ZooKeys 365: 83–104, doi: 10.3897/zookeys.365.6104
DNA barcodes and phylogenetic affinities of the terrestrial slugs Arion gilvus and A. ponsi (Gastropoda,  Pulmonata, Arionidae)
Karin Breugelmans 1, Kurt Jordaens 2,3, Els Adriaens 4, Jean Paul Remon 4, Josep Quintana Cardona 5, Thierry Backeljau 1,3
1 Royal Belgian Institute of Natural Sciences, OD Taxonomy and Phylogeny (JEMU), Vautierstraat 29, B-1000 Brussels, Belgium
2 Royal Museum for Central Africa (JEMU), Leuvensesteenweg 13, B-3080 Tervuren, Belgium
3 Evolutionary Ecology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
4 Laboratory of Pharmaceutical Technology, University of Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium
5 Institut Catala de Paleontologia Miquel Crusafont, Universitat Autònomo de Barcelona, edifici ICP Campus de la UAB, sln 08193 Cerdanyola del Vallès, Barcelona, Spain

Corresponding author: Thierry Backeljau (thierry.backeljau@naturalsciences.be)

Academic editor: M. de Meyer

received 14 August 2013 | accepted 6 December 2013 | Published 30 December 2013

(C) 2013 Karin Breugelmans. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

For reference, use of the paginated PDF or printed version of this article is recommended.

Citation: Breugelmans K, Jordaens K, Adriaens E, Remon JP, Cardona JQ, Backeljau T (2013) DNA barcodes and phylogenetic affinities of the terrestrial slugs Arion gilvus and A. ponsi (Gastropoda, Pulmonata, Arionidae). In: Nagy ZT, Backeljau T, De Meyer M, Jordaens K (Eds) DNA barcoding: a practical tool for fundamental and applied biodiversity research. ZooKeys 365: 83–104. doi: 10.3897/zookeys.365.6104


The Iberian Peninsula is a region with a high endemicity of species of the terrestrial slug subgenus Mesarion. Many of these species have been described mainly on subtle differences in their proximal genitalia. It therefore remains to be investigated 1) whether these locally diverged taxa also represent different species under a phylogenetic species concept as has been shown for other Mesarion species outside the Iberian Peninsula, and 2) how these taxa are phylogenetically related. Here, we analysed DNA sequence data of two mitochondrial (COI and 16S) genes, and of the nuclear ITS1 region, to explore the phylogenetic affinities of two of these endemic taxa, viz. Arion gilvus Torres Mínguez, 1925 and A. ponsi Quintana Cardona, 2007. We also evaluated the use of these DNA sequence data as DNA barcodes for both species. Our results showed that ITS did not allow to differentiate among most of the Mesarion molecular operational taxonomic units (MOTUs) / morphospecies in Mesarion. Yet, the overall mean p-distance among the Mesarion MOTUs / morphospecies for both mtDNA fragments (16.7% for COI, 13% for 16S) was comparable to that between A. ponsi and its closest relative A. molinae (COI: 14.2%; 16S: 16.2%) and to that between A. gilvus and its closest relative A. urbiae (COI: 14.4%; 16S: 13.4%). Hence, with respect to mtDNA divergence, both A. ponsi and A. gilvus, behave as other Mesarion species or putative species-level MOTUs and thus are confirmed as distinct ‘species’.


DNA barcoding, terrestrial slugs, Gastropoda, taxonomy, Iberian Peninsula, Arion ponsi, Arion gilvus


The genus Arion Férussac, 1819 is the most species rich genus of the terrestrial slug family Arionidae (Mollusca, Pulmonata, Gastropoda). It comprizes approximately 40 species, grouped into four subgenera, viz. Arion s.s. Férussac, 1819, Kobeltia Seibert, 1873, Carinarion Hesse, 1926 and Mesarion Hesse, 1926. Species of the subgenus Mesarion (type species: Limax subfuscus Draparnaud, 1805) are characterized by 1) a medium body-size (up to 75 mm when extended), 2) an orange to dark brown dorsum, 3) two dark bands on the sides of the mantle, 4) (usually) yellow to orange body mucus, and 5) an enlarged free-oviduct with a long and V-shaped ligula (Kerney et al. 1983). Many Mesarion species are highly polymorphic with respect to body colour and genital anatomy. As a consequence, the species limits and phylogenetic relationships of taxa within this subgenus have been debated for decades (e.g. Garrido et al. 1995, Castillejo 1997, 1998, Pinceel et al. 2004, 2005a, b, Quinteiro et al. 2005). Arion subfuscus (Draparnaud, 1805) (type locality: Montagne Noire, France) is probably the most problematic “species” within Mesarion as it shows an overwhelming amount of variation in body pigmentation, genital anatomy, and reproductive behavior [see Garrido et al. (1995) and the references listed in their table 1]. This variation often has been interpreted as indicating reproductive isolation between geographically isolated populations, and Arion subfuscus thus is considered a species complex (Wiktor 1973, Waldén 1976, De Winter 1986, Backeljau 1989, Altonaga et al. 1994, Backeljau et al. 1994, Garrido et al. 1995). Especially in the Pyrenees and the coastal regions of Spain there are local, morphologically diverged populations (e.g. Garrido et al. 1995, Castillejo 1998). Several of these have been described as endemic species on the basis of where the epiphallus, oviduct and pedunculus of the bursa copulatrix open into the atrium, in combination with differences in the relative lengths of the vas deferens and the epiphallus (e.g. Castillejo 1998, Garrido et al. 1995, Quintana Cardona 2007). Two of these endemic taxa occur in the eastern coastal region of Spain or the Balearic Islands, viz. Arion gilvus Torres Mínguez, 1925 and Arion ponsi Quintana Cardona, 2007.

Arion ponsi (Figure 1) was described from Menorca (Balearic Islands, type locality: Barranc d’Algendar). The species has a medium body size (range: 54–66 mm), an orange to beige dorsal body colour with dark lateral bands that can be blurry in the posterior parts, a foot sole that is cream coloured with a greyish hue, and a transparent body mucus (Quintana Cardona 2007). Its genital anatomy is very similar to that of Arion gilvus, Arion iratii Garrido, Castillejo & Iglesias, 1995, Arion molinae Garrido, Castillejo & Iglesias, 1995 and Arion lizarrustii Garrido, Castillejo & Iglesias, 1995, but its epiphallus is shorter than the vas deferens (as in Arion molinae) and opens into the genital atrium in between the oviduct and the pedunculus of the bursa copulatrix (unlike in Arion molinae, where the pedunculus is positioned in between the epiphallus and oviduct) (figures 3–5 in Quintana Cardona 2007).

Figure 1.

Arion ponsi Quintana Cardona, 2007 from Menorca (Balearic Islands, Spain).

Arion gilvus (Figure 2) was described from ‘Mandol’ in the Spanish Province of Tarragona. However, the toponym ‘Mandol’ seems to be erroneous (e.g. Bech 1990) and therefore Castillejo (1990) assigned eight specimens with an Arion gilvus morphology from Serra de Pandóls near Gandesa (Province of Tarragona) as topotypes [see also Castillejo and Rodríguez (1991)]. Subsequently, Arion gilvus was redescribed by Garrido (1992). Afterwards, the species has also been found in the Provinces of Valencia, Teruel and Albacete [Borredà (1994), figure 15 in Castillejo (1997), figure 1 in Quinteiro et al. (2005)]. Arion gilvus reaches a length of up to 65 mm when extended. It has a yellowish to brown dorsum that gets lighter downwards at the sides and dark lateral bands that have a yellowish grey line on their upper side (Figure 1). The sole is white or evenly yellowish and the mucus is pale yellow (Torres Mínguez 1925, Bech 1990, Garrido 1992, Castillejo 1997). The epiphallus, the pedunculus of the bursa copulatrix, and the free oviduct join the atrium on a single line with the pedunculus of the bursa copulatrix in the middle, as in Arion molinae, but in contrast to the latter, the epiphallus is longer than the vas deferens (Torres Mínguez 1925, Borredà 1994, Castillejo 1997, and figures 26–28 in Garrido et al. 1995).

Figure 2.

Arion gilvus Torres Mínguez, 1925 from Serra de Pandóls (Valencia, Spain). A dorsal view B lateral view C ventral view.

As illustrated by Arion ponsi and Arion gilvus, the alleged species-specific genital differences among the Iberian species of the Arion subfuscus complex are very subtle and little is known about their intraspecific variation. Moreover, genital differences among arionid taxa do not necessarily imply reproductive isolation (Dreijers et al. 2013). Hence, if alleged species-specific phenotypic differences in arionids are to be interpreted under a phylogenetic species concept, then their correlation with reproductive isolation should be corroborated by molecular data. Molecular markers have been very effective in this respect (e.g. Pinceel et al. 2005a, b, Quinteiro et al. 2005, Geenen et al. 2006, Jordaens et al. 2010). As such, Quinteiro et al. (2005) investigated the taxonomic affinities of Iberian Mesarion species using DNA sequence data. Their analysis of the nuclear ribosomal internal transcribed spacer 1 region (ITS1) showed a polytomy of Mesarion species, yet, the analysis of the mitochondrial NADH dehydrogenase I (ND1) gene suggested a strongly bootstrap supported group of Iberian Mesarion species with a continental-Mediterranean distribution (Arion paularensis, Arion baeticus, Arion urbiae, Arion anguloi, Arion wiktori, and Arion gilvus), and an unsupported group of species with an Atlantic distribution (Arion lusitanicus, Arion nobrei, Arion fuligineus, Arion hispanicus and Arion flagellus). In addition, the positions of three Pyrenean species (Arion lizarrustii, Arion iratii, Arion molinae) remained unresolved. More specifically, the ND1 data placed Arion gilvus as sister taxon of Arion urbiae and Arion anguloi. Quinteiro et al. (2005) did not study individuals from the Balearic Islands and thus probably did not include Arion ponsi.

Because DNA sequence data do not only provide phylogenetic information, but can also serve as DNA barcodes for species identification (Hebert et al. 2003, 2004), we here expand on the work of Quinteiro et al. (2005) by 1) characterizing Arion gilvus and Arion ponsi using mitochondrial COI and 16S rDNA gene fragments, and the larger part of the nuclear ITS1 region, 2) exploring the phylogenetic affinities of Arion gilvus and Arion ponsi within the subgenus Mesarion, and 3) providing diagnostic COI barcodes for both species.

Material and methods

Information on the species and specimens included here is provided in Table 1. In total, we screened 45 specimens (Table 1). DNA was extracted from small parts of the foot using a NucleoSpin Tissue Kit (Macherey-Nagel, Düren) following the manufacturer’s instructions. PCR reactions were done in 25 µl reaction volumes that contained 1.5 mM MgCl2 in 1 × PCR buffer (Qiagen), 0.2 mM of each dNTP, 0.2 µM of each primer and 0.5 units of Taq polymerase (Qiagen). A fragment of the mitochondrial COI and 16S genes was amplified using primer pairs LCO1490 and HCO2198 (Folmer et al. 1994) and 16Sar and 16Sbr (Palumbi 1996), respectively. The nuclear ITS1 region (except the ± first 30 bp) was amplified using the primer pair ITS1L and 58C (Hillis and Dixon 1991). The PCR profile was an initial denaturation step of 5 min at 95 °C, followed by 35 cycles of 45 s at 95 °C, 45 s at an annealing temperature of 40 °C (COI), 42 °C (16S) or 55 °C (ITS1) and 1.5 min at 72 °C, and ending with a final extension step of 5 min at 72 °C. PCR products were purified using the GFX PCR DNA Purification Kit (GE Healthcare) following the manufacturer’s instructions. Purified DNA was diluted in 15 µl of sterile water. PCR-products were bidirectionally sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and run on a ABI3130xl Genetic Analyzer. Sequences were assembled in SeqScape v2.5 (Life Technologies) and inconsistencies were checked by eye on the chromatogram. Sequences were submitted to GenBank under accession numbers KF305196KF305225 for COI, KF356212KF356245 for 16S and KF385449KF385469 for ITS1. These datasets were supplemented with DNA sequences from GenBank [including a few species of the other Arion subgenera (Table 1)]. We used those of Carinarion as outgroup.

Table 1.

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 Quinteiro et al. (2005); NA = not assessed. The specimen ID and GenBank accession numbers of newly sequenced specimens are given in bold.

Species/ID Locality, country COI 16S ITS1 Collection number
Subgenus Mesarion Hesse, 1926
Arion anguloi Martín and Gómez, 1988
ang-SU2777 Torralba del Rio, Spain AY987869 AY947348 AY947386 RBINS Brussels, I.G. 32471
ang-115 (topotype) Osma, Álava, Spain KF305196 KF356212 AJ509055 RBINS Brussels, I.G. 32471
AANG 73A* Burgos, Spain NA NA AY316291
Arion baeticus Garrido, Castillejo and Iglesias, 1995
bae-556 (paratype) Malaga, Spain AY987871 AY947350 AJ509054 MNCN Madrid 15.05/6969
Arion flagellus Colligne, 1893
fla-130 Glasgow, UK AY987880 AY947359 AJ509053 RBINS Brussels, I.G. 32471
fla-161 Glasgow, UK AY987881 AY947360 AJ509052 RBINS Brussels, I.G. 32471
fla-SU672 Salamir, Spain AY987882 AY947361 AY947388 RBINS Brussels, I.G. 32471
AFLA 44A* Croydon, UK NA NA AY316278
Arion fuligineus Morelet, 1845
AFUL 43A* São Silvestre, Portugal NA NA AY316277
Arion fuscus (Müller, 1774)
fus-SU155 Grudki, Poland AY987885 AJ786721 AY947390 RBINS Brussels, I.G. 32471
fus-2320 Predel, Bulgaria AY987886 AJ786722 AY947391 RBINS Brussels, I.G. 32471
fus-SU1335 Steinegg, Austria AY987887 AJ786726 AY947392 RBINS Brussels, I.G. 32471
fus-SU2188 Kreuzen, Austria NA KF356221 NA RBINS Brussels, I.G. 32471
Arion gilvus Torres Mínguez, 1925
gil-46 Serra de Pandóls, Valencia, Spain NA NA KF385450 RBINS Brussels, I.G. 32471
gil-47 Serra de Pandóls, Valencia, Spain KF305199 KF356222 KF385451 RBINS Brussels, I.G. 32471
gil-73 Serra de Pandóls, Valencia, Spain KF305200 KF356223 KF385452 RBINS Brussels, I.G. 32471
AGIL 49A* Serra de Pandóls, Valencia, Spain NA NA AY316282
Arion hispanicus Simroth, 1886
AHIS 52B* Cáceres, Spain NA NA AY316285
Arion iratii Garrido, Castillejo and Iglesias, 1995
ira-559 (paratype) Navarra, Spain AY987892 AY947367 AJ509042 MNCN Madrid, 15.05/18705
Arion lizarrustii Garrido, Castillejo and Iglesias, 1995
liz-562 (paratype) Navarra, Spain AY987893 AY947368 AJ509046 MNCN Madrid, 15.05/18706
ALIZ 47C* Lizarrusti, Spain NA NA AY316280
Arion lusitanicus Mabille, 1868
lus-1613 Feitos, Portugal KF305203 KF356224 NA RBINS Brussels, I.G. 32471
lus-1631 Currais, Portugal KF305204 KF356225 NA RBINS Brussels, I.G. 32471
lus-1641 Cacia, Portugal KF305205 NA NA RBINS Brussels, I.G. 32471
lus-1647 Cacia, Portugal KF305206 NA NA RBINS Brussels, I.G. 32471
lus-1652 Forjães, Portugal KF305207 KF356226 NA RBINS Brussels, I.G. 32471
lus-1654 Currais, Portugal NA KF356227 NA RBINS Brussels, I.G. 32471
lus-1655 Forjães, Portugal KF305208 KF356228 NA RBINS Brussels, I.G. 32471
lus-79 Ursel, Belgium AY987894 AY947369 AJ509062 RBINS Brussels, I.G. 32471
lus-181 Terceira, Azores, Portugal NA NA KF385453 RBINS Brussels, I.G. 32471
lus-186 Namur, Belgium AY987895 AY947370 AJ509061 RBINS Brussels, I.G. 32471
lus-465 Görlitz, Germany NA NA AJ509063 RBINS Brussels, I.G. 32471
lus-509 Emptinne, Belgium KF305209 KF356229 NA RBINS Brussels, I.G. 32471
ALUS 42A* Serra de Arrábida, Portugal NA NA AY316273
ALUS 42B* Serra de Arrábida, Portugal NA NA AY316274
ALUS 42C* Serra de Arrábida, Portugal NA NA AY316275
ALUS 42G* Alpi Carniche, Rivolato, Italy NA NA AY316276
ALUS 62E* Montagne Noire, France NA NA AY316289
ALUS 70C* Girona, Spain NA NA AY316290
Arion molinae Garrido, Castillejo and Iglesias, 1995
mol-565 (paratype) La Molina, Spain AY987896 AY947371 AJ509043 MNCN Madrid, 15.05/18707
AMOL 48A* Serra del Cadí, Barcelona, Spain NA NA AY316281
Arion nobrei Pollonera, 1889
ANOB 41A* Luso, Portugal NA NA AY316271
ANOB 41B* Luso, Portugal NA NA AY316272
Arion paularensis Wiktor and Parejo, 1989
pau-121 Sierra de Guadarrama, Spain KF305210 NA KF385454 RBINS Brussels, I.G. 32471
pau-224 Sierra de Guadarrama, Spain AY987899 AY947374 AJ509057 RBINS Brussels, I.G. 32471
pau-226 Sierra de Guadarrama, Spain NA NA KF385455 RBINS Brussels, I.G. 32471
APAU 51A* Sierra de Guadarrama, Spain NA NA AY316284
Arion ponsi Quintana Cardona, 2007
pon-1959 Ciutadella de Menorca, Spain KF305211 KF356230 KF385456 RBINS Brussels, I.G. 32471
pon-1960 Ferreries, Menorca, Spain KF305212 KF356231 KF385457 RBINS Brussels, I.G. 32471
pon-1962 Ciutadella de Menorca, Spain KF305213 KF356232 KF385458 RBINS Brussels, I.G. 32471
pon-1965 Ciutadella de Menorca, Spain KF305214 KF356233 KF385459 RBINS Brussels, I.G. 32471
Arion subfuscus (Draparnaud, 1805)
sub1-2312 Kortrijk, Belgium KF305215 KF356238 KF385461 RBINS Brussels, I.G. 32471
sub1-2318 Ingrandes, France AY987904 AY860678 AY860729 RBINS Brussels, I.G. 32471
sub1-2317 Burnopfield, UK AY987906 AY860672 AY860726 RBINS Brussels, I.G. 32471
sub1-SU87 Barnstable, MA, USA NA KF356235 NA RBINS Brussels, I.G. 32471
sub1-1618 Wilrijk, Belgium KF305216 KF356236 NA RBINS Brussels, I.G. 32471
sub1-1633 Wilrijk, Belgium KF305217 KF356237 NA RBINS Brussels, I.G. 32471
sub2-SU2424 Heppenbach, Belgium NA KF356239 NA RBINS Brussels, I.G. 32471
sub2-SU349 Grootrees, Belgium NA KF356240 NA RBINS Brussels, I.G. 32471
sub2-2309 Gomzé, Belgium KF305218 KF356241 KF385462 RBINS Brussels, I.G. 32471
sub2-2313 Le Landin, France AY987908 AY860687 KF385463 RBINS Brussels, I.G. 32471
sub2-2314 Heppenbach, Belgium AY987909 AY860709 KF385464 RBINS Brussels, I.G. 32471
sub3-2322 Bucholz, Germany AY987910 AY860716 KF385466 RBINS Brussels, I.G. 32471
sub3-2310 La Salle, France AY987911 AY860722 KF385465 RBINS Brussels, I.G. 32471
sub3-SU2401 La Salle, France NA KF356242 NA RBINS Brussels, I.G. 32471
sub4-123 (topotype) Montagne Noire, France AY987913 AY860682 AY860733 RBINS Brussels, I.G. 32471
sub4-568 (neotype) Montagne Noire, France AY987914 KF356244 AJ509044 MNCN Madrid, 15.05/18704
sub4-2341 Oulès, France AY987912 AY860685 AY860731 RBINS Brussels, I.G. 32471
sub4-SU1058 Col de Peyresourde, France NA KF356243 NA RBINS Brussels, I.G. 32471
sub5-2321 Villemont-Baubiat, France AY987915 AY860681 KF385468 RBINS Brussels, I.G. 32471
sub5-2311 Villemont-Baubiat, France AY987916 AY860679 KF385467 RBINS Brussels, I.G. 32471
ASUB 45A* Montagne Noire, France NA NA AY316279
Arion transsylvanus Simroth, 1885
tra-SU1088 Covasna, Romania AY943858 AY860798 AY947393 RBINS Brussels, I.G. 30412
tra-SU1203 Lunca Vişagului, Romania AY943859 AY860805 AY947394 RBINS Brussels, I.G. 30412
tra-SU1296 Holda, Romania AY943860 AY860799 AY947395 RBINS Brussels, I.G. 30412
Arion urbiae De Winter, 1986
urb-SU2755 Saldaña, Spain AY987919 AY947381 AY947396 RBINS Brussels, I.G. 32471
urb-99 Sierra da Urbia, Spain NA NA KF385469 RBINS Brussels, I.G. 32471
AURB 50A* NA NA AY316283
Subgenus Kobeltia Seibert, 1873
Arion distinctus Mabille, 1868
dis-106 Mortsel, Belgium AY987875 AY947354 AJ509040 RBINS Brussels, I.G. 32471
dis-14 AY987874 AY947353 AJ509038 RBINS Brussels, I.G. 32471
Arion hortensis Férussac, 1819
hor-102 Mortsel, Belgium AY987888 AJ518061 AJ509037 RBINS Brussels, I.G. 32471
hor-220 London, UK AY987889 AY947364 AJ509036 RBINS Brussels, I.G. 32471
Arion intermedius Normand, 1852
int-104 Rochefort, Belgium AY987891 AY947366 AJ509031 RBINS Brussels, I.G. 32471
int-52 Flores, Azores, Portugal AY987890 AY947365 AJ509029 RBINS Brussels, I.G. 32471
Arion obesoductus Reischütz, 1973
alp-1610 Žďárské Vrchy, Czech Republic DQ904249 DQ904248 NA RBINS Brussels, I.G. 32471
alp-208 Saxony, Germany AY987867 AY947346 AJ509041 RBINS Brussels, I.G. 32471
Arion owenii Davies, 1979
owe-310 Devon, UK AY987897 AY947372 AJ509033 RBINS Brussels, I.G. 32471
owe-316 Devon, UK AY987898 AY947373 AJ509034 RBINS Brussels, I.G. 32471
Arion wiktori Parejo & Martín, 1990
wik-SU2693 Viniegra de Abajo, Spain AY987921 AY947383 AY947397 RBINS Brussels, I.G. 32471
wik-44 Burgos, Spain AY987920 AY947382 AJ509060 RBINS Brussels, I.G. 32471
wik-94 Burgos, Spain NA KF356245 AJ509059 RBINS Brussels, I.G. 32471
AWIK 58A* Demanda Sierra, Burgos, Spain NA NA AY316287
AWIK 58C* Urbión Mountains, Soria, Spain NA NA AY316288
Subgenus Carinarion Hesse, 1926
Arion circumscriptus Johnston, 1828
cir-151 Aran Island, Kilmurvey, Ireland AY987872 AY947351 AJ509071 RBINS Brussels, I.G. 32471
Arion fasciatus (Nilsson, 1823)
fas-144 Görlitz, Germany AY987877 AY947356 AJ509068 RBINS Brussels, I.G. 32471
Arion silvaticus Lohmander, 1937
sil-142 Poulseur, Belgium AY987917 AY947379 AJ509070 RBINS Brussels, I.G. 32471
Subgenus Arion s.s. Férussac, 1819
Arion ater-rufus complex
ate-SU517 Musland, Norway AY987870 AY947349 AY947387 RBINS Brussels, I.G. 32471
ate/ruf-1602 Manteigas, Portugal KF305219 NA KF385449 RBINS Brussels, I.G. 32471
ate/ruf-1619 Santa Leocádia, Portugal KF305220 KF356213 NA RBINS Brussels, I.G. 32471
ate/ruf-1620 Gortmore, Ireland KF305221 KF356214 NA RBINS Brussels, I.G. 32471
ate/ruf-1624 Oleirinhos, Portugal KF305222 KF356215 NA RBINS Brussels, I.G. 32471
ate/ruf-1638 Portulezo, Portugal KF305223 KF356216 NA RBINS Brussels, I.G. 32471
ate/ruf-1649 Manteigas, Portugal KF305224 KF356217 NA RBINS Brussels, I.G. 32471
ruf-105 St.-Katelijne Waver, Belgium KF305225 KF356234 NA RBINS Brussels, I.G. 32471
ruf-15 Santiago de Compostela, Spain AY987900 AY947375 AJ509066 RBINS Brussels, I.G. 32471
ruf-155 Brussels, Belgium AY987901 AY947376 AJ509064 RBINS Brussels, I.G. 32471
ruf-180 Hoboken, Belgium AY987902 AY947377 AJ509065 RBINS Brussels, I.G. 32471
ruf-182 Brecht, Belgium AY987903 AY947378 AJ509067 RBINS Brussels, I.G. 32471
ruf-624 Nazareth, Belgium NA NA KF385460 RBINS Brussels, I.G. 32471
AATE 39A* Caldas de Gerês, Portugal NA NA AY316268
AATE 39E* Valporquero Cave, Leon, Spain NA NA AY316269
ARUF 40G* Montagne Noire, France NA NA AY316270

Sequences were aligned in ClustalW (Thompson et al. 1994) with default settings and without subsequent manual adjustments. In each alignment sequences were trimmed to equal length. The final alignments had a length of 504 bp (COI), 408 bp (16S) and 587 bp (ITS1), and of 1499 bp after concatenating the three fragments. The COI sequences were translated to amino acid sequences to check for stop codons (but none were found). The ITS1 sequences were also analysed together with those of Quinteiro et al. (2005). In this way we could extend our taxon coverage to Arion hispanicus Simroth, 1886, Arion fuligineus Morelet, 1845 and Arion nobrei Pollonera, 1889 (Table 1). Because Quinteiro et al. (2005) used other ITS1 primers, we had to trim this dataset to a length of 378 bp. For each gene fragment, and for the concatenated dataset, we constructed Neighbour-Joining (NJ) trees (Saitou and Nei 1987) using the Kimura 2-parameter (K2P) model in MEGA v5 (Tamura et al. 2011) with complete deletion of insertions and deletions (indels). Branch support was evaluated with 1000 bootstrap replicates (Felsenstein 1985). Only bootstrap values ≥ 70% were considered as indicating strong support (Hillis and Bull 1993). Uncorrected p-distances (hereafter simply referred to as p-distance) were calculated in MEGA v5 (Tamura et al. 2011). For these calculations we considered the following Molecular Operational Taxonomic Units (MOTUs): 1) the five 16S rDNA clades of Arion subfuscus (S1 to S5) defined by Pinceel et al. (2005a), 2) Arion anguloi and Arion urbiae jointly as a single MOTU (Backeljau et al. 1994, Quinteiro et al. 2005), 3) Arion wiktori and Arion paularensis jointly as a single MOTU (Backeljau et al. 1996, Quinteiro et al. (2005), and 4) Arion lusitanicus from Portugal vs. Arion lusitanicus from elsewhere as two different MOTUs (Davies 1987, Castillejo 1998, Quinteiro et al. 2005). Standard errors of mean p-distances among taxa and MOTUs were calculated on 1000 bootstrap replicates.


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 Carinarion, Kobeltia (excluding Arion wiktori) and Arion s.s., and for a clade of Arion s.s. + Mesarion (including Arion wiktori) (Figure 3). The subgenus Mesarion was not monophyletic but consisted of (1) a clade of Arion flagellus, Arion wiktori, Arion paularensis, Arion baeticus, Arion urbiae, Arion anguloi, and Arion gilvus, (2) two haplotypes of Arion lusitanicus (lus-79 and lus-186) that formed a sister group of Arion s.s. [insofar Arion lusitanicus is, of course, considered as a member of Mesarion; see e.g. Backeljau (1989)], and (3) a number of species/clades among which the relationships were mostly unresolved. Within Arion subfuscus (for which the monophyly was not supported) there were five clades (S1 to S5), with strong support for (S1, S5), S4) and (S2, S3). The mean p-distance (± SE) among the Mesarion OTUs (including Arion ponsi and Arion gilvus) was 0.168 ± 0.011 (range: 0.11–0.22) for COI, 0.134 ± 0.012 (range: 0.058–0.195) for 16S, and 0.022 ± 0.004 (range: 0.000–0.048) for ITS1 (a minimum distance of zero means that the two sequences only differed in a number of indels). The mean p-distances (± SE) excluding Arion ponsi and Arion gilvus were 0.167 ± 0.011 (range: 0.11–0.22) for COI, 0.130 ± 0.012 (range: 0.058–0.195) for 16S, and 0.023 ± 0.004 (range: 0.000–0.048) for ITS1. For the concatenated dataset these values were 0.108 ± 0.006 (range: 0.071–0.137) (including Arion ponsi and Arion gilvus) and 0.107 ± 0.006 (range: 0.071–0.137) (excluding Arion ponsi and Arion gilvus). The phylogenetic trees inferred from the three gene fragments and from the concatenated dataset are shown in Appendix, Supplementary figures 14 and Figure 3, respectively.

Figure 3.

Neighbour-Joining tree (Kimura 2-parameter model) of a 1499 bp concatenated fragment (504 bp of the mitochondrial cytochrome c oxidase subunit I (COI) gene, 408 bp of the mitochondrial 16S rDNA gene, 587 bp fragment of the nuclear internal transcribed spacer 1 (ITS1) region) for the land slug subgenus Mesarion. Bootstrap values ≥ 70% are shown at the nodes. For sample codes see Table 1.

Arion ponsi

The four individuals of Arion ponsi yielded four COI and three 16S haplotypes (Appendix, Supplementary figures 12), yet two 16S haplotypes only differed by an indel of two base pairs at positions 291–292. For both genes Arion molinae showed the smallest p-distance with Arion ponsi (COI: mean p-distance 0.142 ± 0.014; 16S: mean p-distance 0.162 ± 0.019), but a sister species relationship with Arion molinae was only well-supported by 16S. There were three ITS1 haplotypes for Arion ponsi; one of these had a deletion of a poly-T stretch of six base pairs at positions 556–561; the other two differed by a deletion of a G at position 554. These three ITS1 haplotypes of Arion ponsi clustered within a clade of Arion subfuscus S1–5, Arion lizarrustii, Arion molinae, Arion iratii and Arion transsylvanus (Appendix, Supplementary figure 3). The ITS1 analysis with the sequences of Quinteiro et al. (2005), placed the single remaining Arion ponsi haplotype in the same clade (mean p-distance with the other taxa of this clade = 0.046 ± 0.004), but without bootstrap support (Appendix, Supplementary figure 4).

As for 16S, the concatenated tree of the three gene fragments showed a sister species relationship between Arion ponsi and Arion molinae (Figure 3).

Arion gilvus

The three Arion gilvus specimens yielded two COI (one synonymous A-G substitution at position 366) and one 16S haplotypes. For both genes the smallest mean p-distances were observed relative to Arion urbiae and Arion anguloi (COI: mean p-distance = 0.145 ± 0.013; 16S: mean p-distance = 0.134 ± 0.016). The two Arion gilvus ITS1 haplotypes reduced to one when considering the stretch that overlapped with the Quinteiro et al. (2005) sequences. In this stretch it differed from that of Quinteiro et al. (2005) by a deletion of a T at position 349. Separately, none of the three genes provided reliable evidence about the sister group relationships of Arion gilvus (Appendix, Supplementary figures 14). Yet, the concatenated tree showed a well-supported sister species relationship between Arion gilvus and the Arion urbiae / Arion anguloi clade (mean p-distance = 0.021 ± 0.003) (Figure 3). Mean p-distances within this Arion urbiae / Arion anguloi clade (in which Arion anguloi was paraphyletic) were p = 0.041 ± 0.006 for COI, p = 0.023 ± 0.006 for 16S, p = 0.004 ± 0.002 for ITS1 and p = 0.020 ± 0.003 for the concatenated dataset.


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 Carinarion (Geenen et al. 2006), 2) a clade of Arion s.s. and non-Portuguese Arion lusitanicus (Quinteiro et al. 2005), 3) Arion wiktori clustering with Mesarion species, in particular with Arion paularensis (Quinteiro et al. 2005) instead of with Kobeltia species (Castillejo 1998), and 4) the strong differentiation within Arion subfuscus s.s. that consists of, at least, five phylogenetic species (Pinceel et al. 2005a). It therefore seems that the analysis of COI, 16S and ITS1 DNA sequences yields relevant taxonomic information with respect to the characterisation of arionid species that have been described under the morphospecies concept.

Because Arion gilvus and Arion ponsi were originally described on morphological grounds they are to be interpreted as morphospecies. This phenetic morphological distinction, however, correlates well with a phenetic separation based on mtDNA distances. Indeed, the overall mean p-distance among the Mesarion MOTUs (excluding Arion ponsi and Arion gilvus) dealt with in this study is 16.7% for COI and 13% for 16S. As such, the mean p-distances between Arion ponsi and Arion molinae (COI: 14.2%; 16S: 16.2%) or between Arion gilvus and Arion urbiae (COI: 14.5%; 16S: 13.4%) are perfectly comparable with the mean p-distances among the other MOTUs and morphospecies in Mesarion. Hence, with respect to mtDNA divergence, both Arion ponsi and Arion gilvus, behave as other Mesarion species or putative species-level MOTUs.

Obviously, the strong COI differentiation among Mesarion taxa, and of Arion ponsi and Arion gilvus in particular, suggests that DNA barcoding may be a suitable identification tool for these animals. Yet, this may be a too simplistic conclusion, since stylommatophorans may show extremely high intraspecific mtDNA divergences of sometimes up to 27% (K2P-distances, but note the uncorrected p-distances are almost similar) (Thomaz et al. 1996, Chiba 1999). In addition, Davison et al. (2009) showed that in the Stylommatophora the mean interspecific K2P-distances (± 3%) can be substantially lower than the mean intraspecific K2P-distances (± 12%). Under these conditions, it becomes very difficult to define generally applicable thresholds that distinguish between intra- and interspecific sequence divergences. Such thresholds are normally associated with DNA barcoding gaps (Hebert et al. 2003), but Davison et al. (2009) were unable to detect DNA barcoding gaps in the taxa they studied. Nevertheless, Davison et al. (2009) suggested a pragmatic 4% threshold to separate intra- and interspecific values, but at the same time they also concluded that DNA barcoding in itself is insufficient to identify and/or detect stylommatophoran species. Unfortunately, our sample sizes were too small to explore eventual DNA barcoding gaps in Mesarion.

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 Arion ponsi and Arion gilvus, also represent phylogenetic species, since both form well-supported clades that are “significantly” associated with well-defined, but morphologically different sister species. For Arion ponsi, the sister species appears to be Arion molinae, the distribution range of which is located in NE continental Spain (Castillejo 1997), i.e. north of, and facing, the Balearic Islands. Conversely, the sister taxon of Arion gilvus is the “tandem” of Arion urbiae and Arion anguloi, two species that have been synonymized by Backeljau et al. (1994) and that jointly should be referred to as Arion urbiae. Our DNA sequence data on COI, 16S and ITS1 (e.g. Figure 3), as well as those on ND1 and ITS1 of Quinteiro et al. (2005) are in line with this. As such, the distribution range of Arion urbiae is situated northwest of, and probably adjacent to, that of Arion gilvus. Thus, for both the species pairs Arion ponsi / Arion molinae and Arion gilvus / Arion urbiae, the distribution ranges appear at least consistent with the suggested sister group relationships.

In conclusion, the present work shows that Arion ponsi and Arion gilvus clearly differ from Arion subfuscus or any other currently recognized arionid species. As such, former records of Arion subfuscus from Menorca (e.g. Gasull and van Regteren Altena 1970, Mateo 1993, Beckmann 2007) almost certainly refer to Arion ponsi. Similarly, probably all reports of Arion subfuscus in the regions of Valencia and Albacete involve Arion gilvus (e.g. Borredà 1994, Borredà and Collado 1996). Finally, Borredà (1994) wondered about the eventual relationship between Arion subfuscus from Menorca and Arion gilvus. The current data confirm unambiguously that these are two different species, with the former being Arion ponsi. Yet, the overall phylogenetic relationships within Mesarion and many other Arion subfuscus-like taxa remain to be resolved. In this context, one of the main questions is whether Mesarion in its present use is a monophyletic taxon. At the same time one may wonder about the relationships with the subgenus Arion s.s., with which Mesarion seems to form a well-supported clade (Figure 3).


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 Arion gilvus, and to the editor of Spira to give us permission to use the pictures of Arion ponsi. We wish to thank Ben Rowson and one anonymous referee for their helpful comments.

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Supplementary figure 1.

Neighbour-Joining tree (Kimura 2-parameter model) of a 504 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene for the land slug subgenus Mesarion. Bootstrap values ≥ 70% are shown at the nodes. For sample codes see Table 1.

Supplementary figure 2.

Neighbour-Joining tree (Kimura 2-parameter model) of a 408 bp fragment of the mitochondrial 16S rDNA gene for the land slug subgenus Mesarion. Bootstrap values ≥ 70% are shown at the nodes. For sample codes see Table 1.

Supplementary figure 3.

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 Mesarion. Bootstrap values ≥ 70% are shown at the nodes. For sample codes see Table 1.

Supplementary figure 4.

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 Mesarion. This figure also includes the Iberian Mesarion ITS1 sequences of Quinteiro et al. (2005) Bootstrap values ≥ 70% are shown at the nodes. For sample codes see Table 1.