Muscari armeniacum and M. polyanthum – one or two ?

Ylva Heed

Thesis for the degree of Master of Science in Biology Degree course in Systematics and Biodiversity 30 hp

Department of and Environmental Sciences University of Gothenburg

Muscari armeniacum and M. polyanthum - one or two species?

Ylva Heed

Degree course in Botanical Systematics and Biodiversity (BIO 700) 30 hp, which will when completed, fulfil the requirements towards a Master of Science in Biology (Filosofie magisterexamen i biologi). Department of Plant and Environmental Sciences, University of Gothenburg, april 2010. Supervisor: Prof. Bengt Oxelman. Examinator: Director of the Herbarium, Ph.D. Claes Persson.

Abstract Molecular and morphological studies were combined in order to explore the relationships between Muscari armeniacum and M. polyanthum in the monocotyledonous plant Muscari. Muscari polyanthum is in most Floras considered as a synonym to M. armeniacum but differences in ecology, phenology and morphology have raised questions about their relationships. The study revealed correlations between morphological and sequence data. The results does not, however, give sufficiently support for Muscari armeniacum and M. polyanthum being different species, but indicates a need for further investigation and M. armeniacum partitioning.

Background and aim of the study

Muscari Mill., Grape-, is a monocotyledonous plant genus that received its name from the greek word „moschos‟ which means musk and it alludes to the scent of the . Muscari belongs to the family (APG, 1998; APG II, 2003; APG III, 2009) and consists of about 50 species (Kubitzki, 1998; Stevens, 2001). The genus is traditionally divided into four subgenera; Muscarimia Kostel. ex Los.-Los., (Parl.) Zahar., Stuart and Muscari Stuart (syn. Botryanthus Kunth).

The geographical distribution spans from temperate SE Europe to Caucasus, N Africa and SW Asia. Several species have been introduced and naturalized in other areas. Various species and cultivated forms of Muscari are commonly grown for their early spring flowers.

Presently there is a somewhat confusing classification in the genus; morphological characters – from bulb morphology to pistils or – and karyological data are not unambiguous enough to make taxonomic judgements uncontroversial. There are no global taxonomic revisions of the genus, only regional ones (Czerepanov, 1995; Davis & Stuart, 1984; Garbari & Greuter, 1970; Johnson et al., 1996). It is considered as a difficult genus with “a formidable burden of synonymy, much of it based on cultivated material of unknown origin. Most of the widespread species are extremely variable” (Davis & Stuart, 1984). In addition, petal colour changes when specimens are dried, thereby making it difficult to determine herbarium specimens to species.

Suárez-Santiago & al. (2007) sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA of the Muscari subgenus Muscari species in Spain, and concluded that polyploidy is a frequent phenomenon in the subgenus Botryanthus, that M. neglectum has several ploidal levels and that many of these have been recognized as separate species. They describe the pentaploid and hexaploid levels as two new species, and conclude that these are not autopolyploids from M. neglectum but allopolyploids.

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Muscari armeniacum Leichtlin ex Baker belongs to Muscari subgenus Muscari and is very variable in its morphology. If it is dried, it cannot confidently be separated from M. neglectum Guss. ex Ten. without information of the colour of the flowers as this is, according to the litera- ture, the only constant difference between them; M. neglectum being very dark to blackish blue and M. armeniacum sky blue or violet-tinged. Some authors though, as Yıldırımlı & Selvi (2002), describes differences as for instance in the perianth lobes; being erect in M. armeniacum and patent to recurved in M. neglectum. is widely distributed and can be found in the Caucasus, Central Asia, Central and Southern Europe, Northern Africa and the Middle East. Muscari armeniacum is distributed in the Caucasus, on the Balkan Peninsula and in Asia Minor.

Muscari are usually diploid (x = 9) in Anatolian , but elsewhere often polyploid (Johnson & Brandham, 1997). The chromosome number of M. armeniacum was recorded to be 2n = 18, 18+2B and 36 and the variations in M. neglectum from 2n = 18 to 27, 28, 36, 36+1B, 45, 54 and 63 (Davis et al., 1988).

Muscari polyanthum Boiss. is in most Floras considered as being a synonym to M. armeniacum and is not accepted in The World Checklist of (Govaerts, 2004) as a species of its own. The ecology, phenology and bulb morphology of M. polyanthum and M. armeniacum differs (Henrik Zetterlund pers. comm.). The „polyanthum‟-type grows in wet habitats (damp meadows) at the base of valleys and their never have offsets. In contrast the „armeniacum‟- type grows in tufts higher and dryer up on the slopes, often on disturbed ground and their bulbs do have offsets. In addition, Muscari armeniacum flowers earlier.

Defining what is a species and species delimitation is fundamental in biology but this is complex and a difficult task and scientists have failed to agree on a universal species concept. What is considered as species boundaries, depends on the species concept. Some concepts focus on morphological differences, others on ecological differences or genetic diversity and still others focus mainly on reproductive barriers. A combination of molecular, morphological, ecological and phenological data compose species criteria in this study. As molecular data, I use sequences from the ITS (internal transcribed spacer) region of the ribosomal DNA in the nuclear genome and the trnL-F region of the maternally inherited (Birky, 2001) chloroplast DNA (cpDNA), both regions frequently used in phylogenetic studies of plant groups. As a complement to them sequences from a nuclear gene, cythochrome P450, are used.

The main aim of this study is to investigate whether M. polyanthum should be recognized as a species separate from M. armeniacum or whether it should remain a synonym of the latter. The null hypothesis is that M. armeniacum and M. polyanthum are conspecific. Correlation of the proposed diagnostic characters with DNA sequence data and additional morphological characters could constitute a rejection of the null.

Materials and methods

Both cultivated plant material, coming mainly from Turkey (in the Alpine Department of the Botanical Garden of Gothenburg), and herbarium specimens were used for morphological comparision. A priori names are based on the names mainly given by Henrik Zetterlund, horticultural curator in The Botanical Garden of Gothenburg, on the basis of „polyanthums‟ having no vegetative propagation (bulbils), no in autumn, later flowering onset, stronger growing than „armeniacums‟, growing in flat (low) places overwhelmed with water in springtime

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and, as a contrast, „armeniacums‟ having wreaths of loose sitting daughter bulbs in autumn, supplementary breeding by seeds, leaves coming regularily in autumn, growing on more well- drained places (often in slopes close to flat wet areas), having earlier flowering onset. Of the unspecified species („sp.‟) Henrik Zetterlund consider 2004-2466 as being a possible hybrid between „armeniacum‟ and „polyanthum‟; accessions 2005-2246--2248 appears to be related to and eventually are 'species nova' according to Arnis Seisums. 2005-2307 and 2007-2181 were name set by Arnis Seisums and Janis Ruksans and “can be armeniacums” (Henrik Zetterlund pers.comm.).

Photographs of the living were taken at the onset, both of whole plants and of details. length and leaf width (at the widest point) as well as the width and length of the were measured with a ruler. In order to determine location of the anthers in relation to the apex of the perigone, sections through flowers were made.

The plants used for the molecular analyses were 18 accessions of „armeniacum‟, 6 of „polyanthum‟ and 6 of „sp.‟, the latter were included as their appearance resembled either „armeniacums‟ or „polyanthums‟ (Table 1). DNA was extracted from dried tissue (buds) using E.Z.N.A. SP Plant DNA Mini Kit (Omega Bio-tek, Norcross, USA) extraction protocol.

Three potentially unlinked sequence regions were chosen, the trnL-F region (spanning trnL intron, the 3‟ trnL exon, and trnL-trnF intergenic spacer) from the chloroplast genome, the ITS (ITS1 + 5.8S + ITS2) and a cytochrome gene, CytP450 (Mori et al., 2005), both from the nuclear genome. Primer sequences are listed in Table 2. The primers used for CytP450 were designed using Primer3 in Geneious Pro 4.8.4 (Biomatters Ltd., 2005-2009).

PCR reactions were performed under the following conditions; The PCR reactions were conducted in 25 l G-mix (buffer + dNTPs) (for CytP450 the buffer provided by the manufacturer was used) and Taq polymerase (Thermoprime Plus DNA Po 1, conc 5U/l), 2 l unquantified DNA templat and 20 (M) of both forward and reverse primers. All reactions were run on a GeneAmp PCR System 9700 apparatus (PE Applied Biosystems) with an initial denaturation at 95C for 5 min followed by 33 cycles of 95C for 30 s, 57C for 30 s and 72C for 45 s. The cycle ended with an extension of 72C for 7 min. Amplified products were purified using Multiscreen HTS Vacuum Manifold (Millipore Corp., Billerica, USA) and quantified. Sequencing was performed by Macrogen Inc. (Seoul, Korea).

The sequences were edited and assembled into contigs using Geneious Pro 4.8.4 (Biomatters Ltd., 2005-2009). Some sequences could not be aligned due to double peaks or bad quality, and were therefore either excluded from further phylogenetic analyses or used only as single reads. Polymorphic sites in ITS and CytP450 were coded using the IUB codes (Cornish-Bowden, 1985). Alignments were performed manually by using Se-Al v2.0a11 (Rambaut, 1996-2002). As CytP450 is a coding gene the reading frame of the corresponding amino acids were regarded in that alignment process.

The three gene regions were analysed separately. Maximum parsimony (MP) were conducted in PAUP* v.4.0 b10 (Swofford, 2002); with unweighted characters and 1000 random sequence addition replicates, trees bisection-reconnection (TBR) branch swapping, MULTREES off. All shortest trees were saved and strict consensus trees were computed. Support for the nodes resolved in the strict consensus of the most parsimonous tree was achieved with (non-parametric) bootstrap analysis (Felsenstein, 1985; Mort & al., 2000) using PAUP* with TBR branch swapping on 500 bootstrap replicates with 5 random additions per replicate. Bootstrap support (BS) was

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categorized as strong when over 85%, moderate when 70%--85% and weak when 50%--69% (Kress & al., 2002).

Several polymorphic sites were found in the ITS sequences and because of this, the ITS sequence data was represented not only in a tree, but also as a network constructed from uncorrected pairwise distances (NeighborNet Network) using SplitsTree4 (Huson & Bryant, 2006).

Results

The measured morphological sample values for leaf length and length/width for the inflorescenses were overlapping and slightly differing. Very little overlap between the „polyanthum‟ and „armeniacum‟ types was found in leaf width, where the median for „armeniacum‟ was 6 mm and the lower and upper quartile 4 and 7 mm respectively, whereas „polyanthum‟ median was 11 mm and the lower and upper quartile 8.5 and 13 mm respectively (Fig. 1). Cross-sections of flowers showed that the anthers in most flowers were attached around the middle of the perigon though some of the 'armeniacums' had anthers attached extra high, and one of the 'polyanthum' especially low (2005-2137) (Fig. 2).

In the molecular analyses, 26 taxa were sequenced for the ITS region. The total aligned length of the ITS sequence, was 827 base pairs (bp). Of these, 62 were variable (7.5%) and 27 (43.5% of the variable positions) were potentially phylogenetically informative. In the genetic pairwise distances matrix great variation were shown; from none to 0.04840 between 2005-2148 („armeniacum‟) and 2005-2307 („sp.‟) (Table 3). The parsimony analysis of ITS resulted in a tree (Fig. 3) with a length of 69 steps and low homoplasy (consistency index, CI = 0.9420; retention index, RI = 0.9494). Two taxa were placed on a very long branch (2004-2552 „polyanthum‟ and 2005-2307 „sp.‟). Strong support for this branch was found in the bootstrap 50% majority-rule consensus tree and also for the clade consisting of 2004-2466 („sp.‟) and 2005-2231 („armeniacum‟). In the NeighborNet Network conflicting signals are depicted as a tree, thus indicating little conflict in the data (Fig. 3).

Polymorphisms were found in ten sequences, seven had multiple polymorphisms. Additative polymorphisms were found in five gene positions (329, 571, 608, 633 and 719) (Table 4). In phylograms resulting from MP analyses in which sequences with polymorphisms were excluded 2004-2552 („polyanthum‟) was on a very long branch and the topology was congruent but some branches received slightly higher bootstrap support. CI and RI indexes were slightly changed towards less homoplasy (CI = 0.9512, RI = 0.9608).

The length of the total aligned trnL-F data set was 859 bp including 15 variable (1.7%) and 4 (26.7% of the variable positions) potentially phylogenetically informative sites. The nucleotide differences per site differed from 0.000 to 0.00841 [between 2005-2307 („sp.‟) and 2005-2129 („armeniacum‟)]. A parsimony tree was computed but showed only weak support for a few clades. The branch with 2005-2307 („sp.‟) was very long (Fig. 5). Two of the four splits were wholly congruent with the ITS data, the split separating seven „armeniacums‟; 1987-0715, 2004-2553, 2004-2558, 2005-2065, 2005-2076, 2006-2140 and 2006-2141, and the rest, as well as the 2004- 2552 and 2005-2307 accessions, which in the trnL data also was represented by an extra accession.

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Only 5 accessions of the CytP450 were readable and used as the other displayed double signals throughout. The length of the total aligned CytP450 data set was 768 bp including 163 variable (21.2%) and 77 (47.2 % of the variable positions) were potentially phylogenetically informative sites. In a histogram 15 trees were evaluated and the best tree found (only one) had score 172 and the worst tree (ten) had score 246. In the uncorrected pairwise distance matrix great variation was shown; between 2004-2350 („polyanthum‟) and 2004-2422 („polyanthum‟) there was no difference at all, i.e. they were identical, between 2006-2141 („armeniacum‟) and 2004-2552 („polyanthum‟) the distance was 0.17963 (Table 6). The parsimony tree showed strong support for a very long branch with a clade consisting of a 'polyanthum' (2004-2552) and a 'sp.' (2005-2307), and also strong support for an 'armeniacum' (2007-2141) as sister to 2004-2552 and 2005-2307 (Fig. 6).

Discussion

There are several morphological characters that show consistent and correlated differences. The „polyanthum‟-type differs in having broader leaves, being larger and the leaves being more “upright” and thicker. According to Karlén (1984) and Levin (2002) can depend on circumstances and be variable, but the difference in formation of bulbils between „armeniacum‟ and „polyanthum‟ persist in cultivation (Henrik Zetterlund pers. comm.). There is a phenological difference between „armeniacum‟ and „polyanthum‟ in development of the overground parts and flowers. This difference is preserved in cultivation. „Armeniacum‟ and „polyanthum‟ also inhabit different ecological niches, dryer and more wet respectively (Henrik Zetterlund pers. comm.).

Plenty of genetic polymorphisms were found in the ITS sequences. Double signals in chromato- grams are the visible result from different processes. The same alleles at a given locus on a pair of chromosomes in a homozygous gene will not result in double peaks, but for the heterozygous loci (due to for instance point mutations), this will result. Also, if different loci (one or many) are amplified in the PCR they will show up as double signals in the chromatograms. Different loci can be amplified due to the molecular structure of a gene; e.g. the ITS sequences has a very high tandem repeat structure of highly similar loci and polymorphic sites in the sequences are then to be expected. Through recombinations concerted evolution can homogenize genes within (and between) loci, but this process is more or less efficient so some substitutions may remain and give rise to polymorphic sites (Wendel & Doyle, 1998).

When sequences contains additative polymorphisms, as many of the the ITS sequences did, it can be a result from hybridization between homozygous parents. Polymorphisms does not necessarily have to be present in all sites after hybridization if followed by homogenization, but if there are additative polymorphisms in the same site for many accessions it is more likely that hybridization have occured. The multiple additative polymorphisms found in Muscari indicates hybridization events (allopolyploidy), as part of their history.

Double peaks were seen in the CytP450 chromatograms and made the reading impossible in many of the sequences. The extensive occurrence of double peaks in this gene can be an effect of CytP450 belonging to a multigene family. As a consequence of being a member of a gene family, the gene will exist in many copies (clustered or dispersed) throughout the genome, and CytP450 have at least 5 to 6 copies (Mori et al. 2005).

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Another possible explanation for the doubled sequences and the difficulties in reading and assessing homology in some of the ITS and cytochrome sequences may be duplication of chromosomes or genes, e.g. polyploidy. Polyploidy is common in plants and polyploidization is an important factor in the speciation of plants (Otto & Whitton, 2000). Polyploidy involves gene duplication and multiple copies of a gene at different loci may lead to heterogenous PCR products. If the sequences that were duplicated are being separated in re-sequencing with copy- specific primers inferences about autopolyploidy (genome duplication within a species) or allopolyploidy (hybridization and genome duplication) can be made.

The pairwise genetic distances values differed very much between the studied genes; in the chloroplast data set the differences in distance were very small (less than 1%), in the ITS data set the variation were much higher (5%), and in the cytochrome gene a very high difference (about 18%) was recorded.

The molecular clock hypothesis could explain the differences between the distance values if the mutation rate is not constant across genes. If so, and the mutation rate in chloroplast genes is significantly slower than in the nuclear genes (Palmer & Herbon, 1988), then the distance variation in the cytochrome gene can be due to a very high mutation rate in this gene.

The long branch found in the CytP450 sequences can be an effect of this gene belonging to a gene family. The large distance differences in the cytochrome gene may be due to the cytochrome sequences being paralogous; the Arabidopsis P450 genes are known to be members of a gene family with numerous paralogous (Paquette, Bak & Feyereisen, 2000; Werck-Reichhart, Bak & Paquette, 2002.). If Muscari have duplicated cytochrome genes and the primers anneal to more than one paralogue, it leads to problems in sequence interpretation.

It is not appropiate to draw any taxonomical conclusions from the results in this study, but notable is that phylograms showed a structure with a long branch, partly separated distally, with a 'polyanthum' (2004-2552) and a 'sp.' (2005-2307). As the length of a branch represents genetic distance this indicate that they are a group clearly separated from the other Muscari specimens in this study. Furthermore, long branches in parsimony trees tend to underestimate the number of changes that have occured, so the substitution changes, e.g. differences between the taxa in study, potentially are even greater. In the ITS sequences there was strong support for a split between about 3/4 of the sequences and a group (1/4) of 'armeniacums'. A congruent split was also found in the chloroplast data which suggest this split could be of phylogenetic significance at the „organismal‟ level.

In future studies, samples should include more Muscari specimens, especially Muscari neglectum, and more information could be achieved by analyse of more genes or part of genes. The PCR amplification parameters (e.g. annealing temperature, thermal cycle length and/or template concentration) can be adjusted to enhance the PCR output. The utilized DNA Taq polymerase unfortunately has problems amplifying poly-A repeats which leads to dislocations and problems in making the contigs; using a proofreading enzyme could reduce these difficulties. For greater variation in the data a more sensitive fingerprinting technique, the Amplified Fragment Length Polymorphism PCR method (AFLP), might be more suitable.

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Conclusion

Molecular data in this study shows interesting genetic differences among the Muscaris but, as a challenge for the future, it still remains to find out whether there is polyploidy and hybridization involved in their relationships and if „armeniacum‟ and „polyanthum‟ are to be considered as separate species or not.

Acknowledgements

I would like to thank Vivian Aldén, Zeynep Aydin, Bente Eriksen, Roger Eriksson, Elisabeth Gondel, Nils Hallenberg, Ellen Larsson, Stephan Nylinder, Bengt Oxelman, Claes Persson, Karin Persson, Anna Petri, Bernhard Pfeil, Anja Rautenberg, Anne-Cathrine Scheen, Elisabet Sjökvist, Sven Toresson, Mats Töpel, Henrik Zetterlund and my friends and family who have encouraged and supported me in various ways in making this thesis.

The study is a part of a cooperation agreement between the University of Gothenburg and the Botanical Garden of Gothenburg taking advantage of, and utilizing, the skills represented in the staff of the garden and at the university as well as of the plant material in the garden.

Financial support was given from Filosofiska fakulteternas gemensamma donationsnämnd.

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Appendix

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Table 1. Plants used for molecular analyses and selected characters for comparision. The accession number refers to Plant Database number in the Botanical Garden of Gothenburg.

Accession Plant name Collector and place of collection Measuring Scape length/width (cm) Leaf number/ Leaf base width Leaf length number date numbers/ inflorescences (mm) (cm) length (cm)

1987-0715 Muscari Persson, J. & K. 26/04/2009 14--19/6.5 /2 - - - 6--7 41--43 A armeniacum JP 87-76. Turkey: 8 km from Akseki --7.5--11-- U-shaped. towards Beysehir, 1500 m. 15 1987-0715 Muscari Persson, J. & K. 11/04/2009 /12--14.5 5--7.5/2.5 6/3, 7/4 4--6 24--28--30.5 armeniacum JP 87-76. Turkey: 8 km from Akseki U-shaped. towards Beysehir, 1500 m. 2004-2350 Muscari Zetterlund, H. 26/04/2009 /9--15 3.5--4.5/1.5--2 4/3, 12/6 7--11 16.5--19.5 polyanthum Batman 089. Turkey. Perianths with 'shoulders'. 2004-2381 Muscari Zetterlund, H. 13/04/2009 /10.5 3--3.5 (one)/1.5 5/2, 4/1 so far bud 10--11--14 13--13.5 polyanthum Batman 140. Turkey: Van, Muradiye-- Dogubeyazit, 2 km NE Muradiye water- fall. 2004-2421 Muscari Zetterlund, H. 26/04/2009 - - - 3--4/1.5--2 4/2, 4/2, 5/2 12--18 16--18 polyanthum Batman 220. Turkey: Erzurum, Cat-- f. album Bingöl, 7 km before Cirisli Geçidi, 1900 m. 2004-2422 Muscari Zetterlund, H. 26/04/2009 /15--17 - - - 4/1, clustered: 3/1, 3/1, 4/1, 5--10 16.5--19 polyanthum Batman 220A. Turkey: Erzurum, Cat-- Many sterile flowers. 3/1, 3/1 Bingöl, 7 km before Cirisli Geçidi, Perianths furrowed, slightly 1900 m. 'shouldered'. 2004-2466 Muscari sp. Zetterlund, H. 26/04/2009 /6-10-13-- 3--3.5--4.5--5/1.5--2--2.5 - - - 25--6--7--8-- 31--33--36-- Batman 303. Turkey: Ercincan, Sakaltutan 14--15 Perianths furrowed. 10--11 40 Geçidi. 2004-2552 Muscari Zetterlund, H. 26/04/2009 /9.5--12.5 2.5--5/1.5--2.5 6/3, 3/1, 4/1, 3/1, 13/7, 5/3 8--9--12 14--15--16 polyanthum Batman 465. Turkey: Antalya, Beysehir-- Perianths furrowed, lobes recurved. Colour dark. Antalya, 25 km N Teke Geçidi, 1400 m. 2004-2553 Muscari Zetterlund, H. 11/04/2009 /12--16 4/2.5, 8/1.5 6/4, (1)/0, 6/2 + 1 bud, 6/2 + 1 4--6 23--27.5 armeniacum Batman 466. Turkey: Antalya, Beysehir-- bud, (2) narrow/0, 7/2, 6/2, 4/1 Antalya, 25 km N Teke Geçidi, 1400 m. 2004-2558 Muscari Zetterlund, H. 11/04/2009 /12.5--16 4--5.5/1.75--2 5/2, 8/3, 5/2, 3/1, 6 (one very 4--6 25.5--26.5 armeniacum Batman 478. Turkey: Antalya, Beysehir-- Perianths furrowed, lobes recurved. narrow)/2 Akseki, 5 km NW of Teke Geçidi. 2005-2065 Muscari LST 010 26/04/009 14/ 6.5--10.5--12/2.5--3 11/5, 6/2, (1) tiny/0, 7/1, 5/2, 3--4--5--6 32.5--40--44 armeniacum Turkey: Isparta, near lake Egirdir, Colour Colour royal blue. 5/2, 5/2 1000 m. reddish. 2005-2076 Muscari LST 030 12/04/2009 /12.5--13-- 4.5--6/2--2.5 6/3, 4/0, 7/3 2--6--7 23--25.5 armeniacum Turkey: Isparta, between Egirdir and 14.5--15 U-shaped. Aksehir, 1300 m. 2005-2129 Muscari LST 117 29/03/2009 ------armeniacum Turkey: Bolu, on the road from Beypazari to Bolu, 3 km after Bakyrly Yaylasi,

11

1500 m. 2005-2131 Muscari LST 120 11/04/2009 /14--16 4/2.5, 5.5/3 9/1, (2) narrow/0, 7/2, 2/0, 1/0, 7--11 12--24 armeniacum Turkey: Çankiri, on E80 between Gerede Colour royal blue. 7/2, 3/0, 7/2 and Çerkes, 25 km before Çerkes, 1500 m. 2005-2134 Muscari LST 125 11/04/2009 /4--10.5 4.2/2.5, 5/2.5 6/2 + 2 buds, (2), 7/1, 4/3 3--11 11.5--13.5-- armeniacum Turkey: Amasya, 15 km NW of Merzefon, 21.5 Tavsan Dag, 1600 m. 2005-2137 Muscari LST 128 26/04/2009 /11.5--13 4--7/2--2.5 8/5, 6/2, 6/2, 7/3, 11/6 11--15 21--22--23 polyanthum Turkey: Amasya, 17 km on the road from Some looks defect e.g. “bitten off” Merzifon to Veziköprü, Tavsan Dag, and flattened; fasciation. 1600 m. 2005-2148 Muscari LST 144 11/04/2009, /7.5--10-- 3--3.5--6.5/1.75--2 8/3, 5/2, 8/2, 5/2 4--6 19--27.5--33 armeniacum Turkey: Tokat, 61 km on the road from 12/04/2009 11.5--12 Lobes recurved. Pretty wide Amasya to Turhal, 800 m. opening. 2005-2158 Muscari LST 163 11/04/2009 /4-6.5 3--3.5/1.5--2 5/2, 6/1 + 1 bud, 3--8 16--25 armeniacum Turkey: Erzinkan, road between Refahiye Lobes recurved. 6/2 + 1 bud, 6/1 + 1 bud, and Iliç, 1800 m. 6 + (2) very narrow/2, 6/2 2005-2191 Muscari LST 220 12/04/2009 /10--10.5 3.5--4.5/2 14/2, 2/0, 8/3, 6--8 12.5--13.5-- armeniacum Turkey: Erzurum, road from Erzurum to Many and pretty small. (3) narrow/0, (6) narrow/1 19--19.5-- album Bingöl, Yaylasyu Geçidi, 2400 m. 24.5 2005-2211 Muscari LST 241 13/04/2009 - - - 4--6/2.5 6/3, 6/3, (1) narrow/0, 6--8 16.5--17--25 armeniacum Turkey: Erzurum, road from Erzurum to (1) narrow/0 Ispir, Agiacik Geçidi, 2400 m. 2005-2231 Muscari LST 274 12/04/2009 /11.5--14 3.5--4/2--2.5 6 + (2) very narrow/2, 5/2, 7 5.7 8.5--23--33 armeniacum Turkey: Erzurum, between Tortum and yellowish, tiny (not meas.)/0 Low turgor, Narman, Kireçli Geçidi, 1700 m. hanging. 2005-2246 Muscari sp. LST 295 16/04/2009 /5.5--7--8 3--3.5--4/2 3/2, 4/2, 4/1, 4/0, 5/0, 6--7--8--9 11--11.5--12 Turkey: Artvin, Kaçkar Mts., Altiparmak, --10 Colour light blue, lobes pure white. 4/1 --16--17-- vicinity of Niznara village, scree, 2600-- 18.5 3000 m. 2005-2247 Muscari sp. LST 296 16/04/2009 /8--9--11.5 2.5--3.5--4/1.5--2 3/2, 4/1, 4/1, 6/0, 4--6--8--9 9.5--10--11-- Turkey: Artvin, Kaçkar Mts., Altiparnak, --12 (1) narrow/0, 3/0, 5/1, 4/1 Some leaves wi- 13.5--14--15 vicinity of Niznara village, rocky outcrops, der distally -- 2600--3000 m. differing 1.4--9. 2005-2248 Muscari sp. LST 297 13/04/2009 /8.5--10 3.5--4/2--2.5 3/1, 4/1, 7/1 5--6--8--9 10.5--12-- Turkey: Artvin, Kaçkar Mts., Altiparnak, Perianth opening wide. Pretty wide. 12.5--16-- vicinity of Niznara village, alpine turf, 23.5--26.5-- 2600-3000 m. 27 2005-2260 Muscari LST 321 12/04/2009 /8.5--11 3.5--5/1.5--2 5/3, 2/0, 2/1, 5/2, 6/1, 5--7--8 - - - armeniacum Turkey: Erzurum, road from Köphükoy to Periant opening wide. 6/2, 10/4, 6/3 Narrow ones Mus, on pass between Körsu and Akhören, U-shaped. 2100 m. 2005-2307 Muscari sp. LST 401 13/04/2009 /7--9 2.5/1 9/+ 2 buds, 9/+ 2 buds, 2--3 15--17.5--18 Turkey: Denizli, road from Antalya to So far only one raceme. 12/1 + 2 buds, 9/+ 3 buds --22 Denizli, 30 km before Denizli, Kazikbeli Perianth colour very dark. Geçidi. 2005-2657 Muscari LST 220B 12/04/2009 /7.5-9,5 3.5/1.5 3/2, 5/3, 5--8 16--19.5-- armeniacum Turkey: Erzurum, road from Erzurum to (2) narrow/0, (2) narrow/0, 21.5--22.5

12

Bingöl, Yaylasyu Geçidi, 2400 m. (2) narrow/0, 11/6 2006-2136 Muscari Ruksans, J. 11/04/2009 /9--12--14 4.5--6.5/2 5/2, 10/2, 5/2, 4--8 14.5--20 armeniacum : Irashen, Spitak. Royal blue--liliac, lobes white but 5/5 + 2 buds More “upright” f. caeruleum not pure white. fashion. 2006-2140 Muscari Botanicus 402 12/04/2009 /12.5--15 3--4--6.5--9/2--2.5 6/3, 4/1, 5/2, 3/1 2--5 28.5--31 armeniacum Turkey: the road between Akseki and Low Narrow, Yarpuz. turgor. U-shaped. 2006-2141 Muscari Botanicus 410 11/04/2009 /12--13 4--7/2 6/3, 7/3, 7/3, 7/3 4--8 22--25.5 armeniacum Turkey: road 49--50 between Yarpuz and Reddish. Colour liliac blue, small openings, Bozkir. lobes white, recurved. 2007-2181 Muscari sp. Seisums, A. 13/04/2009 /10--11 3--4/1.5 4 + (1) narrow/1, 5/1 2--6 13--14 ASIE 07-02. Turkey: Akyarma Geçidi on Pure white lobes, also white a Gerede--Ankara road. bit further up.

13

Table 2. Primers used for PCR and sequencing.

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ITS Forward: P17: 5‟-CTACCGATTGAATGGTCCGGTGAA-3‟ (24) (Bolmgren & Oxelman, unpublished) Reverse: 26S-82R: 5‟-TCCCGGTTCGCTCGCCGTTACTA-3‟ (23) (Bolmgren & Oxelman, unpublished) trnL-F region Forward: trnLmus_1: 5‟-CTACGGACTTAATTGGATTGAGC-3‟ (23) (Pfosser & Speta, 1999) Reverse: trnLmus_2: 5‟-AGGATTTTCAGTCCTCTGCTC-3‟ (21) (Pfosser & Speta, 1999)

CytP450 Forward: Cyt450_F2: 5‟-AGCACTGGAGGCAAATGCGGA-3‟ (21) (this study) Reverse: Cyt450_R2: 5‟-GGGTGCAGTCGCAAAAGCTCCT-3‟ (22) (this study)

Table 3. ITS uncorrected pairwise ("p") distance matrix

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1 2 3 4 5 6 7 1 1987-0715 a - 2 2004-2350 p 0.01236 - 3 2004-2381 p 0.01237 0.00123 - 4 2004-2421 p 0.01118 0.00124 0.00123 - 5 2004-2422 p 0.00742 0.00249 0.00373 0.00375 - 6 2004-2466 sp 0.01125 0.01116 0.01240 0.01246 0.00620 - 7 2004-2552 p 0.04055 0.03893 0.04072 0.04098 0.03337 0.03921 - 8 2004-2553 a 0.00000 0.01241 0.01242 0.01120 0.00743 0.01128 0.04073 9 2004-2558 a 0.00000 0.01355 0.01365 0.01227 0.00797 0.01219 0.03973 10 2005-2065 a 0.00124 0.01359 0.01360 0.01241 0.00865 0.01249 0.04071 11 2005-2076 a 0.00125 0.01365 0.01366 0.01244 0.00867 0.01253 0.04091 12 2005-2137 p 0.00891 0.00635 0.00760 0.00765 0.00129 0.00765 0.03357 13 2005-2148 a 0.01438 0.00322 0.00489 0.00492 0.00300 0.01281 0.03599 14 2005-2158 a 0.00875 0.00622 0.00745 0.00753 0.00124 0.00752 0.03556 15 2005-2191 a 0.00711 0.00529 0.00532 0.00719 0.00170 0.01247 0.03613 16 2005-2231 a 0.01120 0.01111 0.01234 0.01243 0.00618 0.00249 0.04082 17 2005-2246 sp 0.00886 0.00128 0.00255 0.00253 0.00000 0.00758 0.03548 18 2005-2247 sp 0.00762 0.00128 0.00256 0.00258 0.00000 0.00636 0.03376 19 2005-2248 a 0.00762 0.00128 0.00256 0.00258 0.00000 0.00636 0.03372 20 2005-2260 a 0.01523 0.00635 0.00762 0.00766 0.00508 0.01396 0.03932 21 2005-2307 sp 0.04438 0.04259 0.04389 0.04381 0.03761 0.04294 0.03168 22 2005-2657 a 0.00504 0.00887 0.01011 0.01018 0.00382 0.00758 0.03677 23 2006-2136 a 0.01243 0.00247 0.00371 0.00373 0.00249 0.01119 0.04066 24 2006-2140 a 0.00000 0.01264 0.01264 0.01140 0.00764 0.01147 0.04046 25 2006-2141 a 0.00000 0.01251 0.01254 0.01130 0.00749 0.01134 0.04040 26 2007-2181 a 0.00894 0.01160 0.01287 0.01293 0.00644 0.01032 0.03968

Uncorrected ("p") distance matrix (continued)

8 9 10 11 12 13 14 8 2004-2553 a - 9 2004-2558 a 0.00000 - 10 2005-2065 a 0.00124 0.00000 - 11 2005-2076 a 0.00126 0.00000 0.00000 - 12 2005-2137 p 0.00892 0.00944 0.01018 0.01019 - 13 2005-2148 a 0.01442 0.01420 0.01440 0.01444 0.00598 - 14 2005-2158 a 0.00877 0.00944 0.00999 0.01003 0.00129 0.00623 - 15 2005-2191 a 0.00712 0.00705 0.00712 0.00713 0.00347 0.00350 0.00527 16 2005-2231 a 0.01129 0.01212 0.01243 0.01254 0.00765 0.01279 0.00754 17 2005-2246 sp 0.00888 0.00942 0.01011 0.01015 0.00259 0.00297 0.00131 18 2005-2247 sp 0.00764 0.00805 0.00889 0.00891 0.00130 0.00153 0.00000 19 2005-2248 a 0.00764 0.00805 0.00889 0.00891 0.00130 0.00153 0.00000 20 2005-2260 a 0.01524 0.01665 0.01649 0.01651 0.00646 0.00479 0.00890 21 2005-2307 sp 0.04457 0.04833 0.04558 0.04580 0.03993 0.05177 0.03943 22 2005-2657 a 0.00506 0.00530 0.00631 0.00634 0.00517 0.00906 0.00509 23 2006-2136 a 0.01245 0.01359 0.01366 0.01369 0.00636 0.00488 0.00624 24 2006-2140 a 0.00000 0.00000 0.00000 0.00000 0.00909 0.01431 0.00900 25 2006-2141 a 0.00000 0.00000 0.00125 0.00127 0.00894 0.01574 0.00887

14

26 2007-2181 a 0.00897 0.00939 0.00894 0.00896 0.00783 0.01237 0.00775

15

Uncorrected ("p") distance matrix (continued)

15 16 17 18 19 20 21 15 2005-2191 a - 16 2005-2231 a 0.01249 - 17 2005 2246 sp 0.00171 0.00754 - 18 2005-2247 sp 0.00172 0.00636 0.00000 - 19 2005-2248 a 0.00172 0.00636 0.00000 0.00000 - 20 2005-2260 a 0.00540 0.01399 0.00519 0.00522 0.00522 - 21 2005-2307 sp 0.04346 0.04281 0.03796 0.03755 0.03752 0.04492 - 22 2005-2657 a 0.00345 0.00756 0.00517 0.00387 0.00387 0.01164 0.03987 23 2006-2136 a 0.00707 0.01118 0.00000 0.00000 0.00000 0.00511 0.04021 24 2006-2140 a 0.00716 0.01140 0.00894 0.00774 0.00774 0.01561 0.04468 25 2006-2141 a 0.00714 0.01127 0.00879 0.00767 0.00767 0.01543 0.04415 26 2007-2181 a 0.00659 0.01027 0.00782 0.00651 0.00651 0.01449 0.04460

Uncorrected ("p") distance matrix (continued)

22 23 24 25 26 22 2005-2657 a - 23 2006-2136 a 0.00888 - 24 2006-2140 a 0.00521 0.01269 - 25 2006-2141 a 0.00515 0.01255 0.00000 - 26 2007-2181 a 0.00649 0.01162 0.00911 0.00903 -

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Table 4. ITS data matrix with polymorphic sites.

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1223355666677 8251267034813 Taxon/Node 5857951835793 ------1987 0715 a AACGCTCCCATCC 2004 2350 p AACGACTCTCCCC 2004 2381 p AACGACTCTMCCC 2004 2421 p AACGACTCTACCC 2004 2422 p AACGMCYCCCCCC 2004 2466 sp AACGCCCCCCCCC 2004 2552 p -ACACCCCCCCCA 2004 2553 a AACGCTCCCATCC 2004 2558 a AACGGCTCCCACC 2005 2065 a AACGGCTCCCACC 2005 2076 a AACGGCTCCCACC 2005 2137 p AACGCCCMCCCCC 2005 2148 a AACGACTCCCCCC 2005 2158 a AACGCCCACCCCC 2005 2191 a --CGACCCCCTCC 2005 2231 a AACGCCCCCCCAC 2005 2246 sp AACGACYMYCCCC 2005 2247 sp AACGMCYMYCCCC 2005 2248 a AACGMCYMYCCCC 2005 2260 a MACGACTCYCCCC 2005 2307 sp TACACCCCCCCCM 2005 2657 a AMCGCCCCCCYCC 2006 2136 a AACGACTCTCCCC 2006 2140 a AACGCTCCATTCC 2006 2141 a AACGCTCCATTCC 2007 2181 a ATKSCMCCCTTCC

------

16

Table 5. ITS uncorrected pairwise ("p") distance matrices (constant characters excluded).

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ITS uncorrected pairwise ("p") distance matrix, sequences with polymorphisms

1 2 3 4 5 6 7 1 1987-0715 a - 2 2004-2350 p 0.16393 - 3 2004-2381 p 0.16667 0.01639 - 4 2004-2421 p 0.14754 0.01639 0.01639 - 5 2004-2422 p 0.09988 0.03565 0.05335 0.05509 - 6 2004-2466 sp 0.14754 0.14754 0.16475 0.16393 0.08251 - 7 2004-2552 p 0.47292 0.46131 0.48118 0.48358 0.39152 0.47064 - 8 2004-2553 a 0.00000 0.16393 0.16667 0.14754 0.09988 0.14754 0.47292 9 2004-2558 a 0.00000 0.16750 0.16979 0.15052 0.10323 0.15096 0.47710 10 2005-2065 a 0.01639 0.18033 0.18306 0.16393 0.11663 0.16393 0.48431 11 2005-2076 a 0.01639 0.18033 0.18306 0.16393 0.11663 0.16393 0.48431 12 2005-2137 p 0.11475 0.08197 0.09911 0.09836 0.01613 0.09836 0.38549 13 2005-2148 a 0.16577 0.03704 0.05517 0.05586 0.03926 0.14788 0.43907 14 2005-2158 a 0.11475 0.08197 0.09911 0.09836 0.01675 0.09836 0.41441 15 2005-2191 a 0.12241 0.06557 0.06766 0.10415 0.02292 0.18612 0.47917 16 2005-2231 a 0.14754 0.14754 0.16475 0.16393 0.08251 0.03279 0.47972 17 2005-2246 sp 0.11815 0.01911 0.03796 0.03591 0.00000 0.10068 0.43405 18 2005-2247 sp 0.09945 0.01874 0.03716 0.03786 0.00000 0.08314 0.41184 19 2005-2248 a 0.09945 0.01874 0.03716 0.03786 0.00000 0.08314 0.41184 20 2005_2260 a 0.20648 0.08566 0.10307 0.10479 0.06700 0.18810 0.46386 21 2005-2307 sp 0.57446 0.55753 0.57708 0.57446 0.48996 0.55674 0.38052 22 2005-2657 a 0.07503 0.12131 0.13920 0.13934 0.05371 0.10492 0.44952 23 2006-2136 a 0.16393 0.03279 0.04918 0.04918 0.03403 0.14754 0.46919 24 2006-2140 a 0.00000 0.16750 0.16979 0.15052 0.10323 0.15096 0.47710 25 2006-2141 a 0.00000 0.16410 0.16748 0.14797 0.09677 0.14727 0.46888 26 2007-2181 a 0.11656 0.14910 0.16657 0.16557 0.08345 0.13264 0.46292

Uncorrected ("p") distance matrix (continued)

8 9 10 11 12 13 14 8 2004-2553 a - 9 2004-2558 a 0.00000 - 10 2005-2065 a 0.01639 0.00000 - 11 2005-2076 a 0.01639 0.00000 0.00000 - 12 2005-2137 p 0.11475 0.11840 0.13115 0.13115 - 13 2005-2148 a 0.16577 0.16692 0.16671 0.16671 0.07415 - 14 2005-2158 a 0.11475 0.11791 0.13115 0.13115 0.01639 0.07603 - 15 2005-2191 a 0.12241 0.12353 0.12299 0.12299 0.04590 0.04528 0.09344 16 2005-2231 a 0.14754 0.15096 0.16393 0.16393 0.09836 0.14828 0.09836 17 2005-2246 sp 0.11815 0.12184 0.13499 0.13499 0.03280 0.03635 0.01613 18 2005-2247 sp 0.09945 0.10302 0.11585 0.11585 0.01639 0.01882 0.00000 19 2005-2248 a 0.09945 0.10302 0.11585 0.11585 0.01639 0.01882 0.00000 20 2005-2260 a 0.20648 0.21045 0.22291 0.22291 0.08512 0.05572 0.12121 21 2005-2307 sp 0.57446 0.58233 0.59141 0.59141 0.50719 0.56880 0.50682 22 2005-2657 a 0.07503 0.07755 0.09251 0.09251 0.07030 0.12323 0.07213 23 2006-2136 a 0.16393 0.16735 0.18033 0.18033 0.08197 0.05323 0.08197 24 2006-2140 a 0.00000 0.00000 0.00000 0.00000 0.11840 0.16692 0.11791 25 2006-2141 a 0.00000 0.00000 0.01683 0.01683 0.11290 0.18000 0.11360 26 2007-2181 a 0.11656 0.11771 0.11578 0.11578 0.10033 0.14775 0.09973

Uncorrected ("p") distance matrix (continued)

15 16 17 18 19 20 21 15 2005-2191 a - 16 2005-2231 a 0.18756 - 17 2005-2246 sp 0.02372 0.10068 - 18 2005-2247 sp 0.02314 0.08314 0.00000 - 19 2005-2248 a 0.02314 0.08314 0.00000 0.00000 - 20 2005-2260 a 0.06803 0.19029 0.06638 0.06747 0.06747 - 21 2005-2307 sp 0.53517 0.55674 0.50157 0.49076 0.49076 0.58550 - 22 2005-2657 a 0.04614 0.10656 0.07409 0.05580 0.05580 0.15993 0.52632 23 2006-2136 a 0.08330 0.14754 0.00000 0.00000 0.00000 0.06674 0.52448 24 2006-2140 a 0.12353 0.15096 0.12184 0.10302 0.10302 0.21045 0.58233 25 2006-2141 a 0.12133 0.14727 0.11475 0.09785 0.09785 0.20462 0.56720 26 2007-2181 a 0.08467 0.13264 0.10180 0.08470 0.08470 0.18703 0.56404

17

Uncorrected ("p") distance matrix (continued)

22 23 24 25 26 22 2005-2657 a - 23 2006-2136 a 0.12131 - 24 2006-2140 a 0.07755 0.16735 - 25 2006-2141 a 0.07382 0.16339 0.00000 - 26 2007-2181 a 0.08633 0.14903 0.11771 0.11545 -

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ITS uncorrected pairwise ("p") distance matrix; sequences without polymorphisms.

1 2 4 6 7 8 9 1 1987-0715 a - 2 2004-2350 p 0.16393 - 4 2004-2421 p 0.14754 0.01639 - 6 2004-2466 sp 0.14754 0.14754 0.16393 - 7 2004-2552 p 0.47292 0.46131 0.48358 0.47064 - 8 2004-2553 a 0.00000 0.16393 0.14754 0.14754 0.47292 - 9 2004-2558 a 0.00000 0.16750 0.15052 0.15096 0.47710 0.00000 - 10 2005-2065 a 0.01639 0.18033 0.16393 0.16393 0.48431 0.01639 0.00000 11 2005-2076 a 0.01639 0.18033 0.16393 0.16393 0.48431 0.01639 0.00000 13 2005-2148 a 0.16577 0.03704 0.05586 0.14788 0.43907 0.16577 0.16692 14 2005-2158 a 0.11475 0.08197 0.09836 0.09836 0.41441 0.11475 0.11791 15 2005-2191 a 0.12241 0.06557 0.10415 0.18612 0.47917 0.12241 0.12353 16 2005-2231 a 0.14754 0.14754 0.16393 0.03279 0.47972 0.14754 0.15096 23 2006-2136 a 0.16393 0.03279 0.04918 0.14754 0.46919 0.16393 0.16735 24 2006-2140 a 0.00000 0.16750 0.15052 0.15096 0.47710 0.00000 0.00000 25 2006-2141 a 0.00000 0.16410 0.14797 0.14727 0.46888 0.00000 0.00000

Uncorrected ("p") distance matrix (continued)

10 11 13 14 15 16 23 10 2005-2065 a - 11 2005-2076 a 0.00000 - 13 2005-2148 a 0.16671 0.16671 - 14 2005-2158 a 0.13115 0.13115 0.07603 - 15 2005-2191 a 0.12299 0.12299 0.04528 0.09344 - 16 2005-2231 a 0.16393 0.16393 0.14828 0.09836 0.18756 - 23 2006-2136 a 0.18033 0.18033 0.05323 0.08197 0.08330 0.14754 - 24 2006-2140 a 0.00000 0.00000 0.16692 0.11791 0.12353 0.15096 0.16735 25 2006-2141 a 0.01683 0.01683 0.18000 0.11360 0.12133 0.14727 0.16339

Uncorrected ("p") distance matrix (continued)

24 25 24 2006-2140 a - 25 2006-2141 a 0.00000 -

------

18

Table. 6. CytP450 pairwise distance matrices.

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Uncorrected ("p") distance matrix

1 2 3 4 5 1 2004-2350 - 2 2004-2422 0.00000 - 3 2004-2552 0.15660 0.16032 - 4 2005-2307 0.14860 0.15117 0.07961 - 5 2006-2141 0.04451 0.04996 0.17963 0.17213 -

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Ti/tv ratios for Kimura 2-parameter distance 90 characters are excluded

1 2 3 4 5 1 2004-2350 - 2 2004-2422 0.00000 - 3 2004-2552 2.12969 2.14570 - 4 2005-2307 2.32181 2.27761 1.86802 - 5 2006-2141 1.59057 1.49974 1.77982 1.87016 -

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19

Raceme length Leaf length

14 50 45 12 40

10 35 q1 q1 30 8 min min max 25 max q3 6 q3 20 median median 4 15 10 2 5

0 0 M. armeniacum M. polyanthum M. armeniacum M. polyanthum

Leaf width Raceme width

3 ,5 20 18 3 16 2 ,5 q1 14 2 min q1 12 max min 1 ,5 q3 10 max median 1 8 q3 median 0 ,5 6

0 4 M. armeniacum M. polyanthum 2

0 M. armeniacum M. polyanthum

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Figure 1. Sample charts comparing length and width of leaves and in „armeniacum‟ and „polyanthum‟ respectively.

20

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Figure 2. Cross-sections of flowers.

21

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Figure 3. Phylogram with parsimony bootstrap support values (top) and a Neighbor-Net network (bottom) for the ITS sequences (polymorphisms included). In both representations a long branch with one „polyanthum‟ (2004-2552) and one „sp.‟ (2005-2307) is clearly deviant from the other sequences.

22

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Figure 4. ITS phylogram with bootstrap support values; sequences with polymorphisms are excluded.

23

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Figure 5. Phylogram with parsimony bootstrap support values for the trnL-F sequences.

24

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Figure 6. Phylogram with parsimony bootstrap support values for the CytP450 sequences. A long branch with strong support is shown for a clade with one „sp.‟ (2005-2307) and one „polyanthum‟ (2004-2552). Sister to this clade, also with strong support, is a branch with an „armeniacum‟ (2006-2141).

25

1987-0715 Muscari armeniacum

26

2004-2350 Muscari „polyanthum‟

27

2004-2381 Muscari „polyanthum‟

28

2004-2421 Muscari „polyanthum‟

29

2004-2422 Muscari „polyanthum‟

30

2004-2466 Muscari sp.

31

2004-2552 Muscari „polyanthum‟

32

2004-2558 Muscari armeniacum

33

2005-2065 Muscari armeniacum

34

2005-2076 Muscari armeniacum

34

2005-2129 Muscari armeniacum

35

2005-2131 Muscari armeniacum

36

2005-2134 Muscari armeniacum

37

2005-2148 Muscari armeniacum

38

2005-2158 Muscari armeniacum

39

2005-2231 Muscari armeniacum

40

2005-2246 Muscari sp.

41

2005-2248 Muscari sp.

42

2005-2260 Muscari armeniacum

43

2005-2307 Muscari sp.

44

2005-2657 Muscari armeniacum

45

2006-2136 Muscari armeniacum f. caeruleum

46

2006-2140 Muscari armeniacum

47

2006-2141 Muscari armeniacum

48

2007-2181 Muscari sp.

49

2001-2191 Muscari armeniacum f. album

50

2005-2211 Muscari armeniacum

51