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Received Date: Revised Date: Accepted Date: Article Type: Special Issue Article RESEARCH ARTICLE

INVITED SPECIAL ARTICLE For the Special Issue: The Tree of Death: The Role of Fossils in Resolving the Overall Pattern of Plant Phylogeny

Short Title: Building the monocot tree of death

Building the monocot tree of death: progress and challenges emerging from the macrofossil-rich Zingiberales Selena Y. Smith1,2,4,6, William J. D. Iles1,3, John C. Benedict1,4, and Chelsea D. Specht5

Manuscript received 1 November 2017; revision accepted 2 May 2018. 1 Department of Earth & Environmental Sciences, University of Michigan, Ann Arbor, MI 48109 USA 2 Museum of Paleontology, University of Michigan, Ann Arbor, MI 48109 USA 3 Department of Integrative Biology and the University and Jepson Herbaria, University of California, Berkeley, CA 94720 USA 4 Program in the Environment, University of Michigan, Ann Arbor, MI 48109 USA 5 School of Integrative Plant Sciences, Section of Plant Biology and the Bailey Hortorium, Cornell University, Ithaca, NY 14853 USA 6 Author for correspondence (e-mail: [email protected]); ORCID id 0000-0002-5923-0404 Author Manuscript

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ajb2.1123

Citation: Smith, S. Y., W. J. D. Iles, J. C. Benedict, and C. D. Specht. 2018. Building the monocot tree of death: progress and challenges emerging from the macrofossil-rich Zingiberales. American Journal of Botany 105(8): XXX. DOI: XXXX

PREMISE OF THE STUDY: Inclusion of fossils in phylogenetic analyses is necessary in order to construct a comprehensive “tree of death” and elucidate evolutionary history of taxa; however, such incorporation of fossils in phylogenetic reconstruction is dependent on the availability and interpretation of extensive morphological data. Here, the Zingiberales, whose familial relationships have been difficult to resolve with high support, are used as a case study to illustrate the importance of including fossil taxa in systematic studies. METHODS: Eight fossil taxa and 43 extant Zingiberales were coded for 39 morphological seed characters, and these data were concatenated with previously published molecular sequence data for analysis in the program MrBayes. KEY RESULTS: Ensete oregonense is confirmed to be part of Musaceae, and the other seven fossils group with Zingiberaceae. There is strong support for Spirematospermum friedrichii, Spirematospermum sp. ‘Goth’, S. wetzleri, and Striatornata sanantoniensis in crown Zingiberaceae while “Musa” cardiosperma, Spirematospermum chandlerae, and Tricostatocarpon silvapinedae are best considered stem Zingiberaceae. Inclusion of fossils explains how different topologies from morphological and molecular data sets is due to shared plesiomorphic characters shared by Musaceae, Zingiberaceae, and Costaceae, and most of the fossils. CONCLUSIONS: Inclusion of eight fossil taxa expands the Zingiberales tree and helps explain the difficulty in resolving relationships. Inclusion of fossils was possible in part due to a large morphological data set built using nondestructive microcomputed tomography data. Collaboration between paleo- and neobotanists and technology such as microcomputed Author Manuscript tomography will help to build the tree of death and ultimately improve our understanding of the evolutionary history of monocots.

KEY WORDS: anatomy; digital morphology; Ensete oregonense; Spirematospermum; Striatornata; Tricostatocarpon

Monocot flowering plants represent ca. 22% of flowering plant species, encompassing a large diversity of morphology, habit, and ecologies. This group is economically important, including many of our staple food crops such as grains (maize, wheat, rice, sorghum), coconuts, plantains; pasture feed for animals; materials produced from species such as bamboo or abaca; spices such as ginger, turmeric, and saffron; and many ornamentals such as spring bulbs, irises, and orchids. Monocots are also ecologically important, forming dominant components of grassland, savanna, fynbos, wetland, and seagrass ecosystems, as well as important parts of tropical forest understories. At an ordinal level, the monocot phylogeny has been relatively stable compared to other groups (APG, 1998, 2016), making them useful for broader studies. Most studies find monocots to be ca. 135 Ma (e.g., Janssen and Bremer, 2004: 134 Ma; Magallón et al., 2015: 135.7 Ma). Monocots represent a good model group for elucidating the patterns and processes of evolution, and understanding their evolutionary history is fundamentally important to human nutrition and well-being. Data from fossil taxa need to be included to obtain the most comprehensive results when inferring phylogenetic relationships and investigating trait evolution, geographic histories, and other aspects of evolution for a lineage. In most cases, fossils are simply considered as constraints on the ages of nodes (e.g., Ho and Duchêne, 2014; but see Ronquist et al., 2012a; Heath et al., 2014; Zhang et al., 2016) within a molecular phylogeny. Either the clade(s) including the fossil(s) or even the entire tree is fixed or constrained: in topology, and inferred ages are dependent on the sequence data, calibration priors, and the model of rate variation used but not uncertainty in the tree or the fossil placement per se. In these cases, morphological data and fossils do not inform the topology of the inferred phylogeny, but rather conform to placements dictated by the researcher. Fossil placements among and within lineages are therefore Author Manuscript not tested as part of the tree-building process. However, we know that present-day diversity in all lineages is a result of complex interactions on geological time scales of extinction, speciation, ecology, morphology, and genetics (e.g., Barnosky, 2001; McElwain and Punyasena, 2007;

Escapa and Pol, 2011; Green et al., 2011; Swenson, 2011; Wiens, 2017). Incorporating morphological data from the fossil record is the only objective way of characterizing extinct lineages and determining where they may fit in the evolutionary history of a lineage. With fossils included as terminal units in the phylogenetic analysis, biogeographic patterns, trait evolution, and impact of environmental and ecological changes across lineages can be examined with greater generality compared to studies based only on extant species with molecular sequence data. There are many cases where the fossil record preserves morphological, spatiotemporal (e.g., Prasad et al., 2005, 2011; Smith et al., 2008, 2009b; Wilf and Escapa, 2015) and even climatic/environmental data (e.g., Wing and Greenwood, 1993; Greenwood and Wing, 1995) that could not be predicted, or would not be considered when only extant lineages are evaluated. Rather than only relying on the information present in extant species to understand patterns of evolution and diversification across the entire history of a clade, including fossils will improve our inferences by incorporating a temporal component to studies of trait evolution, biogeographic patterns, and phylogenies.

Reading the fossil record of monocots Numerous reviews regarding the fossil record of monocots (Doyle, 1973; Daghlian, 1981; Collinson et al., 1993; Herendeen and Crane, 1995; Gandolfo et al., 2000; Greenwood and Conran, 2000; Stockey, 2006; Smith et al., 2010; Friis et al., 2011; Smith, 2013) have discussed the challenges of working with monocot fossils, adding complications to building a monocot “tree of death”, i.e., a tree of life that includes both extant and extinct taxa to resolve overall patterns of phylogenetic relationships. Monocots generally have a low preservation potential because they are often small and herbaceous, lacking “woody” and highly lignified tissues, and have persistent senescent organs, so they either rot in place or never contribute to sediments and thus, do not enter the fossil record (Herendeen and Crane, 1995; Smith, 2013). The lineages of monocots that are better represented tend to be those that are more lignified (e.g., palms) or grow in habitats that are near good depositional environments, such as quiet bodies of fresh water. First and foremost, the study and accurate naming of fossil taxa is a key component of Author Manuscript building a reliable and accurate tree of death. Taxonomy is a vital and dynamic process, and assigning a name and rank to a fossil provides a taxonomic and phylogenetic framework for the taxon in question; we recommend not using unnamed fossils for dating phylogenies (e.g., Bell et
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al., 2010; Smith et al., 2010; Zanne et al., 2014; Tank et al., 2015), as the lack of a name suggests the need for careful evaluation of described morphology and/or ambiguity in phylogenetic placement based on characters analyzed. In addition, one must be cognizant of the framework within which taxa were named as this can influence where they are assumed to be placed within a phylogenetic context. There has been a paradigm shift in paleobotany regarding taxonomy. In the early 20th century, attempts were made to place as many fossils as possible into extant genera. Subsequently, thinking of fossils as extinct taxa that may or may not be directly related to (or nested within) modern taxa became more acceptable and encouraged. While some previously described fossils have been transferred to new extinct genera and relationships to other fossils and to extant taxa were subsequently re-evaluated (e.g., “Viburnum” leaves from the Paleogene of North America and Asia that are now classified in the extinct genera Amersinia and Beringiaphyllum; Manchester et al., 1999), many more have not been reinvestigated. Fossils assigned to extant genera should therefore be approached with particular caution, as the evidence for placing them in an extant genus (or even species) needs to be tested; however, these are very good candidates for further study. Additionally, researchers should keep in mind that while presumed affinities and phylogenetic placement may change once re-evaluated, the name of the taxon is often retained and reflects an incorrect relationship. An example of this is the pollen genus Pandaniidites Elsik, which was described from isolated grains that bore a resemblance to Pandanus Parkinson (Pandanaceae), and given a name that reflected those observations, was classified as Pandanaceae. Subsequently, the grains were found in situ in Pandaniidites that had aroid affinities, demonstrating they belong to Araceae and not Pandanaceae (Stockey et al., 1997): the name, however, using the International Code of Nomenclature for algae, fungi, and plants, did not change. Thus, using Pandaniidites to calibrate a Pandanaceae node would be erroneous (Iles et al., 2015). In addition, many fossil localities remain understudied or even undiscovered. These have the potential to contribute significant information, especially those from regions outside of the mid- and high-latitude northern hemisphere, which is overrepresented in paleobotanical collections. Recent work, for example, has dramatically improved the fossil record of Australia Author Manuscript and New Zealand and of many monocot lineages that are otherwise poorly known in the fossil record (see Conran et al., 2015 for a recent summary). As more fossils are found, the potential to find multiple organs of a species in organic connection grows, allowing for more accurate
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development of a whole-plant concept, and subsequently, for building more complete trait matrices and reducing ambiguity in nomenclature.

The undeniable value of morphology Morphology is key to being able to accurately place fossils in a phylogenetic context and to more broadly understand the evolutionary histories of the lineages they represent. By using morphology and incorporating fossils directly into phylogenetic reconstruction, we can address key questions beyond the simple timing of divergences, such as: How have dispersal and vicariance influenced a lineage’s distribution over time? What biotic and abiotic interactions have driven trait evolution? Has morphological or ecological disparity increased, decreased, or remained constant in time? How does tempo and mode of morphological and molecular trait evolution compare? How widespread are homologies within a group, and in which cases are presumed synapomorphies representative of plesiomorphic characters? In some cases, the fossils will help resolve nodes; in others, they can at least provide insight as to why there is conflict among topologies. Monocot fossils are preserved in a variety of different ways, and the amount of data we can obtain to help determine a fossil’s phylogenetic placement relates to both the preservation type and the organ(s) preserved. Some fossils will never be useful for calibrating nodes on phylogenetic trees because they are not preserved well enough to display any distinctive characters for a particular lineage, but others contain a wealth of information (see Iles et al., 2015). A major challenge (but also benefit) to including fossils in a phylogeny is having appropriate morphological data scored for both the fossil and any extant group with which it is being compared. Unfortunately, unlike other plant groups, the growth habits of many monocots mean they do not often preserve as whole plants, but are more likely to be represented in the fossil record by isolated organs. The characters scored in the extant taxa will need to reflect characters represented in putative fossil relatives, and phylogenetic information will depend on which organ is represented. Reproductive structures (flowers, fruits, and seeds) tend to be more Author Manuscript complex and have more phylogenetically informative characters compared to vegetative features; good examples are the seeds of Zingiberales, which arguably have the most complex seeds among all angiosperms (Benedict et al., 2015a, 2015b, 2016). However, many monocot fossils
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represent vegetative stem or leaf organs. While these are often more cryptic in monocots, especially for gross morphology (e.g., leaf shape, venation), there is still value in including as much data on vegetative structures as possible, especially as these vegetative characters may be indicative of climatic or environmental conditions. One of the main challenges to building a monocot tree of death is the paucity of morphological data for both extant and extinct monocots. Progress is being made, however, especially with the development of novel approaches. There is active research on phytolith morphology, for example, as applied to the fossil record of grasses and Cenozoic vegetation (e.g., Prasad et al., 2005, 2011; Strömberg, 2005, 2011; Miller et al., 2012; Strömberg et al., 2013), enhancing our understanding of variation in phytolith morphotypes across extant monocots and determining the characters that can be used to interpret affinities within the fossil record. For groups like Poaceae, the enhanced understanding of phytolith morphologies and their phylogenetic (or functional group) significance has greatly improved our ability to assign taxonomic affinities to fossils. Within some clades, unique phytolith morphotypes have been found (e.g., Piperno, 2006; Prychid et al., 2004; Chen and Smith, 2013), which has led to the identification of the earliest unequivocal grass fossil, in the rice tribe Oryzeae from the latest Cretaceous (Maastrichtian) of India (Prasad et al., 2005, 2011). X-ray microcomputed tomography using either micro- or nanoscale industrial computed tomography machines or synchrotron-based methods (collectively referred to as “µCT” here for simplicity), has the potential to transform our approaches and help build the monocot tree of death much more efficiently. These µCT techniques use X-rays to image a sample based on differing densities of materials, producing a series of 2D images (tomograms; roughly equivalent to digital greyscale versions of histological sections) that can be imported into various computer programs and visualized in three dimensions (see e.g., Sutton, 2008; Smith et al., 2009a; Davies et al., 2017 for more details). These 3D data sets can be “sliced” in any orientation to produce new 2D sections and can be “segmented” or “digitally dissected” to highlight certain structures in 3D. The lack of morphoanatomical data is a significant barrier to building total evidence trees, but µCT opens up the possibility of building large morphological data sets since it is so much Author Manuscript more rapid than traditional histology (minutes to hours vs. days). Further, µCT offers several other advantages: it is useable on small and large objects; it enables visualization of structures with hard or mixed density tissues (especially common in many fruits and seeds) without tearing
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or destruction of adjacent tissues; it is nondestructive, meaning museums and herbaria are more willing to have their specimens studied; and it produces digital data sets that can be shared widely and publicly archived, enhancing researcher’s ability to work collaboratively, to reinterpret data, and to find new features (e.g., Smith et al., 2009a; Davies et al., 2017). For paleobotanists, µCT also enables us to understand what morphological interpretations might result merely from different planes of sections or variation in the preservation of tissues (e.g., Smith et al., 2009a; Adams et al., 2016). For example, fossils are sometimes obliquely sectioned relative to the standard transverse and longitudinal sections in modern plants, which can affect a researcher’s ability to accurately and confidently make comparisons with other taxa that have been preserved differently or may be three-dimensionally complex (e.g., Matsunaga et al., 2018: morphology of Viracarpon fruits was disputed for more than 50 years, but resolved with the application of µCT). Because accuracy of morphological interpretation can affect the scoring of characters in a data matrix, using 3D data can play a positive role in assigning homologies and reconciling relationships. While monocots have a reputation for having cryptic morphologies (e.g., Rudall, 2000), more characterization of fossil and living monocot traits will undoubtedly lead to a better understanding of the subtleties of their structure and enable recognition and identification of characters useful for phylogenetic analysis.

Zingiberales: A case study in integrating fossils into the phylogeny Evaluating the influences of different analytical choices in preparing a tree of death is necessary so that more robust hypotheses can be produced from the outset. Here, we use an extensive morphological data set of seed anatomy of Zingiberales to test the influence of different data partitions and taxon inclusion/exclusion on the phylogenetic placement of fossil taxa. Eight fossil taxa that are Cretaceous to Pliocene in age are represented by permineralized fruits and seeds with a wealth of morphological data (Chandler, 1925; Jain, 1963; Koch and Friedrich, 1970; Friedrich and Koch, 1971; Knobloch and Mai, 1986; Goth, 1986; Friis, 1988; Manchester and Kress, 1993; Rodriguez-de la Rosa and Cevallos-Ferriz, 1994; Fischer et al., 2009). These include the taxon Spirematospermum Chandler, which has been the subject of Author Manuscript much controversy over its familial identity. Spirematospermum was originally considered to be a member of the family Zingiberaceae (Chandler, 1925; Koch and Friedrich, 1970) but subsequently was thought to be more closely related to Musaceae (Manchester and Kress, 1993;
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Rodriguez-de la Rosa and Cevallos-Ferriz, 1994; Fischer et al., 2009) with Fischer et al. (2009) including the taxon in Musaceae in its own subfamily Pariemusinae, based on their study of S. wetzleri (Heer) Chandler. The availability now of extensively sampled seed morphological data from across all eight families of the order and a large molecular data set with broad taxon sampling (Sass et al., 2016) make it ideal for exploring the impact of including fossils in resolving deeper nodes of the phylogeny and for testing the effect of different parameters and types and quality of data on the placement of fossil lineages during phylogenetic reconstruction. Understanding the influence of data and parameter choices can lead to developing best practices for building any lineage-specific tree of death.

MATERIALS AND METHODS

Fossils Eight fossils were included in the analyses: four species in Spirematospermum, and four other taxa placed in separate genera; only one of these is confidently placed to family (Ensete oregonense Manchester & Kress). Specimens were examined directly in all cases, with data used in scoring coming from the literature and our own observations. Material examined is listed for each taxon. Samples were observed from the following repositories (institutional codes for specimen numbers indicated in parentheses): Arizona State University (ASU); Bayerische Staatssammlung für Paläontologie und Geologie in Munich, Germany (SNSB-BSPG); Birbal Sahni Institute of Palaeosciences, Lucknow, India (BSIP); Cleveland Museum of Natural History, Cleveland, Ohio, USA (CMNH); Florida Museum of Natural History, Gainesville, Florida, USA (FLMNH UF); Museum für Naturkunde, Berlin, Germany (NKM); Smithsonian National Museum of Natural History, Washington, D.C., USA (USNM). Spirematospermum chandlerae Friis (1998) is known from the late Campanian of North Carolina, United States (Friis et al., 2011) (material examined: USNM 712537, 712538). Additional Late Cretaceous taxa are Spirematospermum friedrichii Knobloch & Mai (Knobloch and Mai, 1986) from the Maastrichtian of Eisleben, Germany (material examined: NKM 8516A, 8516B, 8516C, 8516D, 8516E, 8516H [holotype]) and an undescribed species of Spirematospermum (Goth, 1986) from Author Manuscript the late Campanian/early Maastrichtian of Kössen, Germany (hereafter referred to as Spirematospermum sp. ‘Goth’; this taxon will be described in a subsequent paper; material examined: SNSB-BSPG 1984 VII 15, 1984 VII 90). The species that has received the most
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attention is Spirematospermum wetzleri, as it is the most spatially and temporally widespread and well preserved (even with fruits) at many localities; it forms up to 16% of Neogene European fossil assemblages (Fischer et al., 2009). This species was described from deposits across Eurasia and ranges in age from Eocene to Pliocene, and we examined representatives from several Oligocene, Miocene, and Pliocene localities in Germany (material examined: ASU-5000a, b, c; SNSB-BSPG 1970 I, 1977 I, 1972 XXI 79, 1972 XXI 19; FLMNH UF19083-049162). Striatornata sanantoniensis Rodriguez-de la Rosa & Cevallos-Ferriz (material examined: ASU- 5001a) and Tricostatocarpon silvapinedae Rodriguez-de la Rosa & Cevallos-Ferriz (material examined: ASU-5002a) were both described by Rodriguez-de la Rosa and Cevallos-Ferriz (1994) from the Campanian of Mexico, while Musa cardiosperma Jain is from the Maastrichtian-Danian Deccan Intertrappean Beds of India (Jain 1963); despite the name, affinities to Musa are doubtful (material examined: BSIP 31654-1 [holotype], 31654-2, 31654-3; FLMNH UF18311-62188, UF18311-70295, UF18311-70296, UF18311-70298, UF18311-9963, UF19279-62144, UF19506-70302; CMNH PM1711, PM1765). Striatornata, Tricostatocarpon, and Musa cardiosperma, plus Spire. wetzleri, are all preserved as both fruits and seeds. Ensete oregonense (Musaceae) is represented by seeds in the Eocene Clarno Formation, Oregon (Manchester and Kress, 1993); we examined the holotype, FLMNH UF 6621. Fossils ascribed to Alpinia Roxb. (Mai, 1999; Mai and Walther, 1985, 1991) were shown to belong to different, non-Zingiberales taxa (Smith et al., 2015) and are not considered further here.

Phylogenetic data matrix Morphological data for extant taxa were coded using the characters from Benedict et al. (2015a, b, 2016) with modifications to remove uninformative characters and minimize character overlap, resulting in 39 morphological characters (Appendix S1, see Supplemental Data with this article). Halopegia azurae (K.Schum.) K.Schum. (MO Boussengui-Nongo et al. 18), Heliconia acuminata A.Rich. (US Kress 91-3242), Heliconia nutans Woodson (US n.c.), and Canna jaegeriana Urb. (US Kress 88-2245) were newly scored and added to this study. Heliconia L. species were previously scored as having opercula, but here are recoded as having no opercula, Author Manuscript reflecting developmental homologies (their operculum is derived from endocarp tissue, not the seed coat). Fossil taxon traits were coded using both published literature and our (S. Y. Smith and J. C. Benedict) observations of specimens. Molecular data were selected from Sass et al.
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(2016), with a subset of taxa chosen to match the species with available morphological data. Where both types of data were not available for a single species, molecular and morphological characters were concatenated from congeneric species, if only one representative of that genus was included (Calathea G.Mey., Ischnosiphon Körn, Chamaecostus C.D.Specht & D.W.Stev., Curcuma L., Elettariopsis Baker; and Distichochlamys M.F.Newman instead of Scaphochlamys Baker, which are sister genera). Our data matrix is available in Appendix S2.

Phylogenetic analyses Several analyses were run to evaluate phylogenetic relationships, with results from the following presented here: (1) total evidence (= molecular plus morphological data sets), all fossils; (2) total evidence, single fossil (resulting in eight analyses); (3) morphology only matrix, all fossils. We ran total-evidence analyses with all the fossils and each fossil individually to evaluate differences in placement and support values. We ran the morphology-only analysis to evaluate whether the morphology data set carried a different phylogenetic signal. Bayesian analyses of molecular and morphological data were run in MrBayes v3.2.6 (Ronquist et al., 2012b). The molecular data are from Sass et al. (2016) and consist of an alignment of 137,748 bp of coding regions from the plastid and nuclear compartments. The nuclear data consist of 308 markers from across the genome. We partitioned the nuclear and plastid components by codon position resulting in six molecular partitions. Each partition was evaluated under the GTR+Γ4 model of sequence evolution. For the morphological data, we used a symmetric model of character state change and a Γ4 parameter to allow site-to-site heterogeneity, and ascertainment bias was set to parsimony informative. Topology and branch lengths were linked across all six molecular partitions and the morphological partition. Two runs consisting of four chains each were run in parallel, the “temperature” was set between 0.01–0.03 because this range resulted in optimal acceptance ratios. Analyses were run for at least 2 million generations (sampling every 500) and until the standard deviation of split frequencies fell below 0.01. The morphology-only analysis was run under the same setting as the total-evidence analysis above except that only the morphological data were considered, and it was run for 5 million generations. Our data matrix is Author Manuscript available in the online supplemental data (Appendix S2). We consider posterior probabilities (PP) over 98% to be strong support, 95–98% to be moderate support, and below 95% to be weak support.
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Character evolution We used maximum parsimony (MP) to map traits and infer character evolution under the total-evidence all-fossils and the morphology-only all-fossils topologies. Maximum parsimony reconstructions (with opt = deltran) and most parsimonious reconstruction sets were inferred with PAUP* version 4.0a159 (Swofford, 2002). The NEXUS file to carry out these analyses is available in the Appendix S3.

RESULTS

Phylogenetic analyses: total evidence Bayesian total-evidence analyses completed between 2 and 34 million generations. Non- topology parameter estimated sample sizes were nearly always over 200 indicating stable parameter estimates. The total-evidence all-fossils analysis generally showed most families receiving moderate (one family) to strong (five families) support with the exception of Zingiberaceae with 93% PP (Fig. 1) (Lowiaceae was represented by a single individual.). Within Zingiberaceae, the subfamily Zingiberoideae was resolved with weak support (84% PP), and support for relationships within the clade varied from 84–97% PP. The subfamily Alpinioideae was not resolved, forming a polytomy at the base of the family with Zingiberoideae and all the fossils except Ensete oregonense. All species of Spirematospermum were placed within Zingiberaceae; Spirematospermum sp. ‘Goth’ and Spire. friedrichii as isolated species arising from the family polytomy, and Spire. chandlerae and Spire. wetzleri forming a clade with Musa cardiosperma, Striatornata sanantoniensis, and Tricostatocarpon silvapinedae with poor support (63% PP) (also arising from the family polytomy). Ensete oregonense was moderately supported in Musaceae (97% PP) but its placement within the family is unresolved. The total-evidence single-fossil analyses resemble the all-fossils analysis (Fig. 2), except that more branches are resolved (e.g., Zingiberaceae subfamily Alpinioideae) and that Zingiberaceae typically also had higher support (96–100% PP compared to 93% PP), with the exception of the Musa cardiosperma analysis where Zingiberaceae had 74% PP and the Spire. chandlerae analysis with Author Manuscript 91% PP.

Phylogenetic analyses: morphology only
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Bayesian morphological analysis resolved five families as monophyletic although support varied (82–100% PP), but neither Zingiberaceae nor Strelitziaceae were resolved (although the latter formed a clade with its sister group, Lowiaceae, with 84% PP; Fig. 3). Relationships within extant Zingiberaceae were completely unresolved, a single species, Etlingera elatior (Jack) R.M.Sm., was resolved as the sister group to Costaceae, with weak support. The fossil Ensete oregonense was well supported in Musaceae (100% PP). The other fossil taxa formed a polytomy with members of Zingiberaceae, of which some formed species pairs with varying support (52–89% PP). A moderately supported bipartition (95% PP) separates the families Cannaceae, Heliconiaceae, Lowiaceae, Marantaceae, and Strelitziaceae from Costaceae, Musaceae, Zingiberaceae, and the fossils (Fig. 3). All phylogenetic trees are available in Appendix S4.

Character evolution Several characters were found that supported the placement of fossils (excluding Ensete oregonense) within Zingiberaceae and as a clade including Musa cardiosperma, Spire. chandlerae, Spire. wetzleri, Striatornata sanantoniensis, and Tricostatocarpon silvapinedae, which we will refer to as the ‘Spirematospermum plus’ clade. Contorted seeds from tight packing in fruit (character 5) was found to be shared by all members of the ‘Spirematospermum plus’ clade and, was lacking in the two fossil members excluded from this clade, Spire. sp. ‘Goth’ and Spire. friedrichii. A similar case was seen in seed coat tapering at the micropyle (character 7) and chalazal mesotestal proliferation of cells (character 22): all members except Musa cardiosperma in the ‘Spirematospermum plus’ clade had these features, but they were lacking in Spire. sp. ‘Goth’ and Spire. friedrichii. The chalazal chamber (character 23) was found to be a mixture of Amomum-type and Alpinia-type chambers in the ‘Spirematospermum plus’ clade but is lacking in Spire. sp. ‘Goth’ and Spire. friedrichii. Another character that separated the ‘Spirematospermum plus’ clade from Spire. sp. ‘Goth’ and Spire. friedrichii was the relative thickness of the mechanical layer (character 25). Spirematospermum sp. ‘Goth’ and Spire. friedrichii had seed coats less than 100 µm wide at the thinnest point of the seed, where the ‘Spirematospermum Author Manuscript plus’ clade had seeds greater than 100 µm wide to more than 200 µm wide at the thinnest point of the seed. Endotestal thickness (character 33) was also found to be mostly uniform in the ‘Spirematospermum plus’ clade with the exception of Tricostatocarpon silvapinedae where it
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was difficult to discern this character from the fossil material. Endotestal thickness was also scored as missing for Spire. sp. ‘Goth’ and Spire. friedrichii because it was too difficult to discern from in the fossil material. A uniform exotesta (character 29) was found in all members of the ‘Spirematospermum plus’ clade, Spire. sp. ‘Goth’, and Spire. friedrichii, and was one of the only characters that united all taxa considered to be Spirematospermum. One exception is Musa cardiosperma, where the exotesta was highly fragmented and difficult to discern whether the exotesta was uniform or not. Finally, a micropylar collar (character 16) was lacking in all fossils except for Ensete oregonense. Several of these character reconstructions are shown on the total evidence all fossils tree (Fig. 1). Several other morphological characters were found that support a close relationship between Costaceae, Musaceae, and Zingiberaceae. A chalazal indentation of the seed coat (character 10) was found in Musaceae, Costaceae, and one Zingiberaceae (Etlingera elatior) but was absent in the other five families. A conical or cylindrical-shaped micropylar region (character 12), endotestal gap (character 34), chalazal pigment group (character 35), and raphe canal (character 38) were also found to be shared by members of Costaceae, Musaceae, and Zingiberaceae and was not found in any of the other families. The presence of a hilar rim (character 19) was found in Musaceae, some Zingiberaceae, and Lowiaceae, but not in any of the other five families. Another feature only seen among Musaceae and Zingiberaceae taxa was highly differentiated seed coats, where the mesotesta is composed of two to three different types of cells (character 32). To show the strong support for a clade consisting of Costaceae, Musaceae, and Zingiberaceae several of these characters are mapped on to the morphology only all fossils tree (Fig. 3). The character reconstructions for all 39 characters using deltran optimization (shown in Figs. 1 and 3) and the most parsimonious reconstruction sets are available in Appendix S5.

DISCUSSION

Total evidence and the resolution of fossil identity Approaches that integrate molecular sequence data from extant taxa with morphological Author Manuscript data from extant and extinct taxa can help our understanding of character evolution and resolve fossil identity in the context of living diversity (e.g., Nylander et al., 2004). The total-evidence approach adopted here uses a large molecular alignment (+100k bp) of the Zingiberales (Sass et
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al., 2016) with recent work characterizing and describing Zingiberales seed morphology (Benedict et al., 2015a, b, 2016) to place newly scored Zingiberales fossils. Bayesian total-evidence analyses considering fossils singly or as a whole were consistent in fossil placement to the family level within Zingiberales. The Eocene fossil Ensete oregonense is strongly supported as belonging to Musaceae in the single-fossil analysis (100% PP) and moderately supported in the all-fossils analysis (97% PP), confirming initial fossil placement (Manchester and Kress, 1993), although no specific relationship within the family is resolved. While Ensete oregonense is not found in a clade with other Ensete, our sampling of Musaceae is not as broad as it might be, and the fossil is found in a polytomy with Musa and Ensete. Thus, it is premature to think Ensete oregonense might not be in Ensete. In contrast, M. cardiosperma from the Cretaceous–Paleogene boundary has no association with Musaceae and instead is placed in Zingiberaceae, although with poor support (76% PP). The remaining fossils (Spirematospermum spp., Striatornata sanantoniensis, and Tricostatocarpon silvapinedae) also group with Zingiberaceae, with support varying from poor to moderate in Spire. chandlerae and T. silvapinedae (91% and 96% PP, respectively) to strong in Spire. friedrichii, Spire. sp. ‘Goth’, Spire. wetzleri, and Striatornata sanantoniensis (98–100% PP). Three fossils, Spire. chandlerae, Spire. wetzleri, and T. silvapinedae are found deeply nested within subfamily Alpinioideae and closely associated with Aframomum and Renealmia (Fig. 2), although strong support is lacking for these placements. The other fossils are in a clade with subfamilies Alpinioideae and Zingiberoideae, again with low support. Furthermore, when fossils are analyzed together, several of these fossils (Spire. chandlerae, Spire. wetzleri, Striatornata sanantoniensis, T. silvapinedae, and M. cardiosperma) form a clade, albeit with poor support (63% PP) (Fig. 1). The dissolution of subfamily Alpinioideae in this latter analysis is probably caused by the combined effects of low phylogenetic signal in the morphology data in that part of the tree (see Fig. 3) and multiple (fossil) samples with no molecular data (i.e., clustered taxa with missing data) to reliably inform their placement. Individual and combined analysis of fossil taxa support placement of Ensete oregonense in Musaceae, and Spire. friedrichii, Spire. sp. ‘Goth’, Spire. wetzleri, and Striatornata Author Manuscript sanantonionensis in Zingiberaceae with strong support (98–100% PP) (Figs. 1–3). The remaining fossils, Musa cardiosperma, Spire. chandlerae, and Tricostatocarpon silvapinedae, are also best placed in Zingiberaceae; however, support is considerably weaker (74–96% PP)
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and, therefore, ultimately these may represent stem lineage Zingiberaceae. Several of the taxa that can be considered crown Zingiberaceae due to their strongly supported placement within the family are Campanian (or early Maastrichtian) in age; thus, Zingiberales were already diverse (both morphologically and taxonomically) by this time.

Morphological and molecular incongruence The Bayesian analysis of morphology and fossils resolves several families with high support (Cannaceae, Heliconiaceae, and Musaceae with 100% PP), and several other families with low to moderate support (Costaceae and Marantaceae, with 96% PP and 82% PP, respectively) and also identifies several family groups (Heliconiaceae, Lowiaceae, and Strelitziaceae, with 51% PP). An unusual feature of the morphology tree is the separation of Costaceae, Musaceae, Zingiberaceae, and fossils from the remaining families with moderate support (95% PP) (Fig. 3). This bipartition is not seen in the total-evidence tree that shows Costaceae and Zingiberaceae (and all fossils except Ensete oregonense) together in a clade with Cannaceae and Marantaceae (96% PP) (Fig. 2), as is consistent with previous analyses of Zingiberales that recognized a “ginger group” (Zingiberaceae, Costaceae, Cannaceae, Marantaceae) and “banana group” (Musaceae, Lowiaceae, Heliconiaceae, Strelitziaceae). Eliminating fossil taxa from consideration in both the total-evidence and morphology-only analysis (see Appendix S4) strengthens this conflict because the total-evidence-supported bipartition is now 100% PP (the tree showing identical family relationships to Sass et al., 2016). It is currently unclear how to interpret this conflict. One possibility is to believe that the morphological data are saying something true about phylogenetic relationships among the families and fossils of Zingiberales that is being masked or mislead by the molecular data. However, the molecular data come from two separate genomic compartments (plastid and nuclear), each consisting of tens of thousands of base pairs that are congruent as to family relationships (see Sass et al., 2016). Previous molecular analyses also tend to recognize that Costaceae and Zingiberaceae form a strongly supported clade with Cannaceae and Marantaceae (ginger group). In addition, morphological analysis based on different sets of vegetative and Author Manuscript reproductive characters never group Musaceae with Costaceae and Zingiberaceae to the exclusion of Cannaceae and Marantaceae (Dahlgren and Rasmussen, 1983; Kress, 1990; Kress et al., 2001).
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The remaining option is that family relationships as reconstructed by the morphological data reflect instead something about the evolution of seed morphology (the basis for the morphological matrix considered here). This interpretation is supported by the fact that at least eight characters unite Musaceae, Costaceae, and Zingiberaceae (or Musaceae+Zingiberaceae) vs. the other families and include features like the presence of a chalazal pigment group, raphe canal, endotestal gap, and chalazal chamber. These features are commonly seen in the fossil taxa as well, suggesting that these features should be interpreted as plesiomorphic (or possibly convergent), present in Musaceae (found as sister to all other Zingiberales), Zingiberaceae, and sometimes Costaceae (which is sister to Zingiberaceae; Kress et al., 2001; Kress and Specht, 2006; Sass et al., 2016), and lost/simplified through time in many of the other lineages.

Core challenges to building the monocot tree of death As with any large group, the primary hindrance to building a monocot tree of death— from which broader evolutionary, ecological, and morphological patterns can be drawn—is the lack of data. Monocots are considered to have essentially an invisible radiation during much of the Cretaceous (Crepet, 2008), as evidenced by the lack of fossil record for many monocot lineages. Some groups of monocots have a good fossil record in part because they inhabit environments with a high preservation potential, such as aquatic and semi-aquatic alismatids (Stockey, 2006; Smith, 2013), or grow abundantly and have organs resistant to decay (e.g., palms, gingers). Other monocots, though, will never have a great fossil record; for example, mycoheterotrophs or achlorophyllous taxa that are generally small forest-dwellers (although there are charcoalified flowers from the Turonian attributed to Triuridaceae; Gandolfo et al., 2002). However, as more work is done to study both old and new localities, it is likely that at least some of these taxonomic biases can be addressed. For example, orchids (family Orchidaceae) would fall into this category of plants with poor preservation potential, but multiple fossils have now been placed with reasonable confidence in this family (Ramírez et al., 2007; Conran et al., 2009; Poinar, 2016a, b; Poinar and Rasmussen, 2017), demonstrating that with perserverance (and the right search image) more fossils for even poorly represented clades can be Author Manuscript found. In addition, the early-divergent lineages cannot be ignored. Although not addressed here, there are putative monocot fossils that may represent early lineages and need further study to confirm (or deny) their affinities (see e.g., Smith, 2013 for discussion), and applying newer
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methodologies such as µCT can help provide evidence for both extinct crown taxa and more enigmatic stem taxa (e.g., Viracarpon fossils; Matsunaga et al., 2018). These kinds of enigmatic fossils are important to place evolutionarily and provide critical insights into disparity and diversity within monocots. Incorporation of morphological data into phylogenetic analyses—permitting the inclusion of fossils—is a vital step for reconstructing phylogeny. Here we used morphological data from seeds, both extant and fossil, to expand the Zingiberales phylogeny. Our analysis illustrates that with further such studies that develop novel character matrices including phylogenetically informative traits coded for extant and fossil taxa, building a monocot tree of death is possible. While morphology and careful homology assessment are critical for incorporating fossils into the tree of death, challenges remain in the lack of available morphological data of sufficient quality, depth, and breadth to be able to place fossil taxa; but this is a tractable problem, if people and resources are available. Increasing use of µCT offers a tool that is complementary to traditional histology and morphological characterization to expand our sampling and build a morphological data set, with the advantage of being able to make a rich data set available simultaneously to multiple researchers who can mine it for informative data (e.g., following open digital morphology guidelines; Davies et al., 2017). Further, paleobotanists and neobotanists need to work together to build these data sets and the tree of death. The tree of life can never be complete without including past life, but we cannot include extinct taxa without synthesizing knowledge of the morphologies for extant species. This task can be daunting, especially for a group such as monocots that has a reputation of having “cryptic” morphological characters. While fruits and seeds are rich in systematically informative characters, vegetative structures including stems and leaves are understudied and likely include more useful features than has been acknowledged to date. Work on developing better ontologies, and even leveraging resources for crowdsourcing and citizen science (e.g., O’Leary et al., 2018), could help to build the necessary data sets for constructing a comprehensive tree of death.

CONCLUSIONS Author Manuscript Eight extinct species represented by fossil seeds record the presence of past lineages and morphologies within Zingiberales. Extinct species record different combinations of characters than seen in extant taxa. What may seem like incongruence between morphological and
This article is protected by copyright. All rights reserved Smith et al.–Building the monocot tree of death molecular data sets in inferring phylogenetic relationships is better understood with incorporation of fossil data. The fact that the familial affinities of Spirematospermum have been debated for over two decades (largely based on study of Spire. wetzleri) but not quantitatively tested points to the need to incorporate fossils in phylogenetic analyses not only to place the fossils, but also to better understand morphological evolution, homoplasy, and apparent conflicts in the data. Here, we demonstrated that all zingiberalean fossil seeds belong to Zingiberaceae, except Ensete oregonense, which is in Musaceae. While some taxa appear to be within crown Zingiberaceae (close to Alpinioideae), others are likely stem Zingiberaceae. Reclassification of “Musa” cardiosperma, which is here placed within Zingiberaceae, seems justified based on these results, and this taxon is currently being revised by coauthors S. Y. Smith and J. C. Benedict. The high level of extinct diversity, including Cretaceous fossils, in a more-derived family suggests that family-level diversification occurred rapidly within Zingiberales. To undertake this study and include fossil taxa in the Zingiberales phylogeny, morphological data were needed. Microcomputed tomography proved to be effective for collecting this data and is promising for facilitating similar studies in the future. Incorporation of fossil taxa in phylogenetic analyses provides context to interpretation of the results and emphasizes the utility and importance of including morphological and fossil data in building not only a tree of life, but a tree of death as well, which ultimately is necessary to better inform the evolutionary history of a group.

ACKNOWLEDGEMENTS The authors thank Steven Manchester (University of Florida) for access to fossils of Ensete oregonense and “Musa” cardiosperma; Sergio Cevallos-Ferriz for access to material of Striatornata and Tricostatocarpon; and Margaret Collinson (Royal Holloway University of London), W. Friedrich (Aarhus University), Else Marie Friis (Swedish Museum of Natural History), Kurt Goth, Michael Krings (Bayerische Staatssammlung für Paläontologie und Geologie, Munich), Stephan Schultka (Museum für Naturkunde, Berlin), Rashmi Srivastava (Birbal Sahni Institute of Palaeosciences, Lucknow), and Scott Wing (Smithsonian National Museum of Natural History) for access to Spirematospermum material. Comments from Jeff Author Manuscript Saarela (Canadian Museum of Nature) and an anonymous reviewer, as well as the editors, greatly improved the paper, and we thank them for their efforts. This work was supported by the U. S. National Science Foundation grants DEB 1257080 (S.Y.S.) and 1257701 (C.D.S.).
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FIGURE 2. Bayesian total-evidence single-fossil phylogenetic analysis of molecular and morphological data showing individual fossil placement. Trees are rooted with Musaceae as indicated by Sass et al. (2016). Each tree represents the analysis of a single fossil as indicated by Author Manuscript the dagger symbol and bold type. Numbers adjacent to nodes represent the Bayesian posterior probability; nodes without a number received full support, 100% posterior probability. Clades removed from the vicinity of the fossil placement were collapsed for clarity.
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FIGURE 3. Bayesian morphology-only all-fossil phylogenetic analysis. Tree is rooted with Musaceae as indicated by Sass et al. (2016). Fossils are indicated by a dagger symbol and bold type. Numbers adjacent to nodes represent the Bayesian posterior probability; nodes without numbers received full support, 100% posterior probability. The scale is in expected number of morphological state changes per site. Thick ticks indicated maximum parsimony character change reconstruction using deltran optimization for selected characters, thin ticks indicate reversals. See text for further details.
Appendix S3. NEXUS file for morphological data and character mapping in PAUP.
Appendix S4. NEXUS tree file containing all inferred trees.
Appendix S5. PAUP log file containing all character mappings. Author Manuscript
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†Ensete oregonense 22 Musa coccinea 97 61 Musa balbisiana 85 7 Musa acuminata Musaceae Musella lasiocarpa 88 5 Ensete ventricosum 93 Ensete superbum 7 Heliconia nutans Heliconiaceae Heliconia acuminata Orchidantha maxillarioides Lowiaceae 7 Ravenala madagascariensis Strelitzia nicolai Strelitziaceae Strelitzia reginae Canna iridiflora Canna indica Cannaceae Thaumatococcus daniellii Donax canniformis Goeppertia roseopicta 25 Ischnosiphon helenae Marantaceae Marantachloa leucantha Stromanthe stromanthoides 25 Halopegia azurea 96 Costus pulveralentus 7 Costus dubius Cheilocostus speciosus 5 Tapeinochilos ananassae Costaceae Chamaecostus subsessilis 7 Dimerocostus strobilaceus Monocostus uniflorus Siphonochilus kirkii Siphonochiloideae 95 Siamanthus siliquosus 93 25 7 25 Riedelia sp. Alpinia zerumbet 97 5 7 Etlingera elatior 99 22 Alpinioideae Alpinia purpurata 93 25 7 Elettariopsis stenosiphon 51 Aframomum angustifolium 7 Zingiberaceae 54 22 Renealmia alpinia 5 7 25 †Spirematospermum sp. ‘Goth’ 7 †Spirematospermum friedrichii 7 †Spirematospermum chandlerae 66 †Striatornata sanantoniensis †Tricostatocarpon silvapinedae Author Manuscript 5 22 92 †Spirematospermum wetzleri †Musa cardiosperma 7 22 Globba winitti 84 22 5 Curcuma longa Hedychium coronarium 87 Zingiberoideae Scaphochlamys sp. 91 92 97 Zingiber officinale This article is protected by copyright. All rights 0.02 Zingiberreserved spectabile †Ensete oregonense †Musa cardiosperma †Striatornata sanantoniensis †Tricostatocarpon silvapinedae †Ensete ajb2_1123_f2.pdf oregonense Musaceae Musaceae Musa Musaceae coccinea Heliconiaceae 58 Musa
balbisiana Musaceae Heliconiaceae Heliconiaceae Lowiaceae 80 Musa acuminata 92 Strelitziaceae Lowiaceae Lowiaceae Musella lasiocarpa 97 Cannaceae 79 Ensete Strelitziaceae Strelitziaceae Marantaceae ventricosum 86 Ensete 98 Costaceae superbum Cannaceae Cannaceae 98 Siphonochiloideae Heliconiaceae 97 82 Marantaceae Marantaceae 96 Zingiberoideae Lowiaceae Zingiberaceae Siamanthus siliquosus Strelitziaceae 96 75 90 Costaceae Costaceae Riedelia sp. Cannaceae Alpinia Siphonochiloideae Siphonochiloideae zerumbet 54 98 Marantaceae 86 Etlingera Alpinioideae elatior †Musa †Striatornata Costaceae 74 Alpinia cardiosperma Zingiberaceae sanantoniensis purpurata Zingiberaceae 53 Elettariopsis Siphonochiloideae 68 stenosiphon 96 Zingiberoideae 76 Zingiberoideae †Tricostatocarpon 57 Zingiberaceae Zingiberoideae 51 silvapinedae Aframomum 61 Alpinioideae 68 Alpinioideae Alpinioideae angustifolium 54 Renealmia alpinia
†Spirematospermum friedrichii †Spirematospermum sp. ‘Goth’ †Spirematospermum chandlerae †Spirematospermum wetzleri
Musaceae Musaceae Musaceae Musaceae Heliconiaceae Heliconiaceae 99 Heliconiaceae Heliconiaceae Lowiaceae Lowiaceae 99 Strelitziaceae Strelitziaceae Lowiaceae Lowiaceae Cannaceae Cannaceae 98 Strelitziaceae Strelitziaceae Marantaceae Marantaceae
95 Costaceae 99 Costaceae Cannaceae Cannaceae Siphonochiloideae Siphonochiloideae 93 99 96 97 Zingiberoideae Zingiberoideae Marantaceae Marantaceae 91 98 Zingiberaceae Siamanthus Zingiberaceae Siamanthus siliquosus siliquosus 88 95 Costaceae Costaceae Riedelia Riedelia
Author Manuscript sp. sp. Alpinia Alpinia Siphonochiloideae Siphonochiloideae zerumbet zerumbet 99 83 Etlingera 92 Etlingera Alpinioideae elatior Alpinioideae elatior 99 †Spirematospermum †Spirematospermum Alpinia Alpinia Zingiberaceae friedrichii Zingiberaceae sp. ‘Goth’ purpurata purpurata 83 92 52 53 Elettariopsis Elettariopsis 69 82 stenosiphon stenosiphon This article is protected by copyright. All rights Zingiberoideae Zingiberoideae reserved Aframomum Renealmia 58 67 82 angustifolium 92 alpinia Aframomum 93 89 Renealmia 91 Alpinioideae Alpinioideae 70 alpinia angustifolium †Spirematospermum 87 †Spirematospermum chandlerae wetzleri Musa balbisiana Musa acuminata ajb2_1123_f3.pdf Musa coccinea †Ensete oregonense Musaceae Ensete superbum 58 Ensete ventricosum Musella lasiocarpa †Tricostatocarpon silvapinedae 89 †Spirematospermum wetzleri 34 †Musa cardiosperma 57 †Spirematospermum sp. ‘Goth’ †Spirematospermum friedrichii 52 †Spirematospermum chandlerae †Striatornata sanantoniensis 38 Siphonochilus kirkii Siphonochiloideae Curcuma longa 38 Hedychium coronarium 38 Zingiber officinale Zingiberoideae 38 Zingiber spectabile Globba winitti Scaphochlamys sp. 38 Aframomum angustifolium Zingiberaceae Siamanthus siliquosus 12 38 Riedelia sp. 38 Alpinia zerumbet Alpinioideae 12 Alpinia purpurata 12 38 Elettariopsis stenosiphon 12 Renealmia alpinia 12 Etlingera elatior 67 Cheilocostus speciosus 12 38 Costus dubius 96 12 Tapeinochilos ananassae 53 12 38 Monocostus uniflorus Costaceae Costus pulveralentus 56 Chamaecostus subsessilis 12 Dimerocostus strobilaceus Heliconia nutans Heliconiaceae Heliconia acuminata 51 Orchidantha maxillarioides Lowiaceae 84 Ravenala madagascariensis 71 Strelitzia nicolai Strelitziaceae 95 Strelitzia reginae 12 34 35 38 Canna iridiflora Canna indica Cannaceae Thaumatococcus daniellii Goeppertia roseopicta
Author Manuscript 82 Stromanthe stromanthoides 91 Donax canniformis Marantaceae Marantachloa leucantha 55 Ischnosiphon helenae 0.08 Halopegia azurea
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