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Phylogeny, diversification rates and species boundaries of Mesoamerican firs(Abies, Pinaceae) in a genus-wide context

Érika Aguirre-Planter a,1, Juan P. Jaramillo-Correa a,b,1, Sandra Gómez-Acevedo a, Damase P. Khasa b,Jean Bousquet b, Luis E. Eguiarte a,⇑a Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Apartado Postal 70-275, México, D.F., Mexicob Canada Research Chair in Forest and Enrionmental Genomics, Centre for Forest Research and Institute for Systems and Integrative Biology, Université Laval, Québec, Canada G1V 0A6

a r t i c l e i n f o

Article history:Received 18 May 2011Revised 26 September 2011Accepted 28 September 2011Available online 10 October 2011

Keywords:BiogeographyChloroplast DNAConiferMexicoTransverse Volcanic BeltMolecular phylogenyDiversification rates

a b s t r a c t

The genus Abies is distributed discontinuously in the temperate and subtropical montane forests of thenorthern hemisphere. In Mesoamerica (Mexico and northern Central America), modern firs originatedfrom the divergence of isolated mountain populations of migrating North American taxa. However, thenumber of ancestral species, migratory waves and diversification speed of these taxa is unknown. Here,variation in repetitive (Pt30204, Pt63718, and Pt71936) and non-repetitive (rbcL, rps18-rpl20 and trnL-trnF) regions of the chloroplast genome was used to reconstruct the phylogenetic relationships of theMesoamerican Abies in a genus-wide context. These phylogenies and two fossil-calibrated scenarios werefurther employed to estimate divergence dates and diversification rates within the genus, and to test thehypothesis that, as in many angiosperms, conifers may exhibit accelerated speciation rates in the sub-tropics. All phylogenies showed five main clusters that mostly agreed with the currently recognized sec-tions of Abies and with the geographic distribution of species. The Mesoamerican taxa formed a singlegroup with species from southwestern North America of sections Oiamel and Grandis. However, popula-tions of the same species were not monophyletic within this group. Divergence of this whole group datedback to the late Paleocene and the early Miocene depending on the calibration used, which translated invery low diversification rates (r0.0 = 0.026–0.054, r0.9 = 0.009–0.019 sp/Ma). Such low rates were a con-stant along the entire genus, including both the subtropical and temperate taxa. An extended phylogeo-graphic analysis on the Mesoamerican clade indicated that Abies flinckii and A. concolor were the mostdivergent taxa, while the remaining species (A. durangensis, A. guatemalensis, A. hickelii, A. religiosa andA. vejari) formed a single group. Altogether, these results show that divergence of Mesoamerican firs coin-cides with a model of environmental stasis and decreased extinction rate, being probably prompted by aseries of range expansions and isolation-by-distance.

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1. Introduction

Diversification and speciation rates seem to be higher in taxafrom tropical or subtropical ecosystems than from temperate orboreal zones (e.g. Wright et al., 2006; Gillman et al., 2010). Suchdifferences can be prompted by contrasting patterns of populationisolation, adaptation speed, extinction rates and/or stochasticevents that cause reproductive isolation (e.g. Wright et al., 2006;Glor, 2010). For instance, in angiosperms, it has been shown that

the fastest speciation rates are usually attained in taxa with highbackground substitution rates and that have recently expandedinto new habitats, while the lowest are most often observed in spe-cies with both low substitution and extinction rates, and which in-habit stable environments (Lancaster, 2010). Conifers are slowlyevolving taxa that should conform to this last low speciation mod-el. However, based on morphology, conifer taxa appear to be morediversified in the subtropical than in the temperate environments(Farjon, 1990), which might suggest some kind of divergence accel-eration towards the equator. In subtropical conifers, speciation re-lated to geographical isolation is expected to be a predominantdriver for diversification, while slow reproductive isolation shouldbe a delaying factor (e.g. Bouille et al., 2011). Thus, it could behypothesized that the higher number of conifer species observedin the subtropics is either the result of accelerated speciation dueto increased historical isolation, or simply an artifact produced

1055-7903/$ - see front matter � 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2011.09.021

⇑ Corresponding author. Fax: +52 55 5616 1976.E-mail addresses: [email protected] (É. Aguirre-Planter), jaramillo@

ecologia.unam.mx (J.P. Jaramillo-Correa), [email protected] (S. Gómez-Acevedo),[email protected] (D.P. Khasa), [email protected] (J.Bousquet), [email protected] (L.E. Eguiarte).

1 These authors contributed equally to this work.

Molecular Phylogenetics and Evolution 62 (2012) 263–274

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by an elevated phenotypic plasticity that has misled experts whendescribing species based solely on morphological variation (e.g. DeKroon et al., 2005; Prada et al., 2008).

The knowledge of diversification rates and speciation pro-cesses are of particular importance in megadiverse countries likeMexico, where the lack of such information collides with thelarge conservation needs (Callmander et al., 2005). Mexico hasapproximately 30,000 plant taxa (Rzedowski, 1993), from whichabout 10% are of conservation concern (SEMARNAT, 2010). Thisspecies richness has been explained by the habitat variation pro-duced by a complex geological history (Espinosa-Organista et al.,2008; Jaramillo-Correa et al., 2009), which provides ideal condi-tions for the diversification of the many taxa that expanded fromboth North and South America during the last 30 million years(Rzedowski, 1978; Graham, 1999). Conifers were among the firstof such temperate elements that arrived into Mexico (i.e. some23 Ma; Graham, 1999), and since then, they have become onethe most diverse and important components of the montane for-ests of this country. However, in spite of this, the taxonomic sta-tus of many Mexican conifers remains dubious and has beenquestioned in different occasions (e.g. Farjon and Rushforth,1989; Farjon, 1990; Strandby et al., 2009).

Firs (Abies Miller; a predominantly temperate northern genus)are among the most abundant and less studied taxa of the mon-tane forests of Mesoamerica (i.e. Mexico and northern CentralAmerica). They are represented by eight threatened species, sixof which are endemic to Mexico (Liu, 1971; Farjon and Rush-forth, 1989; SEMARNAT, 2010). Their first appearance in theMesoamerican fossil record points to a late arrival in the Plio-cene (i.e. about 5 Ma; Graham, 1976, 1999), which implies arather rapid diversification after their establishment. However,it is unclear whether this date correctly reflects their first com-ing into the region, or if there were one or more migratorywaves from North America. Morphologically, Mesoamerican firshave been grouped into three or two different sections accordingto the most widely recognized classifications (i.e. Liu, 1971; Far-jon and Rushforth, 1989. See Table 1), which suggests at leasttwo independent expansions from as many ancestors. Unfortu-nately, recent molecular phylogenetic analyses of this genus(i.e. Suyama et al., 2000; Xiang et al., 2004, 2009) had limitedsampling at the local scale, and thus low resolution to addressthis issue, while previous population studies (Aguirre-Planteret al., 2000; Jaramillo-Correa et al., 2008) lacked the phyloge-netic context necessary to test this hypothesis. Indeed, wheninferring diversification processes in closely related taxa suchas the Mesoamerican firs, a combination of phylogenetic andphylogeographic analyses on samples covering most of the genusrange is necessary (e.g. Liston et al., 2007; Xu et al., 2010).

In the present study, we used the variation of repetitive andnon-repetitive regions of the chloroplast genome (cpDNA) to: (i)determine the putative number of ancestral lineages andmigratory waves that originated the modern MesoamericanAbies, and (ii) test the hypothesis that the subtropical firs haveexperienced an accelerated diversification with respect to theirtemperate counterparts. To do so, we gathered a large samplefrom all Mesoamerican species that covered most of their ranges,which was included in a phylogenetic analysis to evaluate theirrelationships with other Abies of the world. Then, we estimatedthe divergence times and diversification rates of each group ofthe phylogeny. An exhaustive phylogeographic survey, whichcomplements a previous study on southern Mesoamerican firs(Jaramillo-Correa et al., 2008), was used to improve the resolu-tion of the phylogeny for making inferences on the putative evo-lutionary history of the genus since its expansion intoMesoamerica.

2. Materials and methods

2.1. Sampling and Mesoamerican species

Between one and five individuals were collected for 31 Abiesspecies in natural populations, botanical gardens and arboretums(see Table A1). These taxa are naturally distributed in North Amer-ica, Europe and Asia, and represent most of the sections describedby Liu (1971), and Farjon and Rushforth (1989) (Table 1). Sampledoutgroups included Keteleeria davidiana (Bertrand) Beissner (thesister genus of Abies) and Larix kaempferi (Lambert) Carrière.

For the Mesoamerican firs, sampling was far more extensive inan effort to disentangle their evolutionary and phylogeographicrelationships. Needles were collected from between 10 and 30adult cone-bearer trees in 36 populations in both Mexico and Gua-temala, which covered as much as possible the ranges of the eighttaxa currently described for this region (Fig. 1, Table A1). Thesetaxa include:

1. Abies guatemalensis Rehder. It is the most southerly dis-tributed species of the genus. It forms small-scatteredmountain populations between central Mexico and Hon-duras and El Salvador, at altitudes between 2000 and4000 m above the sea level (a.s.l.) (Martínez, 1948; Dona-hue et al., 1985; Andersen et al., 2006).

2. Abies religiosa (Humboldt, Bonpland et Kunth) Schlechten-dal et Chamisso. This taxon is relatively common along theTransverse Volcanic Belt (TVB) in central Mexico, and isdistributed in mostly continuous stands between 2000and 3500 m.a.s.l. (Martínez, 1948). Populations of this spe-cies are the preferred overwintering habitat of the mon-arch butterfly (Danaus plexippus; Anderson and Browler,1996).

3. Abies flinckii Rushforth. Given its discontinuous distribu-tion in two separate clusters, this species was initiallydescribed as two different varieties of A. religiosa and A.guatemalensis (i.e., A. religiosa var. emarginata and A. guate-malensis var. jaliscana, respectively). Nevertheless, mor-phometric (Rushforth, 1989; Strandby et al., 2009) andgenetic surveys (Aguirre-Planter et al., 2000; Jaramillo-Correa et al., 2008) have provided enough evidence togrant it the species status.

4. Abies hickelii Flous et Gaussen. This species is limited to afew stands growing around 2500–3000 m.a.s.l. in the east-ern portion of the TVB and along the Sierra Madre del Sur,in southern México (Martínez, 1948; Farjon, 1990). It hasone of the smallest ranges of the genus.

5 & 6. Abies durangensis Martínez. This taxon has two currentlyrecognized varieties. Var. durangensis (5) has a scattereddistribution along the Sierra Madre Occidental in north-western Mexico, at elevations between 2000 and2900 m.a.s.l, (Martínez, 1948; Farjon, 1990). Var. coahuil-ensis (6) occurs only in a few stands on damp canyons ataltitudes close to 3000 m.a.s.l., in the northern part ofthe Sierra Madre Oriental (Rushforth, 1989). In this study,both varieties were considered as independent taxa (seebelow).

7. Abies vejari Martínez. The range of this species extendsalong the Sierra Madre Oriental, from southeastern Coahu-ila to Nuevo Leon and in western Tamaulipas (Martínez,1948; Farjon, 1990). It is a high mountain taxon foundbetween 2800 and 3300 m.a.s.l. on steep mountain slopes(Farjon, 1990). We are considering as populations of thistaxon stands previously recognized as Abies mexicanaMartínez, then reduced to Abies vejari var. mexicana

264 É. Aguirre-Planter et al. / Molecular Phylogenetics and Evolution 62 (2012) 263–274


(Martínez) (Liu, 1971) and most recently assigned to a sub-species by Farjon (1990). Our field observations suggestthat these populations are indeed morphologically similar.

8. Abies concolor (Gordon) Lindley ex Hildebrand. This species isdistributed in SW and western USA (Oregon, California, andRocky Mountains of Utah, Colorado, Arizona, New Mexico,

Table 1Comparison of the two most used Abies species classifications (Liu, 1971; Farjon and Rushforth, 1989). Mexican taxa included in the present study are underlined.

Liu Farjon and Rushforth

Subgenus Pseudotorreya I. sect. Bracteatasect. Bracteata A. bracteataA. bracteata II. sect. Balsamea

Subgenus Abies A. kawakamii, A. sibirica, A. balsamea, A. lasiocarpa, A. sachalinensis, A. koreana, A. fraseri, A. nephrolepis, A.veitchiisect. MomiIII. sect. AmabilisA. firmaA. amabilis, A. mariesiisect. HomolepidesIV. sect. GrandisA. holophylla, A. homolepis, A. mariesii, A. kawakamii

A. grandis, A. concolor, A. durangensis, A. guatemalensis, A. flinckiisect. ChensiensesV. sect. OiamelA. chensiensis, A. ernestii

A. religiosa, A. vejari, A. hickelisect. ElateopsisVI. sect. NobilisA. delavayi, A. fargesii, A. recurvata, A. squamataA. procera, A. magnificasect. ElateVII. sect. MomiA. koreana, A. nephrolepis, A. sachalinensis, A. veitchiiA. homolepis, A. recurvata, A. firma, A. beshanzuensis, A. holophylla, A. chensiensis, A. pindrow, A. ziyuanensissect. PichtaVIII. sect. PseudopiceaA. sibiricaA. delavayi, A. fabri, A. forrestii, A. chengii, A. densa, A. spectabilis, A. fargesii, A. fanjingshanensis, A.yuanbaoshanensis, A. squamata

sect. Pindrau

IX. sect. AbiesA. spectabilis, A. pindrow

A. alba, A. cephalonica, A. nordmanniana, A. nebrodensis, A. ciclicasect. Abies

X. sect. PiceasterA. alba, A. nebrodensis, A. nordmanniana, A. cephalonica

A. pinsapo, A. numidicasect. PiceasterA. pinsapo, A. cilicica, A. numidicasect. NobilesA. procera, A. magnificasect. Oyamel

A. religiosa, A. hickeli

sect. Vejarianae

A. vejari

sect. Grandes

A. grandis, A. amabilis, A. durangensis, A. guatemalensis, A.

concolorsect. BalsameaA. balsamea, A. lasiocarpa, A. fraseri

A. hickelii

A. religiosa

A. guatemalensis

A. durang v. coah

A. durangensis

A. vejari

A. concolor

Circle color

Gulf of Mexico

Gulf of California

Guatemala

Honduras

U.S.A.

MexicoSierra MadreOccidental

Sierra MadreOriental

TransverseVolcanic Belt

Sierra Madredel Sur

A. flincki

Pacific Ocean

Fig. 1. Geographic location of Mesoamerican Abies populations included in the phylogenetic and phylogeographic surveys. The dashed lines delimitate the main mountainranges in Mexico that are mentioned in the text.

É. Aguirre-Planter et al. / Molecular Phylogenetics and Evolution 62 (2012) 263–274 265


Idaho, and Nevada) and has a few scattered populations innorthern Mexico (Sonora and North Baja California; Martínez,1948; Farjon, 1990). It occurs between 600 and 3350 m.a.s.l.

Further descriptions of the populations and species included inthe present study can be found in Aguirre-Planter et al. (2000, andreferences therein) and with the samples stored in the HerbarioNacional (MEXU).

2.2. DNA extraction and manipulation

Total DNA was extracted from frozen needles by using a cetyl-trimethyl ammonium bromide (CTAB) mini-prep protocol (Váz-quez-Lobo, 1996), or a DNeasy Plant Mini Kit (Qiagen), and itsconcentration was measured with a GeneSpec spectrophotometer(MiraiBio). Three cpDNA regions, rbcL, rps18-rpl20 and trnL-trnF,were amplified via the polymerase chain reaction (PCR) by usingprimer pairs reported elsewhere (Suyama et al., 2000). These threeregions have been shown to be the most useful for making phylo-genetic inferences in the genus Abies (Suyama et al., 2000). The PCRreaction mixture consisted of 3 lL of 10� reaction buffer, 1.5 lL of30 mM/L magnesium chloride solution, 1.2 lL of a 1.25 mM/L dNTPsolution in equimolar ratio, 0.25 mM for each primer, 25–50 ng oftemplate DNA and 1 U of polymerase in a total volume of 30 lL.Amplification was carried out for an initial denaturation of 95 �Cfor 10 min, followed by 30 PCR cycles consisting of 94 �C for 30 s,55 �C for 1 min and 72 �C for 1 min, with a final extension periodof 72 �C for 10 min. After verifying that a single band was amplifiedby examining the PCR products on 2% agarose gels (in TAE), bothDNA strands were sequenced directly in an Applied Biosystems3130xl DNA Genetic Analyser by using the appropriate primers, aSequenase GC-rich kit (Applied Biosystems) and a dideoxynucleo-tide chain termination procedure.

2.3. Phylogenetic analyses

Sequences alignments were made with CLUSTALW (Thompsonet al., 1994), as implemented in BIOEDIT 7.0.0 (Hall, 1999), for eachindependent data set and manually refined after checking individ-ual chromatograms for putative base-calling errors, and to ensurethe correct alignment of conserved bases and avoid redundant in-dels. Initially, all samples (i.e. between one and five individuals perspecies, see Table A1) were included, then identical sequences innearby populations of the same species were omitted from the fi-nal alignment and only one of them was kept. Final alignmentswere submitted to the program MODELTEST (Posada and Crandall,1998) to find the best-fitting model for each independent DNA re-gion with the Akaike Information Criterion (AIC). Phylogeneticanalyses were conducted using a Bayesian approach (MRBAYES ver-sion 3.1.2; Huelsenbeck and Ronquist, 2001) by implementinggene partitioned data, applying the best fit substitution model,and allowing unlinked parameter estimation among partitions.Two independent Markov Chain Monte Carlo (MCMC) runs wereperformed by using random starting trees and a heating chainscheme (temp = 0.2). Each run included five million generations,and samples were taken every 200 generations. Then, a 50% Bayes-ian consensus was created in PAUP⁄ v. 4 (Swofford, 2000) from alltrees sampled after burn-in (i.e. 2500 iterations). A maximum like-lihood phylogeny was also constructed with RAxML (RandomizedAxelerated Maximum Likelihood), a program for sequential andparallel Maximum Likelihood-based inference of large phyloge-netic trees that has the advantage of doing rapid bootstrap heuris-tic searches (Stamatakis et al., 2008). These analyses were initiallyconducted for each independent dataset and then on the combinedset of sequences, after verifying that all three partitions were con-

gruent. In all cases, K. davidiana and L. kaempferi were used asoutgroups.

2.4. Divergence times and diversification rates

Estimates of diversification ages were obtained with thepenalized likelihood method (Sanderson, 2002) implemented inr8s version 1.71 (Sanderson, 2003). We used the consensus treeobtained with the combined set of three cpDNA sequences (rbcL,rps18-rpl20 and trnL-trnF), and the point estimates of ages (min-imum, maximum and mean with standard deviation) were ob-tained using 272 trees topologically identical to this consensustree. The calibration points used were (1) a minimal age of44.4 Ma for K. davidiana (LePage, 2003) and (2) two putativedates for the split between the genera Keteleeria and Abies (nodeA in Figs 2–4), which were used in two independent analyses.The first one (option 1) constrained this node to a minimumage of 100.4 Ma and a maximum age of 113.8 Ma (Kremp,1967; Xiang et al., 2007), and the second one (option 2) consid-ered minimum and maximum ages of 45.5 Ma and 55.0 Ma,respectively (Axelrod, 1976; Erwin and Schorn, 2005). Diversifi-cation rates were determined following Magallón and Sanderson(2001) by using a formula derived from the method-of-momentsestimator of Rohatgi (1976) on crown groups of the Abies phy-logeny (i.e., formula 7 in Magallón and Sanderson, 2001). Thisanalysis allowed us to obtain two estimates of diversificationrates, corresponding to zero extinction (r0.0) and 90% relativeextinction (r0.9) rates, respectively.

2.5. Phylogeographic analysis of Mexican firs

A previous study on southern Mexican species (Jaramillo-Correaet al., 2008) showed that chloroplast microsatellite markers(cpSSR) were more informative than their mitochondrial counter-parts to depict phylogeographical structure across taxa. Thus, in or-der to extend such a survey to the northern Mexican species (i.e. A.durangensis var. durangensis, A. durangensis var. coahuilensis, A. vej-ari, and A. concolor), and to gain a better resolution of interspecificrelationships than the one obtained with the phylogeny (see Sec-tion 3), the three most informative cpSSRs (Pt30204, Pt63718,and Pt71936; Vendramin et al., 1996) were amplified, electropho-resed, genotyped, and sequenced as previously described (Jara-millo-Correa et al., 2008) for 9–18 individuals from 3 to 5populations per taxon (Table A1). Genotypes for the southernmostspecies (i.e., A. guatemalensis, A. hickelii, A. flincki, and A. religiosa)were gathered from the above mentioned study. The remainingnon-Mexican taxa used in the phylogenetic analyses were ex-cluded given the scope of the hypotheses to be tested at the phylo-geographical level.

Multi-locus cpDNA haplotypes (chlorotypes) were defined byassembling all observed polymorphisms. The evolutionary rela-tionships among chlorotypes were depicted with a minimumspanning tree estimated from DNA sequences with the softwareTCS (Clement et al., 2000), and by using a fix connection limit of5 steps (Templeton et al., 1992). Ambiguities induced by reticu-lations were corrected based on the methodology proposed byCrandall and Templeton (1993). The main chlorotype groups ob-served within each population were then plotted onto a mapand compared with the results of a Bayesian analysis of popula-tion structure and the genetic discontinuities estimated with theMontmonier’s maximum difference algorithm. The Bayesiananalysis was performed with the software BAPS 4.13 (Coranderet al., 2003; Corander and Marttinen, 2006) by setting the num-ber of clusters (k) to range between 2 and 40. The optimal par-tition of populations was then determined by repeating theanalysis 100 times with the k-value that exhibited the minimum



log-likelihood. The genetic discontinuities were determined withthe software BARRIER ver. 2.2 (Manni et al., 2004) by using a pair-wise Slatkin’s linearized FST distance matrix between populationsestimated with ARLEQUIN 3.5 (Excoffier and Lischer, 2010). Thenumber of barriers ranged from 1 to 10 and statistical supportfor these genetic discontinuities was determined from 100 boot-strap replicates of the genetic distance matrix, which were ob-tained by resampling individuals within populations. Onlythose barriers with high statistical robustness (i.e. above 80%)were kept.

3. Results

3.1. Phylogenetic analyses

A total of 1284, 541, and 553 nucleotides were sequenced forrbcL, rps18-rpl20 and trnL-trnF, respectively. The final alignmentfor the Mexican Abies taxa included 35 different sequences,which are all available in Genbank (accession nos. JN935603–JN935765). The best fitting substitution models found with theAIC were TIM + I for rbcL and K81uf + G for spacers rps18-rpl20and trnL-trnF, respectively. The monophyly of the genus Abieswas confirmed in both the ML and Bayesian phylogenies, whichexhibited a same main basic topology composed by five groups(Figs. 2–4). These groups were all supported by high posterior

probabilities, especially when the concatenated matrix and thepartitioned models were used. Nevertheless, the Bayesian treehad higher overall support than the ML phylogeny.

The first group, Group I (node D), was composed by all Meso-american species and A. grandis. All these taxa correspond to sec-tions Oiamel and Grandis from Farjon and Rushforth (1989). Somesmaller assemblages could be observed within this large group,which were mainly composed by populations from the northernand southern parts of Mesoamerica, but without showing any clearor well supported assortment of species (Figs. 2–4). The secondgroup (II, node E) was related to the first group, and includedtwo other species from western North America (A. amabilis andA. magnifica) and one from Japan (A. mariesii). These three taxahave been assigned to sections Amabilis and Nobilis by Farjon andRushforth (1989). All the remaining Asian species were found inthe third group (III, node H) together with the taxa from northernand eastern North America (i.e., A. balsamea, A. lasiocarpa and A.fraseri). The species from this group belong to sections Momi andBalsamea, respectively (Farjon and Rushforth, 1989). The Europeanand Mediterranean taxa (A. alba, A. pinsapo, A. nordmanniana and A.numidica), namely those classified in sections A. and Piceaster byFarjon and Rushforth (1989), were found in the fourth group (IV,node I), and the rare and California-endemic A. bracteata formeda distinct basal group (V, node F) that diverged at an early stagefrom all the others (Figs. 2–4).

0.0050

Abies sachalinensis

Abies guatemalensis LC

Abies durangensis 24

Abies sibirica

Abies guatemalensis 2

Abies religiosa 22

Abies concolor 37

Abies hickeli 4Abies guatemalensis 10

Abies vejari 27

Abies hickeli 46

Abies concolor 2

Abies guatemalensis LX


Abies religiosa 12

Abies guatemalensis PBL

Abies concolor 33

Abies fabri

Larix kaempferi

Abies balsamea

Abies kawakami

Abies flinckii 14

Abies fraseri

Abies nordmanniana

Abies alba

Abies hickeli 7

Abies numidica

Abies vejari 29

Abies koreana

Abies hickeli 1

Abies guatemalensis TAJ

Abies religiosa 13


Abies concolor 36


Abies veitchii


Abies religiosa 48

Abies durangensis var. coahuilensis 26

Abies lasiocarpa


Abies fargesii

Abies pinsapo

Abies religiosa 15

Abies homolepis

Abies concolor 1

Abies flinckii 16

Abies mariesii

Abies nephrolepis

Abies flinckii 19


Abies amabilis

Abies vejari 28

Abies bracteata

Abies firma

Abies grandis

Abies guatemalensis SMI

Abies recurvata

Abies magnifica

Abies holophylla

Keteleeria davidiana

A

B

D

C

E

F

G

H

I

Group I (Mesoamerica +Western North America)

Group II (Western North America + Japan)

Group III(Eurasia + Northern North America)

Group IV (Europe + Mediterranean)

Group V (California)

Fig. 2. Maximum-likelihood phylogenetic tree (50% bootstrap consensus) of 33 Abies taxa inferred from three chloroplast markers (rbcL, rps18-rpl20, trnL-trnF). Thickerhorizontal lines represent branches supported by bootstrap values over 95%. Dashed lines represent branches supported by bootstrap values between 50 and 95%. Romannumbers correspond to meaningful groups from an evolutionary or systematic point of view (see Results and Discussion).



3.2. Divergence times and diversification rates

When assuming that the split between Abies and Keteleeriaoccurred between 113.8 and 100.4 Ma (Kremp, 1967; Xianget al., 2007; option 1), the crown age of origin for the genusAbies (node B in Fig. 3) was estimated in 82 ± 13 Ma. When con-sidering that the genus is composed by 48 species, this estimateresulted in diversification rates of 0.0386 and 0.0205, for thezero (r0.0) and 90% extinction (r0.9) rates, respectively. The esti-mated age for Group I, which included the eight Mesoamericanspecies and A. grandis (node D in Fig. 3), was 58 ± 12 Ma (Ta-ble 2). This group had a diversification rate twice as high(r0.0 = 0.0256, r0.9 = 0.0091) as its closely related Group II(r0.0 = 0.0142, r0.9 = 0.0041; node E in Fig. 3), which apparentlyoriginated later, at 48 ± 15 Ma. The Asian–northern North Amer-ican Group III (node H) would have originated 48 ± 13 Ma ago,and exhibited the highest diversification rate of the genus(r0.0 = 0.0542, r0.9 = 0.0256), while the European–MediterraneanGroup IV (node I) would be slightly younger (43 ± 13 Ma) andwould have a lower diversification rate than Group III(r0.0 = 0.0290, r0.9 = 0.0096). The divergence time between GroupsIII, IV and V (node F, Fig. 3) was estimated in 73 ± 13 Ma, whilethe separation between Groups I and II (node C) should have oc-curred about 72 ± 12 Ma ago. On the other hand, when assumingthe less conservative split dates between Abies and Keteleeria (i.e.between 55 and 45.5 Ma; Axelrod, 1976; Erwin and Schorn,2005; option 2), the age estimates obtained were roughly halfof those produced when assuming option 1 (Table 2).

3.3. Phylogeographic structure in Mesoamerican Abies

All three cpSSRs were polymorphic in the northern Mexicantaxa (A. durangensis var. durangensis, A. durangensis var. coahuil-ensis, A. vejari and A. concolor), exhibiting between 2 and 9 size

variants per species. Sequencing of the amplified fragments con-firmed that most of the polymorphisms were due to variation inthe mononucleotide stretches. However, as in a previous surveyin southern Mexican firs (Jaramillo-Correa et al., 2008), someduplications and SNPs were observed in the regions flankingthe SSRs. Only one of these SNPs was new with respect to thisprevious study, and helped define 31 new haplotypes that wereexclusive to A. concolor and A. durangensis var. durangensis(Fig. 5). The total number of chlorotypes, including those fromthe previous study, was 210, and 91.4% of them had frequenciesbelow 0.01 when averaged over the total sample size for Mexi-can firs (data available from the authors upon request). Forty-nine of these cpDNA types were common and shared across spe-cies, thus suggesting that they represent putative ancestral poly-morphisms or that they have been more recently spread throughinterspecific gene flow (Fig. 5).

The chlorotype network (Fig. 5) revealed three significantly dif-ferent groups of haplotypes, exclusively defined by the indels andSNPs flanking the SSRs, which should represent unique and non-homoplastic mutational events (Vachon and Freeland, 2010). Asmentioned above, the first group was entirely formed by individu-als of A. concolor and A. durangensis var. durangensis and distributedin north-western Mexico. The second group was mostly composedby individuals of A. flincki, and was restricted to the westernmostpart of the Transmexican Volcanic Belt in central Mexico. Thisgroup was already observed in the previous survey on southernMexican firs (Jaramillo-Correa et al., 2008), and in the presentstudy, only a few additional trees of the northern A. vejari were as-signed to it. The third group enclosed individuals from all species,and was widely distributed across Mesoamerica. Interestingly, allindividuals of A. durangensis var. coahuilensis were included in thisgroup, while half of the trees of A. durangensis var. durangensiswere assigned to the first group. Both, the partition obtained withBAPS and the genetic breaks disclosed with barrier coincided with

Abies durangensis var. coahuilensis 26Abies vejari 27


Abies hickeli 1

Abies concolor 36Abies hickeli 7

Abies flinckii 19Abies guatemalensis 11


Abies guatemalensis SMI

Abies religiosa 13

Abies guatemalensis LXAbies hickeli 46

Abies vejari 29


Abies religiosa 12Abies religiosa 15

Abies guatemalensis PBLAbies duangensis 35


Abies vejari 28Abies guatemalensis LC

Abies concolor 37

Abies flinckii 14

Abies grandisAbies guatemalensis 41

Abies concolor 1

Abies guatemalensis TAJAbies guatemalensis 42

Abies religiosa 22Abies flinckii 16

Abies concolor 2

Abies guatemalensis 10Abies concolor 33

Abies religiosa 48Abies hickeli 4Abies amabilis

Abies magnificaAbies mariesii

Abies firmaAbies koreana

Abies nephrolepisAbies sachalinensis

Abies veitchiiAbies homolepis

Abies holophyllaAbies balsamea

Abies sibiricaAbies lasiocarpa

Abies fraseri


Abies fabriAbies fargesii

Abies kawakami

Abies nordmannianaAbies numidica

Abies recurvataAbies alba

Abies pinsapo

Abies bracteata

Larix kaempferi

B

CD

E

GH

F I

A

Group I (Mesoamerica +Western North America)

Group II (Western North America + Japan)

Group III(Eurasia + Northern North America)

Group IV (Europe + Mediterranean)

Group V (California)

Fig. 3. 50% Bayesian consensus tree of 33 Abies taxa inferred from three chloroplast markers (rbcL, rps18-rpl20, trnL-trnF). The letters and roman numerals and romannumerals indicate the groups discussed in the text. Thicker horizontal lines represent branches supported by posterior probabilities over 0.95. Dashed lines representbranches supported by posterior probabilities between 0.5 and 0.95. Roman numbers correspond to meaningful groups from an evolutionary or systematic point of view (seeResults and Discussion).



Abies vejari 27Abies durangensis var. coahuilensis 26Abies durangensis 31

Abies hickeli 1

Abies concolor 36Abies hickeli 7Abies flinckii 19Abies guatemalensis 11


Abies guatemalensis SMIAbies religiosa 12Abies religiosa 15Abies religiosa 13

Abies guatemalensis LXAbies hickeli 46

Abies vejari 29


Abies guatemalensis PBLAbies duangensis 35Abies durangensis 24

Abies vejari 28Abies guatemalensis LC

Abies concolor 37

Abies concolor 33Abies religiosa 48Abies hickeli 4


Abies flinckii 14

Abies grandisAbies guatemalensis 41Abies concolor 1

Abies guatemalensis TAJAbies guatemalensis 42

Abies religiosa 22Abies flinckii 16

Abies concolor 2


Abies amabilisAbies magnificaAbies mariesii

Abies firmaAbies koreana

Abies nephrolepisAbies sachalinensis

Abies veitchiiAbies homolepisAbies holophyllaAbies balsameaAbies sibiricaAbies lasiocarpaAbies fraseriAbies fabriAbies fargesiiAbies kawakami

Abies nordmannianaAbies numidica

Abies recurvataAbies albaAbies pinsapo

Abies bracteata

B

CD

E

FG

H

I

Cretaceous Paleocene Eocene Oligocene Miocene Pliocene

65.0 54.8 33.7 23.8 5.3 1.8

Eocene Oligocene Miocene Pliocene

33.7 23.8 5.3 1.8

Ma

Ma

Option 1

Option 2

Fig. 4. Chronogram derived from the consensus of the 272 compatible Bayesian trees. The letters indicate the groups discussed in the text (see Results and Discussion).Thicker horizontal lines represent branches supported by posterior probabilities between 0.95–1.00. Dashed lines represent branches supported by posterior probabilitiesbetween 0.5 and 0.95.

Table 2Mean, minimum and maximum age estimates (standard deviations in parentheses) derived from 272 sampled Bayesian trees that were topologically identical to the consensusphylogenetic tree obtained for the genus Abies, and diversification rates determined for each species group under zero (r0.0) and 90% relative extinction (r0.9) rates by using twodifferent calibration points.

Nodec Option 1a Option 2b

Mean aged Minimumd Maximumd Diversification rate Mean aged Minimumd Maximumd Diversification rate

r0.0 r0.9 r0.0 r0.9

A 111.99 (3.40) 100.86 113.80 – – 53.80 (2.28) 46.25 55.00 – –B 82.21 (12.85) 54.21 107.27 0.0386 0.0205 38.77 (5.59) 24.70 51.10 0.0819 0.0435C 71.88 (12.12) 48.07 105.17 0.0260 0.0102 33.90 (5.72) 22.42 50.02 0.0552 0.0216D 58.58 (12.32) 32.11 100.56 0.0256 0.0091 27.67 (5.84) 15.31 48.52 0.0543 0.0192E 48.55 (14.24) 21.01 96.47 0.0142 0.0041 22.95 (6.68) 9.61 45.84 0.0302 0.0088F 73.50 (12.95) 47.64 105.08 0.0385 0.0191 34.64 (6.08) 22.23 50.03 0.0817 0.0406G 59.86 (13.67) 32.08 99.73 0.0473 0.0234 28.25 (6.39) 15.57 47.39 0.1002 0.0497H 47.98 (12.85) 21.93 89.06 0.0542 0.0256 22.69 (5.97) 11.15 41.03 0.1147 0.0541I 43.06 (13.13) 16.90 88.56 0.0290 0.0096 20.38 (6.12) 8.52 40.80 0.0614 0.0203

a Option 1 assumes a minimal age of 44.4 Ma for Keteleeria (LePage, 2003), and minimum and maximum ages of 100.4 Ma and 113.8 Ma, respectively (Kremp, 1967; Xianget al., 2007), for the split between Keteleeria and Abies (node A).

b Option 2 assumes the same minimal age for Keteleeria, and ages between 45.5 Ma and 55.0 Ma for the split of this genus from Abies (Axelrod, 1976; Erwin and Schorn,2005).

c Letters correspond to nodes in Figs. 2 and 3.d All ages are given in million years.



these three groups. However, the populations from Guatemalawere additionally separated from the Mexican stands in thesetwo analyses (Fig. 5).

4. Discussion

4.1. Monophyly and divergence of the genus Abies

In the present study, we confirmed the monophyly of the genusAbies, and showed that it contains five strongly supported groupsof species. These groups mostly agree with the recognized sectionsfrom the most widely used classifications (Liu, 1971; Farjon andRushforth, 1989), and with the continental location of species(Figs. 2–4). The majority of these concurrences were already ob-served and discussed in previous works relying on chloroplastand nuclear DNA data (Suyama et al., 2000; Kormut’ák et al.,2008; Xiang et al., 2009), which themselves backed earlier resultsof controlled crosses within and across sections (e.g. Critchfield,1988; Kormut’ák et al., 2008).

Although the topologies of the estimated phylogenies do not al-low inferring precisely where Abies first appeared, the basal positionof the morphologically divergent A. bracteata and its extant distribu-tion in southwestern North America (Griffin and Critchfield, 1972)would support an origin in this region of the world. This observationwould support to some degree the less conservative approach (op-tion 2 in Table 2) used as calibration point. Indeed, evidence fromfragmentary organs indicates that Abies was distributed in the conif-erous forests of western North America during the Eocene (Axelrod,1976; Erwin and Schorn, 2005), relatively close to the region wherethe modern range of A. bracteata is located. Nevertheless, a much ear-lier presence of Abies in Eurasia (i.e. between the Cretaceous and thePaleocene) was inferred from pollen deposits in China (Xiang et al.,2007) and Siberia (Kremp, 1967), which would support the most

conservative calibration approach (option 1 in Table 2). However,caution has been advised when relying on pollen without associatedmacroremnants for performing calibrations, due to unknown homo-plasious characters with now-extinct genera (Erwin and Schorn,2005; Willyard et al., 2007).

The two calibrations approaches used yielded a late Cretaceousand an early Eocene crown-group diversification time, respectively,with the final divergence leading to the extant species occurring inthe Miocene (option 1) and the Pliocene (option 2). Such differ-ences between scenarios are rather common in phylogenetic stud-ies in the Pinaceae (e.g. Willyard et al., 2007; Gernandt et al., 2008),given that the choices made for calibrating the phylogenies repre-sent a significant source of uncertainty, especially for the timeselapsed between the origin of taxa and both the earliest recogniz-able fossil, and the divergence that originated the extant species(Willyard et al., 2007). However, irrespective of which scenario isthe most appropriate for calibrating the Abies phylogeny, it seemsthat two periods were key for the diversification of this genus: theEocene and the Miocene.

The Eocene was a period of falling global temperatures (e.g., Za-chos et al., 2001; Miller et al., 2008), favorable to range expansionsof temperate taxa such as most conifers (Farjon, 2005). Similar cal-ibrated phylogenies of Juniperus and Cupressus revealed that theirfirst waves of diversification also occurred during this epoch(Mao et al., 2010), while studies in Pinus suggested that divergencewithin the subgenus Strobus also began at the end of this period(Gernandt et al., 2008). Thus, as proposed for many Tertiary taxa(Milne and Abbot, 2002; Mao et al., 2010), the earlier Abies diver-sification (i.e. of either its basal lineages according to option 1; orthe crown group according to option 2) might have been promptedby isolation-by-distance and adaptation to marginal environmentsafter an expansion during the cooler periods of the Eocene, to thendiminish during the more climatically stable Oligocene.

B

A. hickelii

A. religiosa

A. guatemalensis

A. durang v. coah

A. durangensis

A. vejari

A. concolor

Circle border

Clade I

Clade II

Clade III

A

A. hickeliiA. religiosa

A. guatemalensis

A. durang v. coah

A. durangensis

A. vejari

A. concolor

Clade IClade II

Clade III

Circle filling A. flincki

Circle filling

A. flincki

Fig. 5. Minimum-spanning network (A) and geographic location of the main cpSSR groups and genetic barriers (dashed arrows) (B) in populations of eight MesoamericanAbies taxa. The network represents the most parsimonious relationships among chlorotypes with a fix connection limit of five steps, where each cpDNA-type is represented bya circle whose size is proportional to its abundance. Small white dotted circles represent missing chlorotypes. The map depicts the relative frequency of each of the three maingroups delineated in the network for each population. The barriers were determined with Montmonier’s maximum-difference algorithm on FST-distances derived fromchlorotype frequencies, and coincided with the groups detected with a Bayesian analysis of population structure. Their thickness is equivalent to their statistical robustness.



On the other hand, the Miocene divergence (i.e. of either mod-ern populations: option 1; or major species groups: option 2) coin-cides with the uplift of major mountain ranges in Asia, in NorthAmerica and in Mesoamerica (e.g., Spicer et al., 2003; Wilson andPitts, 2010; see below). This period was accompanied by anotherprogressive decrease of the global temperature (Zachos et al.,2001). Altogether, these factors should have resulted in new habi-tats suitable for further expansions of temperate taxa (Farjon,2005) which, coupled with the isolating effects of the uprisingmountains, should have prompted diversification in Abies andother conifers (e.g. Bouillé and Bousquet, 2005; Willyard et al.,2007; Mao et al., 2010; Xu et al., 2010).

4.2. Monophyly and divergence of Mesoamerican firs

The Mesoamerican species were all located into a single group(I) together with two taxa from western North America (A. concolorand A. grandis), but without forming monophyletic groups withinthis lineage (node D, Figs. 2–4). This large group conforms to awide phylogeographic unit distributed from the southern parts ofthe Vancouver Island and the shores of British Columbia, alongthe coast and down into the northern parts of Baja California,and from the Pacific Northwest along the Rocky Mountains intothe main Mesoamerican cordilleras. Its related Group II (node E)is also distributed in this region, although its southern limit is re-stricted to central California and its north bounds spans further,into southern Alaska. This group additionally appears to have expe-rienced an ancient long distance migration into eastern Asia, prob-ably via the Bering Land Bridge, where it diverged into modern A.mariesii (Suyama et al., 2000; Xiang et al., 2009). According tothe estimated divergence times, Groups I and II separated betweenthe late Cretaceous and the early Eocene (node C, Fig. 4, Table 2),and remained isolated for enough time to develop reproductivebarriers. Indeed, previous controlled crosses between A. concolor(Group I) and A. magnifica (Group II) only produced a few non-via-ble offspring (Critchfield, 1988), while a survey on cpDNA variationfound no haplotype shared between these two widely sympatricspecies (Oline, 2008).

Both the current distribution of species from Group I (node D)and their divergence dates suggest that they originated betweenthe Paleocene and the Miocene, after a unique southward expan-sion that probably followed the mountain chains of western NorthAmerica. These ranges, collectively known as the American Cordil-lera (Dickinson, 2004), formed in two phases that coincide with thekey periods for the Abies diversification: first during the LaramideOrogeny that took place from the late Cretaceous to the late Paleo-cene (English and Johnston, 2004), and then during the Mioceneand the Pliocene, which originated most of the mountain rangesin central and southern Mexico (Hay and Soeding, 2002). The firstappearance of temperate conifers in the fossil record of Mexicodates back to the early Miocene, about 23 Ma (Graham, 1999),which supports the hypothesis of an early southward expansionand divergence (option 1). However, the first appearance of Abiespollen in this record only occurs until the Pliocene (5 Ma; Graham,1976), which coincides with the hypothesis of a much later diver-gence for the Mesoamerican firs (option 2).

However, a detailed phylogeographic analysis of Mesoamericanfirs with cpSSRs suggest multiple southward migrations fromNorth America, as two particular species, A. concolor and A. flinckii,appeared to diverge significantly from the others (Fig. 5). The dif-ferentiation of A. flinckii was previously interpreted as the resultof a first migratory wave that entered Mexico via the Sierra MadreOccidental, which then diverged in the western part of the Trans-Mexican Volcanic Belt (see Jaramillo-Correa et al., 2008 and refer-ences therein). On the other hand, A. concolor might represent a rel-atively late arrival into northwestern Mexico, which could be as

recent as the last glacial maximum. During this period, conifer for-ests reached their highest development in this country (e.g., Loz-ano-García et al., 1993; Vázquez-Selem and Heine, 2004), whichcould have facilitated southward migrations and/or hybridizationwith local related taxa, such as inferred from the chlorotype distri-bution in A. concolor and A. durangensis var. durangensis (Fig. 5).Nevertheless, these phylogeographical hypotheses are yet to betested through more detailed analyses combining coalescent simu-lations on nuclear variation and ecological niche modeling (e.g.Richards et al., 2007; Carstens et al., 2009; Martínez-Méndezet al., unpublished results).

4.3. Diversification rates in subtropical and temperate Abies

The correlation between increased rates of diversification andpast geological or climatic events and/or the occurrence of adap-tive radiations have been usually invoked to explain the higherspecies diversity in the tropics and subtropics than in the temper-ate regions (e.g. Wright et al., 2006; Glor, 2010). However, in spiteof exhibiting more species towards southern latitudes, the overalldiversification rates of Abies were low (0.0386–0.0819 sp/Ma, thisstudy) and virtually identical in the subtropical (i.e. MesoamericanGroup I, node D) and temperate groups (II, node E to IV node I).These rates are indeed comparable to those of other conifers (e.g.Juniperus 0.078 sp/Ma, Mao et al., 2010) and of slowly evolvingangiosperms, such as the Nympheales (0.031 sp/Ma) and the Win-teraceae (0.0358 sp/Ma; Magallón and Sanderson, 2001). In angio-sperms, low diversification rates are usually compatible with amodel of environmental stasis and decreased extinction (Lancaster,2010), which could be further extended to conifers regardless ofthe environment they inhabit.

Low diversification rates are indeed expected in conifers. Thesetaxa, as most wind-pollinated trees, have extensive gene flow, out-crossing mating systems, long generation times, and slow-develop-ing isolation mechanisms that allow hybridization in secondarycontact zones, which decrease the likelihood of extinction anddiversification (e.g. Bousquet et al., 1992; Perron and Bousquet,1997; Verdú, 2002; reviewed by Petit and Hampe (2006)). Conifersfurther exhibit high amounts of repetitive elements (Grotkoppet al., 2004; Morgante, 2006) and reduced rates of recombinationat both the genome-wide and within-gene scale (Jaramillo-Correaet al., 2010), which also hamper rapid speciation processes. Thus,as in slowly evolving angiosperms, diversification in conifers is ex-pected to be driven by stochastic forces and/or variation at numer-ous loci with small phenotypic effects, rather than on rapidadaptation to new environments (e.g. Bousquet et al., 1992; Eckertet al., 2009).

Although a higher proportion of differentially fixed variation atthese loci across isolated montane taxa can account for the highernumber of Abies taxa in the subtropics, it could also be hypothe-sized that these figures are an artifact of phenotypic plasticity thathas mislead taxonomists. Such a possibility is supported by previ-ous analyses on terpene, allozyme, cytoplasmic DNA and morpho-metric variation, which found little evidence for distinguishing somany individual species within the Mesoamerican Abies complex(Aguirre-Planter et al., 2000; Nava-Cruz et al., 2006; Jaramillo-Cor-rea et al., 2008; Strandby et al., 2009). The morphometric study ofStrandby et al. (2009) further reported a significant correlation be-tween anatomical dissimilarities and geographic distance in thesouthern parts of this complex, which was interpreted as the actionof contrasting environmental pressures on a widely distributedgene pool that has significant levels of phenotypic plasticity. Sucha theory has already been invoked to explain anatomical and bio-chemical variation in conifers, including Abies (e.g. Parker et al.,1981; Huber et al., 2004; Baquedano et al., 2008), but then again,the integrative analysis of genomic, phenotypic and ecological



datasets, and common garden experiments seems necessary to testthis hypothesis.

4.4. Hybridization, ancestral polymorphism or lack of taxonomicresolution?

Altogether, the results of this and previous studies (see above)suggest that three main Abies lineages can be distinguished in Mes-oamerica: two of them corresponding to A. flinckii and A. concolor,and a third one formed by the remaining taxa. Such a sub-divisionmight further imply that a redefinition of the taxonomy of theMesoamerican species is necessary, as proposed by as Strandbyet al. (2009). Nevertheless, the lack of genetic differentiation seemsto be a constant feature in Abies complexes across the world. Forinstance, in the eastern Mediterranean, no significant genetic dif-ferences could be observed among A. bornmuelleriana, A. cephalo-nica, A. equi-trojani, and A. nordmanniana with eithermitochondrial or cpDNA markers (Liepelt et al., 2010), and similarresults were obtained for other complexes in North America (Pot-ter et al., 2010) and Asia (Wang et al., 2011). These comparisonsand the low diversification rates detected herein, point that thesegroups have not had sufficient time of physical and genetic isola-tion to fully differentiate, and that most of them have retainedancestral polymorphisms. This phenomenon has been further ob-served in many distantly related and reproductively isolated coni-fers, and it is likely favored by the large ancestral population sizesand extensive gene flow characteristics of these trees (Bouillé andBousquet, 2005; Chen et al., 2010), and/or by recurrent introgres-sion in secondary contact zones between species with incompletereproductive isolation, such as reported for many Mesoamericanspecies complexes (e.g. Matos and Schaal, 2000; Moreno-Letelierand Piñero, 2009; Peñaloza-Ramírez et al., 2010).

On the other hand, the fact that some species, including theMesoamerican A. concolor and A. flinckii, and the Asian A. gracilis,A. holophylla and A. nephrolepis, do appear well delineated from agenetic point of view (Semerikova et al., 2011; Jiang et al., 2011;present study), might imply that the number of DNA regions sur-veyed is actually insufficient to adequately differentiate all sur-veyed taxa. Indeed, a recent Pinus phylogeny exhibited a muchhigher resolution by using a massive sequencing approach on thechloroplast genome (Parks et al., 2009). However, such studieshave to be placed into a more detailed population genetics contextin order to capture all the putative interactions that closely-relatedand putatively hybridizing taxa have experienced during their evo-lution (e.g., Liston et al., 2007; Bouille et al., 2011). Such integrationwould imply the massive sequencing of hundreds or even thou-sands of genomes, which is the standard sample size in phylogeo-graphic surveys, and which would render such studies currentlynon-viable from an economical and bioinformatic point of view.More practical approaches include the one used herein, which inte-grates non-repetitive (i.e. DNA sequences) and repetitive (cpSSR)datasets into both a genus-wide and a geographically restrictedcontext (Liston et al., 2007; Vachon and Freeland, 2010), and theuse of multiple nuclear SSRs, which can be rapidly developed andanalyzed, and provide direct evidence of isolation or hybridizationevents.

4.5. Implications for conservation

The lack of taxonomic consensus often collides with the conser-vation needs of megadiverse countries such as Mexico or Guate-mala (Callmander et al., 2005). For instance, the indiscriminategrouping of species into one single taxon has left endangered spe-cies unprotected (e.g., Rieseberg, 1991), while hybrid or introgres-sant populations that do not have conservation status under manylegislations have also been left unguarded (e.g. Fitzpatrick et al.,

2010). Thus, for threatened species complexes, such as the Meso-american firs, defining evolutionary lineages with restricted geneflow and significant differences in adaptive traits at the populationlevel seems more appropriate (e.g. de Querioz, 1998; Fraser andBernatchez, 2001). Accordingly, each one of the three cpSSR groupsdetected herein should represent independent lines of ancestry,while populations within these lineages correspond to isolatedunits evolving under the action of stochastic forces for the last gen-erations. Each Mesoamerican fir population should be thus moni-tored for demographic treats such as inbreeding and geneticdrift, while conservation programs at a larger scale should bebased, for the time being, on the three main phylogeographicgroups detected herein. However, the integration of additional eco-logical and landscape genetic data into gene-niche association gra-dients are urgently needed to improve the resolution of thesegroups, especially for the more threatened northern taxa.

Acknowledgments

The authors would like to thank J. Beaulieu, I. Gamache, (Cana-dian Forest Service), C. Sayre (VanDusen Botanical Garden), F.T. Le-dig (Univ. of California Davis), P. Delgado, D. Gernandt, A. Keiman,Y. Nava, C. Saenz, and G.R. Furnier (Institute of Biology and Ecologyof UNAM) for valuable help in sampling, and A. Gagné, S. Senne-ville, and S. Gérardi (Centre for Forest Research of Univ. Laval),and J. Hicks and A. Kelly (Oregon State Univ.) for laboratory assis-tance. The authors also thank U. Strandby and M. Sørensen for pro-viding samples from Guatemala. The authors’ gratitude is alsoextended to S. Ramírez-Barahona for his help with some figuresand comments, and to N. Martínez-Méndez, D. Piñero, V. Souza(Institute of Ecology of UNAM), and Susana Magallón (Institute ofBiology of UNAM) for comments and logistic support along theproject. Further valuable comments from two anonymous review-ers helped improve a previous version of the manuscript. Acknowl-edgements are additionally extended to the Ministère dudéveloppement économique de l’innovation et de l’exportationdu Québec, the Natural Sciences and Engineering Research Councilof Canada (Discovery program), the Consejo Nacional de Ciencia yTecnología (CONACYT, Grant SEP-2004-CO1-46475-Q), the Comi-sión Nacional para el Conocimiento y el uso de la Biodiversidad(CONABIO, Grant B138), the Dirección General de Asuntos de Per-sonal Académico, UNAM, Programa de Apoyo a Proyectos de Inves-tigación e Innovación Tecnológica, (PAPIIT Grant IN224309-3), andthe Programa de Apoyo a las Divisiones de Estudios de Postgrado,UNAM (PADEP-UNAM) in Mexico, for financial support. This paperwas written during a sabbatical leave of L.E. Eguiarte at the Univer-sity of California Irvine with support of UC-MEXUS, CONACYT andDGAPA-UNAM.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2011.09.021.

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author's personal copy - cinvestav...transverse volcanic belt (tvb) in central mexico, and is...

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