Corresponding author: Eike Neubert (
Academic editor: Menno Schilthuizen
In this work, the presence of species of the slug family
Schallenberg VM, Heim R, Schneppat UE, Müller P, Rüetschi J, Neubert E (2022) Revision of the family Milacidae from Switzerland (Mollusca, Eupulmonata, Parmacelloidea). ZooKeys 1116: 149–179.
The family
The aim of this paper is to verify the correct identification of the
Most investigated specimens were freshly collected during field trips in Switzerland, Italy, Republic of San Marino, and France. If possible and necessary, juveniles were kept alive until they reached adulthood. To complete and increase the genetic data set also previously inventoried specimens of the
Juvenile and subadult slugs were kept individually in 0.2 L polypropylene boxes (Rondo 4, Rotho Kunststoff AG, Switzerland) perforated with a hot needle for air exchange. The boxes were stored in a wine refrigerator at 16 °C, the bottom was carpeted with several layers of moistened paper towels. Nutrition was provided in form of slices of champignon, cucumber, and high protein cat feed pellets. The slugs were checked and weighed once per week with replacement of the paper towel and food. The maturity was checked by evaluation of the genital pore: fully adult animals have a visible and clearly open pore. All captured adult specimens were photographed alive, and tissue samples and body measurements were taken before preservation. For preservation, the animals were relaxed and killed in sparkling water for ~ 30 min. The dead animals were placed on kitchen paper and frozen in the freezer for several hours. After that they were thawed in cold water and additional slime was removed by placing the specimens into ethanol. The animals were transferred into 96% ethanol, and, for proper body cavity preservation, 96% ethanol was injected through the sole tip into the body cavity with a 1 mL syringe using a fine needle (Terumo Corporation, Philippines). After 24 h, the slugs were transferred in 80% ethanol. The following day, the ethanol was exchanged, and the specimens were stored in the fridge at 5 °C awaiting DNA extraction and dissection. This procedure properly maintains the soft tissue for anatomical studies and keeps the DNA intact for the genetic investigation.
All specimens used for live body measurements were fully adult specimen from the collection of R. Heim. The following measurements were taken:
Live weight (
Total length (
Sole width (
The ratio of mantle length (
Number of tubercle rows (
Before dissection of selected specimens, photographs of the preserved animals were made in dorsal, lateral (left and right), and ventral position. The dissection of the slug genitalia was performed under a binocular (Leica MZ6) using thin forceps (Dumont 0.5; 3.5) and micro scissors. For the anatomical pictures, the genital organs were detached from the body, spread on a wax bed, and properly pinned with minutia pins to visualize and investigate the structures. Additionally, the inner structures of penis, epiphallus and vagina were shown. Pictures were taken with a Leica DVM6 microscope camera (Leica Camera, Wetzlar, Germany) using the image-processing program FIJI for scaling (
Distribution map of collected specimens.
The total DNA extraction was performed following the manufacturers protocol of the Qiagen Blood and Tissue Kit using the QIAcube extraction robot (Qiagen; Hilden, Germany). Approximately 0.5 cm2 of mantle tissue was cut, cleaned with a sterilized scalpel (Schreiber GmbH, Germany) from superficial slime, and placed in 180 µL ATL buffer and 20 µL proteinase K. For small specimen, an additional snippet from the foot or body wall was added to the digestion mix in order to yield enough DNA for sequencing. The tissue was then incubated for 4 h at 56 °C and 40 rpm in a thermo shaker (Labnet, Vortemp 56, witec AG, Littau, Switzerland). After digestion the QIAcube extraction robot did the DNA extraction following the standard protocol 430 (DNeasy Blood Tissue and Rodent tails Standard). The extracted DNA samples were then stored in a -80 °C freezer for long-term storage.
In this study, the sequences of the mitochondrially encoded barcoding marker cytochrome c oxidase I (
For the phylogenetic analyses, sequence
For sequence processing, alignments and calculation of trees, the software package Geneious v. 9.1.8 (Biomatters Ltd) was used. The protein-coding gene fragment of
To calculate the
With the same alignment data, a second maximum likelihood tree was calculated using the IQ Tree web tool (
Bayesian Inference (
Species partitions analysis was performed on the Assemble Species by Automatic Partitioning (
The results of the genetic analysis based on
Phylogenetic tree of some species of the genus
The best two
Horse-shoe shaped groove on the mantle; pneumostome postmedian; keel connecting sole tip and mantle; sole tripartite, central sole field with V-shaped wrinkles, only visible in preserved animals (
No stimulatory organ in the atrium, accessory glands opening into the vagina (
Probably does not exist, after
Epiphallus more than twice as long as the penis (almost the same length in
The general pigmentation of Swiss specimens varies from a warm reddish brown to dark reddish or chestnut brown to very dark brown without any reddish brown hue. This pigmentation normally is not fading downwards to the fringe of the sole. However, in some cases in the less dark pigmented populations in Switzerland, little fading downwards can be observed (Fig.
The whole slug is covered with small black spots on dorsum, flanks, and mantle, but not on the keel. The characteristic black streaks above the pneumostome and on the same location on the left side of the mantle can vary greatly in size and form. Even though all our investigated specimens had spots and the characteristic streaks on the mantle, in some specimens they are almost invisible because of the overall dark pigmentation (Fig.
Head, neck and ommatophores are dark brown, while the tentacles are slightly paler. In many cases, the black pigmented ommatophoran retractor is clearly visible through the integument.
The keel is commonly paler in colour than the dorsum. Rarely the general pigmentation of the keel matches the dorsal colour, this occurs especially in darker specimens.
The tripartite sole in pale coloured slugs is creamy yellowish, while darker slugs have a pale yellowish brown sole. Many darker coloured specimens have a hue of greyish black at the posterior outer margins of the lateral sole fields formed by very little black dots. Isolated single greyish black spots may occur along the outer edge of the sole.
The keel is extended from the posterior margin of the mantle to the end of the dorsum. In some specimens, the keel was observed to be even slightly extending over the fringe of the sole, like a very little terminal thorn or knob. The keel has always an entirely smooth surface structure and is therefore clearly discernible from the crenulated neighbouring dorsal tubercles. Live and preserved specimens do not differ in the extension of the keel and its structure.
Vagina twice the length of the penis, separated from the atrium by a sphincter; accessory glands entering close to the boundary of atrium and vagina; accessory glands digitiform or sac-like, either beige, brown or bright rusty red coloured; vaginal lumen with elongated, waved folds pointing towards the oviduct and the pedunculus of the bursa copulatrix; pedunculus somewhat longer than the vesicle; vesicle may be pointed or rounded.
Our own distribution records are mainly limited to Switzerland, France, northern Italy, and the Republic of San Marino. The species itself is frequently recorded from the western alpine arc, but also from a wide range in Great Britain, Ireland, The Netherlands, Belgium, Luxemburg, France, Germany, Austria, Poland, Czech Republic, Slovakia, and Hungary (
This species is confined to beech forests on limestone talus.
As original name of this species,
When it comes to morphology
Regarding the genital anatomy we found genetically identified
Torus with spikes inside the vagina, epiphallus with a field of nodes on the surface; for other character states, refer to the paragraph under
The mantle generally matches the dorsal colour. For the topotypic specimen, dots at the edges of the mantle are lacking. However, in light colour morphs, the mantle appears slightly darker than the dorsum because of the accumulated dots and blotches. In many of these specimens the highest density is along the sinus groove.
For the topotypical colour morph the flanks are pigmented as the dorsum, but with a narrow, paler greyish stripe just above the edge of the sole with little blackish grey dots and stripes. This also refers to the flanks below the edge of mantle. In the light colour morphs, the dorsum and flanks are covered by very fine dark dots on the top of the tubercles (not along the grove lines; not a reticulation). This pigmentation pattern is found from the dorsum down to the edge of the sole. Some larger, irregularly scattered black spots more posterior on dorsum and flanks add to this remarkable colouration. The flanks below the mantle lack this dark pigmentation and are of a paler colour than the rest.
Neck and head in the dark colour morphs are black as are the ommatophores. The ommatophores are somewhat translucent, and the black ommatophoran retractor can be seen through the integument. No black dotting on the ommatophores is visible. The tentacles are black. In the light colour morphs, the head, neck and the ommatophores are always darker if compared to the body colour.
The keel is of the same colour as the body and thus difficult to see in a crawling animal.
The sole is uniformly creamy yellowish grey in all specimens. In one topotypic specimen, the posterior ends of the lateral sole fields are pigmented with dark grey dots. In two other specimens, the seam of the sole is pale grey with irregularly dispersed, small black dots.
Ratio of
Vagina shorter than penis; accessory glands entering distally on vagina, close to pedunculus and oviduct, formed by a broad truncus with bundles of tubuli attached; the vaginal walls richly covered with folds forming a zig zag pattern; a prominent torus with a row of acute conical spikes running through the vagina from atrium almost reaching the branching point of the pedunculus of the bursa copulatrix (Fig.
In the summit region of Monte Generoso,
So far, all records in Switzerland originate from areas with a good calcium supply. The altitudinal range of its habitats spans from 435 m near Rovio to the highest point at 1700 m.
There are almost no observations on the biology of the species published.
The author of this species is Karl Ludwig Pfeiffer (1874–1952), who usually published under the name Karl L. Pfeiffer = K. L. Pfeiffer (
The most remarkable new finding is the presence of several/additional colour morphs in
Our recent research on the new additional colour morphs of
In each of the Swiss specimens examined, the posterior end of mantle is markedly indented, as it was already described by
For a rather long period,
Unfortunately, it was impossible to extract DNA from the specimen from Laxolo (
Since its description, eight malacologists (
Not researched and not mentioned by
Sole with a dark central field; for other character states, refer to the paragraph under
The mantle sometimes can be darker than the dorsum because of many black dots and irregular black marbling.
The ommatophores are almost black-brown, but sometimes little translucent in good light, so the black ommatophoran retractor can be seen through the integument. The tentacles are of the same colour.
The colour of the keel ranges from dark brown to rusty orange in its full length.
The sole is grey to dark blackish grey, with the central field sometimes being almost black. In all three sole fields, many black, irregularly jagged spots (chromatophores?) exist, which can be seen only under magnification.
In all specimens, the keel extends from the posterior margin of the mantle to the end of the dorsum. It is not projecting but evenly rounded, sometimes almost flat and not much exposed over the dorsal
Ratio of
The vagina is extremely short, separated from the atrium by a muscular ring; accessory glands sac-like attached at centre of the vagina; vaginal walls simple; pedunculus of bursa copulatrix large in diameter, longish vesicle; pedunculus wall with longitudinal internal folds showing a zig zag pattern; oviduct slim and long.
Living specimens of
All Swiss specimen were exclusively found in urban areas (anthropogenic habitats) in house gardens, along an old city wall, and close to a small brook with trees and shrubs. The population along the old city wall of Lucerne was checked several times over the years and seems to be stable.
We found some Bulgarian specimens ranging from unicoloured yellow to almost completely black. Such colour morphs might be expected in the future in Switzerland, too.
Atrial accessory glands present¸ atrium with internal stimulator (
Not investigated and probably do not exist; not mentioned by
The mantle has the same colouration as the dorsum. Above the pneumostome, along the horseshoe-shaped sinus groove, is a prominent grey band.
The neck and head have a darker colouration compared to the body and are uniformly grey coloured without dots.
The keel is also beige, but lighter coloured compared to the rest of the body.
According to
The keel is elevated over the neighbouring tubercules only close to the mantle, and flattens towards the posterior end.
Vagina shorter than penis; pedunculus broad and equal in length to vesicle; vesicle wider than pedunculus, spherical; female oviduct slender and long.
This species is widespread in the western Palaearctic, in Portugal and parts of Spain, France, United Kingdom, etc. (see
Colour variations of
Anatomy of
The specimen from Vechigen is provisionally identified as
The very small specimen from Porrentruy was not only completely dried up and completely bleached but turned out to be a young juvenile without any sign of a genital pore. In fact, juvenile specimens cannot be determined on species level from exterior characters, when genital anatomy is not developed. For this reason, we suggest considering this record as
Finally, there is a specimen housed in the collection of the Muséum d’histoire naturelle de la Ville de Genève, which had been identified as
In this study we discuss four species of
Comparing the anatomical character states between
We could show that the small and dark specimens of
For future research we recommend following the preservation procedures for slugs as described above. Perfect samples should be accompanied by photographs taken in the field. Juvenile and subadult specimens should be raised until reaching maturity prior to preservation.
Barcoding and species delimitation such as
We are grateful to Marianne Cornu, Zurich, and Monika Cornu, St. Gallen, for support during field collections; to Regula Cornu, Chur for long lasting and ongoing support in field collection and consistent support and assistance in the laboratory and collection; Katja Lassauer, and Christian Roggenmoser, Lucerne for their collections and ongoing enthusiasm. Special thanks go to Guiseppe Nardi for his support with specimens from Italy, and Ira Richling, Stuttgart, for information on a specimen of
Table S1
excel file
Genetically and/or morphologically investigated specimens.