Seed micromorphology and anatomy of 36 Muscari (Asparagaceae) taxa from Turkey with notes on their systematic importance

Introduction

The genus Muscari Mill. is found across the European continent, Mediterranean region and northwest Asia (Jafari and Maassoumi 2011). According to the latest checklist study, the genus is represented by 51 species worldwide (Govaerts 2019). According to other recent studies, Muscari includes 40 species belonging to four subgenera as Muscari, Leopoldia and Botryanthus and Pseudomuscari with controversial status in Turkey, 26 of which are endemic (Dizkırıcı et al. 2019, Eker 2019a,b, Demirci-Kayıran et al. 2019). The genus is characterized by its bulbs, basal leaves, inflorescences, pedicels, flower form and colour, filament placement relative to the tube, and capsule shape (Davis and Stuart 1984). Major taxonomic problems of the genus include the many synonyms among taxa, the fact that type specimens are often cultivated material of unknown origin, that widespread taxa show a lot of variation and the color difference between fresh and dried flowers (Davis and Stuart 1984). Moreover, reliable classification is impossible in the genus because morphological characteristics and karyological information are not complete or consistent enough to make uncontroversial taxonomic judgements (Dizkırıcı et al. 2019). The genus was placed in Hyacinthaceae until the Angiosperm Phylogeny Group (APG) re-evaluated its taxonomic position as a result of subsequent studies, and placed it within the family Asparagaceae (Reveal and Chase 2011, Guner et al. 2012, Demirci and Özhatay 2017).

Several morphological, anatomical, cytological, palynological and ecological studies on taxa belonging to various genera of Asparagaceae have been performed previously (Shoub and Halevy 1971, Bentzer et al. 1974, Küçüker 1990, Uysal 1999, Herrmann et al. 2006, Lynch et al. 2006, Uysal et al. 2007, Gürsoy and Şık 2010, Kahraman et al. 2010, Doğu and Bağcı 2009, Doğu et al. 2011, Sezer et al. 2013). However, the morphological and anatomical features of seeds have been largely ignored in the systematics of taxa in the family, except in a few new species descriptions (Yıldırım 2015, 2016, Doğu and Uysal 2019).

The purpose of this study is to: (i) examine the morphological and anatomical characteristics of seeds of 36 taxa in subgenera Muscari, Leopoldia, Pseudomuscari and Botryanthus of the genus Muscari in Turkey, and (ii) debate the taxonomic use of these characters. The study will also serve as a guide to further related studies on various genera in the family.

Material and methods

The plant specimens were collected from various phytogeographical regions of Turkey during the fruiting season and were deposited at VANF (Van Yüzüncü Yıl University Herbarium). Details are provided in Tab. 1.

No Subgenus Taxa Location Voucher
1 Muscari Muscari macrocarpum Sweet C1 Muğla; between Marmaris and Emecik, after Balık Pass, rocky valley, 36° 46' 27" N, 27° 59' 36" E, 324 m, 01.03.2016 H. Eroğlu 1215
2 *M. racemosum Mill. C2 Denizli; Çameli, Denizli-Fethiye road, 5 km to Aliveren Village, Pinus yards, serpentine fields, 37° 13' 39" N, 29° 26' 52" E, 1264 m, 04.05.2017. H. Eroğlu 1317
3 Leopoldia M. caucasicum (Griseb.) Baker B9 Van; Erek Mountain, south of Sarmaç Village, steppe, 38° 29' 16" N, 43° 29' 26" E, 2200 m, 24.05.2016. H. Eroğlu 1281
4 M. weissii Freyn Antalya: Serik, Kumköy, Pinus pinea forest near the sea, dunes under the woood, 36° 52ʹ 07ʺ N, 30° 56ʹ 36ʺ E, 3 m, 02.04.2016. H. Eroğlu 1220
5 M. comosum (L.) Mill. C2 Muğla, Marmaris, between Marmaris and Datça, Hisarönü Bay, roadside, 36° 47' 59" N, 28° 05' 31" E, 70 m, 16.04.2017. H. Eroğlu 1301
6 M. tenuiflorum Tausch B6 Adana; Feke, Esendere Canyon, Pinus yards, 37° 45' 44" N, 35° 55' 03" E, 651 m, 15.06.2016. H. Eroğlu 1288
7 *M. babachii Eker & Koyuncu C6 Hatay, Antakya, Kisecik Village, Radar road, scrub yards, 36° 18' 15" N, 36° 02' 59" E, 1430 m, 12.06.2016. H. Eroğlu 1286
8 *M. erdalii N.Özhatay & S.Demirci C4 İçel; Mut, south of İbrahimli Village, scrub yards, 36° 40' 55" N, 33° 39' 23" E, 900 m, 02.05.2016. H. Eroğlu 1255
9 M. longipes Boiss. B6 Sivas; Hafik, west of Durulmuş Village, marly hills, 39° 50' 08" N, 37° 18' 20" E, 1312 m, 30.05.2017. H. Eroğlu 1327
10 *M. massayanum C.Grunert C5 Adana; Pozantı, upwards of Hamidiye Village, serpentine slopes, 37° 32' 27" N, 35° 00' 51" E, 1357 m, 01.05.2016. H. Eroğlu 1253
11 *M. mirum Speta C2 Denizli; Çameli, Denizli-Fethiye road, 4 km to Aliveren Village, serpentine slopes, 37° 12' 41" N, 29° 26' 17" E, 1475 m, 04.05.2016. H. Eroğlu 1259
12 *M. elmasii Yıldırım C2 Muğla; Dalaman, above Gürleyik Village, Çal Mountain, Pinus yards, 36° 52' 49" N, 29° 07' 10" E, 1271 m, 14.05.2016. H. Eroğlu 1270
13 *M. ufukii E.Kaya & Demirci B9 Van; Çatak, between Çatak-Bilgi Village, steppe, 38° 03' 48" N, 43° 11' 49" E, 1670 m, 17.07.2017. H. Eroğlu 1341
14 Pseudomuscari *M. coeleste Fomin B9 Van; Erek Mountain, side of Keşiş Lake, humid meadows, 38° 27' 43" N, 43° 34' 51" E, 2564 m, 18.05.2017. H. Eroğlu 1319
15 *M. azureum Fenzl C5 Niğde; Ulukışla, Karagöl, humid meadows, 37° 24' 16" N, 34° 33' 38" E, 2599 m, 01.05.2016. H. Eroğlu 1251
16 Botryanthus *M. aucheri (Boiss.) Baker A9 Kars; Sarıkamış to Handere 5. km, meadows, 40° 18ʹ 33ʺ N, 42° 30ʹ 43ʺ E, 2196 m, 08.06.2016. H. Eroğlu 1285
17 M. armeniacum Leichtlin ex Baker C4 Karaman; Sarıveliler, Atmeydanı place, steppe, 36° 41' 44" N, 32° 31' 00" E, 1665 m, 01.05.2017 H. Eroğlu 1306
18 *M. sivrihisardaghlarensis Yıld. & B.Selvi B3 Eskişehir; Sivrihisar, between Kuzuören and Karacaören villages, stony-rocky streamside, 39° 18' 52" N, 31° 42' 42" E, 1416 m, 02.05.2017. H. Eroğlu 1309
19 M. neglectum Guss. ex Ten. B3 Eskişehir; Sivrihisar, Günyüzü cross, steppe, 39° 29' 42" N, 31° 36' 41" E, 1009 m, 17.04.2016. H. Eroğlu 1242
20 *M. anatolicum Cowley & Özhatay C5 İçel; Toroslar, Arslanköy, above Dümbelek Geçidi rocky slopes, 37° 03' 56" N, 34° 17' 53" E, 2212 m, 11.05.2018. H. Eroğlu 1382
21 *M. tuzgoluensis Yıld. B4 Aksaray, Eskil, 1 km towars Tuzgölü from Eskil, steppe, 38° 24' 43" N, 33° 27' 20" E, 922 m, 13.04.2016. H. Eroğlu 1233
22 *M. discolor Boiss. & Hausskn. ex Boiss. C8 Mardin; Artuklu, Mardin-Diyarbakır road, Akresta pass, stony streamside, 37° 22' 57" N, 40° 39' 09" E, 1138 m, 07.04.2017. H. Eroğlu 1297
23 M. inconstrictum Rech.f. C6 Kilis; south of Kocabeyli Village, stony-rocky fields, 36° 48' 07" N, 36° 54' 59" E, 450 m, 28.02.2016. H. Eroğlu 1212
24 *M. latifolium J.Kirk B2 Çanakkale; Bayramiç, Ayazma promenade, under the forest, humid areas, 39° 44' 45" N, 26° 50' 47" E, 476 m, 03.05.2017. H. Eroğlu 1313
25 *M. adilii M.B.Güner & H.Duman A3 Ankara, Beypazarı, above Hırkatepe Village, arounds of Koçahmet Fountain, marly valleys, 40° 11' 43" N, 31° 46' 39" E, 1000 m, 02.05.2017. H. Eroğlu 1308
26 *M. bourgaei Baker C4 Karaman; Sarıveliler, Atmeydanı Place, meadows, streamside, 36° 41' 25" N, 32° 32' 41" E, 1603 m, 01.05.2017. H. Eroğlu 1304
27 *M. sandrasicum Karlén C2 Muğla; Köyceğiz, Sandras Mountain, Sandras Highland, Değirmenbozuğu Place, stony streamside, 37° 05' 36" N, 28° 53' 23" E, 1356 m, 11.04.2016. H. Eroğlu 1226
28 M. microstomum P.H.Davis & D.C.Stuart B5 Kayseri; Bünyan, between Bünyan and Pınarbaşı 4. km, humid meadows, 38° 49' 40" N, 35° 54' 24" E, 1389 m, 19.05.2016. H. Eroğlu 1277
29 *M. macbeathianum Kit Tan B6 Adana; Tufanbeyli, 2 km from Güzelim Village to Tufanbeyli, dune under Pinus, 38° 09' 24" N, 36° 10' 45" E, 1442 m, 09.05.2018. H. Eroğlu 1374
30 *M. vuralii Bağcı & Doğu C4 Karaman; Sarıveliler, Atmeydanı place, meadows, 36° 41' 25" N, 32° 32' 01" E, 1603 m, 14.04.2016. H. Eroğlu 1234
31 M. parviflorum Desf. C5 İçel; Yenişehir, between Emirler and Turunçlu villages, garden edges, 36° 50' 10" N, 34° 28' 42" E, 288 m, 28.09.2016. H. Eroğlu 1291
32 *M. serpentinicum Yıldırım, Altıoğlu & Pirhan C2 Muğla; Köyceğiz, Sandras Mountain, Sandras Highland, Değirmenbozuğu Place, stony streamside, 37° 05' 36" N, 28° 53' 23" E, 1356 m, 11.04.2016. H. Eroğlu 1224
33 M. botryoides (L.) Mill. B9 Ağrı; Tutak, between Aşağıköşk and Doğanüstün villages, meadows, 39° 24' 21" N, 42° 45' 36" E, 1669 m, 10.05.2016. H. Eroğlu 1262
34 *M. artvinense Demirci & E.Kaya A9 Artvin; Murgul, above Korucular Village, meadows, 41° 18' 00" N, 41° 38' 58" E, 762 m, 13.05.2016. H. Eroğlu 1266
35 *M. atillae Yıldırım B7 Malatya, Akçadağ, Levent Canyon, marly-movement slopes, 38° 26' 03" N, 37° 55' 56" E, 1197 m, 07.04.2017. H. Eroğlu 1296
36 *M. turcicum Uysal, Ertugrul & Dural C4 Konya; Bozkır, above Avdan Highland, snowpatches, steppe, 37° 01' 15" N, 32° 10' 41" E, 1978 m, 11.05.2018. H. Eroğlu 1379
Tab. 1.The examined taxa and their locations (*=endemic taxon).

Macromorphological features of the seeds including colour, shape and size were documented for 100 seeds of 10 individuals per species utilizing a Leica EZ4 binocular microscope with a HD camera (On-line Suppl. Fig. 1, Tab. 2). For the micromorphological features of surface ornamentation, anticlinal and periclinal cell walls, and the form of epidermal cells, the samples were studied with a Scanning Electron Microscope (On-line Suppl. Fig. 2, Tab. 3). Seeds were first placed on the stub with silver epoxy and coated with gold, then examined with a Zeiss LEO 440 SEM.

Subgenus Taxa Shape Seed dimensions
L (mm) W (mm) L/W
Muscari Muscari macrocarpum orbicular 2.98 ± 0.32 2.61 ± 0.29 1.14
M. racemosum broadly ovate-orbicular 3.16 ± 0.22 2.63 ± 0.30 1.20
Leopoldia M. caucasicum broadly ovate 2.34 ± 1.18 1.85 ± 0.11 1.26
M. weissii orbicular 2.08 ± 0.14 1.76 ± 0.12 1.18
M. comosum orbicular 2.23 ± 0.13 2.03 ± 0.11 1.10
M. tenuiflorum broadly ovate-orbicular 2.45 ± 1.15 2.17 ± 0.14 1.13
M. babachii broadly ovate 2.70 ± 0.19 2.22 ± 0.15 1.22
M. erdalii broadly ovate 3.21 ± 0.25 2.43 ± 0.15 1.32
M. longipes oblong-elliptic 2.42 ± 0.24 2.01 ± 0.12 1.20
M. massayanum broadly ovate 3.13 ± 0.23 2.56 ± 0.18 1.22
M. mirum oblong-ovate 2.82 ± 0.22 2.34 ± 0.17 1.20
M. elmasii broadly ovate 2.81 ± 0.25 2.25 ± 0.20 1.24
M. ufukii broadly ovate 3.06 ± 0.17 2.61 ± 0.13 1.17
Pseudomuscari M. coeleste orbicular 2.18 ± 0.16 1.52 ± 0.09 1.43
M. azureum orbicular 2.06 ± 0.10 1.35 ± 0.09 1.52
Botryanthus M. aucheri orbicular 2.11 ± 0.13 1.37 ± 0.08 1.54
M. armeniacum broadly ovate-orbicular 1.96 ± 0.16 1.71 ± 0.11 1.14
M. sivrihisardaghlarensis broadly ovate-orbicular 2.18 ± 0.17 1.69 ± 0.12 1.28
M. neglectum broadly elliptic 2.06 ± 0.15 1.64 ± 0.10 1.25
M. anatolicum orbicular 2.09 ± 0.15 1.72 ± 0.19 1.21
M. tuzgoluensis orbicular 1.94 ± 0.13 1.65 ± 0.13 1.17
M. discolor orbicular 2.23 ± 0.26 1.73 ± 0.11 1.28
M. inconstrictum orbicular 1.99 ± 0.12 1.80 ± 0.12 1.10
M. latifolium ovate-orbicular 2.39 ± 0.20 1.98 ± 0.13 1.20
M. adilii orbicular 2.45 ± 0.16 2.20 ± 0.17 1.11
M. bourgaei orbicular 1.82 ± 0.09 1.42 ± 0.11 1.28
M. sandrasicum orbicular 1.94 ± 0.17 1.53 ± 0.16 1.26
M. microstomum orbicular 1.87 ± 0.13 1.55 ± 0.16 1.20
M. macbeathianum broadly elliptic-lanceolate 1.66 ± 0.13 1.12 ± 0.09 1.48
M. vuralii orbicular 2.15 ± 0.14 1.60 ± 0.12 1.34
M. parviflorum orbicular 1.90 ± 0.16 1.62 ± 0.17 1.17
M. serpentinicum orbicular 1.72 ± 0.11 1.51 ± 0.10 1.13
M. botryoides broadly ovate 1.80 ± 0.14 1.36 ± 0.11 1.32
M. artvinense orbicular 1.70 ± 0.12 1.41 ± 0.10 1.21
M. atillae orbicular 2.25 ± 0.10 1.87 ± 0.11 1.20
M. turcicum orbicular 1.76 ± 0.13 1.34 ± 0.09 1.31
Tab. 2.Macromorphological characters of the seeds of the studied taxa (mean values ± standard deviation, L=length, W=width).
Subgenus Taxa Seed surface Anticlinal Periclinal Epidermal
ornamentation cell wall cell wall cell structure
Muscari Muscari macrocarpum reticulate raised concave polygonal cells
M. racemosum reticulate raised concave polygonal cells
Leopoldia M. caucasicum alveolate sunken concave alveolar cells
M. weissii alveolate sunken concave alveolar cells
M. comosum verrucate sunken convex unclear
M. tenuiflorum reticulate-areolate sunken convex polygonal cells
M. babachii reticulate-areolate sunken convex polygonal cells
M. erdalii ruminate unclear unclear unclear
M. longipes ruminate unclear unclear unclear
M. massayanum ruminate unclear unclear unclear
M. mirum reticulate-areolate sunken convex polygonal cells
M. elmasii reticulate-foveate raised convex polygonal and alveolar cells
M. ufukii ruminate unclear unclear unclear
Pseudomuscari M. coeleste ruminate unclear unclear unclear
M. azureum scalariform sunken convex rectangular and polygonal cells
Botryanthus M. aucheri ruminate unclear unclear unclear
M. armeniacum reticulate-areolate sunken concave polygonal cells
M. sivrihisardaghlarensis rugose unclear Unclear unclear
M. neglectum areolate Sunken concave polygonal cells
M. anatolicum rugose Unclear unclear unclear
M. tuzgoluensis ruminate Unclear unclear unclear
M. discolor ruminate Unclear unclear unclear
M. inconstrictum slightly reticulate raised concave polygonal cells
M. latifolium ruminate unclear unclear unclear
M. adilii ruminate unclear unclear unclear
M. bourgaei ruminate unclear unclear unclear
M. sandrasicum ruminate unclear unclear unclear
M. microstomum verrucate sunken convex unclear
M. macbeathianum ruminate unclear unclear unclear
M. vuralii ruminate unclear unclear unclear
M. parviflorum ruminate unclear unclear unclear
M. serpentinicum verrucate sunken convex unclear
M. botryoides rugose unclear unclear unclear
M. artvinense rugose unclear unclear unclear
M. atillae ruminate unclear unclear unclear
M. turcicum ruminate unclear unclear unclear
Tab. 3.Micromorphological characters of the seeds of the studied taxa.

A survey of seed anatomical characters was done with dry herbarium materials. Cross-sections were taken from the middle of the seed with a fully automatic microtome (Thermo Shonda Met Finesse, Thermo). They were brought through a series of alcohol and xylene, dyed with hematoxylin and eosin-Y in a staining device (ASC 720 Medite) and mounted using Entellan (On-line Suppl. Fig. 3, Tab. 4) (Karaismailoğlu 2015, Karaismailoğlu and Erol 2018, Karaismailoğlu and Güner 2019). Anatomical characteristics were examined with an Olympus CX31 light microscope and Kameram Imaging Software (KAMERAM12 CCD, Argenit Micro System Ltd., Turkey).

Subgenus Taxa Epidermis layers Presence/ absence
Epidermis structures Subepidermis structures Thickness (μm) of crystals
Muscari M. macrocarpum 1 layer, scleranchymatic flat cells 6-7 layers, scleranchymatic crushed cells 59.75 ± 2.48 -
M. racemosum 1 layer, scleranchymatic rectangular cells 3-4 layers, scleranchymatic crushed cells 54.23 ± 3.09 -
Leopoldia M. caucasicum 1 layer, scleranchymatic large flat cells 3-4 layers, parenchymatic orbicular or flat cells 105.44 ± 2.37 -
M. weissii 1 layer, scleranchymatic rectangular cells 3 layers, scleranchymatic large flat cells 46.71 ± 1.82 -
M. comosum 1 layer, scleranchymatic large flat cells 1 layer, parenchymatic rectangular or square cells 38.45 ± 3.63 +
M. tenuiflorum 1 layer, scleranchymatic large flat cells 2-3 layers, scleranchymatic crushed cells 116.59 ± 3.88 +
M. babachii 1 layer, scleranchymatic flat cells 2-3 layers, scleranchymatic crushed cells 121.10 ± 5.64 +
M. erdalii 1 layer, scleranchymatic flat cells 3-4 layers, scleranchymatic crushed cells 40.37 ± 4.21 -
M. longipes 1 layer, scleranchymatic flat cells 5-7 layers, scleranchymatic crushed cells 128.46 ± 4.23 -
M. massayanum 1 layer, scleranchymatic flat cells 2 layers, scleranchymatic flat or crushed cells 68.83 ± 3.47 -
M. mirum 1-2 layers, scleranchymatic crushed cells 5-6 layers, scleranchymatic crushed cells 108.54 ± 2.88 -
M. elmasii 1 layer, scleranchymatic rectangular cells 4-5 layers, scleranchymatic crushed cells 44.16 ± 2.72 -
M. ufukii 2-3 layers, scleranchymatic large flat cells 1 layer, scleranchymatic flat cells 85.35 ± 2.41 -
Pseudomuscari M. coeleste 1 layer, scleranchymatic rectangular cells 2-3 layers, scleranchymatic flat cells 33.62 ± 3.13 -
M. azureum 1 layer, scleranchymatic flat cells 2-3 layers, parenchymatic flat or polygonal cells 39.77 ± 2.54 -
Botryanthus M. aucheri 2 layers, scleranchymatic large flat cells 1 layer, parenchymatic polygonal cells 38.76 ± 1.85 -
M. armeniacum 1-2 layers, scleranchymatic flat cells 2 layers, parenchymatic flat or polygonal cells 71.19 ± 4.06 -
M. sivrihisardaghlarensis 1 layer, scleranchymatic flat cells 3-4 layers, scleranchymatic crushed cells 37.84 ± 3.71 -
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Subgenus Taxa Epidermis layers Presence/ absence
Epidermis structures Subepidermis structures Thickness (μm) of crystals
Botryanthus M. neglectum 1 layer, scleranchymatic flat cells 2 layers, parenchymatic flat cells 41.67 ± 3.24 -
M. anatolicum 2 layers, scleranchymatic flat cells 2-3 layers, scleranchymatic crushed cells 23.08 ± 3.92 -
M. tuzgoluensis 2 layers, scleranchymatic flat cells 1 layer, scleranchymatic polygonal cells 48.33 ± 2.18 -
M. discolor 2 layers, scleranchymatic crushed cells - 18.41 ± 2.38 +
M. inconstrictum 2-3 layers, scleranchymatic crushed cells - 22.05 ± 1.14 -
M. latifolium 1 layer, scleranchymatic large flat cells 2-3 layers, parenchymatic flat cells 66.15 ± 3.52 -
M. adilii 1 layer, scleranchymatic large flat cells 2-3 layers, scleranchymatic crushed cells 31.17 ± 1.84 -
M. bourgaei 1 layer, scleranchymatic large flat cells 2 layers, scleranchymatic flat cells 64.26 ± 2.29 -
M. sandrasicum 1 layer, scleranchymatic large flat cells 2-3 layers, parenchymatic flat cells 71.22 ± 2.31 -
M. microstomum 1 layer, scleranchymatic flat or polygonal cells 2-3 layers, scleranchymatic polygonal cells 69.98 ± 3.53 -
M. macbeathianum 2 layer, scleranchymatic large flat or rectangular cells 1 layer, scleranchymatic polygonal cells 41.13 ± 2.36 -
M. vuralii 1 layer, scleranchymatic flat or polygonal cells 2-3 layers, scleranchymatic polygonal cells 44.86 ± 1.71 +
M. parviflorum 1 layer, scleranchymatic large flat cells - 18.08 ± 0.86 -
M. serpentinicum 1 layer, scleranchymatic large rectangular cells 1 layer, scleranchymatic rectangular cells 39.73 ± 3.15 -
M. botryoides 2-3 layers, scleranchymatic flat cells - 25.81 ± 2.03 -
M. artvinense 1 layer, scleranchymatic polygonal cells 8-10 layers, scleranchymatic crushed and orbicular cells 117.46 ± 3.55 -
M. atillae 3 layers, scleranchymatic flat cells 2-3 layers, scleranchymatic polygonal cells 82.19 ± 2.68 -
M. turcicum 1 layer, scleranchymatic crushed cells - 16.64 ± 3.22 -
Tab. 4.Testa anatomical features of the studied taxa (mean values ± standard deviation, + =presence, - =absence).

The terminology used for seed morphological and anatomical characteristics is compatible with Stearn (1985).

Grouping of taxa was performed using the clustering analysis method (UPGMA) in MultiVariate Statistical Package (MVSP) in accordance with the 44 characters in Tables 2, 3 and 4 (Fig. 1). Characters used in statistical analysis were: seed colour (1); shape: orbicular (2), ovate (3), oblong (4), elliptic (5), lanceolate (6); sizes: length (7), width (8), L/W (9); surface ornamentation: reticulate (10), alveolate (11), areolate (12), verrucate (13), ruminate (14), foveate (15), rugose (16), scalariform (17); anticlinal cell walls: sunken (18), raised (19), unclear (20); periclinal cell walls: convex (21), concave (22), unclear (23); epidermal cell structure: polygonal (24), alveolar (25), rectangular (26), flat (27), unclear (28); anatomical structure of the epidermis: flat (29), rectangular (30), crushed (31), polygonal (32), scleranchymatous cells (33), parenchymatous cells (34); anatomical structure of the subepidermis: crushed (35), flat (36), orbicular (37), square (38), polygonal (39), rectangular (40), scleranchymatous cells (41), parenchymatous cells (42); testa thickness (43); presence of crystals (44). The dissimilarity matrix of the studied taxa was created with MVSP (Kovach 2007) (On-line Suppl. Tab. 1). A dendrogram was created. Also, the cophenetic correlation coefficient is designed to explain the relation between the dendrogram and similarity matrix (On-line Suppl. Tab. 1, Fig. 1).

Fig. 1.Cluster analysis of the studied taxa.

Results

This work assesses macromorphologically the seed features of the studied taxa, including colour, shape and dimensions. All of the taxa examined have the same seed color (black) but the shape and size of seeds vary considerably. Seeds examined can be divided into 7 shapes; orbicular, ovate-orbicular, ovate, oblong-ovate, oblong-elliptic, elliptic and elliptic-lanceolate. Orbicular is the most common type (found in 20 taxa). However, oblong-ovate, oblong-elliptic and elliptic-lanceolate are characteristic types for Muscari mirum, M. longipes and M. macbeathianum, respectively. Seed dimensions range from 1.66 mm to 3.21 mm in length, and from 1.12 mm to 2.63 mm in width. While M. erdalii and M. racemosum have the largest seeds, M. macbeathianum has the smallest seeds (Tab. 2, On-line Suppl. Fig. 1).

The surface ornamentation, anticlinal and periclinal cell walls, and epidermal cell structures of the seeds have been micromorphologically evaluated in this study. Seed surface ornamentation is grouped into nine types: ruminate, reticulate, reticulate-areolate, reticulate-foveate, alveolate, scalariform, rugose, verrucate and areolate. The most common form is ruminate, while areolate, reticulate-foveate and scalariform ornamentation forms were found to be taxon-specific (Tab. 3, On-line Suppl. Fig. 2). The reticulate-foveate (in M. elmasii), areolate (M. neglectum), and scalariform (M. azureum) ornamentation types are each displayed by only one taxon. The anticlinal cell walls in the studied taxa are raised, sunken or unclear. While sunken cell walls are widely seen in the alveolate, verrucate, areolate, reticulate-areolate and scalariform ornamentation types, the reticulate and reticulate-foveate ornamentation types are found where epidermal cells are enclosed by raised walls. Rugose and ruminate types are associated with unclear form (Tab. 3). No clear relationship exists between convex or concave periclinal cell walls and surface ornamentation types; however, ruminate and rugose types are found only with unclear periclinal cells. The shape of epidermal cells on the seed surface has also showed diversity and may be grouped into polygonal, alveolar, rectangular and unclear categories. The most common cell type is unclear, while rectangular and alveolar are fairly rare (Tab. 3).

The results of the examination of the anatomical structures of the seeds are indicated in On-line Suppl. Fig. 3 and Tab. 4. Testa structures of the seeds of the examined taxa generally consisted of 2 main layers, the epidermis and the subepidermis, formed in either the scleranchymatous or parenchymatous tissue. The epidermis layer displays important variations in cell form, consisting of flat, rectangular, crushed, or polygonal cells, in 1-3 layers, and has undulated or straight wall structure. The most frequent form is flat, while the rarest ones are the rectangular and polygonal types (Tab. 4, On-line Suppl. Fig. 3). The subepidermis layer consists of crushed, orbicular, rectangular, square, flat or polygonal cells in 1-10 layers. The most commonly seen types are crushed and polygonal, whereas the rarest ones are the orbicular and square types. The subepidermis layer is not found in some of the examined taxa (M. discolor, M. inconstrictum, M. parviflorum, M. botryoides and M. turcicum) (Tab. 4). The thickness of the epidermis layers varies between 16.64 μm (in M. turcicum) and 128.46 μm (in M. longipes). Raphide crystals are seen in the epidermis or subepidermis layers of seeds in M. comosum, M. tenuiflorum, M. babachii, M. discolor and M. vuralii (Tab. 4, On-line Suppl. Fig. 3).

A dendrogram indicating differences and similarities among the studied taxa was created by numerical analyses of the seed morphological and anatomical characters, based on the variation of 44 characteristics in 36 taxa. The cophenetic correlation between the similarity matrix and dendrogram has been computed as 0.59, representing a good match. Cluster A2 includes the highest number of taxa when compared to other clusters. Muscari sandrasicum forms a clade separate from these clusters in the dendrogram (Fig. 1). M. discolor and M. parviflorum are the most closely related taxa (with a dissimilarity coefficient of 1.01), the most distantly related taxa recorded are M. sandrasicum and M. turcicum (with a dissimilarity coefficient of 136.31) (On-line Suppl. Tab. 1).

Discussion

The morphological features of seeds offer valuable information about evolutionary relationships among flowering plants (Corner 1976, Karaismailoğlu and Erol 2018). However, seed morphological and anatomical features have so far not been extensively used to elucidate inter-species relationships within genera of the family Asparagaceae. This is the first study to reveal the morphological and anatomical features of the seeds of a genus in the family, and it will be a model for subsequent studies on various genera.

The macromorphological characters of seeds display variation among the examined Muscari taxa, with the exception of seed colour, which is consistently black. The general appearance among populations, including floristic characters and capsule structures, of M. macrocarpum and M. racemosum in subgenus Muscari, M. caucasicum and M. weissii in subgenus Leopoldia, M. aucheri and M. armeniacum in subgenus Botryanthus are very similar, but they can be easily distinguished using seed shape and size.

Comparison of the surface micromorphological structure of seeds is of taxonomical importance (Karaismailoğlu and Erol 2018). Heywood (1971) discusses the significance and efficiency of scanning electron microscopy in elucidating taxonomic problems and distinguishing taxa. However, there are few studies on the importance of seed micromorphology in the family Asparagaceae (Yıldırım 2015, 2016). This study on 36 Muscari taxa shows that seed microstructures are useful characteristics in separating the taxa within the family. Almost all of the studied taxa have been examined in this way for the first time, with the exceptions of M. elmasii (smooth) and M. atillae (smooth) (Yıldırım 2015, 2016). We recorded nine seed surface ornamentation types in this study. In the genus, the most common seed ornamentation types are ruminate and reticulate. In contrast to this study, reticulate and reticulate-areolate types have been commonly seen among taxa from various angiosperm families (Tantawy et al. 2004, Karaismailoğlu 2015, Karaismailoğlu and Erol 2018). Two closely related taxa in the subgenus Muscari, M. macrocarpum and M. racemosum, have the same reticulate surface ornamentation type; however, M. macrocarpum has different secondary cuticular protrusions. Seed surface ornamentation is a useful character in distinguishing the taxa of the subgenus Leopoldia, which exhibits five ornamentation types in 11 taxa. In the subgenus Pseudomuscari, M. coeleste and M. azureum taxa are very similar in terms of population appearance, flowers and fruit capsule characteristics; however, they are distinctly different in terms of seed ornamentation types: ruminate and scalariform, respectively. In the subgenus Botryanthus, ornamentation types are diverse (seven types), and the distinct surface ornamentation in nearly identical taxa, such as M. armeniacum-M. aucheri, M. armeniacum-M. bourgaei, M. armeniacum-M. microstomum is proof of the taxonomical significance of this characteristic in the subgenus.

Earlier seed surface studies have indicated that the views and structures of anticlinal and periclinal cell walls are good diagnostic characters in the establishment of inter-species relationships (Barthlott 1981, Karaismailoğlu 2015, 2016). The types of anticlinal and periclinal cell walls, and epidermal cell structures of the examined taxa vary among the taxa, except for those of the subgenus Muscari.

Revisions of the anatomy of the testa of the various angiosperm families are influential in solving systematic problems (Vaughan et al. 1976, Karaismailoğlu and Erol 2018). Koul et al. (2000) have shown that testa structures may be utilized as a valuable characteristic in the separation of the taxa and the clarification of their phylogenetic relationships.

The seed anatomical characters are frequently as useful as morphological characters for plant taxonomy, and they are valuable in the discrimination of closely correlated taxa in various families and genera (Karamian et al. 2012, Karaismailoğlu and Erol 2018, Karaismailoğlu et al. 2018). A detailed review of the literature has not found a previous study aiming at the exploration of phylogenetic relationships among the taxa with a comparative investigation of anatomical structures of the testa in members of the family Asparagaceae. This work is the first such study for the family and is the precursor to subsequent investigations. In this study, we found that the testae mostly consist of two layers, the epidermis and the subepidermis, in the sclerotic or parenchymatous structure. The epidermis type differs among the taxa. This 1-3 layered epidermis may consist of flat, rectangular, crushed, or polygonal cells. The most frequent form is flat, while the rarest are the rectangular and polygonal types. The structure of the subepidermis layer, which is mostly a compressed tissue under the epidermis layers, also displays significant differences among the taxa. The subepidermis layer consists of crushed, orbicular, rectangular, square, flat or polygonal cells in 1-10 layers, except for M. discolor, M. inconstrictum, M. parviflorum, M. botryoides and M. turcicum, which do not have a subepidermis layer. Testa characters such as the structures of the epidermis and subepidermis, thickness of the testa, and the presence or absence of crystals are fairly effective and beneficial in discriminating almost all of the studied taxa, especially in the pairs of closely correlated taxa M. macrocarpum-M. racemosum, M. caucasicum-M. weissii, M. coeleste-M. azureum, and M. aucheri-M. armeniacum. This can be interpreted as follows: the anatomy of the testa is a useful additional character in the Muscari, and it can aid in the classification of this huge genus. The results obtained are also in agreement with similar previous studies performed on seed structure of some taxa of the genera Crocus L. and Romulea Maratti in the closely related family Iridaceae, in terms of the differences observed at interspecific level in testa anatomical structures such as epidermis cell types and thickness of the testa (Grilli Caiola et al. 2010, Karaismailoğlu 2015, Karaismailoğlu et al. 2018).

The dendrogram showing two main clusters largely agree with the results of Davis and Stuart (1984). The seed morphological and anatomical variations have been observed at the species level and subgenus level, especially in shapes, ornamentation types, dimensions, and thicknesses and structures of epidermis and subepidermis layers. The proximity between taxa belonging to subgenera Muscari and Pseudomuscari has been preserved; however, there are taxon transitions between Leopoldia and Botryanthus subgenera. While M. atillae, M. latifolium, M. microstomum and M. armeniacum taxa are among the taxa belonging to Botryanthus subgenus, M. mirum and M. caucasicum taxa are located between Leopoldia taxa.

In conclusion, the study of morphological and anatomical seed characteristics of the studied Muscari taxa offers important insights into the systematics of taxa within the genus.

Key to studied Muscari taxa, based on seed characteristics

  1. 1.Seed shape is orbicular……………………………………………...……….……………….2
  2. 1.Seed shape is ovate, ovate-orbicular, oblong-elliptic, oblong-ovate, elliptic, elliptic-lanceolate.…………………………..…………………..……………………..……………...21
  3. 2.Seed ornamentation is reticulate………………………………......………..……………..…3
  4. 2.Seed ornamentation is alveolate, verrucate, ruminate, scalariform or rugose………..…..….4
  5. 3.Outer epidermis of testa consists of crushed cells, with 2-3 layers…………M. inconstrictum
  6. 3.Outer epidermis of testa consists of flat cells, with 1 layers……………..... M. macrocarpum
  7. 4.Seed ornamentation is alveolate or scalariform…………..………….……………...………5
  8. 4.Seed ornamentation is verrucate, ruminate or rugose……………..………………….…...…6
  9. 5.Seed ornamentation is alveolate……...…………………...…….………………….M. weissii
  10. 5.Scalariform…………………………………………………….………...…….… M. azureum
  11. 6.Seed ornamentation is verrucate or rugose…………………………….….………..…..……7
  12. 6.Ruminate………………...……...………………………………….……………………….11
  13. 7.Anticlinal cell walls are sunken……………………………….……...……………………...8
  14. 7.Anticlinal cell walls are unclear……………………………..……………..……………….10
  15. 8.Outer epidermis of testa consists of flat or polygonal cells……………….…..……………..9
  16. 8.Outer epidermis of testa consists of rectangular cells…………..……….....M. serpentinicum
  17. 9.Subepidermis of testa consists of rectangular cells……………………………..M. comosum
  18. 9.Subepidermis of testa consists of polygonal cells………………...…………M. microstomum
  19. 10.Outer epidermis of testa consists of flat cells……………………………..….M. anatolicum
  20. 10.Outer epidermis of testa consists of polygonal cells……………..……..…….M. artvinense
  21. 11.Outer epidermis is 1 layer……………………………..………………………....………..12
  22. 11.Outer epidermis is 2 or 3 layers………………………..…………...……………………..19
  23. 12.Outer epidermis of testa consists of crushed or rectangular cells…….………...………....13
  24. 12.Outer epidermis of testa consists of flat or polygonal……………….………..…...…..….14
  25. 13.Outer epidermis of testa consists of crushed..………………...…………….…..M. turcicum
  26. 13.Rectangular……………………….………………………………….…………..M. coeleste
  27. 14.Subepidermis layer is absent…………………..……………………..……...M. parviflorum
  28. 14.Subepidermis layer is present…………………………………………….……...……......15
  29. 15.Subepidermis is in parenchymatous structure……………...…...……….…M. sandrasicum
  30. 15.Subepidermis is in scleranchymatous structure……………….............……………..…...16
  31. 16.Subepidermis consists of crushed cells………………..…………...………..……..M. adilii
  32. 16.Subepidermis consists of flat or polygonal cells………..…………...……………….……17
  33. 17.Subepidermis consists of flat ………………………………...….……….……..M. bourgaei
  34. 17.Polygonal………………………...……………………………….…………….…………18
  35. 18.Crystals are present in the epidermis or subepidermis layers ………….…………M. vuralii
  36. 18.Crystals are absent…………..……………………………….…………….M. microstomum
  37. 19.Outer epidermis is 3 layers………………...……...……………….……...……….M. atillae
  38. 19.2 layers………………………………………...…………………………….…………….20
  39. 20.Subepidermis is in parenchymatous structure……….......………………....…….M. aucheri
  40. 20.Subepidermis is in scleranchymatous structure…………………….………M. tuzgoluensis
  41. 21.Seed shape is elliptic or oblong…………………..…………………..………...………….22
  42. 21.Seed shape is ovate or ovate-orbicular…………….………...……….………………...….25
    1. Seed shape is elliptic.…………..…………..……………………….…………..………...23
  43. 22.Oblong……………..…………..…………………………………………………..………24
  44. 23.Seed shape is elliptic-lanceolate…………..…………………….……….M. macbeathianum
  45. 23.Broadly elliptic……………………...…………………………..……..………M. neglectum
  46. 24.Seed shape is oblong-ovate…………………………...……..….………………....M. mirum
  47. 24.Oblong-elliptic………………………..………………………………..….…….M. longipes
  48. 25.Seed shape is ovate…………………..………………...………….……………………...26
    1. Seed shape is ovate-orbicular…………………...………………………………………...32
  49. 26.Seed surface ornamentation is alveolate or reticulate……………….…………….…..…..27
  50. 26.Seed surface ornamentation is ruminate or rugose………..………………………..……..29
  51. 27.Seed surface ornamentation is reticulate……………………....……………….…………28
  52. 27.Alveolate…………………………………………………………….……… M. caucasicum
  53. 28.Seed surface ornamentation is reticulate-foveate…….………………..………….M. elmasii
  54. 28.Reticulate-areolate……………………………..…...……………………..…….M. babachii
    1. Seed surface ornamentation is rugose…………………………...……….…...M. botryoides
  55. 29.Ruminate…………………………………………………..……………..………………..30
  56. 30.Outer epidermis of testa is 2-3 layers……………………………….……………...M. ufukii
  57. 30.Outer epidermis of testa is 1 layer…………………………………….…...……………...31
  58. 31.Subepidermis is 3-4 layers…………………...……………….……………...……M. erdalii
  59. 31.Subepidermis is 2 layers…………...………..……………..……………….M. massayanum
  60. 32.Seed ornamentation is ruminate or rugose…………………………….……..……….…...33
  61. 32.Seed ornamentation is reticulate or reticulate-areolate……………….………………..….34
  62. 33.Seed surface ornamentation is ruminate………...……………...………..……..M. latifolium
  63. 33.Rugose………...………………………..……………..…...……..M. sivrihisardaghlarensis
  64. 34.Seed surface ornamentation is reticulate………………...……...……………M. racemosum
  65. 34.Reticulate-areolate…………………………………...………………….………………...35
  66. 35.Crystals are present in the epidermis or subepidermis layers …….…………M. tenuiflorum
  67. 35.Crystals are absent………………...………..…………….…………………M. armeniacum

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