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Bridging the Alps and the Middle East: Evolution, phylogeny and systematics of the genus Wulfenia (Plantaginaceae)

Article in Taxon · August 2014 DOI: 10.12705/634.18

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Bridging the Alps and the Middle East: Evolution, phylogeny and systematics of the genus Wulfenia (Plantaginaceae)

Boštjan Surina,1,2 Simon Pfanzelt,3 Helena J.R. Einzmann3 & Dirk C. Albach3

1 University of Primorska, Faculty of Mathematics, Natural Sciences and Information Technologies, Glagoljaška 8, 6000 Koper, Slovenia 2 Natural History Museum Rijeka, Lorenzov prolaz 1, 51000 Rijeka, Croatia 3 Institute for Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, Carl von Ossietzky-Str. 9–11, 26111 Oldenburg, Germany Author for correspondence: Dirk Albach, [email protected] ORCID: D.C.A., http://www.orcid.org/0000-0001-9056-7382

DOI http://dx.doi.org/10.12705/634.18

Abstract The genus Wulfenia (Plantaginaceae) demonstrates a striking disjunction between the southeastern Alps (Carnic Alps), the southeastern Dinaric Alps (Prokletije Mountains, Balkan Peninsula) and the Amanos mountains of southern Turkey. This puzzling biogeographic pattern has interested botanists for more than 100 years and Wulfenia has been widely regarded as a Tertiary relict of at least Miocene age in southeastern Europe. Specifically, the identity of populations in the Prokletije Moun- tains either referred to as disjunct populations of W. carinthiaca or a separate species, “W. blecicii ”, has been much debated. Here we analyze AFLP, plastid and nuclear ribosomal sequence data in conjunction with a morphometrical analysis to clarify the taxonomy of the genus and the relationships of the populations to one another. Furthermore, we employ a molecular dating strategy to put these results in a time frame to assess the Miocene relict–hypothesis. Our results provide evidence for a new classification of the genus with four species, W. orientalis, W. glanduligera comb. & stat. nov., W. baldaccii and W. carin- thiaca. The split of the last species into populations disjunctly distributed in the southeastern Alps (W. carinthiaca s.str.) and southeastern Dinaric Alps (“W. blecicii ”), is not supported either by molecular or morphological data, while we find enough evidence in DNA sequence data, growth site specifics and morphology for W. orientalis var. glanduligera to be treated at the species rank. Our dating analysis suggests that the extant genus is rather young with a crown node age of only about 1.24 Ma and 0.61 Ma for the European populations despite a stem node age of about 10.69 Ma. Thus, Wulfenia as a genus is likely a Miocene relict but its uninterrupted presence on the Balkan Peninsula cannot be demonstrated.

Keywords Balkan Peninsula; disjunction; Miocene; molecular dating; relict species; Turkey

Supplementary Material Alignment files are available in the Supplementary Data section of the online version of this article at http://www.ingentaconnect.com/content/iapt/tax

INTRODUCTION similar disjunction patterns (Wen, 1999; Donoghue & Moore, 2003; Kadereit & Baldwin, 2012). Disjunctions in plant species have fascinated botanists for The dating of disjunct relationships among conspecific a long time. In the past decades botanists documented almost taxa and populations of a single taxon between the Alps every conceivable sort of disjunct distribution range (Raven, and other European mountain ranges attracted considerable 1972). Various explanations have been proposed for these pat- attention when appropriate methods for dating in molecu- terns, in particular in the context of dispersal versus vicari- lar phylogeographic studies became available (Kropf & al., ance (Crisci, 2001). Especially, recurrent patterns of disjunct 2009; Dixon & al., 2009; Schneeweiss & Schönswetter, 2010; ranges between North America and eastern Asia (reviewed in Surina & al., 2011; Escobar García & al., 2012). According Boufford & Spongberg, 1983; Wen, 1999) and European and to Kadereit & al. (2008), the relationships of the flora of the Asian mountain systems (e.g., Christ, 1867; Vasudevan, 1975, Alps to floras of areas outside Europe and the possibility of 1977; Ozenda, 1985; Kadereit & al., 2008) are evident and have floristic interchange, has received much less attention. These been discussed. Convincing examples for processes, disper- authors noted a considerable lack of molecular phylogenetic sal and vicariance, as the cause of disjunct distribution areas studies for taxa occurring in the Alps and Asia. Nevertheless, have been put forward (Stace, 1989). Nowadays, the distinction based on floristic comparisons they identified one group of between vicariance and dispersal has been blurred by recogniz- Alpine species linked to Asia by a northern connection and ing that events occurring at different time intervals can result in another group linked to Asia by a southern connection. This

Received: 16 Dec 2013 | returned for first revision: 12 Feb 2014 | last revision received: 9 May 2014 | accepted: 9 May 2014 | not published online ahead of inclusion in print and online issues || © International Association for Plant Taxonomy (IAPT) 2014

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latter group was stated to have a likely Mediterranean origin s.l. with long, distinctly narrower and longer corolla tubes (Kadereit & al., 2008). and almost actinomorphic corolla lobes, the other taxa of the One suitable group to analyze such a pattern of south- genus have slightly obconical tubes and short, obscurely two- ern connection between Europe and Asia is the genus Wul- lipped limbs: the upper one entire or emarginated, the lower fenia, which was discovered in 1779 from the Carnic Alps by one shortly three-lobed. All Wulfenia species but the nomi- F.X. Wulfen and described in 1781 (Jacquin, 1781) to include nate variety of W. orientalis grow in montane or subalpine only W. carinthiaca Jacq. Later, P.E. Boissier (1844) found habitats. Alpine and Dinaric populations of W. carinthiaca are another species, W. orientalis Boiss., from the Amanos Mts. found in tall herb, scree, scrub and forest communities with (Mt. Samandağ) in southern Turkey (province Hatay). In the Mountain Pine (Pinus mugo) and Macedonian Pine (P. peuce), late 19th century, Hungarian botanist A. von Degen (1897) respectively, and Green Alder (Alnus viridis) or just above described the third species in northern Albania (Parun moun- the treeline, whereas W. baldaccii thrives exclusively in rock tain range, southernmost part of the Prokletije Mts. in Dinaric crevices within and above subalpine beech forests (Markgraf, Alps, Balkan Peninsula) collected by A. Baldacci, W. baldaccii 1930, 1931; Baldacci, 1934, 1935). In contrary, W. orientalis var. Degen. At the beginning of the 20th century, Bohemian bota- orientalis prefers rock crevices in much warmer sites at lower nist J. Rohlena (1904) found a disjunct locality of W. carin- altitudes in evergreen Mediterranean-submediterranean for- thiaca approximately 700 km SE from the Carnic Alps, on ests (e.g., Düzenli & Çakan, 2001), while W. orientalis var. Mt. Sjekirica in the Prokletije Mts., southeastern Dinaric Alps. glanduligera thrives in more mesophytic rock crevices within Subsequent findings in the Prokletije Mts. showed the plant montane Oriental Beech (Fagus orientalis Lipsky) forests (e.g., to be more widespread and abundant than in the place of its Akman, 1973). first discovery, the Carnic Alps. The remarkable disjunction Growth is almost continuous and only briefly intermitted by between the Carnic and the Dinaric Alps led Lakušić (1971) winter frost. All Wulfenia species have a high light demand and to segregate the populations from Prokletije Mts. into a new depend on high soil moisture and air humidity (Lepper, 1970a). species, “W. blecicii ” (invalidly published, see Infrageneric All species are diploid with 2n = 18 chromosomes (Lepper, 1968, taxonomy). In the 19th century a number of other species have 1970b; Fischer, 1969; reviewed in Albach & al., 2008). been described under Wulfenia with most now considered spe- To summarize, the taxa of the genus Wulfenia represent cies of the related genera Wulfeniopsis D.Y.Hong, Kashmiria remarkable presumably intraspecific disjuncts (between the D.Y.Hong, Paederota L., Synthyris Benth. or Besseya Rydb. southeastern Calcareous Alps and southeastern Dinaric Alps) (all tribe Veroniceae). The last two again have recently been and interspecific disjuncts (between Europe and the Eastern moved to Veronica (Albach & al., 2004b). This restricted cir- Mediterranean; Fig. 1). Explanations of such disjunctions origi- cumscription of Wulfenia has been supported by recent phylo- nating either by dispersal or vicariance have been the subject of genetic analyses (Albach & Chase, 2004; Albach & al., 2004a). heated debates (e.g., Degen, 1897; Derganc, 1906; Scharfetter, However, the phylogenetic relationships of Wulfenia differ 1908; Ginzberger, 1925; Markgraf, 1932; Gilli, 1933; Baldacci, according to the locus analyzed, being sister to the East Asian 1934; Lakušić, 1964; Lepper, 1970a). With its striking disjunct Lagotis and Veronicastrum based on plastid DNA and sister distribution the genus offers the opportunity for more general to the presumably at least ancestrally European Paederota and insights into the evolution of European and Eastern Mediter- Veronica based on nuclear ribosomal DNA (Albach & Chase, ranean plant life. Wulfenia has been considered a relict from 2004; Albach & al., 2004a). Miocene subtropical times in the area (Lepper, 1970a). How- With the generic circumscription resolved, the main taxo- ever, in the absence of fossils, a Miocene age for the genus nomic question in the genus nowadays concerns the status of could not be supported. Similar cases of a Miocene relict on plants in the Prokletije Mts. as included in W. carinthiaca or as the Balkan Peninsula may be the gesneriad genera Haber- a species of its own, “W. blecicii ”, with two subspecies (“subsp. lea, Jankaea, and Ramonda (Meyer, 1970), for which fossils blecicii ” and “subsp. rohlenae”) and three varieties as proposed are not available either, and Picea omorika (Pančić) Purk. by Lakušić (1971). The split into two species was based mainly (Pinaceae), for which no dating has been attempted (Aleksić on the geographic discontinuity and ecology (Lakušić, 1962, & Geburek, 2010), and likely a few other cases (Turrill, 1929; 1964), morphological differences in the leaves, inflorescences, Stevanović, 1996). calyx and fruit (Lakušić, 1971), as well as some cytological Partly due to such lack of fossils, partly due to the lack criteria (Lakušić & Papeš 1971; Lakušić 1978). This taxonomic of appropriate molecular dating methods until recently, such change has been doubted by Wraber and Lepper (both pers. supposed Miocene relicts in the Balkan flora have rarely been comm. in Kosch, 1992). Another taxonomic question in the analyzed in a thoroughly dated framework. Therefore, the aims genus concerns the status of plant populations from the north- of the current study are to test intrageneric relationships, espe- ern and central Amanos Mts. in Turkey, described as Wulfenia cially species limits between W. carinthiaca and “W. blecicii ” orientalis var. glanduligera (Huber-Morath, 1971), which both and in W. orientalis s.l. but also to estimate dates for evolution- morphologically and ecologically stand well apart from the ary events in the genus. In this context we focus on the time and populations in the southernmost Amanos Mts. (var. orientalis). place of origin for the genus testing the hypothesis of a Miocene The species of Wulfenia are rosette plants with obovate, origin of the genus and estimate the dates for the splits between crenate and fleshy leaves. Inflorescences consist of racemes the Eastern Mediterranean and the European and splits within with numerous blue-violet flowers. Apart from W. orientalis Europe using a Bayesian molecular dating method.

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MATERIAL AND METHODS digitalized photographs by means of a Zeiss Stemi 2000 ste- reomicroscope and the ImageJ processing program (Schnei- Plant material. — Our study included a comprehensive der & al., 2012). Whenever possible, stigma position, whether sampling of 18 populations of Wulfenia (three of W. baldacii, exserted or not, was recorded. one Alpine and ten Dinaric populations of W. carinthiaca, four Descriptive statistics were calculated for each character of W. orientalis s.l.) with up to 30 individuals each for the mor- and a multivariate analysis was performed to identify the phological analysis and five each for the AFLP analysis from structure of variability and to measure the distances between throughout the range of the genus (Fig. 1; Appendix 1). Her- populations and taxa. PCoA (principal coordinate analysis) barium specimens and vouchers are deposited at NHMR. Out- and constrained CCA (canonical correspondence analysis) groups for molecular analyses were sampled based on previous ordination techniques were performed using the Monte Carlo phylogenetic analyses of the family (Albach & Chase, 2004; test with 499 permutations with the help of the CANOCO Albach & al., 2005; see Appendix 2 for voucher information). software (Ter Braak & Šmilauer, 2002). This allowed identi- Morphology. — A total of 210 individuals from 12 popula- fication of statistically significant morphological characters tions of the European taxa (W. carinthiaca s.l., W. baldaccii) (P < 0.05) in explaining the variation within and between taxa. were investigated for the morphological analyses (Appendix 1). In order to determine the lengths of gradients, discriminant Wulfenia orientalis s.l. was omitted since plants from the type analyses, detrended by segments, were initially performed locality (Mt. Samandağ; W. orientalis var. orientalis) were not and the models (linear, unimodal) used accordingly. Since encountered in flowering stage. However, the morphological the assumption of normally distributed data and equality of distinctness of this species from the others has never been variance were violated even after data normalization, non- doubted. Individuals sampled covered the entire geographic parametric MANOVA based on Bray-Curtis distances, as distribution of the taxa in order to represent the full extent of implemented in PAST (Hammer & al., 2001), was used to test morphological variation. Up to 30 inflorescences per popula- for differences between all sampled populations in order to tion were fixed in an ethanol-glycerol mixture (50 : 50). Sixteen avoid a priori assumptions on taxon delimitation, and post hoc quantitative morphometric characters (six of vegetative and tests were carried out using Hotelling’s P values (sequential ten of reproductive region) and two indices (comparing repro- Bonferroni significance) pairwise comparisons. The signifi- ductive and reproductive with vegetative characters, respec- cance was computed by permutation of group membership tively) were examined, measured and calculated (Table 1) from with 10.000 replicates.

10°E 15°E 20°E 25°E 30°E 35°E 40°E AUT Mkr

Alpine population SLO

W. carinthiaca 45°N

ITA SRB Sjk MNE Zel Str Dinaric (Balkan) populations Black Sea Vst Hjl Krf Ndz KSV Adriatic Sea SD B M Mj Mk QS W. baldaccii ALB Tyrrhenian Sea 40°N

Ionian Sea TRK

W. glanduligera DI E DII

500 km Mediterranean Sea S W. orientalis SYR 35°N

Fig. 1. Distribution area of the genus Wulfenia (Plantaginaceae). ALB, Albania; AUT, Austria; ITA, Italy; KSV, Kosovo; MNE, Montenegro; SLO, Slovenia; SRB, Serbia; SYR, Syria; TRK, Turkey. For population acronyms see Appendix 1.

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DNA extraction. — DNA was isolated from ca. 20 mg of agarose gel and run for 30 min at 100 V. After 10 min ethid- tissue from silica gel–dried leaves using the DNeasy Plant Mini ium bromide washing slight band formation was visible. The Kit (Qiagen, Hilden, Germany), following the manufacturer’s PA product was diluted again 20-fold (5 µl PA-product and instructions. The quality of the extracted DNA was checked on 95 µl H2O) and frozen until further handling. For the selective 0.8% TBE-agarose gels and the concentration was measured amplification (SA) 2 µl of each diluted PA sample and 8 µl SA spectrophotometrically with a Picodrop RNA/DNA calculator reaction mix were incubated for 2 h 30 min. The SA reaction (Picodrop., Safron Walden, U.K.). mix for one sample contained: 5 μl Multiplex QIA, 7 ng of each AFLP generation. — The AFLP procedure followed Vos primer EcoRI-ACC, EcoRI-AGG and EcoRI-ACA (Applied & al. (1995) with modifications. To test for reproducibility, six Biosystems, Warrington, Cheshire, U.K.), 86.5 ng primer MseI- replicates were included. Genomic DNA was digested with the CAT (metabion international, Martinsried, Deutschland) and two restriction endonucleases EcoRI and MseI and ligated to 0.85 μl H2O. The SA product was analysed with an ABI 3130XL double-stranded EcoRI and MseI adaptors in one step at 37°C sequencer (Applied Biosystems). for 3 h (TProfessional Standard Thermocycler, Biometra, Göt- AFLP analysis. — GeneMarker v.1.91 (Softgenetics LLC) tingen, Germany). The reaction mix for one sample contained: was used to determine the alleles of the sequenced samples. 0.9 μl H2O, 1.1 μl NaCl, 0.55 μl BSA, 1.1 μl T4 ligase buffer, To achieve this, a mask was generated with the panel-editor- 1 μl of each MseI-adapter (50 µM) and EcoRI-adapter (5 µM), function. On the basis of that mask the program detected alleles 1 U MseI enzyme, 5 U EcoRI enzyme and 0.002 μl T4 ligase. in the range of 70–500 base pairs with a minimum intensity The adapters were heated to 95°C for 5 min and slowly cooled of 100. Afterwards the allele assignment was checked manu- down before use. Ligated DNA fragments were diluted 10-fold ally. A 1/0-matrix was created with monomorphic and unique with H2O and frozen until further handling. For the preselec- fragment classes excluded. Fingerprints deviating by more than tive amplification (PA) 5 µl of each diluted sample and 10 µl two standard deviations from the mean number of fragments of the PA reaction mix were incubated for 1 h 43 min. The PA were considered to have had poor amplification and omitted reaction mix for one sample contained: 14.4 μl H2O, 1.25 μl from the analysis (n = 22). An error rate was estimated based MgCl2, 2.5 μl NEB buffer, 0.25 μl of each dNTP (20 mM), E01 on the strategy of Bonin & al. (2004) using four samples (5%). primer and M02 primer (5 µM each, metabion international, Finally, the following number of samples remained for the Martinsried, Germany) and 0.5 U NEB Taq. After preselec- analyses: 17 W. orientalis s.l. samples, 5 Alpine and 49 Dinaric tive amplification 2 µl of each sample was pipetted on a 1.4% samples (incl. 3 repeats) of W. carinthiaca, respectively, and

Table 1. Morphological characters of the investigated populations of the genus Wulfenia employed in the analysis. W. carinthiaca W. baldaccii Alpine population Dinaric populations Morphological character or index N min Me max SD N min Me max SD N min Me max SD Total leaf length 30 48.6 79.5 94.8 11.3 30 128.7 190.2 237.7 21.04 150 118 192.8 309 26.97 Maximal leaf width 30 15.8 30 37.2 4.11 30 34 68.6 80.9 8.22 150 44 66.5 117.2 10.29 Leaf length at maximal leaf width 30 34.0 59.65 78.8 9.84 30 89.7 133.15 166.4 12.33 150 81.4 133 216 20.38 Leaf teeth length 30 5.3 8.5 9.8 1.06 30 6.3 9.15 10.8 1.12 150 5.3 9.2 16.4 1.81 Leaf teeth hight 30 2.5 5.3 8.4 1.25 30 1.5 2.8 3.2 0.45 150 1.6 2.9 5 0.56 Stem length 30 73.6 123 170 15.61 30 290 329.1 397 23.53 150 230 331 464 39.76 Inflorescence length 30 42.8 62.85 82.5 9.40 30 69 79.85 91.3 5.60 150 35 80 130 13.61 Inflorescence length/stem length 30 0.41 0.51 0.67 0.05 30 0.22 0.25 0.28 0.01 150 0.12 0.24 0.42 0.04 Sepal length 30 3.5 4.2 4.8 0.37 30 6 6.6 7.1 0.31 103 4.5 6.4 7.8 0.70 Petal length 30 12.4 14.75 16.2 0.95 30 10.7 12.25 14.3 0.83 105 8.6 12.8 15.9 1.17 Petal tube length 30 10.1 11.95 13.3 0.88 30 7.3 8.8 10.8 0.84 105 5.9 9.3 12.3 1.03 Petal tube width 30 1.5 1.85 2.4 0.21 30 2.3 2.7 3.2 0.21 105 2 2.7 3.7 0.36 Upper lip width 30 3.2 3.8 4.6 0.33 30 4.1 4.7 5.3 0.36 104 3.3 4.5 5.8 0.50 Upper lip length 30 2.3 2.9 3.4 0.25 30 2.9 3.4 3.7 0.22 105 2.5 3.4 4 0.35 Lower lip width 30 6.6 8.35 9.2 0.59 30 7.1 7.75 9.5 0.56 105 6.6 8.4 10.5 0.76 Lower lip length 30 4.2 5.3 6.2 0.44 30 3.3 4.55 5.5 0.50 104 3.8 5 5.8 0.47 Sepal length/petal length 30 0.24 0.29 0.33 0.02 30 0.47 0.53 0.62 0.04 103 0.34 0.5 0.68 0.07 Gynoecium length 30 10.6 13.2 14.5 1.01 26 10.4 12.9 14.7 1.01 97 8.4 13.1 15.9 1.55 N, number of individuals investigated; min, minimum value; Me, median; max, maximum value; SD, standard deviation.

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7 W. baldaccii samples (incl. 1 repeat; Appendix 1). With the In the phylogenetic analysis of the ITS dataset, a maximum remaining samples, a neighbour-net was constructed using the likelihood–based approach as implemented in RAxML v.7.2.6 program SPLITSTREE4 v.4.6 (Huson & Bryant, 2006) and (Stamatakis, 2006) was used. The GTR + Γ (4 Γ categories) Jaccard distances. The number of fixed and private fragments model of nucleotide substitution was selected. Model param- was analyzed using FAMD v.1.23 (Schlüter & Harris, 2006). eters were estimated by RAxML. In a single run, RAxML Additionally, variance in Shannon’s Index was calculated using computed 1000 bootstrap replicates and subsequently searched 1000 bootstrap replicates over loci with the same software. The for the best-scoring ML tree. Globularia repens was defined rarity index and Nei’s gene diversity were estimated in aflpdat as outgroup. (Ehrich, 2006). Estimating divergence times and biogeography. — In DNA sequencing. — The ndhF-rpl32 spacer was ampli- order to obtain a dated phylogeny of the ITS dataset, we used fied using primers given in Shaw & al. (2005). Reactions of BEAST v.1.8.0 (Drummond & Rambaut, 2007). To account 25 µl were performed using 1 U Taq Polymerase (New England for the uncertainty in evolutionary rate and calibration point Biolabs, Beverly, Massachusetts, U.S.A.), 0.6 µl 50 mM MgCl2, estimations, we made use of the probability distribution–based 2.5 µl Taq-Buffer (New England Biolabs), 2 µl bovine serum calibrations in BEAST. albumin (BSA), 0.25 µl 20 mM dNTPs and 1 µl of each 20 µM Model selection was based on the Akaike information primer. PCR reactions were run in a TProfessional thermocy- criterion (Akaike, 1973) as implemented in jModelTest v.2.1.3 cler (Biometra) under the following conditions: 1 min at 94°C, (Guindon & Gascuel, 2003; Darriba & al., 2012). Since prelimi- 35 cycles of 20 s at 94°C, 30 s at 52°C, and 1 min at 72°C, fol- nary BEAST runs using the best-scoring model, GTR + Γ + I, lowed by 1 : 30 min at 52°C and 8 min at 72°C. The PCR products resulted in poor effective sample size (ESS) values of several were cleaned with Exonuclease I and Calf Intestine Alkaline parameters, among them alpha and pInv, considered to be due Phosphatase (Fermentas, St. Leon-Rot, Germany) according to over-parameterization, we chose the simpler GTR + Γ model. to the manufacturer’s instructions. The same primers used for The difference in Akaike’s information criterion (AIC) between PCR amplification were also used for the cycle sequencing GTR + Γ + I and GTR + Γ was 2.0123. For the analysis of small reactions (10 µl) carried out using the Big Dye Terminator datasets, all models with a ΔAIC of less than 4 can still be Ready Reaction kit according to the manufacturer’s instruc- considered of having substantial support (Kelchner, 2009). tions (Applied Biosystems, Foster City California, U.S.A.). Rate evolution was modelled in an uncorrelated lognormal Sequencing was done on an ABI 3130XL sequencer (Applied relaxed clock framework (Drummond & al., 2006) to allow Biosystems). Assembled sequences were edited manually using for rate variation among lineages. The branch rate prior (ucld. Sequencher v.4.1 (Gene Codes, Ann Arbor, Michigan, U.S.A.). mean) was set to follow a normal distribution with a mean of The nrITS locus including ITS1 and ITS2 was amplified 4.1 25 × 10 −3 ± 1.808 × 10−3. This value is the mean ITS substitu- in 25 µl reaction volumes with primers ITS-4 TCC TCC GCT tion rate of several herbaceous taxa (Kay & al., 2006), exclud- TAT TGA TAT GC (White & al., 1990) and ITS-A GGA AGG ing the outlier Gentiana sect. Ciminalis as rate estimation for AGA AGT CGT AAC AAG G (Blattner, 1999). The reaction this clade is based on a single mutation (Hungerer & Kadereit, contained 1× NEB Thermopol Buffer, 1 U NEB Thermopol Taq, 1998). We used a Yule tree prior as recommended for species- 5 pmol dNTPs (Epicentre, Madison, Wisconsin, U.S.A.), 10 level phylogenies in the BEAST manual. pmol of each primer, 10 ng DNA and 10 µg BSA. The reaction Plantago is the closest relative of Veroniceae for which an conditions were 1 min at 94°C, 35 cycles with 18 s at 94°C, 30 s age estimate has been published (7.1 Ma; Rønsted & al., 2002). at 55°C and 1 min at 72°C and finally 8 min at 72°C. The PCR This estimate is based on the Amsterdam Island endemic Plan- product was purified for sequencing reactions with an enzy- tago stauntonii Reichardt. Calibrating phylogenies often relies matic reaction using ExoSAP according to the manufacturer’s on the occurrence of an endemic taxon on an island of known instructions. The locus was sequenced in both directions with age, but this approach may lead to underestimation of true ages the same primers as for the PCR at StarSEQ (Mainz, Germany). (Heads, 2011). Islands developing around island-generating tec- The assembly and editing of sequences was performed with tonic features—as in the case of Amsterdam Island, which is Sequencher v.4.1. Phyde v.0.9971 (Müller & al., 2005) was used situated on the tectonically active Amsterdam–St. Paul Plateau for sequence alignment. Indels were coded using the simple along the Southeast Indian Ridge (Doucet & al., 2004)—should indel coding algorithm of Simmons & Ochoterena (2000) in not be used for calibration, since former, but now submerged Seqstate v.1.4.1 (Müller, 2005). islands may have harboured ancestral taxa (Jønsson & al., Sequence analysis. — The cpDNA sequences revealed 2010). For that reason, we did not consider Rønsted & al.’s little intrageneric variation. The dataset was therefore ana- (2002) age estimate of Plantago for our calibration. Instead, lyzed only using the parsimony criterion in PAUP* v.4.0b10 paleobotanical/geomorphological and fossil data provided (Swofford, 2002) using the heuristic search algorithm with estimates for the Aragoa crown age and the stem age of the simple taxon addition and tree bisection-reconnection branch ancestor of Plantago/Aragoa. In the BEAST analysis, both the swapping. Parsimony bootstrap support was estimated by ana- Aragoa and the Plantago/Aragoa clades were constrained to lyzing 1000 bootstrap replicates with the same search criteria. be monophyletic, which is supported by phylogenetic analyses Additionally, the data matrix was analyzed using statistical (Bello & al., 2002; Rønsted & al., 2002). parsimony to construct a haplotype network in TCS v.1.21 Jiménez-Moreno & al. (2007a, b) reported fossil Plantago (Clement & al., 2000). pollen from the Rubielos de Mora basin in Spain dated to the

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Early Miocene. Nonetheless, pollen of Plantago and its sister A 180 Aragoa are very similar (Bello & al., 2002), so that the Rubielos de Mora pollen might probably be attributed to a Plantago/ 150 Aragoa ancestor. Consequently, we constrained the Plantago/ 120 Aragoa stem to a minimum age of 19.4 Ma, using an expo- nential distribution curve as suggested by Ho (2007) for fossil 90 calibration points. The evolution of the páramo endemic Aragoa is associ- 60 ated with the final phase of the orogeny of the northern Andes 30 (Bello & al., 2002; Sklenár & al., 2011), which created alpine ecosystems above treeline 3–5 Ma ago (Van der Hammen -300 -250 -200 -150 -100 -50 50 100 150 & Cleef, 1986; Graham, 2009). Aragoa was an early constitu-

Component 2 Component -30 ent of páramo vegetation as shown by pollen records dated to the Late Pliocene/Early Pleistocene (Van der Hammen -60 & González, 1960; Van der Hammen & al., 1973). Assuming that the radiation of Aragoa in the páramo started after the -90 time when the northern Andes reached their modern elevation Component 1 (2.7 ± 0.6 Ma, Gregory-Wodzicki, 2000), we set a uniform age prior for the crown age of Aragoa that spans 0–3.3 Ma. Based B 4.8 on wood anatomy, the ancestor of Aragoa may have evolved as an herbaceous montane or subalpine element (Carlquist, 3.2 1970; Mennega, 1975). Thus, using the age of alpine habitats created by Andean uplift for calibrating the ITS phylogeny 1.6 may lead to the underestimation of the true age of the clade. However, the sampled species show very little ITS and rbcL -48 -36 -24 -12 12 24 36 48 sequence divergence (Bello & al., 2002; this study). This indi- cates a young age for Aragoa and is reminiscent of the rapid Component 2 Component -1.6 and recent radiations of Andean Gentianella, Lupinus, and Astragalus during the Late Pleistocene (von Hagen & Kadereit, -3.2 2001; Hughes & Eastwood, 2006; Scherson & al., 2008). The Aragoa samples included in our phylogeny belong to subge- -4.8 nus Aragoa (Fernández Alonso, 1993). Samples of subgenus Luteoaragoa Fern. Alonso (one species) were not available. The -6.4 four included species, however, reflect the ecological variation within the genus as they occupy different habitats that range from dry and exposed to moist and even boggy sites. Like the Component 1 majority of Aragoa species, they grow in the high-elevation C páramo belt, i.e., they are true alpine elements, with the excep- 180 tion of A. cundinamarcensis Fern.Alonso whose lower altitu- dinal limit corresponds to the transition between high-Andean 150 forest and wet páramo (Fernández Alonso, 1993). 120 MCMC chain convergence to the stationary distribution of three independent runs (each 3 × 107 generations, sampling 90 every 1500th generation) was confirmed using Tracer v.1.5.0 (Rambaut & Drummond, 2009). The resulting tree files were 60 combined with LogCombiner v.1.6.1, discarding the first 15% of 30 the trees as burn-in. TreeAnnotator v.1.6.1 was used to compute

-300 -250 -200 -150 -100 -50 50 100 150 Fig. 2. Principal component analyses (PCA) of: A, 210 individuals Component 2 Component -30 based on 6 vegetative characters (eigenvalues 1: 9221.6, 2: 659.3, 3:

-60 98.9; % of explained variance on the first three axes: 1: 91.8, 2: 6.6, 3: 1.0); B, 165 individuals based on 10 reproductive characters (eigenval- -90 ues 1: 198.2, 2: 4.6, 3: 1.8; % of explained variance on the first three C, Component 1 axes: 1: 96.2, 2: 2.2, 3: 0.9); 165 individuals based on vegetative and reproductive characters (eigenvalues 1: 11258.1, 2: 694.5, 3: 103.0; Wulfenia baldaccii Wulfenia carinthiaca Alpine population % of explained variance on the first three axes: 1: 29.3, 2: 5.7, 3: 0.8). Dinaric populations Samples of Wulfenia orientalis s.l. not included.

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the maximum clade credibility tree with node heights being Table 2. MANOVA of the morphological characters for the European the mean of the age estimates. Both programs are part of the taxa of Wulfenia. BEAST package (Drummond & Rambaut, 2007). A subset of W. carinthiaca W. baldaccii the post-burn-in trees from the BEAST analysis was subse- Alpine pop. Dinaric pop. quently used in a biogeographic analysis using the Bayesian W. baldaccii 0 0 0 binary method in RASP v.2.1 (Yu & al., 2011). We employed a Alpine pop. 0 0 0.9307 model with estimated state frequencies and gamma rate het- W. carinthiaca erogeneity and null root distribution. We explored the effect Dinaric pop. 0 0.973 / 0.4322 0 of maximum number of areas by running the analysis either Upper right hand corner contains values for combined analysis (total restricting the results for a particular node to one or to two sum of squares: 3.58; within-group sum of squares: 0.5219; F: 474.6; maximum areas. The widespread Plantago and Veronica were P < 0.0001), whereas the lower left hand corner contains values for coded as occurring in three or two areas respectively since vegetative characters (total sum of squares: 5.115; within-group sum of squares: 0.6797; F: 675.4; P < 0.0001) followed by those for the the ancestral area for these genera are not known yet. Since reproductive characters (total sum of squares: 0.79; within-group sum these two genera are species-rich and widespread and no bio- of squares: 0.537; F: 38.15; P < 0.0001). P values are given according geographical analysis for them is available, no more detailed to sequential Bonferroni significance. analysis within continents is possible at the moment. Informa- tion on the distribution area for the genera is available from Fischer (2004). populations of W. carinthiaca from W. baldaccii but none was able to distinguish morphologically Alpine and Dinaric speci- RESULTS mens of W. carinthiaca (Table 2). In W. baldaccii stigmas were never exserted, whereas in Alpine and Dinaric populations of Morphology. — Among vegetative characters, differences W. carinthiaca stigmas were exserted in 77% and 61% of all in total leaf length, leaf length at maximal leaf width (reflect- observations, respectively. ing different leaf shapes), leaf teeth height, leaf teeth length, AFLP. — We scored 355 AFLP fragments in the range stem length and maximal leaf width proved to be statistically of 70 to 500 base pairs with an average of 153.4 fragments significant (using MANOVA and CCA) and explained the per individual and an error rate of 4.4%. The neighbour-net majority of the variation along the first axis (Fig. 2A). Descrip- analysis (Fig. 3) supported the distinction of W. orientalis s.l., tive statistics for all characters and two indices are found in W. baldaccii and W. carinthiaca s.l. from each other. Wulfe- Table 1. Among reproductive characters, differences in petal nia orientalis further splits into the southernmost population tube length, petal tube width, petal and sepal length (Fig. 2B) from the type locality (Mt. Samandağ—var. orientalis) and the were significant criteria to differentiate Alpine and Dinaric rest of the populations (var. glanduligera). Wulfenia baldaccii

Wulfenia baldaccii

M SD M M M Mkr Mj Alpine samples QS Mj Krf QS Mkr B Mj B Mj Str Vst

Wulfenia orientalis Vst Wulfenia S Hjl carinthiaca Ndz S S Sjk Sjk Hjl S Mk Mk Hjl Mk Sjk Ndz S Zlt B B Zlt Zlt Mk E Mk Zlt DII

DII E DII DII E E Fig. 3. Neighbour-net diagram of AFLP data constructed for all samples of Wulfenia that accom- DI DI DII DI modated quality criteria. Triangles stand for individuals of W. orientalis and W. glanduligera, full squares for W. baldacii, diamonds for Dinaric populations and full circles for Alpine population of Wulfenia glanduligera W. carinthiaca. For population acronyms see Appendix 1.

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seems to be in an intermediate position between W. carinthiaca the strict consensus tree was mostly due to the absence (due s.l. and W. orientalis s.l. The Alpine samples of W. carinthi- to technical reasons) of the single distinguishing site between aca were nested within the Dinaric ones. Although genetic the W. carinthiaca s.l. and the W. baldaccii haplotypes in one diversity indices should be interpreted carefully given the low individual of W. baldaccii. Among the 15 W. baldaccii and 53 number of individuals per population, they indicated a trend W. carinthiaca s.l. samples there was no intraspecific varia- towards higher genetic diversity (more private, fewer fixed tion. In contrast, five different haplotypes were found for the fragments, higher Shannon’s I, higher rarity) in W. orientalis 19 samples of W. orientalis s.l., which generally split into the s.l. (Table 3). Within W. orientalis s.l., the population from Mt. highly supported southernmost population (Mt. Samandağ, var. Samandağ had the highest genetic diversity (significantly dif- orientalis) and the rest of populations (var. glanduligera). The ferent Shannon’s I), whereas in W. carinthiaca s.l., there was resulting topology is depicted in Fig. 4B. a trend towards higher genetic diversity in the southeastern ITS. — In the ITS phylogeny (Fig. 5), both the ML and the populations (significantly different Shannon’s I). The Alpine Bayesian analysis placed Wulfenia as sister to a clade includ- population resembled the southeastern Dinaric populations in ing Veronica and Paederota (ML bootstrap support [BS] 88, these parameters (Table. 3). posterior probability [PP] 1.00). Wulfenia itself split into an cpDNA. — The alignment of the ndhF-rpl32 spacer region “eastern” clade (BS 81, PP 0.99) and a less supported “western” included 904 positions. Thirty gaps were coded according to clade that included W. carinthiaca s.l. and W. baldaccii (BS the method described, six of them variable within Wulfenia. 68, PP 0.87). The resulting dataset contained 71 (19 in Wulfenia) parsimony- Divergence times and biogeography. — The Bayesian informative positions (1.5% of the nucleotide positions in Wul- analysis gave a crown group age of Wulfenia of 0.49–2.12 Ma fenia). The consistency and retention index of the optimal (95% higher posterior density interval, mean 1.24 Ma) and a trees are 0.898 and 0.968, respectively. The parsimony analysis stem group age of 7.11–14.58 (10.69) Ma (Fig. 5). The “east- resulted in eight most parsimonious trees with Lagotis as sister ern” and “western” clades are 0.19–1.11 (0.61) Ma and 0.19–1.23 to Wulfenia and a basal split between W. orientalis s.l. and the (0.65) Ma old, respectively. The biogeographic analysis sug- other samples of Wulfenia (Fig. 4A). The lack of resolution in gested an Asian origin of Veroniceae with either migration to

Table 3. Genetic diversity indices based on AFLP data for the investigated populations of the genus Wulfenia. Population Individuals Shannon’s Rarity index Nei’s gene Sampling localities acronym analyzed Index (Ehrich, 2006) diversity Wulfenia baldaccii ALB: Qafa e Shtogut QS 2 9.27 0.13 ALB: Qafa e Mshitel M 3 11.59 0.18 Wulfenia carinthiaca—Alpine population AUT: Mokrine Mkr 5 5.37±0.20 3.60 0.06 Wulfenia carinthiaca—Dinaric populations KSV: Mt. Nedžinat Ndz 5 4.91±0.24 3.40 0.04 MNE: Mt. Sjekirica Sjk 5 4.61±0.28 3.27 0.03 MNE: Mt. Starac Str 5 4.58±0.28 3.00 0.04 MNE: Mt. Hajla Hjl 5 4.46±0.29 3.06 0.03 MNE: Mt. Maja e Kolata MK 5 5.53±0.19 2.71 0.07 MNE: Mt. Bogičevica B 5 5.54±0.19 2.87 0.06 ALB: Mt. Maja e Jezerce MJ 4 5.66±0.18 3.32 0.08 MNE: Mt. Zeletin Zlt 5 5.56±0.19 3.18 0.07 MNE: Mt. Visitor Vst 4 5.05±0.23 3.39 0.04 MNE: Karanfili Mts Krf 3 3.68 0.05 Wulfenia orientalis var. glanduligera TRK: Dörtyol DI 3 6.49 0.10 TRK: Dörtyol DII 5 6.00±0.16 6.52 0.09 TRK: Erzin E 4 5.53±0.19 6.38 0.07 Wulfenia orientalis var. orientalis TRK: Mt. Samandağ S 5 6.71±0.11 8.35 0.15 ALB, Albania; AUT, Austria; KSV, Kosovo; MNE, Montenegro; TRK, Turkey.

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the European-Mediterranean region in the common ancestor consequently hypothesized a Miocene origin for the genus. of Wulfenia, Paederota and Veronica and subsequent back- Such adaptation to macroclimate fits well to the result of our migration to Asia in Veronica (maximum number of areas per dating analysis and to our biogeographical analyses by explain- node restricted to one) or shared occurrence in Europe and ing why the genus may have occurred more broadly across Asia with subsequent restriction to particular continents in southern Eurasia. Nevertheless, it remains to be explained why the different lineages (maximum number of areas per node extant species continue to have adaptations to Miocene climate restricted to two). despite changes in climate since. Such evolutionary stasis of functional traits has been observed frequently among other herbaceous perennial genera of temperate regions of the North- DISCUSSION ern Hemisphere and has been explained by a greater extent of niche conservatism of these plants compared to woody taxa Wulfenia evolved in the Miocene. — Based on our cali- (Ricklefs & Latham, 1992). bration points and our dating strategy, we are able to substan- Niche conservatism involves a high degree of specializa- tiate the hypothesis of Lepper (1970a) that the stem lineage tion to the occupied niche space (Ricklefs & Latham, 1992) and of Wulfenia is of Miocene origin (mean age 10.69 Ma, 95% requires the continued presence of suitable habitat, at least at a higher posterior density interval 7.11–14.58 Ma). The age of local scale. To support the hypothesis of niche conservatism, the genus coupled with its much more recent diversification we would need to confirm that the functional traits of Wulfe- (0.49–2.12 Ma) fit into the pattern of taxa that experienced nia are adaptive to Miocene environmental condition and that extinction of the majority of their species caused by the Mes- suitable habitat existed in the area since then but is nowadays sinian salinity crisis (Fiz-Palacios & Valcarel, 2013). Despite more restricted. Physiological studies in Wulfenia are therefore the confirmation of Lepper’s hypothesis and fit into the phy- necessary to demonstrate that life history traits in the genus logenetic pattern of a pre-Messinian relict, some aspects of represent adaptations to the current climate of their habitat as the origin and evolution (e.g., dispersal patterns) of the genus well as to a past subtropical climate with less seasonal change remain unexplained, largely due to extinction and the consider- and higher summer precipitation. able geologic, climatic, and vegetational change since the origin Comparison with close relatives supports that growth form of the genus (Rögl, 1999; Zachos & al., 2001). and fleshy leaves are ancestral character states since they are Extant Wulfenia taxa occupy damp habitats with high air shared by all Veroniceae except for the allopolyploid Veroni- humidity, which are nowadays rare in the Mediterranean area. castrum and the ecologically much more variable species of Leaves are fleshy, featuring a thick mesophyll layer. Lepper Veronica. Furthermore, all these morphologically similar gen- (1970a) and Lakušić (1978) considered these traits to repre- era mostly share their occurrence in moist, montane to sub­ sent preserved adaptations to a subtropical environment and alpine habitats except for Veronicastrum (forests and lowland

A 4/96 Wulfenia orientalis s.l., W. glanduligera Fig. 4. Results from the analysis 23/100 of ndhF-rpl32 sequence data of 6/92 Wulfenia carinthiaca s.l., W. baldaccii sampled populations of the genus Wulfenia and some of its related 11 Lagotis angustibracteata genera. A, Topology of the major 41/100 groups and the outgroup in the 18 Lagotis stolonifera parsimony analysis. Numbers above the branches indicate 24 Veronicastrum virginicum 36/93 branch lengths/bootstrap sup- port. B, Haplotype network based 40 Veronicastrum stenostachyum on TCS-analysis. Numbers above B the branches indicate bootstrap W. baldaccii support, those below branch lengths in parsimony analysis. QS5+M5 W. glanduligera +SD5 Numbers within circles cor- root 82 1 respond to population acronyms W. carinthiaca given in Appendix 1, while

E2 E2 numbers in subscript indicate W. orientalis Alpine population - Mkr5 62 61 alternative 1 1 tree & number of individuals with the 99 (1) 96-92 S +DI DII4 corresponding haplotype. 5 1 5 10 (9) Dinaric populations - 63 1 Ndz5+Sjk5+Hjl4+MK5+B5+ Mj5+Krf5+Str4+Zlt5+Vst5

E1+DI4

eastern clade western clade

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meadows) and Veronica, in which there is a trend from shady, Veronica/Paederota­ as sister to Wulfenia (disregarding poly- montane towards open alpine habitats in several groups, espe- ploid Veronicastrum and Picrorhiza) had already been detected cially in early branching groups (Albach & al., 2004a). Nev- and discussed by Albach & Chase (2004). These authors sug- ertheless, the lack of fossil evidence and the long branch lead- gested that the incongruence was derived from independent ing to the extant species suggests considerable extinction and parallel fixation of the same parental ribosomal DNA unit refutes firm conclusions with regards to the ancestral habitat after the polyploid event that gave rise to extant Veroniceae. and character states of the Wulfenia stem group. Although this hypothesis can only be tested with considerable Biogeography of Wulfenia and tribe Veroniceae. — A genomic data, our dating approach can give a temporal frame complicating factor in the reconstruction of characters and for the processes causing incongruence today. habitats of Veroniceae is the firmly supported incongruence As inferred from the RASP analysis, the ancestor of between phylogenetic hypotheses derived from nuclear ITS Wulfenia occurred in the European-Mediterranean region, and plastid DNA. This incongruence with either Lagotis or whereas the ancestor of all Veroniceae originated in Asia.

Europe/Mediterranean Hemiphragma heterophylla Asia Plantago major 1/100 Plantago media Europe+Asia 1/95 Plantago stauntonii 8.34 Plantago debilis America 1/100 Plantago lanceolata 10.02 Plantago uniflora Plantago coronopus 1/100 15.46 Aragoa abietina Aragoa cundinamarcensis 0.83/90 20.41 1/100 Aragoa cupressina 26.37 1.05 Aragoa corrugatifolia Digitalis grandiflora Wulfenia glanduligera BG Wulfenia glanduligera E1 Wulfenia glanduligera DI4 Wulfenia glanduligera DI1 0.99/81 Wulfenia glanduligera DII2

0.61 Wulfenia glanduligera E2 clade eastern Wulfenia glanduligera E1 58 0.99/ Wulfenia orientalis S5 1/100 68 Wulfenia carinthiaca Krf1

22.08 1.24 Wulfenia carinthiaca Sjk5 DIN 0.99/99 Wulfenia carinthiaca BG

Wulfenia carinthiaca BG ALP 1/88 10.69 0.87/68 Wulfenia ×schwarzii BG

Wulfenia baldaccii M5 clade western 29.09 0.65 Wulfenia baldaccii QS2 1/100 Veronica montana 13.91 4.51 Paederota lutea Picrochiza kurrooa 1/99 7.58 Wulfeniopsis nepalensis 1/100 2.18 0.98/82 11.58 Wulfeniopsis amherstiana Veronicastrum virginicum 1/95 1/100 6.77 1/100 Veronicastrum liukiukense 15.62 Veronicastrum stenostachyum Lagotis glauca 1/99 Lagotis integrifolia Lagotis integra 0.91/76 Lagotis praecox 1/100 Lagotis brachystachya 4.17 Lagotis bracteata Globularia repens

35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0

Fig. 5. Dated phylogeny of the ITS dataset of Wulfenia and some of its related genera. Numbers above branches correspond to posterior prob- abilities/ML bootstrap support (> 0.9 for posterior probabilities, > 80 for ML bootstrap support, except for values related to Wulfenia). Grey bars denote the 95% higher posterior probability density intervals of node ages, which are given for selected nodes. Pie charts on nodes reflect results from biogeographic analysis using RASP, whereas pie charts at the tips reflect the initial area coding for the respective taxon. Results from the analysis restricting the maximum number of areas to two are depicted on the left, whereas those to the right are the results from the analysis restricting the maximum number of areas to one. Black indicates European-Mediterranean origin; white, Asian origin; dark grey, shared Eurasian origin; light grey, American origin. Only pies relevant for the discussion are included. ALP, Alpine samples; DIN, Dinaric samples.

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Veronica and Paederota are, similar to Wulfenia, of presum- self-compatibility in W. carinthiaca. These traits may confer a ably ancestrally European-Mediterranean origin since most high competitiveness that may have allowed for the colonization of the early-branching clades are from Europe and Turkey and persistence of the Alpine population after a dispersal event (Albach & Chase, 2004). Given the phylogenetic inference that but also leading-edge dispersal out of a once continuous distri- the Asian Veronicastrum and Picrorhiza are of hybrid origin bution area across the Dinaric Alps where nowadays no suitable from the ancestors of Wul­fenia and Wulfeniopsis and Veronica/ habitat is available anymore. With regard to this latter sce- Paederota and Wulfeniopsis, respectively (Albach & Chase nario, biogeographic analyses of Balkan plants indicate that the 2004), the most likely scenario based on our biogeographical Prokletije Mts., the highest mountain range in the Dinaric Alps, analysis indicates a Himalayan origin of these hybrid genera at constitute the most important refuge for alpine plants in the a time when the ancestors of Wulfenia, Paederota and Veronica Balkan Peninsula nowadays (Stevanović & al., 2009), whereas were still found in Asia. A minimum age for Veronicastrum is the southeastern Calcareous Alps have been identified as a not the stem group age, but, because of the hybrid origin, the glacial refuge for alpine plants (Tribsch & Schönswetter, 2003; age of its crown group (6.77 Ma; Fig. 5), whereas that for Picro- Schönswetter & al., 2005). A similar distribution gap between rhiza is the age of the last common ancestor with Wulfeniopsis the Prokletije Mts. and the southeastern Alps is, for example, (7.58 Ma; Fig. 5). Thus, at that time a member of the Wulfenia found in Hedysarum hedysaroides L. (Fabaceae), Gentiana or Paederota/Veronica stem group, respectively, must still have nivalis L. (Gentianaceae), Viola zoysii Wulf. (Violaceae), and existed in the Himalayan region. This is, however, independent partly in Silene acaulis L. (Caryophyllaceae), Alyssum ovirense of the split of the ancestor of extant Wulfenia and Veronica/ A.Kern. (Brassicaceae), Papaver alpinum L. (Papaveraceae) Paederota from the rest of Veroniceae, which is likely related and Centaurea nervosa Willd. (Asteraceae) as well as in some to recurrent Mediterranean/Paratheys–Indian Ocean transgres- phylogenetically (and ecologically) closely related but vicari- sions of the mid- and late-Miocene (Rögl, 1999). ant taxa such as Androsace komovensis Schönsw. & Schneew. The differentiation within Wulfenia is relatively recent (Primulaceae, vs. eastern Alpine A. hausmanni Leyb.), and (0.49–2.12 Ma). Mechanisms of intrageneric reproductive iso- Valeriana pancicii Halácsy & Baldacci (Valerianaceae, vs. lation have not yet evolved since all possible hybrids between eastern Alpine V. saxatilis L.) (Stevanović & al., 2009). Thus, the three extant Wulfenia species exist (Lepper, 1970c; Kosch, it is possible that all these species either had parallel dispersal 1992). Lepper (1970a) and Lakušić (1978) regarded W. carinthi- events between these two ranges or they once had a continuous aca and “W. blecicii ”, respectively, as evolutionary conservative distribution area across the Dinaric Alps to the southeastern with little morphological differentiation. However, due to the Alps and subsequently became extinct in the northwestern and young age of the species, as inferred by our dating analysis, central Dinaric Alps during the ice ages or with subsequent such a claim can only be made based on comparison with sister warming. genera (see above). Genetic differentiation between the taxa of Infrageneric taxonomy. — Our analyses provide evidence Wulfenia is likewise not very pronounced, as shown by AFLP for a new classification of the genus reflecting our increased variation (Fig. 3; Table 3) and the cpDNA haplotype network understanding of the evolution of the populations called “W. ble- (Fig. 4B). Hence, the European–eastern Mediterranean disjunc- cicii ” and those populations in southern Turkey, ascribed to tion cannot be explained by the range contraction of a formerly W. orientalis var. glanduligera. Neither our morphological nor widespread Wulfenia ancestor caused by climate deterioration our molecular data support the recognition of the Balkan popu- in the mid-Miocene. Rather, the disjunction must have been lations of W. carinthiaca at the species level as “W. blecicii ”, as caused by a more recent process such as climate cooling at the proposed by Lakušić (1971, 1978) who also failed to designate start of the Pleistocene or long-distance dispersal. Unfortu- the type specimen and thus invalidly published the name (see nately, the direction of such dispersal remains unknown given McNeill & al., 2012). The discriminant analysis of the morpho- the problems inferring the ancestral area of the genus in the logical data did not find any character to distinguish between absence of an appropriate outgroup and the lack of fossils. plants from both distribution areas. Furthermore, all Alpine With regards to the Alpine-Dinaric disjunction within (W. carinthiaca s.str.) and Dinaric (“W. blecicii ”) samples share W. carinthiaca, two alternative hypothetic scenarios can explain the same cpDNA haplotype. With regard to the ITS dataset, the pattern. Either the Alpine population originated from long- the relationships within the “western” clade remained unre- distance dispersal as postulated by Scharfetter (1908), Lepper solved in both the ML and the Bayesian approach. Nonethe- (1970a), and Fritz (1976), or it is a remnant northern population less, morphology, cpDNA and AFLP data show a clear genetic of a once more widespread species. The former scenario may structuring within Wulfenia, resulting in four distinguishable be more plausible based on the nested position of the Alpine taxa: W. carinthiaca (including “W. blecicii ”), W. baldaccii, population among the Dinaric ones in the AFLP network W. glandu­ligera comb. & stat. nov. and W. orientalis. Since the (Fig. 3) but the latter should not be totally discarded. Genetic population of Wulfenia orientalis s.str. from Mt. Samandağ diversity of the Alpine populations compared to diversity of (southern Amanos Mts.) was sampled only at the fruiting stage, populations from the Dinaric Alps argues decidedly against a a direct morphological comparison with plants from central recent dispersal event, while after an older event, one would and northern Amanos Mts. (W. glanduligera) was not possible. expect a sister-group relationship between populations of the However, as already observed by Huber-Morath (1971) and con- two ranges. Furthermore, Scharfetter (1929) documented high firmed by our own observation of herbarium material, speci- seed germinability, extensive vegetative reproduction, and mens of W. glanduligera are completely covered with glandular

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or eglandular hairs (in W. orientalis s.str. glandular or eglan- in Prokletije Mts. Fundings by the European Commission (FP7 Marie dular hairs are present only in the inflorescence), have linear Curie grant—MERG-CT-2007-201204), International Association for calyx lobes which are frequently emarginated (in W. orientalis Plant Taxonomy (IAPT grant in 2008) and Ministry of Culture of the s.str. they are linear-lanceolate and entire), significantly lon- Republic of Croatia (all to BS) are gratefully acknowledged. Finally, ger corolla and anthers positioned in the corolla tube 3–4 mm we are grateful to the comments of the anonymous reviewers that below the throat (in W. orientalis s.str. the anthers are posi- helped to improve the manuscript considerably. tioned at the base of the corolla tube). We observed practically no phenological overlap in flowering between populations of the two taxa, since populations of var. glanduligera from cen- LITERATURE CITED tral and northern Amanos Mts. were at their flowering peak while all observed specimens of W. orientalis s.str. were in an Akaike, H. 1973. Information theory and an extension of the maximum advanced fruiting phase. To that end, the last three attributes likelihood principle. Pp. 267–281 in: Petrov, B.N. & Csaki, F. (eds.), may significantly affect the pollination ecology of the two taxa Second international symposium on information theory. Budapest: and hence significantly limit gene flow between the popula- Akademiai Kiado. Akman, Y. 1973. Contribution a l’etude de la flore des montagnes de tions. According to our field observations and examination of l’Amanus (III). Commun. Fac. Sci. Univ. Ankara, Sér. C, Sci. Nat. herbarium material, specimens of W. orientalis s.str. are much 17: 43–70. more robust with the longest basal leaves (up to 40 cm) of all Albach, D.C. & Chase, M.W. 2001. Paraphyly of Veronica (Veroniceae; taxa of the genus. There are significant differences in habitat Scrophulariaceae): Evidence from the internal transcribed spacer preference between the two species, as well. Wulfenia orientalis (ITS) sequences of nuclear ribosomal DNA. J. Pl. Res. 114: 9–18. s.str., which is restricted to the southernmost Amanos Mts., http://dx.doi.org/10.1007/PL00013971 Albach, D.C. & Chase, M.W. 2004. Incongruence in Veroniceae prefers ophiolitic rocks within east Mediterranean macchie, (Plantaginaceae): Evidence from two plastid and a nuclear region. where Quercus coccifera L., Laurus nobilis L., Arbutus and- Molec. Phylogen. Evol. 32: 183–197. rachne L. and Myrtus communis L., among other woody taxa, http://dx.doi.org/10.1016/j.ympev.2003.12.001 prevail (Düzenli & Çakan, 2001). 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Wulfenia glanduligera (Hub.-Mor.) Surina, comb. & stat. Albach, D.C., Meudt, H.M. & Oxelman, B. 2005. Piecing together the nov. ≡ Wulfenia orientalis var. glanduligera Hub.-Mor. in “new” Plantaginaceae. Amer. J. Bot. 92: 297–315. Bauhinia 4(2): 208. 1971 – Holotype: TURKEY. Hatay, http://dx.doi.org/10.3732/ajb.92.2.297 Middle Amanos Mts. near Erzin, in Carpinus forest by Albach, D.C., Martínez-Ortega, M.M., Delgado, L., Weiss-Schnee- the path towards Üçkaz Yaylâ, 15 Apr 1968, Y. Akman 226 weiss, H., Özgökce, F. & Fischer, M.A. 2008. Chromosome num- bers in Veroniceae: Review and several new counts. Ann. Missouri (G barcode G00343603!; isotype: ANK n.v.). Bot. Gard. 95: 543–566. http://dx.doi.org/10.3417/2006094 Additional specimens examined: W. glanduligera: NHMR Aleksić, J. & Geburek, T. 2010. Mitochondrial DNA reveals com- 1180!; NHMR 1181!; Türkmen s.n., 4.6.1992, Herbarium Adana plex genetic structuring in a stenoendemic conifer Picea omorika No. 3380!; Türkmen s.n., 4.6.1992, Herbarium Adana No. 3381!; [(Panč.) Purk.] caused by its long persistence within the refugial W. orientalis s.str.: NHMR 1179! Balkan region. Pl. Syst. Evol. 285: 1–11. http://dx.doi.org/10.1007/s00606-009-0250-0 Baldacci, A. 1934. Osservazioni sulla Wulfenia baldaccii in relazione al genere Wulfenia. Mem. Reale Accad. Sci. Ist. Bologna, Cl. Sci. ACKNOWLEDGEMENTS Fis., ser. 9, 2: 99–112. Baldacci, A. 1935. Note complementari sull’ecologia e biologia della The paper is dedicated to the late Tone Wraber (University Wulfenia baldaccii. Mem. Reale Accad. Sci. Ist. Bologna, Cl. Sci. Ljubljana, Slovenia), botanical mentor of BS and respected colleague, Fis., ser. 9, 3: 3–10. Bello, M.A., Chase, M.W., Olmstead, R.G., Rønsted, N. & Albach, who inspired the start of this project. We thank Halil Çakan (Univer- D. 2002. The páramo endemic Aragoa is the sister genus of Plan- sity of Adana, Turkey), Marash Rakaj (University of Shkodra, Alba- tago (Plantaginaceae, Lamiales): Evidence from plastid rbcL and nia), Fadil Millaku, Elez Kraniqi (University of Prishtina, Kosovo), nuclear ribosomal ITS sequence data. Kew Bull. 57: 585–597. Halil Markašić and Milutin Praščević (Montenegro) for assistance http://dx.doi.org/10.2307/4110987 in the field, Marion Kever (University of Mainz, Germany) for tech- Blattner, F.R. 1999. Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR. BioTech- nical support with the AFLP and ndhF-rpl32 sequencing and Eike niques 27: 1180–1186. Mayland-Quellhorst (University of Oldenburg, Germany) with ITS Boissier, P.E. 1844. Diagnoses plantarum orientalium novarum, no. 4. sequencing, while Dmitar Lakušić and Gordana Tomović (University Lipsiae [Leipzig]: apud B. Herman. of Belgrade, Serbia) gave valuable information on plant distribution Bonin, A., Bellemain, E., Bronken Eidesen, P., Pompanon, F.,

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Appendix 1. Species, sampling localities, latitude and longitude, population acronyms (Acr), number of individuals used in morphometric (Nmorph), AFLP (NAFLP) and sequence analyses (Nseq), voucher numbers, collectors, collecting dates and GenBank accession numbers of all samples used in the analyses (the first number refers to the ITS nuclear ribosomal sequence and the second, separated by comma, to the ndhF-rpl32 spacer). Abbreviations for countries are as follows: ALB, Albania; AUT, Austria; KSV, Kosovo; MNE, Montenegro; TRK, Turkey.

Wulfenia baldaccii Degen, Nmorph 30, NAFLP 7, Nseq 15 ALB: Qafa Shtogut, 1550 m, 42°18′/19°39′, QS, 30, 2, 5, NHMR 1097, Rakaj & Surina s.n. 17.7.2008, KC237774, KC237859–63 ALB: pass Mshitel, 1350 m, 42°18′/19°37′, M, –, 4, 5, NHMR 863, Rakaj & Surina s.n. 18.8.2007, KC237776, KC237869–73 ALB: Shtegu e Dhenvet, 1750 m, 42°23′/19°44′, SD, –, 1, 5, NHMR 844, Rakaj & Surina s.n. 15.08.2007, –, KC237874–9 Wulfenia carinthiaca Jacq.—Alpine population, Nmorph 30, NAFLP 5, Nseq 5 AUT: Mokrine, 1660 m, 46°33′/13°17′, Mkr, 30, 5, 5, NHMR 1191, Wraber & Surina s.n. 27.6.2009, –, KC237835–9 Wulfenia carinthiaca Jacq.—Dinaric populations, Nmorph 150, NAFLP 49, Nseq 48 KSV: Mt. Nedžinat, 1900 m, 42°39′/20°05′, Ndz, 15, 7, 5, NHMR 1115, Millaku, Krasniqi & Surina s.n. 15.7.2008, –, KC237792–6 MNE: Mt. Sjekirica, 1875 m, 42°41′/19°55′, Sjk, 15, 5, 5, NHMR 1051, Praščević & Surina 7.7.2008, KC237777, KC237797–801 MNE: Mt. Hajla, 2130 m, 42°45′/20°08′, Hjl, 15, 5, 4, NHMR 1105, Praščević, Markašić & Surina s.n. 12.7.2008, –, KC237806–9 MNE: Mt. Maja e Kolata, 1860 m, 42°30′/19°53′, MK, 15, 5, 5, NHMR 1069, Surina s.n. 5.7.2008, –, KC237815–9 MNE: Mt. Bogičevica, 2150 m, 42°34′/20°03′, B, 15, 5, 5, NHMR 1168, Praščević s.n. 7.9.2008, –, KC237825–9 ALB: Mt. Maja e Jezerce, 2200 m, 42°27′/19°47′, MJ, 15, 4, 5, NHMR 859, Rakaj & Surina s.n. 16.8.2007, –, KC237830–4 MNE: Mt. Karanfili, 1870 m, 42°30′/19°47′, Krf, 15, 3, 5, NHMR 1054, Praščević & Surina s.n. 6.7.2008, KC237775, KC237864–8 MNE: Mt. Starac, 2115 m, 42°38′/20°02′, Str, 15, 5, 4, NHMR 1049, Praščević & Surina s.n. 7.7.2008, –, KC237802–5 MNE: Mt. Zeletin, 1950 m, 42°38′/19°50′, Zlt, 15, 5, 5, NHMR 1216, Praščević s.n. 20.6.2009, –, KC237820–4 MNE: Mt. Visitor, 1835 m, 42°36/19°53′, Vst, 15, 5, 5, NHMR 1082, Praščević & Surina s.n. 8.7.2008, –, KC237810–4 Wulfenia orientalis var. glanduligera Huber–Morath, Nmorph –, NAFLP 12, Nseq 14 TRK: Dörtyol, 980 m, 36°49′/36°18′, DI, –, 3, 5, NHMR 1180, Çakan & Surina s.n. 6.5.2009, KC237780/ KC237781, KC237849–53 TRK: Dörtyol, 1140 m, 36°49′/36°19′, DII, –, 5, 4, NHMRs 183, Çakan & Surina s.n. 6.5.2009, KC237779, KC237845–8 TRK: Erzin, 900 m, 36°54′/36°18′, E, –, 4, 5, NHMR 1181, Çakan & Surina s.n. 7.5.2009, KC237778/ KC237782/ KC237783, KC237840–4 Wulfenia orientalis var. orientalis Boiss., Nmorph –, NAFLP 5, Nseq 5 TRK: Samandağ, 600 m, 36°09′/35°58′, S, –, 5, 5, NHMR 1179, Çakan & Surina s.n. 5.5.2009, KC237784, KC237854–8

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Appendix 2. Species collecting locality, voucher information (for newly generated sequences only) and GenBank accession numbers of samples not used in the population-level analyses. GenBank accession numbers refer to the ITS nuclear ribosomal sequence unless otherwise stated, individuals are separated by a semicolon. Aragoa abietina Kunth, see Bello & al. (2002) for details, AJ459404; Aragoa cupressina Kunth, see Bello & al. (2002) for details, AJ459402; Lagotis angusti- bracteata P.C.Tsoong & H.P.Yang, see Albach & al. (2001) for details, AF313028, ndhF-rpl32: KC237791; Lagotis brachystachya Maxim., see Albach & al. (2001) for details, AF313027; Lagotis glauca Gaertn., U.S.A., Alaska, Macoun 1897 (WU), KC237785; Lagotis integra W.W.Sm., China, Sichuan, Dickoree 8540 (GOET) KC237786; Lagotis integrifolia (Willd.) Schischk., Russia, upper Zhumaly valley, Tribsch & Essl 10086 1.8.2003 (WU), KC237787; Lagotis praecox W.W.Sm., see Albach & al. (2001) for details, AF313029; Lagotis stolonifera (C.Koch) Maxim., see Albach & al. (2004c) for details, ndhF-rpl32: KC237790; Paederota lutea L.f., see Albach & al. (2001) for details, AF313024; Picrorhiza kurrooa Royle ex Benth., see Albach & al. (2004c) for details, AF509813; Plantago coronopus L., see Rønsted & al. (2002) for details, AY101882; Plantago debilis R.Br., see Rønsted & al. (2002) for details, AY101868; Plantago lanceolata L., see Rønsted & al. (2002) for details, AY101898; Plantago major L., see Rønsted & al. (2002) for details, AY101861; Plantago media L., see Rønsted & al. (2002) for details, AY101865; Plantago stauntonii Reichardt, see Rønsted & al. (2002) for details, AY101870; Plantago uniflora L., see Rønsted & al. (2002) for details, AY101885; Veronica montana L., see Albach & al. (2001) for details, AF313014; Veronicastrum liukiuense (Ohwi) T.Yamaz., see Albach & al. (2004c) for details, AF509815; Veronicastrum stenostachyum (Hemsl.) T.Yamaz., see Albach & al. (2001) for details, AF313031, ndhF-rpl32: KC237788; Veronicastrum virginicum (L.) Farw., see Albach & al. (2001) for details, AF313030, ndhF-rpl32: KC237789; Wulfenia ×schwartzii Lepper, Cult. RBG Kew, donated by R. Jellico (K), KC237773; Wulfeniopsis amherstiana (Benth.) D.Y.Hong, see Albach & al. (2004c) for details, AF515216; Wulfeniopsis nepalensis (T.Yamaz.) D.Y.Hong, Nepal, Arun valley, Stainton, Sykes, & Williamson 530 (BM), GQ150558/9.

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