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Genetic structure and diversity of the selfing model grass Brachypodium stacei (Poaceae) in Western Mediterranean: out of the Iberian Peninsula and into the islands Distributed under Creative Commons CC-BY 4.0


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Annual Mediterranean species of the genus Brachypodium are promising model plants for energy crops since their selfing nature and short-life cycles are an advantage in breeding programs. The false brome, B. distachyon, has already been sequenced and new genomic initiatives have triggered the de-novo genome sequencing of its close relatives such as B. stacei, a species that was until recently mistaken for B. distachyon. However, the success of these initiatives hinges on detailed knowledge about the distribution of genetic variation within and among populations for the effective use of germplasm in a breeding program. Understanding population genetic diversity and genetic structure is also an important prerequisite for designing effective experimental populations for genomic wide studies. However, population genetic data are still limited in B. stacei. We therefore selected and amplified 10 nuclear microsatellite markers to depict patterns of population structure and genetic variation among 181 individuals from 19 populations of B. stacei occurring in its predominant range, the western Mediterranean area: mainland Iberian Peninsula, continental Balearic Islands and oceanic Canary Islands. Our genetic results support the occurrence of a predominant selfing system with extremely high levels of homozygosity across the analyzed populations. Despite the low level of genetic variation found, two different genetic clusters were retrieved, one clustering all SE Iberian mainland populations and the island of Minorca and another one grouping all S Iberian mainland populations, the Canary Islands and all Majorcan populations except one that clustered with the former group. These results, together with a high sharing of alleles (89%) suggest different colonization routes from the mainland Iberian Peninsula into the islands. A recent colonization scenario could explain the relatively low levels of genetic diversity and low number of alleles found in the Canary Islands populations while older colonization events are hypothesized to explain the high genetic diversity values found in the Majorcan populations. Our study provides widely applicable information about geographical patterns of genetic variation in B. stacei. Among others, How to cite this article Shiposha et al. (2016), Genetic structure and diversity of the selfing model grass Brachypodium stacei (Poaceae) in Western Mediterranean: out of the Iberian Peninsula and into the islands.
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Genetic structure and diversity of the
selfing model grass Brachypodium stacei
(Poaceae) in Western Mediterranean:
out of the Iberian Peninsula and into
the islands
Valeriia Shiposha
, Pilar Catala
, Marina Olonova
Isabel Marques
1Department of Agriculture and Environmental Sciences, High Polytechnic School of Huesca,
University of Zaragoza, Huesca, Spain
2Department of Botany, Institute of Biology, Tomsk State University, Tomsk, Russia
Annual Mediterranean species of the genus Brachypodium are promising model
plants for energy crops since their selfing nature and short-life cycles are an
advantage in breeding programs. The false brome, B. distachyon, has already
been sequenced and new genomic initiatives have triggered the de-novo genome
sequencing of its close relatives such as B. stacei, a species that was until recently
mistaken for B. distachyon. However, the success of these initiatives hinges on
detailed knowledge about the distribution of genetic variation within and
among populations for the effective use of germplasm in a breeding program.
Understanding population genetic diversity and genetic structure is also an
important prerequisite for designing effective experimental populations for
genomic wide studies. However, population genetic data are still limited in
B. stacei. We therefore selected and amplified 10 nuclear microsatellite
markers to depict patterns of population structure and genetic variation
among 181 individuals from 19 populations of B. stacei occurring in its
predominant range, the western Mediterranean area: mainland Iberian Peninsula,
continental Balearic Islands and oceanic Canary Islands. Our genetic results
support the occurrence of a predominant selfing system with extremely high levels
of homozygosity across the analyzed populations. Despite the low level of genetic
variation found, two different genetic clusters were retrieved, one clustering all
SE Iberian mainland populations and the island of Minorca and another one
grouping all S Iberian mainland populations, the Canary Islands and all
Majorcan populations except one that clustered with the former group. These
results, together with a high sharing of alleles (89%) suggest different colonization
routes from the mainland Iberian Peninsula into the islands. A recent colonization
scenario could explain the relatively low levels of genetic diversity and low
number of alleles found in the Canary Islands populations while older colonization
events are hypothesized to explain the high genetic diversity values found in
the Majorcan populations. Our study provides widely applicable information
about geographical patterns of genetic variation in B. stacei. Among others,
How to cite this article Shiposha et al. (2016), Genetic structure and diversity of the selfing model grass Brachypodium stacei (Poaceae) in
Western Mediterranean: out of the Iberian Peninsula and into the islands. PeerJ 4:e2407; DOI 10.7717/peerj.2407
Submitted 11 April 2016
Accepted 4 August 2016
Published 8 September 2016
Corresponding author
Isabel Marques,
Academic editor
Levi Yant
Additional Information and
Declarations can be found on
page 18
DOI 10.7717/peerj.2407
2016 Shiposha et al.
Distributed under
Creative Commons CC-BY 4.0
the genetic pattern and the existence of local alleles will need to be adequately
reflected in the germplasm collection of B. stacei for efficient genome wide
association studies.
Subjects Evolutionary Studies, Genetics, Plant Science
Keywords Annual model grass species, Brachypodium stacei, SSRs, Genetic diversity and structure,
Balearic (Gymnesic) and Canarian islands, Isolation, Western Mediterranean
Approximately one third of Earth’s land is covered by grass-dominated ecosystems
comprising 600 genera and more than 12,000 species (Soreng et al., 2015). Besides their
important ecological role, grasses are the core of human nutrition and several genomic
efforts have focused on economically important species (e.g., rice: International Rice
Genome Sequencing Project (2005); sorghum: Paterson et al. (2009)). Among grasses,
the genus Brachypodium, a member of the Pooideae subfamily, has recently been
developed as a new model system to study the evolution of grasses. The genome of the
annual B. distachyon, commonly known as the false brome, has already been sequenced
(International Brachypodium Initiative, 2010). This species has several features suitable
for the development of a model plant for genomic studies such as a small diploid genome
(355 Mbp), a short annual life-cycle, easily amenable to culture, and a selfing nature
(Gordon et al., 2014).
The taxonomic identity of B. distachyon was recently challenged with the recognition
that the three cytotypes attributed to different ploidy levels in this species (e.g., an
autopolyploid series of individuals with x = 5 and 2n = 10 (2x), 20 (4x), 30 (6x)
chromosomes; Robertson, 1981) were in fact three different species: two diploids, each
with a different chromosome base number, B. distachyon (x = 5, 2n = 10) and B. stacei
(x = 10, 2n = 20), and their derived allotetraploid B. hybridum (x = 5 + 10, 2n = 30)
´n et al., 2012;Lo
´pez-Alvarez et al., 2012). This recent taxonomic split has
triggered new genomic initiatives including the re-sequencing of 56 new accessions of
B. distachyon and the de-novo genome sequencing of B. stacei and B. hybridum,a
project undertaken by the Joint Genome Institute and the International Brachypodium
Consortium (
brachypodium/). The forthcoming genomes of B. stacei and B. hybridum will allow the
development of several functional genomic analyses on these diploid and polyploid
species and their potential transfer to other cereals and forage or biofuel crops. A recent
update on phenotypic traits and habitat preferences of the three species has increased the
number of discriminant features that distinguish them and has thrown new insights into
their respective ecological adaptations (Catala
´n et al., 2016a). However, very scarce
genetic information exists for these close relatives of B. distachyon, especially for the rarest
species of this complex, B. stacei (Catala
´n et al., 2016b). It would, therefore, be invaluable
to have more information especially because a collection of germplasm reflecting the
natural diversity of B. stacei is necessary for future genome wide association studies and
the creation of reference lines.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 2/23
Brachypodium stacei is a monophyletic annual diploid species that diverged first
from the common Brachypodium ancestor, followed consecutively by B. mexicanum,
B. distachyon and the clade of the core perennial taxa (Catala
´n et al., 2012;Catala
et al., 2016b). Several studies have revealed it to be distinct from B. distachyon and
B. hybridum: e.g., protein data: Hammami et al. (2011); nuclear SSRs: Giraldo et al.
(2012); DNA barcoding: Lo
´pez-Alvarez et al. (2012); isozymes: Jaaska (2014).A
recent study using environmental niche models predicted a potential distribution of
B. stacei in coastal and lowland areas of the circum-Mediterranean region (Lo
Alvarez et al., 2015), concurrent with its known geographic distribution (Catala
et al., 2016a). However, a large number of those populations occur in the western
Mediterranean region and in Macaronesia (Lo
´pez-Alvarez et al., 2015;Catala
2016a). Population genetic studies conducted in its annual congener B. distachyon
have demonstrated that the genetic structure does not fit a geographic pattern but
rather might have resulted from a combination of factors such as long distance
dispersal of seeds and flowering time isolation (Vogel et al., 2009;Mur et al., 2011;
Tyler et al., 2016).
Here, we studied the patterns of genetic variation in the mainland Iberian Peninsula
and the western island populations (continental Balearic Islands and oceanic Canary
Islands) of B. stacei to unravel the origin and phylogeographic patterns of its populations.
From all its range, this area is the best known due to previous studies (Catala
´n et al., 2012;
´n et al., 2016a;Lo
´pez-Alvarez et al., 2012;Lo
´pez-Alvarez et al., 2015), which can
guarantee the correct identification of B. stacei since it can be misidentified with its close-
relatives (Lo
´pez-Alvarez et al., 2012). We specifically addressed the following questions:
(1) Is genotypic diversity within populations limited by the prevalence of autogamous
pollinations? (2) Do islands (e.g., continental, oceanic) contain less genetic variation
than mainland areas? (3) Is there a signature of geographic genetic structure in this self-
pollinated plant? Finally, we aim to provide recommendations necessary to establish an
efficient germplasm collection of B. stacei, with the aim of helping future genomic
initiatives in Brachypodium.
Population sampling, DNA extraction and nSSR amplification
A total of 181 individuals were sampled from 19 populations of B. stacei covering the
whole distribution range of this species within the Iberia Peninsula, plus the continental
Balearic (Gymnesic) Islands (Majorca, Minorca) and the oceanic Canary Islands
(Gomera, Lanzarote) (Table 1;Fig. 1). Nine populations were sampled in mainland
Iberian Peninsula and ten across the two groups of islands (Fig. 1). In each population, ten
individuals were collected randomly with a minimum sampling distance of 10 m, with the
exception of the Iberian ALI and the Majorcan BANYA populations where only five and
six individuals were respectively found. Sampling sizes, locations and geographic
coordinates of each population sampled are given in Table 1. Fresh leaves were collected
for each individual, dried in silica gel and stored at -20 C until ready for DNA isolation.
The silica samples for all individuals were deposited in the DNA bank of the BioFlora
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 3/23
group at the University of Zaragoza in Spain and voucher specimens were deposited in
the JACA herbarium (Spain).
Total genomic DNA was extracted from fresh leaf tissue or from silica-dried leaf
samples using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the
manufacturer’s protocol. The 181 samples used in this study were genotyped at 10 variable
nuclear microsatellite markers (nSSRs) developed for B. distachyon (ALB006, ALB022,
ALB040, ALB050, ALB086, ALB087, ALB139, ALB165, ALB181 and ALB311;Vogel et al.,
2009). All those microsatellites were selected because during our preliminary studies they
displayed good quality and high transferability success in B. stacei. The forward primer of
each locus was 5-end labeled with a fluorescent dye. Amplifications were carried out in a
final volume of 10 ml containing between 0.1 and 0.2 ml of each 10 m diluted primer
(forward and reverse), 5 ml PCR Master Mix (QIAGEN) and 2.5 ml DNA. The polymerase
chain reactions (PCR) were carried out on a GeneAmp PCR System 9700 thermocycler
with a thermal profile consisting of a 4-min initial denaturation step at 95 C followed by
35 cycles of 30 s at 95 C, 30 s at 55 C and 1 min at 72 C. A final 72 C extension step of
Table 1 Sampled populations of Brachypodium stacei sorted by geographical area. The location, population code, number of plants genotyped
(N), mean observed heterozygosity (H
) and expected heterozygosity (H
), mean number of alleles (N
), allelic richness (A
), inbreeding coefficient
), selfing rate (s), and number of exclusive genotypes (%. between parenthesis) are shown.
Locality Code NLatitude (N) Longitude (W) H
Mainland (Iberian Peninsula)
S Spain: Granada, Moclin GRA 10 371959N3
4659W 0.240 0.155 12 1.126 0.667*0.800 3 (30%)
S Spain: Almeria, Cabo de Gata ALM 10 36442N2
835W 0.170 0.102 11 1.050 0.0001 0.0001 3 (30%)
S Spain: Jaen: Cazorla, Cortijos Nuevos JAE1 10 381131N2
4814W 0.120 0.116 12 1.176 0.723*0.839 4 (40%)
S Spain: Jaen: Quesada, Tiscar JAE2 10 37465N3
123W 0.200 0.100 10 1.000 1 (10%)
SE Spain: Murcia, Portman PORT 10 373457N0
5115W 0.200 0.100 10 1.000 1 (10%)
SE Spain: Murcia, Calblanque CALBN 10 373559N0
4529W 0.140 0.108 14 1.246 0.526*0.689 4 (40%)
SE Spain: Murcia, Cobaticas CALBA 10 373559N0
4530W 0.110 0.105 15 1.339 0.617*0.763 5 (50%)
SE Spain: Murcia, Cala Reona CALREL 10 373656N0
4256W 0.030 0.009 13 1.239 0.520*0.684 5 (50%)
SE Spain: Alicante, Cabo La Nao ALI 5 384522N0
138E 0.300 0.150 10 1.000 1 (20%)
Balearic (Gymnesic) Islands
Spain: Minorca: Es Mercadal, Toro MEN 10 39596N4
647E 0.240 0.173 13 1.203 0.386*0.556 3 (30%)
Spain: Majorca: Sa Dragonera, Gambes DRAG 10 393513N2
1937E 0.111 0.154 16 1.428 0.916*0.956 5 (50%)
Spain: Majorca: Arta, Peninsula de Llevant ARTA 10 394410N3
206E 0.210 0.128 12 1.126 0.666*0.799 3 (30%)
Spain: Majorca: Campanet, Coves CAMPA 10 394731N2
5812E 0.130 0.138 14 1.434 0.486*0.654 6 (60%)
Spain: Majorca: Alcudia, Punta Negra ALCU 10 395248N3
1041E 0.140 0.108 14 1.200 0.0001 0.0001 2 (20%)
Spain: Majorca: Felenitx, San Salvador FELEN 10 39274N3
1117E 0.130 0.109 14 1.200 0.250 0.400 4 (40%)
Spain: Majorca: Petra, Bonany BONA 10 393538N3
510E 0.290 0.391 23 1.992 0.385*0.5555 9 (90%)
Spain: Majorca: Banyalbufar, Ses Animes BANYA 6 39416N2
3036E 0.167 0.239 15 1.496 0.825*0.904 6 (100%)
Canary Islands
Spain: Gomera: Agulo GOM 10 281059N17
1059W 0.150 0.118 11 1.076 0.891*0.942 2 (20%)
Spain: Lanzarote: Teguise LAN 10 2941N13
311W 0.230 0.136 11 1.096 1.000*1 2 (20%)
values deviating from HWE (P> 0.05).
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 4/23
30 min was included to promote non-templated nucleotide addition at the 3end of the
PCR product. Multiplexed PCR products were genotyped on an Applied Biosystems
3130XL Genetic Analyzer using 2 ml of amplified DNA, 12 ml of Hi-Di formamide and
0.4 ml of GeneScan-500 (LIZ) size standard (Applied Biosystem). Allele sizes were
determined using Peak Scanner version 1.0 (Life Technologies). Within each population,
all loci were checked for the presence of null alleles using MICRO-CHECKER v.2.2.3
(van Oosterhout et al., 2004).
Hardy-Weinberg equilibrium, linkage disequilibrium and genetic
Deviation from Hardy-Weinberg Equilibrium (HWE) was tested at both population
and locus levels using FSTAT (Goudet, 2001). To calculate the extent of linkage
disequilibrium between pairs of loci (LD) in each population we set dememorization
numbers at 10,000 and performed 100,000 iterations for all permutation tests (exact tests)
in Genepop v.4.0.10 (Raymond & Rousset, 1995). Significant values were corrected for
multiple comparisons by Bonferroni correction (Rice, 1989).
For each microsatellite locus and population, genetic polymorphism was assessed by
calculating the total number of alleles (N
, allelic diversity), mean expected heterozygosity
), mean observed heterozygosity (H
), allelic richness (A
), and inbreeding
coefficient (F
) using FSTAT (Goudet, 2001). The inbreeding coefficient was
also estimated using the Bayesian procedure (IIM) implemented in INEst 2.0, which is
Figure 1 Location of the study area of Brachypodium stacei.Collection localities of Brachypodium stacei populations in mainland Iberian
Peninsula, the continental Balearic (Gymnesic) Islands (Minorca and Majorca) and the oceanic Canary Islands. Pie-charts indicate the proportion
of ancestry assigned to individuals of each population by Bayesian clustering analysis using STRUCTURE.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 5/23
robust to the presence of null alleles (Chybicki & Burczyk, 2009). Posterior distribution
was based on 300,000 steps, sampling every 100 steps and discarding the first 30,000 steps
as burn-in. In order to infer the statistical significance of inbreeding we compared the
full model (nfb), the model including only the possibility of null alleles and inbreeding
(nf), and the model including only null alleles and genotyping failures (nb). The best
model was chosen based on the Deviance Information Criterion (DIC; cf. Chybicki,
Oleksa & Burczyk, 2011).
GenAlEx 6 software was used to estimate the mean expected heterozygosity (H
) and
mean observed heterozygosity (H
) for each population (Peakall & Smouse, 2006). In
addition, the selfing rate (s) was also estimated as s=2F
/(1 + F
)(Ritland, 1990). Spatial
patterns of allelic quantity were visualized by mapping variation for the locations across
space with the interpolation kriging function in ARCINFO (ESRI, Redlands, CA, USA),
using a spherical semivariogram model.
Population genetic structure, genetic differentiation and isolation
The Bayesian program STRUCTURE v.2.3.4 (Pritchard, Stephens & Donnelly, 2000) was
used to infer the population structure and to assign individual plants to subpopulations.
Models with a putative numbers of populations (K) from 1–10, imposing ancestral
admixture and correlated allele frequencies priors, were considered. Ten independent runs
with 50,000 burn-in steps, followed by run lengths of 300,000 interactions for each K, were
computed. The number of true clusters in the data was estimated using STRUCTURE
HARVESTER (Earl & vonHoldt, 2012), which identifies the optimal Kbased both on the
posterior probability of the data for a given Kand the K(Evanno, Regnaut & Goudet,
2005). To correctly assess the membership proportions (qvalues) for clusters identified
in STRUCTURE, the results of the replicates at the best fit Kwere post-processed
using CLUMPP 1.1.2 (Jakobsson & Rosenberg, 2007). BAPS v.5.2 (Corander, Marttinen &
¨ntyniemi, 2006) was used to explore population structure further. In contrast to
STRUCTURE, BAPS determines optimal partitions for each candidate K-value and
merges the results according the log-likelihood values to determine the best K-value.
Analyses in BAPS were done at the level group of individuals using the models without
spatial information and by selecting 1–10 as possible K-values. Ten repetitions were
performed for each K. POPULATION 1.2 (Langella, 2000) was used to calculate the
Nei’s genetic distance (D
;Nei, Tajima & Tateno, 1983) among individuals and to
construct an unrooted neighbor-joining tree with 1,000 bootstrap replicates. Nei’s genetic
distance among individuals was also visualized by Principal Components Analysis (PCoA)
with GenAlEx6 (Peakall & Smouse, 2006).
We estimated genetic differentiation among locations using an analysis of molecular
variance (AMOVA) with ARLEQUIN 3.11 (Excoffier, Laval & Schneider, 2005). In
addition, molecular variance was also studied (1) between the genetic groups retrieved by
STRUCTURE and BAPS, (2) between mainland and island populations, (3) within
mainland populations, e.g., S Spain vs. SE Spain, and (4) within island populations, e.g.,
Balearic vs. Canary Islands. In each analysis, variance was quantified among groups,
among locations within groups and within sampling locations. Each AMOVA was run
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 6/23
with 10,000 permutations at 0.95 significance levels. Relationships between genetic and
linear geographic distances (isolation-by-distance, IBD) were examined using a Mantel
test (Mantel, 1967) implemented in ARLEQUIN 3.11 (Excoffier, Laval & Schneider, 2005)
with 10,000 permutations.
Hardy-Weinberg disequilibrium, non linkage disequilibrium
Deviations from HWE were common in the selfed B. stacei. From the 19 populations
sampled, only five were at HWE (GRA, MEN, ARTA, FELEN, GOM) at the 5% level after
the sequential Bonferroni correction (Table 2). Pairwise comparisons between loci
revealed no significant linkage disequilibrium at the P= 5% suggesting that alleles are
assorting independently at different loci.
Genetic diversity and mating system of Brachypodium stacei
For each locus, observed heterozygosity values ranged from 0 to 0.058 (respectively for
loci ALB139 and ALB086), and expected heterozygosity ranged from 0 to 0.145
(respectively for loci ALB139 and ALB087). F
values varied between -0.068 and 0.8482
(respectively for loci ALB311 and ALB022;Table 3) across the loci studied. No null
alleles were detected. The results from the Bayesian analyses implemented in INEst
revealed that only inbreeding contributed to the excessive homozogosity, since this
model (DIC
: 3,300.019) was preferred over the alternative ones (DIC
: 4,400.390;
: 4,400.300) based on the DIC criterion.
From the 19 sampled populations of B. stacei, only 37 distinct alleles were found in
the 181 individuals studied (Fig. 2;Table S1). Most of the alleles (27 alleles; 73%) were
shared between populations while the remaining ones were private to mainland, Majorca,
Minorca or the Canary Islands (10 alleles; 27%). Most of the alleles found in the islands
were also found in the mainland since only three alleles out of 27 (11%) were not
found in the mainland: two alleles were shared between Majorca and Minorca and one
allele was shared between Majorca and the Canary Islands (Fig. 2). Out of 37, four alleles
were exclusively found only in the mainland (10%; three in SE Spain and one in S Spain),
six in Majorca (16%) and one in Minorca (2%) while the Canary Islands had no
unique alleles (Fig. 2).
The number of alleles generally increased in the Balearic Islands, most specially in
Majorca (P< 0.0001) as shown when projected into the geographic space (Fig. 3). Overall,
only 38% (69 out of 181) of all genotyped samples exhibited unique multi-locus genotypes,
as a consequence of the rampant homozygosis (fixed alleles) observed for most loci in most
populations. The observed percentage is lower than one might expect under random
mating, where the frequency of multilocus genotypes is expected to be equal to the product
of the allelic frequencies. However, a relatively high number of unique multi-locus
genotypes were generally found in the populations collected in the island of Majorca, where
up to 100% of all the individuals sampled showed unique multi-locus genotypes (Tabl e 1 ).
Mean observed heterozygosity among the populations of B. stacei varied between
0.110 (mainland population CALBA) and 0.290 (Majorcan population BONA) with a
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 7/23
CI of ± 0.03 at the 95% level, while mean expected heterozygosity varied between 0.090
(mainland population CALREL) and 0.239 (Majorcan population BANYA; Table 1) with
a CI of ± 0.04 at the 95% level. The average F
value was 0.558 (CI: 0.141) varying
between 0.0001 (mainland population ALM, Majorcan population ALCU) and 1 (Canary
Table 2 Results of the Hardy Weinberg exact tests retrieved by GENEPOP for 19 populations of
Brachypodium stacei.P-value (0.05) associated with the null hypothesis of random union of gametes
(or ‘–’ if no data were available, or only one allele was present) estimated with a Markov chain algorithm
and the standard error (S.E.) of this estimate.
Population P-value S.E.
GRA 0.0519 0.0011
JAE1 0.0259 0.0007
JAE2 –
CALBN 0.0077 0.0009
CALBA 0.0007 0.0001
CALREL 0.0249 0.0006
MEN 0.1016 0.0015
DRAG 0 0
ARTA 0.053 0.0012
CAMPA 0.0361 0.0014
FELEN 0.0508 0.0028
BONA 0 0
GOM 0.0515 0.0012
LAN 0.0096 0.0005
Table 3 Characteristics and genetic diversity statistics of the nuclear microsatellite markers used in
the genetic study of Brachypodium stacei.For each locus, the total number of alleles (N
), mean
expected heterozygosity (H
), mean observed heterozygosity (H
), and the fixation index (F
) obtained
from the 181 studied individuals are shown.
Locus Repeat motif N
ALB006 (GT)15 2 0.016 0.016 0.003
ALB022 (CT)11 2 0.035 0.005 0.848
ALB040 (CTT)8 4 0.129 0.047 0.632
ALB050 (GT)15 4 0.122 0.032 0.717
ALB086 (AAG)7 6 0.119 0.058 0.486
ALB087 (AGC)7 6 0.145 0.032 0.758
ALB139 (AGA)7 1 0.000 0.000 0
ALB165 (ATA)12 4 0.066 0.049 0.298
ALB181 (AC)9 5 0.049 0.037 0.253
ALB311 (GA)6 3 0.025 0.026 -0.069
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 8/23
population of LAN). Therefore, the average rate of self-fertilization in B. stacei was
estimated to be 71% considering all the populations (Table 1). However, the wide range
of F
values implies that the predicted level of self-fertilization also varies extensively
across populations with a CI of ± 0.25 at the 95% level.
Population genetic structure among geographical areas
The Bayesian clustering program STRUCTURE found the highest LnP(D) and K
values for K=2(P< 0.001) which differentiated all south (S) Iberian mainland
populations from the southeastern (SE) mainland populations. The populations
collected in the island of Minorca clustered with the SE mainland populations,
whereas samples from the Canary Islands and most of the Majorcan populations
were grouped with the S mainland populations, with the exception of the Majorcan
population of BONA were most individuals were assigned to the same genetic
group found only in the SE mainland populations (Fig. 4A). Some individuals
collected in four populations of Majorca showed genetic admixture between the
two genetic groups (DRAG, CAMPA, ALCU, BONA; Fig. 4A). These results were also
Geographical Areas
Canary Islands
Mainland SE Spain
Mainland S Spain
362340 184180
168166 205203189187 190188182180176174
198196194184182180 294 187181157145 240238236232220 250244242
ALB006 ALB022 ALB040 ALB050
ALB139 ALB165 ALB181 ALB311
Figure 2 Distribution of Brachypodium stacei alleles. Frequency of the alleles found in Brachypodium stacei across the geographical area sampled:
mainland Iberian Peninsula (SE Spain and S Spain) and the islands of Minorca, Majorca and the Canary Islands. Colors of areas are indicated in
the chart.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 9/23
corroborated by BAPS, which retrieved similar results and generally differentiated
S mainland populations, Canary Islands and Majorcan populations from all the
remaining populations sampled with the exception of the Majorcan population of
BONA (Fig. 4B).
The PCoA spatially separated SE Iberian mainland and the Minorca island
populations from all remaining populations at both extremes of axis I, which
accumulated 44.3% of variance (Fig. 5), partially supporting the genetic boundaries
assigned by STRUCTURE and BAPS at K= 2. In this two-dimensional plot, the
S mainland populations, as well as the SE ones and the Island populations (Minorca,
Majorca and the Canary Islands) were well differentiated along the axis 2, which
accumulated 26.2% of variance.
The NJ tree separated all SE mainland populations, Minorca and the Majorcan
population of BONA from the remaining populations, in a highly supported group
(78% bootstrap support (BS) value; Fig. 6A). A similar NJ tree was retrieved when the
admixed individuals indicated by STRUCTURE were excluded (Fig. 6B). The remaining
sub-divisions found in the NJ trees correspond mainly to the populations sampled
although BS values were always very low, with or without admixed individuals
(< 43%, Figs. 6A and 6B).
Genetic differentiation and isolation
Overall, genetic differentiation was significantly high (AMOVA FST = 0.748,
P< 0.00001). The analysis performed over the 19 populations sampled indicated that
Figure 3 Overall allelic richness of Brachypodium stacei.Map of overall allelic richness of Brachy-
podium stacei across the geographic range sampled. Dark areas contain higher richness.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 10/23
75 and 25% of the genetic variation was attributed to variation among and within
populations, respectively (P< 0.00001; Table 4 ). When analyzing the two genetic
groups retrieved by STRUCTURE, an independent AMOVA attributed 24, 54 and
22% of the total variation to variation among groups, among populations within
groups and within populations (Tab l e 4 ). Fixation indices of this analysis were
FST = 0.779, FSC = 0.710 and FCT = 0.240. To further investigate genetic
differentiation between mainland and island populations, an independent AMOVA
also attributed the highest percentage of variation among populations within
groups (68% of the total variance; FST = 0.758, FSC = 0.737 and FCT = 0.077;
Tabl e 4 ). However, genetic variation was equally partitioned among groups, among
populations within groups and within groups when analyzing only island populations
(FST = 0.672, FSC = 0.516 and FCT = 0.322), and predominant among groups
and among populations within groups when analyzing only mainland populations
(FST = 0.884, FSC = 0.783 and FCT = 0.464; Table 4 ).
The Mantel’s test did not detect any significant correlation between the genetic
distance [F
/(1 -F
)] and the geographical distance of the populations studied here
= 0.83, y= 1.755x-0.223, P= 0.085).
Minorca Majorca Canary IslandsS Spain SE Spain
Figure 4 Population structure of Brachypodium stacei.Population structure of 181 individuals of Brachypodium stacei based on 10 nSSRs and
using the best assignment result (K= 2) retrieved by STRUCTURE (A) and by BAPS (B) with Kfrom 1 to 10 (replicated 10) under an admixture
model. Each individual is represented by a thin horizontal line divided into Kcolored segments that represent the individual’s estimated mem-
bership fractions in Kclusters. The different geographic areas are labelled below the graph. Abbreviations of populations follow those indicated in
Table 1.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 11/23
Axis 1 (44.3%)
Axis 2 (26.2%)
Canary Islands Minorca
SE Spain
S Spain
Figure 5 Genetic relationships among Brachypodium stacei populations based on Nei’s genetic
distance. Principal Coordinate analysis (PCoA) samples using the scored nSSRs markers. Percentage
of explained variance of each axis is given in parentheses. Population symbols are shown in the chart.
Figure 6 Unrooted neighbor-joining trees of Brachypodium stacei populations based on Nei’s genetic distance. Unrooted neighbor-joining tree
showing relationships among the individuals collected in 19 populations. Numbers associated with branches indicate bootstrap values based on
1,000 replications. Colours followed the ones depicted in Fig. 4 for K= 2. Population codes are indicated in Table 1. (A) Genetic relationships
among all individuals of B. stacei. (B) Genetic relationships without the individuals of B. stacei showing admixture in STRUCTURE. Note that the
Majorcan population of BONA (arrow) is grouped with SE mainland populations in both NJ trees.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 12/23
Incidence of a highly selfing mating system
From our genetic study, selfing rates of B. stacei were estimated as 79% across all
populations, even reaching values as high as 95% in some populations (Table 1 )
although CI values were fairly wide. Even if null alleles have not been detected, the
high variation occurring between loci for many of the genetic parameters estimated
(Table 3 ) might influence the reported genetic values. Nonetheless, there was clearly a
predominance of homozygous individuals, suggesting that B. stacei is primarily a selfing
species like its close congener B. distachyon (Draper et al., 2001;Gordon et al., 2014).
Although the respective ancestors of these two annual species likely split 16.2 (B. stacei)
and 10.2 (B. distachyon) Mya ago (cf. Catala
´n et al., 2016b), species divergence was
not followed by changes in the mating system of B. distachyon and B. stacei.This
analogous mating system is consistent with similarities in floral morphology and floral
structure in the two species since they both bear relatively small (mean 7–9 mm)
cleistogamous or cleistogamous-type florets having minute (< 0.8 mm) non-exerted
anthers (Catala
´n et al., 2016a). Pollination in B. distachyon usually occurs in closed
flowers leading to extremely high levels of homozygosity (Vogel et al., 2009), such as the
ones reported here for B. stacei. Even more recently diverged species, such as the
perennial B. sylvaticum, display a predominantly selfing system, although the levels
of heterozygosity suggest that this species outcross more often than B. distachyon and
B. stacei (Steinwand et al., 2013).
In nature, selfing is thought to be favored due to its inherent transmission
advantage, as well as assuring reproduction when pollinators or available mates are
scarce (Marques, Draper & Iriondo, 2014) and it is expected to evolve whenever these
advantages outweigh the costs of inbreeding depression (Charlesworth & Willis, 2009).
Table 4 Analysis of molecular variance (AMOVA) for 19 populations of Brachypodium stacei.
Source of variance d.f. Variance
% Variance
All populations Among populations 18 1.011 74.88
Within populations 343 0.339 25.12
Between genetic groups
defined by STRUCTURE
and BAPS (K=2)
Among groups 1 0.370 24.05
Among populations within groups 17 0.831 53.94
Within populations 343 0.339 22.01
Mainland vs. islands Among groups 1 0.109 7.77
Among populations within groups 17 0.954 68.04
Within populations 343 0.339 24.19
Within mainland
populations (S Spain vs.
SE Spain)
Among groups 1 0.831 46.41
Among populations within groups 7 0.752 41.99
Within populations 161 0.207 11.60
Within island populations
(Balearic islands vs.
Canary islands)
Among groups 2 0.448 32.26
Among populations within groups 7 0.486 34.97
Within populations 182 0.455 32.77
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 13/23
But contrary to these short-term benefits, selfing might also reduce effective
recombination rate leading to frequent genetic bottlenecks (Goldberg et al., 2010).
Recombination is generally thought to be advantageous because it breaks down
associations between alleles (linkage disequilibrium), which might lead to the fixation
of deleterious mutations (Charlesworth & Charlesworth, 2000). As a result, it has long
been argued that the evolutionary potential of highly selfing species is quite limited as a
result of reduced genetic diversity and recombination rates (Lynch, Conery & Burger,
1995). However, many important crops such as wheat, barley, beans, and tomatoes,
are predominantly self-fertilizing species despite the possibility of linkage drag
(Morrell et al., 2005). Likewise, linkage disequilibrium is absent in B. stacei despite being
a highly selfing species. There are several possible explanations. The first is that the
relatively low levels of linkage disequilibrium results from a recent transition from a
strict outcrossing ancestral mating system to a predominantly selfing one, so that the
recombination events would still be present (Lin, Brown & Clegg, 2001). However,
recent phylogenetic studies indicated that B. stacei is the earliest extant diverging lineage
within the genus and that other early splits also resulted in selfing species (e.g., B.
´n et al., 2016b). The second possible explanation is that in a temporal
time scale of more than 38 Mya to the common ancestor of the Brachypodium stem
node (e.g., MRCA of the Brachypodieae/core pooids split; Catala
´n et al., 2016b), even
a very low number of outcrossing events might be enough to promote a certain level
of recombination. Although plant species might usually mate through selfing, few
are strictly selfing (Igic & Busch, 2013), creating opportunities for recombination that
helps to break down associations between alleles. Large population sizes, which are
not uncommon in B. stacei, might also reduce linkage disequilibrium. For instance,
the near worldwide-distributed Arabidopsis thaliana is predominantly a selfing
annual species but exhibits a rapid decay in linkage drag in several populations
(Nordborg et al., 2005).
Origin of island populations
Many plant phylogeographic studies have concluded that genetic diversity erodes
across colonization steps, but islands usually exhibit high frequencies of endemism
in comparison with large continental areas as a consequence of isolation and habitat
diversity (Kim et al., 1996;Sanmartı
´n, van der Mark & Ronquist, 2008;Vitales et al.,
2014; see review in Caujape
´-Castells (2011)). In our study, the genetic structure retrieved
by the Bayesian analyses of STRUCTURE or BAPS, or by the results retrieved from the
PCoA and the NJ tree suggests a scenario of colonization from the mainland Iberian
Peninsula into the islands. Individuals collected in Minorca clustered with SE mainland
Iberian populations, whereas individuals from the Canary Islands and most of the
Majorcan populations were clustered with S mainland populations (with the exception
of BONA which is more related to the SE mainland populations). The large number
of alleles (89%) shared between the individuals collected in the Canary Islands and
the ones collected in S Spain could support the hypothesis of colonization from
the mainland Iberian Peninsula. A recent colonization scenario from a mainland
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 14/23
Iberian source fits well the plausible origin of the oceanic Canary island populations,
which show low levels of genetic diversity and multilocus genetic profiles that are a
subset of those found in S Spain (Ta b le 1;Fig. 2). Additionally, Canarian populations
of B. stacei have shown to be phenotypically close to S Spain populations (D. Lo
Alvarez, P. Catala
´n, 2016, unpublished data). However, islands could have also been
colonized by North African coastal populations of B. stacei since ecological niche
models predict the existence of conditions suitable for the existence of this species in
that area (Lo
´pez-Alvarez et al., 2015).
Single vs. multiple colonization scenarios from mainland Iberian sources have been
proposed to explain the origins of the Macaronesian plant populations (cf.
et al., 2012); however, most of them gave rise to new species (Kim et al., 1996;Francisco-
Ortega, Jansen & Santos-Guerra, 1996;Francisco-Ortega et al., 1997;Caujape
2011). Even if B. stacei grows preferentially in relatively stable shady coastal and lowland
places along its distribution area (Catala
´n et al., 2016a), seeds of this annual species could
be also occasionally dispersed through long distances, as inferred from genetic studies
´pez-Alvarez et al., 2012). The fact that all the studied individuals of the Canarian
GOM and LAN populations are morphologically similar to those of the remaining
Mediterranean populations (Catala
´n et al., 2016a) indicates that they belong to the same
species, suggesting that the introduction of the plant in the Canary isles was probably a
very recent one.
Contrastingly, the Balearic populations of B. stacei show similar, or even higher
genetic diversity values in the case of the Majorcan populations (e.g., BONA, Majorca;
Table 1 ), than the mainland Iberian populations. This scenario could be explained
by old colonization events from the mainland followed by insular isolation, which
might have favored the appearance and accumulation of new allelic variants and
genotypes along time (Fig. 2). Also, admixture after multiple colonization’s could
have contributed to this scenario, which has been reported to have occurred in other
postglacial recolonizations in Europe (e.g., Lexer et al., 2010;Krojerova
´& Koubek, 2015). The palaeogeographic configuration of the continental
Balearic Islands could have facilitated the migration of coastal SE Spain and S Spain B.
stacei populations into Minorca and Majorca, and the repeated colonization (and
admixture) of the later island from multiple continental sources (Fig. 4). The
southern Iberian region together with its eastern Iberian range, the Balearic isles
and Provence formed a continuous geological region that split into several microplates
during the Oligocene (Cohen, 1980). In the late Oligocene (30–28 Ma) the Balearic
microplate separated from the eastern proto-Iberian peninsula (Cohen, 1980;
Rosenbaum, Lister & Duboz, 2002) but during the Messinian drought and salinity
asinglelandmass(Gautier et al., 1994) and several land bridges re-established the
connection with the eastern Iberian Peninsula (Lalueza-Fox et al., 2005). Even after
the opening of the Gibraltar strait and the refilling of the Mediterranean basin
(c. 5 Ma), several land bridges were again created during Middle-Upper Pleistocene
that connected the Balearic Gymnesian isles between themselves and between those
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 15/23
islands and the mainland eastern Iberian Peninsula (c. 0.40 Ma; Cuerda, 1975),
favoring the colonization of the islands from mainland plant populations stocks
(Garnatje et al., 2013).
Is there a role for ecogeographical isolation in Brachypodium?
High values of genetic differentiation and a signature of strong genetic structure
were found in B. stacei. Though genetic differentiation values obtained for other selfing
but more outcrossing species of Brachypodium are relatively high (e.g., B. sylvaticum:
0.480 ± 0.28 in native Eurasian populations, and 0.446 ± 0.26 in invasive western
North American populations; Rosenthal, Ramakrishnan & Cruzan, 2008), the high
values of genetic differentiation found in B. stacei are puzzling. Lower genetic
differentiation values than the ones found here usually correspond to different grass
species (e.g., Festuca,
´rez et al., 2008). However, all B. stacei individuals
examined in this study are morphologically similar and correspond to what is
considered to be the same species (Catala
´n et al., 2016a). Population turnover is
expected to increase genetic differentiation among populations if colonizers are
dispersed from different sources (Pannell & Charlesworth, 2000). That might only be
true for B. stacei if wind or other vectors are dispersing seeds across populations, as
hypothesized for the also annual and autogamous B. distachyon (Vogel et al., 2009;Mur
et al., 2011). That would probably erase the patterns of genetic structure that we
have found in STRUCTURE, BAPS, the NJ tree and the PCoA analyses (Figs. 35),
though the high rates of selfing observed could explain the high levels of genetic
differentiation and strong population structure of B. stacei, like reported in other
primarily selfing plants (Nybom, 2004).
Because plants are sessile they experience generations of selection that result in
adaptive genetic differentiation to local environmental conditions if there is a
strong pressure (Kremer, Potts & Delzon, 2014). Although we have no empirical
information for B. stacei, the distribution of the close relatives, B. distachyon and
its allopolyploid derivative B. hybridum, indicates that they are geographically
structured in mesic to arid environments, with B. distachyon occurring predominantly
in more mesic sites and B. hybridum in more aridic sites (Manzaneda et al., 2012).
Brachypodium hybridum is also more efficient in its water usage being significantly
more tolerant to drought than B. distachyon and behaving as a drought-escapist
(Manzaneda et al., 2015). Also, environmental niche model analyses indicate a
preference of B. stacei for warm and arid Mediterranean places (Lo
´pez-Alvarez et al.,
2015), though its habitat preferences are for shady places, probably as a protection
from direct insulation in the aridic environment (Catala
´n et al., 2016a). Therefore,
all together, results suggest an important role for ecogeographical differentiation in
these lineages of Brachypodium (Manzaneda et al., 2012;Manzaneda et al., 2015;
´pez-Alvarez et al., 2015;Catala
´n et al., 2016a;Catala
´n et al., 2016b). More detailed
ecological studies are necessary to understand the potential ecological tolerance of
B. stacei to the arid conditions.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 16/23
Perspectives towards new genomics initiatives in B. stacei
The ongoing de-novo genome sequencing of B. stacei led by the Joint Genome
Institute and the International Brachypodium Consortium (
science/science-programs/plant-genomics/brachypodium/) will provide significant
insights into the mechanisms of polyploid hybrid speciation within the complex
B. distachyonB. staceiB. hybridum, also allowing comparative studies of genomics
and development of functional traits in other crop plants. Biological features, such as a
selfing system, a diploid genome and having amenable growing conditions are all
advantages for the development of a model system and for genomic resources.
All seem to be present in B. stacei. Previous studies showed that the species is
diploid (Catala
´n et al., 2012) and can easily grow even in laboratory conditions,
germinating in less than one week like we have seen in our own laboratory (P. Catala
2016, unpublished data).
The results from the present study support the existence of a highly selfing
system, which from a practical perspective is an advantage in a model species
since it simplifies the process of obtaining pure lines under laboratory conditions
(Gordon et al., 2014). But plant breeding requires the presence of genetic variability
in order to increase the frequencies of favorable alleles and genetic combinations.
Populations from SE Spain are genetically different from the ones in S Spain
and further differentiation might occur in the islands especially in Majorca and
Minorca, where several unique alleles were found. Future studies need to test
if population differentiation reflects local adaptation to different environments.
Nonetheless, researchers of GWA studies need to be careful to avoid reporting
false positive signals (i.e., identifying loci that are not responsible for the variation
in the trait), which can be caused by population structure (Platt, Vilhja
´lmsson &
Nordborg, 2010;Brachi, Morris & Borevitz, 2011). In this sense, several efforts have
been raised to address this problem statistically (Pritchard et al., 2000;Price et al.,
2006;Yu et al., 2006) and recent GWAS can detect loci that are involved
in the natural variation of traits even in highly structure plants like Arabidopsis
(Nordborg et al., 2005).
Thus, to help future genomic initiatives involving B. stacei we recommend the
following guidelines: (1) a collection of different accessions reflecting different ecological
pressures should be generated in order to recover the full genomic diversity of B. stacei;
(2) the creation of a gene bank collection of these materials constitutes a practical and
useful reservoir of genetic variation to avoid uniform cultivars and genetic erosion;
(3) collections should be accessible to facilitate the interchange of material useful for
breeding and other studies. Finally, there is a lack of information for other areas of the
Mediterranean, especially the Eastern Mediterranean–SW Asian area, where B. stacei
has been also found (Lo
´pez-Alvarez et al., 2012;Lo
´pez-Alvarez et al., 2015;Catala
´n et al.,
2016a). A comprehensive study including populations from other Mediterranean
areas is compulsory to fully discover the phylogeographic patterns and genetic diversity
of B. stacei.
Shiposha et al. (2016), PeerJ, DOI 10.7717/peerj.2407 17/23
We thank the Spanish Centro de Recursos Fitogene
´ticos (CRF-INIA), Consuelo Soler and
Antonio Manzaneda for providing us some B. stacei seeds, Maria Luisa Lo
and Diana Lo
´pez-Alvarez for laboratory and greenhouse assistance, and William Scott
for linguistic assistance.
The study has been funded by a Spanish Ministry of Science grant project (CGL2012-
39953-C02-01). IM received funding from the People Programme (Marie Curie Actions)
of the European Union’s Seventh Framework Programme (FP7/2007–2013) under REA
grant agreement PIOF-GA-2011-301257. VS was funded by a Tomsk State University
(TSU, Russia) PhD fellowship. PC and IM were partially funded by a Bioflora grant
cofunded by the Spanish Aragon Government and the European Social Fund. The funders
had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Spanish Ministry of Science grant project: CGL2012-39953-C02-01.
European Unions Seventh Framework Programme: FP7/2007–2013.
REA grant agreement: PIOF-GA-2011-301257.
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Valeriia Shiposha performed the experiments, analyzed the data, wrote the paper,
prepared figures and/or tables.
Pilar Catala
´n conceived and designed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Marina Olonova wrote the paper.
Isabel Marques performed the experiments, analyzed the data, wrote the paper,
prepared figures and/or tables, reviewed drafts of the paper.
Data Deposition
The following information was supplied regarding data availability:
The raw data has been supplied as Supplemental Dataset Files.
Supplemental Information
Supplemental information for this article can be found online at
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... Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The 137 samples used in this study were genotyped at 10 polymorphic nuclear simple sequence repeats (nSSRs) previously developed for B. distachyon (ALB006, ALB022, ALB040, ALB050, ALB086, ALB087, ALB139, ALB165, ALB181 and ALB311; [21]) and following the procedures outlined in [26]. Based on an initial survey, we selected these ten nSSR markers since they produced robust highly polymorphic amplified bands among the entire collection of our B. distachyon samples. ...
... Although these next-generation sequencing (NGS) techniques will probably be predominant in next years, SSRs still have advantages if they are genetically informative, like previously reported in Brachypodium distachyon [21] an in its close annual Mediterranean congeners B. stacei [26] and B. hybridum [27], as well as in the Eurasian perennial B. sylvaticum [28,29]. Also, the number of biases in a SSR study might be much lower than using NGS methods since each locus can be manually genotyped reducing errors [30]. ...
... Flowers of B. distachyon rarely open except under specific environmental conditions (warm, humid and full sun), although even in this case anthers dehisce to the stigmas under the fold of the palea causing primarily self-pollinations [21]. Close-related species, such as the sister species B. stacei, are also primarily selfing plants, though genetic diversity values suggest that it might outcross more often than B. distachyon [26]. For instance, selfing rates of Iberian, Balearic and Canarian B. stacei populations were estimated as 79% [26], which is lower than the ones reported here for Iberian B. distachyon populations. ...
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Background Brachypodium distachyon (Poaceae), an annual Mediterranean Aluminum (Al)-sensitive grass, is currently being used as a model species to provide new information on cereals and biofuel crops. The plant has a short life cycle and one of the smallest genomes in the grasses being well suited to experimental manipulation. Its genome has been fully sequenced and several genomic resources are being developed to elucidate key traits and gene functions. A reliable germplasm collection that reflects the natural diversity of this species is therefore needed for all these genomic resources. However, despite being a model plant, we still know very little about its genetic diversity. As a first step to overcome this gap, we used nuclear Simple Sequence Repeats (nSSR) to study the patterns of genetic diversity and population structure of B. distachyon in 14 populations sampled across the Iberian Peninsula (Spain), one of its best known areas. Results We found very low levels of genetic diversity, allelic number and heterozygosity in B. distachyon, congruent with a highly selfing system. Our results indicate the existence of at least three genetic clusters providing additional evidence for the existence of a significant genetic structure in the Iberian Peninsula and supporting this geographical area as an important genetic reservoir. Several hotspots of genetic diversity were detected and populations growing on basic soils were significantly more diverse than those growing in acidic soils. A partial Mantel test confirmed a statistically significant Isolation-By-Distance (IBD) among all studied populations, as well as a statistically significant Isolation-By-Environment (IBE) revealing the presence of environmental-driven isolation as one explanation for the genetic patterns found in the Iberian Peninsula. Conclusions The finding of higher genetic diversity in eastern Iberian populations occurring in basic soils suggests that these populations can be better adapted than those occurring in western areas of the Iberian Peninsula where the soils are more acidic and accumulate toxic Al ions. This suggests that the western Iberian acidic soils might prevent the establishment of Al-sensitive B. distachyon populations, potentially causing the existence of more genetically depauperated individuals. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0996-x) contains supplementary material, which is available to authorized users.
... Southern Alicante has been confidently identified as the ancestral distribution centre of C. dianius based on the combination of BPEC and DIYABC analysis of plastid DNA data; according to these analyses, the haplotype H7 is probably the most ancestral (Fig. 2), with the southern Alicante phylogeographical group as the root (scenario 1) instead of Ibiza (scenario 2; Fig. 3). Previous studies with similar plant distributions in the eastern Iberian Peninsula and Balearic Islands also identify the mainland as the area of origin (Prentice et al., 2003;Juan et al., 2012;Garnatje et al., 2013;Shiposha et al., 2016). ...
... The active history of multiple colonizations in C. dianius could have meant the erosion of part of its genetic diversity at each colonization step (due to founder effects) as reported in other studies (e.g. Shiposha et al., 2016). In addition, the restricted seed dispersal and the reduced interpopulation gene flow (Table S5) would have fostered the predominance of drift, which has been reported to be one of the main evolutionary forces to explain the high plant diversity in the Mediterranean area (Rosselló, 2013, and references therein). ...
To what extent Pleistocene sea-level fluctuations have affected the genetic diversity of species is one of the current topics in biogeographical research. Carduncellus dianius is a Mediterranean narrow endemic species, restricted to < 20 populations distributed along coastal areas in Alicante (mainland eastern Iberian Peninsula) and on the island of Ibiza (Balearic Islands). To get insights into its evolutionary history and its genetic diversity and structure, we combined the analysis of molecular markers (three plastid DNA regions and AFLP) with ecological niche modelling. Results from dated phylogeographical analyses revealed that this species might have originated in the continental region during the early Pleistocene. The colonization of Ibiza could have occurred by a single long-distance dispersal event, with a subsequent back-colonization from the island to the same continental area of origin. These results corroborate the role of islands as sources for mainland colonization (biodiversity reservoirs) and as refugia during glacial periods. Notably, we detected that populations located on stable landmasses (i.e. not affected by sea rising during interglacial cycles) harboured significantly higher genetic diversity than those that were periodically submerged during the periods of marine transgressions. Our results point out sea-level fluctuations as a factor to be considered in phylogeographical studies focused on species distributed along coastlines.
... These ages are coincident with a warmer and wetter period characteristic of the Upper Tortonian [46]. These changing climate conditions could have caused the origin of diverse environments, promoting the successful diversification of several herbaceous lineages such as the Valerianella sectional ancestors and those of other Mediterranean angiosperms [47]. Evolutionary (Figures 2 and 3) and statistical (Table 3) analyses support the phylogenetic value of the diagnostic carpological traits separating the four monophyletic sections of Valerianella. ...
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Valerianella (cornsalad) is a taxonomically complex genus formed by 50–65 annual Holarctic species classified into at least four main sections. Carpological traits (sizes and shapes of achenes and calyx teeth) have been used to characterize its sections and species. However, the potential systematic value of these traits at different taxonomic ranks (from sections to species (and infraspecific taxa)) has not been tested phylogenetically yet. Here, we have assessed the evolutionary systematic value of Valerianella diagnostic carpological traits at different hierarchical ranks and have demonstrated their ability to separate taxa at the sectional level but not at species level for species of several species pairs. A total of 426 individuals (17 species, 4 sections) of Valerianella were analyzed using AFLP and plastid data. Genetic clusters, phylogenetic trees, and haplotype networks support the taxonomic classification of Valerianella at the four studied sectional levels (V. sects. Valerianella, Cornigerae, Coronatae, Platycoelae) but show admixture for ten taxa from five species pairs (V. locusta—V. carinata, V. coronata—V. pumila, V. multidentata—V. discoidea, V. dentata—V. rimosa, V. eriocarpa—V. microcarpa), which are not reciprocally monophyletic. Dating analyses indicate that the Valerianella sections are relatively old (mid-Miocene), while most species diverged in the Pliocene–Pleistocene. A new section Valerianella sect. Stipitae is described to accommodate the highly divergent and taxonomically distinct V. fusiformis type species. Taxonomic treatments that recognize the sectional ranks and that subsume the separate species of each species pair into single species represent a natural classification for Valerianella.
... They are also a cost-effective method for generating data for a large number of individuals if existing genomic resources such as a transcriptome are available (Ellis & Burke, 2007;Hodel et al., 2016). SSRs have proved informative for population level analysis of other Macaronesian plants, including Argyranthemum (Asteraceae; White et al., 2018), Brachypodium (Poaceae; Shiposha et al., 2016), Bencomia (Rosaceae; Gonz alez-P erez et al., 2009), Sambucus (Sambucaceae; Sosa et al., 2010), Pinus (Pinaceae;Navascu es et al., 2006) and Micromeria (Lamiaceae; Puppo et al., 2016). In this paper, we quantify levels of genetic diversity in E. wildpretii and assess how this diversity is partitioned between islands and populations. ...
The sub-alpine zones of oceanic islands are unique and dynamic ecosystems with high levels of endemism, making them particularly suitable model systems in which to investigate evolutionary and biogeographic processes. The sub-alpine flora of the Canary Islands is restricted to the islands of Tenerife and La Palma. Its origins are poorly understood. Echium wildpretii Hook.f. is an iconic species of the subalpine zones of these two islands, with distinct subspecies recognized on each island. This study examines patterns of genetic and morphological diversity in E. wildpretii to investigate the diversity and evolution of the lineage. Nine microsatellite markers were designed and used to investigate population genetic structure and patterns of gene flow within and between islands and populations. Morphological characters were assessed to test the distinctiveness of the two subspecies recognized. Strong genetic differentiation was observed between islands with higher genetic diversity on the younger island of La Palma than on Tenerife. Very low levels of inter-island gene flow were observed indicating that these taxa are reproductively isolated and evolving independently. Morphological analysis confirmed the distinctiveness of plants from the two islands. Given their genetic and morphological distinctiveness the taxa on Tenerife and La Palma merit recognition as distinct species. Higher genetic diversity in the La Palma species is consistent with an origin of the lineage on this island via upslope colonization, followed by dispersal to Tenerife where the plants show lower genetic diversity.
... The values of H o , H e and h ST here reported for populations of P. dilatatum subsp. flavescens are consistent with previously reported values for other highly autogamous species and the same kind of molecular markers (Baek et al. 2003;Le Corre 2005;Siol et al. 2008;Arraouadi et al. 2009;Volis 2011;Leinonen et al. 2013;Shiposha et al. 2016). A highly structured population is expected for reproductive systems that promote inbreeding, favoring differentiation among populations (Loveless and Hamrick 1984;Ellegren and Galtier 2016;Duminil et al. 2003;Hamrick and Godt 1996). ...
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The current increase in climate variability requires new strategies and sources of germplasm to improve the resilience of sown pastures, among other agricultural systems. For an efficient use of wild germplasm in domestication and breeding it is necessary to achieve a thorough understanding of the structure of the germplasm of potentially useful wild species. Paspalum dilatatum subsp. flavescens Roseng., Arrill. & Izag. is a sexual, autogamous member of the species complex containing the highly valued apomictic forms of P. dilatatum (Dilatata group). In spite of its repeated use in genetic analyses, its genetic variability has never been sufficiently explored. In order to understand the geographical structure of variability and the forces shaping it, a representative group of populations was genotyped with molecular markers and phenotyped in a common garden experiment. A remarkable spatial pattern was observed in the distribution of expected heterozygosity, but genetic distance among populations was generally independent of geographic distance. Most genetic and morphological variability can be attributed to genetic drift and only three of the 12 measured traits showed signs of a possible process of selection. Our data suggest that the genetic diversity and its geographical distribution are probably due to recent geographical range changes. Less diverse peripheral populations contain morphologically differentiated genotypes which can be of great value for breeding.
... In this context, we analyzed plant LTR-RT lineage evolution using a Bayesian population genetic structure approach associated with classical phylogenetic tools to generate a more comprehensive understanding of the evolution and relationships within LTR-RTs lineages. To achieve this aim, we used the STRUCTURE software, which is the most widely used Bayesian tool to identify patterns of population genetic structures, population admixture and hybridization events of natural populations [15][16][17][18][19][20][21]. STRUC-TURE implements a Bayesian model-based clustering method using multilocus genotype data to identify genetic structures by assigning individuals to populations (clusters). ...
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Long terminal repeat retrotransposons (LTR-RTs) in plant genomes differ in abundance, structure and genomic distribution, reflecting the large number of evolutionary lineages. Elements within lineages can be considered populations, in which each element is an individual in its genomic environment. In this way, it would be reasonable to apply microevolutionary analyses to understand transposable element (TE) evolution, such as those used to study the genetic structure of natural populations. Here, we applied a Bayesian method to infer genetic structure of populations together with classical phylogenetic and dating tools to analyze LTR-RT evolution using the monocot Setaria italica as a model species. In contrast to a phylogeny, the Bayesian clusterization method identifies populations by assigning individuals to one or more clusters according to the most probabilistic scenario of admixture, based on genetic diversity patterns. In this work, each LTR-RT insertion was considered to be one individual and each LTR-RT lineage was considered to be a single species. Nine evolutionary lineages of LTR-RTs were identified in the S. italica genome that had different genetic structures with variable numbers of clusters and levels of admixture. Comprehensive analysis of the phylogenetic, clusterization and time of insertion data allowed us to hypothesize that admixed elements represent sequences that harbor ancestral polymorphic sequence signatures. In conclusion, application of microevolutionary concepts in genome evolution studies is suitable as a complementary approach to phylogenetic analyses to address the evolutionary history and functional features of TEs.
... occurs in ephemeral aquatic ponds probably further reduces the probability of gene flow between them. The absence of genetic variation within populations observed in this study is not uncommon in highly autogamous plants (e.g., Shiposha & al., 2016;Marques & al., 2017). ...
The flora of the southwestern Australian biodiversity hotspot is rich in endemic species, many of which remain to be discovered or properly described; estimates of species diversity and levels of endemism should take into account the possible occurrence of cryptic species. Here we explore taxonomic diversity in a Western Australian lineage belonging to the primarily Australian genus Trithuria, the sole genus of Hydatellaceae (Nymphaeales). Recent molecular evidence supports the existence of cryptic species in self-pollinating members of section Trithuria. We investigate Western Australian plants currently classified as T. australis s.l., a self-pollinating member of the section Hydatella. Using evidence from microsatellite data (SSRs), an expanded molecular phylogenetic analysis based on four plastid markers, and fruit micromorphology, we suggest that material traditionally classified as T. australis s.l. belongs to at least four species. Two species occur in the northern part of the distribution range of the group (31° S to 33°27′ S), and two in the southern part (33°27′ S to 35° S). Each northern species has distinctive fruit micromorphology not recorded in other members of the genus. The two southern species are well characterized by molecular characters and seem to be allopatric, but lack obvious morphological differences from each other. We describe one of the northern species as T. fitzgeraldii sp. nov. However, clarifying the names of the other three species is currently problematic as T. australis and another available name are based on collections made 117 years ago, from localities distant from any subsequent records of Hydatellaceae. Based on genome size estimations , we also demonstrate two ploidy levels in the T. australis complex. Our study supports the view that species diversity in Hydatellaceae is strongly underestimated.
Background and aims: It is accepted that contemporary allopolyploid species have originated recurrently, but very few cases have been documented using multiple natural formations of the same species. To extend our knowledge, we have investigated the multiple origins, genetic variation, and structure of the allotetraploid grass Brachypodium hybridum with respect to its progenitor diploid species B. distachyon (D genome) and B. stacei (S genome). For this, our primary focus is the Iberian Peninsula, an evolutionary hotspot for the genus Brachypodium. Methods: We analysed 342 B. hybridum individuals from 36 populations using 10 nuclear SSR loci and two plastid loci. The B. hybridum genetic profiles were compared with those previously reported for B. stacei and B. distachyon. In addition, phylogenetic analysis of the plastid data was performed for a reduced subset of individuals. Key results: The nuclear SSR genetic analysis detected medium to high genetic diversity, with a strong south-to-north genetic structure cline, and a high selfing rate in B. hybridum. Comparative genetic analysis showed a close relatedness of current B. hybridum D allelic profiles with those of B. distachyon, but a lack of similarity with those of B. stacei, suggesting another B. stacei source for the B. hybridum S alleles. Plastid analysis detected three different bidirectional allopolyploidization events: two involved distinct B. distachyon-like ancestors and one involved a B. stacei-like ancestor. The Southeastern (SE) Iberian Peninsula B. hybridum populations were more genetically diverse and could have originated from at least two hybridization events whereas Northeaster-Northwestern (NE-NW) Iberian Peninsula B. hybridum populations were less diverse and may have derived from at least one hybridization event. Conclusions: The genetic and evolutionary evidence support the plausible in situ origin of the SE and northern Iberian Peninsula B. hybridum allopolyploids from their respective local B. distachyon and unknown B. stacei ancestors. The untapped multiple origins and genetic variation detected in these B. hybridum populations opens the way to future evolutionary analysis of allopolyploid formation and genomic dominance and expression in the B. hybridum - B. distachyon - B. stacei grass model complex.
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Matteuccia struthiopteris (L.) Tod. – Ostrich fern (Onocleaceae) is the circumboreal species widely distributed in the temperate zone of the northern hemisphere. In the paper, we presented the results of the observations on the developing population of M. struthiopteris on the man-made lawn situated in the University Grove of Tomsk State University (Tomsk, Russian Federation). In the original making of the lawn, the ground was replaced by new rich soil, which was taken in early summer of 2007 from a native site situated 6.65 km towards south-west from Tomsk University. The formation of the plant community on the new lawn occurred mostly naturally, and sporophytes of this fern appeared naturally. The first sporophytes on the new lawn were detected in 2009, occurring initially in the significant numbers, recorded here. During the time of observation (2009–2018) the number of sporophytes decreased from an original 263 to 96, i.e. 63.5% of sporophytes died. The initial population in 2009 included only juvenile sporophytes. No spore-bearing sporophytes appear during the period of observation. For detection of the source of spores, from which the population on the lawn was formed, we selected three populations having fertile sporophytes, and conducted a molecular-genetic analysis. Two of the analyzed source populations were found to be situated at 160–365 m distance from the developing population and one population occurs near the place where the soil for new lawn making was taken. We used the ISSR method for analyzing the genetic diversity of populations and processed the results by software STRUCTURE Version 2.3.4. The result show, that new population, and population from the soil taking point belong to the same group. The coefficient of Nei’s genetic identity between these populations is high (I = 0.931). This confirms the origin of the new population from spores contained in the soil used for the lawn construction.
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Arlequin ver 3.0 is a software package integrating several basic and advanced methods for population genetics data analysis, like the computation of standard genetic diversity indices, the estimation of allele and haplotype frequencies, tests of departure from linkage equilibrium, departure from selective neutrality and demographic equilibrium, estimation or parameters from past population expansions, and thorough analyses of population subdivision under the AMOVA framework. Arlequin 3 introduces a completely new graphical interface written in C++, a more robust semantic analysis of input files, and two new methods: a Bayesian estimation of gametic phase from multi-locus genotypes, and an estimation of the parameters of an instantaneous spatial expansion from DNA sequence polymorphism. Arlequin can handle several data types like DNA sequences, microsatellite data, or standard multilocus genotypes. A Windows version of the software is freely available on
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We present an updated morphological revision of the three annual species of the genus Brachypodium (Poaceae): B. distachyon, B. stacei, and B. hybridum, which were recently segregated as independent species from the single-species complex B. distachyon s.l. These three species have been proposed as a model system for grass polyploid speciation, and their genomes have been sequenced. However, despite the increasing number of genomic and population-genetic studies conducted for each of these species, no taxonomic updating has been done on them since their original descriptions. B. stacei, the rarest species of the complex, has a protologue based only on the study of specimens from its type locality in Torrent (Formentera, Spain). In this study we update the taxonomic descriptions of the three species using morphoanatomical data from specimens collected throughout their respective native circum-Mediterranean distributions as well as in other localities where they are non-autochthonous. We also provide icons for each species and information about their habitat preferences and geographic distributions.
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The small, annual grass Brachypodium distachyon (L.) Beauv., a close relative of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), is a powerful model system for cereals and bioenergy grasses. Genome-wide association studies (GWAS) of natural variation can elucidate the genetic basis of complex traits but have been so far limited in B. distachyon by the lack of large numbers of well-characterized and sufficiently diverse accessions. Here, we report on genotyping-by-sequencing (GBS) of 84 B. distachyon, seven B. hybridum, and three B. stacei accessions with diverse geographic origins including Albania, Armenia, Georgia, Italy, Spain, and Turkey. Over 90,000 high-quality single-nucleotide polymorphisms (SNPs) distributed across the Bd21 reference genome were identified. Our results confirm the hybrid nature of the B. hybridum genome, which appears as a mosaic of B. distachyon-like and B. stacei-like sequences. Analysis of more than 50,000 SNPs for the B. distachyon accessions revealed three distinct, genetically defined populations. Surprisingly, these genomic profiles are associated with differences in flowering time rather than with broad geographic origin. High levels of differentiation in loci associated with floral development support the differences in flowering phenology between B. distachyon populations. Genome-wide association studies combining genotypic and phenotypic data also suggest the presence of one or more photoperiodism, circadian clock, and vernalization genes in loci associated with flowering time variation within B. distachyon populations. Our characterization elucidates genes underlying population differences, expands the germplasm resources available for Brachypodium, and illustrates the feasibility and limitations of GWAS in this model grass.
A new model for grass functional genomics is described based on Brachypodium distachyon, which in the evolution of the Pooideae diverged just prior to the clade of "core pooid" genera that contain the majority of important temperate cereals and forage grasses. Diploid ecotypes of B. distachyon (2n = 10) have five easily distinguishable chromosomes that display high levels of chiasma formation at meiosis. The B. distachyon nuclear genome was indistinguishable in size from that of Arabidopsis, making it the simplest genome described in grasses to date. B. distachyon is a self-fertile, inbreeding annual with a life cycle of less than 4 months. These features, coupled with its small size (approximately 20 cm at maturity), lack of seed-head shatter, and undemanding growth requirements should make it amenable to high-throughput genetics and mutant screens. Immature embryos exhibited a high capacity for plant regeneration via somatic embryogenesis. Regenerated plants display very low levels of albinism and have normal fertility. A simple transformation system has been developed based on microprojectile bombardment of embryogenic callus and hygromycin selection. Selected B. distachyon ecotypes were resistant to all tested cereal-adapted Blumeria graminis species and cereal brown rusts (Puccinia reconditia). In contrast, different ecotypes displayed resistance or disease symptoms following challenge with the rice blast pathogen (Magnaporthe grisea) and wheat/barley yellow stripe rusts (Puccinia striformis). Despite its small stature, B. distachyon has large seeds that should prove useful for studies on grain filling. Such biological characteristics represent important traits for study in temperate cereals.
In a mixed-mating population, the fitness of selfed individuals, relative to outcrossed individuals, can be estimated from observed changes of the inbreeding coefficient F. In general, three measurements of F, or two measurements of F and one of the selfing rate, spanning two generations are needed. If however, adult F is assumed constant among generations, the adult F and selfing rate of one generation are sufficient to estimate relative fitness. Estimates of the relative fitness of selfed individuals for 14 Mimulus populations, assuming equilibrium of adult F, averaged 0.38, which is significantly lower than the 0.50 threshold needed to favor selfing genotypes. However, estimates for some populations showed large variance and over the 14 populations, relative fitness did not correlate with selfing rate. Violations of the equilibrium assumption causes a positive bias of the estimate and a spurious negative correlation of estimates with selfing rate. Estimates of relative fitness based on two generations of F do not suffer these problems. The need for large sample sizes, multiallelic loci, and moderate levels of natural selfing limits the usefulness of using changes of F to infer inbreeding depression.
We present an updated review of the phylogenetic and evolutionary studies conducted on the model genus Brachypodium. The genus, which contains approximately 20 globally distributed taxa (17 species, 1 variety, and 2 undescribed cytotypes) shows an intermediate evolutionary placement within the grass temperate pooid clade, being closer to the basal than to the recent Pooideae lineages. Our comprehensive molecular phylogenetic survey of all the currently known Brachypodium lineages illustrates a complex reticulate scenario of recently evolved diploid and allopolyploid lineages. Haplotypic statistical parsimony networks, multilabelled (multigenic) Minimum Evolution gene tree discordances, and Bayesian dating analysis have provided a testable hypothesis for the reconstruction of the Brachypodium species tree and for the estimation of its nodal divergence times. Our results support the early splits of the annual and short-rhizomatose lineages (B. stacei, B. mexicanum, B. distachyon) in the Holarctic region during the early-Middle Miocene (and B. hybridum in the Pleistocene), and a profusion of rapid splits for the perennial lineages since the late Miocene to the Pleistocene in the Mediterranean and Eurasian regions, with sporadic colonizations of more remote areas. Several perennial allopolyploid species (B. boissieri, B. retusum, B. phoenicoides, B. rupestre 4x, B. pinnatum 4x) showed homeologous copies from both ancestral and recent genome donors. More in-depth studies of the species of the B. distachyon complex have demonstrated the polyphyletic origin of the allotetraploid B. hybridum from bidirectional crosses of its diploid B. stacei and B. distachyon parents. Our niche modeling analysis has also detected distinct adaptations to different ecological tolerances in the diploids and evidence of niche conservatism for B. hybridum and each of its parents in their native Mediterranean region. Future perspectives include ongoing comparative genomics, phylogenomic and genotype-based phylogeographic studies of Brachypodium.
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from