Amber Inclusions

Now dim, now bright, trapped in its amber tear

A bee seems sealed in its own nectar clear;

For a life of endless toil, most fitting pay

Surely a bee would wish to die this way!

-Martial's Epigrams, ca. 89 a.d. (translation by Valerie Krishna)

Amber, sometimes called resinite, is ancient tree resin. Vast deposits from the Baltic region have been collected for charms, amulets, and objets d'art for at least 13 millennia -making Baltic amber the original precious substance. When the resin was sticky, insects and other small organisms became mired on the surface and were gradually engulfed by the flowing resin, preserving them in finer fidelity than perhaps any other kind of fossil. The use of amber in jewelry and decorative objects, and an intrigue with inclusions have created a special popular appeal of the substance (Grimaldi, 1996).

Fossil resins are scattered throughout earth's surface in deposits from the Carboniferous to the Holocene. Unusual filaments of fossil resin are known from vessels of Carbonifer ous tree ferns, but true amber first appears in the Triassic (ca. 235 mya). Amber is usually secreted externally by the plant into oozing masses, believed to be a way trees seal an injury from invading insects and fungi. Resins are complex mixtures of terpenoids and other biomolecules (Mills et al., 1984), with over 100 individual compounds identified from 7 botanically distinct amber deposits alone (Grimalt et al., 1987). Resins (and therefore amber) from different species of tree (and even populations of some species) have unique chemical profiles, usually characterized using pyrolysis-gas chromatography/mass spectroscopy. This feature is commonly exploited to help identify the type(s) of trees that produced an amber deposit, though associating the amber with plant macrofossils is equally important for identification (e.g., Shedrinsky et al., 1991; Anderson and LePage, 1995; Grimaldi et al., 2000a,b). The recent book by Langenheim (2003) comprehensively treats Recent and fossil resins.

Almost all of the amber produced in the Mesozoic, and much of the Cenozoic amber, is from conifers. Rare exceptions involve a few cases of Cretaceous angiosperm amber (Langenheim, 1969, 2003; Grimaldi et al., 2000b). Some large Cenozoic deposits, though, were formed from broad-leaved (dicot) trees, such as Dominican and Mexican amber (Hymenaea: Leguminosae), and Arkansas and Borneo amber, possibly formed from Dipterocarpaceae. Resin production is widespread among conifers but sporadic among angiosperms. The largest deposit of amber in the world occurs on the southern coasts of the Baltic Region and was formed by a conifer, probably a pine (Pinaceae) (Langenheim, 1995). The oldest deposits that contain insects are from the Early Cretaceous of Austria (Schlee, 1984); England, Lebanon, and Jordan (Azar, 2000); and Choshi, Japan (Fujiyama, 1994), 140-120 mya. The taphonomy of fossil insect assemblages in amber, and world amber deposits, has been comprehensively reviewed by Martinez-Delclos et al. (2004).

Because there is such a spectrum in ages of fossil resin, from several hundred years old (generally referred to as copal) to millions, it is often confusing as to when buried resin becomes amber. Resin begins to polymerize and crosslink almost immediately after exuding and will continue to do so for centuries and millennia in the ground, thus rendering amber much more inert than modern resins. But, the degree to which these processes occur depends on many factors: the molecular composition of the original resin, exposure to ultraviolet light, age, and the amount of geothermal energy imposed by overlying sediments and faulting. Dominican amber (approximately 20 myo), for example, is far more inert and reacts less with solvents than many, much older Cretaceous ambers, largely because of its composition.

One proposal has suggested a solution to the ambiguity of amber and resin based entirely on the practical criterion of 14C dating (Anderson, 1996). Organic materials up to 40,000

2.25. A small menagerie of arthropods in Oligocene amber from southern Mexico. Other forms of fossilization can also preserve aggregations, but no form preserves minute and delicate organisms with the fidelity of amber. AMNH A231; greatest length of piece 31 mm.

2.26. A map of the menagerie in amber in Figure 2.25. There are 36 individual arthropods, including insects and spiders, belonging to four insect orders, ten families, and approximately 13 species. Such pieces are small slices of ancient ecosystems.

years old, including amber, can be reliably dated using the technique. In this proposal, material 250 years old or less is modern or recent resin or copal; that between 250 and 5,000 years old is ancient resin; resin 5,000 to 40,000 years old is subfossil resin; and material older than 40,000 years is amber, fossil resin, or resinite. While this is an arbitrary classification, it is at least a very practical way to distinguish between modern and ancient resins (<40,000 years old) and amber (>40,000 years old). In general, too, copals are lighter in color than amber, they readily melt, and the surface forms a system of fine cracks (or crazing) over several years. In the 14C scheme, material from Madagascar; East Africa; Colombia; Mizunami, Japan; and eastern portions of the Dominican Republic (Burleigh and Whalley, 1983) are recent to ancient resins, not amber.

Forests bleed large quantities of resin as a result of damage by storms, fires, and outbreaks of wood-boring insects. Any one or several of these factors contributed to the prolific amounts of amber produced by ancient forests from the Baltic region, the Dominican Republic and Mexico, and several Cretaceous sites (the most significant deposits are discussed individually later). Productive deposits like Baltic and Dominican amber have captured a great variety of organisms (Figures 2.25 to 2.29): leaves, flowers, portions of vines and stems, fungi, myriad arachnids and insects, swarms and mating pairs of insects, hosts with parasites, even scorpions, small lizards, and frogs. So diverse are these amber Lagerstätte that detailed reconstructions have been made of their paleoenvironments (Brues, 1933b; Larsson, 1978; Grimaldi, 1996). A significant proportion of the biodiversity in amber comprises tiny arthropods a millimeter or less in size, like mites; cecidomyiid and ceratopogonid midges; scelionids and mymarid and mymarommatid fairy "flies" (all Hymenoptera); and minute ptiliid ("featherwing") beetles. Indeed, the smallest arthropod fossils are known from amber,

2.28. A cecidomyiid gall midge caught in Dominican amber while laying its eggs. Some insects reflexively exude eggs as they are dying, which probably happened here. Minute scenes like this are commonly preserved in amber. AMNH DR14-704; length of midge 1.8 mm.

and because of the exquisite preservation, minute structures can be observed at the micron scale (e.g., Figures 10.83, 10.84). Many of the insects in amber died on the surface of a resin flow and then were sealed when more resin flowed over them. If the amber is split along the flow line, exposing a cast

2.27. Cicadellid leafhoppers in Miocene Dominican amber, captured while mating. AMNH DR15-5; total length (both) 6.2 mm.

2.29. A chironomid midge with two parasitic mermithid nematodes bursting from its abdomen, preserved in mid-Cretaceous amber from Burma. AMNH Bu320; length of midge 1.2 mm.

2.27. Cicadellid leafhoppers in Miocene Dominican amber, captured while mating. AMNH DR15-5; total length (both) 6.2 mm.

2.29. A chironomid midge with two parasitic mermithid nematodes bursting from its abdomen, preserved in mid-Cretaceous amber from Burma. AMNH Bu320; length of midge 1.2 mm.

2.30. A milichiid fly in Dominican amber with the vivid red pigment of its eyes preserved. Amber rarely preserves such color on insects. AMNH DR14-1316; body length 2.6 mm.

of the insect, scanning electron micrography can reveal extremely fine external details. Color patterns are frequently preserved, and in some cases the original vivid color remains (Figure 2.30).

Microscopic-scale preservation of internal soft tissues

2.31. A small swarm of 11 Proplebeia stingless bees in Dominican amber. They were captured in a fresh runnel of the resin, which was then engulfed by successive flows that formed layers like a stalactite. AMNH DR14-1054; length of amber 43 mm.

within amber-encased insects was known as early as 1903, but electron microscopy has revealed unexpected, lifelike fidelity (Mierzejewski, 1976; Kohring, 1998), even subcellular structure (Henwood, 1992a,b; Grimaldi et al., 1994). The degree of preservation varies greatly, largely as a result of the chemistry of the resin and how quickly the organism was embedded. Small organisms in Baltic amber, for example, commonly have a milky coating, which is actually a froth of microscopic bubbles, probably formed by gases exuding from the decaying body cavity. Organs and tissues of such insects are usually poorly preserved, or the body cavity is merely a void. Insects well preserved in amber, including from the Cretaceous, often have the organs intact, with virtually none of the shrinkage typically seen in specimens that have been merely dehydrated. Muscles lie in their original positions, and the banding is vivid, with the digestive tract, tracheae, even the nerves, brain (Figures 2.33, 2.34), and symbiotic microbes preserved. For example, modern, wood-boring platypodid and other beetles have small pockets under the cuticle ("mycangia") specialized for harboring symbiotic "ambrosia" fungus, which they inoculate into their galleries and then feed upon. The mycangia of platypodids in Dominican amber were likewise found to contain mycelia and spores of the fungus (Grimaldi et al., 1994).

Even more impressive is the cellular and subcellular preservation, including muscle cells and neurons, myo-fibrils and sarcomeres, membranes, and mitochondria (Henwood, 1992a,b; Grimaldi et al., 1994). Most recently, symbiotic bacteria attached to the membrane of symbiotic protists have been observed in hind gut tissue of a termite in

2.32. A Proplebeia stingless bee in Dominican amber preserved with balls of resin on its hind legs. Stingless (meliponine) bees harvest resin to construct their nests and frequently become trapped, which is why Proplebeia is so common in Dominican amber. Their abundance in the amber has made this bee a favored subject for studies of tissue and molecular preservation. AMNH; length of bee 2.9 mm.

2.31. A small swarm of 11 Proplebeia stingless bees in Dominican amber. They were captured in a fresh runnel of the resin, which was then engulfed by successive flows that formed layers like a stalactite. AMNH DR14-1054; length of amber 43 mm.

2.32. A Proplebeia stingless bee in Dominican amber preserved with balls of resin on its hind legs. Stingless (meliponine) bees harvest resin to construct their nests and frequently become trapped, which is why Proplebeia is so common in Dominican amber. Their abundance in the amber has made this bee a favored subject for studies of tissue and molecular preservation. AMNH; length of bee 2.9 mm.

2.33. A Proplebeia bee exhumed from Dominican amber and imaged with an electron microscope. The amber was carefully split in half, exposing the body cavity and internal organs and tissues. Under high magnification (10,000X) even the banding of muscle myofibrils and the folded cristae of mitochondria are preserved. Reports in the early 1990s of DNA sequences from amber fossils now seem to have been a result of contaminants. AMNH.

2.34. Muscle fibers from a beetle preserved in 125 myo amber from Lebanon, preserved with the tube-like tracheoles that carry oxygen and carbon dioxide to and from tissues. Preservation is not necessarily better in younger ambers but mostly depends on the composition of the resin. AMNH.

Dominican amber (Wier et al., 2002). The unparalleled fine structure of insects embalmed in amber inspired studies on their molecular preservation, and no molecule is of greater evolutionary significance than the one that is the basis of inheritance, DNA.

Does Amber Preserve Ancient DNA? Changes in DNA inherited through generations eventually become part of the blueprint of a lineage, so by reconstructing the sequence of changes we can retrace the evolutionary history and relationships of diverse organisms. Unfortunately, estimates of the times and extent of genetic divergence have traditionally been relegated to comparisons among living species. When polymerase chain reaction (PCR) was developed for amplifying minute quantities of DNA, this led to an avid search for DNA that was truly ancient, from fossils millions of years old. Insects in amber played a leading role in that search.

Interest in DNA from fossils seriously began with study of compression fossil leaves in Miocene sediments from Clarkia, Idaho, a site in which insects are also beautifully preserved (discussed later). Dense mudstones at Clarkia sealed the fossils from destruction by oxygen for nearly 18 million years. When unearthed, some leaves are a vivid green, or autumn yellow, like the day they fell, but within minutes they oxidize to a blackish brown. Cellular and even subcellular preservation of Clarkia plants, including chloroplasts, has been well documented (Smiley et al., 1975; Niklas et al., 1985). The first report of DNA millions of years old was a

770-bp fragment of the chloroplast gene rbcL from leaves of an extinct tree at Clarkia, Magnolia latahensis (Golenberg et al., 1990). This gene is of standard use in the molecular sys-tematics of plants, and sequences of it were found to differ by 17 bp (or 2.2% sequence divergence) between the fossil and a closely related living species. The pitfall of PCR techniques is that contaminant DNA is commonly amplified, even under scrupulously clean conditions, which led some to criticize the study (Golenberg, 1991). Soon after this came a report on DNA from another Clarkia fossil plant, this time a bald cypress (Taxodium) (Soltis et al., 1992). Using the same gene, they found 11-bp changes among the 1,320 they sequenced, representing a divergence of less than 1%. These results soon led to popular accounts on the scientific implications of truly ancient DNA (Gould, 1992). For the first time, it seemed, genetic divergence over millions of years could be directly measured.

While work on Clarkia fossils was being discussed, several other labs focused attention on insect fossils in Miocene amber (20 myo) from the Dominican Republic, renowned for their preservation. Two papers were published within a month of each other, one on the common stingless bee in Dominican amber, Proplebeia dominicana (Cano et al., 1992) (Figures 2.31 to 2.33), the other on the relict termite, Mastotermes electrodominicus (DeSalle et al., 1992) (Figure 7.81). Both came on the heels of the best-selling novel, Jurassic Park, wherein dinosaurs are resurrected from cloned DNA extracted from the blood meals of mosquitoes preserved in amber. Amber had never been so popular.

DNA of the fossil bees' 18S rRNA gene was 7% divergent from several living species in the genus Plebeia. The fossil termite sequences were divergent by 10% from the sole living species of Mastotermes (darwiniensis) in the 16S rRNA gene, an unexpectedly large amount. Phylogenetic analysis, though, provided compelling evidence for a termite identity of the fossil DNA. Soon after the publication of these reports, reviewed and unreviewed reports of DNA being extracted from Hymenaea leaves and chrysomelid beetles in Dominican amber appeared, and then a major critique of all reports on ancient DNA was published (Lindahl, 1993). Of all bio-molecules, DNA is perhaps the most labile (Eglinton and Logan, 1991). It is particularly susceptible to destruction by hydrolysis and oxidation, and even spontaneously decays under ideal conditions. No natural space is completely devoid of water and oxygen, including amber, and preservation of the molecule over millions of years appeared implausible.

Despite Lindahl's caution, a paper was published two months later in the same journal (Nature), reporting DNA from a weevil preserved in Lebanese amber, some 125 million years old (Cano et al., 1993). Publication of the paper on the same day that the film version of Jurassic Park publicly debuted (10 June 1993) was not coincidental, and this propelled the popularity of amber even more. The unique weevil specimen had been splintered open, its tissue extracted, and 541 bp of the 18S rRNA gene were sequenced and compared to other insects, including weevils. Similar to the Dominican amber fossils, the DNA of the Lebanese amber weevil was 7% divergent from the living nemonychid weevil that was sequenced, and phylogenetic analysis again indicated authenticity.

The most convincing test for the authenticity of the ancient DNA is reproducibility. Two independent attempts failed to replicate the extraction of DNA from Proplebeia bees in Dominican amber (Austin et al., 1997; Walden and Robertson, 1997). Even attempts to extract DNA from other kinds of insects in fossil resins failed, including platypodid beetles (Howland and Hewitt, 1994) and phorid flies (Austin et al., 1997) in Dominican amber, as well as bees in much younger East African copal (Austin et al., 1997). In fact, these attempts consistently found contaminant DNA. By the time a report was published on the revival of bacterial spores from Dominican amber (Cano and Borucki, 1995), it was justifiably regarded with widespread skepticism. If the contamination of PCR products was notoriously difficult to control, how much more would it be for ubiquitous Bacillus bacteria? Also, if DNA of insects in amber is at best highly fragmented, why should even highly resistant bacterial endospores endure so long, their genomes perfectly intact?

Attempts to replicate extractions of DNA from the Lebanese amber weevil and Domincan amber Mastotermes have not been made. The weevil was a unique specimen, and destructive sampling of it also generated controversy concerning the study of unique and rare amber specimens. Reanalysis of the published sequences from that specimen, though, indicates that they are probably contaminants from another beetle (Gutiérrez and Marín, 1998). A similar fate perhaps awaits the putative DNA sequences of the extinct Mastotermes. Studies made on the racemization of amino acids in amber fossilized tissues, though, support the possibility that DNA is preserved by amber (Bada et al., 1999). Racemization is the formation of equal proportions of D and l enantiomers of a molecule, and certain amino acids racem-ize over steady rates. Apparently, the extent of amino acid racemization in tissues of insects preserved in amber is very similar to that of modern species, suggesting that DNA could be similarly preserved. But, amino acids are particularly durable molecules (Savage et al., 1990; Bada, 1991; Kemp, 2002). Also, the cuticles from Proplebeia bees in Dominican amber contain no trace of chitin or protein (Stankiewicz et al., 1998c). If a molecule as durable as chitin is completely degraded in amber, it is highly likely that DNA will also be degraded. In fact, it is highly unlikely that any DNA is preserved in ancient fossils of any sort.

Despite controversy and serious suspicion over DNA from amber fossils, these studies brought closer attention to the remarkable preservative qualities of amber, which have a fidelity that is far greater and more consistent than any other kind of fossil.

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