Oligocene Limnobiophyllum (Araceae) from the Central Tibetan Plateau and Its Evolutionary and Palaeoenvironmental Implications

Journal of Systematic Palaeontology

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Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau and its evolutionary and palaeoenvironmental implications

Shook Ling Low, Tao Su, Teresa E. V. Spicer, Fei-Xiang Wu, Tao Deng, Yao-Wu Xing & Zhe-Kun Zhou

To cite this article: Shook Ling Low, Tao Su, Teresa E. V. Spicer, Fei-Xiang Wu, Tao Deng, Yao-Wu Xing & Zhe-Kun Zhou (2019): Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau and its evolutionary and palaeoenvironmental implications, Journal of Systematic Palaeontology, DOI: 10.1080/14772019.2019.1611673 To link to this article: https://doi.org/10.1080/14772019.2019.1611673

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Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau and its evolutionary and palaeoenvironmental implications a a,b c d,e b,d a Shook Ling Low , Tao Su , Teresa E. V. Spicer , Fei-Xiang Wu , Tao Deng , Yao-Wu Xing and Zhe-Kun Zhoua,f aCAS Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, China; bUniversity of Chinese Academy of Sciences, Beijing 100049, China; cState Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy Sciences, Beijing 100093, China; dKey Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, China; eCenter for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Beijing 100101, China; fKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China (Received 16 October 2018; accepted 12 April 2019)

The extinct genus Limnobiophyllum (Araceae) has been considered a tentative link between the Aroideae and Lemnoideae subfamilies of Araceae. General understanding of morphological character evolution among these subfamilies has been limited due to the lack of preserved key structures in fossils such as infructescences. In this study, a new fossil species, Limnobiophyllum pedunculatum Low, Su & Xing sp. nov., is reported based on unusually complete specimens with intact leaves, stolon and attached infructescence and seeds from the late Oligocene of central Tibet, China. It represents the first convincing Limnobiophyllum fossil from the Tibetan Plateau and the first well- documented occurrence from east Asia. Its phylogenetic position was inferred using a matrix of 56 morphological characters and 5226 gene sequences of 41 taxa. Phylogenetic inference based on the matrix suggests that Limnobiophyllum is sister to Cobbania, as well as to the remaining extinct and living genera within the Araceae subfamily Lemnoideae. Reconstruction of vegetative and reproductive character evolution confirms that Limnobiophyllum possessed intermediate characters, especially for infructescences, between the subfamilies Lemnoideae and Aroideae. Within Lemnoideae, both vegetative and reproductive characters show clear reduction and simplification from extinct genera to living lemnoids. These findings shed new light on the evolutionary history of the family Araceae. In addition, the discovery of this species, in association with the surrounding plant megafossil assemblage, suggests a warm, humid lowland environment in the central Tibetan Plateau during the late Oligocene, contradicting previous studies that indicated high elevation of the plateau since the early Palaeogene. However, the extinction of Limnobiophyllum might have been due to both global cooling and orogenesis. Keywords: Araceae; extinction; infructescence; Lemnoideae; Limnobiophyllum; Tibetan Plateau

Introduction characterized by having medium-sized pollen, condensed subterranean stems, unilocular ovaries and locules with Araceae sensu lato (Order: Alismatales) comprises 1–2 ovules. Linear leaves with parallel venation are approximately 3800 species within 125 genera, repre- characteristics of the subfamily Gymnostachydoideae, senting a large and early diverging monocot clade while non-linear expanded leaf blades represent the (Boyce & Croat 2011). The modern distribution of this subfamily Orontioideae (Mayo et al. 1997). The family extends from humid tropics to temperate forests Aroideae are characterized by conspicuous or flag-like and from aquatic to arid habitats. However, more than spathes, non-linear expanded leaf blades and basal 95% of the species are in the ever-wet or perhumid placentation (Mayo et al. 1997). In contrast, the tropics. According to the current classification, three Lemnoideae are aquatic and possess small bodies cor- main clades are recognized within Araceae: proto-aroids responding to leaves and stems, with or without ven- (Gymnostachydoideae and Orontioideae), lemnoids ation and roots, and the reproductive structures are (Lemnoideae) and eu-aroids (Aroideae) (Mayo et al. enclosed in ‘pouches’ (Daubs 1965,p.4).However, 1997; Cusimano et al. 2011). These clades are charac- the evolutionary history of morphological characteris- terized by their unique morphologies. Proto-aroids are tics in this family is still unclear. This is probably

Corresponding authors. Emails: [email protected]; [email protected]

Published online 17 Oct 2019 2 S. L. Low et al. because fertile structures have only rarely been pre- study of their morphological characteristics, Pistia is served in the fossil record. basal to other lemnoids (Stockey et al. 1997, 2016; Lemnoideae represents a well-supported monophyletic Gallego et al. 2014), which might simply be because and near-basal divergent lineage compared to the proto- they share several characters adaptive for aquatic life. aroids (Chase 2004; Cabrera et al. 2008; Cusimano Hence, when these morphological studies are comple- et al. 2011; Nauheimer et al. 2012; Henriquez et al. mented with molecular evidence, their phylogenetic pos- 2014). Unlike proto- and eu-aroids, the Lemnoideae are ition is rearranged with a better solution. For instance, in free-floating aroids and are the most widely distributed a study of the extinct genus Cobbania using a combined aroids around the world (Landolt 1986). Furthermore, the matrix of morphological characters and gene sequences, lemnoids are unique due to their simplified and reduced Stockey et al. (2016) successfully resolved the phylogen- overall morphologies, and it is noteworthy that they etic position between Lemnoideae and Pistia. include the smallest angiosperms in the world (Wolffia This study reports fossils of well-preserved free-float- Horkel ex Schleiden, 1844; Simpson 2006). The place- ing Limnobiophyllum leaves coupled with an infructes- ment of Lemnoideae between the proto- and eu-aroids cence from the middle–upper part of the Dingqing based on molecular data has received much attention since Formation from the late Oligocene of the central their unique morphologies are not similar to either of these Tibetan Plateau. The phylogeny was inferred using groups (Mayo et al. 1997). The lack of supportive mor- matrices including both morphological and molecular phological evidence linking these three clades may be due characters. The discovery of this fossil species could to the extinction of the stem species in the relictual line- contribute to a more comprehensive knowledge of char- ages. Therefore, fossils are crucial to understanding the acter evolution in Lemnoideae. evolutionary transitions among the subfamilies. Meanwhile, the extant genera of Lemnoideae are typ- Kvacek (1995) suggested that the extinct genus ically lowland water-associated species, growing only in Limnobiophyllum Krassilov, 1973 might serve as a ten- warm temperate to tropical regions that are below 1500 tative link between Pistia (Aroideae) and Spirodela m above sea level (elevations of certain studied species (Lemnoideae). The habit and vegetative characters, such are shown in Wang et al. [2010] and Tang et al. as high-order venations and root features, are like those [2014]). Therefore, the discovery of this new fossil spe- of Aroideae; and the pigment cells, campylodromous cies from a present-day altitude of 4683 m above sea venation, air chambers and associated seeds of level has great implications for the palaeoelevation of Limnobiophyllum resemble those of Lemnoideae. the central Tibetan Plateau during the late Oligocene. Another extinct genus in Lemnoideae, Cobbania Stockey, Rothwell & Johnson, 2007, also shares some characters with the subfamily Aroideae, such as the spirally arranged scars (which might represent the seeds), Material and methods and the central aerenchymatous zone in leaves with one central and two lateral major abaxial veins, which Geological setting resemble those of Lemnoideae (Krassilov & Kodrul Fossil specimens were collected from Dayu (near 2009; Stockey et al. 2016). Flowering specimens of L. Tangnu village) in the Lunpola Basin on the central ’ ’’ ’ ’’ scutatum (Dawson) Krassilov, 1973 with intact anthers Tibetan Plateau (32 01 29 N, 089 46 19 E; elevation and in situ pollen have been documented (Stockey et al. 4683 m above sea level; Fig. 1). Fossils are preserved 1997), and the associated seeds of L. expansum (Heer) as impressions in a matrix of greyish-green and red Kvacek (Kvacek 1995) were found to resemble both mudstones, interbedded with shale (Fig. 2). Based on a Spirodela Schleiden, 1839 (subfamily Lemnoideae) previous geochronological study (Sun et al. 2014) and and Pistia (subfamily Aroideae). However, other diag- fossil evidence (Deng et al. 2012), the strata were iden- nostic structures, such as attached infructescences, for tified as the middle–upper part of the Dingqing Limnobiophyllum have never been found. This indirectly Formation, which is attributed to the late Oligocene. contributes to the difficulties of taxonomic placement This date was further supported using radiometric dating of fossils, which limits our understanding of character of the Dingqing Formation in the Lunpola and Nima evolution within Lemnoideae. basins (Wu et al. 2017). Additionally, some other plant Convergent evolution might also have occurred species, such as Koelreuteria lunpolaensis (Jiang et al. among these subfamilies. For example, based on the 2019), Cedrelospermum tibeticum (Jia et al. 2019) and molecular evidence, Pistia was recognized as a genus in Sabalites tibetensis (Su et al. 2019) were also reported Aroideae (Rothwell et al. 2004; Cusimano et al. 2011; from the same strata. All of the specimens in this study Nauheimer et al. 2012). However, if based only on the are deposited in the Paleobotanical Collections of Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau 3

Figure 1. Fossil localities and historical distribution of Limnobiophyllum. A, fossil localities for L. scutatum, L. expansum and L. pedunculatum. B, Dayu in the Lunpola Basin from where L. pedunculatum was collected on the central Tibetan Plateau.

Xishuangbanna Tropical Botanical Garden, Chinese were mounted on stubs and coated with 5 Å of gold Academy of Sciences (XTBGPC). using a Quorum Q150R S sputter coater. The scanning electron microscope images were captured using a Zeiss EVO LS10. In our attempt to obtain pollen, small pieces Morphological studies of matrix were isolated from the mega fossil specimens The fossils were photographed using a Nikon D700 using a laboratory needle, then cleaned with a drop of digital camera (Nikon Corporation, Tokyo, Japan). A 50% HNO3 following the procedure in Traverse (2007). Leica S8APO stereomicroscope (Leica Corporation, The matrix samples were then examined using a Wetzlar, Germany) equipped with a DFC295 digital stereomicroscope. camera was used to examine and document the detailed The terminology used in this study follows Den morphologies. For further detailed morphological obser- Hartog & Van der Plas (1970) and several other authors vations, some seeds were isolated from the holotype for the free-floating aroid fossils (Kvacek 1995; Stockey specimen (XTBGPC XZDY2-0124) using a laboratory et al. 1997, 2007, 2016; Krassilov & Kodrul 2009; needle, then cleaned by immersion in 10% HCl and Gallego et al. 2014). The fossil infructescence was 39% HF following the methods of Kerp (1990). Images compared to the lemnaceous species described by were captured using a Smart Digital Microscope (ZEISS Daubs (1965). Smart Zoom 5, Germany) and an Upright Fluorescent Microscope (ZEISS Axio Imager A2, Germany) equipped with a Zeiss AxioCam MRc, and then proc- Cladistic and phylogenetic analysis essed using the ZEN2012 software. The seeds, which To explore the phylogenetic relationships within lem- were dissected to isolate the upper and lower epidermis, noids, cladistic analyses were performed based on two 4 S. L. Low et al.

derived from seeds, while the other four characters were for the stolon (character 14), rhizome (character 15) and leaf venation pattern (characters 24 and 25) (see Supplemental material, Appendix 1). The first data set included 56 morphological characteristics. The second data set consisted of both genes (trnL, trnL-F, trnkandrbcL; see Supplemental material, Appendix 3) and morphological characters, which were concatenated using Mesquite v. 3.2 (Maddison & Maddison 2017). Lemnoid characters were then mapped onto a majority rule consensus tree (MRCT) to further investigate trait evolution within Lemnoideae.

Systematic palaeontology

Order Alismatales Brown ex Berchtold & Presl, 1820 Family Araceae Jussieu, 1789 nom. cons. (total group) Subfamily Lemnoideae Engler, 1889 Tribe Limnobiophylleae (Kvacek) Bogner, 2009 Genus Limnobiophyllum Krassilov emend. Kvacek, 1995 Limnobiophyllum pedunculatum Low, Su & Xing sp. nov. (Figs 3, 4)

Holotype. XTBGPC XZDY2-0124 (designated here) (Fig. 3). Paratypes. XTBGPC XZDY1-0017, XTBGPC XZDY2- 0095, XTBGPC XZDY2-0108, XTBGPC XZDY2-0114, XTBGPC XZDY2-0115 (Fig. 4D), XTBGPC XZDY2- 0117 (Fig. 4B), XTBGPC XZDY2-0122 (Fig. 4F), XTBGPC XZDY2-0123, XTBGPC XZDY2-0130 (Fig. 4A), XTBGPC XZDY2-0131 (Fig. 4E), XTBGPC Figure 2. Stratigraphy of Dayu in the Lunpola Basin, central XZDY2-0132 (designated here) (Fig. 4C). Tibetan Plateau. Type locality. Dayu in the Lunpola Basin of the central data sets. The first data set consisted only of morpho- Tibetan Plateau (Fig. 1). logical characters (Supplemental material, Appendix 1 Type horizon. Middle–upper part of the Dingqing &2), and the second data set consisted of both molecu- Formation, late Oligocene (Chattian); 26 23.5 Ma. lar and morphological characters. Phylogenetic relationships were inferred using maximum parsimony (MP) in Diagnosis. Plant with 1–2 widely ovate to orbicular TNT (Goloboff et al. 2008). Following Nauheimer et al. leaves, inter-connected by stolon. Leaf has numerous (2012), 41 taxa (of which two were fossils) were roots at the base. Leaf entire, with an apical notch. selected to represent each tribe of Araceae. The out- Hairy leaf surface. Leaf venation campylodromous, sec- group selection was Acorus (Acoraceae), which is ondary veins reticulate. Long peduncle arises near leaf regarded as subtending Araceae in monocot phylogeny base. Infructescence bearing about 40 seeds and sur- (Grayum 1987; Chase et al. 1993; Cabrera et al. 2008). rounded by spathe. Seeds are ellipsoid and ribbed. The morphological data matrix includes 56 characters, of Etymology. The specific epithet pedunculatum refers to which 48 were obtained from previous studies (Les et al. the infructescence with a long and visible peduncle. 1997;Stockeyet al. 1997, 2016;Cusimanoet al. 2011; Gallego et al. 2014) and eight new characters were added Description. These aquatic plants are free-floating and (described in Supplemental material, Appendix 1). Of the interconnected by stolons (Fig. 4E, F). The stolon is new characters, four (characters 51, 52, 54 and 55) were approximately 0.1 cm wide, and 1.0–2.3 cm long. The Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau 5

Figure 3. Holotype XTBGPC XZDY2-0124, Limnobiophyllum pedunculatum Low, Su & Xing sp. nov. from Dayu, central Tibetan Plateau. A, D, overview of the morphology of the fossil. B, E, attached fertile infructescence bearing ribbed and ellipsoid seeds. C, F, the infructescence, star indicates the stipe-like structure in the central part of the infructescence, and triangular shapes indicate the ribbed seeds. Abbreviations: in, infructescence; pe, peduncle; sp, spathe; vg, vegetation point. Scale bars: A, D ¼ 1 cm; B, E ¼ 0.3 cm; C, F ¼ 1mm. roots are numerous and unbranched, and extend to up to campylodromous with 6–12 primary veins in mature 8cm(Fig. 4E). The stem is not present, but a vegetation leaves (Fig. 4). Veins radiate from the leaf base, running point exists (Figs 3A, B, D, E, 4A–F). The leaf mostly parallel along the margin and joining below the apical appears as a single lamina on each individual (presence notch (Figs 4D, 5D). Secondary veins are reticulate and of 2 leaves per plant is rare; Fig. 4D), they are nearly anatomizing (Figs 4C, F, 5B). The peduncle is roughly sessile and well developed. The blade is ovate, orbicular 1.2 cm long (1/3 of the leaf length) and 0.2 cm wide, or oblong-ovate. Blade size varies from 3 to 5.5 cm in attached and arising near the base of the leaf (Fig. 3A, B, length and 2 to 6 cm in width (Fig. 4). The leaf margin D, E). Neither the inflorescence nor the pollen has been is entire and the apex has an apical notch (Fig. 5D). found. The infructescence is oblate spheroidal saucer-like, The leaf base is round (Fig. 4). The lamina surface is densely packed with roughly 40 seeds. The seeds are pubescent with extremely short (25–100 lm long), free, spirally arranged, ellipsoid and longitudinally ribbed dense and compact trichomes (Fig. 5E–H). Venation is (Fig.3B,C,E,F). The spathe (dark segment impressed 6 S. L. Low et al. Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau 7

on the rock matrix: Fig. 3C, F) encircles the infructes- vegetation reproduction by fragmentation (character 4), cence. A broken stipe-like structure can be seen in the frond/leaf type (character 5), stem type habit (character central part of the preserved infructescence (Fig. 3C). 8), stolon (character 14), type of leaf blade (character 18) The seeds are ellipsoid, 770–900 lm long and and maximum number of primary veins (character 26). 450–650 lmwide(Figs 3B, C, E, F, 6A, C–E), with an Meanwhile, Limnobiophyllum and extant lemnoids share estimated total of 6–8 ribs (i.e. 3–4 ribs on either side of four characters, namely a sheath-like petiole (character the seeds; triangular shapes as shown in Figs 3C, F, 6A, 16), pattern on the seed coat (character 54), ribbed seed C). Each seed is characterized with a basal chalaza and (character 55) and number of ribs on the seed (character an apical micropyle at opposite ends (Fig. 6C), with the 56). Within fossils, the characters developed for the operculum at the posterior end. The micropyle shows a genus Cobbania are absence of turion (character 30) and distinct micropylar pole (Fig. 6D, F, G). type of placentation (character 43). The characters developed for the genus Limnobiophyllum are the floral position (character 35) and locular-type anthers (character Results 46; Fig. 8). Limnobiophyllum leaves (4 cm) are larger with well- Phylogenetic analyses developed venation and roots, compared to the veinless Cladistic analysis of the first data set, exclusively based and rootless minute fronds present in both Wolfiella and on a matrix of 56 morphological characters, generated Wolffia (Daubs 1965). The peduncle of L. pedunculatum eight most parsimonious trees (MPTs) of length 304 is about one-third of the leaf length (Fig. 3A, B, D, E); steps (consistency index [CI] ¼ 0.30, retention index this character is reduced to just a vegetation point in liv- [RI] ¼ 0.52; Fig. 7A). In the strict consensus tree (SCT), ing lemnoids (Bogner 2009). In Limnobiophyllum, the 36 nodes collapse to form a large polytomy of proto- infructescence is surrounded by a spathe (Fig. 3C, F); Araceae, Lemnoideae and eu-Araceae. The free-floating however, this does not occur in living lemnoids, where aquatic genera, including Pistia, Limnobiophyllum and the reproductive structures are surrounded by a mem- Cobbania, formed polytomies; only the living lemnoids branous envelope and are enclosed within the budding formed a group (MP ¼ 83%). pouches in the tribe Lemnoideae (Bogner 2009)or Phylogenetic analysis of the second data set, including emerging from the furrow on the dorsal surface in the both molecular and morphological characters, revealed tribe Wolffieae (absent with membranous envelope) that the positions of all genera were consistent with the (Den Hartog & Van der Plas 1970; Sree et al. 2015). current classification of Araceae. All described extinct Associated smooth seed coats are reported for Cobbania floating aroids diverged early in Lemnoideae. The analysis (Stockey et al. 2007, 2016), while an attached infructes- ¼ resulted in three MPTs of length 4196 steps (CI 0.61, cence with ribbed seed coats is present in the ¼ RI 0.58; Fig. 7B). A large polytomy formed in the eu- Limnobiophyllum described here (Figs 3C, F, 6A, C). aroids clade of the SCT, where 15 nodes were collapsed. Ribbed seeds are also characteristic of living lemnoids The fossils clearly fall into the genus and Lemnospermum pistiforme Nikitin (Kvacek 2003). Limnobiophyllum, where L. scutatum diverged earlier Cobbania and Limnobiophyllum have numerous seeds than L. expansum and L. pedunculatum (Fig. 7C). that are spirally arranged, whereas living lemnoids only Limnobiophyllum and Cobbania (C. corrugata and C. have a total of 1–4 seeds (Fig. 9; Daubs 1965; Den hickeyi) are sister clades that diverged earliest in Hartog & Van der Plas 1970). Lemnoideae. Both morphological and molecular analyses suggest that these fossils fall under Lemnoideae. Discussion Character evolution The TNT analysis indicated that Cobbania shares seven Systematic assignment characters with Limnobiophyllum and the rest of the The newly described fossils from the late Oligocene taxa in Lemnoideae, i.e. growth habitat (character 1), Dingqing Formation are well preserved. The fossils

3 Figure 4. Paratypes, Limnobiophyllum pedunculatum Low, Su & Xing sp. nov. from Dayu, central Tibetan Plateau. A–C, round base on leaves of L. pedunculatum with campylodromous venations radiating from the base: A, XTBGPC XZDY2-0130; B, XTBGPC XZDY2-0117; and C, XTBGPC XZDY2-0132. D, overlapping of small leaf with large leaf, XTBGPC XZDY2-0115. E, F, plant with single leaf, inter-connected by stolon: E, XTBGPC XZDY2-0131; F, XTBGPC XZDY2-0122. Abbreviations: an, apical notch; rt, roots; rv, reticulate venations; sl, second leaf; st, stolon; vg, vegetation point. Scale bars: A, C ¼ 1 cm; B, D ¼ 2 cm; E ¼ 2.5 cm; F ¼ 1.5 cm. 8 S. L. Low et al.

Figure 5. Leaf morphology of Limnobiophyllum pedunculatum Low, Su & Xing sp. nov. A, primary veins parallel running and curving to the marginal vein of L. pedunculatum. B, reticulate venations of secondary veins. C, radiating secondary veins. D, the primary veins curving and joining below apical notch. E, F, distribution of trichomes on surface of leaves. G, distribution of trichomes between the primary veins. H, dark-coloured trichomes. Abbreviations: an, apical notch; rv, reticulate venations; tr, trichomes. Scale bars: A, B ¼ 1 cm; C, E ¼ 1 mm; D, F, G ¼ 0.5 mm; H ¼ 50 mm. Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau 9 10 S. L. Low et al.

include an attached fertile infructescence with seeds, Supplemental material, Table S1). The fossils in this which have not previously been confirmed for this study differ from previously reported species owing to genus. The infructescence is oblate spheroidal saucer- the unusual presence of a single ovate to oblong leaf like and densely packed with the seeds freely attached per individual, and which are interconnected by a stolon to the spadix. The combination of these characters (Fig. 4E, F). So far, only one fossil (XTBGPC XZDY2- clearly places the fossils within Araceae (Fig. 3C, F; 0115) was observed with two leaves – a small leaf over- Mayo et al. 1997). As the plants are interconnected by lapping a larger leaf (arrow in Fig. 4D). However, in horizontal stolons with roots extending from the vegeta- the Paleocene species, L. scutatum,3–4 ovate leaves per tion point (Fig. 4E, F), the fossil specimens appear to be plant are attached in a small rosette (Stockey et al. free-floating aquatic plants, which fall under the sub- 1997); while only up to two leaves were reported per family Lemnoideae. Within Lemnoideae, there are five plant in the Miocene European species L. expansum extant genera (Lemna, Spirodela, Landoltia, Wolffia and (¼ Hydromystria expansa) (Hantke 1954; Kvacek Wolffiella; Landolt & Kandeler 1987; Les et al. 1997) 1995). The presence of a single leaf per plant and inter- and two extinct genera (Cobbania and connection by a stolon in our fossils (Fig. 4E, F) might Limnobiophyllum; Kvacek 1995; Stockey et al. 2007). reflect the reduction of the stem, which is present in The taxon in Limnobiophyllum (L. expansum)was both L. scutatum and L. expansum. The absent stem is once interpreted as a species in Hiraea and probably reduced to a vegetation point in our fossils Hydromystria (¼ Hiraea expansa Heer, 1859; (Bogner 2009). Overlapping of the small leaf with the Hydromystria expansa [Heer] Hantke, 1954; see Hantke large leaf (Fig. 4D) is likely to reflect the rosette leaf 1954), but at a later stage was assigned to arrangement of L. scutatum (Stockey et al. 1997) and L. Limnobiophyllum due to the distinctly different charac- expansum (Hantke 1954; Kvacek 1995). However, this ters possessed by the two genera (Kvacek 1995). may be a new vegetatively reproduced daughter plant, Limnobiophyllum is described as a distinct genus due to where the stolon has not fully expanded. The expansion its helically arranged rosette of one to three or four of the stolon to separate the daughter plant away from pubescent, entire-margined leaves with an apical notch, the mother plant is comparable with vegetative propaga- and steeply fork-like primary veins arising from each tion in Pistia (SLL pers. obs.). In our fossils the base of leaf base (Krassilov 1973; Kvacek 1995), characters that the leaves is round (Supplemental material, Table S1; are consistent with the fossils reported in this study. The Fig. 4), compared to the more or less cordate leaves in data compiled thus far show that the primary veins aris- L. scutatum (Stockey et al. 1997) and the mostly sub- ing from the base of the leaves is the most unique char- cordate condition in L. expansum (Hantke 1954; Kvacek acter that distinguishes this genus from other lemnoid 1995). Fossils in the current study do not possess inter- genera (Supplemental material, Table S1). primaries, which are typically present in both L. expan- Limnobiophyllum shares seven characters with Cobbania sum and L. scutatum. In addition, the fossils display a (see Results, above). However, the major primary ven- long and downward-bending peduncle subtended from ation of Ccobbania (recognized as petiolar veins in the vegetation point to support the infructescence (Fig. Stockey et al. [2016]) enters from the base of the short 3A, B, D, E). The infructescence contains ribbed ellips- petiole, and then branches into as many as 20 veins near oid seeds enclosed by the spathe (Supplemental mater- the leaf base. Additionally, the vein in the middle part ial, Table S1; Fig. 3C, F). Considering all of these of the leaves forms a rim that encircles the aerenchyma- unique characteristics, these fossils are classified as a tous zone (Stockey et al. 2007, 2016). These three new species, L. pedunculatum sp. nov. (see above). unique characters of Cobbania are found in neither living species nor studied fossil specimens. Hence, our fossils can be confidently assigned to Limnobiophyllum. Phylogenetic position of Limnobiophyllum There are only two species in the genus The MRCT and SCT analysis, with 56 morphological Limnobiophyllum (Kvacek 1995; Stockey et al. 1997; characters, indicated that Pistia is sister to the extinct

3 Figure 6. Infructescence of Limnobiophyllum pedunculatum Low, Su & Xing sp. nov. and living Araceae. A, infructescence showing seed arrangement of L. pedunculatum. B, infructescence showing fruit arrangement of Typhonium. C, ribs on the seed coat of L. pedunculatum. D, overview of seed under smart digital microscope. E, overview of seed under upright fluorescent microscope showing more clearly on the micropylar and micropylar pole. F, the ribbed seeds of Lemna gibba Linnaeus, 1753. G, overview of micropylar and micropylar pole of L. pedunculatum under scanning electron microscope. H, enlargement detail from G, close-up of micropylar pole on the seed of L. pedunculatum. Abbreviations: ch, chalazal cap; mi, micropylar; mp, micropylar pole. Image in E #Zhang Jiaming. Scale bars: A ¼ 1mm; B ¼ 1 cm; C ¼ 0.5 mm; D ¼ 300 mm; E ¼ 200 mm; F ¼ 250 mm; G ¼ 50 mm; H ¼ 4 mm. Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau 11

Figure 7. Cladistic analyses of Araceae. A, majority-rule consensus tree (MRCT) inferred from morphological character analysis. B, MRCT inferred from the combined morphological and molecular characters analysis. C, MRCT of the clade for Limnobiophyllum. Numbers over the nodes represent the bootstrap values when > 80. 12 S. L. Low et al.

Figure 8. Character evolution of free-floating aroids with fossils included.

Figure 9. Evolution of selected morphologies of extinct and extant free-floating aroids. and extant genera within Lemnoideae (Fig. 7A), which is consideration of the convergent evolution that might have substantiated by the findings of Stockey et al. (1997, occurred between Lemnoideae and Pistia. On the other 2016) and Gallego et al. (2014). However, these morpho- hand, the phylogenetic position of Limnobiophyllum logically based findings were rejected based on the based on the combined morphological and nucleotide Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau 13 sequences (Fig. 7B) provided a more systematic overview The findings from this study confirm for the first of the evolutionary relationships of Araceae. Based on time that Limnobiophyllum possessed ribbed seeds (indi- the phylogenetic tree, Limnobiophyllum and Cobbania cated by the triangular shapes in Figs 3C, F, 6A, C), represent the earliest branching genera for the entire sub- which is also a character of modern lemnoids. This sup- family Lemnoideae (Fig. 7B). This divergence was fol- ports Kvacek’s(2003) hypothesis that detached ribbed lowed by the modern lemnoids. The phylogenetic tree is seeds (Lemnospermum pistiforme) found adjacent to largely consistent with the tree topology of Stockey et al. leaves of L. expansum were from the same species. (2016). Another fossil taxon, Aquaephyllum auriculatum Additionally, the symmetrical position of the basal cha- Gallego, Gandolfo, Cuneo & Zamaloa, 2014, suggested laza and apical micropyle of Limnobiophyllum (L. to be related to living lemnoids (Gallego et al. 2014), pedunculatum) suggest that this species might derive was excluded from our analysis as it is poorly known, from an orthotropous ovule, a character present for liv- with only four complete specimens, and also could be ing Lemnoideae (Daubs 1965; Den Hartog & Van der from some other floating aquatic plants or deposited from Plas 1970) and some members of Aroideae (Mayo et al. nearby vegetation (as discussed in Stockey et al. [2016]). 1997), and also suggested for Cobbanicarpites amuren- sis Krassilov & Kodrul, 2009 (fruits and dispersed seeds associated with C. corrugata; Krassilov & Kodrul Character evolution within Lemnoideae 2009). In contrast, the extinct Cobbania is believed to Nauheimer et al. (2012) suggested that the ancestral possess smooth seeds (Stockey et al. 2007, 2016). The habitat of Araceae is likely to be have been swampy for seeds (>20) of Cobbania and Limnobiohpyllum are spir- proto-aroids (rooted) and aquatic for lemnoids (free- ally arranged, whereas the seed arrangements in floating) during the early evolutionary stage of the fam- Lemnoideae and Wolffieae could not be defined as they ily, based on a phylogenetic framework of Araceae only have 1–4 seeds (Daubs 1965). In the earliest using integrated fossils and molecular data. During tran- diverging proto-aroid clade, approximately 20 fruit ber- ‘ ’ sitions from a wet terrestrial habitat to an aquatic habi- ries with 1–4 smooth seeds (testa is absent for tat (Nauheimer et al. 2012; Kvacek & Smith 2015), Gymnostachys at maturity) in each berry were arranged Limnobiophyllum and Cobbania evolved an overall spirally on the spadix (Seubert 1993; Mayo et al. 1997). reduction in their morphologies to adapt better to an While, in the sister family of Araceae, i.e. Acoraceae, aquatic life. For example, their leaves reduced in size to one to several densely arranged berries with foveolate to about 4 cm, as measured from the fossil specimens smooth seed testa is characteristic for Acorus calamus (Figs 3, 4), along with the reduction of the stem into Linnaeus, 1753 and A. gramineus Solander ex Aiton, either an extremely short stem or solely a vegetation 1789, respectively (Bogner & Mayo 1998; Bogner 2011; point as described by Bogner (2009). Besides, a possible Boyce et al. 2012). Therefore, the number of seeds and reduction of morphologies may have occurred within their spiral arrangement in both Cobbania and Lemnoideae (Fig. 9). For a free-floating aquatic life, the Limnobiophyllum may be from an ancestral state for reduction and simplification of different morphologies Araceae. The presence of smooth seeds representing the seen in extant Lemnoideae include reduced leaf types ancestral character state needs to be proven when an with only primary veins and simple roots (tribe intact fossil of Cobbania is found with an attached Lemnoideae) and reduced leaf types without veins and infructescence. The reduction in the number of seeds roots (tribe Wolffieae) (Sculthorpe 1967; Vaughan & among extant lemnoids may be a derived state due to Baker 1994). Larger leaves with well-developed ven- their tendencies towards vegetative propagation (Landolt ation and roots are present in both Cobbania and 1986; Cole & Voskuil 2011;Fuet al. 2017). Limnobiophyllum (Hantke 1954; Kvacek 1995; Stockey The discovery of Limnobiophyllum expansum demon- et al. 1997, 2007, 2016). However, in extant lemnoids strates an evolutionary relationship between the subfa- such as Wolffia and Wolffiella, the leaf size is miniscule milies Lemnoideae and Aroideae (Kvacek 1995). The and the peduncle is also simplified into a vegetation oblate spheroidal saucer-like infructescence of L. pedun- point. A visible attached peduncle only exists in culatum contains spirally arranged ellipsoid and ribbed Limnobiophyllum (fossils in the current study) to support seeds, which are enclosed by a spathe resembling that the infructescence. The reduced reproductive organs in of eu-aroids. The maturity of an infructescence and fruit modern lemnoids (tribe Lemnoideae) are enclosed dispersal are typically accompanied by the downward- within a budding pouch, a structure that is homologous bending peduncle and spathe split-off, revealing the ber- to the petiole sheath in Araceae. In addition, living lem- ries in Araceae (Wong 2013). The downward-bending noids do not have spathes; instead, the inflorescence is peduncle and the spathe, which has burst open in L. surrounded by a membranous envelope (Bogner 2009). pedunculatum, might indicate that the infructescence 14 S. L. Low et al. developed below water. However, it could also be due Late Cretaceous in North America (Kvacek 1995). The to compression that accompanied fossilization. Fruits of species was also found in Russia and west-central North Araceae are normally juicy berries (Mayo et al. 1997) America from the Paleocene (Stockey et al. 1997) and and the very thin pericarp decays upon maturity for dis- the latest Eocene of North America (Kvacek 1995). The persal (Bogner 2009). Cells from the mesocarp are not fossil species in this study, L. pedunculatum from the always preserved in fossils due to their fleshy and par- late Oligocene of the central Tibetan Plateau, represents enchymatous structures. In the current study, the mature the first fossil record of the genus from this region. seeds were found without a conspicuous pericarp. The Aside from the Tibetan occurrence described herein, pericarp probably ruptured upon maturity or decayed. other East Asian occurrences of Limnobiophyllum are However, these are clearly distinct from the fruits for- from the Palaeogene of the Amur region, eastern merly hypothesized to correspond with this plant (e.g. Russia, presented in an unpublished dissertation on the Krassilov 1973), which are now regarded as an unre- Eocene of Raichikhi by Fedotov (1983, pl. 42, figs lated extinct angiosperm fruit, Porosia Hickey, 1977 1–7), and from the Paleocene of Darmakon by Krassilov (Manchester & Kodrul 2014). Limnobiophyllum pedun- (1976, pl. 11, fig. 5: as Hydrocharis sp.). This finding culatum fossils possessed ribbed seeds which resemble underlines the importance of plant fossil records from those of extant lemnoids, while the horizontal stolons the Tibetan Plateau for increasing our knowledge of the that connect individuals and the campylodromous leaf phytogeographic history of the Northern Hemisphere. It venation resemble those of Cobbania and other supports a close floristic similarity between Asia and described species of Limnobiophyllum. Therefore, these other parts of the Northern Hemisphere. additional characters provide more evidence favouring Another species, L. expansum, has been described the scenario presented by Stockey et al. (1997) that from the Neogene of Europe, including the early Limnobiophyllum represents an intermediate link Miocene of the Czech Republic, the middle Miocene of between Lemnoideae and the eu-aroids. An artistic Germany and the late Miocene of Poland (Kvacek 1995; reconstruction of the morphologies of L. pedunculatum Collinson et al. 2001). Limnobiophyllum has not been is presented in Figure 10. reported after the Miocene. The fossil distribution suggests that Limnobiophyllum might have originated in Limnobiophyllum North America and migrated to the Far East during the Biogeographical history of Paleocene via the Bering Land Bridge – a migration To date, only three species of Limnobiophyllum (includ- route also suggested for Cobbania (Krassilov et al. ing L. pedunculatum) have been reported. Before the 2010; Fig. 1). European Limnobiophyllum might have late Neogene, Limnobiophyllum had a much wider dis- migrated from East Asia, as L. pedunculatum and L. tribution in the Northern Hemisphere, including North expansum are more closely related based in our phyl- America, the Far East, Europe and East Asia ogeny (Fig. 7C). The extinction of Limnobiophyllum (Supplemental material, Table S1; Fig. 1). The earliest after the Miocene might have been due to global cooling known species, L. scutatum, was discovered from the (Zachos et al. 2008) and orogenesis (Potter & Szatmari 2009; Molnar et al. 2010). Today, modern lemnoids inhabit stagnant or slow- moving fresh water in low-altitude regions (below 1500 m above sea level) throughout both tropical and temperate zones with warm and humid conditions (Mkandawire & Dudel 2005a, b). The fossils in this study clearly belong to the basal group of Lemnoideae with similar vegetative and reproductive characters, which indicate this species likely required a similar habitat to that of modern lemnoids (Fig. 8). This also agrees with the habitat conditions indicated by the presence of the climbing perch fish, Eoanabas thibetana (Wu et al. 2017), and the hemipteran Aquarius lunpolaensis (Cai et al. 2019) found in the same layer. Araceae are usually considered a good environmental indicator due to their high sensitivity to environmental Figure 10. Reconstruction of the morphologies of conditions (Wong 2013). However, Limnobiophyllum,as Limnobiophyllum pedunculatum Low, Su & Xing sp. nov. an aquatic aroid, is a typical member of azonal Oligocene Limnobiophyllum (Araceae) from the central Tibetan Plateau 15 vegetation that is not suitable for climate estimates. Supplementary material Nevertheless, the plant megafossil assemblage, as published by Wu et al. (2017) and Sabalites tibetensis Su & Supplementary material for this article can be accessed Zhou, 2019 (Su et al. 2019), from the same sediments here: http://dx.doi.org/10.1080/14772019.2019.1611673. further supports the inference of palaeoenvironment of the central Tibetan Plateau. 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