Reducing the Gap Between Fossils and Extant Species

Systematic Entomology (2011), 36, 573–580

METHODS Virtual dissection using phase-contrast X-ray synchrotron microtomography: reducing the gap between fossils and extant species

MICHEL PERREAU1 and PAUL TAFFOREAU2

1Universite´ Paris 7, IUT Paris Jussieu, Paris, France and 2ESRF, Polygone Scientiﬁque Louis Neel,´ 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 across the fossil has been used several times: Kornilowitch (1903) made visible light microscope observations using this Fossils provide an excellent opportunity for bringing to light method. Subsequently, authors combined this method with evolutionary trends and testing phylogenetic hypotheses. Fos- electron microscope techniques: scanning electron microscope sils preserved in amber are often in a fairly good state (SEM) observations (Henwood, 1992); SEM combined with of preservation, and in some cases allow the reconstruction X-ray microanalysis (Kowalewska & Jacek, 2008); and trans- of complete vanished ecosystems (Poinar & Poinar, 1970). mission electron micropscope (TEM) investigations, which In the case of many insects, identification, generic assign- have led to the observation of ultrastructure, including mito- ment, and morphological phylogenetic analysis of species chondria and nuclei (Grimaldi et al., 1994). Extraction of and higher taxa often involve internal characters. This is DNA has been advocated (Golenberg, 1991; Cano et al., especially true for characters based on genital morphology, 1993), but the results are controversial (Walden & Robert- which is often inaccessible in fossils, making any compar- son, 1997). ison with the extant fauna hazardous. Currently, the visu- The main flaw in breaking amber is the irreparable dam- alization of internal structures of fossil insects in amber age made to the sample. Although internal structures are is a crucial goal for descriptions, identifications and phylogenetic studies. The high quality of preservation of exter- accessible, the sample is usually lost for science, and clearly nally visible features suggests that internal structures could this is unsuitable for holotypes and rare species. Inves- be preserved in just as good condition, at least in some tigations made using these methods have been performed cases. on common species with a large series of identified spec- Several attempts have previously been made to look inside imens, which are impossible to obtain when a clear-cut amber fossils. Dissolving the amber has been tested success- identification of taxa requires an examination of internal fully on Lebanese amber (Azar, 1997) using chloroform as structures. the solvent, but the samples obtained are extremely fragile A non-invasive way to look inside fossil specimens is by and difficult to handle. Cutting the sample along a plane using X-ray imaging. The first use of X-ray microradiogra- phy in entomology (not on fossil specimens) was made by Correspondence: Michel Perreau, Universite´ Paris 7, IUT Paris Goby (1912). Goby resorted to the high resolution of pictures Jussieu, case 7139, 5 rue Thomas Mann, 75205 Paris Cedex 13, France. (linked to the short wavelengths of X-rays) rather than visual- E-mail: [email protected] ising internal structures. Schluter¨ & Sturmer¨ (1982) obtained

© 2011 The Authors Systematic Entomology © 2011 The Royal Entomological Society 573 574 M. Perreau and P. Tafforeau radiographies with a three-dimensional stereoscopic render- the contrast for both complete segmentation of the specimen ing. Grimaldi et al. (2000) made the first three-dimensional and good visualization of the internal structures. The volumes tomographic reconstruction of the external morphology with a were reconstructed using the filtered back-projection algorithm pixel size of 100 μm and an actual resolution in the range of in pyhst (European Synchrotron Radiation Facility). Next, 200–300 μm. More recently, microtomographic images with residual ring artefacts were corrected on the slices using an a resolution in the range of the micron have been obtained in-house tool, and the data was converted from the original (Dierick et al., 2007; Penney et al., 2011). Meanwhile, the 32-bit reconstructions to 16-bit tagged image file format application of propagation phase-contrast X-ray synchrotron (TIFF) stacks. The three-dimensional processing of the volume microtomography (PPC-SRμCT) for the observations of fossil was performed using vgstudiomax 2.1 (Volume Graphics, inclusions in amber started at the European Synchrotron Radi- Heidelberg, Germany), and the final three-dimensional images ation Facility (Grenoble, France). The technique dramatically were obtained using the Phong rendering algorithm. improved the possibilities of non-destructive three-dimensional All the microtomographic data linked to these specimens imaging of these fossils. Until now the technique has been (original slices and processed data) used for the present used to visualize the external morphology of fossils in fuzzy analysis are available to the public on the ESRF online or transparent amber (Tafforeau et al., 2006; Lak et al., 2008; palaeontological database http://paleo.esrf.eu. Soriano et al., 2010; Solorzano-Kraemer´ et al., 2011). Here we explore the possibilities of this technique in an investigation of the internal structures of amber fossil species, illustrated Visible light observations here (Figs 1–3) for a new species of Coleoptera belonging to the family Leiodidae, tribe Anemadini: Nemadus microtomo- The conventional visible light microscopy observations graphicus sp.n. (see Appendix). 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 Material and methods microscope after clearing genitalia in a KOH 0.1 N solution for 10 min, cleaning it in distilled water, dehydrating it in 96% Samples ethanol and mounting it in Euparal between glass slides. The specimens studied come from Baltic amber deposits. After scanning several other specimens, they have been chosen because of their well-preserved internal structures. Results and discussion

Leiodidae are typical coleopterans, with male or female Propagation phase-contrast X-ray synchrotron genital structures that need to be examined for species microtomography identification. When the morphology of the aedeagus is accessible, the identification is mostly straightforward. Without The PPC-SRμCT was performed on the beamline BM5 these structures, few external characters allow conclusive of the European Synchrotron Radiation Facility (Grenoble, identification. Moreover, genital structures also bear characters France). We used a monochromatic beam set at an energy essential in assessing phylogenetic relationships in this group. of 20 keV with a double multilayer monochromator. Thanks The PPC-SRμCT technique allows us to visualize the to the small source and to the source–sample distance external morphology with an accuracy similar to that obtained (58 m in this case), the beam has good coherence properties, with a scanning electron microscope at low magnification, making it possible to use the propagation phase contrast but with the possibility of also observing the sample in any effectively, simply by increasing the sample–detector distance orientation. It is possible to visualize external details that are (Tafforeau et al., 2006). To scan the entire specimen, we used difficult to observe accurately with visible light microscopy, a microscope optic coupled with a charge-coupled device fast- even in transparent amber, such as the metasternal suture readout low-noise (CCD FReLoN) camera, giving an isotropic (Fig. 1d). reconstructed voxel size of 0.7 μm. To reduce the final data Details of the anterior head of the male specimen of size, we used a 2 × 2 binning, giving a voxel size of 1.4 μm. N. microtomographicus sp.n. are shown in Fig. 2a–d. The We used a 20-μmthickGd3Ga5O12 (GGG) scintillator to absence of an external epistomal suture (Fig. 2a) is charac- convert X-rays into visible light. Scans were performed on teristic of the subtribe Nemadina (compared with Anemadina). ◦ a 360 scale with 2500 projections in half-acquisition mode However, some traces of a darker line in place of the epis- (centre of rotation on the right side of the field of view), with tomal suture remains in some extant species of Nemadina 1 s of exposure time per frame and continuous rotation. This (such as Micronemadus pusillimus Kraatz, an Asian representa- protocol allows a reconstruction of a field of view twice as tive of the subtribe). PPC-SRμCT allows us to investigate the large as a normal scan, and the continuous rotation optimizes internal structure of this region of the head by transparency the picture quality for local tomography (Lak et al., 2008). The (Fig. 2b, d), or by using a vertical longitudinal clipping plane propagation distance was set to 70 mm in order to optimize (Fig. 2c, d). Internal structures that leave an external mark

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 (PPC- SRμCT). (d) Detail of the ventral side. The arrow shows the metasternal suture (PPC-SRμCT).

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) Moreover, soft tissues that are presumably the remains of the are clearly visible (Fig. 2b–d: es). Actually, the internal struc- brain are visible too (Fig. 2c: br). Thus it is possible to visual- ture of the head of Coleoptera is well known from histological ize not only tough ectodermic structures, which are more likely methods (Matsuda, 1965), but similar investigations can now to be preserved, but soft tissues as well. We can expect that be performed on fossils without any damage to the sample. in appropriate samples preserved muscles and their insertions,

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 Acknowledgements structures (Deuve, 1993), would be also be visible. We focused our attention on the dissection of genital struc- This work has been funded by the European Synchrotron tures, which is necessary for an unambiguous identification of Radiation Facility under the experiment EC530, as well as many species of Coleoptera, as well as for the assessment of by in-house research beamtime on the beamline BM5. Funds phylogenetic relationships (Fig. 3). It should be noted that the have also been granted by the scientific society ‘Speofauna’ relative locations of the morphological structures are observ- (Paris, France). We are greatly indebted to Karin Schwenninger able with a precision that is difficult to obtain for an actual and Gunther Bechli (Staatliches Museum fur¨ Naturkunde, dissection of an extant species (Fig. 3a). Stuttgart, Germany) for providing the two known specimens of N. tomographicus sp.n. We are also grateful to AndreNel´ (Museum´ national d’Histoire naturelle de Paris, France) for useful discussions, to Carmen Soriano for her help during the Conclusions experiment and for discussions, and to Jon Cooter and Heidi Wild for revising the grammar of this article. 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 References extant species (cf. Video clip S1). Azar, D. 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Mémoires du Muséum National d’Histoire Naturelle, Zoologie, 155, 1–184. requirements of finding monophyletic groups, this was the Dierick, M., Cnudde, V., Masschaele, B., Vlassenbroeck, J., Van best compromise for finding the approximate location of Hoorebeck, L. & Jacobs, P. (2007) Micro-CT of fossils preserved fossils in phylogenetic trees (see a further discussion in in amber. Nuclear Instruments and Methods in Physics Research A, Bethoux, 2009). Two main gaps can be recognized that prevent 580, 641–643. the inclusion of fossil species in phylogenetic analysis on Gnaspini, P. (1996) Phylogenetic analysis of the tribe Ptomaphagini, the same grounds as extant species: the incompleteness of with description of new neotropical genera and species (Coleoptera, the morphological description and the presently impossible Leiodidae, Cholevinae, Ptomaphagini). Papéis Avulsos de Zoologia, investigation of the genetic relationships. PPC-SRμCT and 39, 509–556. Goby, P. (1912) La radiographie des insectes realis´ ee´ avec les rayons X especially the possibility of virtual dissection significantly ultra-mous. Bulletin des Naturalistes de Nice et des Alpes Maritimes reduces the gap between morphological descriptions of fossils (Pages not numbered). and extant species. We must now wait for the invention of Golenberg, E.M. (1991) Amplification and analysis of Miocene plant a technique for reconstructing genetic information in order to fossil DNA. Philosophical Transactions of the Royal Society of reduce the genetic gap! London (B), 333, 419–427. Grimaldi, D., Bonwich, E., Delannoy, E. & Doberstein, S. (1994) Electron microscopic studies of mummified tissues in amber fossils. American Museum novitates, 3097, 1–31. Supporting Information Grimaldi, D., Nguyen, T. & Ketcham, R. (2000) Ultra-high-resolution x-ray computed tomography (UHR CT) and the study of fossils in Additional Supporting Information may be found in the online amber. Studies on Fossils in Amber, with Particular Reference to version of this article under the DOI reference: the Cretaceaous of New Jersey (ed. by D. Grimaldi), pp. 77–91. 10.1111/j.1365-3113.2011.00573.x Backhuys Publisher, Leiden. Hennig, W. (1981) Insect Phylogeny. John Wiley & Sons, New York, Video clip S1. Video illustrating the virtual dissection of New York. Henwood, A.A. (1992) Soft-part preservation of beetles in Tertiary the male genital segment of Nemadus microtomographi- amber from the Dominican Republic. Palaeontology, 35, 901–912. cus sp.n. using PPC-SRμCT (required code: xdvi). Jeannel, R. (1936) Monographie des Catopidae. Mémoires du Muséum d’Histoire Naturelle, 1, 1–433. Please note: Neither the Editors nor Wiley-Blackwell Kornilowitch, N. (1903) Is the structure of striated muscles preserved are responsible for the content or functionality of any in fossil amber? 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Lak, M., Neraudeau,´ D., Nel, A., Cloetens, P., Perrichot, V. & Pronotum 1.37 times wider than long, wider at the base, Tafforeau, P. (2008) Phase contrast x-ray synchrotron imaging: posterior angles widely rounded and distinctively prominent opening access to fossil inclusions in opaque amber. Microscopy at the back, encompassing the base of the elytra (Fig. 1b). and Microanalysis, 14, 251–259. Pronotal surface with the punctuation not aligned in transversal Matsuda, R. (1965) Morphology and evolution of the insect head. strigae. Memoirs of the American Entomological Institute, 4, 1–334. Peck, S.B. & Cook, J. (2007) Systematics, distributions, and bio- Elytra with a tiny punctuation aligned in transversal strigae nomics of the Neoeocatops gen. nov. and Nemadus of North Amer- and perpendicular to the suture (about 36 strigae per mm), ica (Coleoptera: Leiodidae: Cholevinae: Anemadini). The Canadian without longitudinal striae except the sutural stria, which is Entomologist, 139, 87–117. further separated from the suture in the middle than at the Penney, D., Mcneil, A., Green, D.I., Bradley, R., Marusik, Y.M., extremity. Withers, P.J. & Preziosi, R.F. (2011) A new species of anapid spider Metasternal suture short and roughly parallel with the body (Aranea: Aranoidea, Anapidae) in Eocene Baltic amber, imaged axis (arrow in Fig. 1d). using phase contrast X-ray computed tomography. Zootaxa, 2742, Male protarsi strongly dilated, 1.25 times wider than the 60–66. Poinar, G. & Poinar, R. (1970) The Amber Forest. A Reconstruction apex of the protibia (Fig. 2g). Male mesotarsi with the first of a Vanished World, pp. 239. Princeton University Press, Princeton, segment strongly dilated (Fig. 2h), the other segments not New Jersey. dilated. Schluter,¨ T. & Sturmer,¨ W. (1982) X-ray examination of fossil Aedeagus long (more than half the length of the abdomen). insects in cretaceous amber of N.W-France. Annales de la Société Basal lamella of the tegmen long and narrow (Fig. 3b). Entomologique de France, 19, 527–529. Parameres extremely dilated and encompassing the median Solorzano-Kraemer,´ M.M., Perrichot, V., Brown, B.V., Tafforeau, P. lobe laterally, more dilated in their apical half and widely & Soriano, C. (2011) A new species of the Cretaceous genus excavated at the apex (Fig. 3c, e). Basal lamella of the median Prioriphora (Diptera: Phoridae) in French amber. Systematic Entomology, 36, 581–588. lobe as long as the apical part. The apical part of the median Soriano, C., Azar, D., Delclos, X. et al. (2010) Synchrotron phase lobe triangular in dorsal view (Fig. 3f). contrast x-ray imaging of amber inclusions. Comptes Rendus Male genital segment (ninth abdominal segment) fully Palevol, 9, 361–368. developed, with a long spiculum gastrale prominent beyond the Tafforeau, P., Boistel, R., Boller, E. et al. (2006) Applications of x- anterior edge of the epipleurites (Fig. 3i, j). The epipleurites ray synchrotron microtomography for non-destructive 3D studies with a slight ventromedial area of overlap. of paleontological specimens. Applied Physics A, Materials Science Female with a similar external morphology, differing by the and Processing, 83, 195–202. unexpanded protarsi and mesotarsi, and the morphology of the Walden, K.K.O. & Robertson, H.M. (1997) Ancient DNA from amber fossil bees? Molecular Biology and Evolution, 14, 1075–1077. genital segment. The anterior apophysis of the eighth ventrite is long and acute (Fig. 3m). Accepted 19 March 2011 Distribution. Species known only by the holotype and the paratype from Baltic amber, without any information on the Appendix deposit.

Description of Nemadus microtomographicus sp.n. Etymology. The species is named after the method used to Holotype ♂: BALTIC amber, without details on the deposit achieve the complete external and internal description. (Staatliches Museum für Naturkunde, Stuttgart, Germany All the microtomographic data linked to these speci- ◦ n BB-1445-K). mens (original slices and processed data) and used for the Paratype ♀: BALTIC amber, without details on the deposit present analysis are available publicly on the European Syn- (Staatliches Museum für Naturkunde, Stuttgart, Germany chrotron Radiation Facility (ESRF) online palaeontological ◦ n BB-1071-K). 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 Museum´ national Description. Length: 1.8 mm (habitus in situ: Fig. 1a; dor- d’Histoire naturelle de Paris, France, and for Nemadus micro- sal view in Fig. 1b; and lateral view in Fig. 1c by microto- tomographicus, at the Staatliches Museum fur¨ Naturkunde, mography). Stuttgart, Germany. 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 Discussion. The lack of visible epistomal suture, the sin- apical one conical. Antennae slender with antennomeres longer gle first mesotarsomere dilated and the wide male protarsi are than wide, except the sixth and the eighth antennomeres characters that support the placement in the subtribe Nemad- (Fig. 2e). The relative length of the antennomeres to the length ina, and in the genus Nemadus. Nemadus microtomographicus of the first antennomere are as follows: 1; 0.95; 0.77; 0.45; is provisionally placed in the ‘colonoides’ group of species, 0.45; 0.36; 0.64; 0.18; 0.5; 0.55; and 1.1. 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 one- and are not enough to establish the specific status of this half of its width) (Jeannel, 1936; Peck & Cook, 2007). How- species. The examination of genital structures is therefore cru- ever, the definition of the species groups of Nemadus has only cial. Figure 3g, h, k, l shows the genitalia of the extant most been made on the basis of the European and North Amer- common European species N. colonoides (Kraatz), which is ican fauna, excluding the Asiatic fauna. The actual position taken here as an example of the extant species of Nemadus for could be reconsidered when performing a (critically needed) comparison with Nemadus microtomographicus sp.n. Differ- revision of the whole genus. According to Peck & Cook ences occur on the morphology of the parameres (Fig. 3e, g), (2007), the ‘colonoides’ species group contains two North and the thickness and the length of the anterior apophysis of American extant species – Nemadus horni Hatch and Nemadus the genital segment (arrows in Fig. 3j, k). Based on a mor- pusio LeConte – one European extant species – N. colonoides phological phylogenetic analysis (Gnaspini, 1996), the thick- (Kraatz) – and now, an extinct species from Baltic amber: ness of the male ninth abdominal segment apophysis has been N. microtomographicus sp.n. interpreted as a plesiomorphy in another tribe of Cholevinae: Differences based on the external morphology between these Ptomaphagini. The finding of a similar character in an Eocene fossil specimens and the extant species are hardly significant, fossil supports this interpretation.