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Systematics and Biodiversity
ISSN: 1477-2000 (Print) 1478-0933 (Online) Journal homepage: http://www.tandfonline.com/loi/tsab20
Towards a comprehensive, integrative analysis
of the diversity of European microplaninid
land flatworms (Platyhelminthes, Tricladida,
Microplaninae), with the description of two
peculiar new species
Ronald Sluys, Eduardo Mateos, Marta Riutort & Marta Álvarez-presas
To cite this article: Ronald Sluys, Eduardo Mateos, Marta Riutort & Marta Álvarez-
presas (2016): Towards a comprehensive, integrative analysis of the diversity of
European microplaninid land flatworms (Platyhelminthes, Tricladida, Microplaninae),
with the description of two peculiar new species, Systematics and Biodiversity, DOI:
10.1080/14772000.2015.1103323
To link to this article: http://dx.doi.org/10.1080/14772000.2015.1103323
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Research Article
Towards a comprehensive, integrative analysis of the diversity of
European microplaninid land flatworms (Platyhelminthes, Tricladida,
Microplaninae), with the description of two peculiar new species
RONALD SLUYS
1
, EDUARDO MATEOS
2
, MARTA RIUTORT
3
& MARTA
ALVAREZ-PRESAS
3
1
Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands
2
Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 643, 08028-Barcelona, Spain
3
Departament de Gen
etica, Facultat de Biologia and Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona,
Av. Diagonal 643, 08028, Barcelona, Spain
(Received 1 June 2015; accepted 22 September 2015)
The present study forms a first and major step towards a comprehensive morphological and molecular analysis of the
species diversity of European microplaninid land planarians by presenting a molecular phylogenetic tree on the basis of
alignments of the mitochondrial Cox1 gene from 158 specimens as well as a concatenated phylogeny (Cox1 and 18S genes)
on the basis of 41 sequences for nine Microplana species included in this study. Genetic distances between and within
known and new species were calculated. Combined morphological and molecular results facilitated an integrative
delimitation of new species as well as the diagnosis of new populations of already known species. An integrative account is
provided of two new and aberrant species from the Iberian Peninsula and southern France. Through the molecular approach
a Confirmed Candidate Species was detected among the newly sampled populations. Further we document samples from
new localities for five already known species as well as new sites for the Confirmed Candidate Species. The new data
considerably expand the European range of several already known species included in the present study. The results of an
in-depth study of the taxonomic literature, as well as original material, are documented since this was required for
appropriate identification of the new materials. This part of the study resulted in a re-evaluation of the taxonomic status of
several nominal species and in the following taxonomic conclusions: Rhynchodemus pyrenaicus is a species of Microplana
and not Rhynchodemus;Rhynchodemus attemsi,R. peneckei, and R. henrici should be transferred to the genus Microplana;
Microplana attemsi and M. peneckei are not junior synonyms of M. henrici;Microplana styriaca is not a synonym of M.
terrestris;Rhynchodemus howesi is not a junior synonym of M. pyrenaica and belongs to the genus Rhynchodemus and not
to Microplana;Microplana richardi is not a synonym of M. terrestris. The current diagnosis of the genus Microplana
should be amended such that it refers to the situation that the species generally have two small eyes but occasionally may
have multiple eyes.
http://zoobank.org/urn:lsid:zoobank.org:pub:22B437AA-9D41-4DA0-AE3C-BD8F636CB96D
Keywords: biodiversity, Europe, integrative taxonomy, Microplana, molecular systematics, Platyhelminthes, Tricladida
Introduction
Study of the European land planarian fauna in general and
the genus Microplana Vejdovsky, 1890 (Platyhelminthes,
Tricladida, Geoplanidae, Microplaninae) in particular had
almost come to a full stop after the Second World War
when the Graz school of Turbellarian scholars (founded
by L. von Graff
1851y1924) had ceased to exist.
Minelli’s few contributions in the late 1970s form an
exception to this rule (Minelli, 1977 and references
therein). It was only more recently that Jones, Webster,
Littlewood, and McDonald (2008), Mateos, Giribet, and
Carranza (1998) and Vila-Farr
e, Sluys, Mateos, Jones &
Romero (2008,2011) contributed new species descrip-
tions for Europe. This implies that our current knowledge
on the European fauna is still very fragmentary.
In a previous study, Mateos, Carbrera, Carranza, and
Riutort (2009) explored the diversity of microplaninid and
rhynchodeminid land planarian species in the Iberian Pen-
insula and estimated that there are at least 15 species, six
of which were only represented by molecular clades for
which no information was available on the anatomy of the
Correspondence to: Ronald Sluys. E-mail: ronald.
sluys@naturalis.nl
ISSN 1477-2000 print/ 1478-0933 online
ÓThe Trustees of the Natural History Museum, London 2016. All Rights Reserved.
http://dx.doi.org/10.1080/14772000.2015.1103323
Systematics and Biodiversity (2016), 123
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species. The present study forms another step towards a
comprehensive morphological and molecular analysis of
the species diversity of European microplaninid land pla-
narians by describing two new and, in certain respects,
aberrant species from the Iberian Peninsula and southern
France, focusing on an integrative determination of the
taxa on the basis of both molecular and morphological
markers. Further we document, also integratively, samples
from new localities for five other, already known species,
as well as one Confirmed Candidate Species that we use
in our molecular and comparative studies.
Our integrative identification of the new materials
necessitated an in-depth study of the taxonomic literature
and thus also resulted in a re-evaluation of the taxonomic
status of several nominal species. This part of our study
not only concerned a detailed analysis of old taxonomic
literature but also involved examination of some of the
original material detailed in these publications. The
results of our taxonomic re-evaluation are detailed prior
to the systematic account of the new materials since these
data reflect on the identification of the new species. In this
way, the present study forms an important and major step
towards a comprehensive morphological and molecular
analysis of the species diversity of European microplani-
nid land planarians.
Materials and methods
Collection of specimens
Land planarians were collected mainly from under rocks
and fallen logs in moist forest floor habitats. All individuals
used in the molecular analyses, as well as information on
their sampling localities, are listed in Supplementary Table.
Specimens used for morphological studies are listed in the
relevant Material Examined sections of the Systematic and
Integrative Section and are deposited in the collections of
the Naturalis Biodiversity Center, Leiden, The Netherlands
(ZMA collection code; Supplementary Table). All speci-
mens that were studied morphologically have also been
used in the molecular analyses (Supplementary Table).
Morphological analysis and species
hypotheses
Colour descriptions of the body of living or preserved
specimens of the new species follow the online RAL pal-
ette (available at: http://www.ralcolor.com/) through com-
parison with digital images of the worms on a computer
screen. Animals for morphological studies were fixed in
Steinmann’s fluid and, subsequently, transferred to 70%
ethanol. Specimens that had been preserved for anatomi-
cal analysis were cleared in clove oil and then embedded
in synthetic wax, sectioned at intervals of 58mm
(depending on the size of the animals) and mounted on
albumen-coated slides. Sections were stained in Mallory-
Cason/Heidenhain (Humason, 1967; Romeis, 1989; Win-
sor & Sluys, in prep.) and mounted in DPX. Reconstruc-
tions of the copulatory complex were obtained by using a
camera lucida attached to a compound microscope.
The species status of the animals from the various locali-
ties was assessed by applying the phylogenetic species con-
cept as formulated by Cracraft (1983,1987; see also Sluys,
1991) and by comparing qualitative features of their repro-
ductive complex, in particular their copulatory apparatus,
with those of known species, as documented in the taxo-
nomic literature and revealed by examination of histological
sections of relevant museum specimens housed in various
collections. Conformity of the relevant characters with those
of known species enabled taxonomic assignment of the pop-
ulations sampled, while divergences of organismal attributes
suggested the presence of a candidate new species.
DNA sequencing, alignment and phylogenetic
inference
We extracted total genomic DNA from specimens pre-
served in 100% ethanol using WizardÒGenomic DNA
Purification Kit (Promega, Madison, WI, USA) following
the same protocol as in
Alvarez-Presas, Carbayo, Rozas,
and Riutort (2011). We amplified an 822bp fragment of
the mitochondrial cytochrome oxidase I (Cox1) gene and
1690bp from the small ribosomal subunit RNA gene (18S)
type II by polymerase chain reaction (PCR). We used the
primers BarS (
Alvarez-Presas et al., 2011) and COIR
(L
azaro et al., 2009) for the Cox1 gene, and 18S1F,
18S7R, 18S4F and 18S9R (Carranza, Giribet, Ribera,
Bagu~
n
a, & Riutort, 1996) for the 18S amplifications.
Amplification products were purified using a vacuum
manifold (MultiscreenÒHTS Vacuum Manifold, Millipore
Corporation, Billerica, MA, USA). We determined the
DNA sequence from both strands using BigDye (3.1,
Applied Biosystems, Foster City, CA, USA) and the reac-
tion products were separated on the ABI Prism 3730 auto-
mated sequencer (Unitat de Gen
omica dels Centres
Cient
ıfics i Tecnol
ogics de la UB, CCiTUB).
Cox1 DNA sequences were aligned using the translated
amino acid. A multiple, unambiguous alignment was
obtained by Clustal W, as implemented in Bioedit v.7.2.5.
program (Hall, 1999). For 18S sequences, the alignment
was obtained with online MAFFT v. 7 (Katoh & Standley,
2013) using the G-INS-i iterative refinement method. We
used the program GBlocks v. 0.91. (Talavera & Castre-
sana, 2007) to exclude ambiguous areas of the 18S align-
ment (minimum number of sequences for a conserved
position D22; minimum number of sequences for a flank-
ing position D22; maximum number of contiguous non
conserved positions D25; minimum length of a block D5
and allowed gap positions Dwith half) and estimated the
2 R. Sluys et al.
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
DNA sequence evolution model that better fits the data by
using jModelTest v. 2.1.4. (Darriba, Taboada, Doallo, &
Posada, 2012), applying the Akaike information criterion
(AIC). We estimated the phylogenetic relationships of
both single-locus genes and combined data by Maximum
likelihood (ML) (using RaxML v8 software; Stamatakis,
2014) and Bayesian inference (BI) (using MrBayes v.
3.2.2.; Ronquist et al., 2012) methods. We ran partitioned
ML and Bayesian analyses for the concatenated dataset.
Bootstrap support (BS) values (Felsenstein, 1985) were
obtained for ML trees from 10,000 replicates. Four mil-
lion generations were conducted using the Markov chain
Monte Carlo (MCMC) analysis with four chains and two
independent runs for the BI. The sampling was every
1,000 generations and 4,000 trees were saved. Likelihood
values of the cold chains were checked and we verified
convergence among runs with the standard distribution of
split frequencies 0.01. The first 25% of the samples
were discarded as burn-in and we estimated a 50% major-
ity rule consensus tree from the remaining trees.
Species delimitation: molecular and stable
hypotheses
In an ideal situation for molecular species delimitation,
one would have information from population genetics and
coalescent theory and when these data suggest lack of
gene flow this will evidence the existence of a barrier,
with its consequences for genetic differentiation (Bickford
et al., 2007; Burbrink et al., 2011; cf. Carew et al., 2005,
Fontaneto et al., 2008; Fujita, Leach
e, Burbrink, McGuire,
& Moritz, 2012; Olson et al., 2004; Vieites et al., 2009
and references therein). Given the situation that from one
of the new species we only had material from a single
individual, this implied that in the present study we could
not use any of these methodologies to molecularly delimit
species based on coalescent theory. For this reason, we
calculated genetic distances as supplementary information
on the differentiation between the species taxa, in addition
to the topology of the phylogenetic tree itself. Kimura-2-
parameters (K2P) Cox1 pairwise genetic distances were
calculated using the software MEGA 6.06. (Tamura,
Stecher, Peterson, Filipski, & Kumar, 2013) to assess
whether the levels of genetic differentiation conform to
those found for different species within this group. First
were calculated pairwise distances for all individuals and
then averages for within and between species comparisons
were obtained. This resulted in the identification of a
genetic distance range within which known species’ popu-
lations varied, and a phylogenetic gap between intra- and
interspecific distances, which allows the molecular delim-
itation of new species.
Our complete methodology in species delimitation con-
sisted of three main steps. First, hypotheses on candidate
species (based on application of the phylogenetic species
concept, see above) and putative species (based on appli-
cation of molecular markers, see above) were formulated.
Second, agreements and divergences between candidate
and putative species hypotheses were identified. Third,
during an iterative process, reciprocal illumination of
morphological and molecular results eventually resulted
in the formulation of stable species hypotheses.
Abbreviations used in the Figures
ca, common atrium; ed, ejaculatory duct; fgd, female
genital duct; gid, genito-intestinal duct; gl, glands; go, gon-
opore; in, intestine; lpm, longitudinal parenchymal muscu-
lature; ma, male atrium; od, oviduct; pb, penis bulb; pl,
penial lumen; pp, penis papilla; sg, shell glands; vd, vas
deferens; vep, ventral epidermis; vnc, ventral nerve cord.
Results
Resolution of taxonomic complexities in some
nominal European species of Microplana and
Rhynchodemus
Preamble. A comparative analysis of new species of
Microplana and species reported in the literature is com-
pounded by the fact that the taxonomic status of several of
the already known species has been interpreted differently
by different authors. These differences in interpretation
concern (1) synonymization of one nominal species with
another, and (2) the fact that some species have been vari-
ously placed in either the genus Microplana or in Rhyn-
chodemus. Since several of these compounding cases
concern European species, we here first attempt to resolve
the complexities for taxa that are directly relevant for our
comparative analyses (see below). Taxonomic complexi-
ties and synonymization of some other nominal species of
Microplana or Rhynchodemus were discussed and
resolved, where possible, by Jones et al. (2008).
Generic assignment. Most of the older European species
of Microplana were originally described as a member of
the genus Rhynchodemus and were only subsequently
placed in Microplana. Eventually, these taxonomic
actions culminated in the comprehensive Index by Ogren
& Kawakatsu (1989), listing all species of Microplana
known at that time. A crucial step in the clarification of
the classification of these animals was the recognition of
two distinct subfamilies residing under the family Rhyn-
chodemidae, viz. Rhynchodeminae and Microplaninae
(see historical review in Ogren & Kawakatsu, 1988). In
the most recent higher classification of the triclads (Sluys,
Kawakatsu, Riutort, & Bagu~
n
a, 2009) these taxa are clas-
sified as the subfamily Microplaninae and the Tribe
Diversity of European microplaninid land flatworms 3
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
Rhynchodemini. The Rhynchodemini is characterized by
subepidermal longitudinal muscles arranged into bundles,
whereas in the Microplaninae there is only a single layer
of longitudinal muscle fibres, not aggregated into bundles
(cf. Minelli, 1977, fig. 1A, B; Sluys & Riutort, in prep.
fig. 17). This implies that species of Microplana in princi-
ple possess the simple condition, with non-aggregated
muscle fibres.
Ogren & Kawakatsu (1988) provided extended diagno-
ses for the genera Rhynchodemus and Microplana. In the
present context, it suffices to note that species of Micro-
plana usually have a genito-intestinal connection that
may either run from the female genital duct to the gut or
the copulatory bursa shows a connection with the intes-
tine. Species of Rhynchodemus lack a genito-intestinal
connection. In addition, Microplana species show a well-
developed penis with both a papilla and a muscular bulb,
whereas Rhynchodemus lacks a penial papilla.
The various characters discussed above allow us to re-
evaluate some of the taxonomic assignments made by pre-
vious workers. In their Index, Ogren & Kawakatsu (1989)
listed the species Rhynchodemus henrici Bendl, 1908,R.
attemsi Bendl, 1909, R. peneckei Meixner, 1921, and R.
pyrenaicus Von Graff, 1893 under the genus Microplana,
following suggestions made by earlier workers. Subse-
quently, Jones (1998) took a more conservative view and
ranked R. henrici,R. attemsi, and R. peneckei again under
Rhynchodemus, but left R. pyrenaicus in the genus
Microplana.
That the last-mentioned species is a microplaninid
seems to be contradicted by a figure in Von Graff (1899,
Pl. 53, fig. 5), depicting a cross-section of the body with
longitudinal body muscles arranged in bundles. However,
re-examination of this material, present in the collections
of the Naturhistorisches Museum Wien (NHMW; inven-
tory number: 2833), revealed this to be incorrect and that
actually the body musculature is typically microplaninid.
Therefore, the species should be classified as a Micro-
plana and does not belong to the genus Rhynchodemus,
which agrees with the presence of a penial papilla and a
genito-intestinal connection, being features that are
unusual for a species of Rhynchodemus.
Bendl (1909a) mentioned in his description of Rhyn-
chodemus attemsi that the strong longitudinal body
muscles occur in 23 layers on top of each other and that
in transverse sections the longitudinal muscles are partly
arranged in a single row and partly as bundles with 24
fibres. This suggests that the species is a rhynchodeminid.
However, re-examination of the type material in NHMW
(inventory number: 2897) revealed that the body muscula-
ture is typically microplaninid and that there is no
arrangement of the longitudinal body muscles in bundles
and that thus the species should be ranked under Micro-
plana. In addition, presence of genito-intestinal ducts in
this species also suggests that it belongs to the genus
Microplana. According to Bendl (1909a, 1909b) the
female genital duct (what he called receptaculum seminis,
or uterus) would open directly into the gut, an intermedi-
ary genito-intestinal duct being absent. After having
examined Bendl’s specimens, Meixner (1921) concluded
that this particular connection had resulted from a preser-
vation artefact and that actually there were four other con-
nections with the gut (cf. Meixner, 1921, fig. A). Only a
few years later, Steinb€
ock (1924) re-examined the same
material and concluded that there are actually 67 con-
nections between female genital duct and intestine. Pres-
ence of genito-intestinal connections and a distinct penis
in R. attemsi, features that are absent in the genus Rhyn-
chodemus, support the notion that this species is best clas-
sified as a member of the genus Microplana.
Meixner (1921) described for R. peneckei that its longi-
tudinal body musculature consists of ’einer Lage dicker
L€
angsmuskeln ...’ [one layer of thick longitudinal
muscles]. Combined with the fact that this species shows
a penis (small papilla but very large bulb) as well as a gen-
ito-intestinal duct, this implies that R. peneckei should be
classified as a Microplana, which agrees with our re-
examination of the type material (NHMW; inventory
number: 2851).
Modern generic assignment of R. henrici is facilitated
by the fact that Bendl (1908) described its longitudinal
body musculature as weak and to consist of only a single
layer. Although Bendl (1908) himself overlooked the
presence of a genito-intestinal duct, Meixner (1921)
observed two of such connections with the gut to be pres-
ent in the original material of R. henrici (see also
Steinb€
ock, 1924, fig. 15). Therefore, there can be little
doubt that it should be named as Microplana henrici.
Synonymization of species. Minelli (1977) argued that
both Microplana attemsi and M. peneckei should be con-
sidered junior synonyms of M. henrici. Although Ogren &
Kawakatsu (1989) mentioned Minelli’s suggestion, they
listed all three taxa as separate entities in their Index, as
did Jones (1998) in his overview of European land
planarians.
For both M. peneckei and M. henrici it was reported
that the animals showed a darker mid-dorsal stripe on an
otherwise brownish-yellow or rust-coloured body, respec-
tively (Bendl, 1908; Meixner, 1921). The colouration of
M. attemsi was described as uniform dirty-white (Bendl
1909a). With respect to M. peneckei, observations on its
external appearance were made on the single, live speci-
men, but for the other two species only preserved material
was available.
We re-examined the original histological sections of M.
henrici,M. attemsi, and M. peneckei, which are preserved
in the collections of the NHMW (inventory numbers
2898, 2897, 2851, respectively). In contrast to what Mine-
lli (1977) stated, the type material of M. attemsi has not
4 R. Sluys et al.
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
been destroyed, since the full set of material of both speci-
mens mentioned by Bendl (1908) and Meixner (1921)is
present at the NHMW. With respect to M. peneckei, Mine-
lli (1977) only examined two slides with transverse sec-
tions of the front end; however, the full set of material,
consisting of sagittal, horizontal, and transverse (5 in total,
not merely 2) sections of various portions of the body is
still preserved at NHMW.
Our detailed re-examination of the material of these
three species revealed that the original descriptions by
Bendl (1908,1909a) and Meixner (1921) and subsequent
additions and corrections by Meixner (1921) and
Steinb€
ock (1924) are basically correct and that there are a
number of structural differences pointing to their status as
separate species.
Although both M. attemsi and M. henrici have one dis-
tinct annular fold in the atrium (a feature not easily
extracted from the original descriptions), the female geni-
tal duct of the former is much longer and broader and
lined with a much taller epithelium than in M. henrici.In
addition, the organization and the number of connections
between female genital duct and the intestine differs
much between both species. Steinb€
ock (1924) already
showed that in M. attemsi the female genital duct, on
either side and along its length, has up to four openings
into the gut branches, whereas in M. henrici the very ter-
minal part of the female genital duct bifurcates before
connecting with the intestine. Clearly, these are two
completely different ways in which the female genital
duct is connected with the gut.
In M. peneckei the posterior end of the female genital
duct also bifurcates before opening into two separate
branches of the intestine (cf. Meixner, 1921), as is the
case in M. henrici. However, in M. peneckei (1) the penis
papilla is very small and stubby, and (2) the atrium shows
three distinct annular folds (Meixner, 1921 mentions that
only the dorsal fold is annular), in contrast to the conical
penis papilla and the single annular atrial fold in M. hen-
rici. In addition, the penis bulb musculature of M.
peneckei consists of only circular muscle, whereas that of
both M. henrici and M. attemsi consists of circular muscle
interspersed with longitudinal muscle fibres.
Minelli (1977) argued, albeit with some reservation,
that M. styriaca is a junior synonym of M. terrestris.He
argued that the absence of a genito-intestinal duct reported
by Freisling (1935) may signal a not fully mature speci-
men. Further, Minelli (1977) argued that the copulatory
bursa of M. styriaca is similar to the one in M. terrestris.
However, Freisling (1935) specifically pointed out an
important structural difference between the copulatory
bursae of both species. In M. styriaca the copulatory bursa
is a true bursa, lined with a vacuolated epithelium,
whereas the bursa of M. terrestris is lined with a ciliated
epithelium and therefore it is basically part of the female
genital duct. Another difference between the two species
concerns an intrabulbar seminal vesicle, which is absent
in M. styriaca.
Our examination of the type material of M. styriaca
(NHMW; inventory number: 3624), which is extant and
not lost as presumed by Minelli (1977), supports
Freisling’s (1935) observations on the histology of the
copulatory bursa and thus his view that it concerns a true
bursa, different from M. terrestris.ThatM. styriaca is not
a synonym of M. terrestris is also supported by the fact
that the innermost pharyngeal musculature of the former
consists of a coat of circular muscle fibres that is not inter-
spersed with longitudinal fibres, in contrast to M. terrest-
ris, in which this zone consists of intermingled circular
and longitudinal muscle fibres.
Minelli (1974,1977) considered Rhynchodemus howesi
to be a junior synonym of M. pyrenaica. In view of resem-
blances in the gross morphology of the copulatory appara-
tus one may argue indeed that the two nominal species in
fact concern one and the same species. Fortunately,
Scharff’s material is still available from the Dublin
museum. Examination of microphotographs taken from
the histological sections by Dr H. Jones, revealed that the
longitudinal body muscles in specimens of R. howesi are
arranged as small bundles. Therefore, the species R.
howesi should be classified indeed under the genus Rhyn-
chodemus, whereas M. pyrenaica belongs to the genus
Microplana (see above).
Further, there are also some other differences. Scharff
(1900) described live specimens of R. howesi as dorsally
being uniform greyish black and reaching a maximum
length of no less than 130 mm. Von Graff (1899) noted
that M. pyrenaica concerns a giant species since the pre-
served specimen available to him measured 53 mm in
length. Von Graff (1899) described the body colouration
of this animal as honey-yellow.
Differences between the copulatory apparatuses of the
two species are as follows. In M. pyrenaica there is a
small and pointed penis papilla (cf. Heinzel, 1929, Pl. 11,
fig. 7), while in R. howesi a clear papilla is absent (cf.
Scharff, 1900, fig. 2). Absence of a distinct and well-
developed penis papilla in R. howesi supports its assign-
ment to the genus Rhynchodemus.
Microplana pyrenaica was described with a genito-
intestinal duct running dorsally to a branch of the gut
from about halfway along the female genital duct (cf.
Heinzel, 1929, Pl. 11, fig. 7), while in R. howesi a simi-
larly located duct would lead to a glandular uterus or cop-
ulatory bursa (cf. Scharff, 1900, fig. 2). However,
examination of microphotographs of the original material
of the latter learned that the presumed bursa is actually a
branch of the intestine (H. Jones, in litt.).
Minelli (1977) synomymized, albeit with a question
mark, Microplana richardi (Bendl, 1909) with M. terres-
tris and stated that the original material seemed to have
been lost. Fortunately, the type material is still available
Diversity of European microplaninid land flatworms 5
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
at the NHMW (inventory number: 2853). Examination of
these histological sections confirmed Bendl’s (1909a)
observation that the vasa deferentia are expanded to well
developed spermiducal vesicles, filled with sperm. The
spermiducal vesicles extend backwards to almost the end
of the histological preparations. However, this presumed
posterior body end (’Hinterende’, cf. Bendl, 1909a, p. 72)
is not the actual posterior margin of the body since it is
clear that this presumed end of the body actually shows an
open ending that is not bounded by epidermis. In other
words, the posterior region of the body is actually missing.
In view of the well developed testes, oviducts and sperm
ducts there can be little doubt that this missing part con-
tained the copulatory apparatus and the gonopore, both of
which are absent, as observed by Bendl (1909a).
Our observations on the type material put into perspec-
tive Bendl’s (1909a) remark that the testes would extend
backwards to almost the posterior end of the body. It turns
out that the testes extend backwards up to the point where
the spermiducal vesicles start to develop. In other words,
the testes extend backwards to well beyond the pharyn-
geal pouch. The fact that in M. terrestris the testes are pre-
pharyngeal, extending backwards to just before the root of
the pharynx, argues against the synonymization of M.
richardi with M. terrestris.
Molecular datasets and species delimitation
We obtained alignments of mitochondrial Cox1 (822bp)
from 158 specimens and of nuclear 18S rDNA (1672bp
after applying GBlocks) from 42 specimens, including
some GenBank sequences, as listed in Supplementary
Table (some of the GenBank sequences were rese-
quenced, when DNA was available, with the aim of
obtaining a longer fragment). Three Bipalium sequences
were used as outgroup in all the phylogenetic analyses,
following the results of
Alvarez-Presas & Riutort (2014).
The best-fit model of sequence evolution for both genes
was GTRCICG. The ML tree obtained with Cox1 (Fig. 1,
Suppl. Fig. 1) (see supplemental material online) shows
that all of the already known species included in the anal-
ysis are monophyletic. Although these monophyletic
groups, suggesting stable species hypotheses, are highly
supported (1 PP, 77100% BS), their connecting nodes
are not well resolved and without statistical support.
Therefore, it is not possible to draw any conclusions about
the evolutionary relationships between these putative spe-
cies, based on the Cox1 dataset. This is also the reason
why some parts of the BI tree, obtained with the same
data (not shown), present inconsistencies in the relation-
ships between species when compared with the topology
of the ML tree.
All individuals assigned morphologically to Micro-
plana plurioculata Sluys, Mateos &
Alvarez-Presas, sp.
nov., form a monophyletic group with maximum support,
while its sister group is the single individual of Micro-
plana polyopsis Sluys,
Alvarez-Presas & Mateos, sp. nov.
This supported clade of two species is differentiated from
the rest of the species included in our molecular analyses.
Regarding the concatenated phylogeny (Fig. 2), Cox1
and 18S sequences from 41 specimens were included in
the analysis. The most basal group is formed by popula-
tions of the species Microplana nana. As in the case of the
Cox1 tree, in the concatenated phylogeny all known spe-
cies are monophyletic with maximum support but the rela-
tionships between species are not well resolved since the
corresponding nodes have little statistical support; the ML
tree for the 18S gene alone shows the same trend (Suppl.
Fig. 2) (see supplemental material online). In the phyloge-
nies inferred from the concatenated and the 18S datasets
separately, M. plurioculata is again monophyletic with
maximum support and forms the sister taxon of M. polyop-
sis, also with high support. Both species are clearly differ-
entiated from the rest of the species included in the trees.
With respect to known, already described species, mean
pairwise genetic distances within species, are in the range
of 05% (822 bp analysed; Table 1), M. nana clade C
presenting the highest values of genetic variation. Further-
more, between-species genetic differences range from
13% to 20%. The genetic distance between the two new
species M. plurioculata and M. polyopsis is 14.9%, which
clearly delimits them as two independent species from a
molecular point of view.
A special case is that of the species M. nana for which
the phylogeny shows three deep clades (A, B and C,
Fig. 1, Suppl. Fig. 1) (see supplemental material online),
while the distance analysis indicates that the genetic dif-
ferentiation between the three clades approaches that
found for the comparisons between other species of the
genus (10.613%).
Systematic and integrative section
Microplana plurioculata Sluys, Mateos &
Alvarez-Pre-
sas, sp. nov.
(Figs 110,18)
Material examined. Holotype: ZMA V.Pl. 7190.1
(357G243), Parc Natural de la Zona Volc
anica de la Gar-
rotxa, Fageda d’en Jord
a forest, Olot, Girona, Spain,
N42.1459690 E2.5139201, 31 March 2011, coll. E.
Mateos and M.
Alvarez-Presas, sagittal sections of the
prepharyngeal part on 9 slides and sagittal sections of the
posterior part on 11 slides.
Other material: ZMA V.Pl. 7191.1 (384G246), Parc
Natural de la Zona Volc
anica de la Garrotxa, Fageda d’en
Jord
a forest, Olot, Girona, Spain, N42.1459690
E2.5139201, 4 April 2011, coll. E. Mateos and M.
Alvarez-Presas, sagittal sections on 14 slides.
6 R. Sluys et al.
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Etymology. The specific epithet is derived from a combi-
nation of the Latin adjectives plures (more, plural) and
oculatus (with eyes), thus alluding to the multiple eyes
present in this species.
Diagnosis. Body maize yellow with not very conspicu-
ous ochre-brown mid-dorsal stripe. Number of eyes
present on either side of the anteriormost end of the
body variable; generally 23 eyes on either side;
Table 1. Mean pairwise genetic distances between and within the nine species included in the study, based on Cox1 data and under
Kimura-2-Parameter correction.
M.
nana (A)
M.
nana (B)
M.
nana (C)
M.
terrestris
M.
robusta
M.
groga
M.
plurioculata
M.
scharffi
M.
kwiskea
Microplana
sp.
M.
polyopsis
M. nana (A) 0.029
M. nana (B) 0.106 0.004
M. nana (C) 0.123 0.130 0.050
M. terrestris 0.169 0.174 0.171 0.016
M. robusta 0.203 0.199 0.185 0.178 0.033
M. groga 0.155 0.162 0.146 0.165 0.197 0.012
M. plurioculata 0.146 0.149 0.146 0.182 0.194 0.136 0.003
M. scharffi 0.145 0.166 0.149 0.171 0.209 0.159 0.171 0
M. kwiskea 0.143 0.171 0.154 0.174 0.189 0.144 0.164 0.148 0.026
Microplana sp. 0.170 0.171 0.162 0.160 0.196 0.170 0.182 0.132 0.162 0
M. polyopsis 0.192 0.204 0.191 0.177 0.195 0.180 0.149 0.172 0.194 0.176 -
Fig. 1. Maximum likelihood (ML) tree inferred from Cox1 dataset. For space reasons monophyletic groups comprising species or
groups within species have been collapsed and the outgroup (two Bipalium species) has been removed (see the complete figure in Suppl.
Figure S1) (see supplemental material online). Values at nodes correspond to Posterior Probability (PP, BI analysis) and Bootstrap Val-
ues (BV, ML analysis); only values over 0.85 PP and 75% BV are displayed, respectively. Green filled circles and numbers denote how
many individuals for that clade were examined histologically. See Supplementary Table and Figure S1 for molecular codes, field codes,
and museum registration codes for all the individuals used in the study. Scale bar: units in nucleotide substitutions per site.
Diversity of European microplaninid land flatworms 7
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number of eyes present per side of the body may be
unequal. Small ovaries located at about 1/5th of the
distance between the brain and the root of the pharynx.
Dorsally displaced opening of the female genital duct
into the genital atrium. At about halfway along its
length the dorsal section of the female genital duct
communicates with a short and narrow genito-intestinal
duct; copulatory bursa absent. Common atrium with a
number of very large, dorsal folds. Molecular diagno-
sis: this species includes all populations that cluster
with sequences of individuals from this study (Cox1
GenBank Accession numbers KR906606KR906618;
18S GenBank Accession numbers FJ970002,
KR906671KR906675), with significant support in an
adequate molecular delimitation model.
Description. Living animals up to 50 mm long and about
2 mm in diameter. Body maize-yellow (RAL 1006) with
darker, ochre-brown (RAL 8001) mid-dorsal stripe that
sometimes may be poorly visible in the anterior half of
the body but is usually clearly present on the posterior
portion; development of this stripe is variable and depends
on the specimen examined. Body shape cylindrical, with
blunt anterior end and pointed posterior end (Fig. 3). The
number of eyes present on either side of the anteriormost
end of the body varies between specimens. Generally
there are 23 eyes on either side. However, the number
of eyes present per side of the body may be unequal, with
2 eyes on one side and 3 on the other side of the head, or 4
versus 5 (Fig. 4;Table 2).
Subepidermal musculature consisting of a single layer
of circular muscle directly underneath the epidermis,
followed by a single layer of longitudinal muscle.
Parenchymal longitudinal mucles well developed, nota-
bly on the ventral side, where some longitudinal
muscles are also present dorsally to the ventral nerve
cords. A layer of strong transverse muscle fibres is pres-
ent between the ventral nerve cords and the layer of lon-
gitudinal muscle located directlyventrallytothenerve
cords (Fig. 5).
Small pharynx, situated at the middle of the body, mea-
suring between 1/10th 1/11th of the body length. Mouth
opening situated at about 1/3rd of the distance between
the posterior wall of the pharyngeal pocket and the root of
the pharynx.
The outer pharynx musculature consists of 23 layers
of circular muscle directly underneath the epithelium.
Entally to this circular muscle layer there is a parenchy-
mal zone, which is followed by a well-developed zone of
intermingled circular and longitudinal muscle fibres. The
inner pharynx musculature consists of a broad zone of
intermingled circular and longitudinal muscle fibres
directly below the epithelium lining the pharynx lumen.
Testes could not be discerned in the specimens exam-
ined. The vasa deferentia are expanded to spermiducal
vesicles, which recurve and narrow before separately
opening into the very distal part of the intrabulbar lumen.
The latter communicates with the wide penial lumen,
which gradually narrows to form an ejaculatory duct that
opens at the tip of the penis papilla. The entire penis
lumen is lined with a nucleated epithelium.
Fig. 2. ML tree inferred from the concatenated dataset (18S and Cox1). Values at nodes correspond to statistical support in Bayesian
Inference/ Bootstrap; only values displayed over 0.85 and 75%, respectively. Scale bar: units in nucleotide substitutions per site.
8 R. Sluys et al.
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Figs. 36. Microplana plurioculata.Fig. 3. Holotype specimen (ZMA V.Pl. 7190.1 (357G243). External features; anterior end at bot-
tom. No scale bar available. Fig. 4. Holotype specimen (ZMA V.Pl. 7190.1 (357G243). Anterior end with eyes. Fig. 5. Holotype (ZMA
V.Pl. 7190.1 (357G243). Ventral subepidermal and parenchymal musculature; sagittal section. Fig. 6. ZMA V.Pl. 7191.1. Sagittal sec-
tion through copulatory apparatus.
Table 2. Microplana plurioculata. Number of eyes on either side of the body present in 14 specimens from different localities. Body
length was determined on live animals.
Specimen code Field code Locality Length (mm) Eyes right Eyes left
051B019 Montnegre, Barcelona 12 2 3
169G017 Fageda d’en Jord
a, Girona 15 2 2
170G017 Fageda d’en Jord
a, Girona 15 3 3
383G243 Fageda d’en Jord
a, Girona 20 3 2
343G017 Fageda d’en Jord
a, Girona 22 2 2
381G243 Fageda d’en Jord
a, Girona 23 2 2
160B016 Montnegre, Barcelona 25 3 2
168G017 Fageda d’en Jord
a, Girona 25 3 3
265B164 Sauva Negra, Barcelona 25 1 2
358G243 Fageda d’en Jord
a, Girona 25 1 2
384G246 RS335-1 Fageda d’en Jord
a, Girona 33 2 3
382G243 Fageda d’en Jord
a, Girona 35 2 2
165B025 Fageda d’en Jord
a, Girona 40 3 3
357G243 RS332-1 Fageda d’en Jord
a, Girona 50 5 4
Diversity of European microplaninid land flatworms 9
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The penis bulb is made up of strong, interwoven muscle
fibres of which only the circular fibres extend over the
lumen of the penis papilla; this circular muscle layer grad-
ually becomes much thinner around the ejaculatory duct.
The penis papilla is cone-shaped and covered with a
nucleated epithelium. In the largely immature specimen
ZMA V.Pl. 7191.1 the shape of the penis papilla is imme-
diately clear (Fig. 6). However, in the holotype specimen
the distal part of the penis papilla is highly folded and
points towards the dorsal body surface (at one point actu-
ally projecting through the epidermis), probably due to a
preservation artefact (Fig. 7).
The small ovaries are located at about 1/5th of the dis-
tance between the brain and the root of the pharynx and
are positioned directly dorsal to the ventral nerve cords.
The anterior end of each oviduct is expanded to an
ampulla, which communicates with the ventral portion of
the ovary via a highly constricted section. The oviducts
Figs. 78. Microplana plurioculata.Fig. 7. ZMA V.Pl. 7190.1. Sagittal reconstruction of the penis papilla; anterior to the right. Fig. 8.
ZMA V.Pl. 7190.1. Sagittal reconstruction of the copulatory apparatus; anterior to the right.
10 R. Sluys et al.
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run posteriorly directly dorsal to the ventral nerve cords
and are lined with a nucleated epithelium.
Posterior to the gonopore the oviducts curve medially
and open separately into the very distal end of a broad
female genital duct (Fig. 8). The latter opens into the post-
ero-dorsal section of the male atrium. The distal section of
the female genital duct is penetrated by the secretion of
erythrophil shell glands. The female genital duct is lined
with cuboidal, nucleated cells and it is only very weakly
muscularized, if at all. At about halfway along its length
the dorsal section of the female genital duct communi-
cates with a short and narrow genito-intestinal duct
(Fig. 6), which is lined with small, nucleated cells (Fig. 9).
The common atrium shows a number of very large, dor-
sal folds; it is lined with a columnar, nucleated, and glan-
dularized epithelium (Fig. 10). The antero-ventral section
of the common atrium leads to the gonopore. Part of the
distal sections of the male atrium receive the secretion of
another kind of erythrophil glands.
Distribution. The species is known from the type locality
(where it was also collected on 13 March 2006 and 15
May 2010) and from Montnegre Mountains (Sant Celoni,
Barcelona, Spain, N41.675771 E2.588769, 12 January
2004 and 18 September 2005) and Sauva Negra forest
(Castellcir, Barcelona, Spain, N41.7921153 E2.1764179,
13 November 2008) (Fig. 18). The latter records are based
on external similarity and phylogenetic proximity in the
Cox1 gene trees that the specimens from these localities
show with the type specimen (suppl. Fig. 1, individuals
051b019 and 265b164; see supplemental material online).
Discussion. The combination of yellowish body colour,
absence of a copulatory bursa, presence of large folds in
the common atrium, and the dorsally displaced opening of
the female genital duct into the male atrium invites com-
parison with only a few known species of Microplana,
viz. M. attemsi,M. peneckei,M. henrici,M. trifuscoli-
neata, and M. tristriata. Unfortunately, the reproductive
apparatus of M. richardi (Bendl, 1909) from Monaco is
not known (see above). However, the species was
described as dark brown-grey and with only one pair of
eyes and therefore it is unlikely to be similar to M.
plurioculata.
In view of their very similar external appearance as
well as their copulatory apparatus, it may well be the case
that M. trifuscolineata from Mauritius is a junior synonym
of M. tristriata from Anjouan (Comores). These two nom-
inal species share a characteristic feature with another
Indian Ocean species, viz. M. mediostriata from Grande
Comore (Comores). In all three taxa the common atrium
consists of a canal traversing a more or less vertically ori-
ented papilla projecting into the gonoduct. Such a papilla
is clearly absent in M. plurioculata, albeit that the latter
shares with M. tristriata and M. trifuscolineata the situa-
tion that the genito-intestinal duct projects dorsally from
about halfway along the female genital duct. All three
Indian Ocean species have a dark body colouration, show-
ing one or three even darker dorsal stripes, a situation that
is clearly different from M. plurioculata.
Although the external features of M. attemsi,M. henrici
and M. peneckei are different from M. plurioculata, these
three species exhibit some anatomical similarities with M.
plurioculata. Notably, in all four species the female geni-
tal duct opens into the dorsal section of the male atrium, a
condition that is uncommon among species of Micro-
plana. Further, in all four species the wall of the common
atrium is thrown into large folds. However, in M. plurio-
culata the folds originate from only the dorsal side of the
atrium, whereas in M. attemsi,M. henrici and M. peneckei
Figs. 910. Microplana plurioculata. Holotype (ZMA V.Pl. 7190.1 (357G243). Fig. 9. Sagittal section through common atrium, female
genital duct, and genito-intestinal duct. Fig. 10. Sagittal section through male atrium, common atrium, and penis papilla.
Diversity of European microplaninid land flatworms 11
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they sit all around. Moreover, in the last-mentioned three
species the folds are actually rings that surround the entire
atrium, with one of such annular folds in both M. attemsi
and M. henrici and three in M. peneckei (see above).
Another difference between M. plurioculata on the one
hand and M. attemsi,M. henrici and M. peneckei on the
other hand, concerns the connection with the gut. In M.
plurioculata a genito-intestinal duct arises from about
halfway along the female genital duct, a situation that is
very different from the genito-intestinal connections in M.
attemsi,M. henrici, and M. peneckei (see above).
The molecular results show that the M. plurioculata
clade (Figs. 1,2and suppl. Fig. 1, 2, see supplemental
material online) is monophyletic and constitutes an inde-
pendent lineage separated, with high support, from the
rest of the species included in the trees. Moreover, genetic
distances between individuals in this lineage are within
the range observed for other microplaninid planarian spe-
cies. Genetic distances between the new species and the
other species included in the analyses also fall within
the range for interspecific differences. These two facts can
be considered as indirect evidence that this lineage can be
regarded as different species at the molecular level.
In conclusion, there are ample differences, both mor-
phological and molecular, between M. plurioculata and
any of the currently known species of Microplana, thus
justifying the description of a new species. The most obvi-
ous difference between M. plurioculata and its congeners
concerns the presence of more than one pair of eyes in the
new species (but see below for another new species with
multiple eyes).
Microplana polyopsis Sluys,
Alvarez-Presas & Mateos,
sp. nov.
(Figs 1,2,1116,18)
Material examined. Holotype: ZMA V.Pl. 7192.1
(740X308), Valcabr
ere, Valcabr
ere, France, N43.03400
E000.58016, 25 March 2013, coll. M.
Alvarez-Presas,
sagittal sections on 16 slides.
Etymology. The specific epithet is derived from the
Greek polys (many) and opsis (eyesight, vision, visual
power) and alludes to the presence of multiple eyes in this
species.
Diagnosis. Dorsal surface pebble grey with a yellow-
olive mid-dorsal stripe from the tip of the tail to close to
the anterior margin of the body. Up to 8 eye cups on either
side of the head, close to the anterior body margin. Sperm
ducts unite to a short common vas deferens shortly after
having penetrated the penis bulb. Bulbar lumen broad,
lined with a columnar, infranucleated epithelium, thrown
into large folds. Thick coat of circular muscle around bul-
bar and penial lumen. Oviducts with a muscularized
constriction at their point of communication with the ova-
ries; an ampulla is absent. Female genital duct opens into
the most postero-dorsal section of the atrium. Copulatory
bursa absent. Molecular diagnosis: this species includes
all populations that cluster with sequences of individuals
from this study (Cox1 GenBank Accession number
KR906619; 18S GenBank Accession number KR906676),
with significant support in an adequate molecular delimi-
tation model.
Description. Living animal 32 mm in length and 2.5 mm
in width. Dorsal surface pebble grey (RAL 7032) with a
yellow-olive (RAL 6014) mid-dorsal stripe, running from
the tip of the tail to close to the anterior margin of the
body (Fig. 11). Apical end clearer; ventral surface pale.
Up to 8 eye cups situated on either side of the head, close
to the anterior body margin (Figs 12,13).
Subepidermal musculature consisting of a single
layer of circular muscle, followed by a single layer of
longitudinal muscle. Parenchymal muscles well devel-
oped, particularly on the ventral side, with longitudinal
muscles both ventrally and dorsally to the ventral
nerve cords (Fig. 14).
Pharynx short, measuring about 400 mm in length in the
sections prepared of the holotype. Mouth opening located
at about 1/4th of the distance between the posterior wall
of the pharyngeal cavity and the root of the pharynx.
Outer pharyngeal musculature consisting of a thin, subepi-
dermal layer of longitudinal muscle followed by a weakly
developed layer of circular muscle. Inner pharyngeal mus-
culature consisting of a broad zone of intermingled circu-
lar and longitudinal muscle fibres.
Testes absent, at least in the section of the animal
extending from the level of the ovaries to the tip of the tail
(the very anterior part of the body having been cut off for
molecular study), but there is sperm in the spermiducal
vesicles.
At the level of the root of the penis papilla the vasa def-
erentia recurve to penetrate the coat of muscles forming
the antero-lateral wall of the penis bulb. Upon the point of
entrance the diameter of the sperm ducts has diminished
greatly, while the two ducts unite shortly after they have
penetrated the penis bulb. Immediately after union, the
short common vas deferens opens into the bulbar lumen.
The latter is quite broad and lined with a columnar, infra-
nucleated epithelium, which is thrown into large folds.
The bulbar lumen turns into a similarly constructed penial
lumen, which communicates with the ejaculatory duct at
about halfway the length of the penis papilla (Fig. 15).
The ejaculatory duct is lined with a cuboidal, nucleated
epithelium and opens at the tip of the penis papilla. The
latter is a short cone with an almost horizontal orientation
that completely fills the atrium, which communicates with
the gonopore via a broad gonoduct. Circular muscle layer
12 R. Sluys et al.
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around the ejaculatory duct only very weakly developed
(Fig. 16).
The penis bulb is composed of strong, intermingled mus-
cle fibres, of which particularly the circular ones surround
the bulbar and penial lumen in the form of a thick coat.
The nucleated, lining epithelium of the penis papilla is
underlain by a thin layer of circular muscle, followed by a
thin layer of longitudinal muscle.
Since the front end of the body is missing from the his-
tological sections (see above), the precise position of the
ovaries along the body axis could not be determined, apart
from the fact that the gonads are located at about 3 mm
anterior to the root of the pharynx. Upon its ventral point
of communication with the ovary, each oviduct, com-
posed of cuboidal, nucleated cells, shows a constriction
that is surrounded by muscles; an ampulla is absent.
Shortly posterior to the level of the gonopore, the oviducts
curve medially and upwards and separately open into the
distal end of a rather long female genital duct. From this
point onwards, the female genital duct curves antero-dor-
sad to open into the most postero-dorsal section of the
atrium (Fig. 15). The female genital duct receives the
openings of abundant erythrophil shell glands from about
halfway its length to the point where it receives the ovidu-
cal openings. The female genital duct is surrounded by a
layer of circular muscle fibres.
A connection with the intestine could not be discerned,
but it must be said that the histology in the region of the
female genital duct is disturbed by folds in the sections
and by the dense staining of the abundantly present shell
glands. Therefore, presence of a very short and narrow
genito-intestinal duct may be obscured.
Figs. 1114. Microplana polyopsis. Holotype (ZMA V.Pl. 7192.1 (740£308). Fig. 11. External features. No scale bar available.
Fig. 12. Front end with eyes. No scale bar available. Fig. 13. Front end with eyes. No scale bar available. Fig. 14. Ventral subepidermal
and parenchymal musculature; sagittal section.
Diversity of European microplaninid land flatworms 13
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Distribution. Known only from the type locality
(Fig. 18).
Discussion. Evidently, all known species of Microplana
differ from M. polyopsis in the fact that they possess only
one pair of eyes, with the exception of M. plurioculata.
However, M. plurioculata differs morphologically from
M. polyopsis in (1) a different type of outer pharynx mus-
culature, (2) presence of strong transverse parenchymal
muscles, (3) presence of an ampulla at the anteriormost
section of the oviducts, (4) presence of a genito-intestinal
canal (albeit that absence of this structure needs to be veri-
fied in new specimens of M. polyopsis), (5) presence of
large folds in the common atrium (see above).
However, presence of a pale body with a dark mid-dor-
sal stripe, a female genital duct that opens into the post-
ero-dorsal part of the atrium, and absence of a copulatory
bursa still requires a comparison with a few known spe-
cies of Microplana that also show such features, viz. M.
henrici,M. mediostriata,M. uniductus, and M. unilineata.
In the Korean species M. unilineata the oviducts open
into a very short common oviduct, which receives the
secretion of shell glands. The common oviduct opens into
the postero-dorsal section of a vertically oriented female
genital duct that opens into the most ventral part of the
common atrium (Frieb, 1923). Evidently, this situation
differs greatly from that in M. polyopsis, in which the
female genital duct opens into the most dorsal portion of
the atrium.
The species status of Indian M. uniductus, and even its
generic status, remains uncertain in view of the fact that
De Beauchamp (1930) originally described it as a variety
of the species Rhynchodemus sholanus. It remains to be
examined whether his uniductus specimens have the micro-
planinid or the rhynchodeminid type of subepidermal body
musculature (see above). However, in M. uniductus there
are two distinct muscular folds projecting into the atrium,
while the animals showed a dark brown colouration, fea-
tures that do not correspond with M. polyopsis.
A similar dark colouration (rust-brown) was mentioned
for M. mediostriata from the Comores (Geba, 1909). But
this species shows a common atrium that consists of a
canal traversing a vertically oriented papilla that projects
into the gonoduct. Such a distinct structure is absent in M.
polyposis.
European M. henrici is characterized by an annular fold
at the distal end of the male atrium, a structure that is
absent in M. polyopsis. In addition, the penis bulb of M.
henrici is much larger than that of M. polyopsis.
In all four species of Microplana discussed above, a
genito-intestinal duct is present; in M. henrici there is
even a double connection with the gut (see above). A gen-
ito-intestinal connection probably is absent in the single,
presently available specimen of M. polyopsis, although
this needs to be verified on new material when this
becomes available.
Moreover, the molecular results (Figs 1,2; suppl. Figs 1,
2) (see supplemental material online) show that the individ-
ual specimen is a singleton (i.e. it does not correspond to
any of the species included in this study) and although it
Figs. 1516. Microplana polyopsis. Holotype (ZMA V.Pl.
7192.1 (740X308). Fig. 15. Sagittal reconstruction of the copula-
tory apparatus. Fig. 16. Sagittal section through penis papilla.
14 R. Sluys et al.
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
groups with the other multiple-eyed species (M. pluriocu-
lata) it is genetically highly differentiated from the latter in
both its nuclear and mitochondrial genes. The genetic dis-
tance of this individual to other species included in our
analysis, with values ranging from 14.9 to 19.5%, also sup-
ports the notion that it is a separate species.
In conclusion, there are ample differences, both mor-
phological and molecular, between M. polyopsis and any
of the currently known species of Microplana, thus justi-
fying the description of a new species. The most obvious
difference between M. polyopsis and its congeners, apart
from M. plurioculata, concerns the presence of more than
one pair of eyes.
Microplana terrestris (M€
uller, 1774)
(Figs 1,2,17)
Material examined. ZMA V.Pl. 7193.1 (465Y263),
Burnham Beeches, Burnham, Berkshire, UK, N51.55519
W0.62547, 30 April 2012, coll. E. Mateos, M.
Alvarez-
Figs. 1718. Distributional records of terrestrial planarians used in this study. Fig. 17. Main map; rectangular inset corresponds to area
shown in Fig. 18; green square, Microplana kwiskea; blue square, Microplana robusta; black filled circle, Microplana scharffi; red filled
circle, Microplana terrestris; asterisk, Microplana sp.Fig. 18. Detail of northern Spain and southern France, corresponding to rectangu-
lar inset in Fig. 17; green cross, Microplana groga; red star, Microplana nana; blue triangle, Microplana plurioculata; black inverted tri-
angle, Microplana polyposis.
Diversity of European microplaninid land flatworms 15
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
Presas & A. Tud
o, sagittal sections on 11 slides. ZMA V.
Pl. 7194.1 (464Y263), Burnham Beeches, Burnham,
Berkshire, UK, N51.55519 W0.62547, 30 April 2012,
coll. E. Mateos, M.
Alvarez-Presas & A. Tud
o, sagittal
sections on 25 slides. ZMA V.Pl. 7195.1 (498Y265),
Greenfield Copse, Greenfield, Oxfordshire, UK,
N51.62299 W0.97618, 1 May 2012, coll. E. Mateos, M.
Alvarez-Presas & A. Tud
o, sagittal sections of anterior
end on 5 slides and sagittal sections of the posterior end
on 6 slides. ZMA V.Pl. 7196.1 (499Y266), Forest of
Dean, Speechhouse area, Coleford, Gloucestershire, UK,
N51.80975 W2.54908, 2 May 2012, coll. E. Mateos, M.
Alvarez-Presas & A. Tud
o, sagittal sections on 8 slides.
ZMA V.Pl. 7197.1 (512Y268), Withbarrow woodland,
Lake District National Park, Levens, Cumbria, UK,
N54.27279 W2.82916, 3 May 2012, coll. E. Mateos, M.
Alvarez-Presas & A. Tud
o, sagittal sections on 4 slides.
ZMA V.Pl. 7198.1 (568Y271), Grove in the countryside,
Underbarrow, Cumbria, UK, N54.31776 W2.80783, 4
May 2012, coll. E. Mateos, M.
Alvarez-Presas & A.
Tud
o, sagittal sections on 5 slides. ZMA V.Pl. 7199.1
(744W309), Puscza Bukowa, Szczecinski Park Krajobraz-
owy, Szcecin, Pomerania, Poland, N53.35309 E14.66239,
23 June 2013, coll. E. Mateos & M.
Alvarez-Presas, sagit-
tal sections on 13 slides. ZMA V.Pl. 7200.1 (745W309),
ibid., sagittal sections on 21 slides. ZMA V.Pl. 7201.1
(771H331), Parque Nacional Ordesa y Monte Perdido,
Valle Ordesa, Torla, Huesca, Spain, N42.65677
W0.09794, 4 October 2013, coll. E. Mateos & M.
Alvarez-Presas, sagittal sections on 11 slides. ZMA V.Pl.
7202.1 (787H340), Parque Nacional Ordesa y Monte Per-
dido, Valle Escua
ın, Puertolas, Huesca, Spain, N42.60373
E 0.11941, 7 October 2013, coll. E. Mateos & M.
Alvarez-Presas, sagittal sections on 8 slides. ZMA V.Pl.
7203.1 (792H341), Parque Nacional Ordesa y Monte Per-
dido, Valle Ordesa, Torla Huesca, Spain, N42.63855
W0.03422, 8 October 2013, coll. E. Mateos & M.
Alvarez-Presas, sagittal sections on 13 slides. ZMA V.Pl.
7204.1 (695X299), Parc Naturel R
egional Chartreuse, cir-
que de St. Meme, Saint Laurent du Pont, D
ept. Is
ere,
R
egion R
odano-Alpes, France, N45.403669 E5.884730, 3
November 2012, coll. E. Mateos, M.
Alvarez-Presas, L.
Leria & S. Agull
o, sagittal sections on 7 slides. ZMA V.
Pl. 7223.1 (315G215), Tolosa forest, Milany mountains,
municipality of Vallfogona de Ripolles, Girona province,
Spain, N42.1861406 E2.2594109, alt. 880 m, 25 August
2009, coll. E. Mateos, sagittal sections on 5 slides. ZMA
V. Pl. 7224.1 (316G215), ibid., sagittal sections on 5
slides.
Comparative discussion. Although the material exam-
ined represents new sampling sites (Fig. 17) for Micro-
plana terrestris, these do not noticeably extend the known
range of the species (cf. Berland, 1968, fig. 2). However,
it is interesting to note that our phylogenetic tree implies
the presence of two distinct biogeographic clades. The
western clade comprises those Iberian populations that
range from Galicia to Navarra, while the eastern clade
comprises localities in the north-east of the Iberian Penin-
sula (Arag
on and Catalunya) and also other European
localities. This agrees with the results obtained by
Alvarez-Presas, Mateos, Vila-Farr
e, Sluys, & Riutort
(2012) but increases the range of the eastern clade since
the previous study did not include sequences from France,
Poland and Germany that in the present study group in
this clade. Nonetheless, the addition of these geographi-
cally distant populations in the eastern clade does not
increase its genetic diversity, which is still lower than that
of the western clade (Fig. 1).
Microplana kwiskea Jones et al., 2008
(Figs 1,2,17,19,20)
Material examined. ZMA V.Pl. 7205.1 (506Y266), For-
est of Dean, Coleford, Gloucestershire, UK, N51.80975
W2.54908, 20 May 2012, coll. E. Mateos, M.
Alvarez-
Presas & A. Tud
o, 20 May 2012, sagittal sections on 6
slides. ZMA V.Pl. 7206.1 (539Y269), Dalton woodland,
Burton-in-Kendal, Cumbria, UK, N54.17667 W2.69342,
4 May 2012, coll. E. Mateos, M.
Alvarez-Presas & A.
Tud
o, sagittal sections on 4 slides. ZMA V.Pl. 7207.1
(907W425), Rezerwat Spa»a, Spa»a, Poland, N51.53872
E20.14036, 20 October 2013, coll. A. Drozd & H. Kappes,
sagittal sections on 7 slides. ZMA V.Pl. 7208.1 (924I447),
Valle di Rio, Italy, N41.73926 E13.73187, 2 May 2014,
coll. E. Mateos, M.
Alvarez-Presas & A. Poch, sagittal
sections on 8 slides; ZMA V.Pl. 7222.1 (PT 923), ibid.,
sagittal sections on 7 slides. ZMA V.Pl. 7209.1 (941I450),
Bassano Romano, Italy, N42.19146 E12.17922, 3 May
2014, coll. E. Mateos, M.
Alvarez-Presas & A. Poch, sag-
ittal sections on 6 slides. ZMA V.Pl. 7210.1 (1204U468),
Central Balkan National Park, Kalofer, Bulgaria,
N42.6617 E24.95629, alt. 636 m, 18 September 2014,
Fig. 19. Microplana kwiskea. External features of specimen
from Poland. No scale bar available.
16 R. Sluys et al.
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coll. E. Mateos & M.
Alvarez-Presas, sagittal sections of
the front end and the posterior part (including the phar-
ynx) on 4 and 5 slides, respectively. ZMA V.Pl. 7211.1
(1190U466), Kalofer, Bulgaria, N42.62682 E24.96614,
alt. 673 m, 17 September 2014, coll. E. Mateos & M.
Alvarez-Presas, sagittal sections on 7 slides.
Comparative discussion. The most interesting and sur-
prising new findings of M. kwiskea are of course the ani-
mals from Poland, Italy, and Bulgaria. In all molecular
phylogenies inferred, the Polish specimen and those from
Bulgaria are part of a well-supported monophyletic group
including British representatives of the species M. kwis-
kea, and are sister to the clade formed by representatives
from Italy (Figs 1,2; suppl. Figs 1, 2) (see supplemental
material online). The K2P genetic distance between indi-
viduals in the British-Polish-Bulgarian clade ranges
between 00.3%, thus being a case similar to that of
M. terrestris, a species that also shows very low genetic
diversity in its wide geographic range. The Italian
M. kwiskea clade has an internal diversity of 00.9%, and
the average genetic distance between individuals from the
two clades is within the observed intraspecific range for
Microplana, albeit quite high (5.1%).
The pale brown colour of the Polish animal (Fig. 19)
corresponds with that described for M. kwiskea (cf. Jones
et al., 2008) and is quite unlike that of M. terrestris.Eyes
could not be discerned in the live Polish specimen and
neither were they observed in the cleared animal, nor in
histological sections. The absence of eyes is unusual since
most species of Microplana possess a pair of eyes, includ-
ing M. kwiskea. Since only one specimen was available
from Poland it remains unknown at present whether
absence of eyes is due to an artefactual condition in ZMA
V.Pl. 7207.1 or is characteristic for this population. How-
ever, it should be noted that the anterior end of the animal
was whitish (Fig. 19), suggesting that the head was in the
process of regeneration.
Further, the histological sections of the copulatory appa-
ratus of the Polish specimen are not fully sagittal but obli-
que. Nevertheless, a reconstruction of part of the
copulatory apparatus complex that is available (Fig. 20)
actually matches the features described for M. kwiskea.
Notably, the penis papilla is traversed by a broad ejacula-
tory duct that only gradually narrows towards the tip of the
papilla. Further, the lining epithelium of the intrabulbar
lumen and the ejaculatory duct receives the highly charac-
teristic coarsely granular, orange-brown secretion of extra-
bulbar glands, as we observed also in the M. kwiskea
specimens from the UK. The genito-intestinal duct of the
Polish animal is strongly ciliated and shows the papillate
opening into the gut that is characteristic for M. kwiskea
(Fig. 20;cf.Jonesetal.,2008). The prepharyngeal, ventral
testes and the rounded ovaries of specimen V.Pl. 7207.1
are similar to the situation in many species of Microplana.
Clearly, the present specimen from Poland cannot pro-
vide any information on the connections between ovi-
ducts, female genital canal and genito-intestinal duct.
This is due to the fact that accidentally because of the
absence of eyes the posterior tip of the body was used
for molecular analyses and that thus only part of the
female copulatory apparatus remained for morphological
study. However, in view of the molecular results and the
fact that the available morphological features fully con-
form to M. kwiskea we do not hesitate to assign the Polish
animal to this species.
Assignment of the Italian and Bulgarian animals to the
species M. kwiskea was easier since in all anatomical
details they closely match the original taxonomic descrip-
tion by Jones et al. (2008).
Evidently, the new findings result in the situation that
the geographic range of M. kwiskea is no longer restricted
to the British Isles but now also includes continental
Europe (Fig. 17).
Microplana nana Mateos et al., 1998
(Figs 1,2,18,2125)
Material examined. ZMA V.Pl. 7212.1 (307H202),
Valle de Bujaruelo, Torla, province of Huesca, Spain,
N42.6840719 W0.1118863, alt. 1300 m, 31 May 2009,
Fig. 20. Microplana kwiskea. ZMA V.Pl. 7207.1. Sagittal and
partially oblique reconstruction of the copulatory apparatus;
anterior to the right. Vertical broken line indicates that the most
posterior end of the body is missing.
Diversity of European microplaninid land flatworms 17
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coll. M. Mateos, sagittal sections on 2 slides; RS 340-1,
ibid., sagittal sections on 4 slides. ZMA V.Pl. 7213.1
(308H202), Valle de Bujaruelo, Torla, Huesca, Spain,
N42.6840719 W0.1118863, coll. E. Mateos, 31 May 6
2009, sagittal sections on 4 slides. ZMA V.Pl. 7214.1
(301B168), Parque Natural del Cad
ı-Moixer
o, Pedraforca
mountain, les Mollers, Saldes, province of Barcelona,
Spain, N42.25903 E1.71311, alt. 1268 m, 14 May 2009,
coll. E. Mateos, sagittal sections on 5 slides. ZMA V.Pl.
7215.1 (797H344), Parque Nacional Ordesa y Monte Per-
dido, Valle de A~
nisclo, Fanlo, Huesca, Spain, N42.60729
E0.05184, 9 October 2013, coll. E. Mateos & M.
Alvarez-
Presas, sagittal sections on 2 slides. ZMA V.Pl. 7216.1
(360G244), Parc Natural de la Zona Volc
anica de la Gar-
rotxa, Fageda d’en Jord
a, Olot, Girona, Spain,
N42.1489839 E2.5145737, 31 March 2011, coll. E.
Mateos & M.
Alvarez-Presas, sagittal sections on 5 slides.
ZMA V.Pl. 7217.1 (361G244), Parc Natural de la Zona
Volc
anica de la Garrotxa, Fageda d’en Jord
a, Olot,
Girona, Spain, N42.1489839 E2.5145736, 31 March
2011, coll. E. Mateos & M.
Alvarez-Presas, sagittal sec-
tions on 4 slides.
Comparative discussion. Mateos et al. (1998) referred
to a video recording available for their new species
but did not provide any figure of the external appear-
ance of M. nana and for the position of the eyes only
Figs. 2124. Microplana nana.Figs. 2123. External features. Scale bars not available. Fig. 14. ZMA V.Pl. 7214.1. Sagittal recon-
struction of the copulatory apparatus.
Fig. 25. Microplana nana. ZMA V.Pl. 7217.1. Sagittal recon-
struction of the copulatory apparatus; anterior to the left.
18 R. Sluys et al.
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
stated that these ’are located at the anterior end …’
(the particular ftp site is no longer active; the video
can still be seen on http://dpc.uba.uva.nl/cgi/t/text/text-
idx?cDctz;sidD85283e29b4851604e28c5c877d3520f1;
rgnDmain;idnoDm6704a04;viewDtext but it is very
coarse). Therefore, it is noteworthy that it was
observed in the present new material that the eyes are
actually positioned at some distance behind the ante-
rior margin. In this respect, the species looks much
more like a member of the genus Rhynchodemus than
a species of Microplana (Figs 2123). However, its
body musculature and copulatory apparatus clearly
indicate that M. nana is indeed a microplaninid.
The reproductive system of the specimens ZMA V.Pl.
7212.1, V.Pl. 7213.1, V.Pl. 7214.1, and V.Pl. 7215.1 con-
form to the accounts of Mateos et al. (1998) and Vila-
Farr
e et al. (2011). Histological sections of these animals
showed that, unfortunately, specimen V.Pl. 7212.1 is not
fully mature, while the sections of V.Pl. 7213.1 are partly
sagittal and partly transversal, due to the orientation of the
preserved animal. The last-mentioned situation prevented
the production of a graphical representation of a sagittal
reconstruction of the copulatory apparatus that provides a
representative picture of the topographical relationships
of its various parts. However, it is clear that (1) the penis
papilla is an elongated cone, (2) the vasa deferentia sepa-
rately open into the proximal section of the ejaculatory
duct, (3) the latter is broad and ventrally displaced, (4) the
ejaculatory duct is surrounded by a thick layer of circular
muscle, (5) a distinct genito-intestinal duct runs dorsal
from the short female genital duct to a branch of the intes-
tine. Specimens V.Pl. 7214.1 and V.Pl. 7215.1 possesses a
fully developed, albeit small, copulatory apparatus
(Fig. 24).
Neither Mateos et al. (1998) nor Vila-Farr
e et al. (2011)
provided a detailed account of the strong muscle fibres
that are arranged in a thick zone around the ejaculatory
duct. Figures 7C, D in Vila-Farr
e et al. (2011) suggest that
a strong musculature pervades the entire parenchyma of
the penis papilla. However, from their figure 7B it can
already be gleaned that this musculature actually belongs
to the ejaculatory duct, as was also evident in the present
material.
It is noteworthy that the genito-intestinal duct of M.
nana is generally broad and lined with large, cuboidal or
columnar, nucleated and ciliated cells.
The specimens V.Pl. 7216.1 and V.Pl. 7217.1 deserve
some special attention since at first glance their anatomy
seems to differ from the other specimens examined as
well as the accounts of Mateos et al. (1998) and Vila-Farr
e
et al. (2011) on the type material. However, our molecular
results unequivocally showed that these two animals
group with the other M. nana specimens (suppl. Fig. 1,
clade A) (see supplemental material online). The penis
papilla of these animals is a highly elongated, horizontally
oriented cone, pointing into a narrow and also highly elon-
gated atrium (Fig. 25). Similar to the other animals, their
ejaculatory duct is ventrally displaced and surrounded by a
thick coat of circular muscle. The most conspicuous differ-
ence between these two animals and others concerns the
place where female genital duct and genito-intestinal duct
communicate with the atrium. In the type material and
other specimens this communication is located at the poste-
rior end of the atrium, as is the case, for example, in our
specimen V.Pl. 7214.1 (Fig. 24). In specimen V.Pl. 7216.1
this communication is located at the postero-dorsal part of
the atrium while in V.Pl. 7217.1 it is even positioned at the
medio/antero-dorsal wall of the atrium (Fig. 25). From
there on, the female genital duct runs backwards for a short
distance and receives at its posterior end the separate open-
ings of the oviducts. From the point of communication
with the atrium the genito-intestinal duct runs dorsal to
open into a branch of the intestine. Characteristically, the
genito-intestinal duct bifurcates shortly before communi-
cating with the gut. Such a double connection with the gut
was not explicitly mentioned by Mateos et al. (1998)or
Vila-Farr
eetal.(2011), albeit that figures 7C, D in the
last-mentioned publication depict a bifurcation at the dorsal
end of the genito-intestinal duct. This bifurcation was not
evident in our other specimens, but this may simply be due
to their less advanced developmental stage.
In our Cox1 tree (Fig. 1, suppl. Fig. 1) (see supplemen-
tal material online) three highly differentiated clades are
apparent within this species, one of these, Clade C, includ-
ing four specimens from Huesca province, one from
Girona (319G216), and one individual from Tarragona
(060T622), while the rest of the Spanish M. nana speci-
mens are included in the other two clades (of which one is
composed exclusively of individuals from Huesca; Clade
B). In addition, the genetic distance between the three
clades (10.613%) is comparable to values found among
species included in the present study (1320%; Table 1).
Since this genetic diversity is not paralleled at the mor-
phological level, we do presently consider this group to
represent a case of a Deep Conspecific Lineage (DCL; cf.
Vieites et al., 2009), as revealed to be present also in other
groups of planarians (cf. Sluys et al. 2013).
Thus far the known distribution of M. nana covered
localities in the provinces of Barcelona and Girona in
north-eastern Spain (Mateos et al. 1998,2009). The data
of the present study extend the range of the species
130 km to the south (province of Tarragona) and 200 km
to the west (Huesca province) (Fig. 18).
Microplana robusta Vila-Farr
e & Sluys, 2011
(Figs 1,2,17)
Material examined. ZMA V.Pl. 7218.1 (441P253),
Fraga de Catas
os, Lal
ın, Pontevedra, Spain, N42.6367940
W8.0938810, coll. E. Mateos, M.
Alvarez-Presas & A.
Diversity of European microplaninid land flatworms 19
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Tud
o, 5 February 2012, sagittal sections on 8 slides. ZMA
V.Pl. 7219.1 (352Z234), Parque Nacional da Peneda
Ger^
es, Valle R
ıo Homem, Ger^
es, Portugal, N41.7914574
W8.1377124, 28 May 201, coll. E. Mateos, M.
Alvarez-
Presas & E. Sol
a, sagittal sections on 9 slides.
Comparative discussion. Thus far the known distribu-
tion of Microplana robusta concerned localities in the
provinces of A Coru~
na (Northwestern Spain) and Ger^
es
(Northern Portugal) (Vila-Farr
e et al., 2011). The new
locality documented in the present study for specimen V.
Pl. 7218.1 (Fig. 17) is situated at about the middle of the
line connecting the already known localities. However, it
appears that individuals belonging to the Galician locali-
ties (Pontevedra and A Coru~
na) are molecularly closer to
each other than to the two individuals of the other locality
in Portugal (Ger^
es; 345Z233 and 352Z234, Fig. 1, suppl.
Fig. 1) (see supplemental material online). The sections of
the animal from the latter locality (V.Pl. 7219.1) are actu-
ally poor but at least the blunt penis papilla was clear as
well as the coat of strong muscles extending on the atrium.
The external appearance of this animal (uniformly dark
yellowish or very pale brownish) very much resembles
that of M. robusta as depicted in Vila-Farr
e et al. (2011,
fig. 5A).
Microplana scharffi (Von Graff, 1896)
(Figs 1,2,17)
Material examined. ZMA V.Pl. 7220.1 (914I443), Parco
Nazionale d’Abruzzo Lazio e Molise, Italy, N41.78117
E13.88890, 1 May 2014, coll. E. Mateos, M.
Alvarez-Pre-
sas & A. Poch, sagittal sections of the anterior end on 6
slides, and sagittal sections of the posterior end on 6
slides.
Comparative discussion. Jones et al. (2008) provided an
extensive analysis of M. scharffi and its junior synonyms,
based on examination of type material as well as newly
obtained specimens from the UK. Our specimen V.Pl.
7220.1 exhibits all characteristic anatomical details of
M. scharffi as discussed by these authors as well as earlier
workers. In particular, the highly muscular penis bulb is
evident, as is the small copulatory bursa that communicates
with the intestine, and the expansion at the posterior sec-
tion of the female genital duct where it receives the open-
ings of the short common oviduct and the bursal canal.
Jones et al. (2008, p. 9) noted that the original descrip-
tion of M. scharffi does not mention the connection
between copulatory bursa and gut, although presence of
this genito-intestinal connection is evident in the type
material, while there is even ‘…a pencil note (author
unknown) on slide III.5 of the holotype series indicating a
genito-intestinal connection’. This note may well have
been made by Heinzel since he re-examined the type
series and thus discerned the genito-intestinal connection
(cf. Heinzel 1929, p. 446).
From a molecular point of view there is no doubt that
all specimens analysed belong to this species, because
they all have the same haplotype for the Cox1 region.
Accepting that the nominal species Microplana britan-
nicus (Percival, 1925), M. hovassei (De Beauchamp,
1934), and M. decennii (Battalgazi, 1945) are synonyms
of M. scharffi, the known distribution of the latter already
included localities in the UK, Ireland, western Turkey,
Belgium, and Madeira (cf. Ball & Reynoldson, 1981;
Jones et al., 2008). Although our new localities more or
less fall within this known distributional range, they do
add two new countries, viz. Italy and Bulgaria (Fig. 11).
Microplana groga Jones et al., 2008
(Figs 1,2,18)
Comparative discussion. The only two published locali-
ties of Microplana groga correspond to the type locality
in Jones et al. (2008) (’Font Groga’, Massis de Collserola,
Sant Cugat del Vall
es, Barcelona, Spain), and another in
Mateos et al. (2009) (Serra del Corredor, Canyamars, Bar-
celona, Spain). In February 2013 Dr Hugh Jones studied
and identified the specimens 359G244 (Natural History
Museum of London specimen code: NHMUK
2014.5.13.10) and 362G244 (NHMUK 2014.5.13.11) as
M. groga (H. Jones pers. comm.). This identification
allows us to assign to this species the specimens 359G244
and 362G244, which are located in the same molecular
clade. The new records expand the range of M. groga to
include some other localities in north-eastern Spain (Bar-
celona and Girona provinces; Fig. 12).
Microplana sp.
(Fig. 11)
Material examined. ZMA V.Pl. 7221.1 (1208U469),
Kalofer, Bulgaria, N42.60978 E24.95172, alt. 483 m, 18
September 2014, coll. E. Mateos & M.
Alvarez-Presas,
sagittal sections of the anterior end on 3 slides, and sagit-
tal sections of the posterior end on 7 slides.
Comparative discussion. Specimen V.Pl. 7221.1 is far
from being mature, its copulatory apparatus being very
small and much underdeveloped. A genito-intestinal con-
nection is evident, but not like that of M. scharffi, with its
small copulatory bursa communicating with the intestine.
Further, the penis papilla is very small and oriented hori-
zontally, whereas that of M. scharffi is more vertically ori-
ented and very big. However, specimen V.Pl. 7221.1 is
much underdeveloped. Since this animal, together with
another specimen (1189U466) for which material for mor-
phological study was not available, forms a separate
molecular clade that is the sister group of the species M.
scharffi (with a K2P distance of 13.2%; Table 1), we do
20 R. Sluys et al.
Downloaded by [Eduardo Mateos] at 07:51 08 January 2016
here assign it to the category of Confirmed Candidate Spe-
cies (CCC; cf. Vieites et al., 2009).
Discussion
Although molecular distance data do not represent a uni-
versally reliable tool for delimiting species (Hey, 2009),
we have nevertheless used this type of information in our
deliberations. This was enforced by the fact that for one of
the two new species herein described we lacked a sufficient
number of individuals to implement analyses of molecular
species delimitation, such as GMYC (Pons et al. 2006)or
BPP (Yang & Rannala, 2010). Therefore, we have resorted
to genetic distances to determine whether the morphologi-
cal uniqueness of M. plurioculata and M. polyposis was
paralleled by genetic differentiation (Handoo et al., 2014;
Vogler, Beltramino, Peso, & Rumi, 2014).
Both Microplana plurioculata and M. polyopsis do not
comply with the current diagnosis of the genus Micro-
plana, in that this mentions the presence of only ‘two small
eyes’ (cf. Ogren & Kawakatsu, 1988, p. 47), whereas these
two new species have multiple eyes. However, this diagno-
sis can be easily emended by inserting the term ‘generally’,
so that it will read: ‘generally two small eyes’. The unique-
ness of multiple eyes within the genus Microplana suggests
that this feature may form a synapomorphy for M. plurio-
culata and M. polyopsis, indicating that they share a unique
common ancestor. This hypothesis is supported by the fact
that also molecularly the two new species share a sister
group relationship (Figs 1,2;suppl.Figs1,2)(seesupple-
mental material online).
The present study underscores once more the power of
an integrative taxonomic approach, notably by combining
molecular and morphological data. In addition, molecular
sequences may reveal differentiation that is not directly
evident at the morphological level, and thus stimulate more
detailed anatomical studies. In other cases, molecular data
may uncover genetic evolution in the absence of morpho-
logical differentiation, as in the case in M. nana (see
above). Further, statistically analysed molecular data may
suggest which morphological characters are best to diag-
nose species or higher taxa (Carbayo et al., 2013). And
finally, inclusion of molecular data in the diagnosis of a
species (see above) ensures the possibility of species identi-
fication of immature specimens or even planarian cocoons.
Acknowledgements
We are grateful to Professor Dr M. Kawakatsu (Sapporo) for
having initiated and published the taxonomic index to the
terrestrial planarians, a publication that greatly reduces the
time-consuming literature searches that form such an impor-
tant part of taxonomic studies. We thank Drs A. Drozd and
H. Kappes for collecting the Microplana kwiskea material
from Poland. E. Sol
a and L. Leria are thanked for their sup-
port during the 2013 sampling trip to southern France. We
are indebted to Dr H. D. Jones (Natural History Museum,
London) for making available to RS numerous microphoto-
graphs of histological sections of a number of nominal land
planarian species housed at several European natural history
museums. R. Sluys is grateful to Dr H. Sattmann (Naturhis-
torisches Museum, Vienna) for granting access to the collec-
tions of the former Graz school of Turbellarian scholars and
to Dr Chr. H€
orweg (Naturhistorisches Museum, Vienna) for
arranging the digital microphotography equipment.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
M.
Alvarez-Presas acknowledges support from Synthesys,
the European Union-funded Integrated Activities grant
(project grant: NL-TAF 3497). This research was sup-
ported by the Ministerio de Ciencia e Innovaci
on of Spain
(CGL2011-23466).
Supplemental data
Supplemental data for this article can be accessed here: http://dx.
doi.org/10.1080/14772000.2015.1103323.
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