Will amber inclusions provide the first glimpse of a Mesozoic proteome?

Figure 1. Spider entombed in 100 million-year-old amber from Myanmar (Burma).

In 1994, Woodward et al. reported the isolation of DNA fragments from a Late Cretaceous dinosaur bone preserved in bituminous strata [1]. However, the failure to authenticate this and other purported Mesozoic DNA samples soon lead to the consensus that DNA could not possibly survive much more than 100,000 years [2,3] and, even then, only under the most extraordinary circumstances.

There are exceptions that might permit molecular preservation over geological time. The first exception comes from fossils that have been preserved, frozen for millions of years in arctic ice masses. Willerslev et al. recovered DNA frozen under 2 km of glacial ice in Greenland that dated back nearly a million years [4]. Bidle et al. isolated microbial DNA from 8-million-year-old Antarctic ice cores, the oldest known ice on earth [5]. While Antarctica shares many fossil species with South America and Africa, to which it was joined until its separation from the supercontinent Pangaea in the Late Cretaceous period, the warmer climate of Antarctica during that period would have provided no unique opportunities for preservation.

A second exceptional circumstance results when crystallization occurs. Salamon et al. showed that DNA occluded within clusters of intergrown bone crystals was highly resistant to degradation [6]. More recently, Vreeland et al. reported the isolation and growth of spores from a halophile from a brine inclusion within a 250 million-year-old salt crystal [7]. Protein residues were recovered from the calciferous matrices of oyster [8], mollusk [9] and scallop [10] shells dating back to the Cretaceous and Jurassic periods. Amino acid contents were analogous to the contemporary species, with a gradual decrease in residues with increasing age.

Recently, Asara et al. identified several peptides of sequence homology to collagen from the skeletal elements of a Tyrannosaurus rex, providing compelling evidence that proteins might endure geological time better [11]. Noncollagenous proteins including osteocalcin were putatively identified from the compact bone of a 120 million-year-old Iguanodon bernissartensis[12,13]. Furthermore, it has been suggested that the preservation of cells, and even soft tissues, may be possible. Structures bearing a striking similarity to osteocytes, erythrocytes and even blood vessels have been enisled from demineralized Tyrannosaurus, Triceratops and Brachylophosaurus bone samples [14,15]. However, Kaye et al. recently challenged these findings, attributing these structures to endocasts formed by more contemporary microbial biofilms [16]. Congruently, Wick et al. showed that the alleged soft tissue found preserved in a 50 million-year-old bat was due to the replacement of the original soft tissue by lawns of bacteria [17].

Perhaps the most promising circumstance enabling the preservation of proteins over millions of years comes from amber, the fossilized resins of trees. The unfossilized resins are comprised largely of diterpenes, which rapidly dehydrate the included specimen, a prerequisite for preservation, and possess antimicrobial and anti-inflammatory properties that intervene with normal decomposition. Organisms impaled on the surface of the resinous flow, precisely like flies on flypaper, would remain exposed to the elements where decomposition would normally proceed. Occasionally, when the specimen was completely engulfed in the resin within seconds, exquisite preservation can be observed. In one spectacular specimen, a soldier beetle is caught in the act of spraying its chemical defense on an attacking predator, also captured in a literal ‘freeze frame’ of this Mesozoic scenario [18]. This is the earliest fossil evidence of chemical defenses in insects, dating back 100 million years.

The oldest fossiliferous amber is 325 million years old, preceding the Permian–Triassic boundary marking the mass extinction of more than 90% of the species on earth. The oldest known amber inclusions were thought to originate from the Triassic era and included bacteria, fungi, spores, algae and protozoans [19–21]. These early deposits have since been reassigned to the Early Cretaceous period. The extraordinary morphological preservation observed at the tissue and cellular levels in amber specimens has facilitated the taxonomy of many previously undiscovered species of extinct microorganisms, plants, arthropods and their parasites, filling some critical gaps in the fossil record, such as the radiation of angiosperms and their pollinators during the Cretaceous period.

The oldest fossil flowers in amber emanate from the Early Cretaceous period approximately 100 million years ago [22,23]. A predominant feature of these early flowers was their very small size, such that encapsulation of inflorescences in resinous flows was sometimes possible. Amber preservation has provided insights into the emergence of pollinating insects relative to the divergence of angiosperms. Ramirez et al. reported an unprecedented finding in which the mesoscutellum of an extinct stingless bee, Proplebeia dominicana, was captured while still intercalated with an orchid pollinarium in Miocene amber from the Dominican Republic [24]. The oldest known bee species, Mellitosphex burmensis, was later discovered in 2006 in Early Cretaceous Burmese amber [25], potentially predating vespid wasps described from Turonian amber by 10 million years [26]. Other pollinators include an extinct riodinid butterfly, the first butterfly taxonomically described in amber [27]. Concurrent with the explosive Cretaceous coradiations of angiosperms and pollinating insects, spiders had already developed the capacity to produce highly specialized webs for the capture of flying pollinating insects, also preserved in detail in amber specimens dating back 115–121 million years (Figure 1)[28,29].

The first report of the earliest ants date from 100 million-year-old Burmese amber [30]. Amber has provided the first fossil record of nematode parasitism in ants, with one 40 million-year-old inclusion containing a nematode emerging from a male ant [31].

The earliest amber record of hematophagous insects comes from Early Cretaceous Lebanese amber 125–135 million years old [32]. The extraordinary preservation of microscopic detail has enabled the taxonomy of these phlebotomines and ceratopogonids from their emergence during the Cretaceous through to the present, leading to the recent identification of numerous new species [33,34] and their parasites [35,36], and advancing parasitology into the realm of paleoparasitology. Provocative images of an encapsulated female biting midge containing a fresh blood meal were published by Poinar in which microscopic examination later provided the earliest record of trypanosome parasitism in these insects [37]. Nucleated blood cells from the gut of a sand fly preserved in Burmese amber were assigned reptilian origin [38], suggesting the high possibility that Cretaceous dinosaurs could have been infected by trypanosomes or other parasites carried by phlebotomine vectors [39].

Transmission electron microscopy revealed that the morphology of cellular organelles, such as nuclei, endoplasmic reticulum, ribosomes and mitochondria, was maintained in a 40 million-year-old fly imbibed in amber [40]. By contrast, Edwards et al. demonstrated that insects conjoined with gas bubbles in the resin were highly degraded [41].

At the molecular level, DNA has been isolated from the 30 million-year-old fossil termite Mastotermes electrodominicus and successfully amplified by PCR [42]. Clones were reportedly chimeric in structure, with half of the clone identical to the termite sequence and the other half identical to contaminating sequences [43]. Logically, contaminating sequences could derive from microbial flora associated with the primary organism. Cano et al. isolated and amplified Bacillus DNA from the abdominal tissues of the extinct stingless bee P. dominicana[44]. Furthermore, bacterial spores from 25–40 million-year-old inclusions have reportedly been revived and cultured [45]. The oldest fossil DNA amplified and sequenced was isolated from a 120–135 million-year-old amber-encapsulated weevil [46]. Whether DNA can survive this long, even if in amber, remains a controversial question [47].

Obviously, the rarity of some amber specimens prohibits their expenditure for biochemical analyses. More common specimens and larger sample quantities need to be interrogated for the presence of residual biopolymers, since these would probably only be present in trace quantities. The search for meaningful biochemical sequences enabling phylogenic relationships to be drawn has recently shifted towards proteins, which are thought to better endure over geological time scales.

Our group has recently begun investigations on the surviving proteins in insects embedded in amber from the Dominican Republic, 25–40 million years old, via modern proteomic tools, including the combinatorial peptide ligand libraries that have enabled the discovery of very low-abundance proteins in present-day biological samples [48,49]. Preliminary data suggest that a number of proteins can indeed be detected in these paleontological samples.

Financial & competing interests disclosure

Pier Giorgio Righetti is supported by grants from Fondazione Cariplo (Milano) and PRIN 2009 (MURST, Rome). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.