ORIGINAL ARTICLE

Genome size in Filago L. (Asteraceae, Gnaphalieae) and relatedgenera: phylogenetic, evolutionary and ecological implications

Santiago Andres-Sanchez • Eva M. Temsch •

Enrique Rico • M. Montserrat Martınez-Ortega

Received: 30 July 2012 / Accepted: 15 October 2012 / Published online: 18 November 2012

� Springer-Verlag Wien 2012

Abstract Recent studies have proposed a monophyletic

circumscription of Filago and a new subgeneric treatment

for this genus. The aim of this study was to analyse the

nuclear genome size in a phylogenetic framework in order

to evaluate the systematic significance of this trait to pro-

vide insights into the dynamics of genome size evolution

and to assess relationships among DNA content, specific

life and ecological features within the study group. A

holoploid genome size of 76 samples corresponding to 27

taxa was determined using flow cytometry, which repre-

sents the first estimates of genome size in Bombycilaena,

Filago, Ifloga and Logfia. Chromosome counts were per-

formed for six species. Parsimony and Bayesian analysis of

ITS, ETS and rpl32-trnL intergenic spacer sequence data

were used to construct molecular phylogenetic trees. The

evolution of genome size was investigated troughout the

Brownian motion model with the three scaling parameters

k, j and d. The mean 2C-value in the Filago group is

relatively low (1.3644 ± 0.0079 pg) and homogeneous

among species. A high degree of congruence was found

between genome size distribution and the major phyloge-

netic lineages obtained. The generally accepted assumption

that annual, ephemeral and autogamous species show low

genome sizes was confirmed. Also the relatively high DNA

contents found for a couple of species could be correlated

with their highly specific ecological requirements. Phy-

logeny seems to represent the most important factor

explaining the pattern of DNA amount variation in the

Filago group. The DNA amount does not seem to be

strongly influenced by selection.

Keywords Asteraceae � Filago � Flow cytometry �Genome size evolution � Logfia � Phylogeny

Introduction

The phylogenetic, evolutionary and ecological significance

of nuclear DNA amount variation in angiosperms is still

not well understood (e.g., Kellogg 1998; Albach and

Greilhuber 2004; Leitch et al. 2005; Garnatje et al. 2007;

Bennett and Leitch 2010a). DNA C-values (the terms

‘C-value’ and ‘genome size’ are used here in the sense of

Greilhuber et al. 2005) have been estimated for only ca.

6,287 angiosperm species. Within the most species-rich

family of the flowering plants (Asteraceae), DNA C-values

have been studied for ca. 3 % (ca. 680) of the species

(Bennett and Leitch 2010b). Despite the availability of

phylogenetic hypotheses for several genera of the Astera-

ceae and of methods for analysing possible connections

between DNA amount and phylogeny (Harvey and Pagel

1991; Pagel and Meade 2007), this limited coverage has

restricted rigorous testing of genome size evolution within

them (e.g., Chrtek et al. 2009; Duskova et al. 2010). Data

on genome size variation together with those coming from

phylogenetic studies can contribute to a better under-

standing of the systematics and evolutionary relationships

within narrow groups of plants as well as aid in delimiting

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00606-012-0724-3) contains supplementarymaterial, which is available to authorized users.

S. Andres-Sanchez (&) � E. Rico �M. Montserrat Martınez-Ortega

Departamento de Botanica, Facultad de Biologıa,

Universidad de Salamanca, 37007 Salamanca, Spain

e-mail: santiandres@usal.es

E. M. Temsch

Department of Systematic and Evolutionary Botany,

University of Vienna, 1030 Vienna, Austria

123

Plant Syst Evol (2013) 299:331–345

DOI 10.1007/s00606-012-0724-3

taxa (Ohri 1998; Bottini et al. 2000; Mishiba et al. 2000;

Zonneveld 2001; Jakob et al. 2004; Zavesky et al. 2005;

Suda et al. 2007; Chrtek et al. 2009; Zonneveld and Dun-

can 2010; Salabert de Campos et al. 2011). Also the DNA

amount can be used as a taxonomic marker in order to

discriminate among related taxa with the same number of

chromosomes but with different genome sizes (Ohri 1998;

Greilhuber et al. 2007).

Although some variation in genome size has been

sometimes found within species, it seems that in most cases

it is caused by several sources of artefactual variation such

as instrumental or methodological errors (Greilhuber and

Obermayer 1997; Greilhuber 1998, 2005), interference of

secondary metabolites (Greilhuber 1986, 1988; Walker

et al. 2006), differences in measurements technique

between different laboratories (Dolezel et al. 1998) and

taxonomic heterogeneity of the material under investiga-

tion (Murray 2005). Thus, the principle of stable genome

size within species remains generally accepted nowadays

(Ohri 1998; Gregory 2001; Dolezel et al. 2007), although

in some cases different DNA contents have been found

within a particular species that are probably due to eco-

logical variation (e.g., Chooi 1971; Jakob et al. 2004;

Baack et al. 2005; Walker et al. 2006; Marhold et al. 2010).

The large differences in DNA content (ca. 2,535-fold)

observed among angiosperms [2C-values ranging from

0.13 pg in Genlisea aurea A.St.-Hil—Lentibulariaceae—to

304.40 pg in Paris japonica Franch—Melanthiaceae sensu

APG III (2009)—according to Bennett and Leitch (2010a)]

can be caused by several mechanisms. Polyploidization

(not present in the study group) and segmental duplication,

including unequal recombination and non-reciprocal

translocation (Bennetzen 2002), differences in intron size

(Petrov 2001) and amplification of transposable elements

(SanMiguel et al. 1998; Kalendar et al. 2000; Bennetzen

2002) have been suggested to contribute to an increase in

genome size. A decrease in DNA content could be caused

by a loss of whole or partial genes after polyploidization,

unequal crossing over, unequal intrastrand recombination,

a higher overall rate of deletions over insertions or selec-

tion against transposable elements (Comeron 2001; Devos

et al. 2002; Petrov 2002; Wendel et al. 2002; Ma et al.

2004; Bennetzen et al. 2005; Morgan 2001). This signifi-

cant diversity in genome size observed among the angio-

sperms shows no relationship with organismal complexity

and is also independent of chromosome number. Gregory

(2001) summarised the theories that have been proposed to

explain this ‘C-value enigma’ (or ‘C-value paradox’,

Thomas 1971). The nucleotypic theory (Bennett 1971)

postulates a causal link between cell volume and bulk DNA

content. Cell proliferation seems to be faster in plants with

lower genome sizes (e.g., Bennetzen and Kellogg 1997)

and consequently larger genomes and larger cells would

lead to slower growth rates and vice versa, which has

important ecological and evolutionary effects.

The correlation between DNA amount and life form and

breeding system has been widely studied, and it is gener-

ally accepted that annuals, ephemerals and autogamous

species present lower genome sizes than perennials and/or

outcrossers (e.g., Bennett 1972; Leitch and Bennett 2007),

a principle that has been repeatedly confirmed in groups of

related species (e.g., Albach and Greilhuber 2004; Labani

and Elkington 1987; Rayburn and Auger 1990; Torrell and

Valles 2001; Bancheva and Greilhuber 2006).

Less clear are the relationships between cell DNA

content and ecological factors (Knight et al. 2005). The

relationship between genome size and geographical distri-

bution (i.e., latitude) has been documented in several

genera or groups of plants (e.g., crop plants, Bennett 1976;

British flora, Grime and Mowforth 1982). It seems that

genome size may be influenced by temperature (Bennett

1972), altitude (Bennett 1972, Rayburn and Auger 1990, in

crops; Cerbah et al. 1999, in Hypochaeris L.; Temsch and

Greilhuber 2001, in Arachis duranensis Krapov & W.

C. Greg.; Torrell and Valles 2001, in Artemisia L.; or

Bancheva and Greilhuber 2006, in Centaurea L.) and

several sources of stress such as aridity (Torrell and Valles

2001). Associations between DNA content and insularity

(Suda et al. 2003) or endemicity (Vinogradov 2003) have

also been documented. Although some of these studies give

contradictory results, much of the confusion may be due to

the fact that generally these studies do not cover the full

ecological ranges possible, use small sample sizes or the

data are analysed by linear regression and the relationships

may be non-linear (Leitch and Bennett 2007). These

shortcomings have been partially overcome by the studies

developed by Knight et al. (2002, 2005), who analysed

DNA amounts in a high number of species looking at

ecological conditions across a wide environmental gradi-

ent. All of these studies—some of them still preliminary—

highlight the trend that species with large genome sizes are

constrained in their life cycle, life strategies and ecological

conditions (Knight et al. 2005; Leitch and Bennett 2007).

Filago L. and related genera (i.e., Filago group sensu

Anderberg 1991) are annual, ephemeral Asteraceae (Gna-

phalieae) that are widely distributed in the Northern

Hemisphere with the largest number of species in the

Mediterranean region. The evident instability in the generic

and subgeneric classification of the Filago group reflects

the general scarcity of morphological characters tradition-

ally considered relevant for classifying the group, and

possibly some degree of homoplasy. Thus, there were

apparently not enough morphological characters to provide

a satisfactory taxonomic treatment for the group. This

mainly affects the generic boundaries and circumscription

within the Filago group, but also the infrageneric

332 S. Andres-Sanchez et al.

123

classification of Filago itself. Recently, based on the first

phylogenetic analysis of the Filago group derived from

DNA sequence data (Galbany-Casals et al. 2010), a gen-

eric and infrageneric rearrangement has been proposed

(Galbany-Casals et al. 2010; Andres-Sanchez et al. 2011)

together with a set of morphological characters useful for

classification. In this new taxonomic treatment, Filago is

considered independent from Logfia Cass. and enlarged to

include Evax Gaertn. Filago is divided into four subgen-

era [Filago subgen. Filago, Filago subgen. Oglifa (Cass.)

Gren., Filago subgen. Crocidion Andres-Sanchez & Gal-

bany, Filago subgen. Pseudevax (DC.) Andres-Sanchez &

Galbany] and also includes two traditionally monotypic

genera—Cymbolaena Smoljan. and Evacidium Pomel—

that had never been included in Filago. This recent

molecular phylogenetic study suggests two major lineages

within the Filago group: one is composed of the Ameri-

can species plus the Old World species of Logfia except

for Logfia arvensis (L.) J. Holub (now Filago arvensis L.),

and the second lineage comprises Old World species of

Filago, Micropus L. and Bombycilaena (DC.) Smoljan.

plus the traditional monotypic genera Evacidium and

Cymbolaena, plus F. arvensis. In the present study esti-

mates of C-values are provided for accessions belonging

to these two well-supported clades with an emphasis on

the second one. Most of the species included in these

clades grow in open, often disturbed, dry habitats. Some

species show a high degree of habitat specificity, such as

Filago hispanica (Degen & Hervier ex Pau) Chrtek &

Holub, a species restricted to small snow beds at altitudes

higher than 1,500 m or Filago mareotica Delile that

grows exclusively in salt marshes. The relatively low

specificity of habitats, the apparent low diversity of

breeding systems and a homogeneous life form within the

study group allows us to test whether the adaptation to

highly specific ecological conditions can lead to changes

in genome size. The selected study group is also suitable

to test the hypothesis that DNA amount can be used as a

taxonomic marker to differentiate among phylogenetically

related taxa sharing the same number of chromosomes

(mostly 2n = 28; Table 1).

In this article the evolution of 2C-DNA value is evalu-

ated in a representative sample of the Filago group with

respect to a phylogenetic hypothesis derived from nuclear

and plastid DNA sequence data. Flow cytometry was used

for the estimation of the nuclear DNA amount. For all

accessions investigated, the ploidy level was determined.

The distribution of 2C-values is used to evaluate existing

taxonomic concepts. The mode and tempo of genome size

evolution were studied by evaluating the Brownian motion

(BM) model. The ancestral character states for the genome

size were inferred from a model whose parameters have

been optimised using maximum parsimony. Furthermore,

relationships among genome size, life and ecological traits

are also discussed.

Materials and methods

Plant material

Seventy-six samples were grown from seeds from 27 taxa

included in the two major lineages that conform to the

Filago group (Table 1): 22 species or subspecies repre-

senting all four subgenera in Filago (16 of the 34 species of

Filago subgenus Filago, the two species of F. subgen.

Crocidion, the two species of F. subgen. Pseudevax and the

two species of F. subgen. Oglifa), one of the two species of

Bombycilaena, three of the four taxa included in Logfia and

Ifloga spicata (Forskal) Schultz Bip., which is a native to

the Mediterranean area. Micropus and the North American

representatives of the group were not included in this study

because no mature seeds were available. All seeds were

collected in the field and grown in either pots at the

experimental greenhouse of the HBV (Hortus Botanicus

Vindobonensis) of the University of Vienna or in petri

dishes at the laboratory of the Department of Botany of the

University of Salamanca. Vouchers are kept at the Her-

barium of the University of Salamanca (SALA). The gen-

era, subgenera and species concepts follow Galbany-Casals

et al. (2010) and Andres-Sanchez et al. (2011).

Genome size estimation and C-value statistics

Genome size was determined by flow cytometry. Approx-

imately 25 mg of young fresh leaf material from seedlings

of the analysed sample was co-chopped with a sharp razor

blade in 1.1 ml of ice-cold Otto’s I buffer (Otto 1990) as a

nuclear isolating solution together with upper stem leaves

of Solanum pseudocapsicum L. (1C = 1.2946 pg, Temsch

et al. 2010) for internal standardisation in petri dishes.

Solanum pseudocapsicum was chosen as the standard in

order to avoid instrumental problems with linearity because

it is different in genome size in comparison with the

members of the Filago group, but not too different

(Greilhuber et al. 2007). The resulting nuclei suspension

was filtered through a 30-lm nylon mesh. Then 50 ll

RNase (final concentration 0.15 mg/ml of nuclei suspen-

sion) were added and the solution was incubated for

45 min in a water bath at 37 �C. After digestion, 2 ml of

propidium iodide solution (PI in Otto’s buffer II, 60 lg/ml)

was added to the suspension to stain the nuclei and was

incubated for at least 1 h at 7 �C in the dark. A CyFlow

ML (Partec, Munster, Germany) flow cytometer equipped

with a green laser (100 mW, 532 nm, Cobolt Samba;

Cobolt AB, Stockholm, Sweden) and the appropriate filter

Genome size evolution in the Filago group 333

123

combination for PI were used for measurement of the

particle fluorescence emission.

Two additional analyses were conducted to evaluate

relative differences in DNA content among genera within

the Filago group and among species within Filago. In order

to look for differences among genera and among

subgenera, young leaves of F. mareotica and Logfia gallica

(L.) Coss. & Germ. on the one hand and of F. mareotica

(the species shows one of the highest DNA amounts within

the genus) and Filago crocidion (Pomel) Chrtek & Holub

(lowest DNA amount values) on the other were chopped

together and both samples run as described.

Table 1 Information on genome size

Taxon 2n 2C DNA

amount (pg)

CV of sample

(%)

Tukey’s

grouping

species

Tukey’s

grouping

genera

Tukey’s

grouping

subgenera

Filago L. A

Subgenus Filago A

Filago mareotica Delile c. 28* 1.6099 ± 0.0028 3.4900 ± 0.5311 B

Filago lutescens subsp. atlantica Wagenitz 1.4762 ± 0.0180 3.2500 ± 0.6425 C

Filago desertorum Pomel 28 1.4696 ± 0.0081 3.5666 ± 0.5450 CD

Filago argentea (Pomel) Chrtek & Holub c. 28* 1.4496 ± 0.0188 4.2133 ± 0.5700 CDE

Filago micropodioides Lange c. 28* 1.4264 ± 0.0073 3.4833 ± 0.2685 DEF

Filago carpetana (Lange) Chrtek & Holub 1.4192 ± 0.0109 2.9766 ± 0.1331 EFG

Filago vulgaris Lam. 28 1.4104 ± 0.0034 2.9300 ± 0.4657 EFGH

Filago congesta Guss. ex Coss. 1.3970 ± 0.0073 3.7466 ± 0.9867 FGH

Filago lutescens Jordan subsp. lutescens 28 1.3852 ± 0.0074 3.1266 ± 0.4006 FGHI

Filago fuscescens Pomel 1.3737 ± 0.0106 3.5800 ± 0.6489 GHIJ

Filago duriaei Lange 1.3644 ± 0.0119 3.4033 ± 0.4554 HIJ

Filago lusitanica (Samp.) P. Silva 1.3458 ± 0.0314 3.8400 ± 1.1056 IJK

Filago gaditana (Pau) Andres-Sanchez & Galbany 26 1.3374 ± 0.0023 3.3236 ± 0.5589 JKL

Filago pyramidata L. 28 1.3397 ± 0.0287 4.4225 ± 0.6264 JKL

Filago pygmaea L. 26–28 1.3330 ± 0.0097 3.6900 ± 0.7637 JKL

Filago ramosissima Lange 1.2950 ± 0.0112 3.1966 ± 0.3709 LMN

Subgenus Crocidion Andres-Sanchez & Galbany B

Filago nevadensis (Boiss.) Wagenitz & Greuter 1.2659 ± 0.0168 3.2600 ± 0.6279 MN

Filago crocidion (Pomel) Chrtek & Holub 1.2598 ± 0.0221 3.6166 ± 0.5132 N

Subgenus Pseudevax (DC.) Andres-Sanchez & Galbany A

Filago hispanica (Degen & Hervier ex Pau) Chrtek &

Holub

1.7002 ± 0.0036 3.1850 ± 0.2757 A

Filago discolor (Guss. ex DC.) Andres-Sanchez &

Galbany

28* 1.3442 ± 0.0068 2.8200 ± 0.0100 IJK

Subgenus Oglifa (Cass.) Gren. A

Filago griffithii (A. Gray) Andres-Sanchez & Galbany 1.4901 ± 0.0218 3.7633 ± 0.3365 C

Filago arvensis L. 28 1.3092 ± 0.0095 3.2333 ± 0.4964 KLM

Bombycilaena (L.) Smoljan. A

Bombycilaena discolor (Pers.) Laınz 28, c.

28*

1.4692 ± 0.0050 4.0200 ± 0.5154 CD

Logfia Cass. B

Logfia clementei (Willk.) Holub c. 28* 1.1578 ± 0.0172 3.8266 ± 0.7938 OP

Logfia minima (Sm.) Dumort. 28 1.1379 ± 0.0091 3.7433 ± 0.1096 OP

Logfia gallica (L.) Coss. & Germ. 28 1.1103 ± 0.0062 3.7966 ± 0.3910 P

Ifloga Cass. B

Ifloga spicata (Forskal) Schultz Bitp. 14 1.1611 ± 0.0221 4.2700 ± 1.0392 O

Chromosome numbers (2n) (* indicates the new estimations), 2C DNA amount in pg. and standard deviation, coefficients of variance (CV) with

standard deviation and Tukey’s grouping for species, genera and subgenera for each taxa. The classification of the Filago group follows Galbany-

Casals et al. (2010)

334 S. Andres-Sanchez et al.

123

For each taxon three individuals were analysed on dif-

ferent days and generally in different weeks, except for

Bombycilaena discolor (Pers.) Laınz, Filago lutescens

Jordan subsp. lutescens and Filago pyramidata L., where

four individuals were measured because the coefficients of

variation of G0/G1 peaks (CV) were relatively high and

F. hispanica because only two individuals were available.

Five thousand particles were measured per run and 3–5

runs per isolate (4 or 5 instead of 3 runs on the samples

with CVs between 3 and 5 %, depending on the availability

of material).

To calculate the 2C-value, the ratio sample/standard

G1-peak was multiplied by the 2C-value of the standard,

assuming a linear relationship between the measured mean

fluorescence intensities of the standard and samples. Means

and standard deviations were calculated for each taxon.

Several ANOVAs were applied to test for differences in

DNA content among species, subgenera and genera. In

conjunction with the ANOVAs the single-step multiple

comparison Tukey’s range test was used to find which

means are significantly different from one another (Tukey

1953). All statistical analyses were carried out with XLS-

ATS (Addinsoft 2009).

Estimation of chromosome numbers

An extensive literature survey confirmed that the number

of chromosomes remained unknown for many of the spe-

cies included in this study. Thus, we have tried to estimate

this parameter for all of them and have also tried to con-

firm previously published chromosome numbers. Several

fixation and staining methods were used, but only partially

successful results were obtained with the method

explained below. The exact chromosome number was very

difficult to count, but our results were always sufficient to

confirm the ploidy level of the populations. All chromo-

some number estimates were conducted using seeds col-

lected in the field from the same population analysed by

flow cytometry and germinated in the laboratory in petri

dishes. Root and shoot tips were fixed in Carnoy0s solution

(3 absolute ethanol:1 glacial acetic acid) and stored at 4 �C

until use. For chromosome counts the fixed material was

stained in 2 % acetic orcein (La Cour 1954). All squash

preparations were made in a drop of 45 % acetic acid.

Chromosome numbers were analysed under light micros-

copy (Nikon HFX-II type 115).

DNA sequence analysis

A phylogenetic hypothesis for the Filago group based on

one chloroplast DNA (rpl32-trnL intergenic spacer) and

two nuclear DNA regions (ITS, ETS) was obtained by

extracting the taxa included in this study from the data set

used in Galbany-Casals et al. (2010). Ifloga spicata was

used as an outgroup in both phylogenetic reconstructions.

Nucleotide sequences were edited using Chromas v.2.0

(Technelysium Pty. Ltd., Tewantin, Australia) and Bioedit

v.7.0.1 (Hall 1999), and aligned with the program Clu-

stalX v.2.0.10 (Thompson et al. 1997) with subsequent

visual inspection and manual revision. Ambiguous regions

in alignments were removed using Gblocks v.0.91 (Cas-

tresana 2000; Talavera and Castresana 2007) with relaxed

conditions in order to preserve as much information as

possible: ‘‘Minimum Number Of Sequences For A Con-

served Position’’ and ‘‘Minimum Number Of Sequences

For A Flank Position’’ were half the number of sequences,

‘‘Minimum Number Of Contiguous Nonconserved Posi-

tions’’ was 5, ‘‘Maximum Number Of Contiguous Non-

conserved Positions’’ was 10, ‘‘Minimum Length Of A

Block’’ was 5, and ‘‘Allowed Gap Positions’’ was ‘‘With

Half’’. The data matrix is available on request from the

corresponding author and the EMBL accession numbers

are included in Appendix 1. Maximum parsimony analy-

ses (MP) involved heuristic searches conducted with

PAUP* v.4.0b10 (Swofford 2002) using ‘‘tree bisection

reconnection’’ (TBR) branch swapping with character

states specified as unordered and unweighted. The indels

were coded as missing data. To locate other potential

islands of MP trees (Maddison 1991), 1,000 replications

were performed with random taxon addition and also with

TBR branch swapping. Bootstrap analyses (Felsenstein

1985) were conducted with TNT 1.1 (Goloboff et al.

2003) and were performed with 10,000 replicates, random

taxon addition and using TBR branch swapping. Bootstrap

support (BS) values are shown for nodes with

BS C 60 %. For the MP analyses, the consistency index

(CI) and retention index (RI) were calculated excluding

uninformative characters.

Bayesian inference (BI) estimation was calculated using

MrBayes v.3.1.2 (Huelsenbeck and Ronquist 2001; Ron-

quist and Huelsenbeck 2003). The best fitting model of

molecular evolution was determined using the Akaike

information criterion (Akaike 1974; TIM3 ? G; AIC

value = 0.2920) as implemented in the software jModel-

Test 0.1.1. (Posada 2008). Two simultaneous and inde-

pendent parallel runs were performed; for each analysis

four Markov Monte Carlo chains were run simultaneously

starting from random trees. Each analysis was run for 2

million generations, sampling one out of every 200 gen-

erations. The first 4,000 trees (burn-in) of each analysis

were discarded to avoid trees that might have been sampled

prior to the convergence of the Markov chains before

computing the majority-rule consensus tree. Posterior

probability support (PP) was estimated to be significant for

nodes with PP C 0.95.

Genome size evolution in the Filago group 335

123

Evolution of genome size

In order to evaluate the phylogenetic and evolutionary

significance of genome size in the Filago group, the data

on DNA content were coded for all taxa and mapped on the

phylogram resulting from the Bayesian analysis (branch

length information included) using unordered MP as

implemented in Mesquite 2.74 for continuous characters

(Maddison and Maddison 2010).

The evolution of genome size was investigated in the

Filago group by evaluating the Brownian motion (BM)

model as implemented in the GEIGER package (Harmon

et al. 2009) of the software R. Three scaling parameters

were estimated to characterise the tempo of genome size

evolution: (1) lambda (k), which detects whether the shared

evolutionary histories as specified by the phylogeny pro-

duce the patterns of similarity observed in the data (Pagel

1999); k takes the value 1 when the trait was evolved

according to the tree topology; (2) kappa (j), which tests

for a punctual versus gradual mode of trait evolution;

j[ 0 suggests gradualism to some extent, while j = 0

means that trait evolution is independent of the length of a

branch (i.e., punctual mode) (Pagel 1999); (3) delta (d),

which scales the total path lengths in the tree and is

therefore used to test for adaptive radiation; d\ 1 suggests

that the evolution of a trait is disproportionately influenced

by earlier evolution in the phylogeny (shorter paths) and

indicates adaptive radiation, while d[ 1 indicates accel-

erated evolution because trait evolution is more influenced

by longer paths (Pagel 1997, 1999). The four models (BM,

BM ? k, BM ? j, BM ? d) were evaluated in 1,000 trees

sub-sampled from the Bayesian analysis including branch

length values according to Escudero et al. (2010). For this

analysis genome sizes for each taxon were also considered

as mean 2C-values.

Results

2C-values. Estimates of DNA content

Flow cytometry analysis gave high-resolution histograms

with CVs of G0/G1 peaks lower that 5 % for all samples

(Table 1), ranging from 2.8200 ± 0.0100 in Filago dis-

color (Guss. ex DC.) Andres-Sanchez & Galbany to

4.4225 ± 0.6264 in F. pyramidata. The average CV-value

in the standard (S. pseudocapsicum) was 2.86 ± 0.6499,

ranging from 1.67 to 4.86.

DNA content variation was assessed in the 27 species

included in this study. Variation within accessions (popula-

tions) was markedly low (low standard errors; Table 1).

Mean values with standard errors of 2C-values for each

species are summarised in Table 1 and representative

histograms are shown in Fig. 1. The genome size in the

Filago group is relatively low (mean value 1.3644 ±

0.0079 pg) and homogeneous among species. It differed up

to 1.53-fold between F. hispanica (1.7002 ± 0.0036 pg)

and L. gallica (1.1103 ± 0.0062 pg).

Within the genus Filago the average 2C-value is

1.4001 ± 0.1055 pg, ranging from 1.2598 ± 0.0221 pg in

F. crocidion to 1.7002 ± 0.0036 pg in F. hispanica. The

2C-value varied from 1.1103 ± 0.0062 pg in L. gallica to

1.1578 ± 0.0172 pg in Logfia clementei (Willk.) J. Holub

with a mean value of 1.1353 ± 0.0238 pg. The only spe-

cies of Bombycilaena (B. discolor) and Ifloga (I. spicata)

included in this study showed respectively average 2C-

values of 1.4692 ± 0.0050 pg and 1.1611 ± 0.0221 pg.

The analysis of variance (ANOVA) found significant

differences among species (F = 241.308; P \ 0.0001),

among genera (F = 29.158; P \ 0.0001) and among sub-

genera within Filago (F = 6.554; P \ 0.001). Two clear

peaks were found in the running of F. crocidion and

F. mareotica (Fig. 1h) and also in the running of the latter

species together with L. gallica (Fig. 1i).

The Tukey’s range test revealed 16 groups and highly

significant differences (P \ 0.0001) in ca. 80 % of the 350

possible combinations of species pairs (data not shown),

although some species can be ascribed to more than one

group. Two groups were found among the four studied

genera (Table 1), one composed of Bombycilaena and

Filago and the other one made up of Logfia and Ifloga.

Also two groups (Table 1) are found when the four sub-

genera within Filago are considered; one corresponds to

F. subgen. Crocidion and the other three subgenera are

included by this test in the same group.

Estimation of chromosome numbers

We have tried to count the number of chromosomes for all

the species included in this study. Although several

methods were assayed, only that of La Cour (1954) allowed

us to estimate it in six cases (i.e., Filago argentea (Pomel)

Chrtek & Holub (Online Resource 1a), F. discolor (Online

Resource 1b), F mareotica (Online Resource 1c), Filago

micropodioides Lange (Online Resource 1d), L. clementei

(Online Resource 1e) and B. discolor (Online Resource 1f);

all of them were 2n = 2x = ca. 28).

DNA sequence analysis

The analysis of rpl32-trnL intergenic spacer, ITS and ETS

regions exclusively for the species included in this study

were extracted from the data set used in Galbany-Casals

et al. (2010). Although some degree of incongruence was

found among these DNA regions, they were combined in

our analyses in order to preserve the information available

336 S. Andres-Sanchez et al.

123

about the hypothetical hybridisation events involved in the

origin of the Filago group (Smissen et al. 2011). On the

whole our analyses included 2,167 characters, of which

237 are variable and 189 potentially parsimony informa-

tive. The resulting consistency index (CI) is 0.75 and the

retention index (RI) is 0.85. Phylogenetic relationships

inferred from MP analysis (50 % majority rule consensus

of the six MP trees) are almost identical to those obtained

using BI; therefore here only the BI topology with addi-

tion of bootstrap (BS) values (Fig. 2) is shown. In contrast

with Galbany-Casals et al. (2010), the species Filago

vulgaris Lam. clusters within F. subgen Crocidion instead

of within F. subgen. Filago. This position is due to

incongruence between the nuclear and chloroplast phy-

logenies in this particular case (see supplementary mate-

rials in Galbany-Casals et al. 2010) and to the fact that

only one chloroplast region is used here instead of three

as the original authors did. Otherwise the present results

do not differ from the most recent and complete phylo-

genetic analysis.

Fig. 1 Representative histograms of relative nuclear DNA content.

First peak, from left to right, nuclei population of sample (G1) and secondpeak nuclei population of standard (G0). The histograms have been

obtained using flow cytometry analysis of propidium iodide-stained

nuclei for: a Filago pyramidata versus Solanum pseudocapsicum,

b F. hispanica versus S. pseudocapsicum, c F. crocidion versus

S. pseudocapsicum, d F. griffithii versus S. pseudocapsicum, e Bombyc-ilaena discolor versus S. pseudocapsicum, f Logfia clementei versus

S. pseudocapsicum, g Ifloga spicata versus S. pseudocapsicum,

h F. mareotica versus F. crocidion, i F. mareotica versus L. gallica

Genome size evolution in the Filago group 337

123

Evolution of genome size in the Filago group

and in the genus Filago

Analyses of the Brownian motion (BM) model together with

the three scaling parameters are reported in Appendix 2. The

parameter k is very close to 1 (0.934 ± 0.003; BIC

weight = 0.1626 ± 0.0022). Kappa (j) takes a value of

0.8306 ± 0.0038 (BIC weight = 0.1279 ± 0.0009). Finally,

delta (d) is also very close to 1 (1.0550 ± 0.0087; BIC

weight = 0.1030 ± 0.0005).

Figure 3 shows the character genome size mapped on

the consensus tree resulting from the BI analysis using the

parsimony approach for continuous characters in Mesquite

(Maddison and Maddison 2010). Appendix 3 shows the

assignment of ancestral character states for the nodes a–f

(Fig. 3). For the Filago group 1.4069 pg is identified as the

ancestral condition, node a. It is also suggested that the

ancestral genome size for the genus Filago is 1.4249 pg,

node b. Finally the value of the ancestral conditions for the

subgenera Oglifa, node c, Pseudevax, node d, Crocidion,

node e and Filago, node f are 1.4432 pg, 1.4683 pg,

1.2658 pg and 1.4164 pg, respectively.

Discussion

Genome size and chromosome numbers

The 27 estimates of nuclear DNA content are the first to be

published for the genera Filago, Bombycilaena, Logfia and

Ifloga. They represent a significant contribution to the

knowledge of genome sizes in the Gnaphalieae as, to date,

only a single estimation is available for a species of the

tribe (Phagnalon umbelliforme DC., Suda et al. 2003). The

genome size in the Filago group is relatively low, 5.71-fold

lower than the mean value of the parameter for the family

Asteraceae (8.09 ± 6.4 pg) (Bennett and Leitch 2010a).

These low values of DNA amount in Filago and related

genera could support the hypothesis proposed by Leitch

et al. (1998) that, although angiosperms have a very large

range in nuclear DNA amounts, most of them have very

small C-values. Chromosome number estimates for five

species are published here for the first time and for B. dis-

color a previously published chromosome count has been

confirmed (Watanabe 2010).

The general pattern of ploidy already known for Filago,

Bombycilaena and Logfia, that all species throughout the

genera are diploid [2n = (26) 28] and based on x = (13) 14,

seems to have been confirmed here, as this is the unique

ploidal level found for all the populations included in our

analysis. Considering that this single ploidal level has been

estimated by us for six additional samples, this could be in

correspondence with the fact that 2C-values are highly

homogeneous among the species included in the mentioned

genera, as they vary only 1.53-fold between F. hispanica

(1.7002 ± 0.0036 pg) and L. gallica (1.1103 ± 0.0062 pg).

Further chromosome counts are necessary to confirm this

hypothesis.

Genome size, taxonomy and phylogeny

A high degree of congruence was found between genome

size distribution and the phylogenetic lineages obtained by

Galbany-Casals et al. (2010). This most recent phyloge-

netic study of the Filago group suggests two major lineages

within it. As already stated, the first one is composed of the

American species of the group plus the Old World species

of Logfia except for F. arvensis, and the second one of the

Old World species of Filago (including Evax), Micropus

and Bombycilaena, plus F. arvensis and the traditional

monotypic genera Evacidium and Cymbolaena. The gen-

ome size distribution basically matches with these major

phylogenetic lineages (Fig. 3), although this conclusion

Fig. 2 Bayesian consensus tree from the analysis of ITS, ETS and

rpl32-trnL intergenic spacer sequence data. Posterior probabilities are

below branches. The strict consensus of the six most parsimonious

trees has the same topology; bootstrap values above branches

338 S. Andres-Sanchez et al.

123

may be partially limited by the fact that no American

representative of the Filago group was included in our

analysis.

The new taxonomic treatment proposed by Andres-

Sanchez et al. (2011) for the Filago group is based on the

previously mentioned DNA sequence analysis and,

accordingly, gets support in many cases from DNA amount

data. As regards generic boundaries within the Filago

group, one of the most controversial points in the classifi-

cation of the group, the 2C-value, clearly supports the

enlargement of Filago to include Evax and the monotypic

traditional genera Cymbolaena [i.e., Filago griffithii

(A. Gray) Andres-Sanchez & Galbany] and Evacidium

(i.e., F. discolor), and the independence of Logfia. Filago,

Evax, Cymbolaena and Evacidium show overlapping gen-

ome sizes, while Logfia has considerably lower 2C-values

(\1.16 pg). The Tukey’s range test performed (Table 1)

found significant differences (P \ 0.0001) between the

mean 2C-values corresponding to each group—Filago

group sensu stricto sensu Galbany-Casals et al. (2010) and

the genus Logfia. Interestingly, the species F. arvensis,

which in contrast with Anderberg’s (1991) delimitation of

Logfia was definitely placed in Filago based on DNA

sequence data (Galbany-Casals et al. 2010), shows a DNA

amount close to that shown by the members of the genus

Filago; in fact the Tukey’s range test (Table 1) found that

the mean 2C-values corresponding to F. arvensis and to the

three species of Logfia included in this analysis are sig-

nificantly different (P \ 0.0001) from one another. Last,

the genome size of Bombycilaena lies well within the range

of Filago (Table 1), which is also in agreement with the

phylogenetic relationships found by Galbany-Casals et al.

(2010) (Fig. 3), with Bombycilaena showing sister-group

relationships with Filago and both included together with

Micropus L. in the clade Filago group sensu stricto.

The previously mentioned infrageneric classification of

Filago proposes the division of the genus into four sub-

genera (Fig. 3) corresponding to highly supported lineages.

Also, the ANOVA applied to test for differences in DNA

content among the four subgenera within Filago found

significant differences (F = 6.554; P \ 0.001). Particu-

larly, the genome size distribution matches well (Fig. 3)

with the phylogenetic lineage identified as F. subgen.

Crocidion, a newly described subgenus that includes a

couple of species previously classified under Evax. This is

also supported by Tukey’s range test (Table 1), which

Fig. 3 Ancestral character state reconstructions of genome size. The DNA amount has been mapped on the phylogram resulting from the

Bayesian analysis of ITS, ETS and rpl32-trnL intergenic spacer sequence data using unordered maximum parsimony for continuous characters

Genome size evolution in the Filago group 339

123

found significant differences (P \ 0.001) between the

average DNA content of F. subgen. Crocidion (1.2628 ±

0.0043 pg) and the branch corresponding to the remaining

three subgenera (1.4138 ± 0.1005 pg) (Table 1; Fig. 3).

Evolution of genome size in the Filago group

and within the genus Filago

The availability of methods for analysing possible con-

nections between DNA content and phylogeny (Harvey and

Pagel 1991; Pagel and Meade 2007) has allowed rigorous

testing of genome size evolution against the previously

mentioned phylogenetic hypothesis for the Filago group.

This has provided insights into the dynamics of genome

size evolution and made reconstruction of ancestral C-

values possible. The maximum likelihood estimate of the

transformation parameter k is 0.9335 ± 0.0025 (Appendix

2), very close to 1, thus indicating that the phylogeny

correctly predicts the patterns of covariance among species

belonging to the Filago group on the trait DNA content

(Pagel 1999). This high phylogenetic signal could indicate

that the DNA content is not strongly influenced by selec-

tion, because it would be expected that taxon-specific

responses would obscure phylogenetic signal if selective

pressures had directly influenced genome size (Lysak et al.

2009). This is also supported by the fact that the narrow

range of small genome size that characterises the group is

most likely due to a passive tempo of gradual evolution of

this trait than to a scenario where selection or adaptive

radiation have played significant roles.

Additionally it seems that genome size evolved more

rapidly in earlier phases of evolution of the complex than in

later ones (j = 0.8306 ± 0.0038) as might occur in

adaptive radiations (Pagel 1999). The data indicate that

genome size evolved in a gradual rather than in a punctual

mode in the group (d = 0.10550 ± 0.0087), i.e., genome

size has changed linearly with branch length (Pagel 1997,

1999). A similar distribution of trait evolution together

with an early accelerated evolutionary rate in this case

would fit a scenario of phylogenetically grouped differen-

tial susceptibility of the Filago group toward genome size

changes (e.g., via indel formation or retrotransposon

activities, Gregory 2003). These results could be correlated

with the early divergence of the Old World species of

Logfia and the American genera within the Filago group

and the differentiation—connected with an increase of

genome size—of the lineage comprising Filago, Micropus

and Bombycilaena. Evidently this conclusion calls for

caution given that the American genera have not been

sampled and the inclusion of these species would be nec-

essary to corroborate this trend. Therefore, the genome size

would have increased or decreased several times along the

evolutionary history of the Filago group (Fig. 3). From an

ancestor with 2C-value close to 1.4069 pg (Table 1), the

genome size would have increased in the evolution of the

genus Bombycilaena or in F. subgen. Pseudevax, but it

would have decreased in F. subgen. Crocidion and within

the subgenus Filago.

Genome size, life traits and ecological features

The correlation of low genome size with annual life history

is one of the earliest cited relationships of DNA content

and ecological or evolutionary features. Chooi (1971)

suggested it, and Bennett (1972) demonstrated that short

meiotic and mitotic cycles are related to annual life cycles;

likewise low DNA contents and annual life histories are

also correlated. Many authors have discussed this (Grime

and Mowforth 1982; Torrell and Valles 2001; Barow and

Meister 2003; Bancheva and Greilhuber 2006; Weiss–

Schneeweiss et al. 2006) and have stressed that such cor-

relations must be interpreted with caution, as phylogenetic

information is in many cases lacking and many studies

reach different conclusions depending on which statistical

analysis is used (Albach and Greilhuber 2004; Chrtek et al.

2009). The genome size in the Filago group is much lower

than the mean value of the parameter for the angiosperms

(11.88 ± 19.49 pg) and for Asteraceae (8.09 ± 6.4 pg). In

principle, these data would support the idea that annuals

usually have low genome sizes, but the hypothesis that an

upper boundary in DNA amount exists for annual species

that is lower than that for perennials within a given group

cannot be tested in this case study. Strikingly, the 2C-value

for I. spicata, the only representative of the genus Ifloga in

the western Mediterranean area, also a diploid, but with

half the most common chromosome number within the

study group (2n = 14), does not differ from that found in

the remaining species included in this study, being very

close to that shown by the species of Logfia. Although we

were not able to estimate the chromosome number of

I. spicata, there are several counts published for this spe-

cies (Dalgaard 1986; Malallah et al. 2001). A detailed

review of the microphotographs available for I. spicata did

not uncover differences in chromosome size among Ifloga

and the remaining genera included in this study. While the

latter genus comprises exclusively annual species, Ifloga

includes both annuals and perennials species. Although

I. spicata is an annual, the fact that Ifloga lodges also

perennials (Bergh et al. 2011) may condition a relatively

high DNA content in I. spicata. A denser sampling of the

Gnaphalieae would be obviously necessary to evaluate this

hypothesis.

The taxa belonging to the Filago group are mostly

ephemeral and selfers, although in many cases, at least in

Filago, it is not clear whether they are autogamous or

geitonogamous—internal hermaphrodite flowers pollinate

340 S. Andres-Sanchez et al.

123

external female ones (Wagenitz 1965). It is also generally

assumed (Labani and Elkington 1987; Albach and Greilh-

uber 2004) that short life cycles and selfing breeding sys-

tems are correlated with lower DNA contents. The reason

for this is that larger DNA amounts involve longer division

cycles and growth times (Bennett 1972). Again these

hypotheses would be supported in principle by the rela-

tively low genome size found within the Filago group, but

the lack of variation regarding life cycle lengths and

breeding systems within the group prevents further

hypothesis testing.

The principal characteristics of weeds are rapid estab-

lishment and completion of reproductive development,

short generation time and fast production of many small

seeds, and all of them seem to be correlated with DNA

amount (Leitch and Bennett 2007). Within Filago, several

species are considered as weeds (Randall 2007), particu-

larly, F. pyramidata, F. arvensis and Filago pygmaea L.,

and they all show the previously mentioned characteristics.

Additionally, these three species show the widest distri-

bution areas within the genus, and apparently they do not

show any dependence on the kind of soil, climate or

moisture, which makes them well-adapted weeds. Inter-

estingly, with the exception of the two species from

F. subgen. Crocidion and F. ramosissima Lange, the three

of them show the lowest 2C-values within the genus

(Table 1).

The highest DNA contents within the genus Filago are

those shown by two phylogenetically unrelated species,

F. mareotica (1.6099 ± 0.0028 pg) and F. hispanica

(1.7002 ± 0.0036 pg), both with particular ecological

requirements (salt marshes in the first case and small snow

beds and seasonally flooded patches at high altitudes in the

second). The correlation between altitude and genome size

has been a matter of discussion in recent years as divergent

conclusions have been reached by different authors. No

correlation between both factors has been found in Aster-

aceae such as Artemisia (Garcıa et al. 2004) or Tripleuro-

spermum Schultz Bip. (Garcıa et al. 2005), among others,

but an increase in the DNA amount is apparently related to

higher altitudes, for example in Centaurea s.s. (Bancheva

and Greilhuber 2006). For other families even a negative

correlation between altitude and genome size has been

demonstrated (e.g., Reeves et al. 1998; Walker et al. 2006).

As summarised by Knight et al. (2005), it seems that this

correlation depends on the taxonomic group analysed.

Within Filago, although species such as F. arvensis or

F. pyramidata can grow from sea level to high altitudes,

F. hispanica, with a relatively high DNA content, is a

unique taxon within the genus that can be strictly consid-

ered a subalpine species. The positive correlation between

genome size and altitude could be related to the capacity

for growth at low temperatures and frost resistance

(MacGillivray and Grime 1995; Albach and Greilhuber

2004). According to Grime and Mowforth (1982), a higher

genome size would allow growth by cell division during

the preceding favourable season and expansion early in the

season at low temperatures. With phosphate often being a

limiting factor for DNA synthesis, Hanson et al. (2001) and

Albach and Greilhuber (2004) have proposed that a posi-

tive correlation between DNA content and altitude may be

due to a higher availability of phosphate in the soils at

higher altitudes. Filago hispanica grows in a limited range

of altitude and Suda et al. (2003) demonstrated a positive

correlation with altitude for species with such characteris-

tics from Macaronesia, while for taxa from regions with

large altitudinal ranges, this correlation was negative. Also,

Bancheva and Greilhuber (2006) argued that this relation

could be more important in Mediterranean or semi-arid

regions than in temperate or boreal regions.

Many authors have also discussed that environmental

stress, such as aridity (Torrell and Valles 2001), could

influence genome sizes. Kalendar et al. (2000) noted that in

Hordeum spontaneum K. Koch, higher genome sizes are

present when the species grows in dry areas. Similarly, in

Artemisia (Torrell and Valles 2001), large genomes seem

to be associated with arid environments. However, Baack

et al. (2005) proposed several hypotheses to explain

increases of genome size in hybrid sunflowers with respect

to their parents. One of them was that extreme environ-

mental conditions (deserts, sand dunes or salt marshes)

favour high DNA contents. This could also be the case of

the species within Filago that shows the second highest

2C-value, F. mareotica, which grows in salt marshes along

the Mediterranean coast from Egypt to Algeria and in

semideserts from southeastern Spain.

Conclusions

Distribution of genome size is congruent with phylogenetic

lineages identified by analyses of nuclear and chloroplast

DNA sequences in the Filago group. DNA content and

sequence data support the monophyletic circumscription of

Filago to include Evax, Cymbolaena and Evacidium, the

independence of Logfia and the subgeneric treatment

recently proposed for Filago. It seems that, although a lack

of a clear trend toward a decrease or increase in DNA

content was observed, within the Filago group genome size

evolved more rapidly in earlier phases of evolution of the

complex than in later ones, which could be related to the

early divergence of the Old World species of Logfia and

the American genera.

DNA amount does not seem to be strongly influenced by

selection in the Filago group, and the narrow range of

small genome size that characterises the group is most

Genome size evolution in the Filago group 341

123

likely due to a passive tempo of gradual evolution of this

trait than to a scenario where selection or adaptive radia-

tion have played significant roles. The generally accepted

assumption that annual, ephemeral and autogamous species

show low genome sizes was confirmed, although the lack

of variation regarding life history characters and breeding

systems within the group prevents further hypothesis test-

ing. The relatively high DNA contents found in F. hispa-

nica and F. mareotica could be related to their highly

specific ecological requirements (subalpine conditions in

the first case and aridity and soil salinity in the latter).

Acknowledgments We would like to express our deep gratitude to

Prof. J. Greilhuber for generous help at the early stages of this work

and for comments that have improved this manuscript. Many thanks

to Dr. M. Galbany-Casals for her constant support, enthusiastic dis-

cussions and help with phylogenetic analyses. Also our acknowl-

edgement goes to Dr. A.M. Escudero Lirio, who helped with the

statistical analyses using R software, and Dr. F. Gallego and Dr.

L. Delgado for advice regarding chromosome counts. Thanks are also

due to our friend Dr. J. Penas de Giles for his collaboration in the field

work. This work was supported by the Spanish Ministerio de Ciencia

e Innovacion (www.micinn.es) through projects CGL2008-02982-

C03-02/CLI, CGL2011-28613-C03-03 and CGL2009-07555. SAS

was also supported by a research grant financed by MICINN.

Appendix 1

Taxa, location, date and collectors of all samples included

in the analyses of genome size. The EMBL accession

numbers (ITS, ETS, rpl32-trnL intergenic spacer) of each

taxon from Galbany-Casals et al. (2010) are included in

parentheses.

Bombycilaena discolor (Pers.) Laınz, Morocco, Orien-

tal, Taurirt, Narguechoum, Za river, 550 m, 14-IV-2006,

S. Andres–Sanchez et al., SALA134284 (FN645843,

FN645560, FN649364); Filago argentea (Pomel) Chrtek &

Holub, Morocco, Oriental, mouth of Moulouya, 5 m,

16-IV-2006, S. Andres–Sanchez et al., SALA134245

(FN645859, FN645569, FN649373); Filago arvensis

L., Morocco, Atlas Mountains, Adrar-n-Oukaımeden,

3,000 m, 29-VI-2006, A. Herrero et al., SALA134334

(FN645885, FN645605, FN649361); Filago carpetana

(Lange) Chrtek & Holub, Spain, Teruel, Albarracın, Fuente

del Cabrerizo, 1,303 m, 18-VI-2009, S. Andres–Sanchez

et al., SALA135591 (FN645858, FN645568, FN649372);

Filago congesta Guss. Ex DC., Spain, Albacete, Elche de

la Sierra, road to Riopar, 830 m, 13-VI-2007, S. Andres–

Sanchez et al., SALA134211 (FN645848, FN645577,

FN649382); Filago crocidion (Pomel) Cortek & Holub,

Morocco, Taza, Daya Chiker (plain Chiker), 1,365 m,

23-VI-2008, S. Andres–Sanchez et al., SALA139146

(FN645864, FN645601, FN649403); Filago desertorum

Pomel, Spain, Almerıa, Filabres Mountains, 611 m,

31-IV-2007, S. Andres–Sanchez et al., SALA134350

(FN645874, FN645591, FN649391); Filago discolor (DC.)

Andres–Sanchez & Galbany, Morocco, Beni-Mellall, Col

de Tanout ou Fillal, 2,070 m, 6-VII-2006, A. Quintanar

et al., SALA134338 (FN645853, FN645564, FN649368);

Filago duriaei Lange, Morocco, Taza, Daya Chiker (plain

Chiker), 1,370 m, 23-VI-2008, S. Andres–Sanchez et al.,

SALA139174 (FN645881, FN645587, FN649389); Filago

fuscescens Pomel, Spain, Murcia, El cantal, near to

Aguilas, 78 m, 3-IV-2009, S. Andres–Sanchez et al.,

SALA141935 (FN645846, FN645580, FN649394); Filago

gaditana (Pau) Andres–Sanchez & Galbany, Spain, Pont-

evedra, Cangas del Morrazo, Playa del Limens, 20 m,

18-V-2009, S. Andres–Sanchez et al., SALA139193

(FN645869, FN645576, FN649380); Filago griffithii

(A. Gray) Andres–Sanchez & Galbany, Armenia, Ararat,

Hadis Mountains, 1,220 m, 1-VII-2005, C. Navarro et al.,

SALA134833 (FN645888, FN645608, FN649405); Filago

hispanica (Degen & Hervier) Chrtek & Holub, Spain, Jaen,

Pontones, Laguna de la Canada Cruz, 1,590 m, 12-VII-

2007, S. Andres–Sanchez et al., SALA134387 (FN645855,

FN645565, FN649370); Filago lusitanica (Samp.) P. Silva,

Spain, Caceres, Malpartida de Caceres, Los Barruecos,

350 m, 23-IV-2009, E. Rico, SALA110253 (FN645866,

FN645572, FN649376); Filago lutescens subsp. atlantica

Wagenitz, Spain, Cadiz, Tarifa, Facinas, 85 m, 15-VI-

2008, S. Andres–Sanchez et al., SALA139215 (FN645877,

FN645595, FN649399); Filago lutescens Jordan subsp.

lutescens, Spain, Zamora, Almaraz de Duero, Valdedores,

730 m, 3-VII-2008, L. Delgado et al., SALA134827

(FN645876, FN645594, FN649395); Filago mareotica

Delile, Spain, Murcia, Aguilas, road to Vera, 7 m, 24-IV-

2009, S. Andres–Sanchez, SALA139217 (FN645879,

645593, FN649393); Filago micropodioides Lange,

Morocco, Oriental, between Guercif and Saka, 455 m,

17-III-2008, S. Andres–Sanchez et al., SALA134397

(FN645850, FN645582, FN649385); Filago nevadensis

(Boiss.) Wagenitz & Greuter, Spain, Granada, near Las

Sabinas, 2,155 m, 13-VII-2006, J. Penas, SALA134830

(FN645862, FN645599, FN649401); Filago pygmaea L.,

Spain, Salamanca, Calvarrasa de Arriba, Ermita de la Pena,

760 m, 5-VI-2009, S. Andres–Sanchez et al., SALA139211

(FN645868, FN645574, FN649379); Filago pyramidata L.,

Spain, Tarragona, between Poblet Monastery and

Prades, 547 m, 16-VI-2009, S. Andres–Sanchez et al.,

SALA110287 (FN645873, FN645590, FN649383); Filago

ramosissima Lange, Spain, Granada, Izbor, road to

Lanjaron, 534 m, 4-IV-2009, S. Andres–Sanchez et al.,

SALA139149 (FN645880, FN645563, FN649367); Filago

vulgaris Lam., Spain, Gerona, La Jonquera, between La

Jonquera and Cantallops, 220 m, 17-VI-2009, S. Andres–

Sanchez et al., SALA135586 (FN645878, FN645604,

FN649406); Ifloga spicata (Forskal) Schultz Bitp.. Spain,

342 S. Andres-Sanchez et al.

123

Almerıa, Tabernas, 544 m, 22-II-2004, M. Martınez-Orte-

ga et al., SALA134835 (FN645825, FN645627,

FN649356); Logfia clementei (Willk.) Holub, Spain, Al-

merıa, Nıjar, Monsul, beach of Genoveses, 12 m, 13-IV-

2005, M. Santos et al., SALA134322 (FN645837,

FN645612, FN649342); Logfia gallica (L.) Coss. & Germ.,

Spain, Barcelona, between Breda and San Celoni, near to

Batlloria, 92 m, 17-VI-2009, S. Andres–Sanchez et al.,

SALA139203 (FN645838, FN645556, FN649339); Logfia

minima (Sm.) Dumort. Spain, Zamora, Canizal, 822 m,

9-V-2005, M. Martınez-Ortega et al., SALA134213

(FN645817, FN645613, FN649347).

Appendix 2

Models of evolution of genome size. For each model:

values of the log-likelihood, Brownian rate parameter (r2),

third parameter (k, j or d) and Bayes information criterion

weight (BIC). The analyses were carried out with 1000

trees subsampled from the phylogenetic Bayesian analysis.

Model Log-

likelihood

r2 3rd parameter BIC weight

Brownian

motion

39.4336 ±

0.0346

0.2378 ±

0.0008

– 0.5007 ±

0.0021

Brownian

motion ? k39.8941 ±

0.0268

0.1785 ±

0.0011

0.9336 ±

0.0025

0.1627 ±

0.0023

Brownian

motion ? j39.70835 ±

0.0271

0.1142 ±

0.0018

0.8306 ±

0.0038

0.1280 ±

0.0010

Brownian

motion ? d39.49939 ±

0.0346

0.2514 ±

0.0024

1.0550 ±

0.0087

0.1030 ±

0.0005

Appendix 3

Reconstruction of ancestral 2C-values (in pg) using unor-

dered maximum parsimony for continuous characters for

nodes a–f (Fig. 3)

Node Ancestral

character state

a 1.4069

b 1.4249

c 1.4432

d 1.4683

e 1.2658

f 1.4164

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