EVOLUTION AND BIOGEOGRAPHY OF CAMPANULACEAE: FROM GLOBAL PATTERNS TO SHALLOW, SPECIES-LEVEL PROCESSES IN THE MEDITERRANEAN BASIN By ANDREW ALAN CROWL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2016
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EVOLUTION AND BIOGEOGRAPHY OF CAMPANULACEAE: FROM GLOBAL PATTERNS TO SHALLOW, SPECIES-LEVEL PROCESSES IN THE
MEDITERRANEAN BASIN
By
ANDREW ALAN CROWL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
1 OVERVIEW OF CAMPANULACEAE AND INTRODUCTORY REMARKS ............. 13
2 A GLOBAL PERSPECTIVE ON CAMPANULACEAE: BIOGEOGRAPHIC, GENOMIC, AND FLORAL EVOLUTION ................................................................. 15
3 PHYLOGENY OF CAMPANULOIDEAE (CAMPANULACEAE) WITH EMPHASIS ON THE UTILITY OF NUCLEAR PENTATRICOPEPTIDE REPEAT (PPR) GENES ........................................................................................................ 46
Results And Discussion .......................................................................................... 55 Phylogenetic Resolution ................................................................................... 56
Plastid loci .................................................................................................. 56 Pentatricopeptide repeat (PPR) loci ........................................................... 60 Combined plastid and PPR loci .................................................................. 62
Plastid–Nuclear Incongruence .......................................................................... 65 Final Conclusions ................................................................................................... 68
4 EVOLUTION AND BIOGEOGRAPHY OF THE ENDEMIC ROUCELA COMPLEX (CAMPANULACEAE: CAMPANULA) IN THE EASTERN MEDITERRANEAN ................................................................................................. 76
Introduction ............................................................................................................. 76 Oceanic and Continental Islands ...................................................................... 77 Geologic and Climatic History of the Eastern Mediterranean Basin ................. 77 Roucela Complex ............................................................................................. 79 Summary .......................................................................................................... 80
Materials and Methods............................................................................................ 81 Taxon Sampling and DNA Amplification ........................................................... 81 Phylogenetic Analysis ...................................................................................... 81 Species Tree .................................................................................................... 82 Molecular Dating .............................................................................................. 83 Biogeographic Analysis .................................................................................... 84 Ecological Niche Modeling ............................................................................... 85 Diversification ................................................................................................... 86
Plastid ........................................................................................................ 88 Nuclear ...................................................................................................... 89 Species tree and combined dataset ........................................................... 90
Divergence Time Estimates and Ancestral Range Estimation .......................... 91 Niche Modeling ................................................................................................. 92 Diversification ................................................................................................... 92
Discussion .............................................................................................................. 93 Evolution and Biogeography of the Roucela Clade .......................................... 93
5 GENE TREE DISCORDANCE PROVIDES EVIDENCE FOR CRYPTIC DIVERSITY AND INSIGHTS INTO THE EVOLUTION OF A POLYPLOID COMPLEX IN A MEDITERRANEAN CAMPANULA (CAMPANULACEAE) CLADE .................................................................................................................. 105
Table page 2-1 Comparison of the three data sets included in this study. .................................. 40
2-2 Summary of the two divergence-time estimate methods. ................................... 40
2-3 Likelihood statistics from ancestral area estimation models implemented in BioGeoBEARS. .................................................................................................. 41
3-1 Chloroplast and nuclear markers used in this study. .......................................... 70
10
LIST OF FIGURES
Figure page 2-1 Comparison of phylogenetic results from the coding-genes-only data set. ......... 41
2-2 Chronogram of the Campanulaceae clade showing ancestral range estimations and hypothesized polyploidy events. ............................................... 42
2-3 Ancestral estimation of floral symmetry in the Campanulaceae. ........................ 43
2-4 Estimation of ancestral character states for characters related to secondary pollen presentation in the Campanulaceae. ....................................................... 44
2-5 Ks plots showing the distribution of synonymous substitutions among gene duplicates. .......................................................................................................... 45
3-1 Plastid phylogeny of the Campanuloideae clade. ............................................... 71
3-2 PPR phylogeny of the Campanuloideae clade. .................................................. 72
3-3 Combined plastid and PPR phylogeny of the Campanuloideae clade. ............... 73
3-4 Support for plastid-only and combined plastid-PPR trees. ................................. 74
3-5 Divergence time estimates for combined plastid and PPR tree. ......................... 75
4-1 Occurrence map for the Roucela complex. ...................................................... 100
4-2 Results from concatenated and species tree analyses. .................................... 101
4-3 Chronogram of the Roucela clade showing ancestral range estimation. .......... 102
4-4 Niche modeling results for selected taxa in the Roucela complex. ................... 103
4-5 Tempo and pattern of diversification of the Roucela clade. .............................. 104
5-1 Sample localities, species distributions, and comparison of concatenated analyses. .......................................................................................................... 120
5-2 Comparison of species-tree analyses. .............................................................. 121
5-4 Analyses of morphology data. .......................................................................... 123
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
EVOLUTION AND BIOGEOGRAPHY OF CAMPANULACEAE: FROM GLOBAL
PATTERNS TO SHALLOW, SPECIES-LEVEL PROCESSES IN THE MEDITERRANEAN BASIN
By
Andrew Alan Crowl
December 2016
Chair: Nicoletta Cellinese Major: Botany
The Campanulaceae are a diverse clade of flowering plants, encompassing more
than 2300 species, inhabiting myriad habitats from tropical rainforests to arctic tundra.
Using a phylogenetic framework, I inferred the placement and timing of major
biogeographic, genomic, and morphological changes in the history of the group. This
study highlights the diversity and complexity of historical processes driving evolution
within the Campanulaceae from broad, global patterns to shallow, species-level
processes in the Mediterranean Basin.
First, a robust, multi-gene phylogeny, including all major lineages, is presented to
provide a broad, evolutionary perspective of this clade across six continents. Ancestral
range estimation supports an out-of-Africa diversification following the KPg extinction
event. Chromosomal modeling provides evidence for numerous genome-wide
duplication events prior to movements into new, and often harsh, habitats.
Morphological reconstructions support the hypothesis that switches in floral symmetry
and anther dehiscence were important in the evolution of secondary pollen presentation
mechanisms.
12
The Mediterranean Basin is among the most biologically diverse areas in the
world, harboring enumerable poorly understood, species-rich groups. Here, I focus on
the Roucela complex (Campanula subgenus Roucela), 12 annual species found
primarily in the eastern Mediterranean Basin. Plastid and low-copy nuclear markers
were employed to reconstruct evolutionary relationships and provide insights into
patterns of endemism and diversification through time. Diversification of the Roucela
clade appears to have been primarily the result of vicariance driven by the break-up of
an ancient landmass. Contrary to past studies, my findings suggest the onset of the
Mediterranean climate has not promoted diversification in the Roucela complex and, in
fact, may be negatively affecting these species.
Because cryptic diversity was detected within the currently recognized,
widespread species, Campanula erinus, I used a Hyb-Seq approach to obtain two
genomic datasets (nuclear and plastome) across 105 C. erinus individuals, representing
27 populations spanning the Mediterranean Basin. Two lineages were recovered: a
western Mediterranean tetraploid lineage and an eastern Mediterranean octoploid
lineage. Nuclear gene tree topologies and network analyses indicate a hybrid origin for
the octoploid. The Cretan endemic C. creutzburgii (tetraploid) and the western
Mediterranean C. erinus (tetraploid) are implicated as parental lineages.
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CHAPTER 1 OVERVIEW OF CAMPANULACEAE AND INTRODUCTORY REMARKS
With over 2300 species found across six continents (Lammers, 2007b), the
angiosperm clade, Campanulaceae, offers an opportunity to illuminate the historical
mechanisms responsible for the distribution of widespread, pan-tropical and pan-
holarctic biota, two questions that have long been of interest to botanists (Croizat, 1958;
Raven and Axelrod 1974; Sanmartin and Ronquist, 2004; Beaulieu et al., 2013).
Campanulaceae include five major lineages, variously treated as separate
families or subfamilies (e.g., Lammers, 1992, 2007a, 2007b). Little is known about the
early evolution of this group, primarily due to a poor fossil record and unresolved
relationships among the major lineages. Decades of systematic and phylogenetic
investigations into this diverse group have failed to satisfactorily resolve deep
phylogenetic relationships (see Ch. 2 for further discussion and citations). A robust,
comprehensive phylogeny is necessary to generate hypotheses regarding when, where,
and how this clade came to inhabit such a diverse array of habitats and exhibit
significant morphological variation.
Because many relationships at low phylogenetic scales remained unclear, two
low-copy nuclear loci from the pentatricopeptide repeat (PPR) gene family were
developed for use within the Campanuloideae clade (Ch. 3). This study represents the
first inclusion of low-copy nuclear genes for phylogenetic reconstruction in
Campanuloideae.
The Mediterranean Basin is among the most biologically diverse areas in the
world, harboring enumerable poorly understood, species-rich groups. Of the
approximately 25,000 plant species found in the Mediterranean, ca. 50-60% are found
14
nowhere else in the world (Cowling et al., 1996; Thompson et al., 2005). Understanding
evolutionary processes and species diversity is of special interest in this region given
the exceptionally high degree of endemism and high proportion of rare and endangered
taxa. Here I focus on the Roucela complex (Campanula subgenus Roucela). The
current distribution and high level of endemism in this clade appears to be the result of
allopatric speciation driven by the break-up of an ancient landmass (Ch. 4) with more
recent climatic fluctuations negatively affecting these sub-tropically adapted taxa. Gene-
tree discordance and ploidy estimates suggest allopolyploidy has created cryptic
diversity within the currently recognized, widespread species, Campanula erinus. (Ch.
5).
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CHAPTER 2 A GLOBAL PERSPECTIVE ON CAMPANULACEAE: BIOGEOGRAPHIC, GENOMIC,
AND FLORAL EVOLUTION
Introduction
Historical mechanisms responsible for the distribution of widespread, pan-tropical
or pan-holarctic biota have long been of interest to botanists (Croizat, 1958; Raven and
Axelrod 1974; Sanmartin and Ronquist, 2004; Beaulieu et al., 2013). The
Campanulaceae are a clade that has diversified across both areas almost equally well.
The Campanulaceae are a large angiosperm group encompassing over 2300
species found on six continents (Lammers, 2007b). Little is known about its early
evolution, primarily due to a poor fossil record and unresolved relationships among the
major lineages. A robust, comprehensive phylogeny is necessary to generate
hypotheses regarding when, where, and how this clade came to inhabit such a diverse
array of habitats and exhibit significant morphological variation.
The Campanulaceae include five major lineages, variously treated as separate
families or subfamilies (e.g., Lammers, 1992, 2007a, 2007b): (i) Campanuloideae, a
group encompassing ca. 1000 species distributed worldwide with a center of diversity in
the holarctic, include primarily small perennials and represents the only lineage in the
Campanulaceae with radial floral symmetry; (ii) Lobelioideae, encompassing ca. 1200
species worldwide and a center of diversity in the New World tropics, include taxa with a
diverse array of habits from small herbs to tree-like ‘giant lobelias’; (iii) Cyphioideae
include 65 perennial herbs restricted to Africa; (iv) Nemacladoideae are a group of 19 Reprinted with permission from American Journal of Botany, Inc. Original publication: Crowl A.A., Miles N.W., Visger C.J., Hansen K., Ayers T., Haberle R., & Cellinese N. (2016) A global perspective on Campanulaceae: Biogeographic, genomic, and floral evolution. American journal of botany, 103, 233–245. Online access: http://www.amjbot.org/content/103/2/233
16
species known as ‘thread-plants’ and distributed in the southwestern United States and
northern Mexico; (v) Cyphocarpoideae include three poorly known species of small
annuals endemic to the Atacama Desert of Chile.
Phylogenetics
Early attempts to reconstruct phylogenetic relationships within the
Campanulaceae relied on morphology and single-gene datasets (Cosner et al., 1994;
Gustafsson and Bremer, 1995; Gustafsson et al., 1996; Haberle, 1998). These studies
included all five major lineages and confirmed the monophyly of the Campanulaceae
but recovered differing relationships within the family.
Phylogenetic analysis of morphological data resulted in a single clade including
taxa with bilateral floral symmetry sister to the radially-symmetric Campanuloideae
(Gustafsson and Bremer, 1995). Early molecular studies using the rbcL plastid gene
suggested a different topology: Cyphioideae was inferred as the earliest diverging clade
with a successive sister relationship of Cyphocarpoideae, Lobelioideae,
Nemacladoideae, and Campanuloideae (Cosner et al., 1994; Gustafsson et al., 1996).
Haberle (1998) and Ayers and Haberle (1999), using the nrITS region, found
Nemacladoideae sister to Campanuloideae and Cyphioideae sister to Lobelioideae plus
Cyphocarpoideae. To our knowledge, a study including all five Campanulaceae
lineages has not been conducted since.
The combined molecular and morphological analysis of Lundberg and Bremer
(2003) suggested Nemacladoideae sister to Lobelioideae and Campanuloideae sister to
Cyphioideae. These relationships have also been suggested by more recent, multi-gene
studies (Tank and Donoghue, 2010; Knox, 2014), though with low statistical support.
17
Recent molecular dating analyses of the Campanulaceae have shown the
divergence of these major lineages occurred within a relatively short time period
(Beaulieu et al., 2013; Knox, 2014), providing a potential explanation for the difficulty
previous studies have encountered in accurately resolving relationships within the
clade.
Biogeography
Accurate estimates for the age of the Campanulaceae and its individual lineages
within the clade have important implications for uncovering the biogeographic history of
this group and understanding the historical processes responsible for the current,
cosmopolitan distribution. Past estimates suggest an origin for the Campanulaceae in
the Cretaceous to the Paleogene (67-40 million years ago [Ma]; Wikstrom et al., 2001;
Bell et al. 2010; and Knox, 2014). Diversification of the Campanuloideae has been
estimated to begin at 56-23 Ma (Cellinese et al., 2009; Roquet et al., 2009; Mansion et
al., 2012; Crowl et al., 2014; Crowl et al., 2015) and the Lobelioideae at 88-50 Ma
(Antonelli, 2009).
With improved species sampling we are able to estimate divergence dates for all
five major lineages and infer putative dispersal events, which have led to the
cosmopolitan distribution of this clade. A Southern Hemisphere origin has been
hypothesized for the Asterales (Bremer and Gustafsson, 1997; Beaulieu et al. 2013),
agreeing with the out-of-Africa scenario seen in past analyses of Campanulaceae (Knox
et al., 2006; Antonelli, 2009). However, these studies focused mainly on the tropical
clades, Lobelioideae and Cyphioideae. Our sampling allows for more accurate
estimations regarding the ancestral area of this group and biogeographic patterns within
the clade.
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Genome Evolution
Whole-genome duplication (WGD; polyploidy) events have been found to
facilitate movement into new environments and changes in morphology (see Stebbins,
1985; Leitch and Leitch, 2008; Flagel and Wendel, 2009; Parisod, 2012; te Beest et al.
2012). A comprehensive understanding of the relationships within Campanulaceae will
provide the basis to elucidate what role genomic evolution has played in the
evolutionary history this group. A large variation in chromosome number exists across
the clade, with every base number between 6 and 30 (and as high as 40) reported
(Lammers, 2007a). A long-standing debate exists regarding polyploidy in the group, with
little resolution about how to interpret chromosome counts in relation to genomic history
(Lammers, 1993; Stace and James, 1996). To date, the question of whether the high
chromosome numbers found in Campanulaceae are the result of an ancestral
polyploidy event, followed by descending disploidy or numerous, recent polyploidy
events, has not been answered.
Secondary Pollen Presentation
Secondary pollen presentation involves the relocation of pollen from the anthers
to disparate floral organs (Howell et al., 1993; Yeo, 1993). This feature is found across
many clades of angiosperms and throughout the Campanulaceae, with a variety of
mechanisms present (Erbar and Leins, 1989, 1995; Leins and Erbar, 1990, 2005; Yeo,
1993). However, in all cases, pollen presentation occurs via introrse anthers. In the
Campanuloideae and Nemacladoideae the anthers are connivent, forming an ‘anther
ring’ (or a ‘pollen box’ in the case of Cyphioideae), while they are connate in the
Lobelioideae, forming an ‘anther tube’ (Leins and Erbar, 1990; 2003).
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Multiple, specialized mechanisms have evolved within the group. The
Campanuloideae exhibit a simple “deposition mechanism” where pollen is released onto
stylar hairs within the anther ring prior to anthesis (Leins and Erbar, 1990). The stamens
then quickly wither while the style elongates, exposing the pollen to the outside
environment. This is followed by invagination of the hairs, causing pollen to be released
(Leins and Erbar, 1990) and the style to become characteristically pitted. Within the
Campanuloideae, Phyteuma species have evolved a more complex ‘brushing
mechanism.’ This mechanism allows for deposition of pollen onto stylar hairs with the
aid of the corolla, which remains fused at the apical tip, and elongation of the style. The
growing style eventually ruptures through the corolla hood, presenting pollen to
pollinators (Leins and Erbar, 2006). The simple deposition mechanism of the
Cyphioideae is characterized by a pollen box in which the five connivent anthers form a
wall and the stylar tip forms the base of the box (Leins and Erbar 2003). Pollen is shed
into the box and onto the stylar tip prior to anthesis. Unlike in the Campanuloideae, the
simple deposition mechanism of Cyphioideae does not include style elongation or
invagination of the stylar hairs (Leins and Erbar, 2003, 2005). The pump mechanism of
the Lobelioideae is similar to that found in the Asteraceae (often referred to as a plunger
mechanism): following release of the pollen into the anther tube, stylar growth forces
pollen out of the top of the anther tube (Erbar and Leins, 1989; Leins and Erbar, 1990).
Very little is known about secondary pollen presentation in the Nemacladoideae, though
a preliminary study (C. Erbar and P. Leins, University of Heidelberg, personal
communication) suggests this mechanism is similar to that found in the
20
Campanuloideae with pollen deposition onto stylar hairs and retraction of these hairs at
anthesis. The mechanism of pollen presentation within Cyphocarpoideae is unknown.
Leins and Erbar (2006) presented an evolutionary interpretation of secondary
pollen presentation within the Campanulaceae, suggesting the pump mechanism of
Lobelioideae as the ancestral state for the clade. However, the precise phylogenetic
position of all lineages was unknown at the time. A robust, complete phylogeny of the
Campanulaceae has important implications for understanding how secondary pollen
presentation mechanisms may have evolved within this group. Furthermore,
understanding the genomic history and floral character evolution will allow us to uncover
potential floral development and genomic changes associated with secondary pollen
presentation.
Summary
Previous studies of the Campanulaceae have relied on either a small number of
gene regions or limited taxon sampling, resulting in incomplete hypotheses. We have
constructed the largest dataset to date, including 16 gene regions (plastid and nuclear)
and representatives (over 900 species) from all five major lineages to better understand
the evolutionary history of this diverse, cosmopolitan clade of flowering plants.
Methods
Sampling
Custom python scripts were used to mine GenBank
(http://www.ncbi.nlm.nih.gov/genbank) for all available Campanulaceae sequence data.
Sixteen loci were obtained: 15 plastid and one nuclear locus (atpB-rbcL Spacer, atpB,
Table 2-2. Summary of the two divergence-time estimate methods. Crown ages are
given for the major Campanulaceae clades in millions of years before present. Parenthetical values represent 95% highest posterior densities of ages for that node.
Crown clade r8s BEAST MRCA of Rousseaceae and Campanulaceae Campanulaceae
Table 2-3. Likelihood statistics from ancestral area estimation models implemented in
BioGeoBEARS. Model
lnL
AIC
Likelihood Ratio Test P-value
BAYAREA -636.4641 1277 4.5e-82
BAYAREA+J -452.3424 910.7
DEC -515.1424 1034 3.6e-25
DEC + J -461.4439 928.9
DIVALIKE -550.9410 1106 1.6e-33
DIVALIKE+J -478.1609 962.3
Figure 2-1. Comparison of phylogenetic results from the coding-genes-only data set. A)
Maximum likelihood analysis. B) Bayesian analysis. Each of the five clades has been collapsed to a single branch for clarity and an illustration of a representative floral morphology is provided for each. Note the inconsistent placement of Cyphocarpoideae. Numbers on nodes are bootstrap support values (A) and posterior probability values (B). Values on the tips indicate support for the monophyly of the major lineages. Branch lengths are drawn proportional to time.
42
Figure 2-2. Chronogram of the Campanulaceae clade showing ancestral range
estimations and hypothesized polyploidy events. Branches are colored relative to geographic area indicated on the map. Gold circles on branches represent placement of hypothesized polyploidy events. Histogram at the tips of the tree represent chromosome count data used in this study. Lobelioideae clade names based on Antonelli (2008), Campanuloideae clade names refer to those of Mansion et al. (2012).
43
Figure 2-3. Ancestral estimation of floral symmetry in the Campanulaceae. Radial
symmetry is shown in black and bilateral symmetry is shown in red. Pie charts indicate the marginal likelihoods of the character reconstruction at that node. Node highlighted in green indicates ancestral state for the Campanulaceae.
44
Figure 2-4. Estimation of ancestral character states for characters related to secondary
pollen presentation in the Campanulaceae. Character states are given for each reconstruction. Pie charts indicate the marginal likelihoods of the character reconstruction at that node. Node highlighted in red indicates ancestral state for the Campanulaceae. Branching order and clade positions are identical to those in Figure 2-3.
45
Figure 2-5. Ks plots showing the distribution of synonymous substitutions among gene
duplicates. A) Platycodon grandiflorus. B) Lobelia siphilitica. C) Phelline lucida. Black lines indicate inferred peaks, representing signatures of past duplication events.
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CHAPTER 3 PHYLOGENY OF CAMPANULOIDEAE (CAMPANULACEAE) WITH EMPHASIS ON
THE UTILITY OF NUCLEAR PENTATRICOPEPTIDE REPEAT (PPR) GENES
Introduction
Campanulaceae Jussieu are a nearly cosmopolitan group of flowering plants
comprising five subfamilies (Campanuloideae, Lobelioideae, Nemacladoideae,
Cyphioideae, and Cyphocarpoideae), approximately 84 traditionally circumscribed
genera, and more than 2300 species (Lammers, 2007). Historically, there has been
much disagreement as to intrafamilial classification (de Candolle, 1839; Fedorov, 1957;
Takhtajan, 1987), primarily due to the polyphyly of the largest genera, Campanula and
Wahlenbergia (Cellinese et al., 2009; Haberle et al., 2009; Prebble et al., 2011; Mansion
et al, 2012; Prebble et al., 2012).
The heterogeneous Campanuloideae Burnett (approximately 1054 species) are
found primarily in the Northern Hemisphere and are most abundant in temperate areas
of the Old World (Lammers, 2007). They are found from temperate to sub-tropical
areas and occupy a wide variety of habitats, from steppes to high elevation
mountainous regions. Some species have wide distribution ranges, spanning entire
continents, while others are narrow endemics, e.g., restricted to single islands.
Previous studies of the Campanulaceae and Campanuloideae have typically
focused on a few chloroplast genes (Cellinese et al., 2009; Haberle et al., 2009;
Antonelli, 2008; Roquet et al., 2008), one chloroplast marker with expanded taxon
Reprinted with permission from Public Library of Science. Original publication: Crowl A.A., Mavrodiev E., Mansion G., Haberle R., Pistarino A., Kamari G., Phitos D., Borsch T., & Cellinese N. (2014) Phylogeny of Campanuloideae (Campanulaceae) with Emphasis on the Utility of Nuclear Pentatricopeptide Repeat (PPR) Genes. PLoS ONE, 9, e94199. Online access: http://dx.doi.org/10.1371/journal.pone.0094199
47
sampling (Mansion et al, 2012; Borsch et al., 2009), or gene order in the highly
rearranged chloroplast genome (Cosner et al., 1991; Cosner et al., 1993; Cosner et al.,
2004). These studies have made significant progress toward a robust phylogenetic
hypothesis of the group and have highlighted the high level of paraphyly and polyphyly
of many traditionally circumscribed genera, especially Campanula and Wahlenbergia.
However, species-level relationships are yet to be understood and the most widely used
plastid markers within the family (i.e., atpB, matK, and rbcL) have not been able to
provide a significant level of resolution. Furthermore, focusing solely on maternally
inherited markers may obscure the role that hybridization may have played in the
evolutionary history of this group.
Additional studies have attempted to use nuclear data by including the internal
transcribed spacers (ITS) sequences of nuclear ribosomal DNA (Prebble et al., 2011;
Prebble et al., 2012; Roquet et al., 2008; Eddie et al., 2003; Park et al., 2006; Antonelli,
2009; Wendling et al., 2011). Although potentially informative at the species level, this
region is considerably difficult to align with confidence in positional homology across
wide phylogenetic distances in the Campanuloideae and is further complicated by
potential concerted evolution and high levels of homoplasy (for further discussion and
concerns see (Alvarez and Wendel, 2003), but see (Feliner and Rossello, 2007)).
Ultimately, past studies including ITS have shown its significant limitation in resolving
species level relationships and providing accurate information on the placement of
several genera (e.g., Jasione and Musschia).
Inferring robust phylogenies for species-rich clades is of great importance for
understanding processes of speciation, hybridization, and patterns in historical
48
biogeography while posing a major challenge to systematists (Mansion et al, 2012;
Borsch et al., 2009; Avis, 1994). Resolving relationships at low taxonomic levels can be
difficult for taxa that are closely related and/or recently diverged. Furthermore,
relationships at the interspecific level can be complicated by hybridization and
introgression. Thus, multiple rapidly evolving, independent nuclear markers may be
useful, and even necessary, to accurately reconstruct species-level phylogenies (Sang,
2002).
Current molecular and phylogenetic methods allow researchers to obtain large,
multi-gene datasets for phylogenetic studies. However, because of highly conserved
genome organization, gene order, and gene content of the chloroplast genome across
much of angiosperm diversity (but see Cosner et al., 1991; Cosner et al., 1993; Cosner
et al., 2004; Haberle, 2008 for exceptions in the Campanuloideae) and the relative ease
of developing universal primers for both chloroplast and nuclear ribosomal DNA, these
have been the most widely used sources of molecular data for plant phylogenetics
(Small et al., 2004). Although universal markers are more labor-intensive to develop
due to gene duplications and deletions (Sang, 2002), under-utilized low-copy nuclear
genes can be of great value to molecular phylogenetic studies.
Low-copy nuclear genes have a number of advantages over plastid regions: they
are unlinked (Steele et al., 2008), possess increased sequence variation (Gaut, 1998),
and are bi-parentally inherited (Sang, 2002; Small and Wendel, 2004; Sang, 2000).
Unlinked nuclear genes allow for multiple, independent datasets and, therefore,
independent estimates of phylogenetic relationships. This affords researchers with the
ability to utilize coalescent-based species tree approaches, the results of which may be
49
more accurate in the presence of incomplete lineage sorting. In contrast, the
chloroplast and mitochondrial genomes provide single markers due to gene linkage
(Steele et al., 2008; Moore, 1995). Furthermore, the often higher rate of sequence
evolution of low-copy nuclear genes (Sang, 2002; Small and Wendel, 2004) may allow
for greater phylogenetic resolution in clades containing slowly evolving or recently
diverged taxa.
However, working with nuclear markers has its limitations. Successfully
designing primers and amplifying target sequences can be quite difficult and labor-
intensive steps such as cloning are often necessary. In order to confidently reconstruct
species relationships it is of great importance to compare orthologous loci rather than
paralogous copies (Sang, 2002). Because most nuclear genes belong to multi-gene
families with different lineages containing losses or duplications the search for orthology
is a crucial limitation of working with nuclear genes (Sang, 2002; Doyle et al., 2003) and
great care must be taken. Focusing on single- or low-copy nuclear genes, however,
can alleviate this limitation.
In this study we compare results based on five chloroplast markers and 2 single-
copy nuclear loci from the pentatricopeptide repeat (PPR) gene family. The
phylogenetic utility of these nuclear loci for plant phylogenetic reconstruction has been
previously demonstrated by Yuan et al. (Yuan et al., 2009). The PPR loci were found to
have a single orthologue in both Oryza sativa and Arabidopsis thaliana and a rapid rate
of evolution, useful at the intergeneric and interspecific levels (Yuan et al., 2009).
Following empirical studies on Verbenaceae (Yuan et al., 2010), recent studies have
50
demonstrated the utility of these genes in plant phylogenetics (Drew and Sytsma, 2013;
Lu-Irving and Olmstead, 2013).
One of the strengths of using PPR loci is that orthology has been previously
assessed (Yuan et al., 2009). Additionally, because they are intronless, issues with
highly polymorphic introns are avoided (Yuan et al., 2009; Yuan et al., 2010).
In this study we evaluate the utility of two PPR loci to resolve evolutionary
relationships within the Campanuloideae. Our results suggest that these markers, when
considered separately and in combination with plastid data, can be informative tools for
phylogenetic reconstruction and for the detection of putative hybridization events.
Methods
Taxon Sampling, Amplification, & Sequencing
Taxa spanning the Campanuloideae clade were included in this study to test the
utility of two single-copy nuclear genes for reconstructing relationships across this large,
taxonomically diverse group as well as resolving relationships between closely related
species. Cyphia elata (Cyphioidae) and Solenopsis minuta (Lobelioideae) were used as
outgroup taxa based on previous studies (Haberle et al., 2009; Gustafsson and Bremer,
1995; Lundberg and Bremer, 2003).
A number of chloroplast (atpB, matK, petD, rbcL, and trnL-F) and ITS sequences
were taken from previously published works available from Genbank; additional taxa,
including all PPR sequences, were amplified as described below. Total genomic DNA
was extracted from silica dried leaf tissue and herbarium specimens following a
modified cetyltrimethyl ammonium bromide (CTAB) extraction protocol (Doyle and
Doyle, 1987).
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Nuclear (PPR) primers were designed after Yuan et al. (Yuan et al., 2010). We
screened the primer pairs discussed in this study and found AT1G09680 and
AT3G09060 to give clean results when PCR products were directly amplified, with very
few polymorphic sites, suggesting a single copy of these loci within all tested
individuals. Following Lu-Irving & Olmstead (2013), these will hereafter be referred to
as PPR11 (AT1G09680) and PPR70 (AT3G09060), from the order in which Yuan et al.
(Yuan et al., 2009) list them. We found that for the PPR11 locus, 320F and 1590R
primers were the most successful within the Campanuloideae (Table 3-1). For the
PPR70 locus, we used the 930F and 2080R primers. Both of these primer pairs were
used for PCR amplification and sequencing. Chloroplast primer sequences used in this
study are also shown in Table 3-1.
In order to further verify orthology, we screened eight taxa across the
Campanuloideae for multiple copies of both PPR loci. Cloning followed the StratClone
PCR Cloning Kit protocol (Stratagene) following the manufacturer’s instructions.
Between two and eight colonies were picked, amplified, and sequenced using T7 and
T3 primers. An initial phylogeny included directly sequenced PCR products as well as
cloned sequences. Only a single sequence type of PPR70 was found in all individuals.
However, two distinct fragments were amplified using the PPR11 primers for
Campanula pelviformis, C. glomerata, and C. tubulosa. These two distinct sequence
types differed greatly in nucleotide composition and size (approximately 300 bp and 850
bp). We were able to easily distinguish between the ‘large-copy’ and ‘small-copy’ by
simple gel electrophoresis, suggesting cloning was unnecessary and paralogy was not
an issue in this dataset if all amplified fragments were of appropriate length. Given a
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single band was visualized using gel electrophoresis for all subsequent taxa, we
proceeded with direct sequence following amplification.
All new sequences were amplified in 50 μl PCR reactions containing: 1 μl DNA,
10 μl 5X buffer, 5 μl of 25 mM MgCl2, 10 μl Betain, 4 μl of 0.1 μM dNTPs, 5 μl of 5 μM
primers, 1.25 units Taq polymerase (produced in the lab from E. coli), and water was
added to bring to volume. Amplification reactions for nuclear loci were run on an
automated thermal cycler under the following conditions: (1) initial denaturation was
carried out at 95°C for 2 min; (2) five cycles of 95°C for 1 min, 53°C for 1 min, and 72°C
for 2 min; (3) 32 cycles of 95°C for 1 min, 48°C for 1 min, and 72°C for 2 min; (4) a final
elongation step at 72°C for 12 min. Plastid regions were amplified following Haberle et
al. (Haberle et al., 2009) and Borsch et al. (Borsch et al., 2009).
Sequencing was carried out on an ABI Prism 3700 automated sequencer
(Applied Biosystems). Sequences were inspected, assembled, and edited using
Sequencher 4.9 (Gene Codes Corp., Ann Arbor, MI, USA). Initial alignments were
carried out using Muscle (Edgar, 2004) and manually adjusted in Se-Al v2.0 (Rambaut,
2002). Polymorphic sites in heterozygotes were coded using standard IUPAC
ambiguity codes. All sequences have been deposited in GenBank.
Phylogenetic Analyses
JModelTest (Posada, 2008) was used to determine appropriate models of
molecular evolution for all datasets using the Akaike Information Criterion (AIC) and
comparing –ln likelihood scores. The best-fitting models for each dataset are given in
Table 3-1.
All individual gene datasets were analyzed independently (atpB, matK, rbcL,
trnL-F, PPR11, and PPR70) before we analyzed the concatenated chloroplast and PPR
53
datasets. Because the individual datasets recovered largely congruent results, we
combined the PPR and chloroplast loci, using the plastid dataset as a ‘guide’ and
including only PPR accessions for which plastid data was also available. The combined
plastid-PPR matrix included 124 ingroup taxa and 7727 characters. All datasets were
analyzed using maximum likelihood and Bayesian Inference.
Maximum likelihood analyses were run in RAxML (version 7.0.4; Stamatakis,
2006) using the most appropriate model for each dataset. One thousand bootstrap
replicates were generated to measure clade support. Bayesian analyses were
conducted with MrBayes (version 3.1.2; Huelsenbeck and Ronquist, 2001; Ronquist and
Huelsenbeck, 2003) with the following settings. The maximum likelihood model
employed 6 substitution types (nst=6), with rate variation across sites modeled using a
gamma distribution, as well as a proportion of sites being invariant (rates=invgamma).
The Markov Chain Monte Carlo search was run with 4 chains for 5000000 generations,
with trees sampled every 1000 generations. We visually assessed convergence using
AWTY (Nylander et al., 2008).
Although a multi-species coalescent approach is likely to give more accurate
results for multiple unlinked partitions when compared to analyses of concatenated
datasets (e.g., Maddison and Knowles, 2006), our sampling of a single to a few
individuals per species and only three independent loci is likely insufficient to accurately
infer the species tree for Campanuloideae (Knowles and Kubatko, 2010).
Dating Analyses
Dating analyses were carried out with BEAST v1.7.4 (Drummond et al., 2012)
under an uncorrelated lognormal model. Twenty million generations were run logging
parameters every 1000 generations. Tracer v.1.5 (Drummond et al., 2012) was used to
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visualize log files, assess success of runs, and calculate “burn-in” for each analysis.
Post burn-in trees were summarized with TreeAnnotator v.1.7.4 (Drummond et al.,
2012).
Although fossils for calibrating the Campanulaceae tree are limited,
Campanuloideae fossil seeds are available. These fossils, identified as Campanula sp.
and Campanula paleopyramidalis, date to the Miocene of the Nowy Sącz Basin in
Poland (Lancucka-Srodoniowa, 1977; Lancucka-Srodoniowa, 1979). Geological and
palynological studies have dated freshwater deposits of this formation to the Karpatian,
approximately 17-16 MYA (Oszczypko and Stucklik, 1972; Oszast and Stuchlik, 1977;
Nemcok et al., 1998).
Following Cellinese et al., 2009, we used the age of the well-determined C.
paleopyramidalis fossil as a constraint for the most recent common ancestor of C.
pyramidalis and C. carpatica. A lognormal prior distribution was applied to the fossil
constraint with a mean of 5.0, stdev of 1.0, and offset of 16. This gave a minimum age
constraint of 16 MYA for the node where the fossil was assigned, placing most of the
prior probability on this younger age, but still allowing older ages for this constrained
node. Placing this constraint on the most recent common ancestor of all Campanula
species gave marginally younger ages, as expected (Crowl, unpublished data), without
significantly changing our conclusions. Therefore, we restrict our discussion to the
former analysis because we agree with the identification provided by Lancucka-
Srodoniowa (1977) and Lancucka-Srodoniowa (1979).
We used two additional calibrations for the root of the tree based on a recent
study (Bell et al., 2010), which estimated dates for a number of major angiosperm
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clades. Date ranges from the 95% highest posterior densities from this study were used
to constrain the split between the Campanuloideae and the Lobelioideae (41-67 MYA)
and the root of the Campanuloideae (28-56 MYA). Normal distribution priors were
placed on each of these nodes using the mean from each range reported in Bell et al.
(2010) as the mean for the prior distribution: 54 MYA for the Campanuloideae-
Lobelioideae split and 42 MYA for the Campanuloideae root and a stdev of 5.0 for both.
Results And Discussion
We generated 137 PPR11 sequences and 203 PPR70 sequences. The ITS
matrix included 209 taxa. The final chloroplast matrices consisted of 119 atpB, 120
matK, 183 petD, 125 rbcL, and 185 trnL-F sequences (Table 3-1). Results from the
Figure 3-1. Plastid phylogeny of the Campanuloideae clade. Best tree from maximum
likelihood analysis of combined plastid dataset: atpB, matK, petD, rbcL, and trnL-F. Numbers above branches are bootstrap values >50%. Numbers below branches indicate posterior probabilities from Bayesian analysis. Letters A-K refer to nodes and clades discussed in the text.
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Figure 3-2. PPR phylogeny of the Campanuloideae clade. Best tree from maximum
likelihood analysis of combined PPR dataset: PPR11 and PPR70. Numbers above branches are bootstrap values >50%. Numbers below branches indicate posterior probabilities from Bayesian analysis. Letters refer to nodes and clades discussed in the text.
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Figure 3-3. Combined plastid and PPR phylogeny of the Campanuloideae clade. Best
tree from maximum likelihood analysis of combined plastid-PPR dataset: atpB, matK, petD, rbcL, trnL-F, PPR11, and PPR70. Numbers above branches are bootstrap values >50%. Numbers below branches indicate posterior probabilities from Bayesian analysis. Letters refer to nodes and clades discussed in the text. Nodes for which bootstrap values are increased compared to the plastid-only analysis are highlighted in blue.
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Figure 3-4. Support for plastid-only and combined plastid-PPR trees. Maximum-
likelihood trees from the plastid dataset (left) and the combined plastid-PPR dataset (right) with taxon names removed. Branches are shaded relative to BS support with darker branches indicating higher support. Letters correspond to clade/node names in text and in Figure 3-1 and Figure 3-3. Support for many clades is increased with the inclusion of PPR loci while other areas of the tree remain poorly supported.
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Figure 3-5. Divergence time estimates for combined plastid and PPR tree.
Chronogram from BEAST analysis of the combined plastid-PPR dataset. Scale bar is in millions of years before present. Green star indicates placement of fossil constraint. Green diamonds indicate age constraints obtained from Bell et al. (2010). Numbers above branches indicate mean age estimates for clades (in millions of years). Error bars around nodes correspond to 95% highest posterior distributions of divergence times for clades discussed in the text.
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CHAPTER 4 EVOLUTION AND BIOGEOGRAPHY OF THE ENDEMIC ROUCELA COMPLEX
(CAMPANULACEAE: CAMPANULA) IN THE EASTERN MEDITERRANEAN
Introduction
Spatial patterns of biological diversity are shaped by numerous factors, including
biotic interactions, habitat heterogeneity, area, climatic constraints, isolation, and
anthropogenic events (Huston, 1994). Uncovering the relative contributions of these
factors and evolutionary dynamics responsible for driving endemism is essential to
understanding plant diversity and may have important implications for conservation.
Endemic species are non-randomly distributed across terrestrial habitats and
appear to be concentrated in specific regions, or ‘hotspots’ of biodiversity (de Candolle,
1875; Kruckeberg and Rabinowitz, 1985; Myers et al., 2000), such as the
Mediterranean Basin (e.g., Médail & Quézel, 1997; Thompson, 2005). The complex, but
well understood, climatic and geologic history of this region provides an ideal setting for
studying endemism, evolution, and biogeography.
While the western Mediterranean Basin has been relatively well studied (e.g.,
Mansion et al., 2008; Mansion et al., 2009), the eastern basin remains poorly
understood. With a high degree of endemism and both oceanic and continental islands
present, this region affords a unique opportunity to better understand the processes
leading to endemism on these distinctly different classes of islands within the same
geographic area.
Reprinted with permission from John Wiley & Sons, Inc. Original publication: Crowl A.A., Visger C.J., Mansion G., Hand R., Wu H.-H., Kamari G., Phitos D., & Cellinese N. (2015) Evolution and biogeography of the endemic Roucela complex (Campanulaceae: Campanula) in the Eastern Mediterranean. Ecology and Evolution, 5, 5329–5343. Online access: http://onlinelibrary.wiley.com/doi/10.1002/ece3.1791/full
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Oceanic and Continental Islands
Historically, islands were viewed as fragments of continents until Charles Darwin
and Alfred Russell Wallace made a distinction between continental islands, which have
had a past connection with the mainland, and oceanic islands - those that have arisen
from the ocean and have no history of continental connection (Darwin, 1859; Wallace,
1902).
These two types of islands are fundamentally different, both geologically and
biologically. Oceanic islands are formed by volcanic activity or tectonic events and arise
from the ocean, never having been in contact with an organismal source. They,
therefore, have initially empty ecological niche space. Continental islands, in contrast,
are formed by tectonic events or rising sea levels causing the break-up or isolation of a
fragment from the continent and contain a balanced flora and fauna at the time of
isolation. Crete, Kasos, Karpathos, Rhodes, and the numerous small islands off the
west coast of Turkey represent continental systems included in this study while Cyprus
is of oceanic origin.
Geologic and Climatic History of the Eastern Mediterranean Basin
The geologic and climatic history of the eastern Mediterranean Basin since the
Miocene is a complex combination of tectonic events, sea level changes, volcanism,
and a trend toward summer drought and increased seasonality. All of these events have
had a profound effect on the flora and fauna of the area (Thompson, 2005). Below we
lay out those that had the largest impact on biogeographic patterns in the eastern
Mediterranean and are, thus, potential drivers of diversification and current distribution
patterns in this region.
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A continuous landmass (termed Ägäis) stretched from present day Turkey to
present day Greece, until approximately 12 Ma, when rising sea levels and tectonic
activity caused it to break up (Creutzburg, 1963; Dermitzakis, 1990; Triantis & Mylonas,
2009). This began the formation of the Aegean Archipelago and formed many of the
continental islands in the eastern Mediterranean. During this time (12-9 Ma), the Mid-
Aegean Trench (MAT) formed, causing a tectonic split between Crete and Karpathos,
Figure 4-1. Occurrence map for the Roucela complex. Occurrence maps for taxa in the
Roucela complex based on field observations and herbarium collections. Light blue: Campanula drabifolia. Orange: C. creutzburgii. Purple: C. delicatula. Black: C. simulans. Green: C. rhodensis. Pink: C. pinatzii. Dark blue: C. raveyi. Yellow: C. podocarpa. Red: C. kastellorizana. White: C. lycica. ‘X’: C. veneris. Occurrences of the widespread C. erinus not shown. Dashed line indicates approximate location of the Mid-Aegean Trench.
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Figure 4-2. Results from concatenated and species tree analyses. Maximum
Likelihood tree (left) and species tree from *BEAST analysis (right) for the Roucela clade. Bootstrap support (>50%) and posterior probability values (>0.50) given above branches. Photo of C. podocarpa by Charalambos Christodoulou. Remaining photos by AA Crowl.
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Figure 4-3. Chronogram of the Roucela clade showing ancestral range estimation.
Summary chronogram from Bayesian dating analysis (BEAST). Outgroups not shown. Monophyletic species have been reduced to a single lineage, C. erinus excluded. Current distribution of each taxon is indicated on the terminals of the tree. Cr: Crete, TuEA: Turkey and East Aegean islands, Gr: mainland Greece, Cy: Cyprus, Ro: Rodos, Ka: Karpathos and Kasos, E Aeg: east Aegean landmass. Internal colored squares indicate most likely ancestral area recovered by BioGeoBEARS under the DEC+J model. Corners represent ranges immediately following a speciation event. Circles with an arrow denote dispersal events while circles with a line denote vicariance. Horizontal grey bars represent 95% HPD confidence intervals. Maps are paleogeographic reconstructions of the Aegean area through time re-drawn from Kasapidis et al., 2005 and Parmakelis et al., 2006 with water depicted as white and land shaded. Areas are colored as coded in the biogeographic analysis, showing connections and isolation through time.
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Figure 4-4. Niche modeling results for selected taxa in the Roucela complex. Results
from climatic niche modeling analyses for four representative Roucela taxa: Campanula creutzburgii, C. delicatula, C. pinatzii, and C. simulans. Colors represent inferred fundamental climatic niche space for each species. Black dots indicate representative occurrence points to indicate approximate, current distributions. Results suggest realized distributions represent a subset of fundamental climatic niche space for all taxa tested.
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Figure 4-5. Tempo and pattern of diversification of the Roucela clade. Results from
diversification analyses. A) Log lineage-through-time plot (LTT). LTT plots for the posterior distribution of trees from BEAST analysis (post burn-in) shown in grey. Dotted line indicates hypothetical constant diversification. B) Chronogram for the Roucela clade from BEAST analysis. C. erinus reduced to a single accession to approximate the species tree topology. C) Diversification rate through time using the sliding window approach of Simpson et al. (2011). Diversification rate is calculated as the number of nodes over the sum of all branch lengths within a window of given length.
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CHAPTER 5 GENE TREE DISCORDANCE PROVIDES EVIDENCE FOR CRYPTIC DIVERSITY
AND INSIGHTS INTO THE EVOLUTION OF A POLYPLOID COMPLEX IN A MEDITERRANEAN CAMPANULA (CAMPANULACEAE) CLADE
Introduction
Cryptic species are those for which a lack of morphological differentiation has
hindered recognition of genetically distinct lineages. Accurate species delimitation is a
difficult but critical aspect of systematics because, although the subject of much debate,
species are regarded as fundamental units of biogeography, ecology, and conservation.
As threats to biodiversity mount, accurate assessments are increasingly important as
inaccurate delimitation of species may hinder biological inferences and conservation
efforts (Wiens, 2007). In this study we focus on a group of endemic Mediterranean
plants, in which putative cryptic diversity has been postulated (Crowl et al., 2015).
The Mediterranean Basin is among the most biologically diverse areas in the
world, harboring innumerable poorly understood, species-rich groups (Myers, 1990;
Médail & Quézel, 1997). Understanding evolutionary processes and species diversity is
of special interest in this region given the exceptionally high degree of endemism and
number of rare and threatened taxa (Greuter, 1991; Médail & Quézel, 1997).
The Roucela clade (Campanulaceae) comprises 12 currently recognized, mostly
narrowly endemic species of bellflowers found primarily in the eastern Mediterranean
Basin (Carlström, 1986; Crowl et al., 2015). These taxa exhibit a high degree of
endemism within this region, often confined to a single or a few islands, with the
exception of Campanula erinus. As currently recognized, this taxon is distributed across
the Mediterranean Basin. Baker’s Law, which states that self-compatible individuals are
more likely to be successful colonizers following a long-distance dispersal event than
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self-incompatible individuals (Baker 1955), may provide insights into this pattern. The
ability to self-pollinate (Carlström, 1986) likely explains the abnormally broad distribution
pattern of C. erinus within an otherwise highly endemic clade of plants.
The recent phylogenetic analyses of Crowl et al. (2015) recovered strong support
for evolutionary relationships of these taxa with the exception of one clade. These
analyses, based on five plastid and two nuclear loci, failed to disentangle the group
containing the Cretan endemic, C. creutzburgii, C. drabifolia, endemic to the mainland
of Greece, C. simulans from southwestern Turkey, and the most widespread taxon, C.
erinus. Of these, only C. simulans was strongly supported as monophyletic. To increase
phylogenetic resolution within this clade and test hypotheses regarding hybridization
and cryptic speciation, we increased population sampling of each taxon and constructed
a genomic dataset comprising 130 nuclear loci and near-complete plastomes for 105
individuals.
We used these datasets to test hypotheses concerning the nature of previously
unrecognized diversity within the Roucela clade in the Mediterranean Basin, as
suggested by a previous study (Crowl et al., 2015). Specifically, results from numerous
phylogenetic analyses, including concatenation and species-tree approaches, suggest
two cryptic lineages within the currently recognized, widespread species, C. erinus.
These lineages are consistent with both geography and ploidy, with a tetraploid clade
consisting of populations found in the western Mediterranean Basin and an octoploid
lineage restricted to the eastern portion of the basin (Figure 5-1). Network analyses,
corroborated by nuclear gene-tree topologies, indicate a hybrid origin for the octoploid.
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The narrow Cretan endemic, C. creutzburgii (a tetraploid) and the western
Mediterranean C. erinus (also a tetraploid) are implicated as parental lineages.
The target enrichment approach taken here provided a powerful genomic dataset
to uncover previously overlooked diversity in the eastern Mediterranean Basin. Accurate
species assessments in this hotspot of biodiversity are especially critical in the face of
future climate change, which is projected to increase aridity in the Mediterranean Basin
(e.g., Gao and Giorgi, 2015) and, thus, further impact subtropically adapted taxa such
as the Roucela clade (Crowl et al., 2015).
Methods
Sampling
Taxon sampling included 105 representatives from six species in the Roucela
clade. We sampled 2-5 individuals from 27 populations spanning the distribution of
Campanula erinus from the Azores to Cyprus (Figure 5-1). Four individuals of C.
creutzburgii, four individuals of C. drabifolia, and two individuals of C. simulans were
also included. On the basis of recent phylogenetic results (Crowl et al. (2015), two
additional Roucela species were used as outgroups: C. rhodensis and C. lycica. DNA
was extracted from silica-dried and herbarium material following a modified CTAB
extraction protocol (Doyle and Doyle 1987).
Molecular Data
Previous analyses using plastid data and a small number of nuclear loci indicated
difficulty in resolving phylogenetic relationships of Campanula erinus, C. creutzburgii, C.
drabifolia, and C. simulans (Crowl et al., 2015). We, therefore, obtained a large, multi-
locus nuclear dataset and plastome dataset to disentangle species relationships. This
was achieved using a sequence capture approach (Cronn et al., 2012; Mandel et al.,
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2014) for the purpose of target enrichment prior to sequencing the reduced-
representation libraries.
MarkerMiner (Chamala et al., 2015) was used to discover and develop probes for
single-copy nuclear loci. We used four assembled Campanulaceae transcriptomes
(Lobelia siphilitica, Platycodon grandiflorus, Campanula delicatula, and Campanula
erinus; available through the 1KP project; onekp.com). This method uses reciprocal
BLAST (Altschul et al., 1997) searches, a database of single-copy nuclear genes (De
Smet et al., 2013), and clustering steps to identify putative orthologous and single-copy
loci across input sequences. Arabidopsis thaliana was used as a reference to estimate
intron/exon boundaries.
Probes were designed for 246 nuclear loci, ranging from 120-3,680 bp in length,
conserved across the Roucela clade. In-solution biotinylated probes were synthesized
using a custom MYbaits target enrichment kit (MYcroarray, Ann Arbor, MI;
http://www.microarray.com). 120mer probes (10,000 total baits) were used with 2x tiling
density. Additionally, we isolated 90 putatively single-copy nuclear genes conserved
across the Campanulaceae clade, useful for future studies in the family. Library building
and capture reactions were carried out by RAPiD Genomics (Gainesville, FL;
http://www.rapid-genomics.com). Samples were sequenced using the Illumina HiSeq
3000 platform (2x100 reads).
Data Processing
Quality filtering of Illumina reads was carried out using cutadapt (Martin, 2011)
and sickle (Joshi, 2011) to remove adapter sequences and trim low-quality nucleotides.
Default parameters were used. Custom python scripts were then used in combination
with those available from the HybPiper pipeline (Johnson et al., 2016). This pipeline
uses BWA (Li and Durbin, 2009) to align reads to target sequences and SPAdes
(Bankevich et al., 2012) to assemble these reads into contigs. If multiple contigs that
contained sequences representing at least 75% of the original bait length were found,
these were flagged as potential paralogs and removed from all downstream analyses. A
further filtering step was conducting by manual inspection of gene trees to remove
paralogous loci.
Consensus contigs were aligned to the original probe sequences. The resulting
loci were not trimmed to the original probe length, however, allowing the sequences to
extend into putative intronic regions. After quality filtering and removal of potential
paralogous loci, 130 orthologous loci contained sequences for all 109 sampled taxa (no
missing data).
Plastomes were assembled in a similar way, using Trachelium caeruleum
(Haberle et al., 2008) as a reference. Aligned contigs were trimmed to the plastome
reference length.
Phylogenetic Analysis
Individual gene and plastome alignments were constructed using MAFFT
(v.7.245; Katoh et al., 2002; 2013). Plastomes were considered as a single marker for
all subsequent analyses. We estimated individual nuclear gene trees as well as a
concatenated phylogeny using maximum likelihood (ML) with the program RAxML
(v.7.3.2; Stamatakis, 2006). The ML search was run using 10 distinct starting trees and
1000 bootstrap replicates to measure support. PartitionFinder (v.2.0.0; Lanfear et al.,
2012) was used to infer the optimal partitioning schemes and models of molecular
evolution for the alignments using the rcluster search option.
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Initial results indicated rogue taxa were present in the nuclear dataset. We used
Rogue NaRok (Aberer et al., 2013) to identify such OTUs. This analysis, optimized for
support using a majority rule consensus threshold, identified four individuals of C. erinus
as rogue samples. These accessions were removed from all datasets and the ML
analyses were re-run as above.
Coalescent Species-Tree Analyses
The relatively young age of the C. erinus complex suggests lineage sorting has
the potential to confound results from concatenation approaches (Crowl et al., 2015).
We, therefore, utilized recently developed coalescent methods to estimate a species
tree for the clade in this study.
ASTRAL-II (v.4.10.0; Mirarab and Warnow, 2015), which estimates the species
tree that maximizes the number of shared quartet trees given a set of gene trees, has
been found to be consistent and accurate in simulations compared to alternative
coalescent approaches (Mirarab et al., 2014). The 130 ML gene trees (best trees)
inferred using RAxML were used as input, and local posterior probabilities were
estimated to provide support for relationships. With respect to individuals being
assigned to species, C. erinus populations were assigned to eastern-Mediterranean
(octoploid) and western-Mediterranean (tetraploid) lineages while C. drabifolia and C.
creutzburgii populations were kept separate, as suggested by the ML analyses.
Additionally, we used SVDquartets (Chifman and Kubatko, 2014) implemented in
PAUP* (v.4.0a147; Swofford, 2002) to verify results generated by ASTRAL-II. A
coalescent approach originally intended for SNP data, SVDquartets has been shown to
perform well on multilocus datasets despite violating the assumption that sites are
independent (Chifman and Kubatko, 2014). We used the concatenated nuclear data
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matrix as input, evaluated 100,000 random quartets, and assessed support using 100
bootstrap replicates.
Bayesian Concordance Analysis
Though current species-tree methods assume no migration between populations
(Heled and Drummond 2010; Bryant et al. 2012), concordance analyses can still
recover primary phylogenetic signal in the presence of gene flow (Larget et al., 2010).
We, therefore, summarized topological concordance among loci using BUCKy (v.1.4.4;
Baum, 2007; Ane et al., 2007; Larget et al., 2010). Due to computational constraints, it
was necessary to reduce our molecular dataset to the eight major lineages recovered in
previous analyses. To test the impact of the discordance parameter (alpha),
independent analyses were run using alpha=1, alpha=10, and alpha=1000. All analyses
were run with four Markov chain Monte Carlo (MCMC) chains for 1 million generations.
Burn-in was set to 10%.
Network Analysis
We further explored the possibility of hybridization using the program SNAQ
(Solís-Lemus and Ané, 2016). This approach estimates a phylogenetic network under
the coalescent model to account for incomplete lineage sorting, while allowing for
reticulation events within a pseudo-likelihood framework. In order to infer the species
network, we obtained a table of quartet concordance factors from our previous BUCKy
analysis using the bucky.pl script provided in the BUCKy v.1.4.4 package. Tetraploid
and octoploid C. erinus populations were regarded as separate lineages as in all
previous species-tree estimations. We ran two separate analyses, allowing one
(hmax=1) and two (hmax=2) hybridization events. Both were executed with 10
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independent runs on a starting tree (reduced topology from nuclear concatenated
analysis).
Ploidy Estimation
Chromosome counts were obtained from the literature (Carlström, 1986). To infer
ploidy of the 27 C. erinus populations included in our phylogenetic analyses, we used a
modified flow cytometry method described in Roberts et al. (2009). Approximately 4mg
of silica-dried sample material was combined with 2mg of Pisum sativum as an internal
standard in a 1.5-ml Eppendorf tube containing 2-3 zirconia beads and placed in a bead
mill for 1-3 seconds. Cold lysis buffer (500 ul) was added to the ground material and
filtered through cell culture tubes. RNaseA (1 ul) was then added. Finally, 35 ul of
Propidium Iodide staining solution was added to each tube of suspended nuclei.
Samples were run on a BD Accuri C6 flow cytometer (BD Biosciences, San Jose,
California) at the University of Florida. Two to five individuals per population were used
to confirm ploidy estimates.
Morphology
To determine if there were measurable morphological differences between the
two lineages of C. erinus identified by molecular data, we focused on bract teeth, a
morphological feature found to be taxonomically informative within the Roucela
complex. Carlström (1986) showed that the length of the bract teeth unambiguously
distinguishes C. creutzburgii from C. erinus. This feature has the added advantage of
being well preserved in herbarium specimens, regardless of specimen age or
preservation method, as opposed to floral characteristics, which do not preserve well in
this group. Using ImageJ (v.2.0.0), we measured bract length, bract area, and bract
tooth length for 22 digitized specimens of C. creutzburgii and 252 specimens of C.
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erinus across their ranges. To account for tooth length differences due to confounding
factors such as age of the plant or bract age on individual plants, we corrected tooth
measurements by dividing these values by the length of the entire bract and the area of
the bract.
Results
Phylogenetic Analyses
Though plastid regions were not targeted in our probe design, we obtained a
significant amount of data from the plastid genome as a byproduct of the Illumina
sequencing run. We recovered between 83.1% and 99.7% (avg. = 95.7%) plastome
coverage for all samples. Maximum likelihood analysis of the plastome dataset found
strong support for two Campanula erinus clades (Figure 5-1). Tetraploid populations
were maximally supported as monophyletic and sister to C. drabifolia. Octoploid
populations were moderately supported (BS=86) as monophyletic and sister to two
individuals of C. creutzburgii from western Crete. The placement of the remaining C.
creutzburgii samples was not supported but inferred to be sister to a monophyletic C.
simulans.
ML analysis of the concatenated nuclear dataset recovered maximal support for
all relationships discussed below. A monophyletic C. creutzburgii was nested within the
octoploid C. erinus populations, rendering the octoploids paraphyletic. A tetraploid clade
was inferred to be sister to this octoploid assemblage. Campanula drabifolia was found
to be non-monophyletic. Campanula simulans was again recovered as monophyletic
and sister to the rest.
Individual nuclear gene trees showed high levels of phylogenetic discordance.
Close inspection of gene trees indicated two major topologies were being recovered.
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Approximately 30% of nuclear loci indicated tetraploid and octoploid populations of C.
erinus were reciprocally monophyletic (consistent with the ASTRAL-II and SVDquartets
species-tree analyses; see below). The second topology, recovered with approximately
34% of genes, indicated the octoploid lineage was sister to C. creutzburgii. This was
consistent with the plastome dataset and BUCKy analyses (see below). The remainder
(36%) of the gene trees did not fall into these strictly defined categories. However, when
we accounted for low statistical support for relationships of the C. erinus lineages in
these gene trees, we found that nearly all of them approximated one of the two
previously discussed topologies. This more relaxed assignment of gene trees
suggested that nearly half of the sampled genome approximated the plastome-like
topology (close association of the octoploid lineage with C. creutzburgii; 43%), while the
other half (49%) indicated a close association between the octoploid and tetraploid C.
erinus populations.
Species-Tree and Network Analyses
Species-tree analyses of the nuclear dataset recovered relationships consistent
with the individual gene trees but differed from the ML concatenation results. Both
ASTRAL-II and SVDquartets recovered species trees in which tetraploid and octoploid
C. erinus lineages were reciprocally monophyletic when individuals were assigned to
lineages (Figure 5-2, panels A and B). Support for the C. erinus sister relationship,
however, was low (PP=0.79 in ASTRAL-II; BS=72 in SVDquartets). Bayesian
concordance analysis, as implemented in BUCKy, inferred a primary concordance tree
in which the octoploid C. erinus lineage was sister to C. creutzburgii, while the tetraploid
C. erinus lineage was sister to C. drabifolia (Figure 5-2, panel C), consistent with the
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plastome phylogeny. These relationships had concordance factors of CF=0.342 and
CF=0.329, respectively (see also results from manual inspection of gene trees above).
Interestingly, ASTRAL-II and SVDquartets differed in the reconstruction of the
specie-tree topology when individuals were assigned to populations, rather than
assigning them to the lineages discussed above. When we used the population
assignments, ASTRAL-II estimated a topology similar to the concatenation analyses,
with C. creutzburgii populations nested within octoploid C. erinus populations (Figure 5-
2, panel E). There was, however, very low support for this relationship. The
SVDquartets analysis using population assignments strongly supported a monophyletic
octoploid C. erinus, and C. creutzburgii sister to C. simulans (Figure 5-2, panel D).
SNAQ analyses suggested a single hybridization event (Figure 5-3). The best
network (-loglik = 129.62) included a single hybrid edge between C. creutzburgii and the
tetraploid C. erinus, indicating these as the parental lineages of the octoploid C. erinus.
A paraphyletic C. drabifolia was found to be sister to the tetraploid C. erinus, and a
monophyletic C. creutzburgii sister to C. simulans. These analyses estimated a gamma
value of 0.466 for tetraploid C. erinus and gamma=0.534 for C. creutzburgii parental
lineages. Our manual inspection of gene trees yielded similar results with 49% of gene
trees indicating a close association of the octoploid lineage with the tetraploid lineage
and 43% indicating an association with C. creutzburgii.
Ploidy
Flow cytometry results provided easy-to-interpret ploidy estimates for all
individuals tested. Both tetraploid (2n=28) and octoploid (2n=56) populations were
found within C. erinus. The two ploidal levels correspond to geography, with all
identified tetraploid populations occurring west of Greece and all octoploid populations
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occurring in the eastern Mediterranean Basin (Figure 5-1). All C. creutzburgii and C.
drabifolia populations were verified as tetraploid. Carlström (1986) cited an earlier
chromosome count for C. creutzburgii as 2n=56, but warned that this record needs
confirmation as this same count had been reported for C. erinus, which is sympatric on
the island of Crete. Our results validate this concern and we propose 2n=28 may be a
more accurate count for C. creutzburgii based on genome size estimations of five
individuals from three populations, though this should be further verified.
Morphology
Though molecular data and genome size estimates suggest multiple lineages
within C. erinus, previous morphological work failed to distinguish these two groups. Our
morphological dataset agrees with this, indicating no measurable difference between
the two lineages based on bract tooth length (Figure 5-4), suggesting these lineages
represent cryptic diversity. Measurements of the bract tooth showed a clear difference
between C. erinus (both tetraploid and octoploid populations) and C. creutzburgii
(Figure 5-4).
Discussion
Phylogenetic analyses, with corroboration from genome-size estimates and
morphologic data, suggest cryptic diversity is present within Campanula erinus, as
currently recognized. Due to the highly similar morphologies of the two lineages
recovered here, this diversity had, until now, been overlooked. Flow cytometry
estimates of genome size found evidence for tetraploid and octoploid populations.
These populations are geographically distinct, with octoploids occurring in the eastern
Mediterranean – Greece, Aegean islands, and Cyprus – while the tetraploids inhabit a
wide area in the western Mediterranean (Figure 5-1). Unfortunately, our population
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sampling was insufficient to determine the precise geographic location (approximately
mainland Greece or the Balkans) that demarcates the eastern distribution limit of
tetraploid and the western limit of octoploid populations. More in-depth sampling would
provide this boundary and indicate whether or not the tetraploid and octoploid lineages
have overlapping distributions in this region.
Conflicting phylogenetic signal between plastid and nuclear datasets, and within
the nuclear dataset, appears to be the signature of hybridization. The sister relationship
of the octoploid C. erinus lineage with the tetraploid C. erinus and tetraploid C.
creutzburgii, suggests a hybrid origin for this taxon and implicates these tetraploid taxa
as parental lineages. Network analyses, estimated under the coalescent while allowing
for hybridization, confirm this assertion (Figure 5-3). These analyses, with corroboration
from our manual inspection of gene trees, suggest the octoploid lineage of C. erinus
was likely the result of an allopolyploid event (or events) with near-equal contributions
from the two parental lineages.
The non-monophyly of octoploid populations recovered in our phylogenetic
analyses based on the concatenated nuclear dataset (Figure 5-1, panel C) may be
evidence of multiple polyploid origins or simply the result of incomplete lineage sorting.
The results from our species-tree analyses, unfortunately, do not satisfactorily resolve
this issue. Our SVDquartets analysis, in which individuals were assigned to populations,
recovered a maximally supported octoploid C. erinus clade (Figure 5-2, panel D) –
suggesting ILS is the cause for the non-monophyly in the concatenation analyses, while
this same assignment of individuals analyzed with ASTRAL-II (Figure 5-2, panel E)
confirms the non-monophyly of octoploid populations recovered in the concatenation
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analyses. A more in-depth investigation is necessary to confirm whether a single event
led to the octoploid lineage, or if polyploid formation has been recurring.
Dating analyses of Crowl et al. (2015), in which age estimates for the Roucela
clade were inferred within the broader Campanuloideae, suggest the timing of the C.
erinus–C. creutzburgii hybridization event may have coincided with the Messinian
Salinity Crisis. The closure of the Mediterranean Basin’s connection with the Atlantic
Ocean (5.96-5.33 Ma) led to a significant desiccation of the Mediterranean Sea (Hsu et
al., 1973; Krijgsman et al., 1999). This event led to the connection of previously isolated
islands to each other and the mainland, potentially facilitating range-expansion and
sympatry of C. erinus and C. creutzburgii. Crowl et al. (2015) found that the relatively
recent onset of the Mediterranean climate may have caused extinction in the Roucela
complex. This provides another possible explanation for the formation of a hybrid taxon
from two currently non-sympatric species, as it is conceivable that these taxa had wider
distributions in the past. A more in-depth survey of ploidal levels within populations
found in Crete and mainland Greece would provide further insights into the precise
mechanism underlying this apparent allopolyploid event, or events.
Though past researchers have argued that polyploidy may allow for broad
ecological tolerance and, thus, broad geographic ranges (see Stebbins 1950), the
narrow endemic polyploid taxa in the Roucela clade indicate it clearly is not polyploidy
per se that is responsible for the wide distribution of C. erinus. Our results suggest that
the distribution pattern observed within C. erinus is the result of two factors. First, what
has been historically considered C. erinus is, in fact, composed of two lineages. The
assertion that a single species is distributed across the Mediterranean Basin is,
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therefore, erroneous. Second, because the octoploid C. erinus appears to have retained
the ability to self pollinate from the parental tetraploid C. erinus, this cytotype was likely
able to rapidly and widely disperse across the eastern part of the basin, in contrast to
the numerous narrowly distributed taxa in this same region – a result that corroborates
Baker’s Law (Baker 1955).
This study provides evidence for cryptic speciation in a small clade of flowering
plants in the Mediterranean Basin. Our results highlight the utility of target enrichment
approaches for obtaining multilocus, genomic datasets for thorough assessments of
species diversity and the need to carefully consider gene-tree discordance within such
datasets. Much work needs to be done in regards to species assessments across the
Tree of Life in this biodiversity hotspot. With the help of genomic data, future studies will
surely uncover further cryptic diversity, as has been done here, providing more accurate
assessments of biodiversity in this fragile region of the world.
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Figure 5-1. Sample localities, species distributions, and comparison of concatenated
analyses. A) Occurrence map of lineages under investigation. Colors are consistent with those in panels B and C, with tetraploid and octoploid Campanula erinus lineages labeled as such on the map. Circles indicate sampled populations of C. erinus, diamonds indicate sampling localities of C. creutzburgii populations, and squares indicate sampling localities of C. drabifolia populations included in this study. B) Results from maximum likelihood analysis of plastome dataset. Numbers at nodes indicate bootstrap support for relationships and clades discussed in the text. C) Results from maximum likelihood analysis of concatenated nuclear dataset. Numbers at nodes indicate bootstrap support for relationships and clades discussed in the text. Branch colors are consistent with panel B.
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Figure 5-2. Comparison of species-tree analyses. A) Species-tree topology estimated
with SVDquartets when individuals were assigned to the major lineages recovered with concatenation analyses (see Figure 5-1). Numbers at nodes indicate bootstrap support values of relationships. B) Species-tree topology estimated with ASTRAL-II when individuals were assigned to lineages, as in panel A. C) Primary concordance tree from Bayesian Concordance Analysis in Bucky. Numbers indicate concordance factors and are, thus, estimates of the proportion of the genome for which a relationship is true. Branch lengths are in coalescent units. D) Topology estimated with SVD quartets when individuals were assigned to separate populations, rather than the major lineages as in panel A. Numbers indicate bootstrap support values. E) Topology estimated with ASTRAL-II when individuals were assigned to separate populations. Numbers indicate local posterior probability support values. Branch lengths shown in coalescent units.
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Figure 5-3. Hypothesized hybridization scenario. A) Phylogenetic network estimated
with SNAQ showing the tetraploid C. erinus and C. creutzburgii as putative parental lineages of the octoploid C. erinus (dashed lines). Numbers on dashed lines indicate inheritance probabilities from SNAQ analysis. These values represent the proportion of the genome estimated to have been contributed by each parental lineage. To simplify the figure, we have collapsed the C. creutzburgii lineages (found to be reciprocally monophyletic) to a single lineage. B) Depiction of the hypothesized hybridization event indicating maternal and paternal genomes involved in the allopolyploid event. Large circles represent nuclear genomes while smaller squares represent plastomes. Red and purple colors indicate parental contributions from each tetraploid.
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Figure 5-4. Analyses of morphology data. A) Graph of bract tooth length -vs- total bract
length for C. creutzburgii (red), C. erinus 4x (purple), and C. erinus 8x (pink). Solid lines are linear regressions for each species, grey bars indicate 95% confidence intervals. B) Box-plot of bract tooth length corrected for total bract length for the three lineages. Colors consistent with those in (A). Illustrations of the different morphologies shown in upper left corner.
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CHAPTER 6 PHYLOGENETIC DEFINITION OF THE ROUCELA CLADE
Introduction: Roucela
Several phylogenetic studies (Cellinese et al., 2009; Haberle et al., 2009;
Mansion et al., 2012; Crowl et al., 2015) have consistently recovered a highly supported
clade that includes 12 annual Campanula species (Campanulaceae) found in the
Mediterranean Basin (Carlström, 1986). This clade has previously been referred to as
Campanula subg. Roucela (Dumort.) Damboldt or the Roucela complex, and in-depth
studies of its evolutionary history motivated the establishment of a formal phylogenetic
definition for this group.
Phylogenetic Definition: Roucela
ROUCELA (Dumort.) Damboldt 1976 [A. A. Crowl & N. Cellinese], nomen cladi
conversum.
Node-Based Definition. The least inclusive clade containing Campanula erinus
L. 1753, Campanula rhodensis A. DC. 1830, and Campanula pinatzii Greuter & Phitos
1967.
Etymology: Roucela
The name Roucela has previously been used at the rank of genus (Roucela
Though non-monophyletic in Crowl et al. (2015; see below), the type, Campanula erinus
L. 1753 was found to fall within a clade containing other traditionally recognized taxa in
this group. Here, we repurpose Roucela to be used as a clade name approximating
Campanula subg. Roucela (Dumort.) Damboldt.
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Reference Phylogeny: Roucela
Crowl et al. (2015; Figure 4-2).
Composition: Roucela
In the most recent taxonomic revision, Carlström (1986) recognized 12 species in
Campanula subg. Roucela (Dumort.) Damboldt: Campanula creutzburgii Greuter , C.
delicatula Boiss., C. drabifolia Sm., C. erinus L., C. kastellorizana Carlström, C. pinatzii
Greuter & Phitos, C. podocarpa Boiss., C. raveyi Boiss., C. rhodensis A. DC., C.
scutellata Griseb., C. simulans Carlström, and C. veneris Carlström. More recently, one
additional species, C. lycica Kit Tan & Sorger, was described and added to this group
by Tan and Sorger (1987). According to the phylogenetic analyses of Mansion et al.
(2012) and Crowl et al. (2015), however, C. scutellata Griseb. does not appear to
belong to this clade.
Diagnostic Apomorphies: Roucela
The Roucela clade includes dichotomously branched annual species of
Campanula with an unappendaged calyx (Carlström, 1986, Lammers, 2007).
Synonyms: Roucela
None.
Comments: Roucela
The name Roucela has previously been used at the ranks of genus and
subgenus. The group was initially recognized on the basis of morphology: small,
dichotomously branched annuals with unappendaged calyx lobes (Carlström, 1986;
Lammers, 2007). See Crowl et al. (2015; Figure 4-2) for the primary reference
phylogeny.
126
The distribution of the Roucela clade spans the Mediterranean Basin. As
traditionally recognized, the most widespread taxon, C. erinus, is found from the Azores,
southern Europe, northern Africa, and the Arabian Peninsula, an area broadly
corresponding to the Mediterranean climate zone but extending as far east as Iran.
However, see Crowl et al. (in prep.; Ch. 5) and discussion below for a revised view of C.
erinus. The remaining species occupy more restricted distributions – many, narrow
island endemics in the Aegean Archipelago, western Turkey, and Cyprus.
The phylogenetic analyses of Crowl et al. (2015) included all 13 previously
recognized species in the Roucela complex. Utilizing both plastid and nuclear markers,
this study recovered strong support for the monophyly of the group within the broader
Campanuloideae clade, with the exception of Campanula scutellata. Carlström (1986)
pointed out the morphological divergence of C. scutellata as compared to other species
in the group. Phylogenetic analyses have confirmed this observation and suggest this
species is more closely related to other annual taxa in the Megalocalyx clade (Mansion
et al., 2012; Crowl et al., 2015).
Past studies suggest the Roucela clade as being closely related to a clade
containing Northern African and Western Mediterranean taxa (Cellinese et al., 2009;
Haberle et al., 2009; Crowl et al., 2015), though increased taxon sampling indicates that
this clade may also contain Asian campanuloids (Mansion et al., 2012; Crowl et al.,
2016). A more detailed study with increased molecular and taxonomic sampling is
necessary to test these hypotheses.
Within the Roucela clade, the geologic history of the Eastern Mediterranean
appears to have played an important role in the diversification of many species, while
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the climatic history – specifically, the shift from a subtropical climate – may have
adversely affected diversification (see Crowl et al., 2015 for an in-depth discussion).
Because Campanula erinus L. 1753 (synonym: Roucela erinus [L.] Dumort.) was
the type for the previously recognized genus and subgenus, we have included two
specimens (representing the two lineages recovered by Crowl et al. [in prep.; Ch. 5];
see below) of this taxon as internal specifiers in our Roucela clade definition.
Introduction: Holoerinus
The phylogenetic analyses of Crowl et al. (2015) recovered the taxon Campanula
erinus L. as non-monophyletic. Statistical support for this result, however, was
insufficient to draw meaningful conclusions. The more recent, phylogenomic analyses of
Crowl et al. (in prep.; Ch. 5), which increased both population and genomic sampling,
verified this assertion and suggested two cryptic clades within this currently recognized
species. Though seemingly indistinguishable on the basis of morphology, populations
belonging to these groups are recognized on the basis of geography and ploidy:
western Mediterranean tetraploids and eastern Mediterranean octoploids.
Phylogenetic Definition: Holoerinus
HOLOERINUS L. [A. A. Crowl & N. Cellinese], nomen cladi novum.
Branch-modified node-based definition. The most inclusive crown clade
containing Crowl #67 [Campanula erinus L.], 2-Jun-2012, Italy: 3 km west of Baia della
Zagare on Strada Provinciale N. 53, dirt road just before entrance to tunnel, FLAS
260389; but not Crowl #7 [Campanula creutzburgii Greuter], 8-May-2011, Western
Crete, Balos Lagoon, near Kissamos, FLAS 240137; or Cellinese #NC2000 [Campanula
drabifolia Sibth. & Sm.], 13-May-2010, Greece: Kefalonia Island, Pyloros, Village
Agonas, along the road, open phrygana.
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Etymology: Holoerinus
To indicate the inclusion of both tetraploid and octoploid lineages, we combine
the Greek prefix ‘holo’ with the specific epithet of the currently recognized species,
Campanula erinus.
Reference Phylogeny: Holoerinus
Crowl et al. (in prep.; Ch. 5; Figure 5-3).
Introduction: Tetraerinus
Phylogenetic analyses consistently recovered strong support for a clade of
tetraploid Campanula erinus L. populations. This tetraploid clade, found throughout the
western Mediterranean Basin, occurs from the Azores to the Balkans and includes the
type specimen of Campanula erinus. The phylogenetic definition presented here
includes herbarium specimens as specifiers.
Phylogenetic Definition: Tetraerinus
TETRAERINUS L. [A. A. Crowl & N. Cellinese], nomen cladi novum.
Apomorphy-modified node-based definition. The most inclusive crown clade
exhibiting a ploidal level (tetraploid) synapomorphic with that of Crowl #67 [Campanula
erinus L.], 2-Jun-2012, Italy: 3 km west of Baia della Zagare on Strada Provinciale N.
53, dirt road just before entrance to tunnel, FLAS 260389.
Etymology: Tetraerinus
To reflect the tetraploid ploidal level of this clade, we have combined the specific
epithet of Campanula erinus with the Latin prefix, tetra.
Reference Phylogeny: Tetraerinus
Crowl et al. (in prep.; Ch. 5; Figure 5-1). See also Crowl et al. (in prep.; Ch. 5;
Figure 5-2) for results from species tree analyses.
129
Composition: Tetraerinus
The Tetraerinus clade is composed of tetraploid populations of the currently
recognized species, Campanula erinus, occurring in the western Mediterranean Basin
from the Balkans to the Azores.
Diagnostic Apomorphies: Tetraerinus
Tetraploid ploidal level, chromosome count of 2n=28.
Synonyms: Tetraerinus
None.
Comments: Tetraerinus
A genomic dataset consisting of 130 nuclear loci and near-complete plastomes
across 27 populations of Campanula erinus, spanning its distribution, provides strong
evidence for two lineages within this currently recognized taxon (Crowl et al., in prep.;
Ch. 5). Ploidy estimates suggest a strongly supported clade consisting of tetraploid
populations distributed throughout the western Mediterranean Basin.
Introduction: Octoerinus
Phylogenetic Definition: Octoerinus
OCTOERINUS A. A. Crowl & N. Cellinese, nomen cladi novum.
Apomorphy-modified node-based definition. The most inclusive crown clade
exhibiting a ploidal level (octoploid) synapomorphic with that of Crowl #2 [Campanula
erinus L.], 4-May-2011, Greece: southern Crete, small canyon 1km south of Matala,
FLAS 240140.
Etymology: Octoerinus
To reflect the octoploid ploidal level of this clade, we have combined the specific
epithet of Campanula erinus with the Latin prefix, octo.
130
Reference Phylogeny: Octoerinus
Crowl et al. (in prep.; Ch. 5; Figure 5-1). See also Crowl et al. (in prep.; Ch. 5;
Figure 5-2) for results from species-tree analyses.
Composition: Octoerinus
The Octoerinus clade is composed of octoploid individuals of the currently
recognized species, Campanula erinus, occurring in the eastern Mediterranean Basin.
Diagnostic Apomorphies: Octoerinus
Octoploid ploidal level, chromosome count of 2n=56.
Synonyms: Octoerinus
None.
Comments: Octoerinus
A phylogenetic definition for Octoerinus has been constructed to distinguish
morphologically similar octoploid and tetraploid lineages within the currently recognized
species, Campanula erinus. Though these terminal taxa are not ‘independent’ in that
the octoploid appears to be the result of an allopolyploid event in which the tetraploid
Tetraerinus (see above) and tetraploid Campanula creutzburgii are parental lineages,
the phylogenetic analyses of Crowl et al. (in prep.; Ch. 5) showed that populations of the
different ploidal levels form separate clades, consistent with geography.
Octoerinus can be distinguished from Tetraerinus based on ploidal level:
octoploid (2n=56) and tetraploid (2n=28), respectively.
131
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BIOGRAPHICAL SKETCH
Andrew Crowl’s Ph.D. studies focused on the evolution and biogeography of the
Campanulaceae. He is particularly interested in understanding evolutionary patterns
and processes in the Mediterranean Basin and applying genomic data to address
questions related to biogeography, cryptic speciation, and endemism in this biodiversity
hotspot. Andrew received his doctoral degree from the Department of Biology at the