Systematic Entomology (2011), 36, 573–580
M E T H O D S
Virtual dissection using phase-contrast X-ray
synchrotron microtomography: reducing the gap
between fossils and extant species
1
2
M I C H E L P E R R E A U and P A U L T A F F O R E A U
1
Université Paris 7, IUT Paris Jussieu, Paris, France and 2 ESRF, Polygone Scientifique Louis Néel, Grenoble, France
Abstract. Fossils provide excellent opportunities for bringing to light evolutionary
trends, and testing phylogenetic hypotheses. However, the difficulty in accessing
internal structures limits the provision of accurate descriptions, and thus limits
the comparison of fossil specimens with extant fauna. The virtual dissection of
amber fossils by propagation phase-contrast X-ray synchrotron microtomography
(PPC-SRμCT) allows incomparable possibilities for the visualization of genital
structures, which are of prime importance in assessing the taxonomic status and
phylogenetic relationships in many groups of insects. The method is illustrated on
one new species of Coleoptera Leiodidae Anemadini in Baltic amber: Nemadus
microtomographicus sp.n.
Introduction
Fossils provide an excellent opportunity for bringing to light
evolutionary trends and testing phylogenetic hypotheses. Fossils preserved in amber are often in a fairly good state
of preservation, and in some cases allow the reconstruction
of complete vanished ecosystems (Poinar & Poinar, 1970).
In the case of many insects, identification, generic assignment, and morphological phylogenetic analysis of species
and higher taxa often involve internal characters. This is
especially true for characters based on genital morphology,
which is often inaccessible in fossils, making any comparison with the extant fauna hazardous. Currently, the visualization of internal structures of fossil insects in amber
is a crucial goal for descriptions, identifications and phylogenetic studies. The high quality of preservation of externally visible features suggests that internal structures could
be preserved in just as good condition, at least in some
cases.
Several attempts have previously been made to look inside
amber fossils. Dissolving the amber has been tested successfully on Lebanese amber (Azar, 1997) using chloroform as
the solvent, but the samples obtained are extremely fragile
and difficult to handle. Cutting the sample along a plane
Correspondence: Michel Perreau, Université Paris 7, IUT Paris
Jussieu, case 7139, 5 rue Thomas Mann, 75205 Paris Cedex 13, France.
E-mail: [email protected]
© 2011 The Authors
Systematic Entomology © 2011 The Royal Entomological Society
across the fossil has been used several times: Kornilowitch
(1903) made visible light microscope observations using this
method. Subsequently, authors combined this method with
electron microscope techniques: scanning electron microscope
(SEM) observations (Henwood, 1992); SEM combined with
X-ray microanalysis (Kowalewska & Jacek, 2008); and transmission electron micropscope (TEM) investigations, which
have led to the observation of ultrastructure, including mitochondria and nuclei (Grimaldi et al., 1994). Extraction of
DNA has been advocated (Golenberg, 1991; Cano et al.,
1993), but the results are controversial (Walden & Robertson, 1997).
The main flaw in breaking amber is the irreparable damage made to the sample. Although internal structures are
accessible, the sample is usually lost for science, and clearly
this is unsuitable for holotypes and rare species. Investigations made using these methods have been performed
on common species with a large series of identified specimens, which are impossible to obtain when a clear-cut
identification of taxa requires an examination of internal
structures.
A non-invasive way to look inside fossil specimens is by
using X-ray imaging. The first use of X-ray microradiography in entomology (not on fossil specimens) was made by
Goby (1912). Goby resorted to the high resolution of pictures
(linked to the short wavelengths of X-rays) rather than visualising internal structures. Schlüter & Stürmer (1982) obtained
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574 M. Perreau and P. Tafforeau
radiographies with a three-dimensional stereoscopic rendering. Grimaldi et al. (2000) made the first three-dimensional
tomographic reconstruction of the external morphology with a
pixel size of 100 μm and an actual resolution in the range of
200–300 μm. More recently, microtomographic images with
a resolution in the range of the micron have been obtained
(Dierick et al., 2007; Penney et al., 2011). Meanwhile, the
application of propagation phase-contrast X-ray synchrotron
microtomography (PPC-SRμCT) for the observations of fossil
inclusions in amber started at the European Synchrotron Radiation Facility (Grenoble, France). The technique dramatically
improved the possibilities of non-destructive three-dimensional
imaging of these fossils. Until now the technique has been
used to visualize the external morphology of fossils in fuzzy
or transparent amber (Tafforeau et al., 2006; Lak et al., 2008;
Soriano et al., 2010; Solórzano-Kraemer et al., 2011). Here we
explore the possibilities of this technique in an investigation
of the internal structures of amber fossil species, illustrated
here (Figs 1–3) for a new species of Coleoptera belonging to
the family Leiodidae, tribe Anemadini: Nemadus microtomographicus sp.n. (see Appendix).
Material and methods
Samples
The specimens studied come from Baltic amber deposits.
After scanning several other specimens, they have been chosen
because of their well-preserved internal structures.
Propagation phase-contrast X-ray synchrotron
microtomography
The PPC-SRμCT was performed on the beamline BM5
of the European Synchrotron Radiation Facility (Grenoble,
France). We used a monochromatic beam set at an energy
of 20 keV with a double multilayer monochromator. Thanks
to the small source and to the source–sample distance
(58 m in this case), the beam has good coherence properties,
making it possible to use the propagation phase contrast
effectively, simply by increasing the sample–detector distance
(Tafforeau et al., 2006). To scan the entire specimen, we used
a microscope optic coupled with a charge-coupled device fastreadout low-noise (CCD FReLoN) camera, giving an isotropic
reconstructed voxel size of 0.7 μm. To reduce the final data
size, we used a 2 × 2 binning, giving a voxel size of 1.4 μm.
We used a 20-μm thick Gd3 Ga5 O12 (GGG) scintillator to
convert X-rays into visible light. Scans were performed on
a 360◦ scale with 2500 projections in half-acquisition mode
(centre of rotation on the right side of the field of view), with
1 s of exposure time per frame and continuous rotation. This
protocol allows a reconstruction of a field of view twice as
large as a normal scan, and the continuous rotation optimizes
the picture quality for local tomography (Lak et al., 2008). The
propagation distance was set to 70 mm in order to optimize
the contrast for both complete segmentation of the specimen
and good visualization of the internal structures. The volumes
were reconstructed using the filtered back-projection algorithm
in pyhst (European Synchrotron Radiation Facility). Next,
residual ring artefacts were corrected on the slices using an
in-house tool, and the data was converted from the original
32-bit reconstructions to 16-bit tagged image file format
(TIFF) stacks. The three-dimensional processing of the volume
was performed using vgstudiomax 2.1 (Volume Graphics,
Heidelberg, Germany), and the final three-dimensional images
were obtained using the Phong rendering algorithm.
All the microtomographic data linked to these specimens
(original slices and processed data) used for the present
analysis are available to the public on the ESRF online
palaeontological database http://paleo.esrf.eu.
Visible light observations
The conventional visible light microscopy observations
of the genitalia of the most common European species of
Nemadus colonoides (Kraatz) (in comparison with N. microtomographicus sp.n.) were performed on a Leica Diaplan
microscope after clearing genitalia in a KOH 0.1 N solution
for 10 min, cleaning it in distilled water, dehydrating it in 96%
ethanol and mounting it in Euparal between glass slides.
Results and discussion
Leiodidae are typical coleopterans, with male or female
genital structures that need to be examined for species
identification. When the morphology of the aedeagus is
accessible, the identification is mostly straightforward. Without
these structures, few external characters allow conclusive
identification. Moreover, genital structures also bear characters
essential in assessing phylogenetic relationships in this group.
The PPC-SRμCT technique allows us to visualize the
external morphology with an accuracy similar to that obtained
with a scanning electron microscope at low magnification,
but with the possibility of also observing the sample in any
orientation. It is possible to visualize external details that are
difficult to observe accurately with visible light microscopy,
even in transparent amber, such as the metasternal suture
(Fig. 1d).
Details of the anterior head of the male specimen of
N. microtomographicus sp.n. are shown in Fig. 2a–d. The
absence of an external epistomal suture (Fig. 2a) is characteristic of the subtribe Nemadina (compared with Anemadina).
However, some traces of a darker line in place of the epistomal suture remains in some extant species of Nemadina
(such as Micronemadus pusillimus Kraatz, an Asian representative of the subtribe). PPC-SRμCT allows us to investigate the
internal structure of this region of the head by transparency
(Fig. 2b, d), or by using a vertical longitudinal clipping plane
(Fig. 2c, d). Internal structures that leave an external mark
© 2011 The Authors
Systematic Entomology © 2011 The Royal Entomological Society, Systematic Entomology, 36, 573–580
Virtual dissection of fossils using PPC-SRμCT
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Fig. 1. Habitus and external morphology of Nemadus microtomographicus sp.n. (holotype). (a) Visible light micrography of the sample performed
with a binocular microscope coupled with a digital camera. (b) Dorsal view of the habitus (PPC-SRμCT). (c) Lateral view of the habitus (PPCSRμCT). (d) Detail of the ventral side. The arrow shows the metasternal suture (PPC-SRμCT).
© 2011 The Authors
Systematic Entomology © 2011 The Royal Entomological Society, Systematic Entomology, 36, 573–580
576 M. Perreau and P. Tafforeau
Fig. 2. Head and external details of Nemadus microtomographicus sp.n. (holotype) by PPC-SRμCT. (a) Frontal view of the head showing the
absence of external traces of the epistomal suture. (b) Frontal view of the head with transparency, showing the internal structure (es), which in some
groups gives an external epistomal suture, but not in Nemadus species. (c) Transversal cut of the head showing internal structures corresponding
to an ancient epistomal suture (es), and presumably the remains of the brain (br). (d) The same view with a transparency effect. (e) Right antenna.
(f) Right maxillary palpus. (g) Right protibia and protarsus. (h) Right mesotibia and mesotarsus.
(epistomal suture) in some other genera (but not in Nemadus)
are clearly visible (Fig. 2b–d: es). Actually, the internal structure of the head of Coleoptera is well known from histological
methods (Matsuda, 1965), but similar investigations can now
be performed on fossils without any damage to the sample.
Moreover, soft tissues that are presumably the remains of the
brain are visible too (Fig. 2c: br). Thus it is possible to visualize not only tough ectodermic structures, which are more likely
to be preserved, but soft tissues as well. We can expect that
in appropriate samples preserved muscles and their insertions,
© 2011 The Authors
Systematic Entomology © 2011 The Royal Entomological Society, Systematic Entomology, 36, 573–580
Virtual dissection of fossils using PPC-SRμCT
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Fig. 3. Genital structures of the extinct species Nemadus microtomographicus sp.n. (a–f, i, j, m) and the extant species Nemadus colonoides
(Kraatz) (g, h, k, l). (a) Lateral view of habitus and genital structures with a transparency effect. (b) The whole genital segment, including the
aedeagus. (c) Lateral view of the aedeagus. (d) Lateral view of the median lobe of the aedeagus. (e) Lateral view of the right paramere. (f) Dorsal
view of the median lobe of the aedeagus. (g) Dorsal view of the aedeagus. (h) Lateral view of the aedeagus. (i, k) Ventral view of the male
genital segment. (j, l) Lateral view of the male genital segment. (m) Dorsal view of the ventral apophysis of the female eighth abdominal sternite
(paratype). Abbreviations: gsa, male genital segment apophysis (spiculum gastrale); pr, parameres. False colours in Fig. 3a–c are added to make
the different structures clearer: the genital segment (ninth abdominal segment) is shown in blue; the tegmen of the aedeagus is shown in brown;
and the median lobe of the aedeagus is shown without colour. Images are obtained by PPC-SRμCT (b–f, i, j) and by conventional visible light
microscopy (g, h, k, 3l).
© 2011 The Authors
Systematic Entomology © 2011 The Royal Entomological Society, Systematic Entomology, 36, 573–580
578 M. Perreau and P. Tafforeau
which are important for assessing homology in metameric
structures (Deuve, 1993), would be also be visible.
We focused our attention on the dissection of genital structures, which is necessary for an unambiguous identification of
many species of Coleoptera, as well as for the assessment of
phylogenetic relationships (Fig. 3). It should be noted that the
relative locations of the morphological structures are observable with a precision that is difficult to obtain for an actual
dissection of an extant species (Fig. 3a).
Conclusions
The PPC-SRμCT technique enables an incomparable visualization of internal structures. In the investigated specimens, these
observations were critical to give a relevant formal description of this taxon, and to discuss the affinities with the related
extant species (cf. Video clip S1).
Hennig (1981) discussed in detail the problems generated
by including fossils in phylogenetic analysis. When fossil
specimens cannot clearly be placed in a monophyletic group
because of the lack of information available on their characters,
he introduced the concept of the stem group of a group:
a set of fossils that are more closely related to recent
species of the group than to the recent species of its sister
group. Even if unsatisfactory with regards to the phylogenetic
requirements of finding monophyletic groups, this was the
best compromise for finding the approximate location of
fossils in phylogenetic trees (see a further discussion in
Bethoux, 2009). Two main gaps can be recognized that prevent
the inclusion of fossil species in phylogenetic analysis on
the same grounds as extant species: the incompleteness of
the morphological description and the presently impossible
investigation of the genetic relationships. PPC-SRμCT and
especially the possibility of virtual dissection significantly
reduces the gap between morphological descriptions of fossils
and extant species. We must now wait for the invention of
a technique for reconstructing genetic information in order to
reduce the genetic gap!
Supporting Information
Additional Supporting Information may be found in the online
version of this article under the DOI reference:
10.1111/j.1365-3113.2011.00573.x
Video clip S1. Video illustrating the virtual dissection of
the male genital segment of Nemadus microtomographicus sp.n. using PPC-SRμCT (required code: xdvi).
Please note: Neither the Editors nor Wiley-Blackwell
are responsible for the content or functionality of any
supporting materials supplied by the authors. Any queries
(other than missing material) should be directed to the
corresponding author for the article.
Acknowledgements
This work has been funded by the European Synchrotron
Radiation Facility under the experiment EC530, as well as
by in-house research beamtime on the beamline BM5. Funds
have also been granted by the scientific society ‘Speofauna’
(Paris, France). We are greatly indebted to Karin Schwenninger
and Gunther Bechli (Staatliches Museum für Naturkunde,
Stuttgart, Germany) for providing the two known specimens
of N. tomographicus sp.n. We are also grateful to André Nel
(Muséum national d’Histoire naturelle de Paris, France) for
useful discussions, to Carmen Soriano for her help during the
experiment and for discussions, and to Jon Cooter and Heidi
Wild for revising the grammar of this article.
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Pronotum 1.37 times wider than long, wider at the base,
posterior angles widely rounded and distinctively prominent
at the back, encompassing the base of the elytra (Fig. 1b).
Pronotal surface with the punctuation not aligned in transversal
strigae.
Elytra with a tiny punctuation aligned in transversal strigae
and perpendicular to the suture (about 36 strigae per mm),
without longitudinal striae except the sutural stria, which is
further separated from the suture in the middle than at the
extremity.
Metasternal suture short and roughly parallel with the body
axis (arrow in Fig. 1d).
Male protarsi strongly dilated, 1.25 times wider than the
apex of the protibia (Fig. 2g). Male mesotarsi with the first
segment strongly dilated (Fig. 2h), the other segments not
dilated.
Aedeagus long (more than half the length of the abdomen).
Basal lamella of the tegmen long and narrow (Fig. 3b).
Parameres extremely dilated and encompassing the median
lobe laterally, more dilated in their apical half and widely
excavated at the apex (Fig. 3c, e). Basal lamella of the median
lobe as long as the apical part. The apical part of the median
lobe triangular in dorsal view (Fig. 3f).
Male genital segment (ninth abdominal segment) fully
developed, with a long spiculum gastrale prominent beyond the
anterior edge of the epipleurites (Fig. 3i, j). The epipleurites
with a slight ventromedial area of overlap.
Female with a similar external morphology, differing by the
unexpanded protarsi and mesotarsi, and the morphology of the
genital segment. The anterior apophysis of the eighth ventrite
is long and acute (Fig. 3m).
Accepted 19 March 2011
Appendix
Distribution. Species known only by the holotype and the
paratype from Baltic amber, without any information on the
deposit.
Description of Nemadus microtomographicus sp.n.
Holotype ♂: BALTIC amber, without details on the deposit
(Staatliches Museum für Naturkunde, Stuttgart, Germany
n◦ BB-1445-K).
Paratype ♀: BALTIC amber, without details on the deposit
(Staatliches Museum für Naturkunde, Stuttgart, Germany
n◦ BB-1071-K).
Description. Length: 1.8 mm (habitus in situ: Fig. 1a; dorsal view in Fig. 1b; and lateral view in Fig. 1c by microtomography).
Head lacking the external epistomal suture (visible only
through transparency: Fig. 2a, b). Maxillary palpi dilated
(Fig. 2f), the two apical palpomeres of subequal lengths, the
apical one conical. Antennae slender with antennomeres longer
than wide, except the sixth and the eighth antennomeres
(Fig. 2e). The relative length of the antennomeres to the length
of the first antennomere are as follows: 1; 0.95; 0.77; 0.45;
0.45; 0.36; 0.64; 0.18; 0.5; 0.55; and 1.1.
Etymology. The species is named after the method used to
achieve the complete external and internal description.
All the microtomographic data linked to these specimens (original slices and processed data) and used for the
present analysis are available publicly on the European Synchrotron Radiation Facility (ESRF) online palaeontological
database http://paleo.esrf.eu. Three-dimensional prints of the
holotypes have been deposited in collections, for the three
described species at the ESRF and at the Muséum national
d’Histoire naturelle de Paris, France, and for Nemadus microtomographicus, at the Staatliches Museum für Naturkunde,
Stuttgart, Germany.
Discussion. The lack of visible epistomal suture, the single first mesotarsomere dilated and the wide male protarsi are
characters that support the placement in the subtribe Nemadina, and in the genus Nemadus. Nemadus microtomographicus
is provisionally placed in the ‘colonoides’ group of species,
defined by the following set of characters: short length (less
© 2011 The Authors
Systematic Entomology © 2011 The Royal Entomological Society, Systematic Entomology, 36, 573–580
580 M. Perreau and P. Tafforeau
than 2 mm) and short eighth antennomere (shorter than onehalf of its width) (Jeannel, 1936; Peck & Cook, 2007). However, the definition of the species groups of Nemadus has only
been made on the basis of the European and North American fauna, excluding the Asiatic fauna. The actual position
could be reconsidered when performing a (critically needed)
revision of the whole genus. According to Peck & Cook
(2007), the ‘colonoides’ species group contains two North
American extant species – Nemadus horni Hatch and Nemadus
pusio LeConte – one European extant species – N. colonoides
(Kraatz) – and now, an extinct species from Baltic amber:
N. microtomographicus sp.n.
Differences based on the external morphology between these
fossil specimens and the extant species are hardly significant,
and are not enough to establish the specific status of this
species. The examination of genital structures is therefore crucial. Figure 3g, h, k, l shows the genitalia of the extant most
common European species N. colonoides (Kraatz), which is
taken here as an example of the extant species of Nemadus for
comparison with Nemadus microtomographicus sp.n. Differences occur on the morphology of the parameres (Fig. 3e, g),
and the thickness and the length of the anterior apophysis of
the genital segment (arrows in Fig. 3j, k). Based on a morphological phylogenetic analysis (Gnaspini, 1996), the thickness of the male ninth abdominal segment apophysis has been
interpreted as a plesiomorphy in another tribe of Cholevinae:
Ptomaphagini. The finding of a similar character in an Eocene
fossil supports this interpretation.
© 2011 The Authors
Systematic Entomology © 2011 The Royal Entomological Society, Systematic Entomology, 36, 573–580