C and Fcomprise only sinistral taxa, whereas clade E includesonly the dextral taxon Alopia meschendorferi (Bielz,1858). By contrast, clades B and D contain multipleclosely related taxa with both coil directions, includ-ing all enantiomorph taxon pairs. In clade A the twosubgroups A1 and A2 correspond to the invariablydextral Alopia bielzii (Pfeiffer, 1848) and sinistralAlopia bogatensis (Bielz, 1856) forms, respectively.
An intriguing result of the molecular phylogeneticanalysis was that it revealed very high levels ofsequence identity between the enantiomorph taxaAlopia glorifica deceptans Deli & Szekeres, 2011 andAlopia glorifica intercedens (Schmidt, 1857) (100%),
Alopia lischkeana boettgeri (Kimakowicz, 1883),Alopia lischkeana cybaea (Kimakowicz, 1894), Alopianefasta helenae Kimakowicz, 1928, Alopia nefastazagani Szekeres, 1969, Alopia nixa fussi (Kimakow-icz, 1894), and Alopia nixa nixa (Kimakowicz, 1894)(99.8%), Alopia grossuana grossuana Nordsieck, 1977and Alopia grossuana nemethi Deli & Szekeres, 2011(99.6%), as well as Alopia mariae hildegardae Kima-kowicz, 1931 and Alopia mariae mariae Kimakowicz,1931 (98.6%). These data indicate close evolutionaryrelationships within these taxon pairs of oppositechirality but otherwise identical morphology (Fig. 3).
The COI-based phylogram implies that the dextraltaxa did not evolve as a monophyletic lineage that
Figure 3. Shell morphology and COI-based phylogenetic relationships of the enantiomorph taxon pairs. The schematictree follows the topology of the phylogram shown in Figure 2.
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stemmed from an inversion early in Alopia phylogeny.Instead, they appeared polyphyletically as the resultsof several independent inversions, rendering dextralcoiling a homoplastic trait in this genus. The dextrallineages of A. bielzii (clade A1) and A. meschendorferi(clade E) show deep divergence (Fig. 2), whereas someothers reveal only minor or no changes of theCOI sequence relative to the closest related sinistraltaxa.
COI PHYLOGENY ELUCIDATES EVOLUTIONARY
RELATIONSHIPS
We found that clusters of the COI phylogram showgood correspondence with some of the morphologicaltraits shared by subspecies that have been classifiedwithin the same species or species assigned to speciesgroups, especially when these characters have beenconsidered unique for those taxa. Such featuresare, for instance, the rugose shell wall of A. bielzii(clade A1), the characteristic lump behind the peris-tome of A. bogatensis (clade A2), or the elongatedmale genital structures of Alopia glauca (Bielz, 1853),Alopia maciana Badarau & Szekeres, 2001, andAlopia pomatias (Pfeiffer, 1868), which constitutethe subgenus Alopia (Kimakowiczia) Szekeres, 1969(clade F). The correlation between the molecularclade positions of the subspecies and species withtheir shared morphological characters and geographi-cal ranges is shown in Appendix S5.
The molecular data also revealed hitherto unrecog-nized phylogenetic relationships between Alopia formsseparated by large geographical distances. The resultsindicated that the subspecies vranceana belongs toclade D2, corresponding to the species A. glorifica,despite the 110 km that separates it from the PiatraCraiului Mountains where all other glorifica subspe-cies are limited (Fig. 1, localities 21 versus 15–20).Likewise, in clade C1 the subspecies microstoma,nordsiecki, and petrensis cluster together with themorphologically very similar subspecies of regalis(clade C1), although the aforementioned three taxawere traditionally classified with other species (Appen-dix S1) because of their occurrence 120–190 km west ofthe Postavaru and Piatra Mare Mountains, the diver-sity centre of Alopia regalis Bielz, 1851 (Fig. 1, locali-ties 59, 61, and 62 versus 58, 60, 63, 64, and 65).Furthermore, the COI phylogram justifies the classi-fication of the subspecies julii of central Transylvaniawithin the species Alopia livida (Menke, 1828) (Soós,1943; Grossu, 1981; Nordsieck, 2008), as opposed tothat of Wagner (1914) and Szekeres (1976), assumingthat the striking shell similarity of julii and livida(clade D1), occurring 170 km apart (Fig. 1, localities 32versus 31, 33, and 34), resulted from the convergentreduction of distinctive shell structures.
Sequence data also necessitate synonymizing theforms peregrina Kimakowicz, 1943 of the LotruMountains (Fig. 1, locality 44) and soosiana Agócsy &Pócs, 1961 (Fig. 1, locality 55) of the Fagaras Moun-tains (Table 1), which used to be considered valid taxa(Szekeres, 1976; Grossu, 1981; Nordsieck, 2008). Theextensive COI homology and the apparent morpho-logical correspondence reveal that these Alopia formsare merely disjunct populations of Alopia monacha(Kimakowicz, 1894) (99.6% identical with peregrina)and A. pomatias (99.8% identical with soosiana) thatare native to the Bucegi Mountains.
Although COI data elucidated phylogenetic rela-tionships, they also revealed distinct origins of certaintaxa with similar shell morphology and overlapp-ing distribution. Although originally the Alopiaforms from the Postavaru and Piatra Mare mountainswere divided as subspecies of Alopia plumbea(Rossmässler, 1839) or A. regalis, respectively (Kima-kowicz, 1894; Soós, 1943), later the morphologicalsimilarity lead to merging all of these withinA. plumbea (Szekeres, 1976; Grossu, 1981; Nordsieck,2008). Our molecular phylogram, however, indicatesthat the subspecies formerly belonging to A. plumbea(bellicosa and plumbea in clade B2) and those ofA. regalis (deubeli, glabriuscula, mutabilis, proclivis,regalis, and wagneri in clade C1) represent two dis-tinct evolutionary lineages. In another case, morpho-logically similar Alopia taxa of the Piatra CraiuluiMountains were found to harbour two distinct typesof COI sequences. This implies that the subspeciesbelonging to clades B3 (A. lischkeana) and D2 (A. glo-rifica) evolved independently.
DISCUSSION
The 655-bp segment of the COI sequence, which hasbeen widely used for inferring phylogenetic informa-tion in various animal groups, represents one of thebest-studied molecular markers in the subfamilyAlopiinae. In Alopia the COI phylogenies deduced byvarious methods gave consistent topologies withineach of the major clades, indicating that the analysedsequence information was sufficient for reliable recon-struction of the evolutionary relationships (Nguyen,Gesell & Haeseler, 2012). Tree topologies in taxonomi-cally unambiguous groups (e.g. clades A and F) werein full agreement with the morphology-based classi-fication; therefore, we assumed similar good agree-ment between the COI phylogeny and speciation ingroups with less distinctive morphological features.Accordingly, the taxonomic division we propose isbased on the evaluation of both molecular data andmorphological characters, relying on the same princi-ple as applied for defining the systematic status of
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Carinigera species (Gittenberger & Uit de Weerd,2006).
When correlating COI clade positions with sharedmorphological traits and common patterns of geo-graphical distribution (Appendix S5), the taxonomicpositions in two cases proposed here seem to requirefurther clarification. One is the subgenus Alopia(Kimakowiczia), which includes three species (clade F)showing shell and genital structures that markedlydiffer from those of other Alopia groups (Badarau &Szekeres, 2001). But, despite the unique morphology,the subgeneric separation of this group is not sup-ported by the COI phylogeny, which shows the samedepth of divergence for the Alopia (Kimakowiczia)group as for the other major clades of the genus(Fig. 2). In the second case, the relationship betweenA. glorifica and A. lischkeana, both native to the PiatraCraiului Mountains, requires better insight. AlthoughCOI sequences suggest only a distant relationshipbetween these species, their morphological similarityraises the question whether indeed they are of distinctorigin, with nuclear genes showing similar descent asthat of COI. Addressing these questions will requirefurther molecular phylogenetic studies, involving bothnuclear and mitochondrial DNA sequences.
Unlike close evolutionary relationships, the topol-ogy of the major clades had relatively weak support inthe COI phylogram (Appendix S2). A similar resultwas obtained by Uit de Weerd, Piel & Gittenberger(2004), who found that in four Alopiinae generanuclear DNA segments showed greater support fordeeper nodes than COI and other mitochondrialsequences. Uit de Weerd et al. (2004) proposed thatfor genes with a high divergence rate (Pons et al.,2010), this might result from a saturation effect. Butin Alopia, COI sequences show only modest (up to13.0%) maximum intrageneric pairwise divergence, sosaturation alone cannot explain the weaker resolutionof deep branching points and the ambiguous rootposition. We therefore interpret these as likely con-sequences of radiation: a lineage-splitting burstwithin a relatively short period of time (Wilke et al.,2010) that might have happened early during thediversification of the genus.
The only available fossil finds of Alopia were recov-ered from non-layered cave deposits of the Holoceneperiod (Ložek, 1964; Szekeres, 2007). In the absenceof dated fossils we calculated the divergence timeof the main Alopia clades on the basis of the 8.6%per Myr COI clock rate determined in beetles (Ponset al., 2010), considering that an appropriate choice ofthis parameter can lead to realistic estimates evenwhen geological calibration is not possible (Wilke,Schultheiss & Albrecht, 2009). With the applicationof this rate the divergence time was placed at about1.2 Mya, much more recently than the roughly
10.4 Mya calculated with the canonical ~1% rateused previously for mitochondrial DNA sequences(Wilke et al., 2009; Appendix S4). As a group of south-east Mediterranean origin, the ancestors of Alopiacould have colonized the Carpathians via the landcontact formed between the Southern Carpathiansand the Balkan Mountains, disrupting the continuityof the receding Paratethys. This geological transfor-mation took place around the Pliocene–Pleistoceneboundary, dated to 2.6 Mya (Olteanu & Jipa, 2006;Andreescu et al., 2011), suggesting that the appear-ance and expansion of Alopia must be more recentevents. This assumption is in line with the lack ofAlopia in the Early Pleistocene cave deposits of theSprenghi Hill at Brasov (Soós, 1916), and also withthe Late Pleistocene dating of fossil Mastus venera-bilis (Pfeiffer, 1853), a species of the Enidae arrivingat the Carpathians from the same direction, andpreferring the same habitats as Alopia, found inloess samples of south-eastern Hungary (Soós,1943). The modest (13.0%) intrageneric pairwisedivergence of COI sequences in Alopia, as comparedwith those of the Balkan genera Albinaria Vest, 1867(18.2%), Carinigera (19.9%) and Inchoatia Gitten-berger & Uit de Weerd, 2006 (17.8%), also impliesthat this is a relatively young genus in the Alopiinae.Accordingly, the 8.6% clock rate-based 1.2 Myadating of early Alopia divergence, placing this eventat the middle of the Pleistocene, appears to be arealistic estimate.
Mountains of the Southern and southernmostEastern Carpathians were colonized by Alopia fromdifferent directions, leading to the presence of morethan one abundant species in the Piatra Craiului(A. glorifica and A. lischkeana), Bucegi (A. livida andA. monacha), and Ciucas [Alopia canescens (Charpen-tier, 1852) and Alopia nefasta (Kimakowicz, 1894)]ranges. These examples show that although infraspe-cific differentiation was usually confined to a particu-lar mountain, co-occurrence in the same mountaindoes not necessarily mean close phylogenetic relation-ship. Though the current distribution of Alopiaspecies seems to have been formed mostly by waves ofgradual range expansion, disjunct occurrences (e.g.those shown in Fig. 4) also indicate a role for passivelong-range dispersal, possibly mediated by birds (Uitde Weerd et al., 2005; Maciorowski, Urbanska &Gierszal, 2012).
Because of their rock-dwelling character, Alopiaspecies tend to be highly endemic, often dividedwithin the same mountain complex into locally iso-lated subspecies (Soós, 1928). But the present distri-bution of certain groups reveals multiple wavesof range expansion, which were probably facilitatedby major climatic changes in the Late Pleistocene.For instance, the massive westwards expansion of
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alpine A. livida could have been possible during a coldperiod, whereas its displacement by thermophilicA. bielzii over much of its range in the Bihor-Vladeasa, Metaliferi, and Trascau mountains (Fig. 4)must have taken place at stages of warmer climate.Likewise, cooler periods could have been favourablefor the wide distribution of the cold-hardy species ofclade F, whereas during subsequent warming theirhabitats became fragmented and, in the cases ofA. maciana and A. pomatias, increasingly overtakenby more thermotolerant A. bielzii and A. livida,respectively. Disjunct occurrences of four A. regalissubspecies, far away from the main range of thespecies in the Piatra Mare Mountains, also attests toan earlier westwards expansion (Fig. 4).
In the exposed limestone habitats, climatic cyclescould have had a substantial influence on diversifica-tion (Scheel & Hausdorf, 2012). Range expansionsat favourable periods were followed by the breaking-upof the ranges during epochs of harsher climate. Thisled to the severe reduction of population sizes, asseen today in the case of alpine A. pomatias and ofsome species or subspecies occurring at low (below1000 m a.s.l.) altitudes (Badarau & Szekeres, 2001;Szekeres, 2007). Colonization by passive dispersal, acommon method of radiation among rock-dwellingsnails, also generated very small populations. In suchcases unbalanced gene pools result in increased phe-
notypic variability, which can lead to multiple localforms within relatively short periods of time (Gitten-berger, 1991; Rundell & Price, 2009). Such genetic drifteffects may be behind the apparent discrepancybetween the more diverse morphological traits inAlopia and the less diverse indispensable COI genes,compared with other genera of the Alopiinae.
Speciation in Alopia is further enhanced by anincreased tendency for chiral reversal. The anatomicaldifference between the individuals of opposite coilingconsiderably restricts interchiral mating, therebyleading to reproductive isolation and, in most cases,selection-based disappearance of reversed snails inthe population (Gittenberger, 1988; Asami, Cowie &Ohbayashi, 1998). But because of the relatively highfrequency of inversions in this genus, occasionallysuch individuals of the same offspring succeed inestablishing stable subpopulations, a process facili-tated by the low dispersal rate of rock-dwelling snails(Schilthuizen & Lombaerts, 1994). As the reproductivesuccess of each chiral morph is ensured by the avail-ability of surrounding mating partners of the samechirality, this gradually leads to territorial separationand, in time, the formation of isolated sinistral anddextral populations (Ueshima & Asami, 2003). At thislevel of speciation, exemplified by the enantiomophpairs of Alopia, further gene flow between neighbour-ing populations of the opposite chiral forms is severely
Figure 4. Alopia species of disjunct distribution. Main ranges (encircled by broken lines) and isolated occurrences ofAlopia glorifica, Alopia livida, and Alopia regalis forms. Symbols correspond to A. glorifica subspecies of the PiatraCraiului Mountains (gl), A. g. vranceana (vr), A. livida subspecies of the Bucegi Mountains (li), A. l. deaniana (de), A. l.julii (ju), A. regalis subspecies of the Piatra Mare and Postavaru Mountains (re), A. r. doftanae (do), A. r. microstoma (mi),A. r. nordsiecki (no), and A. r. petrensis (pe). Arrows show the assumed directions of range expansion. Major mountainsare identified.
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restricted (Schilthuizen & Lombaerts, 1994). Withtheir clearly different morphology and reproductiveisolation they represent divergent evolutionary line-ages, and can be regarded as distinct subspecies.
Our molecular phylogenetic study revealed thatdextral taxa of Alopia did not evolve monophy-letically, but via multiple independent inversions.Although the establishment and stabilization ofreversed populations happened rarely, this was muchmore common than in any other genera of clausiliids(Gittenberger, Hamann & Asami, 2012). Based onthe COI tree (Fig. 2) we calculate that, consideringa parsimonious scenario of only sinistral to dextralinversions, chirality changes gave rise to new phylo-genetic lineages on at least 13 occasions during theevolution of the genus. This clearly indicates thedecreased stability of chiral fixation in the species ofclades B and D. Because of this unique feature andthe availability of enantiomorph pairs, these Alopiataxa can become valuable subjects of genetic studiesaimed at clarifying the molecular background ofchiral determination and its fixation.
ACKNOWLEDGEMENTS
The authors are thankful to Gheorghe Ban, EdmundGittenberger, Henrik Gyurkovics, Bas Kokshoorn,Harry G. Lee, Mariana Pascu, Endre Sárkány-Kiss,Ioan Sîrbu, and Dennis R. Uit de Weerd for theirsupport and valuable discussions, Tamás Deli, TamásDomokos, Zoltán Eross, András Hunyadi, KornélKovács, Barna Páll-Gergely, Péter Subai, and Cata-lina Trif for their help with fieldwork, and to twoanonymous reviewers for their helpful comments. Z.F.was supported by the Hungarian Scientific ResearchFund (OTKA-NNF 78185) and a János BolyaiResearch Scholarship of the Hungarian Academy ofSciences.
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SUPPORTING INFORMATION
Additional supporting information may be found in the online version of this article:
Appendix S1. COI-based topology and nomenclatural status of the Alopia taxa studied.Appendix S2. Branch support values of the COI clades in Alopia.Appendix S3. Putative root positions in the COI phylogram of Alopia.Appendix S4. Comparison of divergence times in the genera Carinigera and Alopia, based on their COIsequences.Appendix S5. Correlations between COI topology, morphology, and geographical distribution of Alopia species.
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Appendix S1. COI-based topology and nomenclatural status of the studied Alopia taxa __________________________________________________________________________________________________________________________________________________________________________________________________________ Proposed in this study Nordsieck, 2008 Grossu, 1981 Szekeres, 1976 Soós, 1943 __________________________________________________________________________________________________________________________________________________________________________________________________________
CLADE A1 bogatensis angustata (E.A. Bielz, 1859) bogatensis angustata (E.A. Bielz, 1859) bogatensis angustata (E.A. Bielz, 1859) bogatensis angustata (E.A. Bielz, 1859) bogatensis angustata (E.A. Bielz, 1859) bogatensis bogatensis (E.A. Bielz, 1856) bogatensis bogatensis (E.A. Bielz, 1856) bogatensis bogatensis (E.A. Bielz, 1856) bogatensis bogatensis (E.A. Bielz, 1856) bogatensis bogatensis (E.A. Bielz, 1856) CLADE A2 bielzii bielzii (L. Pfeiffer, 1849) bielzii bielzii (L. Pfeiffer, 1849) bielzii bielzii (L. Pfeiffer, 1849) bielzii bielzii (L. Pfeiffer, 1849) bielzii bielzii (L. Pfeiffer, 1849) bielzii clathrata (E.A. Bielz, 1856) bielzii clathrata (E.A. Bielz, 1856) bielzii clathrata (E.A. Bielz, 1856) bielzii clathrata (E.A. Bielz, 1856) bielzii clathrata (E.A. Bielz, 1856) bielzii madensis (Fuss, 1855) bielzii madensis (Fuss, 1855) bielzii madensis (Fuss, 1855) bielzii madensis (Fuss, 1855) bielzii madensis (Fuss, 1855) bielzii tenuis (E.A. Bielz, 1861) bielzii tenuis (E.A. Bielz, 1861) bielzii tenuis (E.A. Bielz, 1861) bielzii tenuis (E.A. Bielz, 1861) bielzii tenuis (E.A. Bielz, 1861)
CLADE B1 subcosticollis (A. Schmidt, 1868) subcosticollis subcosticollis (A. Schmidt, 1868) subcosticollis jickelii (M. Kimakowicz, 1894) occidentalis subcosticollis (A. Schmidt, 1868) occidentalis jickelii (M. Kimakowicz, 1894) vicina fortunata R. Kimakowicz, 1931 hildegardae fortunata R. Kimakowicz, 1931 hildegardae fortunata R. Kimakowicz, 1931 occidentalis fortunata R. Kimakowicz, 1931 n. m. vicina occulta R. Kimakowicz, 1931 subcosticollis occulta R. Kimakowicz, 1931 subcosticollis occulta R. Kimakowicz, 1931 occidentalis occulta R. Kimakowicz, 1931 n. m. vicina tamasorum Szekeres, 2007 subcosticollis tamasorum Szekeres, 2007 n. m. n. m. n. m. vicina vicina (M. Kimakowicz, 1894) subcosticollis vicina (M. Kimakowicz, 1894) subcosticollis vicina (M. Kimakowicz, 1894) occidentalis vicina (M. Kimakowicz, 1894) n. m. CLADE B2 grossuana grossuana Nordsieck, 1977 subcosticollis grossuana Nordsieck, 1977 subcosticollis grossuana Nordsieck, 1977 n. m. n. m. grossuana nemethi Deli & Szekeres, 2011 n. m. n. m. n. m. n. m. plumbea bellicosa (M. Kimakowicz, 1894) plumbea bellicosa (M. Kimakowicz, 1894) plumbea schmidti (M. Kimakowicz, 1894) plumbea bellicosa (M. Kimakowicz, 1894) plumbea bellicosa (M. Kimakowicz, 1894) plumbea plumbea (Rossmässler, 1839) plumbea plumbea (Rossmässler, 1839) plumbea plumbea (Rossmässler, 1839) plumbea plumbea (Rossmässler, 1839) plumbea plumbea (Rossmässler, 1839) CLADE B3 lischkeana boettgeri (M. Kimakowicz, 1883) glorifica boettgeri (M. Kimakowicz, 1883) glorifica boettgeri (M. Kimakowicz, 1883) glorifica boettgeri (M. Kimakowicz, 1883) fussiana boettgeri (M. Kimakowicz, 1883) lischkeana cybaea (M. Kimakowicz, 1894) lischkeana cybaea (M. Kimakowicz, 1894) glorifica obesa (M. Kimakowicz, 1883) glorifica obesa (M. Kimakowicz, 1883) fussiana boettgeri (M. Kimakowicz, 1883) lischkeana galbina R. Kimakowicz, 1943 glorifica galbina R. Kimakowicz, 1943 glorifica galbina R. Kimakowicz, 1943 glorifica galbina R. Kimakowicz, 1943 fussiana intercedens (A. Schmidt, 1857) lischkeana lischkeana (Charpentier, 1852) lischkeana lischkeana (Charpentier, 1852) lischkeana lischkeana (Charpentier, 1852) glorifica lischkeana (Charpentier, 1852) fussiana fussiana (E.A. Bielz, 1852) lischkeana livens (E.A. Bielz, 1853) lischkeana livens (E.A. Bielz, 1853) lischkeana livens (E.A. Bielz, 1853) glorifica livens (E.A. Bielz, 1853) fussiana fussiana (E.A. Bielz, 1852) lischkeana sarkanyi Szekeres, 2007 lischkeana sarkanyi Szekeres, 2007 n. m. n. m. n. m.
CLADE C1 regalis deubeli (Clessin, 1890) plumbea deubeli (Clessin, 1890) plumbea deubeli (Clessin, 1890) plumbea deubeli (Clessin, 1890) regalis deubeli (Clessin, 1890) regalis doftanae Nordsieck, 1977 plumbea doftanae Nordsieck, 1977 plumbea doftanae Nordsieck, 1977 n. m. n. m. regalis glabriuscula (Rossmässler, 1859) plumbea glabriuscula (Rossmässler, 1859) plumbea glabriuscula (Rossmässler, 1859) plumbea glabriuscula (Rossmässler, 1859) regalis adventicia (M. Kimakowicz, 1894) regalis microstoma (M. Kimakowicz, 1883) petrensis Nordsieck, 1995 subcosticollis microstoma (M. Kimakowicz, 1883) occidentalis microstoma (M. Kimakowicz, 1883) occidentalis microstoma (M. Kimakowicz, 1883) regalis mutabilis (M. Kimakowicz, 1894) plumbea mutabilis (M. Kimakowicz, 1894) plumbea mutabilis (M. Kimakowicz, 1894) plumbea mutabilis (M. Kimakowicz, 1894) regalis mutabilis (M. Kimakowicz, 1894) regalis nordsiecki Grossu & Tesio, 1973 subcosticollis nordsiecki Grossu & Tesio, 1973 subcosticollis nordsiecki Grossu & Tesio, 1973 occidentalis nordsiecki Grossu & Tesio, 1973 n. m. regalis petrensis Nordsieck, 1995 petrensis Nordsieck, 1995 subcosticollis subcosticollis (A. Schmidt, 1868) occidentalis occidentalis (O. Boettger, 1879) occidentalis occidentalis (O. Boettger, 1879) regalis proclivis (M. Kimakowicz, 1894) plumbea proclivis (M. Kimakowicz, 1894) plumbea proclivis (M. Kimakowicz, 1894) plumbea proclivis (M. Kimakowicz, 1894) regalis proclivis (M. Kimakowicz, 1894) regalis regalis (M. Bielz, 1851) plumbea regalis (M. Bielz, 1851) plumbea regalis (M. Bielz, 1851) plumbea regalis (M. Bielz, 1851) regalis regalis (M. Bielz, 1851) regalis wagneri (M. Kimakowicz, 1894) plumbea wagneri (M. Kimakowicz, 1894) plumbea wagneri (M. Kimakowicz, 1894) plumbea wagneri (M. Kimakowicz, 1894) regalis wagneri (M. Kimakowicz, 1894) CLADE C2 mafteiana mafteiana Grossu, 1867 glorifica mafteiana Grossu, 1867 glorifica mafteiana Grossu, 1867 glorifica mafteiana Grossu, 1867 n. m. mafteiana valeriae Szekeres, 2007 glorifica valeriae Szekeres, 2007 n. m. n. m. n. m. monacha (M. Kimakowicz, 1894) straminicollis monacha (M. Kimakowicz, 1894) straminicollis monacha (M. Kimakowicz, 1894) livida monacha (M. Kimakowicz, 1894) n. m. subcosticollis vicina (M. Kimakowicz, 1894) subcosticollis vicina (M. Kimakowicz, 1894) occidentalis vicina (M. Kimakowicz, 1894) n. m. straminicollis (Charpentier, 1852) straminicollis straminicollis (Charpentier, 1852) straminicollis straminicollis (Charpentier, 1852) livida connectens Soós, 1928 livida connectens Soós, 1928
CLADE D1 alpina R. Kimakowicz, 1933 glorifica alpina R. Kimakowicz, 1933 glorifica alpina R. Kimakowicz, 1933 livida alpina R. Kimakowicz, 1933 livida alpina R. Kimakowicz, 1933 canescens canescens (Charpentier, 1852) canescens canescens (Charpentier, 1852) canescens canescens (Charpentier, 1852) canescens canescens (Charpentier, 1852) lactea lactea (E.A. Bielz, 1853) canescens haueri (E.A. Bielz, 1859) canescens haueri (E.A. Bielz, 1859) canescens haueri (E.A. Bielz, 1859) canescens haueri (E.A. Bielz, 1859) lactea haueri (E.A. Bielz, 1859) canescens striaticollis (M. Kimakowicz, 1894) canescens striaticollis (M. Kimakowicz, 1894) canescens striaticollis (M. Kimakowicz, 1894) canescens striaticollis (M. Kimakowicz, 1894) lactea striaticollis (M. Kimakowicz, 1894) livida bipalatalis (M. Kimakowicz, 1883) livida sororcula Soós, 1928 livida bipalatalis (M. Kimakowicz, 1883) livida bipalatalis (M. Kimakowicz, 1883) livida bipalatalis (M. Kimakowicz, 1883) livida deaniana Cooke, 1922 livida deaniana Cooke, 1922 livida deaniana Cooke, 1922 glorifica maxima (A. Schmidt, 1856) livida maxima (A. Schmidt, 1856) livida julii A.J. Wagner, 1914 livida julii A.J. Wagner, 1914 livida julii A.J. Wagner, 1914 julii A.J. Wagner, 1914 livida livida (Menke, 1828) livida livida (Menke, 1828) livida livida (Menke, 1828) livida livida (Menke, 1828) livida livida (Menke, 1828) livida livida (Menke, 1828) nefasta helenae R. Kimakowicz, 1928 helenae helenae R. Kimakowicz, 1928 helenae helenae R. Kimakowicz, 1928 valachiensis helenae R. Kimakowicz, 1928 n. m. nefasta mauritii R. Kimakowicz, 1928 canescens mauritii R. Kimakowicz, 1928 canescens mauritii R. Kimakowicz, 1928 canescens mauritii R. Kimakowicz, 1928 lactea mauritii R. Kimakowicz, 1928 nefasta nefasta (M. Kimakowicz, 1894) nefasta (M. Kimakowicz, 1894) nefasta (M. Kimakowicz, 1894) canescens nefasta (M. Kimakowicz, 1894) lactea nefasta (M. Kimakowicz, 1894) nefasta zagani Szekeres, 1969 zagani Szekeres, 1969 valachiensis zagani Szekeres, 1969 valachiensis zagani Szekeres, 1969 n. m. nixa fussi (M. Kimakowicz, 1894) fussi (M. Kimakowicz, 1894) fussi (M. Kimakowicz, 1894) livida livida (M. Kimakowicz, 1828) livida minima (A. Schmidt, 1868) nixa nixa (M. Kimakowicz, 1894) nixa (M. Kimakowicz, 1894) nixa (M. Kimakowicz, 1894) nixa (M. Kimakowicz, 1894) nixa (M. Kimakowicz, 1894) CLADE D2 glorifica deceptans Deli & Szekeres, 2011 n. m. n. m. n. m. n. m. glorifica elegantissima Nordsieck, 1977 glorifica elegantissima Nordsieck, 1977 glorifica elegantissima Nordsieck, 1977 glorifica elegans (E.A. Bielz, 1852) n. m. glorifica glorifica (Charpentier, 1852) glorifica glorifica (Charpentier, 1852) glorifica glorifica (Charpentier, 1852) glorifica glorifica (Charpentier, 1852) fussiana fussiana (E.A. Bielz, 1852) glorifica intercedens (A. Schmidt, 1857) glorifica intercedens (A. Schmidt, 1857) glorifica intercedens (A. Schmidt, 1857) glorifica intercedens (A. Schmidt, 1857) n. m. glorifica magnifica R. Kimakowicz, 1962 glorifica magnifica R. Kimakowicz, 1962 glorifica magnifica R. Kimakowicz, 1962 glorifica magnifica R. Kimakowicz, 1962 n. m. glorifica subita (M. Kimakowicz, 1894) glorifica subita (M. Kimakowicz, 1894) glorifica glorifica (Charpentier, 1852) glorifica subita (M. Kimakowicz, 1894) fussiana subita (M. Kimakowicz, 1894) glorifica vranceana Grossu, 1967 vranceana Grossu, 1967 vranceana Grossu, 1967 valachiensis vranceana Grossu, 1967 n. m. CLADE D3 mariae coronata R. Kimakowicz, 1943 subcosticollis vicina (M. Kimakowicz, 1894) subcosticollis coronata R. Kimakowicz, 1943 occidentalis coronata R. Kimakowicz, 1943 n. m. mariae hildegardae R. Kimakowicz, 1931 hildegardae hildegardae R. Kimakowicz, 1931 hildegardae hildegardae R. Kimakowicz, 1931 occidentalis hildegardae R. Kimakowicz, 1931 n. m. mariae mariae R. Kimakowicz, 1931 subcosticollis mariae R. Kimakowicz, 1931 subcosticollis mariae R. Kimakowicz, 1931 occidentalis mariae R. Kimakowicz, 1931 n. m. mariae soosi Brandt, 1961 hildegardae soosi Brandt, 1961 hildegardae soosi Brandt, 1961 occidentalis soosi Brandt, 1961 n. m.
CLADE E meschendorferi (E.A. Bielz, 1858) meschendorferi (E.A. Bielz, 1858) meschendorferi (E.A. Bielz, 1858) meschendorferi (E.A. Bielz, 1858) meschendorferi (E.A. Bielz, 1858)
CLADE F1 glauca (E.A. Bielz, 1853) glauca (E.A. Bielz, 1853) glauca (E.A. Bielz, 1853) glauca (E.A. Bielz, 1853) glauca (E.A. Bielz, 1853) CLADE F2 maciana Bădărău & Szekeres, 2001 maciana Bădărău & Szekeres, 2001 n. m. n. m. n. m. pomatias (L. Pfeiffer, 1865) pomatias (L. Pfeiffer, 1865) cyclostoma (E.A. Bielz, 1858) cyclostoma (E.A. Bielz, 1858) n. m. soosiana Agócsy & Pócs, 1961 soosiana Agócsy & Pócs, 1961 soosiana Agócsy & Pócs, 1961 n. m. __________________________________________________________________________________________________________________________________________________________________________________________________________ n. m.: not mentioned __________________________________________________________________________________________________________________________________________________________________________________________________________
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