Tracing the Fossil Pollen Record of Hedyosmum (Chloranthaceae), an Old Lineage with Recent Neotropical Diversiﬁcation

Home , Hedyosmum, Hedyosmum mexicanum, Hedyosmum racemosum

Grana, 2013 http://dx.doi.org/10.1080/00173134.2012.760646

Tracing the fossil pollen record of Hedyosmum (Chloranthaceae), an old lineage with recent Neotropical diversiﬁcation

CAMILA MARTÍNEZ1,2, SANTIAGO MADRIÑÁN2, MICHAEL ZAVADA3 & CARLOS ALBERTO JARAMILLO1

1Smithsonian Tropical Research Institute, Ancón, Panamá, 2Laboratorio de Botánica y Sistemática, Universidad de los Andes, Bogotá, Colombia, 3Department of Biology, Seton Hall University, South Orange, New Jersey, USA

Abstract Chloranthaceae represent one of the oldest angiosperm lineages. Hedyosmum, with 45 species, is the only Neotropical genus in the family. The first appearance of Hedyosmum-like pollen was in the Early Cretaceous (∼112 Ma). The next unequivocal record of Hedyosmum-like pollen (Clavainaperturites microclavatus) occurred in the early Miocene. The main objective of this study was to determine the relationship between the fossil C. microclavatus and extant representatives of Hedyosmum. Pollen was examined using light, scanning and transmission electron microscopy. Based on a phylogenetic analysis of pollen traits of Chloranthaceae, we concluded that C. microclavatus is related to Hedyosmum. The abundant Neogene fossil evidence of C. microclavatus from South America showed that the ancestor of extant Hedyosmum migrated to tropical South America during the early Miocene and occupied initially lowlands. A comparison of the C. microclavatus fossil record from both Panama and Colombia/Venezuela suggests that the first Neotropical migration of Hedyosmum was from South America to Central America. The abundant Plio-Pleistocene C. microclavatus from Andean regions supports the hypothesis of a recent radiation of the genus as a consequence of the uplift of the tropical Andes. The biogeographic history of Hedyosmum provides an example of recent enrichment of the Neotropical flora.

Keywords: Asteropollis, Clavainaperturites microclavatus, Miocene, Neotropics

Evidence from the fossil record suggests that angiosperms except for the three basal ‘ANITA’ angiosperms appeared c. 140 Ma during the Early lines, along with Ceratophyllum, magnoliids, mono- Cretaceous (Hughes, 1994; Brenner, 1996; Zavada, cots and eudicots (Qiu et al., 2006; Cantino et al., 2007). Some of the earliest known fossil ﬂowers, 2007; Moore et al., 2007). Studies of whole chloro-

Downloaded by [Yale University Library] at 07:22 05 April 2013 in deposits from Portugal, Australia and China, are plast genomes suggest that Chloranthaceae is proba- 127–120 Ma in age (Friis et al., 2011). Some of these bly sister to magnoliids (Hansen et al., 2007; Jansen flowers are related to Chloranthaceae (Eklund et al., et al., 2007; Moore et al., 2007; Chase et al., 2009). 1997, 2004; Friis et al., 1999, 2011). This result is not necessarily the final word, since Chloranthaceae has 75 extant species with a dis- only one species of Chloranthaceae was sampled by junct tropical amphi-Pacific distribution. The fam- Hansen et al. (2007) and Moore et al. (2007), and ily comprises four genera: Ascarina, Chloranthus, analyses of mitochondrial genes by Qiu et al. (2006) Hedyosmum and Sarcandra (Todzia, 1988). The found different results. phylogenetic position of the Chloranthaceae within Hedyosmum comprises 45 species of shrubs and the angiosperms is still uncertain. Previous studies small trees (Todzia, 1988, 1993; Zhang et al., 2011). based on molecular data have suggested that Most species have a fairly broad distribution; only Chloranthaceae is one of five major lines in 14 species are local endemics, and more than 50% the Mesangiospermae clade that comprises all of the species occur in the northern Andes (Todzia,

Correspondence: Carlos A. Jaramillo, Smithsonian Tropical Research Institute, Roosvelt Ave., Tupper Building – 401, Balboa, Ancón, Apo AA 34002-0948, Panamá, Republic of Panama. E-mail: [email protected]

Figure 1. Distribution of the modern genus Hedyosmum and its fossil pollen record in the form of Asteropollis and Clavainaperturites microclavatus.

1988). Hedyosmum is primarily found from cen- irregularly reticulate with beaded to spinulose muri tral Mexico to central Bolivia along the American (Walker & Walker, 1984). Although morphologi- cordillera, with some species east to Guyana and the cally Clavatipollenites closely resembles Ascarina,it West Indies, and a single species in southeast Asia is still unclear whether it is indeed closely related (H. orientale; Figure 1). The genus is mainly located to it (Eklund et al., 2004). Clavatipollenites has in wet habitats of cool montane cloud forest between been described in Cretaceous strata of all continents 600 and 3000 m above sea level (a.s.l.), although (Eklund et al., 2004). some species are found at sea level (e.g. H. brasiliense Asteropollis, a pollen genus from the Early and H. scaberrimum; Todzia, 1988). Cretaceous, was first thought to be related to The vast majority of pollen grains of extant Hedyosmum on the basis of the branched aperture Chloranthaceae have a relatively constant reticulate and exine structure (more tentatively Doyle, 1969; sculpture, whereas the aperture configuration dis- Walker & Walker, 1984). Later on, it was found plays considerable variation (Endress, 1986; Eklund attached to a Hedyosmum-like flower from Portugal et al., 2004). Hedyosmum has a star-shaped monosul- (Friis et al., 1999). Asteropollis is a pollen grain with

Downloaded by [Yale University Library] at 07:22 05 April 2013 cate aperture with four to six branches (Eklund et al., a (4–)5(–6) branched monosulcate aperture, of glo- 2004). Ascarina has a monosulcate aperture with bose shape and medium size, irregularly reticulate trichotomosulcate variants (Eklund et al., 2004). and with weakly spinulose muri (Walker & Walker, Chloranthus has polycolpate and polyforate pollen 1984). Asteropollis has been found mostly in Laurasia grains, and Sarcandra has polyforate pollen with scat- with a few records from southern Gondwana but tered pores, the reticulate exine pattern is almost it is not known from tropical latitudes (Figure 1). uninterrupted over the apertures, so that the aper- It has been found in the Albian–Cenomanian of tures are camouﬂaged (Endress, 1986; Eklund et al., Greenland (Koppelhus & Pedersen, 1993), Europe 2004). (Azéma et al., 1972; Laing, 1975; Friis et al., The fossil record of Chloranthaceae has been stud- 1999, 2000; Heimhofer et al., 2007), North America ied intensively. Clavatipollenites, an early angiosperm (Hedlund & Norris, 1968; Doyle, 1969; Schwab, fossil pollen genus from the Early Cretaceous, 1977; Nichols & Jacobson, 1982; Walker & Walker, has close similarity to pollen of Chloranthaceae 1984), Australia (Burger, 1990, 1993), Morocco (Couper, 1958; Doyle, 1969; Walker & Walker, (Kotova, 1978), Antarctica (Dettmann & Thomson, 1984; Chapman, 1987; Pedersen et al., 1991) and 1987) and Asia (Li & Liu, 1994; Figure 1). The is thought to be the oldest fossil representative pre-Albian records associated with Asteropollis from of the family. The pollen grains are monosul- Portugal (Friis et al., 1999, 2000) and Argentina cate, slightly boat-shaped to globose, medium-sized, (Prámparo, 1994; Llorens, 2003; Archangelsky et al., Fossil pollen record of Hedyosmum 3

2009) are questionable (J. Doyle, pers. comm., Additionally, six species of Hedyosmum (H. bonplandi- August 2010). There is no clear evidence for a anum, H. racemosum, H. goudotianum, H. costaricense, pre-Albian age of Portugal localities and pollen of H. correanum and H. scaberrimum) and the fossil Argentinian records presents a sulcus but only with C. microclavatus from Panama were examined using three branches. SEM and TEM. Surprisingly, Asteropollis has not been reported Samples were collected from different herbaria fol- after the Campanian (c. 73 Ma; Dettmann & Thom- lowing the procedure of Jarzen and Jarzen (2006). son, 1987; Eklund et al., 2004). It is not until after LM slides were prepared using acetolysis. All pollen the Miocene that fossil pollen grains thought to be samples were treated first with acetic acid and then related to extant Hedyosmum are reported in the macerated to extract the pollen grains. The acetolytic fossil record in the tropics of South and Central solution (9:1) was added for five minutes under low America (Graham, 1976; Hoorn, 1994; Van der heat. The samples were washed with three series of Hammen & Hooghiemstra, 2000; da Silva-Caminha alcohol and mounted in a glycerine-gelatine medium et al., 2010; Figure 1). These pollen grains were to be observed and photographed using a Nikon named Clavainaperturites microclavatus by Hoorn DXM1200F light microscope. (1994), and the original description was based on examination of the grains with light microscopy (LM). A more detailed description requiring scan- Pollen preparation for SEM and TEM ning electron microscopy (SEM) and transmission Samples prepared for SEM were dehydrated in an electron microscopy (TEM) is needed to assess alcohol series of 30, 50, 75, 95 and 100%, fol- natural affinities of pollen grains (Ferguson et al., lowed by 100% acetone and finally with xylene. The 2007). SEM stubs were coated with a thin layer of the In this paper, we perform a detailed analysis of high vacuum wax Apiezon W-100 (Wachtel, 1980). Clavainaperturites microclavatus in order to establish A few drops of the pollen suspension were added on its natural affinities and understand its process of the prepared stubs and then dried for one day and diversification in the Neotropics. Based on molecu- sputter coated with gold-palladium. Specimens for lar evidence and two fossil records associated with SEM were viewed on ZEISS DSM940 and ZEISS Chloranthaceae (Chloranthistemon and Asteropollis), EVO40 scanning electron microscopes. recent studies have estimated ages of nodes in phy- The residues containing fossil pollen in 100% logenies for the family (Zhang & Renner, 2003; xylene were mixed with the polystyrene-based Antonelli & Sanmartín, 2011; Zhang et al., 2011). mounting medium DePex, smeared on a micro- The results of these studies indicate a pattern of early scope slide and allowed to polymerise without a origin but recent diversification for all four extant cover slip. Subsequently, the slides were scanned; genera. individual pollen grains were cut out of the hard- The main goals of this study are to determine if ened polystyrene and placed on a 0.05 µm pore Clavainaperturites microclavatus belongs to the extant Millipore filter on several sheets of filter paper. genus Hedyosmum, to suggest an historical expla- Xylene was then dripped over the polystyrene square nation for the distribution and migration, and to containing the pollen grain until all the polystyrene

Downloaded by [Yale University Library] at 07:22 05 April 2013 propose a more detailed description for this fossil. was removed. On the prepared stubs, a grid was The results could improve our understanding of the inscribed and numbered. The pollen grain, which biogeographic history of Hedyosmum and provide an was already isolated, was transported carefully to the example of recent enrichment of the Neotropical grid coated with gold-palladium and viewed with an ﬂora. scanning electron microscope. The mounted pollen grains were then transferred to a small agar block, dehydrated in alcohol series, embedded in Spurr’s Material and methods low viscosity resin, sectioned on a MT-2b ultra- microtome, stained with uranyl acetate (UA) and Pollen samples of modern and fossil Hedyosmum lead citrate (Pb), and viewed with a Philips Tecnai Pollen grains of 56 individuals from 39 extant species 10 transmission electron microscope. of Chloranthaceae were examined (see ‘Specimens investigated’ section). Specimens of the fossil pollen Morphological analysis of pollen data Clavainaperturites microclavatus from two different localities in Brazil and Panama were also anal- Pollen characters were divided into two categories: ysed. All specimens were observed using transmitted qualitative and quantitative. The qualitative charac- LM. Approximately 50 pollen grains were mea- ters were pollen unit, polar shape, polar symme- sured, depending on the availability of each species. try, exine layers, exine variations, sculpture type, 4 C. Martínez et al.

supratectal sculpturing, infrastructural layer and pollen characters that were observed with LM and endexine presence. The quantitative characters were demonstrated variation between species were used pollen diameter, nexine, sexine, exine and tectum to construct a morphological matrix (Table I) for thickness, aperture type, proportion of lumen/muri posterior phylogenetic analyses. area (in 25 µm2), number of supratectal ornamen- tations (in 6.25 µm2), and foot layer and endexine Phylogenetic analysis of pollen data thickness at the non-apertural region. The morphological characters that could only be observed with In order to establish the phylogenetic position of SEM and TEM were used to emend the diagnosis of the Neogene pollen fossil records of Hedyosmum we the pollen fossil Clavainaperturites microclavatus.The combined a morphological matrix of 40 species of SEM and TEM characters were measured using the Chloranthaceae (Table I), a published phylogeny for ImageJ 1.45 software (Abràmoff et al., 2004). The Chloranthaceae (Zhang et al., 2011) and a set of

Table I. Morphological matrix of pollen characters of fossil and extant Chloranthaceae.

Taxa Diameter Nexine thickness Sexine thickness Exine thickness Sculpture type Aperture type

Hedyosmum angustifolium 31.60 ± 0.65 1.4 1.9 3.3 0/11 Hedyosmum anisodorum 33.79 ± 0.81 1.0 1.6 2.6 0/11 Hedyosmum arborescens 43.65 ± 0.89 1.3 1.4 2.8 1 1 Hedyosmum bonplandianum 38.18 ± 1.31 1.7 2.2 3.9 0/11 Hedyosmum brasiliense 31.53 ± 0.95 1.5 1.5 3.1 0/11 Hedyosmum brenesii 39.18 ± 0.81 1.2 1.4 2.7 0/11 Hedyosmum colombianum 32.90 ± 3.87 2.3 0.9 3.3 1 1 Hedyosmum correanum 40.35 ± 0.99 1.4 2.1 3.5 1 1 Hedyosmum costaricense 34.23 ± 0.75 1.8 2.0 2.8 0 0 Hedyosmum crenatum 38.20 ± 0.81 1.6 1.6 3.2 0 1 Hedyosmum cuatrecazanum 30.38 ± 0.78 1.4 1.6 3.2 1 1 Hedyosmum dombeyanum 30.33 ± 0.74 2.3 1.2 3.5 0/11 Hedyosmum gentryi 31.13 ± 0.86 1 1 2.3 0/11 Hedyosmum goudotianum 42.10 ± 0.74 2.2 2.6 4.8 0 0 Hedyosmum intermedium 20.73 ± 1.10 1.4 1.0 2.3 1 1 Hedyosmum lechleri 31.93 ± 0.59 1.6 1.3 2.9 0/11 Hedyosmum luteynii 30.50 ± 0.25 1.2 1 2.4 0/11 Hedyosmum maximum 33.00 ± 0.52 2.4 1.6 4.0 0/11 Hedyosmum mexicanum 48.55 ± 1.06 ? ? ? 1 1 Hedyosmum orientale 29.34 ± 0.85 1.04 1 2.3 1 1 Hedyosmum parvifolium 35.67 ± 1.03 2.4 2.0 4.5 1 1 Hedyosmum pungens 39.70 ± 1.43 1.2 2.0 3.0 0/11 Hedyosmum racemosum 44.10 ± 1.31 1.9 2.6 4.5 0/11 Hedyosmum scaberrimum 36.65 ± 1.61 1.7 2.0 3.7 0 0 Hedyosmum scabrum 36.03 ± 0.81 ? ? ? 0/11 Hedyosmum spectabile 30.00 ± 0.62 1 1.15 2.6 0/11 Downloaded by [Yale University Library] at 07:22 05 April 2013 Hedyosmum sprucei 37.93 ± 0.71 ? ? ? 0/11 Hedyosmum steinii 38.60 ± 0.56 1.9 1.4 3.3 0/11 Hedyosmum strigosum 44.72 ± 0.89 ? ? ? 0/11 Hedyosmum tepuiense 19.00 ± 0.45 0.8 1 2 1 1 Hedyosmum translucidum 38.35 ± 0.70 ? ? ? 0 1 Clavainaperturites microclavatus 34.73 ± 1.20 1.8 2.0 3.8 1 1 Clavainaperturites microclavatus 21.4 ± 0.72 0.83 0.9 1.73 1 1 Ascarina lanceolata 40.51 1.2 1.7 4.0 1 2 Ascarina lucida 34.67 ± 3.68 1.7 1.1 3.7 1 2 Ascarina philippinensis 21.71 0.8 0.7 1.8 1 2 Chloranthus henryi 37.04 1.4 1.1 2.6 1 3 Chloranthus japonicus 33.98 0.7 0.9 1.8 1 3 Chloranthus serratus 40.66 0.2 0.1 0.4 1 3 Sarcandra chloranthoides 20–50 ? ? ? 1 4 Sarcandra glabra 20–50 ? ? ? 1 4 Hypothetical outgroup ? ? ? ? ? 2

Note: In the column Diameter the symbol ± represents standard deviation. For the trait Sculpture type, the character states are 0, psilate to micro pitted and 1, reticulate. For the trait Aperture type the character states are 0, inaperturate; 1 = sulcus 4–7 branched; 2, sulcus unbranched or with three branches; 3, polycolpate; 4, polyforate; 5, disulcate; 6, ulcerate. For the traits Nexine, Sexine and Exine thickness, and Diameter, the value is equivalent to the average measurements. Fossil pollen record of Hedyosmum 5 Downloaded by [Yale University Library] at 07:22 05 April 2013

Figure 2. SEM, LM and TEM micrographs of ﬁve modern species of Hedyosmum. A–D. Hedyosmum costaricense. E–H. Hedyosmum correanum. I–L. Hedyosmum goudotianum. M–P. Hedyosmum scaberrimum. Q–T. Hedyosmum racemosum.Scalebars–2.5µm(B,F,I,K,M,O, S), 2 µm(L),1µm (G), 500 nm (A, C–E, H, J, N, R, P), 250 nm (T). 6 C. Martínez et al. Downloaded by [Yale University Library] at 07:22 05 April 2013

Figure 3. SEM, LM and TEM micrographs of Clavainaperturites microclavatus (A–C, G)andHedyosmum bonplandianum (D–F, H–J). Scale bars – 5 µm (A, B, D, E, H, I), 2.5 µm(J),2µm(C),1µm (F), 500 nm (G).

palynological records for Hedyosmum and our own Brazil, Colombia, Panama and Venezuela (Appendix, information (Hoorn, 1994; da Silva-Caminha et al., Table AI). 2010; Jaramillo et al., 2011; geographical location, The ﬁrst step in performing the phylogenetic age and abundance of each sample is provided in analyses was to determine, which pollen morpho- the Appendix, Table AI). The phylogeny from Zhang logical characters were phylogenetically informative. et al. (2011) was estimated under Bayesian and We did not assume, apriori, that a given charac- maximum likelihood (ML) analyses using a com- ter was phylogenetically useful. The phylogenetic bined data set of three plastid DNA regions of information of a character was measured follow- 44 species of Chloranthaceae. The tree included ing Pagel’s λ-model (Pagel, 1999), using the Geiger branch lengths. The palynological data set comprised package (Harmon et al., 2008) in R 2.14.0 GUI Neogene records associated with Hedyosmum from 1.42 (R Development Core Team, 2008). Lambda Fossil pollen record of Hedyosmum 7 Downloaded by [Yale University Library] at 07:22 05 April 2013

Figure 4. Chronogram of Chloranthaceae from Zhang et al. (2011). Pollen aperture trait is traced on the phylogeny. The branch colour indicates the type of aperture, which is speciﬁed in the box with pollen images below. The SEM pollen images, representing sulcus unbranched or with three branches, and polycolpate and polyforate apertures, were taken from Eklund et al. (2004). Red branch of Drimys granadensis indicates an ulcerate aperture. Yellow branch of Calycanthus ﬂoridus indicates a disulcate aperture. White branches indicate pollen that is inaperturate or nearly so. Little square before the species name indicates the character state(s) of each taxon. Light grey branches indicate that the character state on the branch is equivocal. The grey shaded area indicates the time range of fossil evidence of Clavainaperturites microclavatus in Brazil, Colombia, Mexico, Panama and Venezuela. 8 C. Martínez et al.

(λ) scales all internal branches of a phylogeny, in be inaperturate or nearly so (indistinct polygonal area which λ = 0 corresponds to no phylogenetic signal on one pole) or aperturate with (4–)5–7 branched and λ = 1 indicates that a trait is evolving accord- sulcus (Figure 2B, F, K, O, S). The aperture length ing to the random walk model (Pagel, 1999; Walls, and width are widely variable even within a sin- 2011). Discrete and continuous traits were evaluated gle individual (e.g. varying between 10 and 15 µm with the ‘fit-Discrete’ and ‘fit-Continuous’ functions in H. bonplandianum). The exine is semitectate, in Geiger, respectively. The ML values from the irregularly reticulate to rugulate (Figure 3H). The models (when λ was set to 0 and to 1) were com- thickness of the exine and its layers is also vari- pared using a likelihood ratio test. The following step able even within individuals of the same species (e.g. was to trace the character history of pollen traits H. correanum, ∼1–2.5 µm thick). The supratectal that presented phylogenetic signal using Mesquite ornamentation is composed of microspinules on the (Maddison & Maddison, 2008) in order to infer muri of the reticulum, which are regularly distributed the synapomorphies of each clade and finally deter- (Figure 2A, E, J, N, R). The lumen area is very mine the phylogenetic position of Clavainaperturites variable (0.7–4.4 µm2/25 µm2) and its shape is irreg- microclavatus. ular (Figure 2A, E, J, N, R). The exine structure To evaluate temporal and biogeographical impli- of the aperture is somewhat irregular; the exine is cations of Clavainaperturites microclavatus, we com- modified near the apertural region (Figure 2H). The pared the molecular dating of Zhang et al. (2011) footlayer thickness is uniform at the apertural region, and the palynological record of C. microclavatus in but the endexine is not (Figure 2H). In the non- Brazil, Colombia, Venezuela and Panama (Appendix, apertural region, the nexine shows a difference in Table AI). Additionally, we mapped the altitudinal density due to the staining process. The less dense range of modern Hedyosmum species (Todzia, 1988) layer is the foot layer and the denser layer is the onto the phylogeny to infer the possible scenery endexine, which is thinner but also continuous in this of migration history, using Mesquite. The elevation region (Figure 2C, D, G, L, P, T). The diameter of ranges were broken into equal intervals from 700 to the pollen grain is widely variable 20–(28–41)–48 µm 3225 m a.s.l. The elevation assigned to each species (Table I). was the midpoint of the range of altitudinal distribu- Within species of Hedyosmum variation in pollen tion based on the monograph of Hedyosmum (Todzia, morphology is minimal. All TEM images for the 1988). six Hedyosmum species analysed revealed the same pattern of layer stratification (Figures 2C, H, L, P, T, 3J). The thickness of the endexine and the Results footlayer remain very constant (0.8 µm thick) at the non-apertural region in all species. The apertural Pollen morphological descriptions for extant Hedyosmum region, on the contrary, shows a gradual thickening Hedyosmum pollen grains are monads with a of the endexine that changes to a granulate texture, spherical-globose shape, radial symmetry and het- rather than being a solid layer. The footlayer and the eropolar (Figure 2B, F, K, O, S). Pollen grains can columellae rapidly decrease in thickness until they Downloaded by [Yale University Library] at 07:22 05 April 2013 Table II. Pollen measurements of six species of Hedyosmum and Clavainaperturites microclavatus using SEM and TEM.

Dia Ex Se Ne Te Lu Mu En F.l. Tax o n (µm) (µm) (µm) (µm) (µm) (µm2) (µm2) (µm) (µm) M/L

Hedyosmum 38.18 ± 1.3 2.09 0.90 1.19 0.55 3.22 21.78 0.41 0.78 1.91 bonplandianum Hedyosmum correanum 40.35 ± 0.9 1.09 0.56 0.53 0.30 1.57 23.43 0.19 0.34 1.75 Hedyosmum 34.23 ± 0.7 2.47 1.30 1.17 0.67 0.70 24.30 0.46 0.71 1.56 costaricense Hedyosmum 42.10 ± 0.7 2.08 1.07 1.01 0.47 1.49 23.51 0.33 0.68 2.05 goudotianum Hedyosmum 44.10 ± 1.3 1.27 0.65 0.62 0.37 4.43 20.57 0.23 0.39 1.71 racemosum Hedyosmum 36.65 ± 1.6 0.90 0.45 0.45 0.20 0.90 24.10 0.09 0.36 3.89 scaberrimum Clavainaperturites 20.83 ± 0.9 1.82 0.88 0.93 0.49 3.47 21.54 0.34 0.60 1.77 microclavatus

Note: Dia, diameter; Ex, exine; Se, sexine; Ne, nexine; Te, tectum; Lu, lumen; Mu, muri; En, endexine; F.l., foot layer; M/L, proportion muri/lumen. Fossil pollen record of Hedyosmum 9 Downloaded by [Yale University Library] at 07:22 05 April 2013

Figure 5. Micrographs of Clavainaperturites microclavatus. A–C. Late Miocene (6.2 Ma); Falcon State, northwestern Venezuela; Codore- Jebe Member Formation: A. Low focus; B. Mid focus; C. High focus. D–F. Late Miocene to Early Pliocene; Tamanduá-Jutaí rivers, Amazonas, Brazil; Solimões Formation: D. High focus; E. Mid focus; F. Low focus. G, H. Late Miocene (7.5 Ma); Saltarín 1A, Vichada, Colombia; Guayabo 5 Formation: G. High focus; H. Low focus. Scale bars – 5 µm.

disappear (Figure 2H). Following, we present the Pollen grains are inaperturate or nearly so, or aper- specific morphological descriptions for six species of turate with a 4–7 branched sulcus. Exine, sexine, Hedyosmum: nexine and tectum mean thicknesses are 3.9, 2.2, 1.7 and 0.5 µm, respectively. Supratectal microspin- Hedyosmum bonplandianum (Figure 3D–F, H–J). — ules on the muri of the reticulum are regularly Two hundred and fifty pollen grains from five distributed and moderately frequent (42/6.2 µm2). individuals were measured (Table II). Average pollen Lumen area (3.2 µm2/25 µm2) is large but smaller diameter is 36.2 µm [standard deviation (SD) = 6.9]. than that of the muri (21.7 µm2/25 µm2). 10 C. Martínez et al.

Hedyosmum correanum (Figure 2E–H). — Fifty pollen grains from one individual were measured (Table II). Average pollen diameter is 33.7 µm (SD = 1.9). Pollen grains are inaperturate or nearly so, and aperturate; when the aperture is present, it is a 5–6 branched sulcus. Exine, sexine, nexine and tectum mean thicknesses are 3.5, 2.1, 1.4 and 0.3 µm, respectively. Supratectal microspinules of the muri are regularly distributed and moderately frequent (39/6.2 µm2). Lumen area (1.5 µm2/25 µm2) is smaller than that of the muri (23.4 µm2/25 µm2).

Hedyosmum costaricense (Figure 2A–D). — Fifty pollen grains from one individual were measured (Table II). Average pollen diameter is 29.4 µm (SD = 3). Pollen grains are inaperturate or nearly so, or aperturate with a 5–6 branched sulcus. Exine, sexine, nexine and tectum mean thicknesses are 2.8, 2.0, 1.8 and 0.6 µm, respectively. Supratectal microspinules of the muri are regularly distributed and abundant (59/6.2 µm2). Lumen area (0.7 µm2/25 µm2) is smaller than that of the muri (24.2 µm2/25 µm2).

Hedyosmum goudotianum (Figure 2I–L). — Fifty pollen grains from one individual were measured (Table II). Average pollen diameter is 34.7 µm (SD = 2.1) µm. Pollen are inaperturate or nearly so, or aperturate with a 4–6 branched sulcus. Exine, sexine, nexine and tectum mean thicknesses are 4.8, 2.6, 2.2 and 0.4 µm, respectively. Supratectal microspinules from the muri of the reticulum are regularly distributed and moderately fre- Figure 6. Palynological fossil records from Colombia, Panama quent (34/6.2 µm2). Lumen area (1.4 µm2/25 µm2) and Venezuela associated with Hedyosmum. A. Map of northern is smaller than that of the muri (23.5 µm2/25 µm2). South America and part of Central America showing the distribution of the localities, where the fossils of Clavainaperturites microclavatus have been found. B. Abundance of fossil records Hedyosmum racemosum (Figure 2Q–T). — One associated with Hedyosmum. This abundance diagram is a com- hundred and ﬁfty pollen grains from three indi- posite of the information from several sites. The composite is

Downloaded by [Yale University Library] at 07:22 05 April 2013 viduals were measured (Table II). Average pollen derived from the lines of correlation used in Jaramillo et al. (2011). diameter is 42.7 µm(SD= 4.8). Pollen grains are Samples from Panama and their corresponding ages were reported by Collins and Coates (1999). Table AI in the Appendix provides inaperturate or nearly so, or aperturate with a the geographical coordinates of each site, and the abundance of 5–6 branched sulcus. Exine, sexine, nexine and C. microclavatus and age of each sample used in the graphic. tectum mean thicknesses are 4.5, 2.6, 1.9 and

Table III. Phylogenetic signal results based on Pagel’s λ model.

Character ML Lambda (λ)MLλ = 0MLλ = 1LRTλ = 1LRTλ = 0

Diameter − 79.00 0.00 − 79.00 − 83.94 0.00 1.00 Nexine − 5.05 0.00 − 5.05 − 18.31 0.00 1.00 Sexine − 12.62 0.15 − 12.74 − 18.36 0.00 0.63 Exine − 28.86 0.00 − 28.86 − 34.95 0.00 1.00 Aperture typea − 29.62 0.96 − 40.90 − 30.33 0.23a 0.00a Scultpture type − 3.76 1.00 − 5.04 − 3.76 0.11 1.00

Note: λ = 0 (no phylogenetic signal); λ = 1 (phylogenetic signal); ML, maximum likelihood; LRT, likelihood ratio test. aCharacter that presents phylogenetic signal. Fossil pollen record of Hedyosmum 11

Figure 7. Chronogram of Chloranthaceae from Zhang et al. (2011). Average elevation is traced on the phylogeny. The branch colours are speciﬁed in the legend. The range of elevation of each species is shown in the bar diagram. In the tree, the grey shaded area indicates the time range, and in the bar diagram, it indicates the elevation range of fossil records of Clavainaperturites microclavatus.

0.3 µm, respectively. Supratectal microspinules from The preliminary placement of the fossil in the the muri of the reticulum are regularly distributed tree was based solely on one synapomorphy, i.e. and moderately frequent (37/6.2 µm2). The lumen the branched sulcus. Our morphological analy- area (4.4 µm2/25 µm2) is very wide but smaller than sis indicates that at the species level, Hedyosmum the width of the muri (20.5 µm2/25 µm2). cannot be differentiated by pollen morphology. The molecular estimated age of splitting between Hedyosmum scaberrimum (Figure 2M–P). — Two H. orientale and the Neotropical Hedyosmum clade hundred pollen grains from four individuals were is the early Eocene. The compilation of fossil measured (Table II). Average pollen diameter is palynological records provides evidence of regular 30.8 µm(SD= 3.7). Pollen grains are inaperurate or presence of Clavainaperturites microclavatus in Brazil, nearly so, or aperturate with a 5–6 branched sulcus. Colombia and Venezuela from 21.5 to 0.13 Ma; Exine, sexine, nexine and tectum mean thicknesses and in Panama from 10.2 to 1.8 Ma (Figure 6,

Downloaded by [Yale University Library] at 07:22 05 April 2013 are 3.7, 2.0, 1.7 and 0.2 µm, respectively. Supratectal Appendix, Table AI). The molecular age of the crown microspinules of the muri of the reticulum are regu- group of Neotropical Hedyosmum (late Oligocene, larly distributed and abundant (67/6.2 µm2). Lumen ∼28 Ma) is supported by the age of the oldest area (0.8 µm2/25 µm2) is irregular but smaller than palynological record in Central and South America that of the muri (24.1 µm2/25 µm2). (early Miocene, ∼21.5 Ma), while fossil records for stem branches from the Campanian (∼76 Ma) through the end of the Oligocene (∼28 Ma) are still Phylogenetic analysis of pollen data unknown (Figure 4). The phylogenetic signal analysis based on the model Mapping altitudes on the phylogeny of Hedyosmum of Pagel’s λ and a resultant value of a likelihood showed an altitudinal range of approximately 1800 m ratio test showed that the only character that pre- a.s.l. for the ancestor of Neotropical species of sented phylogenetic signal was the aperture type, a Hedyosmum (Figure 7). In contrast, our palynological discrete trait (Table III). The character history trace data set comes from localities below 600 m a.s.l. was done only for the aperture type trait (Figure 4). All clades show a wide range of altitude, from sea Given that no more phylogenetic information was level to high mountain ecosystems, with the excep- obtained in the analysis, the morphological data only tion of the clade from the West Indies and Central support a relationship of the fossil Clavainaperturites America, which does not have high elevations microclavatus (Figure 5) to Hedyosmum as a whole. (Figure 7). 12 C. Martínez et al.

Emended diagnosis for the pollen fossil Discussion Clavainaperturites microclavatus The morphological comparison of pollen of extant Family Chloranthaceae Hedyosmum species and the fossil Clavainaperturites microclavatus suggests a close affinity that is highly Morphogenus Clavainaperturites Van der Hammen et supported by the aperture and sculpture types, Wijmstra, 1964. pollen wall stratification and supratectal sculpturing. Aperture type was particularly informative, and per- Morphospecies Clavainaperturites microclavatus mitted us to use pollen morphology to identify genera Hoorn, 1994 within Chloranthaceae (Figure 4). Nevertheless, it Illustrated here in Figure 3A–C; see Table II for SEM was not possible to distinguish Hedyosmum species on and TEM measurements. the basis of pollen morphology even at the ultrastruc- tural level. The emended diagnosis of C. microclava- Natural affinity. — Hedyosmum. tus was necessary to improve its description as well as to evaluate properly the natural affinities of the fossil. The phylogenetic placement of Clavainaperturites Original diagnosis of Clavainaperturites microclavatus was based on its morphological affinity microclavatus from Hoorn (1994) to the entire genus Hedyosmum and its spatiotem- Derivatio nominis. — The name is derived from the poral distribution. The comparison of fossil records very small size of the clavae. with molecular time estimations shows that age of splitting of the most basal member of the Hedyosmum Diagnosis. — Inaperturate, microclavate pollen clade, the Asian species H. orientale (∼46 Ma), is grain. Medium sized with subspheroidal shape. much older than the age of first appearance of Apolar, asymmetric. Tectum perforate. Clavae thin the American fossil C. microclavatus (∼21.5 Ma; and very small (1–1.5 µm). Figure 4). The spatial distribution of C. microclavatus (Figures 1, 6) shows that Hedyosmum had Variability in size. — Lt, +=22–26 µm; ex-M, += already been present in Central and South America 1–1.5 µm. (Brazil, Colombia, Panama, Peru, Venezuela) since the Miocene and in Mexico since the Pliocene Remark. — This species differs from the type- (Graham, 2010). No records of C. microclavatus for species, Clavainaperturites clavatus Van der Hammen the West Indies have been reported (Graham & & Wijmstra, 1964, because of its subspheroidal shape Jarzen, 1969; Graham et al., 2000). and the smaller size of the clavae. The phylogenetic position of the Asian species, Hedyosmum orientale, has varied between studies. Emended diagnosis. — Monad, radial, heteropolar, Eklund et al. (2004) and Antonelli and Sanmartín globose; inaperturate or nearly so, or aperturate, (2011) placed H. orientale as nested among aperture is a sulcus with five or six branches. Exine Caribbean species, whereas Zhang and Renner semitectate, irregularly reticulate. Supratectal orna- (2003) and Zhang et al. (2011) placed it as sis- mentation is composed of microspinules on the muri ter to the remaining species of Hedyosmum.For Downloaded by [Yale University Library] at 07:22 05 April 2013 of the reticulum. our phylogenetic analysis, we used the Zhang et al. (2011) topology, given that their study com- Description. — Monads, shape spherical-globose, prised the most inclusive molecular sampling of the symmetry radial and heteropolar. Average pollen Chloranthaceae to date. diameter is 29 µm(SD= 6.9, N = 25; Figure 5). Concerning the phylogenetic placement of Pollen grains are aperturate or inaperturate or Clavainaperturites microclavatus, we propose that it nearly so (Figure 5). The aperture is a sul- could be either a persisting extinct stem relative of cus with five or six branches when it is present the Neotropical clade or, alternatively, a member (Figure 5). The exine is semitectate, irregularly retic- of the Neotropical clade. These hypotheses do not ulate to regulate (Figure 5F). Exine, sexine and contradict the assumption that Hedyosmum orientale nexine mean thicknesses are 3.8, 2.0, and 1.8 µm, is a relic from a past widespread distribution (Todzia, respectively (Figure 3C, G). The supratectal orna- 1988; Antonelli & Sanmartín, 2011). mentation is composed of microspinules on the The hypothesis that Chloranthaceae had a muri of the reticulum regularly distributed but Laurasian origin (Raven & Axelrod, 1974; Todzia, infrequent (30/6.2 µm2). Lumen area is irregular 1988; Antonelli & Sanmartín, 2011) is neither sup- (3.4 µm2/25 µm2), and smaller than that of the muri ported nor contradicted by the stem fossil record (21.5 µm2/25 µm2; Figure 3A). of Hedyosmum: Asteropollis. The distribution of Fossil pollen record of Hedyosmum 13

Asteropollis was not restricted to Laurasia (Figure 1), thousands of kilometres away (Muller, 1959). it was rather also present during the early to mid- However, these occurrences are rare (Muller, 1959). Cretaceous in Australia, Antarctica and possibly In contrast, C. microclavatus is abundant and found Argentina. Therefore, the stem lineage of the fam- in many different sites and depositional systems ily could have been either Asian or Australasian, and (e.g. ﬂoodplains), suggesting that long-distance thus either Laurasian or Gondwanan (Eklund et al., deposition is unlikely. Other records described in 2004). the literature of C. microclavatus from the Miocene Records of Asteropollis have been widely reported are from the lowlands of Brazil (da Silva-Caminha from the Albian to the Campanian, c.77Ma et al., 2010) and Peru (Hoorn, 1994). In the latter (Dettmann & Thomson, 1987; Eklund et al., 2004). case, Hoorn (1994) studied a stratigraphic section Subsequently, there is a long gap (∼55 myr) in the named Santa Teresa, which is located at lowland record with no fossil pollen of Hedyosmum, in spite elevation, and found C. microclavatus but assumed of its being a genus with distinctive pollen morphol- that the pollen had been transported from the Andes. ogy and easy to recognise. Antonelli and Sanmartin Younger records of Hedyosmum from the Pliocene (2011) have explained its absence as a result of punc- and Pleistocene have been abundantly reported from tual or constant extinction rates, perhaps caused by the highlands of the Andes (Hooghiemstra, 1989; the cooling of global climates since the Eocene– Hooghiemstra & Ran, 1994; Veer & Hooghiemstra, Oligocene, and the K/T event. However, neither of 2000). The spatiotemporal record and abundance these two events explains the absence of Asteropollis of C. microclavatus suggest that the ancestor of the in the Late Cretaceous. crown group of Hedyosmum was from the lowlands, Given the fossil distribution of Asteropollis,the but today the genus is mainly located in wet habitats temporal gap in the fossil record (Late Cretaceous– of cool montane cloud forest between 600 and Oligocene), the Miocene–Pliocene records of 3000 m a.s.l. (Todzia, 1988). The ancestral charac- Clavainaperturites microclavatus in the Neotropics, ter reconstruction of the elevation of Hedyosmum is the modern distribution of Hedyosmum (Figure 1), based on extant distribution and on an estimation and the molecular age estimation (Figure 4), it is of altitude derived only from the average value of plausible that the genus migrated from Asia to the the altitudinal range of each species (Figure 7). The Neotropics during the early Miocene. Similar dis- entire range of elevation shows that some species persal events have been inferred in other extant plant of Hedyosmum, such as H. racemosum,havebroad groups, such as Dracontomelon (Anacardiaceae), ecological tolerances, occurring from low-elevation Leea (Vitaceae), Nypa (Arecaceae), Hernandiaceae, tropical wet forest (300 m a.s.l.) to montane cloud Monimiaceae, and possibly Menispermaceae forest (2700 m a.s.l.; Todzia, 1988). This ecological (Gomez-Navarro et al., 2009; Michalak et al., tolerance could have facilitated the migration or 2010; Renner et al., 2010; Herrera et al., 2011, colonisation of Hedyosmum to higher elevations of 2012). Thousands of palynological samples from the Andean Cordillera after the northern Andes the Neotropics have been studied over the past developed elevations > 2000 m a.s.l. during the late 50 years (e.g. Germeraad et al., 1968; Regali et al., Miocene (Van der Hammen et al., 1973). 1974; Jaramillo et al., 2006, 2011) and pollen of C. It has been suggested that Hedyosmum spread

Downloaded by [Yale University Library] at 07:22 05 April 2013 microclavatus, in spite of being easily recognisable, to South America via Mesoamerica during the has never been found in sediments older than early Late Neogene (Raven & Axelrod, 1974; Todzia, Miocene. 1988; Eklund et al., 2004). Nevertheless, the It seems probable that the ﬁrst Miocene fossil record of Clavainaperturites microclavatus Hedyosmum in tropical America inhabited the low- from South America is older than the records lands, given that all the records of Clavainaperturites from Panama (Colombia and Venezuela: 21.5 Ma; microclavatus displayed on Figure 6 were collected Panama: 10.2 Ma). Consequently, it would be more from either low-elevation sites (< 600 m a.s.l.), or consistent with the record to assume that the sites that are now high in the Andes but accumulated migration occurred from South America to Central before the rise of the Andes. The abundant and America during the Late Miocene, prior to the com- widespread fossil record of Miocene C. microclavatus plete emergence of the Panama land bridge at 3.5 Ma in the lowlands contradicts a previous hypothesis that (Coates et al., 2004), although new geologic evidence proposed that these pollen grains from low-elevation has suggested an earlier uplift of the Panamanian areas were produced in the Andean foothills and Isthmus (∼20 Ma; Farris et al., 2011). The abundant were subsequently transported by wind or water record of C. microclavatus in South America from the currents to their ﬁnal deposition site (Antonelli & Pliocene to the Holocene (Figure 6; Hooghiemstra, Sanmartín, 2011), as was shown for Andean-derived 1989; Hooghiemstra & Ran, 1994; Van der Hammen pollen found in the Orinoco delta and transported & Hooghiemstra, 2000) supports the idea that the 14 C. Martínez et al.

recent radiation of the genus, compared to the much Specimens investigated older age of its stem lineage, was a consequence COL: Herbario Nacional Colombiano; INPA: of the uplift of the tropical Andes (Todzia, 1988; National Institute of Amazonian Research. MO: Antonelli & Sanmartín, 2011). Missouri Botanical Garden Herbarium. PMA: University of Panama Herbarium; SCZ: Smithsonian Tropical Research Institute’s Herbarium; US: Conclusion United States National Herbarium, Smithsonian The study of the pollen morphology of the Institution; STRI: Smithsonian Tropical Research Miocene fossil Clavainaperturites microclavatus per- Institute. mitted its association with the genus Hedyosmum. Hedyosmum angustifolium (Ruiz et Pavón) Solms-Laubach US The phylogenetic analysis of morphological data, 2956779. together with the spatiotemporal distribution of C. Hedyosmum anisodorum Todzia US 3024811. microclavatus, suggests phylogenetic placement of Hedyosmum arborecens Swartz Graham Reference Collection, the fossil as nested within the crown group of STRI, Panamá. Neotropical Hedyosmum. The abundant Miocene Hedyosmum bonplandianum Kunth US 2817542. Hedyosmum brasiliense Martius ex Miquel INPA 112562, US fossil evidence of C. microclavatus from the low- 1776017. lands of Brazil, Colombia, Panama and Venezuela Hedyosmum brenesii Standley Graham Reference Collection, indicates that the ancestor of extant Hedyosmum STRI, Panamá. was from the lowlands, although today the genus is Hedyosmum colombianum Cuatrecasas US 1903932. mainly distributed in wet habitats of cool montane Hedyosmum correanum D’Arcy et Liesner PMA 11872. Hedyosmum costaricense Burger PMA 11882. cloud forest. The younger records of C. microclava- Hedyosmum crenatum Occhioni COL Rangel & Cleef 926. tus from Panama compared to those from Colombia Hedyosmum cuatrecazanum Occhioni US 3434105. and Venezuela suggests that the ﬁrst migration of Hedyosmum dombeyanum Solms-Laubach US 3380267. crown group species of Hedyosmum occurred from Hedyosmum gentryi D’Arcy et Liesner US 3178843. South America to Central America. The abundance Hedyosmum goudotianum Solms-Laubach US 3331031. Hedyosmum intermedium Todzia US 3073795. of fossils from the region associated with the Andes Hedyosmum lechleri Solms-Laubach US 3434091. supports the hypothesis of a recent radiation of the Hedyosmum luteynii Todzia US 2817538. genus as a consequence of the uplift of the tropical Hedyosmum maximum (Kuntze) Schumann US 2533472. Andes. Hedyosmum mexicanum Cordemoy Graham Reference Collection, STRI, Panamá. Hedyosmum orientale Merr. et Chun MO Meijer, W 9899. Hedyosmum parvifolium Solms-Laubach US 2556379. Acknowledgements Hedyosmum pungens Todzia US 3244660. Hedyosmum racemosum (Ruiz et Pavón) G. Don US 3253637. The authors thank James Doyle (University of Hedyosmum scaberrimum Standley PMA 11890. California, Davis) for detailed reviews of sev- Hedyosmum scabrum (Ruiz et Pavón) Solms-Laubach US eral versions of this manuscript; James Richardson 3434069. (Royal Botanic Garden, Edinburgh) and Alexandre Hedyosmum spectabile Todzia US 3422127. Hedyosmum sprucei Solms-Laubach INPA 164251. Antonelli (Gothenburg Botanic Garden) for valuable Hedyosmum steinii Todzia Stein et al. 3292, COL. Downloaded by [Yale University Library] at 07:22 05 April 2013 comments; Fabio Gonzales (Universidad Nacional Hedyosmum strigosum Todzia US 3244661. de Colombia, Bogotá), Fabiany Herrera (STRI), Hedyosmum tepuiense Todzia US 3506867. Mónica Carvalho (STRI) and Silane da Silva (INPA) Hedyosmum translucidum Cuatrecasas INPA 156414. for sending samples; the herbaria COL, US, MO, Ascarina lanceolata Hook. f. Graham Reference Collection, STRI, Panamá. PMA, COAH and INPA for providing samples; Ascarina lucida Hook. f. Graham Reference Collection, STRI, Hong-Zhi Kong for providing the dated phylogeny of Panamá; Eklund et al., 2004. Chloranthaceae; and Mayandi Sivaguru, Alejandra Ascarina philippinensis C.B. Rob. Graham Reference Collection, Restrepo and Surangi Punyasena from the University STRI, Panamá; Eklund et al., 2004. of Illinois, Urbana, and I. Romero, for sharing Chloranthus henryi Hemsl. Graham Reference Collection, STRI, Panamá. photographs of fossil pollen samples. This project Chloranthus. japonicus Siebold Graham Reference Collection, was supported by the Smithsonian Paleobiology STRI, Panamá; Eklund et al., 2004. Endowment Fund, the Unrestricted Endowments Chloranthus serratus (Thunb.) Roem. et Schult. Graham Reference SI Grants, the University of Yale Edward P. Bass Collection, STRI, Panamá; Eklund et al., 2004. Distinguished Visiting Environmental Scholar, and Sarcandra chloranthoides Gardner. Graham Reference Collection, STRI, Panamá; Eklund et al., 2004. a Short Term Fellowship from the Smithsonian Sarcandra glabra (Thunb.) Nakai. Graham Reference Collection, Tropical Research Institute. STRI, Panamá; Eklund et al., 2004. Fossil pollen record of Hedyosmum 15

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Appendix Table AI. Geographic location, age and abundance of Clavainaperturites microclavatus for each sample used in the analysis.

Country Formation Site Age Count Latitude Longitude Source

Colombia Colorado Agata-1 17.01 1 5.83 − 74.32 our own data Colombia Colorado Agata-1 17.19 2 5.83 − 74.32 our own data Colombia Colorado Agata-1 17.2 2 5.83 − 74.32 our own data Colombia Colorado Agata-1 19.25 1 5.83 − 74.32 our own data Colombia Colorado Agata-1 19.36 1 5.83 − 74.32 our own data Colombia Colorado Agata-1 19.5 1 5.83 − 74.32 our own data Colombia Colorado Agata-1 19.57 1 5.83 − 74.32 our own data Colombia Mugrosa Agata-1 21.57 1 5.83 − 74.32 our own data Colombia C1 Gibraltar Ideal 14.53 1 7.04 − 72.18 our own data Colombia C1 Gibraltar Ideal 16.02 1 7.04 − 72.18 our own data Colombia C1 Gibraltar Ideal 16.29 1 7.04 − 72.18 our own data Colombia C1 Gibraltar Ideal 16.67 1 7.04 − 72.18 our own data Colombia C1 Gibraltar Ideal 16.98 1 7.04 − 72.18 our own data Colombia C1 Gibraltar Ideal 17.16 1 7.04 − 72.18 our own data Colombia C1C2 Gibraltar Ideal 17.29 1 7.04 − 72.18 our own data Colombia Honda Higueron-1 14.08 1 3.10 − 75.27 our own data Colombia Caja Medina 7.26 1 4.50 − 73.10 our own data Colombia C1 Medina 16.61 4 4.50 − 73.10 our own data Colombia C3 Medina 18.54 1 4.50 − 73.10 our own data Colombia C3 Medina 18.78 1 4.50 − 73.10 our own data Colombia C3 Medina 19.05 1 4.50 − 73.10 our own data Colombia C5 Medina 19.86 1 4.50 − 73.10 our own data Colombia Molino deViento-1 17.32 1 11.30 − 72.49 our own data Colombia Molino deViento-1 18.48 1 11.30 − 72.49 our own data Colombia Real Montoyas-1A 0.14 4 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.19 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.22 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.24 6 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.25 2 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.28 3 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.43 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.74 7 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.77 3 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.78 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.79 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 0.97 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.03 2 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.11 5 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.18 6 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.21 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.25 4 6.83 − 74.04 our own data Downloaded by [Yale University Library] at 07:22 05 April 2013 Colombia Real Montoyas-1A 1.28 3 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.39 3 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.46 2 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.49 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.53 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 1.81 4 6.83 − 74.04 our own data Colombia Real Montoyas-1A 2.26 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 5.68 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 5.84 3 6.83 − 74.04 our own data Colombia Real Montoyas-1A 5.99 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 6.98 15 6.83 − 74.04 our own data Colombia Real Montoyas-1A 7.07 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 10.27 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 10.69 2 6.83 − 74.04 our own data Colombia Real Montoyas-1A 10.84 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 11.97 2 6.83 − 74.04 our own data Colombia Real Montoyas-1A 12.11 2 6.83 − 74.04 our own data Colombia Real Montoyas-1A 13.05 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 14.04 1 6.83 − 74.04 our own data

(Continued) Fossil pollen record of Hedyosmum 19

Tab le A I. (Continued).

Country Formation Site Age Count Latitude Longitude Source

Colombia Real Montoyas-1A 14.53 1 6.83 − 74.04 our own data Colombia Real Montoyas-1A 14.77 4 6.83 − 74.04 our own data Colombia Real Montoyas-1A 16.11 1 6.83 − 74.04 our own data Colombia Guayabo Palomas-1-ST1 12.12 2 4.43 − 73.36 our own data Colombia Leon Palomas-1-ST1 12.65 3 4.43 − 73.36 our own data Colombia Leon Palomas-1-ST1 13.17 1 4.43 − 73.36 our own data Colombia Leon Palomas-1-ST1 13.84 1 4.43 − 73.36 our own data Colombia Leon Palomas-1-ST1 14.6 1 4.43 − 73.36 our own data Colombia Leon Palomas-1-ST1 15.26 1 4.43 − 73.36 our own data Colombia Leon Palomas-1-ST1 15.91 1 4.43 − 73.36 our own data Colombia C1 Palomas-1-ST1 16.19 1 4.43 − 73.36 our own data Colombia C3 Palomas-1-ST1 19.11 1 4.43 − 73.36 our own data Colombia Pebas Puerto Leguizamo 15 3 − 0.09 − 74.63 our own data Colombia Leon San Juan-1 15.72 1 3.38 − 73.86 our own data Colombia Guayabo Santa Maria-1 11.91 1 6.01 − 71.65 our own data Colombia Leon Santa Maria-1 12.02 1 6.01 − 71.65 our own data Colombia Leon Santa Maria-1 12.08 1 6.01 − 71.65 our own data Colombia Leon Santa Maria-1 12.28 1 6.01 − 71.65 our own data Colombia Guayabo Tame-2 12.23 1 6.57 − 71.76 our own data Colombia Guayabo Tocoragua-1 12.7 1 6.28 − 71.80 our own data Colombia Guayabo Tocoragua-1 12.96 1 6.28 − 71.80 our own data Colombia Leon Tocoragua-1 14.56 1 6.28 − 71.80 our own data Colombia Leon Tocoragua-1 14.93 1 6.28 − 71.80 our own data Colombia Leon Tocoragua-1 15.31 1 6.28 − 71.80 our own data Colombia C4 Tocoragua-1 19.98 1 6.28 − 71.80 our own data Colombia C4 Tocoragua-1 20.02 1 6.28 − 71.80 our own data Venezuela La Vela Urumaco East 4.52 1 11.47 − 69.55 our own data Venezuela La Vela Urumaco East 5.51 1 11.47 − 69.55 our own data Venezuela La Vela Urumaco East 5.52 1 11.47 − 69.55 our own data Venezuela La Vela Urumaco East 5.61 1 11.47 − 69.55 our own data Venezuela Codore Algodones Urumaco West 3.46 1 11.18 − 70.26 our own data Venezuela Codore Jebe Urumaco West 6.27 4 11.18 − 70.26 our own data Venezuela Codore Jebe Urumaco West 6.6 1 11.18 − 70.26 our own data Venezuela Urumaco Urumaco West 7.53 1 11.18 − 70.26 our own data Venezuela Urumaco Urumaco West 7.98 1 11.18 − 70.26 our own data Venezuela Urumaco Urumaco West 8.59 5 11.18 − 70.26 our own data Venezuela Urumaco Urumaco West 11.97 1 11.18 − 70.26 our own data Venezuela Urumaco Urumaco West 12.4 2 11.18 − 70.26 our own data Venezuela Socorro Urumaco West 13.57 1 11.18 − 70.26 our own data Venezuela Socorro Urumaco West 13.78 1 11.18 − 70.26 our own data Venezuela Socorro Urumaco West 14.09 1 11.18 − 70.26 our own data Panama Gatun 01-Sabanita to Payardi 5.6 1 9.35 − 79.81 our own data Panama Gatun 01-Sabanita to Payardi 5.6 3 9.35 − 79.81 our own data Downloaded by [Yale University Library] at 07:22 05 April 2013 Panama Escudo Veraguas 10-North Coast 2.05 1 9.10 − 81.56 our own data Panama Escudo Veraguas 10-North Coast 2.05 1 9.10 − 81.56 our own data Panama Escudo Veraguas 10-North Coast 2.05 1 9.10 − 81.56 our own data Panama Escudo Veraguas 10-North Coast 2.05 1 9.10 − 81.56 our own data Panama Escudo Veraguas 10-North Coast 2.05 5 9.10 − 81.56 our own data Panama Escudo de Veraguas 10-North Coast 2.75 1 9.10 − 81.56 our own data Panama Escudo de Veraguas 10-North Coast 2.75 1 9.10 − 81.56 our own data Panama Escudo de Veraguas 10-North Coast 3.55 2 9.10 − 81.56 our own data Panama Escudo de Veraguas 10-North Coast 3.55 4 9.10 − 81.56 our own data Panama Shark Hole Point 12-North Valiente 3.45 1 9.04 − 81.74 our own data Panama Shark Hole Point 12-North Valiente 4.6 1 9.04 − 81.74 our own data Panama Shark Hole Point 12-North Valiente 4.6 2 9.04 − 81.74 our own data Panama Nancy Point 12-North Valiente 6.05 1 9.04 − 81.74 our own data Panama Unnamed 15-South Valiente 1.8 3 9.11 − 81.91 our own data Panama Nancy Point 15-South Valiente 1.8 3 9.11 − 81.91 our own data Panama Nancy Point 15-South Valiente 6.95 1 9.11 − 81.91 our own data Panama Cayo Agua 16-North Point West 4.25 1 9.18 − 82.06 our own data Panama Cayo Agua 16-North Point West 4.25 7 9.18 − 82.06 our own data Panama Cayo Agua 17-Piedra Roja Point west 4.25 2 9.14 − 82.01 our own data Panama Cayo Agua 17-Piedra Roja Point west 4.25 2 9.14 − 82.01 our own data

(Continued) 20 C. Martínez et al.

Tab le A I. (Continued).

Country Formation Site Age Count Latitude Longitude Source

Panama Cayo Agua 17-Piedra Roja Point west 4.25 4 9.14 − 82.01 our own data Panama Cayo Agua 17-Piedra Roja Point west 4.25 4 9.14 − 82.01 our own data Panama Cayo Agua 20-Nispero Point South 3.55 1 9.17 − 82.02 our own data Panama Cayo Agua 20-Nispero Point South 3.55 5 9.17 − 82.02 our own data Panama Tuira Rio Chico 6.95 4 8.22 − 77.57 our own data Panama Pucro? Rio Chico 6.95 1 8.22 − 77.57 our own data Panama Tuira Rio Chico 6.95 1 8.22 − 77.57 our own data Panama Tuira Rio Chico 6.95 1 8.22 − 77.57 our own data Panama Chucunaque Rio Chucunaque 7.05 1 8.33 − 77.76 our own data Panama Tuira Rio Tuira 10.15 1 8.24 − 77.54 our own data Panama Tuira Rio Tuira 10.15 2 8.24 − 77.54 our own data Panama Tuira Rio Tupisa 6.95 4 8.28 − 77.61 our own data Panama Tuira Rio Tupisa 6.95 1 8.28 − 77.61 our own data Panama Chucunaque Rio Turquesa 6.95 1 8.40 − 77.76 our own data Panama Chucunaque Rio Turquesa 6.95 1 8.40 − 77.76 our own data Panama Gatun Seccion 1 Sabanita to Payardi 9.6 1 9.35 − 79.81 our own data Panama Gatun Seccion 1 Sabanita to Payardi 9.6 3 9.35 − 79.81 our own data Panama Gatun Seccion 1 Sabanita to Payardi 9.6 4 9.35 − 79.81 our own data Panama Gatun Seccion 1 Sabanita to Payardi 9.6 1 9.35 − 79.81 our own data Panama Escudo de Veraguas Seccion 11 South Coast 3.55 1 9.09 − 81.54 our own data Panama Escudo de Veraguas Seccion 11 South Coast 3.55 2 9.09 − 81.54 our own data Panama Cayo Agua Seccion 19 North-Tiburon Point 3.55 2 9.15 − 82.02 our own data Panama Cayo Agua Seccion 19 North-Tiburon Point 3.55 2 9.15 − 82.02 our own data Panama Cayo Agua Seccion 19 North-Tiburon Point 3.55 2 9.15 − 82.02 our own data Panama Cayo Agua Seccion 19 North-Tiburon Point 3.55 4 9.18 − 82.04 our own data Panama Cayo Agua Seccion 19 North-Tiburon Point 4.25 4 9.18 − 82.04 our own data Panama Cayo Agua Seccion 19 North-Tiburon Point 3.55 1 9.18 − 82.04 our own data Panama Gatun Seccion 2 Margarita to Gatun 8.9 1 9.26 − 79.92 our own data Panama Gatun Seccion 2 Margarita to Gatun 8.9 2 9.26 − 79.92 our own data Panama Gatun Seccion 2 Margarita to Gatun 8.9 2 9.26 − 79.92 our own data Panama Gatun Seccion 2 Margarita to Gatun 8.9 6 9.26 − 79.92 our own data Panama Gatun Seccion 2 Margarita to Gatun 10.05 1 9.26 − 79.92 our own data Panama Gatun Seccion 2 Margarita to Gatun 10.05 1 9.26 − 79.92 our own data Panama Unnamed Seccion 23 Bastimentos Island 2.65 1 9.32 − 82.11 our own data Panama Unnamed Seccion 23 Bastimentos Island 2.65 9 9.32 − 82.11 our own data Panama Unnamed Seccion 24 Solarte Cay 3.55 3 9.33 − 82.21 our own data Panama Unnamed Seccion 24 Solarte Cay 3.55 4 9.33 − 82.21 our own data Panama Unnamed Seccion 24 Solarte Cay 3.55 8 9.33 − 82.21 our own data Panama Unnamed Seccion 24 Solarte Cay 3.55 8 9.33 − 82.21 our own data Panama Gatun Seccion 3 Toro Point 10.2 3 9.26 − 79.94 our own data Panama Gatun Seccion 3 Toro Point 10.2 5 9.26 − 79.94 our own data Panama Gatun Seccion 9 Calzones River 8.6 1 9.04 − 80.64 our own data Panama Gatun Seccion 9 Calzones River 8.6 1 9.04 − 80.64 our own data Downloaded by [Yale University Library] at 07:22 05 April 2013 Brazil Acre 1AS-27-AM-5 7 1 − 4.28 − 67.91 da Silva-Caminha et al 2010 Brazil Acre 1AS-27-AM-21 7 1 − 4.28 − 67.91 da Silva-Caminha et al 2010 Brazil Acre 1AS-27-AM-26 7 1 − 4.28 − 67.91 da Silva-Caminha et al 2010 Brazil Acre 1AS-27-AM-2 7 5 − 4.28 − 67.91 da Silva-Caminha et al 2010 Brazil Acre 1AS-27-AM-23 7 5 − 4.28 − 67.91 da Silva-Caminha et al 2010 Brazil Acre 1AS-19-AM19-4 7 1 − 4.55 − 69.16 da Silva-Caminha et al 2010 Peru Pebas Pijuayal 15 5 − 3.34 − 71.80 Hoorn 1994 Peru Pebas Santa Teresa 17 4 − 3.50 − 73.10 Hoorn 1994 Colombia Pebas Santa Soﬁa 10 3 − 3.90 − 70.10 Hoorn 1994 Colombia Pebas Mocagua 10 5 − 3.79 − 70.30 Hoorn 1994 Colombia Pebas Los Chorros 1E 10 4 − 3.76 − 70.40 Hoorn 1994 Colombia Pebas Los Chorros 1W 10 6 − 3.74 − 70.40 Hoorn 1994 Mexico Paraje Solo Veracruz 3.6 1 18.30 − 95.00 Graham 2010

Note: Only Pre-Quaternary data are included here.