THE EVOLUTION OF SELFING, INBREEDING DEPRESSION, AND POLYPLOIDY IN
THE CLAYTONIA PERFOLIATA COMPLEX (PORTULACACEAE)
By
JOSEPH HOWARD RAUSCH
A dissertation submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY School of Biological Sciences
MAY 2008 To the Faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of JOSEPH HOWARD RAUSCH find it satisfactory and recommend that it be accepted.
______Chair
______
______
______
ii ACKNOWLEDGEMENTS
I would first like to thank Washington State University and the School of Biological
Sciences for the opportunity to attend graduate school. Many thanks to my advisor Richard
Gomulkeiwicz and the members of my graduate committee, Larry Hufford, Brian Husband, and Scott Nuismer. Also, I would like to thank my first advisor, Martin Morgan.
I would like to give special thanks to Eric Roalson and Scott Nuismer for the use of laboratory facilities, and Chuck Cody for greenhouse assistance. I would like to thank my fellow graduate students for their many discussions, especially John Clark, Josh Brokaw, Matt
King, Devin Drown, Robin O’Quinn, John Schenk, K. Marlowe, and Josh Neely. I would also like to acknowledge Steve Novak, Jim Smith, Patrick McIntyre, Brian Barringer, Ken
Chambers, John Miller, Atsushi Yamauchi, and Benji Oswald.
For financial support I would like to thank Martin Morgan and Richard Gomulkeiwicz for the opportunity of research assistantships, and Mike Tatum and Jerry McGaughran for the opportunity of the USDA Forest Service SCEP program. Additional financial support was provided by the WSU Graduate School Scholar Award Program, Betty W. Higinbothan
Fellowship, and the National Science Foundation grants DEB 0128896 to Martin Morgan and
DEB 0209916 to Richard Gomulkeiwicz.
Finally, I would like to thank my wife Lindsay and my two boys Leif and Eric, for their encouragement and emotional support.
iii THE EVOLUTION OF SELFING, INBREEDING DEPRESSION, AND POLYPLOIDY IN
THE CLAYTONIA PERFOLIATA COMPLEX (PORTULACACEAE)
Abstract
By Joseph Howard Rausch, Ph.D. Washington State University May 2008
Chair: Richard Gomulkiewicz
Polyploidy, the doubling of chromosomes within a species (autopolyploidy) or chromosome doubling in conjunction with hybridization (allopolyploidy), is accepted as having a major role in plant evolution. The ubiquity of polyploidy in plants indicates that polyploidization is a common form of speciation with significant ecological and genetic consequences. The objective of this research is to investigate the evolution of polyploidy; both theoretical and empirical approaches are considered.
First, theoretical models are developed to examine the role of selfing, inbreeding depression, and population size on autopolyploid establishment in mixed cytotype populations. This research is presented in Chapter One. The Second Chapter represents a response to a technical comment of Chapter One. Results from both of these chapters show that autopolyploid establishment is facilitated by increased selfing and/or lower inbreeding depression in polyploids relative to diploids. Second, molecular and quantitative population- level approaches are used to empirically examine the relationship between polyploidy, selfing, and inbreeding depression in populations of the Claytonia perfoliata polyploid complex. This
iv is presented in Chapter Three. Results show that inbreeding depression is greatest in outcrossing populations and that ploidy has no significant effects on the level of inbreeding depression. Third, DNA-based data is used to infer phylogenetic relationships and polyploid origins among diploid and polyploidy entities in the Claytonia perfoliata polyploid complex.
This is presented in Chapter Four. Results show that both autopolyploidy and allopolyploidy are common within the Claytonia perfoliata complex and that many cytotypes have multiple independent polyploid origins.
v TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... iii
ABSTRACT ...... iv
LIST OF TABLES ...... viii
LIST OF FIGURES ...... ix
DEDICATION ...... xi
ATTRIBUTION ...... xii
CHAPTER ONE. THE EFFECT OF SELF-FERTILIZATION, INBREEDING DEPRESSION, AND
POPULATION SIZE ON AUTOPOLYPLOID ESTABLISHMENT ...... 1
ABSTRACT ...... 2
INTRODUCTION ...... 3
THE MODEL ...... 6
RESULTS ...... 10
DISCUSSION ...... 14
ACKNOWLEDGEMENTS ...... 20
LITERATURE CITED ...... 21
FIGURE LEGENDS ...... 28
CHAPTER TWO. FORMULATION OF COMPETITION BETWEEN SEXUAL AND
SELFING FORMS: A RESPONSE TO YAMAUCHI ...... 32
LITERATURE CITED ...... 37
vi CHAPTER THREE. SELF-FERTILIZATION AND INBREEDING DEPRESSION IN DIPLOID
AND POLYPLOID POPULATIONS OF THE CLAYTONIA PERFOLIATA (PORTULACACEAE)
POLYPLOID COMPLEX ...... 39
ABSTRACT ...... 40
INTRODUCTION ...... 41
METHODS ...... 47
RESULTS ...... 59
DISCUSSION ...... 64
ACKNOWLEDGEMENTS ...... 71
LITERATURE CITED ...... 72
FIGURE LEGENDS ...... 88
CHAPTER FOUR. PHYLOGENETIC RELATIONSHIPS AND POLYPLOID ORIGINS WITHIN
THE CLAYTONIA PERFOLIATA POLYPLOID COMPLEX (PORTULACACEAE) ...... 91
ABSTRACT ...... 92
INTRODUCTION ...... 93
MATERIALS AND METHODS ...... 98
RESULTS ...... 103
DISCUSSION ...... 108
TAXONOMIC TREATMENT ...... 140
ACKNOWLEDGEMENTS ...... 144
LITERATURE CITED ...... 145
FIGURE LEGENDS ...... 155
vii LIST OF TABLES
CHAPTER ONE.
Table 1. Variables, parameters, and critical values influencing
tetraploid establishment ...... 27
CHAPTER THREE.
Table 1. Taxa and populations of the Claytonia perfoliata s.l. complex
used for the inbreeding depression study ...... 79
Table 2. Mating system parameters of populations ...... 81
Table 3. Wilcoxon signed-rank tests and paired t-tests for seeds per fruit,
percent germination, percent survival, and number of flowers
per plant ...... 82
Table 4. Analyses of variance tables for relative performance of
cumulative fitness ...... 86
Table 5. Results of multiple comparison tests ...... 87
CHAPTER FOUR.
Table 1. Taxa and cytotypes of Claytonia perfoliata s.l.
and Claytonia washingtoniana ...... 153
Table 2. Revised taxonomy of Claytonia perfoliata s.l.
and Claytonia washingtoniana ...... 154
viii LIST OF FIGURES
CHAPTER ONE.
Figure 1. Frequency of tetraploids as a function of unreduced gamete
production under random mating ...... 29
Figure 2. Frequency of tetraploids as a function of unreduced gamete
production under variable relative fitness, selfing, and
inbreeding depression ...... 30
Figure 3. Mean time to tetraploid fixation due to stochastic processes
as a function of unreduced gamete production ...... 31
CHAPTER THREE.
Figure 1. Maximum likelihood tree of Claytonia perfoliata s.l. populations
used for the inbreeding depression study ...... 89
Figure 2. Bivariate plot of populations for relative performance of cumulative
fitness by primary selfing rate and inbreeding coefficient ...... 90
CHAPTER FOUR.
Figure 1. Maximum likelihood trees of matK/trnK, ITS, and combined data set,
for diploid Claytonia perfoliata s.l.; and map of diploid specimens . . . . . 159
Figure 2. Maximum likelihood tree of matK/trnK for diploid and
polyploid Claytonia perfoliata s.l...... 160
ix Figure 3. Maximum likelihood tree of ITS for diploid and polyploid
Claytonia perfoliata s.l...... 161
Figure 4. Geographic distribution of diploid and polyploid specimens
with identical sequences to diploid Claytonia perfoliata
subspecies mexicana ...... 162
Figure 5. Tetraploid and hexaploid cytotypes of Claytonia perfoliata s.s. linked
with identical sequences on maximum likelihood trees
of matK/trnK and ITS ...... 163
Figure 6. Octaploid and decaploid cytotypes of Claytonia perfoliata s.s. linked
with identical sequences on maximum likelihood trees
of matK/trnK and ITS ...... 164
Figure 7. Tetraploid and hexaploid cytotypes of Claytonia paviflora linked
with identical sequences on maximum likelihood trees
of matK/trnK and ITS ...... 165
Figure 8. Tetraploid Claytonia rubra subspecies rubra and hexaploid
Claytonia rubra subspecies depressa linked with identical sequences
on maximum likelihood trees of matK/trnK and ITS ...... 166
x DEDICATION
This dissertation is dedicated to my mother Nancy Mae (1951-2007).
xi ATTRIBUTION
For Chapters One and Two, Martin T. Morgan helped with writing and aided in the formulation of the stochastic calculations. Chapters Three and Four were written entirely by myself. I collected and analyzed data for all Chapters.
xii CHAPTER ONE
THE EFFECT OF SELF-FERTILIZATION, INBREEDING DEPRESSION, AND
POPULATION SIZE ON AUTOPOLYPLOID ESTABLISHMENT
JOSEPH H. RAUSCH AND MARTIN T. MORGAN
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236
[Formatted for Evolution, Allen Press]
1 Abstract.--- The minority cytotype exclusion principle describes how random mating between diploid and autotetraploid cytotypes hinders establishment of the rare cytotype. We present deterministic and stochastic models to ascertain how selfing, inbreeding depression, unreduced gamete production, and finite population size affect minority cytotype exclusion and the establishment of autotetraploids. Results demonstrate that higher selfing rates and lower inbreeding depression in autotetraploids facilitate establishment of autotetraploid populations. Stochastic effects due to finite population size increase the probability of polyploid establishment and decrease the mean time to tetraploid fixation. Our results extend the minority cytotype exclusion principle to include important features of plant reproduction and demonstrate that variation in mating system parameters significantly influence the conditions necessary for polyploid establishment.
2 Polyploidy is a significant force in plant evolution (Grant 1981; Stebbins 1947; Winge
1917); estimates of the number of angiosperm taxa that are of polyploid origin range from 30 to 70% (see review in Soltis et al. 2004). Autopolyploids undergo chromosome doubling within a species and thus have more that two sets of homologous chromosomes (Stebbins
1947). Autopolyploid plants were once considered extremely rare in nature (Grant 1981;
Stebbins 1950), but are now recognized as more common (Soltis et al. 2004). Various mechanisms may account for the formation of polyploids (e.g., somatic chromosome doubling of a zygote or early embryo, polyspermy, see Ramsey and Schemske 1998), but the production and union of unreduced diploid gametes (Bretagnolle and Thompson 1995; Harlan and de Wet 1975; Ramsey and Schemske 1998; Thompson and Lumaret 1992) is thought to be the major mechanism of autotetraploid formation.
Newly occurring autotetraploids are subject to ‘minority cytotype’ negative frequency- dependent selection (Levin 1975). Minority cytotypes have reduced fitness because they have fewer compatible mates available. In a random mating population and in the absence of other fitness differences, minority cytotype disadvantage eliminates tetraploids starting at frequencies less than 50% from the population. A number of factors may compensate for the minority cytotype disadvantage. Felber (1991) stressed the importance of continual unreduced gamete production by diploids as a mechanism for forming and maintaining new tetraploids at low frequency. Other factors that can compensate for the minority cytotype disadvantage include the triploid bridge (Felber and Bever 1997; Husband 2004; Ramsey and Schemske
1998), higher relative fitness of tetraploids (Felber 1991; Fowler and Levin 1984), niche separation between cytotypes (Fowler and Levin 1984; Rodriguez 1996a), prezygotic
3 isolation mechanisms (Husband and Sabara 2004), iteroparity (Rodriguez 1996b), immigration (Levin 1975), and stochastic processes (Li et al. 2004).
Self-fertilization can ameliorate the minority cytotype disadvantage by increasing the frequency of compatible matings (Levin 1975; Rodriguez 1996a; Rodriguez 1996b; Stebbins
1971). A major consequence of selfing, though, is inbreeding depression. Differences in selfing rates between polyploids and their diploid progenitors are often assumed to exist, although strong empirical evidence is lacking (Soltis and Soltis 2000). Polyploids may have lower selfing rates than diploids because of increased herkogamy (Webb and Lloyd 1986) due to the larger size of polyploid flowers (Stebbins 1971). Alternatively, polyploidy may be associated with a loss of self-incompatibility (de Nettancourt 2000; Mable 2004; Miller and
Venable 2000), and lower inbreeding depression in tetraploids (due to multiple alleles at each locus masking deleterious mutations), which should facilitate evolution of increased selfing rates (Lande and Schemske 1985). However, the relationship between inbreeding depression and chromosome doubling may be complex (Bever and Felber 1992; Charlesworth and
Charlesworth 1987; Ronfort 1999), and under certain circumstances (e.g. due to a reduction in heterotic interactions among alleles, Bennett 1976) inbreeding depression may increase with ploidy. Since differences in selfing and inbreeding depression among cytotypes are likely, understanding the formation and persistence of polyploidy is incomplete without considering these factors.
To understand further the formation and persistence of autotetraploids, we (1) develop a deterministic model to ascertain how relative fitness, selfing, inbreeding depression, and unreduced gamete production affect minority cytotype disadvantage; and (2) develop a stochastic version of the model to investigate the effects of population size on the
4 establishment of tetraploids. We show that higher selfing rates and/or lower inbreeding depression in polyploids are ways to overcome the minority cytotype disadvantage and facilitate establishment of polyploid populations. Small population size also reduces the level of unreduced gametes needed to establish polyploid populations. Our model differs from previous investigations (Baack 2005; Levin 1975; Rodriguez 1996b) by considering selfing rates and inbreeding depression which vary between cytotypes, and by considering how inbreeding depression and unreduced gamete production alter the effects of selfing. Although stochastic models of polyploid establishment account for unreduced gamete production (e.g.
Li et al. 2004), ours is the first to include the effects of selfing, inbreeding depression, and population size.
5 THE MODEL
Deterministic Calculations
We start by developing recursion equations for the frequency of diploids and tetraploids; triploids are assumed to be lethal and are not included in the model. Table 1 summarizes parameters and variables. Consider an infinite population of hermaphroditic diploid dt and tetraploid tt =1 - dt individuals with equal generation times and non-overlapping generations. Time t begins with the mating of adults and ends before mating in the next generation t + 1. Diploid individuals produce a fraction u of unreduced (2n) gametes and 1 - u reduced (haploid) gametes. We assume that 2n micro- and mega-gametophytes are produced with the same frequency, and tetraploid individuals produce only reduced (2x) gametes.
Diploids and tetraploids reproduce through selfing sd, st or outcrossing 1 - sd, 1 - st.
Offspring resulting from self-fertilization suffer inbreeding depression δd, δt. Diploids arise
2 2 2 from the union of reduced (x) gametes through outcrossing dt (1 - u) (1 - sd) or self-
2 fertilization dt (1 - u) sd (1 - δd). Tetraploids form by outcrossing through the union of two
2 2 reduced (2x) gametes from tetraploid parents (1 - dt) (1 - st) , or one reduced gamete from a tetraploid and one unreduced (2n) gamete from a diploid 2dt (1 - dt)(1 - sd)(1 - st)u, or two
2 2 2 unreduced gametes from diploid parents dt (1 - sd) u . Tetraploids also form by selfing of a
2 diploid through the union of 2n gametes dt sd u (1 - δt), or of a tetraploid through the union of reduced gametes (1 - dt) sd (1 - δt). Tetraploids have fitness (the product of tetraploid fertility and viability, relative to diploids) of w. This represents fitness effects related to adaptation to the ecological setting, rather that such factors as ability to find a mate. Recursion equations describing the change in diploid and tetraploid frequencies over a single generation are then:
6 2 2 2 2 dt +1 = {dt (1- sd ) (1- u) + dt sd (1- u) (1- äd )}/ k 2 2 = {[dt (1- sd )] + dt sd (1- äd )}(1- u) / k
2 2 2 2 2 (1) tt +1 = {(1- dt ) (1- st ) + 2dt (1- dt )(1- sd )(1- st )u + dt (1- sd ) u 2 + (1- dt )st (1- ät ) + dt sdu (1- ät )}w/ k 2 2 = {[(1- dt )(1- st ) + dt (1- sd )u] +[(1- dt )st + dt sdu ](1- ät )}w/ k
The factor k
2 2 k = {[dt (1- sd )] + dt sd (1- äd )}(1- u) 2 2 +{[(1- dt )(1- st ) + dt (1- sd )u] +[(1- dt )st + dt sdu ](1- ät )}w ensures that diploid and tetraploid frequencies sum to one. We solve the recurrence equations to determine equilibria (when dt+1 = dt) and their stability, using Mathematica (Wolfram
1996) to arrive at analytic solutions. Expressions are complicated in general, and are summarized numerically below.
We use the deterministic model to investigate how relative fitness, selfing, and inbreeding depression affect cytotype frequencies, as a function of unreduced gamete production. We focus on critical values that represent the minimum tetraploid frequency tcrit and the minimum frequency of unreduced gametes ucrit needed for tetraploids to spread to fixation in the population. Populations with the following conditions were analyzed: (1) random mating, but relative fitness differences among cytotypes, w ≠ 1; (2) equal and unequal selfing rates of cytotypes in the absence of inbreeding depression; and (3) equal and unequal inbreeding depression among cytotypes (mathematical expressions allow selfing and inbreeding depression to simultaneously differ among cytotypes, but we restrict our attention to each factor acting alone).
7 Stochastic Calculations
A stochastic version of our model investigates how population size affects the establishment of tetraploids. Our focus is on the average time for tetraploids to become fixed in an initially diploid population. We treat the deterministic frequencies calculated in equation
(1) as expected outcomes, with actual frequencies following a binomial distribution around this expectation. The expected frequency of diploids in generation t + 1, given the frequency of diploids in the previous generation dt, is E[dt+1|dt] = dt+1. Suppose that there are i diploid individuals in generation t. The probability of drawing j tetraploids out of N total individuals in the population is
N a (E[d | d ]) N − j (1 E[d | d ]) j (2) ij = t+1 t − t+1 t j
Let the ith element in a vector pt of length N be the probability that a population has i = 0,
1,…, N - 1 tetraploid individuals. If the N x N matrix A has entries aij given by equation (2), then the recurrence equation
pt+1 = A pt (3) describes how the probability distribution that a population has i tetraploid individuals changes over one generation. The sum of the elements of pt, ||pt||, is the probability that a population has at least one diploid, so 1-||pt|| is the frequency of populations fixed for tetraploids. Let p0 be a vector with zeros everywhere except at i = 0; this corresponds to a population consisting entirely of diploid individuals. Repeated application of equation (3)
t indicates that the probability distribution after t generations is pt = A p0. In this scenario, ||pt||
8 is the probability that the original population contains at least one diploid after t time periods.
The average time until all populations lose diploid individuals is
∞ ∞ t p A t p (I A) −1 p (4) = ∑ t = ∑ o = − o t=0 t=0
The final step relies on convergence of At to 0, which occurs because of the unidirectional and positive production of unreduced gametes u > 0: introduction of tetraploids into diploid populations is certain, and by chance will eventually reach fixation; once fixed, the formulation does not allow reintroduction of diploids. Stochastic calculations explore how fitness, self-fertilization, and inbreeding depression interact with population size to determine the time required for tetraploid fixation.
9 RESULTS
Figure 1 sketches general results of our analysis. There are up to three equilibria, found by solving equation (1) when dt+1 = dt = d (Felber 1991; Levin 1975). An upper, stable, equilibrium occurs when the population consists entirely of tetraploids (d = 0). Two additional equilibria exist when the frequency of unreduced gametes u is greater than 0 but less than a critical value ucrit. The lower of these equilibria is stable, and corresponds to a balance between introduction of unreduced gametes and selection against tetraploids because of rare cytotype disadvantage. An unstable equilibrium lies between the two stable equilibria. In the absence of unreduced gamete production, u = 0, the unstable equilibrium is
2 1− sd (1+ δ d ) − st w(1− δt ) + sd tcrit = 2 2 (5) (1− sd ) + (1− st ) w
This corresponds to the minimum tetraploid frequency required for tetraploids to spread to fixation, and is Levin’s minority cytotype disadvantage. Levin (1975), however, only considered populations with equal selfing rates (st = sd ≥ 0), and equal fitnesses of diploid and tetraploid types (w = 1). Increasing the frequency of unreduced gametes u decreases the unstable equilibrium while increasing the lower, stable, equilibrium. The equilibria converge when u = ucrit. Increasing unreduced gamete production above ucrit always results in populations evolving to fixation of tetraploids. Felber (1991) shows that
ucrit = 3 − 2 2 ≈ 0.1716 in random mating populations. Analysis following the methods of
Levin and Felber result in general expressions for ucrit with partial self-fertilization and inbreeding depression. These expressions are complicated and provide no additional heuristic insight, and thus are not presented in full here.
10 Figure 2 summarizes how cytotype fitnesses, selfing rates, and inbreeding depression influence tcrit and ucrit. Figure 2a shows that higher fitnesses of tetraploids relative to diploids in random mating populations create less restrictive conditions for tetraploid establishment.
Analysis shows that, under these conditions, tcrit = 1 / (1 + w) and ucrit = 1+ 2w − 2 w(1+ w) .
Decreasing the fitness of tetraploids relative to diploids w increases both the minimum frequency required for tetraploids to spread to fixation tcrit and the minimum rate of unreduced gamete production required for tetraploids to spread to fixation ucrit (Fig. 2a and Felber 1991).
Figures 2b and 2c show that greater selfing in tetraploids, in the absence of inbreeding depression, facilitates tetraploid establishment. With equal selfing rates in the two cytotypes
(st = sd; Fig. 2b) and in the absence of unreduced gamete production, selfing does not influence tcrit (Levin 1975). With unreduced gamete production, greater selfing rates increase the lower equilibrium because a lower degree of ineffective pollinations among cytotypes are occurring. Greater selfing rates also decrease the threshold for tetraploids to spread to fixation tcrit, and reduce the minimum rate of unreduced gamete production required for tetraploids to spread to fixation ucrit. Greater self-fertilization in tetraploids compared to diploids, st > sd, further reduces the unstable equilibrium and rate of unreduced gamete production required for tetraploid fixation (Fig. 2c). The reverse effect occurs when st < sd. In general, greater self- fertilization in tetraploids facilitates tetraploid establishment. This occurs because a greater proportion of tetraploid ovules are being fertilized by n = 2x pollen (from tetraploids and unreduced diploid 2n pollen) than are ovules of diploids being fertilized by n = 1x pollen
(only from diploids).
Figures 2d and 2e show that inbreeding depression acts to counter the effects of self- fertilization. When selfing and inbreeding depression are equal in both cytotypes δd = δt, and
11 the level of inbreeding depression increases, it reduces the effect of selfing on the value of ucrit. Thus, as inbreeding depression approaches one the population appears more like a random mating population (Fig. 2d). The effect is more complex when inbreeding depression differs δd ≠ δt between cytotypes. When selfing rates are equal, the cytotype with the higher inbreeding depression suffers an effectively lower selfing rate. For instance, higher inbreeding depression in the tetraploid increases both critical values tcrit and ucrit (Fig. 2e).
Simultaneous differences between cytotypes in selfing rates and inbreeding depression further complicate cytotype exclusion. If there are high but unequal rates of selfing in both cytotypes, and the cytotype with the highest selfing rate has the highest inbreeding depression, that cytotype experiences more severe effects of exclusion. For example, higher selfing and inbreeding depression in diploids reduces the unstable equilibrium for tetraploids, and reduces the critical values tcrit and ucrit (not shown). If the values of diploid selfing and inbreeding depression are large enough, the critical value of tetraploid frequency decreases below zero, tcrit ≤ 0. In this case, any frequency of tetraploids eventually leads to exclusion of all diploids regardless of the frequency of unreduced gametes. In contrast, tetraploid selfing and inbreeding depression much greater than in diploids result in drastic increases in the unstable equilibrium and critical values (not shown). In some cases, when the production of unreduced gametes is not considered, tetraploid critical values can exceed one, tcrit ≥ 1. In this situation and in the absence of unreduced gamete production, cytotype exclusion eventually eliminates tetraploids regardless of their original frequency.
Figure 3 summarizes effects of finite population size on the mean time to fixation of tetraploids when cytotypes have equal fitnesses. Calculations use equation (4), with the initial population consisting entirely of diploids. Figure 3a shows that the mean time to tetraploid
12 fixation increases with population size and decreases with unreduced gamete production. Low frequencies of unreduced gamete production (e.g., < 0.05) can result in very long times
(>10,000 generations) to fixation, even in very small populations. Small population size is particularly important at lower levels of unreduced gamete production. Recognizing three stages helps to understand the process of tetraploid fixation. Unreduced gamete production and frequency-dependant selection drive populations to the lower, stable equilibrium. Drift then moves tetraploid frequencies from the lower, stable equilibrium, to above the middle, unstable equilibrium. Finally, the population moves more-or-less deterministically toward tetraploid fixation. Population size primarily affects the second stage (drift from the lower stable equilibrium to above the higher unstable equilibrium), with small population size facilitating this transition.
The remaining parts of Figure 3 illustrate effects of selfing and inbreeding depression like those in Figure 2. Figure 3b shows that equal cytotype selfing rates without inbreeding depression hastens tetraploid establishment; increasing population size moves the curves up and to the right, as in Figure 3a. Similar results occur when st ≥ sd, whereas when st < sd, the mean time to fixation of the tetraploid increases (Fig. 3c). Also similar to the deterministic model, inbreeding depression reduces the effects of selfing (Fig. 3d and 3e), and higher inbreeding depression in one cytotype disproportionately negates the effects of selfing.
Increasing population size once again moves curves up and to the right in each panel, as in
Figure 3a.
13 DISCUSSION
The minority cytotype exclusion principle (Felber 1991; Levin 1975) plays a very important role in understanding conditions for polyploid establishment. Our results extend this principle to partially selfing populations with inbreeding depression, and to finite populations where stochastic effects are important.
Direct evidence for minority cytotype exclusion includes reduced seed production in mixed compared with pure stands of Dactylis (Maceira et al. 1992), between-year changes in rye cytotype frequencies (Hagberg and Ellerström 1959), and a negative relationship between diploid percentage seed set per fruit (though not total seed set) and tetraploid frequency
(Husband 2000) in Chamerion angustifolium. Chamerion angustifolium is the only example of an autopolyploid where selfing rates (sd = 0.45, st = 0.43) and inbreeding depression values
(δd = 0.67 and δt = 0.95) are known (Husband and Schemske 1997). With these parameter values, the unstable equilibrium with no unreduced gamete production is tcrit = 0.292. Diploid plants of C. angustifolium have a significantly larger mean number of ovules per individual plant than tetraploids (Husband 2000). This elevates the ‘effective’ frequency of diploids, and may explain why Husband (2000) finds that tetraploids had significantly lower levels of seed production relative to diploids in an experimental population with equal cytotype frequencies.
The frequency of unreduced gamete production is key to autotetraploid establishment
(Figs. 2, 3). Ramsey and Schemske (1998) estimate the mean frequency of unreduced gametes in non-hybrid angiosperms to be 0.56%, but this varies greatly between species, populations, individuals, flowers, and even genders (e.g., Bretagnolle and Thompson 1995; Kaul and
Murthy 1985; Maceira et al. 1992; Parrott and Smith 1984). Substantial genetic variation and response to environmental stresses (e.g., nutrients, pathogens, water, herbivory, and especially
14 temperature, see Bretagnolle and Thompson 1995; Felber 1991; Ramsey and Schemske 1998) also influence unreduced gamete production. It is thus very difficult to identify values of u relevant to natural populations; <10% seems very likely, and at this range, the selfing rate and degree of inbreeding depression (Fig. 2), and finite population size (Fig. 3) play important roles in tetraploid establishment.
Selfing in tetraploids equal to or greater than that in diploids reduces the rate of unreduced gamete production needed for establishment of tetraploids (Figs. 2b,c, 3b,c). Many researchers suggest that tetraploids should have higher selfing rates than diploids (Soltis and
Soltis 2000; Stebbins 1971), but empirical evidence does not consistently support this assertion. Chromosome doubling may break down some forms of gametophytic self- incompatibility (de Nettancourt 2000; Levin 2002), but there is no strong association between self-compatibility and polyploidy at the family or species level (Mable 2004; see Miller and
Venable 2000 for counter-examples). Polyploids often differ morphologically from their diploid progenitors, particularly in flower size (Stebbins 1971). Larger flowers can increase herkogamy and hence decrease selfing rate (Barrett and Eckert 1990; Webb and Lloyd 1986).
However, in one of the few evaluations of this hypothesis (in C. angustifolium, Husband and
Schemske 1997), flower size is larger in tetraploids but electrophoretically-estimated selfing rates do not differ between cytotypes. In addition, there are numerous cases (e.g., Claytonia parviflora, Miller and Chambers 1993; Tarasa, Tate and Simpson 2004) where polyploid flowers are smaller and apparently more selfing than diploid progenitors.
Reduced inbreeding depression in tetraploids makes tetraploid establishment easier
(Figs 2d,e, 3d,e). Inbreeding depression is often due to homozygosity of recessive, deleterious alleles, and in this case higher autotetraploid heterozygosity reduces inbreeding depression
15 (Lande and Schemske 1985). The situation is more complicated with partial dominance of deleterious alleles (Ronfort 1999) or with inbreeding depression due to heterotic allelic interactions (Bever and Felber 1992). In these cases, tetraploid inbreeding depression can be greater than in diploids, and does not always decrease with the selfing rate. The meiotic timing of unreduced gamete production (Bretagnolle and Thompson 1995) and the degree of double reduction (Butruille and Boiteux 2000) affect gamete and offspring heterozygosity and the inbreeding depression of new polyploid lineages (Charlesworth and Charlesworth 1987).
Empirical research from agricultural systems generally indicates lower inbreeding depression in diploids (e.g. Dewey 1966), whereas the only known example of diploid and autotetraploid inbreeding depression estimates from the same species complex (C. angustifolium, Husband and Schemske 1997) found inbreeding depression in tetraploid (0.67) less than in diploids
(0.95). Results from tetraploid populations of Campanula americana indicate low inbreeding depression (0.26), yet high outcrossing rates (0.938) (Galloway et al. 2003); diploids were not sampled.
Our results give some insight into the evolution of mating systems in polyploids. As we demonstrate, lower inbreeding depression in tetraploids will facilitate tetraploid establishment with equal cytotype selfing rates (Fig. 2e). Lande and Schemske (1985) predicted that inbreeding depression should be lower in tetraploids than in diploids. If inbreeding depression is the primary barrier to the evolution of selfing, low inbreeding depression in newly created polyploids may encourage the evolution of higher selfing rates in polyploid lineages and further promote polyploid establishment. Conversely, high inbreeding depression in polyploids following a loss of self-incompatibility may be a mechanism responsible for the evolution of gender dimorphism in polyploid plants (Miller and Venable
16 2000). Our model shows that when some degree of selfing is present only in tetraploids (as with a loss of self-incompatibility), and inbreeding depression is high in tetraploids, it creates circumstances that favor tetraploid exclusion (not shown). Thus, evolution of mechanisms that prevent inbreeding in tetraploids (i.e. dioecy, Miller and Venable 2000) may restore relatively less-restrictive conditions for polyploid establishment present during random mating.
Mixed cytotype populations are abundant (e.g. Baack 2004; Burton and Husband
1999; Hardy et al. 2000; Husband and Schemske 1998). Our model (Fig. 1) and the results of
Felber (1991) show that tetraploids can be maintained at low frequency as long as unreduced gametes are produced. The long time required for diploid exclusion in finite populations (Fig.
3) primarily reflects time spent in fluctuations about the lower stable equilibrium when tetraploids are relatively rare, so finite population size does not provide significant additional insight into mixed cytotype populations with intermediate tetraploid frequencies. In contrast, environmental heterogeneity (Li et al. 2004) and niche separation between cytotypes (Fowler and Levin 1984; Rodriguez 1996a) can maintain both cytotypes at an arbitrary frequency.
An important assumption of our model is that triploids are lethal (i.e., triploid block).
Triploid lethality is a regular assumption of models investigating polyploidy establishment
(e.g. Felber 1991; Fowler and Levin 1984; Levin 1975; Rodriguez 1996a). However, the occurrence of a triploid bridge may be a significant force in tetraploid formation and establishment (Ramsey and Schemske 1998). Felber and Bever (1997) demonstrated that if triploid fitness is high enough, there is a decrease in the threshold of 2n gametes necessary to deterministically fix tetraploids in the population. Furthermore, empirical evidence indicates that a triploid bridge may play an important role in the formation and persistence of tetraploids in diploid populations (Husband 2004). Inclusion of triploids would significantly
17 increase the complexity of our model (e.g. triploid selfing rates, inbreeding depression, fitness, production of aneuploid, x, 2x, and 3x gametes). The effect of selfing on the formation of tetraploids via the triploid bridge, would likely depends on the relative fitness of triploids and the degree to which triploids produce euploid (x, 2x, 3x) gametes.
Assortative mating may also play a large role in reproductive isolation of diploid and tetraploid cytotypes in the same population. Differences in phenology, pollinators, pollinator fidelity, and population structure have all been shown to contribute to reproductive isolation between diploid and autotetraploid cytotypes (Husband and Sabara 2004; Segraves and
Thompson 1999). In essence, assortative mating has a similar effect on polyploid establishment as selfing; both processes reduce the number of inter-cytotype pollinations and lessen the effects of minority cytotype disadvantage. However, offspring produced by selfing incur the fitness disadvantage of inbreeding depression while assortative mating depends on reliable pollinators. Thus, there is a trade-off between the reproductive assurance provided by selfing (and negative effects of inbreeding depression) and the reliability of pollinators required for assortative mating.
Though not explicitly modeled here, a metapopulation dynamic might greatly facilitate polyploid establishment. Tetraploid selfing (e.g., for reproductive assurance; Figs. 3b,c), reduced inbreeding depression (Figs. 3d,e), non-random dispersal (Li et al. 2004) and greater production of unreduced gametes in stressful environments (Bretagnolle and Thompson 1995;
Ehrendorfer 1980; Stebbins 1971) enhance this process. This could help explain the association between weedy habit and polyploidy (Barrett and Richardson 1986; Brown and
Marshall 1981) and why polyploids are often on the fringe of related diploid ranges (Levin
2002). A metapopulation dynamic does not require that tetraploids exhibit superior adaptation
18 to newly colonized environments, as sometimes suggested (Soltis and Soltis 2000; Stebbins
1950).
Levin (1975) and Felber (1991) show that minority cytotype exclusion is an obstacle to tetraploid establishment when unreduced gamete production is rare. Our results show that higher tetraploid selfing rates, lower tetraploid inbreeding depression, and small population size help to overcome minority cytotype exclusion. These results add to a growing body of evidence suggesting that the conditions necessary for polyploid establishment are less restrictive than originally thought.
19 ACKNOWLEDGEMENTS
B. Husband and two anonymous reviewers provided insightful comments. This work was encouraged by opportunities provided by R. Gomulkiewicz, and supported by the
National Science Foundation (DEB 0128896).
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26 Table 1. Variables, parameters, and critical values influencing tetraploid establishment
Symbol
Variables
Cytotype frequency at time t (diploid, tetraploid) dt, tt
Parameters
Frequency of 2n gamete production u
Selfing rate (diploid, tetraploid) sd, st
Inbreeding depression (diploid, tetraploid) δd, δt
Relative fitness of tetraploid w
Population size N
Critical values
Frequency of tetraploids needed for tetraploids to spread tcrit
to fixation when u = 0
Frequency of u needed for tetraploids to spread to ucrit
fixation when dt = 1
27 FIGURE LEGENDS
Figure 1. Frequency of tetraploid as a function of unreduced gamete production under random
mating. Tetraploid frequencies t above the unstable equilibrium (dashed line) will
eventually exclude diploid cytotypes from population, while t below the unstable
equilibrium move to the lower stable equilibrium (solid line). Unreduced gamete
production greater than ucrit will eventually exclude diploids from the population.
Figure 2. Frequency of tetraploid as a function of unreduced gamete production under
variable relative fitness, selfing, and inbreeding depression. Effect of relative fitness
differences (a) among cytotypes, equal (b) and unequal (c) selfing among cytotypes in
the absence of inbreeding depression, and equal (d) and unequal (e) inbreeding
depression among cytotypes.
Figure 3. Mean time to tetraploid fixation due to stochastic processes as a function of
unreduced gamete production. Effect of population size (a) on the mean time to
fixation of tetraploids in a population with an initial population entirely composed of
diploids, the horizontal dashed line represents critical value of u in an infinitely sized
population where tetraploids will eventually exclude diploids from population (right
side of line) or will remain at the frequency of the lower stable equilibrium. Effect of
equal (b) and unequal (c) selfing among cytotypes in the absence of inbreeding
depression, and equal (d) and unequal (e) inbreeding depression among cytotypes.
28 Figure 1:
29 Figure 2:
30 Figure 3:
31 CHAPTER TWO
FORMULATION OF COMPETITION BETWEEN SEXUAL AND SELFING FORMS:
A RESPONSE TO YAMAUCHI
JOSEPH H. RAUSCH AND MARTIN T. MORGAN
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236
[Formatted for Evolution, Allen Press]
32 The model of autotetraploid establishment presented in Rausch and Morgan (2005)
extends the minority cytotype exclusion of Levin (1975) and Felber (1991) to include variable
rates of selfing and inbreeding depression among cytotypes. We agree with Yamauchi that
Rausch and Morgan (2005) implicitly assume pollen limitation. We offer here additional
commentary on our original formulation, the extension offered by Yamauchi, and the
biological context of self-pollination.
In our model, a fraction of the ovules available for outcrossing remains unfertilized,
and this fraction increases with the selfing rate. Yamauchi proposes that this occurs when
there is both pollen discounting (Harder and Wilson 1998) and outcross pollen limitation
(Knight et al. 2005). Our interpretation, however, is that a fraction of the outcross pollen pool
is available for within-cytotype outcrossing (e.g., dt / (dt + tt) = dt for the diploid cytotype; no
pollen discounting) but only some of this results in successful outcross ovule fertilization
(e.g., 1 - sd for the diploid cytotype; pollen limitation in proportion to the selfing rate).
Yamauchi’s extension assumes complete pollen discounting (cytotype representation in the
outcross pollen pool inversely proportional to cytotype selfing rate), and no pollen limitation.
Many other formulations are possible. For instance, using the symbols defined by Rausch and
Morgan (2005) and used by Yamauchi, the equations
2 2 2 (1a) dt +1 = [dt (1− sd )(1− u) + dt sd (1− u) (1−δd )]/k
€ t (1 d )2 (1 s ) (1 d )(1 s )d u (1 d )(1 s )d u d 2(1 s )u2 t +1 = [{ − t − t + − t − t t + − t − d t + t − d (1b) (1 d )s (1 ) d s u2(1 ) w /k + − t t −δt + t d −δt } ]
€ 33 describe a model with neither pollen limitation nor pollen discounting. More generally, the full diversity of approaches used to investigate diploid self-fertilization can be employed to model autotetraploid establishment.
The model present in Rausch and Morgan (2005) implicitly assumes that pollen limitation of outcrossed ovules increases with cytotype selfing rate. The magnitude of pollen limitation across species can be high (Burd 1994), although the relationship between selfing rate and pollen limitation of outcross ovules is not known. Nonetheless, floral morphologies resulting in high self-fertilization (e.g., reduced flower size and pollinator reward, increased proximity and hence interference between anthers and stigmas) may often contribute to myriad changes in reproductive biology (Schoen et al. 1996) that promote pollen limitation.
Knight et al. (2005) provide indirect support for greater outcross pollen limitation with selfing, finding that pollen limitation increased with decreasing flower size (when autogamous species are included). The results presented in Rausch and Morgan (2005) may be particularly appropriate at moderate to high levels of selfing, where floral morphological changes are most likely to restrict outcross pollen receipt.
Rausch and Morgan’s (2005) model assumes pollen limitation without pollen discounting, whereas Yamauchi’s model assumes complete pollen discounting without pollen limitation. Reasonable mixed mating models often assume incomplete pollen discounting (e.g.
Porcher and Lande 2005) or pollen discounting that can vary with the selfing rate (e.g. Harder and Wilson 1998; Holsinger et al. 1984; Nagylaki 1976; Wells 1979). Available empirical research indicates an effective absence of pollen discounting in mixed mating species
(Holsinger and Thomson 1994; Kohn and Barrett 1994; Rausher et al. 1993) or high levels of pollen discounting in highly selfing species (Holsinger 1992; Ritland 1991).
34 Consequences of different assumptions are reflected in model outcomes. To illustrate, higher selfing provides an ‘automatic’ advantage (analogous to Fisher 1941) by reducing ineffective matings among cytotypes (assuming triploid lethality, Levin 1975): with no pollen limitation or discounting, more highly selfing cytotypes contribute more viable offspring to the next generation than less selfing cytotypes. Outcross pollen limitation provides additional benefit to selfing, because selfing increases fertilized ovule production. This consequence of pollen limitation is easily illustrated when there is no unreduced gamete production (u = 0). In this case tcrit measures the threshold frequency above which autotetraploids must rise to eliminate diploids from the population. Figure 2c of Rausch and Morgan (2005) shows that tcrit depends on diploid and autotetraploid selfing rates, with greater diploid selfing increasing tcrit, i.e., increasing the threshold required for autotetraploid establishment. In contrast to pollen limitation, pollen discounting erodes the advantage to selfing (analogous to Holsinger et al. 1984; Nagylaki 1976; Wells 1979). Our analysis of Yamauchi’s model shows that, in his model, tcrit always equals _: complete pollen discounting exactly eliminates the reproductive advantage to higher selfing, regardless of cytotype selfing rates.
Our model and the modification provided by Yamauchi anticipate that selfing facilitates autotetraploid establishment, especially when higher selfing and/or lower inbreeding depression are present in autotetraploids. Rausch and Morgan’s (2005) model is appropriate when pollen limitation occurs, and provides conditions that are conducive to autotetraploid establishment. Yamauchi’s formulation is appropriate when complete pollen discounting occurs, and makes autotetraploid establishment more difficult. Other formulations of self-fertilization are possible and indeed appropriate depending on biological context; the
35 roles of pollen discounting and pollen limitation in autotetraploid establishment deserve further treatment.
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38 CHAPTER THREE
SELF-FERTILIZATION AND INBREEDING DEPRESSION IN DIPLOID AND
POLYPLOID POPULATIONS OF THE CLAYTONIA PERFOLIATA (PORTULACACEAE)
POLYPLOIDY COMPLEX
JOSEPH H. RAUSCH
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236
[Formatted for Evolution, Blackwell Publishing]
39 ABSTRACT– Inbreeding depression is the reduction in fitness due to inbreeding (e.g. selfing or mating among relatives). Inbreeding increases homozygosity and the subsequent expression of recessive deleterious alleles. Self-fertilization is an extreme form in inbreeding and one of the most common forms of diversification in flowering plants. While inbreeding depression is thought to be the primary force preventing the evolution of selfing in plants, selfing populations can eventually purge deleterious alleles from the population. This process will result in relatively low levels of inbreeding depression in selfing populations. Polyploidy has also been proposed to influence levels of inbreeding depression; most researchers suggest that polyploids are expected to exhibit less inbreeding depression than diploids due to the buffering effect of multiple alleles per locus. Here the relationship between cumulative inbreeding depression and self-fertilization was studied in eleven populations of the Claytonia perfoliata s.l. polyploid complex. Selfing rates and inbreeding depression were measured for six diploid populations, three tetraploid populations, and two hexaploid populations.
Inbreeding depression was significantly greater in three diploid outcrossing ( s = 0.09, δ =
0.72) populations than in diploid and polyploid selfing populations ( s = 0.96, δ = 0.23). € € Among the selfing populations, diploids (δ = 0.26) and tetraploids (δ = 0.32) were not € significantly different. One hexaploid population had significantly€ lower inbreeding € € depression (δ = -0.03) than either diploids or tetraploids; however this may be the result of the colonizing history rather than effects from polyploidy. Results indicate that the selfing rate was€ the most important factor for explaining levels of inbreeding depression and that the effect of polyploidy on inbreeding depression is generally inconclusive for selfing C. perfoliata populations.
40 Introduction
The evolution of self-fertilization is one of the most prevalent evolutionary transitions in flowering plants (Schemske and Lande 1985). Self-fertilization (selfing) is favored over cross-fertilization (outcrossing) due to a 50% transmission bias (Fisher 1941). All else being equal, selfing types will contribute gametes to the next generation as an ovule parent, a selfed pollen parent, and an outcrossed pollen parent, while outcrossing types will only contribute copies as an ovule parent and an outcrossed pollen parent. However, while a great deal of plants are selfing, the majority of plants generally outcross (Goodwillie et al. 2005). This suggests that there are strong forces opposing the evolution of selfing. For example, pollen discounting, the reduction in pollen available for outcrossing as a result of increased selfing
(Harder and Wilson 1998), can reduce the transmission bias of selfing.
Most theoretical models (e.g Lande and Schemske 1985) and empirical research (see
Husband and Schemske 1997) consider inbreeding depression as the major force counteracting the evolution of selfing. Inbreeding depression is often defined as the reduction in fitness of selfed progeny relative to outcrossed progeny and, in the absence of other forces, inbreeding depression values greater than 0.5 are generally considered sufficient to prevent the evolution of selfing (Charlesworth and Charlesworth 1987). A significant amount of research, both theoretical and empirical, has been applied to explain the joint evolution of selfing and inbreeding depression (e.g. Husband and Schemske 1996; Lande and Schemske
1985). Selfing populations have been shown empirically to have lower inbreeding depression
(Husband and Schemske 1997). Theory has shown that selfing increases homozygosity and the subsequent purging of deleterious alleles from the population results in lower genetic load and inbreeding depression (Lande and Schemske 1985).
41 Polyploidy, the duplication of chromosomes within a species (autopolyploidy) or chromosome doubling in conjunction with hybridization (allopolyploidy), is accepted as having a major role in plant evolution. Despite the ubiquity of polyploidy in plants (estimates range from 35-100%, Cui et al. 2006; Soltis et al. 2004; Wolfe 2001), relatively few investigators have explicitly considered the evolution of selfing and inbreeding depression in polyploids relative to diploids (however, see Barringer and Gerber unpublished data; Husband et al. 2008; Husband and Schemske 1997; Lande and Schemske 1985; Ronfort 1999).
Intuitively, it seems that inbreeding depression would be lower in polyploids due the masking of deleterious recessive alleles associated with carrying multiple alleles per locus.
For example, in an initially random mating diploid population, selfing will increase homozygosity by 50% per generation (Wright 1922), compared to ~17% in an autotetraploid population (Kempthorne 1957). For this reason, it has been suggested that polyploids should exhibit higher selfing rates than diploids due to lower levels of inbreeding depression (Rausch and Morgan 2005; Soltis and Soltis 2000). However, available theoretical research on the evolution of selfing and inbreeding depression in polyploids vary in their predictions.
Inbreeding depression in polyploids may be less than, equal to, or greater than that of diploids. This will depend on the genetic basis of inbreeding depression (i.e. overdominance or partial dominance, Charlesworth and Charlesworth 1987), dominance coefficients associated with multiple alleles per locus (i.e. allelic dosage effects, Ronfort 1999), the age of the system (i.e. neopolyploidy vs. paleopolyploidy), and the type of polyploidy (i.e. allopolyploidy vs. autopolyploidy).
Lande and Schemske (1985) show lower inbreeding depression in tetraploids compared to diploids if inbreeding depression is caused by completely recessive lethal and
42 sub-lethal mutations. However, they show that if inbreeding depression is caused by partially recessive alleles there will be no difference between tetraploids and diploids. These results apply equally to disomic (allopolyploid) or tetrasomic (autopolyploid) species. Lande and
Schemske (1985) also showed that inbreeding depression due to quantitative variation should be approximately equal for diploids and allotetraploids.
Using a mutation-selection equilibrium approach, Ronfort (1999) extended the diploid case of Ohta & Cockerham (1974) to autotetraploidy with dominance coefficients that differ among heterozygous genotypes (i.e. h1, h2, h3, for AAAa, AAaa, Aaaa, respectively). Ronfort’s model also allows for variable rates of selfing that can purge mutational load from the population. In contrast to Lande and Schemske (1985), this model predicts equal inbreeding depression in diploids and tetraploids under complete recessivity (h1 = h2 = h3 = 0).
Inbreeding depression caused by partially recessive mutations (e.g. h1 << h2 < h3, duplex- mutation effect) generally results in lower inbreeding depression in tetraploids relative to diploids for a given selfing rate. However, the results are complex and often analytically intractable, and under a few scenarios tetraploids can have higher inbreeding depression than diploids. An unexpected result of Ronfort’s model shows that if a ‘dosage-like’ dominance effect (i.e. h2 = 2h1 and h3 = 3h1; Lande and Schemske 1985; Wright 1934) is assumed, inbreeding depression can vary non-monotonically, increasing and then decreasing as the selfing rate increases. This is contrary to the diploid case and the results described above, which show that inbreeding depression always decreases as the selfing rate increases.
In contrast to the models described above, which consider inbreeding depression caused by partial dominance and generally lower inbreeding depression in polyploids, other models show significantly higher inbreeding depression in polyploids. Busbice and Wilsie
43 (1966) and Bever and Felber (1992) considered inbreeding depression upon selfing in initially random mating autotetraploid populations. They anticipate greater inbreeding depression in polyploids relative to diploids. In these models, selection does not purge deleterious mutations from the population through increased selfing, and it is assumed that inbreeding depression is caused by loss of heterotic interactions (i.e. overdominance) among three or more alleles per locus. In a similar model, Bennett (1976), considered a two-allele system in random mating equilibrium. He showed that inbreeding depression will be greater upon selfing in an outcrossing autopolyploid if inbreeding depression is caused by a loss of interactions among loci that combine additively; however, Bennett notes that inbreeding depression in newly created polyploids may be less than diploids. All of these cases (Bennett 1976; Bever and
Felber 1992; Busbice and Wilsie 1966) may be particularly applicable to inbreeding depression in outcrossing paleopolyploids, as partial selfing is not included in their models.
Empirical studies investigating inbreeding depression in diploid-polyploid pairs from both agricultural and natural systems show no clear trend in the association of polyploidy and inbreeding depression. Early experiments were primarily limited to agricultural plants; diploid and autotetraploid types of Medicago sativa (Busbice and Wilsie 1966) and diploid, autotetraploid, and autohexaploid types of Agropyron cristatum (Dewey 1966) support the view that inbreeding depression (as measured by yield) increases with ploidy. However, in a separate investigation of Agropyron cristatum, Dewey (1969) found lower inbreeding depression in synthetic autotetraploids than in diploids. This supports Bennett’s (1976) suggestion that inbreeding depression in neopolyploids will be less than that of diploids and
Otto and Whitton’s (2000) finding that genetic load will increase with the age of the polyploid system.
44 Empirical evidence in natural populations is also scarce. Husband and Schemske
(1997) measured outcrossing rates and inbreeding depression in diploid and autotetraploid populations of Chamerion (Epilobium) angustifolium. They found lower cumulative inbreeding depression in autotetraploids (0.67) compared to diploids (0.95); primary outcrossing rates were not significantly different. However, subsequent research of synthesized C. angustfolium neotetraploids indicates not detectable levels of inbreeding depression (Husband et al. 2008). Rosquist (2001) compared early acting inbreeding depression (i.e. seed set and germination) in diploid and allotetraploid populations in the genus Anthericum; allopolyploids had lower inbreeding depression than the closely related diploids.
Recently, Barringer and Gerber (unpublished data) compared inbreeding depression in selfing and outcrossing diploid/allopolyploid pairs within the genus Clarkia. They found that mating system was more important than ploidy for explaining inbreeding depression.
Outcrossing populations had higher inbreeding depression than selfing populations and ploidy was not a significant effect. However, polyploid populations did have slightly lower inbreeding depression. In contrast, Johnston and Schoen (1996) compared inbreeding depression in closely related Amsinckia populations; selfing allotetraploids had higher inbreeding depression than selfing diploids. Other research investigating inbreeding depression in polyploids alone shows low levels of inbreeding depression (Belaoussoff and
Shore 1995; Galloway et al. 2003).
Collectively, these studies strongly suggest that more evidence is needed to uncover major trends in the joint evolution of selfing and inbreeding depression in polyploids. To that end, the objectives of this paper are: 1) estimate selfing rates using allozyme electrophoresis
45 of six diploid and five polyploid populations of the C. perfoliata polyploidy complex, and 2) measure inbreeding depression in these eleven populations. The C. perfoliata polyploidy complex is a good system to study these questions due to labile nature of eupolyploidy within the complex and the variability of mating systems within and among cytotypes.
46 Methods
STUDY SYSTEM
Claytonia perfoliata s.l. (Portulacaceae) is a morphologically diverse annual species complex composed of a variable array of diploids and eupolyploids (from tetraploid to decaploid). The currently accepted circumscription of this complex suggests that it is based upon three morphologically distinct diploid (2n = 12) taxa: C. perfoliata ssp. mexicana, C. parviflora ssp. grandiflora, and C. rubra ssp. rubra (Miller 2003; Miller and Chambers 1993;
Miller and Chambers 2006). Many of the tetraploid varieties and subspecies within the complex are morphologically indistinguishable from their respective diploid conspecifics and are assumed to be intraspecific autopolyploids (Miller 1978; Miller and Chambers 1993); other cytotypes (tetraploids and higher) have intermediate morphologies which suggest allopolyploid origins (Miller 1978; Miller and Chambers 1993). To date, all chromosome counts within the C. perfoliata complex (> 1,000 throughout the range) have shown normal and complete bivalent formation during meiosis, regardless of ploidy (Miller 1976; Miller
1978; Miller 1984b; Miller and Chambers 2006; Swanson 1964). This suggests that most polyploids are either of allopolyploid origin or diploidization is ubiquitous among autopolyploid cytotypes.
Recent investigations (Rausch; unpublished data, Chapter 4) suggest that the relationships among the species, subspecies, and cytotypes of C. perfoliata s.l. are more complicated than originally thought. For example, tetraploid cytotypes of C. parviflora ssp. parviflora are morphologically similar (e.g. linear leaves) to diploid C. parviflora ssp. grandiflora. Yet, evidence from nrDNA and cpDNA sequences suggest that tetraploid C. parviflora ssp. parviflora is derived from diploid C. perfoliata ssp. mexicana with possible
47 input from diploid C. parviflora ssp. grandiflora. This, along with disomic inheritance at allozyme loci, suggests that tetraploid C. parviflora ssp. parviflora are allotetraploids.
Hexaploid C. perfoliata ssp. perfoliata has been traditionally assumed to be autopolyploid based on morphological similarity to diploid C. perfoliata ssp. mexicana.
Results from DNA sequences do not contradict this assertion: all hexaploid cytotypes are nested within C. perfoliata ssp. mexicana. However, some allozyme loci show disomic inheritance and other enzyme systems are suggestive of possible “fixed” heterozygosity
(Gottlieb 1982a; Soltis and Soltis 2000). Thus, hexaploid C. perfoliata are equivocally allopolyploid in origin.
Sequences of C. parviflora ssp. grandiflora form a monophyletic group within the C. parviflora / C. perfoliata clade (fig. 1). Thus, diploid and polyploid C. perfoliata and polyploid C. parviflora are paraphyletic to C. parviflora ssp. grandiflora. Additional results indicate that diploid and tetraploid C. rubra ssp. rubra consistently form a monophyletic group sister to C. parviflora / C. perfoliata. Tetraploid C. rubra also show some allozyme banding patterns consistent with tetrasomic inheritance. Based on these data, and the morphological similarity among cytotypes, tetraploid C. rubra are presumed autopolyploid.
Although, this paper will utilize nomenclature of Miller and Chambers (2006), their taxonomy does not necessarily reflect phylogenetic relationships; therefore, statistical analyses will incorporate relationships described above (fig. 1) and by Rausch (unpublished data, Chapter
4).
Apparent mating system differences within the complex have also been identified
(Miller 1976; Miller 1978; Miller and Chambers 1993). For example, diploid C. parviflora ssp. grandiflora is considered to be a facultative outcrosser (i.e. protandrous flowers and high
48 pollen-ovule ratios). Most other taxonomic and cytotypic entities are either obligate selfers
(i.e. cleistogamous flowers) or selfers that occasionally outcross (i.e. small flowers and low pollen-ovule ratios); including some with larger flowers and delayed selfing mechanisms.
POPULATION SAMPLING AND PRELIMINARY ANALYSES
Forty-two populations were collected from California, Idaho, Oregon, Nevada, and
Washington from April-July, 2005. Locations for population sampling were primarily based on the work of Swanson (1964), Fellows (1971), and Miller (Miller 1976; 1978; 1981; 1984a) who all characterized the spatial distribution of cytotypes in the C. perfoliata polyploid complex in western North America. Additional locations for population sampling were compiled from herbarium records (UC, JEPS, OSC, WS). Historical collection information
(e.g. collection dates, phenologies, etc.) was used to determine when populations should be sampled to maximize seed collection. Forty individual plants and their seeds were collected haphazardly at approximately two-meter intervals from forty-two populations of various species and subspecies within the complex. Seeds were placed in paper envelopes and multiple representative voucher specimens for each population were deposited in the WSU
Ownbey Herbarium (WS). Approximately five young field-collected inflorescences per population were fixed in a mixture of chloroform:ethanol:glacial acetic acid (4:3:1 , v/v/v), and were stored in a 70% solution of aqueous ethanol at < 0° C. Staining of gametophytic microspore mother cells were accomplished with the ethanol-hydrochloric acid-carmine methods of Snow (1963) and Miller (1976).
Thirty-six populations were grown in the greenhouse in December 2005. These populations were selected from the original 42 populations on the basis of subspecific
49 identification, successful chromosome counts, and insight from J. M. Miller and K. L.
Chambers (personal communication, 2006). For example, several taxa and cytotypes were overrepresented (e.g. nine populations of C. parviflora ssp. grandiflora) and thus some of their populations were not selected. Plants were grown in a greenhouse common garden environment and plant positions were rotated every 2-3 weeks to minimize maternal effects and greenhouse micro-environmental effects. Germination rates for four populations were very low and these populations were excluded from the experiment. The remaining 32 populations were preliminarily sampled for allozyme variability to determine suitable populations for inclusion in the final experiment.
Populations variable for one or more allozyme loci were desired so that population outcrossing rates could be estimated and compared to inbreeding depression levels of populations. Variability was assayed using cellulose acetate electrophoresis for the enzyme systems ADH, MDH, IDH, LAP, PGI, PGM, and TPI by the methods of Herbert and Beaton
(1993) and Soltis et al. (1983) (additional methods given below). Populations were preliminarily sampled until one or more enzyme systems were found to be variable for each population. Allozyme variability was found in at least one enzyme system (either ADH, PGI,
PGM, or MDH) in 15 populations. Eleven of these populations (Table 1) were selected for the inbreeding depression experiment: diploid C. parviflora ssp. grandiflora populations include
American River Canyon, CA (ARC1); San Antonio Creek, CA (SAC1); and Stanislaus River
Canyon, CA (SRC); tetraploid C. parviflora ssp. parviflora populations include Kern River
Canyon, CA (KRC) and Pine Flats Reservoir, CA (PFR); diploid C. perfoliata ssp. mexicana include Oat Mountain, CA (OM) and Las Cruces, CA (LC); hexaploid C. perfoliata ssp. perfoliata include American River Canyon, CA (ARC2), and San Antonio Creek, CA (SAC2)
50 (these populations were allopatric with the two C. parviflora ssp. grandiflora populations of the same name); and C. rubra ssp. rubra include the diploid population French Cabin Creek,
WA (FCC) and the tetraploid population Steens Mountain, OR (SM).
MATING SYSTEM
Analysis of selfing rates in each population was accomplished by assessing allozyme variability of open-pollinated progeny arrays using cellulose acetate electrophoresis (Herbert and Beaton 1993). Field-collected seeds were either placed on moistened filter paper in a petri dish and harvested approximately seven days after germination for immediate use or leaf material was harvested from potted plants growing in the greenhouse and frozen for later use.
Plant material from the seedlings or frozen leaves was ground in a tris-HCl grinding buffer-
PVP solution (pH 7.5), having a PVP (MW 40,000) concentration of 12% w/v (Soltis et al.
1983). Gel and electrode buffers included a tris-glycine buffer (27.5 mM Trizma base, 213.1 mM Gycine, pH = 8.5) for alcohol dehydrogenase (ADH, EC 1.1.1.1), phosphoglucoisomerase (PGI, EC 5.3.1.9), and phosphoglucomutase (PGM, EC 5.4.2.2); and a citric acid buffer (54 mM anhydrous citric acid, 0.013 % 4-[3-aminopropyl] morpholine, pH
= 7.0) for malate dehydrogenase (MDH, EC 1.1.1.37). Staining solutions and protocols followed those of Herbert and Beaton (1993). Interpretation of allozyme variability is inferred by known information on enzyme cellular compartmentalization and subunit structure
(Gottlieb 1982b; Herbert and Beaton 1993; Weeden and Wendel 1989).
Enzyme variability among progeny is used to estimate single-locus and multi-locus outcrossing rates (t), inbreeding coefficients (F), and standard errors using the program
MLTR (Ritland 2002) for disomic loci and MLTET (Ritland 1990) for tetrasomic loci (C.
51 rubra 4x only). These programs estimate mating system parameters via a maximum likelihood
method that infers the maternal genotypes and the allelic frequency of the pollen pool to
determine the likelihood of an outcrossing event. Standard errors of the outcrossing rates and
inbreeding coefficients were determined by a non-parametric bootstrap procedure with 1000
replicates, using progeny arrays (family) as the re-sampling unit. Progeny arrays from field-
collected seeds included 4-8 individuals per family (20-32 families per population). Progeny
from the same maternal plants used for crosses in the inbreeding depression experiment were
used for the mating system analysis.
Measured selfing rates (sm) were determined by subtracting the outcrossing rates (t)
from unity, sm = 1 – t. Since the measured selfing rate excludes any progeny that were lost to
early acting inbreeding depression (i.e. seed abortion and lack of germination), the primary
selfing rate (sp) was determined as
sm s p = , 1−δe + sm δe
where δe is early acting inbreeding depression (here measured as the RP of SEED*GERM,
€ see below). Thus, the primary selfing rate estimates the selfing rate at the time of fertilization
rather than the selfing rate of the seedling stage (Husband and Schemske 1996; Maki 1993).
MEASUREMENT OF INBREEDING DEPRESSION
Population level inbreeding depression was estimated by comparing selfed and
outcrossed offspring in a greenhouse common garden environment. Family level inbreeding
depression was not included as a factor in the analysis, as the goal of this research is to
compare population level differences, and comparisons of inbreeding depression among
52 families are often confounded (see Fox 2005; Johnston and Schoen 1994). In addition, theoretical models that make predictions on the evolution of selfing and inbreeding depression in plants do not consider differences in family-level inbreeding depression. Family structured data, however, were used to determine the level of inbreeding depression within each population (i.e. selfed offspring were compared to outcrossed offspring from the same maternal plant).
For each population (Table 1), two flowers from each maternal plant were emasculated under a dissecting scope 1-2 days before the flower fully opened. After the flower and stigma opened (2-3 days later), each flower was either cross-pollinated by randomly selecting another plant from the same population, or self-pollinated by selecting a mature anther from the same plant. Approximately 25-35 maternal plants per population were pollinated. Pollinations occurred over an eight-week period from April 30 to June 27, 2006.
Additionally, a sub-sample of five plants from each population was selected for emasculation of one flower (unpollinated) to determine if any unintentional pollen transfer had occurred.
None of these flowers resulted in fertilization or seed set. After flowers were successfully pollinated (i.e. fruits began to develop), a loose covering of breathable athletic tape was applied to each fruit to prevent seed loss. Upon maturation, Claytonia fruits explosively eject seeds; so, the tape prevented loss of seed by containing it within the taped fruit. Mature seeds were harvested and placed in labeled envelopes, and number of seeds per fruit was noted.
Seeds were subjected to cold treatment for one month before planting to enhance germination. Seeds were planted in 4x4 inch containers with standard potting mix and placed in the greenhouse under conditions that resembled external ambient temperature. Data from four ontogenetic stages were recorded for each treatment (i.e. selfed or outcrossed): number of
53 seeds per fruit (SEED), percentage of seeds germinating (GERM), survival of an individual to reproduction (SURV), and number of flowers produced (FLWR). Germination date was recorded for each individual. After SEED and GERM were recorded, a randomly chosen offspring for each treatment (i.e. selfed or outcrossed) was selected to grow for the remainder of the experiment. Survival of an individual to reproduction (SURV) was determined for each treatment and either coded as 1 for survival or 0 for death. Plants were harvested 120 days after germination and the number of flowers produced (FLWR) was measured for each plant.
Inbreeding depression has been shown to differ among different ontogenetic stages (Husband and Schemske 1996). Thus, the best measure to compare the overall effects of inbreeding is cumulative fitness (Husband and Schemske 1996). Cumulative fitness (CFIT) was calculated as the product of SEED, GERM, SURV, and FLWR.
Inbreeding depression (δ) is commonly computed as δ =1− wS wO , where wS is the fitness of selfed offspring and wO is the fitness of outcrossed offspring (Charlesworth and
Charlesworth 1987; Johnston and Schoen 1994). This€ method of estimating inbreeding depression is appropriate when outcrossed individuals consistently outperform selfed individuals. However, if selfed individuals often have higher fitness than outcrossed individuals (i.e. outbreeding depression, Lynch 1991), it can easily be shown that this method results in a biased, asymmetrical distribution of inbreeding / outbreeding depression values around zero (Agren and Schemske 1993; Barringer and Gerber unpublished data; Dudash et al. 1997). Thus, for experiments that observe a significant degree of outbreeding depression - such as the results presented here - an unbiased estimate of inbreeding / outbreeding depression is preferred.
54 The method of “relative performance” (Agren and Schemske 1993) is an unbiased
measurement that creates a symetrical distribution of inbreeding/outbreeding depression
values around zero. Thus, to compare selfed and outcrossed individuals from the same
maternal plant, relative performance (RP) was calculated for each trait and each maternal
family as
wS wO RP =1− , if wS ≤ wO , and RP = −1, if wS > wO , wO wS
where wO is the fitness (or the phenotypic value of a fitness trait) of outcrossed offspring and
€ wS is the fitness of selfed€ offspring. Values range from -1 to 1; positive values of RP indicate
inbreeding depression (i.e. outcrossed progeny preformed better than selfed progeny), and
negative values indicate outbreeding depression (i.e. selfed progeny outperformed outcrossed
progeny). Mean population RP was calculated as the average of family-level RP.
When outcrossed individuals outperform selfed individuals, relative perfomance
estimates are equal to the traditional estimates of inbreeding depression. In the case where
selfed individuals outperform outcrossed individuals, relative performance will apply equal
weight to estimates of outbreeding depression as to inbreeding depression estimates
(Barringer and Gerber unpublished data; Dudash et al. 1997). Hereafter, the terms inbreeding
depression and outbreeding depression will be used interchangeably with positive relative
performance and negative relative performance, respectively.
STATISTICAL ANALYSES
Two types of statistical analyses were used to evaluate inbreeding depression. Within
populations, parametric and non-parametric paired tests were used to determine if the trait
55 differences among selfed and outcrossed individuals of the same family were significantly different than zero. Analysis of variance (ANOVA) was used to determine if there were significant differences for relative performance among phylogenetic groups and among cytotypes and populations within phylogenetic groups.
Paired tests were used for comparing selfed and outcrossed half-sibs within populations to determine if population level inbreeding depression was significantly different than zero. One trait, FLWR, was distributed normally (normality was assessed with the
Shapiro-Wilk test in all cases); CFIT had a slightly skewed distribution but was normally distributed after a logarithmic transformation (lnCFIT). Both of these were analyzed using a paired t-test for each population.
The three remaining traits, SEED, GERM, SURV, were not normally distributed; the assumption of normality could not be met with any standard data transformation. This necessitated the use of the non-parametric equivalent of a paired t-test, the Wilcoxon signed- rank test. The paired t-tests and Wilcoxon signed-rank tests were analyzed using the
MATCHED PAIRS procedure of JMP IN v.5.1.2 (SAS Institute, Cary, NC). To control for experiment-wide type-I error rate a sequential Bonferroni test (Holm 1979; Rice 1989) was used to evaluate statistical significance for all populations and traits.
A nested ANOVA was used to determine if relative performance of cumulative fitness
(CFIT RP) was significantly different among phylogenetic groups. For this analysis, population was considered a random effect nested within phylogenetic group (e.g. population
[phylogenetic group]). Additionally, three separate ANOVAs, one for each phylogenetic group (Table 1 and fig. 1), were conducted to determine if there were significant differences for CFIT RP among cytotypes and/or populations within phylogenetic groups. For the
56 phylogenetic group GRAND (i.e. diploid C. parviflora ssp. grandiflora), a one-factor
ANOVA was used with population as a random effect; ploidy was not considered as a factor since all populations are diploid. A nested ANOVA was used for the phylogenetic group
PERF, with population as a random effect nested within ploidy. The phylogenetic group
RUBRA (i.e C. rubra ssp. rubra) had only one population for each cytotype; thus, a one- factor ANOVA was used with ploidy as a fixed effect. In this case ploidy and population represent the same dataset. Significant effects were analyzed with the post-hoc multiple comparison Tukey test (Zar 1996). The nested and one-way ANOVAs and Tukey test were analyzed using the standard least squares FIT MODEL and the least squares means TUKEY
HSD procedures of JMP IN, respectively.
Finally, the correlation between population selfing rates (sp) and cumulative relative performances (CFIT RPmean) was evaluated by two different methods. First, the Spearman rank correlation was used by the MULTIVARIATE and SPEARMAN’S RHO procedure of
JMP IN. Second, phylogenetically independent contrasts (PICs, Felsenstein 1985) were used by the CONTRAST method in PHYLIP v3.66 (Felsenstein 1989; Felsenstein 2004) in combination with the BIVARIATE procedure of JMP IN. However, selfing rates may not be the best measure of inbreeding history (Husband and Schemske 1996; Latta and Ritland
1994). The inbreeding coefficient (F) may be a more appropriate measure because estimates of F should also reflect inbreeding that may arise for reasons other that the mating system
(e.g. genetic bottlenecks, population substructure). Additionally, the selfing rates estimated by
MLTR only reflect the progeny generation, while F will include any effects from historical selfing. Thus, the correlation between population inbreeding coefficient (F) and cumulative
57 relative performances (CFIT RPmean) was also evaluated with the Spearman Rank Correlation and PICs.
58 Results
MATING SYSTEM ESTIMATES
Mating system parameters (selfing rates and inbreeding coefficients, Table 2) were estimated by inferring progeny genotypes at four variable loci (Adh-1, Pgi-2, Pgm-2, and
Mdh-3). All of these loci exhibited disomic inheritance except for the tetraploid C. rubra population (SM), which demonstrated tetrasomic inheritance (Pgi-2) in preliminary artificial crosses (data not shown). Field-collected seeds from SM were variable for Pgi-2, but all progeny were homozygous; thus, this population was entirely selfing. Multi-locus and single- locus measured selfing rates were not significantly different (± 0.01) for all populations, so only single-locus selfing rates are reported (Table 2).
The three populations of C. parviflora ssp. grandiflora were primarily outcrossing (sp
= -0.01 to 0.15). One population (SRC) had a negative selfing rate, however, it was not significantly different than zero. Since C. parviflora ssp. grandiflora is protandrous, the measured selfing levels are most likely due to geitanogamous selfing. Inbreeding coefficients for these populations ranged from 0.24 to 0.45.
All other populations were highly selfing (sp ≥ 0.93), with the exception of one hexaploid population, ARC2 (sp = 0.79). The other hexaploid population (SAC2) was completely selfing (sp = 1, F = 1). Both diploid C. perfoliata ssp. mexicana populations (LC and OM) were entirely selfing. These populations were observed to be almost exclusively cleistogamous in the field, but many individuals had partially opened flowers while growing in the greenhouse. One tetraploid population (PFR) of C. parviflora ssp. parviflora showed low levels of outcrossing (sp = 0.92) in the progeny generation and, as suggested by the parental inbreeding coefficient (F = 0.84), has probably had occasional historical outcrossing
59 events. The other tetraploid population (KRC) was completely selfing. Both the diploid and tetraploid populations of C. rubra showed complete inbreeding in prior generations (F = 1).
However, the diploid population (FCC) showed outcrossing events in the progeny generation
(sp = 0.96).
INBREEDING DEPRESSION WITHIN POPULATIONS
All traits measured, with the exception of survival to reproduction, show some level of inbreeding depression in at least one population. Table 3 summarizes the descriptive statistics for all populations and traits, the results of the paired tests, and the values for RP.
Number of seeds per fruit (SEED) had significant inbreeding depression for the three diploid C. parviflora ssp. grandiflora populations (0.49, 0.44, and 0.20 for ARC1, SAC1,
SRC, respectively). Five populations (ARC2, FCC, KRC, OM, and SM) showed outbreeding depression for SEED, though none were significantly different than zero. Two populations,
SM (4x C. rubra) and SRC (2x C. parviflora ssp. grandiflora), had significant inbreeding depression for percentage of seeds germinating (GERM). No populations showed any significant inbreeding depression for survival to reproduction (SURV). For the most part, all individuals survived to reproduction after germination. The exceptions were a few self- fertilized individuals from C. parviflora ssp. grandiflora populations. Number of flowers produced (FLWRS) had the highest levels of inbreeding depression for the four measures of fitness. Nine of the eleven populations had inbreeding depression for FLWRS. The exceptions were the hexaploid ARC (C. perfoliata) and the diploid FCC (C. rubra), which had non- significant positive values for RP of FLWRS.
60 Measured relative performance for cumulative fitness (RP CFIT) values ranged from -
0.03 for hexaploid ARC2 to 0.78 for diploid SAC1. The three populations of C. parviflora ssp. grandiflora had the highest levels of inbreeding depression (0.76, 0.78, and 0.63 for
ARC1, SAC1, and SRC, respectively). The diploid C. perfoliata ssp. perfoliata populations were almost identical for RP CFIT; OM was 0.32 and LC was 0.33. Tetraploid C. parviflora had the largest range of values with RP CFIT for PFR being 0.22 and KRC having a value of
0.42; PFR was not significantly different than zero and KRC was the highest of all selfing populations and polyploidy populations. For C. rubra, the diploid population (FCC) had lower cumulative inbreeding depression (RP CFIT = 0.14, not significantly different than zero) than the tetraploid population (SM, RP CFIT =0.27). One hexaploid population, SAC2, had low inbreeding depression (RP CFIT = 0.20), and the other, ARC2, was the only population to exhibit measured outbreeding depression (RP CFIT = -0.03). Neither of these values were significantly different than zero. Paradoxically, ARC2 had the lowest selfing rate
(sp = 0.79) of all selfing populations. Overall, four of the five measures of fitness (SEED,
GERM, FLWRS, and CFIT) had some degree of outbreeding depression in all populations.
Approximately 10% of the families across all populations showed outbreeding depression (RP
< 0) for CFIT.
INBREEDING DEPRESSION AMONG AND WITHIN HIERARCHICAL LEVELS
Cumulative inbreeding depression as measured by relative performance of cumulative fitness (RP CFIT) was analyzed among and within phylogenetic groups with nested and one- factor ANOVAs (Table 4). The nested ANOVA among phylogenetic groups had statistically significant results for both the primary factor of phylogenetic groups and nested factor of
61 populations [phylogenetic groups]. These were further analyzed with a Tukey test to determine which phylogenetic groups and populations were significantly different. For phylogenetic groups, PERF (RP CFIT = 0.24) and RUBRA (RP CFIT = 0.20) were not significantly different from each other, but both were significantly different from GRAND
(RP CFIT = 0.72). This result conforms to the prediction of lower in breeding depression in selfing populations (e.g. PERF and RUBRA) compared to outcrossing populations (e.g.
GRAND). For populations nested in phylogenetic groups, differences are summarized in
Table 5.
Nested and one-factor ANOVAs within phylogenetic groups had statistically significant results for the PERF group but not for GRAND or RUBRA (Table 4). The nested
ANOVA for PERF resulted in no significant differences among ploidy; significant effects for population [ploidy] were detected. Population [ploidy] was further analyzed with a Tukey test to determine which populations were significantly different. Results are congruent with those found for the populations [phylogenetic groups] analysis described above and presented in
Table 5. Hexaploid C. perfoliata populations had the lowest levels of RP CFIT (ARC2 = -
0.03, SAC2 = 0.19). One hexaploid population (ARC2) was significantly lower than both of the diploid C. perfoliata populations (LC = 0.33, OM = 0.32) and one of the tetraploid C. parviflora populations (KRC = 0.43); the other hexaploid population (SAC2) was not significantly different from any other population within PERF. Tetraploid C. parviflora populations were not significantly different than diploid C. perfoliata populations.
62 CORRELATION OF INBREEDING AND INBREEDING DEPRESSION
Neither of the Spearman’s rank correlation analyses between the primary selfing rate
(sp) and RP CFIT, nor the inbreeding coefficient (F) and RP CFIT were significant.
Spearman’s rank correlation coefficient between sp and RP CFIT was ρs = -0.353 (df = 10, p =
0.29) and between F and RP CFIT was ρs = -0.491 (df = 10, p = 0.13). This is likely, in part, due to the high variance in RP CFIT of completely selfing populations. The PICs analyses, however, resulted in a significant correlation between the primary selfing rate (sp) and RP
CFIT (df = 9, r = 0.96, p < 0.001); yet the PICs correlation between the inbreeding coefficient
(F) and RP CFIT was not significant (df = 9, r = 0.59, p < 0.073). A graphical depiction of the result for sp, F, and RP CFIT is shown in fig. 2.
63 Discussion
This study is one of the first to explicitly consider the effect of polyploidy and selfing rate on inbreeding depression (also see Barringer and Gerber unpublished data; Husband and
Schemske 1997; Johnston and Schoen 1996). Here it is found that inbreeding depression is lower in highly selfing populations, and that among the selfing populations, inbreeding depression is lowest for hexaploids. Diploid and tetraploid selfing populations, however, showed no difference in the levels of inbreeding depression.
EFFECT OF MATING SYSTEM
Theoretical investigations (e.g. Charlesworth et al. 1990; Lande and Schemske 1985) predict relatively low levels of inbreeding depression in populations that predominantly self- fertilize in comparison to outcrossing populations; selfing increases homozygosity and the subsequent purging of deleterious alleles from the population results in lower genetic load and inbreeding depression (Lande and Schemske 1985). The research presented here supports this expectation and shows that mating system was the most important factor for explaining levels of cumulative inbreeding depression; the three outcrossing populations had significantly higher inbreeding depression than all selfing populations. This is not surprising, as this trend has been observed across a large number of species and studies (Husband and Schemske
1996).
Inbreeding depression was primarily observed in two ontogenetic stages: SEED and
FLWR. Most of the populations had significant inbreeding depression for FLWRS (Table 3), and the three outcrossing C. parviflora ssp. grandiflora populations showed significant inbreeding depression for SEED. This is in agreement with the findings of Husband and
64 Schemske (Husband and Schemske 1996); they found that inbreeding depression of seed production is often very high in outcrossing plant populations. Species that predominantly outcross, yet which are self-compatible, may have reduced seed set after artificial selfing due to cryptic or partial self-incompatibility (Eckert and Allen 1997). This makes it difficult to differentiate between developing seed mortality due to early-acting inbreeding depression vs. cryptic self-incompatibility. It is possible that the high levels of inbreeding depression for
SEED in C. parviflora ssp. grandiflora may actually be due to partial self-incompatibility.
However, self-incompatibility has not previously been reported in Claytonia or any other
Portulacaceous species. Additionally, many self-pollinated treatments in C. parviflora ssp. grandiflora populations resulted in presence of visible aborted seeds; this is indicative of early acting inbreeding depression rather than self-incompatibility. In any event, exclusion of SEED from the data set would still result in high cumulative inbreeding depression for C. parviflora ssp. grandiflora.
EFFECT OF PLOIDY
The relationship between inbreeding depression and polyploidy is very complex and theoretical investigations vary in their conclusions: inbreeding depression in polyploids may be higher, lower, or equal to that of a diploid (Bennett 1976; Bever and Felber 1992; Busbice and Wilsie 1966; Lande and Schemske 1985; Otto and Whitton 2000; Ronfort 1999). Models that consider inbreeding depression in a random mating (i.e. outcrossing) population generally predict that inbreeding depression will be greater in autopolyploids if inbreeding depression is caused by overdominance (e.g. Bennett 1976; Bever and Felber 1992; Busbice and Wilsie
1966), lower if inbreeding depression is cause by partial dominance in auto- or allopolyploids
65 (Lande and Schemske 1985; Ronfort 1999), and equal if inbreeding depression is caused by quantitative variation in allopolyploids (Lande and Schemske 1985). Unfortunately, this paper was unable to include outcrossing polyploid populations, so a test of predictions from these models is beyond the scope of this paper.
In contrast to the random mating models above, Ronfort (1999) assumed mutation- selection equilibrium and inbreeding depression due to partial dominance in populations of partially selfing autotetraploids. For highly selfing autopolyploid populations, Ronfort (1999) showed that autopolyploids will generally have lower inbreeding depression than diploids.
However, in some specific cases there will either be no difference between diploids and autotetraploids, or higher inbreeding depression in autotetraploids. Inbreeding depression is predicted to be higher in tetraploids when the dominance coefficient for the diploid is much larger than the dominance coefficients for the tetraploid (i.e. h >> h1, h2, h3) or equal under complete recessivity (h = h1 = h2 = h3 = 0). Alternatively, autotetraploids could also have higher inbreeding depression if the mutation rates in the tetraploid are much higher than in the diploid.
In this paper, the only diploid-autopolyploid pair is the two C. rubra populations; both were highly selfing. While the diploid (FCC) and tetraploid (SM) populations were not significantly different than one another, the diploid did have lower cumulative inbreeding depression than the polyploid (Table 5). Thus, based on the statistical analysis, the results for
C. rubra support a case where alleles contributing to inbreeding depression primarily exhibit complete recessivity. Alternatively, there may not be enough power to detect a significant difference between the two populations (which would possibly indicate large differences between dominance coefficients, Ronfort 1999).
66 Currently, formal theoretical models examining the joint evolution of self-fertilization and inbreeding depression in allopolyploids do not exist. Lande and Schemske (1985) provide the only treatment of the relationship between inbreeding depression and allopolyploidy; yet, their results are limited to inbreeding depression upon selfing in random mating populations and are thus not appropriate for predicting results in highly selfing populations. Heuristically, however, it may be fitting to assume that inbreeding depression will be relatively lower in selfing allopolyploids due to the phenomenon of “fixed” heterozygosity (i.e. different alleles among homeologous loci regardless of homozygosity within homologous loci).
Empirical evidence has produced conflicting results. For example, in a study of mixed- mating Anthericum species, Rosquist (2001) found that allopolyploids had lower inbreeding depression than the closely related diploids for traits that measure early acting inbreeding depression. Similarly, Barringer and Gerber (unpublished data) found that inbreeding depression in Clarkia was lower in selfing allopolyploids than in selfing diploids. In contrast,
Johnston and Schoen (1996) reported higher inbreeding depression in selfing allotetraploids than selfing diploids in Amsinckia.
The results reported here also provide conflicting results. Tetraploid C. parviflora ssp. parviflora, a putative allopolyploid, had one population (KRC) with higher inbreeding depression than diploid C. perfoliata (a putative parent species) and another population (PFR) with lower inbreeding depression. As a group, the inbreeding depression levels were approximately equal to the diploids. Hexaploid C. perfoliata, which has putative allopolyploid origins, provides the best evidence for lower inbreeding depression in allopolyploids. The hexaploid populations had lower inbreeding depression than either the diploids or the tetraploids.
67 Autogamous hexaploid populations of C. perfoliata ssp. perfoliata, along with hexaploid C. parviflora ssp. parviflora (not sampled here), are the most widespread cytotypes within the complex (Miller 1976; Miller 1978; Swanson 1964). These hexaploids primarily colonize and occupy highly disturbed habitats (e.g. roadsides, ephemeral drainages, landslides, etc.). This suggests that ruderal populations can often be founded by only a few individuals. Lande and Schemske (1985) suggest that small populations, perhaps those which have recently undergone a genetic bottleneck, may result in relatively lower levels of inbreeding depression. Explicit investigations of populations size and bottlenecks
(Charlesworth et al. 1992; Kirkpatrick and Jarne 2000) often suggest that this will have a negligible effect on the degree of inbreeding depression. However, Bataillon and Kirkpatrick
(2000) found that inbreeding depression will have a positive relationship with populations size (i.e. inbreeding depression increases as population size increases). The reason for this is that as small populations become fixed for alleles (both beneficial and deleterious) inbreeding depression is low because individuals are effectively mating with relatives (i.e. all individuals share the same fixed alleles). In the case of highly selfing Claytonia populations with low inbreeding depression (e.g. hexaploid C. perfoliata ssp. perfoliata), cross-pollination treatments may have actually been among closely related individuals descended from the same initial colonizers and thus not represent a case where polyploidy has a significant lowering effect on inbreeding depression.
CONCLUSIONS
The research presented here is one of a few studies (Barringer and Gerber unpublished data; Husband and Schemske 1997; Johnston and Schoen 1996) to explicitly consider
68 inbreeding depression and mating system of closely related diploid and polyploid populations.
This study found that inbreeding depression is lower in selfing populations than in outcrossing populations, regardless of ploidy; a finding that is in agreement with a large body of theoretical and empirical research.
Theoretical investigations of the relationship between polyploidy and inbreeding depression vary in their predictions: inbreeding depression may be less than, equal to, or greater than that of diploids depending on the assumptions of the models. The research presented in this paper considers inbreeding depression in six diploid populations, four putative allopolyploid selfing populations, and one putative autotetraploid selfing population.
Only one model has explicitly considered inbreeding depression in selfing autopolyploids:
Ronfort (1999) showed that inbreeding depression is generally predicted to be lower in selfing autopolyploids, but under some cases it may be equal or greater than diploids. The one autotetraploid population in this study showed no significant difference in inbreeding depression when compared to a related diploid population.
Inbreeding depression in the two putative allotetraploid selfing populations were also not significantly different than the related diploids. However, the two putative allohexaploid populations had the lowest inbreeding depression of all populations; they were significantly different than the related diploids. This may be a case where inbreeding depression is lower due to the effects of ploidy. Then again, as described above, this observation may also be due to the colonizing nature of this cytotype. It may be that there is no true difference between diploids and allopolyploids. Unfortunately, formal theoretical predictions of inbreeding depression in selfing allopolyploids do not exist at this time. Thus, there is a need for
69 theoretical investigation of the joint evolution of selfing and inbreeding depression in allopolyploids (perhaps considering the effect of fixed heterozygosity).
The effect of polyploidy on inbreeding depression is inconclusive for the C. perfoliata complex. This study highlights the need for further empirical and theoretical research of the joint evolution of selfing and inbreeding depression in polyploid plants.
70 Acknowledgements
Thanks to R. Gomulkiewicz, L Hufford, B. Husband, and S. Nuismer for discussions and comments on earlier versions of this manuscript. Thanks to E. Roalson for the use of laboratory facilities and C. Cody for help with greenhouse operations. This research also benefited from discussions with J. Brokaw, B. Barringer J. Smith, S. Novak, P. McIntyre, K.
Chambers, J. Miller, and M. Morgan. Funding was provided by the Betty W. Higginbothan
Fellowship and the National Science Foundation grants DEB 0128896 to M. Morgan and
DEB 0209916 to R. Gomulkeiwicz
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78 Table 1. Taxa and populations of the C. perfoliata complex used in this study. Population abbreviations (see text for full population names), chromosome number of sporophytes (2n), phylogenetic group designation from
Rausch (unpublished data, Chapter 4), and approximate locations of the populations given as county, state, latitude (W), and longitude (N).
Phylogenetic Location Species / subspecies 1 Population 2n = group 2 County Latitude Longitude C. parviflora ssp. grandiflora ARC1 12 GRAND El Dorado, CA 38.914 ° -121.040 °
SAC1 12 GRAND Calaveras, CA 38.198 ° -120.444 °
SRC 12 GRAND Tuolumne, CA 38.061 ° -120.378 °
C. parviflora ssp. parviflora KRC 24 PERF Kern, CA 35.531 ° -118.663 °
PFR 24 PERF Fresno, CA 36.907 ° -119.299 °
C. perfoliata ssp. mexicana LC 12 PERF Santa Barbara, CA 34.508 ° -120.225 °
OM 12 PERF Ventura, CA 34.459 ° -118.903 °
C. perfoliata ssp. perfoliata ARC2 36 PERF El Dorado, CA 38.915 ° -121.039 °
SAC2 36 PERF Calaveras, CA 38.203 ° -120.452 °
79 Table 1. continued.
C. rubra ssp. rubra FCC 12 RUBRA Kittitas, WA 47.351 ° -121.116 °
SM 24 RUBRA Harney, OR 42.663 ° -118.988 °
1. As per Miller and Chambers (1993; 2006)
2. From Rausch (unpublished data, Chapter 4)
80 Table 2. Mating system parameters of populations used in this study. Given are the variable allozyme loci used to infer selfing rates and parental inbreeding coefficients, and the number of families and individuals examined per population.
Selfing rate Number of Parental Variable Population 2n = families measured, primary, inbreeding coefficient, Enzyme (progeny) sm (s.e.) sp (s.e.) F (s.e.)
ARC1 12 Pgm-2, Mdh-3 20 (98) 0.13 (0.08) 0.20 (0.03) 0.45 (0.17)
SAC1 12 Pgm-2 31 (149) 0.15 (0.12) 0.22 (0.04) 0.24 (0.17)
SRC 12 Pgm-2, Mdh-3 24 (115) -0.01 (0.05) -0.02 (0.02) 0.40 (0.16)
KRC 24 Adh-1 28 (160) 1.00 (0) 1.00 (0) 1.00 (0)
PFR 24 Pgi-2 26 (130) 0.92 (0.03) 0.93 (0.01) 0.84 (0.12)
LC 12 Pgm-2 20 (81) 1.00 (0) 1.00 (0) 1.00 (0)
OM 12 Pgi-2 20 (96) 1.00 (0) 1.00 (0) 1.00 (0)
ARC2 36 Mdh-3 28 (140) 0.79 (0.03) 0.78 (0.01) 0.81 (0.13)
SAC2 36 Pgi-2 32 (160) 1.00 (0) 1.00 (0) 1.00 (0)
FCC 12 Pgi-2 30 (150) 0.96 (0.02) 0.96 (0.01) 1.00 (0)
SM 24 Pgi-2 25 (120) 1.00 (0) 1.00 (0) 1.00 (0)
81 Table 3. Wilcoxon signed-rank tests, T, and paired t-tests for seeds per fruit (SEED), percent germination
(GERM), percent survival (SURV), number of flowers per plant (FLWRS). Other information includes, number of individuals N, mean population value for selfed individuals X S , mean population value for outcrossed individuals X O , standard errors (s.e.), and mean relative performance RPmean. € Trait Taxa C. parviflora C. parviflora C. perfoliata € ssp. grandiflora ssp. parviflora ssp. mexicana Ploidy 2x 4x 2x Population ARC1 SAC1 SRC KRC PFR LC OM SEED N 27 24 32 28 29 29 24
X S (s.e.) 1.4 (0.17) 1.6 (0.23) 2.2 (0.16) 2.6 (0.16) 2.6 (0.16) 2.6 (0.12) 2.3 (0.20)
X O (s.e.) 2.9 (0.06) 2.8 (0.08) 2.8 (0.09) 2.5 (0.17) 2.7 (0.13) 2.8 (0.09) 2.5 (0.19) € RPmean 0.49 0.44 0.20 -0.02 0.05 0.05 -0.08
€ T Wilcoxon 144 * 81.0 * 72.0 * -5.0 6.5 13.0 12.5 GERM N 22 19 30 24 27 29 22
X S (s.e.) 0.71 (0.07) 0.58 (0.07) 0.61 (0.07) 0.61 (0.05) 0.64 (0.06) 0.61 (0.06) 0.67 (0.04)
X O (s.e.) 0.82 (0.06) 0.79 (0.06) 0.87 (0.05) 0.81 (0.05) 0.70 (0.05) 0.78 (0.05) 0.76 (0.05)
€ RPmean 0.09 0.19 0.28 0.20 0.06 0.20 0.08
€ T Wilcoxon 13.0 44.0 55.0 * 63.5 36.0 52.0 25.5
82 Table 3. continued
SURV N 19 16 24 23 25 26 21
X S (s.e.) 0.89 (0.07) 0.88 (0.10) 0.88 (0.07) 1 (0) 1 (0) 0.96 (0.04) 1 (0)
X O (s.e.) 1 (0) 0.96 (0.04) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) € RPmean 0.11 0.13 0.13 0 0 0.04 0
€ T Wilcoxon 1.5 1.5 3.0 0 0 0.5 0 FLWR N 17 14 21 23 25 26 21
X S (s.e.) 57.0 (6.0) 54.6 (5.5) 74.4 (8.1) 83.7 (4.0) 79.0 (5.9) 99.3 (6.6) 118.4 (4.8)
X O (s.e.) 150.0 (9.6) 145.0 (9.7) 147.2 (6.3) 122.2 (5.8) 114.9 (6.0) 135.0 (7.4) 134.4 (5.6)
€ RPmean 0.60 0.61 0.50 0.31 0.32 0.26 0.11
€ t-ratio 9.21 *** 9.98 *** 9.66 *** 8.76 *** 7.06 *** 5.38 *** 3.12 * lnCFIT N 17 14 21 22 25 25 21
X S (s.e.) 4.1 (0.13) 4.2 (0.12) 4.6 (0.16) 4.9 (0.10) 4.8 (0.15) 5.1 (0.09) 5.2 (0.10)
X O (s.e.) 5.8 (0.11) 5.8 (0.13) 5.8 (0.07) 5.6 (0.10) 5.2 (0.09) 5.6 (0.11) 5.7 (0.07)
€ RPmean 0.76 0.78 0.63 0.42 0.22 0.33 0.32
€ t-ratio 9.36 *** 14.3 *** 8.04 *** 4.74 ** 2.5 4.31 * 4.87 ***
* p < α adj = 0.05, ** p < α adj = 0.01, *** p < α adj = 0.001
83 Table 3. continued
C. perfoliata C. rubra ssp. perfoliata ssp. rubra 6x 2x 4x ARC2 SAC2 FCC SM 30 32 26 29
2.8 (0.03) 2.8 (0.07) 2.9 (0.11) 3.0 (0.03)
3.0 (0.14) 3.0 (0.03) 2.8 (0.12) 2.7 (0.15)
-0.07 0.06 -0.01 -0.09
-3.5 10.5 -0.5 -5.0
28 32 24 27 0.86 ± 0.04 0.70 ± 0.05 0.78 ± 0.05 0.69 ± 0.06 0.79 ± 0.05 0.80 ± 0.04 0.75 ± 0.06 0.94 ± 0.03 -0.07 0.10 -0.03 0.27 -22.0 39.0 < 0.1 64.5 *
84 Table 3. continued
28 31 23 26
1 (0) 1 (0) 1 (0) 1 (0)
1 (0) 1 (0) 1 (0) 1 (0)
0 0 0 0
0 0 0 0
28 31 23 26
196.5 (5.3) 181.6 (5.8) 63.8 (4.3) 70.3 (3.4)
207.3 (5.8) 202.5 (7.9) 79.4 (4.6) 76.3 (3.6)
0.05 0.09 0.20 0.08
2.62 4.08 * 3.14 * 1.51
28 31 23 26
6.2 (0.07) 5.8 (0.09) 4.9 (0.12) 4.9 (0.09)
6.1 (0.08) 6.1 (0.07) 5.1 (0.12) 5.3 (0.06)
-0.03 0.20 0.14 0.27
-0.50 2.61 1.46 3.68 *
85 Table 4. Analyses of variance for relative performance of cumulative fitness (RP CFIT) within and among phylogenetic groups of fig. 1.
Sum of Mean Source DF F Ratio Squares Square
Among Phylogenetic Groups
Phylogenetic Group 2 9.73 4.87 11.12 **
Population [Phylogenetic Group] (r) 8 3.69 0.46 3.48 ***
Error 243 32.22 0.13
Total 253 45.75
Within GRAND
Population (r) 2 0.23 0.12 2.87
Error 49 1.98 0.04
Total 51 2.21
Within PERF
Ploidy 2 2.12 1.06 2.56
Population [Ploidy] (r) 3 1.25 0.42 2.70*
Error 147 22.63 0.15
Total 152 25.88
Within RUBRA
Ploidy 1 0.20 0.20 1.25
Error 47 7.61 0.16
Total 48 7.81
* p < 0.05, ** p < 0.01, *** p < 0.001
86 Table 5. Results of the Tukey test for multiple comparisons of population nested in phylogenetic group and population nested in ploidy for PERF. Given are the populations, ploidy, phylogenetic group, and mean relative performance of cumulative fitness (RPmean
CFIT) in descending order. Values connected by the same letter are not significantly different.
Population Ploidy Group RPmean CFIT
SAC1 2x GRAND 0.78 a
ARC1 2x GRAND 0.76 a
SRC 2x GRAND 0.63 a b
KRC 4x PERF 0.42 a b c
LC 2x PERF 0.33 b c
OM 2x PERF 0.32 b c
SM 4x RUBRA 0.26 c d
PFR 4x PERF 0.22 c d
SAC2 6x PERF 0.19 c d
FCC 2x RUBRA 0.14 c d
ARC2 6x PERF -0.03 d
87 Figure Legends
Figure 1. Maximum likelihood tree of the combined cpDNA and nrDNA data set (Rausch,
unpublished data, Chapter 4) showing the phylogenetic relationships among the
populations used in this study. Bootstrap values are given below the branches. Shaded
areas correspond to the phylogenetic groups used in the nested ANOVA and presented
in Tables 1 and 5.
Figure 2. Bivariate plot of populations for relative performance of cumulative fitness by
primary selfing rate (left pane) and inbreeding coefficient (right pane).
88 Figure 1:
89 Figure 2:
90 CHAPTER FOUR
PHYLOGENETIC RELATIONSHIPS AND POLYPLOID ORIGINS
WITHIN THE CLAYTONIA PERFOLIATA POLYPLOIDY COMPLEX
(PORTULACACEAE)
JOSEPH H. RAUSCH
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236
[Formatted for Systematic Botany]
91 ABSTRACT. The Claytonia perfoliata polyploid complex is a morphologically diverse group that includes diploid through decaploid cytotypes. Here, nrDNA (ITS) and cpDNA
(matK/trnK) are used to determine the phylogenetic relationships among diploid and polyploid taxa and to infer polyploid origins. This study provides the first molecular approach that considers all taxa and cytotypes within this well studied polyploidy complex. Results from ITS indicate three primary clades of diploids corresponding to C. perfoliata ssp. mexicana, C. parviflora ssp. grandiflora, and C. rubra ssp. rubra. Results from matK/trnK show a well-supported C. rubra ssp. rubra clade that is sister to a C. perfoliata ssp. mexicana and C. parviflora ssp. grandiflora clade. Polyploids have two principle geographic regions of genetic, cytological, and morphological diversity: the Sierra Nevada Mountains of California and the Columbia River Gorge of Washington and Oregon. The majority of polyploids share identical sequences with diploids, yet others possess unique polyploid sequences. By comparing incongruence among gene trees, multiple independent origins of allopolyploids are found to be common and numerous, especially for tetraploids of C. parviflora and hexaploids of C. perfoliata. Evidence for autopolyploidy is less common, but it does occasionally occur.
The implications for taxonomy of this morphologically confusing group are discussed.
92 INTRODUCTION
Claytonia perfoliata s.l. (Portulacaceae: Montieae) is a morphologically diverse species complex composed of a variable array of annual diploids and polyploids (from tetraploid to decaploid). The complex is typified by a single perfoliate leaf that subtends the inflorescence. Taxonomic treatments of the C. perfoliata complex have variably included it within either Montia (e.g. Hitchcock 1964; Howell 1893; Pax and Hoffman 1934), Limnia
(Rydberg 1932) or Claytonia (e.g. McNeill 1975; Miller and Chambers 1993; Swanson 1966).
Recent molecular work by O’Quinn and Hufford (2005) clearly shows that the C. perfoliata complex is a monophyletic group within McNeill’s (1975) Claytonia section Limnia.
Additionally, they found support for the inclusion of C. washingtoniana in the C. perfoliata complex; C. washingtoniana is a putative allotetraploid of C. perfoliata and C. sibirica
(Fellows 1971).
The most recent revision of the C. perfoliata complex by Miller and Chambers (1993;
2006) recognize three species and nine subspecies (table 1). Their treatment is primarily based on growth habit, ontogenetic morphology of basal leaves, and the geographic distribution of cytotypes. Claytonia parviflora includes erect plants with linear, filiform, or narrowly spatulate basal leaves, C. perfoliata includes erect to sub-erect plants with linear juvenile leaves and obovate, rhombic, deltate, or reniform basal leaves at maturity, and C. rubra includes plants with flattened rosettes and spatulate, rhombic, or deltate juvenile and mature leaves. Miller and Chambers (1993; 2006) describe Claytonia perfoliata s.l. as a polyploid
93 complex based upon three morphologically distinct diploid (2n = 12) taxa: C. perfoliata ssp. mexicana, C. parviflora ssp. grandiflora, and C. rubra ssp. rubra (table 1).
Polyploids of the C. perfoliata complex are common, morphologically diverse, and geographically widespread. They well exceed the geographic range of diploids and are also successful invaders of disturbed habitats in temperate regions of western North America,
Europe, South America, and New Zealand (Miller and Chambers 2006). Polyploid cytotypes range from tetraploid to decaploid, and early research of polyploidy within the complex by
Miller (1976) and Swanson (1964) identified many potential cytological / morphological types which have since been placed within various subspecies within the complex (table 1;
Miller and Chambers 1993; Miller and Chambers 2006). The subspecies are defined by unique combinations of geography and morphology (as described above), and thus do not necessarily reflect common ancestry. Six of the nine subspecies are entirely polyploid (table
1, Miller and Chambers 2006).
Published accounts of polyploidy in the C. perfoliata complex are copious and extensive (Fellows 1976; Heiser and Whitaker 1948; Lewis 1962; Lewis 1963; Lewis 1967;
Miller 1976; Miller 1978a; Miller 1978b; Miller 1978c; Miller 1984; Miller and Chambers
2006; Miller et al. 1984; Nilsson 1966; Nilsson 1967; Raven 1962; Swanson 1964; Taylor and
Taylor 1977); however, only one study (Miller 1978b) has attempted to determine the origins of polyploid taxa and cytotypes within the complex. Miller (1978b) used morphology to postulate a hypothetical scheme of polyploid origins that included autopolyploids, allopolyploids, and homoploid hybrids. He noted that many cytotypes, principally tetraploids, are morphologically indistinguishable from their respective diploid conspecifics and are assumed to be autopolyploids. Other cytotypes have unique or intermediate morphologies that
94 suggest allopolyploid origins or homoploid hybridization (Miller 1976; Miller 1978b; Miller
1978c; Miller and Chambers 1993).
Traditionally, morphology and cytology have been used to infer polyploid origins (e.g.
C. perfoliata described above, Miller 1978b; Tragopogon, Ownbey 1950); however, the last two decades have seen an increase in research of polyploid origins using molecular genetic techniques (e.g. allozymes and DNA-sequencing, as reviewed in Soltis and Soltis 1993; Soltis and Soltis 1999; Soltis et al. 2004b). Allozymes are often used to infer polyploid origins by assessing the segregation patterns and dosage effects of electrophoretically different alleles.
Autopolyploids often show tetrasomic inheritance (e.g. Hardy et al. 2001), while allopolyploids typically show disomic inheritance and/or “fixed” heterozygosity (e.g. Arft and
Ranker 1998). However, older polyploid systems can become diploidized and exhibit strict disomic inheritance in both autopolyploids (e.g. Widen and Widen 2000) and allopolyploids
(Soltis et al. 2004b). Thus, incorrect inferences are possible, for example, if one assumes that disomic inheritance always indicates allopolyploidy, when in fact diploidized autopolyploids may also show disomic inheritance (e.g. Widen and Widen 2000).
The use of plastid and nuclear DNA sequences to evaluate polyploid origins can provide corroborating or contradictory evidence to that of allozymes and morphology. This approach can also show if multiple, recurrent polyploid origins have occurred. Numerous researchers have demonstrated that multiple independent origins of both allopolyploids (e.g.
Brochmann and Elven 1992; Soltis et al. 1995) and autopolyploids (e.g. Segraves et al. 1999;
Van Dijk and Bakx-Schotman 1997) are a common form of diversification. In fact, multiple origins appear to be the rule rather than the exception (Soltis and Soltis 1999; Soltis et al.
2004a).
95 Parentage of polyploids (specifically allopolyploids) can be determined by considering congruence among independent gene trees (Linder and Rieseberg 2004). In the vast majority of angiosperms chloroplasts are maternally inherited (Corriveau and Coleman 1988;
Mogensen 1996). This makes chloroplast DNA (cpDNA) ideal for inferring the maternal parentage of polyploids. A number of researchers (Campbell et al. 1997; Ge et al. 1999; Popp et al. 2005; Popp and Oxelman 2001; Sang et al. 1995) have used cpDNA in conjunction with the internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA) to investigate origins of polyploidy taxa.
The internal transcribed spacer is generally shown to exhibit “concerted evolution”: a homogenizing process where a family of genes (in this case ITS) comes to have the same genetic sequence (Alvarez and Wendel 2003). While this is not a problem for determining the parental origins of autopolyploids, several studies (e.g. Brochmann et al. 1996; Popp et al.
2005; Soltis et al. 2008; Wendel et al. 1995) have demonstrated that homoeologous ITS sequences in allopolyploids can be homogenized so that one of the parental sequences is lost.
Indeed, rapid concerted evolution in allopolyploids can occur over a relatively short period of evolutionary time (e.g. < 80 years, Kovarik et al. 2005; Soltis et al. 2008). If concerted evolution homogenizes the paternal sequence, the incongruence between the ITS and cpDNA gene tree can reveal allopolyploid origins. If, however, the maternal sequences are homogenized, the ITS sequences will not reveal the hybrid origins of the polyploid. Thus, autopolyploidy serves as the null hypothesis when inferring polyploid origins from congruence among gene trees (Popp and Oxelman 2007).
The objectives here are to use ITS and the matK/trnK region of the chloroplast genome to 1) determine the phylogenetic relationships among diploid taxa of C. perfoliata
96 complex, 2) determine the phylogenetic relationships among diploid and polyploid subspecies, cytotypes, and morphologically ambiguous entities within the C. perfoliata complex, 3) infer putative polyploid origins within the C. perfoliata complex by comparing topological congruence of gene trees, and 4) provide a revised taxonomic treatment based on the results of objectives one through three. This is the first molecular study to consider all taxa within this well-studied polyploidy complex.
97 MATERIALS AND METHODS
Taxon Sampling and Chromosome Counts. A total of 283 specimens of the C. perfoliata complex were collected from approximately 200 populations in the western United
States (California, Oregon, and Washington) during the spring and summer of 2005, 2006, and 2007. Populations sampling was designed to maximize the collection of morphologically and geographically diverse specimens. Specimens were pressed and leaf tissue samples were dried in silica gel for DNA extraction. Young inflorescences were also sampled and fixed in a mixture of chloroform: ethanol: glacial acetic acid (4:3:1, v/v/v) for chromosome counts.
Pressed specimens were deposited at the Marion Ownbey Herbarium (WS) at Washington
State University; collection numbers are the author’s (JHR) unless otherwise noted.
Of the 283 collected specimens, 124 were selected for chromosome counts. These were chosen to emphasize geographic and morphological diversity, and to ensure that all 11 taxa (including subspecies) were represented in the analyses. Fixed inflorescences were transferred and stored in a 70% solution of aqueous ethanol at < 0° C. Staining of gametophytic mother cells was accomplished with the ethanol-hydrochloric acid-carmine methods of Snow (1963) and Miller (1976). Visualization of chromosomes was accomplished under 1000x (oil-emersion) magnification.
Claytonia sibirica was selected as the outgroup, as this species and related taxa (e.g.
C. exigua, C. palustris) have previously been found to be sister to the C. perfoliata complex based on molecular data (Hershkovitz and Zimmer 2000; O'Quinn and Hufford 2005) and
98 morphological / cytological / phytochemical data (Miller and Chambers 2006). Additionally,
C. sibirica is a putative parent species of the tetraploid C. washingtoniana.
DNA Extraction, Amplification, and Sequencing. Total genomic DNA was isolated from silica-dried material of field-collected plants using DNeasy Plant Mini DNA Extraction
Kits according to the manufacturer’s instructions (Qiagen Inc., Valencia, California, USA).
The primers matK-1470R (5'-AAG ATG TTG ATT GTA AAT GA-3') and trnK-3914F (5’-
TGG GTT GCT AAC TCA ATG G-3’, Johnson and Soltis 1994) were used to amplify the 5’ end of the matK plastid coding region and the trnK intron (matK/trnK) using the polymerase chain reaction (PCR). Polymerase chain reaction reactions for matK/trnK contained 2 µL of forward and reverse primers in 10 µM concentrations, 2 µL of 4 µM dNTPs, 0.3 µL of 5
U/µM Taq DNA polymerase (cat. # M0267L, New England BioLabs, Ipswich, MA), 5 µL 1X
ThermoPol Buffer (New England BioLabs), 37.7 µL H2O, and 1 µL of ~50 ng/µL genomic
DNA, for a total reaction volume of 50 µL. Amplifications were accomplished with the following PCR profile: 3 min. at 95° C, followed by 35 cycles of 1 min. at 95° C, 1 min. at
48° C, and 2 min. at 72° C, and a final extension of 72° C for 7 min.
The internal transcribed spacer (ITS) and the 5.8S coding region of the nuclear ribosomal tandem repeat were amplified using the primers N-nc18S10 (5’-AGG AGA AGT
CGT AAC AAG-3’) and C26A (5’-GTT TCT TTT CCT CCG CT-3’, Wen and Zimmer
1996). Reaction concentrations and volumes for PCR were the same as matK/trnK. The PCR amplification profile was 3 min. at 94° C, followed by 35 cycles of 1 min. at 94° C, 1 min. at
48° C, and 2 min. at 72° C, and a final extension of 72° C for 15 min.
Double stranded PCR products were purified using ExoSAP type enzymatic incubation. ExoSAP reactions included 8 µL PCR product, 0.2 µL Exonuclease I (cat. #
99 M0293S, New England BioLabs), 0.5 µL Antarctic Phosphotase (cat. # M0289S, New
England BioLabs), and 1.8 µL reaction buffer (50 mM Bis Tris-Propane, 1mM MgCl2, 0.1 mM ZnCl2, pH = 6.0). Reactions were incubated at 37° C for 45 min. followed by a enzymatic denaturing incubation of 80° C for 15 min.
Direct cycle-sequencing of cleaned template DNA was accomplished using the ABI
PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems) following manufacturer’s instructions. The primers used for the initial PCR were also used for cycle-sequencing of ITS. In addition to the primer pair used for PCR of the ~1300 base-pair matK/trnK region, two internal primers (360F [5'-CGG GAA AGG CTT CTC CCA CG-3'] and 670R [5'-GGA ATT TCC ACA ATG ACT GC-3'], O'Quinn and Hufford 2005) were used for cycle-sequencing. Purification of cycle-sequencing products used PERFORMA DTR
Gel Filtration Cartridges (cat. # 42453, Edge BioSystems, Gaithersburg, MD) or
PERFORMA DTR Ultra 96-Well Plate Kits (cat. # 55373, Edge BioSystems) following manufacturer’s specifications. Sequencing was preformed using an ABI PRISM 377 DNA
Sequencer (Applied Biosystems, Foster City, CA). Sequencing chromatograms were assembled, proofed, and edited with Sequencher 4.0 (Gene Codes Corporation, Ann Arbor,
MI), and consensus sequences were manually aligned in Se-Al v2.0a11 (Rambaut 1996).
Phylogenetic Analyses. Maximum likelihood (ML) analyses were performed on reduced data sets of diploid taxa only for matK/trnK, ITS, and combined data, and on complete data sets that included all unique sequences from both diploids and polyploids for matK/trnK and ITS. For all analyses, indels were treated as missing data and redundant identical sequences were removed to simplify the analysis. DT-ModSel (Minin et al. 2003), as implemented in PAUP* v4.0b10 (Swofford 2002), was used to select one of 56 models of
100 evolution. The DT-ModSel analysis employs Bayesian information criterion using a decision theory framework to select the most likely model based on error of branch lengths as a performance measure and penalties for model over-parameterization. The TIM + I + Γ, and
HKY + Γ models of molecular evolution were selected for the reduced diploid matK/trnK and
ITS data sets, respectively. The combined analysis of diploid matK/trnK and ITS sequences employed the GTR + I + Γ model. Model selection for the complete data sets of matK/trnK and ITS was the same as the reduced diploid data sets.
Maximum likelihood (ML) analyses were accomplished using PHYML (Guindon and
Gascuel 2003). The algorithm utilized by PHYML simultaneously adjusts the tree topology and the branch lengths to rapidly arrive at an optimal tree, and in the process avoids getting trapped near local optima. Neighbor-joining trees were constructed and used as the starting trees for the ML analyses following the BIONJ methods of Gascuel (1997). Model parameters determined by DT-ModSel were utilized for each analysis. Topological robustness of the ML tree was tested with 1000 bootstrap replicates (Felsenstein 1985)
In addition to bootstrap replicates, the approximate likelihood ratio test (aLRT) was used to infer branch support. The approximate likelihood ratio test is a relatively new approach to assess branch support for maximum likelihood trees (Anisimova and Gascuel
2006). The aLRT assesses the likelihood gain of a specific internal branch in comparison with the null hypothesis that the inferred branch does not exist if the remaining tree topology is maintained. Anisimova and Gascuel (2006) show through simulation that aLTR is generally more conservative than Bayesian posterior probabilities and more liberal than Felsenstein’s
(1985) bootstrap values. Branch support was estimated from the minimum value of either an aLTR parametric χ2–based branch support or an aLRT non-parametric branch support based
101 on a Shimodaira-Hasegawa-like procedure using aLRT-PHYML v1.1 (Anisimova and
Gascuel 2006; Guindon and Gascuel 2003). Clades with bootstrap and/or aLRT values >70% are considered to have support.
102 RESULTS
Chromosome Counts. All chromosome counts of the C. perfoliata complex showed normal meiosis and complete bivalent chromosome formation. Of the 124 attempted chromosome counts, 101 were successful; diploid (2n = 12), tetraploid (2n = 24), hexaploid
(2n = 36), octaploid (2n = 48), and decaploid (2n = 60) cytotypes were observed (table 1).
Chromosome counts were obtained from 47 individuals of C. perfoliata (6 ssp. mexicana; 29 ssp. perfoliata; 12 ssp. intermontana), 34 C. parviflora individuals (11 ssp. grandiflora, 19 ssp. parviflora, 2 ssp. utahensis. and 2 ssp. viridis), 18 C. rubra individuals (15 ssp. rubra; 3 ssp. depressa), and 2 individuals of C. washingtoniana. All of these specimens were sequenced for matK/trnK and ITS.
Phylogenetic Data. Of the 101 individuals sequenced for both matK/trnK and ITS, 27 of these were diploid (see fig. 1 caption for collector numbers), and 74 were polyploid (see figs. 2 and 3 for collector numbers). Sequences of matK/trnK had 29 variable sites (out of
1323 base pairs, 2.2% variability) with a total of 17 unique sequences (or cpDNA haplotypes).
Twelve unique sequences were obtained from diploid individuals (with some of the sequences also present in polyploids) and five were unique to polyploid individuals. Hereafter,
“matK/trnK sequence” will be used interchangeably with “matK/trnK haplotype”
Internal transcribed spacer (ITS) sequences were variable for 34 sites (out of 605 base pairs, 5.6% variability). Three ITS sequences showed two chromatographic peaks at one base pair, remaining sequences were unambiguous at all sites, indicating that homogenization of parental sequences has likely occurred in allopolyploid taxa (Alvarez and Wendel 2003).
103 There were a total of 22 unique ITS sequences. Twelve were present in diploid individuals
(and some polyploids), and ten were unique to polyploid individuals.
Phylogenetic Analyses. The phylogenetic analyses were divided into two major data sets: reduced analyses that consider diploids alone, and complete analyses that consider both diploids and polyploids together. Three ML analyses examined the combined diploid set
(unique diploid combinations of matK/trnK and ITS) of 16 samples, the reduced diploid set of
12 matK/trnK sequences, and the diploid set of 12 ITS sequences. Two ML analyses examined the complete (diploid + polyploid) matK/trnK and ITS data sets.
DIPLOIDS ONLY. Analysis of the combined diploid data set resulted in a single ML tree (fig. 1a, ln likelihood = -3382.76). Diploid C. rubra ssp. rubra forms a well-supported clade sister to the rest of the complex. All diploid specimens of Claytonia perfoliata ssp. mexicana and C. parviflora ssp. grandiflora form a supported clade, in which the latter is monophyletic and C. perfoliata ssp. mexicana is paraphyletic.
The separate analyses of diploid matK/trnK and ITS sequences resulted in trees with similar topologies as the combined diploid analyses. Both the matK/trnK tree (fig. 1b, ln likelihood = -2020.39) and ITS tree (fig. 1c, ln likelihood = -1255.95) have diploid C. rubra ssp. rubra clades as sister to the rest of the complex. Similarly, diploid C. parviflora ssp. grandiflora are monophyletic in both analyses. Incongruence among the trees arises from the placement of C. perfoliata ssp. mexicana. For matK/trnK, subspecies mexicana is paraphyletic to C. parviflora ssp. grandiflora (fig. 1b), but for ITS, subspecies mexicana forms a clade sister to C. parviflora ssp. grandiflora (fig. 1c).
The results displayed in figure 1a (and subsequent figures) show unique combinations of diploid matK/trnK and ITS sequences that are identified by the subspecific epithet and a
104 number corresponding to the unique genetic combination (e.g. rubra 1, rubra 2, …; mexicana
1, mexicana 2, …; see fig. 1). However, in some cases two or more diploid specimens have identical sequences for one gene but different sequences for another. For example, mexicana 4 and mexicana 5 have different matK/trnK haplotypes (fig. 1b), yet they share identical ITS sequences and are thus identified as mexicana 4/5 in the ITS tree (fig. 1c). This naming scheme represents unique combinations of sequences for a single diploid specimen, groups of diploid specimens with identical sequences and similar morphology, or a specific unique sequence that is shared by a group of diploids and polyploids. The following sections and figures will utilize this naming scheme.
DIPLOIDS AND POLYPLOIDS. The complete data set (diploids + polyploids) of matK/trnK contained 12 diploid sequences (from the reduced diploid data set) and five unique polyploid sequences. For ITS, there were 12 diploid sequences and 10 unique polyploid sequences. Unique polyploid sequences sometimes occur in multiple subspecies and cytotypes; for simplification, these are identified by the lowest ploidy and subspecies in which they occur (e.g. figs. 2 and 3). Furthermore, subspecific epithets will occasionally be used hereafter as a proxy for the complete Latin polynomial (e.g. C. perfoliata ssp. mexicana = subspecies mexicana = mexicana).
The complete analyses resulted in one ML tree for matK/trnK (fig. 2, ln likelihood = -
2046.11) and ITS (fig. 3, ln likelihood = -1355.92) with similar topologies as the reduced diploid analyses (fig. 1b and 1c, respectively). Diploid C. rubra ssp. rubra are sister to the rest of the complex, and include tetraploid C. rubra ssp. rubra (figs. 2 and 3). Similarly, monophyly of diploid C. parviflora ssp. grandiflora also received support from matK/trnK
105 and ITS, with the ITS tree also including one unique tetraploid C. parviflora ssp. parviflora sequence within the grandiflora clade (fig. 3).
Diploid C. perfoliata ssp. mexicana are again paraphyletic for matK/trnK (fig. 2).
Haplotypes mexicana 2-5 form a well-supported clade with tetraploid C. washingtoniana, diploid C. parviflora ssp. grandiflora, and tetraploid C. parviflora ssp. parviflora. Haplotype mexicana 1 is positioned deeper in the tree than the other mexicana haplotypes and is not included in the clade with the other diploid mexicana and grandiflora haplotypes (fig. 2). For
ITS, diploid C. perfoliata ssp. mexicana are included in a monophyletic group that includes unique polyploid sequences of hexaploid C. perfoliata ssp. perfoliata, hexaploid C. rubra ssp. depressa, and octaploid C. perfoliata (fig. 3).
Most of the polyploid sequences for both genes are identical to specific diploid sequences (see superscripted annotations to the right of figs. 2 and 3). This implies that these polyploids are derived (at least in part) from the diploids with which they share identical sequences. For the most part, the geographic distribution of polyploids parallels that of the diploids with which they share identical sequences. For example, polyploids that possess sequences from C. parviflora ssp. grandiflora are all found in the Sierra Nevada Mountains of
California, where subspecies grandiflora is endemic. Similarly, polyploids with sequences identical to diploid C. rubra ssp. rubra are generally found in the same geographic region as diploid subspecies rubra, albeit at lower elevations. However, the majority of polyploids with identical sequences as C. perfoliata ssp. mexicana do not occur within subspecies mexicana’s current geographical range.
Sixty-two of the 74 polyploids share identical matK/trnK sequences with diploid C. perfoliata ssp. mexicana, regardless of species designation or geographic distribution, the
106 majority of which are shared with mexicana 1 and mexicana 4 (fig. 2). Thirty-seven polyploids share the same haplotype with mexicana 1 and these were widely distributed throughout California, Oregon, and Washington (fig. 4a). Haplotype mexicana 4 is present in
20 polyploid individuals, primarily from the southern Sierra Nevadas as well as other scattered locations in California, Oregon, and Washington (fig. 4a). Similarly, 32 of the 74 polyploid individuals share identical ITS sequences with diploid mexicana 4/5 (fig. 3). This
ITS sequence is primarily distributed in the Columbia River Gorge of Washington and
Oregon, with other locations scattered in California, Oregon, and Washington (fig. 4b).
107 DISCUSSION
Polyploidy in many species of Claytonia is highly labile (namely C. exigua, C. lanceolata, C. sibirica, and C. virginica, Miller and Chambers 2006). The dynamic nature of polyploidy in the Claytonia perfoliata complex confirmed here has been demonstrated in previous investigations (e.g. Miller 1978c; Swanson 1964). This study, however, is the first to explicitly use DNA-sequence data to determine phylogenetic relationships among diploids and polyploids, and to infer putative polyploid origins in the C. perfoliata complex (however, see Miller 1978b for a morphological treatment). The following sections will discuss cytology, the relationships among diploid taxa, the influence of specific diploid entities on the geographic distribution of polyploids, and putative origins of polyploid taxa.
Cytology. All chromosome counts showed normal meiosis and complete bivalent chromosome formation. While this is expected in diploids, bivalents in polyploids often suggests high rates of allopolyploidy or diploidization of older autopolyploids (Sybenga
1969). However, Ramsey and Schemske (2002) found that bivalent formation can also be common in newly created autopolyploids. No occurrences of aneuploidy were observed, and despite the occurrence of mixed-ploidy populations, no triploids were found, nor were there any other odd-numbered ploidy levels. Previous investigations (described above) have also not found any odd-numbered polyploids.
Diploid Taxa and the Geographic Distribution of Diploid Sequences in Polyploids.
The three diploid cytotypes analyzed here correspond to the three diploid taxa described by
Miller and Chambers (1993; 2006). Diploids are morphologically distinct and occur in
108 different geographic ranges, habitats, and elevations. Diploid C. perfoliata ssp. mexicana is distributed from the coastal mountains and beaches of California and the highlands of
Arizona, Mexico and Central America. It is characterized by an erect habit, deltate or reniform mature basal leaves with conspicuous mucronate tips, and small autogamous flowers
(Miller and Chambers 2006). Diploid C. parviflora ssp. grandiflora is characterized by linear basal leaves and elongated racemes with flowers adapted to outcrossing. It is endemic to mid- elevations in the Sierra Nevada Mountains of California. Diploid C. rubra ssp. rubra is common at mid- to high elevations in the mountains of western North America, and is identified by deltate-shaped juvenile leaves, flattened rosettes, and small autogamous flowers.
Miller and Chambers (2006) used cytological, morphological, and phytochemical data to infer phylogenetic relationships in Claytonia. Their analysis placed C. parviflora as sister to the polytomous clade of C. perfoliata + C. rubra + C. washingtoniana. In contrast, the results here show that C. rubra is sister to the C. perfoliata + C. parviflora + C. washingtoniana clade, and that diploid C. parviflora ssp. grandiflora forms a supported clade nested within the C. perfoliata + C. parviflora + C. washingtoniana clade.
CLAYTONIA PERFOLIATA SSP. MEXICANA. Diploid C. perfoliata ssp. mexicana consistently forms a paraphyletic group for both matK/trnK analyses and for the combined analysis of diploids. All diploid specimens of subspecies mexicana are included within the well-supported C. perfoliata + C. parviflora clade, except mexicana 1 (217a, figs. 1b, and 2).
In contrast, both ITS analyses place all diploid specimens of subspecies mexicana in a single clade (including some unique polyploid sequences). Consequently, the results from the matK/trnK and combined analyses bring into question the treatment of C. perfoliata
(specifically ssp. mexicana) and C. parviflora as a distinct species. Many taxonomists
109 consider paraphyletic taxa as illegitimate (e.g. Ebach et al. 2006); yet contrary views also exist (e.g. Nordal and Stedje 2005).
The geographical distribution of diploid subspecies mexicana found here is in agreement with previous reports (Miller 1978c; Swanson 1964). However, Miller (1978a;
1978c) also found diploids in Arizona, Mexico, and Guatemala. Unfortunately, samples from these locations are not included in this study. All diploid C. perfoliata specimens here were collected in the southern California coastal mountains, except for mexicana 1 which was collected at a coastal beach/dune location in northern California.
The mexicana 1 specimen is also morphologically different from the other C. perfoliata ssp. mexicana diploids. The mexicana 2 through 5 specimens all have variegated foliage (caused by subepidermal gas pockets, K. L. Chambers, personal communication) that often becomes lightly red-tinged at maturity, and they have racemes that are exserted up to one decimeter above the perfoliate leaves. In contrast, mexicana 1 has congested racemes that rarely exert more than a few centimeters above the perfoliate leaves, and foliage that is entirely lime-green to pinkish in coloration. Thus, mexicana 1 is genetically, geographically, and morphologically distinct from the other mexicana individuals in this study.
As noted above, 37 polyploid specimens share the same matK/trnK haplotype with mexicana 1. Thus, the mexicana 1 haplotype appears to be the maternal (i.e. chloroplast) donor for exactly half (37/74) of all of the polyploid specimens examined here, regardless of specific, subspecific, or cytotypic designation. For example, many tetraploid specimens of C. perfoliata ssp. mexicana, C. parviflora ssp. viridis, and C. parviflora ssp. parviflora all shared the mexicana 1 matK/trnK haplotype (see fig. 2 for more examples). The mexicana 1 matK/trnK haplotype is found in the Columbia River Gorge of Washington and Oregon, the
110 Blue Mountains of Oregon, the coastal beaches and mountains of Oregon and California, and the central Sierra Nevadas (see fig. 4a).
The matK/trnK haplotype of mexicana 4 is also common in many polyploids. It is present in 20 polyploid individuals from the southern Sierra Nevadas and other scattered locations in California, Oregon, and Washington (see fig. 4a). Similarly, 31 of the 74 polyploid individuals share identical ITS sequences with the mexicana 4/5 ITS sequence (fig.
3). These were distributed in the Columbia River Gorge of Washington and Oregon, the Blue
Mountains of Oregon, and the coastal beaches and mountains of Oregon and California (see fig. 4b).
Two plausible hypotheses can explain the current distribution of the matK/trnK mexicana 1 and 4 haplotypes and the mexicana 4/5 ITS sequences: 1) diploids with mexicana sequences may have had historically larger ranges and these were subsequently replaced by related polyploids, or 2) related polyploids may have formed from diploid mexicana populations and subsequently colonized areas where they are currently distributed. For the
Columbia River Gorge populations, geological evidence may tentatively support the second hypothesis, as this area was repeatedly scoured with numerous, cataclysmic floods at the end of the Pleistocene (i.e. the glacial Lake Missoula floods, Baker and Bunker 1985). For Sierran populations, mexicana diploids may have contributed to polyploids in the Transverse Ranges of California (not sampled here) and the polyploids subsequently spread north through the
Sierra Nevadas. Alternatively, polyploids with mexicana sequences could have colonized the
Sierra Nevadas from diploid or polyploid populations in the Central Valley of California, which are now extinct due to human activities (i.e. agricultural and urban development). Both of these scenarios would also suggest the second hypothesis. In any case, there is a
111 disproportionate contribution of plastid and nuclear sequences of diploid subspecies mexicana in polyploids, regardless of the specific and/or subspecific designation of the polyploid cytotypes.
CLAYTONIA PARVIFLORA SSP. GRANDIFLORA. The diploid taxon C. parviflora ssp. grandiflora is morphologically distinct from all other taxa and cytotypes within the C. perfoliata complex. Subspecies grandiflora is easily identified by its erect habit, linear or filiform basal leaves, elongated racemes that are well exserted above the perfoliate leaves, large protandrous flowers (> 12 mm wide) with relatively long pedicels (> 1 cm long), and pink or white petals (Miller and Chambers 1993). It is limited in distribution to mid-elevations in the Sierra Nevadas where it is often found growing with hexaploid C. perfoliata ssp. perfoliata.
Diploid C. parviflora ssp. grandiflora share sequences with various polyploids in the
Sierra Nevadas. Two tetraploid individuals possess the matK/trnK grandiflora 2/3/4 haplotype (fig. 2), and eight polyploid individuals share ITS sequences with grandiflora 3, 4, and 5 (fig. 3). Subspecies grandiflora was likely the pollen donor for polyploids that shared grandiflora ITS sequences; its flowers are adapted for outcrossing (e.g. protandry) and it produces more pollen than either of the other diploid taxa. For instance, subspecies grandiflora has pollen/ovule ratios approximately five times that of diploid subspecies mexicana and four times that of diploid C. rubra (Miller 1978c). Thus, there is a greater probability that subspecies grandiflora may have served as the pollen donor for putative allopolyploids with which it shares sequences.
CLAYTONIA RUBRA SSP. RUBRA. Diploids of C. rubra ssp. rubra have previously been reported in the mountains and foothills of southern British Columbia, Washington, Oregon,
112 northern Idaho, western Montana, Nevada, and California (Miller 1978c). It commonly occupies dry coniferous forests (primarily Pinus ponderosa, Pinus jeffreyi, and Pseudotsuga menziesii) and is also common under riparian shrubs and trees. Claytonia rubra ssp. rubra is characterized by flattened rosettes, small autogamous flowers, cauline leaves that are separate to perfoliate, and deltate basal leaves in juvenile and mature plants. It often has beet-red to reddish-brown pigmentation.
In all analyses, diploid specimens of C. rubra ssp. rubra form a well-supported clade
(including some polyploid C. rubra) that is sister to the C. parviflora + C. perfoliata + C. washingtoniana clade. This is consistent with the treatment of C. rubra as a distinct species.
However, all of the diploid C. rubra specimens analyzed here were from the mountains and foothills of Washington and Oregon, and the Siskiyou Mountains of northern California. As noted, C. rubra ssp. rubra is also widely distributed in the intermountain west and the Rocky
Mountains, and is common in the Sierra Nevadas. Thus, any inferences made here about the taxonomic status of diploid C. rubra are limited to the populations of the Pacific Northwest.
Inference of Polyploid Origins. The majority of polyploids share identical sequences with diploids, yet many sequences were possessed exclusively by polyploids. The existence of unique polyploid sequences can be explained by three different hypotheses. First, it could be that they share their sequences with a diploid entity that was not sampled here. Second, the associated diploid taxa may be extinct. Third, the diploid parental lineages might still exist and be present in the analysis, but a sufficient amount of time has passed that allowed the polyploid sequences to diverge from the diploids.
As discussed above, data from DNA sequences can be used to infer polyploid origins by comparing congruence among gene trees. For example, since cpDNA (such as matK/trnK)
113 is uniparentally inherited from the ovule, it can be used to infer a particular polyploid’s maternal donor. In contrast, biparentally inherited nuclear genes that undergo concerted evolution, such as ITS, can reveal the pollen donor of an allopolyploid if concerted evolution homogenizes the paternal sequence. If, however, the maternal sequences are homogenized, the ITS sequences will not reveal allopolyploid origins. Thus, ITS sequence homogenization from the maternal parent of an allopolyploid cannot be differentiated from unilateral inheritance of both markers through genome duplication within a species (i.e. autopolyploidy). Therefore, as Popp and Oxelman (2007) suggest, autopolyploidy serves as the null hypothesis when comparing congruence or incongruence among plastid and nuclear gene trees.
Some unique polyploid sequences can present a problem for determining diploid parent taxa. For example, the supported ITS mexicana clade (fig. 3) includes all diploid specimens of subspecies mexicana, polyploids that share identical sequences to diploid mexicana, and polyploids with unique sequences (e.g. 6x depressa, 6x perfoliata, and 8x perfoliata). In this example, the unique polyploid sequences are likely derived from diploid subspecies mexicana. In contrast, some unique sequences exclusive to polyploids (e.g. 6x perfoliata k and 6x perfoliata l in fig. 3) are not included within any supported clade that also includes diploids. In this case, diploid parentage cannot be determined under the current concept of diploid taxa within the complex. However, allopolyploidy can be inferred if, for example, these polyploids share a matK/trnK sequence with one of the diploids in a supported clade.
Morphology and additional genetic data (e.g. allozymes) are used in conjunction with
DNA sequence data to support or contradict inferred polyploid origins. Below, three cases of
114 autopolyploidy, numerous cases of allopolyploidy, and a few equivocal cases are described.
These are discussed in relation to the morphological species and subspecies described by
Miller and Chambers (1993; 2006).
Claytonia perfoliata Polyploids. Of the four species in the C. perfoliata complex, polyploid cytotypes are most common in C. perfoliata s.s. (Miller and Chambers 2006). The results here support this: 41 of the 74 polyploid specimens were cytotypes of C. perfoliata s.s.
Tetraploids through decaploids were observed, with hexaploids being the most common cytotype.
TETRAPLOID SUBSPECIES MEXICANA. Claytonia perfoliata ssp. mexicana tetraploids
(and decaploids, not sampled here) are morphological indistinguishable from their diploid counterparts: both have linear basal leaves in juvenile plants and deltate or reniform basal leaves with conspicuous mucronate tips in mature plants (Miller and Chambers 2006). Based on phenotypic and geographic evidence, polyploid cytotypes of subspecies mexicana have previously been suggested to have autopolyploid origins (Miller 1978b).
One tetraploid C. perfoliata ssp. mexicana individual (218) was found in this study
(fig. 5a). This specimen was collected in a coastal beach/sand dune habitat of northern
California. It has identical sequences for both matK/trnK and ITS to sympatric diploid mexicana 1 (fig. 5a). Thus, the null hypothesis of autopolyploidy cannot be rejected. This, in conjunction with the nearly identical morphology to sympatric diploid mexicana 1, strongly suggests that this tetraploid is an autopolyploid derived from mexicana 1.
Tetraploids attributable to C. perfoliata ssp. mexicana are common in Quercus woodlands and chaparral of the Santa Cruz Mountains and the Coastal Ranges of southern
California, the Santa Catalina Mountains of southeast Arizona, and the coastal beaches of
115 California (Miller 1978c). Miller (1978c) noted that the coastal distribution of tetraploid C. perfoliata ssp. mexicana follows that of its diploid counterpart. He also found an occurrence of a tetraploid C. perfoliata ssp. mexicana (JMM 364, Miller 1976) at a coastal beach location in Humboldt County, California, presumably collected within kilometers of the individual sampled in this paper.
TETRAPLOID SUBSPECIES PERFOLIATA. Contrary to previous investigations, this study found two tetraploid individuals of C. perfoliata in the Sierra Nevadas. These specimens (182,
183) ambiguously key to subspecies perfoliata (e.g. robust plants with deltate mature basal leaves) (e.g. robust plants with deltoid mature basal leaves, Miller and Chambers 2006), yet they have purple abaxial leaf pigmentation that is suggestive of subspecies intermontana.
Furthermore, many other features are consistent with C. parviflora spp. grandiflora morphology. Inflorescences of these tetraploids are very similar to subspecies grandiflora: they have elongated racemes that were well exerted above the perfoliate cauline leaves and large pink flowers (~15 mm wide) with relatively long pedicels (> 1.4 cm on lowermost flowers). Additionally, these specimens possess protandrous flowers, a characteristic that has previously been thought to only occur in subspecies grandiflora.
These tetraploids (4x perfoliata in fig. 5a) were allopatric with two diploid specimens of C. perfoliata ssp. grandiflora (JHR 170 and 175) with which they share identical sequences for matK/trnK (grandiflora 2/3/4 in figs. 2 and 5a) and ITS (grandiflora 3 in figs. 3 and 5a); thus, the null hypothesis of autopolyploidy cannot be rejected. The tetraploids were found at slightly higher elevations in than the parapatric diploid C. parviflora ssp. grandiflora.
The molecular, geographic, morphological evidence suggest autopolyploid origins of these tetraploid cytotypes from diploid C. parviflora ssp. grandiflora. However, allopolyploid
116 origins cannot be ruled out; the abaxial purple pigmentation of deltate mature basal leaves are implicative of C. perfoliata ssp. perfoliata and/or ssp. intermontana. Allopolyploidy could therefore explain the presence of these leaf characteristics. However, if autopolyploidy is evoked, the leaf characteristics could also be a consequence of genome duplication (i.e. 'gigas' effect, Stebbins 1971), the result of subsequent ecological diversification, and/or the result of genetic drift.
HEXAPLOID SUBSPECIES PERFOLIATA. Hexaploids of C. perfoliata ssp. perfoliata (and ssp. intermontana) are the most common and widely distributed cytotypes in the C. perfoliata complex (Miller 1978c; Swanson 1964). Subspecies perfoliata hexaploids are a morphologically diverse group, sharing only broad basal leaves, an erect growth habit, and green to pinkish pigmentation as common traits. The range of this cytotype extends from western British Columbia through Washington, Oregon, and western Idaho, to California, including the Central Valley, Sierra Nevada foothills, and Coastal Ranges (Miller 1978c;
Miller and Chambers 2006; Swanson 1964). It is primarily found at low to mid-elevation in a variety of different habitats.
Hexaploids of subspecies perfoliata either possess unique polyploid sequences or share their sequences with diploid C. perfoliata ssp. mexicana. Twenty-two of the 23 hexaploid specimens share identical matK/trnK sequences with diploid C. perfoliata ssp. mexicana (fig. 5b), and one has a unique polyploid haplotype (310, 6x perfoliata 5 in fig. 5b).
In contrast, four unique polyploid ITS sequences occur in 15 of the 23 hexaploid specimens; the other eight have the mexicana 4/5 ITS sequence. In total, there are nine combinations of matK/trnK and ITS (6x perfoliata 3-11 in fig. 5b)
117 Claytonia perfoliata ssp. perfoliata hexaploids that possess the mexicana 1 matK/trnK haplotype are highly variable and occur in the Columbia River Gorge and the coastal mountains and beaches of Oregon and California (fig. 5b). As noted above, mexicana 1 is morphologically and genetically distinct. Hexaploid specimens that have the mexicana 1 matK/trnK haplotype key to C. perfoliata ssp. perfoliata, but are highly variable for inflorescence structure, pigmentation, and overall size. For example, contrary to most other hexaploids of subspecies perfoliata, all of these specimens have relatively small seeds (< 3 mm long, e.g. subspecies mexicana) and include robust erect plants with open flowers and greenish-yellow (206, 244) or greenish-red foliage (109); vigorous sub-erect plants with large perfoliate cauline leaves, compact racemes that barely surpass the cauline leaves, and small cliestogamous flowers (248); or small sub-erect plants with small open flowers and brownish- green pigmentation (225).
Hexaploids that have the mexicana 1 matK/trnK haplotype have either the mexicana
4/5 ITS sequence (6x perfoliata 4 in fig. 5b) or a unique polyploid ITS sequence sister to mexicana 4/5 (6x perfoliata 3 in fig. 5b). Since all three of these sequences are either shared with diploid mexicana or are within the mexicana ITS clade, autopolyploidy cannot be rejected. However, the high degree of variability in pigmentation and morphology suggest that some of these types may have input from taxa other than C. perfoliata ssp. mexicana.
Two hexaploids of C. perfoliata ssp. perfoliata collected from the northern Sierra
Nevadas are characterized by an erect habit, greenish-yellow foliage, pink stems, elongated racemes having primarily autogamous flowers, and large seeds (3-5 mm long) that are typical in the majority of C. perfoliata ssp. perfoliata hexaploids (Miller and Chambers 2006;
Swanson 1964). Similar hexaploid individuals sampled from the same populations as these
118 two specimens (34, 38) show disomic allozyme banding patterns (Rausch, unpublished data,
Chapter 3), which is often indicative of allopolyploidy. These individuals (6x perfoliata 11 in fig. 5b) had the matK/trnK sequence for mexicana 5, and shared identical ITS sequences with
4x C. parviflora ssp. parviflora, a putative allopolyploid (see below). Thus, these hexaploids, are most likely allopolyploid in origin.
Three hexaploids from the eastern Columbia River Gorge and the Blue Mountains possess the matK/trnK mexicana 4 haplotype and the ITS sequences from mexicana 4/5 (6x perfoliata 6 in fig. 5b). Autopolyploidy cannot be rejected since these individuals have sequences for both genes that are shared by mexicana 4. Two of these hexaploids (273, 283) have morphology typical of putative allohexaploid C. perfoliata ssp. perfoliata described above: they have elongated racemes, large seeds, deltate basal leaves, and an erect habit. The other (301) is a robust plant collected in a mixed population with hexaploid C. rubra ssp. depressa; it had a unique suite of morphological characters reminiscent of diploid subspecies mexicana (cauline and basal leaves), hexaploid subspecies perfoliata (habit and stems), and hexaploid subspecies depressa (racemes and flowers).
Six hexaploid perfoliata specimens (45, 50, 70, 164, 168, 172) from low to mid elevations in the Sierra Nevadas share comparable morphology and pigmentation. These specimens all share the mexicana 4 matK/trnK sequence and are robust upright plants with green foliage, pink stems, deltate basal leaves, elongated racemes, large open flowers (8-12 mm wide), and large sepals (> 5 mm long) and seeds (> 3 cm long). Five of these (6x perfoliata 7 and 8 in fig. 5a) have unique ITS sequences that form a monophyletic hexaploid group, and one (6x perfoliata 9 in fig. 5b) shares its ITS sequence with tetraploid C. parviflora ssp. parviflora. Since none of the ITS sequences are present in the mexicana ITS
119 clade, autopolyploidy origins are not likely. However, based on the information found here, these polyploids cannot be categorically linked to any of the diploid taxa other than subspecies mexicana.
Based on morphological evidence, Miller (1978b) proposed that hexaploids of subspecies perfoliata contain both autopolyploid and allopolyploid cytotypes; he postulated that allohexaploids are the result of hybridization between C. perfoliata and C. parviflora.
The results here generally support this claim. For example, some hexaploids (e.g. 225, 248,
301) possess identical sequences to diploid subspecies mexicana for both genes, and have morphology congruous with subspecies mexicana. Other hexaploids (e.g. 45, 50, 70) have matK/trnK haplotypes from subspecies mexicana and unique polyploid sequences for ITS.
These specimens, primarily from the Sierra Nevadas, have characteristics typical of both C. perfoliata and sympatric C. parviflora ssp. grandiflora (i.e. larger flowers and elongated racemes). However, hexaploids do not share any sequences with subspecies grandiflora. In any case, the results here demonstrate that hexaploid C. perfoliata ssp. perfoliata is a geographically and morphologically diverse, polyphyletic group, with many independent polyploid origins (fig 5b).
HEXAPLOID SUBSPECIES INTERMONTANA. Claytonia perfoliata ssp. intermontana is primarily distributed within the Intermountain Regions of Idaho, Nevada, and Utah, the
Columbia Basin of Oregon and Washington, and in various mountain ranges of western North
America (e.g. Sierra Nevada, Bighorn, and Klamath Mountians). Here, two distinct hexaploid types of subspecies intermontana were collected. These two types differ in morphology, geography, and genetic composition.
120 The first type consisted of two specimens collected in sagebrush-steppe ecosystems: one (281) from the eastern Columbia River Gorge and the other (130) from the Steens
Mountain area of southeast Oregon (6x intermontana 1 in fig. 5b). Both of these fit the classical description of subspecies intermontana (Miller and Chambers 1993). Plants were small, sub-erect, with pigmentation grey-green adaxially and beet-red abaxially, and mature basal leaves that are elliptic-rhombic to subdeltate.
Miller and Chambers (1993) indicate that the sub-erect habit and beet-red pigmentation of many subspecies intermontana populations is suggestive of genetic influence from C. rubra. Additionally, Miller (1978b) describes hexaploids (presumably subspecies intermontana) that are putative allopolyploids of C. rubra and C. parviflora. Results here (fig.
5b) find no support of genetic input from either C. rubra or C. parviflora; however, both individuals (130, 281) were sympatric with C. rubra. These hexaploid individuals have the same matK/trnK haplotype as diploid mexicana 1, and a unique polyploid ITS sequence that is shared (fig. 3) with hexaploid subspecies depressa (also from sagebrush-steppe habitat). In this case, autopolyploidy can be tentatively rejected. However, this should viewed with caution, as the unique ITS sequence are sister to the mexicana ITS clade that contains mexicana 1 (figs. 3 and 5b).
Specimens of the second hexaploid type of subspecies intermontana were collected at mid-elevation in the central Sierra Nevadas. These individuals (178, 189, 190) were characterized by broadly erect habits, relatively long inflorescences, rhombic basal leaves with reddish-green pigmentation on the adaxial surfaces and reddish-purple pigmentation on the abaxial surface.
121 These Sierran hexaploids (6x intermontana 2 in fig. 5b) are clearly allopolyploid in origin as they share matK/trnK sequences with mexicana 1 and ITS sequences with grandiflora 2/3/4. The grandiflora 2/3/4 ITS sequence has one diploid C. parviflora ssp. grandiflora individual (163) that was parapatric with the hexaploid specimens of subspecies intermontana. This same combination of matK/trnK and ITS sequences also exists in sympatric C. parviflora ssp. parviflora tetraploids (4x parviflora 8 in fig. 7a). While the pigmentation of the parviflora tetraploids and the intermontana hexaploids were similar, these cytotypes were morphologically very different from each other. Given their close geographic proximity and shared genetic ancestry, it is entirely likely that these tetraploids contributed, in part, to the Sierra Nevadan intermontana hexaploids.
OCTAPLOID SUBSPECIES PERFOLIATA. Two independently derived octaploids of subspecies C. perfoliata ssp. perfoliata were observed in this study. One group was collected from the coastal mountain forests of Northern California and the other from the Kern River
Canyon of the southern Sierra Nevadas. The two types are also morphologically distinct from each other and from other cytotypes of C. perfoliata ssp. perfoliata.
Miller (1976) identified some northern California specimens (JMM: 361, 362, 367,
369) of C. perfoliata ssp. perfoliata as the “Klamath Mountains octaploids”. These were described as robust ruderal plants with small pink flowers (~ 5 mm wide), variegated sepals, and deltate basal leaves. It is uncertain if Miller’s Klamath Mountains octaploids are the same taxonomic entity as the Northern California octaploids analyzed in this paper. First, the octaploid specimens described here (209, 212, 214) were located at low elevations within the
Eel River watershed, approximately 90 km south of Miller’s collections; Miller’s specimens were located on serpentine soils at higher elevations in the Klamath Mountains. Second, while
122 the octaploids described here share many traits in common with the Klamath Mountains octaploids, they differ in having conspicuously dark-purple sepals, slightly larger flowers, and rhombic basal leaves.
Miller (1978b) hypothesized that octaploids within C. perfoliata ssp. perfoliata are allopolyploids of C. parviflora and C. perfoliata. The results here do not explicitly contradict this; however, autopolyploid origins of the northern California octaploids (8x perfoliata in fig.
6a) cannot be ruled out. All three specimens (209, 212, 214) have the diploid mexicana 1 matK/trnK haplotype and a unique polyploid ITS sequence. This ITS sequence is only possessed by these octaploids and is nested within the ITS mexicana clade (fig. 3).
The Kern River Canyon octaploids provide partial support for Miller’s (1978b) suggestion of allopolyploid origins. This cytotype (8x perfoliata 4 in fig. 6a) shares matK/trnK sequences with mexicana 4, and possess a unique ITS sequence found only in these two octaploid specimens (86, 145). Placement of this octaploid ITS sequence is uncertain; it is not included in either the mexicana ITS clade or the grandiflora ITS clade (fig.
3). Consequently, autopolyploidy can be rejected with certainty. These octaploids are robust plants with relatively large perfoliate cauline leaves, large seeds, small open flowers, and are similar to previous reports of octaploids in the Kern River Canyon (Miller 1978c),
OCTAPLOID SUBSPECIES INTERMONTANA. Octaploids attributable to C. perfoliata ssp. intermontana are endemic to the south side of the Columbia River Gorge (Oregon) where they primarily occupy habitat at the base of north-facing basalt cliffs and talus (Miller 1976).
Miller (1976) was the first to discover this cytotype, which he referred to as the “Columbia
Gorge Octaploids”, and later these were annotated as C. perfoliata ssp. intermontana (e.g.
JMM: 313, 401, 402; OSC accession #’s 152638, 152637, 152643, respectively).
123 Overall, specimens (271, 238, 249, 250, 278; JMM: 313, 401, 402) of the Columbia
Gorge Octaploids fall under the description of subspecies intermontana; however, these specimens share distinctly similar morphology and pigmentation that are unique to these octaploids. These specimens all have a sub-erect habit, broadly rhombic mature basal leaves with purple abaxial coloration, red-tinged sepals, and open flowers with pinkish petals approximately 6 cm long.
The Columbia Gorge Octaploids (8x intermontana 1 and 2 in fig. 6a) share all ITS sequences with mexicana 4/5. Four of the five specimens share matK/trnK haplotypes with mexicana 1; the other specimen shares a unique polyploid sequence with a hexaploid subspecies intermontana and hexaploid subspecies depressa, which were also collected from the Columbia River Gorge. Miller (1978b) suggested that octaploids of the C. perfoliata complex are allopolyploids that originate by various combinations of diploid and polyploid C. perfoliata and C. parviflora. The phylogenetic results here do not refute this, nor do the result refute the possibility of autopolyploidy. However, given the unique morphology and habitat of these octaploids, it is entirely possible that they are composed of combinations of disparate
C. perfoliata s.l. cytotypes.
DECAPLOID SUBSPECIES PERFOLIATA. Previous reports (Miller 1978c; Miller and
Chambers 2006) of decaploids in the C. perfoliata complex were from plants collected in the
Columbia River Gorge of Oregon and Washington (termed “Columbia Gorge decaploid” by
Miller 1976) and from subspecies mexicana in Guatamala. Here, the Columbia Gorge decaploids are relocated and a new decaploid is reported from the Kern River Canyon in the
Sierra Nevadas. Results indicate that these have geographically separate, independent origins.
124 Miller (1976) identified numerous decaploid specimens in the Columbia River Gorge attributable to subspecies perfoliata. He described these individuals as robust plants with perfoliate cauline leaves up to 11 cm in diameter and deltate mature basal leaves. These were often found at roadside locations or other ruderal habitats. The morphology and habitat of the
Columbia Gorge decaploids used in this study are in agreement with Miller’s (1976) observations.
Here all four decaploid individuals sampled from this region had diploid mexicana 1 sequences for matK/trnK and diploid mexicana 4/5 sequences for ITS. This matK/trnK - ITS sequence combination was also observed in 16 other polyploid specimens, including tetraploid subspecies viridis (fig. 7a), hexaploid subspecies perfoliata (fig. 5b), hexaploid subspecies parviflora (fig. 7b), and octaploid subspecies intermontana (fig. 6a). With the exception of subspecies viridis, all of these had at least one specimen that was also found in the Columbia River Gorge.
Miller (1978b) suggested that decaploids of the C. perfoliata complex are autoallopolyploids. Based on morphology, he claimed that they primarily have input from C. perfoliata s.s., as well as some input from C. parviflora. The results from the matK/trnK and
ITS analyses show input only from diploid subspecies mexicana. Thus, the null hypothesis of autopolyploidy cannot be rejected for the Columbia Gorge decaploids; however allopolyploid origins cannot be ruled out either.
One decaploid specimen was also found in the Kern River Canyon of the southern
Sierra Nevadas. This is the first known report of a decaploid from this region. It was collected with sympatric tetraploids of C. parviflora ssp. parviflora and octaploids of C. perfoliata ssp. perfoliata, with which it shares the diploid mexicana 4 haplotype for matK/trnK. However,
125 the decaploid shared a unique ITS sequence with hexaploid C. perfoliata ssp. perfoliata from the southern Sierra Nevadas (see 10x perfoliata 2 in fig. 6b). Thus, this decaploid most likely had allopolyploid origins and evolved independently from the Colombia Gorge decaploids.
Claytonia parviflora Polyploids. There are three distinct polyploid subspecies within
C. parviflora: ssp. parviflora, ssp. utahensis, and ssp. viridis. Each of these is characterized by a combination of morphology, cytology, and geographic distribution, but all share linear or spatulate basal mature leaves as a diagnostic trait (Miller and Chambers 1993; Miller and
Chambers 2006).
TETRAPLOID SUBSPECIES PARVIFLORA. Tetraploid Claytonia parviflora ssp. parviflora are characterized by linear basal leaves at all ontogenetic stages and perfoliate cauline leaves
2-5 cm in diameter, which are suggestive of diploid C. parviflora ssp. grandiflora.
Inflorescences are composed of small self-compatible flowers borne on compact to moderately elongate (1 dm) racemes, which is similar to some diploid C. perfoliata ssp. mexicana specimens. Miller (1978b) suggested that tetraploid C. parviflora ssp. parviflora may have autopolyploid origins due to its morphological similarity to C. parviflora ssp. grandiflora, but that some morphologically intermediate forms may be the result of allopolyploid hybridization of historically sympatric diploid populations of C. perfoliata or C. rubra. The specimens collected here are highly variable and show no natural morphological grouping of specimens within this supspecies and cytotype.
Tetraploid C. parviflora ssp. parviflora generally parallels the geographic distribution of diploid C. parviflora ssp. grandiflora (fig. 7a). These tetraploids are common in mid- elevation mixed coniferous forests and Quercus chaparral of the Sierra Nevada. Tetraploid specimens attributable to C. parviflora ssp. parviflora have also been found sympatric with
126 diploid C. perfoliata ssp. mexicana in the San Jacinto Mountains of southern California and the Coastal Ranges of central California (Miller 1978c), however these were not included in the present study.
Results from the phylogenetic analysis show that all tetraploid Claytonia parviflora ssp. parviflora share identical matK/trnK sequences with diploid C. perfoliata ssp. mexicana
(mexicana 1, 2, and 4). For ITS, two specimens had identical sequences to grandiflora 4 and grandiflora 5 (fig. 7a). One specimen (187b) had a unique ITS sequence nested in the grandiflora clade (figs. 3 and 7a) and four specimens had unique ITS sequences that were sister to the grandiflora clade (fig. 3 and 7a). These data show that tetraploid Claytonia parviflora ssp. parviflora share haplotypes with diploid subspecies mexicana and subspecies grandiflora, while also possessing unique sequences sister to subspecies grandiflora. Thus, based on molecular data, autopolyploidy for C. parviflora ssp. parviflora tetraploids can be rejected.
Additionally, tetraploid Claytonia parviflora ssp. parviflora individuals sampled from the same populations as specimen 138 (4x parviflora 5 in fig. 7a) have show disomic allozyme banding patterns (Rausch, unpublished data, Chapter 3), which is often indicative of allopolyploidy. Disomic inheritance, along with the molecular evidence outlined above and the morphological similarities to both diploid C. perfoliata ssp. mexicana and C. parviflora ssp. grandiflora, strongly suggest allopolyploid origins of tetraploid C. parviflora ssp. parviflora.
Based on the incongruence between the matK/trnK and ITS data sets (fig. 7a), there are a maximum of eight possible multiple independent origins of C. parviflora ssp. parviflora.
Maternal contribution in all cases appears to be from C. perfoliata ssp. mexicana (fig. 7a).
127 Evidence from ITS suggest that C. parviflora ssp. grandiflora probably served as the pollen donor in most cases. As mentioned above, diploid C. parviflora ssp. grandiflora produces about five times more pollen per ovule than C. perfoliata ssp. mexicana.
TETRAPLOID SUBSPECIES UTAHENSIS. Claytonia parviflora ssp. utahensis is morphologically similar to subspecies parviflora, but is distinguishable by its diminutive growth habit, spatulate mature basal leaves, and geographic distribution. It ranges from the
Peninsular and Desert ranges of southern California, north to the eastern slopes Sierra Nevada
(including the Kern River Canyon) and eastward to the mountain and range systems of
Nevada, southern Utah, and northern Arizona (Miller and Chambers 1993). Subspecies utahensis is found in ephemerally mesic locations of arid habitats often in association with
Pinus monophylla, Yucca brevifolia, Purshia glandulosa, Larrea divaricata, or Acacia plant communities (Miller and Chambers 2006). To date, all cytotypic accessions of subspecies utahensis are consistently tetraploid (Miller and Chambers 2006), including those presented here.
Only two specimens of this taxon were obtained for this study, both of these were from the Kern Canyon area of the Sierra Nevada. Unfortunately, specimens from the majority of subspecies utahensis’ range were not analyzed. So, generalizations to the rest of this group should be viewed with caution. The two specimens of subspecies utahensis included in this study have identical matK/trnK and ITS sequences as two specimens of C. parviflora ssp. parviflora (4x parviflora 5 in fig. 7a). The two subspecies parviflora individuals were also collected from the Kern River Canyon area of the Sierra Nevada; however, the C. parviflora ssp. utahensis populations were found at higher elevations.
128 Based on the DNA sequence data, the null hypothesis of autopolyploidy can be rejected for subspecies utahensis. Given the morphological similar to tetraploid C. parviflora ssp. parviflora and the finding of identical matK/trnK and ITS sequences to subspecies parviflora, C. parviflora ssp. utahensis may not be the result of a unique evolutionary origin, but instead are the result of ecomorphological adaptations to the habitats that it occupies.
TETRAPLOID SUBSPECIES VIRIDIS. Claytonia parviflora ssp. viridis is the most morphologically distinct entity within the C. perfoliata complex and is easily distinguishable from all other taxa by the presence of linear, completely separate cauline leaves 1-6 cm long
(Miller and Chambers 2006). Both tetraploid and hexaploid cytotypes have been found.
Tetraploids, which were sampled here, are primarily found in chaparral and oak woodlands of the Californian Peninsular and Transverse Ranges (Miller 1976; Miller 1978c). Tetraploids are often sympatric with diploid C. perfoliata ssp. mexicana and tetraploid C. parviflora ssp. parviflora in the Santa Lucia Mountains of southern California (Miller 1978c). Hexaploid cytotypes of this subspecies, which were not included in this study, occur in Pinus coulteri forests of the Peninsular Mountains of California and in mesic montane locations of the
Mojave Desert, where they are often sympatric with C. parviflora ssp. utahensis (Miller
1978c; Miller and Chambers 2006).
Miller (1978b) postulated that polyploids of subspecies viridis may have been derived from a fourth diploid taxon, distinct from diploid C. parviflora, C. perfoliata, and C. rubra.
However, diploids of C. parviflora ssp. viridis have never been found, and the molecular evidence shows no support for this: subspecies viridis sequences were identical to diploid subspecies mexicana 1 for matK/trnK (figs. 2 and 7a) and mexicana 4/5 for ITS (figs. 3 and
7a). Thus, autopolyploid origins for subspecies viridis cannot be rejected. However, the
129 distinct morphology of subspecies viridis, and lack of diploid cytotypes assignable to subspecies viridis, suggests that it may have allopolyploid origins.
Miller (1976) also observed tetraploids with intermediate morphology of cauline and basal leaves between tetraploid C. perfoliata s.l. (or possibly C. rubra) and tetraploid C. parviflora ssp. viridis in southern California. He suggested that these may be homoploid hybrids between these two tetraploid entities (Miller 1976). Alternatively, intermediate morphological types may represent transitional forms from a continuum of ecotypes within C. perfoliata tetraploids. In this scenario, tetraploid C. perfoliata s.l. would have had morphological diversification (due to drift or ecological selection) to forms attributable to C. parviflora ssp. viridis.
HEXAPLOID SUBSPECIES PARVIFLORA. Hexaploid cytotypes with an erect habit and filiform, linear, or narrowly spatulate basal leaves are identified as C. parviflora ssp. parviflora. These, like the hexaploid subspecies perfoliata, are common, widespread, and exist in a variety of habitats in British Columbia, Washington, Oregon, and California.
All of the C. parviflora hexaploids found in this study possess the mexicana 4/5 ITS sequence and one of three different matK/trnK haplotypes. One specimen (119), collected from the Palouse Prairie, has a unique matK/trnK polyploid sequence whose phylogenetic placement is unresolved (6x parviflora 1 in fig. 7b). This specimen has narrowly spatulate basal leaves, lime-green pigmentation, and a small number of pink flowers borne on short racemes. Six specimens share the mexicana 1 matK/trnK haplotype (6x parviflora 2 in fig.
7b). These all have similar morphology to the Palouse Prairie specimen described above, and were collected in the Columbia River Gorge and Blue Mountains of Oregon and the Coastal
Ranges of Southern California. Two other specimens share the mexicana 4 matK/trnK
130 haplotype (6x parviflora 3 in fig. 7b), yet are very different morphologically. One of these hexaploids (201) was also collected from the Coastal Ranges; it has a suberect habit, filiform leaves, and small autogamous flowers. The other (159a), from the Sierra Nevadas, are robust plants with variegated foliage, longer racemes with small pink flowers, and spatulate basal leaves.
Miller (1978b) proposed that parviflora hexaploids were allopolyploids between C. parviflora and C. perfoliata or C. rubra. While this hypothesis cannot be rejected, no evidence of allopolyploidy exists here, except for the one specimen with the unique matK/trnK sequence, and its placement is equivocal. With the exception of this one specimen, the hypothesis of autopolyploidy cannot be rejected.
Swanson (1964) referred to two different types of weedy hexaploids in C. perfoliata s.l. He named these two types the “linear-leaved hexaploid” and the “deltate-leaved hexaploid” and considered them extreme ends of a continuum of variable leaf shapes; Miller and Chambers (1993) later classified these as hexaploid C. parviflora ssp. parviflora and C. perfoliata ssp. perfoliata, respectively. The results here suggest that Swanson’s (1964) description may be more appropriate than Miller and Chambers’ (1993) nomenclature. For example, eight of the nine parviflora hexaploids share identical combinations of matK/trnK–ITS sequences with heaxaploid C. perfoliata ssp. perfoliata (e.g. matK/trnK mexicana 1 or 4 and ITS mexicana 4/5, fig. 5b). Additionally, as previously noted, leaf-shape is highly variable and intermediate leaf shapes are common.
Claytonia rubra Polyploids. Polyploids assignable to C. rubra are divided into two groups: subspecies rubra, which consists of tetraploids (and diploids), and subspecies depressa, which has tetraploid and hexaploid cytotypes (Miller and Chambers 2006).
131 Tetraploid C. rubra ssp. rubra are somewhat uncommon and are morphologically indistinguishable from their diploid counterparts (Miller and Chambers 2006).
Claytonia rubra ssp. rubra is characterized by deltate juvenile and mature basal leaves, flattened basal leaf rosettes, small flowers, beet-red to reddish-brown pigmentation, and occasional one-sided fusion of cauline leaves (Miller and Chambers 2006). Subspecies depressa has a strongly flattened growth habit, light-green pigmentation, spatulate to rhombic mature basal leaves, and an otherwise similar morphology to subspecies rubra. Additionally, subspecies depressa is primarily limited to coastal beaches and lower inland elevations and subspecies rubra is commonly found in montane habitats at higher elevations.
TETRAPLOID SUBSPECIES RUBRA. Two C. rubra ssp. rubra tetraploids were found: one from the Steens Mountain area of eastern Oregon, and the other from the Sierra Nevadas.
Both of these clearly fit the description of C. rubra ssp. rubra. However, the Steens Mountain sequences are consistent with diploid C. rubra, while the Sierra Nevada shares sequences with diploid C. perfoliata and diploid C. grandiflora.
The Steens Mountain tetraploid has a unique matK/trnK sequence nested within the diploid C. rubra haplotypes (figs. 2 and 8), and the ITS sequence is shared with rubra 3/4
(figs. 3 and 8). Based on molecular evidence, the null hypothesis for autopolyploid cannot be rejected. Furthermore, tetraploid C. rubra ssp. rubra individuals sampled from the same populations as specimen 133 (4x rubra 1 in fig. 8) show allozyme banding patterns consistent with tetrasomic inheritance in artificial crosses (Rausch, unpublished data, Chapter 3); as noted, tetrasomic inheritance is generally indicative of autopolyploidy. Thus, given the morphological similarity to diploid C. rubra ssp. rubra, the congruence of DNA sequences,
132 and the observation of tetrasomic inheritance in individuals of the same population, this specimen is unequivocally an autotetraploid derived from diploid C. rubra ssp. rubra.
In contrast, the tetraploid collected from the central Sierra Nevadas shows a very different phylogenetic signal. This specimen possesses the mexicana 1 matK/trnK haplotype and the grandiflora 4 ITS sequence, yet it has classic C. rubra ssp. rubra features: flattened rosettes, deltate basal leaves in juvenile and mature plants, and reddish-purple pigmentation
(slightly different than other beet-red colored C. rubra). The simplest explanation of this paradoxical result is that allopolyploid hybridization occurred between diploid subspecies mexicana and grandiflora, followed by convergent evolution to a rubra-like form. Similar to many populations of C. rubra ssp. rubra, this specimen was collected from a monomorphic population at a relatively high elevation (> 2200 m); thus, increased pigmentation and flattened growth form may be adaptations to high elevations. Indeed, plants that are native to high elevations often have flattened rosettes (Korner 2003) and increased pigmentation to deal with higher levels of UV radiation (Barnes et al. 1987).
Miller (1978c) describes numerous occasions where C. rubra in the Sierra Nevadas is sympatric with tetraploid C. parviflora ssp. parviflora. The Sierran C. rubra tetraploid described here (229) was collected approximately 1 km away from a tetraploid C. parviflora ssp. parviflora individual (187a, see 4x parviflora 8 in fig. 7a), with which it shares identical matK/trnK (mexicana 1) and ITS (grandiflora 4) sequences. An alternative explanation could be that gene flow or introgression has occurred between C. parviflora tetraploids and the
Sierran rubra tetraploid, and C. rubra has maintained these sequences within the population while also maintaining morphology of C. rubra.
133 A third hypothesis can also account for the paradoxical result of the Sierran C. rubra tetraploid possessing grandiflora and mexicana sequences. This combination of sequences is also observed in three nearby specimens of hexaploid C. perfoliata ssp. intermontana (see 6x intermontana 2 in fig. 5b) that also have reddish-purple pigmentation. The mating of a hexaploid cytotype with a diploid can produce tetraploid offspring. In fact, Miller (1978b) hypothesized that the formation of some tetraploids was due to the interbreeding of diploid C. rubra with allohexaploid C. perfoliata s.l. Thus, intercytotype mating between Sierran diploid
C. rubra and hexaploid C. perfoliata ssp. intermontana is another plausible hypothesis for the sequence pattern seen in the Sierra rubra tetraploid.
HEXAPLOID SUBSPECIES DEPRESSA. Both tetraploid and hexaploid cytotypes are included in C. rubra ssp. depressa. Miller (1976) and Swanson (1964) described various geographical cytotypes that are currently recognized as C. rubra ssp. depressa. Such types include the “Scott’s Valley tetraploid” of northern California (Swanson 1964), the “coastal tetraploid” of Oregon, Washington, and British Columbia (Fellows 1976; Miller 1976), and the “sagebrush hexaploid” of the Columbia Basin (Miller 1976). Subsequent chromosome counts indicate that coastal types also include hexaploids (e.g. JMM 501, OSC accession #
152776).
This study found two genetically distinct hexaploid cytotypes that correspond to
Miller’s (1976) early descriptions of subspecies depressa. One of these, the “sagebrush hexaploid” type, shows maternal input from diploid and autotetraploid C. rubra. The other type fits the description of the “coastal” hexaploid; yet it has sequences in common with diploid C. perfoliata ssp. mexicana for both markers.
134 The “sagebrush hexaploid” specimens were collected in arid regions of the eastern
Columbia River Gorge within sagebrush steppe habitat. All three specimens (284, 262, 265) have a unique ITS polyploid sequence that is sister to the mexicana ITS clade (fig. 8) and is shared with hexaploid intermontana. For matK/trnK, two of the specimens share identical sequences with the diploid rubra 4 haplotype (see 6x depressa 3 in fig. 8), while the other specimen shares an identical haplotype with autotetraploid C. rubra ssp. rubra (see 6x depressa 2 in fig. 8). This suggests that the “sagebrush hexaploids” of subspecies depressa, are allopolyploids with the maternal donor being diploid (and possibly autotetraploid) C. rubra ssp. rubra and other donor being an unknown type with possible relation to diploid C. perfoliata ssp. mexicana.
Hexaploid specimens (JHR 229, 232) attributable to the “coastal” subspecies depressa variety were collected from sand-dunes on the Oregon coast. These specimens share an identical matK/trnK haplotype with diploid mexicana 1, and possess a unique polyploid genotype nested within the C. perfoliata ssp. mexicana ITS clade, that is shared with hexaploid C. perfoliata ssp. perfoliata (figs. 3 and 8). Autopolyploidy is not rejected because the unique polyploid ITS sequence differs from the mexicana 1 ITS sequence at only one of
34 variable base pairs.
Additionally, the morphology and pigmentation of these specimens are suggestive of the coastal C. perfoliata ssp. mexicana populations. The diagnostic differences being that the subspecies depressa specimens have a strongly flattened growth habit (rather than sub-erect) and an ontogenetic sequence of spatulate basal leaves (rather than linear to deltate). Thus, given the data presented here, it is entirely possible that the coastal subspecies depressa variety is an autohexaploid of C. perfoliata ssp. mexicana.
135 Claytonia washingtoniana Tetraploids. Fellows (1971) determined that the annual taxa C. washingtoniana is the allotetraploid hybrid of C. perfoliata s.s. and C. sibirica. He used traditional light microscopy methods to compare camera lucida images of putative parental genomes in western Claytonia species with chromosome preparations of experimentally produced backcross hybrids involving C. washingtoniana and other Claytonia species. Claytonia washingtoniana is distinguished from C. perfoliata s.l. by its multi- bracteate inflorescence and separate cauline leaves and from C. sibirica by having multiple small autogamous flowers that arise at each bract (compared to one large flower per bract in
C. sibirica) and its annual lifecycle (compared perennial C. sibirica). Claytonia washingtoniana is distributed along the northern California coast and the Puget Sound of
Washington, where it is sympatric with both C. perfoliata s.l. and C. sibirica (Miller and
Chambers 2006).
Claytonia washingtoniana has unique matK/trnK and ITS sequences. The results presented here show that C. washingtoniana is consistently included within the C. perfoliata
+ C. parviflora clades for both matK/trnK and ITS, albeit it has significantly longer branch lengths than any other taxa within these clades (figs. 2 and 3). This result was also observed by O’Quinn and Hufford (2005), however they showed C. washingtoniana as sister to C. parviflora ssp. grandiflora. This provides some support for Fellow’s (1971) hypothesis of C. washingtoniana being a allopolyploid between C. perfoliata s.l. and C. sibirica. Clearly, more molecular work is needed to determine if the genome of C. washingtoniana has genetic input from C. sibirica.
Morphological variability and convergence in polyploids. Miller and Chambers
(1993; 2006) primarily based their treatment of the C. perfoliata complex on geographical
136 distribution, growth habit, and ontogenetic morphology of basal leaves. As noted above,
Claytonia parviflora includes erect plants with linear, filiform, or narrowly spatulate basal leaves, C. perfoliata includes erect to sub-erect plants with linear juvenile leaves and obovate, rhombic, deltate, or reniform basal leaves at maturity, and C. rubra includes plant with flattened rosettes and spatulate, rhombic, or deltate juvenile and mature leaves.
Identification of taxa within the complex is often difficult. Field botanists familiar with the overall variability in the C. perfoliata complex can testify to the difficulty of identifying widespread, sympatric polyploid subspecies (e.g. subspecies perfoliata, intermontana, parviflora, and utahensis). If fact, Miller and Chambers (1993; 2006) repeatedly mention that polyploids of the C. perfoliata complex include numerous intermediate morphological forms, with leaf shapes and growth habits varying within and among subspecies, cytotypes, and populations. The DNA-based evidence presented here suggests that leaf morphology and growth habit are either variable traits within and among cytotypes or the result of morphological convergence (i.e. homoplasy) of independently derived polyploids. Thus, leaf morphology and growth habit do not represent diagnostic characters for differentiating polyploid species.
For example, specimens 243 and 244 key to C. parviflora ssp. parviflora and C. perfoliata ssp. perfoliata, respectively. Both of the specimens were hexaploid (6x parviflora in fig. 7b and JHR 244: 6x perfoliata in fig. 5b) and shared identical sequences for matK/trnK
(mexicana 1) and ITS (mexicana 4/5). Additionally, both were robust hexaploid plants with similar pigmentation and inflorescence morphology; and they were collected in the Columbia
River Gorge approximately 10 m from each other. This suggests these two specimens (and all similarly related individuals), have the same origin and belong in the same
137 taxonomic/cytotypic entity. Leaf shape in this taxon would thus be a variable trait, and not represent a diagnostic character differentiating the same cytotype of two different species.
Indeed, individuals were observed in the same population with intermediate leaf shapes and otherwise similar overall morphology and pigmentation to specimens 243 and 244.
Evidence for convergence in growth habit is seen in numerous specimens. For example, the C. rubra autotetraploid from eastern Oregon shares matK/trnK and ITS sequences with diploid C. rubra, while the C. rubra tetraploid from the Sierra Nevadas has matK/trnK and ITS sequences from diploid C. perfoliata ssp. mexicana and C. parviflora ssp. grandiflora, respectively (fig. 8). Thus, the Sierran rubra tetraploid most likely represents a case of convergent evolution to a rubra-like form, adapted to high elevations. Similarly, C. rubra ssp. depressa are divided into two distinct hexaploid (and sometimes tetraploid) varieties with different origins: the “coastal” type that shares sequences with subspecies mexicana, and the inland “sagebrush” type that shares matK/trnK sequences with C. rubra and has a unique ITS sequence that is sister to subspecies mexicana (fig. 8). Further examples could be discussed ad nauseam, especially within tetraploid C. parviflora and hexaploid C. perfoliata. Clearly, species and subspecies of the C. perfoliata complex, as defined by Miller and Chambers (1993; 2006), are morphological types based on homoplasious characters.
Conclusions. This study highlights the dynamic nature of polyploidy in the Claytonia perfoliata complex, and is the first to explicitly use DNA-based methods to determine phylogenetic relationships among diploids and polyploids, and to infer polyploid origins within the complex. Polyploid subspecies and cytotypes within the C. perfoliata complex are genetically complex. Results show that polyploids have multiple independent origins and that allopolyploidization is the most common form of diversification. Additionally, polyploid taxa
138 under the current treatment represent morphological types based on homoplasious characters
(e.g. growth habit and basal leaf shape).
The results of this paper suggest numerous cryptic polyploid species. This has implications for conservation and sensitive species status of taxa within the C. perfoliata complex, as many of these polyploid species are uncommon and occur in habitats that are currently being developed and/or altered by human activities (e.g. Columbia Gorge intermontana octaploids). Indeed, the California Native Plant Society and the California
Department of Fish and Game are currently considering the diploid C. parviflora ssp. grandiflora for sensitive species status (McIntyre et al. 2006). This study highlights the need for further molecular and morphological work in this confusing polyploid complex.
139 TAXONOMIC TREATMENT
The results of this study, as well as those described by Miller and Chambers (1993;
2006), show that C. perfoliata s.l. is a morphologically diverse polyploid complex. Diploid taxa are morphologically, genetically, and geographically distinct. However, polyploid taxa often have reticulate phylogenies, multiple independent origins from diploid taxa, and unique or intermediate morphologies compared to the diploids.
This taxonomic treatment presents new and previously published combinations of the
C. perfoliata complex at the infraspecific level of variety. Similar to other published accounts
(reviewed in Hamilton and Reichard 1992), variety is defined as an infraspecific taxon that is morphologically or geographically distinct from other varieties, but which can (and does) interbreed easily with other varieties when sympatric. Thus, as defined here, varieties do not necessarily reflect common ancestry.
The following varieties (also presented in table 2) generally parallel Miller and
Chambers’ (1993; 2006) descriptions and keys of species and subspecies within the C. perfoliata complex. Justification for this treatment is summarized when necessary (e.g. taxa formerly in C. parviflora and C. rubra).
CLAYTONIA PERFOLIATA Donn ex Willdenow. var. PERFOLIATA. Sp. pl. 1: 1186. 1798. TYPE:
specimen prepared from cultivated plants (lectotype, designated by Miller and
Chambers, 1993: B-W-4984, photocopy: OSC!).
140 CLAYTONIA PERFOLIATA Donn ex Willdenow var. INTERMONTANA (J.M. Miller & Chambers).
Dorn. Vasc. Pl. Wyoming, ed. 3: 377. 2001. TYPE: U.S.A. Nevada: Churchill County,
Hwy. 50, 8 mi. east of Eastgate, 7 mi. west of Carroll Summit. 19 May 1960, H.K.
Sharsmith 4767 (holotype OSC!).
Claytonia perfoliata Donn ex Willdenow var. mexicana (Rydberg) J.H. Rausch, comb. nov.
BASIONYM: Limnia mexicana Rydberg. N. Amer. Fl. 21: 309. 1932. TYPE: MEXICO.
Mèxico: Nevada de Toluca, 15 Oct. 1903, Rose & Painter 7924 (holotype: US, photo:
OSC!).
In all analyses, the specimens of C. parviflora ssp. grandiflora (under Miller and
Chambers’ 1993 nomenclature) form supported clades within the C. parviflora + C. perfoliata + C. washingtoniana clades (figs. 1-3). This suggests that C. parviflora ssp. grandiflora should be treated as an infraspecific taxon of C. perfoliata s.s., and that the use of the morphologically defined C. parviflora should be discontinued at the species level. The revised taxonomy reflects this: C. parviflora and its subspecies (under Miller and Chambers’
1993 treatment) are specified as varieties of C. perfoliata s.s.
Claytonia perfoliata Donn ex Willdenow var. grandiflora (J.M. Miller & Chambers) J.H.
Rausch, comb. nov. BASIONYM: Claytonia parviflora Douglas ex Hooker ssp.
grandiflora J. M. Miller & Chambers. Novon 3: 270. 1993. TYPE: U.S.A. California:
Calaveras County, San Antonio Creek south of Sheep Ranch on the road to Murphy’s,
8 May 1977, J. M. Miller 666 (holotype: OSC!; isotypes: CAS, RSA, SD).
141 CLAYTONIA PERFOLIATA Donn ex Willdenow var. PARVIFLORA (Douglas ex Hooker) Torrey.
Pacif. Railr. Rep. 4(5): 71. 1857. TYPE: U.S.A. “near Indian villages or where wood
has been destroyed by fire” [probably along the course of the Columbia River of
Oregon or Washington], 1825, D. Douglas s.n. (holotype: K, photos: OSC!, US;
isotype: BM).
CLAYTONIA PERFOLIATA Donn ex Willdenow var. UTAHENSIS (Rydberg) Poellnitz. Repert. Sp.
Nov. Regni Veg. 30: 302. 1932. TYPE: U.S.A. Utah: St. George, 1877, Palmer 56
(holotype: NY, photos: OSC!; isotype: NY).
Claytonia perfoliata Donn ex Willdenow var. viridis (Davidson) J.H. Rausch, comb. nov.
BASIONYM: Montia spathulata (Douglas ex Hooker) Howell var. viridis Davidson.
Bull. So. California Acad. Sci. 5: 61. 1907. TYPE: U.S.A. California: Los Angeles
County, San Gabriel Mountains, Big Rock Creek, 7 June 1906, Hasse & A. Davidson
1507 (holotype: LACM [at RSA]; isotype: GH).
Diploids of C. rubra (under Miller and Chambers’ 1993 nomenclature) are morphologically distinct and consistently form well-supported clades sister to the other taxa within the complex. However, Miller (1978b) and Miller and Chambers (2006) also note the occurrence of diploids with intermediate morphology between rubra and mexicana and/or parviflora (not sampled here). Furthermore, tetraploids and hexaploids often possess sequences from the other diploid taxa. For example, the Sierran tetraploid has sequences
142 identical to those possessed by grandiflora and mexicana, and depressa hexaploids share identical ITS sequences with mexicana. Given this degree of genetic and morphological variability, the revised taxonomy presented here treats C. rubra and its subspecies (under
Miller and Chambers’ 1993 treatment) as varieties of C. perfoliata s.s.
CLAYTONIA PERFOLIATA Donn ex Willdenow var. RUBRA (Howell) Poellnitz. Repert. Sp.
Nov. Regni Veg. 30: 301. 1932. TYPE: U.S.A. Washington: Cimcoe [Simcoe]
Mountains, June 1880 [1881], T. Howell s.n. (lectotype, designated by J.M. Miller &
Chambers, 1993: ORE!, photo: OSC!; isolectotype: US).
CLAYTONIA PERFOLIATA Donn ex Willdenow var. DEPRESSA (A. Gray) Poellnitz. Repert. Sp.
Nov. Regni Veg. 30: 301. 1932. TYPE: U.S.A. Washington: San Juan Island, 1858,
Lyall s.n. (lectotype, designated by J.M. Miller & Chambers, 1993: GH, photo: OSC!).
Claytonia washingtoniana is the putative allotetraploid hybrid of C. perfoliata s.s. and
C. sibirica (Fellows 1971). Morphologically, C. washingtoniana is most similar to C. sibirica.
The results of this study show that C. washingtoniana is consistently within the same clade as
C. perfoliata. Given this information, C. washingtoniana is kept as a distinct species.
CLAYTONIA WASHINGTONIANA (Suksdorf) Suksdorf. Werdenda 1: 10. 1923. TYPE: U.S.A.
Washington: King County, damp mossy woods, Lake Washington, 5 Aug. 1890, W. N.
Suksdorf 957 (holotype: WS!; isotypes: F, GH, ISC, MIN, MO, US, WIS).
143 ACKNOWLEDGEMENTS
Thanks to R. Gomulkiewicz, L Hufford, B. Husband, and S. Nuismer for discussions and comments on earlier versions of this manuscript. Thanks to E. Roalson for the use of laboratory facilities and C. Cody for help with greenhouse operations. This research also benefited from many discussions with R. O’Quinn, J. Brokaw, J. Smith, S. Novak, M. King, J.
Clark, E. Roalson, P. McIntyre, K. Chambers, J. Miller, and M. Morgan. Funding was provided by the Betty W. Higginbothan Fellowship and the National Science Foundation grants DEB 0128896 to M. Morgan and DEB 0209916 to R. Gomulkeiwicz
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152 TABLE 1. Taxa and cytotypes considered in this study. Nomenclature follows the treatment of Miller and Chambers (1993;
2006). Chromosome counts are those reported here and in Miller (1976; 1978c) and Miller and Chambers (2006). Cytotypes marked with x are used in this study, and those marked with have been reported elsewhere.
2n = Species Subspecies 12 24 36 48 60
C. perfoliata Donn ex Willdenow subsp. perfoliata x x x x
subsp. mexicana (Rydberg) J.M. Miller & Chambers x x
subsp. intermontana J.M. Miller & Chambers x x
C. parviflora Douglas ex Hooker subsp. parviflora x x
subsp. grandiflora J.M. Miller & Chambers x
subsp. utahensis (Rydberg) J.M. Miller & Chambers x
subsp. viridis (Davidson) J.M. Miller & Chambers x
C. rubra (Howell) Tidestrom subsp. rubra x x
subsp. depressa (A. Gray) J.M. Miller & Chambers x
C. washingtoniana (Suksdorf) Suksdorf x
153 TABLE 2. Revised taxonomic treatment for the C. perfoliata complex and C. washingtoniana. Infraspecific (variety) epithets here correspond to infraspecific (subspecies) epithets of Miller and Chambers (1993, 2006) presented in Table 1. Infraspecific taxa in bold represent new combinations.
Species Variety
C. perfoliata Donn ex Willdenow var. perfoliata
var. depressa (A. Gray) Poellnitz
var. grandiflora (J.M. Miller & Chambers) J.H. Rausch, comb. nov.
var. intermontana (J.M. Miller & Chambers) Dorn
var. mexicana (Rydberg) J.H. Rausch, comb. nov.
var. parviflora (Douglas ex Hooker) Torrey
var. rubra (Howell) Poellnitz
var. utahensis (Rydberg) Poellnitz
var. viridis (Davidson) J.H. Rausch, comb. nov.
C. washingtoniana (Suksdorf) Suksdorf
154 FIGURE LEGENDS
FIGURE 1. Maximum likelihood trees of a) combined analysis of matK/trnK and ITS, b)
matK/trnK only, and c) ITS only, for diploid C. rubra, C. perfoliata, and C.
parviflora; and map showing locations of diploid specimens used in this study. Branch
support (bootstrap / aLRT) given below or to the left of branches with >70% bootstrap
or > 0.70 aLRT (as a percentage). Superscripts refer to the following author collection
numbers: C. rubra ssp. rubra [1] 289, 295; [2] 302; [3] 112, 115, 116, 287; [4] 122,
126, 311, 320. C. perfoliata ssp. mexicana [1] 217a; [2] 101; [3] 100; [4] 104; [5] 199.
C. parviflora ssp. grandiflora [1] 30, 32; [2] 69; [3] 170, 175; [4] 163; [5] 60; [6] 312;
[7] 41, 43, 49.
FIGURE 2. Maximum likelihood tree of matK / trnK. Terminal nodes indicate lowest ploidy of
identical sequences shared among other taxa and cytotypes; taxa are indicated by
subspecific designation and diploid taxa are bold. Thickened branches are those with
branch support >70% (bootstrap / aLRT ); support values are given to the left of the
branch. Superscripts refer to identical sequences shared by other cytotypes and taxa
(on right); italicized numbers indicate the author’s specimen collection numbers.
FIGURE 3. Maximum likelihood tree of ITS. Terminal nodes indicate lowest ploidy of
identical sequences shared among other taxa and cytotypes; taxa are indicated by
subspecific designation and diploid taxa are bold. Thickened branches are those with
branch support >70% (bootstrap / aLRT ); support values are given to the left of the
155 branch. Superscripts refer to identical sequences shared by other cytotypes and taxa
(on right); italicized numbers indicate the author’s specimen collection numbers.
FIGURE 4. Geographic distribution of specimens (regardless of ploidy) with identical
sequences to diploid C. perfoliata ssp. mexicana specimens (mexicana 1 through
mexicana 5 correspond to the those indicated on figures 1-3). Open circles represent
specimens with identical matK/trnK sequences and gray stars represent specimens
with identical ITS sequences. Solid squares represent the location of diploid C.
perfoliata ssp. mexicana specimens, with the exception of one of the solid squares
within the mexicana 1 pane, which represents a putative autopolyploid of mexicana 1.
FIGURE 5. Tetraploid (A) and hexaploid (B) cytotypes of C. perfoliata linked with identical
sequences on ML trees of matK/trnK (from fig. 2) and ITS (from fig. 3). Taxa are
indicated by subspecific designation. Bold taxa on ML trees indicate diploid
taxa/specimens shown in figures 1-3. Polyploid taxa are connected to diploid
sequences with solid lines and to unique polyploid sequences with dashed lines.
Circled numbers correspond to geographic location of the author’s numbered
specimens for A) tetraploid C. perfoliata: [1] 218; [2] 182, 183; and B) hexaploid C.
perfoliata: [1] 130, 281; [2] 178, 189, 190; [3] 206; [4] 109, 225, 244, 248; [5] 310;
[6] 273, 283, 301; [7] 164, 168, 172; [8] 45, 50; [9] 70; [10] 157; [11] 34, 38.
FIGURE 6. Octaploid (A) and decaploid (B) cytotypes of C. perfoliata linked with identical
sequences on ML trees of matK/trnK (from fig. 2) and ITS (from fig. 3). Taxa are
156 indicated by subspecific designation. Bold taxa on ML trees indicate diploid
taxa/specimens shown in figures 1-3. Polyploid taxa are connected to diploid
sequences with solid lines and to unique polyploid sequences with dashed lines.
Circled numbers correspond to geographic location of the author’s numbered
specimens for A) octaploid C. perfoliata: [1] 271; [2] 238, 249, 250, 278; [3] 209,
212, 214; [4] 86, 145; and B) decaploid C. perfoliata [1] 254a, 258, 260, 267; [2] 141.
FIGURE 7. Tetraploid (A) and hexaploid (B) cytotypes of C. parviflora linked with identical
sequences on ML trees of matK/trnK (from fig. 2) and ITS (from fig. 3). Taxa are
indicated by subspecific designation. Bold taxa on ML trees indicate diploid
taxa/specimens shown in figures 1-3. Polyploid taxa are connected to diploid
sequences with solid lines and to unique polyploid sequences with dashed lines.
Circled numbers correspond to geographic location of the author’s numbered
specimens for A) tetraploid C. parviflora [1] 194, 195; [2] 65; [3] 154; [4] 146; [5] 95,
138; [6] 135, 153; [7] 84, 85; [8] 187a; [9] 55; [10] 187b and B) hexaploid C.
parviflora [1] 119; [2] 193, 235, 241, 243, 291, 294; [3] 201, 159a.
FIGURE 8. Tetraploid C. rubra and hexaploid C. rubra ssp. depressa linked with identical
sequences on ML trees of matK/trnK (from fig. 2) and ITS (from fig. 3). Taxa are
indicated by subspecific designation. Bold taxa on ML trees indicate diploid
taxa/specimens shown in figures 1-3. Polyploid taxa are connected to diploid
sequences with solid lines and to unique polyploid sequences with dashed lines.
157 Circled numbers correspond to geographic location of the author’s numbered specimens: [1] 133; [2] 284; [3] 262, 265; [4] 229, 232; [5] 185.
158 FIGURE 1:
159 FIGURE 2:
160 FIGURE 3:
161 FIGURE 4:
162 FIGURE 5:
163 FIGURE 6:
164 FIGURE 7:
165 FIGURE 8:
166