Annales de la Société entomologique de France (N.S.), 2016 http://dx.doi.org/10.1080/00379271.2016.1230477

When phylogeny meets geology and chemistry: doubts on the dating of Ethiopian amber David Cotya*, Matthieu Lebonb & André Nela aInstitut de Systématique, Évolution, Biodiversité, ISYEB – UMR 7205 – CNRS, MNHN, UPMC, EPHE, Muséum national d’Histoire naturelle, Sorbonne Universités, 57 rue Cuvier, CP 50, Entomologie, F-75005 Paris, France; bÉquipe ‘Histoire Naturelle de l’Homme Préhistorique’ (HNHP), UMR 7194 – CNRS Département de Préhistoire du Muséum national d’Histoire naturelle, Sorbonne Universités, Université Perpignan Via Domitia, Musée de l’Homme, 17 Place du Trocadéro, F-75116 Paris, France (Accepté le 26 août 2016)

Summary. The first fossil species of a Melissotarsus ant is described (Melissotarsus ethiopiensis n. sp.), suggesting that Ethiopian amber could be cenozoic rather than cretaceous. This hypothesis is supported by a Fourier transform infrared spectroscopy (FTIR) analysis of the amber itself, suggesting a weak maturation of the fossil resin, and a revision of the pyrolysis–gas chromatography–mass spectrometry (PY-GC-MS) analysis done by Schmidt and his collaborators in 2010, in an article in which the deposit was described for the first time.

Résumé. Quand la phylogénie rencontre la géologie et la chimie: doutes sur l’âge de l’ambre éthiopien. La première espèce fossile du genre de fourmi Melissotarsus est décrite (Melissotarsus ethiopiensis n. sp.), ce qui laisse suggérer que l’ambre éthiopien pourrait être cénozoïque plutôt que crétacé. Cette hypothèse est appuyée par une analyse Spectroscopie infrarouge à transformée de Fourier (FTIR) de l’ambre lui-même, qui suggère une faible maturation de la résine, et une réinterprétation des analyses Pyrolyse-chromatographie en phase gazeuse-spectrométrie de masse (PY-GC-MS) effectuée par Schmidt et ses collaborateurs en 2010, dans un article où le gisement était décrit pour la première fois. http://www.zoobank.org/urn:lsid:zoobank.org:pub:64BF56C3-1F35-4125-A683-0997F56028DF Keywords: Ethiopian amber; Insecta; Formicidae; Melissotarsus; Fourier transform infrared spectroscopy

2013). It has been suggested that they also satisfy their African record of fossil insects is still scarce and poorly energetic needs by absorbing host tree tissues (Mony documented. Any discovery of a new deposit can therefore et al. 2013). Another fascinating aspect of these ants is be considered of great importance to improve our knowl- that workers walk with their mesothoracic legs pointing edge of ancient ecosystems. Up to date, the Cretaceous upwards, in contact with the ceiling of the galleries, but Lebanese, Jordanian, and Ethiopian ambers were the they stagger on flat surfaces, being unable to walk correctly. unique records from the Arabo-African palaeocontinent. The ages of these ambers need to be accurately determined to avoid biases in the studies of associated fossils, with Material and methods possible consequences in palaeobiogeography and phylo- The original external surface of the amber piece was removed by genetic analyses. Schmidt et al. (2010) described the first polishing; final lustration was done using diatomite powder. The amber deposit of Ethiopia, supposedly Cretaceous in age specimen was examined under Nikon SMZ1000 (Shinagawa, (Cenomanian). Here, we report the discovery in this amber Tokyo, Japan) and Olympus SZX9 (Shinjuku, Tokyo, Japan) fi – stereomicroscopes. Photos were taken with an Olympus E-3 of the rst fossil Melissotarsus ant (Figure 1A D), putting digital camera. Several digital pictures were reconstructed using in doubt this dating, and suggesting a Cenozoic age on the Helicon Focus software. basis of the recent phylogenetic dating of this genus. This Acronyms for measurements result is supported by a Fourier transform infrared spectro- BL: body length; BLW: body length with wing; FWL: forewing scopy (FTIR) analysis of the amber itself, and a revision of length; FWW: forewing width; HL: head length; HW: head width; – – the pyrolysis gas chromatography mass spectrometry CI: cephalic index (HW/HL); SL: scape length; SW: scape width. (PY-GC-MS) analysis done in Schmidt et al. (2010). FTIR was performed on a Vertex 70 spectrometer (Bruker, Melissotarsus is a cryptic modern myrmicine genus, Billerica, MA, USA) in attenuated total reflexion (ATR) mode. with highly specialized behaviour. Workers and gynes dig Less than 1 mg of sample was powdered and pressed on a diamond ATR accessory (Quest, Specac, Orpington, Kent, UK). Infrared galleries in their host tree bark and raise Hemiptera spectra were collected by accumulation of 64 scans in the range of Diaspididae, supposedly for their flesh (Schneider et al. 4000–370 cm−1 with a spectral resolution of 2 cm−1.

*Corresponding author. Email: [email protected]

© 2016 Société entomologique de France 2 D. Coty et al.

Figure 1. Melissotarsus ethiopiensis n. sp. A, Photograph of general habitus (scale bar: 0.4 mm). B, Frontal view of head (scale bar: 0.2 mm). C, Drawing of forewing (scale bar: 2.7 mm). D, Photograph of detail of basitarsus (scale bar: 0.1 mm).

Locality of amber as well as the dimensions of this specimen when it was Our fossil ant was found in a batch of crude amber acquired in alive. Consequently, many measurements are not given Ethiopia by DC to a craftsman, close to the locality where the here, even if they could have been taken, and even those amber was extracted, in the surrounding of the hamlet named given here have to be taken with caution. However, for- Jema (10°06ʹ59.60ʺN 39°08ʹ00.11ʺE), near the village of Merany, North Shewa, Amhara. tunately, all the diagnostic characters of the genus are Midda, given as type locality for the amber outcrop in clearly visible. Schmidt et al. (2010) is not a village or a locality, but more like a ‘district’; however, it is impossible to find on topographical maps. Local people also reported to DC that some amber mines Order Hymenoptera Linné, 1758 have also been dug during the two or three recent years close to Family Formicidae Latreille, 1809 the town of Mahal Meda, even if their access was apparently prohibited by the Ethiopian government when DC went there in Subfamily Myrmicinae Lepeletier de Saint-Fargeau, February 2014. 1835 Genus Melissotarsus Emery, 1877

Results Type species. Melissotarsus beccarii Emery, 1877. Systematic palaeontology The whole body of the specimen has been strongly dehy- Other species. Melissotarsus emeryi Forel, 1907, drated and deformed during diagenesis, so that the Melissotarsus weissi Santschi, 1910, Melissotarsus description cannot reflect with accuracy the appearance insularis Santschi, 1911. Annales de la Société entomologique de France (N.S.) 3

Melissotarsus ethiopiensis n. sp. posterodistal margin of wing ciliate. Hind wing small, Holotype. Ethiopia, surrounding of the hamlet named hardly visible. Jema (10°6ʹ59.60ʺN 39°8ʹ0.11ʺE), near the village of Comparison with modern species. Our fossil differs Merany, North Shewa, Amhara. Specimen ETH 001, from the Western African Melissotarsus beccarii in the collection David Coty, stored at the Muséum national more open angle between M and Rs and in the more d’Histoire naturelle, Paris. pronounced spine on the basal part of the anteroventral side of the petiole (Delage-Darchen 1972). The Ethiopian Measurements in mm. BL: 2.10; BLW: 3.30; FWL: 2.74; (but also known from Western, Central and Southern FWW: 0.98; HL: 0.34; HW: 0.43; CI: 1.26; SL: 0.20; SW: Africa) M. emeryi seems to have this spine more 0.06. pronounced than in our fossil (Weber 1952; Delage- Description. Head subtriangular. Eyes seem to have been Darchen 1972). The Western African M. weissi has no large and ovoid, certainly somehow similar to the ones of spine on the basal part of ventral side of petiole and a M. insularis gynes, but unfortunately completely forewing cell 2R1 shorter than in our fossil, even if it dehydrated, visible under some angles and light axes, shares with our fossil a rather open angle between M and distinguishable from the head cuticle as they have Rs (Santschi 1910). It seems that the female of the ‘silver’ reflects (See Figure 1B). Frontal lobes present, Madagascan M. insularis has never been described confluent, situated centrally and high on dorsum of head (Santschi 1911). Considering those comparisons, and the fi with antennal sockets not fully exposed, somewhat fact that it is the rst fossil representative of this genus, we elevated. Antennal scrobes absent. Antennae with six decided to describe a new species, Melissotarsus segments, apical and preapical ones much larger than ethiopiensis n. sp. preceding funicular segments and forming a distinctive two-segmented club; scape short and broad; pedicel small and moderately thin, as long as wide; 3rd antennal ATR-FTIR analyses segment very short, much wider than long; 4th segment as ATR-FTIR is a well-known technique used to obtain spec- long as 2nd and 3rd segment together, and slightly wider tra from a very wide range of solids, liquids and gases, in than long; 5th segment 1.5 time longer than broad, slightly order to identify and characterize them (Hind et al. 2001). shorter than segment 1, 2, 3, 4 together; mandibles well In the studied Ethiopian amber, most intense absorption − developed, with four teeth; basal tooth moderately bands are observed between 2800 and 3000 cm 1, corre- developed, dull and blunt; second tooth well developed, sponding to C-H vibrations bands. Two main components dull and blunt, third tooth small, denticle-like, apical tooth are observed at 2925 and 2967 cm−1. Alkyl groups are sharp, long and well developed; presence of a dull and also characterized by bands at 1454 cm−1(CH2, CH3) and blunt prominence on outer margin of mandible, and of few 1393 cm−1 (CH3). Carbonyl vibration bands from ester, half-long, semi-erected setae on inner part of mandibles. ketone and carboxylic acid groups are mainly observed −1 Anterior coxae much smaller than the massively devel- between 1680 and 1750 cm , and C-O vibration bands oped middle and hind coxae; petiole and postpetiole from ester and carboxylic acid are observed at 1240 and −1 strongly deformed, a distinct spine on basal part of ante- 975 cm . Weaker bands observed at 3082, 1642 and −1 roventral side of petiole. 887 cm can be attributed respectively to C-H, C = C Forewing 2.74 mm long, 0.98 mm wide; pterostigma and C-H vibration of exocyclic methylene groups (Beck strongly reduced, almost absent; venation as in Figure 1C; et al. 1965; Guiliano et al. 2006). A high relative absor- Forewing hyaline, with microtrichia present on all wing, bance of these bands is indicative of a weak maturation of subcostal vein and radius touching, both ending 1.72 mm ambers (Guiliano et al. 2006; Onoratini et al. 2009). from base; M separating from radius very close to wing Absorption band at 1642 cm-1 could also result from base; Rs separating from radius 0.90 mm from wing base, O-H bending (Tappert et al. 2011). aligned with M and fused with it into Rs+M for 0.28 mm, then separating again; Rs distally weakly sigmoidal, cross- vein r-rs between Rs and R 0.07 mm, reaching apex of R; Discussion pterostigma extremely reduced at apex of R; cell [R] The new species described here falls within Melissotarsus 0.81 mm long and 0.22 mm wide; cell [1R1] 0.86 long because of the following characters (Bolton 1982, 1994): and 0.15 mm wide; cell [2R1] 0.67 mm long and 0.18 mm body with a petiole and a postpetiole between alitrunk and wide; vein M weakly sigmoidal distal of its separation gaster, frontal lobes present, with antennal sockets not from Rs, reaching apical wing margin; Cu separating fully exposed, promesonotal suture completely absent; from M 0.69 mm from wing base; crossvein 1cu-a basitarsal segment of each leg strongly swollen with apical 0.27 mm from wing base, vein 1A weak and distally circlets of teeth; spongiform or lamellate appendages evanescent; distal part of wing without any close cell, absent from petiole and post-petiole; mandible with four 4 D. Coty et al.

Figure 2. ATR-FTIR spectra. A, Spectrum of Ethiopian amber with attribution on main vibration bands. B, Comparison of Ethiopian amber features with Eocene Baltic amber, and Cretaceous amber from Archingeay (France) and Salignac (France). Annales de la Société entomologique de France (N.S.) 5 teeth; antennae six-segmented; frontal lobes confluent, mentioned by the authors, it would be the first example of situated centrally and high on dorsum of head; anterior a Cretaceous Class Ic amber. We rather consider that the coxae much smaller than mid and hind coxae. fact that this amber is a Class Ic one can be considered as After the phylogeny of Myrmicinae proposed by Ward a new argument for a Cenozoic origin of this amber. et al. (2015), the genus Melissotarsus falls in the tribe Ethiopian amber (Figure 2A) shows similar features Crematogastrini, forming a clade (Melissotarsus + with other fossil resin spectra from various ages and Rhopalomastix). The divergence between these two gen- localities (Guiliano et al. 2007; Wolfe et al. 2009; era is estimated to have occurred between 50 and 30 Murillo-Barroso & Martinón-Torres 2012). If the unusual million years before present. Furthermore, Ward et al. wave scale used by Schmidt et al. (2010) in their FTIR (2015) also estimated that Terataner Emery, 1912, sister analysis makes difficult a complete comparison of their genus of this clade, is Cenozoic, with dating between 65 amber spectra with our Ethiopian amber spectra, some and 48 Ma. These datings are therefore strongly incon- clear common points can be found, rendering them simi- gruent with a late Cenomanian age (93–95 Ma) for the lar: both Ethiopian amber spectra display in this region an − Ethiopian amber as proposed by Schmidt et al. (2010). intense C-O band that is observed at 1240 cm 1. Three Several further arguments put doubt on the conclu- well-resolved bands are at 1172, 1140 and 1105 cm−1. sions of Schmidt et al. (2010). The arguments favouring Both differ from the other types of amber previously a Cretaceous age in Schmidt et al. (2010) were a combina- analysed by this technique, especially in the range tion of stratigraphic data, sporomorphs of the amber bear- 1250–1100 cm−1 (Guiliano et al. 2006, 2007; Wolfe ing layers, physico-chemical properties of the amber itself, et al. 2009; Murillo-Barroso & Martinón-Torres 2012). and pollens found in some amber samples. However, none This spectral range is considered as a fingerprint region of these data is unequivocal. The geological map of the for the identification of amber origins and corresponds to region (Belay et al. 2009) mentioned only two sedimen- C-O single bonds in aromatic esters and phenols (Beck tary sections in the area: some Cretaceous sandstones et al.1965; Langenheim & Beck 1968; Vávra & Vycudilik referred as Debre Libanos sandstone (Assefa 1991)and 1976). We therefore compared the Ethiopian amber spec- Upper sandstone or Amba Aradom Formation (Tefera trum to the Eocene Baltic amber, to the Cretaceous ambers et al. 1996) plus a Cenozoic sandstone unit intercalated from Archingeay and Salignac (France) (Figure 2B), and between Quaternary and upper Miocene basalts. The latter to the amber spectra proposed in Guiliano et al. (2007). comprises sandstone, conglomerate and interbedded dia- The relative intensity of exocyclic methylene bands tomite, and its precise age has not been determined. observed at 3082, 1642 and 887 cm−1 in the Ethiopian However, even if not reported in this geological map, amber is more similar to the Cenozoic one than to the other sedimentary sections may also be present in this Cretaceous amber samples, as a high relative absorbance area, intercalated between Cenozoic basalts. As our of these bands is indicative of a weak maturation of amber has been bought locally and not directly collected, ambers (Guiliano et al. 2006; Onoratini et al. 2009). The − it is hard to be sure from which exact strata it has been bands at 1240 and 1172 cm 1 were also reported in extracted. Nevertheless the geological data show that the Simetite, a Sicilian Cenozoic amber (Murillo-Barroso & Cenozoic sediments can be candidate as well as the Martinón-Torres 2012). The ATR-FTIR analysis of Cretaceous ones. However, even if the amber truly Ethiopian amber therefore suggests a weak maturation of comes from the Debre Libanos sandstone, it could also Ethiopian samples, as already shown by Schmidt et al. be possible that the age of this formation is erroneous, as (2010). no unequivocal element is given for its age (Assefa 1991; Schmidt et al. 2010). A complete geological study is therefore needed to give a precise age to this amber. Conclusion Our purpose here is not to revise the entire data of the The ant species described herein, together with an ATR- paper of Schmidt et al. (2010), but, as an example of FTIR analysis, and the revision of the PY-GC-MS analysis ambiguous arguments, we can mention the interpretation done in Schmidt et al. (2010), suggest that the Ethiopian and the result of the PY-GC-MS done on this fossil resin. amber could not be of Cretaceous age, but is more likely It is mentioned (Schmidt et al. 2010) that this amber is a Cenozoic. mature Class Ic: ‘Although we don’t have conclusive information regarding its botanical source, conceivably the resin could have been produced by a previously unknown Cretaceous gymnosperm, or possibly even Acknowledgements from a mid to late Cretaceous angiosperm. Its chemistry DC thanks Dereje Kura, Jamber Aberra, Nidaw Kefelegn, and Belayneh Geto for the great help they gave him in his trip in is analogous to that of less mature Miocene angiosperm Ethiopia. We thank the Muséum national d’Histoire naturelle for ambers found in Mexico and the Dominican Republic that providing access to the ‘Plateau de Spectrométrie Infrarouge du were produced by members of the genus Hymenaea.’ As MNHN’. 6 D. Coty et al.

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