Microcnemum coralloides (Chenopodiaceae- Salicornioideae ... · Microcnemum coralloides...

Microcnemum coralloides (Chenopodiaceae-Salicornioideae): an example of intraspecific East-West

disjunctions in the Mediterranean region

by

Gudrun Kadereit1 & Ahmet Emre Yaprak2

1 Institut für Spezielle Botanik und Botanischer Garten, Johannes Gutenberg-Universität Mainz, 55099 Mainz, Germany. Corresponding author: [email protected]

2 Ankara University, Science Faculty, Department of Biology, Besevler/Ankara, Turkey. [email protected]

Anales del Jardín Botánico de MadridVol. 65(2): 415-426

julio-diciembre 2008ISSN: 0211-1322

Abstract

Kadereit, G. & A.E. Yaprak. 2008. Microcnemum coralloides(Chenopodiaceae-Salicornioideae): an example of intraspecificEast-West disjunctions in the Mediterranean region. Anales Jard.Bot. Madrid 65(2): 415-426.

Microcnemum is a monotypic genus of Salicornioideae compris-ing rare, annual, hygrohalophytic herbs growing in hypersaline in-land lagoons and salt pans. Microcnemum coralloides shows anEast-West disjunction in the Mediterranean region: M. coralloidessubsp. coralloides occurs in central and eastern Spain while M.coralloides subsp. anatolicum grows in Turkey, Syria, Armenia andIran. We studied the phylogeny, biogeography and morphologicaldifferentiation of M. coralloides. Molecular analyses, using fivewestern and eight eastern accessions of the species, were basedon three different markers (nuclear ITS and plastid atpB-rbcL spac-er and trnT/F region) analysed with Maximum Parsimony andMaximum Likelihood. Estimates of divergence times were calcu-lated using a Likelihood Ratio Test (LRT) and the Penalized Likeli-hood (PL) method. The two subspecies can be clearly distin-guished by their different seed testa surface. Other diagnosticcharacters were not found. The molecular data (ITS and ML analy-sis of the trnT/F region) indicate that M. coralloides subsp. coral-loides originated from within M. coralloides subsp. anatolicumwhich implies an East Mediterranean origin and subsequent west-ward dispersal. Age estimates for the split of the two subspeciesrange from 2.8–0.5 million years ago. Considering the relativelylow genetic differentiation and the low crown group age (0.7–0.1mya) of M. coralloides subsp. coralloides in comparison to M.coralloides subsp. anatolicum we favour the hypothesis that theIberian part of the species range was established during cold peri-ods of the Early Pleistocene and that the range of the species wasfragmented during a warmer period soon after its arrival in Iberia.

Keywords: Biogeography, disjunction, Microcnemum, molecu-lar clock, East Mediterranean, Spain.

Resumen

Kadereit, G. & A.E. Yaprak. 2008. Microcnemum coralloides(Chenopodiaceae-Salicornioideae): un ejemplo de las disyuncio-nes intraespecíficas Este-Oeste en la región mediterránea. AnalesJard. Bot. Madrid 65(2): 415-426 (en inglés).

Microcnemum es un género monotípico de Salicornioideae queconsiste en hierbas higrohalófilas, anuales, raras, que crecen encuencas endorréicas hipersalinas del interior y salares. Microcne-mum coralloides muestran una disyunción Este-Oeste en la regiónmediterránea: M. coralloides subsp. coralloides aparece en el cen-tro y el levante español, mientras que M.coralloides subsp. anato-licum crece en Turquía, Siria, Armenia e Irán. Estudiamos la filoge-nia, la biogeografía y la diferenciación morfológica de M. coralloi-des. Los análisis moleculares, empleando cinco adquisiciones occi-dentales y ocho orientales de la especie, se basaron en tresmarcadores distintos (ITS nuclear, espaciadores plástidos atpB-rbcL y región trnT/F) analizados con Máxima Parsimonia y MáximaVerosimilitud. Las estimaciones de tiempos divergentes se calcula-ron empleando una Prueba de Verosimilitud (LRT) y el método deVerosimilitud Penalizada. Las dos subspecies se distinguen clara-mente por la diferencia en la superficie de su envoltura. No se en-contraron otras características de diferenciación. Los datos mole-culares (ITS y análisis ML de la región trnT/F) indican que M. cora-lloides subsp. coralloides originó de dentro de M. coralloidessubsp. anatolicum, lo cual implica un origen en el este del Medite-rráneo y su posterior dispersión hacia el oeste. Las estimaciones deedad para la separación de las dos subespecies data desde hace2,8 a 0,5 millones de años. En vista de la diferenciación genética,relativamente baja y la reducida edad del grupo terminal (0,7-0,1millones de años) de M. coralloides subsp. coralloides en compa-ración con M. coralloides subsp. anatolicum, favorecemos la hipó-tesis de que la parte ibérica de la gama de la especie se estableciódurante periodos fríos del Bajo Pleistoceno y que la gama de la es-pecie se fragmentó durante un periodo más cálido no mucho des-pués de su llegada a la Península.

Palabras clave: Biogeografía, disyunción, Microcnemum, relojmolecular, Mediterráneo oriental, España.

Introduction

Plant species that show an extreme East-West dis-junction in the Mediterranean region have been no-ticed for a long time (Engler, 1879; Willkomm, 1896;Braun-Blanquet & Bolòs, 1957; Davis & Hedge,1971). More recently, also some examples of disjunct-ly distributed lichens were reported in the literature(Barreno, 1991; Egea & Alonso, 1996). Additionalstriking examples come from studies of cave-dwellingCrustaceae (Brehm, 1947), freshwater zooplankton(Miracle, 1982), Coleoptera (Sanmartin, 2003) andfrom a detailed survey of the insect fauna of the cen-tral Monegros region of Spain which listed 62 speciesshowing a disjunct distribution between NE Spainand the steppes in the eastern Mediterranean area orCentral Asia (Ribera & Blasco-Zumeta, 1998). Al-though molecular and analytical tools are now avail-able to study the origin and age of disjunct distribu-tion patterns, the list of molecular studies which focuson extreme disjunctions of plant species in theMediterranean is relatively short (but see: Castro &al., 2002; Carine & al. 2004; Rosselló & al., 2007;Thompson, 1999).

Three main explanations have been put forward forextreme East-West disjunctions in the Mediterraneanregion: 1. The extant populations have been inter-preted as relict populations of a formerly (pre-Pleis-tocene) continuous or at least wider distribution area,and probably survived in isoclimatic areas of theMediterranean (Taberlet & al., 1998) from wheresome of them again expanded. 2. The extant popula-tions in one part of the disjunct distribution area arethe result of colonization through long distance dis-persal. The direction of dispersal is usually inferredfrom the asymmetrical distribution of the specific tax-on with the newly colonized area having a smaller ex-pansion (Davis & Hedge, 1971; Ribera & Blasco-Zumeta, 1998; Thompson, 1999). 3. Some taxa are in-terpreted to be early and undocumented introduc-tions by man (Willkomm, 1896).

Microcnemum is a monotypic genus of Chenopo-diaceae subf. Salicornioideae that shows an extremeEast-West disjunction in the Mediterranean region.Like most members of Salicornioideae it grows in hy-grohalophytic habitats and has succulent, apparentlyleafless, articulated stems and spike-like inflores-cences. Among salicornioidean genera, Microcnemumis characterised by comprising small annual herbs.The bracts subtending three flowers that are free fromeach other are connate and form a cup-shaped struc-ture. The perianth is reduced to a tiny membranouslobe and the seeds are ovoid, flattened and have agranulose or papillose surface (Aellen, 1967; Molero,1986, 2000). Of these characters only the minute peri-

G. Kadereit & A.E. Yaprak

anth rudiment which disappears in the fruiting stagerepresents an autapomorphy and is unique in Sa-licornioideae (Kadereit & al., 2006).

Microcnemum occurs along the shores of flat, step-pic lagoons in extremely saline sites. It often grows onthick salt crusts together with a few other species thatare salt-tolerant and able to cope with extreme hyper-saline conditions during the dry season. Associatedwith Microcnemum coralloides in Turkey are, for ex-ample, Kalidium foliatum Moq., K. wagenitzii(Aellen) Freitag & G. Kadereit, Halocnemum strobi-laceum M. Bieb., Halimione verrucifera (M. Bieb.)Aellen and Salicornia L. spp. (all Chenopodiaceae),while M. coralloides in Spain often is associated withHalopeplis amplexicaulis Ung.-Sternb., Halocnemumstrobilaceum M. Bieb., Arthrocnemum macrostachyum(Moric.) K. Koch and several species of SarcocorniaA.J. Scott and Salicornia (also all Chenopodiaceae).The presence of Microcnemum seems to fluctuatefrom year to year probably in response to seasonalfluctuations in rainfall (E. Yaprak, pers. obs.; B.M.Crespo, pers. comm.).

Microcnemum coralloides comprises two subspe-cies. Microcnemum coralloides subsp. coralloides is re-stricted to inland salt lakes or salt pans in central andeastern Spain (Molero, 1986; Blanché & Molero,1990). For some time after its discovery in 1959, M.coralloides subsp. anatolicum was only known fromthe type locality or nearby (Aellen, 1967). Later it wasalso recorded from central and NW Iran, the SyrianDesert, Armenia and several new localities in Turkey(Mouterde, 1966; Botschantzev & Barsegjan, 1972;Gabrielian, 1981; Akhani, 1988; Akhani & Ghorban-li, 1993; Hamzaoglu & al., 2005; Yaprak, 2008). Apartfrom their disjunct distribution, the two subspeciesdiffer only in the structure of the seed testa, which isdensely and distinctly papillose in M. coralloides sub-sp. anatolicum (Wagenitz, 1959) but only granulose orslightly papillose in M. coralloides subsp. coralloides(compare Figs. 1-4 in Molero, 1986). Other differ-ences between the two subspecies have never beenidentified. The type collection of M. coralloides subsp.anatolicum, how-ever, was incomplete because theplant was collected in the fruiting stage only (Wa-genitz, 1959).

A recent molecular systematic study of subf. Sa-licornioideae demonstrated that, based on nuclearDNA (ITS), Microcnemum is most closely related toArthrocnemum Moq. (Kadereit & al., 2006). Arthroc-nemum contains low shrubs that differ from Microc-nemum in several characters (e.g., growth form, num-ber of stamens) but also shows some similarities (e.g.,shape of the connate bracts, free flowers, membra-nous perianth; Kadereit & al., 2006). It is a ditypicgenus that is distributed in inland and coastal halo-

416

Anales del Jardín Botánico de Madrid 65(2): 415-426, julio-diciembre 2008. ISSN: 0211-1322

phytic habitats throughout the Mediterranean area,SW Asia and North America. Statistical support forthe sister group relationship of these two genera waslow, and the topology of chloroplast DNA based trees(atpB-rbcL spacer; Kadereit & al., 2006) suggests thatthe closest living relatives of Microcnemum are mem-bers either of the Salicornia/Sarcocornia lineage or ofthe Australian Salicornieae. However, the branches ofthese four lineages (Arthrocnemum, Microcnemum,Salicornia/Sarcocornia lineage, Australian Salicor-nieae) are long and diverged more or less simultane-ously in the Middle Miocene (19.6-14.6 mya; Kadereit& al., 2006).

The finding that the two subspecies of Microcne-mum represent a lineage that originated during theMiddle Miocene, show an extremely disjunct distrib-ution but, at the same time, are morphologically verysimilar, leads to the following questions: 1. Are thetwo subspecies supported by molecular evidence? 2.Does flower morphology provide additional charac-ters to distinguish the two subspecies? 3. When didthe two subspecies separate? 4. Is the distributionfound today the result of recent dispersal or is it betterexplained as a relict of a formerly wider distributionarea?

To address these questions we sequenced three dif-ferent markers from the chloroplast and nucleargenome for five accessions of M. coralloides subsp.coralloides and eight accessions of M. coralloidessubsp. anatolicum. Where a likelihood ratio test(LRT) rejected a model of strict clock-like evolution,we applied a Penalized Likelihood (PL) approach toobtain divergence dates of interest. Additionally, westudied the morphology of the poorly known and rareM. coralloides subsp. anatolicum.

Biogeography of Microcnemum coralloides

Material and methods

Plant material for molecular and morphological studies.–The plant material included in the molecularstudy is listed in Table 1 and the accessions of Microc-nemum included in the molecular analyses aremapped in Fig. 1. Altogether 13 accessions of Microc-nemum were sampled for ITS and the atpB-rbcL spac-er, five from Spain, six from Turkey, one from Iran andone from Armenia. For the trnT/F region, one Spa-nish Microcnemum sample and the Armenian samplefailed to amplify and for two samples only a partial se-quence was obtained (compare Table 1). Additionally,two accessions of Arthrocnemum macrostachyum,Halosarcia indica (Willd.) Paul G. Wilson, Sarcocorniautahensis (Tidest.) A.J. Scott and Halopeplis amplexi-caulis were included in all analyses. According to theresults of Kadereit & al. (2006), Halopeplis served asoutgroup in this study. The other lineages included inthe analysis are most closely related to Microcnemum.

The material of M. coralloides subsp. anatolicumlisted in Table 1 was also used for morphological ex-amination.

DNA isolation, amplification, and sequencing.– To-tal genomic DNA was either isolated from silica geldried stems or young herbarium material using 20-50mg. For DNA extraction the NucleoSpin plant DNAextraction kit (Macherey-Nagel) was used followingthe manufacturer’s specifications.

Standard PCR amplification was performed usingstandard reaction mixes (e.g., Kadereit & al., 2003) in-cluding 0.25-1% DMSO and c.1 ng/µl DNA with aGrant Autogene II-Thermocycler programmed as de-scribed in Kadereit & al. (2005). Amplification pro-ducts were checked on 0.8% agarose gels. PCR pro-

417


Fig. 1. Localities of Microcnemum coralloides included in the molecular analyses (numbers refer to population numbers listed in Table1). Microcnemum coralloides subsp. coralloides shows a scattered distribution in central and eastern Spain (compare alsohttp://www.programanthos.org) and M. coralloides subsp. anatolicum has been recorded from a few localities in Turkey, Syria, Ar-menia and central, eastern and northwestern Iran.

G. Kadereit & A.E. Yaprak418


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ducts were directly purified with the PCR product pu-rification kit (Macherey-Nagel). PCR primer se-quences used for ITS and the atpB-rbcL spacer can befound in Kadereit & al. (2006). The trnT/F region wasamplified in three fragments (primer sequences afterTaberlet & al. (1991) if not indicated otherwise): 1.trnT-f (= ucp-a, forward) and trnL-b (modified forthis study, reverse): 5’ AAT TCC AGG GTT TCTCTG AAT TTG –3’; 2. ucp-c (forward) and ucp-d(reverse); 3. trnL-460 (after Worberg & al., 2007) andtrn-F (= ucp-f, reverse).

The purified PCR products were sequenced usingthe ABI Prism Dye Terminator Cycle SequencingReady Reaction Kit (Perkin Elmer) and five-fold di-luted PCR primers as sequencing primers. For cyclesequencing, thermocyclers were programmed as de-scribed in Kadereit & al. (2005). Extension productswere sequenced on ABI 373 or 377 automated se-quencers.

Alignment.–Forward and reverse sequences wereedited and assembled to consensus sequences whichthen were aligned using SequencherTM 4.1. Thealignments of all markers were straightforward andrequired little manual correction. Indels were unam-biguous and coded in a 0/1 matrix for all markers(compare Table 2). Mononucleotide microsatellites inthe cp markers were excluded from the analyses.

Maximum Parsimony analyses.–The matrices wereanalysed under maximum parsimony (MP) criteriaseparately and combined. A partition homogeneity


test (Farris & al., 1994; implemented in PAUP*4.0b10) was conducted with 1000 homogeneity repli-cates and the same heuristic search conditions as de-scribed below. All analyses (markers separately andcombined) included heuristic searches with 1000replicates of random taxon addition and tree-bisec-tion-reconnection (TBR) branch swapping. MP Boot-strap was performed under the same conditions as theheuristic search with 1000 replicates of 100 randomtaxon additions.

Maximum Likelihood analyses.–Maximum likeli-hood (ML) analyses were conducted for all three datasets and the combined data set using the best-fit sub-stitution model determined by Modeltest 3.06 (Posa-da & Crandall, 1998). Maximum likelihood searcheswere performed with the settings outlined in Table 3.The heuristic search was performed with TBR branchswapping and 100 random sequence additions. Boot-strap support values were calculated from 100 repli-cates of heuristic searches again using TBR swappingand 100 random additions of sequences.

Molecular clock analysis.–Age estimates for Sali-cornioideae published in Kadereit & al. (2006) wereused to calibrate the basal node as indicated in Fig. 2.The age estimates calculated in Kadereit & al. (2006)used Salicornites massalongoi from Oligocene de-posits (35.4-23.3 Mya, Chiavu, Italy; Principi, 1926)as a fossil calibration point and was based on a broadsample of Salicornioideae. Because the study ofKadereit & al. (2006) was based on ITS and the atpB-rbcL spacer, the age estimates obtained from thesetwo markers were used for the corresponding datasets here. For the calibration of the trnT/F tree andthe tree resulting from the combined analysis the ageestimates for ITS and the atpB-rbcL spacer in Kade-reit & al. (2006) were used in combination (22.2-17.8mya; compare Fig. 2C,D,G,H).

For the estimation of divergence times we used twodifferent approaches: 1. Likelihood ratio test (LRT;Felsenstein, 1988) and 2. Penalized Likelihood (PL)as implemented in “r8s” (Sanderson, 2002). The latterwas conducted twice: 1. based on the tree resultingfrom the MP analyses and 2. based on the ML trees.

LRT: A likelihood ratio test (Felsenstein, 1988)was conducted for all three data sets and the com-bined data set. Rate-constancy was tested by calcu-lating log-likelihood scores for trees with and withouta molecular clock enforced using PAUP. Non-signifi-cance at the 0.01 level between tree topologies, indi-cating that a molecular clock can not be rejected, wasassessed with Modeltest 3.06 (Posada & Crandall,1998).

PL: The MP and ML tree topologies shown in Fig. 2 (except the ML tree of the atpB-rbcL spacer

419


Table 2. Sequence characteristics of the molecular markers usedand tree statistics of the Maximum Parsimony analyses.

*1 No amplification results for sample 2002/15 of Microcnemum coralloidessubsp. anatolicum for the trnT/F region, but sample in the ITS data set identi-cal with sample 642.

Marker ITS atpB-rbcL trnT/F combinedspacer region data

Taxa includedMicrocnemum/other Salicornioideae 12/5 12/5 11*1/5 11*1/5

Aligned bp 663 744 1876 3283

Within Microcnemum∑ variable sites 10 8 31 49Indels/ point mutations 1/9 3/5 13/18 17/32Parsimony informative 4 6 19 29

All taxa analysed∑ variable sites 157 52 178 387Parsimony informative 79 21 78 177Tree length 207 54 204 473Number of shortest trees 1 1 21 18Tree indices CI 0.9 CI 0.96 CI 0.90 CI 0.89

RI 0.89 RI 0.97 RI 0.91 RI 0.89

G. Kadereit & A.E. Yaprak420


Fig. 2. Phylograms derived from Maximum Parsimony (MP) analyses of the ITS (A), atpB-rbcL spacer (B), trnT/F region (C) and com-bined data sets (D) as well as Maximum Likelihood (ML) analyses of the ITS (E), atpB-rbcL spacer (F), trnT/F region (G) and combineddata sets (H). Number of character changes are indicated above branches in A-D, MP bootstrap values for A-D and ML bootstrap values for E-H below branches. The calibrated node is marked with a grey box, age estimates derived from the Penalized Likelihoodmethod are shown in bold face (except 2F, here age estimates are derived from the clock-enforced ML tree). The analysis details aresummarized in Table 2 and 3, the age estimates are summarized in Table 4.

which was clock-like) were implemented in the r8scommand block together with the following settings:method Penalized Likelihood (PL), algorithm Trun-cated-Newton (TN) method. For each analysis weperformed two runs: 1. a preliminary run estimatingthe optimal smoothing parameters with cross valida-tion start set to 0, cross validation increase set to 0.2and number of steps k = 50; 2. the second run imple-mented the optimal smoothing parameters (MP trees:1 000 000 each for ITS, atpB-rbcL spacer and thetrnT/F region and 4 for the combined analysis; MLtree: 4 for ITS and combined data set and 10 for thetrnT/F region) and estimated the ages of the nodes us-ing the constraints (minimum age, maximum age) asindicated in Fig. 2 (shaded box).

Confidence intervals were calculated for the age es-timates of the combined ML tree using r8s (Sander-son, 2002) and the r8s bootstrap kit (Eriksson, 2002).Relative branch length from 100 bootstrapped treeswere transformed into 100 absolutes ages. This agedistribution was then used to calculate 95% confi-dence intervals for the crucial nodes in Microcnemum.

Results

Morphological data.–Comparison of the morpholo-gy of the two subspecies revealed a great overall simi-larity. After the survey of more material from Turkey(listed in Table 1), the seed testa remains the only un-ambiguous character to distinguish the two sub-


species (Fig. 3, see also Molero, 1986: Figs. 1-4). TheIranian and Armenian specimens included here alsoshow a distinctly papillose seed testa as typical for M. coralloides subsp. anatolicum. Whether the popu-lations growing in Syria have papillose seeds, too, hasnot been investigated. A more delicate and lessbranched habit of the eastern subspecies does notconsistently distinguish the two taxa because dwarfforms of M. coralloides subsp. coralloides can also beobserved in Spain (G. Kadereit, per. obs.; Molero,2000: 535, Fig. 160a). We found that fertile segmentsof “well-grown” individuals are usually larger (2.5-3.5 mm long) in M. coralloides subsp. coralloides thanin M. coralloides subsp. anatolicum (1.5-2.2 mm long).Although we also saw flowering material from Turkey,which was unavailable to Wagenitz (1959), we did notfind any additional character to delimitate the twosubspecies. The number of stamens is one in both. Re-garding coloration, we found that although M. coral-loides subsp. anatolicum never turns crimson, col-oration is variable and can not be used as a diagnosticcharacter.

Molecular data.–The partition homogeneity testshowed a p-value of 0.03 which means that only 3% ofthe artificial partitions resulted in shorter or equallylong trees than the original partitions. Therefore, thethree data sets do not show a significantly differentphylogenetic signal when a level of 0.01 is used.

Sequence characteristics of the molecular markersused and tree statistics of the Maximum Parsimony

421


Table 3. Settings and results of the Maximum Likelihood analyses and results of a Likelihood ratio test (LRT) conducted for all data setand the combined data set (df = degrees of freedom).

Marker ITS region atpB-rbcL spacer trnT/F region combined data sets

Substitution model GTR+I GTR GTR+G GTR+I

Nucleotide frequencies A 0.19 A 0.394 A 0.383 A 0.346C 0.296 C 0.139 C 0.140 C 0.171G 0.315 G 0.115 G 0.151 G 0.184

T 0.2 T 0.322 T 0.327 T 0.299

Rate matrix AC 1.052 AC 0.467 AC 1.158 AC 0.932AG 6.703 AG 0.917 AG 0.903 AG 01.943AT 1.748 AT 0.082 AT 0.203 AT 0.251CG 0.422 CG 0.994 CG 0.645 CG 0.703CT 2.597 CT 0.917 CT 0.903 CT 1.261GT 1.0 GT 1.0 GT 1.0 GT 1.0

Fraction invariable sites or gamma distribution 0.6368 0 0.239 0.76

No of trees/ML score 1/1738.89 1/1189.69 1/3363.37 1/6576.38

ML score with clock enforced 1754.714 1200.05 3393.0 6606.53

Results of LRT df = 15 df = 15 df = 14 df = 14ratio 31.65 ratio 20.73 ratio 59.34 ratio 66.3p = 0.0072 p = 0.146 p < 0.0000001 p < 0.0000001

not clock-like clock-like not clock-like not clock-like

analyses are summarized in Table 2. The trees areshown in Fig. 2A-D. The ITS and atpB-rbcL spacerdata sets each resulted in only one shortest tree (Fig. 2A,B), and the analysis of the trnT/F region re-sulted in 21 shortest trees that differed in those cladeswithin the subspecies of Microcnemum which did notreceive bootstrap support (Fig. 2C). The combinedanalysis resulted in 18 equally parsimonious treetopologies, and again differences in topology wasfound in those branches within the subspecies of Mi-crocnemum that did not receive bootstrap support(Fig. 2D). The results of the Maximum Likelihoodanalyses are summarized in Table 3. The trees areshown in Fig. 2E-H.

The main results of the molecular clock analyses areindicated in Fig. 2 and summarized in Tables 3 (LRT)and 4 (age estimates for Microcnemum and confi-dence intervals for age estimates in the combinedML). Only the atpB-rbcL spacer data set was clock-


like. Therefore, the PL method was not conducted forthe ML tree for this marker. Generally ML tree basedage estimates and MP tree based age estimates did notdiffer strongly except for the atpB-rbcL spacer (Fig.2B,F).

Discussion

Morphological differentiation between the two subspecies of Microcnemum

In 1957 Wagenitz (1959) discovered a Turkish po-pulation of Microcnemum along the shores of TuzGölü (Nigde prov., Turkey). He compared his collec-tions with some Spanish specimens of Microcnemumcoralloides and found a great overall similarity. Theonly differences Wagenitz noted were: 1) a more deli-cate and less branched habit in the Turkish speci-mens, 2) papillose seeds in the Turkish versus granu-late seeds in the Spanish specimens (Fig. 3), and 3) ayellow coloration of the specimens versus crimson orblue-greenish in the Spanish ones. Although Wa-genitz collected his material mainly in the fruitingstage, he was able to observe mature anthers whichalso did not show any differences to the Spanish ma-terial. Despite the great similarity of the Turkish andSpanish specimens Wagenitz regarded the differencesfound sufficient to describe the Turkish material as anew subspecies, M. coralloides subsp. anatolicum.

The results of our own morphological study of M.coralloides subsp. anatolicum which included severalnew populations from Turkey and material from Iranand Armenia (Fig. 1, Table 1) fully support the find-ings of Wagenitz (1959). We also found great overallmorphological similarity of M. coralloides subsp. ana-tolicum with M. coralloides subsp. coralloides, includ-ing flower morphology. The seed testa is the only con-sistent character to distinguish them (Fig. 3; Molero,1986: Figs. 1-4). Slight differences in habit (height,succulence and branching) and coloration do not dis-tinguish the two subspecies reliably and are probablyplastic and strongly influenced by seasonal fluctua-tions of rainfall and salinity. Both subspecies of M.coralloides show variation, and grow to only very smallsize under unfavourable conditions.

Molecular phylogeny of Microcnemum

The molecular analyses presented here recover thesame conflicting phylogenetic relationships of Microc-nemum as found by Kadereit & al. (2006). The ITSdata set shows a sister group relationship to Arthroc-nemum, albeit with very low support (MP: BS 57%,ML: BS 52%), while the atpB-rbcL spacer data setmoderately supports a sister group relationships ofMicrocnemum to a clade comprising Sarcocornia uta-

422


Fig. 3. SEM pictures of seeds of Microcnemum coralloides sub-sp. coralloides (a, seed taken from sample 2 in Table 1) and M.coralloides subsp. anatolicum (b, seed taken from sample 8 inTable 1). Scale bars in a and b: 100 µm.

hensis (representing the Sarcocornia/Salicornia lin-eage) and Halosarcia indica (representing the Aus-tralian Salicornieae). The analysis of the trnT/F regionsupports a sister group relationship of Microcnemumto Sarcocornia (MP: BS 61%; ML: BS 83%) and thetwo clades are sister to Halosarcia indica (MP: BS78%; ML: BS 96%). In the combined analysis thephylogenetic signal of the atpB-rbcL spacer data setprevails (Fig. 2D,H). This conflict in the relationshipsof Microcnemum is smaller than it seems. Bootstrapsupport is low in all three data sets and Microcnemumis best regarded as part of a polytomy including theSarcocornia/Salicornia lineage, Arthrocnemum andAustralian Salicornieae. All four clades originatedduring the Middle Miocene (Fig. 2) probably in theTethys area, where three of the four clades are stillfound today. The molecular data presented here show that Microcnemum originated 20.9-13.8 mya(Table 4).

The relationship between the two subspecies of M. coralloides is resolved in three different ways(Table 4): 1. They are sister to each other [combinedanalysis (ML; MP), trnT/F region (MP) and atpB-rbcL spacer (MP)], 2. M. coralloides subsp. anatolicumis paraphyletic in relation to M. coralloides subsp.coralloides [ITS (ML, MP), trnT/F region (ML)], 3.relationships are not resolved [atpB-rbcL spacer(ML)]. Considerable genetic diversity among the ac-cessions of M. coralloides subsp. coralloides can onlybe found in the trnT/F region while the accessions ofM. coralloides subsp. anatolicum show genetic diversi-ty in all three markers. Therefore, the crown groupage of M. coralloides subsp. anatolicum is distinctlyolder (4.1-0.7 mya) than that of M. coralloides subsp.coralloides (0.7-0.08 mya, Fig. 2, Table 4). Consider-ing the geographical isolation of the two taxa, theirmolecular differentiation and the presence of at leastone distinct diagnostic character, their recognition assubspecies is fully justified.

Biogeography of Microcnemum

A major aim of this study was to infer the time ofseparation between M. coralloides subsp. coralloidesand M. coralloides subsp. anatolicum. This node re-ceived age estimates ranging from 2.8-0.5 mya basedon the ML and MP trees and different dating methods(Fig. 2, Table 4). The age of 7.9 mya calculated for theatpB-rbcL spacer based on the MP tree (Fig. 2B) is aclear outlier compared to the other estimates obtainedand may well have resulted from the low number ofcharacters of this data set (Tables 2, 4). The ITS ageestimates are the youngest, and the ML tree datedwith the PL method of the trnT/F region yielded theoldest estimates. Recent intersubspecific introgres-


sion of ITS as a possible explanation of the young ageestimates obtained from this marker seems unlikely inview of the great geographical distance between thetwo subspecies.

The young age estimate for the ITS data set mayalso result from an unusual mutation rate. However,comparison with the ITS mutation rates (average:4.13 × 10–9 subs/site/year; range: 1.72-8.34 × 10–9

subs/site/year) found for herbaceous plants (Kay &al., 2006) shows that the estimated rates for all Mi-crocnemum samples (3.0-4.3 × 10–9 subs/site/year) liewell within this range and close to the average rate.

Probably the most objective and possible accurateestimate with the present data is that of the combinedanalysis, which incorporates all characters availableand balances marker specific rate variation amongclades. In the two combined trees the split is dated to2.5 (MP) and 1.7 (ML) mya, respectively. In the com-bined ML analysis the split of the two subspecies isdated to 1.7 mya with the 95% confidence intervalranging from 2.17-1.28 mya (Table 4).

A split between the two subspecies around 2.5-1.5mya as indicated by the combined analyses would im-ply a separation shortly before, or at the onset of, thePleistocene. Like Wagenitz (1959), we therefore ex-clude recent long distance dispersal or anthropoge-nic introduction to Spain as explanations for the dis-junction of Microcnemum. Both explanations can berejected because of the genetic distinctness of M. co-ralloides subsp. coralloides (12 mutations in the com-bined analysis, Fig. 2D) and its intrasubspecific di-versification at least in the trnT/F region (Fig. 2C,G).

Microcnemum coralloides shows an extremely scat-tered distribution in both parts of its range. As out-lined in the introduction, the species is restricted toinland lagoons with extreme salinity regimes where itis associated with a few other plant species (mostlyChenopodiaceae) able to tolerate hypersaline condi-tions. The habitat of Microcnemum is likely to havebeen available only in isolated localities throughoutthe past, although it may have been more widespreadduring the Messinian crisis of the Late Miocene (Kri-jsman & al., 1999; Duggen & al., 2003; Thompson,2005) and during cooler and drier periods of thePleistocene (Shackleton & al., 1984; Lang, 1994; VanAndel & Tzedakis, 1996).

Two explanations can be offered for the disjunctdistribution of Microcnemum:

1) Microcnemum was widely and more or less con-tinuously distributed in the Mediterranean area sincethe Late Miocene. Such distribution range was frag-mented by the onset of the climatic oscillations at thebeginning of the Pleistocene. Together with other nonfrost-tolerant plants Microcnemum was restricted toareas with suitable climatic conditions. Such areas ex-

423


isted in southern Spain, southern Sicily, southernAnatolia, SW Asia and N Africa (Van Andel & Tze-dakis, 1996). This would imply that extant sites of thespecies in central and eastern Spain were re-colonizedfrom localities in southern Spain.

2) Alternatively, it seems possible that the Iberianpart of the distribution range of Microcnemum was es-tablished only at the onset of the Pleistocene. In coldperiods of the Pleistocene the sea level fell and largecoastal plains emerged (Shackleton & al., 1984; Lang,1994; Van Andel & Tzedakis, 1996). At such timessaline lagoons probably were frequent along the low-er and shallow coast lines and could have allowed mi-gration of hygrohalophytic taxa such as Microcne-mum.

Considering the paraphyly of M. coralloides subsp.anatolicum in relation to M. coralloides subsp. coral-loides in the ITS and trnT/F trees (Fig. 2A,E,G), thestronger genetic differentiation of subsp. anatolicumin comparison to subsp. coralloides in all three mark-ers, and the higher crown group age of the former


(Fig. 2), we prefer the second of the above two hy-potheses and postulate that the Iberian part of therange of M. coralloides was established at the onset ofthe Pleistocene. Such explanation requires that al-though conditions during cold parts of the early Pleis-tocene allowed migration over long distances, geneflow between different parts of the distribution rangewas interrupted soon after the arrival of the species inIberia. The alternative explanation, Pleistocene frag-mentation of a wider distribution area, fails to explainthe asymmetrical distribution of genetic diversity seentoday, unless additional factors such as populationbottlenecks are advocated.

Although Microcnemum is a rather extreme exampleof an intraspecific East-West disjunction in the Medi-terranean region, it is not a singular case. More than 70examples in c. 25 different plant families can be foundin the literature (Engler, 1879: 53-57; Willkomm 1896:103-104; Tab. 1 “oriental plants”; Davis & Hedge,1971; Greuter & al., 1984, 1986, 1989).

However, only few examples have been analysed

424


1. Marker ITS region atpB-rbcL spacer trnT/F region combined data sets

Relationships of the M. coralloides subsp. Sister group relationship Sister group relationship Sister group relationshiptwo subspecies of coralloides nested within in MP analysis not in MP; M. coralloidesM. coralloides (see M. coralloides subsp. resolved in ML analysis subsp. coralloides nested also Fig. 2) anatolicum within M. coralloides

subsp. anatolicum in ML

ML clockLRT (Table 3) not clock-like clock-like not clock-like not clock-likestem age genus 18.1-16.8stem age subsp. both 2.2-2.0split betw. subsp. 2.2-2.0

PL method with ML treestem age genus 18.3 *1 16.2 20.9stem age subsp. anat. 1.5 anat. 4.1 both 1.7

cora. 0.8 cora. 2.3split betw. subsp. 0.8 2.2 1.7

PL method with MP treestem age genus 16.6 18.4 13.8 17.2stem age subsp. anat. 0.7 both 7.9 both 2.8 both 2.5

cora. 0.5split betw. subsp. 0.5 7.9 2.8 2.5

2. Confidence Intervals (CI) for the combined ML tree (Fig. 2H)

Microcnemum stem CI: 19.0-22.5 (mean = 20.8)subsp. coralloides crown CI: 0.33-0.72 (mean = 0.53)subsp. anatolicum crown CI: 0.71-1.26 (mean = 0.98)split of the two subspecies CI: 1.28-2.17 (mean = 1.73)

Table 4. Age of Microcnemum and dating the split of the two subspecies (in my): 1. Results of molecular clock analyses [ML clock =age estimates are derived from the clock-enforced ML tree (LRT = Likelihood Ratio Test), PL method = Penalized Likelihood method asimplemented in r8s, anat. = M. coralloides subsp. anatolicum, cora. = M. coralloides subsp. coralloides]; 2. Confidence intervals for thecombined ML tree (Fig. 2H).

*1 PL estimate not needed because LRT was clock-like

with molecular tools. Sanmartin (2003) concludedthat the Pachydeminae (Coleoptera, Scarabaeoidea),a group of beetles disjunctly distributed in the Eastand West Mediterranean area, originated in the south-east Mediterranean region and initially diversified inthe Middle East and Iran-Afghanistan region. Thepresence of the group in the Iberian Peninsula was in-terpreted as a result of migration through NorthAfrica and Gibraltar during the Late Miocene. A mol-ecular study of Buxus balearica Lam. (Rosselló & al.,2007) using ITS sequence data found a clear geo-graphical split between western and eastern acces-sions of the species. The molecular data and the ecol-ogy of B. balearica as a non cold-tolerant shrub re-stricted to mesic environments support the hypo-thesis that the few extant populations in Anatolia(fomerly separated as Buxus longifolia Boiss.) are re-licts of a fomerly wider distribution area. Unfortu-nately, this study does not estimate the date of the splitbetween western and eastern populations.

Acknowledgements

We are grateful to J. Akopian (Yerevan), M. Assadi (Teheran),P. Catalán (Zaragoza), M.B. Crespo (Alicante), H. Freitag (Kassel)D. Gómez (Jaca) and J. Mehregan (Mainz) for supplying the rareand hard to find material of M. coralloides. We thank N. MunevverPinar (Ankara), B. Niethard (Mainz), D. Franke (Mainz) and E. Westberg for various technical help. We thank J.J. Aldasoro(Madrid), F. Blattner (Gatersleben), P. Catalán (Zaragoza), H.Freitag (Kassel), J. W. Kadereit (Mainz), G. Schneeweiss (Vienna)and P. Vargas (Madrid) for critically reading and substantially improving the manuscript. A research visit of A.E. Yaprak toMainz in 2005 was supported by the DAAD (German AcademicExchange Service).

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Associate Editor: P. CatalánReceived: 4-III-2008

Accepted: 21-VII-2008

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