Zoologica Scripta

Non-destructive imaging to describe a new species of Obama

land planarian (Platyhelminthes, Tricladida)
FERNANDO CARBAYO, TIAGO M. FRANCOY & GONZALO GIRIBET

Submitted: 14 July 2015
Accepted: 27 January 2016
doi:10.1111/zsc.12175
Carbayo, F., Francoy, T.M., Giribet, G. (2016). Non-destructive imaging to describe a new
species of Obama land planarian (Platyhelminthes, Tricladida). —Zoologica Scripta, 00, 000–
000.
The land planarians in the genus Obama include the largest species of the Neotropical
Geoplaninae. Morphological discrimination of Obama species can be difficult, as many spe-
cies are morphologically similar and some present asymmetric – difficult to interpret –
copulatory organs. New techniques are thus welcomed to provide faster species description
and identification. Here, we study several specimens of Obama by means of mainly 2D
and 3D lCT-based images obtained through X-ray microcomputed tomography (lCT) of
a paratype, and, complementarily, histological sections of the holotype and a second para-
type, which were digitized into virtual slides. Comparison of these images with traditional
histological sections and descriptions of the known species of the genus allows us to con-
clude that our specimens constitute a new species, which we describe here. We further
evaluate the phylogenetic placement of the new species using gene fragments from the
mitochondrial cytochrome c oxidase subunit I and the nuclear elongation factor-1a. Raw
and derivative lCT data and virtual histological sections were deposited in an open repos-
itory (GigaDB) and are freely available. This work leads us to conclude that lCT consti-
tutes a relatively fast, inexpensive non-destructive method that produces results
comparable to those of traditional histology, and is thus amenable for describing flatworm
species.
Corresponding author: Fernando Carbayo, Laborat
orio de Ecologia e Evolucß~ao, Escola de Artes,
Ci^encias e Humanidades, Universidade de S~ao Paulo – USP, Av. Arlindo Bettio, 1000, CEP
03828-000 S~ao Paulo, SP, Brazil E-mail: baz@usp.br
Fernando Carbayo, Laborat
orio de Ecologia e Evolucß~ao, Escola de Artes, Ci^encias e Humanidades,
Universidade de S~ao Paulo – USP, Av. Arlindo Bettio, 1000, CEP 03828-000 S~ao Paulo, SP,
Brazil and Programa de P
os-Graduacß~ao em Zoologia, Departamento de Zoologia, Instituto de Bio-
ci^encias, Universidade de S~ao Paulo, Rua do Mat~ao, Trav. 14, 321, Cidade Universit
aria, CEP
05508-900 S~ao Paulo, SP, Brazil. E-mail: baz@usp.br
Tiago M. Francoy, Laborat
orio de Ecologia e Evolucß~ao, Escola de Artes, Ci^encias e Humanidades,
Universidade de S~ao Paulo – USP, Av. Arlindo Bettio, 1000, CEP 03828-000 S~ao Paulo, SP,
Brazil. E-mail: tfrancoy@usp.br
Gonzalo Giribet, Museum of Comparative Zoology, Department of Organismic and Evolutionary
Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02139, USA. E-mail:
ggiribet@g.harvard.edu

Introduction
Most Neotropical land planarians belong to Geoplaninae
(Platyhelminthes, Tricladida, Geoplanidae), a group of
flatworms comprising ca. 265 species. One of the most
diverse genera is Obama Carbayo et al., 2013; with 36
named species. The genus is mainly distributed along the
coastal ranges of the Brazilian Atlantic forest and includes
the largest species of Geoplaninae, ranging between 3 and
30 cm in length. These large, leaf-shaped land planarians
have both mono- and trilobed eyes, their sensory pits are
frequently arranged in multiple rows, and their penis
papilla is laterally displaced. Discriminating species of
Obama using morphology can be difficult, as many species


are morphologically similar (Alvarez-Presas et al. 2015;
Carbayo et al. 2016). In addition, the fact that male
organs may be asymmetrical along the sagittal plane
makes it difficult to determine their actual shape by only
examining conventional histological slides. However, due

ª 2016 Royal Swedish Academy of Sciences 1

Flatworms off the knife edge  F. Carbayo et al.
to DNA sequencing, morphological traits that otherwise
would have been considered just intraspecific variation are


currently recognized as species-specific (Alvarez-Presas
et al. 2015).
In the pre-histological era, land planarians were summar-
ily described based only on their external aspect (e.g. Dar-
win 1844; Schultze & M€ uller 1857). With few exceptions
(e.g. Schirch 1929), following the development and
improvement of tissue processing and the accurate histo-
logical work of von Graff (1899), histology became the pre-
ferred technique to describe planarians, up to the present
time. Notwithstanding the advantages of histology, prepa-
ration of large specimens may require hundreds of histo-
logical slides, is laborious and time-consuming and may
produce uneven results (Winsor 2001; Metscher 2009).
There is also an inherent risk of tissue damage or loss of
paraffin serial sections during histological preparation
(Ziegler et al. 2010; Sluys et al. 2013) because of the very
thin and delicate resulting paraffin sections. Additionally,
specimens can only be sectioned along a single plane, and
each specimen, including types, is intrinsically unique. In
addition, difficulties in accessing type material for taxo-
nomic revisions often slow down progress (Wheeler et al.
2012). The above-mentioned limitations of histological
techniques may be overcome by creating digitized cyber-
types (Godfray 2007) or e-types (Wheeler et al. 2012). For
the huge prospective numbers of unknown species still to
be described and named, it would be even more effective
to produce two-dimensional (2D) and three-dimensional
(3D) e-types (Godfray 2007; Wheeler et al. 2012; Faulwet-
ter et al. 2014) by means of non-destructive techniques.
Indeed, there are commercially available instruments and
techniques that can now overcome these limitations (i.e.
destructive sampling) and non-availability of type material
by providing 3D morphological visualization non-destruc-
tively, such as confocal laser scanning microscopy (cLSM),
X-ray microcomputed tomography (lCT), synchrotron
lCT, optical projection tomography (OPT) or magnetic
resonance imaging (MRI) (see reviews of these techniques
in Sharpe 2004; Betz et al. 2007; Ziegler et al. 2010;
Laforsch et al. 2012; Ziegler 2012). These non-destructive
techniques are especially valuable when studying rare speci-
mens (Ziegler et al. 2012). Furthermore, 2D and 3D digi-
tized images can be widely disseminated online and be
made broadly available, thus avoiding constraints on bor-
rowing physical specimens or having to visit natural history
collections (Balke et al. 2013; Faulwetter et al. 2013).
Virtual imaging-based approaches have accurately
revealed morphological aspects of a variety of fossil (e.g.
Sutton et al. 2001; Molineaux et al. 2007; Garwood et al.
2009; Rodrigues et al. 2012; Le Cabec et al. 2015) and
extant mineralized and soft-bodied organisms (e.g.
2 ª 2016 Royal Swedish Academy of Sciences
Metscher 2009; Dinley et al. 2010; Fern
andez et al. 2014;
Akkari et al. 2015). Yet in practice, use of virtual sections
has been little explored for describing new extant taxa (see
Penney et al. 2007; Stoev et al. 2013).
In this study, we describe a new species of a large soft-
bodied land flatworm from the genus Obama by examining
both conventional histological slides of two specimens and,
mainly, 2D and 3D lCT-based images of a third specimen.
We also establish the uniqueness of the new species follow-
ing DNA sequencing and assess the reliability of lCT-
based images as compared with conventional histological
preparations.
Materials and methods
Specimens
Specimens were sampled in the Reserva Biol
ogica do Alto
da Serra de Paranapiacaba (Santo Andr
e, State of S~ao
Paulo, Brazil). The area is an isolated patch of Atlantic
rainforest, at ~800 m in altitude, and rich in biodiversity.
Using a lamp, we conducted searches on soil litter and
trails at night (20 h–04 h). Specimens were photographed,
and euthanized in boiling water, and a small tissue sample
was preserved in 96% ethanol for DNA extraction, while
the main part of the body was fixed in 10% formalin and,
subsequently, transferred to 80% ethanol for permanent
preservation. Colour descriptions of the body of living and
fixed specimens follow the online palette RAL colours
(RAL Gemeinn€ utzige GmbH, available at https://www.ral-
farben.de/uebersicht-
ral-classic-farben.html?&L=1) by comparison with digital
pictures of the specimens on a computer screen.
Type specimens are deposited in the Museu de Zoologia
da Universidade de S~ao Paulo (MZUSP) and in the
Museum of Comparative Zoology (MCZ), Harvard
University. Specimens included in the molecular analysis
are deposited in the MZUSP.
Histology and digitization of histological sections
First a traditional approach was followed, generating histo-
logical slides for two specimens in order to examine their
internal anatomy and describe details of the epithelium, ori-
entation of single muscle fibres and the nature of glandular
secretions and to use these for comparative purposes with
the new approach, that is, lCT-based images. For histologi-
cal preparation, parts of the body were embedded in paraf-
fin, sectioned at 7 lm, transferred to glass slides coated
with albumen–glycerine and stained with the Mallory
method as modified by Cason (1950). The slides were then
examined with a compound microscope. Among all histo-
logical slides prepared, those including structures of interest
were digitized with a Zeiss Axio Scan Z1 and the resulting
virtual slides were uploaded on GigaDB and are freely avail-

able (Carbayo & Lenihan 2016a). Additionally, further
details on data acquisition, availability, quality, and require-
ments can be found in Carbayo & Lenihan (2016b).
Microcomputed tomography
Virtual 2D sections and virtual 3D reconstructions were
undertaken for paratype MCZ 59100. The specimen was
submerged in a solution of 0.3% phosphotungstic acid
(PTA) and 3% dimethyl sulfoxide (DMSO) in 95%
hydrated ethanol for 90 days. Before scanning, the specimen
was rinsed in 95% ethanol and placed inside a plastic straw
filled with 95% ethanol. The specimen was scanned with an
X-ray lCT SkyScan 1173 scanner (Bruker MicroCT, Kon-
tich, Belgium) equipped with a Hamamatsu 130/300 tung-
sten X-ray source and a flat panel sensor camera detector
with 2240 x 2240 pixels. Preliminary scans were conducted
to assess the quality of the staining technique. As contrast
could seemingly be enhanced, the specimen was restained
with 1% iodine in absolute ethanol (I2E). To do that, we
rinsed the PTA stained specimen with 95% ethanol and
placed it in I2E for 48 h. Shortly, before scanning, the spec-
imen was rinsed in absolute ethanol for 5 min. Scan settings
were as follows: 34–35 KV; source current: 160–190 lA;
image pixel size: 6.04–9.95 lm; Filter = No Filter; Depth
(bits) = 16; exposure (ms) = 1050–1200; rotation step
(deg) = 0.1–0.16; frame averaging = ON (6–14). Scans took
2.5–11.5 h. Final scans included the entire body (5 scans),
anterior end (3 scans), pharynx (1 scan) and copulatory
apparatus (1 scan) and were stored as tiff files. The recon-
structions were carried out as bmp formatted images using
the software Nrecon version 1.6.6.0 (Bruker MicroCT,
Kontich, Belgium) under the following settings: smooth-
ing = 0; ring artefact correction = 15; Beam Hardening
Correction = 63%; HU Calibration = OFF.
Sets of virtual sections were reconstructed with the soft-
ware package Data viewer (http://www.skyscan.be/prod-
ucts/downloads.htm) and a selection of sets with the
original grey scale or with false colour rendering were
saved. Contrast of greyscale virtual sections was enhanced
with GIMP (GNU Image Manipulation Program 2.8.10;
The GIMP team www.gimp.org, 1995-2014). CTvox
(http://www.skyscan.be/next/CTvox64.zip) was utilized to
obtain virtual 3D images. The original grey scale colour of
the models was preserved or adjusted with false colour ren-
dering using the R, G, B and opacity channels combined
with different shadow and light position options available
in the software.
A 3D model of the copulatory apparatus was recon-
structed with the software Amira 5.6 (FEI Co., Hillsboro,
Oregon, USA). For this, we manually segmented and then
surface-rendered, from the virtual sections, selected struc-
ª 2016 Royal Swedish Academy of Sciences
F. Carbayo et al.  Flatworms off the knife edge
tures that would be visible in the final figure. We then
exported the 3D reconstruction image as a tiff formatted
figure and corrected minor sharp angles with the help of
GIMP.
lCT-based images are deposited in the repository
GigaDB (Carbayo & Lenihan 2016a) and MCZbase under
accession number MCZ 59100 (http://mczbase.mcz.har-
vard.edu/guid/MCZ:IZ:59100). Type specimens are depos-
ited in the Museu de Zoologia da Universidade de S~ao
Paulo (MZUSP) and in the Museum of Comparative Zool-
ogy (MCZ), Harvard University (see details in the section
Type material below).
Molecular methods
To determine the phylogenetic position of the newly
described species within the genus Obama, we sequenced
two markers for two individuals of the new species and
compared them to other species of Obama and species of
its sister group, that is, Cratera Carbayo et al., 2013
(Table S1). For most of the specimens, genomic DNA was
extracted using the Wizard Genomic DNA Purification kit
(Promega, Madison, WI, USA) following the manufac-
turer’s instructions. A fragment of the mitochondrial pro-
tein-encoding gene cytochrome c oxidase subunit I (COI)
was amplified using conditions described in earlier flat-


worm work (Sunnucks et al. 2006; Alvarez-Presas et al.
2011). A partial region of the nuclear encoding gene elon-
gation factor-1a (EF-1a) was amplified as in our previous
work (Carbayo et al. 2013). Complementary strands of each
of the two gene fragments were sequenced at the Centro
de Estudos do Genoma Humano, Universidade de S~ao
Paulo. The remaining sequences were obtained from Gen-
Bank. The forward and reverse sequences of each individ-
ual were paired using Chromas Pro version 1.5
(Technelysium Pty, Ltd.; http://www.technelysium.com.au/
ChromasPro.html) and manually checked for possible
errors.
Multiple sequence alignment of each gene was conducted
with MUSCLE (Edgar 2004). We then concatenated both
genes to be used in subsequent analyses. The best-fit model
of nucleotide substitution of each individual gene and the
concatenated matrix were assessed under the Akaike infor-
mation criterion (AICc) using jModeltest (Darriba et al.
2012). The individual gene sequences and the concatenated
matrix were used to search for the optimal tree under max-
imum likelihood. A 10 000 bootstrap replicates was run in
MEGA 6 (Tamura et al. 2013).
Systematic account
Order Tricladida Lang, 1884 Suborder Continenticola
na & Riu-
Carranza, Littlewood, Clough, Ruiz-Trillo, Bagu~
3
Flatworms off the knife edge  F. Carbayo et al.
tort, 1998 Family Geoplanidae Stimpson, 1857 Subfamily
Geoplaninae Stimpson, 1857 Genus Obama Carbayo,


Alvarez-Presas, Olivares, Marques, Froehlich & Riutort,
2013
Obama otavioi Carbayo, sp. n
Type material. All specimens were collected by F. Carbayo
et al. at ~800 m altitude in areas covered with Atlantic
forest in the Reserva Biol
ogica do Alto da Serra de
Paranapiacaba, Santo Andr
e, State of S~ao Paulo, Brazil
(-23.77 697, -46.31 212), on 22.ix.2012. Holotype MZUSP
PL 1574 (field number, F5470): anterior end: transverse
sections on 40 slides; anterior region 2, containing ovaries:
sagittal sections on 35 slides; anterior region 3: horizontal
sections on 24 slides; pharynx: sagittal sections on 28 slides;
copulatory apparatus: sagittal sections on 49 slides. Para-
type MZUSP PL 1573 (field number, F5435): pharynx:
sagittal sections on 46 slides; copulatory apparatus: sagittal
sections on 61 slides. Paratype MCZ 59100 (field number,
F5434): Preserved in 75% ethanol.
Etymology. Named after the late Brazilian zoologist
Ot
avio Froehlich (1958–2015).
Type locality. Reserva Biol
ogica do Alto da Serra de
Paranapiacaba, Santo Andr
e, State of S~ao Paulo, Brazil.
Diagnosis. Species of Obama ca. 10 cm in length fully
extended. Dorsum greenish dark brown or dark brown;
ventral side greyish brown. Male atrium with a sheath-like
fold at the right side, encircling the basis of the penis
papilla; numerous glands piercing this fold. Penis papilla
obliquely projecting sideways to the left side of the body,
occupying almost 75% of the total length of the male–
female atria and with its dorsal insertion posterior to the
gonopore level. Diverticulum of the female atrium dorso-
posteriorly oriented.
Description
Living animals flat, lanceolate, with anterior half progres-
sively narrowing and posterior end obtusely pointed. About
90 x 7 mm when creeping. General colour of the dorsum
greenish dark brown or dark brown, caused by blackish
spots on either a greyish green or brownish beige ground,
respectively (Fig. 1 A). Anterior region darker, with the tip
brownish. Ventral side greyish brown, with numerous min-
ute darker dots; body margins greyish (Fig. 1 B). During
fixation, one paratype contracted strongly and a bump dor-
sal to the copulatory apparatus appeared (Fig. 1 C).
Clear halos, inconspicuous in plain sight, encircle the
eyes. Being marginally arranged at the anterior end (Fig. S1
C), the eyes are spreading progressively onto the back to
4 ª 2016 Royal Swedish Academy of Sciences
occupy a band of one-fourth of the body width at the end of
the first third of the body. Eyes 50 lm in diameter; of the
single-cup type in the anterior fourth of the body, otherwise
they are constituted of three pigmented lobes.
Sensory pits are simple invaginations, ventromarginally
distributed (Fig. 1 E) in a single row, and limited to
approximately the anterior third of the body. There is a
glandular margin contouring the sides along the entire
body (Fig. 1 D–E). This is constituted by three types of
glands, producing cyanophilic, erythrophilic or xanthophilic
granules. Five millimetres from the anterior end, the nerve
plate acquires a two-ganglion shape, but the transition to a
plate along the body axis is very gradual, so they are not
clearly delimited in the antero-posterior axis.
The three typical cutaneous muscle layers of Geoplani-
nae are present: one circular followed by a diagonal one
with decussate fibres, and then a longitudinal layer with
fibres arranged into bundles, both dorsally and ventrally.
The most external longitudinal fibres, that is, those laying
immediately under the decussate ones, run at a slight angle
to the longitudinal axis (Fig. 1 H, S1 B), as in Cratera cuar-
assu Carbayo & Almeida, 2015. Thickness of cutaneous
musculature relative to body height at the pre-pharyngeal
region (CMI): 10.5%. The three most common parenchy-
mal muscle layers are also present, that is, a dorsal layer of
decussate diagonal fibres, a supraintestinal layer of trans-
verse fibres, and a subintestinal layer, the latter above the
ventral nerve plate.
The mouth lies at a distance from the anterior end that
is equivalent to 69-72% of the body length and opens
approximately in the middle of the pharyngeal pocket
(Fig. 1 F, G). Pharynx cylindrical, 3.0–3.5 mm in length,
occupying nearly two-thirds of the pharyngeal pouch.
Oesophagus conspicuous, about 1 mm in length. Outer
epithelium of the pharynx underlain by a thin layer of lon-
gitudinal muscle, followed by a thin layer of circular fibres
with interspersed longitudinal fibres. Inner pharyngeal
epithelium and that of the oesophagus underlain by a thick
layer (100–150 lm thick) of circular fibres with longitudi-
nal fibres interspersed.
Testes mature, located between the supraintestinal
parenchymal muscle layer and the intestine. They extend
from the level of the ovaries to nearly the dorsal root of
the pharynx. Sperm ducts dorso-internal to the ovovitelline
ducts dilated distally and containing sperm near the copula-
tory apparatus; curved medially to communicate with the
two short branches of the prostatic vesicle. Prostatic vesicle
C-shaped in lateral view located outside of the common
muscle coat (Fig. 2 I, Fig. S1 D, E); lined with a columnar,
ciliated epithelium, pierced by glands producing fine ery-
throphilic granules, and surrounded by a layer (15 lm
thick) of interwoven muscle fibres.
 F. Carbayo et al.  Flatworms off the knife edge

A B

E F

G H

Fig. 1 A—H. —A. Habitus of living holotype in dorsal view. Anterior to the right. About 60 mm in length. —B. Habitus of living paratype
MZUSP PL 1573 in ventral view. Anterior to the right. —C. Dorsal view of a 3D rendering of the lCT dataset of paratype MCZ 59 100. Note
bump (arrow) dorsal to the copulatory apparatus. Anterior to the right. —D. Virtual transverse section at the level of the ovaries of paratype
MCZ 59100. —E. Microphotograph of the glandular margin of holotype in a transverse section of the anterior region of the body. —F. Virtual
sagittal section of the pharynx of paratype MCZ 59100. Anterior to the right. —G. Microphotograph of the pharynx of the paratype MZUSP
PL 1573 on a sagittal histological section. Anterior to the right. —H. Microphotograph of a horizontal histological section of the ventral
cutaneous musculature of paratype MCZ 59100 at the right side of the body. Anterior topside. Abbreviations: c, circular cutaneous muscles; d,
diagonal cutaneous muscles; de, dorsal epidermis; e, eye; gm, glandular margin; i, intestine; in, insertion of the pharynx; l, longitudinal cutaneous
muscles; m, mouth; o, ovary; ov, ovovitelline duct; ph, pharyngeal pouch; sp, sensory pit; ve, ventral epidermis; vi, vitellaria.

ª 2016 Royal Swedish Academy of Sciences 5

Flatworms off the knife edge  F. Carbayo et al.

I J

K L

Fig. 2 I—M. Paratype MCZ 59100. —I. lCT-based image of a sagittal section of the copulatory apparatus. Arrowheads point to glandular
ring of female atrium. —J. lCT-based image of a coronal section of the male atrium showing the asymmetric penis papilla point to the
left. Anterior at the top. —K. lCT-based image of a transverse section of penis papilla levelled at the opening of the ejaculatory duct. Left
margin of the body is damaged. —L. lCT-based image of maximum intensity projection rendering of the copulatory apparatus. See
glandular ring of the female atrium (arrow). —M. 3D reconstruction of the copulatory apparatus. Abbreviations: co, common glandular; de,
dorsal epidermis; ej, ejaculatory duct; f, male atrium fold; fa, female atrium; g, gonopore; gl, mass of gland cells of male atrium; i, intestine;
ma, male genital atrium; mc, muscular coat; ov, ovovitelline duct; pp, penis papilla; pv, prostatic vesicle; sd, sperm duct; sg, shell glands; ve,
ventral epidermis; y, dorso-ventral body axis; x, transverse body axis; z, antero-posterior body axis.

6 ª 2016 Royal Swedish Academy of Sciences

 F. Carbayo et al.  Flatworms off the knife edge

P Q R

Fig. 3 N—R. —N. Diagrammatic reconstruction of the copulatory apparatus of the holotype from sagittal sections —O. Microphotograph
of the copulatory apparatus of the holotype on a sagittal histological section. —P. Microphotograph of a sagittal section of paratype
MZUSP PL 1573 showing ventral insertion of the penis papilla and epithelium of male atrium pierced by gland cells. —Q.
Microphotograph of a sagittal section of holotype at the level of the ovaries. —R. Microphotograph of a sagittal section of the female
atrium holotype. Anterior to the right in all figures. Abbreviations: co, common glandular; de, dorsal epidermis; ej, ejaculatory duct; f, male
atrium fold; fa, female atrium; g, gonopore; gl, mass of gland cells of male atrium; i, intestine; l, longitudinal cutaneous muscles; ma, male
genital atrium; mc, muscular coat; o, ovary; ov, ovovitelline duct; pp, penis papilla; pv, prostatic vesicle; sd, sperm duct; sg, shell glands; t,
testis; ve, ventral epidermis; vi, vitellaria

Ejaculatory duct running acentrally through the ventral
portion of the penis papilla (Fig. 2 J, K; S1 D, E), lined
with a ciliated epithelium. Penis papilla tongue-shaped,
projecting obliquely sideways from the dorsal-right wall of
the male atrium, so that its tip lays at the left side of the
body (Fig. 2 J, K, M). Its dorsal insertion is at the same
level as the gonopore posterior to it, so that it can occupy
up to three quarters of the total extension of the male and
female atria (MZUSP PL 1573, MZUSP PL 1574), but in
the strongly contracted paratype MCZ 59100, it is anterior.
Stroma of the penis papilla traversed by variously arranged
muscle fibres and by numerous glands discharging ery-
throphilic granules through the epithelium of the penis
papilla. Penis papilla lined with a non-ciliated, nucleated,
cuboidal epithelium, whose free surface is erythrophilic
except a relatively extensive area around the opening of the
ejaculatory duct. Thin layer of circular muscle fibres under
the penial epithelium, followed by an equally thin layer of
longitudinal fibres. Male atrium smooth, excepting for a
sheath-like fold at the right side, encircling the basis of the
penis papilla. This fold extends backwards beyond the
gonopore and separates the male from the female atrium.
Lining epithelium of the male atrium cuboidal, non-
ciliated, while that of the longitudinal fold is pierced by
very numerous glands producing a fine granular secretion
with undetermined staining affinity (Fig. 2 I-K, Fig. 3 P),
because this secretion may stain either pinkish or bluish. At
high magnification, the granules seem to have an ery-
throphilic central core and a cyanophilic peripheral part.
Epithelium of the male atrium underlain by a thin layer
(5 lm thick) of circular muscle fibres, followed by another
layer (5 lm thick) of longitudinal fibres. This muscular
organization is different under the epithelium traversed by
the undetermined secretion: the circular fibres are scattered
and the longitudinal ones are absent.
Ovaries elongate, ca. 0.9 mm in length and 0.15 mm in
diameter (Fig. 3 Q). They are ventral to the anteriormost
testes, located at a distance of 24% from anterior end of the
body, lying immediately above the ventral nerve plate. Vitel-
laria distributed around the intestine and between the main
intestinal branches (Fig. 3 Q). Ovovitelline ducts emerging
laterally from the dorsal side of the ovaries. In the holotype,

ª 2016 Royal Swedish Academy of Sciences 7

Flatworms off the knife edge  F. Carbayo et al.
the very initial anterior portion of the oviducts is full of sperm.
Ovovitelline ducts ascending laterally to the female atrium
and joining dorsally to its median region. Female atrium
strongly contracted in paratype MCZ 59100, the ducts joining
dorsal to the posterior region. Common glandular ovovitel-
line duct running posteriorly and entering an ample female
genital canal, which is formed by an anteriorly oriented pro-
jection from the postero-dorsal region of the female atrium,
thus showing the proflex condition. This atrium is an irregu-
lar cavity (Fig. 2 I, L, M; Fig 3, R), lined with a non-ciliated,
columnar epithelium, 30–70 lm high. The cytoplasm of the
epithelial cells possesses numerous fine erythrophilic gran-
ules, and their nuclei are located at different heights, so giving
a stratified aspect. Scarce glands secreting fine erythrophilic
granules are present under the muscularis of the female
atrium. An annular-shaped portion of the female atrium is tra-
versed by glands in its posterior region (Fig. 2 I, L). Towards
the gonopore, the epithelium is also traversed by scattered
glands, likely producing amorphous xanthophilic secretion.
Female atrium surrounded by a 15-lm-thick layer of circular
muscle fibres, interspersed with longitudinal ones. In para-
type, MZUSP PL 1573 sperm is present in the lumina of the
ascending portion of the ovovitelline ducts, the common glan-
dular ovovitelline duct, and the female atrium.
Biological note. Specimens MCZ 59100 and MZUSP PL
1573 were found mating at midnight in spring (September)
(Fig. S1 A).
Molecular characterization
The best-fit substitution model for the individual genes
and also for the concatenated dataset was GTR+G+I. The
same phylogenetic pattern was found in the analysis of the
two gene fragments and for the concatenated genes
(Figs. S2, S3). Maximum likelihood analysis found the two
individuals of Obama otavioi deeply nested within Obama, as
sister group to a clade including O. marmorata (Schultze &
M€ uller 1857), O. anthropophila Amaral, Leal-Zanchet &


Carbayo, 2015 (Alvarez-Presas et al. 2015), O. decidualis


Amaral & Leal-Zanchet, 2015 (Alvarez-Presas et al. 2015)
and O. josefi (Carbayo & Leal-Zanchet, 2001) (Fig. 4). This
clade receives high bootstrap support in all three analyses
and is sister group to O. ladislavii (von Graff, 1899), a clade
also with high bootstrap support. These results indicate
that the two individuals from O. otavioi belong to the clade
of Obama marmorata and that their sequences are different
from those of all other specimens studied.
Discussion
Taxonomic remarks
On the basis of the shape of the male copulatory organ,
the 36 named species of Obama may be divided into two
8 ª 2016 Royal Swedish Academy of Sciences
groups. One group, which includes the type species of the
genus, O. fryi (von Graff, 1899), comprises 28 species. All
members of this clade have a penis papilla that is symmetri-
cal in sagittal plane, albeit that this clade is unsupported in
the molecular analysis (Fig. 4), and thus, this character may
constitute a symplesiomorphy. The remaining nine species
have an asymmetric penis papilla, and those represented in
the phylogenetic analysis show that the group is mono-
phyletic. The new species thus is here proposed to belong
to the second group, which includes Obama anthropophila,
O. carbayoi Oliveira & Leal-Zanchet, 2012 (do Amaral et al.
2012), Obama carrierei (von Graff, 1897), Obama decidualis,
Obama josefi, Obama ladislavii, Obama marmorata, Obama
ruiva (Froehlich & Froehlich, 1972) and a new species of
the genus found in Europe and Brazil, described by Car-
bayo et al. (2016). Future work should attempt to include
all of these species in a molecular analysis.
Regarding body coloration, only three species resemble
the new species in having dark spots over a clearer back-
ground, viz. O. marmorata, O. josefi and the European spe-
cies of Obama. Nevertheless, in the two former species, the
spots are striated, whereas in the new species, they are
roughly circular. The dorsum of adult O. josefi is clearer
than that of the new species, while it displays three longitu-
dinal dark stripes. These nine species present a copulatory
apparatus similar in the general shape, but they differ from
that of O. otavioi in its details.
One remarkable feature of the new species is the long
penis papilla relative to the total length of the male+female
atria. It reaches 75% of the atrium in two of the specimens
(but 55% in the strongly contracted paratype MCZ 59100).
This relative size is only comparable with that of O. mar-
morata, in which the penis papilla occupies 80% of the
male + female atria, and O. ruiva, occupying 65% of the
male + female atria. This relative length drops to nearly
50% or less in other species of the group. Apart from the
different body coloration, each of these latter two species
presents additional features differentiating them from the
new species: (i) in O. marmorata, the male atrium is not
folded; (ii) in Obama ruiva, there are pits located on the
epithelium of the basal half of the penis papilla, receiving
the secretion of glands, as does the crescentic fold of the
male atrium. Furthermore, the penis papilla of O. ruiva is
conical in contrast to the tongue-shaped penis papilla of
the new species.
Further characteristics differentiating the species with a
short penis papilla relative to the total length of the
male+female atria are as follows. In O. carrierei and
O. carbayoi, the diverticulum of the female atrium receiv-
ing the common ovovitelline duct is conspicuous and not
dorso-posterior as in the new species. In O. ladislavii and
O. josefi, the dorsal insertion of the penis papilla is ante-
 F. Carbayo et al.  Flatworms off the knife edge

Fig. 4 Maximum likelihood tree of the concatenated data set. Numbers on branches indicate bootstrap proportions above 50%.
logL = 3623.39. Arrowhead indicates the asymmetrical penis papilla clade. With the exception of O. decidualis, all species are illustrated.
Figures at different scales.

ª 2016 Royal Swedish Academy of Sciences 9

Flatworms off the knife edge  F. Carbayo et al.
rior to the level of the gonopore, giving it a more conical
aspect. Obama otavioi also differs from the European
Obama, O. anthropophila and O. decidualis in that they dis-
play the dorsal insertion of the penis papilla anterior to
the gonopore level; furthermore, in the European species
of Obama, the a massive mixed fine granular secretion is
discharged into the male atrium produced by glands that
pierce a ring surface of the epithelium.
Recognition of the taxonomic characters of Obama otavioi
sp. n. on the lCT-based images
lCT images provided enough detail for identification of the
new species, therefore constituting a relatively fast, inexpen-
sive non-destructive method for identifying whole land pla-
narian specimens. With the exception of body colour, all
external features are visible in the lCT-based images, that is,
size and general shape of the body, as well as mouth, gono-
pore, eyes, glandular margin, eyes and sensory pits, with the
advantage of being able to produce 3D reconstructions of
the specimens that can be embedded in portable document
format (pdf) for publication (Murienne et al. 2008; Ziegler
et al. 2011). Nevertheless, eyes and sensory pits can only be
observed with some difficulty in the lCT digital images, and
although they do occur at the most anterior portion of the
animal – as seen in histological slides – we were unable to
detect them. Nonetheless, these characters are easily
observed in specimens immersed in alcohol or clove oil.
Internal anatomy is well outlined in the lCT images,
especially those structures that are rich in secretions,
although the nature of the secretion is only discernible as
subtle nuances in brightness. The nerve plate, alimentary
system and the copulatory apparatus are well outlined.
Height of the epithelia is measurable on the lCT-based
images, but their nuclei and cilia are hardly visible at the
resolution we used. Muscle fibres are easily discerned when
grouped into bundles.
To summarize, we identified two limitations of lCT
scanning for taxonomic studies of land planarians, that is,
observation of small structures such as sensory pits and
single muscle fibres, and recognition of the nature of the
gland secretions. However, visualization of small structures
can seemingly be overcome with the use of higher resolu-
tion X-ray computed tomography instruments already
available, which can provide resolution down to 0.5 lm or
less. Regarding the nature of the gland secretion, this char-
acter has hardly been used as a diagnostic feature for geo-
planids and thus should not hamper further descriptions of
new taxa using new technologies.
Conclusions
To our knowledge, this is among the first taxonomic
descriptions of a new extant soft-bodied invertebrate spe-
10
cies with the help of virtual sections and 3D images
obtained by non-destructive techniques (see also Tessler
et al. 2016). In addition, we have been able to identify diag-
nostic characters using these techniques for a species
belonging to a genus containing species that are morpho-


logically very similar (see Alvarez-Presas et al. 2015).
A raw data set of lCT-based images provides a number
of possibilities to create 2D and 3D views, an ever-grow-
ing field in zoology and palaeontology (e.g. Garwood
et al. 2010; Ziegler et al. 2010). It also provides more
comprehensive descriptions of species (Raupach et al.
2015) and, due to relatively fast processing time, lCT is
a potentially powerful tool for studies on intra- and inter-
specific phenotypic variation (Sombke et al. 2015).
Although lCT-based data sets are very large is size, there
is already available a variety of repositories (see Deans
et al. 2015). As for constraints, the handling and actual
access to lCT-based images remains costly, as lCT
images are very large and thus require high performance
computers. However, some natural history museums are
beginning to offer access to such data sets and a database,
GigaDB, can now provide a citable digital object identifier
for such datasets (Lenihan et al. 2014a,b). In turn, lCT
digital imaging has the virtue of being non-destructive
(Giribet 2010; Ziegler 2012) and facilitates remote access
to virtual e-types (Faulwetter et al. 2013). It also has the
potential to accelerate acquisition of zoo-anatomical data
in unprecedented ways (e.g. Ziegler & Menze 2013).
Coupled with novel developments in the use of ontologies
and standards for anatomical descriptors (Ram
ırez et al.
2007; Vogt 2009; Vogt et al. 2012), virtual morphology
should further the use of anatomical data for taxonomy
and systematics.
We believe that the virtues of lCT and related tech-
niques clearly outweigh the above-mentioned few taxo-
nomic limitations. Current limitations in resolution will be
overcome by improved equipment, as will limitations of
data storage as a result of growing image repositories and
faster computing. Structures visible in lCT-based images
are comparable to those observable in conventional images
produced by dissection and histology.
Acknowledgements
The Instituto de Bot^anica da Secretaria do Meio Ambiente
do Estado de S~ao Paulo provided permits for the fieldwork.
Marcos Santos Silva, Ana Laura Almeida and Amanda Cseh
helped with fieldwork. Ana Cristina Vasconcellos made his-
tological sections. J

ulio Pedroni conducted molecular bench
work. Brian D. Metscher provided valuable comments on
PTA and iodine staining. Jennifer Lenihan helped with scan-
ning. Andy Williston helped with the Amira software. Pedro
Dias is acknowledged for kindly providing use of the work-
ª 2016 Royal Swedish Academy of Sciences
 F. Carbayo et al.  Flatworms off the knife edge

station facility (Proc. FAPESP 2014/18002-2). Jos
e Ernesto Carbayo, F. & Lenihan, J. W. (2016a). Micro-computed tomogra-
Belizario and Beatriz Viana dos Santos for scanning work phy scans and virtual histological slides of a recently described
with Axio Scan Z1 (Proc. USP 2012.1.17660.1.0). S~ao Paulo land planarian species of Obama otavioi (Platyhelminthes).
GigaScience Database, doi: 10.5524/100190.
Research Foundation (FAPESP, process nr. 2014/13661–8)
Carbayo, F. & Lenihan, J. W. (2016b). Micro-computed tomogra-
provided financial support to FC. Alexander Ziegler and an phy scan and virtual histological slide data for the land planarian
anonymous referee are deeply thanked for their constructive Obama otavioi (Platyhelminthes). GigaScience, in press.
comments. This work was supported by the Museum of Cason, J. E. (1950). A rapid one-step Mallory-Heidenhain stain for
Comparative Zoology, Harvard University. connective tissue. Stain Technology, 25, 225–226.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Specimens used for the molecular analysis
with their sampling sites and basis for identification. Gen-
Bank accession numbers for COI and EF-1a are provided.
ª 2016 Royal Swedish Academy of Sciences
F. Carbayo et al.  Flatworms off the knife edge
Fig. S1. A—E. —A. Paratypes MCZ 59100 and F5435
under natural conditions, mating ventral to ventral side (in-
set: outline of the two individuals mating). Anterior to the
right. —B. Virtual horizontal section of the ventral epider-
mis and longitudinal cutaneous musculature of paratype
MCZ 59 100 on the left side of the body. Anterior topside.
—C. False-colour 3D rendering of the dorsal anterior end
of paratype MCZ 59100. Note tip of body accidentally bro-
ken during handling. —D. Diagrammatic reconstruction of
copulatory apparatus of paratype MZUSP PL 1573 from
sagittal sections. —E. Microphotograph of a sagittal section
of the copulatory apparatus of paratype MZUSP PL 1573.
Fig. S2. Maximum likelihood tree based on the COI
fragment gene.
Fig. S3. Maximum likelihood tree based on the EF-1a
fragment gene.
13