Proc. Natl. Acad. Sci. USA Vol. 94, pp. 7367–7372, July 1997 Evolution Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation (molecular evolutionyrelative rates testyrbcL pseudogeneyparallel bootstrap) CLAUDE W. DEPAMPHILIS*, NELSON D. YOUNG, AND ANDREA D. WOLFE† Department of Biology, Vanderbilt University, Nashville, TN 37235 Communicated by M. T. Clegg, University of California, Riverside, CA, May 2, 1997 (received for review February 27, 1996) ABSTRACT The plastid genomes of some nonphotosyn- of feeding cells that reside solely within the photosynthetic host thetic parasitic plants have experienced an extreme reduction plant. in gene content and an increase in evolutionary rate of Despite the profound morphological and physiological remaining genes. Nothing is known of the dynamics of these changes associated with the parasitic lifestyle, all parasitic events or whether either is a direct outcome of the loss of plants examined to date retain plastids (3, 4) and plastid photosynthesis. The parasitic Scrophulariaceae and Oroban- genomes (5–9, reviewed in ref. 10). Parasites, therefore, pro- chaceae, representing a continuum of heterotrophic ability vide an outstanding opportunity to learn about gene and ranging from photosynthetic hemiparasites to nonphotosyn- genome evolution and function in the absence of the plastid thetic holoparasites, are used to investigate these issues. We genome’s primary physiological function, photosynthesis (10). present a phylogenetic hypothesis for parasitic Scrophulari- For example, the holoparasite Epifagus [Orobanchaceae (11)] aceae and Orobanchaceae based on sequences of the plastid has a greatly reduced plastid genome lacking photosynthetic gene rps2, encoding the S2 subunit of the plastid ribosome. and purported chlororespiratory genes (8), RNA polymerase Parasitic Scrophulariaceae and Orobanchaceae form a mono- genes, and some components of the plastid translation appa- phyletic group in which parasitism can be inferred to have ratus (12, 13). Conopholis, another Orobanchaceae, shares evolved once. Holoparasitism has evolved independently at many of these plastid gene deletions (6, 7, 10) and has least five times, with certain holoparasitic lineages represent- additionally lost one copy of the inverted repeat (ref. 7 and ing single species, genera, and collections of nonphotosyn- C.W.D., S. R. Downie, and J. D. Palmer, unpublished data). thetic genera. Evolutionary loss of the photosynthetic gene Despite the loss of plastid-encoded transcriptional genes, the rbcL is limited to a subset of holoparasitic lineages, with Epifagus and Conopholis plastid DNAs are transcribed (8, 14, several holoparasites retaining a full length rbcL sequence. In 15), and normal plastid intron processing and RNA editing contrast, the translational gene rps2 is retained in all plants have been demonstrated in Epifagus (15). These observations investigated but has experienced rate accelerations in several and compelling evolutionary arguments (16) have suggested hemi- as well as holoparasitic lineages, suggesting that there strongly that the Epifagus plastid genome is functional and may be substantial molecular evolutionary changes to the serves to express a small number of retained genes of non- plastid genome of parasites before the loss of photosynthesis. photosynthetic function (8, 16). It is intriguing that nearly all Independent patterns of synonymous and nonsynonymous of the remaining plastid genes in Epifagus appear to have rate acceleration in rps2 point to distinct mechanisms under- evolved at an accelerated pace compared with tobacco, a lying rate variation in different lineages. Parasitic Scrophu- related photosynthetic plant (12, 13). lariaceae (including the traditional Orobanchaceae) provide These observations demonstrate that the normally conser- a rich platform for the investigation of molecular evolutionary vative plastid genome has the potential for rapid structural process, gene function, and the evolution of parasitism. evolution when coding requirements are decreased; these observations also have other important implications for plastid Among the most remarkable features of flowering plants, gene evolution and function (reviewed in ref. 10). However, we including aquatic and carnivorous plants, epiphytes, and plants know nothing about the dynamics of the events that led to the tolerant of extreme desiccation, is their many adaptations to extraordinary plastid DNAs seen in parasitic plants. For varied physical and biotic environments. One extraordinary example, is there some predictable order to the genetic plant adaptation is parasitism, in which modified roots (haus- changes associated with the loss of photosynthesis? Have the toria) are used to transfer water, minerals, and a diverse profound alterations to the plastid genome seen in Epifagus collection of carbon compounds from a host plant to the and Conopholis all followed the loss of photosynthesis, or parasite (1, 2). The evolution of parasitism has probably might some have begun before its loss? Are the similarities occurred at least eight times within the angiosperms (1). On between the plastid genomes of these plants due to conver- several occasions this has proceeded to an extreme form gence or to shared ancestry? How rapidly do the structural and known as holoparasitism, in which the parasite obtains virtu- other changes to the plastid genome come about in parasitic ally all of its reduced carbon from its host. In addition to the plants? loss of photosynthesis and associated pigments (2), holopara- The parasitic ScrophulariaceaeyOrobanchaceae is uniquely sites exhibit reduction or loss of leaves and loss of nonhaus- suited to investigate these issues. Within this group are facul- torial roots, and, in one extreme group (Rafflesiaceae) (1), vegetative tissues have been reduced to a mycelium-like mass Abbreviation: ML, maximum likelihood. Data deposition: The sequences reported in this paper, including plant The publication costs of this article were defrayed in part by page charge collection and voucher data, have been deposited in the GenBank database (accession nos. U48738-U48777). payment. This article must therefore be hereby marked ‘‘advertisement’’ in *To whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. © 1997 by The National Academy of Sciences 0027-8424y97y947367-6$2.00y0 †Present address: Department of Plant Biology, Ohio State University, PNAS is available online at http:yywww.pnas.org. Columbus, OH 43210. 7367 Downloaded by guest on September 28, 2021 7368 Evolution: dePamphilis et al. Proc. Natl. Acad. Sci. USA 94 (1997) tative as well as obligate parasites, hemiparasites of widely Phylogenetic Analysis. Parsimony analyses used PAUP 3.1.1 varying photosynthetic ability, and a diverse collection of fully (25) on an Apple Quadra 950. Options were 500 random taxon heterotrophic holoparasites (1, 2, 17–19). No other group of addition sequences, tree-bisection reconnection branch swap- parasitic plants displays this continuum of parasitic abilities. ping, mulpars, and steepest descent. Bootstrap analyses (26) Comparative analysis of the plastid genomes of these plants were performed in parallel on 28 Apple Quadra 650 computers should provide a powerful tool for the interpretation of with different random number seeds. Settings were identical to molecular evolution relative to photosynthetic ability (10); above except that 10 random taxon input orders were used for however, no explicit phylogeny has been proposed. each of 400 bootstrap replicates. Bremer support analyses (27) In this paper, we develop a phylogenetic framework for the were carried out for all trees up to two steps longer than the study of evolutionary changes associated with parasitism using most parsimonious trees. Bremer support values (27) for rps2, a plastid gene new to plant phylogenetic analysis. rps2, remaining nodes were estimated using inverse constraints (28). encoding ribosomal protein CS2, is one of the more rapidly Maximum likelihood (ML) phylogenetic analyses (29) were evolving plastid genes known to be retained in all parasitic performed using PHYLIP 3.5 (30) and FASTDNAML 1.0.6 (31) ScrophulariaceaeyOrobanchaceae (refs. 12 and 16 and run on a Sun Microsystems SparcStation 10. Ten random input C.W.D., unpublished data), so sequences provide many char- orders were run using empirical base frequencies and a acters for phylogenetic analysis. First, we show that parasitism transitionytransversion ratio of 2.0. has evolved just once in Scrophulariaceae and Orobanchaceae, rbcL Gene Assays. PCR and Southern blot assays (8) were but we trace the loss of photosynthesis to a minimum of five used to provide complementary information about the pres- independent lineages within this group. The phylogeny is then ence or absence of rbcL in each plant. PCR amplifications used used to demonstrate that the photosynthetic gene rbcL is often primers RH1 (59-ATGTCACCACAAACAGAAACTA- retained in nonphotosynthetic lineages, sometimes long after AAGC) and Z1352R (59-CTTCACAAGCAGCAGCTAG- the loss of photosynthesis. In addition, the translational gene GTCAGGACTCC). Amplifications were as for rps2, except a rps2 had experienced rate accelerations in several hemi- as well 50-ml reaction used 0.32 mM for each primer and annealing was as holoparasitic lineages. These patterns