Tanya Renner

PhD Student, University of California, Berkeley

Dissertation Research at the University of California, Berkeley

My first encounter with a carnivorous plant was at the Huntington Botanical Gardens in San Marino, California. I was only about 4 feet tall and stood as high as I could to see the deadliest plant to all known insects: the tropical pitcher Nepenthes. A peek inside this plant’s traps revealed a pool dotted with bugs. After experiencing Nepenthes at the botanical garden, I began to wonder how such a plant could have come to exist. Fifteen years later, I continue to question how organisms diversify. I am interested in mutations that occur at the nucleotide level, and I hope to use evolutionary biology as a method to promote preservation by showing what contributes to the uniqueness of a species. For my dissertation project in Chelsea D. Specht’s lab at the University of California, Berkeley, my dissertation research includes a study of relationships between genera within the carnivorous plants of the Caryophyllales and a molecular evolutionary analysis of their digestive enzymes.

The Carnivorous Caryophyllales
The carnivorous plants of the angiosperm order Caryophyllales (core eudicot) have a unique evolutionary history, encompassing taxa that have diverse morphologies and include genera that have lost the ability to digest prey. One project that is part of my dissertation research at the University of California, Berkeley is to create a more robust phylogeny for the carnivorous Caryophyllales that includes greater taxon sampling from each genera and focuses on the evolution of gland morphology within this group.

In 1960, Léon Croizat proposed that carnivorous plants comprised a single group at the base of the Angiosperms based upon similarities in trap type [1]. More recently, however, carnivory has been hypothesized to have arisen independently at least five times in the plant kingdom, within orders Ericales, Lamiales, Oxalidales, Poales, and Caryophyllales as a result of convergent evolution [2, 3]. Based upon recent phylogenetic studies of angiosperms, four families, comprising in total more than 300 species, have been placed as a monophyletic group within the order Caryophyllales: Droseraceae, Drosophyllaceae, Nepenthaceae and Dioncophyllaceae. Included within these families are the carnivorous genera Drosera (sundews), Dionaea (Venus flytrap), Nepenthes (tropical pitcher plants), Aldrovanda (aquatic flytrap), Drosophyllum (Portuguese sundew) and Triphyophyllum, along with non-carnivorous genera Ancistrocladus, Habropetalum, and Dioncophyllum.

Outside of this monophyletic group are the families Polygonaceae and Plumbaginaceae, which together share a number of synapomorphies with the carnivores including vascularized, multicellar glands [4]. In Polygonaceae and Plumbaginaceae, these glands function in the secretion of mucilage or salt for protection in halophytic conditions, epizoochory and to deter herbivory [5]. In the carnivorous plants, homologous glands have evolved to function in the secretion of digestive enzymes as well as to absorb amino acids and other organic nutrients [5].

Within the carnivorous Caryophyllales, a variety of glands that can both exude and absorb exist. In the genera Drosera, Drosophyllum and Triphyophyllum, two types of glands are present: (1) vascularized, stalked, multicellular glands like those of Polygonaceae and Plumbaginaceae, and (2) non-vascularized, sessile glands. In Dionaea, marginal multicelluar glands have been reduced to “teeth” and trigger hairs held perpendicular to the lamina, although sessile glands that secrete enzymes are still present. Within Nepenthaceae, glandular pits partially covered by epidermis are positioned at the base of the pitcher, while vascularized, multicellular stalked glands are absent. In the non-carnivorous genera sister to Nepenthaceae (Ancistrocladus, Habropetalum, Dioncophyllum), glandular pits are present on the abaxial surface of the leaf, but function in the secretion of epicuticular waxes rather than digestive enzymes[6]. The current phylogeny supports the hypothesis that embedded glands in Nepenthaceae, Ancistrocladaceae and Dioncophyllaceae are derived from glands found in Droseraceae [7].

Chitinolytic Enzymes in the Carnivorous Caryophyllales
My second project in the Specht lab is to determine the degree of homology among chitinases in carnivorous plants and to elucidate the method by which these enzymes have become specifically adapted for the carnivory.

The isolation and characterization of carnivorous plant digestive enzymes secreted from glands began in the nineteenth century, when Joseph Hooker discovered the first protease in Nepenthes sp. trap fluid cir. 1874 [8]. In 1875, Charles Darwin published his accounts on Drosera rotundifolia and the ability of Drosera to digest nitrogenous and phosphate-containing compounds [9]. However, it was not until the 1970s that the basic enzyme composition in carnivorous plant mucilage was characterized. Among the various enzymes identified to be important in plant carnivory, chitinases have been one of the most thoroughly studied [10-14].

Active production of chitinolytic enzymes were first demonstrated in Drosera and Nepenthes by Amagase et al. [10]. Several years later, Robins and Juniper found Dionaea muscipula traps to exhibit chitinase activity [14]. Twenty years later, two types of Nepenthes khasiana chitinases (class Ia and Ib) involved in digestion were sequenced and described, only one of which was found to be upregulated in response to the colloidal chitin [11]. During the same year, Matusikova et al. demonstrated the localized expression of chitinase mRNA within the tentacles of Drosera rotundifolia via in situ hybridization [12].

Based on their on the structure of their domains and percent sequence homology, class I chitinases involved in plant carnivory may have descended from class I chitinases that play a role in pathogen defense. Changes in selectional pressure may be evident if protein function has shifted from defense to digestion. My goal is to compare selectional pressure between chitinases that are induced in response to chitin and those that are constitutively expressed. Preliminary studies of class Ib chitinases in Nepenthes have shown a noticeable decrease in the overall number of amino acids under positive selection, which may be an indicator of a shift from positive to purifying selection during the evolution of chitinase enzymes in carnivorous plants.

Acknowledgements: This project was funded in part by the UC Berkeley College of Natural Resources & the NSF Graduate Research Fellowship Program. Thank you to the entire Specht Lab for their insight & suggestions, especially my advisor Dr. Chelsea D. Specht and undergraduate researcher Sarah Starkey. In addition, the following sources for specimen donation are integral to this project: The following sources for specimen donation are integral to this project: UC Berkeley Botanical Garden, Missouri Botanical Garden, Bogor Botanical Garden, University of Würzburg (Dr. Gerhard Bringmann and Andreas Irmer) & California Carnivores in Sebastopol, CA (Peter D’Amato). Many of the carnivorous plant photos for this page are used with thanks and by permission of www.sarracenia.com (Dr. Barry Rice, UC Davis).

Visit the Specht lab website at http://pmb.berkeley.edu/~specht/.

Figure 1: An introduction to the carnivorous plants and the five origins of carnivory. The Caryophyllales holds the largest number of genera and is a unique in that there has been a gain and loss of carnivory. Figure adapted from the Angiosperm Phylogeny Group II, 2003[3] and pictures from Dr. Barry Rice (UC Davis, www.sarracenia.com).

Figure 2: Bayesian inference of phylogeny (MrBayes v3.1.2, Huelsenbeck, Larget, can der Mark, Ronquist) constructed using a partitioned analysis of two concatenated nuclear and chloroplast molecular markers (GTR+gamma, ITS and GTR+invgamma, psbA-trnH) ran for 10 million generations. In this phylogenetic reconstruction, solid lines represent genera that are not carnivorous, whereas dashed lines represent carnivorous and part-time carnivorous genera. Throughout the evolution of the carnivorous Caryophyllales and their gland types, a general trend exists: a gain and loss of multicellular stalked glands that can exude and absorb. Micrograph drawings of glands: [15], [16], [17].]

Figure 3: Class I chitinases have been shown to be under strong positive selection in Arabis, with adaptive replacements occurring disproportionately in the active cleft[18]. This is thought to be due to an arms-race between chitinases and chitinolytic inhibitors produced by pathogenic fungi. However, in carnivorous plants, class Ib chitinases that induced in response to chitin might be instead undergoing purifying selection, as the main constituents in plant traps are insects, which do not produce any known chitinolytic inhibitors. Class Ia chitinases that are constitutively expressed in carnivorous plants might be undergoing positive selection as in non-carnivorous plants. Image adapted from the following images: crystal structure of barley chitinase[19], mushroom (Hygrocybe sp., and tenebrionid beetle (Nhu Nguyen, UC Berkeley).

References Cited
1. Croizat, L., Principia Botanica. 1960, Caracas, Venezuela: published by the author.
2. APG, An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden, 1998. 85: p. 531-553.
3. APGII, An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society, 2003. 14: p. 399-436.
4. Judd, W.S.C., C.S., Kellogg, E.A, Stevens, P.F., Donoghue, M.J., Plant Systematics: A Phylogenetic Approach. III ed. 2007, Sunderland, MA: Sinauer Associates, Inc.
5. Juniper, B.E., Robins, R.J., Joel, D.M., The Carnivorous Plants. 1989, London, UK: Academic Press.
6. Kaplan, D., PB 107, Plant and Microbial Biology Reader 1997. II: p. 33.
7. Heubl, G., Bringmann, G., Meimberg, H., Molecular phylogeny and character evolution of carnivorous plant families in Caryophyllales-revisted. Plant Biology, 2006. 8: p. 821-830.
8. Lönnig, W.E., Becker, H.A., Carnivorous Plants. Nature Encyclopedia of Life Sciences, 2004. doi: 10.1038/npg.els.0003818.
9. Darwin, C.R., Insectivorous Plants. 1875, London: John Murray.
10. Amagase, S., et al., Digestive enzymes in insectivorous plants IV. Enzymatic digestion of insects by Nepenthes secretion and Drosera peltata extract: proteolytic and chitinolytic activities. J. Biochem. , 1972. 72(765-767).
11. Eilenberg, H., et al., Isolation and characterization of chitinase genes from pitchers of the carnivorous plant Nepenthes khasiana. J. Exp. Bot. , 2006. 57(11): p. 2775-2784.
12. Matusikova, I., et al., Tentacles of in vitro-grown round-leaf sundew (Drosera rotundifolia L.) show induction of chitinase activity upon mimicking the presence of prey. Planta, 2005. 222: p. 1020–1027.
13. Hatano, N., Hamada, T., Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata. J. Proteome Re., 2008. 7: p. 809-816.
14. Robins, R., Juniper, B., The secretory cycle of Dionaea muscipula Ellis. V. The absorption of nutrients. New Phytologist 1980. 86(413-422).
15. Wilson, J., The mucilage and othr glands of the Plumbagineae. Annuals of Botany, 1896. IV(XIV).
16. Williams, S.E., Comparative sensory physiology of the Droseraceae-the evolution of a plant sensory system. Proceedings of the American Philosophical Society, 1976. 120(3): p. 187-204.
17. Pitcher of Nepenthes distillatoria, in The Encyclopædia Britannica: A Dictionary of Arts, Sciences, Literature and General Information 1910-1911.
18. Bishop, J.G., Dean, A.M., Mitchell-Olds, T., Rapid evolution in plant chitinase: molecular targets of selection in plant pathogen coevolution. Proc. Natl. Acad. Sci. USA, 2000. 97(10): p. 5322-5327.
19. Robertus, J.D., Monzingo, A.F., Marcotte, E.M., Hart, P.J., Structural analysis shows five glycohydrolase families diverged from a common ancestor. The Journal of Experimental Zoology, 1998. 282: p. 127-132.

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