Population Genetics and Identity of an Introduced Terrestrial Slug: Arion subfuscus s.l. in the North-east USA (Gastropoda, Pulmonata, Arionidae)

Several European species of the terrestrial slug genus Arion have been introduced into North America. A case in point is the species complex A. subfuscus s.l. which has become one of the most abundant slug taxa in North America. In Europe this complex consists of at least two cryptic species, viz. A. fuscus and A. subfuscus s.s., the latter of which is further subdivided in five strongly divergent mtDNA lineages (A. subfuscus S1–S5). In order to determine which of these A. subfsucus s.l. taxa are present in the NE USA and in order to assess their population genetic structure, we compared mtDNA, nDNA and allozyme variation between populations from the NE USA and Europe. Our results show that (1) at least A. subfuscus S1 has become successfully established in the NE USA, (2) founder effects are the most likely explanation for the loss of a large amount of molecular genetic variation in populations from the NE USA (i.e. a loss of 96% of the 16S rDNA haplotypes, 67% of the ITS1 alleles and 46% of the alleles at polymorphic allozyme loci), and (3) part of the remaining genetic variation in NE USA populations was probably due to multiple introductions from the British Isles and the European mainland, and the hybrid structure of most of these source populations. Apparently, the extreme loss of molecular genetic variation in this introduced species has not prevented it from successfully establishing and spreading in novel environments.     

Phylogeny of slug species of the genus Arion: evidence of monophyly of Iberian endemics and of the existence of relict species in Pyrenean refuges

The Iberian Peninsula contains the majority of the Paleartic land slug species of the genus Arion, which exhibits diverse taxonomic problems. The present study investigated Arion taxonomy on the basis of analyses of the mitochondrial ND1 gene and nuclear internal transcribed spacer 1 (ITS1) sequences. The Iberian endemic species were monophyletically clustered in two divergent sister clades. The topotype specimens of Arion lusitanicus and the closely related species Arion nobrei and Arion fuligineus, as well as Arion hispanicus and Arion flagellus, were grouped into an ‘Atlantic’ clade, whereas Arion baeticus, Arion gilvus, Arion anguloi, Arion wiktori and Arion paularensis were included in a ‘Continental–Mediterranean’ clade. Calibration of mutation rate in the ND1 gene suggested that the divergence of these two clades occurred around the Pliocene–Pleistocene boundary, with subsequent speciation events during the Pleistocene. A group of ancestral and divergent endemic species with distribution centred in the Pyrenean mountain range (Arion molinae, Arion lizarrusti, Arion antrhacius and Arion iratii) arose in the Pliocene and survived through the Pleistocene in geographically confined small populations. Arion lusitanicus showed up to be polyphyletic: specimens, sampled outside the geographic range of the topotype in the north‐western Iberian Peninsula, were included in a non‐monophyletic clade together with the widely distributed species Arion ater and Arion rufus. The divergent species with a wide European distribution (Arion subfuscus, Arion hortensis, Arion fagophilus and Arion intermedius) were located in basal positions in all topologies. The evolutionary history of these slug species (highly sensitive to climatic factors, with capacity for both outcrossing and selfing, and with low dispersal ability) appears to have been moulded by Pliocene–Pleistocene climate events and by the rugged topography of southern Europe, giving rise to repeated cycles of population isolation during periods of glaciation alternating with interglacial expansions limited by geographic barriers.     

The effects of food and density of the movement ofArion ater andAriolimax columbianus (Pulmonata: Stylommatophora) between habitats

Experiments were conducted to determine if slug density, or food quantity and quality could cause dispersal by slugs. Arion ater dispersed quickly from overcrowded areas, where high mortality was incurred by this species. Poor food conditions, caused more dispersal than good food conditions, but food had no effect on the mortality of A. ater. Neither food nor density factors had significant effects on the dispersal of Ariolimax columbianus. However, more A. columbianus died in the high- than low-density treatments. A. columbianus mortality was unaffected by the food factor. Dispersing Arion were heavier than non-dispersers, but there was no difference between the weights of dispersing and non-dispersing Ariolimax. There were significant seasonal changes in the dispersal rate, mortality and weight of dispersing Arion, whereas only the mortality of Ariolimax changed significantly during the experiments. Since Arion ater did not markedly reduce its activity during unsuitable weather it was able to leave areas of poor food and high slug density fairly rapidly. Ariolimax columbianus' large size allowed it to remain inactive during unfavourable weather and permitted this species to be relatively unresponsive to the experimental treatments.     

An electrophoretic characterisation of three paratypes of Arion fagophilus De Winter, 1986, with notes on the subgeneric division of the genus Arion Férussac, 1819 (Mollusca, Pulmonata)

Three paratypes of Arion fagophilus were electrophoretically compared with nine other West European species of the genus Arion. Esterases, glutamate-oxaloacetate-transaminase, a-amylase, superoxide dismutase, phosphoglucomutase, fumarase, α-glycerophosphate dehydrogenase and phosphogluconate dehydrogenase were screened in the hepatopancreas and general proteins in the albumen gland. A. fagophilus appears to be a very well defined species, which may be assigned to the subgenus Kobeltia. The subgeneric division of the genus Arion is preliminary discussed with respect to electrophoretic and chromosomal data. It is concluded to retain four subgenera, which may be grouped two by two: Mesarion and Arion s. s. on one hand, and Kobeltia and Carinarion on the other. It is remarked that the electrophoretic differences between Norwegian and Belgian A. ater suggest a separation into two taxa.     

The genetic dynamics of the rapid and recent colonization of Denmark by <Emphasis Type="Italic">Arion lusitanicus</Emphasis> (Mollusca,Pulmonata, Arionidae)

We describe the genetic dynamics of the recent establishment of the ‘Iberian slug’, Arion lusitanicus J. Mabille 1868, in Denmark and compare its population structure to two other members of the ‘large Arion complex’, Arion ater ater, native to Denmark, and Arion ater rufus, introduced into Denmark in the early 1900s. Assaying allozyme polymorphism at seven enzyme loci, we found that: (1) None of the three taxa reproduce primarily by self-fertilization. Differences among loci and colonies in the pattern of deviation from Hardy–Weinberg equilibrium are most consistent with isolate mixing and perhaps with low amounts of selfing. (2) For both A. lusitanicus and A. a. rufus, gene diversity is lower in Danish colonies than in southern German colonies, implying population bottlenecks in the establishment of Danish colonies. (3) Significant linkage disequilibrium values usually involve the same three loci, viz. PGI, MDH-1 and MDH-2, suggesting physical linkage among these loci. (4) For both A. a. rufus and A. lusitanicus, the overall gene frequencies from Denmark and southern Germany are homogeneous, while variation among colonies within these regions ranges from around 15 to 28% for the three taxa. This indicates strong, local population genetic subdivision but with little restriction to gene flow from possible source areas. The heterogeneity in measures of diversity and differentiation indicates that population structure for all three taxa is dominated by ongoing founder effects, local extinction/colonisation dynamics, and genetic drift processes.     

The effects of food and density on the nocturnal behaviour ofArion ater andAriolimax columbianus (Pulmonata: Stylommatophora)

The effects of food supply and population density on the nocturnal behaviour of Arion ater and Ariolimax columbianus were investigated. Density did not significantly affect A. ater's level of activity or short-term movement, resting, or feeding. A. columbianus was more active and moved, rested, and fed more frequently when slug density was high. A. ater foraged and rested more often, but fed less when good food was unavailable. Ariolimax's only response to the food regime was to feed more when good food was available. Seasonal changes in the level of activity and behaviour of Arion were evident, whereas Ariolimax's activity and behavioural repertoire were not similarly affected. Arion ater's nightly activity appeared to be mainly food oriented, while Ariolimax columbianus seemed most responsive to slug density during its nocturnal activity periods.     

Thermal Compensation of Respiration in Pulmonate Snails (Pulmonata) of Arion and Deroceras Genera Living in Polar and Temperate Climatic Zone

Comparison of respiration rate in pulmonate snails living in various climatic zones demonstrated higher constant a in representatives of Arion genus (A. subfuscus and A. fasciatus) from Polar Area (Murmansk Region) as compared to inhabitants of temperate latitudes (Moscow Region). The snails of Deroceras genus (D. reticulatum) from these two climatic zones were indistinguishable by relative standard metabolism. Different effects of climatic thermal conditions on respiration rates in representatives of these two snail genera can be due to their specific biology. Representatives of Deroceras genus are short-cycle synanthropic species, while the snails of Arion genus are long-cycle species living mostly in the forest zone.     

The influence of weather upon the activity of slugs

Summary The activity of slugs was measured by a catch per unit effort sampling system based upon night searching, and was related to the microclimatological conditions in the habitat by regression analysis. The activity of Arion hortensis Fér., Arion subfuscus (Drap.), Arion lusitanicus Mab., Milax budapestensis (Hazay), and of all slugs irrespective of species, was found to be best related to temperature and vapour pressure deficit. The relevance of these results to the application of effective methods for the control of slugs is discussed.     

Arion transsylvanus (Mollusca,Pulmonata, Arionidae): rediscovery of a cryptic species

Jordaens, K., Pinceel, J., Van Houtte, N., Breugelmans, K. & Backeljau, T. (2010). Arion transsylvanus (Mollusca, Pulmonata, Arionidae): rediscovery of a cryptic species. —Zoologica Scripta, 39, 343–362. Cryptic species are abundant among invertebrates and are often hard to recognise. Molecular markers are an extremely useful tool to delineate cryptic taxa, although they should be applied with caution because different genes and techniques may yield different outcomes. We illustrate how cross‐validation by molecular and morphological data can be applied to optimise taxonomic interpretations when cryptic species are involved. This is performed for the terrestrial slug Arion subfuscus species complex which represents a historical ‘taxonomic garbage can’. Gonad morphology, allozymes and mtDNA data consistently showed that slugs from Romania and a location in E Poland represent a strongly differentiated taxon within this complex. These slugs are therefore formally redescribed and assigned to A. transsylvanus Simroth 1885 ; a forgotten nominal taxon from Transylvania. Diagnostic characters, including DNA sequences for the mitochondrial 16S rDNA are presented. Animals with the morphology of A. brunneus Lehmann 1862 , a nominal taxon which has also been reported from Romania, have the gonad type, allozyme alleles and 16S rDNA haplotypes of either A. fuscus, A. subfuscus or A. transsylvanus. Therefore, A. brunneus is regarded as a colour morph shared by several species and hence, A. transsylvanus is probably the only A. subfuscus‐like species in the Romanian Carpathians.     

Immunocytochemical Localisation of Glycosaminoglycan-Like Activity in the Mucus and Associated Tissues of Terrestrial Slug Species (Gastropoda: Pulmonata)

Indirect immunofluorescence assays were conducted on cryotome sections of four terrestrial slug species from three distinct phylogenetic groups, Arion ater (L.), Arion hortensis (Férussac), Tandonia (Milax) budapestensis (Hazay), and Deroceras reticulatum (Müller) using monoclonal antibodies for two glycosaminoglycans (GAGs), heparan sulphate, and chondroitin sulphate. Specific staining for a heparan sulphate-like component was demonstrated in the foot and tail regions of each species and was particularly intense in A. ater and A. hortensis, notably in the epidermis and associated mucus-like material, and in mucus-like material from the pedal gland region of the latter species. Subsequent studies with A. ater confirmed the presence of heparan-sulphate–like activity in the caudal gland duct region. No evidence of specific staining for chondroitin sulphate-like activity was found in any of the slug species. This work suggests that a specific GAG, or a group of closely related GAGs, is a common component of mucus in a range of slug species and of different types of mucus, including trail (pedal) mucus and the more viscous mucus produced by the caudal gland.     

Genetic differentiation of disjunct populations of the antsFormica aquilonia andFormica lugubris in Europe

Summary The speciesFormica aquilonia andF. lugubris of the mound-building red wood ants have a disjunct boreoalpine distribution in Europe. The populations ofF. aquilonia in Finland, Switzerland and the British Isles show little genetic differentiation, whereas the populations ofF. lugubris show considerable differentiation. The Central European populations morphologically identified asF. lugubris can be genetically divided into two groups (here called types A and B). Type B is found in the Alps and the Jura mountains, and is genetically inseparable fromF. aquilonia. Type A lives sympatrically with type B in the Jura mountains and is also found in the British Isles. Sympatry of the two types in the Jura shows that these are separate species. It remains open whether type B is morphologically atypicalF. aquilonia or whether it is a separate species, perhaps with a past history of introgression betweenF. aquilonia andF. lugubris. The gene frequencies in the Finnish populations ofF. lugubris differ from those of both types A and B. Genetic differences withinF. lugubris indicate that the populations have evolved separately for a long time. The social structure ofF. lugubris colonies also shows geographic variation. The nests in Finland and the British Isles seem to be mainly monogynous and monodomous, whereas the nests in Central Europe are polygynous and form polydomous colonies.F. aquilonia has polygynous and polydomous colonies in all populations studied.     

Genetic differentiation across the social transition in a socially polymorphic sweat bee,Halictus rubicundus

Eusociality is widely considered a major evolutionary transition. The socially polymorphic sweat bee Halictus rubicundus, solitary in cooler regions of its Holarctic range and eusocial in warmer parts, is an excellent model organism to address this transition, and specifically the question of whether sociality is associated with a strong barrier to gene flow between phenotypically divergent populations. Mitochondrial DNA (COI) from specimens collected across the British Isles, where both solitary and social phenotypes are represented, displayed limited variation, but placed all specimens in the same European lineage; haplotype network analysis failed to differentiate solitary and social lineages. Microsatellite genetic variability was high and enabled us to quantify genetic differentiation among populations and social phenotypes across Great Britain and Ireland. Results from conceptually different analyses consistently showed greater genetic differentiation between geographically distant populations, independently of their social phenotype, suggesting that the two social forms are not reproductively isolated. A landscape genetic approach revealed significant isolation by distance (Mantel test r = 0.622, P < 0.001). The Irish Sea acts as physical barrier to gene flow (partial Mantel test r = 0.453, P < 0.01), indicating that geography, rather than expression of solitary or social behaviour (partial Mantel test r = −0.238, P = 0.053), had a significant effect on the genetic structure of H. rubicundus across the British Isles. Although we cannot reject the hypothesis of a genetic underpinning to differences in solitary and eusocial phenotypes, our data clearly demonstrate a lack of reproductive isolation between the two social forms.     

Molecular identification of the exotic slug <Emphasis Type="Italic">Arion subfuscus</Emphasis> sensu stricto (Gastropoda: Pulmonata) in California,with comments on the source location of introduced populations

Arion subfuscus sensu lato (s. l.) refers to a cryptic slug species complex that is native to Europe and which, from there, has been introduced into the northeastern U.S. The species complex was detected in California for the first time in 2005 and recently 12 specimens were collected during statewide surveys. The genital morphology of these specimens suggested that only A. subfuscus sensu stricto (s. s.) was present and partial sequences of mitochondrial 16S rDNA (443 bp) showed that they all belonged to a single haplotype of the mitochondrial lineage, S1 sensu Pinceel et al. (Genetica 125: 155–171, 2005). This result was corroborated by a parallel analysis of a 655 bp COI DNA barcode. The 16S rDNA S1 haplotype (S1-03) of the Californian specimens is hitherto known only from the northeastern U.S. Hence, it is likely that populations may have been introduced to California from the northeastern states, rather than directly from the native area of the S1 haplogroup in Europe.     

<Emphasis Type="Italic">Angiostoma norvegicum</Emphasis> n. sp. (Nematoda: Angiostomatidae) a parasite of arionid slugs in Norway

Angiostoma norvegicum n. sp. (Angiostomatidae) is described from the oesophagus, crop and the buccal mass of five species of slugs of the family Arionidae, Arion vulgaris (Moquin-Tandon), Arion ater (L.), Arion fasciatus (Nilsson), Arion fuscus (Müller) and Arion rufus/Arion ater hybrid), collected throughout Norway. Angiostoma norvegicum n. sp. was found parasitising arionids at seven of the 30 sample sites examined (23.3%), and 9.9% of all Arion spp. were infected with this nematode. The new species is characterised by its large size (4.0–8.6 mm long) and in having: lateral alae; 6 + 6 papillae at the cephalic end; a large circular mouth aperture; a spacious stoma; a pharyngeal basal bulb without valvular apparatus; an excretory pore near the base of bulb; a distal part of posterior ovary always outstretched; an anterior ovary distally nearly always outstretched; a vulva situated anterior to mid-body; long, nearly straight spicules and a small gubernaculum; three circumcloacal papillae and caudal genital papillae (GP) arranged in a pattern 1+2/3+3 with GP 5 and GP 8 opened on dorsal side of narrow bursa not reaching tail tip; short conical tails in both sexes with tips supplied by 4 short, unequal denticles. Morphologically, A. norvegicum n. sp. is similar to Angiostoma limacis Dujardin, 1845, which diagnostic characteristics are given based on examination of specimens from Norway and the UK. Conversely, the phylogenetic analyses based on D2D3 large subunit (LSU) rRNA gene sequences performed in the present study did not support the morphological affinity of these two species. Phylogenetic analyses demonstrated that although Angiostoma spp. cluster together, A. norvegicum n. sp. forms a tight monophyletic clade with the milacid nematode parasites Angiostoma margaretae Ross, Malan & Ivanova, 2011 and Angiostoma milacis Ivanova & Wilson, 2009.     

INBREEDING AND THE RATE OF SELF-FERTILIZATION IN A GRAPE FERN,BOTRYCHIUM DISSECTUM

Botrychium dissectum is a homosporous fern with bisexual, subterranean gametophytes. Because of these features, B. dissectum would be suspected of displaying a very high frequency of self-fertilization. Sporophytes collected from three populations of this species were assayed for heterozygosity by determining the electrophoretic mobility patterns displayed by two polymorphic enzymes. Extreme deviations from Hardy-Weinberg expectations were observed in each population and analyzed by means of F-statistics. The average inbreeding coefficient was found to be 0.951. A population genetic model is derived that demonstrates that the rate of intragametophytic self-fertilization in homosporous ferns is equal to the inbreeding coefficient calculated from deviations from Hardy-Weinberg expectations. It is therefore concluded that B. dissectum outcrosses about 5% of the time.     

Phylogeographic past and invasive presence of Arion pest slugs in Europe

Arion vulgaris (syn. A. lusitanicus) is the most destructive pest slug in Europe. The species has been regarded a classic case of an ongoing biological invasion with negative economic and ecological impact in many European countries, but this status has recently been contested. In this study, we assessed mitochondrial and autosomal genetic diversity in populations of A. vulgaris across the entire distribution range in order to characterize its evolutionary history. Mitochondrial diversity in A. vulgaris was strongly reduced compared with the closely related and largely codistributed noninvasive species A. rufus and A. ater, indicating a very rapid spread of A. vulgaris through Europe. Autosomal diversity assessed in 632 individuals from 32 populations decreased towards eastern and northern Europe which is consistent with the reported expansion of the species towards these regions in the last decades. Demographic simulations supported very recent population founding events in most of the European range. The short periods between the first detection of A. vulgaris in different countries and only a very weak association of genetic structuring among populations with geographical distances suggest a human contribution in the ongoing expansion of the slug. We propose that this contribution may ultimately prevent the exact localization of the debated region of origin of A. vulgaris. However, the reclassification of A. vulgaris as noninvasive would be premature. Without counter measures, the Eastern and Northern European countries can expect to see this biological invasion continued in the future.     

Population size and habitat quality affect genetic diversity and fitness in the clonal herb <Emphasis Type="Italic">Cirsium dissectum</Emphasis>

Remaining populations of plant species in fragmented landscapes are threatened by declining habitat quality and reduced genetic diversity, but the interactions of these major factors are rarely studied together for species conservation. In this study, the interactions between population size, habitat quality, genetic diversity and fitness were investigated in 22 populations of the clonal herb Cirsium dissectum throughout the British Isles. Regression analysis was used to identify significant factors, and a structural equation model was developed to illustrate and integrate these interactions. It was found that smaller populations (measured as the total number of plants) had lower genetic diversity (proportion of polymorphic loci), and that reduced genetic diversity (allelic richness) had a negative impact on the survival of seedlings grown under standard conditions. Habitat quality also had a large effect on C. dissectum. Unmanaged sites with tall vegetation, no bare soil and higher nutrient levels had smaller populations of C. dissectum, but flowering was promoted. Flowering was suppressed in heavily grazed sites with short vegetation. Higher levels of bare soil and phosphorus both had a positive relationship with genetic diversity, but probably for distinctly different reasons: bare soil provides safe sites for establishment, whilst phosphorus may promote flowering and improve seed germination. In order to conserve C. dissectum, management needs to maintain site heterogeneity so that C. dissectum can flower and establishment gaps are still available for seedlings; when either component is reduced, negative feedbacks through reduced genetic diversity and individual fitness can be expected. This study therefore highlights the importance of considering both conservation genetics and habitat quality in the conservation of plant species.     

Conservation of genetic uniqueness in remaining populations of red squirrels (Sciurus vulgaris L.) in the South of England

The Eurasian red squirrel (Sciurus vulgaris) is an emblematic species for conservation, and its decline in the British Isles exemplifies the impact that alien introductions can have on native ecosystems. Indeed, red squirrels in this region have declined dramatically over the last 60 years due to the spread of squirrelpox virus following the introduction of the gray squirrel (Sciurus carolinensis). Currently, red squirrel populations in Britain are fragmented and need to be closely monitored in order to assess their viability and the effectiveness of conservation efforts. The situation is even more dramatic in the South of England, where S. vulgaris survives only on islands (Brownsea Island, Furzey Island, and the Isle of Wight). Using the D‐loop, we investigated the genetic diversity and putative ancestry of the squirrels from Southern England and compared them to a European dataset composed of 1,016 samples from 54 populations. We found that our three populations were more closely related to other squirrels from the British Isles than squirrels from Europe, showed low genetic diversity, and also harbored several private haplotypes. Our study demonstrates how genetically unique the Southern English populations are in comparison with squirrels from the continental European range. We report the presence of four private haplotypes, suggesting that these populations may potentially harbor distinct genetic lineages. Our results emphasize the importance of preserving these isolated red squirrel populations for the conservation of the species.     

Rural–urban gradients and the population genetic structure of woodland ground beetles

Genetic diversity and differentiation of two carabid beetle species were examined in woodlands along rural–urban gradients in two cities (Brussels, Belgium, and Birmingham, UK), based on allozymes, studied in more than 1000 beetles. Compared to Abax ater, Pterostichus madidus showed higher levels of genetic diversity but lower genetic differentiation, probably because of its ability to survive in non-forest habitats. Higher genetic diversity in both species was observed in Brussels as compared to Birmingham. However, genetic differentiation among sites was higher in Birmingham corresponding to the more extreme degree of fragmentation and isolation between the Birmingham woodlands. The isolation-by-distance model did not explain genetic differentiation among sites within the two regions. Gene diversity in P. madidus Birmingham populations was higher in smaller urban forests with a small perimeter. A similar absence of genetic erosion in smaller and more highly modified (urban) populations was also obtained for A. ater populations from Brussels, with a higher genetic diversity in sites closer to woodland edges. This unexpected result is hypothesised to be the result of an adaptive increase of genetic diversity in more heterogeneous landscapes, closer to woodland edges and in smaller and more perturbed forests.