Disrupted phylogeographical microsatellite and chloroplast DNA patterns indicate a vicariance rather...

ORIGINALARTICLE

Disrupted phylogeographicalmicrosatellite and chloroplast DNApatterns indicate a vicariance rather thanlong-distance dispersal origin for thedisjunct distribution of the Chileanendemic Dioscorea biloba (Dioscoreaceae)around the Atacama Desert

Juan Viruel1, Pilar Catalan1 and Jose Gabriel Segarra-Moragues2*

1Departamento de Agricultura y Economıa

Agraria, Escuela Politecnica Superior de

Huesca, Universidad de Zaragoza, C/Carretera

de Cuarte Km 1, E22071, Huesca, Spain,2Centro de Investigaciones sobre Desertificacion

(CIDE-CSIC-UV-GV), C/Carretera Moncada-

Naquera Km 4.5, E-46113, Moncada,

Valencia, Spain

*Correspondence: Jose Gabriel Segarra-

Moragues, Centro de Investigaciones sobre

Desertificacion (CIDE-CSIC-UV-GV), C/

Moncada-Naquera Km 4.5, E-46113, Moncada,

Valencia, Spain.

E-mail: [email protected]

ABSTRACT

Aim The Chilean endemic Dioscorea biloba (Dioscoreaceae) is a dioecious

geophyte that shows a remarkable 600 km north–south disjunction in the

peripheral arid area of the Atacama Desert. Its restricted present-day distribution

and probable Neogene origin indicate that its populations have a history linked to

that of the Atacama Desert, making this an ideal model species with which to

investigate the biogeography of the region.

Location Chile, Atacama Desert and peripheral arid area.

Methods Two hundred and seventy-five individuals from nine populations were

genotyped for seven nuclear microsatellite loci, and plastid trnL–F and trnT–L sequences

were obtained for a representative subset of these. Analyses included the estimation of

genetic diversity and population structure through clustering, Bayesian and analysis of

molecular variance analyses, and statistical parsimony networks of chloroplast

haplotypes. Isolation by distance was tested against alternative dispersal hypotheses.

Results Microsatellite markers revealed moderate to high levels of genetic

diversity within populations, with those from the southern Limarı Valley showing

the highest values and northern populations showing less exclusive alleles. Bayesian

analysis of microsatellite data identified three genetic groups that corresponded to

geographical ranges. Chloroplast phylogeography revealed no haplotypes shared

between northern and southern ranges, and little haplotype sharing between the

two neighbouring southern valleys. Dispersal models suggested the presence of

extinct hypothetical populations between the southern and northern ranges.

Main conclusions Our results are consistent with prolonged isolation of the

northern and southern groups, mediated by the life-history traits of the species.

Significant isolation was revealed at both large and moderate distances as gene flow

was not evident even between neighbouring valleys. Bayesian analyses of

microsatellite and chloroplast haplotype diversity identified the southern area of

Limarı as the probable area of origin of the species. Our data do not support recent

dispersal of D. biloba from the southern range into Antofagasta, but indicate the

fragmentation of an earlier wider range, concomitant with the Pliocene–Pleistocene

climatic oscillations, with subsequent extinctions of the Atacama Desert populations

and the divergence of the peripheral ones as a consequence of genetic drift.

Keywords

Desert, Dioscorea gr. Epipetrum, dispersal models, genetic diversity, genetic structure,

nuclear microsatellite, plastid haplotypes, South America, vicariance, yams.

Journal of Biogeography (J. Biogeogr.) (2012) 39, 1073–1085

ª 2011 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/jbi 1073doi:10.1111/j.1365-2699.2011.02658.x

INTRODUCTION

The geological and climate changes caused by two major events

of the Cenozoic in South America, the separation of Antarctica

from this subcontinent (Oligocene–Miocene) and the uplift of

the Andes (Miocene), strongly affected the distribution of

biomes in Chile (Gayo et al., 2005; Hinojosa & Villagran, 2005;

Hinojosa et al., 2006). The Andean uplift [between 40 and 8

million years ago (Ma)] also created a barrier to the easterly

tropical humid air masses, leading to the establishment of a

mediterranean-type climate in central Chile (Reynolds et al.,

1990). The composition and distribution of the Chilean

mediterranean flora and fauna have evolved under these new

climatic conditions, which are characterized by winter rain and

summer drought (Villagran, 2001; Hinojosa & Villagran, 2005;

Amico & Nickrent, 2009). Quaternary volcanism and glacia-

tion also affected this region and shaped the distribution of the

present flora (Villagran & Armesto, 1980; Villagran, 2001). The

occurrence and impact of the glaciers were variable along the

central Andes, and apparently favoured the existence of

different plant and animal refugia (Markgraf et al., 1995;

McCulloch et al., 2000).

The last great uplift of the Andean Cordillera in the Pliocene

(c. 4 Ma) isolated a variety of lowland taxa on both sides of the

Andes, and created new corridors for both temperate and

xerothermic organisms running in parallel to this mountain

range (Palma et al., 2005). The existence of parallel east Andes

and west Andes corridors has been documented for several

temperate and arid-adapted organisms that colonized the

southern latitudes from more northerly ones (Webb, 1991;

Rundel & Dillon, 1998; Katinas & Crisci, 2000; Palma et al.,

2005; Katinas et al., 2008), and also for migrations that

occurred in the opposite direction, as documented for some

Atacama plants (Hershkovitz et al., 2006; Luebert et al., 2009).

Due to their complex phylogeographical patterns, the migra-

tion pathway followed by some taxa is uncertain (e.g. the

central Chilean parasitic plants Tristerix spp., Amico &

Nickrent, 2009). The orogeny of the central Andes and

oscillation of Pleistocene glacial cycles further contributed to

the expansion of the Atacama and coastal deserts on the

western side of the Andean divide (Palma et al., 2005).

However, palaeoclimatic evidence supports the existence of

both Pleistocene and Holocene pluvial phases in the Atacama

Desert (Sylvestre et al., 1999; Betancourt et al., 2000; Latorre

et al., 2003), which may have facilitated the dispersal or

survival of organisms less adapted to current arid ecological

conditions.

The Atacama Desert in northern Chile extends for more

than 1000 km along the western coast of South America (Pinto

et al., 2006) and is considered one of the most arid and

inhospitable places on earth (Houston & Hartley, 2003; Clarke,

2006). The hyper-aridity of this area dates from the Miocene

(24 Ma; Clarke, 2006) or the Pliocene (Placzek et al., 2009),

although the aridity may have been alleviated by periodical

Pleistocene and Holocene rains (Sylvestre et al., 1999; Placzek

et al., 2009). This aridity shows a gradient from the most arid

northern areas (Arica, 0.5 mm annual precipitation) to the

more humid southern range (Copiapo, 12 mm) (Clarke,

2006), but with some areas showing drought periods of more

than 20 years (Rundel et al., 1991). The hyper-aridity pre-

cludes most vegetation, although coastal mountain ranges may

benefit from extra moisture provided by sea mists (Pinto et al.,

2006), favouring the development of patchy and highly diverse

plant communities that include a large number of local

endemics (Dillon et al., 2009).

Several hypotheses have been proposed to explain the

biogeography of plant species along the Atacama aridity

gradient. One widely accepted theory proposes a south-to-

north latitudinal evolutionary sequence in which species

tolerant of aridity evolved from ancestors in temperate

southern or central Chile [e.g. Nolana (Solanaceae) Dillon

et al., 2009; Oxyphyllum (Asteraceae) Luebert et al., 2009;],

with such taxa probably taking advantage of foggy coastal areas

during a northward expansion. However, the distributions of

other taxa, especially those showing north–south disjunctions

across the Atacama Desert, require alternative explanations.

Long-distance dispersal from central Chile to the northern

edge of the Atacama Desert and onwards may have occurred in

some cases [e.g. Larrea (Zygophyllaceae) Lia et al., 2001;

Hoffmannseggia (Leguminosae) Simpson et al., 2005;]; but

other taxa with disjunct distributions may be the result of

fragmentation of an ancient, more widespread distribution

range following the establishment of arid conditions (Pen-

nington et al., 2004; Lopez et al., 2006; Albrecht et al., 2010).

The central Chilean endemic Dioscorea gr. Epipetrum (=

Epipetrum Phil.) is composed of two species: Dioscorea biloba

(Phil.) Caddick & Wilkin and Dioscorea humilis Colla (Viruel

et al., 2008, 2010a). Dioscorea biloba has a disjunct north–

south distribution, with two population clusters separated by

more than 600 km, each located close to the southern and the

northern peripheries of the Atacama Desert (Fig. 1). These

populations have been recognized as two subspecies by Viruel

et al. (2010a): the northerly D. biloba subsp. biloba is currently

known from three populations all located in the narrow coastal

mountain range of Tal-Tal (Antofagasta region; Table 1,

Fig. 1), while the southerly D. biloba subsp. coquimbana

comprises six populations distributed in two adjacent valleys at

Limarı and Choapa (Coquimbo region; Table 1, Fig. 1). Both

southern valleys show a more temperate and humid climate

than Antofagasta.

Dioscorea biloba is a diploid dioecious geophyte (Viruel

et al., 2008, 2010a). The very few male and female flowers

produced are very inconspicuous, and the pollination mech-

anism is unknown. Fruit capsules develop in rock crevices due

to the plant’s sprawling habit and, unlike the majority of

Dioscorea species, seeds are wingless.

Current biogeographical research using various molecular

markers and broad taxon sampling has revealed that Dioscorea

gr. Epipetrum is an early branching lineage within the South

American Dioscoreaceae, with an origin at the end of the

Miocene (c. 5.5 Ma; J.V. et al., unpublished data), and so most

likely pre-dating the establishment of the Atacama Desert.

J. Viruel et al.

1074 Journal of Biogeography 39, 1073–1085ª 2011 Blackwell Publishing Ltd

Using spatial structure and dispersal modelling analyses of

nuclear microsatellite markers, we aimed to determine whether

the disjunct distribution of D. biloba is the result of fragmen-

tation of a wider ancient distribution or the result of more

recent long-distance dispersal events. We also used Bayesian

estimation of population structure and parsimony analysis of

chloroplast (cp) haplotype networks to infer a probable place

of origin for the species.

Cluster 1

Cluster 2

Eb8

Cluster 1

Cluster 2

Cluster 3

(a) (b)

At

Co

Val

0°

10°S

20°S

30°S

40°S

80°W

70°W

60°W

50°W

40°W

30°W

90°W

50°S

10°N

Db7

Db9Db8

Db5

Db6

Db1Db2

Db3Db4

100 km

Ant

Paci

fic O

cean

Arg

entin

a

25°S

29°S

Figure 1 Geographical distribution of sam-

pled populations of Dioscorea biloba

(Table 1) in Chile and Bayesian analyses of

the genetic structure of nine populations

of D. biloba based on nuclear microsatellite

data. The mean proportion of membership of

each predefined population to each inferred

genetic cluster is shown. (a) K = 2 and

(b) K = 3 clusters. Chilean administrative

regions: Ant, Antofagasta; At, Atacama; Co,

Coquimbo; Val, Valparaıso.

Table 1 Population data and genetic diversity indices in nine Chilean populations of Dioscorea biloba for seven microsatellite loci.

Populations Latitude Longitude

Elevation

(m a.s.l.)

Population

size n A HO HE FIS

Southern range

Db1: Coquimbo. Limarı valley,

road from Ovalle to La Aguada,

before Chalinga

30�43.964¢ S 71�22.836¢ W 250 < 500 26 5.86 0.661 0.651 )0.016***


road from Chalinga to La Aguada

30�47.875¢ S 71�27.056¢ W 240 < 200 30 6.00 0.676 0.691 +0.022ns


road from Parral de Quiles to San

Pedro de Quiles

30�57.630¢ S 71�28.417¢ W 650 < 200 30 6.43 0.686 0.706 +0.030**


road from Quenen to Maqui de

Quiles

31�03.920¢ S 71�31.534¢ W 550 < 100 26 7.29 0.617 0.651 +0.054*

Db5: Coquimbo. Choapa valley,

road from Los Vilos to

Illapel km10

31�51.938¢ S 71�23.895¢ W 90 < 300 41 5.14 0.345 0.458 +0.248***

Db6: Coquimbo. Choapa valley,

Canela Alta, next to Choapa river

31�23.249¢ S 71�24.975¢ W 330 < 500 28 5.86 0.371 0.483 +0.242***

Northern range

Db7: Antofagasta. Tal-Tal, Perales

Hill, road to the antenna

25�25.631¢ S 70�25.515¢ W 800 < 100 25 5.43 0.531 0.469 )0.135***



25�25.623¢ S 70�25.616¢ W 780 < 100 33 5.57 0.494 0.491 )0.006***



25�25.691¢ S 70�25.672¢ W 700 < 100 36 5.71 0.571 0.489 )0.171***

n, sample size; A, mean number of alleles per locus; HO, HE, observed and expected heterozygosity, respectively; FIS, inbreeding coefficient.

ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

Phylogeography of Dioscorea biloba in Atacama

Journal of Biogeography 39, 1073–1085 1075ª 2011 Blackwell Publishing Ltd

MATERIALS AND METHODS

Sampled populations, microsatellite genotyping

and plastid sequencing

All nine known populations of D. biloba were sampled (Fig. 1),

including 275 individuals [181 individuals from six southern

populations (Db1–Db6, D. biloba subsp. coquimbana) and 94

individuals from the three northern populations (Db7–Db9, D.

biloba subsp. biloba)].

Fresh leaves were dried on silica gel and later reduced to a

fine powder on a Mini-BeadBeater-8 cell disrupter (BioSpec,

Bartlesville, OK, USA). DNA was extracted using the DNeasy

Plant Minikit (Qiagen, Barcelona, Spain) and eluted in 50 lL

in TRIS-EDTA 0.1· buffer. Seven unlinked microsatellite loci

were amplified following Viruel et al. (2010b) and polymerase

chain reaction (PCR) products were analysed on an ABI 3730

DNA Analyzer automated sequencer (Applied Biosystems,

Madrid, Spain) using the LIZ500 (Applied Biosystems) inter-

nal lane size standard. Allele sizing was performed using

GeneMarker 1.80 software (Softgenetics, State College, PA,

USA).

Plastid trnL–F and trnT–L regions were analysed from a

subset of 52 and 25 individuals, respectively, representing all

populations. The trnL–F and trnT–L regions were amplified

using the ‘c’/’f’ and ‘a’/’b’ primer pairs and procedures of

Taberlet et al. (1991), respectively, and sequenced on an ABI

3730xl DNA Analyzer (Applied Biosystems) by Macrogen Inc.

(Seoul, Korea). Forward and reverse sequences were compared,

assembled and corrected using Sequencher 4.1.4 (GeneCodes

Corp., Ann Arbor, MI, USA). Sequences were deposited in

GenBank under the accession numbers HQ638381–HQ638395

for trnL–F and HQ638374–HQ638380 for trnT–L.

Microsatellite data analysis

Allele frequencies, mean number of alleles per locus (A), and

observed (HO) and unbiased expected (HE) heterozygosities

(Nei, 1978) were calculated for each population using genetix

v. 4.05 (Belkhir et al., 2004). Wright’s F-statistics were

estimated according to Weir & Cockerham (1984) using

genepop¢007 (Rousset, 2008) and departures from Hardy–

Weinberg (HW) equilibrium were tested for significance by

Fisher’s exact tests.

Population genetic structure was investigated using a

Bayesian clustering method implemented in structure 2.1

(Pritchard et al., 2000); this is an individual-based approach

that does not assume a priori membership to a population, and

which permits elucidation of the optimal number of genetic

clusters (K) and the assignment of individuals to the different

clusters. Analyses were based on an admixture ancestral model

with correlated allele frequencies (because of high FIS values in

some populations; see Results), for a range of K values from 2

to 11. The proportion of membership of each individual and

population to the K clusters was calculated. We performed 20

runs for each K with a burn-in period and a run length of the

Markov chain Monte Carlo (MCMC) of 1 · 105 and 1 · 106

iterations, respectively. The number of K clusters was esti-

mated with the ad hoc parameter (DK) of Evanno et al. (2005).

Mean FST values corresponding to the divergence of each

inferred K cluster from the hypothetical ancestral population

were also calculated.

We assessed whether population diversity indices differed

across the geographical ranges of D. biloba and among the

genetic groups revealed by structure analyses. Average allelic

richness per locus (A*) was estimated by applying the

rarefaction method of Hurlbert (1971) adapted by El Mousa-

dik & Petit (1996). Observed heterozygosity (HO) and genetic

diversity within populations (HS), inbreeding coefficient (FIS)

and average pairwise population differentiation estimates (FST)

were compared among populations groups using fstat 2.9.3.2

(Goudet, 2001) and tested for significance using 10,000

permutations.

Analyses of molecular variance (AMOVA; Excoffier et al.,

1992) were performed to partition the total genetic variance

into variance components, within and among populations and

within and among predefined groups of populations, using

Arlequin 3.11 (Excoffier et al., 2005). These analyses were

conducted for D. biloba s.l., and at several hierarchical levels

according to their north–south distribution, and to the

number of genetic clusters detected with structure. The

significance of the variance components was obtained with

1000 permutations.

An exploratory analysis of the relative contributions of

mutation and genetic drift to differentiation between popula-

tions revealed that genetic drift and migration contributed

more to differentiation than mutation, and thus genetic

distances preferentially based on such factors [e.g. those based

on the infinite allele model (IAM), Kimura & Crow, 1964]

were better suited for describing genetic relationships among

populations (see Appendix S1 in Supporting Information).

Pairwise genetic distances between populations were calcu-

lated using the Nei et al. (1983) DA genetic distance with

populations 1.2.31 (Langella, 2000) and used to construct

neighbour-joining (NJ) trees using mega 4.0 (Tamura et al.,

2007). The robustness of the groupings was assessed by 1000

replicate bootstrap analyses.

Dispersal model analysis

Analyses of alternative dispersal models, each of them

supporting migrations along different specific pathway net-

works (Buckler et al., 2006), were tested against the null

hypothesis of isolation by distance (IBD), defined as the

shortest pairwise linear distance between populations.

Although the disjunct geographical range of D. biloba suggests

that IBD may have shaped the genetic differentiation of its

populations, other processes related to past climatic changes

might have provided opportunities for alternative dispersals.

The strength of such alternative colonization hypotheses was

tested through simple and partial correlations between genetic

distances (DA) and pairwise geographical (IBD) or dispersal

J. Viruel et al.


(dispersal models) matrices (Dietz, 1983; Smouse et al., 1986)

using Phylogeographer 1.1 (Buckler, 1999; Buckler et al.,

2006), and significance was assessed with 10,000 permutations.

Dispersal model testing was restricted to those models that

represented all the potential colonization patterns between the

three main genetic groups detected within D. biloba (e.g.

Limarı, Choapa and Tal-Tal, see Results). For this, populations

within the same geographical area were either connected on

the basis of their geographical proximity or to a node

intermediate between them. Some dispersal models required

the creation of an additional virtual node to connect

geographically adjacent or separate population groups, or to

test for potential dispersals northward or southward to give the

present distribution range of the species. Finally, a simple

dispersal model connecting all populations by their shortest

distances was computed to test for congruence or departure

from the expected IBD model.

Plastid DNA data analyses

Haplotype polymorphism was estimated within populations

and within genetic and geographical groups through the

analysis of the number of segregating sites (S), the number of

haplotypes (h), the haplotype diversity index (Hd) and the

average number of pairwise nucleotide differences between

DNA sequences hp (Tajima, 1983). Genetic diversity between

populations and genetic groups was estimated through the

number of shared mutations (shm) and the average number of

nucleotide differences (d) among haplotypes. All indices were

computed with DnaSP 5 (Librado & Rozas, 2009). Statistical

parsimony haplotype networks were created with tcs 1.21

software (Clement et al., 2000) using the default settings of the

software. AMOVA was used to estimate the partitioning of

nucleotide diversity within and among population, and

Arlequin was used to compare within and between geo-

graphical regions.

RESULTS

Microsatellite genetic diversity and structure

in Dioscorea biloba populations

All seven microsatellite loci were polymorphic in all nine

populations (Table 1 and data available upon request). The

number of alleles ranged from five (loci B628 and B322) to 39

(locus B204) with a mean of 14.29 ± 12.33 (±SD) alleles per

locus. The mean number of alleles per locus and population

ranged from 5.14 ± 4.22 (Db5) to 7.29 ± 4.11 (Db4, Table 1).

From the 100 microsatellite alleles scored, 40% were shared by

all populations, while 50% and 10% were exclusive to southern

and northern populations, respectively. However, allelic rich-

ness was not significantly different between northern

(A = 5.26) and southern (A = 5.91) groups (Table 2) nor

when populations were separated into three groups (Tal-Tal,

Limarı and Choapa). However, significant differences

(P = 0.029) were obtained when the Limarı populations were

compared with Tal-Tal + Choapa (Table 2). Observed hetero-

zygosities ranged from 0.345 ± 0.306 (Db5) to 0.686 ± 0.156

(Db3), and unbiased expected heterozygosities from

0.458 ± 0.307 (Db5) to 0.706 ± 0.137 (Db3) (Table 1). The

Limarı populations showed significantly higher heterozygosity

in independent comparisons of populations from the three

geographical areas and when compared with the Tal-

Tal + Choapa group (Table 2). Four of the nine populations

showed HW deviations towards heterozygote deficiency, one

showed non-significant departure from HW equilibrium, and

the remaining four, including all the Tal-Tal ones, showed a

significant heterozygote excess (Table 1). Differences among

populations between northern and southern groups were only

marginally significant (Table 2). Nevertheless, the populations

from Limarı showed overall FIS values close to HW equilib-

rium, whereas those from Choapa and Tal-Tal showed positive

and negative deviations from HW, respectively, with signifi-

cant differences among the three groups (P = 0.002, Table 2).

Moderate but significant (P < 0.05) levels of population

differentiation were observed among populations, except for

the neighbouring Db8 and Db9. Pairwise FST values ranged

from a minimum of FST = 0.007 (Db8–Db9; Tal-Tal), to a

maximum of FST = 0.322 (Db5–Db7, Choapa–Tal-Tal) (data

not shown). Average pairwise FST (mean, 95% CI) was almost

seven times higher between southern populations (FST = 0.155,

0.095–0.233) than between northern ones (FST = 0.024, 0.006–

0.054), this difference being only marginally significant

(Table 2). Changes in FST values were obtained when the

southern Choapa populations were joined with the northern

Tal-Tal ones (FST = 0.192 versus Limarı FST = 0.050), and

Table 2 Comparison of mean genetic polymorphism and

inbreeding and fixation coefficient values between geographical

and genetic groups of populations of the Chilean endemic Dios-

corea biloba.

Groups/indices A HO HS FIS FST

Northern (Tal-Tal) 5.265 0.534 0.483 )0.104 0.024

Southern

(Limarı+Choapa)

5.912 0.548 0.600 +0.086 0.155

P-value 0.2266 0.9030 0.1372 0.0505 0.0607

Tal-Tal+Choapa 5.268 0.459 0.477 +0.037 0.192

Limarı 6.233 0.662 0.678 +0.023 0.050

P-value 0.0285 0.0170 0.0081 0.8774 0.0056

Tal-Tal 5.265 0.534 0.483 )0.104 0.024

Limarı 6.233 0.662 0.678 +0.023 0.050

Choapa 5.271 0.355 0.469 +0.244 0.057

P-value 0.2067 0.0024 0.0021 0.0017 0.9319

A, allelic richness calculated after the rarefaction method of El

Mousadik & Petit (1996) and based on a minimum sample size of 25

individuals (corresponding to the smallest Db7 population).

HO, HS, average observed and expected heterozygosities within pop-

ulations, respectively. FIS, inbreeding coefficient; FST, average pairwise

population differentiation.

P-values are based on 10,000 permutations. Northern, n = 3 popula-

tions; southern, n = 6 populations. Significant values are indicated in

bold.



these were statistically significant (P = 0.0056, Table 2). Con-

sidering the three areas as independent groups resulted in non-

significant FST differences (P = 0.9319, Table 2), suggesting

homogeneity among populations within each geographical

area.

Bayesian analysis of population structure showed a maxi-

mum DK = 867.72 value for K = 2. In this clustering,

individuals from southern Limarı populations showed a high

proportion of membership to cluster 1 and those of northern

Tal-Tal populations and the southernmost Choapa popula-

tions showed a high proportion of membership to cluster 2

(Fig. 1a). Mean FST values corresponding to the divergence

between clusters 1 and 2 from the hypothetical ancestral

population were 0.086 and 0.106, respectively, indicating that

cluster 1 populations were less diverged from the ancestral

population. A further maximum DK = 526.20 value was

obtained for K = 3. This clustering separated the Limarı,

Tal-Tal and Choapa populations into three clusters (1, 2 and

3), with individuals from two Limarı populations showing

some degree of admixture with populations of Choapa

(Fig. 1b). Mean FST values corresponding to the divergence

between clusters 1, 2 and 3 from the hypothetical ancestral

population were 0.096, 0.182 and 0.224, respectively, indicat-

ing again that the Limarı populations were closer to the

hypothetical ancestor.

Non-hierarchical AMOVA attributed 20.85% of the total

variation among populations (Table 3). AMOVA based on a

geographical hierarchy attributed 17.06% of the variation to

differences between northern and southern groups and 10.43%

to differences among populations within groups. A similar

result was obtained in the AMOVA based on K = 2 genetic

hierarchy (Limarı versus Choapa + Tal-Tal, Table 3). Finally,

AMOVA for the K = 3 genetic hierarchy revealed the highest

proportion of variance among groups (21.95%) and also the

lowest genetic variance among populations within groups

(3.44%, Table 3). All variance components were significantly

different from zero (P < 0.001).

Analyses using SPAGeDi 1.2 g (Hardy & Vekemans, 2002)

provided evidence of a preponderant role of genetic drift and

migration in population differentiation in D. biloba (Appen-

dix S1). The NJ tree (Fig. 2) revealed three clusters of

populations corresponding to their geographical membership:

the four southern populations from the Limarı Valley, the two

populations from the southern Choapa Valley (99% BS) and,

the three northern populations from Tal-Tal (72% BS).

Plastid trnL–F and trnT–L sequence variation

The trnL–F region of D. biloba ranged from 724 to 726 bp and

contained 21 polymorphic sites excluding indels, five of which

were parsimony informative, producing 15 haplotypes. Indels

were found in the southern haplotypes I, II, VI, VII, VIII, IX

and in northern haplotype XI (1-bp deletion each), and in

southern haplotypes III, IV and V (2-bp deletion each)

corresponding to a chloroplast simple sequence repeat (cpSSR)

poly-T track. The trnT–L region of D. biloba ranged from 825

to 827 bp. Three out of four polymorphic sites were parsimony

informative, indicating seven haplotypes. Indels were found in

the southern haplotype II (1-bp deletion), and in the northern

haplotypes VI and VII (two 1-bp deletions each).

According to the trnL–F region, the northern group of

populations showed higher S and hp diversity values (S = 13,

hp = 1.453) than the southern group of populations (S = 12,

hp = 0.780). This is a consequence of the highest within-

Table 3 Analyses of molecular variance

(AMOVA) of Chilean Dioscorea biloba

populations using nuclear microsatellite

data.

Source of variation

(groups)

Sum of

squared

deviations (SSD) d.f.

Variance

components

% of the

total

variance

1. Dioscorea biloba s.l.

Among populations 246.794 8 0.47678 20.85

Within populations 979.183 541 1.80995 79.15

2. Geographical membership: southern (Db1–Db6) versus northern (Db7–Db9)

Among groups 123.722 1 0.42593 17.06

Among populations

within groups

123.072 7 0.26045 10.43


3. Genetic membership: two clusters of structure analysis; cluster 1 (Db1–Db4) versus cluster 2

(Db5–Db9)

Among clusters 123.804 1 0.40027 16.21

Among populations

within clusters

122.989 7 0.25864 10.48


4. Genetic membership: three clusters of structure analysis; cluster 1 (Db1–Db4) versus cluster

2 (Db5–Db6) versus cluster 3 (Db7–Db9)

Among clusters 206.039 2 0.53253 21.95

Among populations

within clusters

40.755 6 0.08345 3.44


J. Viruel et al.


population diversity parameters (S = 10, h = 4) in northern

population Db9, and the lowest such parameters in the

southern populations Db1–Db4 (S = 0–2, h = 1–2) (Appen-

dices S2 & S3). Within the southern range, the southernmost

Choapa populations showed higher diversity (S = 8, h = 5,

Hd = 0.57, hp = 1.403) than those from the Limarı (S = 4,

h = 4, Hd = 0.26, hp = 0.378). Diversity values of the south-

ernmost Choapa and northernmost Tal-Tal ranges were

comparable, and showed the highest number of shared

mutations (shm = 4), reflecting the closer genetic affinities

between the Tal-Tal and Choapa groups. This is in contrast

with other pairwise comparisons, including the Limarı group,

which did not share mutations, and the high divergence of the

Choapa group (Table 4) measured by nucleotide differences

(d = 1.43 Choapa–Tal-Tal; d = 0.93 Choapa–Limarı; Tal-Tal–

Limarı d = 0.90).

The largest outgroup probability obtained for haplotypes I

of the trnL–F (0.27) and I of trnT–L (0.43), both from the

southern range of D. biloba, indicate a probable ancestry of the

species in this area.

Southern cp-haplotypes of both trnL–F and trnT–L datasets

clustered together in the parsimony network and were fairly

differentiated from those found in the northern area. AMOVA

analyses revealed 41.9% and 78% of the total variance among

northern and southern ranges for trnL–F and trnT–L, respec-

tively. Marked geographical structure was detected within the

southern range despite the relatively short distance separating

the Limarı and Choapa valleys (Fig. 3). Populations from

Choapa showed an exclusive trnT–L haplotype that linked the

cp-haplotypes from northern and southern areas (Fig. 3b).

AMOVA analyses attributed 80.85% of the total variance of

trnT–L between Limarı and Choapa (Fig. 3b).

Hypothesis testing of isolation-by-distance

and dispersal models

Ten out of 20 tested models showed higher simple and multiple

correlation values than the IBD model (Fig. 4, Table 5). Model

1, which included the presence of a hypothetical, geographically

intermediate node between the southern and northern groups

of D. biloba in the foothills of the Andes (Fig. 4), showed the

highest correlation value (r = 0.9180). Model 2, which incor-

porated a hypothetical, geographically intermediate coastal

node, showed the next highest correlation (r = 0.9159). Addi-

tional models, which included the presence of other hypothet-

ical geographically located intermediate nodes (models 3–8),

showed decreasing correlation values (r = 0.8257 to r = 0.7093,

Choapa

Southern range

6980

72

Limarí

Tal-Tal

Northern range

99

72

Figure 2 Neighbour-joining tree based on DA genetic distance

(Nei et al., 1983), showing the genetic relationships among

populations of the Chilean endemic Dioscorea biloba. Bootstrap

support values (BS) ‡ 50% obtained from 1000 permutations

over populations are shown above the branches.

Table 4 Estimates of the number of shared mutations (shm; above diagonal) and the average number of nucleotide differences (d; below

diagonal) between nine populations and three geographical ranges of Dioscorea biloba based on analysis of trnL–F sequences. Analyses were

conducted with DnaSP 5 (Librado & Rozas, 2009). Bold numbers indicate the highest shm and d values between southernmost and

northernmost populations and ranges of Choapa and Tal-Tal. See Table 1 for details of populations.

Population/group Db1 Db2 Db3 Db4 Db5 Db6 Db7 Db8 Db9 Limarı Choapa Tal-Tal

Db1 – 0 0 0 0 0 0 0 0

Db2 0.33 – 0 0 0 0 0 0 0

Db3 0.17 0.50 – 0 0 0 0 0 0

Db4 0.17 0.50 0.33 – 0 0 0 0 0

Db5 0.67 1.00 0.83 0.83 – 1 1 0 1

Db6 0.83 1.17 1.00 1.00 1.44 – 1 0 1

Db7 0.33 0.67 0.50 0.50 0.94 1.11 – 0 0

Db8 0.17 0.50 0.33 0.33 0.83 1.00 0.50 – 0

Db9 0.17 2.00 1.83 1.83 2.28 2.44 2.00 1.83 –

Limarı – 0 0

Choapa 0.93 – 4

Tal-Tal 0.90 1.43 –



Table 5). Models 9 and 10 that did not imply the presence of

hypothetical intermediate nodes had lower correlation values

(r = 0.6769–0.6520).

Partial correlations of, respectively, model 1 and model 2 and

the IBD model indicated that the dispersal distances of these

models explained a significant part of the variation that was not

explained by the IBD model alone, whereas partial correlations

of the IBD model and model 1 and model 2 were non-

significant, indicating that all the variation explained by the

IBD model was already explained by those models. Also, the

values of the coefficients of simple (r2) and multiple (R2)

determinations in models 1 and 2 were similar, whereas the r2

value of the IBD model was lower than the multiple coefficients

of both models, indicating that substantial information was

gained by adding other sources of variation (models 1 or 2)

once the first source (IBD model) was fitted (Table 5).

The correlation analyses selected models 1 and 2 as the

optimal dispersal models (Table 5, Fig. 4). Both hypotheses

depicted dispersal scenarios in which the migration route

included a hypothetical, geographically intermediate node

between the two Coquimbo groups and the Antofagasta group

that could have been located either at the western side of the

Andes or in coastal areas, which historically extended across

much of the present Atacama Desert.

Dispersal models do not infer the ancestries of the nodes so

additional sources need to be invoked to clarify the most likely

directions of migrations. Ancestral Bayesian microsatellite

groups and the outgroup probability of plastid haplotypes both

indicated that the southern Coquimbo populations may be

older than the northern Antofagasta ones (see above). Thus, a

single configuration would be compatible with the dispersal

routes of models 1 and 2. These models indicated that the

colonization most likely originated from the area of the

southern Limarı group with migration towards the interme-

diate hypothetical Atacama node. This initial phase was

probably followed by two colonizations: one back-colonization

to the southern Choapa region and one northward migration

to the Tal-Tal region.

Db7

Db9Db8

Db5

Db6

Db1Db2

Db3Db4

IV (4)

V (5)

VI VII

I (12) II

III

Db1

Db2

Db3Db4

Db6

Db5

Db7

Db8Db9

(a) (b)

I (22)

II

VIIVI

V

IV

III(5)

VIII

X

IX

XIII

XI

XIV

XII(13)

XV

Figure 3 Statistical parsimony networks

and geographical distribution of chloroplast

haplotypes of the Chilean endemic Dioscorea

biloba. (a) Fifteen trnL–F haplotypes.

(b) Seven trnT–L haplotypes. Pie charts

indicate relative frequencies of each

haplotype in each population. Chloroplast

haplotypes, from the statistical parsimony

network obtained with tcs (Clement et al.,

2000), are denoted by roman numerals. Black

dots indicate unsampled haplotypes. The

sizes of the circles or squares are proportional

to the number of sequences representing each

haplotype and these are indicated in brackets

when larger than one.

Models 1 2 3 4 5 6 7 8 9 10

Figure 4 Dispersal models analysed in the Chilean endemic

Dioscorea biloba. Model 1: (1,2,3,4)//(1,2,3,4)-Node1//Node1-

(5,6)//Node1-(9-8-7); Model 2: (1,2,3,4)//(1,2,3,4)-Node2//

Node2-(5,6)//Node2-(9-8-7); Model 3: (1,2,3,4)//(1,2,3,4)-

Node3//Node3-(5,6)//Node3-(9-8-7); Model 4: (1,2,3,4)//

(1,2,3,4)-Node4//Node4-(5,6)//Node4-(9-8-7); Model 5:

(1,2,3,4)//(1,2,3,4)-Node5//Node5-(5,6)//Node5-(9-8-7); Model 6:

(1,2,3,4)//(1,2,3,4)-Node6//Node6-(5,6)//Node6-(9-8-7); Model

7:(1,2,3,4)//(1,2,3,4)-Node7//Node7-(5,6)//Node7-(9-8-7); Model

8: (1,2,3,4)//(1,2,3,4)-Node8//Node8-(5,6)//Node8-(9-8-7); Model

9: 5//5-6-4-3-2-1//5-(9-8-7) and Model 10: 5-6-4-3-2-1-(9-8-7).

The numbers represent abbreviations of population code numbers

given in Table 1. All connective paths are read from left to right,

beginning with the southernmost population(s). Populations in

brackets were joined by a proximal geographical hypothetical node

(grey dots). Double slashes represent a principal bi- or trifurca-

tion, and white dots hypothetical geographically intermediate

nodes.

J. Viruel et al.


DISCUSSION

Influence of life-history traits and historical factors

on the genetic diversity of Dioscorea biloba

Life-history and reproductive traits have important effects on

levels of genetic diversity and their distribution within and

among wild plant populations (Hamrick et al., 1991; Hamrick &

Godt, 1996). The observed high levels of genetic diversity within

populations of D. biloba are consistent with the dioecious

breeding system of this species and are comparable to values for

other dioecious Dioscorea species analysed with microsatellite

markers, such as the wild Dioscorea tokoro Makino (A = 6.2,

HO = 0.54, HE = 0.68; Terauchi & Konuma, 1994) and the

cultivated Dioscorea trifida L. f. (A = 6.0, HE = 0.60; Hochu

et al., 2006), but they are higher than levels found in Dioscorea

(Borderea) chouardii Gaussen (A = 1.70, HO = 0.14,

HE = 0.13–0.14; Segarra-Moragues et al., 2005) and Dioscorea

(Borderea) pyrenaica Bubani & Bordere ex Gren. (A = 1.56–

3.22, HO = 0.122–0.232, HE = 0.129–0.257; Segarra-Moragues

et al., 2007). The latter two species share similar life-history

traits with D. biloba but have more restricted distributions.

The heterozygote deficiency found in four of the nine southern

Choapa populations (Table 1) is surprising for a dioecious

perennial herb in which self-fertilization is prevented by dioecy.

This result may suggest that plants in small populations may have

experienced biparental inbreeding processes over a long period

of time. Nonetheless, similar patterns of heterozygote deficiency

have been found in other dioecious dwarf yams such as the two

Dioscorea gr. Borderea species (Segarra-Moragues et al., 2005,

2007). These latter species are similar to D. biloba in having

flowers that are unattractive to flying insects and limited

dispersal distance of the seeds. Moreover, although the pollina-

tion mechanism of D. biloba has not been studied, ant-

pollination, as reported in the Dioscorea gr. Borderea (Garcıa

et al., 1995), would imply short-distance dispersal of pollen,

resulting in mating among spatially close and probably related

individuals, thus increasing inbreeding. The significant hetero-

zygote excess shown by northernmost populations (Table 1)

does not contradict this hypothesis as individuals and popula-

tions from this range are spread over much shorter geographical

distances which could be covered by both ant pollination and

passive seed dispersal mechanisms. Other explanations for the

high FIS values may be related to the presence of null alleles and

can only be confirmed by progeny analyses.

At a larger geographical scale, high levels of inbreeding as

the consequence of short-distance dispersal of pollen and seeds

may contribute to strengthening the among-population struc-

ture, as shown for other organisms with low migration rates

(Zattara & Premoli, 2005).

Phylogeography and dispersal routes of Dioscorea

biloba

Dispersal model analyses have been successfully applied to

infer the most likely colonization routes of oceanic island and

continental plant populations (Buckler et al., 2006; Dıaz-Perez

et al., 2008). In D. biloba they assisted the selection of the best

migration models that provide alternatives to the intuitive IBD

model based solely on the shortest geographical distances.

In this study we have shown that the northern Tal-Tal

populations of D. biloba are genetically more related to the

southernmost populations from the Choapa Valley than to the

less distant southern Limarı ones (Figs 1a & 3b). This could be

the result of long-distance dispersal from the southernmost

area to the north, or, alternatively, it may represent an ancient

vicariant fragmentation of a broader area.

Here, models 1 and 2, which include a hypothetical

geographically intermediate step between the southern and

northern groups (Table 5, Fig. 4), provided the best coloniza-

tion scenarios. Both models support evidence of a continuous

ancestral distribution area of D. biloba from Coquimbo to

Table 5 Simple and partial correlation analyses between DA genetic distances and geographical or dispersal distances for Dioscorea biloba.

Dispersal models correspond to those described in Fig. 4.

Model r P r2 r M ŒG P r G ŒM P R2

Geographical 0.6516 0.0023 0.4246

Model 1 0.9180 < 0.001 0.8428 0.8526 0.0012 0.0210 0.4197 0.8428

Model 2 0.9159 < 0.001 0.8388 0.8534 0.0012 0.1725 0.1270 0.8435

Model 3 0.8257 < 0.001 0.6818 0.8628 0.0010 0.7334 < 0.001 0.8529

Model 4 0.8135 < 0.001 0.6617 0.8618 0.0010 0.7499 < 0.001 0.8519

Model 5 0.8051 < 0.001 0.6482 0.8706 0.0014 0.8607 < 0.001 0.9088

Model 6 0.7542 < 0.001 0.5687 0.6913 0.0053 0.6996 0.9887 0.7797

Model 7 0.7374 < 0.001 0.5438 0.7577 < 0.001 )0.6804 0.9989 0.7549

Model 8 0.7093 0.0015 0.5031 0.6301 0.0029 )0.5494 0.9902 0.6530

Model 9 0.6769 < 0.001 0.4582 0.2551 0.0458 )0.0844 0.7620 0.4620

Model 10 0.6520 0.0021 0.4252 0.0673 0.3500 )0.0593 0.6278 0.4272

r, simple correlation coefficient between genetic and geographic or dispersal model distances. P, probability for a random r higher than observed r

after 10,000 permutations. rM ŒG, partial correlation coefficient between genetic and dispersal model distances once geographic distance was fixed.

rG ŒM, partial correlation coefficient between genetic and geographic distance once dispersal model distance was fixed. r2, coefficient of determination.

R2, coefficient of multiple determination.



Antofagasta that probably pre-dated the establishment of the

Atacama Desert. Nuclear microsatellite and cpDNA data

indicate that the Tal-Tal individuals do not represent a subset

of the genetic pools of the Choapa individuals. The presence

of 10 exclusive microsatellite alleles and totally exclusive

cp-haplotypes in the northern range rejects the possibility of a

recent long-distance dispersal founder effect from southern

sources, and favour a long in situ persistence. Life-history traits

of D. biloba also suggest that northern populations are unlikely

to result from recent long-distance dispersal from the southern

range. Although dispersal models 9 and 10, which did not

include a hypothetical intermediate population, were concor-

dant with this dispersal hypothesis, they were less convincing

than other models, as were models that predicted connective

paths through areas far away from the current distribution area

of D. biloba (e.g. models 5 and 6), and even less than the

optimal models 1 and 2 (Table 5, Fig. 4).

While dispersal models that include a hypothetical geo-

graphically intermediate step between the southern and

northern groups (models 1 and 2) support evidence of a

continuous distribution area of D. biloba from Coquimbo to

Antofagasta, they also provide support for alternative putative

dispersal routes by which D. biloba could have expanded

northwards during somewhat less arid conditions. Model 1

favours evidence for a migration route along the western

foothills of the Andes, whereas model 2 favours a coastal

colonization pathway. Although currently D. biloba is located

mostly in coastal areas, the western lowland Andes could also

have provided suitable habitats for the migration of this species

as this route seems to have supplied suitable corridors for the

expansion of diverse plants and animals in Plio-Pleistocene

times (Latorre et al., 2003; Palma et al., 2005). Climatic

fluctuations, including the recent (< 3 Ma) rainfall events,

have been documented to impact the arid landscape of the

Atacama (Placzek et al., 2009). Range expansions and connec-

tions between northern and southern areas could have ended

in the complete isolation of such populations following

extinction of any putative intermediate populations in the

late Holocene, when the current hyper-arid conditions of the

Atacama Desert were established (Betancourt et al., 2000;

Latorre et al., 2003). This kind of fragmentation of a previ-

ously extended range could explain why the most geograph-

ically separated extant groups from Choapa and Tal-Tal still

share an important set of microsatellite alleles.

Geographical isolation and origin of Dioscorea biloba

Geographical isolation is considered a primary process in

diversification (Perret et al., 2007). When geographical barriers

that limit or prevent gene flow among populations are

imposed, the isolated gene pools may evolve independently,

ultimately leading to speciation. Yet genetic differentiation

among populations within a given species may be seen as the

initial step to speciation (Coyne & Orr, 2004). Our analyses

have revealed two highly divergent Antofagasta and Coquimbo

genetic groups within D. biloba located within and at the

southern boundary of the Atacama Desert. This result is

expected for two groups that are geographically separated by

more than 600 km and by an inhospitable hyper-arid area that

establishes a significant geographical barrier to reproduction

between both population groups, as revealed by the IBD

analysis. While morphological (Viruel et al., 2010a) and

molecular data lend support for the recognition of both

population groups as two subspecies of D. biloba, deciphering

their origins can further contribute to understanding the

evolutionary history of this endemic lineage of Dioscorea and

its contribution to the biodiversity of Chilean mediterranean

and arid areas.

Interestingly, our analyses revealed that the most likely area

for the origin of D. biloba was the southern Coquimbo area.

This is supported by the lower genetic differentiation from

the hypothetical ancestral gene pool of the southern popu-

lations (FST = 0.086) compared with northern ones

(FST = 0.106) based on nuclear microsatellite markers and

by cpDNA data, which show trnL–F (Fig. 3a) and trnT–L

(Fig. 3b) plastid haplotypes I nested in the central positions

of the respective networks and being predominant in the

populations, a fact that is interpreted as indicative of ancestry

(Jakob & Blattner, 2006). The distribution of Dioscorea gr.

Epipetrum encompasses four taxa, three of which are

restricted to mediterranean climate areas of Chile and which

are distributed as far south as the Atacama Desert in central

Chile but which are absent from Argentina (Viruel et al.,

2010a). This is consistent with the diversification of this

group post-dating the Andean uplift in the Miocene.

However, the isolation of the northern populations after

the establishment of the Atacama Desert would have favoured

the diversification of northern D. biloba in more recent times,

probably during the transition from the Pliocene to the

Holocene after the adaptation of southern Chilean palaeoen-

demic lineages to northern areas of increased aridity. Similar

patterns have been reported for other plant groups that

radiated into northern Chilean and arid areas from southern

ancestors (Katinas & Crisci, 2000; Hershkovitz et al., 2006;

Schmidt-Jabaily & Sytsma, 2010) and strengthen the desig-

nation of the Atacama Desert as a biodiversity hotspot of

neoendemic radiation (Luebert et al., 2009).

ACKNOWLEDGEMENTS

We thank E. Perez-Collazos and L. Villar for their help during

fieldwork. A. Dıaz-Perez for his valuable comments and

discussion on dispersal model analysis, P. Gibbs and T. Langdon

for their linguistic assistance, and the editor P. Ladiges and two

anonymous referees for their valuable comments that have

improved the manuscript. This study has been supported by a

Fundacion BBVA BIOCON 05-093/06 project grant. J.V. was

supported by a Fundacion BBVA PhD grant, an Instituto de

Estudios Altoaragoneses project, and two research stays at

Royal Botanic Gardens, Kew, funded by SYNTHESYS GB-TAF

and the Diputacion General de Aragon-Caja de Ahorros de la

Inmaculada (DGA-CAI). J.G.S.-M. was supported by two

J. Viruel et al.


consecutive Spanish Aragon Government ‘Araid’ and Ministry

of Science and Innovation ‘Ramon y Cajal’ post-doctoral

contracts.

REFERENCES

Albrecht, E., Escobar, M. & Chetelat, R.T. (2010) Genetic

diversity and population structure in the tomato-like

nightshades Solanum lycopersicoides and S. sitiens. Annals of

Botany, 105, 535–554.

Amico, G.C. & Nickrent, D.L. (2009) Population structure and

phylogeography of the mistletoes Tristerix corymbosus and

T. aphyllus (Loranthaceae) using chloroplast DNA sequence

variation. American Journal of Botany, 96, 1571–1580.

Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N. & Bonhomme, F.

(2004) GENETIX 4.05, logiciel sous WindowsTM pour la

genetique des populations. Laboratoire Genome, Populations,

Interactions, CNRS UMR 5171, Universite de Montpellier

II, Montpellier, France.

Betancourt, J.L., Latorre, C., Reach, J.A., Quade, J. & Rylander, K.A.

(2000) A 22000-year record of monsoonal precipitation from

northern Chile’s Atacama Desert. Science, 289, 1542–1546.

Buckler, E.S., IV (1999) Phylogeographer: a tool for developing

and testing phylogeographic hypotheses, 0.3 ed. Available at:

http://www.maizegenetics.net/phylogeographer.

Buckler, E.S., IV, Goodman, M.M., Holtsford, T.P., Doebley,

J.F. & Sanchez, G. (2006) Phylogeography of the wild sub-

species of Zea mays. Maydica, 51, 123–134.

Clarke, J.D.A. (2006) Antiquity of aridity in the Chilean Ata-

cama Desert. Geomorphology, 73, 101–114.

Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a

computer program to estimate gene genealogies. Molecular

Ecology, 9, 1657–1659.

Coyne, J.A. & Orr, H.A. (2004) Speciation. Sinauer Associates,

Sunderland, MA.

Dıaz-Perez, A., Sequeira, M., Santos-Guerra, A. & Catalan, P.

(2008) Multiple colonizations, in situ speciation, and vol-

canism-associated stepping-stone dispersals shaped the

phylogeography of the Macaronesian red fescues (Festuca L.,

Gramineae). Systematic Biology, 57, 732–749.

Dietz, J. (1983) Permutation tests for association between two

distance matrices. Systematic Zoology, 32, 21–26.

Dillon, M.O., Tu, T., Xie, L., Quipuscoa-Silvestre, V. & Wen, J.

(2009) Biogeographic diversification in Nolana (Solana-

ceae), a ubiquitous member of the Atacama and Peruvian

Deserts along the western coast of South America. Journal of

Systematics and Evolution, 47, 457–476.

El Mousadik, A. & Petit, R.J. (1996) High level of genetic

differentiation for allelic richness among populations of the

argan tree (Argania spinosa (L.) Skeels) endemic to Mor-

occo. Theoretical and Applied Genetics, 92, 832–839.

Evanno, G., Regnaut, S. & Goudet, J. (2005) Detecting the

number of clusters of individuals using the software

structure: a simulation study. Molecular Ecology, 14,

2611–2620.

Excoffier, L., Smouse, P.E. & Quattro, J.M. (1992) Analysis of

molecular variance inferred from metric distances among

DNA haplotypes: application to human mitochondrial DNA

restriction data. Genetics, 13, 479–491.

Excoffier, L., Laval, G. & Schneider, S. (2005) Arlequin (ver-

sion 3.0): an integrated software package for population

genetics data analysis. Evolutionary Bioinformatics Online, 1,

47–50.

Garcıa, M.B., Antor, R.J. & Espadaler, X. (1995) Ant pollina-

tion of the palaeoendemic dioecious Borderea pyrenaica

(Dioscoreaceae). Plant Systematics and Evolution, 198, 17–

27.

Gayo, E., Hinojosa, L.F. & Villagran, C. (2005) On the per-

sistence of tropical paleofloras in central Chile during the

Early Eocene. Review of Palaeobotany and Palynology, 137,

41–50.

Goudet, J. (2001) FSTAT v. 2.9.3.2, a program to estimate and

test gene diversities and fixation indices. Available at: http://

www2.unil.ch/popgen/softwares/fstat.htm.

Hamrick, J.L. & Godt, M.J.W. (1996) Effects of life history

traits on genetic diversity in plant species. Philosophical

Transactions of the Royal Society B: Biological Sciences, 351,

1291–1298.

Hamrick, J.L., Godt, M.J.W., Murawski, D.A. & Loveless, M.D.

(1991) Correlations between species traits and allozyme

diversity: implications for conservation biology. Genetics

and conservation of rare plants (ed. by D.A. Falk and K.E.

Holsinger), pp. 76–86. Oxford University Press, New York.

Hardy, O.J. & Vekemans, X. (2002) SPAGeDi: a versatile

computer program to analyse spatial genetic structure at the

individual or population levels. Molecular Ecology Notes, 2,

618–620.

Hershkovitz, M.A., Hernandez-Pellicer, C.C. & Arroyo, M.T.K.

(2006) Ribosomal DNA evidence for the diversification of

Tropaeolum sect. Chilensia (Tropaelaceae). Plant Systematics

and Evolution, 260, 1–24.

Hinojosa, L.F. & Villagran, C. (2005) Did South American

mixed paleofloras evolve under thermal equability or in the

absence of an effective Andean barrier during the Cenozoic?

Palaeogeography, Palaeoclimatology, Palaeoecology, 217, 1–

23.

Hinojosa, L.F., Armesto, J.J. & Villagran, C. (2006) Are Chilean

coastal forests pre-Pleistocene relicts? Evidence from foliar

physiognomy, palaeoclimate, and phytogeography. Journal

of Biogeography, 33, 331–341.

Hochu, I., Santoni, S. & Bousalem, M. (2006) Isolation,

characterization and cross-species amplification of micro-

satellite DNA loci in the tropical American yam Dioscorea

trifida. Molecular Ecology Notes, 6, 137–140.

Houston, J. & Hartley, A.J. (2003) The central Andean west-

slope rainshadow and its potential contribution to the origin

of hyper-aridity in the Atacama Desert. International Journal

of Climatology, 23, 1453–1464.

Hurlbert, S.H. (1971) The nonconcept of species diversity: a

critique and alternative parameters. Ecology, 52, 577–586.



Jakob, S.S. & Blattner, F.R. (2006) A chloroplast genealogy of

Hordeum (Poaceae): long-term persisting haplotypes,

incomplete lineage sorting, regional extinction, and the

consequences for phylogenetic inference. Molecular Biology

and Evolution, 23, 1602–1612.

Katinas, L. & Crisci, J.V. (2000) Cladistic and biogeographic

analyses of the genera Moscharia and Polyachyrus (Astera-

ceae, Mutisieae). Systematic Botany, 25, 33–46.

Katinas, L., Crisci, J.V., Schmidt-Jabaily, R., Williams, C.,

Walker, J., Drew, B., Bonifacio, J.M. & Sytsma, K.J. (2008)

Evolution of secondary heads in Nassauviinae (Asteraceae,

Mutisieae). American Journal of Botany, 95, 229–240.

Kimura, M. & Crow, J.F. (1964) The number of alleles that can

be maintained in a finite population. Genetics, 49, 725–738.

Langella, O. (2000) Populations 1.2.31. Population genetic

software (individuals or populations distances, phylogenetic

trees). Available at: http://www.bioinformatics.org/~tryphon/

populations/

Latorre, C., Betancourt, J.L., Rylander, K.A., Quade, J. &

Matthei, O. (2003) A vegetation history from the arid pre-

Puna of northern Chile (22–23� S) over the last 13500 years.

Palaeogeography, Palaeoclimatology, Palaeoecology, 194, 223–

246.

Lia, V.V., Confalonieri, V.A., Comas, C.I. & Hunziker, J.H.

(2001) Molecular phylogeny of Larrea and its allies (Zygo-

phyllaceae): reticulate evolution and the probable time of

creosote bush arrival to North America. Molecular Phylo-

genetics and Evolution, 21, 309–320.

Librado, P. & Rozas, J. (2009) DnaSP v5: a software for

comprehensive analysis of DNA polymorphism data. Bio-

informatics, 25, 1451–1452.

Lopez, R.P., Alcazar, D.L. & Macıa, M.J. (2006) The arid and

dry plant formations of South America and their floristic

connections: new data, new interpretation? Darwiniana, 44,

18–31.

Luebert, F., Wen, J. & Dillon, O. (2009) Systematic placement

and biogeographical relationships of the monotypic genera

Gypothamnium and Oxyphyllum (Asteraceae: Mutisioideae)

from the Atacama Desert. Botanical Journal of the Linnean

Society, 159, 32–51.

Markgraf, V., McGlone, M. & Hope, G. (1995) Neogene

paleoenvironmental and paleoclimatic change in southern

temperate ecosystems – a southern perspective. Trends in

Ecology and Evolution, 10, 143–147.

McCulloch, R.D., Bentley, M.J., Purves, R.S., Hulton, N.R.J.,

Sugden, D.E. & Clapperton, C.M. (2000) Climatic inferences

from glacial and palaeoecological evidence at the last glacial

termination, southern South America. Journal of Quaternary

Science, 15, 409–417.

Nei, M. (1978) Estimation of average heterozygosity and

genetic distance from a small number of individuals.

Genetics, 89, 583–590.

Nei, M., Tajima, F. & Tateno, Y. (1983) Accuracy of estimated

phylogenetic trees from molecular data. Journal of Molecular

Evolution, 19, 153–170.

Palma, E.R., Marquet, P.A. & Boric-Bargetto, D. (2005) Inter-

and intraspecific phylogeography of small mammals in the

Atacama desert and adjacent areas of northern Chile. Journal

of Biogeography, 32, 1931–1941.

Pennington, R.T., Lavin, M., Prado, D.E., Pendry, C.A., Pell, S.

& Butterworth, C.A. (2004) Historical climate change and

speciation: neotropical seasonally dry forest plants show

patterns of both Tertiary and Quaternary diversification.

Philosophical Transactions of the Royal Society B: Biological

Sciences, 359, 515–538.

Perret, M., Chautems, A., Spichlger, R., Barradouch, T.G. & Sa-

volaien, V. (2007) The geographical pattern of speciation and

floral diversification in the neotropics: the tribe Sinningieae

(Gesneriaceae) as a case study. Evolution, 61, 1641–1660.

Pinto, R., Barrıa, I. & Marquet, P.A. (2006) Geographical

distribution of Tillandsia lomas in the Atacama Desert,

northern Chile. Journal of Arid Environments, 65, 543–552.

Placzek, C., Quade, J., Betancourt, J.L., Patchett, P.J., Rech,

J.A., Latorre, C., Matmon, A., Holmgren, C. & English, N.B.

(2009) Climate in the dry central Andes over geologic,

millennial, and interannual timescales. Annals of the Mis-

souri Botanical Garden, 96, 386–397.

Pritchard, J.K., Stephens, M. & Donnelli, P. (2000) Inference of

population structure from multilocus genotype data.

Genetics, 155, 945–959.

Reynolds, J.H., Jordan, T.E., Johnson, N.M., Damanti, J.F. &

Tabbutt, K.D. (1990) Neogene deformation of the flat-

subduction segment of the Argentine–Chilean Andes: mag-

netostratigraphic constraints from Las Junta, La Rioja

province, Argentina. Geological Society of America Bulletin,

102, 1607–1622.

Rousset, F. (2008) genepop’007: a complete re-implementa-

tion of the genepop software for Windows and Linux.

Molecular Ecology Resources, 8, 103–106.

Rundel, P.W. & Dillon, M.O. (1998) Ecological patterns in the

Bromeliaceae of the lomas formations of Coastal Chile and

Peru. Plant Systematics and Evolution, 212, 261–278.

Rundel, P.W., Dillon, M.O., Palma, B., Mooney, H.A., Gul-

mon, S.L. & Ehleringer, J.R. (1991) The phytogeography and

ecology of the coastal Atacama and Peruvian Deserts. Aliso,

13, 1–49.

Schmidt-Jabaily, R. & Sytsma, K.J. (2010) Phylogenetics of

Puya (Bromeliaceae): placement, major lineages, and evo-

lution of Chilean species. American Journal of Botany, 97,

337–356.

Segarra-Moragues, J.G., Palop-Esteban, M., Gonzalez-

Candelas, F. & Catalan, P. (2005) On the verge of extinction:

genetics of the critically endangered Iberian plant species,

Borderea chouardii (Dioscoreaceae) and implications for

conservation management. Molecular Ecology, 14, 969–982.

Segarra-Moragues, J.G., Palop-Esteban, M., Gonzalez-

Candelas, F. & Catalan, P. (2007) Nunatak survival vs.

tabula rasa in the Central Pyrenees: a study on the endemic

plant species Borderea pyrenaica (Dioscoreaceae). Journal of

Biogeography, 34, 1893–1906.

J. Viruel et al.


Simpson, B.B., Tate, J.A. & Weeks, A. (2005) The biogeogra-

phy of Hoffmannseggia (Leguminosae, Caesalpinioideae,

Caesalpinieae): a tale of many travels. Journal of Biogeogra-

phy, 32, 15–27.

Smouse, P., Long, J. & Sokal, R. (1986) Multiple regression and

correlation extensions of the Mantel test of matrix corre-

spondence. Systematic Zoology, 35, 627–632.

Sylvestre, F., Servant, M., Servant-Vildary, S., Causse, C.,

Fournier, M. & Ybert, J.P. (1999) Lake-level chronology on

the Southern Bolivian Altiplano (18�–23� S) during Late

Glacial time and the early Holocene. Quaternary Research,

51, 54–66.

Taberlet, P., Gielly, L., Pautou, G. & Bouvet, J. (1991) Uni-

versal primers for amplification of three non-coding regions

of chloroplast DNA. Plant Molecular Biology, 17, 1105–1109.

Tajima, F. (1983) Evolutionary relationship of DNA sequences

in finite populations. Genetics, 105, 437–460.

Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4:

Molecular Evolutionary Genetics Analysis (MEGA) soft-

ware version 4.0. Molecular Biology and Evolution, 24,

1596–1599.

Terauchi, R. & Konuma, A. (1994) Microsatellite polymor-

phism in Dioscorea tokoro, a wild yam species. Genome, 37,

794–801.

Villagran, C. (2001) Un modelo de la historia de la vegetacion

de la Cordillera de la Costa de Chile central-sur: la hipotesis

glacial de Darwin. Revista Chilena de Historia Natural, 74,

793–803.

Villagran, C. & Armesto, J.J. (1980) Relaciones florısticas entre

las comunidades relictuales del norte chico y la zona central

con el bosque del sur de Chile. Boletın del Museo Nacional de

Historia Natural de Chile, 37, 87–101.

Viruel, J., Segarra-Moragues, J.G., Perez-Collazos, E., Villar, L.

& Catalan, P. (2008) The diploid nature of the Chilean

Epipetrum and a new base number in the Dioscoreaceae.

New Zealand Journal of Botany, 46, 327–339.

Viruel, J., Segarra-Moragues, J.G., Perez-Collazos, E., Villar, L.

& Catalan, P. (2010a) Systematic revision of the Epipetrum

group of Dioscorea (Dioscoreaceae) endemic to Chile. Sys-

tematic Botany, 35, 40–63.

Viruel, J., Catalan, P. & Segarra-Moragues, J.G. (2010b) New

microsatellite loci in the dwarf yams Dioscorea gr. Epipetrum

(Dioscoreaceae). American Journal of Botany, 97, e121–e123.

Webb, S.D. (1991) Ecogeography of the Great American

Interchange. Paleobiology, 17, 266–280.

Weir, B.S. & Cockerham, C.C. (1984) Estimating F-statistics

for the analysis of population structure. Evolution, 38, 1358–

1370.

Zattara, E. & Premoli, A.C. (2005) Genetic structuring in

Andean landlocked populations of Galaxias maculatus: effects

of biogeographic history. Journal of Biogeography, 32, 5–14.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Appendix S1 SPAGeDi analysis and summary for pairwise

population differentiation statistics, FST, pRST and RST between

populations of Dioscorea biloba.

Appendix S2 Distribution of trnL–F and trnL–T haplotypes

in the populations of Dioscorea biloba.

Appendix S3 Plastid trnL–F and trnT–L haplotype diversity

analysis of Dioscorea biloba populations and geographical

groups.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such mate-

rials are peer-reviewed and may be re-organized for online

delivery, but are not copy-edited or typeset. Technical support

issues arising from supporting information (other than

missing files) should be addressed to the authors.

BIOSKETCHES

Juan Viruel is a PhD student at the University of Zaragoza,

Spain. His work deals with the study of the endemic Epipetrum

group of Dioscorea that includes population genetics, phylo-

geography and phylogenetics.

Pilar Catalan (University of Zaragoza) works on the popu-

lation genetics and phylogeny of a variety of plant families

with a particular interest in grasses.

Jose Gabriel Segarra-Moragues (CIDE) focuses on pop-

ulation genetics of endangered species, and on ecological

processes driving plant evolution.

Author contributions: J.G.S.-M. and P.C. conceived the

project. J.G.S.-M. and J.V. provided material. J.V. collected

the data. J.V., J.G.S.-M. and P.C. analysed the data. J.G.S.-M.,

P.C. and J.V. wrote the manuscript. J.G.S.-M. and P.C.

contributed equally to this paper as senior authors.

Editor: Pauline Ladiges



Disrupted phylogeographical microsatellite and chloroplast DNA patterns indicate a vicariance rather...

Documents

Transcript of Disrupted phylogeographical microsatellite and chloroplast DNA patterns indicate a vicariance rather...