Detección de poliovirus mediante western blot

Unusual Late Cretaceous to Early Tertiary Angiosperms

Despite the modest rise of the angiosperms during the earliest Cretaceous (Crane et al. 1995), only coniferous origins for ambers have been suggested until the very late Cretaceous. Ambers representing two angiosperm fami-lies, Dipterocarpaceae and Altingiaceae, occur in sediments reported as late Cretaceous and early Tertiary (Paleocene and Eocene). Those of

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paceae have a longer authenticated Cretaceous record than Altingiaceae, but because there are more problems associated with positive identification of the dipterocarp source, Altingiaceae are discussed first. Additionally, there is indirect evidence that a member of the Clusiaceae was producing floral resin in the late Cretaceous, related to resin-collecting bees preserved in the amber.

Liquidambar (Plate 13 and Figure 2-8; Altingiaceae, including some for-mer hamamelids) or its immediate ancestor is considered to be the source of amber from several localities along the North American Atlantic Coastal Plain, close to the Cretaceous–Tertiary boundary but primarily in the Paleo-cene. The chemical evidence is based on the presence of polystyrene (Figure 4-1) and its derivatives, determined through IR (Langenheim and Beck 1968), FTIR and Py-GC (Grimaldi et al. 1989), and Py-GC-MS (K. Anderson et al.

1992). Cinnamic acid (Figure 1-8) and its esters, major constituents of Liquid-ambar trunk resin (Chapter 8), are readily decarboxylated to styrene, which can polymerize and then remain stable for millions of years. Hamamelids apparently occurred in warm temperate to subtropical mixed coniferous for-ests, which included taxodiaceous amber-producing trees during the late Cre-taceous (Graham 1999). However, the genus Liquidambar has not been pos-itively identified from plant fossils until the Paleocene. IR of the amber from the late Cretaceous Atlantic Coastal Plain is virtually indistinguishable from Montana amber (Langenheim and Beck 1968), and Py-GC shows it to be almost pure polystyrene (Shedrinsky et al. 1989–91). Authentication of amber as the source of polystyrene is essential, of course, given the occurrence of synthetic polystyrene in the environment. A Liquidambar source is further supported for this amber by Muller’s (1981) report of Liquidambar pollen from Paleocene sediments in the Rocky Mountains. Thus, by inference, amber from the Cretaceous–Tertiary boundary could well be from Liquidambar or at least a close relative. In any event, the appearance of Liquidambar in North America essentially begins during the Tertiary. The Atlantic Coastal Plain amber is similar chemically to siegburgite, which had been related to Liquid-ambar by isolation of cinnamic acid and styrene. Siegburgite occurs in Eocene–

Miocene Rhineland brown coals; Teichmüller (1958) considered Liquid-ambar to be a prominent component in the Myrica-Cyrilla marshes and Sequoia woods that formed these coals (Appendix 4).

More than 200 angiosperms have been recovered from clay in central New Jersey late Cretaceous deposits, which have also yielded a rich

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blage of insects preserved in amber. Flowers in the clay have been charcoali-fied, that is, it is assumed that the intense heat of forest fires hardened cell walls, making them impermeable to water. Furthermore, the flowers re-mained three-dimensional because they were charcoalified before sediments covered them. Although these flowers are not directly related to the amber, an interesting discovery was made in a fossil flower, Paleoclusia, a close relative of Clusia (Clusiaceae) that occurred in the clay (Figure 4-8). Using scanning electron microscopy, Crepet and Nixon (1998) found secretory cavities or canals in the receptacle, sepals, and petals in these 90-million-year-old flow-ers that are similar to those of modern Clusiaceae (Curtis and Lflow-ersten 1990).

Clusia is dioecious, bearing male and female flowers on separate plants. In some present-day Clusia species, the female flowers have modified anthers that open up and release resin rather than pollen (Plate 30). Stingless meli-ponid bees collect pollen from male flowers and resin for nest construction from female flowers (Chapter 5). Although no amber was actually found, the Paleoclusia female flowers have resin-like material stretched across the open-ings in modified anthers. Thus Paleoclusia is a rare example, suggesting the presence of floral resin relatively early in angiosperm evolution. The

occur-Figure 4-8. Paleoclusia, Cretaceous relative of Clusia, which produces resin in female flowers from modified anthers, ×68. Note the petal, subtend-ing a fascicle of long and short modi-fied stamen filaments and the five-lobed stigma. Strands, thought to be resin, fill staminodial anthers in other specimens. Courtesy of W. L. Crepet.

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rence of meliponid stingless bees in amber in nearby Cretaceous deposits, similar to those from Dominican amber (Figure 4-9), suggests the possibility of a relationship between these bees and clusiaceous flowers, mediated by resin, spanning many millions of years (Crepet 1999).

The Cretaceous and Tertiary Dipterocarp Amber Enigma

Considering the copious production of resin by genera in the tropical family Dipterocarpaceae (Chapters 7 and 8), one might expect coal deposits with massive amounts of dipterocarp amber. Large accumulations of amber in late Cretaceous coal deposits (e.g., Blind Canyon coals in the Blackhawk forma-tion on the Wasatch Plateau, Utah) have been shown by Py-GC-MS to be composed of sesqui- and triterpenoids very similar to those of dipterocarp resins (Meuzelaar et al. 1991). These deposits have as much as 15% resin content, which is very high compared to most coal beds. An associated pollen flora dominated by Sequoia (Parker 1976) was deposited in a swamp with a warm temperate to subtropical climate. There is no dipterocarp pollen to support the chemistry, but Sequoia neither produces large amounts of resin nor has a chemistry similar to that of the amber. Py-GC-MS shows that amber

Figure 4-9. Stingless bee (Proplebeia) in Dominican amber. Scale, 1 mm. This meliponid bee is relatively common in Hymenaea amber; it becomes entangled when attempting to collect resin balls for nest construction (Chapter 5). It also belongs to the same group of bees that probably collected resin from Paleoclusia. From Carmago et al. (2000) with permission of the American Museum of Natural History.

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in late Cretaceous beds from the Hanna Basin, Wyoming, also has chemical characteristics of Dipterocarpaceae (Grimaldi et al. 2001). The trees pro-ducing this amber are thought to have grown under environmental condi-tions different from most of the Hanna Basin amber, which may be of taxo-diaceous origin. Additionally, IR and Py-GC-MS of some amber from the mid-Eocene Claiborne lignites in central Arkansas show that its chemistry is similar to that of resin from Shorea (Dipterocarpaceae). Again, dipterocarps were not represented among the primarily pinaceous pollen in these Arkansas lignites (Saunders et al. 1974).

Dipterocarp pollen is not present in the pollen that occurs in the Blind Canyon coals and Claiborne lignites, but that does not indicate that diptero-carps were not present. In fact, the combination of pollen walls that do not preserve well and a lack of distinctive sculpturing limits the value of carp pollen as fossils (Muller 1970). Moreover, the percentage of diptero-carp pollen is always low, even in present-day forests on peat completely dominated by Shorea (Ashton 1982).

Another hurdle for a dipterocarp source for these North American ambers is that the family today occurs predominantly in Southeast Asia, with the sub-family Dipterocarpoideae, in which resin producers occur, restricted there (Chapter 2). There is evidence, however, that the subfamily was not restricted to Southeast Asia in the past. Winged fruits in late Cretaceous sediments on the eastern coast of the United States and the early Cretaceous in England have been compared to the extinct dipterocarp Woburnia. Wolfe (1972) reported leaf impressions of Parashorea and Anisoptera from North Ameri-can Eocene subtropical forests, genera that today grow in Asian peat swamp forests. In fact, Wolfe (1975) asserted that much of the present Southeast Asian flora can be thought of as a relict of an early Tertiary boreotropical flora that has been largely but not completely eliminated from the New World.

The discovery that the polycadinene polymer structure (Figure 4-1, and Appendix 3, Class II), thought to be distinctive for Dipterocarpaceae, also occurs in resin canals of fruits of an extinct member of the Mastixiaceae (included in Cornaceae) from Germany and England (van Aarssen et al. 1994) initially seemed to provide a tantalizing alternative source for the North Amer-ican amber. Mastixiaceous plants occurred in the boreotropical flora through-out the northern hemisphere and apparently were common in mid-Tertiary European coal deposits (Jung et al. 1971). Like the resin-producing

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carpaceae, however, resin-producing species of Mastixia today are restricted to Indomalesia, and neither mastixiaceous macro- nor microfossils are asso-ciated with the extensive North American amber deposits. Furthermore, since mastixiaceous amber is associated with fruits, it is not known if the plants produced the large amounts of resin that dipterocarps do today. Thus mastixi-aceous resin clouds the issue and is not more convincing than that from Dip-terocarpaceae as the source of North American amber deposits. Similarity in the polymer chemistry of the ambers suggests the possibility of convergent evolution of this polymer, as Dipterocarpaceae belong to the eurosids II where-as mwhere-astixiaceous plants belong to euwhere-asterids I (Appendix 2, Cornaceae).

In sum, the botanical source of the amber from the North American Blind Canyon, Hanna Basin, and Claiborne deposits remains something of an enigma. From the available evidence, replete with caveats, these ambers appear to have been derived from a dipterocarp source rather than any other.

Certainly, they are derived from an angiosperm rather than any known coni-fer such as Pinus or Sequoia, as suggested by pollen found in the deposits with the amber.

In contrast to the mystery regarding the amber from the three North American localities, some Miocene coal deposits in Southeast Asia have large quantities of resin chemically similar to that of present-day dipterocarps.

Lacking the biogeographic problems presented by the North American deposits, it is reasonable to assume that these deposits are from dipterocarps even though there is no supporting botanical evidence. Various dipterocarps produce enormous amounts of resin, which could easily accumulate in appro-priate deposition sites (Chapter 8). In fact, the largest piece of amber in the world, which is similar to dense coal impregnated with resin, comes from Sarawak, Malaysia (Figure 4-10).

The copiously resin-producing genus Shorea (Plate 44 and Figure 9-1) has been suggested as the source of Miocene amber from two localities on Sumatra (Appendix 4) based on the similarity of IR of Shorea resin and the amber at one site (Langenheim and Beck 1965) and ¹³C NMR of the amber, indicating characteristic sesqui- and triterpenoids, at the other (Brackman et al. 1984). Using several chemical components to characterize a taxon is more chemosystematic than depending on a single polymer, which may, of course, be important in the fossilization process. Brackman and coworkers investi-gated this fossil resin because it occurs in sufficiently large amounts, in finely

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dispersed veins and lenses, that they thought it could be produced in bulk quantity as a sideline of coal processing (Chapter 8). As in coal deposits, fos-sil resin of probable dipterocarp source, as evidenced by the sesqui- and triter-penes, is a common constituent of Southeast Asian petroleum source rocks (S.

Stout 1995). For example, Miocene amber from the Mahakam Delta, Indone-sia, is related to Miocene petroleum there and at numerous other Southeast Asian sites (Chapter 9). Although geochemists have begun to recognize the contribution that plant resin can make to crude oil, this role for resin is most prominent in the apparently dipterocarp accumulations in Southeast Asian Tertiary basins.

Figure 4-10. Excavation of the largest single piece of amber in the world, in Sarawak, Malaysia.

Mid-Miocene in age, it is assumed to be derived from a member of the Dipterocarpaceae. At more than 68 kg, the piece had to be sawed into several sections to transport it to the Museum für Naturkunde, Stuttgart, where it is on display. From Grimaldi (1996) with permission of Harry N. Abrams, Inc.

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Mid-Tertiary Leguminous Amber

Ambers from several mid-Tertiary deposits in the New World, and from the Eocene and Pleistocene in Africa, have been either demonstrated or suggested as having a tropical leguminous source (Figure 4-7 and Appendix 4). Concom-itant chemical and botanical evidence from Mexican and Dominican amber as well as the depositional conditions of the Mexican amber definitely sup-port Hymenaea as the source tree, unlike the puzzlement over the sources of Baltic amber and some ambers of possibly dipterocarp origin. First, there is compelling chemical evidence from IR (Langenheim and Beck 1965, Langen-heim 1969), CP/MAS ¹³C NMR (Cunningham et al. 1983; Lambert et al.

1985, 1989), and Py-GC-MS (K. Anderson et al. 1992) that Hymenaea is the source of amber in large deposits in Mexico and the Dominican Republic.

Shedrinsky et al. (1989) and K. Anderson (1995), using Py-GC and Py-GC-MS, further showed that the polymers in the Mexican and Dominican ambers are essentially indistinguishable, both consisting of labdatriene skeletons with an enantiomeric configuration (Appendix 3, Class Ic). Inclusions of plant parts in these ambers also support Hymenaea as the source, but possibly dif-ferent species in the two areas.

Major portions of Dominican amber were once thought to come from deposits of different ages: Eocene, Oligocene, Miocene, and Pleistocene (re-viewed by Grimaldi 1995). Although ambers of varying age may exist (Dilcher et al. 1992), Iturralde-Vinent and MacPhee (1996) clearly demon-strated that the primary deposits were formed in a single sedimentary basin during the latter part of the early Miocene to mid-Miocene (15–20 Ma).

Deposition apparently occurred near the shore, probably in coastal lagoons adjacent to low, densely forested hills (Figure 4-3) with little evidence of any extensive redeposition. Thus, according to biostratigraphic data based on invertebrate and vertebrate fossils, the major deposition of Dominican amber occurred over a 5-million-year period. This short interval, in contrast to the 20 million years hypothesized for the redeposited Baltic deposits, provides a temporal benchmark that can be used to calibrate rates of molecular evolu-tion in amber-embedded taxa.

All floral parts of Hymenaea are included in Dominican amber, with petals characteristic of the African H. verrucosa type particularly abundant in the major deposits (Plate 26; Hueber and Langenheim 1986). Poinar (1991) described this Dominican amber species, with its African affinities, as H.

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tera. If some of the smaller deposits of Dominican amber in the western part of the country are Pleistocene in age, they were not produced by H. protera but could have been produced by H. torrei. That species has characteristics of both H. verrucosa and H. courbaril but occurs only in Cuba at present (Lee and Langenheim 1975). If the amber is even younger than Pleistocene, the resin was probably produced by H. courbaril, the only Hymenaea species that occurs in the Dominican Republic today. However, Hymenaea floral parts are not known from Pleistocene or younger deposits; hence, the rela-tionships of the Hymenaea species producing that amber are unknown.

Although the only large deposits of amber in the Greater Antilles occur in northern areas of the Dominican Republic, there are trace and small amounts from the early to mid-Miocene in Haiti and northeastern Puerto Rico (Itur-ralde-Vinent and Hartstein 1998). It is assumed that these ambers are also probably derived from Hymenaea.

Amber from Chiapas, the southernmost state of Mexico, has been dated as late Oligocene to early Miocene based on foraminiferal zonal sequences (Frost and Langenheim 1974) and radiometry (Berggen and van Couvering 1974). The age range, 22.5–26 Ma, indicates 3–4 million years between the beginning and end of the amber accumulation period. That interval is some-what smaller than that of the Dominican amber, with Mexican deposition starting and ending earlier. The first reported plant remains of Hymenaea were parts of the calyx and numerous stamens, the latter possibly resulting from bat pollination, which occurs in the taxonomic section to which H.

courbaril belongs (Lee and Langenheim 1975). Stamens could easily have become trapped in the sticky resin exuding from the tree, as abundant sta-mens fall to the ground under trees following a night of pollination by bats.

Leaflets and sepals of Hymenaea in the Mexican amber share characteristics with H. courbaril, now widespread, and H. intermedia, which presently is very restricted in Amazonia (Langenheim 1966).

Floral parts in both Dominican and Mexican amber, suggesting relation-ship to extant species in the primitive section Trachylobium of Hymenaea, bring up the difficult question of whether Hymenaea originated in Africa or the Americas. An understanding of such phytogeographic history depends on an adequate fossil record and some knowledge of the phylogeny of the taxa involved as well as geologic history (particularly plate tectonics) to assess the relative roles of oceanic dispersal and vicariance. Africa and South

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ica were joined until the early Cretaceous but were split by the mid-Creta-ceous. Unfortunately, the first unequivocal evidence for caesalpinioid legumes is from the non-resin-producing genus Crudia in Paleocene sediments from Nigeria. Because of the high diversity of genera as well as the high percentage of genera endemic to Africa, legume systematists (e.g., Brenan 1966, Raven and Polhill 1981, Herendeen and Dilcher 1992) have assumed that Africa was the primary site for radiation and evolution of tropical legumes, with a secondary center in Amazonia.

Although Hymenaea today is restricted to the eastern coast of Africa and adjacent islands, Langenheim and Lee (1974) assumed it once occurred in moist forests across central Africa but was restricted by a progressively drying climate and major uplifts in central Africa during the Tertiary and Pleistocene.

This view is supported by the distributions of a close relative, Guibourtia, as well as Tessmannia and Oxystigma, which occur in the same East African coastal belt as Hymenaea but are also abundant in West Africa with other re-lated resin-producing caesalpinioids (Chapter 2). Furthermore, there is possi-ble Eocene evidence of Guibourtia in Tanzania (Herendeen and Jacobs 2001).

Langenheim and Lee (1974) suggested that an ancestor of Hymenaea ver-rucosa was dispersed to eastern Brazil via favorable ocean currents following the separation of the African and American continents. During the early Ter-tiary, the continents were still relatively close and rain forests were widely distributed on both, increasing the chance of successful dispersal. Hymenaea pods are known to float great distances in the ocean (Gunn and Dennis 1976), and seeds can germinate rapidly following transport. Herendeen and Dilcher (1992) stressed the importance of oceanic dispersal in interpreting the distri-bution of fossil legumes in general.

Poinar (1991), on the other hand, proposed vicariance (plants carried to present positions on a landmass) to explain the geographic origin of the genus. Poinar thought that Hymenaea originated in the Americas during the Cretaceous when South America and Africa formed a common landmass.

Poinar (1992a) further elaborated the view that “Hymenaea expanded and speciated during the late Mesozoic from a distributional center located along the equator. Diversification resulted in the genus extending from Mexico across South America and into the adjoining African continent.” This alter-native view is not supported by either the fossil record or relationship to other resin-producing legumes. Although evidence from petals places both the

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Dominican and Mexican Hymenaea amber species in the primitive section Trachylobium, understanding the evolution of the Mexican species appears to be more complex than that of the Dominican one. All specimens of the

Dominican and Mexican Hymenaea amber species in the primitive section Trachylobium, understanding the evolution of the Mexican species appears to be more complex than that of the Dominican one. All specimens of the