Slugs of Britain and Ireland: identification, understanding and control.

Full text

The Slugs of Britain and Ireland: Undetected and
Undescribed Species Increase a Well-Studied,
Economically Important Fauna by More Than 20%
Ben Rowson
1
*, Roy Anderson
2
, James A. Turner
1
, William O. C. Symondson
3
1National Museum of Wales, Cardiff, Wales, United Kingdom, 2Conchological Society of Great Britain & Ireland, Belfast, Northern Ireland, United Kingdom, 3Cardiff
School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom
Abstract
The slugs of Britain and Ireland form a well-studied fauna of economic importance. They include many widespread
European species that are introduced elsewhere (at least half of the 36 currently recorded British species are established in
North America, for example). To test the contention that the British and Irish fauna consists of 36 species, and to verify the
identity of each, a species delimitation study was conducted based on a geographically wide survey. Comparisons between
mitochondrial DNA (COI, 16S), nuclear DNA (ITS-1) and morphology were investigated with reference to interspecific
hybridisation. Species delimitation of the fauna produced a primary species hypothesis of 47 putative species. This was
refined to a secondary species hypothesis of 44 species by integration with morphological and other data. Thirty six of these
correspond to the known fauna (two species in Arion subgenus Carinarion were scarcely distinct and Arion (Mesarion)
subfuscus consisted of two near-cryptic species). However, by the same criteria a further eight previously undetected
species (22% of the fauna) are established in Britain and/or Ireland. Although overlooked, none are strictly morphologically
cryptic, and some appear previously undescribed. Most of the additional species are probably accidentally introduced, and
several are already widespread in Britain and Ireland (and thus perhaps elsewhere). At least three may be plant pests. Some
evidence was found for interspecific hybridisation among the large Arion species (although not involving A. flagellus) and
more unexpectedly in species pairs in Deroceras (Agriolimacidae) and Limacus (Limacidae). In the latter groups,
introgression appears to have occurred in one direction only, with recently-invading lineages becoming common at the
expense of long-established or native ones. The results show how even a well-studied, macroscopic fauna can be vulnerable
to cryptic and undetected invasions and changes.
Citation: Rowson B, Anderson R, Turner JA, Symondson WOC (2014) The Slugs of Britain and Ireland: Undetected and Undescribed Species Increase a Well-
Studied, Economically Important Fauna by More Than 20%. PLoS ONE 9(3): e91907. doi:10.1371/journal.pone.0091907
Editor: Donald James Colgan, Australian Museum, Australia
Received August 1, 2013; Accepted February 17, 2014; Published March 19, 2014
Copyright: ß2014 Rowson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by a Research Grant from the Leverhulme Trust to the authors (RPG-068). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ben.rowson@museumwales.ac.uk
Introduction
Slugs are among the invertebrates most readily encountered by
people in north-west Europe, with many species occurring in
gardens and in or around buildings. The British and Irish slug
fauna of 36 species [1] includes important pests, post-glacial relicts,
indicators of ancient woodland and even putative endemics [2].
However, most are widespread European species, many of them
introduced in other parts of the world. Almost all the introduced
slugs of other temperate regions also occur in Britain, including
those of South Africa (11 species [3]) and New Zealand (14 species
[4]). At least 18 species (half the currently recognised British fauna)
are established in the USA and/or Canada [5,6,7]. Some of these
species have a long history of study. The early depictions of British
slugs in Lister (1685) [8] were a source for the descriptions by
Linnaeus (1758) [9] and other early European workers. After
Scharff’s (1891) Irish monograph [10], the British fauna was
monographed twice in the 20
th
century [11,12], establishing a
benchmark for identification guides [13,14], population genetic
studies (e.g. [15,16,17]) and applied works (e.g. [18,19]). In Britain
and Ireland, slugs have been included in a pioneering mollusc
distribution mapping scheme since the 1880s [2] so have been
subject to careful public-participatory recording and study,
resulting in updated and comprehensive checklists (most recently
in 2008 [1]). As a result, the British and Irish slug fauna must rank
among the world’s best studied.
However, additional species in the fauna have nevertheless been
recognised relatively recently either by examining ‘‘aggregates’’ of
superficially similar species [20,21] or direct detection [22,23,24].
Wide-ranging phylogeographic work has also demonstrated the
presence of additional taxa in Britain [25]. In order to detect such
taxa they must be distinguished from those already known to be
present, which must themselves be adequately characterised. Slugs
present particular problems in identification due to overlapping
external morphology and the need to examine internal characters,
so despite their importance and conspicuousness they are often
neglected during biodiversity assessments and also by amateur
malacologists. In 2011 we began producing a new comprehensive
identification guide to the British and Irish slugs, aimed at non-
specialists (the most recent being [14]). Such guides depend upon
correctly identified reference specimens, ideally vouchered in an
accessible museum collection. In the case of slugs these should
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preferably be photographed alive since some diagnostic features
are lost or obscured on preservation. We took this opportunity to
sequence mitochondrial DNA (mtDNA) from these and other
specimens. This provided an independent corroborator of
identifications where conspecific reference sequences already
existed, or a potential future reference where they did not.
The survey also allowed us to use species delimitation
techniques to test the contention that there are currently 36 slug
species established in Britain and Ireland [1]. ‘‘Established’’ refers
to reproducing populations (whether native or introduced) as
opposed to ‘‘adventive’’ populations that do not sustain themselves
by reproduction. Invertebrate ‘‘species’’ in a checklist such as [1]
have usually been delimited, studied and recorded by non-
molecular techniques (Arion (Mesarion) fuscus (Mu¨ller) is an
exception [26]). In general, they have been considered species
under a biological species concept invoking reproductive isolation.
This species concept is sometimes difficult to apply in European
slugs, which are typically simultaneous hermaphrodites with a
varying tendency to self-fertilise [16]. In the family Arionidae in
particular, a flexible species concept may have to be adopted as a
consequence of mixed breeding systems and variable evolutionary
rates [25,26,27,28,29]. The phylogenetic species concept, where
species are delimited as genetically distinct monophyletic lineages,
and evolutionary species concept, where a species is a lineage with
a separate evolutionary fate, have been discussed or invoked in
these studies as being more appropriate. This approach is followed
here. We stress that our study is an exercise in the species
delimitation of the fauna of Britain and Ireland and not a
phylogenetic or phylogeographic study of wide-ranging European
species. However, this does not preclude species being distin-
guished first in an island fauna that has, through natural
establishment and introduction, sampled that of the continent
(e.g. [20,23,24]).
Sequence data from mtDNA have proven effective in the
diagnosis and delimitation of European slugs as phylogenetic
species, showing at least some concordance with the morphology
and behaviour of species recognised by non-molecular methods
and sometimes supporting their recognition as biological species
also (e.g. [30,31,32,33] in addition to those cited above). However,
sequence data alone do not always provide sufficient objective
criteria for species delimitation [34], and mtDNA has its own
limitations as an evolutionary marker [35]. It is also evident that
intraspecific mtDNA divergences vary considerably across terres-
trial mollusc families [36]. A number of methods have recently
been developed to make species delimitation from sequence data
more consistent, and more independent of delimitation based on
non-molecular data to which it can then be compared. Pre´vot et
al. [37] recently applied and compared several of these in a
European terrestrial mollusc with a mixed breeding system. Each
delimited slightly different numbers of species, all of which were
readily recognised as monophyletic on phylogenetic trees, leading
them to question how a particular delimitation method might
justifiably be favoured a priori. One method tested, Automatic
Barcode Gap Discovery (ABGD) [38] has several advantages. It
requires no prior knowledge of the likely intraspecific distances
and is robust to their variation within a dataset. Being distance-
based it does not use monophyly (allowing it to be investigated
independently from distinctness) and is computationally very
efficient [38]. In gastropod studies [37,39] it delimited as few or
fewer species than the coalescent-based General Mixed Yule
Coalescent (GMYC) delimitation method [40], most ABGD
species being a subset of those delimited by GMYC. This contrast
was more marked where some species were represented by many
fewer sequences than others [39]. This is a sampling problem
considered an inescapable consequence of biological rarity in
delimitation studies [41]. As our aims were to test a hypothesis of
36 species and to attribute specimens to these whenever justified,
we used the distance-based and conservative ABGD method in
our study.
To best aid future identification by confirming whether external
morphological variation was intraspecific, our sampling paid more
attention to the more conspicuously variable slug species,
especially the ‘‘larger Arionidae’’ (Arion subgenus Arion). This
group includes A. (A.) ater (Linnaeus), A. (A.) rufus (Linnaeus), and
the notorious pest A. (A.) vulgaris Moquin-Tandon (called A.
lusitanicus auct. non Mabille in some publications). These are
morphologically similar species that either hybridise (e.g.
[42,17,43,44,45]) or overlap morphologically to give the impres-
sion of having done so [46]. It has been suggested, at least in the
British media, that they may also hybridise with Arion (A.) flagellus
Collinge, a common species in Ireland and Britain but not
elsewhere outside Iberia. The potential for hybridisation further
complicates the application of species concepts in the larger
Arionidae, so we sought evidence of hybridisation between these
species as well as aiming to clarify their genetic identity. In the
event, the data suggested that hybridisation occurs in other
families.
Materials and Methods
Ethics statement
All necessary permits were obtained for the described study,
which complied with all relevant regulations. The only protected
species in this study is Geomalacus maculosus Allman, which occurs in
the Republic of Ireland but not the UK. For this species, the
necessary Licence under the Wildlife Act 1976-2010 and
Derogation Licence under the European Communities (Natural
Habitats) Regulations 1997 were obtained from the Department of
Arts, Heritage and the Gaeltacht.
Samples
Slugs were collected by hand, often at night and/or in wet
weather, from over 200 sites across Britain (England, Scotland and
Wales) and Ireland (Northern Ireland and the Republic of Ireland)
between 2010 and 2012. Attempts were made to maximise the
range of latitude, longitude, altitude, habitat and soil types
covered. As native species were the initial focus, most collecting
was done in less disturbed areas including National Nature
Reserves and National Parks but we also included agricultural,
urban and brownfield sites. Some known sites for the rarer species
were visited repeatedly. Additional specimens were collected by
members of the Conchological Society of Great Britain and
Ireland and members of the public, or taken from the collections of
the National Museum of Wales, Cardiff, UK, where all specimens
are now deposited. The single exception was a Testacella specimen
from the Natural History Museum, London, UK (Table S1).
Specimens for sequencing were selected to cover the widest
possible range of morphology, geography and habitat. This meant
one or at most a few individuals from each population were
selected, increasing the number of populations sampled. Adult
specimens were dissected where necessary and identified with
reference to the taxonomic literature and museum collections.
Inevitably, a small number of juvenile slugs could only be
identified tentatively by morphology and were identified a posteriori
from the DNA results.
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Sequencing
In Arionidae, the modified universal primers 16SAR and
16SBR ([7], modified from [47]) were used to amplify an approx.
440 bp region of the 16S large subunit mitochondrial ribosomal
DNA. This region has been established as the mitochondrial
marker of choice in arionid studies (e.g. [27,28,30,48]). For other
families, the universal primers LCO1490 and HCO2198 [49]
were used to amplify an approx. 650 bp region of the COI gene.
Approximately 2 mm
3
of tail or other tissue from each specimen
was incubated in 1 ml 0.16Tris EDTA (‘‘low TE’’) at 20uC for
30 min to replace preservatives. DNA was extracted with the
Qiagen DNEasy kit with a single elution with 200 ml Buffer AE.
Two ml of this extract was used as template in PCRs using GE
Healthcare illustra PuReTaq PCR beads with 0.25 ml of each
primer (10 mM) and ultra-pure water to a volume of 25 ml. Cycling
conditions (Eppendorf Mastercycler) for COI and 16S were: 94uC
for 2 min 30 s, (94uC for 30 s, 47uC for 45 s, 72uC for 1 min
15 s640 cycles), 72uC for 10 min. Conditions for ITS-1 were:
94uC for 3 min, (95uC for 1 min, 55uC for 1 min, 72uC for
2 min635 cycles), 72uC for 5 min. After visualisation and
quantification on agarose gels, products were cleaned with GE
Healthcare illustra ExoStar 1-Step and sequenced by Euro-
fins|MWG Operon (www.eurofinsgenomics.eu). Sequences were
edited and compiled in BioEdit 7.1.3 [50] and submitted to
GenBank (accession numbers KF894075-KF894388, details in
Table S1).
Assembling datasets
The data were divided into subsets for analysis because: i) two
different mitochondrial fragments were used; ii) several arionid
subgenera or species groups have been the target of previous
genetic work; iii) particular sampling attention had been paid to
the larger Arionidae; and iv) several of the relevant families are not
closely related to one another [51]. For 16S analysis the subsets
were 1) Arion subgenus Arion, including A. flagellus;2)Arion subgenus
Mesarion, in which great intraspecific variability has been found
[25,26] and 3); the remaining, smaller Arionidae. Preliminary
analysis indicated that A. flagellus could have been included in
either dataset 1 or 2; the former was chosen given the suggestion
that this species may hybridise with others in this dataset. Analyses
were repeated on a dataset including all Arionidae. For COI data
the subsets included: 4) Limax; and 5) Other Limacidae. Analyses
were repeated on a dataset including all Limacidae. The
remaining subsets were: 6) Agriolimacidae; 7) Milacidae; 8)
Testacellidae; 9); Boettgerillidae; and 10) Trigonochlamydidae.
All relevant sequences available from members of each family in
GenBank were downloaded and incorporated into each subset (7
Oct 2013; details in Table S2) with the exception of native North
American and Asian arionid genera and the eastern European
limacid genus Bielzia. Some short Limax sequences showing limited
overlap with our dataset, and a possibly misidentified Deroceras
sequence (FJ917286), were excluded from further analysis.
Sequences in each subset were aligned by CLUSTALW in
MEGA 5.1 [52] using default parameters and checked by eye.
Identical sequences were collapsed to haplotypes. Sequences were
realigned for the whole family datasets for Arionidae and
Limacidae.
Analysis
Species were delimited, refined and identified in a three-stage
process. Firstly, putative species were delimited statistically using
the 16S and COI data to produce a primary species hypothesis for
each dataset (PSH). This required no a priori information on the
number of species expected, or how genetically different such
species might be. It used a consistent approach across families and
simultaneously dealt with existing GenBank data from Europe and
beyond. Next, those putative species that included individuals
from Britain and/or Ireland were assessed with other data to
refine each PSH to a secondary species hypothesis (SSH). Finally,
the species accepted in the SSH were identified by comparing
them to the currently known species of the fauna [1].
To examine whether hybridisation has occurred between
species-level lineages in Arion subgenus Arion, a rapidly evolving
nuclear marker was also sequenced from these species. The
primers ITS1 and 5.8C [25] were used to amplify an approx-
imately 570 bp region of the internal transcribed spacer (ITS-1).
These authors considered topological conflict (non-monophyly)
between ITS-1 and 16S data an indicator of hybridisation and
introgression between species-level lineages in subgenus Mesarion
(see also [28,29]).
Table 1. Datasets and species delimitation by ABGD.
Dataset Region Sequences Haplotypes
Max. prior intraspecific K2P
distance over which stable
Number of putative
species (total)
Number of putative species
(Britain/Ireland only)
Arion (Arion) 16S 134 60 0.050 9 6
Arion (Mesarion) 16S 190 160 0.115 15 5
Other Arionidae 16S 185 96 0.038 11 9
All Arionidae 16S 509 316 0.038 34 20
Limax COI 203 179 0.012 27 3
Other Limacidae COI 72 70 0.066 6 6
All Limacidae COI 275 249 0.028 27 9
Agriolimacidae COI 102 80 0.022 9 7
Milacidae COI 30 28 0.007 5 5
Testacellidae COI 22 17 0.200 4 4
Trigonochlamydidae COI 6 2 n/a n/a n/a
Boettgerillidae COI 4 3 n/a n/a n/a
doi:10.1371/journal.pone.0091907.t001
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Table 2. Summary of primary species hypotheses (PSH) and secondary species hypotheses (SSH). Haps., number of haplotypes; Inds., number of individuals.
PSH
PSH generated by
ABGD Haps. Inds. Mean K2P distances Monophyly Morphologically
unique? SSH Name applied to SSH
(whole family ABGD analyses) Whole family Subset
Whole family Subset Intraspecific
Interspecific
(minimum) NJ BI NJ BI External Internal
1 (1A +1B +1C
form a single
PSH)
- 23 46 0.02 0.08 66 0 0 0 X (Split PSH 1) (no SSH)
1A ‘‘ X 20 36 n/a n/a 99 100 100 99 X X 1A Arion (Arion) ater (Linnaeus, 1758)
1B ‘‘ X 2 8 n/a n/a 93 63 93 59 X 1B+1C Arion (Arion) cf. vulgaris Moquin-
Tandon, 1855
1C ‘‘ X 1 2 n/a n/a n/a n/a n/a n/a ? ? ‘‘ ‘‘
2 X X 9 22 0.00 0.07 100 100 100 100 X 2 Arion (Arion) rufus (Linnaeus, 1758)
3 X X 7 8 0.01 0.07 99 100 100 100 ‘‘ 3 Arion (Arion) cf. empiricorum A.
Fe
´russac, 1819
4 X X 14 26 0.00 0.11 100 100 100 100 X X 4 Arion (Arion) vulgaris Moquin-Tandon,
1855
5 X X 3 24 0.00 0.10 100 100 100 100 X X 5 Arion (Arion) flagellus Collinge, 1893
6 X X 51 54 0.01 0.17 100 100 100 100 ? X 6 Arion (Mesarion) fuscus (O. F. Mu
¨ller,
1774)
7 X X 21 47 0.01 0.10 96 100 95 100 X X 7 Arion (Mesarion) subfuscus
Draparnaud, 1805 (two, currently
indistinguishable evolutionary species
[25])
8 X X 44 45 0.01 0.14 100 100 100 100 ‘‘ ‘‘ 8 ‘‘
9 X X 2 2 0.02 0.13 100 100 100 100 X 9+10 Arion (Mesarion) cf. iratii Garrido,
Castillejo & Iglesias, 1995
10 X X 2 2 0.03 0.17 100 100 100 100 ‘‘ ‘‘ ‘‘
11 X X 48 76 0.04 0.20 100 100 100 90 X X 11 Arion (Carinarion) circumscriptus
Johnston, 1828 (including A. (C.)
silvaticus Lohmander, 1937)
12 X X 13 20 0.01 0.20 100 100 100 100 X X 12 Arion (Carinarion) fasciatus (Nilsson,
1823)
13 X X 8 22 0.01 0.22 100 100 100 100 X X 13 Arion (Kobeltia) distinctus J. Mabille,
1868
14 X X 4 10 0.00 0.18 100 100 100 100 X X 14 Arion (Kobeltia) hortensis A. Fe
´russac,
1819
15 X X 7 22 0.01 0.20 100 100 100 100 X X 15 Arion (Kobeltia) intermedius Normand,
1852
16 X X 1 3 0.01 0.13 100 97 100 98 ? X 16 Arion (Kobeltia) occultus Anderson,
2004
17 X X 3 12 0.00 0.13 100 100 100 100 X X 17 Arion (Kobeltia) owenii Davies, 1979
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Table 2. Cont.
PSH
PSH generated by
ABGD Haps. Inds. Mean K2P distances Monophyly Morphologically
unique? SSH Name applied to SSH
(whole family ABGD analyses) Whole family Subset
Whole family Subset Intraspecific
Interspecific
(minimum) NJ BI NJ BI External Internal
18 X X 4 4 0.00 0.14 100 100 100 100 ? X 18 Arion (Kobeltia) cf. fagophilus de
Winter, 1986
19 X - 1 4 0.00 0.07 n/a n/a n/a n/a X X 19 Geomalacus maculosus Allman, 1843
20 X X 23 25 0.01 0.06 99 100 99 100 X X 20 Limax cinereoniger Wolf, 1803
21 X X 21 24 0.00 0.06 99 100 99 100 X X 21 Limax maximus Linnaeus, 1758
22 X X 10 10 0.03 0.08 99 100 99 100 X X 22 Limax cf. dacampi Menegazzi, 1854
23 X X 9 9 0.00 0.11 100 100 100 100 X X 23 Limacus flavus (Linnaeus, 1758)
24 X X 14 14 0.01 0.11 100 100 100 100 X 24 Limacus maculatus (Kaleniczenko,
1851)
25 X X 26 26 0.01 0.14 100 100 100 100 X X 25 Lehmannia marginata (O. F. Mu
¨ller,
1774)
26 X X 8 9 0.01 0.08 100 100 100 100 X 26 Ambigolimax valentianus (A. Fe
´russac,
1822)
27 X X 8 8 0.02 0.08 83 99 97 91 X 27 Ambigolimax nyctelius (Bourguignat,
1861)
28 X X 5 5 0.01 0.13 100 100 100 100 X X 28 Malacolimax tenellus (O. F. Mu
¨ller,
1774)
29 X n/a 8 8 0.02 0.05 99 100 n/a n/a X 29 Deroceras agreste (Linnaeus, 1758)
30 X n/a 11 14 0.00 0.05 99 84 n/a n/a X 30 Deroceras reticulatum (O. F. Mu
¨ller,
1774)
31 X n/a 21 26 0.02 0.09 99 100 n/a n/a X 31 Deroceras invadens Reise et al., 2011
32 X n/a 9 11 0.01 0.08 99 100 n/a n/a X 32 Deroceras panormitanum (Lessona &
Pollonera, 1882)
33 X n/a 7 10 0.01 0.05 99 100 n/a n/a X 33+34+35+36 Deroceras laeve (O. F. Mu
¨ller, 1774)
(multiple lineages; further study
required)
34 X n/a 4 5 0.00 0.06 99 100 n/a n/a ‘‘ ‘‘ ‘‘
35 X n/a 4 4 0.01 0.05 99 100 n/a n/a ‘‘ ‘‘ ‘‘
36 X n/a 12 20 0.02 0.05 57 0 n/a n/a ‘‘ ‘‘ ‘‘
37 X n/a 4 4 0.01 0.11 100 100 n/a n/a X X 37 Milax gagates (Draparnaud, 1801)
38 X n/a 10 11 0.00 0.10 100 91 n/a n/a X 38 Tandonia budapestensis (Hazay, 1881)
39 X n/a 3 3 0.04 0.11 99 97 n/a n/a X 39 Tandonia cf. cristata (Kaleniczenko,
1851)
40 X n/a 10 10 0.03 0.10 98 100 n/a n/a X X 40 Tandonia sowerbyi (A. Fe
´russac, 1823)
(may include T. marinellii Liberto et al.,
2012)
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Primary species hypotheses
PSHs were generated with the distance-based method ABGD
[38]. Briefly, this divides a set of aligned sequences into groups,
statistically inferring the groups most likely to correspond to
species. It does so recursively, using a range of potential maximum
pairwise intraspecific distances inferred from the data. It produces
an array of PSHs with different numbers of groups delimited using
different distance values. For example, very small or large values
inevitably delimit unrealistically many or few species [38,39]. To
avoid having to discuss all PSHs produced, some criterion is
required to select which of them to investigate further. We chose
the PSH corresponding to the smallest number of groups that was
stable over three or more successive distance values. When the
initial and recursive partitions indicated different numbers of
groups at the same distance value, we chose the smaller of the two.
This criterion had the advantages of being i) independent of the
expected number of species; ii) consistent across different datasets;
and iii) conservative in terms of the numbers of putative species
recognised.
ABGD analyses were run at http://wwwabi.snv.jussieu.fr/
public/abgd/abgdweb.html on aligned haplotype sequences using
K2P distances. To ensure a wide range of distance values were
investigated, Pmin was kept at 0.001 but the maximum potential
Pmax was raised from 0.1 to 0.2. To allow comparison between a
greater number of alternative distance values, the number of steps
was raised from 10 to 20. Other settings were as default (relative
gap width, X = 1.5; number of bins = 20).
Secondary species hypotheses
The SSH for each dataset consisted of an equal or smaller
number of species, some putative species being combined in order
to remain conservative. In the sole case where ABGD delimited a
putative species in one analysis (Arionidae) but split it in the subset
analysis (Arion (Arion)), both alternatives were considered (see
Results and Discussion).
As a result of the ABGD analysis, putative species were by
definition genetically distinct from their close relatives. To be
considered a species under the SSH, each putative species had also
to show evidence of reciprocal monophyly with respect to a sister
group, so being at least a phylogenetic species. This was
investigated and visualised using neighbour-joining trees based
on the K2P distance, with support for monophyly quantified with
1000 bootstrap pseudoreplicates in MEGA. Gaps were treated by
pairwise deletion. As a comparison using a character-based
method involving a model of sequence evolution, Bayesian
Inference (BI) was also used, with support for monophyly
quantified with posterior probabilities. FindModel (www.hiv.lanl.
gov/content/sequence/findmodel/findmodel.html) was used to
select the most appropriate evolutionary model (GTR +I+Cin
each case) according to log likelihood and the Akaike information
criterion. BI was implemented in MrBayes v3.1.2 [53] with two
parallel runs of 5,000,000 generations, sampling trees every 1000
generations, with the first 25% of the trees discarded as burn-in
and other settings as default. Convergence on a stable log
likelihood before the burn-in period was evident in all analyses.
These methods were also used to analyse the ITS-1 data from the
Arion (Arion) species.
Morphological distinctness was also considered as a criterion for
the recognition of species in the SSH. The external and internal
(adult genital) morphology of each putative species was investi-
gated using standard techniques. Putative species that were closely
related (i.e. showed evidence of joint monophyly) but did not differ
in morphology were generally not recognised as separate species in
the SSH. However, it became clear that in two cases (interpreted
Table 2. Cont.
PSH
PSH generated by
ABGD Haps. Inds. Mean K2P distances Monophyly Morphologically
unique? SSH Name applied to SSH
(whole family ABGD analyses) Whole family Subset
Whole family Subset Intraspecific
Interspecific
(minimum) NJ BI NJ BI External Internal
41 X n/a 1 2 0.00 0.19 n/a n/a n/a n/a X X 41 Tandonia rustica (Millet, 1843)
42 X n/a 5 7 0.01 0.22 100 100 n/a n/a X X 42 Testacella haliotidea Draparnaud, 1801
43 X n/a 5 8 0.00 0.22 100 100 n/a n/a X X 43 Testacella maugei (A. Fe
´russac, 1819)
44 X n/a 2 2 0.00 0.20 100 100 n/a n/a X X 44 Testacella scutulum G. B. Sowerby I,
1821
45 X n/a 5 5 0.00 0.20 100 100 n/a n/a X X 45 Testacella cf. scutulum G. B. Sowerby I,
1821
n/a n/a n/a 2 4 0.00 n/a n/a n/a n/a n/a X X (46) Boettgerilla pallens Simroth, 1912
n/a n/a n/a 2 6 0.00 n/a n/a n/a n/a n/a X X (47) Selenochlamys ysbryda Rowson &
Symondson, 2008
doi:10.1371/journal.pone.0091907.t002
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as evidence of introgression, see below) there was conflict between
putative species assignment and morphology. Thus the criterion of
morphological distinctness, though useful, could not be consis-
tently applied. To do so would also preclude the recognition of
genuinely cryptic species ([54]) as SSH species, should either
occur.
Refinement of PSHs to SSHs by this method is an example of
integration of molecular with other data, here specifically of
‘‘integration by congruence’’ in the sense of Padial et al. [55]. This
approach requires congruence of (for example) mtDNA and
morphology for species recognition. According to Padial et al.
[55], integration by congruence is less likely to overestimate species
numbers and better promotes future taxonomic stability than the
alternative, integration by cumulation, which requires no initial
congruence between datasets.
Comparison of the SSH to the known fauna
The species in the SSH were then compared to the species
making up the known fauna of Britain and Ireland [1]. For most
SSH species this was straightforward from morphological identi-
fications already made, and/or the inclusion of GenBank
sequences in each PSH. Others required recourse to museum
collections and the wider taxonomic literature.
Results and Discussion
450 sequences were obtained from 388 individuals (Table S1)
and compared with 659 sequences from GenBank (Table S2),
representing 695 haplotypes in all. For all three gene regions, some
haplotypes were found at more than one site. The three most
Figure 1. Larger Arionidae (
Arion
subgenus
Arion
). Midpoint-rooted NJ tree based on 16S data; values above branches are % bootstrap support
($75), those below are Bayesian posterior probabilities, expressed as % ($80). Grey bars indicate clades. Species new to the fauna of Britain and/or
Ireland are indicated by *.
doi:10.1371/journal.pone.0091907.g001
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Figure 2. Arionidae:
Arion
subgenus
Mesarion
.Midpoint-rooted NJ tree based on 16S data; values above branches are % bootstrap support (NJ),
those below are Bayesian posterior probabilities, expressed as %. Grey bars indicate clades. Species new to the fauna of Britain and/or Ireland are
indicated by *.
doi:10.1371/journal.pone.0091907.g002
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Figure 3. Other Arionidae. Midpoint-rooted NJ tree based on 16S data; values above branches are % bootstrap support (NJ), those below are
Bayesian posterior probabilities, expressed as %. Species new to the fauna of Britain and/or Ireland are indicated by *.
doi:10.1371/journal.pone.0091907.g003
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Figure 4. Limacidae (genus
Limax
). Midpoint-rooted NJ tree based on COI data; values above branches are % bootstrap support (NJ), those below
are Bayesian posterior probabilities, expressed as %. Species new to the fauna of Britain and/or Ireland are indicated by *.
doi:10.1371/journal.pone.0091907.g004
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extreme examples were 16S haplotypes attributed to A. (A.)
flagellus,A. (A.) ater, and A. (A.) vulgaris (22, 11 and 9 individuals
respectively) that were found throughout Britain and Ireland.
Primary species hypotheses
The PSHs generated by ABGD analyses and selected
according to our criteria are summarised in Table 1. Histograms
Figure 5. Other Limacidae. Midpoint-rooted NJ tree based on COI data; values above branches are % bootstrap support (NJ), those below are
Bayesian posterior probabilities, expressed as %. Species new to the fauna of Britain and/or Ireland are indicated by *.
doi:10.1371/journal.pone.0091907.g005
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and graphs illustrating the full range of PSHs generated
are given in Figs. S1–S3. Variation was too limited in
Boettgerillidae and Trigonochlamydidae to conduct the
analysis (mean intraspecific K2P distance 0.001 and 0.000
respectively) so each was considered to comprise a single
species.
Figure 6. Agriolimacidae. Midpoint-rooted NJ tree based on COI data; values above branches are % bootstrap support (NJ), those below are
Bayesian posterior probabilities, expressed as %. Species new to the fauna of Britain and/or Ireland are indicated by *.
doi:10.1371/journal.pone.0091907.g006
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The maximum prior intraspecific K2P distance over which the
number of putative species was stable varied considerably between
taxa (0.038–0.115 in Arionidae, 0.012–0.200 in other families)
(Table 1). Above this value, the number of putative species
delimited fell rapidly (Figs. S1–S3). The exception was Milacidae
where it rose from 5 to 8 and was again stable up to K2P 0.038,
although our conservative criteria selected the former value.
Compared to the whole family analysis, subset analyses generated
more putative species in total in Arionidae (34 versus 35), and
fewer putative species in total in Limacidae (27 versus 33).
However, these differences were due to the differential splitting of
continental putative species (e.g. several provisionally recognised
Limax species). In both families, the total number of putative
species occurring in Britain and Ireland was identical in the whole
family and subset analyses (Table 1). The overall PSH, comprising
the selected PSH from each of the subset analyses, delimited 45
putative species for Britain and/or Ireland, plus one species each
from Boettgerillidae and Trigonochlamydidae.
Secondary species hypotheses
Each of the 45 putative species was represented by at least two
sequences and most comprised several haplotypes and/or
sequences from more than one locality (Table 2). Almost all were
identically delimited, genetically distinct, and monophyletic
(Figs. 1–8; whole family trees available on request). All but two
putative species were identically delimited in the whole family and
subset analyses. One was PSH 19, G. maculosus, in which the Irish
and Spanish haplotypes formed a single putative species in the
whole family analysis, and two in the subset. The other was in the
Arion (Arion) dataset, where PSH 1 was delimited in the ABGD
analysis of all Arionidae, but split into PSH 1A, PSH 1B, and PSH
1C in the subset.
Figure 7. Milacidae & Testacellidae. Midpoint-rooted NJ trees based on COI data; values above branches are % bootstrap support (NJ), those
below are Bayesian posterior probabilities, expressed as %. Species new to the fauna of Britain and/or Ireland are indicated by *.
doi:10.1371/journal.pone.0091907.g007
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Figure 8. Arionidae. Midpoint-rooted BI tree based on ITS-1 data; values above branches are % bootstrap support ($75), those below are Bayesian
posterior probabilities, expressed as % ($85).
doi:10.1371/journal.pone.0091907.g008
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All putative species were genetically highly distinct. Where more
than one haplotype was found the minimum mean interspecific
distance was always 2.5 or more times greater, often tens of times
greater, than the mean intraspecific distance. The intraspecific
distance was lower than the prior value given by ABGD in all cases
except two putative species in the Milacidae (PSH 39 and PSH 40)
in which it slightly exceeded it (Table 2). This may be a result of
the limited data available on this family, whose ABGD analysis
was also unique in offering an alternative, yet larger, stable
number of putative species. Under this alternative PSH, for
example, Italian sequences including Tandonia marinellii Liberto et
al. [56] would be separated from T. sowerbyi (Fe´russac). However,
our criterion favours more the conservative PSH.
Almost all putative species with more than one haplotype were
strongly supported as monophyletic in all NJ and BI analyses.
Again the exception was PSH 1: there was very weak support for
the monophyly of PSH 1 as a whole, yet support for the
monophyly of PSH 1A and a clade comprising PSH 1B +PSH
1C.
Most (35) of the putative species were morphologically unique,
externally and/or (in adult or near-adult) specimens, internally.
Most of the remainder were in Arionidae: PSH 1, whose
constituents PSH 1A and PSH 1B were each unique; PSH 1C,
represented by a single GenBank haplotype from the USA (and
discussed because of its delimitation as part of PSH 1); PSH 3,
represented in Britain only by a juvenile; PSH 7 and PSH 8, which
both corresponded to A. (M.) subfuscus (Draparnaud) but could not
be satisfactorily distinguished from one another even using the
genital features of [28]; and PSH 9 and PSH 10 which both
corresponded to A. (M.) iratii Garrido et al. but could not be
satisfactorily distinguished from one another. In Agriolimacidae,
the four PSHs into which Deroceras laeve (Mu¨ller) was split could not
be satisfactorily distinguished, partly because most specimens were
aphallic. More detailed discussion of morphological features,
geographical distribution, and identification of certain putative
species is given below.
In consequence, 36 of the 45 putative species in the PSH were
readily accepted as species in the SSH we propose. One of the
remainder, PSH 1, was split into two, SSH 1A and SSH 1B +1C,
because of its lack of monophyly and morphological heterogeneity.
PSH 7 and PSH 8, although morphologically indistinguishable,
were maintained as separate because together they were not
monophyletic in any analysis. In contrast, and to remain
conservative, two sets of other morphologically indistinguishable
putative species were combined (PSH 9 +PSH 10, and PSH 33 +
PSH 34 +PSH 35 +PSH 36). The SSH we propose thus
recognises a total of 42 species, or 44 including Boettgerillidae and
Trigonochlamydidae.
Comparison of the SSH to the known fauna
Of the 44 SSH species, 32 included one or more GenBank
sequence. The remaining 12 had presumably not previously been
sequenced for the gene region in question. As expected, many
species in each category (24 in the former and 9 in the latter,
totalling 33) could readily be considered equivalent to known
species in the British and Irish fauna [1] (Table 2, and names on
Figs. 1–8). This was consistent with the morphological features we
used to identify specimens and, in general, the names of previously
identified GenBank sequences. For example, PSH 4 consists of
sequences of A. (A.) vulgaris (or ‘‘A. lusitanicus’’ non Mabille) from six
continental countries, and from specimens from around Britain
and Ireland whose morphology conforms to that species (e.g.
[21,42]). This confirms it is widespread in Britain and Ireland,
including in SW England where it has been recorded since at least
the 1960s [2,21]). Most of the remaining 32 British and Irish SSH
species correspond to other widespread and relatively well-
characterised species. Two species described from Ireland, G.
maculosus and A. (A.) flagellus, are each moderately closely related to
Spanish haplotypes (Fig. 1). We note however that unless the Irish
haplotypes are detected in Spain, neither native status or ancient
introduction can be ruled out (e.g. see [57,58]). For brevity, we do
not discuss the currently known SSH species further, except for
those which appear to show evidence of hybridisation (see below).
This leaves 11 SSH species needing further discussion. In one
case, a single SSH species corresponded to more than one known
species: A. (Carinarion) circumscriptus Johnston and A. (C.) silvaticus
Lohmander. Together these formed a single putative species, PSH
11 which was monophyletic (Fig. 3). Both names were thus
associated with the single species SSH 11, although within it, the
British and Irish haplotypes identified as A. (C.) circumscriptus and A.
(C.) silvaticus clustered in separate monophyletic groups. This is
consistent with the findings of Geenen et al. [27] who suggested
the three widespread Carinarion taxa be considered a single
biological species. However, like them we found that the third of
these, A. (C.) fasciatus (Nilsson) (SSH 12) was considerably more
distinct than the others and more reliably identifiable morpho-
logically. Given this and the evidence of habitat separation
between A. (C.) circumscriptus and A. (C.) silvaticus in Britain and
Ireland [2] we suggest these two be treated as a single species,
perhaps with two recognised subspecies. As A. (C.) fasciatus is more
distinct and formed a separate PSH we retain it as a species.
In another case, two morphologically indistinguishable SSH
species corresponded to a single known species. SSH 7 and SSH 8
each corresponded to specimens identified as A. (M.) subfuscus,it
being uncertain to which (if either) the name should be
preferentially applied [26]. Indeed Pinceel and others [25,26,28]
found this taxon to consist of multiple deeply divergent 16S
lineages which they attributed to allopatric divergence and an
accelerated rate of mutation. This included two present in Britain
and Ireland (their S1 and S2 [25]) that correspond to our SSH 7
and SSH 8. As there was some evidence of interbreeding between
these they treated them as evolutionary species requiring further
revision before being named, as was possible with A. (M.)
transsylvanus Simroth, endemic to Romania and Poland [28]. We
found limited agreement between SSH 7 and SSH 8 and the
subtle morphological characters of S1 and S2 offered by [28]. All
S2 specimens had genitalia corresponding to these in that the
epiphallus joined the atrium between the bursa and oviduct, but
this pattern was also seen in some S1 specimens. S2 was found
only in northern, western and upland areas (Table S1) although
has also been found sympatric with S1 in central and southeast
England by [25]. Until further data are available we treat both as
part of the still-enigmatic taxon A. (M.) subfuscus.
This left eight SSH species that did not correspond to any of the
36 species in [1], 35 of which were themselves successfully
delimited using the same criteria. This represents an increase of
22% on the known fauna, a striking and unexpected finding that
led to further investigation of these ‘‘additional’’ species.
Additional species
Of the eight additional SSH species, four were in Arionidae and
one each in Limacidae, Agriolimacidae, Milacidae and Testacelli-
dae. Up to four of the species appear previously undescribed, but
we refrain from formal description here until further data are
available. We provide brief diagnoses and use the term ‘‘cf.’’
(confer) to indicate a nominal species to which the species should
be compared.
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Figure 9. Morphology of four potentially new species. External appearance and salient parts of genitalia shown alongside those from
sequenced similar species for comparison. Abbreviations: at, atrium; bc, bursa copulatrix; ep, epiphallus; ov, oviduct; pe, penis; pr, penial retractor
muscle.
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We note that none of the eight additional species are
morphologically cryptic, at least not by the strictest of definitions,
i.e., they are not impossible to reliably identify based on
morphology alone [54]. They could therefore have been
recognised without sequence data and so can be considered
previously overlooked. We also note that six of them were found at
more than one widely separated site, with two occurring in both
Britain and Ireland. The introduced or native status of each
remains open to question. The remaining two, as yet known only
from single established populations, are almost certainly accidental
introductions.
SSH 1B
+
1C: Arion (Arion) cf. vulgaris Moquin-Tandon,
1855. Potentially a new species. Each of the SSH species in Arion
subgenus (Arion) (with the exception of A. (A.) flagellus) was
approximately equally genetically distant from one another. SSH
1B was found at four sites in the east of England, from Yorkshire to
Kent, with another haplotype near London (a museum specimen
collected in 2004). They were mainly from disturbed habitats.
Externally the animal is variable and readily confused with either
A. (A.) vulgaris (SSH 4) or A .(A.) rufus (SSH 2). It is generally greyish
brown, with faint lateral colour bands persisting in some adults,
and the sole is often paler than typical A. (A.) vulgaris. It lacks the
rocking response of A. (A.) ater (SSH 1A) and A. (A.) rufus (e.g. [12]).
Internally it is very distinct from both A. (A.) ater and A. (A.) rufus,
having an elongate corrugated ligula in the oviduct, generally like
that of A. (A.) vulgaris. However, the ligula is delicately fringed for
all or part of its length as well as being corrugated (Fig. 9). These
delicate fringes were not found in the sequenced A. (A.) vulgaris
specimens. The correct name for this species is difficult to establish
at present (see also SSH 3, and the section on hybridisation below)
and more data on whether other populations can be consistently
distinguished from A. (A.) vulgaris is desirable. It appears not to
correspond with any of the four species discussed in the arionid
review by Jordaens et al. [29] whose ‘‘non-Iberian A. lusitanicus’’
presumably corresponds to A.(A.) vulgaris. Neither ITS-1 nor 16S
data suggest a close relationship between our species and A. (A.)
flagellus, or to the true Portuguese A. lusitanicus Mabille (Fig. 8). It is
possible that some of the British ‘‘A. lusitanicus’’ populations studied
by Davies [21] in her initial discrimination of these from A. (A.)
flagellus may belong to this species, especially given inconsistencies
in the mating behaviour recently highlighted by Dreijers et al.
[45]. However, our data suggest that A. (A.) vulgaris itself (SSH 4),
being much more widespread in Britain and Ireland, is also likely
to have been present in Surrey and the other areas discussed by
Davies [21]. It is unknown where else in Europe our species occurs
and whether it is native to Britain, there being a lack of data from
central and southern France in particular. Noble & Jones [17]
discussed a ‘‘non-pest’’ form of A. (A.) vulgaris (again as ‘‘A.
lusitanicus’’) from southern France while Noble [42] discussed
alternative Pyrenean forms that might correspond to this species.
The most similar available 16S sequences are those recognised in
some analyses as PSH 1C (Fig. 1). These are from ‘‘Arion rufus’’
from the west coast of the USA (Washington State: [30]), whose
morphology may deserve further investigation.
SSH 3: Arion (Arion) cf. empiricorum A. Fe
´russac
1819. Sequences of this SSH species have been identified as
‘‘Arion rufus’’ from Belgium, Denmark, and (as introduced) Canada
and the USA. In Britain it was represented by a single juvenile
from a cemetery in central London identified as (and sympatric
with) A.(A.) vulgaris, whose juveniles can look very similar. It has
been proposed [59] that the name A. (A.) rufus Linnaeus be applied
to the species occurring in Britain (presumably the widespread and
common SSH 2 in our study). Alternative names available for
similar continental species include Arion empiricorum Fe´russac, [1],
but this cannot be resolved here. The London population is likely
to be an introduction and as with SSH1B +1C, should be
considered a potential plant pest.
SSH 9
+
10: Arion (Mesarion) cf. iratii Garrido, Castillejo
& Iglesias, 1995. This SSH combines PSH 9 and PSH 10, both
from upland forestry areas in the Brecon Beacons National Park
and South Wales valleys. They could not be distinguished
morphologically but both differ from all sampled British and Irish
A. (M.) subfuscus and A. (M.) fuscus in having dark spots or speckles
on the back in at least some individuals. This feature is shared with
three species described by Garrido et al., 1995 [60] from the
Pyrenees which differ from each other only very subtly: A. (M.)
iratii,A. (M.) lizarrusti, and A. (M.) molinae. Given the accelerated
rate of 16S evolution posited for Mesarion species [25] it is not clear
whether the constituent PSHs should be combined in one SSH or
not, but analyses suggest both are related to A. (M.) iratii and A.
(M.) lizarrusti rather than to A. (M.) subfuscus, A. (M.) fuscus,A. (M.)
transsylvanus etc. (Fig. 2). Neither Wales nor the Pyrenees were
represented in the analysis of Pinceel et al. [28,29]; indeed they
noted that sampling in Iberia had yielded none of the A. (M.)
subfuscus lineages S1–S5. This SSH is therefore provisionally
associated with A. (M.) iratii. Its introduction to Britain with
forestry or industry cannot be ruled out, but neither can a native
distribution that includes the highest uplands in southern Britain.
SSH 18: Arion (Kobeltia) cf. fagophilus (de Winter,
1986). Potentially a new species. Genetically it is distinct from
all British and Irish Kobeltia and from the central European A. (K.)
obesoductus Reischutz (formerly A. (K.) alpinus Pollonera) (Table 2,
Fig. 3). Morphologically it is most similar to A. (K.) occultus
Anderson from Northern Ireland and A. (K.) fagophilus de Winter
from the western Pyrenees. Externally, the coarser tubercles
distinguish the new species from all British or Irish Kobeltia except
A. (K.) occultus. The latter is yellower and flatter, with even coarser
tubercles. Both resemble A. (K.) fagophilus externally except in
tentacle colour, which is bright red in A. (K.) fagophilus [61] versus
cold blue-black in these species. Davies [20] considered tentacle
colour one of the most reliable external features distinguishing
Kobeltia species. Internally, this species lacks an epiphallus process
and differs from A. (K.) fagophilus and A. (K.) occultus in having a
donut-like swelling at the entrance of the bursa copulatrix to the
atrium, rather than a thickening as in the other species (Fig. 9)
[23,61]. It is common at several sites in lowland river valleys in
South Wales, in wet Alnus woodland. It is probably an introduction
from the Pyrenees, although this small and indistinct species could
be an overlooked native restricted to this relatively natural habitat
type.
SSH 22: Limax cf. dacampi Menegazzi, 1854. Potentially
a new species. Genetically it is clearly not part of the two known
British and Irish species which form separate PSHs (Table 2,
Fig. 4). It forms a clade (Fig. 4) with sequences from the Apennines
of central Italy to which no species name is applied, and another
group comprising sequences from Italy, Switzerland and Croatia
that depositors identified as Limax cf. dacampi Menegazzi [32]. In
the whole family ABGD analysis, both clades form a single PSH
but form two PSHs in the analysis of Limax only. In Britain it was
found at a single site in North-east Yorkshire, in an ornamental
mixed woodland in the grounds of a school. Adults and juveniles
have been found there on several occasions over the past two years
(A. Norris & T. Crawford, pers. comm.). The species is externally
and internally distinct from the other British species (e.g. as
monographed by Quick [12]), notably in having a much longer
penis than either (both in absolute terms and in being longer than
the body even when retracted) (Fig. 9). It is almost certainly an
introduction from central Italy.
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SSH 32: Deroceras panormitanum (Lessona & Pollonera,
1882). An established population in a public city garden in
South Wales has the internal anatomy of this Sicilian/Maltese
species (one blunt penial appendage, one tapering) and an almost
identical COI haplotype (Fig. 6). This species and its relatives were
recently thoroughly revised based on molecular, morphological
and behavioural data [33]. Its external morphological variation
overlaps with that of the widely introduced D. invadens Reise et al.,
so it may have been overlooked elsewhere. In Britain, it is
probably a recent introduction.
SSH 39: Tandonia cf. cristata (Kaleniczenko,
1851). Found in South Wales and western Ireland in allotments,
adjacent woodlands and a churchyard. Museum collections
suggest that it is more widespread than our data indicate (although
we could not amplify DNA from these). It has evidently been
misidentified as juvenile T. budapestensis (Hazay), T. sowerbyi or
Milax gagates (Draparnaud) in the past but is genetically distinct
from these (Fig. 7). It differs from these in its smaller size, generally
paler colour, plain sole, and in having dark pigment confined to
the spaces between tubercles. Using Wiktor’s key [62] it keys to
Tandonia cristata (Kaleniczenko), a species of countries bordering
the Black Sea. There is some discrepancy between accounts of the
internal anatomy of this species [62,63], and there are many
similar species. Until these can be better studied we consider the
British and Irish species likely to represent T. cristata, which would
be introduced. It is potentially a root crop pest like other Tandonia
species.
SSH 45: Testacella cf. scutulum G. B. Sowerby I,
1821. Potentially a new species. Genetically it is as distinct from
T. scutulum Sowerby as the other two Testacella species are from one
another (Fig. 7), and forms a PSH separate to them in delimitation
analysis (Table 2). Morphologically, it is close only to T. scutulum
with which it keys out in the synoptic key of Nardi & Bodon [64].
However, the two differ in the width of the penis (apparently
independently of the size of the animal) and in whether or not the
penis tapers (Fig. 9). Externally, they differ in body colour and in
the shape formed by the meeting of the two dorsal grooves,
although these features might not be consistent. The subterranean
Testacellidae are rarely collected so only limited material is
available and intraspecific morphological variation remains poorly
understood [65] (although there is abundant evidence that T.
haliotidea Draparnaud and T. scutulum are distinct, contrary to [2]).
It also remains to be established to which of the two similar species
Sowerby’s name ought to be restricted. Early descriptions of the
anatomy of T. scutulum from near the type locality in London
[11,66] show a broad, tapering penis. Both species are probably
introductions from the Mediterranean [2]; recent figures suggest
both may occur in Italy [67].
Hybridisation
Here, intraspecific hybridisation is considered the exchange of
genes between PSHs that would otherwise be considered
phylogenetic species, and would otherwise correspond precisely
to morphologically distinct and widely-recognised species (e.g.
‘‘A.(A.) vulgaris’’). Although limited, our data suggest that hybrid-
isation may have occurred in the larger Arionidae. More
unexpectedly, contrasts between mtDNA and morphology also
suggest hybridisation in Agriolimacidae and Limacidae.
Interspecific hybridisation has long been suspected, if not fully
proven, among the large Arionidae, in particular between A. (A.)
ater and A. (A.) rufus and between one or both of these and A. (A.)
vulgaris (e.g. [15,42,43,44,68,69]). Recently, Dreijers et al. [45]
showed experimentally that German A. (A.) vulgaris (as A. lusitanicus)
and German A. (A.) rufus can reciprocally exchange sperm. ITS-1
sequences showed far less variability than mtDNA sequences. NJ
and BI analyses show that all five SSHs in the larger Arionidae
(SSHs 1A, 1B, 2, 3 and 4) form a single clade without internal
structure, to the exclusion of A. (A.) flagellus (SSH 5) and all other
Arionidae (Fig. 8). Similar conflicts between mtDNA and ITS-1
data have been interpreted as evidence of hybridisation or a
shared nuclear gene pool in arionid studies [25,27]. By these
criteria, hybridisation might well have occurred between any
combination of the five SSHs in the larger clade, either before or
since their arrival in Britain or Ireland. In contrast, A. (A.) flagellus
is genetically distinct from any of them in both mtDNA and ITS-1.
We conclude that evidence for its hybridisation with other British
or Irish species is lacking.
Morphologically, we found that all adult specimens we
sequenced could be referred to one of the known British species
in the large Arionidae, or the newly-discovered SSH 1B, and were
unconvinced that any individual was indisputably hybrid. We
found no specimens whose species assignment by morphology was
incompatible with their assignment by mtDNA (unlike the non-
arionid examples below). It has been suggested that hybrids are
morphologically recognisable [17,42,68] but this was not clarified
by our data. It remains possible that hybrids in this group
(especially after the F1 generation) could resemble either parent
species and be effectively impossible to distinguish from them
morphologically [46]. Elsewhere in Europe, such hybrids might
account for the inclusion of GenBank haplotypes of Belgian,
Polish, Spanish and Czech ‘‘A. rufus’’, and Polish ‘‘A. lusitanicus’’ in
our PSH 1A. We nonetheless remain confident that this SSH be
called A. (A.) ater, it being the only SSH that included jet-black
individuals from remote western and upland regions, considered
typical of A. (A.) ater [11,17,42,68] (Table S1). The identities of the
other SSHs in the group are discussed above. While the taxonomy
may be revised in future, we provisionally recognise five species in
this group in the British fauna, three of which occur in Ireland (not
including A. (A.) flagellus).
In Agriolimacidae, in ABGD, NJ and BI analyses (Fig. 6)
Deroceras reticulatum (Mu¨ller) and D. agreste (Linnaeus) formed two
PSHs/clades. Using the internal morphological criteria of Wiktor
[70] however, neither morphospecies was monophyletic and so
they cannot be accepted as separate SSHs here unless some
individuals are considered hybrids. All individuals with D. agreste
anatomy had a D. agreste mtDNA sequence, but some individuals
with D. reticulatum anatomy had a D. agreste mtDNA sequence.
These were largely from the same sites as the D. agreste individuals,
or nearby. The pattern could be explained by one-way introgres-
sion of D. reticulatum genes into D. agreste populations. As mtDNA is
maternally inherited in pulmonates, the D. reticulatum individuals in
the D. agreste clade would be introgressed hybrids descended from a
D. agreste mother whose eggs were fertilised by D. reticulatum sperm.
That no D. agreste individuals with D. reticulatum mtDNA were
found may indicate that fertilisation in the opposite direction has
not occurred, is rarer, or results in sterile offspring. One-way
interspecific sperm transfer is known in other Deroceras species [33].
In Britain and Ireland D. agreste is a rare, northerly distributed
post-glacial relict species characteristic of uplands and other wild
habitats, while D. reticulatum is a common, widespread and often
synanthropic species [2,13]. If the speculation is correct, this
interaction between the species could partly explain their habitat
and range discrepancies, with the post-glacial D. agreste in long-
term retreat exacerbated by hybridisation with D. reticulatum.
Similarly in Limacidae, in ABGD, NJ and BI analyses (Fig. 5)
Limacus flavus (Linnaeus) and L. maculatus (Kaleniczenko) formed
two PSHs/clades. Again, using internal morphological criteria
[71] neither morphospecies was monophyletic.The only morpho-
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logically typical adult L. flavus (i.e. one with the bursa copulatrix
duct originating high up on the vagina; [71]) was in PSH 23 in
which all other specimens either had immature genitalia or were
typical of L. maculatus (i.e. with the bursa duct obtaining from the
base of the vagina, atrium, or base of the penis). Many of them
resembled L. maculatus externally [13,14]. All specimens in PSH 24
resembled L. maculatus internally and externally, including one
from near the type locality in Crimea, Ukraine. GenBank
sequences attributed to L. flavus were split between the two clades,
with continental European sequences [31] in the L. flavus clade and
British sequences in the L. maculatus clade. The pattern can be
interpreted in a similar way to D. agreste/reticulatum. Neither Limacus
species is considered native although L. flavus has been known in
Britain since at least 1685 [2,8] and was the only Limacus known in
Ireland in 1891 [10]. Anecdotal evidence and the increasing
frequency of L. maculatus being recorded in Britain and Ireland
(e.g. [72], also personal observations) suggest it is becoming
common and widespread while L. flavus becomes rarer. One-way
introgression from the more recent invader could explain this
trend.
Conclusion
This study demonstrates how a simple, geographically extensive
yet morphologically informed approach to DNA sampling can be
effective in screening a fauna for additional taxa and other changes
resulting from past invasions. The detection of so many additional
species, and the evidence for hybridisation in Agriolimacidae and
Limacidae, were unexpected. The proportion (22%) of additional
species revealed is remarkably high compared to other recent
studies and given the long history of study of this fauna. For
example, no additional taxa were found in DNA barcoding
surveys of the complete Irish solitary bee fauna of 55 species [73]
or the Welsh native and archaeophyte angiosperm and conifer
flora of over 1000 species [74]. Our estimates also exceed the
proportion of cases of deep intraspecific divergence (i.e. potential
additional species) found and discussed among Bavarian myria-
pods (3% of 122 species [75]), Romanian butterflies (4% of 180
species [76]) or Bavarian geometrid moths (5% of 400 species
[77]). With the exception of [75] these studies were largely based
on existing museum collections so may have overlooked additional
taxa in the field, but as we found such taxa may also be present in
existing collections as in the case of Tandonia cf. cristata. Only
among British and Irish earthworms [78] and Scottish tardigrades
[79] have similar studies reported as high a proportion of
additional putative taxa. These are groups with a much less rich
history of taxonomic description, participatory study and record-
ing than slugs, and do not include as many potentially serious
pests. Indeed, further undetected slug species may already be
lurking, particularly in large urban areas which our survey could
not thoroughly cover. We conclude that although the British and
Irish slug fauna is well-studied, it was far from fully-known, even as
recently as 2008 [1]. If further invasions are to be detected or
controlled, this and other slug faunas worldwide will need to be
watched more closely in future.
Supporting Information
Figure S1 ABGD analysis for Arionidae. Arrow indicates
selected PSH and its corresponding position on the distribution of
pairwise K2P distances.
(TIF)
Figure S2 ABGD analysis for Limacidae and Agriolima-
cidae. Arrow indicates selected PSH and its corresponding
position on the distribution of pairwise K2P distances.
(TIF)
Figure S3 ABGD analysis for Milacidae and Testacelli-
dae. Arrow indicates selected PSH and its corresponding position
on the distribution of pairwise K2P distances.
(TIF)
Table S1 Specimen data, haplotypes, and GenBank
accession numbers.
(XLS)
Table S2 GenBank data and haplotypes included in
analyses.
(XLS)
References S1 Additional references cited in Table S2.
(DOC)
Acknowledgments
Among the many people who assisted with fieldwork or provided
specimens we thank in particular Chris du Feu, Adrian Norris, Simon
Allen, Adrian Sumner, Ron Carr and Christian Owen. We thank Nico
Puillandre for advice on ABGD and two reviewers for comments on the
manuscript.
Author Contributions
Conceived and designed the experiments: BR RA JAT WOCS. Performed
the experiments: BR RA JAT WOCS. Analyzed the data: BR RA JAT
WOCS. Contributed reagents/materials/analysis tools: BR RA JAT
WOCS. Wrote the paper: BR RA JAT WOCS.
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