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TWO STUDIES IN EVOLUTIONARY HISTORY: THE ORIGIN OF THE NONPHOTOSYNTHETIC ALGAE POLYTOMA AND THE REPRODUCTIVE MECHANISM OF ACANTHAMOEBA

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

By

Robert Rumpf, M. S.

The Ohio State University 1997

Dissertation Committee: Approved By Dr. C. William Birky, Jr., Adviser

Dr. Tom Byers

Dr. Daniel Crawford Advis Dr. Amanda Simcox Department of Molecular Genetics UMI Number: 9731704

UMI Microform 9731704 Copyright 1997, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT

The evolutionary history of two disparate organisms, each with their own unique evolutionary question, was studied using phylogenetic analysis of DNA sequences. Polytoma, a non-photosynthetic alga of the family

Chlamydomonadaceae, is most closely related to the photosynthetic genus

Chlamydomonas based on morphological evidence. Complete sequences of the Rrn18 genes were obtained from 13 strains of Polytoma. Phylogenetic analysis showed that these strains formed two clades. One clade shows modest sequence diversity but is represented by strains collected at widely dispersed sites in Europe and America. The other clade consists of a single isolate from the Canary Islands. Both clades lie well within the extended clade that includes all species of Chlamydomonas for which sequence data are available. The two Polytoma clades are separated from each other by several green species, indicating that the extant nonphotosynthetic

Chlamydomonadaceae arose from photosynthetic ancestors at least twice.

These results further suggest that nonphotosynthetic mutants are capable of establishing lineages that can spread widely but have a higher probability of extinction than their photosynthetic congeners.

Acanthamoeba castellanii is the agent responsible for Acanthamoeba keratitis, a potentially vision-threating opportunistic parasitic infestation of the cornea in humans. It has generally been assumed that Acanthamoeba reproduces asexually, primarily from lack of any evidence indicating otherwise. Since the method of reproduction can affect the etiology and treatment of the infection, we decided to use molecular sequence data to determine the method of reproduction. Ancient asexual lineages will accumulate allelic sequence divergence (ASD), and unless this divergence is masked by frequent convergence events such as mitotic recombination, gene conversion, or ploidy cycles, measurement of ASD can be used diagnostically to determine the mode of reproduction of an organism. The IBP gene was PCR amplified, cloned, and sequenced from Acanthamoeba castellanii Neff and Acanthamoeba griffini S7. Divergences between alleles within a clonal culture of each species were within the expected range for sexually reproducing species; alternately

Acanthamoeba might be an asexual species with a high frequency of sequence convergence events. A phylogenetic tree constructed from the data failed to show the allele-specific topology expected of an obligate asexual species in the absence of convergence. Southern blotting showed IBP to be a single-copy gene; thus the minimum ploidy of Acanthamoeba can be determined by the number of unique alleles found. Acanthamoeba griffini S7 had eleven unique alleles, suggesting a minimum ploidy for Acanthamoeba of eleven. ASD was greater in A. griffinithan in A. castellanii, implying that there are significant differences in the effective population size, frequency of sex, or the frequency of convergence events between the two species. Additional study is required to resolve this issue.

Ill Dedicated to my wife, Deborah, in thanks for her support, understanding, and patience...

IV ACKNOWLEDGMENTS

First and foremost, I’d like to thank my adviser, Dr. 0. William Birky, for all of his help, support, encouragement, and for just plain being a nice guy.

Without his scientific and intellectual input, my work would not be as complete as it is; it certainly wouldn’t have been as much fun.

I’d like to thank the members of the Birky lab who helped me learn techniques and tricks, helped with analysis and discussion, and kept the alpha- geek in his place; Dawne Vernon, David Schreiber, Julie Gordon, Doug Byers,

Pam MacKowski, and Stacey Seibert-Harley.

I’d like to thank the Fuerst lab and the Byers labs for their contributions of data, primers, DNA, and the occasional chuckle.

I’d also like to thank my wife, Deborah, who has beyond a doubt earned her application to sainthood. Long roads. Deb, but we’re traveling together...

I’d like to thank my parents, who instilled their values of hard work and an education in me, and were always there for me.

And to all the rest who provided me with support, hope, help, inspiration, and sanity checks: The Haven Gaming group (Steve Schwartz, Carole

McCallister, Chris Nickel, Kathy Hamilton, and Bruce Coulson), The W ednesday

Gaming Group (Will Ray, Jim Emrick, Dan Schreiber, Chris Myers), Gary

Grumbling, and the kids. Merlin and Mi-ke Rumpf. Special thanks to Sting and

Vangelis for setting the mood, and Apple Computer for making the Macintosh. VITA

October 17, 1966 Bom - Dayton, Ohio

1988 B.S. Biology, University of Dayton

1990 M. S. Biology, University of Dayton

1990 - present Graduate Teaching and Research Associate,

The Ohio State University Department of Molecular Genetics

PUBLICATIONS Research Publication

1. Rumpf, R., Vemon, D., Schreiber, D, and Birky, C. W. Jr, “Evolutionary Consequences of the Loss of Photosynthesis in Chlamydomonadaceae: Phylogenetic Analysis of Rrn18 (18S rDNA) in 13 Polytoma strains (Chlorophyta).” J. Phycol., 32,119, (1996).

2. Gordon, J., Rumpf, R., Shank, S. L., Vemon, D., and Birky, C. W. Jr., "Sequences of the RRN18 Genes of Chlamydomonas humicola and 0. dysosmos are identical, in agreement with their combination in the species C. applanata (Chlorophyta).” J. Phycol., 31, 312, (1995).

FIELDS OF STUDY Major Field: Molecular Genetics

VI TABLE OF CONTENTS Page

Abstract...... ii

Dedication ...... iv

Acknowedgments ...... v

Vita...... vi

List of Tables ...... ix

List of Figures ...... x

Chapters:

1. Introduction

Introduction to Polytoma...... 1

Goals of the Polytoma project ...... 2

Introduction to Acanthamoeba...... 4

Goals of the Acanthamoeba Project ...... 6

Introduction to Phylogenetic Methods ...... 8

2. Published Paper: Evolutionary Consequences of the Loss o f 10 Photosynthesis in Chlamydomonadaceae: Phylogenetic Analysis oiRrnlS (188 rDNA) in 13 Polytoma strains (Chlorophyta)

3. Allelic Sequence Divergence, Reproduction, and Ploidy in Acanthamoeba

Introduction ...... 18

Materials & Methods ...... 22 vii Results & Discussion ...... 31

Conclusions ...... 48

Bibliography ...... 50

VIII LIST OF TABLES

Table P age

1 List of Polytoma stocks ...... 11

2 List of complete Rm18 sequences and their sources ...... 12

3 Numbers of substitution and indel differences between ...... 13 complete sequences of Rrn18 genes from pairs of strains

4 Organisms used in TBP alignment for primer design ...... 24

5 Table of TBP Clones ...... 30

6 Distance matrix of TBP clones ...... 32

7 Distance matrix of TBP clones (introns excluded) ...... 33

IX LIST OF FIGURES

Figures Page

1 Phylogram of complete Rm7S sequences ...... 13

2 Cladograms based on partial Rm 7 8 sequences ...... 14

3 Schematic tree depicting the implications of ASD accumulation ... 21

4 Alignment of TBP used in designing degenerate primers ...... 25

5 Map of the A. castellanii TBP g e n e ...... 28

6 Neighbor-joining distance tree of Acanthamoeba TBP alleles 34

7 Southem blots of Acanthamoeba genomic DNA probed with 35 TBP

8 Alignment of Acanthamoeba TBP clones ...... 39 CHAPTER 1

INTRODUCTION

Polytoma

The family Chlamydomonadaceae consists primarily of photosynthetic green algae. Eight genera within the Chlamydomonadaceae, however, are nongreen obligate heterotrophs which lack the ability to photosynthesize

(Pringsheim 1963). One of the best characterized of these is Polytoma, which is believed to be closely related to Chlamydomonas based on morphological data. Polytoma are ovoid biflagellated unicells similar to Chlamydomonas except that they are salmon pink rather than green, as they produce carotenoids but not chlorophyll (Links et al. 1963). Over a dozen Polytoma isolates are readily available from culture collections, making the genus amenable to study. Nonphotosynthetic chlorophytes can arise by mutation from photosynthetic ancestors; this occurs under laboratory conditions and is assumed to occur in the wild (Harris 1989). The nonphotosynthetic genus

Polytoma is capable of metabolizing acetate as its primary carbon and energy source, whereas many chlamydomonads are facultative heterotrophs capable of acetate metabolism, photosynthesis, or both. The growth rate of Polytoma is comparable to that of Chlamydomonas (Boynton et al., 1972), suggesting that loss of photosynthesis may be selectively neutral, or nearly neutral, especially in a nutrient-rich environment.

A Polytoma species can be postulated to arise from a nonphotosynthetic

Chlamydomonas mutant in a minimum of three steps: (1) the mutation is fixed 1 in the population, or establlishes a new population in a new habitat; (2) the heterotrophic population becomes isolated from the photosynthetic members of the same species by geographical and/or reproductive isolation; and (3) additional mutations in the photosynthetic pathway accumulate in the absence of selective pressure, making the loss of photosynthesis irreversible (Rumpf et al., 1996). After chlorophyll synthesis is lost, the species would be classified as a Polytoma.

Loss of photosynthesis removes selective pressure from the majority of plastid genes involved in photosythesis and protein synthesis (Wolfe et al.

1992a & 1992b), which will increase the probability of fixation of additional mutations in genes required for photosynthesis, making the loss of photosynthesis irreversible. Additionally, these nonphotosynthetic species are less versatile than the facultative heterotrophs from which they evolved, and presumably are more limited in the habitats they can occupy. It seem s logical that nonphotosynthetic lineages would become established and speciate infrequently at best, and have a higher probability of extinction than their photosynthetic congeners. Chapter 2 of this work details my investigations into the evolutionary relationship of Polytoma and its nearest photosynthetic relative,

Chlamydomonas, in attempts to characterize its origin and extract information regarding the frequency with which nonphotosynthetic lineages arise, how long they survive, and the extent to which they speciate.

Project Goal: Polytoma

The goal of the first half of my project has been to investigate the evolutionary relationship between 13 Polytoma species and their photosynthetic relatives among the Chlamydomonads using the sequence of the gene encoding the nuclear small subunit RNA of the ribosome {Rm18). The

Rrn18 gene is useful in this analysis because it is essential for protein synthesis and therefore ubiquitous and highly consen/ed. The rRNA product forms a complex secondary structure containing loops and stems, both of which contribute to the information content of the molecule. The gene has been used extensively in phylogenetic studies (e.g. Femholm et al., 1989), providing a large database of sequences and phylogenetic trees for comparison.

By reconstructing the phylogeny of Polytoma, I hope to answer several questions: (1) how many times has this nonphotosynthetic lineage arisen from photosynthetic ancestors; (2) how speciose is each of these lineages; and (3) how old is each lineage.

Nonphotosynthetic mutants frequently arise spontaneously under laboratory conditions, and, by extrapolation, in nature. These mutants can and do establish species of colorless algae. Over time, additional mutations arise in the pathway responsible for photosynthesis, which is no longer under selection, making reversion unlikely. Why hasn't this happened to all photosynthetic lineages, rendering them incapable of photosynthesis? There are two approaches to answering this question: individual selection and species selection. Selection working at the level of the individual would prevent the nonphotosynthetic lineage from being established, whereas selection at the level of the species would lead to the extinction of the nonphotosynthetic lineage after it had already been established.

It is conceivable that the nonphotosynthetic mutants are infrequently fixed in the population, as a direct result of the mutation itself. Losing a major nutritional pathway would seem intuitively to be detrimental. It has been shown, however, that the growth rates of photosynthetic and nonphotosynthetic species is similar in nutrient-rich environments (Boynton et al. 1972), so at least under these conditions the loss of photosynthesis is not detrimental, and individual selection would not come into play. It is also likely that nonphotosynthetic species become extinct more readily or speciate less frequently, or both. Again this sounds reasonable as these species would be less adaptable to changing environmental conditions than their facultative heterotrophic competitors, and would thus occupy a narrower niche.

Phylogenetic trees of photosynthetic and nonphotosynthetic lineages could shed light on this by way of topology: if the nonphotosynthetic lineages were few in number but deeply rooted, this would suggest a low spéciation rate; alternately if the non photosynthetic lineages were abundant but short- branched, this would suggest a high rate of extinction. Both of these would indicate that selection was working at the level of the species. A third alternative, in which all lineages were approximately equal in branch length, with an origin near the time of one of the major extinctions, such as the

Cretaceous-Tertiary (KT) boundary, would suggest strong individual selection against nonphotosynthetic mutants except at times of low light and high nutrient availability, both of which were presumed present at the times of the major extinctions.

Acanthamoeba

Acanthamoeba is the causative agent of granulomatous amebic encephaitis, a generally fatal opportunitistic infection of the central nervous systems common in immunocompromised individuals (Visvesvara et al., 1983;

Ma et al., 1990; John, 1993), and Acanthamoeba keratitis, a potentially vision- threating parasitic invasion of the cornea in contact lens wearers (Dart, 1988). This genus of free living protozoans has been divided into three morphological groups based on cyst morphology (Pussard and Pons, 1977). A. castellanii and A. griffini, the objects of this study, fall into Group II, which is characterized by cysts of < 18 pm in diameter, and stellate, polygonal, triangular, oval, or round endocysts (Visvesvara, 1991; Rivasi et al., 1995). Acanthamoeba alternates between trophozoite and cyst stages in its life cycle, making treatment of infections difficult. The cyst stage is highly resistant to a wide variety of conditions and antimicrobial compounds (Osato et al., 1991; Hay et al., 1994;

Elder et al., 1994), allowing the organism to survive in this state until treatment is discontinued.

It is believed that Acanthamoeba reproduces asexually; this is due primarily to the lack of convincing evidence for genetic recombination (Akins,

1981). The observation of identical mitochondrial 16S genes in association with differing nuclear 18S genes suggests that reproduction in Acanthamoeba castellanii may not be entirely clonal (Ledee 1995). The method of reproduction affects the etiology of the diseases caused by an infectious parasite; an asexual organism makes an excellent rapid colonizer, whereas sexual reproduction of the infectious agent facilitates the quick and efficient dissemination of drug resistance genes throughout the population. The resulting diseases should be treated accordingly.

Another question in Acanthamoeba genetics is that of ploidy.

Measurement of nuclear DNA in A. casteilanii Neff suggests a total nuclear DNA content at log phase of 10^ bp (King, 1980). Kinetic complexity m easurements suggest a haploid genome size of 4-5 x 10^ bp (Bohnert & Heermann, 1974), whereas pulse field electrophoresis has suggested that the genome size is on 5 the order of 2.3-3.S x 10^ bp (Rimm et al., 1988). Based on these data, the ploidy may range anywhere from 10 to 22. Chapter 3 of this work details my investigation into both the method of reproduction and the ploidy of Acanthamoeba.

Project Goal: Acanthamoeba

The primary goal of my project regarding Acanthamoeba is to determine the method of reproduction of two species of Acanthamoeba, A. castellanii and A. grifinii. To accomplish this goal, I utilized the measurement of allelic sequence divergence (ASD), a technique pioneered by Dr. Matthew Mesleson, and for which the theory was fully developed by Birky (1996).

In a sexual species, the two alleles of any gene will have a most recent common ancestor, referred to as the coalescent of the alleles, within a relatively short span of time (an average of 2N@ generations where N@ is equal to the effective population size). The expected neutral sequence divergence between two different alleles in a sexual population equals 4 N e |i , where p. is the mutation rate. In a lineage where the capability for sexual reproduction has been lost, however, the distance to the coalescent will be significantly greater; not only will the alleles have the existing 2N@ generations of divergence time from when the organism was sexual, but time t (the time since the loss of sexual reproduction) will allow for additional divergence of some pairs of alleles, most notably those in one individual or clone. Thus the expected neutral sequence divergence between two different alleles in an asexual population equals 4N@p + 2tp, where t = the time since the loss of sexual reproduction. This effect was clearly seen in the bdelloid rotifers, which had an ASD as high as 40% (M. Meselson and D. Welch, personal communication), which is significantly higher than the 6 maximum value found for sexual species (Avise, 1994; Moriyama and Powell,

1996).

Allelic sequence divergence will be limited by selection; in a protein coding gene, however, synonymous substitutions will be subject to weak or no selection. Thus using a protein coding gene and concentrating on divergence at the third codon position will largely avoid the problem of selection.

It is possible that, although a species is asexual, sequence divergence can be reduced in one of several ways; this is called sequence convergence.

Mitotic recombination, a ploidy cycle, or rare sex (in which case the description of the species as asexual is a matter of semantics) can reduce or eliminate allelic sequence divergence. In such cases, measurement of sequence divergence could lead to an asexual species being mistaken for a sexually reproducing one. This effect can be partially countered by sequencing the alleles of several genes or the alleles of one gene in a polyploid; only a ploidy cycle will affect all genes in an organism or all genes on a single chromosome. Another interesting result of the loss of sexual reproduction is that the alleles within an organism could be more divergent to one another than to alleles in another organism; this was also seen in the bdelloid rotifers (M. Meselson and

D. Welch, personal communication). This results in allele-dependent phylogenetic trees, in which different sets of alleles have different tree topologies (See Chapter 3 and Figure 3).

By amplifying, cloning, and sequencing all possible alleles of a given protein coding gene from Acanthamoeba, I will be able to measure the alleleic sequence divergence, and possibly determine whether or not Acanthamoeba reproduces sexually. Additionally, by counting the number of unique alleles cloned I can determine a minimum ploidy number for the organism. Phylogenetic Methods

A phylogenetic tree based on any data, be it sequence data or morphological characteristics, is at best an approximation of the true state of the universe. The level of agreement between the inferred tree and the true tree depends on the nature and quality of the data as well as the method of phylogenetic reconstruction employed (Gray, 1992). Strong correlation between trees derived from the same data by different reconstruction methods based on different evolutionary models suggests that the inferred trees are close to the true tree (Woese et al., 1980). Three reconstruction methods were used for my analysis of the Rm18 gene in Polytoma: neighbor-joining, parsimony, and maximum likelihood.

Distance methods employ a specific evolutionary model to first calculate the number of substitutions between sequences in a pairwise fashion, generating a distance value for all possible pairs of taxa. Distances are generally corrected for multiple hits using one of a variety of methods. These values are used to construct a tree based on the neighbor-joining algorithm, which optimizes the tree based on observed distances (seen after tree construction) and actual distances (from the pairwise calculations). By adjusting the separation of pairs of nodes based on their divergence from all other nodes, the neighbor-joining algorithm counteracts the effects of differing rates of evolution in the various taxa (Saitou and Nei, 1987). The neighbor- joining trees in this work were constructed using the PHYLIP package (Felsenstein, 1993 version 3.5).

Parsimony algorithms construct trees so that the minimum possible number of base substitutions are required for the resulting tree topology. No correction is made for multiple hits, and only informative sites are taken into 8 consideration. Parsimony trees in this work were constructed using PAUP 3.5 (Swofford 1993).

Maximum-likelihood algorithms construct trees by recursively calculating

the likelihood of node topologies for each position within the alignment,

beginning at the top of the tree and moving downwards one node at a time,

testing the likelihood of the topology against a specific evolutionary model. The

likehood calculations derived at each step are used in subsequent steps to find

the likelihood of the next node. This process is repeated until all nodes are

calculated; the overall likelihood is then calculated by summing the products of

the root likelihoods with the prior likelihoods at each node; the best tree is the

one with the highest likelihood. Maximum likelihood assum es that each site

evolves independently following either the Poisson or some other probability

process; thus the likelihood of a phylogeny can be calculated separately for

each site (Felsenstein, 1981).

All trees in this work were bootstrapped. Bootstrapping is a resampling technique in which a dataset of n positions is resampled n times at a random

position within the array, creating a new data set of the same size as the original. Since the resampling is random, any given site within the array may be represented not at all, once, or in multiple. The bootstrap values which are placed at nodes of the resulting phylogenetic trees represent the proportion of bootstrap datasets which supported that particular node (Felsenstein, 1988).

These values, although not statistical measures themselves, can be used to infer which nodes are more strongly supported by the given dataset. J. PkyaL SX, 119-126 (1996)

EVOLUTIONARY CONSEQUENCES OF THE LOSS OF PHOTOSYNTHESIS IN CHLAMYDOMONADACEAE: PHYLOGENETIC ANALYSIS OF R m I8 (IBS rDNA) IN 13 POLYTOMA STRAINS (CHLOROPHYTA)*

Robert Rumpf, Daume Vemon, David Schreiber,* and C. William Birky, Jr.* Department of Molecular Cenetia, The Ohio State University. 484 West Twelfth Avenue, Columbus, Ohio 43210

ABSTRACT et al. 1989); here we show that 12 additional isolates Complete sequences of the RrnlS genes were obtained of Polytoma are also members of the Chlamydomonas from 13 strains of the nmphotosynthetic algal genus Po­ clade. lytoma. Phylogenetic analyses showed that these strains It is generally assumed that nonphotosynthetic formed two clades. One clade shows only modest sequence chlorophytes arose from photosynthetic ancestors. diversity but is represented by strains collected at widely Polytoma species are obligate heterotrophs capable dispersed sites in Europe and America. The other clade of utilizing acetate as their sole carbon and energy consists of a single isolate from the Canary Islands. Both source, while many chlamydomonads are facultative clades lie well within the extended clade that includes all heterotrophs that can utilize acetate, photosynthe­ species of Chlamydomonas for which sequence data are sis, or both. Nonphotosynthetic mutants are easily available. The two Polytoma clades are separated from obtained in C. reinhardtii and can result from mu­ each other by several green species, suggesting that the tations in any of a number of different genes in the extant runphotosynthetie Chlamydomorutdaceae arosefrom chloroplast or nucleus (Harris 1989). Many of them photosynthetic ancestors at least twice. These results suggest are easily maintained on acetate in the light or dark, that runphotosynthetie mutants are capable of establishing but detailed comparisons of growth rates to wild- lineages that can spread widely but have a higher prob­ type cells have been made for only two mutants ability of extinction than their photosynthetic congeners. (Boynton et al. 1972). One of these, ac-20, grows only slightly slower than wild type under heterotro­ Key index words: 18S rRNA; Chlorophyta; Chlamy­ phic conditions with no light and acetate, in spite of domonas; heterotrophic algae; loss of photosynthesis; mo­ having a reduced rate of plastid protein synthesis. lecular evolution; Polytoma; phylogenetics; Rm lS gene Thus, it is reasonable to suppose that some non­ photosynthetic mutants (perhaps a small minority) Most chlorophyte algae are photosynthetic and might be selectively neutral or nearly neutral in na­ contain chlorophyll in their plastids, as suggested by ture, especially in an environment rich in organic the common appellation "green algae.” However, nutrients and under less intense and discontinuous a number of nongreen heterotrophic species are also illumination. Such a mutant could give rise to a Po­ classified in the Chlorophyta on the basis of mor­ lytoma species by the following steps: 1 ) the mutation phological and biochemical similarities to various is fixed in the population (probably by random drift), photosynthetic groups. One of the best-known gen­ or establishes a new population in a new habitat; 2) era of heterotrophic chlorophytes is Polytoma, oval the heterotrophic population becomes isolated from to elongated bifiagellate unicells with a single large photosynthetic members of the same species by ge­ plastid (leucoplast) partly surrounding the centrally ography and/or the acquisition of genetic isolation located nucleus (Lang 1963, Pringsheim 1963). T he mechanisms; and 3) additional mutations in the pho­ leucoplast contains ribosomes and DNA (Scherbel tosynthetic pathway accumulate in the absence of et al. 1974, Siu et al. 1975a, b, c, Vemon-Kipp et selective pressure, making the loss of photosynthesis al. 1989). Polytoma is believed to be closely related irreversible. The population will be classified as Po­ to Chlamydomonas on morphological grounds lytoma when chlorophyll synthesis is lost, due to ei­ (Pringsheim 1963). T he genus Chlamydomonas is di­ ther the initial mutation or, more often, a subse­ verse in terms of morphology, habitat, and metab­ quent mutation. olism and has been classified into over 450 species T he loss of photosynthesis is likely to have im­ on the basis of morphology and sensitivity to cell portant consequences for evolution of the plastid wall autolysins (Ettl 1976, Schlôsser 1984, Ettl and genome. These include the loss of most or all genes Schldsser 1992). We used DNA sequence data to coding for proteins involved in photosynthesis (Wolfe verify the close relationship between one isolate o f et al. 1992b, but see Siemeister and Hachtel 1990) Polytoma and Chlamydomonas reinhardtii (Vemon-Kipp and the accelerated evolution of genes coding for ribosomal RNAs (rRNAs) and proteins involved in protein synthesis (Wolfe et al. 1992a). In addition, ' Received 28 June 1995. Accepted 26 October 1995. ' Present address: USDA, Forest Service, 359 Main Road, Del­ it is likely that selection operating at the level of aware, Ohio 43015. species or populations favors photosynthetic over ' Author for reprint requests. nonphotosynthetic organisms. Although an obligate

10 T able 1. List o/Polytoma slodts.

Stock member" Sixties* Where eoDcaed Collector No.' UTEX 19 P. uvelta Ehrenberg ? E. G. Pringsheim U22940 UTEX 964 P. uvelta Ehrenberg Woods Hole, Mass , USA F. Moewus U2294S ATCC 30963 P. obtusum ? ? U22938 SAG 195.80 ITetrablepharis sp.) South Africa E. G. Pringsheim U229S7 CCAP 62/2M P. uvelta Ehrenberg Leiden, Holland E. G. Pringsheim U22942 SAG 62-2c P. uvelta Ehrenberg Brixen, Tirol, Italy E. G. Pringsheim U2294I SAG 62-13 IP. mirum Pringsheim) Soiling, Germany E. G. Pringsheim U22934 SAG 62-16 (P. difficile Pringsheim)Heidelberg, Germany E. G. Pringsheim U22932 SAG 62-18 {p. eilipticum Pringsheim) Klasen-Brixen, Tirol, Italy E. G. Pringsheim U22933 SAG 62-20 Polytoma sp. Prutz, Tirol, Italy E. G. Pringsheim U22939 SAG 62-21 {P. anomale Pringsheim) California, USA E. G. Pringsheim U2293I SAG 62-27 (P. oviforme Pringsheim) Tenerife, Spain E. G. Pringsheim U229S6 DHl P. obtusum ? ? U229S5 • ATCC - American Type Culture Collection, CCAP ” Culture Collection of Algae and Protozoa, SAC “ Sammlung von Algen- kulturen Gottingen, UTEX - University of Texas Culture Collection of Algae; DH1 was supplied by David Herrin and came originally from the collection of L. Provasoli at Yale University; strain designation Is that of the authors. * Names in parentheses are provisional or former names of strains now designated Polytoma sp. ' Gen Bank Accession number for R m iS (IBS) gene sequence.

heterotroph might reproduce as rapidly as a pho­ MATERIALS AND METHODS tosynthesizer in an environment rich in organic nu­ trients, it will be unable to occupy the nutrient-poor All itrains were obuined from icock centers or other labora­ tories (Table 1) and subcloned (with the exception of SAG 62- environments that are open to photosynthesizers. 2C, which we were unable to subclone successfully). Some strains As part of our investigations of the evolutionary have never been assigned specific names, whereas others were consequences of the loss of photosynthesis in algae, given provisional names that appear to have been dropped sub­ we determined the evolutionary relationships of a sequently; therefore, we will refer to them by their strain num- large sample of the available strains of Polytoma to ben. Stocks were grown in 10 mL liquid PM media (2.0 g sodium species of Chlamydomonas. The nuclear R m l8 gene acetate (hydrate), 1.0 g yeast extract, 1.0 g tryptone, doubly dis­ tilled water to I L) and transferred monthly usinga lilOdilution and the small subunit rRNA for which it codes have of existing stock into fresh medium. been widely used to reconstruct evolutionary his­ DNA prtparation. Cells w ere grown to log phase in 1-2 L PM. tories. The sequences of these genes have been used Cells were harvested by centrifugation (Beckman JAIO, 14 x g, to clarify the phylogeny of 28 Chlamydomonas species 10 min), and the resulting pellet was frozen in liquid nitrogen and 12 species from closely related genera (Jupe et for storage at —80* C. T h e cell pellet was ground with sand in a al. 1988, Buchheim et al. 1990, Buchheim and Chap­ cold mortar, transferred into 5 roLCTAB buffer(IOO mM Tris- man 1991,1992, Chapman and Bucheim 1991, Lar­ HCI, pH 8, 1.4 M NaCI, 20 mM EDTA, 2% cetyltrimethyl am­ monium bromide, and 0.2% /3-mercaptoethanol) in a mortar son et al. 1992). The maximum sequence divergence warmed to 65* C, and incubated at 65* C for 45-60 min. The within this group is about 10%, greater than that resulting lysate was extracted once with 24:1 chloroformiisoamyl between gymnosperms and angiosperms. Parsimony alcohol, and the DNA was precipitated with ethanol and resus­ trees generated with these data consistently group pended in I mL TE (10 mM Tris-HCI, pH 8.0, I mM EDTA, these organisms into six or seven clades. To which, pH 8.0). if any, of the members of this group are the various Potjourast chain Ttaction amplifications. T h e R m lS gene was am­ plified from the crude lysates with primers Icxated at the ends of species of Polytoma related? the gene. The 5' and S' primers were, respectively, SSUIA - 5'- We have now sequenced the complete R m l8 gene TGGTTGATCCTGCCAGTAG-S" (sense strand sequence) and from 13 Polytoma strains and two strains of Chlam­ SSU2 - 3 -CACTTGGACGTCTTCCrrAGT-5' (antisense se­ ydomonas. We constructed phylogenetic trees using quence). The optimal amplification conditions for each lysate our sequences and those of other species of Chlam­ were determined empirically. Then multiple amplifications under ydomonas and closely related green algae provided these conditions were performed and pooled for use as template in subsequent sequencing reactions. Tlte pooling of multiple in­ by other researchers. Our goals were to determine dividual amplifications minimizes sequencing errors due to mis- 1) the number of distinct heterotrophic lineages, i.e. incorporation of nucleotides by TAQ polymerase during indi­ the number of times extant nonphotosynthetic spe­ vidual amplifications. The pooled DNA was purified via the cies arose from photosynthetic ancestors; 2) the age CeneClean kit (Bio 101). of the heterotrophic lineages; and 3) the extent of Accidental amplification of DNA from the wrong organism spéciation and dispersion of the nonphotosynthetic was extremely unlikely because I) algal DNA molecules greatly lineages. Our results show only two origins, one rep­ oumumbered any contaminating DNA molecules because mul­ tiple amplifications were done, each using about I nU of the crude resented by a single isolate, the other by 12 widely lysate from a 2-L culture, and 2) all sequences clearly belong to distributed isolates that probably represent a single the Chlamydomonas clade, of which the only cultures in the lab species. were the Polytoma isolates, Chlamydomonas reinhardtii, C. humicola.

11 MOLECULAR PHYLOGENETICS OF POLYTOMA

T a b u 2. List of eompltte R m li stqvences ajid Iheinouras. For a second dataset of partial rRNA sequences from 29 ad­ ditional Chlamydomonadaceae and SO other chlorophytes (kindly Tmmoo CcnBanà* Reference provided by Mark Buchheim), our complete sequences were trun­ Asteromonas gracilis M956I4 Unpublished cated to match the partial sequences and aligned as earlier. This CMamyiomonas humicola^ UIS984 Gordon et al. 1995 data set had 96S total sites. Chlamyiomonas rtinhanltii Gunderson et al. Phylogenetic analysis. Three types of phylogenetic analysis pro­ 1987 grams were run on Macintosh and Iris Indigo computen. The Chlortlla vulgaris XIS688 Huss and Sogin 1989 DNAboot, DNADist. and neighbor-joining algorithms of Phylip Dunaliilla parva M62998 Unpublished V . S.56 (Felsemtein 1989,1993) were used to construct neighbor- DunalitUa satina M84S20 Wilcox et al. 1992 joining trees; the Kimura two-parameter method with a transi­ Glycine max M200I7 Eckenrode et al. 1985 tion/transversion ratio of 2.0 was used to correct for multiple Oryza sativa X00755 Takaiwa et al. 1984 hits. PAUP V . 4.0d25 (Swofford 199S, results published with Saccharmyces eerevisiae M27607 Mankin et al. 1986 permission of the author) was used to generate maximum par­ Volvox carteri X53904 Rausch et al. 1989 simony trees with three kinds of searches: exhaustive and branch- Zamia pumila M20017 Naim and Perl 1988 and-bound heuristic searches, which give the most parsimonious • Accession numbers. tree, and a full heuristic search, from which the 50% majority ** Combined with other strains under C. applanata by Ettl and consensus tree was used. All trees (except the one from the ex­ Schlôsser (1992). haustive search maximum parsimony) were bootstrapped 100 times. Branch swapping by tree-bisection-reconnection (TB) was used with accelerated transformation (ACCTRAN); IQ random sequence addition replicates were used for each of the 100 boot­ C. d}tosmos, and three isolates of Poljltmella that have different straps in the heuristic searches, for a total of 1000 replicates. sequences from those shosvn here (unpubl.). There were 9 1 informative sites in the complete sequences and Dirnt utpuTtdngof doublt-slranded Df/A. The pooled polymerase 157 in the partial sequences. Finally, fastONAmI (Olsen et al. chain reaction (PCR) products were directly sequencÂl using the 1994) was used to construct maximum likelihood trees. Two data double^tranded DNA Cycle Sequencing kit and protocols (BRL), sets were used in the phylogenetic analysis: I) the complete se­ with two modifications: 200-500 ng of PCR product and 1.5-2 quences from six green Chlamydomonadaceae, IS Polytoma strains, aL of 10 U-aL~' TAQ DNA polymerase were used for each and Chlorella, and 2) a subset of the partial sequences including reaction. Primers for both strands were constructed for con­ 24 green Chlamydomonadaceae, Polytoma strains 964 and 62-27, served sites at approximately 200-bp intervals along the gene and and Chlorella. Chlorella vulgaris was used as an outgroup because used for consecutive sequencing reactions. Each strain was se­ Chlorella species are consistently separated from Chlamydomonas quenced at least twice on one strand; several were sequenced species in trees based on 5S and IBS rRNA sequences (Chapman twice on both strands. The high level of conservation of this gene and Bucheim 1991). Distance matrices (Table S) were constructed allowed for an additional proofreading step: sequences from two with the aid of The Indel/Substitution Machine, a Hypercard- closely related strains were compared and sites of divergence were based program (R. R., unpubl.). The program constructs matrices checked on the autoradiographs to positively identify differences, o f pairwise base substitutions and indels (gaps due to either in­ which were then sequenced again for verification. All sequence sertions or deletions. autoradiograms were read independently by at least two people. Other OS A sequences. The complete R m l8 sequences of other algae, yeast, and plants were obtained from Gen Bank; accession RESULTS AND DISCUSSION numbers and references are given in Table 2. Partial sequences Choice o f genes. Several factors make the R m l8 of IBS rRNAs from additional Chlamydomonadaceae species gene an excellent candidate for phylogenetic anal­ shown in Figure 2 were provided by Mark A. Buchheim (Uni­ versity of Tulsa). ysis. The gene product (18S rRNA) is essential for Sequence alignment. DNA sequences were entered directly in the protein synthesis and therefore can be found in all SeqApp computer application package (Gilbert 1992) running on organisms. The rRNA product forms a secondary Macintosh computers. SeqApp, through links to CAP (Contig structure rich in stems and loops; the structure is Assembly Program, Huang 1992) and ClustalV (Higgins and Sharp sufficiently conserved that it can be used to improve 1988, 1939, Higgins et al. 1992), was also used to assemble the alignments. Finally, this gene has already been used sequencing fragments of each strain into a contig and then to to determine the phylogenetic relationships of nu­ produce the initial alignment of six green Chlamydomonadaceae and IS PMjtoma strains. We refined this alignment by hand, in­ merous organisms (e.g. Fernholm et al. 1989), and fluenced by primary sequences from three land plants {Zamia a large database of 18S sequences and phylogenetic pumila, CIjcsne max, and Oryzn sativa) and a yeast (Saecharomjces trees is available for comparison (Maidak et al. 1994). eerevisiae). We further relink the alignment using conserved pat­ For the photosynthetic Chlamydomonadaceae in terns in the secondary structures for this gene in one land plant particular, complete sequences of the R rnl8 gene (Zea mays, Gutell et al. 1985), three green algae {Chlorella vulgaris. are available for 8 species, and partial sequences of Huss and Sogin 1989; Volvox carteri, Rausch et al. 1989: Chlam­ ydomonas reiniutrdtii, Gutell et al. 1985), and S. eerevisiae (Gutell the 18S rRNA are available for 18 additional species et al. 1985). (Vemon-Kipp et al. 1989, Buchheim et al. 1990, We were able to align 98.9% of the R m I8 gene from 12 of Buchheim and Chapman 1991,1992, Chapman and the IS Polytoma strains (the strains in the P. uvella clade), leaving Bucheim 1991; Mark A. Buchheim, pers. commun.). out only the first 4 5' positions and the last 16 S' positions for Aligntnenls. The secondary structure of the 18S lack of data in all species. For the entire array of complete algal rRNA is highly conserved, in part by compensating sequences for the taxa shown on the accompanying trees, we were able to align 96.6% of the gene, omitting only two segments base substitutions (Hillis et al. 1994). This conser­ of 6 and S5 bases where the alignment was ambiguous, as well vation can be used to verify the accuracy of align­ as the short sections at the 5' and S' ends for lack of data; this ments: if a region in one sequence has a particular left 1775 base pain. secondary structure, the same region in another se­

12 ROBERT RUMPF ET AU

T able S. Numbers o f subsdtution and indel differences between complete sequences o R f m I8 genes from pairs of strains^ Indels are above the diagonal, substitutions below.

c. a AO. C fêly IVy Pêty W r M r M r M r M r t y r M r M r M r Cktor- Vêt­ hard’ D. Mi­ Mac Smmi. ■w lorn. MM P#M Mm M form 6%.*7 aU M4 19 62.1c 6 M 6M ( DHl I9 5 J0 6116 61-11 61-10 30966 611m Cklonlla is 10 11 ll 14 11 11 19 14 15 13 13 19 14 16 16 19 15 12 Vulvox 110 — 7 16 16 19 19 16 22 17 18 16 16 22 17 19 19 22 18 16 C. reinhardlii 105 14 — 15 14 18 15 15 19 14 15 13 13 20 14 16 16 19 15 13 D. parva 88 88 81 — 0 3 8 4 12 7 8 6 6 13 7 9 9 12 8 6 D. salina 90 90 84 12 — 3 8 4 12 7 8 6 6 13 7 9 9 12 8 6 Asleromonas 106 102 94 40 43 — 11 7 15 10 11 9 9 16 10 12 12 15 11 9 Polytoma 62-27 91 87 81 53 54 69 — 18 26 19 19 18 18 26 19 23 21 24 20 20 C. humieola 80 78 70 29 28 49 51 — 12 9 10 8 8 12 9 9 9 14 8 8 Polytoma 964 88 82 75 32 31 54 55 18 — 9 10 8 8 4 7 9 7 12 8 6 Polytoma 19 89 83 75 32 31 54 55 18 0 — 3 1 1 9 2 6 4 7 3 3 Polytoma 62-2c 89 82 75 32 31 54 55 18 0 0 — 2 2 10 2 7 5 8 3 4 Polytoma 62-3 89 82 75 32 31 54 55 18 0 0 0 — 0 8 1 5 3 6 2 2 Polytoma 62-18 89 82 75 32 31 54 55 18 0 0 0 0 — 8 1 5 3 6 2 2 Polytoma D H l 89 82 75 32 31 54 55 18 0 0 0 0 0 — 7 7 7 12 8 6 Polytoma 195.80 90 82 76 33 32 55 56 19 1 1 1 1 1 1 — 6 2 5 3 1 Polytoma 62-16 88 83 76 32 31 54 54 19 1 1 1 1 1 1 0 — 6 11 5 5 Polytoma 62-21 90 83 76 33 32 55 56 19 1 1 1 1 1 1 0 0 — 7 3 3 Polytoma 62-20 89 83 76 33 32 55 56 19 1 1 1 1 1 1 2 2 2 — 8 6 Polytoma 30963 89 83 76 33 32 55 56 19 1 1 1 1 1 1 2 2 2 2 — 4 Polytoma 62-2m 91 84 77 34 33 56 57 20 2 2 2 2 2 2 3 3 3 3 3 —

quence should have a similar structure. For exam­ ple, Polytoma strain 62-27 exhibited a large number of base substitutions compared to the other Chla- mydomonadaceae; nevertheless, the secondary structure in all regions of the rRNA was conserved. ■«cutup Sequence differences. The numbers of pairwise base substitutions and indels in the R m l8 gene were tab­ ulated for the species used in tree reconstruction (Table 3). Strains 19, 964, 62-2c, 62-3, 62-18, and DHl do not differ in base substitutions and were combined for the purpose of phylogenetic analysis as ihe Polyloma 964 clade. Strains 195.80,62-16, and 62-21 are also identical (except for indels) and were combined as the Polytoma 195.80 clade. Most of the Polytoma strains can be differentiated by indels; only strains 62-3 and 62-18 have R m l8 sequences that are identical in all respects. Phylogenetic trees. Neighbor-joining, maximum graciiii parsimony, and maximum likelihood algorithms eotytama S2-Z7 were used to reconstruct the phytogeny of these — Vfl/w* emuri species. Each of these methods has distinct advan­ tages and disadvantages and is prone to different sources of error (Hillis and Moritz 1990, Miyamoto and Cracraft 1991, Stewart 1993, Hillis et al. 1994), FtC. I. Phylognun of complete R m l8 lequences. Numbers so that the strongest conclusions can be drawn when on the branches are branch lengths from the neighbor-joining all methods produce the same tree topology. For the algorithm: phylogram branch lengths are proportional to these complete-sequence data set, all three methods pro­ numbers. T h e tree topology is the same as that of the most par­ simonious tree obtained with an exhaustive or branch-and-bound duced the same cladogram (Fig. 1). For these trees search, the 50% majority consensus tree obtained with a full we used the default weighting of 2 transitions : 1 heuristic search, and the maximum likelihood tree. Numbers at transversion. We determined that the actual ratio the nodes are numbers of bootstrap replicas (out of 100) con­ in this data set was 1.726:1; when we used this ratio, taining the corresponding clade; from top to bottom, these are the same parsimony and maximum likelihood trees from the neighbor joining, maximum parsimony, and maximum likelihood algorithms. The most parsimoniotis tree had a tree were obtained (not shown). Twelve of the Polytoma length of 265, a consistency index of 0.8528, and a rescaled species form a clade that we refer to as the P. uvella coiuistency index of 0.6436.

13 MOLECULAR PHYLOGENETICS OF POLYTOMA

Nllak(wMlnlii( have at least one green lineage between Polyloma 62- .ic CAimw# 27 and the P. uvella clade. For the maximum par­ simony trees, this includes all of the most parsimo­ nious trees produced by bootstrap resampling, plus the 38 trees with lengths no more than 10 changes longer than that of the most parsimonious tree, i.e. from 265 to 275. In the complete sequence tree, a üj- PoytonviMte •®—} monophyletic origin o f all Polyloma species from a single heterotrophic muunt would require a mini­ mum of two reversions of the loss of photosynthesis (the partial sequence trees would require three or ■Oysmorphocoeaa - -* four reversions). We can assess the probability of r C wM ri* m ir ) 762 ■ Cvfnê laOota ^ two reversions from the branch lengths on the max­ ■ Volmfâunu* j". imum parsimony tree of complete sequences. Be­ tween the original loss of photosynthesis and the second of these hypothetical reversions (on the C. humieola branch), the R m l8 gene accumulated a minimum of 18 base substitutions (data not shown) and six insertions or deletions (estimated by multi­ plying the number of base substitutions times the Poiynnil 62-27 - C tiuri» erudiÊ n n ratio of substitutions to insertions or deletions be­ _r*nli «upanwiat-' tween strain 62-27 and C. humieola). During this time, each of the photosynthetic genes, which would not Fic. 2. Cladogrami based on partial R m I8 sequences. The be subject to selection after photosynthesis was first cladograms were obtained with the neighbor-joining (left) or lost, must have incurred many additional substitu­ maximum parsimony (right) method. Numbers in boxes at the tions, insertions, and deletions. Reactivating pho­ nodes are numbers of bcwtstrap replicas (out of 100) containing the clade. Clades with nodes marked by dark triangles on the tosynthesis would then require the reversion of mul­ nodal box were supported by the maximum likelihooid tree. tiple base substitutions, insertions, and deletions in one lineage, which is extremely unlikely. The P. uvella elade may represent a single speeies or several elosely related speeies. T h e topology o f the phy­ clade; it includes strains 62.20 and 195.80 and the logenetic trees demonstrates that Polyloma species 10 strains in the 964 and 195.80 clades. The thir­ arose at least twice. However, it does not eliminate teenth Polytoma strain, 62-27, was consistently sep­ the possibility that the P. uvella clade is polyphyletic, arated from the P. uvella clade by two green lineages i.e. that there are more than two origins overall. (C. humieola and Asleromonas, thinaliella parva, D. That this is unlikely is indicated by the low genetic salina). variability within this clade. Within the P. uvella Trees were also constructed using the larger data clade, 62-3 and 62-18 are identical. The pairwise set of partial sequences (Fig. 2). Many bootstrap val­ sequence divergence within the remaining P. uvella ues were low, but the neighbor-joining, maximum clade is less than 0.17% base substitutions (0-3 sub­ parsimony, and maximum likelihood trees agreed stitutions/1775 bp). This can be compared to the in several important features. First, these trees are divergence of the R m l8 genes of Chlamydomonas similar to those of Buchheim et al. (Buchheim et al. moewusii GerlofF (UTEX 97) and C. eugamelos Moe- 1990, Buchheim and Chapman 1991, 1992, Chap­ wus (UTEX 9), which are considered to be conspe- man and Bucheim 1991) in that they divide the Chla- cific on the basis of their ability to interbreed in the mydomonadaceae into six major clades: 1) Carteria laboratory (Gowans 1963) and of their similar mor­ crucifera and C. lunzensis, 2) Carteria sp. UTEX LB phology (Ettl and Schlosser 1992). Partial sequences 762 and C. radiosa, 3) Chlamydomonas peterfii and C. of the small-subunit rRNA of C. moewusii and C. nuxicana, 4) Chlamydomonas zebra, C. reinhardlii, and eugamelos, provided by Mark Buchheim (pers. com­ Volvox aureus, 5) Chlamydomonas geitleri, C. pitsch- mun.), differ by 1 base substitution out of 737 base manii, and C. moewusii UTEX 9, and 6) C. humieola pairs (approximately 50% of the gene); this corre­ with Dunaliella, Asleromonas, Chlorogonium, Slephan- sponds to a sequence divergence o f 0.13% base sub­ osphaera, and Haemaloeoeeus species. Second, the P. stitutions. All die substitution differences within the uvella clade consistently falls within the Haematoeoe- P. uvella clade occur at only four positions, none of eus clade. Third, Polyloma 62-27 is a sister-group of which are phylogenetically informative. Thus, it is the geitleri clade. Fourth, Polyloma 62-27 is always not surprising that the clade was not convincingly separated from the P. uvella clade by one or more resolved by any of the three computer algorithms. photosynthetic lineages. There is more variation within the P. uvella clade Polytoma is polyphylelie. All trees supported a min­ with respect to indels than base substitutions. Mem­ imum o f two origins of the genus Polytoma. All trees bers of the clade differ by 0-12 indels, compared to

14 ROBERT RUMPF ET AL.

2 in the partial sequences from C. moewusii and C. heterotrophic lineage that is dying out. Of course eugamelos. All are insertions or deletions of a single we cannot rule out the possibility that it represents base pair. The pairwise comparisons show no ap>- a clade that is successful in nature but is rarely col­ (nrent correlation between the number of base sub­ lected by Pringsheim’s methods or does not thrive stitutions and the number of indels between a pair in stock collections. of sp>ecies. Although there are many more indels These observations are compatible with the hy­ than substitutions within this clade, all the indels pothesis that heterotrophic mutants rarely give rise occur at just 25 sites, of which only 8 are phyloge­ to heterotrophic spocies, presumably because the netically informative. Parsimony analysis of the P. mutants are at a selective disadvantage relative to uvella clade using indels placed P. uvella strain 964 their photosynthetic cohorts. The data are also com­ with P. oblusum DHl in a separate clade from the patible with the hypothesis that heterotrophic spe­ other strains, which form a pwlytomy (not shown). cies are at a disadvantage relative to photosynthetic This agrees with sequence data from the leucoplast spocies. This might be manifested as a higher ex­ rmI6 gene (Dawne Vernon, unpubl.) tinction rate, lower spéciation rate, or both. This Estimating ages q/'Polytoma clades. T h e loss of pho­ disadvantage could be expocted on the basis that the tosynthesis that gave rise to the P. uvella clade must heterotrophs are more limited in the range of en­ have occurred after the common ancestor of the P. vironments they can occupy than are their photo- uvella clade and C. humieola, but before the last com­ trophic congeners, which are also facultative het­ mon ancestor of all members of the P. uvella clade. erotrophs. They may also be at a disadvantage in The first px>int can be estimated from the minimum environments rich in organic nutrients where both divergence between a member of the P. uvella clade can grow. Normal photosynthetic strains of both andC. humieola, which is approximately 1% (18 base Chlamydomonas reinhardtii and C. humieola show high­ substitutions/1775). The second point can be esti­ er growth rates with mixotrophic growth, where mated from the maximum divergence between two they have both sunlight and acetate as carbon and members of the?, uvella clade, which is 0.17%. Wil­ energy sources, than with strictly heterotrophic son et al. (1987) presented evidence indicating that growth (on acetate in the dark) (Boynton et al. 1972, the small-subunit rRNA gene evolves at an approx­ Laliberte and de la Noue 1993). Similarly, photo­ imately constant rate of lO"'" base pair substitutions synthetic C. reinhardtii grow faster under mixo­ par site pier year in organisms ranging from bacteria trophic conditions than do nonphotosynthetic mu­ to vertebrates. This would place the loss of photo­ tants that are obligately heterotrophic. Only in the synthesis in the lineage leading to the P. uvella clade dark do some mutants grow at nearly the same rate somewhere between 50 and 8.5 mya. For 62-27, we as the wild typje (Boynton et al. 1972). A higher can only estimate the maximum time since the loss extinction rate and/or lower spieciation rate of non­ of photosynthesis, because this nonphotosynthetic photosynthetic lineages is an attractive explanation lineage has only one member. The average diver­ for the failure o f nonphotosynthetic spiecies o f Chla- gence between 62-27 and members of the C. humi- mydomonadaceae to replace the photosynthetic cola-P. uvella clade is approximately 3.1%, which forms. gives us a maximum divergence time o f roughly 155 If heterotrophic mutants frequently gave rise to million years between 62-27 and its nearest green heterotrophic spiecies, we would exp>ect to see many relatives. Photosynthesis could have been lost at any independent origins of Polytoma, not just two. And time since then. if there were no species-level selection favoring pho­ tosynthetic over heterotrophic species, we would ex­ pect to see more heterotrophic lineages branching CONCLUSIONS from deep within the inclusive Chlamydomonas clade, Our data show only two origins for the 13 Polytoma some of which might show substantial divergence strains we examined. One of these gave rise to the within the lineage. However, conclusive evidence P. uvella clade, which may represent a single spiecies. for or against selection at the individual and spxcies It may have arisen recently, as indicated by the lim­ level would require the phylogenetic analysis of a ited genetic divergence within the clone. Neverthe­ much larger sample of sequences, preferably from less, it spread widely across both continents from a random sample of recent isolates of photosynthetic which samples have been taken. The example of P. and nonphotosynthetic species, preferably coupled uvella shows that a heterotrophic clade can be very with breeding analysis to identify biological species successful for at least several million years. This con­ and competition experiments under as nearly nat­ clusion assumes that the collection sites in Table 1 ural conditions as pessible. are correct, but some may be erroneous due to con­ The earliest possible origin of the P. uvella het­ tamination or other mistakes in the long mainte­ erotrophic clade, about 50 mya, falls close to the nance o f the strains in culture. T h e second Polytoma time of the K.T (Cretaceous-Tertiary) boundary. clade is represented by a single strain (62-27). It The time of the KT boundary coincides with the might have arisen more recently and had less time time of mass extinctions, probably brought about to spread, or it might be the remnant of an ancient by an extraterrestrial impact (Alvarez et al. 1980).

15 MOLECULAR PHYLOGENETICS OF POLYTOMA

The large quantities of dust placed in the atmo­ Felsenstein.J. 1989. Phylogeny Inference Package, Version 3.2. sphere by such an event might significantly reduce Cladistics 5:164—6. 1993. P H YU P (Phylogeny Infirtnct Pachogt), Version 3.5p. the available sunlight. It is interesting to speculate Distributed by the author. Department of Genetics, Univer­ that a nonphotosynthetic mutant might be least dis­ sity of W ashington. 8eattle. advantaged relative to its green relatives and most Femholm, B., Bremer, K. tjom vall, H. 1989. The Hierarchy of likely to become establish^ as an independent lin­ Life. Molecules and Morphology in Phylogenetie Analysis. In Pro­ ceedings from Nobel 8ymposium 70. Excerpta Medica, Am­ eage, under these conditions. Genes from more sterdam, 499 pp. strains of P. uvella would have to be sequenced in Gilbert. D. G. 1992. SeqApp, a Biological Sequence Editor and Anal- order to test this hypothesis by accurately dating the ysis Program for Macintosh Computers. Published electronically origin of this nonphotosynthetic lineage. on the Internet, available via gopher or anonymous ftp to In addition to illuminating the evolutionary his­ ftp.bio.indiana.edu. Gordon, J., Rumpf, R„ 8hank, 8. L., Vernon, D. Ic Birky, C. W., tory of nonphotosynthetic algae, our data provide Jr. 1995. Sequences of the rm lS genes of Chlamydomonas the necessary historical background for studies of humieola and C. dysosmos are identical, in agreement with their the effects of the loss of photosynthesis on the mo­ combination in the species C. applanata (Chlorophyta).]. Phy- lecular evolution of plastid genes. These studies show col. 31:312-3. Gowans, C. 8. 1963. The conspecificity of Chlamydomonas euga- that the sequence evolution of several plastid genes metot and Chlamydomonas momusii: an experimental ap­ is accelerated in P. uvella 964 but not in Polyloma proach. Phycalogia 3:37-44. strain 62-27 (Dawne Vernon, unpubl.). Accelerated Gunderson, J. H., Elwood, H., Ingold, A., Kindle, K. Ic 8ogin, evolutionary rates also have been reported for plas­ M. 1987. Phylogenetic relationships between chlorophytes, tid rRNA and ribosomal protein genes in the non­ chrysophytes and oomycetes. Proc. Nat. Acad. Sci U.S.A. 84: 5823-7. photosynthetic angiosperm Epifagus (Wolfe et al. Gutell, R. R., Weiser, B., Woese, C. R. Ic NoUer, H. F. 1985. 1992a). The lack of detectable acceleration in strain Comparative anatomy of 16-S-like ribosomal RNA.Prvg. NucL 62-27 suggests that photosynthesis may have been Acids Res. MoL BioL 32:155-216. lost more recently in this lineage than in the P. uvella Harris, £. H. 1989. The Chlamydomonas Sourcebook. Academic lineage. Press, New York, 780 pp. Higgins, D. G., Bleasby, A. J. Ic Fuchs, R. 1992. Clustal V: im­ proved software for multiple sequence alignment. CABIOS 8:189-91. We thank Mark A. Buchheim for providing us with his rRNA Higgins, D. G. Ic 8harp, P. M. 1988. CLU8TAL: a package for sequence data: Paul Fuersi and his collaborators for primers; and performing multiple sequence alignment on a microcom­ Mark A. Buchheim, Russell L. Chapman, Nicholas W. Glllham. puter. Gene 73:237-44. and Jeffrey A. Palmer for helpful suggestions with the manu­ 1989. Fast and sensitive multiple sequence alignment on script. The Ribosome Data Base was an invaluable source of a microcomputer. CABIOS 5:151-3. information. This research was supported by NSF research grant Hillis, D. M., Huelsenbeck, J. P., t Cunningham, C. W. 1994. BSR-9107069 and an REU supplement. Authors may be con­ Application and accuracy of molecular phylogenies. Science tacted by e-mail as follows: C. W. Birky, Jr.: [email protected]; (Wash. D.C) 264:671-7. R. Rumpf: [email protected]; 0. Schreiben fswa/s-djchreiber/ Hillis, D. M. Ic Moritz, C., Eds. 1990. Molecular Systematics. 8in- [email protected]; and D. Vernon: vernon.l6@ auer Associates, 8underland, Massachusetts, 588 pp. osu.edu. Huang, X. 1992. A contig assembly program based on sensitive detection of fragment overlaps. Genomics 14:18-25. Alvarez, L. W., Alvarez, W., Asaro, F. Ic Michel, H. V. 1980. Huss, V. A. Ic Sogin, M. L. 1989. Primary structure of the Extraterrestrial cause for the Cretaceous-Tertiary extinc­ Chlorella vulgaris small subunit ribosomal RNA coding re­ tion. Science (Wash. D.C.) 208:1095-108. gion. Nucl. Adds Res. 17:1255. Boynton, J. E., Gillham, N. W. & Chabot, J. F. 1972. Chloroplast Jupe, E. R., Chapman, R. L. & Zimmer, E. A. 1988. Nuclear ribosome deficient mutants in the green alga Chlamydomonas RNA genes and algal phylogeny—the Chlamydomonas ex­ reinhardi and the question of chloroplast ribosome function. ample. BioSystems 21:223-30. J. CellSci 10:267-305. Laliberte, G. & de la Noue, J. 1993. Auto-, hetero-, and mixo­ Buchheim, M. A. Ic Chapman, R. L. 1991. Phylogeny of the trophic growth of Chlamydomonas humieola (Chlorophyceae) colonial green flagellates: a study of 188 and 268 rRNA on acetate./ PhycoL 29:612-20. sequence data. BioSystems 25:85-100. Lang, N.J. 1963. Electron-microscopic demonstration of plastids 1992. Phylogeny of Carteria (Chlorophyceae) inferred m Polytoma. J. PrototooL 10:333-9. from molecular and organismal data./ PhycoL 28:363-74. Larson, A., Kirk. M .M .jc Kirk. D.L. 1992. Molecular phylogeny Buchheim, M. A., Turm el. M., Zimmer, E. A. & Chapman, R. L. of the volvocine flagellates. MoL BioL EvoL 9:85-105. 1990. Phylogeny of Chlamydomonas (Chlorophyta) based on Maidak, B. L., Larsen, N„ McCaughey, M. J., Overbeek, R., cladistic analysis of nuclear 188 rRNA sequence d ata./ Phy­ Olsen, G.J., Fogel, K., Blandy, J. Ic Woese, C. R. 1994. T he coL 26:689-99. Ribosomal Database Project. NucL Acids Res. 22:3485-7. Chapman, R. L.& Bucheim, M. A. 1991. Ribosomal RNA gene Mankin, A. 8., 8kryabin, 1. G. & Rubtsov, P. M. 1986. Identi­ sequences: analysis and significance in the phylogeny and fication of the additional nucleotides in the primary structure taxonomy of green algae. Cn’l. Rev. Plant ScL 10:343-68. of yeast 188 rRNA. 44:143-5. Eckenrode, V. K., Arnold. J. Ic Meagher, R. B. 1985. Compar­ Miyamoto, M. M. Ic Cracraft. J., Eds. 1991. Phylogenetic Analysis ison of the nucleotide sequence of soybean 188 rRNA with of DNA Sequences. New York, Oxford University Press, 358 the sequences of other small-subunit rRN As./ Mol. EvoL 21 ; PP 259-69. Naim, C. J. 1c Perl, R. J. 1988. The complete nucleotide se­ Ettl, H. 1976. Die Cattung Chlamydomonas Ehrenberg. Brih. quence of the small-subunit ribosomal RNA coding region Nova Htdtvigia 49:1-1122. for the cycad Zamia pumUa: phylogenetic implications./ MoL Ettl, H. Ic 8chl6sser, U. G. 1992. Towards a revision of the EvoL 27:133-41. systematks of the genus Chlamydomonas (Chlorophyta). I. Olsen, G. J.. Matsuda, H., Hagstrom, R. Ic Overbeek, R. 1994. Chlamydomonas applanata Pringsheim. Bot Xrto 105:323-30. fastDNAmI: a tool for construction of phylogenetic trees of

16 ROBERT RUMPF ET AL.

ONA lequences using maximum likelihood. CompuL AppL Stewart, C-B. 1993. The powers and pit&lls of parsimony. Nature BioscL 10:41-8. (Lond.) 361:603-7. Pringsheim, E. G. 1965. Farilau Algm. Gustav Fischer Verlag, Swoffbrd, D. L. 1993. PAUP: Phylogmthc Analysis Using Parsi­ Stuttgart, 471 pp. mony, Version 3.5p. Computer program distributed by the Rausch, H., Larsen, N. & Schmitt, R. 1989. Phylogenetic rela­ Illinois Natural History Survey, Champaign, Illinois. tionships oF the green alga Ve/vox cartni deduced from small- Takaiwa, P., Oona, K. Ic Sugiura, M. 1984. The complete nu­ subunit ribosomal RNA comparisons./. MoL EvoL 29:255- cleotide sequence o f a rice 17S rRNA gene. Nucl. Acids Rts. 65. 12:5441-8. Scherbel, G., Behn, W. So Arnold, C. G. 1974. Untersuchungen Vemon-Kipp, D., Kuhl, S. A. & Birky, C. W , Jr. 1989. Molecular zur genetischen Funktion des farblosen Plastiden von Poly­ evolution o f Polytoma, a non-green chlorophyte. In Boyer, C, loma mirum. Arch. MicroUoL 96:205-22. T., Shatuion, J. C. Ic Hardison, R. C. [Eds.] Physiology, Bio- Schlosser, U. G. 1984. Species-specific sporangium autolysins dumistty, and Centtia of Nongum Plastids. American Society (cell-wall-dissolving enzymes) in the genus Chlamydomonas. of Plant Physiologists, Rockville, Maryland, pp. 284-6. In Irvine, D. E. G. & John, D. [Eds.] SysUmatics of Iht C u m Wilcox, L. W., Lewis, L. A., Fuerst, P. A. Ic Floyd, G. L. 1992. Alga*. Cambridge University Press, Cambridge, pp. 409-18. Group 1 introns within the nuclear-encodôl small-subunit Siemeister, G. & Hachtel, W. 1990. Structure and expression of rRNA gene of three green algae. MoL BioL EvoL 9:1103-18. a gene encoding the large subunit of ribulose-1,5-bisphos- Wilson, A. C., Ochman, H. k Prager, E. M. 1987. Molecular plute carboxylase (ricL) in the colourless euglenoid flagellate time scale for evolution. TIG 3:241-7. Astasia longa. Plant MoL BioL 14:825-53. Wolfe, K. H., Morden, C. W., Ems, S. C. k Palmer, J. D. 1992a. Siu, C-H., Chiang, K. S. & Swift, H. 1975a. Characterization of Rapid evolution of the plastid translational apparatus in a cytoplasmic and nuclear genomes in the colorless alga Poly­ nonphotosynthetic plant: loss or accelerated sequence evo­ toma. V . Molecular structure and heterogeneity of leucoplast lution of tRNA and ribosomal protein genes. /. MoL EvoL D N A ./. MoL BioL 98:369-91. 35:304-7. Siu, C-H., Swift, H. & Chiang, K. S. I975h. Characterization of Wolfe. K. H., Morden, C. W. k Palmer, J. D. 1992b. Function cytoplasmic and nuclear genomes in the colorless alga Poly­ and evolution of a minimal plastid genome from a nonpho­ toma. 1. Ultrastructureal analysis of organelles. /. Coll. BioL tosynthetic parasitic plant. Proc. N at Acad. ScL U.S.A. 89: 69:362-70. 10648-52. 1975c. Characterization o f cytoplasmic and nuclear ge­ nomes in the colorless alga Polytoma. II. General character­ ization of organelle nucleic acids./. OIL BioL 69:371-382.

17 CHAPTER 3

ALLELIC SEQUENCE DIVERGENCE, REPRODUCTION.

AND PLOIDY IN ACANTHAMOEBA

Introduction

Acanthamoeba is a genus of ubiquitous free-living amoebae which are the causative agent of Acanthamoeba keratitis, a potentially vision-threatening invasion of the cornea, and granulomatous amebic encephalitis, a potentially fatal invasion of the central nervous sytem (Ma et al. 1990). It is believed that

Acanthamoeba reproduces exclusively asexually, primarily because there is no convincing evidence for genetic recombination (Akins, 1981); however, the observation of identical mitochondrial 168 genes in association with differing nuclear 188 genes suggests that reproduction in Acanthamoeba castellanii may not be entirely clonal (Ledee 1995). It is possible that Acanthamoeba has rare or cryptic sex; for example, exchange of genetic information between individuals may occur only every hundred or every thousand generations, or in a manner which we are unable to detect. The method of reproduction affects the etiology of the diseases caused by an infectious parasite; an asexual organism is an excellent rapid colonizer, whereas sexual reproduction of the infectious agent faciiitates the quick and efficient dissemination of drug resistance genes throughout the population. Consequently it is important to

18 know the mode of reproduction for the prognosis, treatment, and epidemiology of the disease.

Another unanswered question in Acanthamoeba genetics is that of ploidy. M easurement of nuclear DNA in A. castellanii Neff suggests a total nuclear DNA content at log phase of 10^ bp (King, 1980). Kinetic complexity measurements suggest a haploid genome size of 4-5 x 10^ bp (Bohnert &

Heermann, 1974), whereas pulse field electrophoresis has suggested that the genome size is on the order of 2.3-3.S x 10^ bp (Rimm et al., 1988). Based on these data, the ploidy may range anywhere from 10 to 22.

In order to address the questions of sexuality and ploidy in

Acanthamoeba, I used a technique pioneered by Dr. Matthew Meselson and theory developed by Birky (1996) that uses allelic sequence divergence (ASD) to identify ancient asexual lineages. Allelic sequence divergence refers to the sequence differences among alleles of a given gene within an individual. In organisms that reproduce sexually, the alleles of a gene are shared among the members of the species and random drift eliminates most mutations one at a time. In an asexual species, however, the different copies of a gene within a lineage will accumulate different mutations from the moment that sexual reproduction is lost; thus in an asexual lineage the amount of ASD can be much higher than that found in a sexual species. This has been seen in two species of the asexual bdelloid rotifers, which have ASD values ranging from 10% to

45%, with an average of 29%, in 4 genes from 3 species (Meselson, personal communication). In contrast, in sexual species ASD ranges from 0% to 5.7% with an average of less than 1% (Avise, 1994; Moriyama and Powell, 1996).

19 Another interesting result of the loss of sexual reproduction is that the structure of phylogenetic trees can differ depending on which specific alleles

are used to represent each taxonomic unit. In particular, two alleles in one individual or clone may differ more from each other than either does to an allele

in another strain or species (see Figure 3); this was also seen in the bdelloid

rotifers (Meselson, unpublished). By constructing a phylogenetic tree of allele

sequences between two or more strains of an organism, it is possible to test

whether or not the species are asexual by analyzing the resulting tree topology. Allelic sequence divergence can be reduced or eliminated by any of

several phenomena, collectively called allelic sequence convergence; these are (1) mitotic crossing-over and gene conversion; (2) a ploidy cycle; or (3) rare

sex (in which case the description of the species as asexual is a matter of

semantics) (Birky, 1996).

If two homologous chromosomes have a crossover during G2, 50%

of the time the loci distal to the point of crossover will be homozygous after cell division. If two homologs undergo mitotic gene conversion during G1, the

region involved in the conversion will become homozygous unless the event is reciprocal; if it occurs during G2 half of the progeny will be homozygous. In any

of these cases ASD will be reduced in segments of chromosomes. Only small

segments on the order of a few kilobases will be involved in a single gene

conversion event, while mitotic recombination can affect segm ents as large as

an entire chromosome arm. Mechanisms by which the ploidy of an organism is

decreased and subsequently restored (ploidy cycle) have been described (Kondrashov 1994). A ploidy cycle will reduce ASD in each chromosome that is

involved; for example if ploidy is reduced to one, ASD will be reduced to zero and will begin to accumulate again after the ploidy is increased. If the ploidy

20 (a) 13 2 4

Spéciation Event

M r Loss of Sex

(b)

Figure 3. Diagramatic representation of the consequences of the loss of sexual reproduction.

(a) A phylogenetic tree representing a sexual lineage which has speciated (left) and a lineage which has lost the ability to reproduce sexually (right). The alleles of the gene of interest begin to diverge following the loss of sex, and the lineage speciates; alleles 1 and 3 are in one species; alleles 2 and 4 in the second. By tracing backwards to the coalescent of the individual alleles, it can be seen that alleles within either species are less closely related to one another than they are to alleles in the other species.

(b) Phylogenetic tree from (a) showing allelic relationships in the asexual lineage.

21 were cycled via aneuploid intermediates, convergence would affect all loci on a

chromosome to the same extent, and different chromosomes would have different levels of ASD.

If sequence convergence is frequent, measurement of sequence divergence could lead to an asexual species being mistaken for a sexually

reproducing one. This effect can be partially countered by sequencing the

alleles of several genes; only a ploidy cycle affecting all chromosomes at the

same time will reduce the ASD to the same extent in all genes in the organism. Selecting a target gene is crucial for the measurement of ASD.

Detrimental mutations will accumulate more slowly than neutral mutations and

reduce the observed value of ASD. This problem can be largely avoided by

using a protein coding gene and concentrating on divergence at the third codon

position, where most mutations are neutral or nearly so. Additionally, the gene

must be unique and not a member of a multi-gene family; otherwise it is

possible that an allele of a related gene might be mistakenly identified as an allele of the gene of interest. This would inflate the resulting ASD values and

could lead to an unfounded conclusion of asexuality in the species in question.

We selected the TATA-binding protein (TBP) gene because it is a protein

coding gene for which the sequence of an allele from Acanthamoeba castellanii

was available from GenBank,and Southern hybridization indicates that TBP is a

single-copy gene in Acanthamoeba casfe//an/7 Neff (Wong et al. 1992).

Materials and Methods Strains and DNA Acquisition

Purified whole-cell DNA from Acanthamoeba casteiianii strain Neff and

Acanthamoeba griffini strain S7 was obtained from Jill Schreoder-Diedrich in the lab of Dr. Tom Byers at The Ohio State University.

22 PCR Amplification

Non-degenerate primers for the 5’ and 3’ ends of the GenBank A. castellanii TBP sequence were designated ACTFIID5’ (sequence: gga tcc cat ctt cga cca gc) and ACTFIID3’ (sequence: aac tga tea get gat gat gt) respectively. Initial PCR amplification using nondegenerate primers resulted in clones which, when sequenced, were identical. To ensure that all possible alleles of TBP were amplified, including those which might have diverged significantly from the GenBank TBP sequence of Acanthamoeba casteiianii Neff, degenerate primers were used for all subsequent PCR amplifications.

Degenerate primers were designed using an alignment of TBP genes from the organisms listed in Table 4. Conserved regions of the gene were chosen for primer locations (Figure 4; see also Figure 5). Degenerate primers for the 5’ and 3’ ends of the amplified region of TBP were designated dACTFIIDS’

(sequence: cac caa gca ccc xtc xgg xa) and dACTFIID3’ (sequence: xcg xac xtt xgc xcc c) respectively. PCR amplification used one pi of DNA (-1 pg/pl) in a PCR reaction mix containing 50 pmol of each primer, 1.25 mM concentrations of dATP, dCTP, dGTP, and dTTP, 10 pi 10XTAQ buffer containing 15mM magnesium (Perkin-Elmer Cetus), 10 units TAQ polymerase (Life

Technologies), and 10 pi triple-distilled water. Amplifications involving degenerate primers differed only in using 1000 pmol of primer. Amplifications used a cycle of 95° C for 4 minutes, 55° C for 1 minute, and 72° C for 1 minute for the first cycle, and 95° C for 1 minute, 55° C for 1 minute, and 72° C for 1 minute for an additional 29 cycles. The degenerate amplification yielded a smear in all lanes. The amplification products of approximately 700 to 800 bp in size were band-isolated, purified by the QIAquick PCR purification protocol

(Qiagen), and used as the template for a second degenerate amplification,

23 Organism Genbank Accession Number

Saccharomyces cerevisiae M27135

Schizoaccharomyces pombe X53415

Dictyostelium discoideum M64861

Triticum aestivum X59874

Acetabularia cliftonii Z28644

Acanthamoeba castellanii M93340

Table 4. Organisms used in TBP alignment for primer design.

24 . SA GGA G^-'- -"G^Jj^" 1aA 0 Scerevisiae | TT - • - - C| CB- - -“ )@TCG Spombe cc - - t a c a t c Ha Ddiscoidiiun c c g b g g c g g t g g a - - g g t g SO g I GAB Taestivum ...... 0 Acliftonii t t a BDc t a c g g a | A0TGTcBI|c AGAGC@GGG Acastellanii

I t t^P ! I I I I. ■ 85 --TTGATCG- aSmHCC AGAHA.''- Scerevisiae c — - -C T T - TA T- - - - 0 g A T 0 - - - A 0TG TTC - Spombe ...... Ddiscoidium Bn G@ B g C .Q...... GGG|^0GGGG- ...... Taestivum A B g T Acliftonii CAG0GCGACC GGTGGGGAGG GGG03gG0CC CAG0TAGGGG Acastellanii

■ ■ .10^ ■ ■ ■ - H5 . ■ . 135 ...... 145 G0AAAC ----- GAAT H G ...... A GATGGTAMESA Scerevisiae - - - G HT ------G TTGTGGG^^gA Spombe - T ^ 0 T G ...... ^ - - Ddiscoidium iGGG GT^QtGGGAr - ...... GAG ...... 0 0 G Taestivum ...... AGTG ...... Q ...... Acliftonii GACTTCATGA CATC TcBBHacaGC 0CTGGTGGCA GGGTCGT00G Acastellanii

155. . . . .195 AACGAGGAAl TAAA AAGA - HG Scerevisiae TGCCAATAA CA- A AIHA-G AC GATT CCGG- gA Spombe - - Ddiscoidium GA - - GGAGGG GGT g SB g TGA B 3 g 0 A G G0G 0- - - Taestivum ...... Acliftonii CCTTCAC AGTaŒgTGA GCCCGcSc0A TcSaStGCTG CCAGcSaC0A Acastellanii

TGAAAA Scerevisiae TTGAAA GAA- Spombe Ddiscoidium G Taestivum Acliftonii GAGGT Acastellanii 5’ Primer (continued)

Figure 4. Alignment of TBP used in designing degenerate primers. DNA sequences were obtained from Genbank and aligned using ClustalW. Conserved regions indicated by the brackets at the 5’ and 3' ends of the gene were used as a basis for designing degenerate primers.

25 Figure 4 (continued)

c a c TgCAAAA Scerevisiae CCC tR c a a a a Spombe CGC TgCAAAA AACAGA A Ddiscoidium ce TgCA^AA Taestivum AA C T 5ÎA a a a Acliftonii B t cT v r A A A A Acastellanii

... 315 . . 335 . . . . t.Pc TAC A TOC GAA TaÏjAACC Scerevisiae c 9 c TAC A T(.C (^AA T ACAACC Spombe CAA Ta |j A a [j(. Ddiscoidium (.A? TAC A ACC Taestivum (■A? TAC A ACC Acliftonii g a P t a c a a c c Acastellanii 365^ A ICA TC.ScflA Scerevisiae A TllA K^aGlJA Spombe A T ||A TCjAC.A A Ddiscoidium A rCA TC.AG^A Taestivum A tQ a TG^GAA Acliftonii ATCATGAGAA Acastellanii

. . . .435 A A A ATGGTT TACMGGTGBCAAAAAGI jGA Scerevisiae AAAA TGGT T Spombe AAGA AAAA T CjGT T AAGT Ddiscoidium AA^A T G G 1 Taestivum AAAA TC^GT Acliftonii A a P a TGGT Acastellanii

.455 AAGClPocj A ÜAAAA T A Scerevisiae a a c c t S g c ; AGAAA^TA Spombe a 3SÎTTGCTG AGAAAA TA Ddiscoidium AACiCTTGCTÜ CCAC^gAA^TA Taestivum CGAAC TGCTG GcSgAAAA TA Acliftonii c g t P P g c t g ccacîPa aP ta Acastellanii

515 Ac Q g a S t TCA a Q a tQ c a a a a Scerevisiae ACGGATTTjÿjA AGA TTCA^AA Spombe ACOcAT TTQA AGA TTGAAAA Ddiscoidium aQcgaBttca AGA tS c a ^AA Taestivum ACGGAaTTQA a Qat SBaaaa Acliftonii CTC GA TTTCA AGA TTC a P a A Acastellanii

26 (continued) Figure 4 (continued)

Scerevisiae Spombe Ddiscoidium Taestivum TAGTAA Acliftonii CAACCAC Acastellanii

A A TC Scerevisiae 3 a Tl Spombe A A T l Ddiscoidium ACi(.C T Q a t c Taestivum AC.CX.gUACjCT AA Acliftonii ACicr.AnAr.c T Acastellanii

Scerevisiae Spombe Ddiscoidium Taestivum Acliftonii Acastellanii

Scerevisiae Spombe Ddiscoidium Taestivum Acliftonii Acastellanii 3’ Pnmer 775i . . ■ ■ ■ ■ . ,785i ■ ■ , Scerevisiae Spombe TTAATGM ATTAC Ddiscoidium T C T - - Taestivum AA TTTACIAAA Acliftonii Acastellanii

27 5’ Primer 3 ’ P rim er 589-609 1285-1270

0 250 500 750 1000 1250 1500 1772

_ 1 E X : n 2

TATA signal 270 - 276 PolyA signal: 1501 - 1508 J

CAAT signal lO 00 280 - 287

Figure 5. Map of the A. castellanii TBP gene. Arrows above sequence map indicate amplification primer positions. which routinely produced a distinct band of the proper size. Degenerate

amplification products from A. castellanii were 681 bases in length, as expected

based on the GenBank sequence; degenerate amplification products from A.

griffini were 795 bases in length. The size difference between the two species

is the result of additional bases in the introns in A. griffini. Cloning of PCR Products

The TBP amplification products were either cloned into pUCIS (Life

Technologies) or cloned by the TA cloning technique (Invitrogen). These clones were sequenced either by direct double stranded cycle sequencing (Life

Technologies) in our laboratory or by automated sequencing via an Applied Biosystems Model 373A version 1.2.1 automated sequencer at the Molecular

Genetics Sequencing Facility at the University of Georgia. All clones were

sequenced on both strands; sequences will be deposited into GenBank. Table

5 gives the amplification method, vector, and sequencing technique used for each clone. Southern Blotting

Whole cell DNA was digested overnight with SstEII, Xba\, Kpn\, and

HindlW (all enzymes from Life Technologies), run out on an a 0.7% agarose gel,

and transferred to a nitrocellulose membrane for Southern analysis (Maniatis,

1989). The membrane was prehybridized at 65° C for 1 hour with a

prehybridization solution (30 ml 20xSSC, 5 ml lOOx Denhardts, 5 ml 10% SDS,

and 1 ml of 10 mg/ml salmon sperm DNA, boiled for 10 minutes prior to use).

The membrane was then hybridized at 68° C for 12 hours after adding the probe solution consisting of 150ul water, full-length TBP amplification fragment from A. castellanii Neff labelled with Redi-Prime (Amersham), and 0.5 ml salmon sperm DNA, boiled for 10 minutes prior to use. After hybridization the

29 Clone * Species Cloned By: Sequenced By: V e c to r

TBP1 A. castettani Neff Standard Manual pUCIS TBP2 A. casteUanS Neff Standard Manual p u c ts TBP3 A castellanu Neff Standard Manual pUCtS TBP4 A castellans Neff Standard Manual pUC18 TBP5 A casteSanS Neff Standard Manual pUCIS TBP6 A castellans Neff Standard Manual pUCIS dTBPI A castellans Neff CloneAmp Automated pAMPIO dTBP2 A. castellanii Neff CloneAmp Automated pAMPIO dTBP3 A castellans Neff CloneAmp Automated pAMPtO dTBP4 A. castellans Neff CloneAmp Automated pAMPtO dTBPS A. castellanii Neff CloneAmp Automated pAMPIO dTBPS A. castellanii Neff CloneAmp Automated pAMPIO dTBP7 A. castellanii Neff CloneAmp Automated pAMPtO dTBPS A. castellanii Neff CloneAmp Automated pAMPIO ACgTBPI A. griffini 87 TA Automated pCRl 2.1 ACgTBP2 A. griffini 37 TA Automated pCR1 2.1 ACgTBPS A. griffini 57 TA Automated pCRI 2.1 ACgTBP4 A. griffini 87 TA Automated pCRI 2.1 ACgTBPS A. griffini 87 TA Automated pCRI 2.1 ACgTBPS A. griffini 87 TA Automated pCRI 2.1 ACgTBPT A. griffini 87 TA Automated pCRI 2.1 ACgTBPS A. griffini 87 TA Automated pCRI 2.1 ACgTBPS A. griffini 87 TA Automated pCRI 2.1 ACgTBPI0 A. griffini 87 TA Automated pCRI 2.1 ACgTBPII A. griffini 87 TA Automated pCRI 2.1

Table 5. Cloning methods and vectors used in cloning TBP from A. castellanii and A. griffini. Standard cloning was performed as in Sambrook (1989). CloneAmp cloning indicates Life Technologies CloneAmp System. TA cloning was done with the invitrogen TA PCR Cloning System. pAMP and pCRI 2.1 were obtained from Life Technologies and Invitrogen respectively.

30 membrane was washed as per Sambrook (1989) and visualized via phosphoimager. Sequence Alignment and Analysis

Sequences were aligned using ClustalW (Higgens et al. 1992) on an Iris

Indigo computer and SeqApp (Gilbert 1992) on a Power Macintosh. Of the 795

bases sequenced, 472 were in exons and could be unambiguously aligned; the

remainder were in introns and, although highly conserved in the majority of

clones, could not be unambiguously aligned in all clones. Painm/ise sequence differences were determined from the alignment using the DNADIST algorithm

of Phylip 3.5 (Felsenstein 1989, 1993) and corrected for multiple hits by the Nei

2-parameter method (Tables 6 and 7). These distances were used to construct

a Neighbor-Joining distance tree (Figure 6) with the DNADIST algorithm of Phylip 3.5.

Results & Discussion

Southern blotting was performed on A. castellanii and A. griffinii to verify

that the TBP gene is unique and not a member of a multi-gene family. The

Southern blot, probed at low stringency with the TBP product amplified from A.

castellanii, showed that TBP homology was limited to a single restriction fragment for most enzymes in both species. The fact that the TBP probe from A.

castellanii hybridized to A. griffini showed that the hybridization conditions were

of sufficiently low stringency to detect sequences differing by as much as 25%. We conclude that the TBP gene is unique in the A. castellanii and A. griffinii genomes (Figure 7), in agreement with the results of Wong et al. (1992) for A.

castellanii. Consequently the different clones of TBP we identified probably

31 GB TBPl TBP2 TBPS TBP4 TBPS TBPS dTBPI dTBP2 dTBPS dTBP4 dTBPS dTBPS dTBP7 dTBPS gTBPI gTBP2 gTBP3 gTBP4 gTBPS gTBPS gTBPT gTBPS gTBPS gTBPItgTBP11

GB • 0.0015 0.0015 0.0015 0.0015 0.0015 0.0015 0.0075 0.0120 0.0075 0.0090 0.0075 0.0120 0.0105 0.0090 02453 02507 02473 0.2549 02432 02478 0.2445 02453 02624 02453 02498

TB Pl 1 • 0.0015 0.0015 0.0015 0.0015 0.0015 0.0073 0.0118 0.0073 0.0088 0.0074 0.0118 0.0103 0.0086 0.2447 02500 02467 0.2541 02426 0.2471 0.2439 02447 02615 0.2447 02491

TBP2 1 1 • 0.0000 0.0000 0.0000 0.0000 0.0058 0.0103 0.0059 0.0073 0.0059 0.0103 0.0088 0.0073 02447 02500 02467 02541 02426 02471 0.2439 02447 0.2615 0.2447 02491

TBP3 1 1 0 0.0000 0.0000 0.0000 0.0058 0.0103 0.0059 0.0073 0.0059 0.0103 0.0088 0.0073 02447 02500 02467 02541 02426 02471 0.2439 02447 02615 02447 02491

TBP4 1 1 0 0 . 0.0000 0.0000 0.0058 0.0103 0.0059 0.0073 0.0059 0.0103 0.0088 0.0073 0.2447 02500 02467 0.2541 02426 02471 0.2439 02447 02615 0.2447 02491

TBPS 1 1 0 0 0 ■ 0.0000 0.0058 0.0103 0.0059 0.0073 0.0059 0.0103 0.0088 0.0073 0.2447 02500 02467 0.2541 02426 02471 0.24% 02447 02615 0.2447 02491

TBPS 1 1 0 0 0 0 - 0.0058 0.0103 0.0059 0.0073 0.0059 0.0103 0.0068 0.0073 02447 02500 02467 0.2541 02426 02471 0.2439 02447 02615 02447 02491

dTB PI 5 5 4 4 4 4 4 0.0088 0.0059 0.0103 0.0088 0.0133 0.0188 0.0188 02508 02541 02487 0.2603 02487 02532 02500 02506 02677 02506 02512

dTB P2 9 9 8 8 8 8 8 7 - 0.0118 0.0118 0.0088 0.0088 0.0148 0.0133 0.2512 02525 02512 0.2587 02553 02516 0.2525 02512 02619 0.2491 02516

dTBPS6 6 5 5 5 5 5 5 10 - 0.0059 0.0074 0.0073 0.0103 0.0044 02512 02546 02491 ,2608 02491 02537 0.2504 02512 02682 0^12 02516

dTBP4 7 7 6 6 6 6 6 8 10 6 - 0.0044 0.0103 0.0118 0.0073 0.2512 02566 0.2512 0.2606 02491 02537 0.2504 02512 02640 0.2532 02537

dTBPS 9 9 8 8 8 8 8 10 11 8 6 - 0.0074 0.0104 0.0059 02464 02497 02464 0.2538 02464 02468 0.2476 02464 02591 02464 02468 03 dTBPS 10 10 9 9 9 9 9 11 9 6 10 7 - 0.0133 0.0044 0.2521 02513 02521 0.2596 02562 02525 0.2534 02521 02649 02480 02525 K3 dTBPT 11 11 10 10 10 10 10 12 15 10 13 11 11 - 0.0118 02529 02564 02509 0.2605 02529 02555 0.2522 02529 02680 0.2529 02534

dTBPS 7 7 6 6 6 6 6 9 11 5 7 9 6 13 - 0.2491 02525 02512 0.2587 02532 02516 0.2504 02491 02661 0.2471 02537

ACgTBPI ISO 143 143 143 143 143 143 146 147 147 147 147 148 ISO 146 • 0.0638 0.0026 0.0595 0.0079 0.0066 00026 0.0013 0.0701 0.0066 0.0053

ACgTBP2 163 146 146 146 146 146 146 148 148 149 160 149 148 152 148 47 • 0.0638 0.0308 0.0653 0.0668 0.0641 0.0638 0.0437 0.0609 0.0639

ACgTBPS 161 144 144 144 144 144 144 145 147 146 147 147 148 149 147 2 47 • 0.0595 0.0079 0.0066 0 .0 ^ 0.0013 0.0715 00066 0.0039

ACgTBP4 166 146 148 148 148 148 148 151 151 152 152 151 162 154 151 44 26 44 - 0.0663 0.0596 0.0583 0 .0 ^ 0.0579 0.0609 0.C610

ACgTBPS 159 142 142 142 142 142 142 145 149 146 146 148 150 160 148 6 48 6 48 - 0.0119 0.0066 0.0079 0.0744 0.0105 0.0092

ACgTBPS 162 145 145 145 145 145 145 148 148 149 149 148 149 152 148 6 SO 6 45 10 • 0.0053 0.0053 0.0731 0.0079 0.0079

ACgTBPT 162 145 145 145 145 145 145 148 150 149 149 150 151 152 149 5 50 5 46 8 8 - 0.0013 0.0704 0.0053 0.0063

ACgTBPS 160 143 143 143 143 143 143 146 147 147 147 147 148 150 146 1 47 1 43 6 5 4 - 0.0701 0.0052 0.0053

ACgTBPS 170 152 152 152 152 152 152 155 153 156 154 154 155 158 155 52 34 53 44 55 55 55 52 - 0.0701 0.0716

ACgTBPIO 160 143 143 143 143 143 143 146 146 147 148 147 146 ISO 145 5 45 5 45 8 7 7 4 52 • 0.0079

ACgTBPII 163 146 146 146 146 146 146 147 148 148 149 148 149 151 149 5 48 4 46 8 6 8 5 54 7 •

Table 6. Distance Matrix of Acanthamoeba TBP clones. Values above the diagonal represent distances for the entire cloned region, corrected for multiple hits via the Jukes-Cantor method. Values below the diagonal represent the absolute number of substitutions found. 0 8 TB Pl TBP2 TBPS TBP4 TBPS TBPS dTBPI dTBP2 dTBPS dTBP4 dTBPS dTBPS dTBPT dTBPS gTBPt gTBP2 gTBPS gTBPS gTBPS gTBPS gTBPT gTBPS gTBPS gTBPICgTBPII

GB . 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0064 0.0129 0.0064 0.0086 0.0086 0.0129 0.0086 0.0086 0.0850 0.0852 0.0873 0.0850 0.0826 0.0650 0.0832 0.0850 0.0897 0.0873 0.0897

TB Pl 0 • 0.0000 0.0000 0.0000 0.0000 0.0000 0.0064 0.0129 0.0064 0.0086 0.0086 0.0129 0.0086 0.0086 0.0854 0.0855 0.0877 0.0854 0.0830 0.0854 0.0835 0.0854 0.0901 0,0877 0.0901

TBP2 0 0 - 0,0000 0.0000 0.0000 0.0000 0.0064 0.0129 0.0064 0.0086 0.0086 0.0129 0.0086 0.0086 0.0854 0.0855 0.0677 0.0854 0.0830 0.0854 0.0835 0.0854 0.0901 0.0877 0.0901

TBP3 0 0 0 • 0.0000 0.0000 0.0000 0.0064 0.0129 0.0064 0.0086 0.0086 0.0129 0.0086 0.0086 0.0854 0.0855 0.0877 0.0854 0.0830 0.0854 0.0835 0.0854 0.0901 0.0877 0.0901

TBP4 0 0 0 0 0.0000 0.0000 0.0064 0.0129 0.0064 0.0086 0.0086 0.0129 0.0086 0.0086 0.0854 0.0855 0.0877 0.0854 0.0830 0.0854 0.0835 0.0854 0.0801 0.0877 0.0901

TBPS 0 0 0 0 0 0.0000 0.0064 0.0129 0.0064 0.0086 0.0086 0,0129 0.0086 0.0086 0.0854 0.0855 0.0877 0.0854 0.0830 0.0854 0.0835 0.0854 0.0901 0.0877 0.0901

TBP6 0 0 0 0 0 0 ■ 0.0064 0.0129 0.0064 0.0086 0.0086 0.0129 0.0086 0.0086 0.0854 0.0855 0.0877 0.0854 0.0830 0.0854 0.0835 0.0854 0.0901 0.0877 0.0901

dTB PI 3 3 3 3 3 3 3 • 0.0086 0.0043 0.010T 0.0108 0.0151 0.0108 0.0129 0.0901 0.0879 0.0877 0.0901 0.0877 0.0901 0.0883 0.0901 0.0849 0.0925 0.0901

dTBP2 T T 7 7 7 7 7 5 - 0.0129 0.0173 0.0108 0.0086 0,0151 0.0151 0.0903 0.0857 0.0903 0.0879 0.0952 0.0879 0.0910 0.0903 0.0927 0.0903 0.0809

dTBPS4 4 4 4 4 4 4 3 8 • 0.0043 0.0086 0.010T 0.0086 0.0064 0.0903 0.0881 0.0879 0.0903 0.0879 0.0903 0.0685 0.0903 0.0952 0.0927 0.0903

dTBP4 5 5 5 5 5 5 5 6 10 4 ■ 0.0043 0.0108 0.0108 0.0064 0.0903 0.0906 0.0903 0.0903 0.0879 0.0903 0.0685 0.0903 0.0852 0.(652 0.0927

dTBPS T T 7 7 7 7 7 8 9 6 4 - 0.0086 0.0108 0.0065 0.0859 0,0837 0.0859 0.0835 0.0883 0.0835 0.0665 0.0859 0.0883 0.0883 0.0859

dTBPST T 7 7 7 7 7 8 6 5 7 6 • 0.0129 0.0064 0.0905 0.0835 0.0905 0.0881 0.0954 0.0881 0.0912 0.0805 0.0829 0.0881 0.(605

dTBPT 6 6 6 6 6 6 6 7 10 5 a 8 6 • 0.0108 0.0907 0.0885 0.0883 0.0907 0.0907 0.0907 0.0889 0.0907 0.0856 0.0832 0.0907 CO CO dTBPS5 5 5 5 S 5 5 T 9 5 5 7 5 8 • 0.0879 0.0857 0.0903 0.0879 0.0927 0.0879 0.0885 0.0879 0.0827 0.0879 0.0927

ACgTBPI SB 38 38 38 38 38 38 40 41 41 41 41 41 42 40 - 0.0188 0.0041 0.0166 0.0124 0.0083 0.0041 0.0021 0.0208 0.0083 0.0083

ACgTBPS 39 39 39 39 39 39 39 40 40 41 42 41 39 42 40 10 - 0.0188 0.0041 0.0209 0.0209 0.0189 0.0188 0.0062 0.0166 0.0188

ACgTBPS 39 39 39 39 39 39 39 39 41 40 41 41 41 41 41 2 10 - 0.0166 0.0124 0.0083 0.0041 0.0021 0.0229 0,0083 0.0062

ACgTBP4 38 38 38 38 38 38 38 40 40 41 41 40 40 42 40 8 3 8 - 0.0251 0.0166 0.0146 0.0145 0.0062 0.0166 0.0187

ACgTBPS 37 37 37 37 37 37 37 39 43 40 40 42 43 42 42 6 11 6 12 • 0.0166 0.0104 0.0124 0.0272 0.0145 0.0145

ACgTBPS 38 38 38 38 38 38 38 40 40 41 41 40 40 42 40 4 11 4 8 8 • 0.0062 0.0062 0.0229 0.0083 0.0103

ACgTBPT 40 40 40 40 40 40 40 42 44 43 43 44 44 44 43 5 13 5 10 8 6 • 0.0021 0.0210 0.0062 0.0083

ACgTBPS 38 38 38 38 38 38 38 40 41 41 41 41 42 42 40 1 10 1 7 6 3 4 . 0.0208 0.0062 0.0063

ACgTBPS 40 40 40 40 40 40 40 42 42 43 43 41 41 44 42 to 4 11 3 13 11 13 10 • 0.0229 0.0229

ACgTBPIO 39 39 39 39 39 39 39 41 41 42 43 42 40 43 40 4 9 4 8 7 4 6 3 11 • 0.0103

ACgTBPII 40 40 40 40 40 40 40 40 41 41 42 41 41 42 42 4 10 3 9 7 5 7 4 11 5 •

Table 7. Distance Matrix of Acanthamoeba TBP clones. Values above the diagonal represent distances for only exons within the cloned region, corrected for multiple hits via the Jukes-Cantor method. Values below the diagonal represent the absolute number of substitutions found. s. cerevisae r TBPgb 70 TBPl

TBP2

TBP3

TBP4 87 TBP5

TBP6

• dTBPI

93 dTBPS dTBP4

dTBPS 100 • dTBPS 64 ■ dTBPS

I - dTBP7

dTBP2

ACgTBPI

' ACgTBPS ACgTBPS

" ACgTBPS MOO ACgTBP7

ACgTBPS

-100' ACgTBPIO ACgTBPII

ACgTBP2 1177 '— ACgTBP4 97 ■ ACgTBPS

Figure 6. Neighbor-joining tree of Acanthamoeba TBP alleles. Tree was constructed using the DNABoot (100 bootstrap replicates), DNADIst (Nei 2- parameter correction for multiple hits), and Neighbor algorithms of Phylip 3.5p. Bootstrap values at nodes represent the proportion of trees supporting that particular node topology. Bootstrap values of 100 strongly support the two clades, one containing the A. castellanii clones (TBP 1-6 and dTBP 1-7) and another containing the A. griffini clones (ACgTBP 1-11).

34 Digested Genomic DNA A. castellanii A. griffini Marker + Control I 1 ÎI nil

2 kb - i

1 kb —

0.5 k b -

Figure 7. Southern blot showing copy-number of TBP genes in A. castellanii and A. griffini genomes. Genomic DNA from both species was digested with Bstell, Hindlll, Kpnl, and Xbal. Digested DNA was run out on a 0.7% agarose gel and probed with the TBP PCR product. Unlabelled probe was used as a control. Presence of a single band in three of the four A. castellanii lanes and all four of the A. griffini lanes confirms that TBP is single-copy in both species.

35 occupy the same locus on different chromosomes, and thus represent true

alleles.

It is of interest that when degenerate amplification was used to derive

cloning inserts, no duplicate clones (as determined by sequence) were obtained. This suggests that the population of different clones has not been

exhausted and that additional alleles exist.

The sequence divergence data for Acanthamoeba castellanii show that

the clones are extremely similar to one another. The minimum divergence was

0 substitutions out of 455 bases in exons; the maximum divergence was 10.

This translates to a maximum divergence of approximately 0.0219 or 2.2%.

When the introns are included, the divergence reaches a maximum of 15

substitutions out of 681 bases, which is equal to 0.022 or 2.2% divergence.

This is well within the range of 0% - 5.7% diversity found between alleles of a

sexually reproducing species (Avise 1994; Moriyama & Powell 1996), and far

short of the 10-45% divergence Meselson and Welch found in the asexual bdelloid rotifers. Of the 9 polymorphic sites within A. castellaniis TBP coding

region, 5 were in the first or second codon position, and 4 were in the third codon position.

The sequence divergence data for A. griffinii shows similar results; the

minimum divergence was 1 substitution out of 455 coding bases; the maximum divergence was 13. This translates to a maximum distance of approximately

0.0285 or 2.9% divergence. When the introns are included, the divergence reaches a maximum of 55 substitutions out of 795 bases, which is equal to 0.0691 or 6.9% divergence. Again, this is well within the range expected for sexually reproducing species. Of the 49 polymorphic sites within A. griffiniis

TBP coding region, 6 were in the first or second codon position, and 43 were in

36 the third codon position. Table 6 shows the distance calculations and raw

substition numbers for all of the cloned TBP alleles as well as the GenBank A.

castellanii TBP sequence.

Nucleotide diversity (tc) was also calculated for each set of clones, k, a

measure of polymorphism in a population, is the average proportion of

nucleotide differences per site between any two randomly chosen sequences

and is calculated using the formula

TC = X ij XjXjTCjj

where X,- and Xj are the frequencies of the ilh and yth type of DNA sequences

and TCij is the proportion of different nucleotides between these sequences (Li

and Grauer 1991). tc is usually calculated by sampling alleles from a population

rather than from an individual as in our case; if we assume that Acanthamoeba

is reproducing sexually under conditions of random mating, then the alleles within an individual will be as random a s if we had taken them from multiple

individuals in a population. We can then compare our estimates of tc to known

values of tc for sexual species to test whether or not Acanthamoeba could be

truly sexual. In cases of identical clone sequences (i.e. A. caste/Zan//TBP 2-6),

where it was unclear whether we had cloned separate alleles that happened to

be identical in sequence, or had cloned the same allele multiple times, only one

of the duplicate sequences was used in determining tc. This could lead to a

slight overestimation of the true value of tc in the population if the duplicates are

in fact separate alleles. The clones from A. castellanii have a tc of 2.3 x 10-s,

and the clones from A. griffini hawe a tc of 9.1 x 10-5. These values are lower than the maximum t c of 9.7 x 10-3 seen for several species of Drosophila 37 (Moriyama and Powell 1996) and the average k of 4.4 x 1 (H seen for humans

(Li and Sadler 1991). Since k can be equated with ASD in a randomly mating population, this suggests that Acanthamoeba may be reproducing sexually at least part of the time.

An alignment made using the cloned TBP alleles from Acanthamoeba castellanii, Acanthamoeba griffini, the GenBank A. castellanii TBP sequence, and the S. cerevisiae TBP sequence is shown in Figure 8. This alignment was used to construct the neighbor-joining (distance) tree shown in Figure 6. This tree clearly separates the A. castellanii clones and the A. gr/Wn/clones into separate clades. Thus there is no indication that any allele in one species is more closely related to any allele in the other species, as would be expected if

Acanthamoeba has been reproducing strictly asexually since before the divergence of these two species and if their convergence is rare. We conclude that eWherAcanthamoeba reproduces sexually occasionally or it is undergoing frequent sequence convergence. The TBP alleles in A. griffinii fall into two clades, as shown on the tree: one containing alleles 2, 4, and 9; and the other containing the rest of the alleles. Within these clades the divergences are comparable to those seen in

A. castellanii. The branches leading to the genes in A. griffini are longer than those leading to the A. castellanii genes, suggesting that the rate of evolution may be greater in the lineage leading to A. griffinii. An increase in the rate of base pair substitution between two lineages must be due to a higher mutation rate, or a higher probability of fixation (decreased selection), or both. If mutation rates in these two species are similar, other factors might contribute to the observed difference in divergence. The effective population size of the two strains may vary; since this affects fixation of alleles, it can contribute to

38 3T AGTCATBGAG TBPgb TBPI TBP2 TBP3 TBP4 TBPS TBP6 iTBPI dFBP2 dTBPS dTBP4 dTBPS dTBP6 dTBP7 dTBPS a t H o t I :TGC TA C G T G CACC G G Q Q G 6G ACgTBPI a t D g t I E c # A CTGC TA C G T G CACC GGGiQQGG ACgTBP2 CTGC TA C G T G 3 A C C GGGGGGG ACgTBPS TA C G T G 3A C C GGGGGGG ACgTBP4 TA C G T G CACC GGGGGGG ACgTBPS CTGC TA C G T G CACC GGGGGGG ACgTBPô CTGC TA C G T G CACC GGGGGGG ACgTBPJ CTGC TA C G T G CACC GGGGGGG ACgTBPS 3TGCGGTA CNTG CACC G G G G G C fl ACgTBP9 :TGCgGTA CGTG CACC GGGGGGG ACgTBPlO :TG c B g TA C G T G CACC GGGGGGG ACgTBPU

I I A (,. [ C TBPgb UjUU TBPI M , I L 1(1 TBP2 I ( j T (. r u(, TBPS T (. r c: r c c TBP4 TACTL TiiTLT t'-C TBPS TTACrc T(jTC I C I TBP6 T T A C T C T G T L T Ll dTBPI T T AC rc rCjTC Î ('( dTBPl f T AC. T(, dTBPS dTBP4 dTBPS dTBP6 dTBP7 dTBPS AATTTGGGAA FGGCCA AA GGAGGGTGAA ACgTBPI AATTTGGGAA TGGCCAAA GGAGGGTGAA ACgTBP2 AATTTGGGAA TGGCCAAA GGAGGGTGAA ACgTBPS AATTTGGGAA FGGCCAAA GGAGGGTGAA ACgTBP4 AATTTGGGAA FGGCCAAA GGAGGGTGAA ACgTBPS AATTTGGGAA FGGCCAAA GGAGGGTGAA ACgTBPS AATTTGGGAA FGGCCAAA GGAGGGTGAA ACgTBP7 AATTTGGGAA _FGGCCAAA GGAGGGTGAA ACgTBPS B t t g g g g a t c GAAT a c a t c t I g a a t OD g a OI g I c ACgTBP9 AATTTGGGAA G gF G G C C A A A GGAGGGTGAA ACgTBPlO AATTTGGGAA m GBFGGCCAAA GGAGGGTGAA ACgTBPU

(Continued)

Figure 8. Alignment of TBP Clones from A. castellanii (AcTBP1-6 and dTBPI- 7) and A. gr/Wn/(ACgTBPI-11). The Genbank sequence (AcTBPgb) is included as a reference.

39 Figure 8. (continued)

A... A AC A 5a Sj 3u

ATCC A C gTBPl TCCTTCT ATCC ACgTBPZ TCCTTCT ATCC ACgTBPJ TCCTTCT ATCC ACgTBP4 TCCTTCT ATCC ACgTBPS TCCTTCT ATCC ACgTBPô TCCTTCT ATCC ACgTBP7 iT C C T T C T ATCC ACgTBPS c c D c t B: IVTTCCT TCTTGAT | a | a ACgTBPÇ TCCTTCT ATCC ACgTBPlO Fi [TCCTTCT ATCC A C gTBPU .155 J6 5 S î 4'- ' CACl QTAC TBPgb T T T l : l il TBPl T T T I ) ' 1 i ;l TBP2 TBP3 TBP4 TBP5 TBP6 dTBPl dTBP2 dTBPJ efTBP4 dTBPS dTBPô dTBP7 dTBPS g r C T A G G T T G C A A G C T C ACCT A C gTBPl H t c t a GGT T G C A A G C T C ACCT A C gTB P l D t c t a GGT T G C A A G C T C ACCT ACgTBPJ H t c t a GGT T G C A A G C T C ACCT ACgTBP4 H t c t a GGT T G C A A G C T C ACCT ACgTBPS R t c t a GGT T G C A A G C T C ACCT ACgTBPô H t c t a GGT T G C A A G C T C ACCT ACgTBP7 B t c t a GGT T G C A A G C T C ACCT ACgTBPS G G g g A A T GGCTGCAAGC ACgTBPÇ T G C A A G C T C A C C T ACgTBPlO TGCAAGCTC ACCT A C gTBPU

(Continued) 40 Figure 8. (continued)

.^2#. TTTTQ g t c a B c /ATQA A G A C G TBPgb TBPl TBP2 TBP3 TBP4 TBPS TBP6 dTBPl dTBP2 dTBPJ dTBP4 dTBPS dTBPô dTBP7 dTBPS SC O TA ACGCCGAG CGTTTCG T G ACgTBPl 3C G T A ACGCCGAG CGTTTCG T G ACgTBP2 GCGTA ACGCCGAG CGTTTCG T G ACgTBPJ SC O TA ACGCCGAG CGTTTCG T G ACgTBP4 SC O TA ACGCCGAG CGTTTCG T G ACgTBPS 3C G T A ACGCCGAG C G T T if lG T G ACgTBPô 3C G T A ACGCCGAG CGTTTCG T G ACgTBP7 SC O TA ACGCCG^ CGTTTCG T G ACgTBPS TC G C C ttgca H* G TA A I tac B^c C T ACgTBPÇ CGTA A CG CCG A G f CGTTTCG T G ACgTBPlO CGTA ACGCCGAG CGTTTCG T G A C gTB Pll

■ « » 27S 1 t . . _ LI .^ .25-1-1w A CGC T C T T l GGG||AAGAT(G ATG G T s g c I c c a a g Q g TBPgb TBPl TBP2 TBPJ TBP4 TBPS TBP6 dTBPl dTBP2 dTBPJ dTBP4 dTBPS dTBPô (TTBP7 dTBPS IT G A T T A T GAGGATTCGC AAGH CGACCGCCCT CATCTOTGCA ACgTBPl T C A T T A T GAGGATTCGC a a g B c g a c t g c c c t c a t c t b t g c B ACgTBP2 T G A T T A T GAGGATTCGC AAgB CGACCGCCCT CATCTHTGCA ACgTBPJ T G A T T A T GAGGATTCGC AAGB CGACTGCCCT CATCTnTGC] ACgTBP4 IT G A TTA T GAGGATTCGC AAgB CGACCGCCCT CATCTDTGCA ACgTBPS T G A T T A T GAGGATTCGC AAgB CGACCGCCCT CATC tB t GCA ACgTBPô T G A T T A T GAGGATTCGC AAgB CGACCGCCCT CATCTBTGCA ACgTBP7 lîG A T T A T GAGG^CGC ,__AAgB CGACCGCCCT CATCTBTGCA ACgTBPS T T T C G I tg BBtI aQ AGGATT CGC| a GCC c B a B a c I a c B g | ACgTBPÇ T G A T T A T GAGGATTCGC AAGB CGACCGCCCT CATCTOTGCA ACgTBPlO TGATTAT GAGGATTCGC CAAg B CGACCGCCCT CATCTBTGCA A C gTB Pll

(Continued) 41 Figure 8. (continued)

TCTCQTCT ATCA TBPgb G TAC TBPl G TAC TBP2 SCTG G TAC TBP3 3CTG G TAC TBP4 G TAC TBPS G TAC TBP6 tfTBPl OTBPl dTBPS dTBP4 dTBPS dTBPô dTBP7 dTBPS ÎAAG ACgTBPl 3AAG ACgTBPS GTCT AAG 3AAG ACgTBPS GTCT ÎAAG ACgTBP4 GTCT ÎAAG ACgTBPS G T C T CAC ÎAAG ACgTBPô GTCT ÎAAG ACgTBP? GTCT ÎAAG ACgTBPS AGATGGTCTG ACgTBP9 GGGTCflCAAG ACgTBPlO g g g t g B c a a a AC gTB P ll

— m -- t c a a g a t t c B GAACATCGTC TBPgb r c G TBPl TBP2 TBPS TBP4 TBPS TBPô ATCA dTBPl TCA dTBPS ATCA dTBPS ATCA dTBP4 TCA dTBPS ATCA dTBPô ATCA dTBPS ATCA dTBPS ACgTBPl GCAT ACgTBPS G CA T ACgTBPS ACgTBP4 CCAT ACgTBPS GCAT ACgTBPô GCAT ACgTBPS __ GG GCAT ACgTBPS t c g | g AGGAAGTATG (\TCAT ACgTBPÇ » GG GCAT mcG ACgTBPlO GG GCAT U g C G A C gTB Pll

(Continued) 42 Figure 8. (continued)

GGCTCGTGCG CTCGAGG TBPgb TCA GB a CATC GATT TBPl CATC GATT TBP2 GATT TBP3 CATC GATT TBP4 CATC GATT TBPS CATC GATT TBPÔ dTBPl dTBP2 dTBPS dTBP4 dTBPS dTBPô dTBP7 dTBPS AGTTC ACgTBPl AGTTC ACgTBPl AGTTC CGAC ACgTBPS AGTTC ACgTBP4 AGTTC CGAC ACgTBPS AGTTC AGACGGTT ACgTBPô AGTTC ACgTBP7 AGTTC TCgAGA CGGTT ACgTBPS CCAAGTTCCT ATTCAGAACA ACgTBP9 AGTTC TC CGAC ACgTBPlO AGTTC TC CGAC ACgTBPU

■ I I 1 ■ 1 t ■ ■ TACGTCCTCC ATCj T C T | TBPgb GCTGAC a g I ATf TBPl QC T GAC ATI TBPl GC TGAC AT TBPS GC TGAC AT TBP4 GC TGAC AT TBPS GC TGAC 1 ^ AT TBPÔ GTACGT b e l IT dTBPl GTACGT cc !t dTBPl GTACGT cc| |T dTBPS GTACGT cc T dTBP4 GTACGT cc T dTBPS GTACGT cc T dTBPô GTACGT cc| T dTBP7 GTACGT cc |t dTBPS g c c I a c E A l TG AG ACgTBPl a I TG AG ACgTBPl a I TG AG ACgTBPS a I TG AG ACgTBP4 a I TG AG ACgTBPS A l TG AG ACgTBPô a B TG AG ACgTBP7 aJ TG AG ACgTBPS 3ACG 1 & GTCTTGA GGGTCTCGJ TTH ACgTBP9 GCC t Q t G C C lA C f aP ACgTBPlO g c c t B t g c c I a c E a I ACgTBPU

(Continued)

43 Figure 8. (continued)

: a g a g g C A G C T AATC ITGCTGTG raPgâ *\CT G a I ivg TBPJ TBP2 TBP3 TBP4 TBPS TBPÔ dTBPl SO dTBP2 dTBPS dTBP4 30 dTBPS SO dTBPô T O T NTA sc dTBPl 30 dTBPS ACgTBPJ T T C T O I T [TAAO ACgTBPl OOTOTTO B g B ACgTBPS SOKCT TOTOOTOTTC [TAGN ACgTBP4 OOTOTTO ~ ACgTBPS OOTOTTO ACgTBPô GOAOOT OOTOTTO ACgTBPl ACgTBPS OATC ACgTBP9 GOAOOT OOTOTTO ACgTBPlO GOAOOT AC gTBPll

1 1 1 1111 I 11 I > I w OATGOAOAOC A O G T G O A G T g TBPgb o c a IBIc t g o B g t g o a t g t TBPl T G O A T G T TBPl T G O A T G T TBPS T G O A T G T TBP4 T G O A T G T TBPS TGOATGT TBPÔ dTBPl dTBPl dTBPS dTBP4 dTBPS dTBPô dTBPl dTBPS ACgTBPl GOTACWCUA OACA ACAA ACgTBPl ACgTBPS TGTAC* TGAAOOOOl ACgTBP4 AnGSTOO ACgTBPS AgGOTOO ACgTBPô A g G U T O O ACgTBPl 3 T D 0 T A 0 a QBg Btoo I ACgTBPS c t t o c o t c B a TAGTGTTA AAAT ACgTBP9 " 3 T A 0 AgkBTOOl ACgTBPlO 3 T A 0 AUGaTOO ACgTBPll

(Continued) 44 Figure 8. (continued)

■ * * « . ■ t TCGGGAAAGA TBPgb T TBPl T TBP2 T TBP3 T TBP4 T TBPS T TBPÔ \ dTBPJ dTBP2 dTBPS dTBP4 dTBPS dTBPô dTBP7 dTBPS Bc ATCACAAAAT GDAGDACGAI ACgTBPl AGCGCCGGCT ATCACAAAAC g H a g d a c g a ACgTBP2 ATpTggGCT ATCACAAAAT gBagBacgaI ACgTBPS c a | c g c | g g | c B t c a c a a a a 3TA G TA C G A ACgTBP4 ATCACAAAAT G dA G B A C G A l ACgTBPS ATCACAAAAT g Oag ^ cga ACgTBPô ATCACAAAAT g H a g d a c g a ACgTBPJ ATCACAAAAT g Qag Bacga I ACgTBPS a c t g c a g c g I ACACACACAC ACCTCATCACIVMIvmv ACgTBPÇ ATKT##GCT ATCACAAAAT bD a g B a c g a I ACgTBPlO a t R t D g c t ATCACAAAAT :lsQag Bacga B ACgTBPU

45 observed divergence of alleles. If A. castellanii is asexual, the time since the last convergence event (or the frequency of convergence) will affect the obsen/ed divergence. Alternately the time since the most recent sexual episode (or the frequency of sexual reproduction) may vary between the two strains, which would also contribute to the observed difference in divergence.

Further study is required before this question can be conclusively answered.

The neighbor-joining tree also points out a peculiarity within the A. griffini clones. As with the A. castellanii clones, the majority of the A. griffini clones are similar in sequence to one another and have short branch lengths separating them from one another. A. griffini clones 2,4, and 9, however, are not only set apart in a separate clade within the primary A. griffini clade, but have branch lengths and corresponding bootstrap values that distinctly separate them from one another and the other A. griffini clones. This suggests that A. griffini clones

2, 4, and 9 were not affected by a recent convergence event or events that affected the other 8 clones. Chromosomal rearrangements such as reciprocal translocations can render chromosomes immune to sequence convergence in several ways (Birky 1996):

(1 ) Chromosomal rearrangements function as crossover supressors in that they may prevent the necessary alignment of chromosomes from occuring.

(2) Improper pairing of rearranged chromosomes during meiosis is likely to yield inviable progeny.

(3) Chromosomal rearrangements may prevent ploidy reduction if the reduction causes the resulting cells to be homozygous for a deletion.

(4) If a chromosome differs sufficiently from its homolog(s), then during sexual reproduction it will effectively function as a separate chromsome,

46 ensuring transmission of its particular allele to all of its offspring and eliminate sequence convergence by inbreeding.

The observation that the overwhelming majority of substitutions in the A.

griffinii TBP clones occurs at synonymous sites suggests that the alleles are all

under selection and functional. The number of polymorphic sites in the A.

castellanii strain is too small to make any statistically significant conclusions

regarding this, although the low divergence from the GenBank sequence

suggests that all the A. castellanii alleles are probably functional. Additional

evidence supporting the functionality of these genes can be derived from the

probability of that no stop codons are introduced into the gene by substitution in

the absence of selection. There are 35 sense codons within the A. castellanii

clone dTBPS which can be changed to a stop codon (a nonsense mutation) by

the substitution of a single base (i.e. UCG to UAG), and 6 which can be

converted to a stop by the substitution of one of two bases (i.e. UAC to UAA or

UAG), The expected number of nonsense (stop) mutations can be determined by the formula:

Sip/S + 2S2|i/3

where Si is the number of codons which could be made into a stop codon by

one substitution, 82 is the number of codons which could be made into a stop

codon by one of two substitutions, and p, is the expected number of synonymous

substitutions per site in the allele in question if it became a pseudogene when it diverged. A minimum estimate of p can be obtained from the divergence

between clone dTBPS and A. griffini clone AcgTBP7. In third codon positions, which are mostly synonymous, these two clones diverge by 0.216%. From this

47 it can be calculated that 3.38 nonsense mutations should have occurred in this

clone. The probability that no nonsense mutations would occur is given by

applying the first term of the Poisson distribution to the formula for the expected

number of nonsense mutations:

e-Si(p)/3 + 2S2(p )/3

which in this case gives us 0.034 or 3.4%. The absence of stop codons in this clone is significant and strengthens the argument that these alleles are functional.

Since TBP is a single-copy gene in Acanthamoeba, we can use these data to determine the minimum ploidy number of Acanthamoeba. A. castellanii had 10 unique alleles, whereas A. griffinii had 11 alleles. This suggests that the minimum ploidy for A. casteilanii is 10, and the minimum ploidy for A griffini is

11. These estim ates are in line with other estim ates of ploidy in Acanthamoeba (Byers 1986).

Conclusions

Allelic sequence divergences among TBP alleles in A. castellanii and A. griffini are well within the range for alleles in a sexual species. My data suggest that either A. castellanii is sexual, or it is undergoing frequent sequence convergence. The fact that sexual reproduction has never been observed in

Acanthamoeba in spite of years of laboratory study suggests that the species is having sex infrequently if at all. Based on the number of unique alleles of TBP

48 cloned, the minimum ploidy of A. castellanii must be 10, and the minimum ploidy of A. griffini must be 11.

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56