The identity of the invasive yellow-striped terrestrial planarian found recently in Europe: Caenoplana variegata (Fletcher & Hamilton, 1888) or Caenoplana bicolor (Graff, 1899)?

Abstract

Terrestrial planarians with a dorsal yellow stripe and dark lateral surfaces and up to 15-20 cm long have been found in several countries in Europe, the earliest in 2008. They are similar to two species originally from Australia, Caenoplana variegata (Fletcher & Hamilton, 1888) and C. bicolor (Graff, 1899), both described on external characters only, with no anatomical information. Careful reading suggests that there is no significant difference between the original descriptions. Further: observations on live specimens show considerable variation between individuals and in individuals over time and before and after feeding, negating any distinction between descriptions. Examination of three sectioned specimens shows considerable difference in sexual maturity, though one seems almost fully mature and the reproductive system is described. Molecular results show that specimens from the United Kingdom and Spain are of the same species. It is concluded that the planarians should be referred to as C. variegata, C. bicolor being a junior synonym.

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ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Accepted by W. Sterrer: 12 Sept. 2019; published: 5 Feb. 2020 193
Zootaxa 4731 (2): 193–222
https://www.mapress.com/j/zt/
Copyright © 2020 Magnolia Press Article
https://doi.org/10.11646/zootaxa.4731.2.2
http://zoobank.org/urn:lsid:zoobank.org:pub:BC349407-17A3-4FAF-AB01-38772AD5BF51
The identity of the invasive yellow-striped terrestrial planarian found recently
in Europe: Caenoplana variegata (Fletcher & Hamilton, 1888)
or Caenoplana bicolor (Graff, 1899)?
HUGH D JONES1,4, EDUARDO MATEOS2, MARTA RIUTORT3 & MARTA ÁLVAREZ-PRESAS3
1Scientific Associate, Life Sciences Department, Natural History Museum, London, SW7 5BD, UK.
2Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Universitat de Barcelona, Barcelona 08028, Spain.
3Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Barcelona 08028, Spain.
4Corresponding author. E-mail: flatworm@btopenworld.com.
Abstract
Terrestrial planarians with a dorsal yellow stripe and dark lateral surfaces and up to 15-20 cm long have been found in
several countries in Europe, the earliest in 2008. They are similar to two species originally from Australia, Caenoplana
variegata (Fletcher & Hamilton, 1888) and C. bicolor (Graff, 1899), both described on external characters only, with no
anatomical information. Careful reading suggests that there is no significant difference between the original descriptions.
Further: observations on live specimens show considerable variation between individuals and in individuals over time
and before and after feeding, negating any distinction between descriptions. Examination of three sectioned specimens
shows considerable difference in sexual maturity, though one seems almost fully mature and the reproductive system
is described. Molecular results show that specimens from the United Kingdom and Spain are of the same species. It is
concluded that the planarians should be referred to as C. variegata, C. bicolor being a junior synonym.
Key words: Feeding, Geoplana, predation, molecular identification, alien species, invasive species
Introduction
Land planarians (Fig. 1) with a longitudinal mid-dorsal yellowish band, itself with two mid-brown narrow lines,
predominantly dark brown, almost black but variable lateral surfaces and pale underside have been found in the
United Kingdom (UK) since 2008 (Jones, unpublished records), Spain (Álvarez-Presas et al., 2014), France (Justine
et al., 2014), The Netherlands (de Waart, 2016) and Greece (Vardinoyannis & Alexandrakis, 2019). The species was
named as Caenoplana bicolor (Graff, 1899) by Álvarez-Presas et al. (2014), de Waart (2016), Sluys (2016) and
Vardinoyannis & Alexandrakis (2019). Graff (1899) distinguished C. bicolor from a similar species, Caenoplana
variegata (Fletcher & Hamilton, 1888), both originally found in Australia and initially placed in the genus Geoplana
Stimpson, 1857. Both species were described on external characteristics only but Graff (1899) made no definitive
distinction between the two and without ever having examined a living specimen. However, careful reading of the
original and other descriptions, and examination of living specimens, the colour pattern of which can vary consider-
ably, throws doubt on any distinction between these two named species.
Central to the generic and specific definitions of land planarians is the anatomy of the muscular system and the
reproductive system but there has been no published anatomical account of any specimen under either name. The
type specimen of C. bicolor in the Berlin Zoological Museum (ZMB 3506) has been sectioned and shows partial
development of the reproductive system, enabling Winsor (1991) to re-assign it to the genus Caenoplana Moseley,
1877. For comparative purposes, two further specimens have been partially sectioned, a specimen collected in Aus-
tralia in 1897 deposited in the Natural History Museum, London (NHMUK), and one collected in the UK in 2015,
both identified as C. variegata (details follow). The three show considerable differences in the development of
their copulatory apparatus which makes firm conclusions difficult, though other anatomical details are similar and
do not suggest specific differences. In addition, taking into account the advantages of species identification using

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194 · Zootaxa 4731 (2) © 2020 Magnolia Press
molecular tools, some individuals collected in Spain and in the UK have been sequenced in order to identify them
in an integrative approach.
Materials and methods
Observations on living animals. Specimens received from various localities were kept individually in transparent
plastic containers with well-fitting, sealed lids, and small pieces of moistened wood were placed in each container
to maintain humidity and to provide refuge. The containers were placed on a shaded windowsill at ambient tempera-
ture. At intervals, specimens were weighed and photographed, and potential food items added.
Anatomy. A mature specimen from the NHMUK.97 collection and a specimen collected in the UK in 2015 (Cv7,
NHMUK.2018.8.31.1) have been partially sectioned to allow comparison with the Berlin specimen, ZMB3506.
Winsor (1973) deposited a specimen as G. variegata (but see below) in the National Museum of Victoria
(NMV), Australia (reg. no G2275) which also has earlier material but none of the NMV material has been examined
for this paper.
Specimens selected for sectioning were divided as below and sectioned portions embedded in wax, serially
sectioned at 12 µm, stained in Harris’s haematoxylin and eosin and mounted in Canada balsam.
Locations are given as decimal latitude and longitude.
Figure abbreviations: c1, first, dorsal, cavity in the male system; c2, second cavity in the male system; cf,
common female duct; cm, circular muscle; ed, ejaculatory duct; ep; epidermis; fo, female opening into atrium;
gp, gonopore; i, intestine; jod, junction of the ovovitelline ducts; jsd, junction of the sperm ducts; lm, longitudinal
muscle bundle; LS, longitudinal section(s); mo, male opening into atrium; mp, melanic pigment; nc, nerve cord; o,
ovary; od, ovovitelline duct; pm, parenchymal muscle; rh, rhabdites; sd, sperm duct; t, testis; tn, transverse nerve
commissure; TS, transverse section(s); vf, ventral fold in male atrium.
Molecular methods
DNA extraction, gene amplification and sequencing. A small portion of tissue fixed in absolute alcohol was
digested with Wizard Genomic DNA Purification Lysis Buffer (Promega, Madison, WI, USA) and Proteinase K
overnight at 37ºC. The DNA extraction was then completed according to the manufacturer’s instructions.
We amplified an approximately 1 kb fragment of the mitochondrial Cytochrome c oxidase I (Cox1 gene) by
PCR reaction in a volume mixture of 25 μl. We used primers BarS (Álvarez-Presas et al., 2011) and COIR (Lázaro
et al., 2009) and conditions were as in Álvarez-Presas et al. (2011). Amplification products were purified with a
vacuum manifold (MultiScreen®PCR96 Vacuum Manifold from Millipore Corporation or AcroPrep™ Advance
96 Filter Plate 30K Omega™ from Pall Corporation). Purified PCR products were sequenced from both strands at
Macrogen Inc. (Madrid, Spain).
Geneious v.10.2.2 (https://www.geneious.com) was used to revise the chromatograms and obtain the consensus
definitive sequences.
Molecular phylogenetic analyses. Table 2 lists the sequences used for this study. We obtained new sequences
from four individuals collected in the UK, provisionally identified as C. variegata, for comparison with a GenBank
sequence from a specimen previously identified as C. bicolor by Álvarez-Presas et al. (2014). DNA sequences were
translated into aminoacids using the genetic code number 9 corresponding to mitochondrial genome of Echino-
derms and Platyhelminthes and aligned manually in Bioedit v.7.0.9.0. (Hall, 1999). All sequences were unambigu-
ously aligned. We estimated the DNA sequence evolution model that best fits the data for the Cox1 gene sequences
using jModelTest 2.1.10. (Darriba et al., 2012), applying the Akaike information criterion (AIC). Phylogenetic re-
lationships were estimated by Maximum Likelihood (ML) using IQtree v.1.6.10 software (Nguyen et al., 2015) and
Bayesian inference (BI) using MrBayes v. 3.2.6. (Ronquist et al., 2012) at the CIPRES Science Gateway (Miller et
al., 2010). Bootstrap support (BS) values were obtained for ML trees from 1,000 replicates. In the BI analyses we
ran four chains to allow heating and used default priors, five million generations were run using the Markov Chain
Monte Carlo (MCMC) analysis in two independent runs. Sampling was every 1000 generations. The stationarity
and convergence of the runs were checked by plotting Log likelihood values versus number of generations and in-
specting when the standard deviation of split frequencies had reached < 0.01, respectively.
Species delimitation analyses. After the traditional phylogenetic analyses, we inferred the putative species

THE YELLOW-STRIPED LAND PLANARIAN IN EUROPE Zootaxa 4731 (2) © 2020 Magnolia Press · 195
boundaries using two exploratory methods: a) the Poisson Tree processes model (PTP) implemented in the bPTP
web server (Zhang et al., 2013) and b) the Automatic Barcode Gap Discovery (ABGD; Puillandre et al., 2012) us-
ing the ABGD web-based interface (http://www.abi.snv.jussieu.fr/public/abgd/). We ran the bPTP analysis with the
default parameters based on the Bayesian Inference tree obtained with MrBayes using the Cox1 data, specifying
that the outgroup is constituted of all specimens included in the tree that do not belong to the Caenoplana genus.
For the ABGD analysis we used the default parameters for P-values (Pmin = 0.001 and Pmax = 0.10), default value
of relative gap width X=1.5 and K80 distance metrics.
Published descriptions
Fletcher & Hamilton (1888) described specimens found in New South Wales, Australia as Geoplana variegata (at
that time most species with many eyes were placed in the genus Geoplana). Their description follows. Comments
in square brackets […] are of the present authors.
Geoplana variegata, n. sp.
(Plate V, figs 3 and 3’)
[reproduced here as Fig. 2a].
Undersurface white or cream-coloured in the centre, changing to greenish yellow at the margins. In the median
line of the dorsal surface is a very narrow linear longitudinal stripe of pale yellow or greenish-yellow, bordered
on either side by a slightly wider but still narrow linear stripe of dark brown or greenish-brown, its inner margin
the straighter and better defined; external to each of which again is a stripe of pale or greenish-yellow, twice or
three times the width of the median one; these in turn are each bounded externally by a very broad band extend-
ing outwards nearly to the lateral margin of the body, which band consists of an inner very dark and well-defined
portion in width about 1/3 of the whole, an outer marginal portion well defined but less intensely coloured, and
an intermediate portion consisting of numberless fine irregular wavy lines and streaks, with blotches and patches
of the yellowish ground colour shewing through; beyond each of the broad bands is a narrow band of pale or
greenish yellow. The median stripe, except for a short anterior portion where its bounding lines fuse, is very
well defined throughout; its bordering dark lines are lost quite anteriorly in the red or bright sienna colour of the
extreme tip, while just posteriorly they become confluent with the corresponding dark bands.
Length of the largest specimen when living and crawling 17 cm; breadth 5 mm; the same in spirit 13.8 cm
long, 7 mm broad; length of smaller specimen 2.6 cm, breadth 2 mm; we have had various intermediate sizes. In
a specimen 7.5 cm long the oral aperture is 25 mm behind the anterior extremity [33% of body length], and the
generative aperture 13 mm posterior to the mouth [51% of body length].
[Note: is this a mistake? Was anterior and posterior confused? In which case mouth would be 37 mm, 49%,
and the gonopore 50 mm, 67%. These are much more typical proportions in land flatworms generally and for
other, similar, specimens (Table 1).]
It is difficult to express accurately the exact tints of the dark bands in living specimens; they appear of vari-
ous shades of brown yet tinged with dark green; sometimes they are almost sage green. In spirit specimens all
the yellow and green tints are lost; the ground colour becomes almost whitish or cream color, and the dark bands
various shades of brown.
Hab.- County of Cumberland, Springwood, Mt. Wilson, Hartley Vale, Capertree, Burrawang (all NSW, Australia).
This fine species resembles C. subviridis in the general character of the markings, but differs in their ar-
rangement, the narrow median stripe with its narrow bordering dark stripes in the one case, markedly contrasting
with the broad median stripe with its intensely dark and relatively broader stripes in the other. The new species
has also the dark inner margin of the broad bands wider.
[Note particularly the comment “It is difficult to express accurately the exact tints of the dark bands in living
specimens” etc.]
Dendy (1892) identified similar specimens from Queensland as Fletcher & Hamilton’s species, G. variegata,
and gave a further description and a colour illustration:

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196 · Zootaxa 4731 (2) © 2020 Magnolia Press
TABLE 1. Body length, position of the mouth, M (pharyngeal aperture) and gonopore, Gp, from the anterior end (mm)
and position of mouth and gonopore as % of body length of specimens named as either Caenoplana variegata (Cv) or C.
bicolor (Cb) taken from literature or measured from museum specimens. Also shown are dimensions taken from Winsor
(1973) and of the sectioned UK specimen CV7. Fletcher & Hamilton (1888) possibly confused anterior and posterior (see
text), * indicates corrected values.
Sp Length M Gp M% Gp%
Fletcher & Hamilton 1888 Cv 75 25 38 33 51
Fletcher & Hamilton corrected Cv 75 37* 50* 49* 67*
NHMUK97.11.1 Cv 80 42 60 52.5 75
NHMUK97.11.1 (sectioned) Cv 113 58 80 51 71
NHMUK97.11.1 Cv 115 58 80 50.4 69.5
Median Cv excl F&H 1888 50.7 70.25
Dendy 1892 Cv 115 50 75
Graff 1899 Cb 75 36 55 48 73
Graff 1899 Cb 72 40 56 55 78
ZMB Vermes 3506 Cb 66 39 52 59 79
NHMUK1924.8.15 Went Falls Cv 80 35 48 44 60
NHMUK1924.8.15 Avoca Cv 134 63 88 47 66
NHMUK1924.8.15 Avoca Cv 70 37 - 53 -
NHMUK1924.8.15 Avoca Cv 60 26 38 43 63
NHMUK1924.8.15 Bundanoon Cv 130 60 84 46 65
Median Cb 48 69.5
Overall median 50 70.25
Winsor 1973 live (preserved) ? 90 (30) 62 68 69 76
CV7 live ? 84 46 61 55 73
CV7 preserved ? 52 32 39 61.5 75
Geoplana variegata, Fletcher & Hamilton
(Plate XI, Fig. 2)
[reproduced here as Fig. 2b].
This very handsome species was obtained by Professor Spencer in large numbers and would seem to be the
commonest species in the district visited by him. The body is long and narrow, even when lying still and coiled
into a knot; tapering a good deal more gradually in front than behind. The shape of the dorsal surface varies
from more or less flattened to strongly ridged, according to the position of the animal; it may be said to be
characteristically ridged, as shown in the figure. The worm reaches a very large size. The largest specimen,
after preservation in spirit, measured 115 mm in length by 6 mm in greatest breadth; I could not get it to crawl
about actively so as to measure it when crawling. A smaller specimen, however, in which also a portion of the
posterior extremity was broken off; measured about 163 mm in length by 5 mm in breadth when crawling. The
peripharyngeal aperture (in spirit) is in about the middle of the ventral surface [50%] and the genital aperture
about half-way between the peripharyngeal and the posterior extremity [75%]. The eyes are not very numerous,
in two patches one on each side of the anterior end of the body and continued in close-set single series all round
the anterior margin. The predominant tint of the dorsal surface varies from green or violet to rich reddish-brown
or brownish-red. Running down the mid-dorsal line is a very narrow stripe of bright yellow. On each side of this
is a slightly broader stripe of dark brown, whose outer edge is ill-defined. Then a still slightly broader stripe of
bright yellow with a few very minute flecks of brown. Then a very broad, dark band of the predominant tint,
most commonly dark greenish-brown or grey, almost black, gradually fading outwards into a narrow band of
pale greyish or greenish-blue. Then a narrow stripe of a very dark brownish colour only slightly wider than the
narrow blue band, and, lastly, another narrow blue band but wider than the first and extending to the margin
of the ventral surface. The anterior extremity is pinkish. In the mid-ventral line there is a rather narrow, almost

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white band gradually merging on each side into a pale purplish-grey band which, in turn, gradually merges into
the pale greenish-blue band at the margin of the dorsal surface.
All things considered, I have little hesitation in identifying this species with Messrs. Fletcher & Hamilton’s
G. variegata, especially as the latter appears to vary somewhat in tint. As this very handsome species has not
before been figured of the natural colours, I have thought it desirable to do so now. Localities- Gympie (Mary
River); Burnett River; Cooran [all Queensland].
[Note the comment that this “appears to vary somewhat in tint”.]
Dendy mentions this species in three further publications. Dendy (1893a):
Geoplana variegata, Fletcher & Hamilton.
A single specimen of this common New South Wales and Queensland species was obtained by Professor Spen-
cer at Bedlam Heights [Tasmania] in January 1893, and, together with coloured drawings of the living animal,
placed by him in my hands. I have no hesitation in making the identification, although, curiously enough, the
species has not yet been found in the intervening colony of Victoria. The general shape of the body (in spirit),
the position of the external apertures, and, above all, the very characteristic arrangement of the coloured stripes,
are identical. The general ground tint of the body in life was brown or bluish-brown, and the three narrow
stripes, usually of “pale-yellow” or “greenish-yellow”, lying in and near the mid-dorsal line, were decidedly
green. When crawling, the specimen measured 44 mm long by 3 mm broad.
Dendy (1893b): notes this specimen from Tasmania, without further description, and comments: “It is re-
markable that, although a common Queensland and New South Wales species, it has not yet been recorded from
Victoria.
Dendy (1894): “This beautiful and well-characterised species was found at both Wentworth Falls and Black-
heath [New South Wales], and I received numerous specimens both alive and in spirit. The prevailing tint of the
markings was brown on a yellow ground.”
Specimens found by Spencer (see the Dendy 1892 extract above) were sent by Dendy to Graff at the University
of Graz, Austria, and Graff (1899) described the specimens as a new species, G. bicolor, retaining G. variegata as a
separate species. In his description of G. bicolor, Graff (1899) reproduces Dendy’s (1892) G. variegata text in full
(above), in English, followed by:
Nach dieser Beschreibung und der vortrefflichen Abbildung Dendy’s kann keine Rede davon sein, die vor-
stehende Form mit der G. variegata Fletch. Ham, zu identificiren. Ich habe mich davon ausserdem durch Unter-
suchung von vier ausgezeichnet conservirten Exemplaren überzeugt, die ich der Güte des Herrn W. B. Spencer
verdanke. Sie sind sämmtlich von querovalem Durchschnitte, der Bauch schwach, der Rücken stärker convex und
die Seiten abgerundet und fast in ganzer Länge gleichbreit. Im Gegensatze zum lebenden Objekte ist das Hinterer-
ende allmählich zugespitzt, während sich das Vorderende rasch verjüngt und abrundet, ein Beweis, dass letzteres
sich in Spiritus stärker contrahirt als ersteres. Die Haut ist auf dem ganzen Rücken bis zum Bauchrande mit gel-
bröthlichem (hell-testaceus oder mehr luteus) Pigmente erfüllt, das mittlere Drittel der Rückenbreite hat daher diese
Farbe und erscheint nur dort lebhaft gelb, wo das Epithel fehlt. Im Reste der Oberseite bis zum Bauchrande ruft
das durchscheinende Parenchympigment eine blaugrüne (aerugineus) Färbung hervor, die überall dort, wo dasselbe
reichlicher angehäuft ist, mehr in’s Grüne, an anderen Stellen dagegen (so z. B. nahe dem Bauchrande) mehr in’s
Hellblaue spielt.’ Die dichte Anhäufung desselben Pigmentes ruft die sechs Streifen hervor.: zwei feine mediale
(die bei meinen Spiritusexemplaren aus einzelnen dunkelblau-grün durchscheinenden Flecken zusammengesetzt
erscheinen), die die blaue Seitenzone nach innen begrenzenden Lateralstreifen und die Marginalstreifen. Die later-
alen sind nach aussen allmählich verwaschen und so breit wie die Medialstreifen mitsammt der von ihnen einge-
schlossenen Medianzone. Die Marginalstreifen, etwa doppelt so breit wie die Medialstreifen, liegen in der Mitte
zwischen Lateralstreifen und Bauchrand. Die Grenze der blauen Farbe gegen die Bauchfläche ist zwar mit der Lupe
deutlich zu sehen, aber deshalb nicht scharf in die Augen springend, weil die ganzen Seitentheile des Bauches von
durchscheinendem Pigmente hellviolett (fumosus) gefärbt erscheinen und erst allmählich und ohne Grenze in die
weissliche (cremeus) Bauchmitte übergehen. Am Vorderende werden die violetten Bauchseiten erheblich heller und
es nimmt hier die ganze Bauchfläche gegen die von der weissen Sinneskante umsäumte Spitze hin einen gelben
(sulphureus) Ton an. Eine Concavität ist indessen hier auch nicht einmal andeutungsweise vorhanden.
Die beiden grössten Individuen waren 75 (resp. 72) mm lang, bis 4,8 (3,6) mm breit und 2,7 (2,5) mm dick;

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198 · Zootaxa 4731 (2) © 2020 Magnolia Press
der Mund liegt. 36 (40) mm, die Geschlechtsöffnung 55 (56) mm vom Vorderende entfernt. Dass bei dem etwas
kleineren, aber erheblich schlankeren Thiere die Öffnungen weiter nach hinten liegen, hat offenbar in einer ger-
ingeren Contraction des Vorderkörpers desselben seinen Grund.
Die zwei kleinsten Exemplare sind durch einen viel dunkleren Ton der Seitentheile des Bauches (Ventral-
zone) ausgezeichnet, sowie dadurch, dass hier die Medialstreifen unvollständig sind. Bei dem einen (von 59mm
Länge) fehlen sie im letzten Drittel ganz und sind vorne wiederholt unterbrochen, bei dem anderen (von 51mm
Lange) sind sie nur am äussersten Vorderende deutlich und am übrigen Körper nur in kümmerlichen Spuren
vorhanden.
Translation of Graff’s German text [comments by HDJ]:
According to this description [Dendy’s 1892, see above], and Dendy’s excellent illustration, there can be
no question of identifying the above form with G. variegata Fletch. Ham. Furthermore, I have obtained, thanks
to Herr W. B. Spencer, four excellently preserved specimens, and my examination of them has also convinced
me that this is the case. They are all “queroval” [oval, width greater than height] in cross section; the belly is
slightly convex and the back more strongly convex; the sides are rounded and the same width for almost the
whole length. Unlike in the living specimens [he only examined preserved specimens], the posterior end of these
specimens is tapered gradually but the anterior end tapers and rounds quickly. This shows that when preserved
in alcohol the latter contracts more strongly than the former. On the whole of the back, up to the belly edge, the
skin is filled with yellow-reddish pigments (pale testaceus) [brick-coloured: brownish red, brownish yellow or
reddish brown] or more luteus [yellowish]. Therefore the middle third of back-width is of this colour and only
appears vivid yellow where the epithelium is absent. On the rest of the dorsal surface as far as the belly edge,
the parenchymal pigment shines through, producing a blue-green (aerugineus) [verdigris, copper-green] colour-
ing; in the places where the pigment is most concentrated it tends more to green, and in other places e.g. near
the belly edge, it tends more towards pale blue. The strong concentration of this same pigment produces the six
stripes; two thin medial stripes (in my preserved specimens these appear to be made up of individual dark blue-
green flecks/spots); the lateral stripes which border the blue side zone on the inside; and the marginal stripes. The
lateral stripes fade gradually to the outside and they are as broad as the medial stripes together with the median
zone enclosed by these. The marginal stripes, roughly twice as wide as the medial stripes, lie midway between
the lateral stripes and the belly edge. The border of the blue colour with the belly-surface can be clearly seen
with a hand lens but is not otherwise obvious. This is because all the side parts of the belly appear violet coloured
(fumosus) [smoky?] from the pigments shining through, and they (the side parts) shade into the whitish (cremus)
[cream] middle of the belly gradually and without a border. At the anterior (front) end, the violet belly sides be-
come considerably lighter, and here towards the head, which is surrounded by the white sensory edge, the whole
belly surface takes on a yellow (sulphureus) shade. But here also there is no trace of concavity.
The two biggest specimens were 75 (resp 72) mm long, up to 4.8 (3.6) mm wide and 2.7 (2.5) mm thick.
The mouth is 36 (40) mm [48% and 56% respectively] and the reproductive opening 53 (50) mm [71% and 69%]
away from the anterior end. In the smaller but considerably narrower specimen the openings are further towards
the back and this is obviously caused by the lesser contraction of the front of the body.
The two smallest specimens are characterized by a much darker shade on the side parts of the belly (ventral
zone), also by medial stripes which are incomplete. In one specimen (59 mm long) they are completely absent in
the last third, and at the front they are repeatedly interrupted. In the other specimen, (51 mm long) they are only
clearly present at the very end of the anterior extremity, and on the rest of the body only present in faint traces.
Winsor (1973) recorded a specimen identified as G. varigata (sic) from Victoria. Winsor’s description:
Geoplana varigata, Fletcher & Hamilton, 1887.
Diagnosis: With four dark longitudinal stripes, the lateral stripes broader than the median stripes, marginal
zones mottled, dorsal ground colour yellow to green. Geoplana varigata was differentiated from G. subviridis
(Moseley), the median zone in the latter broad and the paired innermost stripes lateral rather than median in po-
sition, from G. howitti (Dendy), in which the lateral stripes were narrower than the median stripes and from G.
bicolor (Graff), which had six dark stripes dorsally.
The Victorian specimen had a dorsal median zone of blue-green ground colour 0.25 mm wide bordered on
either side by faint brown-flecked paired median stripes 0.25 mm wide. External to these were 0.5 mm wide

THE YELLOW-STRIPED LAND PLANARIAN IN EUROPE Zootaxa 4731 (2) © 2020 Magnolia Press · 199
lateral zones of ground colour which merged into the marginal brown-flecked paired stripes 0.6 mm wide, the
inner margins of which were well defined. These were bordered by a fine zone of ground colour continuous with
the ventral surface. The dorsal stripe pattern is illustrated in Figure 1 [reproduced here as Fig. 2c].
The anterior tip was pink and the ventral surface a blue-white colour, pale over the creeping foot. Eyes were
multiple, concentrated in three rows on the anterior third of the body with a single row around the anterior tip.
The specimen measured 90 mm long and 2 mm wide when crawling, the pharyngeal aperture 62 mm from
the anterior end [69%] and the genital aperture 4 mm posterior to the pharyngeal aperture [73%]. The dimensions
of the worm following fixation were 30 mm long and 3 mm wide. This single specimen was lodged as a voucher
specimen with the National Museum of Victoria (Reg. No. G2275.)
Three spirit specimens of Geoplana varigata labelled “typical”, collected by T. Steel from Bundanoon, New
South Wales, in the N.M.V. collections, were also examined and compared to the Victorian specimen. The above
description of the dorsal stripe pattern and the relative positions of the pharyngeal and genital apertures agreed
closely with those of previous authors, and with the spirit specimens examined. Difficulty was encountered in
the description of colour as it was a mixture of yellow and green, and comparison to faded spirit specimens was
pointless. Geoplana varigata had been recorded from localities in the Blue Mountains, Bundanoon and Bur-
rawang, New South Wales, and from Bedlam Heights, Tasmania. The Victorian specimen was collected from
beneath a rotting snow gum at a locality 4,900 feet A.S.L., 1.2 miles from the Moroka Road, on the Tarli Karng
track via McFarlane saddle, Wellington Plains, Gippsland. Geoplana varigata was associated with G. howitti, G.
spenceri, G. lucasi, G. sanguinea and the nemertine worm Geonemertes australiensis, all normally found in wet
mountainous areas. Some specimens of G. spenceri, normally a blue-green planarian, were collected, in which
the dorsal ground colour was a clear yellow in one specimen, and in two others a yellow green. Typical speci-
mens were also found in the same locality. This colour variation observed in G. spenceri may explain the similar
tints in G. varigata, there perhaps being certain environmental factors influencing pigmentation.
At present, Wellington Plains, Gippsland, is the only known Victorian locality of G. varigata, and the status
of this land planarian is considered rare.
Winsor (pers. comm.) now considers that, due to its small size and mature state, this specimen could be a fur-
ther, undescribed, species.
Winsor (1991), after sectioning and examining the type specimen of G. bicolor (Berlin Museum, ZMB Vermes
3506, originating from the collection of Graff) placed G. bicolor and also G. variegata in the genus Caenoplana
Moseley, 1877, hence Caenoplana bicolor (Graff, 1899) and C. variegata (Fletcher & Hamilton, 1888). This ge-
neric placement is here accepted.
So there are a number of descriptions of external features of generally similar worms with, according to Graff
(1899), two species, possibly three species if Winsor’s (1973) specimen should be different. Graff’s (translated)
phrase “there can be no question of identifying the above form with G. variegata”, is unequivocal. However, in
the subsequent text he makes no detailed comparison and gives no points of differentiation between C. bicolor and
C. variegata. Moreover, Graff was only able to examine specimens preserved in spirit (alcohol) and the colour of
specimens can be affected by preservation in alcohol—note Winsor’s (1973) comment “comparison to faded spirit
specimens was pointless”. Further, the specimens received by Graff had been collected by Spencer, sent to Dendy
who forwarded them to Graff. But Dendy (1892) himself had ‘little hesitation’ in identifying them as G. variegata.
So how and why did Graff distinguish the two supposed species?
Winsor (1973, 1991 and 2008 pers. comm.) suggests that the two species are distinguished by the number of
dark stripes, four in C. variegata and six in C. bicolor, and Graff (1899) states that his specimens had six dark
stripes. However, it is perfectly possible to interpret Fletcher & Hamilton’s (1888) description as having six dark
stripes, three on each side as follows (Fletcher & Hamilton text in quote marks): ‘narrow linear stripe of dark brown
or greenish-brown, its inner margin the straighter and better defined;’ This applies to the European specimens- the
narrow dark lines either side of the midline, the median stripes of Winsor (1973), the medial stripes of Graff (1899)
[stripe 1]; ‘very broad band extending outwards nearly to the lateral margin of the body,’…The whole of this band
would be considered the second, lateral stripe of Winsor (1973), in which case there would be four stripes, but
Fletcher & Hamilton go on: ‘which band consists of an inner very dark and well-defined portion [stripe 2], in width
about 1/3 of the whole, an outer marginal portion well defined but less intensely coloured’ [stripe 3], ‘and an in

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TABLE 2. List of samples used in the molecular analysis with code used in the tree and GenBank accession number.
Species/Morphotype Tree Code Locality / Source GenBank Code
Cox1
Family Geoplanidae
Subfamily Rhynchodeminae
Tribe Caenoplanini
Artioposthia sp. Artioposthia sp. Aus Australia / * MN990642*
Artioposthia testacea Artioposthia testacea - / * MN990643*
Arthurdendyus triangulatus KP195028_Arthurdendyus triangulatus_UK United Kingdom / Roberts,D.M. et al. (GenBank) KP195028
Caenoplana sp. 1 Caenoplana sp. 1 - / Alvarez-Presas et al. 2008 DQ666031
603_Caenoplana morph Ca1 Townsville (Australia) / Álvarez-Presas et al. 2014 KJ659643
605_Caenoplana morph Ca1 KJ659644
658_Caenoplana morph Ca2 Bordils (Girona, Spain) / Álvarez-Presas et al. 2014 KJ659650
Caenoplana sp. 4 Caenoplana sp. 4 - / Alvarez-Presas et al. 2008 DQ666032
Caenoplana variegata 654_Caenoplana bicolor Bordils (Girona, Spain) / Álvarez-Presas et al. 2014 KJ659648
PT1600_Caenoplana variegata_Cv7 Coventry, UK / * MN990645*
PT1602_Caenoplana variegata_Sn Southampton, UK / * MN990646*
PT1603_Caenoplana variegata_Cf Cardiff, UK / * MN990647*
PT1604_Caenoplana variegata_CvP MN990648*
Caenoplana coerulea C.coeruleaTallaganda1 Tallaganda (Australia) / Sunnucks et al. 2006 DQ227621
CcoTal2 DQ227625
CcoTal3 DQ227627
CcoTal4 DQ227629
CcoTal8 DQ227635
CcoTal9 DQ227634
Caenoplana morph Ca1 415_Caenoplana morph Ca1 Vall de’n Bas (Girona, Spain) / Álvarez-Presas et al. 2014 KJ659618
416_Caenoplana morph Ca1 KJ659619
423_Caenoplana morph Ca1 KJ659626
445_Caenoplana morph Ca1 Badalona (Barcelona, Spain) / Álvarez-Presas et al. 2014 KJ659635
649_Caenoplana morph Ca1 Granollers (Barcelona, Spain) / Álvarez-Presas et al. 2014 KJ659646
Caenoplana morph Ca2 426_Caenoplana morph Ca2 Bordils (Girona, Spain) / Álvarez-Presas et al. 2014 KJ659628
427_Caenoplana morph Ca2 KJ659629
428_Caenoplana morph Ca2 KJ659630
430_Caenoplana morph Ca2 KJ659631
431_Caenoplana morph Ca2 KJ659632
PT655 MN990644*
657_Caenoplana morph Ca2 KJ659649
...continued on the next page

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TABLE 2. (continued)
Species/Morphotype Tree Code Locality / Source GenBank Code
Cox1
OUTGROUP: Tribe Rhynchodemini
Dolichoplana sp. Dolichoplana sp. - / Alvarez-Presas et al. 2008 DQ666037
D. striata D.striata Bra Igreginha (Brazil) / Carbayo et al. 2013 KC608226
Platydemus manokwari Platydemus manokwari Aus Townsville (Australia) / Alvarez-Presas et al. 2008 AF178320
Rhynchodemus sylvaticus Rhynchodemus sylvaticus_Canyamars Canyamars (Barcelona, Spain) / Mateos et al. 2009 FJ969946
*Sequences obtained in this study

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termediate portion consisting of numberless fine irregular wavy lines and streaks, with blotches and patches of the
yellowish ground colour shewing through’. In that case there would be six stripes. This description applies to the
specimens shown in Fig. 1b and c.
Winsor (1973, 1991 and 2008 pers. comm.) is of the opinion that the two species are distinct: ‘The external fea-
tures that readily distinguish the species are the presence of a fine reticulate pattern, yellow-green colour (fugitive
in spirit) and plain pale ventrum in C. variegata/C. subviridis compared to more solid greenish-blue-brown colour
(spirit stable) and pattern, with pale blue ventrum with median pale zone on C. bicolor.” However, Fletcher & Ham-
ilton (1888) write that the ventral surface is ‘white or cream-coloured in the centre, changing to greenish yellow at
the margins’, so the ventrum of C. variegata is not plain pale.
After careful interpretation of these descriptions, it is our opinion that there is no substantive difference between
them and consequently there is no justification for distinguishing between the species C. variegata and C. bicolor.
Do observations on living specimens and/or anatomical comparison of specimens suggest any support or oth-
erwise for this conclusion?
Results
Observations on living specimens
Colour and stripes. One of us, HDJ, has been maintaining specimens, found in various UK locations, in captivity,
some individuals for over five years. In living specimens, the degree of pigmentation can be seen to vary consider-
ably both between individuals (Fig. 1), and within individuals over time (Fig. 3) and even along the length of the
same individual.
Figure 1a shows a specimen (CV7, NHMUK.2018.8.31.1, subsequently sectioned for anatomical study) with
two prominent brown lines within the yellowish dorsal band, and the lateral bands appear uniformly almost black.
This could be interpreted as having four dark lines—thus C. variegata under Winsor’s (1973) distinction. Figure 1b
shows a macrophotograph of part of a different coiled specimen. The medial brown lines are clear within the yel-
lowish dorsal band, but there is some difference in the intensity of the brown colour along the body. The lateral band
consists of a black stripe next to the yellowish dorsal band, fading to a broader blue-green zone speckled with brown
(left part of photograph) before the dark marginal stripe. But on the right of the photograph there appear to be two
dark stripes within the blue-green zone, but only on one side of the body. Figure 1c shows an individual with paler
medial brown lines and fewer lateral dark lines. Figure 1d shows an individual with very little brown pigment in the
dorsal medial lines and the lateral band shading from a dark blue-green to a paler blue-green with no dark pigment
laterally. Figure 1e shows a specimen with no brown medial lines and just feint dark pigment at the border of the
yellow mid-band, but not along its whole length.
Figure 3 shows photographs of a single specimen, originally from Southampton, UK, held in captivity for
nearly five years. In 2013 the medial brown stripes and the lateral band appear uniformly very dark, though the
lateral band can be seen in close-up to be resolved into several stripes. However, in 2018, the medial brown stripes
are much paler, and the lateral band shows several dark lines over a greenish ground colour. This specimen fis-
sioned soon after capture with two ca 1 cm portions breaking off the rear (one shown in Fig. 3c). They had partially
regenerated after about 3 weeks (Fig. 3e). The larger portion (Fig. 3d, f, g) has never, after nearly five years, shown
any sign of maturity evidenced by a gonopore. By October 2018 the larger single portion had undergone fission into
three portions (Fig. 3h).
Figure 4 shows photographs of another specimen (the specimen shown in Fig. 1e) taken in March, May, August
and December 2018. After March it was fed ad lib on woodlice, Oniscus asellus L., and consumed four specimens
before the photograph in May. Initially it was pale with little dark pigment, but after feeding both the median brown
stripes and the darker dorso-lateral and lateral stripes are present.
Thus the intensity and shade of the mid-dorsal yellowish stripe and of the two narrow brown medial lines within
the yellow stripe can vary, the latter may be almost invisible in some. Pigmentation from the margin of the dorsal
yellowish band to the lateral margin of the body appears to vary within and between individuals from fairly uniform
dark brown/black, in which case they can be considered to have four stripes, to a dorsal and ventral darker region
separated by ground colour and random dark areas or stripes of dark colour, as described by Fletcher & Hamilton
(1888), in which case they might be considered to have six stripes, to almost no dark pigment (Fig. 1d, e). Thus it
seems certain that intensity and colour of the various stripes are affected by diet, feeding and starvation.

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Thus there is intraspecific and temporal individual variation. Consequently the precise number of dark stripes,
which Winsor (1973, 1991 and 2008 pers. comm.) has suggested distinguishes the two species, is, at best, a weak
character to separate two species. Consequently there is no justification for separating C. variegata and C. bicolor
on this basis.
Feeding and reproduction
The flatworms seem to feed on almost any arthropod. In captivity they have been fed on woodlice (Isopoda) and
mealworms (larvae of Tenebrio molitor L.). Prey is captured by means of sticky mucus that covers the flatworm’s
body and then manipulated to the region of the pharyngeal aperture. After feeding on woodlice, the exoskeleton
of the prey is left empty except for the gut and its contents which appear intact (Fig. 4e). They do not touch earth-
worms, slugs or snails. In the field they have been seen feeding on a spider (Fig. 4f). Brittlebank (1888) noted a
“banded, leech-like worm… or Planarian worm” (later he uses the phrase “striped leech-like”) feeding on a wood-
louse in Spring Vale, Victoria, Australia: “It caught this insect (sic) by means of the mucous coating with which
these worms are covered … and after crawling over it a short time, it protruded an organ from the under side of the
body ... and inserted it between the segments on the underside of the slater”; “...it was not long before the empty
shell was all that remained of what had once been a slater or wood-louse”. He also found one “devouring the larva
of a ground beetle”. As he gave no further description of the worms, their species is a matter of speculation but they
could have been C. variegata.
The worms readily reproduce by fission, portions 1-3 cm long breaking off the rear and regenerating. In one
damaged specimen two small portions about 1 cm long broke off and regenerated (Fig. 3b, c, d, h).
NHMUK preserved specimens
Three collections of specimens labelled Geoplana variegata have been deposited in the Natural History Mu-
seum, London (NHMUK) in 1877, 1897 and 1924. (NHMUK accession numbers take the form of year, month, day
and specimen number. Hence NHMUK.77.11.2 was deposited on 2 November 1877.)
NHMUK.77.11.2.9. label reads: “Geoplana variegata Fl & Ham. = das zweite der beiden in vorn Glase” [the
second of the two in front vial]. Followed by a boxed section reading: “Caenoplana subviridis Moseley (types).
Hab. Parramatta N. S. Wales. Challenger Exped. 77.11.2.9”. Then: “enthalen gewesenen Exemplare [contained
copies]. No 30. Von Graff”. The vial contains one entire worm ca 7 cm long, mouth at about 4 cm, but no gonopore
visible.
NHMUK.77.11.2.11 has several labels: “Caenoplana sp? Parramatta NSW. Challenger exped. Caenoplana
nondescribed? = var of subviridis. Geoplana variegata Fl & Ham. No 31, von Graff.” Inner labels read “Geoplana
sp? = Caenoplana var subviridis 77.11.2.11. Geoplana variegata”. The vial contains four portions, apparently of
two broken specimens, one about 7 cm long, mouth about 3.5 cm, the other about 5 cm long, mouth 2.5 cm. No
gonopore visible in either.
The labels of both the 1877 items are in the handwriting of Graff who also signed both labels. The 1877 speci-
mens were collected by H. N. Moseley in the course of the Challenger Expedition (1872-1876). Thus they pre-date
the naming of either species by Fletcher & Hamilton (1888) and Graff (1899) but were subsequently examined by
Graff, presumably in the mid 1890s, who has identified the specimens as far as possible and written and signed the
labels. How Graff was able to suggest their species is problematic since there is now no sign of striations on any
of them, unless at the time striations were visible. However, he did consider them to be G. variegata, which seems
inconsistent with his subsequent description of the new species G. bicolor for similar specimens. Currently these
specimens, like most NHMUK specimens, are stored in 80% industrial methylated spirits (IMS) in which some
pigmentation can sometimes be preserved, but previously they may have been in formalin which tends to remove
pigmentation.
NHMUK.97.11.1.26-28. “Geoplana variegata F&H, Bundanoon NSW, Typical, T. Steel, 1897: three speci-
mens.” One of these specimens that does show faint dark striations has been selected and partially sectioned—de-
tails in the next section.
NHMUK.1924.8.15.486-495. Large jar with 7 vials and painted red lid (indicating type material). “Geoplana
variegata F&H, Wentworth Falls, Feb 1894, T. Steel. Received with letter from T. Steel dated 25 March 1894”, six
specimens; “Geoplana variegata F&H, Avoca NSW, T. Steel, XI 05 [November 1905?]”, four specimens; “Geoplana
variegata F&H, Bundanoon NSW, Typical, T. Steel, 1897”, one specimen.

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FIGURE 1. Caenoplana variegata. Specimens from the UK showing variation in pigmentation. a, specimen from Coventry, UK, as received
(collected January 2015, specimen Cv7, sectioned for anatomical studies). Note the lateral surfaces appear uniformly black. Scale bar 1 cm. b–d,
specimens after many months in captivity and fed at occasional intervals: b, a coiled specimen showing varied dark pigmentation laterally; c,
another specimen with limited dark pigment laterally; d & e, a specimen with almost no dark pigment laterally though a little anteriorly bordering
the dorsal yellow band.
The NHMUK.1924 specimens are part of the extensive collection of material from Australia and New Zealand
deposited by Dendy shortly before his death.

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Thus all the 1924 specimens may be considered co-types of G. variegata. Who made this designation is un-
known, possibly Dendy. However, if Graff’s (1899) conclusion is accepted, the 1924 collection, at least, might be G.
bicolor, even though some were collected prior to publication of Graff’s (1899) monograph! Of course it is possible
that Dendy simply did not re-label his specimens prior to deposition, though he was well aware of Graff’s (1899)
monograph.
FIGURE 2. a, Plate V, figs 3 and 3’ of Fletcher & Hamilton (1888) as Geoplana variegata. b, Plate XI, Fig. 2 of Dendy (1892),
as Geoplana variegata (but see text) original in colour. c, Figure 1 of Winsor (1973) as Geoplana varigata (but see text).
Taxonomic section
Order TRICLADIDA Lang, 1884
Suborder CONTINENTICOLA Carranza, Littlewood, Clough, Ruiz-Trillo, Baguñà & Riutort, 1998
Family GEOPLANIDAE Stimpson, 1857
Subfamily RHYNCHODEMINAE Graff, 1896
Tribe CAENOPLANINI Ogren & Kawakatsu, 1991
Genus Caenoplana Moseley, 1877

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FIGURE 3. Caenoplana variegata. Photographs of a single specimen from Southampton, UK, collected April 2013, taken on
the dates and times shown: a & b, as received. Note the varied lateral pigmentation in (a), but that in (b) the lateral band seems
uniformly black (grooves on the background stone are 1 cm apart, scale bars where shown 1 cm). The white patch near the
posterior end was a wound and the worm fissioned at this point shortly afterwards into three portions, a long anterior portion
about 5 cm long and two posterior portions about 1 cm long, one shown in (c); d, the two posterior portions and e, the anterior
fissioned portion, all three portions showing regeneration; f, g and h, the anterior portion (1 mm graph paper scale), by October
2018 (h) it had fissioned into three portions. The dark lineation varies considerably over time.

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FIGURE 4. Caenoplana variegata. a–d; photographs of the same specimen taken on the dates shown. Between 7 March and 7
May it had consumed four woodlice (Oniscus asellus L.) and several more before 6 August and more before 12 December; e, a
woodlouse after being fed upon by a specimen; f, a specimen feeding on a spider (photo Sally Ann Hurry).
Caenoplana variegata (Fletcher & Hamilton, 1888)
Geoplana variegata Fletcher & Hamilton, 1888
Geoplana variegata Dendy, 1892
Geoplana variegata Dendy, 1893a
Geoplana variegata Dendy, 1893b
Geoplana variegata Dendy, 1894
Geoplana bicolor Graff, 1899
Geoplana variegata Graff, 1899
Geoplana variegata Steel, 1900

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Geoplana varigata Winsor, 1973
Geoplana bicolor Winsor, 1973
Geoplana varigata Winsor, 1977
Australopacifica bicolor Ogren & Kawakatsu, 1991
Australopacifica variegata Ogren & Kawakatsu, 1991
Caenoplana bicolor Winsor, 1991
Caenoplana variegata Winsor, 1991
Caenoplana bicolor Winsor, 1997
Caenoplana bicolor Álvarez-Presas et al. 2014
Espèce “rayée jaune” Justine et al., 2014
Caenoplana bicolor De Waart, 2016
Caenoplana bicolor Sluys, 2016
Caenoplana bicolor Vardinoyannis & Alexandrakis, 2019
Material examined. NHMUK.97.11.1.26-28, as Geoplana variegata, Bundanoon, NSW, T. Steel, 1897 [34.66S,
150.28E]. The selected specimen (henceforth referred to as NHMUK.97) had a prominent gonopore and faint vis-
ible dark lines dorsally (Fig. 5a, b). It was cut into six portions: anterior portion 42 mm long and 30 mm posterior
portion, neither sectioned (subsequently the posterior 5 mm severed and used for attempted molecular analysis); 4
mm pre-pharyngeal portion, TS, 7 slides; 13 mm long pharyngeal portion, LS, 30 slides; 10 mm portion, not sec-
tioned; 11 mm portion including the copulatory apparatus, LS, 27 slides.
Zoological Museum, Berlin (ZMB) Vermes 3506; Geoplana bicolor, syntype (hereafter referred to as
“ZMB3506”). This is one of the specimens found by W.B. Spencer in Queensland (Gympie; Burnett River; Cooran),
Australia, mentioned in Dendy (1892) and sent by Dendy to Graff (Winsor, pers. comm). It has been sectioned by
L. Winsor. 143 slides: Anterior, LS, 48 slides, H&E, 8μm; Mid-portion LS and TS, 47 slides, H&E (except 2 slides
trichrome); genital portion, LS, 48 slides, H&E (except one trichrome).
NHMUK.2018.8.31.1. Collected near Coventry, UK [52.385449N, 1.648287W], 28 January 2015, J. Tomnie,
(slides labeled and hereafter referred to as “Cv7”). It had a prominent gonopore thus mature. Killed in warm water
for 30 s, then in 100% ethanol. Cut into five portions: the anterior 12 mm and posterior 8 mm (Fig. 5, c & d) not
sectioned and remain preserved in ethanol (the posterior tip was subsequently removed for molecular analysis); a 4
mm pre-pharyngeal portion, TS, 7 slides; an 8 mm pharyngeal section, LS, 13 slides; a 12 mm portion containing
the copulatory apparatus, LS, 16 slides.
FIGURE 5. Caenoplana variegata. Photographs of remaining preserved portions of the sectioned specimens, taken 10 August
2018. NHMUK97: dorsal (a) and ventral (b) surfaces, preserved in 1897, now in 80% IMS. Cv7: dorsal (c) and ventral (d) sur-
faces, killed and preserved 29 January 2015, now in 100% ethanol. 1 mm graph paper background, scale bar 5 mm.

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Anatomical descriptions. External dimensions and the relative positions of the pharyngeal aperture (mouth)
and gonopore of the three sectioned specimens, other available specimens and also taken from the literature are
shown in Table 1.
NHMUK.97 (Figs 6, 7): The epidermis is damaged or missing over large areas but is a monolayer of columnar
cells, about 40 μm thick dorsally and about 20 μm ventrally. Many rhabdites are present both dorsally and ventrally.
The creeping sole, measured from TS, is about 75% of the width.
The preserved specimen showed faint longitudinal dark lines (Fig. 5) but corresponding pigment cannot be
detected in TS (it can be detected in ZMB3506 and Cv7—see later).
A prominent ring zone of parenchymal mixed circular and longitudinal muscle is present, about 130 μm thick
ventrally and 300 μm thick dorsally.
There is a pair of ventral nerve cords with transverse commissures and a sub-epidermal muscle plexus. The
nerve cords are about 1.3 mm apart.
The digestive system is typically triclad. The pharynx is cylindrical, about 6 mm long by 2 mm in diameter,
the dorsal insertion a little more posterior than the ventral. The pharyngeal aperture (mouth) is about 67% along the
pharyngeal pouch. The pharyngeal musculature of consists of an outer layer of circular fibres 100 μm wide, a central
layer of longitudinal and radial fibres respectively about 300 μm wide and an inner layer of alternating longitudinal
and circular fibres about 100 μm wide.
Numerous mature testes are present lateral to the ventral nerve cord on both sides. They are present in the TS
and the LS of the pharyngeal portion, both anterior, alongside and posterior to the pharyngeal pouch, but there
are none in the portion of the copulatory apparatus. Typically their maximum dimension is about 250 μm. Each is
slightly elongated towards the ventral side of the nerve cord, presumed to indicate the position of the efferent duct.
Sperm ducts cannot be distinguished with certainty.
Unfortunately the excised sectioned body portion is too short to show the full antero-posterior extent of the
copulatory apparatus so that the entry of any sperm ducts anteriorly cannot be determined. Measured from the
gonopore the male atrium is at least 7 mm long. The male atrium contains a large fold projecting from the ventral
surface (Fig. 7d, e).
Immature oviducts 15 μm wide with no lumen visible are present just dorsal to the nerve cord on either side.
They run the full length of the sectioned portion containing the copulatory apparatus and are presumed to open into
the copulatory apparatus at the posterior, female, end. Measured from the gonopore the female portion is at least 5
mm. The female atrium is simple with little differentiation.
Both male and female atria open into the gonopore with a simple opening.
ZMB3506 (Figs 8, 9): The epidermis is damaged or missing over large areas but is a monolayer of columnar
cells. The creeping sole, measured from TS, is about 85% of the width.
Brown sub-epidermal pigment, probably melanic, is visible concentrated in dorsal and lateral regions (Fig.
8c, d). The concentrations are: two near-medial, each about 100 μm wide and 100 μm apart, lightly pigmented and
restricted to the sub-epidermal circular muscle and longitudinal muscle bundles; two dorsally but more lateral,
more heavily pigmented, about 1 mm apart and about 400 μm wide and extending through the circular muscle and
longitudinal muscle bundles through to the rhabditogen layer; two laterally, less heavily pigmented, each about 200
μm wide also extending through to the rhabditogen layer. The pigment concentrations are assumed to coincide with
the dark stripes in living animals.
A prominent parenchymal ring zone of mixed circular and longitudinal muscle is present, about 130 μm thick
ventrally and 300 μm thick dorsally.
There is a pair of ventral nerve cords with transverse commissures and a sub-epidermal muscle plexus. The
nerve cords approximately 1.75 mm apart. The ventral nerve cords expand near the anterior end.
The digestive system is typically triclad. The pharynx is cylindrical, about 5 mm long by 1.7 mm, the dorsal
insertion a little more posterior than the ventral insertion. The pharyngeal aperture (mouth) is about 30% along the
pharyngeal pouch. The pharyngeal musculature of consists of an outer layer of circular fibres 100 μm wide, a central
layer of longitudinal and radial fibres about 400 μm wide and an inner layer of alternating longitudinal and circular
fibres about 200 μm wide.
Numerous immature testes, 60-70 μm in diameter, are present lateral to the nerve cord on either side. Sperm
ducts cannot be discerned in TS or LS. The copulatory apparatus appears to be in the very early stages of develop-
ment with little differentiation. The male part is about 4.5 mm long measured from the gonopore. At the anterior of
the male part there is a sinuous narrow duct, internal diameter about 15 μm and about 500 μm long from anterior

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to posterior (longer when straightened) though no afferent sperm ducts are visible entering the anterior end of this
duct. This duct opens into the male atrium, about 70 μm wide and 4 mm long, which is more or less linear with little
differentiation.
Possible early-stage ovaries are present immediately dorsal to the ventral nerve cord on each side about 1.4 mm
from the anterior end. Immature ovovitelline ducts, with no lumen visible, run posteriorly on the dorsal surface of
the ventral nerve cord on either side. They can be traced posterior to the pharynx but not to the rear of the female
copulatory system. No ovovitelline ducts can be seen entering the rear of the female atrium. The female atrium is
about about 8.5 mm long from posterior to gonopore and 70 μm wide, also with little differentiation.
Both male and female atria open into the gonopore with a simple opening.
Cv7 (NHMUK.2018.8.31.1) (Figs 10, 11, 12): The epidermis is a monolayer of columnar cells, about 50 μm
thick dorsally and about 20 μm ventrally. Many rhabdites are present both dorsally and ventrally. The ventral epi-
thelium is heavily ciliated, the cilia are about 5 μm long. The creeping sole, measured from transverse sections, is
about 83% of the width.
Brown sub-epidermal pigment, probably melanic, is present concentrated in regions of the dorsal and lateral
circumference (Fig. 10c, d). The concentrations are: two near-medial, each about 100 μm wide and 100 μm apart,
lightly pigmented and restricted to the sub-epidermal circular muscle and longitudinal muscle bundles; two dor-
sally but more lateral, more heavily pigmented, about 1 mm apart and about 400 μm wide and extending through
the circular muscle and longitudinal muscle bundles through to the rhabditogen layer; two laterally, less heavily
pigmented, each about 200 μm wide also extending through to the rhabditogen layer. The pigment concentrations
would appear to coincide with the dark stripes in living animals.
A prominent parenchymal ring zone of mixed circular and longitudinal muscle is present, about 130 μm thick
ventrally and 300 μm thick dorsally.
There is a pair of ventral nerve cords with transverse commissures and a sub-epidermal muscle plexus. The
nerve cords are 1.75 mm apart.
The digestive system is typically triclad. The pharynx is cylindrical, about 3.5 mm long by about 1.5 mm, the
dorsal insertion a little more posterior than the ventral insertion. The pharyngeal aperture (mouth) is about 33%
along the pharyngeal pouch. The pharyngeal musculature consists of an outer layer of circular fibres 40-60 μm
wide, a central layer of longitudinal and radial fibres 300 μm wide, and an inner layer of alternating longitudinal and
circular fibres 100 μm wide.
No mature testes are visible in any sectioned portion. However, empty cavities are present lateral to the nerve
cord on each side in the position of the testes of other specimens (Fig. 11a, b) and these are interpreted as spent tes-
tes. A sperm duct is present running along the inner, medial, side of the ventral nerve cord on each side, on the dorsal
margin of the parenchymal muscle (Fig. 11b, c) and can be traced to the anterior of the male copulatory apparatus.
Each is dorso-ventrally flattened, about 20 μm by 10 μm, the lumen is about 5 μm. No sperm are visible in any part
of the sperm ducts. The sperm ducts approach the copulatory apparatus ventrally, turn dorsally, join about half way
up the body, continue dorsally and open into a dorsal cavity (Fig. 12, “c1”) via a complex diffuse opening (Fig. 12j).
This dorsal cavity is roughly circular though has an angular point ventrally, perhaps indicating an outlet though none
is discernable. It measures about 350 μm anterior-posterior, by 350 μm dorso-ventral, by 440 μm laterally. Ventral
and slightly posterior to this is another cavity (Fig. 12, “c2”) measuring about 290 μm anterior-posterior, by 200 μm
dorso-ventral, by 280 μm laterally. From the ventral side of this is what appears to be the thickened wall of a possible
ejaculatory duct though no lumen is visible. This turns dorsally and then posteriorly to open into the base of a blunt
projection into the broad male atrium. The atrium is about 2 mm long with prominent ventral folds (Fig. 11d).
The anterior portion of the worm containing the ovaries (if present) has not been sectioned. A prominent mature
ovovitelline duct (Fig. 11b & c) is present dorsal to the ventral nerve cord on each side, external diameter about 50
μm, internal about 20 μm. The lumen is flagellated. The female copulatory apparatus appears to be fully developed.
The ovovitelline ducts run to the posterior of the copulatory system join ventrally and the combined ovovitelline
duct runs dorsally for about 250 μm, turns anteriorly and widens to form the female genital canal. The posterior
part of the canal, about 400 μm long, is surrounded by copious eosinophilic glands presumed to secrete the cocoon.
The anterior part has none of these glands and opens into the female atrium via what appears to be a small projec-
tion. The female atrium has some transverse folds at its posterior but broadens anteriorly before opening into the
gonopore.
In the region of the gonopore, there are numerous small invaginations both dorsally and ventrally (Fig. 12l).

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FIGURE 6. Caenoplana variegata. NHMUK97: a, mid-dorsal and, b, mid-ventral TS showing the epithelium and cutaneous
muscle, scales 50 µm; c, TS enlargement of dorsal portion of (d), scale 200 µm; d, whole TS, scale 500 µm. Note in (c) lack of
sub-cutaneous melanic pigment in regions corresponding with the dark stripes of living animals, cf. Figs 8 & 10; e, LS of the
pharynx, * indicates the position of the pharyngeal aperture, scale 2 mm (anterior to the right).

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FIGURE 7. Caenoplana variegata. NHMUK97: a, LS showing several mature testes, scale 200 µm; b, TS showing the ventral
nerve cord, ovovitelline duct and testis on one side, scale 100 µm; c, LS showing the immature ovovitelline duct immediately
dorsal to the ventral nerve cord, scale 200 µm; d & e, LS of the portion containing the copulatory apparatus: d, the extent of the
male atrium (right); e, the extent of the female atrium (left). Both extend beyond the sectioned portion. Scale 2 mm.

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FIGURE 8. Caenoplana bicolor. ZMB3506: mid-dorsal (a) and mid-ventral (b) TS showing the epithelium and cutaneous
muscle, scales 50 µm; c, TS, enlargement of dorsal portion of (d), scale 200 µm; d, whole TS, scale 500 µm. The double-ended
arrows above c and d indicate regions of sub-cutaneous melanic pigment presumed to correspond with the dark stripes of living
animals, cf. Figs 6 & 10; e, LS of the pharynx, * indicates the position of the pharyngeal aperture, scale 2 mm (anterior to the
right).

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FIGURE 9. Caenoplana bicolor. ZMB3506: a, LS showing several immature testes, scale 100 µm; b, TS showing ovovitelline
duct, ventral nerve cord and immature testis on one side, scale 100 µm; c, longitudinal section showing the immature ovovitel-
line duct immediately dorsal to the ventral nerve cord, scale 100 µm; d, longitudinal section showing the immature ovovitelline
duct and probable immature ovary, scale 100 µm; e, LS showing the maximum extent of the male atrium (right); f, showing the
maximum extent of the female atrium (left), scale bar 2 mm; g and h, enlargement of the extremities of the female and male atria,
scale bar 250 µm; e, diagram of the copulatory apparatus, male to the right.

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FIGURE 10. Caenoplana variegata. Cv7: mid-dorsal (a) and mid-ventral (b) TS showing the epithelium and cutaneous muscle,
scales 50 µm; c, TS enlargement of dorsal portion of (d), scale 200 µm; d, whole TS, scale 500 µm. The double-ended arrows
above c and d indicate regions of sub-cutaneous melanic pigment presumed to correspond with the dark stripes of living animals,
cf. Figs 6 & 8; e, LS of the pharynx, * indicates the position of the pharyngeal aperture, scale 2 mm (anterior to the right).

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FIGURE 11. Caenoplana variegata. Cv7: a, LS showing several empty testes, scale 100 µm; b, TS showing the sperm duct,
ventral nerve cord, ovovitelline duct and empty testis on one side, scale 100 µm; c, LS showing the ovovitelline duct on the
dorsal margin of the ventral nerve cord, and the sperm duct on the ventral margin of the ventral nerve cord, scale 100 µm; d, LS
of the copulatory apparatus, scale 2 mm.

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FIGURE 12. Caenoplana variegata. Cv7: diagram and selected LS micrographs of the copulatory apparatus: a, overlapping
images through the gonopore, female atrium to the left, male to the right; b–e, sections of the female atrium and ducts; f–j, sec-
tions through the male atrium and ducts (i is an enlargement of the entry of the combined sperm duct into cavity c1); k, diagram-
matic reconstruction. All (except i) to the same scale, 1 mm scale bars shown in a and k (scale bar in i = 200 µm). Vertical lines
link the same feature.

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Molecular results
The best-fit model of sequence evolution for Cox1 was GTR+I+G. The ML topology tree obtained from the
Cox1 dataset is shown in Fig. 13, with support value for bootstrap replicates and Posterior Probability (Bayesian
Inference) indicated at the nodes.
Tree topologies are congruent among the different methods used, although some of the basal nodes are not well
supported in any of them. All trees, however, show the monophyletic position of all specimens of C. variegata, and
the specimen of C. bicolor from GenBank clustering together.
FIGURE 13. Maximum Likelihood tree inferred using the Cox1 dataset. Values at nodes correspond to BP support values (left)
and PP from the Bayesian analysis (right). Vertical bars at right correspond to the molecular species delimitation methods as-
signations (purple: ABGD; orange: bPTP).
Discussion
Anatomy. The position of the mouth and gonopore is similar in the available specimens identified either as C.
variegata or C. bicolor (Table 1). Had there been differences, they might have indicated different species but this
does not confirm that they are all of the same species since many land flatworms show similar proportions. These
dimensions are from specimens with both a visible mouth and gonopore, thus presumably at least partially mature,
though a gonopore was present in two of the sectioned specimens (NHMUK.97 and ZMB3506) that clearly were
not fully mature. Obviously, in immature specimens the position of the gonopore cannot be determined. The species
seems to reproduce mostly by fission, at least in captivity, so the position of the mouth is likely to vary considerably
according to the stage of regeneration and whether it was an anterior or posterior regenerating portion.
Transverse sections of the three specimens are very similar in proportions and general appearance, and the sub-
epidermal and parenchymal musculature is similar in all three (Figs 6, 8, 10).
When the Cv7 specimen was received its lateral surfaces appeared almost uniformly black (Fig. 1a), and it
could be considered to have four dark stripes (two near-medial in the yellow stripe, and two broad dark lateral
stripes). Thus on Winsor’s (1973) definition it would be C. variegata. It was preserved within 1 day of receipt and
the remaining preserved portions still show dark striations (Fig. 5c, d). Transverse sections of the same specimen
clearly show sub-epidermal melanic pigment but in six distinct regions round the circumference (Fig. 10c, d). Thus
on Winsor’s (1973) definition it would be C. bicolor. The Berlin specimen also shows some melanic pigment in the
same six regions, though less intense (Fig. 8c, d). No pigment can be seen in sections of the NHMUK.97 specimen
(Fig. 6c, d) even though stripes can be seen on the remaining preserved portions (Fig. 5a).

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The pharynx is cylindrical in all three, though of different lengths. The pharyngeal musculature is similar. The
position of the pharyngeal opening along the pharyngeal pouch varies between the three specimens. Unless condi-
tions were identical and carefully controlled at the time of preservation, the degree of contraction is likely to vary,
and whether such differences are of value in distinguishing species must be questionable unless confirmed across
many more specimens.
All three sectioned specimens had a visible gonopore. Two (NHMUK.97 and Cv7) were selected for sectioning
precisely for this reason, the assumption being that they were mature. Yet, the sections show that the state of matu-
rity of the three is very different. It is unfortunate that ZMB3506, the type specimen of Graff’s species Geoplana
bicolor, appears to be in the very early stages of maturation. Had it been in a more advanced state of maturation,
more closely approaching that in Cv7, it would have been much simpler to draw conclusions one way or another.
NMHMUK.97 has mature testes with seemingly mature sperm, yet sperm ducts are not distinguishable nor does
the male copulatory apparatus seem to be fully developed, at least compared with Cv7, though a prominent ventral
fold is present in both. NMHMUK.97 also has underdeveloped ovovitelline ducts which can be traced to the region
of the copulatory apparatus, though not right to its posterior, female end, and the female copulatory apparatus is
underdeveloped.
Numerous early stage testes are present in ZMB3506 though no sperm ducts are discernable. Possible early
stage ovaries and underdeveloped ovovitelline ducts are present, the latter can be traced to behind the pharynx but
not as far as the copulatory apparatus. The copulatory apparatus is minimally developed and neither sperm ducts nor
oviducts can be discerned entering the male and female extremities.
Cv7 has apparently empty testes but clear sperm ducts, and the male ducts in the copulatory apparatus do not
seem to be fully developed in that a connection between the dorsal cavity (c1) and the second cavity (c2) and be-
tween c2 and the ejaculatory duct cannot be distinguished, nor is there a lumen in the ejaculatory duct. It does have
mature ovovitelline ducts and the common female duct appears fully mature with shell gland cells present.
So, ZMB3506 is in the early stages of maturity, both male and female systems; the testes of NHMUK.97 are
mature, the ovovitelline duct seems immature; the male system of Cv7 appears spent but the female system seems
fully mature. Assuming that the three specimens are of the same species, this might indicate protandrous maturation,
though that would require confirmation by examination of several more specimens in various stages of maturity.
Winsor’s (1991) generic definition for Caenoplana includes: “testes and sperm ducts ventral; penis of the ever-
sible type, without papilla; vagina enters ventrally or horizontally; ovovitelline duct enters vagina ventrally; copula-
tory organs without adenodactyls or adenomuralia.” As far as can be determined from specimens not fully mature,
all three specimens conform to this and thus are of the genus Caenoplana Moseley, 1877, as amended by Ogren &
Kawakatsu (1991) and Winsor (1991).
But are they the same species? The proportions of the transverse sections, the sub-epidermal and parenchymal
musculature, and the ventral nerve cords are similar in all three.
However, the copulatory apparatus in both NHMUK.97 and ZMB3506 is underdeveloped and relatively simple
but that of Cv7 is more developed so that firm conclusions are difficult. In the one specimen that has mature testes,
NHMUK.97, the testes are ventral, as are the immature testes in ZMB3506 and the presumed spent testes in Cv7. The
male ejaculatory duct of Cv7 opens into the atrium through a blunt projection, interpreted as an eversible penis.
There is considerable difference between the male ducts in the copulatory apparatus of ZMB3506 and of Cv7.
The former shows a basal sinuous duct widening into the atrium, the latter is much more complex though incom-
plete. Both NHMUK.97 and Cv7 do have a prominent ventral fold in the male atrium. How significant this might be
is unclear given the different degree of development and requires confirmation from further mature specimens.
Thus there are anatomical differences between the three specimens. However, the likely different preservation
protocols and the different stage of maturity in each makes it dangerous to ascribe these differences as specific dif-
ferences.
Molecular discussion. Both bPTP and ABGD methods delimit 7 lineages within the genus Caenoplana. The
results of the species delimitation analysis coincide in the two methods used in the assignment of Caenoplana vari-
egata and Caenoplana bicolor to a single species. For the rest of the assignments, there are some discrepancies, such
as the delimitation of C. coerulea from Tallaganda (Sunnucks et al., 2006) and the Ca2 morphotype of the genus
Caenoplana (Álvarez-Presas et al., 2014), which according to ABGD would be a single species, and according to
bPTP would be two species instead; and the case of individuals PT605 and PT658 (both from the study by Álvarez-
Presas et al., 2014), classified as a single species by bPTP and as two separate species by ABGD.

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General discussion. Graff (1899) separated Geoplana bicolor from G. variegata, apparently on the grounds
of colour and striation, though he did not clearly distinguish them by listing differences. The description of Graff
(1899) is based on that of Dendy (1892) and Graff was only able to examine preserved specimens provided by
Dendy, even though he described colour! We have shown that living specimens can show a range of colour and stria-
tion, even in the same individual over time, a least partly due to feeding. Thus these flatworms can match either or
both original species definitions, or neither if they lose much of their dark striation. Given also that Graff identified
specimens from New South Wales that had been found in 1877 (NHMUK.77.11.2.9 & 11) as Geoplana variegata,
it is not clear on what grounds he distinguished G. bicolor from these specimens.
One implication of Graff’s (1899) distinction is that all specimens found in both Australia and Europe since
1892, including those reported by Dendy (1892), should be identified as C. bicolor. A major question arises if this
is accepted: why is there apparently no record of C. variegata from anywhere since Fletcher & Hamilton’s (1888)
description? It seems improbable that Fletcher & Hamilton’s (1888) record of C. variegata, collected in New South
Wales, should be the only known one of that species, whereas similar specimens, found in Queensland, New South
Wales, possibly Victoria, Tasmania and also Europe are C. bicolor. Of course, this is no proof of anything but it
seems strange.
The three sectioned specimens above do show differences but due to the very different states of maturity of the
three specimens it is not possible to state for certain that they are of the same species, nor that they are of different
species. However, the differences are readily attributable to the different states of maturity and we do not believe
that the differences are significant as far as possible species difference. The anatomical descriptions of the three
specimens above should at least allow their identity to be confirmed or otherwise if and when a full comparison is
made of similar specimens.
The molecular analyses show that all the specimens belong to a monophyletic group (Fig. 13). The results of
the two methods of molecular species delimitation that we have applied in this study show that the specimens are of
the same species. Moreover, the new individuals analysed, here identified as C. variegata, belong to the same group
as a specimen identified in a previous work as C. bicolor, so we are dealing not with two distinct species, but with
only one. What we need to know is which one.
Possibly the only way to resolve for certain the question of what species these flatworms may be, would be
to make systematic collections across the range of distribution, particularly from original 19th Century localities in
Australia (New South Wales, Queensland, Victoria and Tasmania) and localities in Europe or elsewhere, though it
is likely that original Australian localities have been considerably altered by human activities in the intervening 100
plus years. Specimens, preferably mature, should be photographed alive, preserved and processed using a standard
protocol and anatomical and molecular comparisons made.
It is also important to correctly identify invasive or potentially invasive species. This is the case of the taxon
with which we are dealing here, which has already been found in several countries in Europe, presumably intro-
duced and distributed inadvertently through horticultural activity.
However, the central problem remains, namely, how would the species be determined given that they were
originally described only on external descriptions and that those descriptions are not substantially different? On the
evidence above, though incomplete, we believe there is no current justification for distinguishing between Geopla-
na variegata Fletcher & Hamilton, 1888 and Geoplana bicolor Graff, 1899. The former specific name has priority
and the species should now be referred to as Caenoplana variegata (Fletcher & Hamilton, 1888).
Acknowledgements
Leigh Winsor is thanked for his thoughts and opinions though he may not agree with our conclusions. Mrs Ann
Fox is thanked for the translation of Graff’s (1899) German text. HDJ would like to thank Jackie Tomnie, Deborah
Cole, Gethin Jones, Mike Lole and several other collectors in the UK who have sent specimens or photographs.
Specimens were embedded and stained in the Histology Laboratory, University of Manchester and Peter Walker is
thanked for his help.

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