horticulturae Article Phylogenetics of Taxus Using the Internal Transcribed Spacers of Nuclear Ribosomal DNA and Plastid trnL-F Regions Patricia Coughlan 1, James C. Carolan 2, Ingrid L. I. Hook 3, Lisa Kilmartin 1,3 and Trevor R. Hodkinson 1,* 1 Botany Building, School of Natural Sciences, Trinity College Dublin, the University of Dublin, Dublin D2, Ireland; [email protected] (P.C.); [email protected] (L.K.) 2 Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland; [email protected] 3 School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, the University of Dublin, Dublin D2, Ireland; [email protected] * Correspondence: [email protected]; Tel.: +353-1896-1128 Received: 17 October 2019; Accepted: 26 February 2020; Published: 12 March 2020 Abstract: Taxus is a genus of trees and shrubs with high value in horticulture and medicine as a source of the anticancer drug paclitaxel. The taxonomy of the group is complex due to the lack of diagnostic morphological characters and the high degree of similarity among species. Taxus has a wide global geographic distribution and some taxonomists recognize only a single species with geographically defined subgroups, whereas others have described several species. To address these differences in taxonomic circumscription, phylogenetic analyses were conducted on DNA sequences using Maximum Likelihood, Bayesian Inference and TCS haplotype networks on single and combined gene regions obtained for the nuclear ribosomal ITS region and the plastid trnL intron and trnL-F intergenic spacer. Evidence is presented for the sister group status of Pseudotaxus to Taxus and the inclusion of Amentotaxus, Austrotaxus, Cephalotaxus and Torreya within Taxaceae. Results are consistent with the taxonomic recognition of nine species: T. baccata, T. brevifolia, T. canadensis, T. cuspidata, T. floridana, T. fuana, T. globosa, T. sumatrana and T. wallichiana, but evidence is found for less species distinction and considerable reticulation within the T. baccata, T. canadensis and T. cuspidata group. We compare the results to known taxonomy, biogeography, present new leaf anatomical data and discuss the origins of the hybrids T. media and T. hunnewelliana. × × Keywords: biogeography; hybridization; Taxus; nrITS; paclitaxel; phylogenetics; taxaceae; taxol; trnL-F 1. Introduction Morphological differences between species of Taxus L. are slight and individuals within species have high levels of phenotypic plasticity, their morphology varying considerably with the environment [1]. This often leads to mis-identification and problems with classification using traditional taxonomic methods, which rely solely on morphological characters. Indeed, the delimitation of species in Taxus has been a long-standing taxonomic problem. Pilger [2] and Elwes and Henry [3] classified Taxus as monotypic with several geographical subspecies, a view supported by Dempsey and Hook [4] who provided evidence based on needle morphological variation and chemical characteristics. Furthermore, Dempsey [5] conducted a morphological and phytochemical analysis to investigate intra- and inter-generic relationships of Taxus and related genera, and concluded that few characters could be used to distinguish among Taxus species, but found that a clear distinction between the sister taxa Cephalotaxus and Torreya could be resolved. Horticulturae 2020, 6, 19; doi:10.3390/horticulturae6010019 www.mdpi.com/journal/horticulturae Horticulturae 2020, 6, 19 2 of 16 Horticulturae 2020, 6, x FOR PEER REVIEW 2 of 16 Contrary to this monotypic view of Taxus, numerous authors have recognized multiple species Contrary to this monotypic view of Taxus, numerous authors have recognized multiple species including Pilger [2], Cope [6], and Farjon [7] who resolved 7–12 species, while Spjut [8] recognized including Pilger [2], Cope [6], and Farjon [7] who resolved 7–12 species, while Spjut [8] recognized 24 species and 55 varieties. Currently nine species are listed in the Plant List [9] namely T. baccata L. 24 species and 55 varieties. Currently nine species are listed in the Plant List [9] namely T. baccata L. (Europe, Canada), T. brevifolia Nutt. (Western North America), T. canadensis Marshall (eastern Canada, (Europe, Canada), T. brevifolia Nutt. (Western North America), T. canadensis Marshall (eastern USA), T. cuspidata Siebold and Zucc. (Japan), T. floridana Nutt. ex Chapm. (Florida, south United Canada, USA), T. cuspidata Siebold and Zucc. (Japan), T. floridana Nutt. ex Chapm. (Florida, south States), T. fuana Nan Li & R.R. Mill, T. globosa Schltdl (Mexico), T. sumatrana (Miq.) de Laub. (China) United States), T. fuana Nan Li & R.R. Mill, T. globosa Schltdl (Mexico), T. sumatrana (Miq.) de Laub. and T. wallichiana Zucc. (Eastern India) (Figure1). (China) and T. wallichiana Zucc. (Eastern India) (Figure 1). Figure 1. Distribution of Taxus speciesspecies andand itsits sistersister genusgenus PseudotaxusPseudotaxus.. Adapted and modifiedmodified from Li etet al.al. [10[10]].. GroupingsGroupings of of ITS ITS sequences sequences in in relation relation to theto the biogeography biogeography of Taxus of Taxusare also are shown.also shown. Blue, redBlue, and red purple and purple ellipses ellipses represent represent phylogenetic phylogenetic groupings groupings from the TCSfrom network the TCS analysis network (Figure analysis 4). (Figure 4). The importance of species delimitation and classification of Taxus goes beyond taxonomy, taxonomicThe importance identification of forspecies horticulture delimitation and comparative and classification biology. Theof Taxus genus goes comprises beyond members taxonomy, that synthesizetaxonomic theidentification anti-cancer for taxane horticulture drug paclitaxel and comparative (Taxol) and incorrectbiology. The species genus identification comprises can members hinder cultivationthat synthesize and the drug anti production-cancer taxane efforts drug [4, 11paclitaxel]. All Taxus (Taxol)species and inincorrect this study species produce identification paclitaxel can in varyinghinder cultivation amounts [4 ,11and]. 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AccordingAccurate to thespecies International identification Union is also for Conservationrequired for conservation of Nature (ICUN) of Taxus Red species List, and several forests species [12]. ofAccordingTaxus are to threatened the International to different Union degrees for Conservation [13–18]. Taxus of Nature brevifolia (ICUN)and Taxus Red List, mairei several (=T. wallichiana species of var.Taxusmairei are threatened(Lemée and to H.Ldiffereév.)nt L.K.Fu degrees and [13 Nan–18] Li). Taxus are classified brevifolia asand Near Taxus Threatened, mairei (=T. andwallichiana Vulnerable, var. respectively,mairei (Lemée with and theirH.Lév.) numbers L.K.Fu currentlyand Nan decreasing.Li) are classified A major as Near part Threatened of this decline, and is Vulnerable, attributed torespectively, logging [17 with] and their the harvestingnumbers currently of bark fordecreas paclitaxeling. A production, major part althoughof this decline this exploitation is attributed has to largelylogging stopped, [17] and following the harvesting the development of bark for of paclitaxel a semi synthetic production process, although to produce this paclitaxelexploitation from hasT. baccatalargely. stoppedTaxus wallichiana, following, Taxus the development globosa, Taxus of chinensisa semi synthetic (=T. wallichiana process tovar. producechinensis paclitaxel(Pilg.) from Florin), T. Taxusbaccata. fauna Taxusand wallichiana,Taxus contorta TaxusGri globosaff.(=,T. Taxus wallichiana chinensisssp. (=T. contorta wallichiana (Griff var..) Silba) chinensis are all (Pilg.) classified Florin) as, EndangeredTaxus fauna and [13, 15Taxus,16,19 contorta]. This Griff. is due (= toT. thewallichiana over exploitation ssp. contorta of T. (Griff.) walliachiana Silba) andare allT. chinensisclassifiedfor as paclitaxelEndangered production. [13,15,16,19 Deforestation]. This is due has to caused the over populations exploitation of Taxus of T. globosawalliachianato decline; and T. globosachinensisis notfor yetpaclitaxel exploited production. for paclitaxel Deforestation production. hasTaxus caused fuana populationsaccording of to theTaxus Red globosa List has to becomedecline; endangeredT. globosa is duenot yet to over exploited exploitation for paclitaxel associated production. with medical Taxus use fuana along according with over-collection to the Red for List fuel has and become fodder. Shahendangered et al. [20 due] observed to over thatexploitation the populations associated of T. with fuana medicalwere declining, use along mainly with over due to-collection human pressure for fuel and fodder. Shah et al. [20] observed that the populations