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] (Received 8 June 2012; accepted 9 October 2012) © 2013 Collegium Palynologicum Scandinavicum 2 C. Martínez et al. 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