Journal of Plant Research

, Volume 131, Issue 1, pp 125–141 | Cite as

Diversity and evolution of leaf anatomical characters in Taxaceae s.l.—fluorescence microscopy reveals new delimitating characters

  • Christoph Elpe
  • Patrick Knopf
  • Thomas Stützel
  • Christian Schulz
Regular Paper


Taxaceae s.l. comprise six genera (including Cephalotaxus) and about 35 species; The present study aims to give new insights into the evolution of this family, especially into the phylogenetic position of Cephalotaxus. Moreover, only little is known about comparative leaf anatomy of this family and this study aims to expose and interpret the diversity and evolution of leaf anatomical characters and to assess their applicability to identify taxa at the generic and species level. A detailed phylogeny was reconstructed, using both maximum likelihood and Bayesian inference, with a combined dataset of four molecular markers from the plastid and nuclear genomes. Leaf sections from 132 specimens, representing 32 species and four varieties (fresh and herbarium material) were inspected, using fluorescence microscopy. Ancestral characters were reconstructed using Mesquite. The phylogenetic analyses provided full support for Cephalotaxus as sister group to Taxaceae s.str. Within the latter, two monophyletic tribes Taxeae (comprising Austrotaxus, Pseudotaxus, and Taxus) and Torreyeae (comprising Amentotaxus and Torreya) were fully supported. Fluorescence microscopy was shown to be very useful for identifying leaf tissues and their constitution. We were able to show that particularly sclerified tissues have highest potential for the discrimination of both freshly collected samples and rehydrated herbarium specimens at the generic and species level. A correlation between the presence of different sclereid types could be shown and sclereids were hypothesized to pose a primitive trait in the evolution of Taxaceae s.l. New identification keys were generated on the basis of leaf anatomical characters. The microscopic method presented here is applicable for further studies within gymnosperms and probably in angiosperms, as well.


Character evolution Conifers Fluorescence microscopy Leaf anatomy Phylogeny Taxaceae s.l. 



We would like to thank all botanical institutions and Botanical Gardens (BG) (Atlanta BG, Pinetum Blijdenstein, BG Bochum, BG Bonn, Plantentuin Esveld, BG Marburg, Royal BG Edinburgh, and the Jardin des Plantes Paris), as well as several private collections (Mr. Hubertus Nimsch and Mr. Albrecht Weiss) for their support in providing access to living material. Likewise, we thank the Nationaal Herbarium Nederland in Leiden and the Museum National d’ Historie Naturelle in Paris for their generous access to herbarium material.

Supplementary material


  1. Ayensu ES (1967) Aerosol ot solution—an effective softener of herbarium specimens for anatomical study. Biotech Histochem 42:155–156. doi: 10.3109/10520296709115000 Google Scholar
  2. Brodribb TJ, Feild TS, Jordan GJ (2007) Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiol 144:1890–1898. doi: 10.1104/pp.107.101352 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Brodribb TJ, Feild TS, Sack L (2010) Viewing leaf structure and evolution from a hydraulic perspective. Funct Plant Biol 37:488. doi: 10.1071/FP10010 CrossRefGoogle Scholar
  4. Buchholz JT, Gray NE (1948) Taxonomic revision of Podocarpus. I. The sections of the genus and their subdivisions with special reference to leaf anatomy. J Arnold Arbor 29:49–63Google Scholar
  5. Burrows GE, Bullock S (1999) Leaf anatomy of wollemi pine (Wollemia nobilis, Araucariaceae). Aust J Bot 47:795. doi: 10.1071/BT98019 CrossRefGoogle Scholar
  6. Chamberlain CJ (1935) Gymnosperms, structure and evolution. The University of Chicago Press, ChicagoGoogle Scholar
  7. Chaw S, Long H, Wang B, Zharkikh A, Li W (1993) The phylogenetic position of Taxaceae based on 18 S rRNA sequences. J Mol Evol 37:624–630CrossRefPubMedGoogle Scholar
  8. Cheng Y, Nicolson RG, Tripp KE, Chaw S (2000) Phylogeny of Taxaceae and Cephalotaxaceae genera inferred from chloroplast matK gene and nuclear rDNA ITS Region. Mol Phyl Evol 14:353–365. doi: 10.1006/mpev.1999.0710 CrossRefGoogle Scholar
  9. Christenhusz M, Reveal JL, Farjon A, Gardner MF, Mill RR, Chase MW (2011) A new classification and linear sequence of extant gymnosperms. Phytotaxa 19:55–70CrossRefGoogle Scholar
  10. Collins D, Mill RR, Möller M (2003) Species separation of Taxus baccata, T. canadensis. cuspidata (Taxaceae) and origins of their reputed hybrids inferred from RAPD and cpDNA data. Am J Bot 90:175–182. doi: 10.3732/ajb.90.2.175 CrossRefPubMedGoogle Scholar
  11. Cope E (1998) Taxaceae: The genera and cultivated species. Bot Rev 64:291–322CrossRefGoogle Scholar
  12. Dörken VM, Nimsch H (2014) Morpho-anatomical investigations of cones and pollen in Cathaya argyrophylla Chung & Kuang (Pinaceae, Coniferales) under systematical and evolutional aspects. Feddes Rep 125:25–38. doi: 10.1002/fedr.201400035 CrossRefGoogle Scholar
  13. Dörken VM, Zhang Z, Mundry I, Stützel T (2011) Morphology and anatomy of male cones of Pseudotaxus chienii (Cheng WC) Cheng WC (Taxaceae). Flora 206:444–450. doi: 10.1016/j.flora.2010.08.006 CrossRefGoogle Scholar
  14. Eckenwalder JE (2009) Conifers of the world: the complete reference. 1st edn. Timber Press, PortlandGoogle Scholar
  15. Farjon A (2001) World checklist and bibliography of conifers, 2nd edn. Royal Botanic Gardens, KewGoogle Scholar
  16. Farjon A (2010) A handbook of the World’s Conifers. BRILL, LeidenCrossRefGoogle Scholar
  17. Farjon A, Filer D (2013) An atlas of the world’s conifers: an analysis of their distribution, biogeography, diversity and conservation status. BRILL, LeidenCrossRefGoogle Scholar
  18. Ferguson DK (1985) A new species of Amentotaxus (Taxaceae) from Northeastern India. Kew Bull 40:115. doi: 10.2307/4108483 CrossRefGoogle Scholar
  19. Florin R (1931) Untersuchungen zur Stammesgeschichte der Coniferales und Cordaitales. In: I. Morphologie und Epidermisstruktur der Assimilationsorgane bei den rezenten Koniferen. 10, vol I. K Svenska Vetensk Akad Handl, StockholmGoogle Scholar
  20. Florin R (1948a) On Nothotaxus, a new species of the Taxaceae, from Eastern China. Acta Horti Bergiani 14:385–395Google Scholar
  21. Florin R (1948b) On the morphology and relationships on the Taxaceae. Bot Gaz 110:31–39CrossRefGoogle Scholar
  22. Florin R (1958) On Jurassic taxads and conifers from north-western Europe and eastern Greenland. Acta Horti Bergiani 17:257–402Google Scholar
  23. Fu L, Li N, Mill RR (1999a) Cephalotaxaceae Fl China 4:85–88Google Scholar
  24. Fu L, Li N, Mill RR (1999b) Taxaceae Fl China 4:89–96Google Scholar
  25. Gerlach D (1977) Botanische Mikrotechnik: Eine Einführung, 2., überarb. u. erw. Aufl. Thieme, StuttgartGoogle Scholar
  26. Ghimire B, Lee C, Heo K (2014) Leaf anatomy and its implications for phylogenetic relationships in Taxaceae s. l. J Plant Res 127:373–388. doi: 10.1007/s10265-014-0625-3 CrossRefPubMedGoogle Scholar
  27. Gosling PG, McCartan SA, Ives LM, Cunningham VJ, Squirrell J, Thomas P (2008) Preliminary observations on fruit handling, seed germination and chloroplast inheritance of an Amentotaxus hybrid arising at the Royal Botanic Garden Edinburgh from A. argotaenia (F) x A. formosana (M). Sibbaldia 6:101–115Google Scholar
  28. Hamidipour A, Radjabian T, Charlet DA, Zarrei M (2011) Leaf anatomical investigation of Cupressaceae and Taxaceae in Iran. Wulfenia 18:95–111Google Scholar
  29. Hao DC, Xiao PG, Huang B, Ge GB, Yang L (2008) Interspecific relationships and origins of Taxaceae and Cephalotaxaceae revealed by partitioned Bayesian analyses of chloroplast and nuclear DNA sequences. Plant Syst Evol 276:89–104. doi: 10.1007/s00606-008-0069-0 CrossRefGoogle Scholar
  30. Hart JA (1987) A cladistic-analysis of conifers - preliminary-results. J Arnold Arbor 68:269–307Google Scholar
  31. Hu Y, Wang H, Wang F (1992) Leaf anatomy of Austrotaxus in relation to its systematic position. Cathaya 4:69–77Google Scholar
  32. Janchen E (1949) Das System der Koniferen. Oesterr Akad Wiss Math-Naturwiss. Kl Sitzungsber Abt 1 Biol 158:155–262Google Scholar
  33. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucl Acid Res 30:3059–3066. doi: 10.1093/nar/gkf436 CrossRefGoogle Scholar
  34. Kausik S (1975) The leaf structure in Podocarpus brevifolia (Stapf.) Foxw. Proc Ind Acad Sci B 81:197–206Google Scholar
  35. Kausik S, Bhattacharya S (1977) Comparative foliar anatomy of selected gymnosperms: leaf structure in relation to leaf form in Coniferales and Taxales. Phytomorphology 27:146–160Google Scholar
  36. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. doi: 10.1093/bioinformatics/bts199 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Knopf P, Nimsch H, Stützel T (2007) Dacrydium × suprinii, sp. nova – a natural hybrid of Dacrydium araucarioides × D. guillauminii. Feddes Rep 118:51–59. doi: 10.1002/fedr.200711126 CrossRefGoogle Scholar
  38. Knopf P, Schulz C, Little DP, Stützel T, Stevenson DW (2012) Relationships within Podocarpaceae based on DNA sequence, anatomical, morphological, and biogeographical data. Cladistics 28:271–299. doi: 10.1111/j.1096-0031.2011.00381.x CrossRefGoogle Scholar
  39. Lang X, Su J, Zhang Z, Lu S (2013) A taxonomic revision of the genus Cephalotaxus (Taxaceae). Phytotaxa 84. doi: 10.11646/phytotaxa.84.1.1
  40. Leslie AB (2012) Branching habit and the allocation of reproductive resources in conifers. Ann Bot 110:915–921. doi: 10.1093/aob/mcs150 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Leslie AB, Beaulieu JM, Rai HS, Crane PR, Donoghue MJ, Mathews S (2012) Hemisphere-scale differences in conifer evolutionary dynamics. Proc Natl Acad Sci USA 109:16217–16221. doi: 10.1073/pnas.1213621109 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Li H (1952) The genus Amentotaxus. J Arnold Arbor 33:192–198Google Scholar
  43. Little DP, Knopf P, Schulz C, Hajibabaei M (2013) DNA barcode identification of Podocarpaceae—the second largest conifer family. PLoS One 8:e81008. doi: 10.1371/journal.pone.0081008 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Liu J, Möller M, Gao LM, Zhang D, Li D (2011) DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species. Mol Ecol Resour 11:89–100. doi: 10.1111/j.1755-0998.2010.02907.x CrossRefPubMedGoogle Scholar
  45. Locosselli GM, Ceccantini G (2012) Plasticity of stomatal distribution pattern and stem tracheid dimensions in Podocarpus lambertii: an ecological study. Ann Bot 110:1057–1066. doi: 10.1093/aob/mcs179 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Lu Y, Ran J, Guo D, Yang Z, Wang X, Buerki S (2014) Phylogeny and divergence times of gymnosperms inferred from single-copy nuclear genes. PLoS One 9:e107679. doi: 10.1371/journal.pone.0107679 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Maddison W, Maddison D (2016) Mesquite: a modular system for evolutionary analysis. Version 3.03. Accessed 24 Oct 2016
  48. Möller M, Gao LM, Mill RR, Li D, Hollingsworth ML, Gibby M (2007) Morphometric analysis of the Taxus wallichiana complex (Taxaceae) based on herbarium material. Bot J Linn Soc 155:307–335. doi: 10.1111/j.1095-8339.2007.00697.x CrossRefGoogle Scholar
  49. Möller M, Gao LM, Mill RR, Liu J, Zhang D, Poudel RC, Li D (2013) A multidisciplinary approach reveals hidden taxonomic diversity in the morphologically challenging Taxus wallichiana-complex. Taxon 62:1161–1177. doi: 10.12705/626.9 CrossRefGoogle Scholar
  50. Mundry I, Mundry M (2001) Male cones in Taxaceae s. l.—an example of Wettstein’s pseudanthium concept. Plant Biol 3:405–416CrossRefGoogle Scholar
  51. Napp-Zinn K (1966) Anatomie des Blattes: I. Blattanatomie der Gymnospermen. Gebrüder Bornträger, Berlin-NikolasseeGoogle Scholar
  52. Orr MY (1944) The Leaf Anatomy of Podocarpus Trans Bot Soc Edinburgh 34:1–54. doi: 10.1080/13594864409441551 CrossRefGoogle Scholar
  53. Page CN (1990) Taxonomic concepts in conifers and ginkgoids. In: Kramer KU, Green PS (eds) Pteridophytes and gymnosperms. Springer, Berlin HeidelbergGoogle Scholar
  54. Peterson RL, Hersey RE, Brisson JD (1978) Embedding softened herbarium material in Spurr’s resin for histological studies. Biotech Histochem 53:1–9. doi: 10.3109/10520297809111436 Google Scholar
  55. Pilger R (ed) (1903) Taxaceae. Das Pflanzenreich, IV.5. W. Engelmann, Leipzig and BerlinGoogle Scholar
  56. Pilger R (1916) Die Taxales. Mitt Dtsch Dendrol Ges 25:1–30Google Scholar
  57. Posada D, Buckley T (2004) Model selection and model averaging in phylogenetics: advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Syst Biol 53:793–808. doi: 10.1080/10635150490522304 CrossRefPubMedGoogle Scholar
  58. Poudel RC, Möller M, Gao LM, Ahrends A, Baral SR, Liu J, Thomas P, Li D, Joly S (2012) Using morphological, molecular and climatic data to delimitate yews along the Hindu Kush-Himalaya and adjacent regions. PLoS One 7:e46873. doi: 10.1371/journal.pone.0046873 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Price RA (1990) The genera of Taxaceae in the southeastern United States. J Arnold Arbor 71:69–91CrossRefGoogle Scholar
  60. Quinn CJ, Price RA, Gadek PA (2002) Familial concepts and relationships in the conifer based on rbcL and matK sequence comparisons. Kew Bull 57:513. doi: 10.2307/4110984 CrossRefGoogle Scholar
  61. Rambaut A (2012) FigTree v1.4.0: tree figure drawing tool. Accessed 26 Oct 2016
  62. Rao A (1965) Studies on foliar sclereids in gymnosperms. Proc Ind Acad Sci B 61:196–203Google Scholar
  63. Rao A (1977) Morphogenesis of sclereids in Gnetum gnemon. Ann Bot 39:973–974CrossRefGoogle Scholar
  64. Rao A, Bhupal OP (1972) Topology of sclereids. Bot Surv India 14:41–55Google Scholar
  65. Rao A, Malaviya M (1963) The peculiar sclereids of Cephalotaxus drupacea Sieb. et Zucc. Proc Ind Acad Sci B 59B:228–236Google Scholar
  66. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574. doi: 10.1093/bioinformatics/btg180 CrossRefPubMedGoogle Scholar
  67. Sahni B (1920) On certain archaic features in the seed of Taxus baccata, with remarks on the antiquity of the Taxinae. Ann Bot 34:117–132CrossRefGoogle Scholar
  68. Saxton WT (1930) Notes on conifers: IV. Some points in the leaf anatomy of Fokienia hodginsii Henry and Thomas and Libocedrus macrolepis B. and H. Ann Bot 44:167–171CrossRefGoogle Scholar
  69. Schulz C, Stützel T (2006) Variability of male cones in Chamaecyparis as an example for Cupressaceae male cones. Feddes Rep 117:146–157. doi: 10.1002/fedr.200511085 CrossRefGoogle Scholar
  70. Schulz C, Jagel A, Stützel T (2003) Cone morphology in Juniperus in the light of cone evolution in Cupressaceae s.l. Flora 198:161–177. doi: 10.1078/0367-2530-00088 CrossRefGoogle Scholar
  71. Schulz C, Knopf P, Stützel T (2005) Identification key to the Cypress family (Cupressaceae). Feddes Rep 116:96–146. doi: 10.1002/fedr.200411062 CrossRefGoogle Scholar
  72. Schulz C, Klaus KV, Knopf P, Mundry M, Dörken VM, Stützel T (2014) Male cone evolution in conifers: not all that simple. Am J Plant Sci 5:2842–2857. doi: 10.4236/ajps.2014.518300 CrossRefGoogle Scholar
  73. Spjut R (2007) Taxonomy and nomenclature of Taxus (Taxaceae). J Bot Res Inst Texas 1:203–289Google Scholar
  74. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690. doi: 10.1093/bioinformatics/btl446 CrossRefPubMedGoogle Scholar
  75. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313. doi: 10.1093/bioinformatics/btu033 CrossRefPubMedPubMedCentralGoogle Scholar
  76. Stefanoviac S, Muriel J, Deutsch J, Broutin J, Masselot M (1998) Phylogenetic relationships of conifers inferred from Partial 28 S rRNA gene sequences. Am J Bot 85:688–697CrossRefPubMedGoogle Scholar
  77. Stützel T, Röwekamp I (1999) Female reproductive structures in Taxales. Flora 194:145–157CrossRefGoogle Scholar
  78. Swafford DL (2003) PAUP∗: Phylogenetic analysis using parsimony (∗and other methods). Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts. Accessed 24 Oct 2016
  79. Tripp KE (1995) Cephalotaxus: the plum yews. Arnoldia 55:25–39Google Scholar
  80. Vaidya G, Lohman DJ, Meier R (2011) SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27:171–180. doi: 10.1111/j.1096-0031.2010.00329.x CrossRefGoogle Scholar
  81. Wang T, Su Y, Zheng B, Li X, Zeng Q, Qu L, Gu H (2003) Cladistic analysis of chloroplast rbcL gene and trnL-trnF intergenic spacer sequences in Taxaceae and related taxa. In: Sharma AK, Sharma A (eds) Plant genome: biodiversity and evolution, vol 1, part A: Phanerogams. Scientific Publications, Enfield, pp 103–116Google Scholar
  82. Wilson P, Buonopane M, Allison TD (1996) Reproductive biology of the monoecious clonal shrub Taxus canadensis. Bull Torrey Bot Club 123:7. doi: 10.2307/2996301 CrossRefGoogle Scholar
  83. Worsdell WC (1897) VIII. On transfusion-tissue: its origin and function in the leaves of Gymnospermous Plants. // VIII. On “Transfusion-tissue”. Trans Linn Soc London Bot 5:301–319. doi: 10.1111/j.1095-8339.1897.tb00205.x Google Scholar
  84. Wu H, Hu Z (1997) Comparative anatomy of resin ducts of the Pinaceae. Trees 11:135. doi: 10.1007/s004680050069 CrossRefGoogle Scholar
  85. Zou J, Sun Y, Li L, Wang G, Yue W, Lu Z, Wang Q, Liu J (2013) Population genetic evidence for speciation pattern and gene flow between Picea wilsonii, P. morrisonicola and P. neoveitchii. Ann Bot 112:1829–1844. doi: 10.1093/aob/mct241 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan KK 2017

Authors and Affiliations

  1. 1.Department for Evolution and Biodiversity of PlantsRuhr-Universität BochumBochumGermany
  2. 2.Botanischer Garten RombergparkDortmundGermany

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