Corresponding author: Thierry Backeljau (
Academic editor: M. de Meyer
The Iberian Peninsula is a region with a high endemicity of species of the terrestrial slug subgenus
The genus
As illustrated by
Because DNA sequence data do not only provide phylogenetic information, but can also serve as DNA barcodes for species identification (
Information on the species and specimens included here is provided in
List of specimens used in this study with specimen ID, sampling locality, GenBank accession numbers for the COI, 16S and ITS1 sequences, and collection number at the museums (if available). Neo-, para- and topotypes have been indicated. Specimen codes with an asterisk are data taken from
Species/ID | Locality, country | COI | 16S | ITS1 | Collection number |
---|---|---|---|---|---|
|
|||||
ang-SU2777 | Torralba del Rio, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
Osma, Álava, Spain |
|
|
|
RBINS Brussels, I.G. 32471 | |
AANG 73A* | Burgos, Spain | NA | NA |
|
|
bae-556 (paratype) | Malaga, Spain |
|
|
|
MNCN Madrid 15.05/6969 |
fla-130 | Glasgow, UK |
|
|
|
RBINS Brussels, I.G. 32471 |
fla-161 | Glasgow, UK |
|
|
|
RBINS Brussels, I.G. 32471 |
fla-SU672 | Salamir, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
AFLA 44A* | Croydon, UK | NA | NA |
|
|
AFUL 43A* | São Silvestre, Portugal | NA | NA |
|
|
fus-SU155 | Grudki, Poland |
|
|
|
RBINS Brussels, I.G. 32471 |
fus-2320 | Predel, Bulgaria |
|
|
|
RBINS Brussels, I.G. 32471 |
fus-SU1335 | Steinegg, Austria |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Kreuzen, Austria | NA |
|
NA | RBINS Brussels, I.G. 32471 |
|
Serra de Pandóls, Valencia, Spain | NA | NA |
|
RBINS Brussels, I.G. 32471 |
|
Serra de Pandóls, Valencia, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Serra de Pandóls, Valencia, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
AGIL 49A* | Serra de Pandóls, Valencia, Spain | NA | NA |
|
|
AHIS 52B* | Cáceres, Spain | NA | NA |
|
|
ira-559 (paratype) | Navarra, Spain |
|
|
|
MNCN Madrid, 15.05/18705 |
liz-562 (paratype) | Navarra, Spain |
|
|
|
MNCN Madrid, 15.05/18706 |
ALIZ 47C* | Lizarrusti, Spain | NA | NA |
|
|
|
Feitos, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Currais, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Cacia, Portugal |
|
NA | NA | RBINS Brussels, I.G. 32471 |
|
Cacia, Portugal |
|
NA | NA | RBINS Brussels, I.G. 32471 |
|
Forjães, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Currais, Portugal | NA |
|
NA | RBINS Brussels, I.G. 32471 |
|
Forjães, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
lus-79 | Ursel, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Terceira, Azores, Portugal | NA | NA |
|
RBINS Brussels, I.G. 32471 |
lus-186 | Namur, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
lus-465 | Görlitz, Germany | NA | NA |
|
RBINS Brussels, I.G. 32471 |
|
Emptinne, Belgium |
|
|
NA | RBINS Brussels, I.G. 32471 |
ALUS 42A* | Serra de Arrábida, Portugal | NA | NA |
|
|
ALUS 42B* | Serra de Arrábida, Portugal | NA | NA |
|
|
ALUS 42C* | Serra de Arrábida, Portugal | NA | NA |
|
|
ALUS 42G* | Alpi Carniche, Rivolato, Italy | NA | NA |
|
|
ALUS 62E* | Montagne Noire, France | NA | NA |
|
|
ALUS 70C* | Girona, Spain | NA | NA |
|
|
mol-565 (paratype) | La Molina, Spain |
|
|
|
MNCN Madrid, 15.05/18707 |
AMOL 48A* | Serra del Cadí, Barcelona, Spain | NA | NA |
|
|
ANOB 41A* | Luso, Portugal | NA | NA |
|
|
ANOB 41B* | Luso, Portugal | NA | NA |
|
|
pau-121 | Sierra de Guadarrama, Spain |
|
NA |
|
RBINS Brussels, I.G. 32471 |
pau-224 | Sierra de Guadarrama, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
pau-226 | Sierra de Guadarrama, Spain | NA | NA |
|
RBINS Brussels, I.G. 32471 |
APAU 51A* | Sierra de Guadarrama, Spain | NA | NA |
|
|
|
Ciutadella de Menorca, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Ferreries, Menorca, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
pon-1962 | Ciutadella de Menorca, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Ciutadella de Menorca, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Kortrijk, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
sub1-2318 | Ingrandes, France |
|
|
|
RBINS Brussels, I.G. 32471 |
sub1-2317 | Burnopfield, UK |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Barnstable, MA, USA | NA |
|
NA | RBINS Brussels, I.G. 32471 |
|
Wilrijk, Belgium |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Wilrijk, Belgium |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Heppenbach, Belgium | NA |
|
NA | RBINS Brussels, I.G. 32471 |
|
Grootrees, Belgium | NA |
|
NA | RBINS Brussels, I.G. 32471 |
|
Gomzé, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Le Landin, France |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Heppenbach, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Bucholz, Germany |
|
|
|
RBINS Brussels, I.G. 32471 |
|
La Salle, France |
|
|
|
RBINS Brussels, I.G. 32471 |
|
La Salle, France | NA |
|
NA | RBINS Brussels, I.G. 32471 |
sub4-123 (topotype) | Montagne Noire, France |
|
|
|
RBINS Brussels, I.G. 32471 |
sub4-568 (neotype) | Montagne Noire, France |
|
|
|
MNCN Madrid, 15.05/18704 |
sub4-2341 | Oulès, France |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Col de Peyresourde, France | NA |
|
NA | RBINS Brussels, I.G. 32471 |
|
Villemont-Baubiat, France |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Villemont-Baubiat, France |
|
|
|
RBINS Brussels, I.G. 32471 |
ASUB 45A* | Montagne Noire, France | NA | NA |
|
|
tra-SU1088 | Covasna, Romania |
|
|
|
RBINS Brussels, I.G. 30412 |
tra-SU1203 | Lunca Vişagului, Romania |
|
|
|
RBINS Brussels, I.G. 30412 |
tra-SU1296 | Holda, Romania |
|
|
|
RBINS Brussels, I.G. 30412 |
urb-SU2755 | Saldaña, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Sierra da Urbia, Spain | NA | NA |
|
RBINS Brussels, I.G. 32471 |
AURB 50A* | NA | NA |
|
||
|
|||||
dis-106 | Mortsel, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
dis-14 |
|
|
|
RBINS Brussels, I.G. 32471 | |
hor-102 | Mortsel, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
hor-220 | London, UK |
|
|
|
RBINS Brussels, I.G. 32471 |
int-104 | Rochefort, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
int-52 | Flores, Azores, Portugal |
|
|
|
RBINS Brussels, I.G. 32471 |
alp-1610 | Žďárské Vrchy, Czech Republic |
|
|
NA | RBINS Brussels, I.G. 32471 |
alp-208 | Saxony, Germany |
|
|
|
RBINS Brussels, I.G. 32471 |
owe-310 | Devon, UK |
|
|
|
RBINS Brussels, I.G. 32471 |
owe-316 | Devon, UK |
|
|
|
RBINS Brussels, I.G. 32471 |
wik-SU2693 | Viniegra de Abajo, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
wik-44 | Burgos, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Burgos, Spain | NA |
|
|
RBINS Brussels, I.G. 32471 |
AWIK 58A* | Demanda Sierra, Burgos, Spain | NA | NA |
|
|
AWIK 58C* | Urbión Mountains, Soria, Spain | NA | NA |
|
|
|
|||||
cir-151 | Aran Island, Kilmurvey, Ireland |
|
|
|
RBINS Brussels, I.G. 32471 |
fas-144 | Görlitz, Germany |
|
|
|
RBINS Brussels, I.G. 32471 |
sil-142 | Poulseur, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
|
|||||
ate-SU517 | Musland, Norway |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Manteigas, Portugal |
|
NA |
|
RBINS Brussels, I.G. 32471 |
|
Santa Leocádia, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Gortmore, Ireland |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Oleirinhos, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Portulezo, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
Manteigas, Portugal |
|
|
NA | RBINS Brussels, I.G. 32471 |
|
St.-Katelijne Waver, Belgium |
|
|
NA | RBINS Brussels, I.G. 32471 |
ruf-15 | Santiago de Compostela, Spain |
|
|
|
RBINS Brussels, I.G. 32471 |
ruf-155 | Brussels, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
ruf-180 | Hoboken, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
ruf-182 | Brecht, Belgium |
|
|
|
RBINS Brussels, I.G. 32471 |
|
Nazareth, Belgium | NA | NA |
|
RBINS Brussels, I.G. 32471 |
AATE 39A* | Caldas de Gerês, Portugal | NA | NA |
|
|
AATE 39E* | Valporquero Cave, Leon, Spain | NA | NA |
|
|
ARUF 40G* | Montagne Noire, France | NA | NA |
|
Sequences were aligned in ClustalW (
The alignments comprized 504 bp for COI (196 variable sites), 408 bp for 16S (121 sites with alignment gaps, 122 variable sites) and 587 bp for ITS1 (277 sites with alignment gaps, 64 variable sites). For the concatenated dataset, there was strong support for the subgenera
Neighbour-Joining tree (Kimura 2-parameter model) of a 1499 bp concatenated fragment (504 bp of the mitochondrial cytochrome
The four individuals of
As for 16S, the concatenated tree of the three gene fragments showed a sister species relationship between
The three
The NJ-tree of the concatenated dataset confirms the major outcomes of previous phylogenetic studies, viz. 1) a strong support for the monophyly of the subgenus
Because
Obviously, the strong COI differentiation among
Because DNA barcoding on its own may be unreliable for identifying and detecting species-level taxa in stylommatophorans, it its necessary to backup this sort of data with, amongst others, phylogenetic analyses. As such, our phylogenetic trees of the DNA sequence data show that the morphospecies
In conclusion, the present work shows that
This work was supported by BELSPO Action 1 project MO/36/017 and FWO Research Network W0.009.11N “Belgian Network for DNA Barcoding”. We are indebted to Dr. Ramón Martín (Bilbao) for providing us with specimens of
Neighbour-Joining tree (Kimura 2-parameter model) of a 504 bp fragment of the mitochondrial cytochrome
Neighbour-Joining tree (Kimura 2-parameter model) of a 408 bp fragment of the mitochondrial 16S rDNA gene for the land slug subgenus
Neighbour-Joining tree (Kimura 2-parameter model) of a 587 bp fragment of the nuclear internal transcribed spacer 1 (ITS1) region for the land slug subgenus
Neighbour-Joining tree (Kimura 2-parameter model) of a 378 bp fragment of the nuclear internal transcribed spacer 1 (ITS1) region for the land slug subgenus