A systematic revision of the genus Manekia (Piperaceae)

1. A systematic revision of the genus Manekia (Piperaceae) Heather Schubert

2. Outline (Bornstein) • Introduction of Piperaceae • Taxonomic history of Piperaceae • Introduction of Manekia • Materials and methods • Results • Discussion Piper obliquum

3. Piperaceae (Agardh) • Pantropical family • Consists of 5 genera • 2,400-3,600 species (Callejas 2001; Bornstein 2005; Wanke et al. 2007; Smith et al. 2008) • Largest genera Peperomia and Piper

4. Piperaceae (Bornstein) • Aromatic • Perennial • Shrubs, trees or vines (pipers) • Herbs (peperomias) • Leaves • Alternate, opposite, whorled or basal • Sessile or petiolate P. arboretum

5. Piperaceae • Inflorescence • Spicate or racemose • Terminal or leaf-opposed (Bornstein) P. arboreum (Bornstein)

6. Piperaceae (Bornstein) • Flower • Small, perfect (Neotropical) or imperfect (Old World tropics), lack a perianth • Subtended by a glabrous to pubescent bract • Unilocular ovary with single, basal ovule • Fruit • drupe P. abalienatum

7. (Bornstein) (Bornstein) P. schiedeanum P. pseudofuligineum

8. (Bornstein) P. arboreum

9. P. arboreum (Bornstein)

10. Piper nigrum • Black pepper • Spice/seasoning (Bornstein)

11. Piper betle • “Betle leaf” chewed with the areca nut • Used as herbal medicine and for narcotic properties (Bornstein)

12. The Problem in Piperaceae • Circumscription • Large number of described species • Based on characters of questionable taxonomic value, or fragmented material lacking flowers • Based on occurrence in a distinct political distributional unit

13. Circumscription in Piper • Several generic segregates established (Kunth 1839; Miquel 1843-33) • Reduced to synonymy or recognized at sectional level (C. de Candolle 1869) • Additional genera created (Trelease and Yuncker 1950; Yuncker 1958) • Phylogenetic analysis confirmed Manekia generic status (Jaramillo et al. 2004)

14. Circumscription: ManekiavsSarcorhachis • Sarcorhachis and Manekia recognized (Trelease 1927) • Placed in tribe Pothomorpheae • Single species of Manekia, M. urbanii • Haiti • Six species and two varieties of Sarcorhachis (Steyermark 1971) • Costa Rica to Colombia, Ecuador, Peru, Venezuela, Brasil and the Lesser Antilles

15. Sarcorhachis and Manekia • Shared a multitude of features • Taxonomic synonyms (Bornstein 1996) • Principle III of the International Code of Botanical Nomenclature • Earlier name deserves priority • Bornstein (1996) proposed conserving the name Sarcorhachis • Arias et al. (2006) provided most new name combinations

16. Manekia • Scandent habit • Alternate, palmately veined leaves • Petiole vaginate • Spikes usually axillary, occasionally terminal (solitary or paired) • Stamens 4 • Stigmas 3-5 • Fruits laterally compressed and immersed in fleshy rachis • Rachis pubescent

17. M. urbanii M. incurva M. venezuelana M. naranjoana M. sydowii M. obtusa (http://images.search.yahoo.com/central and southamerica)

18. Systematic revision of Manekia • Morphological study • Examination of type specimens and other herbarium material • Molecular investigation • Examined three DNA regions • Internal transcribed spacer (ITS) • psbj-petAintergenicintron • rpl32 spacer • Phylogenetic analysis pending

19. ITS = a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) http://www.google.com/images

20. Circular structure of the chloroplast genome of Loliumperenne Diekmann, K. et al. DNA Res 2009 0:dsp008v1-8; doi:10.1093/dnares/dsp008

21. Herbarium Materials • A, BM, C, ILL, F, G, GH, K, MO, NY, P, RB, US and WIS • Destructive sampling • MO, NY, US • Collected after 1980 • Wide distribution

22. Morphological Characteristics Vegetative Reproductive Inflorescence/Infructescence Position Rachis Bract Stamen position Anther Carpel Stigma Fruit • Leaf • Shape • Base • Apex • Venation • Pubescence • Texture/Color • Stipule • Prophyll • Stem

23. Morphological Measurements • Internode length • Prophyll length • Leaf blade length and width • Petiole length • Petiole vagination distance • Inflorescence/ Infructescence length • Peduncle length • Anther length • Fruit length

24. Manekia naranjoana Petiole vagination

25. Methods: Extraction and amplification • QuiagenDneasy extraction kit • PCR/Amplification • ITS • psbj-petA • rpl32 • Amplifications run in MJ Research programmable thermal cyclers PTC-100-60 and PTC-100-96

26. Methods: PCR and cloning • Visually verified PCR products on 0.7% agarose gels • Gel purification using Millipore Montage DNA Gel Extraction kit • Cloned using a Promega PGEM T-easy cloning kit

27. Methods: Sequencing • Li-CorLongreadIR 4200 automated sequencer • Verified with BLAST searches • Edited using Geneious Pro 4.7.6 • Aligned using CLUSTAL 2.0.12 • Submitted to Genbank upon publication

28. Statistical and Phylogenetic Analyses • Canonical Discriminant Analysis • Maximum Likelihood Phylogenetic Analysis with Bootstrap values (Bornstein) P. friedrichsthalii

30. 7 main veins 5 main veins Petiole vagination stops prior to blade Bract partly ciliate Petiole vagination extends onto blade Bract ciliate

31. Leaf base obtuse, deltoid, oblique 5 or 9 main veins Leaf base cordate 7 main veins 5 main veins 7 main veins

32. naranjoana (Costa Rica) 67 sydowii (Peru) rpl32 spacer 550 nucleotides sydowii (Peru) sydowii (Peru) sydowii (Colombia) 92 sydowii(Ecuador) naranjoana (Panama) 52 venezuelana (Venezuela) naranjoana (Costa Rica) 86 venezuelana (Venezuela) sydowii (Peru) sydowii (Peru) venezuelana (Venezuela) incurva (Dominica) 100 incurva (Dominica) obtusa (Brasil) 69 obtusa (Brasil) 100 naranjoana (Panama) 75 0.5 substitutions/site sydowii (Peru) sydowii (Colombia) naranjoana (Costa Rica) obtusa (Brasil) obtusa (Brasil) naranjoana (Costa Rica)

33. Discussion • 4 distinct taxa • M. incurva • M. obtusa • M. urbanii • M. naranjoana • Basionym: Piper naranjoanum C. DC., Linnaea 37: 363. 1872. • Basionym:Sarcorhachis sydowiiTrel., Repert. Spec. Nov. Regni Veg. 48: 16. 1940. • Basionym: SarcorhachisvenezuelanaSteyermark, Pittiera 3: 33. 1971.

34. Discussion • Better resolution • Use ITS, psbj-petAand rpl32 individually and combined • M. venezuelana- morphological analysis • M. urbanii– genetic analysis • Future Studies • Establish phylogenetic and biogeographic relationships between Manekia and Zippelia

35. Acknowledgements I thank Southeast Missouri State University for allowing me to conduct this research. I acknowledge Dr. Allan J. Bornstein and Dr. James F. Smith for obtaining specimens. I extend thanks to Boise State University (BSU), especially Dr. James F. Smith, for allowing me to use the necessary equipment and teaching me the techniques needed for this study. Also, I thank Dr. Michael S. Taylor for allowing me to use the necessary equipment in his lab. I thank Dr. Bornstein, Dr. Smith and Dr. Taylor for comments and discussions; Dr. John S. Scheibe for assistance with statistical analyses; my committee members, Dr. Bornstein, Dr. Taylor and Dr. Michael Aide, for assistance and encouragement throughout this study; and Jay Zimmers from BSU for helping with molecular work. This study was funded by the College of Graduate Studies, the College of Science and Mathematics, the Department of Biology, Dr. Allan J. Bornstein, Dr. Michael S. Taylor, and Dr. James F. Smith (BSU).

36. References Arias, T., R. Callejas, and A. Bornstein. 2006. New combinations in Manekia, an earlier name for Sarcorhachis (Piperaceae). Novon 16: 205-208. Bornstein, A. 1989. Taxonomic studies in the Piperaceae—I. The pedicellate pipers of Mexico and Central America (Pipersubg. Arctottonia). Journal of the Arnold Arboretum 70: 1-55. . 1991. The Piperaceae in the southeastern United States. Journal of theArnold Arboretum, Supplementary Series 1: 349-366. . 1996. Proposal to conserve the name Sarcorhachis against Manekia (Piperaceae). Taxon 45: 323-324. . 2005. Piperaceae. Pp. 453-457 in A Tropical Garden Flora, eds. G. Staples & D.R. Herbst. Honolulu: Bishop Museum Press. Callejas, R. 2001. Piperaceae. Pp. 1928-1984 in Flora de Nicaragua, Tomo III, eds. W. D. Stevens, C. Ulloa, A. Pool, and O. M. Montiel. St. Louis: Missouri Botanical Garden Press. Candolle, C. de. 1869. Piperaceae. DC. Prodr. Syst. Nat. Veg. 16(1): 235-471. Committee for Spermatophyta. 1998. (1223) Conserve SarcorhachisTrel. Against ManekiaTrel. (Piperaceae). Taxon 47: 870. Holmgren, P. K., N. H. Holmgren and L. C. Barnett (editors). 1990. Index Herbariorum Part I: The Herbaria of the World, 8th edition. New York Botanical Garden, Bronx, NY. Jaramillo, M. A., Callejas, R., Davidson, C., Smith, J. F., Stevens, A. C. and E. J. Tepe. 2008. A phylogeny of the tropical genus Piper using ITS and the chloroplast intronpsbj-petA. Systematic Botany 33(4): 647-660. Jaramillo, M. A., P. S. Manos, and E. A. Zimmer. 2004. Phylogenetic relationships of the perianthlessPiperales: Reconstructing the evolution of floral development. International Journal of Plant Science 165(3): 403-416. Kunth, K. S. 1839. Bemerkungenüber die Familie des Piperaceen. Linnaea 13: 561-726. McNeill. 2007. Miquel, F. A. G. 1843-44. SystemaPiperacearum. Rotterdam. Möller, M and QCB Cronk. 1997. Origin and relationships of Saintpaulia H. Wendl. (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences. American Journal of Botany 84: 956-965. Shaw, J., Lickey, E. B., Schilling, E. E. and R. L. Small. 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: The tortoise and the hare III. American Journal of Botany 94(3): 275-288. Smith, J. F., A. C. Stevens, E. J. Tepe and C. Davidson. 2008. Placing the origin of two species-rich genera in the late cretaceous with later species divergence in the tertiary: a phylogenetic, biogeographic and molecular dating analysis of Piper and Peperomia (Piperaceae). Plant SystEvol 275: 9-30. Standley, P. C. and J. A. Steyermark. 1952. Piperaceae. In: Flora of Guatemala. Fieldiana, Bot. 24(3): 228-337. Steyermark, J. A. 1971. Notes on the genus SarcorhachisTrel. (Piperaceae). Pittieria 3: 29-37. Trelease, W. 1927. The Piperaceae of Panama. Contributions from the United States National Herbarium 26(2): 15-17. Trelease, W. and T. G. Yuncker. 1950. The Piperaceae of northern South America. 2 vols. Urbana. Wanke, S., L. Vanderschaeve, G. Mathieu, C. Neinhuis, P. Goetghebeur, and M. S. Samain. 2007. From forgottontaxon to a missing link? The position of the genus Verhuellia (Piperaceae) revealed by molecules. Annals of Botany 99: 1231-1238. Wen, J and EA Zimmer. 1996. Phylogeny and biogeography of Panax L. (the ginseng genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. MolecPhylogenetEvol 6: 167-177. Yuncker, T. G. 1958. The Piperaceae—A family profile. Brittonia 10: 1-7.

37. Questions? (Bornstein) P. bredemeyeri

38. Herbarium codes for herbaria loaned from for this study.