Genome size increases in recently diverged hornwort clades

Abstract

As our knowledge of plant genome size estimates continues to grow, one group has continually been neglected: the hornworts. Hornworts (Anthocerotophyta) have been traditionally grouped with liverworts and mosses because they share a haploid dominant life cycle; however, recent molecular studies place hornworts as the sister lineage to extant tracheophytes. Given the scarcity of information regarding the DNA content of hornworts, our objective was to estimate the 1C-value for a range of hornwort species within a phylogenetic context. Using flow cytometry, we estimated genome size for 36 samples representing 24 species. This accounts for roughly 10% of known hornwort species. Haploid genome sizes (1C-value) ranged from 160 Mbp or 0.16 pg (Leiosporoceros dussii) to 719 Mbp or 0.73 pg (Nothoceros endiviifolius). The average 1C-value was 261 ± 104 Mbp (0.27 ± 0.11 pg). Ancestral reconstruction of genome size on a hornwort phylogeny suggests a small ancestral genome size and revealed increases in genome size in the most recently divergent clades. Much more work is needed to understand DNA content variation in this phylogenetically important group, but this work has significantly increased our knowledge of genome size variation in hornworts.

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Genome size increases in recently diverged hornwort clades
1
Jillian D. Bainard and Juan Carlos Villarreal
Abstract: As our knowledge of plant genome size estimates continues to grow, one group has continually been neglected: the
hornworts. Hornworts (Anthocerotophyta) have been traditionally grouped with liverworts and mosses because they share a
haploid dominant life cycle; however, recent molecular studies place hornworts as the sister lineage to extant tracheophytes.
Given the scarcity of information regarding the DNA content of hornworts, our objective was to estimate the 1C-value for a range
of hornwort species within a phylogenetic context. Using flow cytometry, we estimated genome size for 36 samples representing
24 species. This accounts for roughly 10% of known hornwort species. Haploid genome sizes (1C-value) ranged from 160 Mbp or
0.16 pg (Leiosporoceros dussii) to 719 Mbp or 0.73 pg (Nothoceros endiviifolius). The average 1C-value was 261 ± 104 Mbp (0.27 ± 0.11 pg).
Ancestral reconstruction of genome size on a hornwort phylogeny suggests a small ancestral genome size and revealed increases
in genome size in the most recently divergent clades. Much more work is needed to understand DNA content variation in this
phylogenetically important group, but this work has significantly increased our knowledge of genome size variation in horn-
worts.
Key words: genome size, DNA content, hornworts, Anthocerotophyta, polyploidy, evolution, phylogeny.
Résumé : Alors que notre connaissance de la taille des génomes chez les plantes s’accroît sans cesse, un groupe s’avère constamment
négligé, celui des anthocérotes. Les anthocérotes (division Anthocerotophyta) ont traditionnellement été groupés avec les hépatiques
et les mousses, mais ils ont une morphologie différente et il a été suggéré qu’ils seraient proches des trachéophytes. Compte tenu du
peu d’information sur le contenu en ADN chez les anthocérotes, l’objectif de ce travail était d’estimer la valeur C chez une gamme
d’anthocérotes dans le cadre d’analyses phylogénétiques. Au moyen de la cytométrie en flux, les auteurs ont estimé la taille du génome
chez 36 échantillons représentant 24 espèces. Ceci représente environ 10% des espèces connues d’anthocérotes. La taille des génomes
haploïdes variait entre 160 Mb ou 0,16 pg (Leiosporoceros dussii) et 719 Mb ou 0,73 pg (Nothoceros endiviifolius). La taille moyenne était de
261 ± 104 Mb (ou 0,27 ± 0,11 pg). La reconstruction de la taille du génome ancestral a
`l’aide d’une phylogénie des anthocérotes suggère
que le génome ancestral était petit et que des augmentations seraient survenues au sein des clades récemment apparus. Beaucoup plus
de travail sera nécessaire pour mieux connaître la variation du contenu en ADN au sein de ce groupe phylogénétique important, mais
le présent travail a déja
`contribué a augmenter considérablement l’état des connaissances sur la variation de la taille des génomes chez
les anthocérotes. [Traduit par la Rédaction]
Mots-clés : taille du génome, contenu en ADN, anthocérotes, Anthocerotophyta, polyploïdie, évolution, phylogénie.
Introduction
Understanding genome size variation has proven to be useful in
many aspects of organismal biology, particularly in comparative
studies using various morphological and ecological traits (Bennett
and Leitch 2011). Unfortunately, our knowledge of genome size
variation in hornworts is nearly nonexistent, as few reliable esti-
mates for hornworts have been published to date (Kew C-values
Database; Bennett and Leitch 2012). Hornworts share a haploid
dominant life cycle with mosses and liverworts but are distin-
guished by a gametophyte associated with cyanobacteria, the
presence of pyrenoids in the chloroplasts of haploid cells, and
asynchronous meiosis of their spore mother cells along an acropetal
gradient of spore maturation in the sporophyte with indeterminate
acropetal growth (Renzaglia et al. 2009;Villarreal and Renner 2012).
Hornworts may only include 200–250 species (Cargill et al. 2005;
Villarreal et al. 2010); however, they hold an important position in
the phylogeny of land plants, as they are likely to be the sister group
to extant tracheophytes (Qiu et al. 2006;Chang and Graham 2011).
Leitch and Leitch (2013) emphasized the need for genome size
estimates for hornworts to strengthen our understanding of ge-
nome size evolution in land plants. Renzaglia et al. (1995) esti-
mated the genome size for Notothylas orbicularis (1C-value = 0.17 pg)
and Phaeoceros laevis (1C-value = 0.27 pg), but confirmation from
flow cytometric measurements have been called for (Voglmayr
2000). Nevertheless, cytological data suggest small genome sizes
for hornworts, as they have low chromosome numbers (n= 4–6
with a number of accessory chromosomes), small chromosome
sizes, and an apparent rarity of polyploidy (e.g., Rink 1935;
Proskauer 1957;Przywara and Kuta 1995).
Given the lack of knowledge of DNA content variation in horn-
worts, our research objective was to estimate genome size from a
range of taxa. We also reconstructed the evolutionary history of ge-
nome size variation in hornworts within a phylogenetic context.
Materials and methods
Sample collection
Hornwort specimens were collected from a wide range of loca-
tions (Table 1) and were stored in sealed petri dishes with moist
Received 4 March 2013. Accepted 11 July 2013.
Corresponding Editor: Ryan Gregory.
J.D. Bainard.* Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
J.C. Villarreal. Systematic Botany and Mycology, University of Munich (LMU), Menzinger Straße 67, Germany.
Corresponding author: Jillian D. Bainard (e-mail: jillian.bainard@gmail.com).
*Present address: Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada.
1This article is one of a selection of papers published in this Special Issue on Genome Size Evolution.
431
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paper towel at 4 °C until flow cytometry was performed. Addition-
ally, several samples were cultured on agarose or vermiculite (for
culturing methods see Villarreal and Renzaglia 2006). Species
were identified by comparing to type material. Herbarium acces-
sions of all samples can be found at the George Safford Torrey
Herbarium (University of Connecticut), the Munich Herbarium (M),
and the Australian National Herbarium (CANB) (see supplementary
data, Tables S1 and S2
2
).
Genome size estimation
Fresh hornwort tissue was used for all flow cytometric analyses.
Several herbarium specimens were tested but none produced us-
able data. The hornwort tissue was first prepared alone to deter-
mine the relative fluorescence of the hornwort nuclei to choose
an appropriate standard. Thallus tissue was gently cleaned with
deionized water to remove any soil or other contaminants. For all
flow cytometric analyses, plant tissue was finely chopped with a
razor blade in LB01 buffer (Doleˇ
zel et al. 1989) with 150 ␮g/mL
propidium iodide (Sigma) and 50 ␮g/mL RNase A (Sigma). The
buffer and propidium iodide concentration were chosen based
on preliminary studies (Bainard et al. 2010). For most samples,
1% polyvinylpyrrolidone (PVP) was also added to the buffer to
improve the quality of the flow data. The resulting homogenate
was filtered through a 30 ␮m filter (Partec CellTrics) and incu-
bated on ice for 20–40 min. The relative fluorescence of the horn-
wort nuclei allowed selection of the appropriate standard. Given
the very small DNA content of the hornworts, the standards used
were Raphanus sativus L. ‘Saxa’ (1.11 pg/2C; Doleˇ
zel et al. 1998) and a
tetraploid derivative (0.64 pg/2C) of the diploid Arabidopsis thaliana
‘Columbia’ (0.32 pg/2C; Bennett et al. 2003). We confirmed the
tetraploid 2C-value of 0.64 pg by repeated comparisons to
R. sativus. Standards were also analyzed independently on each day
of testing to look for inhibition effects (sensu Price et al. 2000).
2
Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/gen-2013-0041.
Table 1. List of hornwort genome size estimates organized alphabetically within families.
Family Species
Mean
1C-value
(pg) ± SE
Mean
1C-value
(Mbp)
a
General collection location
Leiosporocerotaceae Leiosporoceros dussii (Steph.) Hässel 0.19±0.004 184 Chiriquí, Panama
Leiosporoceros dussii (Steph.) Hässel 0.16±0.004 160 Coclé, Panama
Anthocerotaceae Anthoceros fusiformis Aust. 0.19±0.003 186 Oregon, United States
Anthoceros lamellatus Steph. 0.19±0.003 185 Bucaramanga, Colombia
Anthoceros lamellatus Steph. 0.20±0.007 193 Chiriquí, Panama
Anthoceros punctatus L. 0.18±0.007 178 Australian Capital Territory,
Australia
Folioceros fuciformis (Mont.) D.C. Bhardwaj 0.18±0.007 176 Queensland, Australia
Dendrocerotaceae Dendroceros crispus (Sw.) Nees 0.27±0.007 268 Chiriquí, Panama
Megaceros gracilis (Reichardt) Steph. 0.30±0.002 297 Victoria, Australia
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.32±0.009 308 Morelos, Mexico
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.33±0.001 319 Mexico
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.36±0.003 348 Tennessee, United States
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.32±0.007 310 Tennessee, United States
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.32±0.001 313 Tennessee, United States
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.32±0.009 308 North Carolina, United States
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.33±0.000 319 North Carolina, United States
Nothoceros aenigmaticus (R.M. Schust.) J.C. Villarreal & McFarland 0.33±0.003 326 North Carolina, United States
Nothoceros endiviifolius (Mont.) J. Haseg. 0.73±0.012 719 Patagonia, Chile
Nothoceros fuegiensis (Steph.) J.C. Villarreal 0.34±0.001 335 Patagonia, Chile
Nothoceros vincentianus (Lehm. & Lindenb.) J.C. Villarreal 0.41±0.004 405 Chiriquí, Panama
Nothoceros vincentianus (Lehm. & Lindenb.) J.C. Villarreal 0.35±0.004 345 Chiriquí, Panama
Nothoceros vincentianus (Lehm. & Lindenb.) J.C. Villarreal 0.34±0.013 337 Veraguas, Panama
Phaeomegaceros coriaceus (Steph.) Duff et al. 0.18±0.005 174 Pohangina Valley, New Zealand
Notothyladaceae Notothylas orbicularis (Schwein.) Sull. ex A. Gray 0.19±0.002 188 Chiriquí, Panama
Phaeoceros carolinianus (Michx.) Prosk. 0.18±0.011 174 Australian Capital Territory,
Australia
Phaeoceros carolinianus (Michx.) Prosk. 0.19±0.004 184 Chiriquí, Panama
Phaeoceros dendroceroides (Steph.) Hässel 0.19±0.009 186 Chiriquí, Panama
Phaeoceros engellii Cargill & Fuhrer 0.21±0.005 202 Victoria, Australia
Phaeoceros flexivalvis (Nees & Gott.) Hässel 0.18±0.010 176 La Vega, Dominican Republic
Phaeoceros inflatus (Steph.) Cargill & Fuhrer 0.18±0.003 172 Victoria, Australia
Phaeoceros laevis (L.) Prosk. 0.24±0.002 233 Portugal
Phaeoceros (Paraphymatoceros) pearsonii (M.A. Howe) Prosk. 0.24±0.003 232 California, USA
Phaeoceros (Paraphymatoceros) proskauerii Stotler et al. 0.23±0.004 223 California, USA
Phaeoceros sp. 0.26±0.004 253 Chile
Phymatocerotaceae Phymatoceros bulbiculosus (Brot.) Stotler et al. 0.28±0.006 270 Coimbra, Portugal
Phymatoceros phymatodes (M.A. Howe) Duff et al. 0.22±0.002 217 California, USA
Note: General collection location is provided; for more collection details see the supplementary data, Table S1.
a
Mega base pairs (Mbp) (1 pg = 0.978 × 10
9
base pairs; Doleˇ
zel et al. 2003).
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Depending on the amount of tissue available, 1–2 cm
2
of hornwort
thallus was co-chopped with 1 cm
2
of leaf standard, and repli-
cates were analyzed on separate days to have a total of three
estimates per sample (one sample had four replicates and three
samples only had two; 106 accessions total). Genome size was
calculated as follows:
1C-value ⫽1C sample peak mean
2C standard peak mean × standard 2C-value (pg)
All flow cytometric measurements were conducted on a Partec
CyFlow SL (Partec GmbH, Münster, Germany). The flow cytometer
utilized a blue solid-state laser tuned at 20mW operating at
488 nm. Before each use, the instrument was calibrated using
3␮m calibration beads (Partec, Münster, Germany). Instrument
settings were optimized to ensure the sample and standard peak
were both visible and as far from the low-end debris as possible.
Fluorescence intensity was measured at 590 ± 25 nm (“FL2”) on a
linear scale to estimate genome size. Additional parameters re-
corded included “FL3” (fluorescence intensity at 630 nm, observed
on a log scale), forward scatter (a measure of particle size), and
side scatter (a measure of particle surface complexity). These pa-
rameters were used in combined scattergrams with FL2 to gate the
data after acquisition, by drawing polygon gates around the nu-
clei of interest. Given the very small size of the nuclei, a consid-
erable amount of gating was necessary to separate the particles of
interest from debris. All analyses were completed using FloMax
Software (v. 2.52; Partec, Münster, Germany).
Phylogenetic reconstruction
DNA was sampled from 23 hornwort accessions (Table S2). As
outgroups, we included 10 taxa representing early land plants
(mosses) and seedless vascular plants (lycopods, ferns) to root the
phylogeny but not coded for the ancestral reconstruction (see
below). To infer phylogenetic relationships we used the plastid
gene rbcL and second exon of the mitochondrial gene nad5. Se-
quence editing and alignment were carried out in Geneious v. 5.6.6
(created by Biomatters, available from http://www.geneious.com/).
Phylogenetic analyses were performed under likelihood (ML) op-
timization and the GTR + CAT substitution model, using RAxML
(Stamatakis et al. 2008). Statistical support was assessed via
100 ML bootstrap replicates and the same substitution model. All
1C-values (Mbp) were distributed into six evenly spaced bins and
mapped onto the phylogeny using parsimony reconstruction in
the “trace character state” application in Mesquite v. 2.75 (http://
mesquiteproject.org;Maddison and Maddison 2011; Fig. S1). When
species had multiple estimates, we used the average. The results
were mapped and summarized onto a simplified chronogram of
the hornworts from Villarreal and Renner (2012) (Fig. 1).
Results
We estimated genome size for 36 samples, representing
24 species (Table 1). This accounts for roughly 10% of known
Fig. 1. Simplified chronogram from Villarreal and Renner (2012) with genome sizes (in Mbp) showing increased genome size in the genera
Megaceros,Dendroceros, and Nothoceros. The mapping is a simplified version of the genome size values with four arbitrary categories to illustrate
the diversity of genome sizes in hornworts. All accepted genera are represented. Genera in paler green branches were not sampled for the
study. The lowest genome sizes seem to be the ancestral condition in the hornworts (see supplementary data, Fig. S1). Ovals are color-coded to
indicate the putative genome size classes identified in analyzed samples. A blue tick mark in Nothoceros shows a genome increase in Nothoceros
endiviifolius with the largest genome size (⬃719 Mbp or 0.73 pg) at least twice the typical genome size in other species of the genus (see text).
Exemplars of hornwort species are illustrated (clockwise from top left): Nothoceros endiviifolius (Mont.) J. Haseg. (Chile); Nothoceros vincentianus
(Lehm. & Lindenb.) J.C. Villarreal (Costa Rica); Phaeomegaceros coriaceus (Steph.) Duff et al. (New Zealand); Leiosporoceros dussii (Steph.) Hässel
(Panama). Pictures courtesy of L. Lewis (N. endiviifolius) and J. Duckett (P. coriaceus).
Bainard and Villarreal 433
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hornwort species and includes representatives from most of the
extant hornwort genera (Table 2). Haploid genome sizes (1C-
value) ranged from 160 Mbp (0.16 pg) for Leiosporoceros dussii to
719 Mbp (0.73 pg) for Nothoceros endiviifolius. The average 1C-value
was 261 ± 104 Mbp (0.27 ± 0.11 pg). Flow cytometry worked rela-
tively well for DNA content estimation, although many samples
required multiple attempts to achieve discernible nuclei peaks.
Several samples did not work at all including Anthoceros venosus
and Anthoceros angustus. The hornwort tissue generally had low
nuclei counts (as compared with other bryophytes), resulting in
the need for significant amounts of tissue. This was not always
possible with such small plants and also likely contributed to
higher levels of debris. Most flow cytometry accessions had on
average over 1500 nuclei in both the sample and standard peak.
The small DNA content of the hornwort nuclei neared the resolu-
tion capacity of the flow cytometer, making peak detection diffi-
cult and increasing the coefficients of variation (CV) (Voglmayr
2007). For all samples the standard CV was less than 5%, but the
hornwort peak CV ranged from 5% to 10%. However, having repli-
cates of each sample (with low standard errors) and replicates for
several species allowed us to confirm the repeatability of our mea-
surements (Table 1). As noted, dried herbarium samples did not
work at all, though dried tissue has previously worked for mosses
and several angiosperm species (Bainard et al. 2010,2011). Cul-
tured samples were also less successful than the fresh field-
collected tissue, possibly due to interference from ingredients in
the media. All flow histograms had only one peak of hornwort
nuclei; there was no evidence of endopolyploidy.
Genome size appears to have increased in the most recently
divergent hornwort clades, as revealed by mapping the estimates
onto a phylogenetic tree (Fig. 1). Low genome sizes are likely the
ancestral condition in hornworts (Fig. S1). The lowest genome size
is found in the sister taxon to all other hornworts, Leiosporoceros
(160 Mbp or 0.16 pg). The species sampled from Anthocerotaceae
have constrained genome sizes (176–193 Mbp or 0.18–0.20 pg);
however, few representatives were sampled from this family
(Table 2). The Notothyladaceae had the highest sampling coverage
(⬃15%, Table 2) and has a wider range in genome sizes (172–
253 Mbp or 0.18–0.28 pg). Both species of the Phymatocerotaceae
have genome sizes similar to some Phaeoceros species. The high-
est genome sizes are found in the most nested clades in the
Dendrocerotaceae (subfamily Dendrocerotoideae), namely Megaceros
(297 Mbp or 0.30 pg) and Nothoceros (308–719 Mbp or 0.32–0.73 pg).
The Austral American species N. endiviifolius has the largest genome
size of the hornworts sampled, whereas the closely related Austral
species Nothoceros fuegiensis is more similar to other Nothoceros species
(Table 1). In comparison to the Leiosporocerotaceae and Anthocerota-
ceae, most representatives in the Dendocerotaceae have genomes
roughly twice as large (Table 1). This increase in genome size likely
took place ⬃75 mya in the ancestor of Dendrocerotoideae (Fig. 1).
Discussion
This research contributes to the need for hornwort genome size
estimates (Leitch and Leitch 2013) and to our overall understand-
ing of land plant DNA content. Hornworts appear to have con-
strained genome sizes, similar to mosses (Voglmayr 2000;Bainard
2011). The very small genome sizes confirm the expectations based
on small chromosome size and number. Additionally, our esti-
mates for N. orbicularis and P. laevis closely match those obtained by
Renzaglia et al. (1995), differing by less than 3% and confirming
the accuracy of those measurements. Further species coverage is
required to determine if all hornwort taxa have small genomes
and whether an increase in genome size is restricted to the most
recently divergent clades.
There was some evidence of intraspecific variation in genome
size (Table 1). Whereas slight variations could be due to estimation
error, some may also be true intraspecific variation (e.g., in
Nothoceros vincentianus). One possible source for these differences
could be the presence of accessory chromosomes. Proskauer
(1957) found that accessory chromosomes often varied between
populations of N. vincentianus and P. laevis subsp. carolinianus, but
stayed constant within a clone. These accessory chromosomes
were not present in antheridial tissue, suggesting maternal trans-
mission (Proskauer 1957).
Interestingly, our data show clear increases in DNA content as
hornwort species diverge (Fig. 1). In general, several lineages show
independent increases in genome size and there also appears to
be an increase in the most recent common ancestor of Dendroc-
erotoideae. The largest genome size obtained belongs to N. endi-
viifolius, which is part of the most recently diversified clade
(Villarreal and Renner 2012). As this genome size is considerably
larger than the sister taxa analyzed, this could be an example of a
recent polyploid event. Even though existing hornwort chromo-
some numbers rarely exhibit evidence of polyploidy (Przywara
and Kuta 1995), the possibility of auto- or allopolyploidy cannot be
ruled out. A less likely scenario could include ancient polyploid
events followed by chromosome rearrangements and subsequent
haploidization (Rensing et al. 2007; or “diploidization” in diploid
dominant plants, Wolfe 2001). Further research including chro-
mosome counts and map-based analyses will provide insight into
whether or not polyploidy has shaped hornwort genomes. Alter-
natively, DNA content can change rapidly through variation in
repetitive DNA (such as long terminal repeat retrotransposons;
Vitte and Panaud 2005).
One of the unique features of hornworts is that they form asso-
ciations with cyanobacteria (Villarreal and Renzaglia 2006). Cya-
nobacteria living within the hornwort tissues may have led to
erroneous genome size estimates. However, previous genome size
estimates for cyanobacteria are all well under 10 Mbp or 0.01 pg
(Herdman et al. 1979;Larsson et al. 2011). These values were esti-
mated using considerably different methodology from that used
here (i.e., reassociation kinetics and genome sequencing, respec-
tively), which could lead to large discrepancies between estimates.
Regardless, it seems unlikely that any cyanobacteria associating
with the hornworts would have the quantity or genome size to
interfere with the results.
As this research on hornworts contributes to our knowledge of
genome size variation in land plants, there is still much more that
can be done. Broader species coverage representing unsampled
hornwort genera is necessary. Chromosome counts are vital to
Table 2. Percent coverage of hornwort genera analyzed for genome
size in this study listed by family.
Family Genus
No. of
species
analyzed
No. of
known
species
Coverage
(%)
Leiosporocerotaceae 1 1 100
Leiosporoceros 1 1 100
Anthocerotaceae 4 99 4.04
Anthoceros 3 80 3.75
Folioceros 1 17 5.88
Sphaerosporoceros 020
Dendrocerotaceae 7 67 10.45
Dendroceros 1 43 2.33
Megaceros 1425
Nothoceros 41040
Phaeomegaceros 11010
Notothyladaceae 10 66 15.15
Notothylas 1 21 4.76
Phaeoceros 7 41 17.07
Paraphymatoceros 2450
Phymatocerotaceae 2 2 100
Phymatoceros 2 2 100
Note: Numbers of known hornwort species taken from Villarreal and Renner
(2012).
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determine if chromosome numbers are correlated with genome
size and whether differences in genome size involve polyploidy.
Beyond learning more about patterns of DNA content variation in
hornworts, there is much to be discovered regarding the biologi-
cal significance of this variation. Renzaglia et al. (1995) put forth a
compelling hypothesis relating genome size to sperm size and
complexity in a sample of hornworts, mosses, and liverworts.
Other morphological features that could be explored include cell
size and spore size. DNA content may also be correlated with
sexual systems, as, traditionally, a transition to combined sexes
(monoicy) in bryophytes has been associated with genome dou-
bling (Wyatt and Anderson 1984). However, this theory was
recently challenged in mosses (Jesson et al. 2011) and also does
not appear to apply to liverworts (Bainard et al. 2013). Our data
also does not seem to correlate the doubling of genome size
with any sexual systems. For example, within Nothoceros, the
monoicous N. vincentianus and N. fuegiensis have genome sizes
similar to the dioicous N. aenigmaticus, and they are smaller
than the dioicous N. endiviifolius. Continued work on such mor-
phological and ecological traits will be necessary to learn if
there is any adaptive significance to the small genome sizes
found in hornworts.
Acknowledgements
We would like to thank D.C. Cargill for sending Australian
hornwort samples, B. Shaw for Chilean samples, and C. Garcia for
P. bulbiculosus; S.G. Newmaster for providing laboratory facilities;
B. Goffinet for field and lab materials and for helpful comments
on the manuscript; and L.L. Forrest and B. Doyle for providing
field support. We are grateful to two anonymous reviewers for
critical feedback on an earlier draft of the manuscript. Funding
support was provided to J.D.B. (Natural Sciences and Engineering
Research Council of Canada PGS-D) and J.C.V. (Deutsche For-
schungsgemeinschaft Grant RE-603/14-1 and National Science
Foundation Award DDIG-0910258).
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