Universidad De Los Andes Facultad De Ciencias
UNIVERSIDAD DE LOS ANDES FACULTAD DE CIENCIAS,
DEPARTAMENTO DE CIENCIAS BIOLÓGICAS
TRACING BACK THE POLLEN FOSSIL RECORD OF HEDYOSMUM
(C HLORANTHACEAE ), A BASAL ANGIOSPERM
CAMILA MARTÍNEZ AGUILLÓN
Trabajo de grado para optar al título de Biólogo
Director: Carlos Jaramillo, STRI
Co-director: Santiago Madriñán, Universidad de los Andes
Bogotá D.C., 2009 TRACING BACK THE POLLEN FOSSIL RECORD OF HEDYOSMUM
(C HLORANTHACEAE ), A BASAL ANGIOSPERM
ABSTRACT
The Chloranthaceae family has been described as one of the oldest lineages within the angiosperms. Hedyosmum is the only Neotropical genus and is composed of approximately 45 species. The fossil record shows that the first apparition of the genus was in the Early Cretaceous (~120 Ma), followed by a time gap of 70 Ma during the
Paleogene, where no fossil records of Hedyosmum were found. It is only until the Early
Miocene when a new record associated with Hedyosmum: Clavainaperturites microclavatus is found. The association was established using only transmitted light microscopy. The aim of this study was to determinate the relationship between the fossil
Clavainaperturites microclavatus from the Miocene and the extant genus Hedyosmum using transmitted light microscopy, scanning and transmitted electron microscopy. The quantitative characters were evaluated with a non-metric multidimensional scaling. Given the high morphological affinity of the pollen fossil with the extant Hedyosmum it is concluded that C. microclavatus belongs to Hedyosmum, and that the radiation of the genus could have occurred in Central and South America in the Early Miocene, before to the emergence of the Panamanian Isthmus and the rising of the Andean cordillera. Two hypotheses were suggested to explain the gap in the fossil record of the genus, a change in the pollination syndrome or/and a dramatic population decrease after the K/T event.
Finally as a consequence of the last hypothesis, the divergence point of the Hedyosmum could have occurred ~ 30 Ma, instead of ~ 45 Ma as was suggested before. INTRODUCTION
Evidence from the fossil record indicates that angiosperms could have appeared first approximately 140 Ma in the Early Cretaceous (Brenner 1996, Wills & MacElwain 2002,
Zavada 2007). Some of the earliest known fossil flowers were found approximately 127–
120 Ma in deposits from Portugal, Australia and China (Taylor & Hickey 1990, Friis et al. 1999, Sun et al. 2002). Chloranthaceae has been suggested as one of the extant families that can be related to these flowers (Walker & Walker 1984, Crane et al. 1989,
Herendeen et al. 1993, Eklund et al. 1997, 2004, Friis et al. 1999:). Additionally,
Clavatipollenites , a fossil-pollen genus from the early Cretaceous has close similarity to pollen of Chloranthaceae, specifically the genus Ascarina (Doyle 1969, Chapman 1987,
Pedersen et al. 1991, Traverse 2007).
Chloranthaceae has 75 extant species with a disjunct Old and New World tropical distribution. It is composed by four genera: Sarcandra, Chloranthus, Ascarina and
Hedyosmum (Todzia 1988). The phylogenetic position of Chloranthaceae within the angiosperms is uncertain. Early studies related the family to Piperales and Laurales
(Takhtajan 1980), and then separated it on a monotypic order called Chloranthales
(Dahlgren, 1983). Recent analyses based on morphological homologies and molecular data, suggest that Chloranthaceae could have diverged in different positions together with
Ceratophyllum, the eudicots and monocots, above the ANITA grade, and below the eumagnoliids (Graham & Olmstead 2000, Mathews & Donoghue 2000, Soltis et al. 2000,
Zanis et al. 2002, Eklund et al. 2004, Qiu et al. 2006). Chloranthaceae is a monophyletic family. Molecular and morphological studies show the genus Hedyosmum to be monophyletic and sister to other Chloranthaceae, and Ascarina to be sister to Sarcandra and Chloranthus (Ekund et al. 2004) . Todzia (1988) divided the genus Hedyosmum in two subgenera: Hedyosmum Solms–Laubach and Tafalla (Ruiz &
Pavón) Solms–Laubach, and five sections. New molecular studies indicate that the infrageneric classification must be recircumscribed in order to become monophyletic
(Antonelli 2008).
Hedyosmum comprises 44 species of shrubs and small trees (Todzia 1993) . Most species have a fairly broad distribution range, only 14 species are local endemics, and more than
50% of the species occur in the northern Andes (Todzia 1988). Hedyosmum ranges from central Mexico to central Bolivia, east to Guyana, the West Indies and has a single species in Southeast Asia ( H. orientale ). The genus is mainly located in wet habitats of cool montane cloud forest between 600 and 3,000 m (Todzia 1988).
The vast majority of pollen grains of Chloranthaceae have a relatively constant reticulate sculpture, whereas, the aperture configuration displays considerable variation (Eklund et al. 2004). Hedyosmum has a star-shaped monosulcate aperture with four to six branches
(Eklund et al. 2004). Ascarina has a monosulcate aperture with trichotomosulcate variants (Eklund et al. 2004). Chloranthus has polycolpate and polyforate pollen grains, and Sarcandra has polyforate pollen with scattered pores (Eklund et al. 2004).
Asteropollis, a pollen genus from the Early Cretaceous was related to Hedyosmum because it was found attached to Hedyosmum -like flower from the Portuguese Flora (Friis
1994, 1999). Asteropollis is a reticulate pollen grain, with a star-shaped aperture, that was originally described in Oklahoma and was later found in central Atlantic North America,
Portugal and Australia (Walker & Walker 1984, Friis 1999). After the Campanian (~73
Ma), Asteropollis has not been reported. It is only until the Miocene, Pliocene and
Quaternary that fossil pollen grains referred to Clavainaperturites microclavatus are assumed to be related to extant Hedyosmum, they are reported in South America and
Panama (e.g. Hoorn 1994, van der Hammen & Hooghimiestra 2000). Clavainaperturites microclavatus was described by Hoorn (1994) as a pollen grain with polar, asymmetric, inaperturate, microclavate, with medium size and subespheroidal shape. Nevertheless, the description was done using only transmitted light microscopy. A deeper evaluation of pollen characters requires scanning electron and transmitted electron microscopes to reveal minute characters and firmly establish affinities with extant species (Ferguson et al. 2007).
Li-Bing Zhang and Susanne Renner (2003) used the extensive fossil record of
Chloranthaceae to determinate multiple calibration points of genetic distances for the tree of the family. Hedyosmum -like flower bearing Asteropollis pollen from the Barremian-
Aptian (120 Ma) and Chloranthus -like androecia from the Turonian (90 Ma) were the fossils used as alternative calibration points to reconstruct the phylogeny. The ages of the topology of the tree vary greatly depending on the calibration point used. The midpoint of these ages indicates that the initial divergence among species in the crown group of
Hedyosmum started 45 Ma, whereas the crown group of Chloranthus diverged 17 Ma, and the crown group of Ascarina had its initial divergence 14 Ma.
The pollination syndrome of Hedyosmum has been another matter of discussion. Given the stamen and floral morphology of the Hedyosmum -like flower from the Barremian– Aptian, wind pollination has been suggested (Friis et al. 2000). Furthermore, Asteropollis pollen occurs abundantly in dispersed palynofloras suggesting high pollen production and high dispersal pollen potential of the plant, this is often seen in wind-pollinated taxa.
Nectaries or specialized food bodies were not observed in this flower (Friis et al. 2000).
Extant Hedyosmum also has been described as wind pollinated (Todzia 1988) although fieldwork to test this hypothesis has not been carried out.
The temporal and spatial pattern of distribution of Hedyosmum suggest that the genus could have a Laurasian and Gondwanan origin in the Early Cretaceous (Eklund et al.
2004), then spreading to North America and finally to South America in the Late
Neogene (Raven & Axelrod 1974, Todzia 1988). Its radiation in South America could have been a consequence of Panama’s land bridge and/or the uplift of the Andes cordillera (Todzia 1988). The Chloranthaceae fossil record indicates that today’s genera represent the survivors of a group that during the Early Cretaceous was much more widespread and diverse in both Laurasia and Gondwana (Todzia 1988, Doyle & Donogue
1993, Kong et al. 2002, Zhang & Renner 2003, Eklund et al. 2004). This may have contributed to the incredible morphological diversity among the four extant genera, and may have made difficult to resolve the phylogenetic placement of Chloranthaceae (Zhang
& Renner 200, Hansen et al. 2007).
The main goal in this study is to test if Clavainaperturites microclavatus is related to extant Hedyosmum. Six extant species of Hedyosmum will be compared to the grains from Clavainaperturites microclavatus using SEM, TEM and light microscopy. Another
22 species of Hedyosmum will be analyzed using only light microscopy. The morphological variation within and among species will be quantified to assess the degree of similarity of the fossil taxa versus the putative living relatives.
METHODS
Pollen of 47 individuals of 28 extant species of Hedyosmum was examined for the study.
A pollen-fossil, Clavainaperturites microclavatus, from three different locations was analyzed (Table 1). All the specimens were observed first using transmitted light microscopy (LM); around 30 pollen grains were measured depending on the availability of each species. Then, six species of Hedyosmum, H. bonplandianum, H. racemosum, H. goudotianum, H. costaricense, H. correanum and H. scaberrimum, and the fossil C. microclavatus from Panama, were examined using scanning electron microscopy (SEM) and transmitted electron microscopy (TEM).
Samples were collected from different herbaria following the Jarzen & Jarzen procedure
(2006). Light microscopic slides were prepared using the acetolysis procedure. For this, all pollen samples were treated first with acetic acid and then macerated to extract the pollen grains. The acetolytic solution (9:1) was added for five minutes under low heat.
The samples were washed with three series of alcohol and mounted in a glycerin–gelatin medium to be observed and photographed in a Nikon DXM1200F light microscope.
Preparation of both, recent and fossil samples for SEM and TEM was performed following the Zavada’s (2007) procedure at the East Tennessee State University. The scanning electron microscopes used were a ZEISS DSM940 and a ZEISS EVO40. The transmission electron microscope used was a LIBRA® 120 Plus. The stubs required for
SEM were prepared melting a thin coat of the high vacuum wax Apiezon W-100. The SEM process for recent and fossil pollen started with the dehydration of the samples with alcohol series of 30, 50, 75, 95 and 100%, followed by acetone and finalizing with xylene. The samples of extant species were submerged in alcohol, while the fossils were kept in xylene. To finish, few drops of the pollen solution were added on the prepared stubs and then dried for one day. Then, the residues of the fossil samples were mixed with the polystyrene-based mounting medium (DePex), smeared on a microscope slide, and allowed to polymerize without a cover slip. Subsequently, the slides were scanned; individual pollen grains were cut out on the hardened polystyrene and placed on a
0.05 !m pore Millipore filter on several sheets of filter paper. After that, xylene was dripped over the polystyrene square containing the pollen grain until all the polystyrene was removed. On the prepared stubs, a grid was inscribed and numbered. The pollen grain that was already isolated was transported carefully to the grid using a hair attached to a stick. For TEM, additionally, the pollen grains were transferred to a small agar block, dehydrated in alcohol series, embedded in Spurr’s Low Viscosity Resin, sectioned on a
MT-2b ultramicrotome, visualized and photographed. Finally the SEM and TEM stubs were both coated with a gold-palladium mix during 3 minutes approximately.
The characters evaluation was done first, making two categories: variable and not variable characters. The not variable characters were: pollinic unit, polar shape, polar symmetry, exine layers, exine variations, sculpture type, aperture shape, sculpture type, supratectal sculpturing, infrastructural layer, endexine presence, endexine variations, and endexine/footlayer proportion. The variable characters were compiled (Table 2) and analyzed with a Non-metric Multidimensional Scaling (NMS), to determinate if the pollen fossil C. microclavatus was falling within the set of data of the extant Hedyosmum. All statistical analyses were done using R 2.8.0 GUI 1.26 (R-development- Core Team,
2007). The NMS was performed using the Community Ecology Package, Vegan version
1.15-0. The data for the NMS was coded and organized in a matrix. To find two dimensions and use metric scaling, the analysis needs dissimilarities as input. A dissimilarity matrix was constructed for all the species and characters that presented some variability. The coding of the data was done, based on Zavada’s method (2007) for discrete data. For continuous light microscopy measurements, the coding was done with the cladistic treatment for morphometric data of Thiele (1993).
The variable characters were coded as is listed bellow: pollen diameter: (0) < 22 !m; (1)
22.1-29 !m; (2) 29.1-31 !m; (3) 31.1-37 !m; (4) < 37.1 !m. Nexine: (0) < 1 !m; (1) 1.1-
1.50 !m; (2) 1.51-1.99 !m; (3) > 2 !m. Sexine: (0) < 1 !m; (1) 1.10-1.40 !m; (2) 1.41-
1.90 !m; (3) > 1.91 !m. Exine: (0) < 0.50 !m; (1) 0.51-2.0 !m; (2) 2.10-3.0 !m; (3) >3.1.
Aperture: (0) Inaperturate; (1) 4(–6)7-chotomosulcate; (2) Inaperturate and 4(–6)7- chotomosulcate. Supratectal ornamentations: (0) microspinules, mid frequency, small size; (1) microspinules, high frequency, small size. Tectum: (1) rugulate-lumina smaller than the width of the muri; (2) reticulate-lumina larger than width of the muri.
Finally, from the variable characters, an analysis was done to compare the interspecific and intraspecific variability, performing a Wilcoxon Rank Sum Test for non-parametric data. This, in order to choose which of the variable characters could be useful taxonomically to define species. RESULTS
Low levels of differentiation within species were found using SEM and TEM. All the
TEM pictures for the six Hedyosmum species analyzed, revealed the same pattern of layer stratification. There was no presence of intine because the acetolysis process degrades the layer. The endexine/footlayer proportion remained very constant at the nonapertural region in all the species. The apertural region, on the contrary, showed a gradual slimming of the endexine until rupture. The footlayer remained continuous and the columellas started becoming smaller until they disappeared (Fig. 3I). The TEM revealed that although the aperture is not totally open there is differentiation of the layer in the region. Is possible that this will be the reason why the aperture is so variable.
The Wilcoxon Rank Sum Test was performed for the variable characters measured with light microscopy. The test demonstrated that the pollen diameter was the only character that could be useful taxonomically at species level because its variation was higher interspecifically than intraspecifically (Table 2). The remaining characters present high intraspecific variation; therefore its utility is limited at an intrageneric level, but stills being useful for the description of Hedyosmum pollen .
Hedyosmum species present a constant morphology along most of the taxonomic characters evaluated in this study. The pollinic unit of Hedyosmum is a monad, with a spherical-globose shape, radial symmetry and circular polarity. The pollen grains could be inaperturated or aperturated with a (4 –) 5–7 branch armed or chotomosulcate aperture. The aperture length and width, is widely variable even within a single individual, around 10 to 15 !m of difference. The exine is mostly semitectate, irregularly reticulate to rugulate and formed by columellas. The thickness of the exine and its layers is variable and does not present a specific pattern by species. The supratectal ornamentation is scabrate and composed by microspinules on the muri of the reticulum regularly distributed. The lumen area is very variable and its shape is irregular. The footlayer is regularly constant at the apertural region of the pollen grain, while the endexine is not. The columellas are not frequent in that region. The diameter that was always measured from a polar view at the equatorial axe (Chapman 1987) is widely variable (20 –) 36–48 !m. Below, there are morphological descriptions for six species of
Hedyosmum and for the fossil Clavainaperturites microclavatus .
Hedyosmum bonplandianum (Fig. 4). Measurements were done from five individuals of different locations. The pollen grains are inaperturated or aperturate, when the aperture is present is 4–7 chotomosulcate, although a few are sometimes inaperturated. The mean thickness of the exine, the sexine and the nexine, is 2.76, 1.42 and 1.33 !m respectively.
The supratectal microspinules on the muri of the reticulum are regularly distributed and moderately frequent. The lumen is smaller than the width of the muri. The average pollen diameter is 36.2 ± 2.25 !m, with a standard deviation of 6.9 !m.
Hedyosmum correanum (Fig. 5). Measurements were done from one individual. The pollen grains are mostly 5,6 chotomosulcate, although few of them are inaperturated. The mean thickness of the exine, the sexine and the nexine, is 2.81, 1.28 and 1.53 !m respectively. The supratectal microspinules of the muri are regularly distributed and highly frequent. The lumen area is smaller than the width of the muri. The average of the diameter is 33.7 ± 0.65 !m, with a standard deviation of 1.92 !m. Hedyosmum costaricense (Fig. 6). Measurements were done from four individuals of different locations. The pollen grains are inaperturated or aperturate, when the aperture is present is 5–6 chotomosulcate. The mean thickness of the exine, the sexine and the nexine, is 2.88, 1.31 and 1.57 !m respectively. The supratectal microspinules of the muri are regularly distributed and moderately frequent. The lumen area is wider that the width of the muri. The average of the diameter is 29.42 ± 1.14 !m, with a standard deviation of
3.1 !m.
Hedyosmum goudotianum (Fig. 7). The measurements were done from three individuals of different locations. The pollen grains are mostly 4–6 chotomosulcate, although few of them are inaperturated. The mean thickness of the exine, the sexine and the nexine, is
3.571, 1.692 and 1.879 !m respectively. The supratectal microspinules from the muri of the reticulum are regularly distributed and highly frequent. The size of the lumina is smaller than the width of the muri. The average pollen diameter is 34.7 ± 0.74 !m, with a standard deviation of 2.194 !m.
Hedyosmum racemosum (Fig. 8). The measurements were done from three individuals of different locations. The pollen grains are mostly 5–6 chotomosulcate, although few are sometimes inaperturated. The mean thickness of the exine, the sexine and the nexine, is
3.27, 1.69 and 1.57 !m respectively. The supratectal microspinules from the muri of the reticulum are regularly distributed and highly frequent. The size of the lumina is always wider that the width of the muri. The average pollen diameter is 42.7 ± 1.44 !m, with a standard deviation of 4.8 !m. Hedyosmum scaberrimum (Fig. 9). Measurements were done from four individuals of different locations. The pollen grains are 4–7 chotomosulcate, although a few of them are inaperturated. The mean thickness of the exine, the sexine and the nexine, is 2.746, 1.35 and 1.39 !m respectively. The supratectal microspinules of the muri of the reticulum are regularly distributed and moderately frequent. The lumen area is very variable and the shape is irregular, however, the size of the perforation is always wider that the width of the muri. The average of the diameter is 30.88 ± 1.33 !m, with a standard deviation of
3.7 !m.
Clavainaperturites microclavatus (Fig. 10). Measurements were done from three different locations. The pollen grains are inaperturated or aperturate, when the aperture is present is 5–6 chotomosulcate. The mean thickness of the exine, the sexine and the nexine, is 1.62, 0.99 and 0.86 !m respectively. The supratectal microspinules of the muri of the reticulum are regularly distributed and moderately frequent. The lumen area is very variable and the shape is irregular, however, the size of the perforation is always wider that the width of the muri. The average of the diameter is 20.89 ± 1.36 !m, with a standard deviation of 3.1 !m.
On the other hand the Non-metric Multidimensional Scaling (NMS) shows the distribution of the species based on all the variable characters evaluated (Fig. 1). C. microclavatus is nested close with H. tepuiense and H. intermedium . The character that is driving the differences more clearly is the diameter of the pollen, because the species which C. microclavatus is clustered have the smallest diameter, so as C. microclavatus does (Fig. 2). These results suggest that C. microclavatus is related to Hedyosmum . DISCUSSION
The qualitative morphological description made for the genus Hedyosmum , enclosed the character description obtained for Clavainaperturites microclavatus, which is an indication of the close relationship between these taxa. While comparing and studying the range of morphological variation quantitatively in the pollen of the living Hedyosmum and the fossil Clavainaperturites microclavatus, all pollen grains where treaded in the same category. The NMS showed that all the species, except for Hedyosmum intermedium, H. tepuiense, and C. microclavatus were grouped together . Differences between dimension 1, could be explained because of the differences of the pollen diameter, which was the only character obtained as possibly useful taxonomically.
Nevertheless, it by itself was not enough to recognize species, because it was frequently overlapped with different species (Fig. 2). In the other hand the differences within the dimension 2, cannot be differentiated from stochastic factors due to all the remaining characters presented high intraspecific variation. In conclusion, both qualitative and quantitative morphological descriptions, demonstrates that the fossil C. microclavatus belongs to the extant genus Hedyomum.
Based on the morphological data available for the taxa studied, C. microclavatus was included within the molecular phylogeny (Antonelli, 2008) as part of the crown group, specifically as nested with the Central and South American clade (Fig.3). Asteropollis was included as the sister group of the entire genus Hedyosmum (Fig.3) . Whether or not
Asteropollis corresponds to a stem group or a divergent taxon that became extinct, its morphology shows that no pollen modifications have occurred through time. Close resemblance between the pollen-record from the Early Cretaceous, the Neogene and recent taxa was clearly observed. Moreover, despite the wide altitudinal and habitat range, and the long history of the genus, is possible that none strong selective forces were driving changes in pollen characters. Including the fossil C. microclavatus within the crown group of the genus was easier to explain the high morphological similarity found between them in most of the characters studied.
The pollen size, together with other morphological characteristics, has often been related to the pollination syndrome of a plant. Since it would be truth, certain changes in pollination could have occurred. The long gap in Hedyosmum fossil record (~ 68 Ma) might be a response of a change in the pollination syndrome. Given that the pollen-fossil record is very susceptible to amounts of pollen produced by a plant, is possible that a shift from anemophily to entomophily has occurred during this gap, bringing as a consequence less production of pollen grains and changes in size. Furthermore, is also known, that the probability to find a pollen-fossil record is subjected to the amount of pollen deposited, is possible that while Hedyosmum was absent in the pollen record, animals were pollinating its flowers. Subsequently, after the initial divergence in the
Early Miocene, another change in pollination syndrome could have occurred and the plants became anemophilous just as its early ancestors. Nevertheless, some species that live in very humid and dense understory, included C. microclavatus could have remained being pollinated by animals, this would also explain the wide range of pollen size that the genus has. Although this is not the more parsimonious explanation there is not strict evidence suggesting the actual pollination syndrome of the genus, thus this hypothesis cannot be discarded. In the same way as the magnoliid clade was probably affected by the mass extinction event at the Cretaceous/Tertiary boundary (K/T event) 65.5 Ma, Hedyosmum could have also been influenced. Tracing back the pollen fossil record of Hedyosmum is observed that the last pollen-record of Asteropollis was found until the end of the Campanian in the
Late Cretaceous, close to the K/T event. Then during the Palaeogene no reports of the genus have been found, and it is only until the Early to Mid-Miocene when pollen grains related to Hedyosmum are found again. In this way it is possible that just a few taxa had survived to this event and for a long time the genus became rare or scarce, until a new radiation happened and gave origin to the species that we found today. Evidence from the fossil record in general indicates a major disruption of some ecosystems, usually involving changes in floral composition. Around one third of all angiosperm taxa represented by fossil pollen failed to survive the K/T event in North America (Nichols
2007, Nichols & Johnson 2008). Is also possible that the ancestor of the crown of
Hedyosmum had survived to the K/T event in biological refugees, far away from the site of the impact in Yucatán. Antonelli (2008) suggests that the most recent common ancestor of genus Hedyosmum may have been restricted to Australasia.
The molecular clock proposed by Zhang & Renner (2003) for Chloranthaceae shows that the topology could be extremely sensible to the calibration points used in the analysis.
Thus, even when the fossil record is abundant within a family, age estimations for divergence points has wide error margins. The ages proposed for the divergence point of the crown group of Hedyosmum varied from 60 to 29 Ma. If the hypothesis about the K/T event is accepted, is possible to suggest that the fossil record favor one of the younger ages proposed, around 30 Ma, given that during the Paleogene the fossil record is totally absent, therefore, the divergence point could start just at the end of the Oligocene.
On the other hand, the migration of Hedyosmum has also been puzzling. Cretaceous fossil record indicates that the genus was common and widespread in Laurasia. Then, after the absence gap of the Paleogene, the genus was found first in the Neotropics, with C. microclavatus in the Miocene and Pliocene of Panama, Colombia and Brazil. Is possible then to refute partially the hypothesis proposed by Raven and Alxelrod (1974), who hypothesized that the migration of the genus from Central to South America occurred in the Late Neogene with the formation of the Panama isthmus. This new founding gives evidence to show that the migration to South America occurred at or before the Early
Miocene, ~20 Ma before it was considered. Nonetheless, Hedyosmum migration goes so much further, molecular studies (Antonelli 2008) shows that H. orientale, the only species that is located in Asia is derived from the ancestral clade, that is located in the
West Indies (Fig.9). Thus, the most parsimonious explanation to this event is that the migration occurred by long dispersal. Additionally, the pollen-fossil record indicates that the species radiation of the crown group could have occurred before to the emergence of the Panamanian isthmus and the uplift of the Andean cordillera, because derived species like H. correanum, H. bonplandianum, H. goudotianum and H. mexicanum, have the same distribution as C. microclavatus, hence, the species radiation and migration could have taken place before or at the Early Miocene.
An improved understanding of the pollen fossil record of Hedyosmum allowed a better approximation of the long history of the genus. Although much more information is necessary to explain certainly the gap found during the Paleogene, both a change in the pollination syndrome and a partial extinction at the K/T event are equally probable and nonexclusive hypotheses. The relation between C. microclavatus and Hedyosmum would be hard to test without the high level of detail that the SEM and TEM provide.
Hedyosmum, as one of several examples of migration and radiation of species in the
Neotropics, is useful in our attempts to understand evolution and migration processes.
Given that Chloranthaceae is one of the oldest angiosperm families, the understanding of its fossil record could be clue to get an approximation to the origin of the angiosperms.
ACKNOWLEDGMENTS
We are grateful to Michael Zavada and Enrique Moreno for their help in different stages of this study. Carol Kellof (US), Luis Carlos Jimenez (COL) and Mireya Correa (PMA and SCZ) who facilitated access to their respective herbarium collections. Alexandre
Antonelli and Isabel Sanmartin who let us used unpublished data. Silane da Silva who provides us pollen samples from Brazil. The Smithsonian Tropical Research Institute for providing logistic and financial support for this study, through the Short Term Fellowship for Latin-American students.
REFERENCES
Antonelli, A. 2008. Spatiotemporal Evolution of Neotropical Organisms: New Insights into an Old Riddle. Doctoral Thesis. Department of Plant and Environmental Sciences, University of Gothenburg, Göteborg, Sweden.
Brenner, G. 1996. Evidence for the Earliest Stage of Angiosperm Pollen Evolution: A Paleoequatorial Section from Israel. – Flowering Plant Origin, Evolution & Phylogeny, 91-115.
Chapman, J.L. 1987. Comparison of Chloranthaceae pollen with the Cretaceous Clavatipollenites complex taxonomic implications for Palaeopalynology. – Pollen Spores 29: 249–272.
Crane, P. R., Friis, E. M., & Pedersen, K. R. 1989. Reproductive structure and function in Cretaceous Chloranthaceae. Pl. Syst. Evol. 165. Suppl. 3: 211–226.
Doyle, J.A. 1969. Cretaceous angiosperm pollen of the Atlantic Coastal Plain and its evolutionary significance. Harv. Univ. 50: 1–35.
Doyle, J. A. & Donoghue, M. J. 1993. Phylogenies and angiosperm diversification. Paleobiology. 19. Suppl. 2: 141–167.
Eklund, H., Doyle, J. A. & Herendeen, P. S. 2004. Morphological Phylogenetic Analysis of Living and Fossil Chloranthaceae. Int. J. Pl. Sci. 165. Suppl. 1: S107–S151.
Eklund, H., Friis, E. M. & Pedersen, K. R. 1997. Chloranthaceous floral structures from the Late Cretaceous of Sweden. Pl. Syst. Evol. 207. Suppl. 1: 13–42.
Ferguson, D. K., Zetter, R. & Paudayal, K. N. 2007. The need for the SEM in palaeopalynology. Comptes Rendus Palevol. 6. Suppl. 6–7: 423–430.
Friss, E. M., Pedersen, K. R. & Crane, P. R. 1994. Angiosperms floral structures from the Early Cretaceous of Portugal. Pl. Syst. Evol. Suppl. 8: 31–49.
Friis, E. M., Crane, P. R. & Pedersen, K. R. 1986. Floral evidence for Cretaceous chloranthoid angiosperms. Nature. 320. Suppl. 6058: 163-164.
Friis, E. M., Doyle, J. A., Endress, P. K. & Leng, Q. 2003. Archaefructus - angiosperm precursor or specialized early angiosperm?. Trends Plant Sci. 8. Suppl. 8: 369–373.
Friis, E. M., Pedersen, K. R. & Crane, P. R. 1999. Early Angiosperm Diversification: The Diversity of Pollen Associated with Angiosperm Reproductive Structures in Early Cretaceous Floras from Portugal. Ann. Mo. Bot. Gard. 86. Suppl. 2: 259–296.
Friis, Else Marie, Kaj Raunsgaard Pedersen, and Peter R. Crane. 2000. Reproductive Structure and Organization of Basal Angiosperms from the Early Cretaceous (Barremian or Aptian) of Western Portugal. Int. J. Pl. Sci. 161. Suppl. 6: S169-S182.
Graham, Sean W., and Richard G. Olmstead. 2000. Utility of 17 chloroplast genes for inferring the phylogeny of the basal angiosperms. Am. J. Bot. 87. Suppl. 11: 1712– 1730. van der Hammen, T. & Hooghiemstra, H. 2000. Neogene and Quaternary history of vegetation, climate, and plant diversity in Amazonia. Quaternary Sci. Rev. 19. Supl. 8: 725–742.
Hansen, D. R., Dastidar, S. G., Cai, Z., et al. 2007. Phylogenetic and evolutionary implications of complete chloroplast genome sequences of four early-diverging angiosperms: Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae). Mol. Phylogenet. Evol. 45. Suppl. 2: 547–563.
Herendeen, P. S., Crepet, W. L. & Nixon, K. C. 1993. Chloranthus -like Stamens from the Upper Cretaceous of New Jersey. Am. J. Bot. 80. Suppl. 8: 865–871.
Hoorn, M.C. 1994. An environmental reconstruction of the palaeo-Amazon river system (Middle to late Miocene, N.W. Amazonia). Palaeogeogr. Palaeocl. 112:187–238
Jarzen, D. M. & Jarzen, S. A. 2006. Collecting pollen and spore samples from herbaria. Palynology. 30: 111–119.
Kong, H., Chen, Z. & Lu, A. 2002. Phylogeny of Chloranthus (Chloranthaceae) Based on Nuclear Ribosomal ITS and Plastid trnL-F Sequence Data. Am. J. Bot. 89. Suppl. 6: 940–946.
Mathews, S. & Donoghue, M. J. 2000. Basal Angiosperm Phylogeny Inferred from Duplicate Phytochromes A and C. Int. J. Pl. Sci. 161. Suppl. 6: S41–S55.
Nichols, D. J. 2007. Selected plant microfossil records of the terminal Cretaceous event in terrestrial rocks, western North America. Palaeogeogr. Palaeocl. Paleoeco. Suppl. 255: 22–34
Nichols, D. J & Johnson, K. R. 2008. Plants and the K-T boundary. Cambridge University Press.
Pedersen, K.R., Crane P. R., Drinnan A. N., Friis E. M. 1991. Fruits from the mid- Cretaceous of North America with pollen grains of the Clavatipollenites type. Grana . 30. Suppl. 3–4: 577–590. Qiu, Y., Libo. L., Tory, H. A., Ruiqi, L., Taylor, D. W., Issa, M. J., Alexander, R. J., Mona, V. L., Adam, W.M. 2006. Reconstructing the basal angiosperm phylogeny: evaluating information content of mitochondrial genes. Taxon 55: 837–856.
Soltis, D. E., Soltis, P. S., Chase, M. W., et al. 2000. Angiosperm phylogeny inferred from 18S rDNA, vbcL, and atpB sequences. Bot. J. Linn. Soc. 133. Suppl. 4: 381– 461.
Sun, G., Ji, Q., Dilcher, D. L., et al. 2002. Archaefructaceae, a New Basal Angiosperm Family. Science. 296. Suppl. 5569: 899–904.
Takhtajan, A. L. 1980. Outline of the Classification of Flowering Plants (Magnoliophyta). Bot. Rev . 46. Suppl. 3: 225–359.
Taylor, D.W. & Hickey, L.J. 1990. An Aptian plant with attached leaves and flowers: implications for angiosperm origin. Science. 247: 702–704.
Thiele, Kevin. 1993. The holy grail of the perfect character: the cladistic treatment of morphometric data. Cladistics. 9. Suppl. 3: 275–304.
Todzia, C. A. 1988. Chloranthaceae: Hedyosmum . Flora Neotropica Monograph. 48:1– 139.
Todzia, C. A. 1993. New Species of Hedyosmum (Chloranthaceae) from Northern South America. Novon . 3. Suppl. 1: 81–85.
Traverse A. 2007. Paleopalynology. Second Edition. Springer. The Netherlands. van der Hammen, T., & Hooghiemstra, H. 2000. Neogene and Quaternary history of vegetation, climate, and plant diversity in Amazonia. Quaternary Science Reviews. 19. Suppl. 8: 725–742.
Walker J. W., Walker A. G. 1984. Ultrastructure of Lower Cretaceous angiosperm pollen and the origin and early evolution of flowering plants. Ann. Missouri Bot. Gard. 71: 464–521.
Wills, K. J. & MacElwain, J. C. 2002. The evolution of plants. Oxford University Press Inc. New York.
Zanis, M. J., Soltis, D. E., Soltis, P. S., Mathews, S., Donoghue, M. J. 2002. The Root of the Angiosperms Revisited. Proc. Natl. Acad. Sci. 99. Suppl. 10: 6848–6853.
Zavada, M. 2007. The identification of fossil angiosperm pollen and its bearing on the time and place of the origin of angiosperms. Pl. Syst. Evol. 263. Suppl. 1: 117–134.
Zhang, L. & Renner, S. 2003. The Deepest Splits in Chloranthaceae as Resolved by Chloroplast Sequences. Int. J. Pl. Sci. 164. Suppl. 5: S383–S392. TABLES
Table 1. List of specimens analyzed. US: United States National Herbarium, Smithsonian Institution; COL: Herbario Nacional Colombiano; PMA: University of Panama Herbarium; SCZ: Smithsonian Tropical Research Institute’s Herbarium; Graham reference collection.
Extant taxa Diam (!m) Ex (!m) Se (!m) Ne (!m) Co (!m) Collection Distribution H. angustifolium (Ruiz & Pavón) Solms-Laubach 31.60 ± 0.65 2.250 1.633 1.400 1.333 US Ecuador to Bolivia H. anisodorum Todzia 33.79 ± 0.81 2.300 1.250 1.000 0.950 US Ecuador to Bolivia H. arborecens Swartz 43.65 ± 0.89 3.200 1.800 1.450 1.500 Graham reference collection Jamaica, Puerto Rico, Lesser Antilles H. bonplandianum Kunth 38.18 ± 1.31 1.893 1.727 1.947 1.427 US, PMA, COL Nicaragua, Costa Rica, Panama, Colombia H. brasiliense Martius ex Miquel 31.53 ± 0.95 2.500 1.367 1.600 1.067 US Brazil, Paraguay H. brenesii Standley 39.18 ± 0.81 2.900 1.467 1.350 1.167 Graham reference collection Honduras to Panama H. colombianum Cuatrecasas 32.90 ± 3.87 3.250 1.700 0.933 1.400 US, Colombia H. correanum D’Arcy & Liesner 40.35 ± 0.99 1.436 1.320 1.860 1.020 PMA Panama H. costaricense Burger 34.23 ± 0.75 1.879 1.660 1.873 1.360 PMA, SCZ Costa Rica, Panama H. cuatrecazanum Occhioni 30.38 ± 0.78 3.300 1.767 1.900 1.467 US Colombia to Bolivia H. dombeyanum Solms–Laubach 30.33 ± 0.74 3.550 1.833 1.233 1.533 US Peru, Bolivia H. gentryi D’Arcy & Liesner 31.13 ± 0.86 2.250 1.333 1.433 1.033 US Panama, Colombia, Venezuela H. goudotianum Solms–Laubach 42.10 ± 0.74 2.229 1.973 2.293 1.673 US, COL Nicaragua to Colombia, Venezuela, Ecuador, Peru H. intermedium Todzia 20.73 ± 1.10 2.600 1.050 0.950 0.750 US Venezuelan Guayana H. lechleri Solms–Laubach 31.93 ± 0.59 2.950 1.233 1.333 0.933 US Peru H. luteynii Todzia 30.50 ± 0.25 3.000 1.300 1.300 1.000 US Colombia, Ecuador H. maximum (Kuntze) Schumann 33.00 ± 0.52 3.100 2.025 1.600 1.700 US Bolivia, Peru H. mexicanum Cordemoy 48.55 ± 1.06 0.964 1.267 1.667 0.967 Graham reference collection Mexico to Panama H. parvifolium Solms–Laubach 35.67 ± 1.03 4.150 2.167 2.025 1.867 US Colombia, Venezuela H. pungens Todzia 39.70 ± 1.43 3.300 1.100 1.433 0.800 US Colombia H. racemosum (Ruiz & Pavón) G. Don 44.10 ± 1.31 3.857 1.993 2.133 1.693 US, COAH Colombia to Bolivia H. scaberrimum Standley 36.65 ± 1.61 3.364 1.453 1.680 1.153 PMA, COL Nicaragua, Costa Rica, Panama, Colombia, Ecuador H. scabrum (Ruiz & Pavón) Solms–Laubach 36.03 ± 0.81 2.150 1.100 2.225 0.800 US Colombia to Bolivia H. spectabile Todzia 30.00 ± 0.62 3.100 1.100 1.150 0.800 US Ecuador, Peru H. sprucei Solms–Laubach 37.93 ± 0.71 3.200 1.100 1.600 0.800 Silane Ecuador, Peru H. strigosum Todzia 44.72 ± 0.89 5.050 2.167 1.750 1.867 US Colombia, Venezuela H. tepuiense Todzia 19.00 ± 0.45 3.500 1.167 1.233 0.867 US Venezuelan Guayana H. translucidum Cuatrecasas 38.35 ± 0.70 2.250 1.233 1.625 0.933 Silane Venezuela, Colombia, Peru Fossil taxon Diam (!m) Ex (!m) Se (!m) Ne (!m) Co (!m) Epoch Occurrence Clavainaperturites microclavatus Hoorn 20.83 ± 0.89 1.690 0.992 0.866 0.614 Middle Miocene Central Panama Clavainaperturites microclavatus Hoorn 22.91 ± 092 1.730 0.910 0.830 0.820 Late Miocene Late Miocene, Amazon, Brasil Clavainaperturites microclavatus Hoorn 23.10 ± 0.61 1.981 1.002 0.972 0.950 Early Pliocene Early Pliocene, Chocó, Colombia
Table 2. Results for the Wilcoxon Rank Sum Test for non-parametric data performed to variable characters. P-value = 2 Tailed P-value. N.A.= Normal Approximation.
Character P-value N.A. Sexine thickness 0.9295 0.088 Nexine thickness 0.0643 1.850 Columella thickness 0.4118 0.821 Exine thickness 0.1431 1.464 Aperture presence 0.1511 1.435 Aperture arms 0.2395 1.176 Aperture width 0.7216 0.356 Aperture length 0.8235 0.223 Pollen diameter 0.0011 3.273
FIGURES
Figure 1. Non-metric Multidimensional Scaling for C. microclavatus and 28 species of Hedyosmum studied. The dissimilarity matrix was assembled from all variable characters examined.
Figure 2. Boxplot showing the range of the pollen diameter of 28 species studied, including the fossil. H. intermedium, H. tepuiense and C. microclavatus are the smallest species found. There are significant differences between species t = 143.7248; p-value < 2.2e-16; 95% confidence interval = 36.18-37.18 mean of x= 36.
Figure 3. Distribution of extant and fossil Hedyosmum, modified from the most parsimonious tree with the molecular outgroups arrangement from Antonelli (2008). The outgroup is Cer demersum: Ceratophyllum demersum.
Figure 4. SEM, LM, TEM micrographs of H. bonplandianum. A, Mid-size, collapsed pollen grain. B, Supratectal ornamentation: frequent microechins on the muri of the reticulum. C, Crashed pollen grain showing layer structure: semitectate. D-F, Focal-sectioning showing 6-chotomosulcate aperture, reticulate ornamentation. G-H, Non-apertural region; thick footlayer; endexine present in some regions of the wall, diffused and thin.
Figure 5. SEM, LM, TEM micrographs of H. correanum. A-C, Collapsed pollen grain showing variable lumina areas; supratectal ornamentation composed of frequent microechins on the muri. D-E, Focal- sectioning from polar view, showing 6-chotomosulcate aperture. G-H, Non-apertural region of the wall; thick footlayer; small and less frequent columellas; thin endexine. I, Apertual region of the wall; slimming of the footlayer; discontinued endexine.
Figure 6. SEM, LM, TEM micrographs of H. costaricense. D-F, Focal-sectioning showing an inaperturate pollen grain, with a thick and well defined nexine. G-I, Non apertural region of the wall; very thick footlayer and thin enedexine, irregularly distributed along the pollen grain; relatively frequent columellas.
Figure 7. SEM, LM, TEM micrographs of H. goudotianum. A-B, Small area of the lumina, wide muri. C, Pollen grains showing a 5-chotomosulcate aperture. D-F, Focal-sectioning of an inaperturated pollen grains; reticulate ornamentation. G-I, Non-apertural region showing high frequency of relatively equidistant columellas; thick footlayer; difesed endexine, irregularly distributed along the pollen grain.
Figure 8. SEM, LM, TEM micrographs of H. racemosum. A, 5-chotomosulcate pollen grain. B-C, Wide lumina region; high frequency of microechins on the muri. D-F, Focal-sectioning from a polar view showing a pollen grain showing a 5-chotomosulcate pollen grain. G-I, Non-apertural region of a pollen grain showing columellas with low frequency, small size and irregular disposition; thin footlayer; thin endexine, irregularly distributed along the pollen grain.
Figure 9. SEM, LM, TEM micrographs of H. scaberrimum. A-C, Wide muri and small lumina area; high frequency of microechins. D-F, Focal-sectioning of an inapertured pollen grain; reticulate ornamentation. G-I, Non-apertural region showing regular frequency of columellas of mid size; thin endexine irregularly distributed along the pollen grain.
Figure 10. SEM, LM, TEM micrographs of Clavainaperturites microclavatus. A-C, Wide lumina area in proportion to the muri; low frequency of microechins. D-F, Focal-sectioning of a 5-chotomosulcate pollen grain; reticulate ornamentation. G-I, Non-apertural region showing regular frequency of columellas of mid size; thin endexine irregularly distributed along the pollen grain.