Hidden phylogeographic complexity in the Sierra Madre Oriental: the case of the Mexican tulip poppy...

ORIGINALARTICLE

Hidden phylogeographic complexity

in the Sierra Madre Oriental: the case

of the Mexican tulip poppy Hunnemannia

fumariifolia (Papaveraceae)

Victoria Sosa*, Eduardo Ruiz-Sanchez and Flor C. Rodriguez-Gomez

Departamento de Biologia Evolutiva, Instituto

de Ecologia, Veracruz, Mexico

*Correspondence: Victoria Sosa, Departamento

de Biologia Evolutiva, Instituto de Ecologia,

A. C., Apartado Postal 63, 91000 Xalapa,

Veracruz, Mexico.

E-mail: [email protected]

ABSTRACT

Aim A phylogeographic study of the endemic Mexican tulip poppy

Hunnemannia fumariifolia (Papaveraceae) was conducted to determine: (1) the

historical processes that influenced its geographical pattern of genetic variation;

(2) whether isolation by distance was one of the main factors that caused genetic

divergence in populations of this species; and (3) whether genetic flow still exists

between populations from northern arid zones (Chihuahuan Desert and Sierra

Madre Oriental) and those from southern arid zones (Tehuacan-Cuicatlan

Valley) – populations that are separated by the Transvolcanic Belt.

Location Xerophytic vegetation in Mexico.

Methods Chloroplast DNA (cpDNA) sequences of three regions, trnH-psbA,

rpl32-trnL(UAG) and ndhF-rpl32, were obtained for 85 individuals from 17

populations sampled in the field, covering the entire range of H. fumariifolia. The

evolutionary history of these populations was investigated using a nested clade

phylogeographic analysis and also by conducting various population genetic

analyses.

Results In total, 17 haplotypes were detected, 14 of which were found in the

Sierra Madre Oriental. Differentiation among populations based on cpDNA

variation (GST = 0.787, SE 0.0614) indicated population structure in

H. fumariifolia, corroborated by a fixation index (FST) of 0.907. Results from

analysis of molecular variance found that most of the total variation (90.71%,

P < 0.001) was explained by differences among populations. Three regions were

determined based on geological correspondence – the Chihuahuan Desert, Sierra

Madre Oriental and Tehuacan-Cuicatlan Valley – and the variation between them

was significant (43.39%, P < 0.001). Results of a Mantel test showed a significant

correlation between genetic and geographic distances (r = 0.511; P = 0.0001),

suggesting a pattern of isolation by distance, which was corroborated by nested

clade phylogeographic analysis. Mismatch distribution analysis indicated a

sudden demographic expansion.

Main conclusions Our study found that isolation by distance influenced

genetic divergence in populations of H. fumariifolia. The finding that allopatric

fragmentation influenced genetic divergence in populations in the Sierra Madre

Oriental may be a reflection of the complex geology of the area. Our results

suggest that the areas located in the north of the Sierra Madre Oriental acted as

post-glacial refugia for some populations.

Keywords

Allopatric fragmentation, Chihuahuan Desert, contiguous range expansion,

long-distance dispersal, Mexico, phylogeography, post-glacial refugia, Sierra

Madre Oriental, Tehuacan-Cuicatlan Valley.

Journal of Biogeography (J. Biogeogr.) (2009) 36, 18–27

18 www.blackwellpublishing.com/jbi ª 2008 The Authors

doi:10.1111/j.1365-2699.2008.01957.x Journal compilation ª 2008 Blackwell Publishing Ltd

INTRODUCTION

In Mexico, the Late Eocene, middle to Late Miocene and

Pleistocene were characterized by cooling climates (Graham,

1987). The mid-Pleistocene was drier than the Late Pleistocene

in the desert areas of Mexico, where there were extensive

palaeolakes (Metcalfe, 2006). In contrast, climatic changes

during the Late Pleistocene and Holocene were smaller in

magnitude than those occurring in other parts of the tropics

and subtropics (Metcalfe et al., 2000). However, northern

Mexico was much wetter than it is at present as a result of

winter rain from the mid-latitudes (Metcalfe et al., 2000,

2002).

The centre of Mexico comprises the Mexican Plateau, which

is crossed by mountain ranges and segmented by deep rifts.

The plateau is bordered by two mountain ranges, the Sierra

Madre Oriental and the Sierra Madre Occidental, which

converge south of the plateau (Fig. 1). The Transvolcanic Belt

is a large mountain range running from west to east in central

Mexico (Fig. 1). It harbours 13 of the highest peaks of Mexico.

Many valleys and basins divide the Transvolcanic Belt, which is

considered to be a group of active volcanic mountains of

recent origin.

Topographic diversity, climate change, and access to both

temperate and tropical source areas account for the diverse

flora and vegetation of Mexico (Rzedowski, 1978, 1993).

A great proportion of the endemic flora of Mexico is located in

the country’s arid regions (Rzedowski, 1993). The majority of

desertic vegetation is located in the north, but the southern

Tehuacan-Cuicatlan Valley and the Balsas River Basin also

possess xerophytic habitats (Rzedowski, 1978). The largest

desert is the Chihuahuan Desert, which covers most of

northern-central Mexico and extends northwards across

western Texas and southern New Mexico (Schmidt, 1979).

The Chihuahuan Desert and the Tehuacan-Cuicatlan Valley

are separated by the Transvolcanic Belt (Fig. 1). It has been

argued that the Tehuacan-Cuicatlan flora is phytogeographi-

cally related to that of other semi-arid regions, among them

the Chihuahuan Desert (Smith, 1965; Villasenor et al., 1990).

We selected the only species of the endemic Mexican

genus Hunnemannia (Papaveraceae) and conducted a phy-

logeographic study to investigate the evolutionary history of

plant populations distributed in northern and southern

desertic areas. The Mexican tulip poppy, Hunnemannia

fumariifolia Sweet, is a herbaceous perennial that grows in

xerophytic habitats. Hunnemannia is related to Eschscholzia

Figure 1 Geographical distribution of Hunnemannia fumariifolia in Mexico. Population numbers correspond to those in Table 1; haplo-

types to those in Table 2.

Phylogeography of Hunnemannia fumariifolia

Journal of Biogeography 36, 18–27 19ª 2008 The Authors. Journal compilation ª 2008 Blackwell Publishing Ltd

and Dendromecon and forms part of the Eschscholzioideae, a

group of genera from western North America and Mexico

(Hoot et al., 1997). Hunnemannia fumariifolia has a variable

morphology, varying mainly in the size of the plants and the

depth of the indentation of leaves. Small plants with deeply

incised leaves have been considered a different species

(H. hintoniorum, Nesom, 1992); however, these traits are

variable at the population level and are not limited to a

geographic area.

Hunnemannia is found in the Chihuahuan Desert, in the

Sierra Madre Oriental and also in the arid zone of the

Tehuacan-Cuicatlan Valley, growing on gypsum soils (Fig. 1).

The distribution of H. fumariifolia lies within the morphotec-

tonic provinces proposed by Ferrusquia-Villafranca (1993), i.e.

the Chihuahua-Cohauila Plateau, the Sierra Madre Oriental

and the Transvolcanic Belt.

Among methods to estimate gene flow and genetic popu-

lation divergence, nested clade phylogeographic analysis

(NCPA, Templeton et al., 1995), which takes into account

the processes that cause genetic differences in natural popu-

lations, is one of the most common in phylogeographic

studies. Furthermore, NCPA was intended to discriminate

among a wide array of processes and events that shape species

history, such as allopatric fragmentation, contiguous range

expansion, and restricted gene flow caused by isolation by

distance (Templeton et al., 1995). Despite some criticisms of

this method (Knowles & Maddison, 2002; Petit, 2008), NCPA

is still the only method with the potential for disentangling

multiple and overlying effects of historical and recurrent events

within a given data set (Templeton, 2004). In this phylogeo-

graphic study, NCPA was performed with ANeCA, one of the

most recent programs available (Panchal, 2007; Panchal &

Beaumont, 2007).

The aim of this study was to investigate the evolutionary

history of populations from the entire range of H. fumariifolia

based on three chloroplast DNA regions, namely rpl32-

trnL(UAG), ndhF-rpl32 and trnH-psbA, using a NCPA and

population genetic analyses. Three major questions are

addressed: (1) Which historical processes influenced the

geographical pattern of genetic variation in H. fumariifolia?

(2) Was isolation by distance one of the main factors that

caused genetic divergence in populations of this species?

(3) Does genetic flow still exist between populations from the

southern arid zones and those from the northern deserts –

populations that are separated by the Transvolcanic Belt?

MATERIALS AND METHODS

Sampling

A total of 85 individuals from 17 populations were sampled in

the field, in a north–south 900-km transect, covering the entire

geographical range of H. fumariifolia (Table 1). Three to seven

individuals were collected per population. Fresh leaves and

flowers were gathered from each individual, and dried in silica

gel.

DNA extraction, amplification, and sequencing

Total genomic DNA was isolated from silica-gel-dried leaf

tissue using the modified 2X CTAB method (Rogers &

Bendich, 1985; Doyle & Doyle, 1987). The chloroplast

spacers rpl32-trnL(UAG) and ndhF-rpl32 were amplified and

sequenced using the primers and protocols of Shaw et al.

(2007). The trnH-psbA region was amplified and sequenced

using primers trnH2 (Tate & Simpson, 2003) and psbA (Sang

et al., 1997) based on the protocols of Shaw et al. (2005).

Amplification products and DNA were purified using QIA-

quick columns (Quiagen, Valencia, CA, USA) following the

protocols provided by the manufacturer. Clean products were

sequenced using Taq BigDye terminator cycle sequencing kits

(Perkin Elmer Applied Biosystems, Foster City, CA, USA)

using an ABI 310 automated DNA sequencer (Perkin Elmer

Applied Biosystems). Electropherograms were edited and

assembled using sequencher 4.1 (Gene Codes, Ann Arbor,

MI, USA). Sequences were manually aligned with Se-Al

v. 2.0a11 (Rambaut, 2002).

Nested clade phylogeographic analysis and molecular

variability

Nested clade phylogeographic analysis was performed following

the approach of Templeton et al. (2005) using the program

ANeCA (Panchal, 2007). A statistical parsimony network was

obtained using the program tcs (Clement et al., 2000). Based on

the resulting network, nested clades were defined following the

rules of Templeton et al. (1987) and Templeton & Sing (1993).

Clade (Dc) and nested clade (Dn) distances were estimated to

assess the association between the nested cladograms and

geographic distances among sampled localities (Templeton

Table 1 Localities, studied populations of Hunnemannia

fumariifolia, haplotypes and sample size (in parentheses).

Population Location

Latitude and

longitude

Altitude

(m a.s.l.) Haplotype

1 Galeana N24!44¢ W99!58¢ 1746 A(5) K(1)

2 Bonanza N24!36¢ W101!26¢ 2296 C(4)

3 La Escondida N24!04¢ W99!57¢ 1714 I(4) J(3)

4 Real de Catorce N23!42¢ W100!51¢ 2650 C(4) O(1)

5 Cerro Tahti N20!41¢ W99!24¢ 2020 D(5)

6 Coixtlahuaca N17!43¢ W97!22¢ 1953 N(5)

7 San Isidro N25!21¢ W100!20¢ 1736 G(4) Q(1)

8 La Luz N25!21¢ W100!18¢ 1355 G(4)

9 Cienega N25!22¢ W100!13¢ 1355 G(5) H(1)

10 Altares N24!43¢ W99!53¢ 1420 A(5)

11 Rio de San Jose N24!34¢ W99!55¢ 1480 P(5)

12 Zaragoza N23!59¢ W99!47¢ 1374 I(4) A(1)

13 Ciudad del Maız N22!27¢ W99!40¢ 1420 B(3)

E(1) F(1)

14 Arteaga N21!13¢ W99!49¢ 1248 B(3)

15 Tolantongo N20!36¢ W99!01¢ 1900 L(5)

16 Mezquititlan N20!31¢ W98!38¢ 1408 L(6) M(1)

17 Tequixtepec N17!45¢ W97!20¢ 2002 N(3)

V. Sosa et al.

20 Journal of Biogeography 36, 18–27

ª 2008 The Authors. Journal compilation ª 2008 Blackwell Publishing Ltd

et al., 1995). The program geodis (Posada et al., 2000) was used

to test the null hypothesis of no geographic association of clades

and nested clades, with a 95% confidence level, and with 10,000

permutations. If values were significant, the inference key of

Templeton (2004) was used to recognize probable populational

processes and/or historical events of the clades.

Parameters of population diversity, i.e. average within-

population gene diversity (hS), total gene diversity (hT) and

genetic differentiation over all populations (GST), as well as

equivalent parameters (vS, vT, NST), were also estimated,

taking into account the evolutionary distance of haplotypes

using HaploNst and permut (http://www.pierroton.inra.fr/

genetics/labo/Software/index.html) (Pons & Petit, 1995,

1996). Population structure (FST) was also estimated. Analysis

of molecular variance (amova, Excoffier et al., 1992) based

on sequences was performed to assess genetic differentiation

within and among populations as well as among geographical

regions. Sampling populations were grouped into three

geographical regions – the Chihuahuan Desert, Sierra Madre

Oriental and Tehuacan-Cuicatlan Valley – corresponding to

morphotectonic provinces (Ferrusquia-Villafranca, 1993).

Parameters of demographic and spatial expansion (s, h0,

h1), including a mismatch distribution analysis (MDA), were

estimated with 1000 parametric boostrap replicates, as well as

Harpending’s raggedness index (r) (Rogers & Harpending,

1992; Schneider & Excoffier, 1999; Excoffier, 2004). The

genetic matrix distance was constructed in paup* v. 4.0b10

(Swofford, 2003). The appropriate likelihood model (F81+I)

based on the Akaike information criterion was found with

ModelTest v. 3.07 (Posada & Crandall, 1998). These

analyses and the amova were performed using the program

arlequin v. 3.01 (Excoffier et al., 2005). Significance tests

were conducted with 10,000 permutations. Spatial genetic

structure was also assessed by testing the significance of

isolation by distance (IBD) with a Mantel test with 10,000

random permutations with the matrices of pairwise popula-

tion differentiation statistics (FST) and the natural logarithm

of the geographical distances (Rousset, 1997). The test was

performed using the program tfpga v. 1.3 (Miller, 1997).

GenBank accession numbers of the cpDNA rpl32-trnL(UAG)

sequences are EU169024–EU169030; ndhF-rpl32 sequences

are EU169018–EU169023; and trnH-psbA sequences are

EF464658–EF464664.

RESULTS

Sequence analysis

The length of the three cpDNA regions (rpl32-trnL(UAG),

ndhF-rpl32 and trnH-psbA) was 2163 bp. Fourteen substitu-

tions were detected in positions 83, 297, 389, 491, 723, 1024,

1039, 1137, 1167, 1290, 1590, 1854, 1885 and 1958. Five indels

(in positions 436–444, 446–451, 502–503, 742–744 and 1788–

1804) were coded as a single-mutation change. The 30-bp

inversion near the 3¢ end of trnH-psbA in positions 2100–2129

was included in analyses by manually reversing the region and

then coding it as a single evolutionary event that resulted in a

single mutation. The rest of the mutations included short

indels of 1 or 2 bp counted as a single-mutation site in

positions 2036 and 2037 (Table 2).

Haplotype relationships and geographical distribution

In total, 17 haplotypes were detected (Table 2, Fig. 1).

Parsimony analysis resulted in a resolved network (Fig. 2).

Haplotype A is connected to K by one substitution and to P by

a hypothetical haplotype. Haplotype G is connected to Q and

M. Haplotype L is connected to M and I by one substitution.

Haplotype F is connected to I and B by three and four

hypothetical haplotypes, respectively. Haplotype B is con-

nected to O by one substitution. All these haplotypes are

interior. Haplotypes at the tips of the parsimony network are D

and H, both connected to M by one hypothetical haplotype.

Haplotype E is connected to B by two hypothetical haplotypes,

and, finally, haplotypes C and N are connected to O by one

substitution (Fig. 2).

With regard to the Sierra Madre region, six populations

have one haplotype [La Luz (G); Altares (A); Rio San Jose (P);

Arteaga (B); Tolantongo (L); Cerro Tahti (D)], and six

populations have two haplotypes [Galeana (A, I); La Escondida

(I, J); San Isidro (G, Q); Cienega (H, G); Zaragoza (A, I);

Mezquititlan (L, M)]. Ciudad del Maiz has three haplotypes

(B, E, F). In the Chihuahuan Desert region, Bonanza has a

single haplotype (C) and Real de Catorce has two haplotypes

(C, O). In the Tehuacan-Cuicatlan Valley region, both

Coixtlahuca and Tequixtepec have the same haplotype (N)

(Tables 1 and 2; Fig. 2).

Chloroplast haplotype diversity and population

differentiation

Differentiation among populations based on cpDNA variation

(GST = 0.787, SE 0.0614) indicated population structure in

H. fumariifolia, corroborated by a fixation index (FST) of 0.907.

Within-population gene diversity (hS) was 0.201 (SE 0.0579).

Total gene diversity (hT) was 0.947 (SE 0.0138). Differentiation

for ordered alleles (NST) (0.913, SE 0.0395) was higher than

that for GST (U = 2.85, P < 0.01), indicating phylogeograph-

ical structure of cpDNA in H. fumariifolia. Parameters vS and

vT showed values almost identical to hS and hT (0.083, SE

0.0377; 0.0953, SE 0.0431, respectively). The results of the

amova are presented in Table 3. Most of the total variation

(90.71%, P < 0.001) was explained by differences among

populations. The three regions (Chihuahuan Desert, Sierra

Madre Oriental and Tehuacan-Cuicatlan Valley) showed a

significant (43.39%, P < 0.001) variation among themselves,

suggesting that the three separately distributed groups have a

strong genetic differentiation. Results of the Mantel test

showed a significant correlation between the pairwise estimates

of FST and the natural logarithm of the geographical distances

(r = 0.511; P = 0.0001), suggesting a pattern of isolation by

distance.



Demographic and nested clade phylogeographic

analysis

Mismatch distribution analysis links the number of differences

between haplotypes and haplotype frequency, and in our

analysis was unimodal. It showed a significant deviation from

the sudden expansion model. Estimated parameters from the

total cladogram were s = 9.539, h0 = 0.000 and h1 = 18.496.

Based on Harpending’s raggedness index (r = 0.449,

P = 0.03), the hypothesis of sudden demographic expansion

Table 2 Seventeen haplotypes of Hunnemannia fumariifolia were recognized based on three chloroplast DNA sequences, rpl32-trnL(UAG),

ndhF-rpl32 and trnH-psbA.

Sequence Position Collection Sites

Haplotype

82344444444444445577771111111111111111111111111122222222222222222222222222222222

GBERCXSLNARZMOTQU

39833334444445590024440011257777777777778888888933111111111111111111111111111111

7667890167890112332342336998899999999990000058500000000000011111111112222222222

n Region4977008901234567890123445867012345678901234567890123456789

A CGA------ATATAGTTTG---CACTGCATTAATATGAAATGATTTATA-CCCGCCCTCTTGATAAACAAGAAATTTCGG 50000000050100000 11 SMO

B A.GCTATAT------G...---......-----------------CGG.-****************************** 00000000000033000 6 SMO

C A.G......------G...---......-----------------CGG--.............................. 04040000000000000 8 CD

D .A.......------G--.---..A.T....................G.-.............................. 00005000000000000 5 SMO

E A.G......------G...---......-----------------....-****************************** 00000000000010000 1 SMO

F .A.......------G--T---......-----------------CGG.-****************************** 00000000000010000 1 SMO

G .A.......------G--.AAA.....A...................G.-.............................. 00000044500000000 13 SMO

H .A.......------G--.---.CA......................G.-****************************** 00000000100000000 1 SMO

I .A.......------G--T---...G.....................G.-.............................. 00400000000400000 8 SMO

J .A.......------G--T---...G.....................G.-.............................. 00300000000000000 3 SMO

K ...------ATATAG....---...G.......................-.............................. 10000000000000000 1 SMO

L .A.......------G--.---.........................G.-.............................. 00000000000000560 11 SMO

M .A.......------G--.---...G.....................G.-.............................. 00000000000000010 1 SMO

N A.G......------G...---......-----------------CGG.A.............................. 00000500000000003 8 T-CV

O A.G......------G...---......-----------------CGG.-.............................. 00010000000000000 1 CD

P .........ATATAG....---.........................G.-.............................. 00000000005000000 5 SMO

Q .A.......------G--.AAAT....A...................G.-.............................. 00000010000000000 1 SMO

G: Galeana; B: Bonanza; E: La Escondida; R: Real de Catorce; C: Cerro Tahti; X: Coixtlahuaca; S: San Isidro; L: La Luz; N: Cienega; A: Altares; R: Rio

San Jose; Z: Zaragoza; M: Ciudad del Maiz; O: Arteaga; T: Tolantongo; Q: Mezquititlan; U: Tequixtepec. CD: Chihuahuan Desert; SMO: Sierra Madre

Oriental; T-CV: Tehuacan-Cuicatlan Valley.

Sequence with *: fragment 30-bp inversion; –: indel.

Figure 2 Statistical parsimony network

and resulting set of nested clades of the 17

cpDNA haplotypes found in Hunnemannia

fumariifolia. A–Q: sampled haplotypes.

Solid bars: hypothetical haplotypes.

The number of individuals is given in

parentheses.

V. Sosa et al.



was accepted. We also performed MDA separately for each

clade resulting from NCPA that resulted in contiguous range

expansion and inclusive outcome clades (2-3, 3-2 and 2-4).

The raggedness index in clades 2-4 and 3-2 showed non-

significant values of P (r = 0.178, P = 0.23; r = 0.016,

P = 0.81), but that in clade 2-3 showed significant values

(r = 0.209, P = 0.01).

Nested clade phylogeographic analysis showed a significant

relationship between genetic and geographical distributions in

H. fumariifolia (Table 4). Restricted gene flow with isolation

by distance was detected in clades 1-4 and 3-1. Clade 1-4

included haplotypes C and O from the Chihuahuan Desert,

and haplotype N from the Tehuacan-Cuicatlan Valley. Clade

3-1 had all haplotypes in the Sierra Madre Oriental (SMO).

Clades 1-9 and 2-2 showed allopatric fragmentation. Clade 1-9

had a distribution in the north (haplotype J) and in the south

(haplotype L) of the SMO. Clade 2-2 included haplotypes

A-K, P, distributed in the north of the SMO (Figs 1 & 2).

Contiguous range expansion was detected in clades 2-3 and

3-2. Restricted gene flow with isolation/dispersal but with

some long-distance dispersal over intermediate areas not

occupied by the species, or past gene flow followed by

extinction of intermediate populations was detected in clade

2-1. An inclusive outcome was observed in clade 2-4 and in the

entire cladogram (Table 4).

DISCUSSION

Population differentiation

Hunnemannia fumariifolia showed population differentiation

(GST = 0.787 and FST = 0.907). The GST value was in agree-

ment with other plant phylogeographical studies (GST = 0.60–

0.96) (e.g. Demesure et al., 1996; El Mousadik & Petit, 1996;

Dumolin-Lapegue et al., 1997; Dutech et al., 2000; Cavers

et al., 2003; Huang et al., 2004; Marchelli & Gallo, 2006; Ikeda

& Setoguchi, 2007). Genetic differentiation in plants has been

attributed to limited seed or pollen dispersal (Petit et al.,

2005), and limited genetic structure has been attributed to high

dispersal ability (e.g. Palme et al., 2003; Jones et al., 2006; Wu

et al., 2006; Chung et al., 2007). In our results, NST (0.913) was

significantly higher than GST (0.787), suggesting that pairs of

different haplotypes from the same population have more

similar sequences than pairs of different haplotypes from

Table 3 Results of the analysis of molecular variance for 17

populations of Hunnemannia fumariifolia grouped in three

geographical regions (Chihuahuan Desert, Sierra Madre Oriental

and Tehuacan-Cuicatlan Valley) based on cpDNA sequence data.

Source of variation d.f.

Sum of

squares

Variance

components

Percentage

of variation

(%)

Among populations (total) 16 0.132 0.00162 90.71*

Within populations 68 0.011 0.0017 9.29

(1,3,5,7,8,9,10,11,12,13,14,

15,16) vs. (2,4) vs. (6,17)

Among groups 2 0.042 0.00107 43.39*

Among populations

within groups

14 0.089 0.00123 49.89*

Within populations 68 0.011 0.00017 6.72*

*P < 0.001.

Table 4 Results of the nested clade phylogeographical analysis and interpretations according to the revised inference key of Templeton

et al. (2005) for 17 populations of Hunnemannia fumariifolia from Mexico.

Nested

clade v2 P-value Dc Dn

Inference

chain Inferred pattern

1-4 18.7 0.0000 33.9928S )62.219 1-2-3-4 NO Restricted gene flow with isolation

by distance

1-9 14.0 0.0010 No interior/tip

clades exist

1-19 NO Allopatric fragmentation

2-1 37.8333 0.000 )41.7955L 212.1502L 1-2-3-5-6-7-8 YES Restricted gene flow/dispersal but

with some long-distance dispersal

over intermediate areas not occupied

by the species; or past gene flow

followed by extinction of intermediate

populations

2-2 16.0 0.0000 )14.4461S )9.7687S 1-19 NO Allopatric fragmentation

2-3 27.0 0.0010 )308.4168S )306.4102S 1-2-11-12 NO Contiguous range expansion

2-4 14.5918 0.0000 No interior/tip

clades exist

1-2 IO Inconclusive outcome

3-1 37.0 0.0000 198.4136L 159.9941L 1-2-3-4 NO Restricted gene flow with isolation

by distance

3-2 54.8333 0.0000 )116.4518S )109.1562S 1-2-11-12 NO Contiguous range expansion

Total

cladogram

78.2963 0.0000 No interior/tip

clades

1-2 IO Inconclusive outcome

Dc and Dn are the clade and nested clade distances, respectively.



distinct populations (Pons & Petit, 1996). Similar results, in

which NST is higher than GST, have been reported in a number

of phylogeographical studies of plants (e.g. El Mousadik &

Petit, 1996; Dumolin-Lapegue et al., 1997; Cavers et al., 2003;

Petit et al., 2005; Marchelli & Gallo, 2006; Aizawa et al., 2007).

Isolation by distance and allopatric fragmentation

Isolation by distance can be studied through the correlation of

genetic and geographical distances and also through nested

clade analysis (e.g. Cuenca et al., 2003). Results of our Mantel

test found a significant correlation between genetic and

geographical distances in H. fumariifolia, suggesting isolation

by distance. Furthermore, NCPA found three clades (1-4, 2-1

and 3-1) with evidence for restricted gene flow and isolation by

distance, but clade 2-1 also showed dispersal but with some

long-distance dispersal, or past gene flow followed by extinc-

tion of intermediate populations. Clade 1-4 included individ-

uals of four populations, two of them from the Chihuahuan

Desert (Bonanza and Real de Catorce), and two from the

Tehuacan-Cuicatlan Valley (Coixtlahuca and Tequixtepec).

These two regions are separated by the Transvolcanic Belt and

a distance of c. 800 km (Fig. 1). Clade 3-1 grouped individuals

from nine populations, all of them from the SMO. Some

populations (Galeana, San Isidro, La Luz, Cienega, Altares, Rio

San Jose and Zaragoza) are in the north of the SMO, whereas

two of them (Cerro Tahti and Mezquititlan) are in the south of

the SMO, and the two groups are separated by 400 km

(Fig. 1).

Nested clade phylogeographic analysis also found two clades

(1-9 and 2-2) in which processes such as allopatric fragmen-

tation influenced genetic divergence. Clade 1-9 included

individuals from three populations, namely Mezquititlan and

Tolantongo in the south and La Escondida in the north of the

SMO. Clade 2-2 included individuals from four populations

(Galeana, Altares, Rio San Jose and Zaragoza), all of them in

the north of the SMO. We suggest that allopatric fragmenta-

tion could be a result of the uplift of groups of mountains

within the SMO. The SMO is one of the most convoluted

geological regions of Mexico. In the north, it includes groups

of mountains in southern Nuevo Leon, western Tamaulipas

and northern San Luis Potosi. Another band of mountains

extends westwards to Nuevo Leon across southern Coahuila.

These groups of mountains were lifted in different periods in a

complex way (Ferrusquia-Villafranca, 1993).

The complex processes found in populations of H. fumarii-

folia, such as restricted gene flow with isolation by distance or

long-distance dispersal, allopatric fragmentation and/or con-

tiguous range expansion, have been reported in a number of

other plant phylogeographical studies (e.g. Bittkau & Comes,

2005; Wu et al., 2006; Cornman & Arnold, 2007).

Post-glacial refugia

There is debate over the importance of northerly and southerly

refugia in Europe and over the existence of the so-called

‘cryptic’ northern refugia (Lopez de Heredia et al., 2007;

Bhagwat & Willis, 2008). However, there is agreement over a

common phylogeographical pattern of distribution that has

been detected by Taberlet & Cheddadi (2002) in the vegetation

of the temperate regions of the Northern Hemisphere. During

cold periods, the geographical ranges of most species were

restricted to a single refugium or a few refugia in the south.

During subsequent warming, species expanded their ranges,

mainly northwards, colonizing areas according to their dispersal

abilities and ecological requirements. When the climate became

cooler, northern populations disappeared, leaving no descen-

dants. This agrees with previous studies that determined that

refugia were located in southern areas of north-eastern North

America and of unglaciated western North America (Brunsfeld

et al., 2001; Soltis et al., 2006). Phylogeographical studies have

found that, if genetic diversity is high, areas acted either as a

refugium or as mixing zones of organisms (Huang et al., 2004).

In the first case, haplotypes are closely related, whereas the latter

case may include distantly related haplotypes (Petit et al., 2002;

Huang et al., 2004; Marchelli & Gallo, 2006; Wu et al., 2006).

Our mismatch distribution analysis results suggest that areas in

the north of the SMO acted as post-glacial refugia for some

populations of H. fumariifolia. This coincides with the climate

changes of the Pleistocene, during which the northern portion

of Mexico was wetter (Metcalfe et al., 2000, 2002).

Our results indicated that some populations of the SMO are

the consequence of post-glacial range expansion, as indicated by

parameters for sudden expansion models. The same pattern of

distribution is found in another desert plant, Agave lechuguilla,

for which populations seem to have originated in the north and

more recently to have colonized the south (Silva-Montellano &

Eguiarte, 2003). In contrast, for the creosote bush, Larrea

tridentata, another desert plant, populations recently colonized

the north from southern refugia (Duran et al., 2005).

During the Pleistocene the Tehuacan-Cuicatlan Valley was

moister and cooler, with regular winter frosts (McNeish et al.,

1972). During this period, the valley had extensive grassland.

Many cacti, Agave and other desert plants could not live under

such conditions. The transition to the recent climate regime

occurred in the period 7800–7400 bc (McNeish et al., 1972).

Therefore, we suggest that populations of the Mexican tulip

poppy survived in refugia in the northern SMO before this

climatic transition. Phylogeographical studies of other plant

species with a similar geographical distribution to that of

H. fumariifolia would be valuable for understanding the

evolutionary histories of North American xerophytic plants.

In summary, our study found that the historical processes

that influenced the geographical pattern of genetic variation in

H. fumariifolia were restricted gene flow with isolation by

distance, allopatric fragmentation, and sudden demographic

expansion. In addition, isolation by distance was detected as

the main factor that shaped distributional patterns in popu-

lations of this species. Moreover, there is no current genetic

flow between populations from the SMO and those from the

Chihuahuan Desert, nor between these northern populations

and those from the Tehuacan-Cuicatlan Valley.

V. Sosa et al.



ACKNOWLEDGEMENTS

We thank two anonymous referees for their suggestions, which

improved our paper. We also thank Pablo Carrillo, Arturo De

Nova and Daniel de la Rosa for assistance with fieldwork, Carla

Gutierrez for her suggestions on a previous version of the

manuscript, and Bianca Delfosse for editing the English. DNA

extraction and sequencing were made possible through a grant

to V.S. by CONACYT (P39601526).

REFERENCES

Aizawa, M., Yoshimaru, H., Saito, H., Katsuki, T., Kawahara,

T., Kitamura, K., Shi, F. & Kaji, M. (2007) Phylogeography

of a northeast Asian spruce, Picea jezoensis, inferred from

genetic variation observed in organelle DNA markers.

Molecular Ecology, 16, 3393–3405.

Bhagwat, S.A. & Willis, K.J. (2008) Species persistence in

northerly glacial refugia of Europe: a matter of chance or

biogeographical traits? Journal of Biogeography, 35, 464–482.

Bittkau, C. & Comes, H.P. (2005) Evolutionary processes in a

continental island system: molecular phylogeography of the

Aegean Nigella arvensis alliance (Ranunculaceae) inferred

from chloroplast DNA. Molecular Ecology, 14, 4065–4083.

Brunsfeld, S.J., Sullivan, J., Soltis, D.E. & Soltis, P.S. (2001) A

comparative phylogeography of northwestern North

America: a synthesis. Integrating ecology and evolution in a

spatial context (ed. by J. Silvertown and J. Antonovics),

pp. 319–340. Blackwell Science, Oxford.

Cavers, S., Navarro, C. & Lowe, A.J. (2003) Chloroplast DNA

phylogeography reveals colonization history of a Neotro-

pical tree, Cedrela odorata L., in Mesoamerica. Molecular

Ecology, 12, 1451–1460.

Chung, J.-D., Lin, T.-P., Chen, Y.-L., Cheng, Y.-P. & Hwang,

S.-Y. (2007) Phylogeographic study reveals the origin and

evolutionary history of a Rhododendron species complex in

Taiwan. Molecular Phylogenetics and Evolution, 42, 14–24.

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

computer program to estimate gene genealogies. Molecular

Ecology, 9, 1657–1659.

Cornman, R.S. & Arnold, M.L. (2007) Phylogeography of Iris

missouriensis (Iridaceae) based on nuclear and chloroplast

markers. Molecular Ecology, 16, 4585–4598.

Cuenca, A., Escalante, A.E. & Pinero, D. (2003) Long-distance

colonization, isolation by distance, and historical demo-

graphy in a relictual Mexican pinyon pine (Pinus nelsonii

Shaw) as revealed by paternally inherited genetic markers

(cpSSRs). Molecular Ecology, 12, 2087–2097.

Demesure, B., Comps, B. & Petit, R.J. (1996) Chloroplast DNA

phylogeography of the common beech (Fagus sylvatica L.) in

Europe. Evolution, 50, 2515–2520.

Doyle, J.J. & Doyle, J.L. (1987) A rapid DNA isolation pro-

cedure from small quantities of fresh leaf tissues. Phyto-

chemistry Bulletin, 19, 11–15.

Dumolin-Lapegue, S., Demesure, B., Fineschi, S., Le Corre, V.

& Petit, R.J. (1997) Phylogeographic structure of white oaks

throughout the European continent. Genetics, 146, 1475–

1487.

Duran, K.L., Lowrey, T.K., Parmenter, R.R. & Lewis, P.O.

(2005) Genetic diversity in Chihuahuan Desert populations

of creosote bush (Zygophyllaceae: Larrea tridentata).

American Journal of Botany, 92, 722–729.

Dutech, C., Maggia, L. & Joly, H.I. (2000) Chloroplast diversity

in Vouacapoua americana (Caesalpinaceae), a neotropical

forest tree. Molecular Ecology, 9, 1427–1432.

ElMousadik, A. &Petit, R.J. (1996)Chloroplast phylogeography

of the argan tree of Morocco. Molecular Ecology, 5, 547–555.

Excoffier, L. (2004) Patterns of DNA sequence diversity and

genetic structure after a range expansion: lessons from the

infinite-island model. Molecular Ecology, 13, 853–864.

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

molecular variance inferred from metric distances among

DNA haplotypes: applications to human mitochondrial

DNA restriction data. Genetics, 131, 479–491.

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

3.01. An Integrated software package for population genetics

data analysis. Evolutionary Bioinformatics Online, 1, 47–60.

Ferrusquia-Villafranca, I. (1993) Geology of Mexico: a syn-

opsis. Biological diversity of Mexico: origins and distribution

(ed. by T.P. Ramamoorthy, R. Bye, A. Lot and J. Fa), pp.

3–107. Oxford University Press, New York.

Graham, A. (1987) Tropical American Tertiary floras and

paleoenvironments: Mexico, Costa Rica and Panama.

American Journal of Botany, 74, 1519–1531.

Hoot, S.B., Kadereit, J.W., Blattner, F.B., Jork, K.B., Schwarz-

bach, A.E. & Crane, P.R. (1997) Data congruence and

phylogeny of the Papaveraceae s.l., based on four data sets:

atpB and rbcL sequences, trnK restriction sites, and mor-

phological characters. Systematic Botany, 22, 575–590.

Huang, S.-F., Hwang, S.-Y., Wang, J.-C. & Lin, T.-P. (2004)

Phylogeography of Trochodendron aralioides (Trocho-

dendraceae) in Taiwan and its adjacent areas. Journal of

Biogeography, 31, 1251–1259.

Ikeda, H. & Setoguchi, H. (2007) Phylogeography and refugia

of Japanese endemic alpine plant, Phyllodoce nipponica

Makino (Ericaceae). Journal of Biogeography, 34, 169–176.

Jones, M.E., Shepherd, M., Henry, R.J. & Delves, A. (2006)

Chloroplast DNA variation and population structure in the

widespread forest tree, Eucalyptus grandis. Conservation

Genetics, 7, 691–703.

Knowles, L.L. & Maddison, W.P. (2002) Statistical phylo-

geography. Molecular Ecology, 11, 2623–2635.

Lopez de Heredia, U., Carrion, J.S., Jimenez, P., Collada, C. &

Gil, L. (2007) Molecular and palaeoecological evidence for

multiple glacial refugia for evergreen oaks on the Iberian

Peninsula. Journal of Biogeography, 34, 1505–1517.

Marchelli, P. & Gallo, L. (2006) Multiple ice-age refugia in a

southern beech of South America as evidenced by chloro-

plast DNA markers. Conservation Genetics, 7, 591–603.

McNeish, R.S., Peterson, F.A. & Neely, J.A. (1972) The archae-

ological reconnaissance.The prehistory of the TehuacanValley.



Vol. 5. Excavations and reconnaissance (ed. by R.S. McNeish),

pp. 496–504. University of Texas Press, Austin, TX.

Metcalfe, S.E. (2006) Late Quaternary environments of the

northern deserts and central Transvolcanic belt of Mexico.

Annals of the Missouri Botanical Garden, 93, 258–273.

Metcalfe, S.E., O’Hara, S.L., Caballero, M. & Davies, S.J. (2000)

Records of late Pleistocene–Holocene climatic change in

Mexico – a review. Quaternary Science Reviews, 19, 699–721.

Metcalfe, S.E., Say, A., Black, S., McCulloch, R. & O’Hara, S.

(2002) Wet conditions during the last glaciation in the

Chihuahuan Desert, Alta Babicora Basin, Mexico. Quater-

nary Research, 57, 91–101.

Miller, M. (1997) Tools for Population Genetics Analysis

(TFPGA), version 1.3. A Windows" program for the analysis

of allozyme and molecular population genetic data. Depart-

ment of Biological Sciences, Northern Arizona University,

Flagstaff, AZ.

Nesom, G.L. (1992) A second species of Hunnemannia (Pap-

averaceae) and synopsis of the genus. Phytologia, 73, 330–337.

Palme, A.E., Semerikov, V. & Lascoux, M. (2003) Absence of

geographical structure of chloroplast DNA variation in sal-

low, Salix caprea L. Heredity, 91, 465–474.

Panchal, M. (2007) The automation of nested clade phylo-

geographic analysis. Bioinformatics, 23, 509–510.

Panchal, M. & Beaumont, M.A. (2007) The automation and

evaluation of nested clade phylogeographic analysis. Evolu-

tion, 61, 1466–1480.

Petit, R.J. (2008) The coup de grace for the nested clade

phylogeographic analysis? Molecular Ecology, 17, 516–518.

Petit, R.J., Brewer, S., Bordacs, S., Burg, K., Cheddadi, R., Coart,

E., Cottrell, J., Csaikl, U.M., Dam, B., Deans, J.D., Espinel, S.,

Fineschi, S., Finkeldey, R., Glaz, I., Goicoechea, P.G., Jensen,

J.S., Konig, A.O., Lowe, A.J.,Madsen, S.F.,Matyas, G.,Munro,

R.C., Popescu, F., Slade, D., Tabbener, H., Vries, S.G.M.,

Ziegenhagen, B., Beaulieu, J. & Kremer, A. (2002) Identifi-

cation of refugia and post-glacial colonization routes of

European white oaks based on chloroplast DNA and fossil

pollen evidence. Forest Ecology and Management, 159, 49–74.

Petit, R.J., Duminil, J., Fineschi, S., Hampe, A., Salvini, D. &

Vendramin, G.G. (2005) Comparative organization of

chloroplast, mitochondrial and nuclear diversity in plant

populations. Molecular Ecology, 14, 689–701.

Pons, O. & Petit, R.J. (1995) Estimation, variance and optimal

sampling of gene diversity. I. Haploid locus. Theoretical and

Applied Genetics, 90, 462–470.

Pons, O. & Petit, R.J. (1996) Measuring and testing genetic

differentiation with ordered versus unordered alleles.

Genetics, 144, 1237–1245.

Posada, D. & Crandall, K.A. (1998) Modeltest: testing the

model of DNA substitution. Bioinformatics, 14, 817–818.

Posada, D., Crandall, K.A. & Templeton, A.R. (2000) GeoDis: a

program for the cladistic nested analysis of the geographical

distribution of genetic haplotypes. Molecular Ecology, 9,

487–488.

Rambaut, A. (2002) Se-Al Sequence Alignment Editor, v2.0a11.

Department of Zoology, University of Oxford, Oxford.

Available at: http://tree.bio.ed.ac.uk/software/seal/ (last

accessed 20 April 2008).

Rogers, S.O. & Bendich, A.J. (1985) Extraction of DNA from

milligram amounts of fresh, herbarium and mummified

plant tissues. Plant Molecular Biology, 5, 69–76.

Rogers, A.R. & Harpending, H. (1992) Population growth

makes waves in the distribution of pairwise genetic differ-

ences. Molecular Biology and Evolution, 9, 552–569.

Rousset, F. (1997) Genetic differentiation and estimation of

gene flow from F-statistics under isolation-by-distance.

Genetics, 145, 1219–1228.

Rzedowski, J. (1978) La vegetacion de Mexico. Limusa, Mexico,

D. F.

Rzedowski, J. (1993) Diversity and origins of the phanero-

gamic flora of Mexico. Biological diversity of Mexico: origins

and distribution (ed. by T.P. Ramamoorthy, R. Bye, A. Lot

and J. Fa), pp. 129–144. Oxford University Press, New York.

Sang, T., Crawford, D.J. & Stuessy, T.F. (1997) Chloroplast

DNA phylogeny, reticulate evolution, and biogeography of

Paeonia (Paeoniaceae). American Journal of Botany, 84,

1120–1136.

Schmidt, R.H. (1979) A climatic delineation of the ‘‘real’’

Chihuahuan Desert. Journal of Arid Environments, 2, 243–

250.

Schneider, S. & Excoffier, L. (1999) Estimation of demographic

parameters from the distribution of pairwise differences

when the mutation rates vary among sites: application to

human mitochondrial DNA. Genetics, 152, 1079–1089.

Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W.S., Miller,

J., Siripun, K.C., Winder, C.T., Schilling, E.E. & Small, R.L.

(2005) The tortoise and the hare. II: Relative utility of 21

noncoding chloroplast DNA sequences for phylogenetic

analysis. American Journal of Botany, 92, 142–166.

Shaw, J., Lickey, E.B., Schilling, E.E. & Small, R.L. (2007)

Comparison of whole chloroplast genome sequences to

choose noncoding regions for phylogenetic studies in

angiosperms: the tortoise and the hare. III. American Journal

of Botany, 94, 275–288.

Silva-Montellano, A. & Eguiarte, L.E. (2003) Geographic pat-

terns in the reproductive ecology of Agave lechuguilla

(Agavaceae) in the Chihuahuan Desert. II. Genetic variation,

differentiation, and inbreeding estimates. American Journal

of Botany, 90, 700–706.

Smith, C.E. (1965) Flora, Tehuacan Valley. Fieldiana, 31, 49–

100.

Soltis, D.E., Morris, A.B., McLachlan, J.S., Manos, P.S. & Soltis,

P.S. (2006) Comparative phylogeography of unglaciated

eastern North America. Molecular Ecology, 15, 4261–4293.

Swofford, D.L. (2003) PAUP*: phylogenetic analysis using

parsimony (*and other methods), version 4.0b10. Sinauer,

Sunderland, MA.

Taberlet, P. & Cheddadi, R. (2002) Quaternary refugia and

persistence of biodiversity. Science, 297, 2009–2010.

Tate, J.A. & Simpson, B.B. (2003) Paraphyly of Tarasa

(Malvaceae) and diverse origins of the polyploid species.

Systematic Botany, 28, 723–737.

V. Sosa et al.



Templeton, A.R. (2004) Statistical phylogeography: methods of

evaluating and minimizing inference errors. Molecular

Ecology, 13, 789–809.

Templeton, A.R. & Sing, C.F. (1993) A cladistic analysis of

phenotypic associations with haplotypes inferred from

restriction endonuclease mapping. IV. Nested analyses with

cladogram uncertainty and recombination. Genetics, 134,

659–669.

Templeton, A.R., Boerwinkle, E. & Sing, C.F. (1987) A cladistic

analysis of phenotypic associations with haplotypes inferred

from restriction endonuclease mapping. 1. Basic theory and

an analysis of alcohol dehydrogenase activity in Drosophila.

Genetics, 117, 343–351.

Templeton, A.R., Routman, E. & Phillips, C.A. (1995)

Separating population structure from population history: a

cladistic analysis of the geographical distribution of mito-

chondrial DNA haplotypes in the tiger salamander,

Ambystoma tigrinum. Genetics, 140, 767–782.

Templeton, A.R., Maxwell, T., Posada, D., Stengard, J.H.,

Boerwinkle, E. & Sing, C.F. (2005) Tree scanning: a method

for using haplotype trees in phenotype/genotype association

studies. Genetics, 169, 441–453.

Villasenor, J.L., Davila, P. & Chiang, F. (1990) Fitogeografıa

del Valle de Tehuacan-Cuicatlan. Boletın de la Sociedad

Botanica Mexico, 50, 135–149.

Wu, S.-H., Hwang, C.-H., Lin, T.-P., Chung, J.-D., Cheng,

Y.-P. & Hwang, S.-Y. (2006) Contrasting phylogeographical

patterns of two closely related species, Machilus thunbergii

and Machilus kusanoi (Lauraceae), in Taiwan. Journal of

Biogeography, 33, 936–947.

BIOSKETCHES

Victoria Sosa holds a full-time research position at the

Institute of Ecology in Xalapa, Mexico. Her interests are in the

phytogeography, systematics and conservation of the endemic

flora of Mexico.

Eduardo Ruiz-Sanchez is a graduate student in the PhD

program of Systematics at the Institute of Ecology in Xalapa,

Mexico. His interests are in the phytogeography and system-

atics of the flora of Mexico.

Flor C. Rodriguez-Gomez is a student in the Master’s

program at the Institute of Ecology in Xalapa. Her interest is in

population genetics.

Editor: Jorge Crisci



Hidden phylogeographic complexity in the Sierra Madre Oriental: the case of the Mexican tulip poppy...

Documents

Transcript of Hidden phylogeographic complexity in the Sierra Madre Oriental: the case of the Mexican tulip poppy...