ORIGINAL ARTICLE

Microsatellite markers unravel the population genetic structureof the Azorean Leontodon: implications in conservation

Elisabete F. Dias • Julie Sardos • Luıs Silva •

Maria G. B. Maciel • Monica Moura

Received: 27 November 2012 / Accepted: 20 October 2013

� Springer-Verlag Wien 2013

Abstract The genus Leontodon L. (Asteraceae) com-

prises approximately 50 species with a natural distribution

area covering North America, Europe, northern Africa, and

western Asia. Two of these species are endemic to the

Azores Archipelago: Leontodon filii and Leontodon rigens.

Although both species were targeted with several taxo-

nomic revisions, so far no studies into their genetic

diversity have been carried out. In this research, the pop-

ulation genetic structure and diversity of both taxa were

assessed using five newly developed SSR markers. Four

hundred and thirty-seven individuals collected throughout

the archipelago were included in the study. A total of 98

alleles (25–12 per locus, average = 19.6) and an overall

excess of homozygotes (multilocus Fis = 0.37, range

0.16–0.53) were found for L. rigens populations. For L.

filii, 52 alleles in total (8–13 per locus, average = 10.4)

were found, overall near the HW equilibrium (multilocus

Fis = 0.07, range -0.25 to 0.57). The two species showed

an equivalent proportion of rare alleles (L. rigens 80.6 %;

L. filii 76.9 %). Both a Principal Coordinate Analysis and a

Bayesian analysis proposed the existence of two well-

defined groups, but pooled L. filii populations from Faial

Island with L. rigens populations. The largest proportion of

genetic variability was found within populations (L. rigens

72.6%; L. filii 78.9 %). The highest values of gene flow

were obtained for L. filii within the central group of islands.

Our results update the current distribution given for the

Azorean Leontodon taxa, clearly indicating that conserva-

tion measures should be applied to several populations. The

results also reveal that a revision of the Azorean Leontodon

should be carried out to clarify species delimitation.

Keywords Leontodon � Asteraceae � Conservation �Endemic � Microsatellites � Azores islands

Introduction

The genus Leontodon L. (Asteraceae) comprises about 50

species with a natural distribution area covering North

America, Europe, northern Africa, and western Asia (Bogler

2006). Two species are considered endemic to the Azores

archipelago: Leontodon filii (Hochst. ex Seub.) Paiva &

Ormonde and Leontodon rigens (Aiton) Paiva & Ormond

(Silva et al. 2010). It is thus one of the few genera showing

more than one endemic species in the archipelago. Originally

assigned to Microderis, the Azorean taxa was placed in

Leontodon by Paiva and Ormonde (1974), who considered

that the degree of inflorescence ramification and the number

of capitula were the main traits distinguishing the two spe-

cies. Thus, clear distinction between the species on mor-

phological grounds is problematic whenever the plants are

not flowering (Moura et al. 2010). Historical references

indicated a possible third species [Microderis umbellata

(Hochstetter ined.)] from Flores (Seubert 1844) suggesting

that species delimitation might require a revision.

Lack (1981), in a study of the Azorean Lactuceae

demonstrated that both L. filii and L. rigens have 14

chromosomes and are diploid. Both species are

E. F. Dias (&) � L. Silva � M. G. B. Maciel � M. Moura

CIBIO, Centro de Investigacao em Biodiversidade e Recursos

Geneticos, InBIO Laboratorio Associado, Polo dos Acores,

Departamento de Biologia, Universidade dos Acores, Rua da

Mae de Deus, Apartado 1422, Ponta Delgada 9501-801, Azores,

Portugal

e-mail: elisabetefdias@uac.pt

J. Sardos

Bioversity-France, Parc Scientifique Agropolis II,

34397 Montpellier Cedex 5, France

123

Plant Syst Evol

DOI 10.1007/s00606-013-0937-0

hemicryptophytes with anemochorous and hydrochorous

dispersal (Silva et al. 2009). Natural populations of L. ri-

gens are estimated to include more than 10,000 individuals.

Those of L. filii are estimated to contain 5,000 to 10,000

individuals, and this species is protected by Bern Con-

vention (Silva et al. 2009).

Leontodon filii has been found in steep slopes, ravines,

inland cliffs, wet places in crater margins near water-

courses, juniper woodland and extremely wet bogs, while

L. rigens occurs in wetlands, coastal cliffs, steep slopes,

craters, ravines, natural laurel forest and juniper woodland,

margins of permanent and semi-natural pastures, natural

meadows (Holcus and Festuca), wet meadows, young lava

flows with pioneer vegetation, waterfalls and roadside

slopes. Both species are threatened by habitat degradation

and reduction of available area, resulting from changes in

land use, including expansion of pastureland and plant

invasions. Additionally, some populations currently have a

low density of individuals and are geographically restricted

(Silva et al. 2009).

The distribution of the Azorean Leontodon species

remains unclear. The Azores Biodiversity Portal (2008) lists

as co-habiting the same locations in most of the Azorean

islands, with the exception of Corvo, where only L. rigens is

listed, and Graciosa and Santa Maria Islands where the

species do not occur. However, in the most recent checklist

of Azorean vascular flora (Silva et al. 2010), L. filii is

considered to occur in Pico, Sao Jorge, and Terceira, while

its presence was considered as doubtful in Faial and Sao

Miguel; L. rigens was considered to be geographically

confined to the island of Sao Miguel, as its presence is

doubtful in Pico and Terceira. Recent collections indicated

that the known distribution may contain errors.

To date, no extensive studies on the genetic variability

and population structure Azorean endemic Leontodon have

been made. These are necessary to define effective in situ

and ex situ conservation measures (for list of Azorean

priority species see Silva et al. 2009).

Francisco-Ortega et al. (2000) in a review of allozyme

variation in 69 mainly outcrossing endemic species of the

Canarian flora verified that these studies reported a high

total diversity within species and a relatively high differ-

entiation among populations (Perez de Paz and Caujape-

Castells 2013). The same study also indicated that the

average species-level genetic diversity at the allozyme loci

was unusually high. In a similar study devoted to the

Canarian Tolpis spp. (Crawford et al. 2006), allozymes

revealed low genetic diversity within species, a relatively

high proportion of the diversity among populations, and

low divergence among species. Caujape-Castells et al.

(2008) have hypothesized that the low values of genetic

variability obtained in Gran Canaria’s small subpopula-

tions of another endemic Asteraceae species, Atractylis

arbuscula, were connected to the common family charac-

teristic of possessing a sporophytic self-incompatibility

system.

In this context, this paper presents a comprehensive

population genetic study using newly developed micro-

satellite (SSR) markers.

Our objectives were (1) to determine the correspondence

between current chorological data of the Azorean Leonto-

don species and the major groups of genetic variability

obtained, (2) to estimate intra and inter-population genetic

variability patterns, (3) to identify populations with very

low genetic variability, and (4) to determine the existence

of population units requiring genetic and morphological

studies to confirm taxonomic recognition.

Materials and methods

Study site

The Azores Archipelago is located in the NE of the

Atlantic Ocean, between the 368 and 438 of North latitude

and the 258 and 318 of West longitude. The closest main-

land is the Iberian Peninsula, approximately 2,000 km

East; Nova Scotia is 2,300 km Northwest, Bermuda at

3,500 km southwest, and Madeira 1,200 km southeast. The

Azores are composed by nine islands distributed by three

main groups: western (Flores and Corvo), central (Faial,

Pico, Sao Jorge and Terceira), and eastern (Sao Miguel and

Santa Maria; Fig. 1). The vascular plant flora comprises

approximately 1,110 taxa including 73 endemic taxa (Silva

et al. 2010). Historically, research on Azorean flora was

limited by its remote geographic location and by the low

number of endemic plant taxa documented in early

checklists. This pattern led to the conclusion that the

archipelago’s endemic biodiversity was low, in comparison

with the Canary Islands and Madeira (Carine and Schaefer

2010). However, recent molecular studies have revealed

previously undetected patterns of diversity within the Az-

ores archipelago as well as new endemic taxa (Carine and

Schaefer 2010; Schaefer 2003; Schaefer and Schonfelder

2009; Schaefer et al. 2011; Silva et al. 2011; Martins et al.

2013; Moreira et al. 2013; Moura et al. 2013).

Plant material and sampling

During the summer and the early autumn of 2010, trips to

all the islands of the archipelago were carried out to

complement the samples of endemic Leontodon spp.

already available at the DNA bank collection of the AZB

herbarium (Biology Department, Azores University).

Depending on leaf size, one or two leaves per individual

were collected and immediately stored in a plastic bag with

E. F. Dias et al.

123

silica gel. After drying, the leaves were vacuum sealed in

plastic bags and stored in folders. The number of individ-

uals sampled per population varied: for small population

sizes (n \ 15) all individuals were sampled, and for larger

populations, between 10 and 30 individuals were sampled.

In total, and including already existing and new accessions,

51 different populations and 437 individuals were sampled

(Tables 1, 2). One to two representative voucher specimens

per population were also collected, pressed, mounted, and

stored at AZB. The locations of all populations were geo-

referenced and mapped using ArcView GIS 3.2.

General DNA extraction

DNA was extracted from dry leaves using a modified

Doyle and Dickson CTAB protocol (Doyle and Dickson

1987). Due to the difficulties encountered in obtaining

high-quality DNA, modifications proposed by Borges et al.

(2009) were used. Specifically, 700 ll of 39 CTAB, 50 ll

of Sarcosyl, and 10 ll of Proteinase K were used for the

initial lysis step, and the first washing step was performed

with 500 ll of chloroform–isoamylalcohol (24:1)

(SEVAG) with an additional wash using 500 ll of SEVAG

and 200 ll of 39 CTAB. DNA was then precipitated by

adding 450 ll of isopropanol and re-suspended in 50 ll of

pure water. DNA quality and quantity were measured using

a Nanodrop 2000 (Thermo Fisher Scientific) spectropho-

tometer. Samples were conserved at -20 �C until use.

Microsatellite development

Total DNA from fresh leaves of one individual of L. rigens

was sent to the Savannah River Ecology Laboratory

(University of Georgia), where the enrichment procedure

described in Glenn and Schable (2005), with the exceptions

described in Lance et al. (2010), was followed for micro-

satellite isolation. Sequences were subjected to a 30 quality

trim where only one base in the last 25 bases of the

sequence contains a quality score less than 20 or alterna-

tively contains one ambiguous base. CAP3 was then used

to assemble sequences at 98 % sequence identity using a

minimal overlap of 75 bp. Along with singlets, contigs of

two or three sequences were searched for the presence of

microsatellite DNA loci using the program

Fig. 1 Azores Archipelago and location of the Leontodon populations requiring conservation measures. Squares represent L. filii populations

and triangles, L. rigens. Estimated island ages (Myr) are also represented

Microsatellite markers unravel the population

123

MSATCOMMANDER version 0.8.1 (Faircloth 2008) and

primers designed with Primer3. One primer from each pair

was extended on the 50-end with an engineered sequence

(M13R tag 50-GGAAACAGCTATGACCAT-30) to enable

the use of a third primer identical to the M13R, which

allows for a inexpensive fluorescent labelling of the PCR

product obtained (Schuelke 2000), and a GTTT ‘‘pigtail’’

was added to the 50-end of the untagged primer to facilitate

accurate genotyping (Brownstein et al. 1996).

Out of the 183 sequences of primer pairs provided by the

Savannah River Ecology Lab we selected 24 primer pairs, 12

with expected PCR products ranging between 100–200 bp (A

series) and 12 exhibiting expected PCR products ranging

between 200 and 300 bp (B series) to allow later the multi-

loading of PCR products (Oetting et al. 1995). All the primer

pairs (with the tag sequence included) were selected on criteria

of non-complementarities within and between primers, low

secondary structures and 30-end instability (Rychlik 1995).

Table 1 Geographical distribution of the Leontodon rigens popula-

tions sampled in the Azores archipelago

Island

group

Island (N) Populations Codes n Total

Eastern Sao

Miguel

(118)

Sete Cidades (Caldeira

do Alferes)

SMAL 10 118

Road to Outeiro da

Lomba

SMOL 10

Lagoa do Canario

(road)

SMEC 5

Lagoa do Canario SMLC 10

Lagoas Empadadas SMEM 10

Lagoa do Fogo SMLF 17

Lagoa do Areeiro SMLA 6

Sete Cidades (Lomba

do Pico)

SMLP 12

Monte Escuro SMME 3

Planalto dos

Graminhais

SMPG 3

Pico Bartolomeu SMPB 6

Pico do Carvao SMPR 5

Nordeste (Tronqueira) SMTR 10

Sete Cidades (Vista do

Rei)

SMVR 11

Western Flores

(57)

Burreiro FLBU 10 68

Caldeira Seca FLLS 13

Caldeira Branca (road

to Morro Alto)

FLCB 9

Road to Ponta Delgada FLPD 6

Road to Morro Alto FLMA 10

Miradouro Craveiro

Lopes

FLCL 9

Corvo

(11)

Cumeeiras do

Caldeirao

COMC 11

A total of 186 individuals were collected

N number of individuals per island, n number of individuals per

population, Total number of individuals per island group (Eastern and

Western)

Table 2 Geographical distribution of the Leontodon filii populations

sampled in the Azores archipelago

Island

group

Island (N) Populations Codes n Total

Eastern Sao

Miguel

(3)

Lagoa do Canario SMLC 1 3

Pico da Vara SMPV 2

Central Pico (117) Cabeco do Coiro PICO 15 249

Cabeco dos Misterios PICM 7

Cabeco Redondo PIRE 2

Caldeirao da

Ribeirinha

PIRB 2

Chao Verde PICV 13

Lagoa do Peixinho PILP 16

Lomba do Capitao PILO 12

Miradouro (perto

Cabrais)

PIMI 14

Mountain (Baldio de

S. Mateus)

PIBM 15

Ribeira Funda PIRF 6

Transversal (near

Torrinhas)

PITT 15

Faial (24) Alto do Guarda-Sol FAGS 8

Cabeco do Trinta FATR 10

Caldeira rim FAEC 6

Terceira

(55)

Rocha do Chambre TERC 9

Algar do Carvao TEAC 9

Caldeira da Agualva TECA 1

Caldeira de Santa

Barbara

TESB 8

Serra do Labacal

(Moldes)

TESL 1

Pico da Bagacina TEPB 8

Pico do Gaspar TEPG 10

Cone near Pico do

Gaspar

TEGE 9

Sao Jorge

(53)

Faja do Santo Cristo SJFS 8

Morro Pelado SJMP 13

Pico da Esperanca SJPE 10

Pico do Areeiro SJPA 10

Road to Pico da

Esperanca

SJSE 12

Western Flores (2) Burreiro FLBU 2 2

A total of 254 individuals were collected

N number of individuals per island, n number of individuals per

population, Total number of individuals per island group (Eastern,

Central, and Western)

E. F. Dias et al.

123

Microsatellite selection

In the first phase of the test, all 24 primer pairs were tested

on four samples of L. rigens and four samples of L. filii

using a unlabelled tag primer in a final volume of 25 ll

consisting of 25 ng of DNA, 19 NH4 Buffer, 1.5 mM

MgCl2, 0.4 lM of untagged primer, 0.08 lM of tagged

primer, 0.36 lM of unlabelled tag primer, 200 lM of

dNTPs, 1U of Immolase (Bioline) and using a Biometra

TGradient thermocycler. Touchdown thermal cycling pro-

grammes (Don et al. 1991) encompassing a 10 �C span of

annealing temperatures ranging between 64 and 54 �C were

used for all loci. The PCR program consisted of the fol-

lowing steps: 95 �C for 7 min (hot start); 96 �C for 3 min;

20 cycles of 96 �C for 30 s, the highest annealing temper-

ature of 64 �C (decreased by 0.5 �C per cycle) for 30 s, and

72 �C for 30 s; 20 cycles of 96 �C for 30 s, 54 �C for 30 s,

and 72 �C for 30 s; and finally 72 �C for 10 min for the final

extension of the PCR products. Five ll of PCR products was

then run on a 3.5 % agarose gel, stained with SafeViewTM

Nucleic Acid Stain (abm) and visualized under UV to check

for amplification, polymorphism and scorability of the

bands. Eleven primer pairs exhibited scorable amplified

products of the expected length range and with at least two

alleles. In the second phase, the variability of the eleven

polymorphic loci was assessed in 24 samples: 16 L. rigens

and 8 L. filii, coming from a total of 13 populations.

After analysis of the quality of the PCR products

obtained with the universal primer M13R, five primers with

acceptable to high scorability were selected to run the

complete study (Table 3).

Full-scale genotyping

After optimization, the amplifications for the whole sample

were performed using the protocols presented in Table 4.

The M13R was labeled either with PET, FAM, NED or

VIC. The Taq polymerase used with all the markers was

Immolase (Bioline) with the exception of the primer pair

LR2A02 which used Biotaq (Bioline). Amplification pro-

ducts were diluted, multiloaded, run on an ABI-3130xl

Genetic Analyzer and sized with LIZ500 size standard. The

genotypes obtained were scored using the software Gene-

Marker V.1.97 Demo version (Softgenetics).

Table 3 Description of the five SSR polymorphic loci that exhibited acceptable to high scorability in the Azorean Leontodon

Name Primer Sequences Repeat motif Size range (bp) Dye

LR2A02 Forward aAAGCCGTATGTGAAGAAGAATTC (AC)8 200–250 VIC

Reverse bTGATGCAAACTCAGAACACTC

LR4B01 Forward aACGATATGCCGAATCTGTAC (ACAT)8 200–250 PET

Reverse bTGTTGTAGGAAAGGAAGAGG

LR2A03 Forward bCCATCAAATGTCTCAAATACC (AC)10 150–175 FAM

Reverse aTTAGCCCTCCTTGAAATG

LR4B08 Forward bTGTTGTAGGAAAGGAAGAGG (ACAT)9 240–260 NED

Reverse aACGATATGCCGAATCTGTAC

LR4B10 Forward aTTACGTCTTAACCACCATCTG (AAAC)9 250–300 FAM

Reverse bTTGGAGAAGGTTGAATAAGG

a Indicates ‘‘pigtail’’ tag (50-GTTT-30)b Indicates M13R tag (50-GGAAACAGCTATGACCA-30)

Table 4 PCR conditions for the set of microsatellites selected for the

wide-range genotyping of the Azorean Leontodon

Name PCR mix (Vf = 25

ll-100 ng of DNA)

Cycling program

LR2A02 100 lg/ml of BSA. 19 NH4

Buffer, 2.5 mM MgCl2,

0.4 lL untagged primer,

0.08 lM tagged primer,

0.36 lM Universal tag

M13R., 200 lM dNTP,

1.25 U of Biotaq

95 �C for 4 min; 22 cycles:

95 �C for 30 s, 64 �C

(Touchdown -0.5 �C

each cycle) for 45 s,

72 �C for 45 s; 16 cycles:

95 �C for 30 s. 53 �C for

45 s, 72 �C for 45 s;

72 �C for 10 min

LR2A03 100 lg/ml of BSA, 19

ImmoBuffer, 3 mM

MgCl2, 0.4 lL untagged

primer, 0.12 lM tagged

primer, 0.28 lM

Universal tag M13R,

200 lM dNTP, 1 U of

Immolase

95 �C for 7 min; 95 �C for

3 min; 20 cycles: 95 �C

for 30 s, 65 �C

(Touchdown -0.5 �C

each cycle) for 30 s,

72 �C for 45 s; 20 cycles:

95 �C for 30 s, 55 �C for

30 s, 72 �C for 30 s;

72 �C for 10 min

LR4B01

LR4B08

LR4B10

100 lg/ml of BSA, 19

ImmoBuffer, 3 mM

MgCl2, 0.4 lL untagged

primer, 0.2 lM tagged

primer, 0.2 lM Universal

tag M13R, 200 lM dNTP,

1 U of Immolase

95 �C for 7 min; 95 �C for

3 min; 20 cycles: 95 �C

for 30 s, 65 �C

(Touchdown -0.5 �C

each cycle) for 30 s,

72 �C for 45 s; 20 cycles:

95 �C for 30 s, 55 �C for

30 s, 72 �C for 30 s;

72 �C for 10 min

Microsatellite markers unravel the population

123

Data analysis

Population structure was analysed with a Principal

Coordinate Analysis (PCoA) based on the dissimilarity

matrix between genotypes, using DARwin version 5.0

(Perrier and Jacquemmoud-Collet 2006; Perrier et al.

2003). Dissimilarities were calculated using the simple

matching indices for allelic data. To further understand

the genetic structure, we used a Bayesian approach to

estimate the number of genetic clusters. This model-

based analysis was run with the program STRUCTURE

version 2.3.3 (Pritchard et al. 2000) using a batch-ori-

ented web program package for construction of super

matrices ready for phylogenomic analyses (Kumar et al.

2009). We ran 10 replicates for each K value ranging

from 1 to 10 with a burn-in length of 50,000 followed by

500,000 iterations of each chain using the admixture

model along with the assumption of correlated allele

frequencies between groups (Falush et al. 2003).

STRUCTURE then partitioned individuals of the sample

according to the membership coefficient Q, that ranges

from 0 (lowest affinity to the group) to 1 (highest affinity

to a group), across K groups. The optimal value of

K was determined by examining both Ln P(D) and DK,

an ad hoc quantity related to the second order rate of

change of the log probability of data with respect to the

number of clusters (Evanno et al. 2005). Graphics of

STRUCTURE results were produced using DISTRUCT

1.1 (Rosenberg 2004).

The software GENETIX 4.02 (Belkhir et al. 2002) was

used to calculate the mean number of alleles, the observed

and expected mean heterozygosities (Hobs and Hexp,

respectively) and Wright’s indexes, namely the inbreeding

coefficient (Fis) and the fixation index (Fst) under the null

hypothesis of Hardy–Weinberg (HW) equilibrium. The

total expected heterozygosity or genetic diversity (HT) was

determined using the unbiased estimate method of Nei

(1987); Fst and Fis values were calculated following Weir

and Cockerham (1984).

The number of distinct multilocus genotypes (MLGs)

present within islands and within populations was deter-

mined using the software GenoType (Meirmans and Van

Tienderen 2004). In order to assess the exact number of

MLGs in the dataset, we chose a threshold of zero, i.e. no

difference allowed between clonemates.

Arlequin 3.5 (Excoffier and Lischer 2010; Excoffier

et al. 1992) was used to (1) calculate the partition of

genetic variability within and among populations with

Analysis of Molecular Variance (AMOVA) and (2) esti-

mate gene flow among populations.

The population matrix is available at DEMIURGE

(http://www.demiurge-project.org/) with digest code

D-NMICR-86.

Results

The initial factorial analysis applied to the entire sample of

Leontodon revealed that L. rigens and L. filii clustered

separately, except for the Faial populations of L. filii which

appeared grouped with L. rigens (data not shown).

Due to this unexpected spatial distribution in the fac-

torial analysis, we proceeded with the Bayesian analysis.

The maximum value of Ln P(D), the posterior probabilities

of the data for each K, was reached for K = 10, but the

plateau started at K = 2 (Fig. 2). Variance across runs was

very low for K = 1 and K = 2 and increased for the values

of K C 3. We observed one peak of DK at K = 2. Taking

into consideration the variation across runs, the partitioning

of individuals and the Fst = 0.23, which indicates a high

level of differentiation, we assume K = 2 as the most

likely value for K. Regarding the partitioning of individu-

als, clusters for K = 2 exhibit a pattern that is consistent

with the two currently accepted taxonomic units, i.e. L.

rigens and L. filii, with the exception of L. filii from Faial

Island which clustered with L. rigens (Fig. 3). The results

obtained with STRUCTURE (Fig. 3) were in accordance

with those obtained with DARwin.

Additionally, PCoAs were performed with DARwin

on each of the two main groups identified so as to

further investigate possible subgroups. Within the genetic

cluster comprising all L. rigens populations and popu-

lations of L. filii from Faial (Fig. 4), the samples from

Sao Miguel exhibited considerable variation and were

mainly distributed in the left side of the plot, somewhat

separated from those from Faial. A small number of

individuals from Sao Miguel plotted near the samples

from Corvo and Flores. Other individuals from Flores

are located on the right side of the plot along with a

number of accessions from Faial. However, several of

the samples from Faial plotted separately although they

did not belong to a specific population. Regarding the

genetic cluster including L. filii (except samples from

Faial) most accessions from Sao Jorge plotted together,

along with some populations from Pico and Terceira.

However, Pico accessions from Cabeco do Coiro, a

secluded population in the rim and crater of a parasitic

volcanic cone located in the central-south part of the

island, grouped separately, as did those from Rocha do

Chambre, a narrow valley located in northwestern side

of Terceira Island (Fig. 5).

Only one population of L. filii was found in Sao Miguel

and Flores, both with small number of individuals (three

and two, respectively). Consequently, these populations

were not included in subsequent analysis.

A total of 98 alleles were found for L. rigens and L. filii

from Faial, with a total gene diversity (HT) of 0.65 and an

overall excess of homozygotes (multilocus Fis = 0.37). L.

E. F. Dias et al.

123

filii displayed a total of 52 alleles, HT = 0.53 and a mul-

tilocus Fis of 0.07. A similar proportion of rare alleles was

obtained for both groups (80.6 % for L. rigens and L. filii

from Faial and 76.9 % for L. filii), with the highest values

occurring in the islands of Sao Miguel and Flores for L.

rigens and of Pico for L. filii (Table 5). Populations of L.

rigens from Sao Miguel and those of L. filii from Terceira

displayed the highest numbers of multilocus alleles for each

group (57 and 36 alleles, respectively) with an excess of

homozygotes (Fis = 0.51 and Fis = 0.19, respectively;

Table 5). In Flores Island, the multilocus Fis value obtained

for L. rigens (0.02) also suggests that the populations follow

HW equilibrium, while for the populations from Corvo and

Faial, an excess of homozygotes was calculated (Table 5). In

contrast, values of multilocus Fis for L. filii in Sao Jorge and

Pico islands indicated an excess of heterozygotes (although

very close to equilibrium in Pico), although mixed locus-

specific Fis values were again obtained (Table 5).

The allelic richness varied from an average of 3 alleles

in Sao Jorge up to 10 alleles in Sao Miguel. The island with

the highest percentage of private alleles is Pico (45 %), and

the island with the lowest percentage is Sao Jorge with only

18.8 % (Table 6).

Particular allelic patterns appeared in Corvo in locus

LR4B08 and LR4B01 and in Faial a large range of alleles

is present at locus LR2A02. The dominant alleles were

similar in all the islands for the genetic cluster 2, while in

the genetic cluster 1 the diversity of dominant alleles was

higher (data not shown).

In order to further evaluate the genetic diversity within

populations and within islands, the number of distinct

multilocus genotypes (MLGs) was determined (Table 6).

With our set of markers, L. filii exhibited 168 MLGs out of

226 individuals sampled, while L. rigens exhibited 191 out

of 212 individuals sampled. Generally, for both L. rigens

and L. filii, the distinct MLGs index (MLGs/N) was either 1

or close to it.

AMOVA analyses applied to the groups defined above

showed that the largest proportion of genetic variation was

found within populations (73 and 79 %, for L. rigens with

L. filii from Faial and L. filii, respectively; Tables 7, 8).

Average Fst values calculated for both groups denoted a

considerable overall differentiation among populations

(0.27 and 0.21, respectively). Gene flow values calculated

between islands for L. filii indicate a high number of

migrants within the central group, with the highest value

obtained between Pico and S. Jorge (Table 9).

The values obtained for Fis, private alleles and the dis-

tinct MLGs index were compared to determine which

populations should have priority for conservation, namely

Fig. 2 Methodology from

Evanno et al. (2005) for the

interpretation of STRUCTURE

results. a Median Ln(K),

b Ln0(K), c Ln0 0(K) and

d Median Delta K are presented

for each value of K. The peak of

Median DK at K = 2 indicate

one putative right value for K

Fig. 3 Graphical display of the STRUCTURE output. Individuals are

represented as thin vertical lines partitioned into segments corre-

sponding to their membership in genetic clusters indicated by

different shades of grey, * marks the populations of L. filii that

cluster with L. rigens. All populations starting with LF belong to

Leontodon filii and LR to L. rigens

Microsatellite markers unravel the population

123

the populations that showed (1) low distinct MLGs index;

(2) strongly positive Fis values, resulting in a homozygotes

excess; and (3) a higher proportion of private alleles

reflecting unique genepools were considered to be in need

of restoration measures. The localizations of these popu-

lations were represented on a map of the archipelago

(Fig. 1).

Discussion

Genetic structure

Our study confirmed the existence of two main genetic

groups in the archipelago: one composed of L. rigens

individuals occurring in S. Miguel, Flores and Corvo, plus

all the L. filii from Faial; and the second was composed of

L. filii individuals occurring in S. Miguel, Terceira, Pico, S.

Jorge and Flores.

Please check and confirm the edit in the following:

Leontodon rigens from S. Miguel, possesses the highest

number of alleles; it is, therefore, possible that in the past,

the Azorean archipelago held a single ancestral endemic

species of Leontodon, which later evolved by allopatric

speciation. Sao Miguel is the second oldest island in the

archipelago (4.01 Myr; Franca et al. 2003), and in this

scenario dispersal and colonization are likely to have

occurred from east to west. The absence of L. rigens on

Terceira could be the result of never having dispersed

there, despite the geographic location and the age of the

island. Alternatively, it may reflect the destruction of pre-

vious populations by extensive volcanic activity without

subsequent reestablishment (Calvert et al. 2006). Such

events occurred throughout the archipelago. They

Fig. 4 PCoA performed on

simple matching dissimilarities

matrix obtained from the

molecular data matrix of L.

rigens s. l. Factors 1 and 2

represent 25.2 % of the total

variance observed. Legend: Sao

Miguel black squares, Corvo

white squares, Faial crosses,

and Flores triangles

E. F. Dias et al.

123

contributed to the destruction of large areas of vegetation

and have been hypothesized to explain the intra-archipel-

ago genetic characteristics of other Azorean taxa, namely

Juniperus brevifolia (Silva et al. 2011). Alternatively, both

species could have emerged from a common ancestor, now

extinct.

Human intervention in the native vegetation has also

been highly disruptive throughout time and is the most

probable explanation for the extinction of the endemic

Leontodon species on the islands of Santa Maria and

Graciosa. These islands have suffered an intense human

impact and the general low altitude topography led to a

deficiency of habitat refugia where the species could have

survived until the present days. According to our data, L.

rigens is also present in Flores and Corvo. Flores is the

oldest island of the Western group (2.16 Myr; Franca et al.

2003) and one of the oldest within the archipelago; thus an

early colonization of Flores by L. rigens was possible.

Flores accessions separated into two disjoint subgroups:

one composed mainly of individuals from of Lagoa Seca

caldera in Flores altogether with all accessions from Corvo,

and the second by a mix of Flores and some of Faial’s

accessions (Fig. 4). Accessions from Flores, Corvo toge-

ther with some accessions from Faial appear as showing

individuals that are somewhat intermediate, between

accessions from Sao Miguel at one extreme and other

accessions from Faial at the other. Faial is the westernmost

island of the central group. It is younger than Flores (0.73

Myr; Franca et al. 2003), and might have been colonized

Flores with later gene flow between populations on the two

islands (see Table 9). This could explain the similarities

found with several Flores accessions. The topography of

Faial’s Caldeira and the presence of Pico Mountain, as an

important geographical barrier (2,351 m asl), are some

factors that might explain the uniqueness of the Faial

Island’s populations in the central group. However,

according to our results, gene flow might also exist

between Faial and Terceira (Table 9). Both the population

Fig. 5 PCoA performed on

simple matching dissimilarities

matrix obtained from the

molecular data of L. filii cluster.

Factors 1 and 2 represented

30.41 % of the total variance

observed. Legend: Sao Jorge

dots, Pico squares, and Terceira

triangles

Microsatellite markers unravel the population

123

Ta

ble

5N

um

ber

of

gen

oty

ped

ind

ivid

ual

s(N

),n

um

ber

of

alle

les,

Hobs,

Hexp,

Fis

and

per

cen

tag

eo

fra

real

lele

s(f

req

uen

cyq\

0.0

5),

for

each

locu

so

fth

eL

.ri

gen

sp

lus

L.

fill

ifr

om

Fai

al

(Clu

ster

1)

and

L.

fili

i(C

lust

er2

)p

op

ula

tio

ns

dis

trib

ute

din

the

sev

enis

lan

ds

of

the

Azo

res

arch

ipel

ago

Clu

ster

sIs

lan

dN

LR

2A

02

LR

2A

03

LR

4B

01

All

eles

Hexp

Hobs

Fis

Rar

eal

lele

s(%

)A

llel

esH

exp

Hobs

Fis

Rar

eal

lele

s(%

)A

llel

esH

exp

Hobs

Fis

Rar

eal

lele

s(%

)

1S

.M

igu

el1

18

11

0.3

10

.13

0.5

88

1.8

70

.22

0.1

10

.50

71

.41

40

.83

0.4

70

.44

57

.1

Flo

res

54

40

.09

0.0

9-

0.0

26

6.7

90

.64

0.8

8-

0.3

77

7.8

11

0.6

90

.65

0.0

67

2.7

Co

rvo

11

20

.42

0.5

5-

0.3

30

.03

0.4

50

.55

-0

.21

0.0

90

.89

0.5

50

.40

0.0

Fai

al2

41

20

.80

0.8

8-

0.0

95

0.0

50

.61

0.7

5-

0.2

54

0.0

50

.61

0.4

60

.25

40

.0

Ov

eral

l2

07

18

0.3

40

.22

0.3

69

4.7

16

0.4

80

.41

0.1

68

7.5

24

0.8

40

.53

0.3

77

9.2

2T

erce

ira

55

60

.42

0.2

90

.31

50

.07

0.7

10

.64

0.1

15

7.1

70

.72

0.5

30

.27

28

.6

S.

Jorg

e5

33

0.0

90

.09

-0

.03

66

.76

0.5

90

.83

-0

.41

50

.04

0.5

70

.68

-0

.19

25

.0

Pic

o1

17

50

.29

0.0

50

.82

60

.07

0.5

90

.87

-0

.47

57

.13

0.4

80

.59

-0

.23

33

.3

Ov

eral

l2

25

80

.28

0.1

20

.57

75

.01

00

.64

0.8

0-

0.2

57

0.0

90

.58

0.6

0-

0.0

37

7.8

Clu

ster

sIs

lan

dN

LR

4B

08

LR

4B

10

Mu

ltil

ocu

s

All

eles

Hexp

Hobs

Fis

Rar

eal

lele

s(%

)A

llel

esH

exp

Hobs

Fis

Rar

eal

lele

s(%

)A

llel

esH

TH

obs

Fis

Rar

eal

lele

s(%

)

1S

.M

igu

el1

18

16

0.7

20

.26

0.6

46

2.5

90

.67

0.3

80

.44

55

.65

70

.55

0.2

70

.51

64

.9

Flo

res

54

14

0.7

20

.68

0.0

67

8.6

90

.72

0.5

00

.31

33

.34

70

.57

0.5

60

.02

67

.4

Co

rvo

11

70

.87

0.3

60

.59

0.0

20

.52

0.9

1-

0.8

20

.02

30

.63

0.5

80

.08

0.0

Fai

al2

46

0.7

20

.46

0.3

76

6.7

70

.83

0.7

10

.15

14

.33

50

.71

0.6

50

.09

42

.9

Ov

eral

l2

07

25

0.7

80

.41

0.4

88

4.0

12

0.8

10

.48

0.4

15

0.0

98

0.6

50

.41

0.3

78

0.6

2T

erce

ira

55

90

.75

0.6

00

.20

44

.47

0.7

40

.64

0.1

42

8.6

36

0.6

70

.54

0.1

94

1.7

S.

Jorg

e5

33

0.5

10

.57

-0

.11

33

.34

0.3

20

.13

0.5

90

.02

00

.42

0.4

6-

0.1

12

8.6

Pic

o1

17

50

.49

0.6

0-

0.2

24

0.0

11

0.4

60

.27

0.4

37

2.7

31

0.4

60

.48

-0

.03

58

.1

Ov

eral

l2

25

12

0.5

80

.59

-0

.01

83

.31

30

.54

0.3

20

.40

76

.95

20

.53

0.4

90

.07

76

.9

Clu

ster

sw

ere

defi

ned

reg

ard

less

of

tax

on

om

ictr

eatm

ent

E. F. Dias et al.

123

Table 6 Allelic richness (A) and percentage of private alleles (P) per island, number of individuals (N), number of distinct multilocus genotypes

(MLGs) and clonal richness (MLGs/N) per population, calculated for the two genetic clusters obtained

Clusters Island Taxon A P Populations N MLGs MLGs/N

1 S. Miguel L. rigens 11 38.6 SMLC 10 10 1.00

SMLA 6 5 0.83

SMOL 10 10 1.00

SMEC 5 5 1.00

SMPR 5 5 1.00

SMEM 10 9 0.90

SMLF 17 16 0.94

SMAL 10 10 1.00

SMLP 12 11 0.92

SMME 3 3 1.00

SMPG 3 3 1.00

SMTR 10 7 0.70

SMVR 11 10 0.91

SMPB 6 6 1.00

Flores L. rigens 9 23.9 FLBU 10 6 0.67

FLLS 11 10 0.91

FLCB 9 6 0.67

FLPD 5 4 0.80

FLMA 9 8 0.89

FLCL 10 9 0.90

Corvo L. rigens 4 42.8 COMC 11 10 0.91

Faial L. filii 6 21.4 FAGS 8 8 1.00

FATR 10 10 1.00

FAEC 6 6 1.00

2 Terceira L. filii 6 27.6 TERC 9 9 1.00

TEAC 9 9 1.00

TECA 1 1 1.00

TESB 8 8 1.00

TESL 1 1 1.00

TEPB 8 7 0.88

TEPG 10 10 1.00

TEGE 9 8 0.89

S. Jorge L. filii 3 18.8 SJFS 8 6 0.75

SJMP 13 10 0.77

SJPE 10 8 0.80

SJPA 10 10 1.00

SJSE 12 10 0.83

Pico L. filii 4 45 PICO 15 15 1.00

PICM 7 6 0.86

PIRE 2 2 1.00

PIRB 2 2 1.00

PICV 14 8 0.57

PILO 12 5 0.42

PILP 16 8 0.50

PIMI 14 4 0.29

PIBM 15 5 0.33

PIRF 6 4 0.67

PITT 15 12 0.80

Microsatellite markers unravel the population

123

of Rocha do Chambre in Terceira and all of the populations

sampled from Faial exhibit somewhat different genetic

characteristics. Furthermore, the characteristics of the later

lead to clustering with L. rigens accessions, while the

Terceira population slightly separated from the remaining

L. filii. The restricted locations of the population of Rocha

do Chambre in Terceira and Caldeira in Faial coupled with

a founder effect could have contributed to the convergence

of genetic characteristics, reflected in our results by the

estimation of possible gene flow. In fact, the difference

observed between the dominant alleles present in Cluster 2

(Table 5) could indicate the occurrence of founder effects.

It is thus possible that both locations were colonized by an

early L. filii form which later evolved in less circumscribed

areas but remained as a relict in the caldera of Faial and

Rocha do Chambre valley. Another possibility would be a

stochastic event of genetic convergence derived from

genetic drift.

Regarding L. filii, its origin in the archipelago likely

resulted from a recent speciation event, perhaps in the

oldest islands of the central group already colonized by L.

rigens and due to an easy interisland dispersal became the

dominant taxon in that group. The geographical location of

S. Jorge Island, between Terceira and Pico, is reflected in

our analysis by the high gene flow values and the proximity

in the multifactorial analysis between several accessions

ensuing from these islands, particularly with Pico. As with

Rocha do Chambre in Terceira and Faial’s Caldeira, the

slight differentiation revealed by the population of Cabeco

do Coiro in Pico might result from specific geographical

conditions, namely its location inside the narrow caldera of

a small volcanic cone.

Genetic diversity

The total genetic diversity obtained in our study for L.

rigens and L. filii is much higher than the allozyme

diversity values reviewed by Francisco-Ortega et al.

(2000), for the Canary Islands, and for most of the island

endemics reviewed by Franks (2010). Even if this is

expected since microsatellites are more variable markers

than allozymes (Hedrick 1999), the values obtained can

also be considered high by microsatellite marker standards

for silverswords in Hawaii (Friar and Ladoux 2000; Friar

et al. 2001, 2007).

Since the Azorean islands are much younger than sev-

eral islands of the Canarian archipelago (Schaefer 2003), it

is probable that Leontodon does not constitute an old

lineage that took refuge from Quaternary glaciations that

occurred in Europe, although studies with genus Pericallis

have indicated that the Azorean endemic species P. mal-

vifolia radiated on the Azores about 3 Myr ago, before

other genus radiations occurred in the Canary Islands

(Carine and Schaefer 2010). Another hypothesis put for-

ward by Francisco-Ortega et al. (2000) for the high genetic

diversity of the Canary Islands endemic flora relates to the

proximity of the archipelago to the African mainland which

probably facilitated multiple introductions of continental

taxa. However, the Azores are located in the mid Atlantic

Ocean and thus dispersal from continental land masses is

more difficult (Bellemain and Ricklefs 2008). Therefore, it

is more likely that, as with the Cyprus endemic Cedrus

brevifolia (Eliades et al. 2011), the high genetic diversity

obtained for the Azorean Leontodon indicates that it

Table 7 Analysis of molecular variance applied to 27 populations of

L. rigens from Sao Miguel, Flores, and Corvo and L. filii from Faial

Source of variation df Sum

squares

Variance of

component

Percentage of

variation

Among group 3 81,800 0.26687 Va 15.17

Among populations

within groups

21 100,021 0.2147 Vb 12.2

Within populations 393 502,312 127,815 Vc 72.63

Total 417 684,134 175.971

Groups corresponded to islands

Table 8 Analysis of Molecular Variance applied to 24 populations of

L. filii from Pico, Sao Jorge and Terceira. Groups corresponded to

islands

Source of variation df Sum

squares

Variance of

component

Percentage of

variation

Among group 2 29.545 0.06516 Va 4.78

Among populations

within groups

21 108.090 0.22212 Vb 16.28

Within populations 393 458.719 107.680 Vc 78.94

Total 449 596.353 136.408

Table 9 Gene flow values calculated between all islands of the

archipelago and for the two genetic clusters obtained

Cluster 1 Cluster 2

S. Miguel Flores Corvo Faial Terceira S. Jorge

Cluster 1

Flores 1.5

Corvo 1.0 0.9

Faial 0.9 1.4 0.9

Cluster 2

Terceira 0.8 0.9 0.9 1.0

S. Jorge 0.5 0.5 0.4 0.5 2.5

Pico 0.5 0.5 0.5 0.6 2.2 11.2

E. F. Dias et al.

123

originated from a widespread congener species, which is

supported by the close phylogenetic proximity of L. rigens

to the widely-distributed L. hispidus (Samuel et al. 2006).

On the other hand, we also observed that genetic vari-

ation was mainly found within populations, which is not in

accordance with the results obtained for several Canary

Islands endemic species, where most genetic variability

resided among populations (Francisco-Ortega et al. 2000).

Furthermore, restricted gene flow between islands, a

common pattern found in other population genetics studies

with island endemics (Franks 2010), was not observed,

particularly within the Azorean central sub-archipelago.

Unexpectedly, gene flow was also found between L. filii

from Faial and L. rigens from Flores. These results indicate

that some populations of both species might eventually

produce hybrids. The difficulty in determining the genetic

affinities of Leontodon populations using solely morpho-

logical traits, particularly in the case of non-flowering

individuals, makes it inadvisable to use any individual as a

diaspore source unless molecularly screened by the present

work.

Conservation genetics

Due to the large scale of our study, a wealth of information

on many of the existing Leontodon populations in the

archipelago is now available and it is unquestionable that

the genetic patterns documented should be taken into

consideration for the establishment of species recuperation

plans. The uniqueness of some of the studied populations

and the low heterozygosity values obtained for others

indicate that extreme care should be taken regarding (1) the

populations that should be selected for conservation and (2)

the implementation of augmentation conservation

strategies.

When dealing with genetic conservation, allelic richness

is one of the most relevant criteria for measuring diversity

(Bataillon et al. 1996; Kremer 1994; Petit et al. 1998).

According to our results, allelic richness is higher in the

islands of S. Miguel, Flores, Terceira?? and Faial

(Table 6). Additionally, our analysis established that the

islands with a higher percentage of private alleles are

Corvo, S. Miguel, Terceira and Pico. They should be

considered as a priority for the implementation of conser-

vation measures. We also took into consideration Fis values

which reveal excesses of homozygotes, i.e. the populations

with positive Fis values are subjected to inbreeding which

can increase the possibilities of the next generations being

affected by recessive or deleterious characters (Whitlock

2002). The number of individuals of Leontodon that, with

our set of markers, exhibit identical MLGs is high in some

populations. As sampling was designed to avoid the col-

lection of clones, and notwithstanding the possibility of

infrequent apomixis (Noyes 2007), we assume that the

identical MLGs ensue from different mating events and

that their strong occurrence highlights the small number of

alleles involved in mating in those specific populations.

The compilation of the percentage of private alleles per

population, the rate of MLGs repetition within populations

and of the Fis values, shows that the need to implement

protective conservation measures is crucial for the entire

islands of Corvo and Faial, for five populations in Pico, two

in Terceira, one in Sao Jorge and eight in Sao Miguel

(Fig. 1).

The comparably high genetic specificity of populations

in Corvo and Faial along with the populations of Rocha do

Chambre in Terceira and Cabeco do Coiro in Pico suggests

that seeds or individuals from these islands should not be

used in conservation programmes on other populations as

they correspond to different genetic entities. Seeds or

plantlets issuing from S. Miguel should not be introduced

in Flores or Corvo, as evolutionary mechanisms are likely

to be in action in the western group. Furthermore, diaspores

collected in the central group should not be introduced into

either the western or the eastern groups. The maintenance

of ex situ collections of seeds might also be an important

conservation measure to preserve the unique gene pools of

the most differentiated populations (Moreira et al. 2009;

Martins et al. 2011). However, sampling of seeds should be

conducted with extreme care in all the population area so

that all genotypes are equally represented in the collection.

Reintroduction of the endemic Leontodon spp. in Santa

Maria and Graciosa should not be attempted since using

diaspores from populations existing in other islands might

result in stepping-stone dispersal effects and contamination

of natural populations with different genetic entities.

Considering that Asteraceae are mainly outcrossing

(Caujape-Castells et al. 2008), the existence of restricted

Leontodon spp. populations with a reduced number of

individuals, as in Corvo and Faial islands, might result in a

decrease in compatible mating types and thus increase the

risk of biparental inbreeding and inbreeding depression

(Francisco-Ortega et al. 2000; Caujape-Castells et al. 2008;

Perez de Paz and Caujape-Castells 2013). In these specific

cases, a genetic rescue program might be implemented

through an ex situ seed propagation protocol using seeds

from each population and a later reintroduction of plantlets

in the same populations of origin. Ex situ propagation

should be conducted under laboratory conditions instead of

open air nurseries or gardens to avoid contamination with

local populations.

Besides government imposed restrictions to interpopu-

lation mixture, other management measures should also be

undertaken, similarly to what is presently implemented in

the Canary Islands for endangered species, namely infor-

mation campaigns and training of roadside workers acting

Microsatellite markers unravel the population

123

in natural areas (Martın 2005), and most importantly, the

definition of in situ protection measures through the pres-

ervation of the species natural habitats. Specifically, there

is the need for the urgent eradication of invasive alien

species, which are increasingly common in the Azorean

native vegetation areas, plant species, such as Hedychium

gardnerianum Sheppard ex Ker Gawl., Erigeron karviski-

anus D.C. and Leycesteria formosa Wall. and animal spe-

cies, such as Oryctolagus cuniculus (Linnaeus 1758), all

previously listed as top invasive species in the Azores

(Silva et al. 2008). Government restrictions should also be

implemented to the use of native vegetation areas as

grazing land for cattle and goats. Commercial exploitation

of Azorean Leontodon plants and their use in roadsides and

gardens should also be restricted as it increases the chances

of genetic contamination of nearby natural populations.

In conclusion, this study contributed to generate data

fundamental to implementing an appropriate conservation

plan for Azorean endemic Leontodon by documenting

variability patterns and detecting populations in critical

need of protection. In addition, the improved understanding

of the population genetic diversity and structure and the

recognition of complex sub-structures will help future

morphological, phylogenetic and geographical studies

focused on the taxon circumscription and the evolution of

Leontodon in the Azores.

Acknowledgments This study was funded by DRCTC (Direccao

Regional da Ciencia, Tecnologia e Comunicacoes) and by FEDER

and was part of projects VERONICA and DEMIURGO (MAC PCT).

The Authors would like to thank Dr. Mark Carine for revising the

English version of the manuscript, and the staff at Secretaria Regional

do Ambiente and Servicos Florestais who contributed during the

collection of some of the material used in the study.

References

Azores Biodiversity Portal 2008. http://www.azoresbioportal.angra.

uac.pt

Bataillon TM, David JL, Schoen DJ (1996) Neutral genetic markers

and conservation genetics: simulated germplasm collections.

Genetics 144:409–417

Belkhir K, Borsa P, Chikhi L, Raufasre N, BonHomme F (2002)

Genetix 4.04, Logiciel sous Windows TM pour la genetique des

populations. Laboratoire Genome, Populations, Interations.

CNRS UMR5000. Universite de Montpellier II, France

Bellemain E, Ricklefs RE (2008) Are islands the end of the

colonization road? Trends Ecol Evol 23(8):461–468

Bogler DJ (2006) Leontodon Linnaeus. In: Flora of North America

Editorial Committee (eds) Flora of North America. Magno-

liophyta: Asteridae, part 6: Asteraceae, part 1, vol 19. Oxford

University Press, New York, pp 294–296

Borges A, Rosa MS, Recchia GH, Queiroz-Silva JR, Bressan EA,

Veasey EA (2009) CTAB methods for DNA extraction of

sweetpotato for microsatellite analysis. Sci Agric 66(4):529–534

Brownstein MJ, Carpten JD, Smith JR (1996) Modulation of non-

templated nucleotide addition by Taq DNA polymerase: primer

modifications that facilitate genotyping. Biotechniques

20:1004–1010

Calvert AT, Moore RB, McGeehin JP, Silva AMR (2006) Volcanic

history and 40Ar/39Ar and 14C geochronology of Terceira Island,

Azores, Portugal. J Volcanol Geoth Res 156:103–115

Carine MA, Schaefer H (2010) The Azorean diversity enigma: why

are there so few Azorean endemic flowering plants and why are

they so widespread? J. Biogeogr 37:77–89

Caujape-Castells J, Marrero-Rodrııguez A, Baccarani-Rosas M,

Cabrera-Garcıa N, Vilches-Navarrete B (2008) Population

genetics of the endangered Canarian endemic Atractylis arbus-

cula (Asteraceae): implications for taxonomy and conservation.

Plant Syst Evol 274:99–109

Crawford DJ, Archibald JK, Santos-Guerra A, Mort ME (2006)

Allozyme diversity within and divergence among species of Tolpis

(Asteraceae-Lactuceae) in the Canary Islands: systematic, evolu-

tionary and biogeographical implications. Am J Bot 93(4):656–664

Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991)

‘Touchdown’ PCR to circumvent spurious priming during gene

amplification. Nucleic Acids Res 19:4008

Doyle JJ, Dickson EE (1987) Preservation of plant samples for DNA

restriction endonuclease analysis. Taxon 36:715–722

Eliades N-GH, Gailing O, Leinemann L, Fady B, Finkeldy R (2011)

High genetic diversity and significant population structure in

Cedrus brevifolia Henry, a narrow endemic Mediterranean tree

from Cyprus. Plant Syst Evol 294:185–198

Evanno G, Regnault S, Goudet J (2005) Detecting the number of

clusters of individuals using the software structure: a simulation

study. Mol Ecol 14(8):2611–2620

Excoffier L, Lischer HEL (2010) Arlequin suite ver. 3.5: a new series

of programs to perform population genetics analyses under

Linux and Windows. Mol Ecol Resour 10:564–567

Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular

variance inferred from metric distances among DNA haplotypes:

application to human mitochondrial DNA restriction data.

Genetics 131:479–491

Faircloth BC (2008) MSATCOMMANDER: detection of microsat-

ellite repeat arrays and automated, locus-specific primer design.

Mol Ecol Resour 8:92–94

Falush D, Stephens M, Pritchard JK (2003) Inference of population

structure using multilocus genotype data: linked loci and

correlated allele frequencies. Genetics 164(4):1567–1587

Franca Z, Cruz JV, Nunes JC, Forjaz VH (2003) Geologia dos

Acores: uma perspectiva actual. Acoreana 10(1):11–140

Francisco-Ortega J, Santos-Guerra A, Kim S-C, Crawford DJ (2000)

Plant genetic diversity in the Canary Islands: a conservation

perspective. Am J Bot 87(7):909–919

Franks SJ (2010) Genetics, evolution and conservation of islands

plants. J Plant Biol 53:1–9

Friar E, Ladoux T (2000) Microsatellite analysis of a population crash

and bottleneck in the Mauna Kea silversword, Argyroxiphium

sandwicense ssp. sandwicense (Asteraceae), and its implications

for reintroduction. Mol Ecol 9:2027–2034

Friar EA, Boose DL, LaDoux T, Roalson EH, Robichaux RH (2001)

Population structure in the endangered Mauna Loa silversword,

Argyroxiphium kauense (Asteraceae), and its bearing on rein-

troduction. Mol Ecol 10(7):1657–1663

Friar EA, Cruse-Sanders JM, McGlaughlin ME (2007) Gene flow in

Dubautia arborea and D. ciliolata: the roles of ecology and

isolation by distance in maintaining species boundaries despite

ongoing hybridization. Mol Ecol 16(19):4028–4038

Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci.

Method Enzymol 395:202–222

Hedrick PW (1999) Perspective: highly variable loci and their

interpretation in evolution and conservation. Evolution

53(2):313–318

E. F. Dias et al.

123

Kremer A (1994) Diversite genetique et variabilite des caracteres

phenotypiques chez les arbres forestiers. Gen Sel Evol

26(1):105–123

Kumar S, Skjæveland A, Orr RJS, Enger P, Ruden T, Mevik B, Burki

F, Botnen A, Shalchian-Tabrizi K (2009) AIR: a batch-oriented

web program package for construction of supermatrices ready

for phylogenomic analyses. BMC Bioinformatics 10:357

Lack H (1981) Die Lactuceae (Asteraceae) der azorischen Inseln.

Willdenowia 11:211–247

Lance SL, Light JE, Jones KL, Hagen C, Hafner JC (2010) Isolation

and characterization of 17 polymorphic microsatellite loci in the

kangaroo mouse, genus Microdipodops (Rodentia: Heteromyi-

dae). Conserv Genet Resour 2:139–141

Martın JL (2005) Guıa metodologica para la elaboracion de planes de

especies amenazadas de Canarias. Direccion General del Medio

Natural, Gobierno de Canarias

Martins J, Moreira O, Silva L, Moura M (2011) Vegetative

propagation of the endangered Azorean tree, Picconia azorica.

Arquipelago. Life Mar Sci 28:39–46

Martins J, Moreira O, Sardos J, Maciel MGB, Silva L, Moura M

(2013) Population genetics and conservation of the Azorean tree

Picconia azorica. Biochem Syst Evol 49:135–143

Meirmans PG, Van Tienderen PH (2004) GENOTYPE and GENO-

DIVE: two programs for the analysis of genetic diversity of

asexual organisms. Mol Ecol Notes 4:792–794

Moreira O, Martins J, Silva L, Moura M (2009) Propagation of the

endangered Azorean cherry Prunus azorica using stem cuttings

and air layering.Arquipelago. Life Mar Sci 26:09–14

Moreira O, Martins J, Sardos J, Maciel MGB, Silva L, Moura M

(2013) Population genetic structure and conservation of the

Azorean tree Prunus azorica (Rosaceae). Plant Syst Evol.

doi:10.1007/s00606-013-0829-3

Moura M, Dias EF, Silva L, Schaefer H, Carine MA (2010) Molecular

systematics and morphology of the endemic Leontodon in the

Azores islands. Communication. International Symposium

FloraMAC 2010. September 23–25, Ponta Delgada, Acores

Moura M, Silva L, Caujape-Castells J (2013) Population genetics in

the conservation of the Azorean shrub Viburnum treleasei Gand.

Plant Syst Evol. doi:10.1007/s00606-013-0836-4

Nei M (1987) Molecular evolutionary genetics. Columbia University

Press, New York

Noyes RD (2007) Apomixis in the Asteraceae: diamonds in the rough.

Funct Plant Sci Biotech 1(2):207–222

Oetting WS, Lee HK, Flanders DJ, Wiesner GL, Sellers TA, King RA

(1995) Linkage analysis with multiplexed short tandem repeat

polymorphisms using infrared fluorescence and M13 tailed

primers. Genomics 30:450–458

Paiva JAR, Ormonde J (1974) Sobre Thrincia carreiroi Gandoger e

Thrincia subglabra Gandoger. Bol Soc Brot 2(47):271–291

Perez de Paz J, Caujape-Castells J (2013) A review of the allozyme

data set for the Canarian endemic flora: causes of the high

genetic diversity levels and implications for conservation. Ann

Bot. Oxford University Press

Perrier X, Jacquemmoud-Collet JP (2006) DARwin software. http://

www.darwin.cirad.fr/darwin

Perrier X, Flori A, Bonnot F (2003) Data analysis methods. In:

Hamon P, Seguin M, Perrier X, Glaszmann JC (eds) Genetic

diversity of cultivated tropical plants. Enfield Sciences Publisher,

Montpellier, pp 43–76

Petit RJ, El Mousadik A, Pons O (1998) Identifying populations for

conservation on the basis of genetic markers. Conserv Biol

12:844–855

Pritchard JK, Stephens M, Donnelly P (2000) Inference of population

structure using multilocus genotype data. Genetics 155:945–959

Rosenberg N (2004) Distruct: a program for the graphical display of

population structure. Mol Ecol Notes 4:137–138

Rychlik W (1995) Selection of primers for polymerase chain

reactions. Mol Biotechnol 3:129–134

Samuel R, Gutermann W, Stuessy TF, Ruas CF, Lack HW,

Tremetsberger K, Talavera S, Hermanowsky B, Ehrendorfer F

(2006) Molecular phylogenetics reveals Leontodon (Asteraceae,

Lactuceae) to be diphyletic. Am J Bot 93:1193–1205

Schaefer H (2003) Chorology and diversity of the Azorean flora.

Dissertationes Botanicae 374:1–130

Schaefer H, Schonfelder P (2009) Smilax azorica, S. canariensis

(Smilacaceae) and the genus Smilax in Europe. Monografıas del

Instituto de Estudios Canarios 78:297–307

Schaefer H, Moura M, Maciel MGB, Silva L, Rumsey F, Carine MA

(2011) The Linnean shortfall in oceanic island biogeography: a

case study in the Azores. J Biogeogr 38(7):1345–1355

Schuelke M (2000) An economic method for the fluorescent labeling of

PCR fragments: a poor man’s approach to genotyping for research

and high-throughput diagnostics. Nat Biotechnol 18:233–234

Seubert M (1844) Flora Azorica quam ex collectionibus schedisque

Hochstetteri patris et filii elaboravit et tabulis XV propria manu

aeri incisis illustravit Mauritius Seubert. Adolphum Marcum,

Bonnae

Silva L, Ojeda Land E, Rodrıguez Luengo JL (2008) Invasive

terrestrial flora and fauna of Macaronesia: Top 100 in Azores,

Madeira and Canaries. ARENA, Ponta Delgada

Silva L, Martins M, Maciel G, Moura M (2009) Flora Vascular dos

Acores. Prioridades em Conservacao. Azorean Vascular Flora.

Priorities in Conservation. Amigos dos Acores & CCPA, Ponta

delgada

Silva L, Moura M, Schaefer H, Rumsey F, Dias EF (2010) List of

vascular plants (Tracheobionta). In: Borges PAV, Costa A,

Cunha R, Gabriel R, Goncalves V, Martins AF, Melo I, Parente

M, Raposeiro P, Rodrigues P, Santos RS, Silva L, Vieira P,

Vieira V (eds) A list of the terrestrial and marine biota from the

Azores. Princıpia, Cascais, pp 9–33

Silva L, Elias RB, Moura M, Meimberg H, Dias E (2011) Genetic

Variability and differentiation among populations of the Azorean

endemic gymnosperm Juniperus brevifolia: baseline information

for a conservation and restoration perspective. Biochem Genet

49:715–734

Weir BS, Cockerham CC (1984) Estimating F-statistics for the

analysis of population structure. Evolution 38:1358–1370

Whitlock MC (2002) Selection, load and inbreeding depression in a

large metapopulation. Genetics 160:1191–1202

Microsatellite markers unravel the population

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