Examining the Needle in the Haystack: Evolutionary Relationships in the Mistletoe Genus Loranthus (Loranthaceae

Mohan Prasad Devkota

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Examining the Needle in the Haystack: Evolutionary Relationships in the Mistletoe Genus Loranthus (Loranthaceae) Authors: Nickrent, Daniel L., Su, Huei-Jiun, Lin, Ruo-Zhu, Devkota, Mohan Prasad, Hu, Jer-Ming, et al. Source: Systematic Botany, 46(2) : 403-415 Published By: The American Society of Plant Taxonomists URL: https://doi.org/10.1600/036364421X16231785234748 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Complete website, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/terms-of-use. Usage of BioOne Complete content is strictly limited to personal, educational, and non - commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) Systematic Botany (2021), 46(2): pp. 403–415 © Copyright 2021 by the American Society of Plant Taxonomists DOI 10.1600/036364421X16231785234748 Date of publication August 11, 2021 Examining the Needle in the Haystack: Evolutionary Relationships in the Mistletoe Genus Loranthus (Loranthaceae) Daniel L. Nickrent,1,7 Huei-Jiun Su,2 Ruo-Zhu Lin,3 Mohan Prasad Devkota,4 Jer-Ming Hu,5 and Gerhard Glatzel6 1 School of Biological Sciences, Southern Illinois University, Carbondale, Illinois 62901-6509, USA; nickrent@siu.edu 2 Department of Earth and Life Sciences, University of Taipei, Taipei, 100; hjsu@utaipei.edu.tw 3 Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing; linruozhu@caf.ac.cn 4 Amrit Campus, Tribhuvan University, P.O. Box 102, Kathmandu, Nepal; himalayanforum@gmail.com 5 Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei; jmhu@ntu.edu.tw 6 Institute of Forest Ecology, University of Natural Resources and Applied Life Sciences, Vienna, Austria; gerhard.glatzel@oeaw.ac.at 7 Author for correspondence Communicating Editor: James I. Cohen Abstract—The genus Loranthus (Loranthaceae) consists of ca. nine Old World species distributed from eastern Asia to Europe. Loranthus, the type of the family, has had a complex taxonomic history that continues today, partly because most mistletoes in the family have resided in this genus. For this reason, there are over 1800 Loranthus species names, the vast majority of which are synonyms for mistletoes in other genera. The present work sampled representatives of nine species considered bona ﬁde members of the genus. Using complete plastome sequences, nuclear ribosomal DNA, and mitochondrial 26S rDNA, phylogenetic gene trees were generated to assess interspeciﬁc relationships. The Loranthus plastomes ranged in size from 121 to 125 kb and exhibited the quadripartite structure seen in most Santalales. These plastomes have lost or pseudogenized 24 genes, including all of the NADH dehydrogenase complex, thus reducing the genomes to ca. 90 functional genes. Cladistic analyses of morphological characters were conducted and these trees compared to the molecular trees, thus informing which taxonomic characters best deﬁne clades and characterize species within the genus. Two major clades in Loranthus were identiﬁed. The Europaeus clade includes the deciduous species L. europaeus, L. grewingkii, L. lambertianus, and L. tanakae mostly distributed above 30 N latitude. The Odoratus clade, mostly distributed below 30 N latitude, included L. guizhouensis, L. kaoi, L. odoratus, and L. pseudo-odoratus. The latter four species are genetically closely related, show percurrent (vs. pseudo-dichotomous) branching, and have evergreen leaves. Unisexual ﬂowers have apparently evolved independently in each of the two clades. Future work should focus upon the species delimitation in the Odoratus clade and whether hybridization is occurring among any members. Keywords—Chloroplast, cladistics, deciduous, mitochondrion, phylogenetics, plastome, ribosomal RNA. year as Linnaeus (1762) but four months earlier. Jacquin’s brief Latin description: “simple racemes, all terminal, ﬂowers dioecious; frequently in oak woods; ﬂowering April and May” and illustration clearly refer to this species. Thus, the name Loranthus Jacq. (1762) was conserved at the Edinburgh International Botanical Congress (1964) with the type being L. europaeus Jacq. Despite the fact that over 70% of the generic names in Loranthaceae (as applied today) were proposed prior to 1900, many major works at that time took a broad view and considered most mistletoes in that family to be members of the genus Loranthus. For example, the treatment of Loranthoideae by Engler (1894) recognized Loranthus and only nine other genera. Engler was aware that other authors had placed these mistletoes in various different genera but he considered them sections of Loranthus. Examples include Dendropemon (Blume) Rchb., Dendrophthoe Mart., Loxanthera Blume, Macrosolen (Blume) Rchb., Passovia (as Pasowia) H.Karst., Tapinanthus (Blume) Rchb., and Tolypanthus (Blume) Rchb. Just one year later, Tieghem (1895) published many new generic names in the family, of which 30 are in use today. Overall, the majority of species in Loranthaceae have at one time been considered species of Loranthus. Today, applying the name Loranthus is essentially a “needle in a haystack” problem. A search of that genus on the World Flora Online website returns over 1800 speciﬁc names. Thus, the major difﬁculty encountered by the scientiﬁc community has been to determine which of the many synonyms in the genus apply to other genera and which apply to Loranthus in the strict sense. Statistics from several online databases and herbaria (Table 1) demonstrate that species composition concepts With 76 genera and over 1000 species, the mistletoe family Loranthaceae is the largest in the sandalwood order (Nickrent 2020). This paper focuses on the genus Loranthus Jacq., the type of the family, whose complex taxonomic history has been the source of confusion for over 200 yr. In the ﬁrst edition of Species Plantarum (Linnaeus 1753), two members of Loranthaceae were listed, Loranthus americanus L. and Scurrula parasitica L. In the second edition (Linnaeus 1762–1763), ﬁve Loranthus species were listed: L. americanus, L. scurrula L., L. occidentalis L., L. loniceroides L., and L. stellis L. The Appendix to that edition, published in 1763, added two more species, L. uniﬂorus Jacq. from America and L. europaeus Jacq. from Europe. Following a normal type methodology, the name L. americanus should have been the type for the genus and the family; however, in the years following Linnaeus’ publications, this New World species was being referred to by Martius (1830) as Psittacanthus americanus (L.) Mart. To avoid nomenclatural disruption and confusion, the 1930 International Botanical Congress in Cambridge conserved the name Loranthus L. (1762) with the type being L. scurrula L. Unfortunately, Scurrula was also being considered a genus distinct from Loranthus by G. Don (1834) and subsequent workers. Ironically, Linnaeus (1753) himself treated Scurrula as a separate genus in his ﬁrst edition, and only in the second edition combined it with Loranthus as L. scurrula. As described by Balle et al. (1960), most mistletoe authorities of the late 19th and early 20th century regarded Loranthus to be the name associated with the holarctic species L. europaeus. She also discovered that the chemist and botanist Nicolaus Joseph von Jacquin (1762) published that speciﬁc name in the same 403 Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) 64 104 71 83 151 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 a b, c, e, f 1804 1370 220 c, d, e 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 World Floras Word Flora Online Catalog of Life GBIF Regional Floras Flora China Flora Nepal Flora Pakistan Herbaria Harvard University Herbaria Kew Royal Botanic Gardens Naturalis Biodiversity Center (BioPortal) New York Botanical Garden (C. V. Starr VH) Museum National d'Histoire Naturelle TROPICOS Loranthus europaeus Loranthus grewingkii Loranthus guizhouensis Loranthus kaoi Loranthus lambertianus Loranthus odoratus Loranthus pseudo-odoratus Loranthus tanakae Additional non-Loranthus Loranthus names Collections named Loranthus SYSTEMATIC BOTANY Loranthus delavayi TABLE 1. Loranthus Concepts and Collections. a) L. cleghornii Bedd. 5 Helixanthera cleghornii (Bedd.) Dans. b) L. cordifolius Wall. 5 Scurrula cordifolia (Wall.) G. Don. c) L. longiﬂorus Desr. 5 Dendrophthoe longiﬂora (Desr.) Tiegh. d) L. macrantherus (Eichl.) Hemsl. 5 Psittacanthus macrantherus Eichl. e) L. pulverulentus Wall. 5 Scurrula pulverulenta (Wall.) G. Don. f) L. vestitus Wall. 5 Taxillus vestitus (Wall.) Dans. Websites for each source are as follows: Word Flora Online (http://www.worldﬂoraonline.org/); Catalog of Life (https://www.catalogueoﬂife.org/); GBIF (https://www.gbif.org/); Flora China (http://www.eﬂoras.org/ﬂora_ page.aspx?ﬂora_id=2); Annotated Checklist of the Flowering Plants of Nepal (http://www.eﬂoras.org/ﬂora_page.aspx?ﬂora_id=110); Flora Pakistan (http://www.eﬂoras.org/ﬂora_page.aspx?ﬂora_id=5); Harvard University Herbaria (https://kiki.huh.harvard.edu/databases/specimen_index.html); Kew Royal Botanic Gardens (http://apps.kew.org/herbcat/navigator.do); Naturalis Biodiversity Center (BioPortal) (https://bioportal.naturalis.nl/); New York Botanical Garden (C. V. Starr VH) (https://sciweb.nybg.org/science2/hcol/allvasc/index.asp.html); Museum National d'Histoire Naturelle (https://science.mnhn. fr/institution/mnhn/collection/p/item/search/form); TROPICOS (https://www.tropicos.org/home). 404 Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) [Volume 46 as well as the number of Loranthus collections vary widely (Table 1). For the World Floras, including World Flora Online (superseding the Plant List), Catalog of Life, and GBIF (Global Biodiversity Information Facility), decision making processes are in place that determine which names are valid and accepted and which are invalid or synonyms. This differs from other compilations, such as IPNI (International Plant Names Index), where a comprehensive list of names is available but the subset of accepted names is not indicated. Along with the six species listed in the Flora of China (Qiu et al. 2003) and the type species L. europaeus, two others can be added that were named in the 19th century (L. grewingkii Boiss. & Buhse and L. odoratus Wall.). This results in nine species of Old World mistletoes, a number used here as a working hypothesis (Table 2). None of the three World Flora web sites includes all nine of these species. The Catalog of Life and GBIF report only two accepted species whereas World Flora Online lists seven of the nine species. Danser (1938) believed that Loranthus (Hyphear Danser) and Helixanthera Lour. were congeneric, whereas Barlow (1997) considered the two genera “very closely related,” but he resisted merging the two in his Flora Malesiana treatment. The taxonomic confusion surrounding Loranthus that began over a century ago continues to the present. Following the philosophy of Danser (1938), Rajasekaran (2007) transferred L. lambertianus Schult.f. and L. odoratus into Helixanthera. That this transfer was improper is supported by phylogenetic analyses (Vidal-Russell and Nickrent 2008a; Nickrent et al. 2019) where Loranthus and Helixanthera are shown to be not closely related; indeed they were classiﬁed in different subtribes (Loranthinae and Amyeminae, respectively) within Tribe Lorantheae by Nickrent et al. (2010). Among the three Regional Floras, the Flora of China includes six of the nine species (the status of species outside the region was not addressed). The Flora of Nepal correctly lists L. lambertianus and L. odoratus, two species placed in Helixanthera by Rajasekaran (2007). The online Flora of Pakistan lists four species of Loranthus but all four are synonyms of species in other genera. A species frequently missed by World and Regional Floras is Loranthus grewingkii, this despite being named in 1860 and having been shown to differ from L. europaeus based on a numerical analysis of morphological and anatomical characters (Shahi Shavvon et al. 2012). Similarly, L. odoratus from Nepal was named in the early 19th century (Wallich 1824), but was not recognized as a species on any of the World Flora websites. This may be connected to the merger of this species into Helixanthera by Rajasekaran (2007). Interestingly, L. odoratus is reported for Sumatra and Sulawesi (Indonesia) by Barlow (1997); however, he considered it conspeciﬁc with L. delavayi (Tiegh.) Engl. Whether this taxon is L. odoratus, L. delavayi, or a different species, its presence represents a major geographical disjunction from the other taxa. Although the World Flora Online lists seven species accepted here, it also includes three species that are now in different genera, including the New World species Psittacanthus macrantherus Eichl. (Table 1). Six major herbaria were selected to determine the representation of Loranthus specimens in those collections (Table 1). Most herbaria have specimens with only three or fewer correct Loranthus names, with the remaining being synonyms. The exception is the Museum National d'Histoire Naturelle in Paris where six of the nine species are present among the specimens. In all herbaria there are large numbers of specimens named “Loranthus” that require annotation to other genera. The Catalog of Life website lists 1370 Loranthus synonyms and their 2021] NICKRENT ET AL.: EVOLUTIONARY RELATIONSHIPS IN LORANTHUS accepted name equivalents; however, it uses the genus Hyphear and apparently accepts the transfer of L. odoratus and L. lambertianus to Helixanthera, thus reducing their Loranthus species count to just two. The confusion about what constitutes Loranthus s. s. has generated misinformation on popular websites (e.g. Wikipedia) as well as the scientiﬁc literature. In particular, the biomedical ﬁeld has perpetuated the inappropriate use of Loranthus when in fact the mistletoe is in another genus. Here poor taxonomy has real world consequences because chemicals extracted from mistletoes have been used to treat human diseases such as hypertension, diabetes, epilepsy, and cancer. An example is the use of the name “L. micranthus” for a mistletoe found in Nigeria and India. This name is a synonym of Ileostylus micranthus Tiegh. (from New Zealand) but more likely it is a misspelling of L. micrantherus Engl. which is a synonym of Englerina gabonensis (Engl.) Balle that occurs in Nigeria (Nickrent 2014). Correct identiﬁcation of the plant from which such chemicals are derived is essential for experimental repeatability. Despite being low in number of species, the genus Loranthus sits at a pivotal position with the family phylogenetically and nomenclaturally. Previous phylogenetic work (Vidal-Russell and Nickrent 2008a; Su et al. 2015; Liu et al. 2018; Nickrent et al. 2019) provided strong support for its sister relationship to the monotypic Cecarria obtusifolia (Merr) Barlow that is distributed from the Philippines to Queensland, Australia. Those studies sampled three or fewer species of Loranthus, thus cannot be used to infer relationships among species within the genus. The present work sampled representatives of all nine species and, using nuclear ribosomal DNA and complete plastome sequences, generated gene trees useful in assessing interspeciﬁc relationships. Moreover, relationships gleaned from the trees were used to address the types of morphological characters employed in Loranthus taxonomy. MATERIALS AND METHODS Outgroup Choices and Taxon Sampling—Fourteen accessions of Loranthus were sampled for this study (Appendix 1) as well as three outgroup genera: Cecarria obtusifolia (Australia), Moquiniella rubra (A.Spreng.) Balle (South Africa), and Nuytsia ﬂoribunda (Labill.) G.Don (Australia). Previous molecular phylogenetic analyses in Loranthaceae helped guide outgroup 405 choices. All studies (Vidal-Russell and Nickrent 2008a; Su et al. 2015; Liu et al. 2018; Nickrent et al. 2019) conﬁrm Nuytsia R.Br. ex G.Don as the sister species to the entire family. Moquiniella Balle and Loranthus are both members of tribe Lorantheae but are in different subtribes (Emelianthinae and Loranthinae, respectively). Finally, Cecarria Barlow and Loranthus are the only two genera in Subtribe Loranthinae. Molecular Methodology—The mistletoe samples used here derived from fresh and frozen tissues as well as herbarium specimens and silica gel dried tissue (available in the Dryad Digital Repository, Supplemental File 1, Nickrent et al. 2021). Genomic DNA was extracted following the CTAB method (Doyle and Doyle 1987) or the silica column method outlined in Neubig et al. (2014). A Qubit 3.0 ﬂuorometer (Thermo Fisher Scientiﬁc, Waltham, Massachusetts) was used to determine DNA concentrations and DNA quality was assessed with gel electrophoresis. Sequences were generated using a genome skimming approach (Dodsworth 2015) where 64–100 DNA samples were multiplexed per lane giving reads of 100, 250, or 300 bps in length, depending upon the platform. The different sequencing platforms employed were Illumina HiSeq 2500 (Rapid Genomics, Gainesville, Florida), Illumina MiSeq (VYM Genome Research Center, National Yang-Ming University, Taipei, Taiwan), and NovaSeq SP (Roy J. Carver Biotechnology Center, Urbana, Illinois). The resulting fastq ﬁles were processed using CLC Genomics Workbench (CLC bio, AsiaPac, Taipei, Taiwan) or Geneious Prime 11.0.3 1 7 (Kearse et al. 2012) where paired ends were matched and the ends trimmed using default settings. Paired reads with the adaptor sequences removed were further ﬁltered by Trimmomatic v. 0.39 (Bolger et al. 2014) to remove low quality bases and reads shorter than 36 bp. Reference based assemblies were conducted for both the ribosomal cistron and the plastomes. For the ribosomal cistron, existing small and large subunit sequences from Moquiniella rubra (MH390507, MH390549) were concatenated and used as a reference to assemble the entire 9588 bp cistron. This sequence was then used to assemble Cecarria and Nuytsia. For the Loranthus species, the 6418 bp cistron from L. kaoi (J.M.Chao) H.S.Kiu (Su145) was used as reference. The de novo assemblies of Cecarria, Moquiniella, Nuytsia, and Loranthus kaoi (Su145) were conducted using CLC Genomics Workbench software (v8.5.1, CLC Bio, Qiagen, Aarhus, Denmark). Contigs containing plastid sequences were selected by BLAST searching against the plastome sequences of Nicotiana tabacum L. (NC_001879.2) and Schoepﬁa jasminodora Siebold & Zucc. (NC_034228.1) and were used for building initial scaffolds. Gap ﬁlling was completed by an iterative process of mapping all Illumina reads to the scaffolds using BWA v. 0.7.4 (Li and Durbin 2009), as well as with Samtools (Li et al. 2009), and visual inspection by IGV (Robinson et al. 2011). The complete plastome of Loranthus kaoi was then used as reference to assemble the plastomes of the other Loranthus species. When assembling to a reference, the Geneious mapper was used at medium sensitivity where multiple best matches were mapped randomly. Details about the NextGen sequencing, including platform used, insert sizes, number of reads, and Genbank numbers for all newly generated accessions are given in Supplemental File 1 (Nickrent et al. 2021). The initial annotations of the plastomes were performed using GeSeq (Tillich et al. 2017) and exon boundaries were adjusted TABLE 2. Species of Loranthus Jacq. Additional specimens identiﬁed as L. odoratus were collected in Bhutan, the Chinese mainland, India, Indonesia, Myanmar, Thailand, and Vietnam. Species Authority Year Distribution Foliar Habit Hosts Eastern and southeastern Asia from Chinese mainland to Myanmar and Vietnam Europe Iran, Afghanistan Evergreen Alnus, Pyrus, Carpinus, Persea, Cyclobalanopsis, Keteleeria Deciduous Deciduous Deciduous Quercus, Castanea Prunus, Pyrus, Acer, Cotoneaster, Crataegus, Quercus Platycarya, Quercus Evergreen Deciduous Taxillus, Scurrula, etc. Quercus Evergreen Evergreen Quercus Castanopsis, Quercus Deciduous Prunus, Pyrus, Acer, Betula, Quercus, Ulmus Loranthus delavayi (Tiegh.) Engl. 1897 Loranthus europaeus Loranthus grewingkii Jacq. Boiss. & Buhse 1762 1860 Loranthus guizhouensis H. S. Kiu 1983 Loranthus kaoi Loranthus lambertianus (J. M. Chao) H. S. Kiu Schult. f. 1983 1829 Loranthus odoratus Loranthus pseudo-odoratus Wall. Lingelsh. 1824 1922 Loranthus tanakae Franch & Sav. 1878 Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) Southwestern Chinese mainland Taiwan island Chinese mainland and Nepal Nepal Central portion of Chinese mainland Eastern Asia from the Chinese mainland to Korea and Japan 406 SYSTEMATIC BOTANY by comparing with the plastome sequences of N. tabacum. The presence or absence of orthologous genes in each genome was veriﬁed using BLASTN searches (e-value cutoff 5 1e210) against the plastomes of Nicotiana and Schoepﬁa. The accuracy of the completed plastomes was validated by mapping the ﬁltered raw reads to the draft plastome. The boundaries of the inverted repeats were carefully inspected to ensure the regions had achieved adequate read depth. Mitochondrial large subunit ribosomal DNA (rDNA) sequences were also obtained thus adding a third dataset from another subcellular genome. Molecular Phylogenetic Analyses—Multiple sequence alignments were conducted in Jalview (Waterhouse et al. 2009) using a combination of automated and manual approaches. These were then exported in Phylip format and converted to Nexus format in Mesquite version 3.5 (Maddison and Maddison 2018). The plastome, nuclear rDNA cistron, and mitochondrial 26S rDNA Nexus ﬁles, available from the Dryad Digital Repository (Supplemental Files 2–4, Nickrent et al. 2021) were analyzed separately (not concatenated) and in their entirety, i.e. not partitioned by genes or codons. Tree inference was by maximum parsimony implemented in PAUP v. 4.0 (Swofford 2002) and maximum likelihood with RAxML v. 7.2.8 (Stamatakis 2006) using GTR with gamma. For parsimony analyses, all characters received equal weight (of type “unord”) and gaps were treated as missing data. Maximum parsimony bootstrap (MPBS) heuristic searches used 1000 random stepwise addition replicates with tree bisectionreconnection branch swapping, holding 10 trees of length $ 1 at each step. Morphological Character Scoring—A morphological character matrix was constructed composed of 14 taxa and 21 vegetative, ﬂoral, and fruit characters. All nine Loranthus species shown in Table 2 were included and Cecarria obtusifolia as outgroup. Additional terminals for L. lambertianus and L. odoratus were included to test species cohesiveness. Photographs of living plants and herbarium specimens, veriﬁed as to their speciﬁc identity, were used as sources of data for populating the matrix. A number of categorical and continuous characters were explored and these data summarized in an Excel spreadsheet. The protologues and later literature for the various species were examined with regard to characters and character states. These data were placed in an Excel spreadsheet and compared to empirical observations made from specimens (types and others). Those characters that were found to be parsimony uninformative, or were unavailable from several taxa, were excluded from the matrix that was used for analysis. In general, macromorphological features were scored across all taxa whereas micromorphological features sometimes could not be obtained from the herbarium sheet photos. Measurements from the photographs were made using Adobe Photoshop (v. 21.2.1) by using the ruler tool and setting a measurement scale (unique for each photo) calibrated with the centimeter ruler photographed with the specimen. These data were placed in Excel ﬁles and the ranges and averages determined. Comments on how the 21 characters used in this analysis were scored, as well as comments on 15 other characters that were considered but excluded for various reasons can be seen in Supplemental File 5 (Nickrent et al. 2021). Morphological Character Analysis—Eleven of the 21 characters were categorical whereas 10 were continuous. The categorical characters were treated “as is” whereas for the continuous characters, average values were natural log transformed [ln(x11)] and range-standardized [xs 5 (xmin/max-min) 3 10] as reported in Thiele (1993) using Microsoft Excel. These log transformed averages were gap coded based on their overall distributions. Characters 12, 17, 18, and 19 received three states and the remaining characters two states. The data were entered into Mesquite and exported as a Nexus ﬁle (Supplemental ﬁle 6, Nickrent et al. 2021). Maximum parsimony (MP) analysis was performed with PAUP v. 4 (Swofford 2002) using a branch and bound search with the addition sequence set as furthest. [Volume 46 RESULTS Plastome Structures—The Loranthus and outgroup plastomes conform to the typical quadripartite structure seen in most members of Santalales. These plastomes range in size from 121 kb (L. odoratus) to 125 kb (L. lambertianus) with an average of 123 kb. For the outgroups, Moquiniella is of average size whereas Cecarria is smaller (116 kb) and Nuytsia is larger than average (139 kb). The typical angiosperm plastome contains on average 112 nonredundant genes (Supplemental ﬁle 7, Nickrent et al. 2021). In contrast, most Loranthus have just 90 full-length (supposedly functional) plastome genes. These plastomes have experienced 15 gene losses and 9 cases of pseudogenes. Ten of the 11 ndh genes have been lost and the other (ndhB) is a pseudogene. Other protein-coding gene losses include rpl32, rps15, and rps16. Six additional protein-coding genes are pseudogenes: infA, ndhB, psaI, rpl16, rpl33, and ycf15. For tRNA genes the picture is less consistent across species. All Loranthus have lost trnG-UCC and trnV-UAC and all have trnK-UUU as a pseudogene. For trnA-UGC and trnIGAU, presence, absence, and pseudogenes are seen. The patterns of plastome gene presence and absence in the three outgroup species differ from Loranthus. The longest and most intact plastome is seen in Nuytsia where infA, psaI, rpl16, rpl32, and rpl33 are present and intact. For the latter, a mutation of ATG to CTG has occurred in the start codon, unlike Moquiniella that has a typical codon at this position. Genes present as pseudogenes in Loranthus are intact in Moquiniella and Nuytsia such as psaI and rpl16. Among all the species sampled here, Nuytsia has the most intact complement of tRNA genes, with only trnG-UCC and trnV-UAC being absent or pseudogenized. The smaller genome of Cecarria is reﬂected in a higher degree of gene loss and pseudogenization. Unlike the other species, pseudogenes are seen in matK and psbZ, and apparently, infA has been lost entirely. Molecular Phylogenetics of Loranthus—The statistics obtained from maximum parsimony analysis of the plastome, nuclear rDNA, and mitochondrial 26S rDNA datasets are shown in Table 3. The highest number of parsimony informative characters was directly proportional to the number of characters in the subcellular genomes with plastome . nu rDNA . mt rDNA. This trend is in the reverse direction when the parsimony informative characters are expressed as a percentage of the number of characters. Mitochondrial 26S rDNA also had the highest consistency index of the three genomes. In terms of tree topologies, all analyses strongly support a monophyletic genus Loranthus with Cecarria sister to this clade. The individual plastome and mt 26S rDNA trees were most similar, differing mainly in the placement of Loranthus odoratus. TABLE 3. Gene and genome diversity statistics. Number of taxa Number of characters (alignment length) Constant characters Variable uninformative characters Parsimony informative characters Number of maximum parsimony trees Tree length CI minus uninformative characters Plastome Nuclear rDNA Mitochondrial 26S rDNA 17 148,114 133,075 (89.8%) 10,930 (7.3%) 4109 (2.7%) 1 17,356 0.7807 17 6544 5682 (86.8%) 467 (7.1%) 395 (6.0%) 1 1344 0.6741 17 4507 3977 (88.2%) 223 (4.9%) 307 (6.8%) 3 723 0.7942 Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) 2021] NICKRENT ET AL.: EVOLUTIONARY RELATIONSHIPS IN LORANTHUS 407 FIG. 1. Phylogenetic relationships among Loranthus species from whole plastome and nuclear rDNA cistron sequences. MLBS values above nodes, MPBS below nodes. For the plastome, this taxon was sister to a clade containing L. delavayi, L. kaoi, and L. pseudo-odoratus Lingelsh. For mt rDNA, this species was included within this clade, but with low support (Supplemental ﬁle 8, Nickrent et al. 2021). The trees resulting from maximum parsimony and maximum likelihood analyses for the plastome and nuclear rDNA datasets are compared in Fig. 1. The overall topologies for the two trees are nearly congruent, differing mainly in the placement of Loranthus lambertianus. For the plastome and mitochondrial datasets, this species is strongly supported as sister to a clade containing L. delavayi, L. guizhouensis H.S.Kiu, L. kaoi, L. odoratus, and L. pseudo-odoratus. For the nuclear rDNA dataset, this species is sister to a clade composed of L. europaeus, L. grewingkii, and L. tanakae Franch & Sav., but with moderate to weak support. Loranthus odoratus is strongly supported as sister to the L. delavayi, L. kaoi, and L. pseudo-odoratus clade on the plastome tree. Its position in the nuclear rDNA tree is not strongly supported; indeed if the nodes with low support are collapsed, it becomes part of the polytomy containing the Odoratus complex of species. The tree resulting from concatenating all three partitions was identical to the plastome tree (data not shown). Cladistic Analysis of Morphological Characters—Each of the 21 morphological characters optimized on the overall cladogram are shown in Supplemental ﬁle 9 (Nickrent et al. 2021). A branch and bound analysis of the morphological character matrix yielded 30 equally parsimonious trees of length 48 (Fig. 2). Bootstrap analysis showed strong support for the nodes along the “spine” of the tree but no support for relationships among L. Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) delavayi, L. kaoi, L. odoratus, and L. pseudo-odoratus. Strong support was obtained for a clade containing L. grewingkii and L. tanakae, however, L. europaeus was not part of this clade. Examination of alternate tree topologies as implemented in Mesquite showed that making L. europaeus part of this clade (as strongly indicated by molecular data) added only one step to the tree. Similarly, only one extra step was required to make the two accessions of L. lambertianus monophyletic and two steps to place this clade with L. europaeus, L. grewingkii, and L. tanakae, as indicated by the rDNA tree (Fig. 1). A hypothesis for the organismal phylogeny of Loranthus includes two major clades: Europaeus and Odoratus (Fig. 2). DISCUSSION This is the ﬁrst species level phylogeny of a genus in Loranthaceae utilizing complete plastome sequences. This dataset, complemented with the nuclear rDNA cistron, mitchondrial 26S rDNA, and morphology, are mostly congruent with regard to inferring species relationships within Loranthus. Although plastome genome sequences have been published for 15 Loranthaceae species (Li et al. 2017; Shin and Lee 2018; Yuan et al. 2018; Chen et al. 2019; Yu et al. 2019; Zhao et al. 2019), only eight of the 76 genera are represented and these are all Old World species. The plastome of Loranthus tanakae was reported by Chen et al. (2019) but the identiﬁcation was listed as unveriﬁed. Moreover, its plastome size of 123.4 kb differs signiﬁcantly from the size obtained in this study from two accessions of 408 SYSTEMATIC BOTANY [Volume 46 FIG. 2. Relationships among Loranthus species determined by cladistic (maximum parsimony) analysis of morphological characters. To the right, the majority rule consensus of 30 equally parsimonious trees of length 48. Numbers above nodes indicates the percentage of trees with that topology. The topology of the smaller tree to the left conforms to that of the rDNA tree (Fig. 1) for L. lambertianus and L. europaeus and adds two steps, thus making a monophyletic Europaeus clade. Foliar habit (deciduous vs. evergreen), geographic distribution, and plant sexual mode are plotted on this tree. that species (121.7 kb). For this reason, this and other previously generated sequences were not included in this study. Plastome Features—The pattern of gene loss in Loranthaceae plastomes conﬁrms previous observations, such as the loss (and pseudogenization) of all NADH dehydrogenase complex genes. Based on their limited sampling in the family, Chen et al. (2019) state that Loranthaceae lack the transcription initiation factor I gene (infA). As shown here, both Nuytsia and Moquiniella contain intact infA genes, thus highlighting the importance of robust taxon sampling before making inferences about gene presence/absence trends. The average size for the Loranthus plastome generated in this study (123.2 kb) is very similar to the size averaged across ten other Loranthaceae genera (123.6). The largest plastome in Loranthaceae is Nuytsia (139 kb; Supplemental ﬁle 7, Nickrent et al. 2021) which is similar to the average size from 10 other nonViscaceae Santalales which range from 118 to 156 kb. It is important to state that Nuytsia is one of three root parasitic genera in the family that form a grade at the base of the Loranthaceae phylogenetic tree (Vidal-Russell and Nickrent 2008b; Liu et al. 2018; Nickrent et al. 2019). The larger size of the Nuytsia plastome is signiﬁcant given its phylogenetic position as sister to all other members of Loranthaceae (Vidal-Russell and Nickrent 2008a; Su et al. 2015; Liu et al. 2018; Nickrent et al. 2019). As seen in other clades of Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) Santalales, the trend in nutritional modes is root hemiparasitism . stem hemiparasitism . holoparasitism which is correlated with reductions in plastome size (Petersen et al. 2015; Wicke and Naumann 2018). Chen et al. (2019) state that “There are also some rare events of evolutionary reversions or atavism in which the stem-parasites evolved back into root-parasites (e.g., Nuytsia, Atkinsonia, and Gaiadendron in Loranthaceae).” This statement is not true for the following reasons. The sister group to Loranthaceae is the Schoepﬁaceae/Misodendraceae clade where the former are root parasites and the latter stem parasites (mistletoes). A reconstruction of parasitism for the node joining these two groups yields a root parasitic ancestor. Indeed, the ancestor of Misodendraceae was likely a root parasite and the mistletoe habit evolved at some point along this long branch (Vidal-Russell and Nickrent 2008b). Thus, the early diverging members of Loranthaceae evolved from root parasitic Santalales ancestors and did not revert to this habit from stem parasites. Gene Tree Incongruence—Phylogenetic analyses of the plastome sequences resulted in a fully resolved tree topology using both maximum parsimony and maximum likelihood (Fig. 1). This tree was nearly identical to that obtained from the mitochondrial 26S rDNA (Supplemental ﬁle 5, Nickrent et al. 2021) and with the exception of Loranthus lambertianus, the nuclear ribosomal DNA cistron. Examination of the alignment 2021] NICKRENT ET AL.: EVOLUTIONARY RELATIONSHIPS IN LORANTHUS 409 FIG. 3A. Loranthus species and outgroup. A. Cecarria obtusifolia, the outgroup species; plant with ﬂowers and young fruits. B–D. Loranthus delavayi, a dioecious species. B. Staminate ﬂowers showing central pistillode. C. Pistillate ﬂowers bearing staminodes. D. Plant with fascicles and young infructescences. Inset: Mature fruits. E–H. L. europaeus. E. Habit of plant in fruit. F. Staminate inﬂorescence. G. Pistillate inﬂorescence. H. Possibly bisexual ﬂower, two stamens removed. I–K. L. grewingkii. I. Shoots bearing inﬂorescences. J. Inﬂorescence. Inset showing ﬂower detail. K. Shoot with infructescences showing mature white berries. Inset shows dried fruits that turn red. Photo credits: A. Bruce Gray (no. 5354). B, C. Wevin, Nature Campus web site. D. Rouzhu Lin. Inset Hosan, Nature Campus web site. E. Jaınos Bognaır, Flickr. F. Gerhard Glatzel. G. Robert Videıki, ForestryImages number 5396004, Doronicum Kft., Bugwood.org. H. Roland Aprent. I, J. Seyed Mansour Mirtajadini. K. Gerhard Glatzel. indicates that indeed the rDNA sequence of this species has a number of features more similar to the Europaeus clade than the Odoratus clade (Fig. 2). For example, a unique insertion of 50 bp in ITS-1 is present in L. lambertianus, the other members of the Europaeus clade, and L. guizhouensis. Therefore, it appears that the conﬂict in topology is real and not an artifact of poor or ambiguous alignment. With respect to L. odoratus, the nuclear rDNA and mt 26S rDNA tree topologies are simply less resolved than the plastome tree and are not incongruent with it. A number of explanations have been proposed to account for cytoplasmic and nuclear gene trees incongruences, including chloroplast capture (Rieseberg and Soltis 1991), incomplete lineage sorting (Maddison and Knowles 2006), and androgenesis (Hedtke and Hillis 2011). Natural hybridization Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) in Loranthaceae is apparently rare, although it has been documented in Amyema Tiegh. (Calder et al. 1982). Chloroplast capture requires introgression, which has recently been shown to occur in Psittacanthus schiedeanus (Cham. & Schltdl.) G.Don and P. calyculatus G.Don (Baena-Dıaz et al. 2018). The conﬂicting molecular data, plus the position of L. lambertianus midway between the Europaeus and Odoratus clades on the plastome tree, suggests this species could be composed of mixed cytonuclear genotypes, combining the nuclear genome of the former and the plastome of the latter clades. A ﬁtness advantage (e.g. higher seed production) can occur through interaction of cytoplasmic genes from one species and nuclear genes from another, but this hinges upon partial male sterility (Tsitrone et al. 2003). Although L. lambertianus appears to be synoecious, it is of interest that “experiments” in sexual 410 SYSTEMATIC BOTANY [Volume 46 FIG. 3B. Loranthus species. L–M. L. guizhouensis. L. Plant sample showing pseudo-dichotomous branching. M. Terminal immature inﬂorescence showing decussate, clavate buds. N–Q. L. kaoi. N. Leafy shoot with young fasciculate infructescences. O. Flowering shoot. P. Mature fruits. Q. Seed hanging by viscin threads with adhering fruit wall. R, S. L. lambertianus. R. Habit of mistletoe showing pseudo-dichotomous branching and brachyblasts. S. Leafy shoots with terminal inﬂorescences. T–V. L. odoratus. T. Flowering shoot showing axillary inﬂorescences. U. Inﬂorescence. V. Shoot with young fruits. W, X. L. pseudo-odoratus. W. Inﬂorescences in fascicles. X. Shoot with immature and mature fruits. Y, Z. L. tanakae. Y. Shoot with terminal inﬂorescence. The inset shows a mucronulate leaf apex from an herbarium specimen. Z. Infructescences. Photo credits: L, M. Rouzhu Lin. N–Q. Jer-Ming Hu. R–U. Mohan Devkota. V. Gerhard Glatzel. W, X. Rouzhu Lin. Y. 云中鸟, straybird726.com, Flickr. Z. Chapeng, iNaturalist. Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) 2021] NICKRENT ET AL.: EVOLUTIONARY RELATIONSHIPS IN LORANTHUS mode differences and altered sex ratios (Elias 1997) are occurring in Loranthus. The Loranthus Phenotypes—Discussion of the morphological characters used for the cladistic analyses is presented in Supplemental ﬁle 3 (Nickrent et al. 2021), hence these details will not be repeated here. In some cases the observations recorded 411 from type and other material agreed with the protologue descriptions, in other cases they did not. In that case, observations made from the specimens were used. The critical features that can be used to identify the nine recognized species of Loranthus are included in the following key and can also be seen in photos presented in Fig. 3. KEY TO LORANTHUS 1. 1. Distal branching pseudo-dichotomous; inﬂorescences terminal, not in fascicles, leaves deciduous.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. Leaves oblanceolate, oblance-ovate, or narrowly ellipic, generally less than 3.5 cm long; inﬂorescences less than 2.5 cm long; fruits translucent white, drying dark red or brown; Iran, Afghanistan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. grewingkii 2. Leaves elliptic, lance-ovate, ovate, or obovate, generally greater than 3.5 cm long; inﬂorescences more than 2.5 cm long; fruits yellow. . . . . . . . . . . . 3 3. Anther locules two; eastern Chinese mainland to Japan, Korea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. tanakae 3. Anther locules four . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4. Petioles 6–12 mm long; plant sex subdioecious, ﬂowers per inﬂorescence 6–10; ovary from a raised cupule, not sunken into pit on inﬂorescence axis; Europe to western Iran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. europaeus 4. Petioles 2–4 mm long; plant sex synoecious (all ﬂowers bisexual); ﬂowers per inﬂorescence 8–16; ovaries sunken into pits on inﬂorescence axis;. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5. Flowers greenish; southeastern Chinese mainland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. guizouensis 5. Flowers yellowish; southwestern Chinese mainland, Nepal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. lambertianus Distal branching percurrent, inﬂorescences axillary, in fascicles, leaves evergreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6. Eight (rarely 10) or fewer ﬂowers per inﬂorescence, fruit globose; central to eastern Chinese mainland . . . . . . . . . . . . . . . . . . . . . . . . . L. pseudo-odoratus 6. Eight or more ﬂowers per inﬂorescence, fruit ovoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7. Epiparasitic on other Loranthaceae; anther locules two; Taiwan island . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. kaoi 7. Not epiparasitic; anther locules four . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8. Plants dioecious, female ﬂowers with staminodes; Chinese mainland, Myanmar, Vietnam. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. delavayi 8. Plants synoecious; Nepal (southeast Asia, Malesia?) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. odoratus Europaeus Clade—Loranthus europaeus, L. grewingkii, and L. tanakae are strongly supported as a clade with both the plastome and nuclear rDNA datasets. The geographic distribution of these three species is remarkable, spanning 11,000 km from Europe (L. europaeus), to Iran and Afghanistan (L. grewingkii) and the Chinese mainland, Japan and Korea (L. tanakae) with a 4000 km gap. The Chinese Virtual Herbarium lists over 200 collections of L. europaeus from the Chinese mainland, but most likely many of these are L. tanakae that very much resembles L. europaeus. Although all of these species of the Europaeus clade share synapomorphies such as pseudodichotomous branching, deciduous leaves, terminal inﬂorescences, cupule type ﬂoral/rachis junctions, and greenish petals, they also differ in some categorical characters such as petal reﬂection, anther locule number, and fruit shape. As mentioned in the Introduction, L. grewingkii is seldom included in World and Regional ﬂoras, but it is clearly a distinct species. This is supported by the molecular data reported here as well as the micro- and macromorphological characters reported by Shahi Shavvon et al. (2012). That study mentioned the presence of four bracts at the branching site (node) in L. grewingkii that are not present in L. europaeus. These bracts are likely prophylls (Kuijt 2013), which exist in both species. Shahi Shavvon et al. (2012) describe the fruit of this species as red, but apparently did not precisely translate the Latin from Boissier and Buhse (1860) which said “baccis e sicco globosis rubris,” i.e. “berries spherical, red when dry.” Mature translucent white fruits as well as red desiccated fruits are shown in Fig. 3. Finally, the key in Shahi Shavvon et al. (2012) distinguishes L. grewingkii from L. europaeus by the evergreen (vs. deciduous) leaves in the former species. Collections recently made at two locations in Iran indicates that L. grewingkii is deciduous (G. Glatzel pers. obs.). Loranthus lambertianus and L. guizhouensis—One accession of a taxon hypothesized to be L. lambertianus from Nepal was included in the molecular analyses (number 6752). Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) Morphological data from this specimen, plus a taxon created from the protologue by Schultes and Schultes (1829), were included in the cladistic analysis. As shown in Fig. 2, these two taxa are adjacent, and to place them in a clade adds just one step to the length of the tree. The majority of the character states scored for these two taxa are the same, although the Schultes and Schultes (1829) description gives the ﬂower merosity as ﬁve instead of six. Overall, it is likely that the 6752 specimen corresponds to L. lambertianus as originally described from Nepal. This species is represented by very few collections, probably because it occurs in inaccessible places in the Himalayas and is likely also quite rare in nature. The type of branching, pseudodichotomous vs. percurrent, generally separates the Europaeus clade from the Odoratus clade (Fig. 2). Between these two clades is Loranthus guizhouensis that shows percurrent branching below and pseudodichotomous above. The occurrence of both branching types in one mistletoe species is not unprecedented, as it is also documented in Phoradendron Nutt. (Viscaceae) (Kuijt 2003). Unlike some members of the Odoratus clade with yellow or white petals, L. guizhouensis has green petals like L. europaeus, L. grewingkii, and L. tanakae of the Europaeus clade. This species is closely related to L. lambertianus as evidenced by both molecular and morphological character analyses. Odoratus Clade—As deﬁned here, the Odoratus clade contains the following taxa: Loranthus delavayi, L. kaoi, L. odoratus, and L. pseudo-odoratus. As shown by molecules (Fig. 1) and morphology (Fig. 2), species relationships within this complex are not well resolved. The sampling achieved in this study is insufﬁcient to provide further insight into the interspeciﬁc systematic relationships, however, some observations can be made. Four accessions for L. delavayi were sampled for DNA sequencing and they are not monophyletic. Both the plastome and nuclear rDNA phylogenetic trees (Fig. 1) show that the two accessions from Taiwan island, Su 160 and Wu 033, are on a 412 SYSTEMATIC BOTANY clade with L. kaoi (Su 145) and the two accessions from the Chinese mainland (6748, 6912) are on a clade with L. pseudo-odoratus (6915). One solution to this is to simply combine all four species into one. This was partially done by Barlow (1997) who placed L. delavayi in L. odoratus. But as shown on the molecular tree (Fig. 1), L. odoratus is genetically the most distinct member of this complex, thus taxonomic combination at this time is premature. The Flora of Taiwan currently recognizes two Loranthus species, L. delavayi and L. kaoi, basically following the treatments of Chao (1973) and Kiu (1983), where the former listed them under the genus Hyphear. Chao (1973) named Hyphear kaoi J.M.Chao based on its epiparasitic habit (unique in the genus) and the presence of bisexual ﬂowers. In the same publication he combined two previously recognized Taiwanese Loranthus species, L. owatarii Hayata (Matsumura and Hayata 1906) and L. koumensis Sasaki (Sasaki 1931), placing them in synonymy with L. delavayi from the Chinese mainland. The results of the molecular phylogenetic analyses (Fig. 1), suggest that the samples identiﬁed here as L. delavayi from Taiwan island might well be distinct taxa, thus following the original treatments of Matsumura and Hayata (1906), Sasaki (1931), and Hosokawa (1936). Although Chao (1973) indicated that he compared specimens of Hyphear owatarii Danser from Taiwan island with H. delavayi from the southern Chinese mainland, no data were presented justifying his conspeciﬁc status. A comparison of leaf and inﬂorescences dimensions for 16 mainland Chinese L. delavayi specimens and 13 “L. owatari” specimens from Taiwan showed signﬁcant differences only in petiole length (data not shown). A more detailed systematic study of these taxa is required to properly circumscribe species. Only one accession of Loranthus odoratus was included in the molecular analyses owing to sample availability. This is unfortunate because the morphological analyses indicate this name is being applied to mistletoes with markedly different phenotypes. Four L. odoratus taxa were included in the cladistic analysis, the type specimen (from Nepal, at K), collection 4977 (from Nepal), A. F. G. Kerr 6692 (Thailand, at P), and Grifﬁth 2719 (Bangladesh, at P). The ﬁrst three of these emerge in a polytomy indicating very similar phenotypes. In contrast, the Grifﬁth 2719 collection is sister to L. pseudo-odoratus with strong bootstrap support. Seven other specimens ranging from Tibet, Myanmar, Vietnam, and Indonesia were also analyzed and they resolved in at least four distinct clades (data not shown). Although the limited amount of information available from herbarium specimen images could account for this, the wide variation in macromorphological (mostly vegetative) features suggests these specimens were either misidentiﬁed or they might represent new species. An example of the latter is a collection from Manipur State in India (Bullock 877, at P) that has very large (average 5 mm long) clavate buds that, when open, reveal only stamens (no style) with large anthers (average 1.4 mm long). The only other species in this complex that is dioecious with large anthers is L. delavayi, however, the Bullock specimen does not match other morphological features of that species. Biogeography—The molecular phylogenetic study of Loranthaceae by Vidal-Russell and Nickrent (2008a) provided a general scenario for the dispersion and radiation of the family from Australia to South America, Asia, and Africa. More precise historical biogeographical analyses were conducted by Liu et al. (2018) who showed that the Loranthus/Cecarria clade occupies a central position in the loranth phylogeny, as sister to all of the x 5 9 clade (tribe Lorantheae). The crown group age for Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) [Volume 46 Loranthaceae was estimated to be of Eocene age (53–66 MYA) and the Loranthus/Cecarria clade diverged in the Eocene (ca. 39.6 MYA). Only two species of Loranthus were included, Loranthus europaeus and L. odoratus, and they diverged in the Tortonian of the Miocene (ca. 9.7 MYA). Given that these two species are members of the two major clades (Fig. 2), this age can be used as an estimate of their divergence. It can be hypothesized that after splitting from Cecarria, the Loranthus ancestor migrated from Australasia into southern Asia. It continued spreading westward during the middle Miocene and, after a climatic optimum, began experiencing drier and cooler conditions, especially in central Asia, northwestern Chinese mainland and Mongolia. After the Miocene, most areas of western and central Asia turned into deserts caused by the uplift of the Himalayas, which likely forced the distribution to regions south of this boundary. In addition to the westward spread of the ancestor that generated L. europaeus, L. grewinkii, and L. lambertianus, a member of this clade also spread northward into the Chinese mainland, giving rise to L. tanakae. The ancestor to the Odoratus clade also diversiﬁed, but these derivatives remained mainly south of 30 N latitude and east of 85 N longitude. Foliar Habit—Deciduousness in Loranthaceae is extremely rare, outside of Loranthus being found only in Desmaria mutabilis Tiegh. ex T.Durand & B.D.Jacks of South America. Five species of Loranthus have been reported as being deciduous (Table 2): L. europaeus, L. grewingii, L. guizhouensis, L. lambertianus, and L. tanakae. Because of its rarity, deciduousness is considered an apomorphic state with evergreen leaves being plesiomorphic. Indeed, this polarity was proposed for the genus Loranthus (Glatzel et al. 2017). Loranthus lambertianus was listed as deciduous in Flora of China (Qiu et al. 2003) and this has been conﬁrmed for collection 6752 from Nepal by M. Devkota, thus supporting its placement in the Europaeus clade as seen on the nuclear rDNA tree (Fig. 1). When viewing foliar habit in the context of the phylogeny of the genus (Figs. 1, 2), the outgroups and most members of the Odoratus clade are evergreen. The one exception is Loranthus guizhouensis that is sister to the Odoratus clade on the nuclear rDNA tree (Fig. 1). That topology implies that this species evolved the deciduous habit independently from the Europaeus clade. Although it is listed in Flora of China as being deciduous (Qiu et al. 2003), foliar habit was not speciﬁcally indicated in the protologue for this species by Kiu (1983). The leaves are described as “papery or thinly leathery” and it occurs at elevations of 200–1400 m in subtropical broadleaf evergreen forests in the southern Chinese mainland, as broadly classiﬁed in Song (1995). A more recent vegetation map (Pan et al. 2015) shows that the western portion of the L. guizhouensis distribution (ca. 360 km from central Guizhou to eastern Yunnan) is a complex mosaic of vegetation types, including tropical broadleaf evergreen, temperate needleleaf evergreen, and temperate broadleaf deciduous forest types. Known populations occur south of 30 N latitude in Guandong, Guangxi, Guizhou, Hunan, Jiangxi, and Yunnan Provinces where the mean annual temperature is 18–20 C (Liu et al. 2003), hence they do not experience freezing temperatures in winter. This mistletoe is reported to parasitize Quercus myrsinifolia (Fagaceae) and Platycarya strobilacea (Juglandaceae); the former is evergreen whereas the latter is deciduous. Exactly what environmental or host physiological cues cause this mistletoe to drop its leaves remains to be explained. Finally, it is possible that the information in Flora of China is erroneous and that in fact this mistletoe is evergreen. 2021] NICKRENT ET AL.: EVOLUTIONARY RELATIONSHIPS IN LORANTHUS Sexual Mode in Loranthus—For the whole plant sexual mode, the term synoecious is being used here (following Nickrent et al. 2019) for the condition where all ﬂowers, on all individuals in the population, are bisexual (i.e. no unisexual ﬂowers occur). A desirable feature of this term is that it has the same sufﬁx (-ecious) as other whole plant sexuality terms such as monoecious (staminate and pistillate ﬂowers on one individual) and dioecious (staminate and pistillate ﬂowers on separate individuals). The term bisexual is used here to refer to the presence of a functional androecium and gynoecium in an individual ﬂower. Other terms equivalent to bisexual include perfect, hermaphroditic, and cosexual (Sakai and Weller 1999). The majority of the genera in Loranthaceae (66 of 76) are synoecious, including the outgroup genera Cecarria and Moquiniella (Nuytsia is polygamomonoecious). Flowers in Loranthaceae can be large, brightly colored and bird pollinated, or inconspicuous and pollinated mainly by insects. Two genera (Tupeia and Struthanthus) are entirely dioecious whereas in six genera of subtribe Psittacanthinae, both bisexual and unisexual ﬂowers occur with varying sexual conditions. In tribe Lorantheae, only two genera show bisexual and unisexual ﬂowers: Baratranthus and Loranthus where plant sexuality is synoecious for some species and dioecious for others. Loranthus europaeus is considered subdioecious, i.e. populations have pistillate plants, staminate plants, and some individuals with both staminate and bisexual ﬂowers (Sakai and Weller 1999). Subdioecy was ﬁrst described in L. europaeus by Jacquin (1762) who used the term “hermaphroditus sterilis” for plants with female sterile plus bisexual ﬂowers. One study of L. europaeus from Slovakia (Elias 1997) showed a male biased sex ratio, however, only staminate and pistillate plants were discussed (not bisexuals). The protologue for L. grewingkii by Boissier and Buhse (1860) states “ﬂoribus dioicus,” which, if interpreted literally, means “ﬂowers dioecious,” but photos of this species indicate the ﬂowers are bisexual. Another species of Loranthus with unisexual ﬂowers is L. delavayi which is described (and illustrated) as fully dioecious (Qiu et al. 2003). Because this species and L. europaeus occur on different clades, it is assumed they evolved unisexuality independently (Fig. 2). For the clade of Loranthus from Taiwan, L. kaoi is certainly synoecious (Chao 1973). For the two taxa labeled L. delavayi, the protologue for L. owatarii (Matsumura and Hayata 1906) gives a brief description that pertains to female ﬂowers (with staminodia) but no accompanying description of the male ﬂower was given. This species was included in L. delavayi by Chao (1973). The sexual mode in the Loranthus odoratus complex is not clear. The protologue by Wallich (1824) does not indicate unisexual ﬂowers and describes both the androecium and gynoecium, thus it is assumed that bisexual ﬂowers were being observed. This is in agreement with observations of this species in Nepal by M. Devkota (Fig. 3). In his Flora Malesiana treatment, Barlow (1997) describes a taxon from Sulawesi, Indonesia as Loranthus odoratus. The description includes only information on the androecium, indicating “ﬂowers probably functionally unisexual but often with vestigial organs of the other sex.” The illustration shows a plant with inﬂorescences bearing young fruits. One herbarium specimen (E. F. De Vogel 5486, Naturalis Biodiversity Center, no. L1643709) from Sulawesi, annotated as L. odoratus by Barlow, was examined that shows mostly young ﬂower buds and a note on the label indicates “fruit young, greenish.” Unfortunately, the photograph is insufﬁcient to determine whether this specimen has bisexual or Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) 413 unisexual ﬂowers. Overall, plant sexual mode in Loranthus is much in need of further research. The phylogenetic data reported here have provided answers to some questions surrounding the enigmatic genus Loranthus, but at the same time, have uncovered numerous areas that require further research. The genus appears to be composed of species that fall into two major clades. Distributed mainly north of 30 N latitude are four deciduous species in clade Europaeus: L. europaeus, L. lambertianus, L. grewingkii, and L. tanakae. This clade is strongly supported by nuclear rDNA and (minus L. lambertianus) complete plastome sequences. The second major clade is Odoratus, composed of the remaining ﬁve species that are distributed mostly south of 30 N latitude. Four of these species are evergreen whereas one (L. guizhouensis) has possibly evolved the deciduous habit independently of the Europaeus clade. Unisexuality evolved twice independently in Loranthus, once in the Europaeus and once in the Odoratus clades. ACKNOWLEDGMENTS We wish to thank Prof. Seyed Mansour Mirtajadini, Mr. Ilia Marza, Prof. Ehsan Sayad, and the late Prof. Saeidi-Mehrvarz for providing information on reliable sites for Loranthus grewingkii in Iran. Other collectors of specimens used in this study are Carlos Reif (L. europaeus), Cheng-Chiang Wu (L. delavayi), and Adrienne Markey via Bryan Barlow (Nuytsia ﬂoribunda). Gitte Petersen kindly provided an unpublished sequence of L. europaeus. Funding was provided by the Austrian Academy of Sciences, Dr. Anton Oelzelt-Newin'schen Stiftung, to GG, Botanical Images Activities fund at SIUC to DLN, the Ministry of Science and Technology, Taiwan (106-2311-B-845-001-MY3) to HJS, and by the National Natural Science Foundation of China (31400520). Finally, we dedicate this paper to Nicolaus Joseph von Jacquin (1727-1817) whose insightful work, at an early stage of plant taxonomy, described the type species for the entire family Loranthaceae. AUTHOR CONTRIBUTIONS The project was conceived by GG and discussed at an organizational meeting 6 November 2017 in Vienna, Austria. All authors collected samples of Loranthus used in this study. Unpublished DNA sequence data for L. delavayi and L. kaoi were provided by HJS. Laboratory work and data analyses were performed by DLN and HJS. Morphological character analyses were conducted by DLN who also prepared the graphics and wrote the ﬁrst draft of the paper. 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Characterization of the complete chloroplast genome of Taxillus nigrans. Mitochondrial DNA. Part B, Resources 4: 472–473. APPENDIX 1. The order of information is as follows: species, location, coordinates, date, collector and number, herbarium code and name, DNA accession no., host, Genbank accession numbers for plastome, nuclear rDNA, and mitochondrial 26S rDNA. A dash indicates missing data. Ingroup: Loranthus delavayi (Tiegh.) Engl., the Chinese mainland, Sichuan Province, Muli County, 28 02'15”N, 101 11'72”E, 18 May 2013, R.-Z. Lin LRZ2013011, CAF; Dendrological Herbarium, Chinese Academy of Forestry, DLN 6912, Fagus longipetiolata, MT987626, MT968895, MT968911. Loranthus delavayi (Tiegh.) Engl., the Chinese mainland, Yunnan Province, Near Fuguosi Temple, Lijiang City, 26 57'15”N, 100 11'55”E, 16 August 2016, R.-Z. Lin YN1613, CAF; Dendrological Herbarium, Chinese Academy of Forestry, DLN 6748, –, MT987625, MT968894, MT968910. Loranthus delavayi (Tiegh.) Engl., Taiwan, Qingjing Farm, Nantou County, 24 02'57”N, 121 9'25”E, 10 November 2018, H.-J. Su Su160, TAI, National Taiwan University, Su160, Quercus sp., MT987628, MT968906, MT968913. Loranthus delavayi (Tiegh.) Engl., Taiwan, Mei-Feng, Nantou County, –, 27 February 2003, J.-M. Hu and C.-C. Wu Wu033, TAI, National Taiwan University, Wu 033, –, MT987624, MT968909, MT968914. Loranthus europaeus Jacq., Republic of Austria, Lower Austria, City of Vienna, 48 09'20”N, 16 26'20”E, 17 July 2005, D. L. Nickrent and G. Glatzel 4982, SIU, Southern Illinois University, DLN 4982, Quercus robur, MT987630, MT968896, MT968920. Loranthus europaeus Jacq., Czeck Republic, Prague Region, City of Prague, 50 04'02”N, 1 25'41”E, September 2015, C. Reif 2027, C, The Natural History Museum of Denmark, University of Copenhagen, CR 2027, Quercus sp., MT987629, MT968897, MT968919. Loranthus grewingkii Boiss. & Buhse, Islamic Republic of Iran, Kerman Province, East side of the town of R abor, just west of split in Hwy. 88, 29 17'19.92”N, 56 55'43.90”E, 22 October 2018, G. Glatzel s.n., –, DLN 6892, Prunus dulcis, MT987631, MT968898, Downloaded From: https://bioone.org/journals/Systematic-Botany on 20 Aug 2021 Terms of Use: https://bioone.org/terms-of-use Access provided by Nepal (R4L) 415 MT968921. Loranthus guizhouensis H.S.Kiu, the Chinese mainland, Guizhou Province, Lizhuang village, Kaiyang County, 26 52'07”N, 107 08'03”E, 14 May 2019, R.-Z. Lin GZ1902, CAF; Dendrological Herbarium, Chinese Academy of Forestry, DLN 6925, –, MT987632, MT968899, MT968917. Loranthus kaoi (J.M.Chao) H.S.Kiu, Taiwan, Mei-Feng, Nantou County, 24 05'17”N, 121 10'28”E, 6 August 2018, H.-J. Su Su145, TAI, National Taiwan University, Su145, Taxillus rhododendricolus parasitic on Corylus heterophylla, MT987633, MT968905, MT968915. Loranthus lambertianus Schult.f., Federal Democratic Republic of Nepal, Bagmati Pradesh Province, Gaurishankar Conservation Area (GCA), Dolakha District, Bigu Rural Municipality (Gaupalika), 27 59'00.7”N, 86 12'29.1”E, 2 May 2015, M. Devkota 1421, KATH; National Herbarium and Plant Laboratories, DLN 6752, Quercus semecarpifolia, MT987634, MT968900, MT968918. Loranthus odoratus Wall., Federal Democratic Republic of Nepal, Bagmati Pradesh Province, Dakshinkali Municipality, Chalnakhel Forest, Kathmandu District, 27 37'58”N, 85 16'69”E, 30 November 2019, M. Devkota 1202, KATH; National Herbarium and Plant Laboratories, DLN 4977, Quercus glauca, MT987636, MT968903, MT968916. Loranthus pseudo-odoratus Lingelsh., The Chinese mainland, Zhejiang Province, Azalea Canyon in Fengyang Mountain, Longquan City, 27 52'48”N, 119 10'8”E, 26 February 2019, R.-Z. Lin ZJ1905, CAF; Dendrological Herbarium, Chinese Academy of Forestry, DLN 6915, Cyclobalanopsis stewardiana, MT987635, MT968904, MT968912. Loranthus tanakae Franch & Sav., the Chinese mainland, Shaanxi Province, Wulingzigou, Daohuigou Villeage, Fengxian County, 34 13'N, 106 39'E, 19 May 2012, R.-Z. Lin FX 12050005-3, PE; Herbarium, Institute of Botany, Chinese Academy of Sciences, DLN 6750, Quercus sp., MT987638, MT968907, MT968922. Loranthus tanakae Franch & Sav., the Chinese mainland, Beijing Municipality, Baihuashan Mt. Mentougou District, 39 49'57”N, 115 34'36”E, 27 May 2019, R.-Z. Lin BJ1901, CAF; Dendrological Herbarium, Chinese Academy of Forestry, DLN 6926, Quercus sp., MT987637, MT968908, MT968923. Outgroup: Cecarria obtusifolia (Merr.) Barlow, Commonwealth of Australia, Queensland, McIlwraith Range, ca. 27 air km NE of Coen, 13 45'02”S, 143 20'05”E, 6 December 2001, B. Hyland 16493, CNS, Australian Tropical Herbarium (originally in Queensland Herbarium, MBA), DLN 4562, Syzygium forte, MT987627, MT968893, MT968924. Moquiniella rubra (A.Spreng.) Balle, Republic of South Africa, Western Cape Province, Karoo Desert National Botanical Gardens, Worcester, 33 36'52”S, 19 27'02”E, 29 October 1996, D. L. Nickrent 4087, SIU, Southern Illinois University, DLN 4087, Olea europaea, MT987639, MT968901, MT968925. Nuytsia ﬂoribunda (Labill.) G.Don, Commonwealth of Australia, Western Australia, Perth, 31 57'05”S, 115 51'36”E, 28 November 1994, A. Markey, via B. Barlow, SIU, Southern Illinois University, DLN 3080, –, MT987640, MT968902, MT968926.

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