Diverse fossil Onagraceae pollen from a Miocene palynoflora of north-east China: early steps in resolving the phytogeographic history of the family more

Plant Syst Evol (2012) 298:671–687 DOI 10.1007/s00606-011-0578-0 ORIGINAL ARTICLE Diverse fossil Onagraceae pollen from a Miocene palynoflora of north-east China: early steps in resolving the phytogeographic history of the family ´ Friðgeir Grımsson • Reinhard Zetter • Qin Leng Received: 29 October 2011 / Accepted: 29 November 2011 / Published online: 24 December 2011 Ó Springer-Verlag 2011 Abstract The origin and evolution of angiosperms can be unravelled by using fossil records to determine first occurrences and phytogeographic histories of plant families and genera. Many angiosperm families, for example the Onagraceae, have a poor macrofossil record, but are more common in palynological records. Modern Onagraceae produce pollen clearly distinct from that of other angiosperms. Combined morphological features obtained by use of light and scanning electron microscopy have enabled assignment of fossil Onagraceae pollen to extant genera, and therefore tracing of the origin and past distributions of extant Onagraceae lineages. We studied a Miocene palynoflora from the Daotaiqiao Formation of north-east China. Using the singlegrain technique, we examined individual Onagraceae pollen/ tetrads using both light and scanning electron microscopy. Fossil Onagraceae pollen is more frequent than macrofossil remains, but is still rare, and usually represented by a single taxon in palynological samples. Remarkably, samples from the Miocene of north-east China contain five different species: two of Circaea, one of Epilobium, and two of Ludwigia. Such a large number of Onagraceae taxa from a single palynoflora is unknown elsewhere. Whereas Ludwigia pollen is known from Cenozoic sediments of the northern hemisphere, the Circaea pollen is the first fossil pollen assignable to this extant genus. This is also the first fossil record of Epilobium from China. Although the young geological age of this sample does not enable consideration of time of origin for the genera encountered, the co-occurrence of Circaea, Epilobium, and Ludwigia in the mid to late-Miocene of East Asia sheds some light on their phytogeographic histories. Keywords Biogeography Á Epilobium Á Cenozoic Á Circaea Á Ludwigia Á Northern hemisphere Introduction The Onagraceae (order Myrtales) is composed of approximately 650 species belonging to 17–18 genera (Wagner et al. 2007). The family currently has a cosmopolitan distribution, with most members occurring in temperate regions and with a prominent concentration of species in the Americas. Morphological and molecular analysis suggest that the Onagraceae are monophyletic, with the genus Ludwigia as a basal group (Levin et al. 2003; Ford and Gottlieb 2007). Although the morphological and molecular interrelationship of Onagraceae genera is now relatively clear, the timing of divergence between many of its genera and their paleobiogeographic histories are still poorly understood. On the basis of pollen the fossil record of the family seems to date back to the Upper Cretaceous (Eyde and Morgan 1973; Skvarla et al. 1978; Muller 1981; Martin 2003), but the macrofossil record is composed of only few, and mostly doubtful, findings, except some Circaea-type fruits from the Oligocene to Pliocene (Szafer 1947; Dorofeev 1963, 1969) and Ludwigia fruits/seeds from the Oligocene to Miocene (Friis 1985; Mai 1985). ´ F. Grımsson (&) Á R. Zetter University of Vienna, Department of Palaeontology, Althanstraße 14 (UZA II), 1090 Vienna, Austria e-mail: fridgeir.grimsson@univie.ac.at Q. Leng Bryant University, Department of Science and Technology, 1150 Douglas Pike, Smithfield, RI 02917, USA Q. Leng LPS, Nanjing Institute of Geology and Palaeontology, 39 East Beijing Road, Nanjing 210008, China 123 672 ´ F. Grımsson et al. Because of excellent work on the pollen morphology of extant taxa, including detailed accounts using combined light microscopy (LM), scanning electronic microscopy (SEM), and transmission electron microscopy (TEM), the characteristic features of all major tribes and genera within Onagraceae are now well known (Ting 1966; Brown 1967; Skvarla et al. 1976, 1978; Praglowski et al. 1983, 1987, 1988, 1994; Patel et al. 1984; Keri and Zetter 1992; Punt et al. 2003; Makbul et al. 2008). Combined microscopic observations have also shown that the Onagraceae produce distinct pollen types that cannot be confused with pollen from any other angiosperm family (cf. Patel et al. 1984). The well-documented pollen features of modern Onagraceae, and their distinct morphological characteristics observable using combined LM and SEM studies, now make it possible to readily identify fossil Onagraceae pollen/tetrads to the generic level. In some cases it is also possible to distinguish between closely related species, a situation previously impossible. Therefore, although the fossil record of Onagraceae is very incomplete, any new discovery of fossil pollen from this family provides critical data on the former distribution patterns of particular genera, because each occurrence gives additional information relevant to the phytogeographic history of a genus. Here, we report on our new findings and analyses of several fossil pollen taxa of Onagraceae from a single palynoflora of the Daotaiqiao Formation, north-east China. The detailed morphological features obtained by the single-grain technique (Zetter 1989; Hesse et al. 2009) furnished sufficient morphological details to assign fossil pollen to various extant genera of the family Onagraceae. The studied palynoflora is the only fossil flora which yields several co-occurring Onagraceae species. It contains the earliest records of Circaea pollen in the northern hemisphere, and the earliest fossil record of Epilobium pollen from China. carbonates. After decanting the HCl liquid, the residue was boiled for approximately 10 min in HF (200 ml) and transferred to a 4-l beaker of water. After settling, the liquid was decanted and the residue was again boiled in HCl for 5 min. After settling, the HCl was decanted and the remaining solution was centrifuged and then washed 3–4 times with water. The sample was then acetolysed, centrifuged, and washed up to four times. The remaining organic material was mixed with glycerine and stored in small sample tubes. The single-grain technique Fossil Onagraceae pollen grains/tetrads were investigated under both LM and SEM, using the single-grain technique (Zetter 1989; Hesse et al. 2009). This technique has already proved very useful when studying fossil palynofloras, providing morphological details and accurate systematic ´ ´ identification (Grımsson et al. 2008, 2011a, b; Grımsson and Zetter 2011; Denk et al. 2010, 2012). Drops from the sample tubes were transferred to slides and single Onagraceae grains/tetrads were picked out by use of a preparation needle with a single human nasal hair mounted on it. The Onagraceae pollen grains/tetrads were placed on separate slides with fresh drops of glycerine for photography under LM. The same pollen grains/tetrads were then transferred to SEM stubs with the help of a preparation needle and washed with drops of absolute ethanol to dissolve the remaining glycerine. The stubs with the Onagraceae pollen/ tetrads were sputter-coated with gold, and the pollen/tetrads were photographed under SEM. The same individual tetrads or pollen grains were then turned over, re-sputtered with gold, and re-photographed under SEM. Conservation of material Parts of the original sedimentary rock sample, the treated sample stored in a tube, and SEM stubs of fossil Onagraceae pollen and tetrads are stored in the collection of the Department of Palaeontology, University of Vienna, Austria, under accession numbers IPUW 6904/01-85. Geological outline and age of the sediments studied The geological settings, including the sedimentary succession, from where the sample for this palynological study was collected have been described in detail by Liu et al. (1995, 1996) and Leng (1997, 2000a, b). The sample was taken from a dark-gray and finely laminated claystone which is rich in plant macrofossils and fossilized skeletons of freshwater fish and well-preserved insects. The claystone is approximately 3.5 m thick and belongs to the lower part of the sedimentary succession composing the Materials and methods The sample studied was collected from the Daotaiqiao Formation located at Beipaizi, approximately 4 km north of Sifangtai Village, Huanan County of Heilongjiang Province, north-east China (approximate latitude 46°35N, longitude 131°19E). The outcrop was exposed by placer gold mining, and the sample was obtained from Zhang’s Well, one of several placer gold-mining wells, named after its owner, Mr. Zhang. Preparation of samples Sediments were washed and dried, and hand ground by use of a mortar and pestle. The powder was boiled in concentrated HCl (350 ml) for 5–10 min to remove all 123 Diverse fossil Onagraceae pollen from a Miocene palynoflora 673 Daotiaqiao Formation (Working Group of ‘‘Regional Stratigraphic Chart of Heilongjiang Province’’ 1979; Ni 1992). The age of the Daotaiqiao Formation has been assigned to the Miocene, based on the correlation of plant macrofossils and fossilized fish remains (Working Group of ‘‘Regional Stratigraphic Chart of Heilongjiang Province’’ 1979). Recently the formation has been more precisely assigned to the late middle Miocene–early late Miocene (approximately 12–11 Ma), by correlation with well-dated fossil macro and microfloras in East Asia (Liu et al. 1995, 1996; Liu 1998; Leng 1997, 2000a, b) and by the correlation of vertebrate faunas, including various fossil freshwater fish (Chang et al. 1996) and fossils of terrestrial mammals (Qi 1992). Results Systematic palaeobotany Descriptions of Onagraceae pollen grains and tetrads include diagnostic features observed under LM and SEM. Terminology for description follows mostly Punt et al. (2007) and Hesse et al. (2009). The systematic section starts with the family Onagraceae, and genera appear in alphabetical order. Species descriptions are based on 2–20 grains/tetrads studied under both LM and SEM. Family Onagraceae Juss. Genus Circaea L. Circaea sp. 1 (Fig. 1a–i). Description—Pollen, monad, triporate, shape oblate, outline of central body circular in polar view, elliptic in equatorial view, equatorial diameter 35–43 lm under LM, 29–38 lm under SEM; apertures prominent, markedly protruding, annulate with an atrium, constricted at the base, ectoaperture more or less circular; exine 1.8–2.1 lm thick (LM), nexine thinner or as thick as sexine, sexine markedly thickened at base of apertures (LM), nexine homogeneous, sexine composed of small closely packed globular elements forming a coarse spongy-paracrystalline structure (SEM), sculpturing consisting mostly of globular but also rod-like to elongate elements, elements of similar configuration on both proximal and distal faces of pollen and around apertures, occasional large globular structures seen on surface (see arrow A, Fig. 1c); proximal face with remains of a single or two attachment points (see arrow B, Fig. 1c) of viscin threads (SEM). Remarks—The pollen morphology of extant Circaea species has been described using LM, SEM, and TEM by Skvarla et al. (1978), Patel et al. (1984), Praglowski et al. (1994), and Punt et al. (2003). The general outline and shape of this fossil pollen type, in combination with the prominent apertural protrusions, sculpturing consisting mostly of globular but also rod-like to elongate elements and its very few viscin threads, suggests that this pollen type belongs either to Clarkia or Circaea (compare with Table 2 in Patel et al. 1984). On the other hand, the size ratio of the central body of the pollen to the protruding apertures corresponds to what has been reported by both Patel et al. (1984) and Punt et al. (2003) for Circaea. In Clarkia, the apertural protrusions are much larger and broader at the base. Clarkia pollen usually has more viscin threads than Circaea pollen. Also, the ‘‘large’’ globular structures as noted on the surface of the fossil pollen from China are apparently common on pollen from extant species of Circaea (Skvarla et al. 1978). Fossil record—Before this study no fossil record existed for pollen assigned to Circaea. Zetter and Keri (1987) suggested that their fossil pollen from the Miocene of Austria, assigned to the form-genus Corsinipollenites, might have botanical affinities to Circaea, but our revaluation of the material has shown that it should be placed in the extant genus Ludwigia. Today, there are approximately seven species of Circaea in China (Flora of China Editorial Committee 2008). Our data show that this genus was already present in China during the Miocene. Genus Circaea L. Circaea sp. 2 (Fig. 2a–h). Description—Pollen, monad, tricolporate, shape oblate, convex triangular in polar view, elliptic in equatorial view, equatorial diameter 32–37 lm under LM, 30–34 lm under SEM; apertures prominent, slightly protruding, annulate with an atrium; exine 2.3–2.6 lm thick (LM), nexine slightly thinner than sexine, no thickening of sexine in apertural regions, nexine thickened at base of apertures (LM), nexine homogeneous, sexine composed of small closely packed globular elements forming a coarse spongyparacrystalline structure (SEM), sculpturing consists of globular to rod-like elements, elements of same configuration on both proximal and distal faces of pollen and around the apertures (SEM), occasional large globular structures seen on surface (see arrow, Fig. 2c); single (or few) viscin thread on proximal side, attachment of viscin threads slightly thickened, viscin threads smooth, 0.4–0.5 lm in diameter (SEM). Remarks—The justification for assignment of this pollen type to Circaea is the same as for Circaea sp. 1. Note also the ‘‘large’’ globular structures on the surface of this fossil pollen type. Part of a single smooth viscin thread is also preserved on this pollen. The Circaea sp. 1 differs from Circaea sp. 2 in having a thicker exine, the apertures are less protruding, and the sculpturing consists only of 123 674 ´ F. Grımsson et al. Fig. 1 a–i Circaea sp. 1. a, b LM micrographs. c–i SEM micrographs. a, b Polar view. c, e, g, i Proximal face. d, f, h Distal face. c. Arrow (A) indicating attachment points of viscin threads, and arrow (B) indicating large globular tectal element. e Close-up of aperture, frontal view. f Close-up showing surface around aperture. g Close-up of aperture, polar view. h Close-up showing centre of distal face. i Close-up showing centre of proximal face. Scale bar 10 lm in (a– d), 1 lm in (e–i) 123 Diverse fossil Onagraceae pollen from a Miocene palynoflora 675 Fig. 2 a–h Circaea sp. 2. a LM micrograph. b–h SEM micrographs. a Polar view. b, d, f, h Distal face. c, e, g Proximal face. c Arrow indicating a large globular tectal element. d Close-up of surface around aperture. e Close-up of aperture, frontal view. f Close-up showing centre of distal face. g Close-up of viscin thread. h Brake showing pollen wall. Scale bar 10 lm in (a–c), 1 lm in (d–h) 123 676 ´ F. Grımsson et al. globular to rod-like elements that are not as elongated as in Circaea sp. 1. Genus Epilobium L. Epilobium sp. (Fig. 3a–j). Description—Pollen, monad, triporate, shape oblate, convex triangular in polar view, elliptic in equatorial view, equatorial diameter 89–93 lm under LM, 72–81 lm under SEM; apertures prominent, annulate with an atrium, incorporated into central body of pollen, endoaperture lalongate, ectoaperture more or less circular (LM) with irregular margins (SEM); exine 2.5–2.9 lm thick under LM, 2.2–2.5 under SEM, nexine thinner than sexine, sexine markedly thickened in aperture regions, nexine dividing from sexine at base of aperture (LM), nexine homogeneous and of constant thickness, nexine 0.7–0.8 lm thick (SEM), sexine composed of small closely packed globular elements forming a coarse spongy-paracrystalline structure, sexine 1.4–1.7 lm thick (SEM), sculpturing consisting of rod-like and globular elements (SEM); viscin threads on proximal side, viscin threads smooth, 0.3–0.5 lm in diameter (SEM). Remarks—The pollen morphology of extant Epilobium species has been described using LM, SEM, and TEM by Skvarla et al. (1978), Patel et al. (1984), Praglowski et al. (1994), Punt et al. (2003), and Makbul et al. (2008). The shape and outline of the pollen, and, especially, the configuration of apertures, in combination with sculpturing consisting of rod-like and globular elements and smooth viscin threads (Skvarla et al. 1978) indicate that this pollen type belongs to Epilobium (compare with Table 2 in Patel et al. 1984). The fossil Epilobium pollen from China corresponds well to all pollen within the Epilobium types/ groups as defined by Punt et al. 2003. Fossil record—Previously there were few records of fossil pollen assigned to the genus Epilobium, and most were reported from Quaternary sediments (cf. Martin 2003). Additional records assigned to the fossil pollen form-genus Corsinipollenites (Thiergart) Nakoman, and believed to have botanical affinities to Epilobium, include pollen from the Oligocene of Antarctica (Mildenhall 1989), the Oligocene to Pleistocene of New Zealand (Pocknall 1982; Daghlian et al. 1984; Mildenhall and Pocknall 1989), and the Pleistocene of Central Europe (Krutzsch 1968). Most of these reports were based on LM observation only, making it difficult to compare with our Chinese material, and with pollen of extant taxa. The Epilobium pollen from China is the first fossil pollen assigned to the extant genus based on combined LM and SEM observations. This is also the earliest fossil record of this genus from China. Today, there are approximately 33 species of Epilobium in China (Flora of China Editorial Committee 2008). Genus Ludwigia L. Ludwigia sp. 1 (Figs. 4a–h, 5a–i, 6a–h, 7a–h). Description—Pollen, tetrad, diameter of tetrads 50–67 lm under LM, 42–57 lm under SEM; pollen tricolporate (pororate or with irregular ectoapertures), shape oblate, convex triangular in polar view, elliptic in equatorial view, equatorial diameter 32–42 lm under LM, 27–38 lm under SEM; apertures distally oriented and prominent, apertures annulate with an atrium (LM); exine 2.0–2.3 lm thick, nexine thinner than sexine (LM), nexine homogeneous, sexine composed of small closely packed globular elements forming a coarse spongy-paracrystalline structure, sculpturing consists of rugulate to shortly striate elements (SEM), elements often shorter on proximal face of pollen especially around attachment points of viscin threads, mostly elongated around apertures; viscin threads originating on proximal face of pollen, attachment points are thickened and fused by three or more elongated elements, viscin threads smooth, diameter of threads frequently varying, generally 3.5–7.0 lm, narrower threads sometimes originating from thickened parts, threads sometimes twisted together (SEM); pollen in tetrads, pollen mainly connected in apertural areas with elongated sexine elements that form bridges between adjacent apertures (see arrow, Fig. 4e), short internal bridges between pollen also visible outside apertural regions (see arrows, Figs. 4g, 5i) (SEM). Remarks—The pollen morphology of extant Ludwigia species has been described using LM, SEM, and TEM by Skvarla et al. (1978), Praglowski et al. (1983), Patel et al. (1984), and Punt et al. (2003). Onagraceae pollen tetrads occur only in Camissonia, Ludwigia, Boisduvalia, and Epilobium (Table 2 in Patel et al. 1984). The tetrads, in combination with the configuration of the apertures, the type of viscin threads and their way of attachment, the rugulate to shortly striate sculpturing, and the arrangement of connections between pollen of the tetrads, show that these tetrads belong to Ludwigia (Praglowski et al. 1983; Patel et al. 1984). This Ludwigia type pollen frequently occurs as tetrads in the Chinese sample. Until now we have only found a single monad that we can assign to this fossil pollen type. Fossil record—Fossil Onagraceae pollen assigned to the extant genus Ludwigia (and to the former genus Jussiaea that was merged into Ludwigia; Hara 1953) is relatively frequent compared with that of all other genera of the family. Fossil Ludwigia pollen has been reported from the Paleocene and Eocene of China (Song et al. 2004), the Eocene to Oligocene of Canada (Rouse 1962, 1977; Piel ´ ´ 1971), the Eocene of Columbia (Gonzales-Guzman 1967) and Russia Far East (Brattseva 1969), the Oligocene to Pliocene of the USA (Rachele 1976; Traverse 1955), the 123 Diverse fossil Onagraceae pollen from a Miocene palynoflora 677 Fig. 3 a–j Epilobium sp. a–d LM micrographs. e–j SEM micrographs. a, b Polar view. c, d Equatorial view. c Focus through main body of pollen. d Focus on aperture. e, g, i Proximal face. f, h, j Distal face. g Close-up showing centre of proximal face. h Brake showing pollen wall. i Close-up of viscin thread. j Close-up of surface around aperture. Scale bar 10 lm in (a–f), 1 lm in (g–j) 123 678 ´ F. Grımsson et al. Fig. 4 a–h Ludwigia sp. 1. a LM micrograph. b–h SEM micrographs. a, b Tetrad. c Other side of same tetrad as in (b). d Close-up of surface around aperture. e Close-up of two adjacent apertures showing connecting bridge (note arrow). f Close-up showing centre of distal face of a pollen. g Close-up of viscin thread, arrow indicating internal bridges between central bodies of adjacent pollen grains. h Close-up showing centre of distal face of a pollen. Scale bar 10 lm in (a–c), 1 lm in (d–h) 123 Diverse fossil Onagraceae pollen from a Miocene palynoflora 679 Fig. 5 a–f Ludwigia sp. 1. a LM micrograph. b–f SEM micrographs. a, b Tetrad. c Close-up of two adjacent apertures showing connecting bridge. d Close-up showing many long viscin threads. e Close-up of crossing viscin threads. f Close-up showing centre of distal face of a pollen. g–i Ludwigia sp. 1. g LM micrograph. h, i SEM micrographs. g, h Tetrad. i Close-up showing equatorial regions of pollen grains, arrow indicating an internal bridge between central bodies of adjacent pollen grains. Scale bar 10 lm in (a, b, g, h), 1 lm in (c–f, i) 123 680 ´ F. Grımsson et al. Fig. 6 a–h Ludwigia sp. 1. a LM micrograph. b–h SEM micrographs. a–c Tetrad. c Other side of same tetrad as in (b). d Close-up of viscin threads, showing narrow parts. e Close-up of several viscin threads. f Close-up showing normal twisted viscin threads and narrower threads originating from thickened parts. g Close-up of two adjacent apertures showing connecting bridge. h Close-up of equatorial region of a pollen. Scale bar 10 lm in (a–c), 1 lm in (d–h) 123 Diverse fossil Onagraceae pollen from a Miocene palynoflora 681 Fig. 7 a–h Ludwigia sp. 1. a LM micrograph. b–h SEM micrographs. a Polar view. b, d, f Distal face. c, e, g, h Proximal face. d Close-up of surface around aperture. e Close-up showing centre of proximal face. f Close-up showing centre of distal face. g Arrow indicating attachment point of viscin thread. h Close-up of surface around aperture. Scale bar 10 lm in (a–c), 1 lm in (d–h) 123 682 ´ F. Grımsson et al. Fig. 8 a–h Ludwigia sp. 2. a LM micrograph. b–h SEM micrographs. a Polar view. b, d, f Distal face. c, e, g, h Proximal face. d Close-up of surface around aperture. e Close-up of surface around aperture. f Close-up showing centre of distal face. g Close-up of viscin thread. h Close-up showing centre of proximal face. Scale bar 10 lm in (a–c), 1 lm in (d–h) 123 Diverse fossil Onagraceae pollen from a Miocene palynoflora 683 Miocene of Mexico (Graham 1976a, b, 1987), and the Miocene/Pliocene of Guatemala (Graham 1998). There are also several fossil pollen records assigned to the formgenera Corsinipollenites and Jussitriporites that are thought to have botanical affinities to Ludwigia. These include pollen from the Paleocene to Oligocene of China (Zheng et al. 1999), the Paleocene/Eocene of Argentina (Quattrocchio and Volkheimer 1990; Quattrocchio et al. 1997), the Eocene of California (Frederiksen 1983) and ´ ´ Columbia (Gonzales-Guzman 1967), and the Eocene to Pliocene of Europe (Krutzsch 1968, 1970). Most of these determinations were based on LM observations only, and some reports lack micrographs and/or descriptions, making further comparison with our fossil Ludwigia type from China and other extant taxa impractical. There are currently approximately nine extant species of Ludwigia in China. Of these, three species disperse their pollen in tetrads; L. octovalvis (Jacquin) P. H. Raven, L. perennis L., and L. prostrate Roxburgh (Flora of China Editorial Committee 2008). The tetrads presented here are the first Ludwigia tetrads reported from the fossil record of China (Zheng et al. 1999; Song et al. 2004; Wang 2006), which confirms that this pollen dispersal type was already present in Chinese Ludwigia species during the Miocene. Ludwigia sp. 2 (Fig. 8a–h). Description—Pollen, monad, tricolporate, shape oblate, convex triangular in polar view, elliptic in equatorial view, equatorial diameter 42–56 lm under LM, 33–44 lm under SEM; apertures semi-prominent, apertures annulate with an atrium, apertures incorporated into general outline, endoaperture lalongate (LM); exine 1.3–1.6 lm thick, nexine thinner than sexine (LM), nexine dividing from sexine at base of aperture, sexine not thickened in apertural region (LM); sculpturing consisting of short rod-like to rugulate elements, elements shorter on proximal face of pollen especially around attachment points of viscin threads, also elongated in apertural regions (SEM); viscin threads occurring on proximal face, attachment points clearly thickened and fused by three or more elongated elements, viscin threads are smooth, 0.2–0.4 lm in diameter (SEM). Remarks—The justification for the assignment of this pollen type to Ludwigia is more or less similar to that for Ludwigia sp. 1. The main difference between the two Ludwigia types is that Ludwigia sp. 2 does not occur in tetrads, as does the Ludwigia sp. 1. In addition, the Ludwigia sp. 2 pollen is larger than those forming the Ludwigia sp. 1 tetrads, their exine is also much thinner, and the form/ shape of their apertures is different from those of Ludwigia sp. 1. The sculptural elements in the aperture regions of Ludwigia sp. 2 are more compact and less elongated than those of Ludwigia sp. 1. Discussion The pollen record of Onagraceae in China Most of the Chinese fossil pollen believed to have botanical affinities to Onagraceae have been placed under the fossil pollen form-genus Corsinipollenites (Thiergart) Nakoman. Over the past years, several different formspecies have been established for individual pollen records from China, but Zheng et al. (1999) summarized all these findings and grouped Chinese Cenozoic Onagraceae pollen into four fossil form-species. According to Zheng et al. (1999), Corsinipollenites triangulus (Zakl.) Ke et Shi occurs throughout most of the Paleogene and Neogene of China, C. ludwigioides Krutzsch occurs in the Paleocene to the Oligocene, C. hungaricus subsp. hungaricus (Kedves et ´ Adorjan) Sun et Li occurs only in the Eocene, and C. tetraporus Ke et Shi is confined to the Oligocene (Zheng et al. 1999). Based on available pollen records from China (Zheng et al. 1999; Song et al. 2004; Wang 2006), it is clear that Onagraceae pollen grains have their first appearance in the Paleocene in this part of Asia. Despite this, the generic diversity and divergence of Cenozoic Onagraceae pollen in China remains unknown. The validity of various fossil Onagraceae pollen groups and form species In the palaeo-palynological literature, three-pored pollen with a botanical affinity to Onagraceae has been assigned to various artificial form-groups, particularly fossil pollen form-genera. These include, among others, Pollenites oculus noctis Thiergart (Thiergart 1940), Corsinipollenites (Nakoman 1965; Zetter and Keri 1987), Onagra´ ceaepollis M. Kedves et A. M Adjorjan (Kedves and ´ ´ ´ Adorjan 1966), Jussitriporites A. E. Gonzales-Guzman ´ ´ (Gonzales-Guzman 1967; Pares Regali et al. 1974; Muller 1981), Crassiorites Zamaloa et Romero (Zamaloa and Romero 1990; Zetter et al. 1999), and Colombipollis ´ ¨ Sarmiento Peres in Jansonius, Hills et Hartkopf-Froder ´ rez 1992; Jansonius et al. 2002; Pocknall (Sarmiento Pe and Jarzen 2009). Of these form-genera comprising three-pored Onagraceae pollen, most authors assigned their fossil pollen to Corsinipollenites, while some also assigned the pollen to extant genera (Nakoman 1965; Eyde and Morgan 1973; Skvarla et al. 1978; Muller 1981; Zetter and Keri 1987; Martin 2003). Two-pored pollen believed to have a botanical affinity to Onagraceae has been assigned to the fossil pollen form-genus/ species Diporites aspis Pocknall et Mildenhall (Pocknall and Mildenhall 1984; Berry et al. 1990). As most of the reports on fossil Onagraceae pollen are based on LM studies only, it is difficult to compare 123 684 ´ F. Grımsson et al. individual findings, either with each other or with pollen of extant taxa. Although fossil pollen has been assigned to a particular fossil group or form-species, it is not certain if these ‘‘species’’ are in fact composed of pollen from a single taxon or several true biological taxa. It is also uncertain whether fossil pollen from the same biological species has been assigned to different form-species. These uncertainties have mainly resulted from the use of LM observation alone, which often cannot distinguish Onagraceae pollen grains/tetrads into genera. These uncertainties can only be resolved by applying additional methods, e.g. the single-grain technique, by which a single Onagraceae pollen/tetrad is observed under both LM and SEM. As a result, most of the previously reported palynological record of Onagraceae yields reliable information at the family level only, until new studies using SEM, or both LM and SEM, are carried out on the same material. The earliest fossil records of Onagraceae pollen and a summary of the macrofossil record The earliest definite fossil Onagraceae pollen is known from the Upper Cretaceous (Maastrichtian) of Venezuela and Colombia, South America (Colombipollis; Sarmiento ´ Perez 1992; Pocknall and Jarzen 2009). It is uncertain whether or not other Upper Cretaceous records from western and eastern North America (Stover 1964; Drugg 1967; Chmura 1973) really belong to Onagraceae. In Eurasia, Onagraceae pollen has its first occurrence in the Paleocene of Central Europe and eastern Asia (Krutzsch 1968; Zheng et al. 1999). Further south, in Australasia, Onagraceae pollen are known to have occurred from the Eocene (Martin 2003). Following the first occurrence of Onagraceae pollen on the above-mentioned continents, pollen grains belonging to this family apparently occur, though rarely, from the Eocene or the Oligocene to the Holocene (Eyde and Morgan 1973; Skvarla et al. 1978; Muller 1981; Martin 2003). The macrofossil record of Onagraceae is far from as ‘‘rich’’ as the pollen record, and is composed solely of fruits and seeds. Until now only three of approximately 17 extant genera have been reported as macrofossils from Cenozoic sediments of the Northern Hemisphere. Many of these fossils have been assigned to Ludwigia, most of which occur in European sediments, including the Oligocene to Pliocene of Germany (Mai 1985, 1989, 1997, 1998, ¨ 2000, 2001; Mai and Walther 1988; Mai and Wahnert 2000), the Miocene to Pliocene of Poland, the Czech Republic, and Slovakia (Buzek et al. 1985; Knobloch 1988; Zastawniak 1992), the Miocene of Denmark (Friis 1985), and the Pliocene of Italy (Mai 1995). Circaea fruits/seeds are known from the Oligocene to Miocene of Russia, east of the Urals (Dorofeev 1963, 1969), and the Pliocene of Poland (Szafer 1947) and western Russia (Nikitin 1957). Until now, Epilobium fruit/seeds have only been reported from the Pliocene of western Eurasia (Mai 1985). Interestingly, all three genera represented by the Chinese fossil pollen are also the only known Onagraceae genera represented by macrofossils in Eurasia, providing further support of their taxonomic identification. Early steps in tracing the phytogeographic history of Onagraceae genera Of extant Onagraceae, only a few genera have a wide geographical distribution. Epilobium is the only cosmopolitan genus. Chamerion and Circaea are both north temperate genera, being most diverse in Asia, and Ludwigia is pantropical, but extending into temperate North America and Asia. Most other genera are confined to parts of North America and/or Central America (Calylophys, Gaura, Gongylocarpus, Hauya, Lopezia, Stenosiphon, and Xylonagra), or have a distribution range extending from North America to South America (Camissonia, Clarkia, Gayophytum, and Oenothera). One exception is Fuchsia, which is dispersed from southern North America to South America, but also occurs in New Zealand and on Tahiti (Levin et al. 2003; Wagner et al. 2007). Most of the genera are unknown from the fossil record (see above), and morphological and molecule-based phylogenetic studies have shown that many of the extant southern North American, Central American and South American genera are situated far from the base of the family tree (e.g. Oenothera, Stenosiphon, Gaura, Calylophus, and Camissonia; Levin et al. 2003; Ford and Gottlieb 2007). This suggests that they may have evolved late in the evolutionary history of the family, and would therefore not be found in Upper Cretaceous or Paleogene sediments. Katinas et al. (2004) have also shown that many of the endemic North American Onagraceae genera (e.g. tribe Onagreae) evolved and diversified under special local environmental (often arid) conditions, reaching a climax point in post-Oligocene times, especially during the Miocene and Pliocene. This could also explain the complete absence of Onagreae genera from Eurasian Cenozoic sediments. Of the three genera recorded in this study from the Miocene of north-east China (Circaea, Epilobium, Ludwigia), phylogenetic studies seem to agree that Ludwigia (tribe Jussiaeeae) has the most basal position (most ancient) in the family tree, and is a sister group to the remaining Onagraceae (Levin et al. 2003; Ford and Gottlieb 2007). It would therefore not be surprising for Ludwigia to have the earliest fossil record of all extant Onagraceae. The LM-based palynological record of Ludwigia (see above) suggests that it already had a wide geographical distribution in the Paleocene, and was most 123 Diverse fossil Onagraceae pollen from a Miocene palynoflora 685 likely growing in the Americas and East Asia at this time, but occurring first in Europe in the Eocene. Previous pollen records also suggest that Ludwigia prevailed on these continents until the present day, but this needs to be supported further by SEM or combined LM and SEM investigations. The genus Circaea (tribe Circaeeae) is thought to be the sister group of Fuchsia (tribe Fuchsieae), and the Circaea– Fuchsia clade occurs early/low in the family tree (Levin et al. 2003; Ford and Gottlieb 2007). Based on molecular clock analyses, the clade was believed to have already diverged from Hauya (tribe Hauyeae) by the early Eocene, between 53–52 Ma, with subsequent divergence between Fuchsia and Circaea in the middle Eocene, at approximately 41 Ma (Berry et al. 2004; Xie et al. 2009). Until now, there have been no records of fossil Circaea pollen, and the scarce macrofossil record is confined to the Oligocene of Russia (east of the Urals) and the Pliocene of Europe (see above). The American Eocene origin of Circaea, as hypothesized by Xie et al. (2009), needs testing by investigation of fossil records. However, subsequent dispersal of Circaea to Eurasia via the Bering land bridge, as also hypothesized by Xie et al. (2009), would correspond to the earliest records of the family in the Oligocene of Russia (east of the Urals). Epilobium (tribe Epilobieae) is part of the Epilobieae– Onagreae clade, positioned in the most recently divergent branch of the family tree (Levin et al. 2003; Ford and Gottlieb 2007). If its phylogenetic position is correlated with molecular clock analyses of more ‘‘primitive’’ or older clades, for example the Jussiaeeae, Hauyeae, Fuchsieae, and Circaeeae (Berry et al. 2004; Xie et al. 2009), it is likely that the first expected fossil record of Epilobium would not be older than the early Oligocene. The fossil record of Epilobium however remains scarce, and the few pollen records are mostly ‘‘uncertain’’ LM-based studies (for SEM see Daghlian et al. 1984) from the Oligocene to Pleistocene of New Zealand, the Oligocene of Antarctica, and the Pleistocene of Europe. the Miocene Epoch these genera were already co-occurring in a single flora in north-east China. Before this study, Ludwigia pollen were reported from palynological records using LM only. This is the first study describing fossil Ludwigia pollen using a combination of LM and SEM for single pollen/tetrads. The Epilobium pollen reported here is the first pre-quaternary pollen assigned to the extant genus, and is also the first fossil Epilobium pollen recorded from China. The Circaea pollen is the first fossil pollen assigned to this extant genus. Furthermore, we also confirm that Onagraceae pollen can be accurately identified at least to the generic level, and can therefore be used to trace readily the origin and past distribution patterns of extant genera. This warrants future studies on ambiguous fossil Onagraceae pollen. With this and future studies on fossil Onagraceae pollen, the origin and evolution of the family, including earliest records of particular genera, time of divergence between genera, distribution patterns, migration routes, and diversification and extinction events, will finally become clear. Acknowledgments This study was funded by the FWF (Austrian Science Fund) with a grant to FG (project number M 1181-B17), and with a grant to QL from the CAS/SAFEA International Partnership Program for Creative Research Teams, the Pilot Project of Knowledge Innovation, CAS (project number KZCX2-YW-105). References Berry PE, Skvarla JJ, Partridge AD, Macphail MK (1990) Fuchsia pollen from the tertiary of Australia. Aust Syst Bot 3:739–744 Berry PE, Hahn WJ, Sytsma KJ, Hall JC, Mast A (2004) Phylogenetic relationships and biogeography of Fuchsia (Onagraceae) based on noncoding nuclear and chloroplast DNA data. 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