Morphological, Anatomical, Palynological and Karyological Studies on Endemic Alyssum kaynakiae (Brassicaceae) from Southwest Region of Turkey Author Aktürk, Ceren 0000-0003-0927-7308 Department of Pharmaceutical Botany, Faculty of Pharmacy, Biruni University, Istanbul, Turkey. & Department of Biology, Faculty of Art and Science, Bursa Uludağ University, Bursa, Turkey. & cerenakturk 5 @ gmail. com; https: // orcid. org / 0000 - 0003 - 0927 - 7308 cerenakturk5@gmail.com Author Yilmaz, Özer 0000-0003-1498-5827 Department of Biology, Faculty of Art and Science, Bursa Uludağ University, Bursa, Turkey. & ozery @ uludag. edu. tr; https: // orcid. org / 0000 - 0003 - 1498 - 5827 ozery@uludag.edu.tr text Phytotaxa 2022 2022-07-26 555 3 231 240 http://dx.doi.org/10.11646/phytotaxa.555.3.2 journal article 107631 10.11646/phytotaxa.555.3.2 d08af674-5cf7-47c0-8188-c535b47cfd44 1179-3163 6911061 Alyssum kaynakiae Yılmaz (sect. Gamosepalum (Hausskn.) T. R. Dudley) ( Fig. 1 ) Holotype :— TURKEY . C2 Denizli : Çameli , Üzümlü-Çameli , Kirazlıyayla 36° 58′ 35″ N , 29° 14′ 20″ E , 890 m a.s.l. , 9 May 2010 , Ö . Yılmaz 87/10-1 ( BULU ). FIGURE 1. General view of A. kaynakiae —Locality: Denizli: Üzümlü—Çameli, around Kirazlıyayla (10/17 BULU). Biennial with short, erect, or ascending, branched fertile stems, 1.5–13 cm tall. Indumentum of dense silvery-canescent, lepidote hairs, 0.2–0.6 mm in diameter. Leaves 3–10.5 × 0.5–2.5 mm , linear, broadly acute, or obtuse, slightly conduplicate, with dense indumentum, cauline leaves increasing in size upwards. Basal leaves sometimes present, oblong, 4–12 × 1.5–3.5 mm . Inflorescence raceme, usually short and sparse. Pedicels 1–3.5 mm in fruit, divergent. Sepals 2.5–5.5 × 1–3 mm , dimorphic, elliptic, narrowly oblong, broadly acute, or obtuse, margins membranous, upper surface with dense lepidote hairs, inner surface with sparse stellate hairs, appearing connate because of interlocking indumentum. Petals 3–8 × 1–2 mm , clearly exceeding the sepals, spatulate, claw margins entire, usually emarginate or occasionally truncate, hairy with dense lepidote hairs or glabrous, yellow. Siliculae 2–4.5 × 1.5–4 mm , oval or orbicular, valves equally inflated, hairy with minute dense lepidote hairs 0.2–0.3 mm in diameter, dehiscent, loculi 2 ovulate with apical placentation. Style 0.7–3.5 mm with dense lepidote hairs. Seed 1.5—2.0 mm, surfaces reticulatepapillate ( Fig. 2 ). Root anatomy. In the roots of A. kaynakiae , the epidermis consists of single-layered cells. Collenchyma is located below the epidermis with thinner lateral walls. It has 2 layers. Cortex is composed of 3-4 rows of parenchyma cells. Xylem tissue is surrounded by phloem tissues ( Fig. 5 , A and B ). Stem anatomy. In A. kaynakiae , the epidermis consists of single-layered, oblong cells surrounded by a cuticle layer. A small number of stomata were found between the epidermis cells. There are also lepidote hairs on the epidermis. Cortex is composed of 5–7 rows of parenchyma cells. Vascular bundles are collateral and surrounded by endodermis. Collenchyma is located below the endodermis. Phloem and xylem members are apparent. The pith of the stem is formed of large, round parenchyma cells ( Fig. 5 , C , and D). FIGURE 2. Micromorphology of A. kaynakiae . A. Lepidote hairs on cauline surface, B. Lepidote hairs on leaves, C. Lepidote hairs on the upper surface of leaves, D. Lepidote and stellate hairs on calyx surface, E. Fruit surface, F. Stylus, G. Seed, H. Seed surface—(10/17 BULU). Leaf anatomy. In the cauline leaves of A. kaynakiae , the epidermis consists of single-layered cells and is covered by lepidote hairs on the surface. The mesophyll layer consists of 2–3 palisade parenchyma cells and spongy parenchyma cells in the middle of leaves. Stomata are present on both sides of the leaf and located at the epidermis level. The vascular bundle is surrounded by bundle sheath cells. In the basal leaves, palisade and spongy parenchyma cells could not be differentiated ( Fig. 5 , E , and F ). A. kaynakiae has tricolpate pollen grains. The polar axis ( P ) is 36.34 ± 3.1 μm, and the equatorial axis ( E ) is 23.32 ± 2.1 μm. The ratio of P / E is 1.56 μm. The pollen grains are prolate in shape. Colpus length is 31.13 ± 3.25 μm, and colpus width is 1.18 ± 0.4 μm. The exine thickness is 1.5 ± 0.25 μm, and the intine thickness is 0.56 ± 0.1 μm. The exine ornamentation is reticulate ( Fig. 3 ). The chromosome number of A. kaynakiae is 2n=16 ( Fig. 4 ). FIGURE 3. The pollen morphology of A. kaynakiae . A. Reticulate ornamentation of the pollen grain, B. Colpus of the pollen grain (10/17 BULU). FIGURE 4. A–B. The chromosomes of A. kaynakiae (×1000). In this study, morphological, anatomical, palynological, and karyological characteristics of A. kaynakiae were analysed. In terms of micromorphological properties, A. kaynakiae has densely lepidote hairs. The stem, leaves, and fruits surfaces of A. kaynakiae are covered by lepidote hairs, whereas sepals have stellate hairs along with lepidote hairs. The seed surface of A. kaynakiae is reticulate-papillate. Karabacak et al. (2016) reported in their study that seed surfaces of Alyssum species are reticulate, reticulate-papillate, and papillate. In the roots of A. kaynakiae , the primary structure was observed. The epidermis is undivided, and collenchyma tissue takes part just below the epidermis. The cortex is composed of several rows of parenchyma cells under the collenchyma. There is no cavity between the parenchyma cells that create the cortex tissue. Xylem is surrounded by phloem tissue. In the stem of A. kaynakiae , the epidermis is single-layered. The cortex layer is composed of 5–7 rows of parenchyma cells. Single-layer collenchyma and endodermis are located under the cortex parenchyma. The number of vascular bundles varies from 9 to 11. The pith region is formed entirely of parenchyma cells. Cauline and basal leaves of A. kaynakiae were analysed. For the cauline leaves, the surface is covered by lepidote hairs, and the epidermis consists of single-layered cells. Cauline leaves include the mesophyll layer with 2–3 palisade parenchyma and spongy parenchyma cells in the middle of the leaves. Both sides of the leaves have stomata located at the epidermis level. Bundle-sheath cells surround the vascular bundle. Palisade and spongy parenchyma cells could not be differentiated in the basal leaves. Nazari et al. (2013) underlined that some Iranian species of A . sect. Gamosepalum could be distinguished not only morphologically but also by anatomical characters (anatomy of peduncle). In the study of Yılmaz (2012) , it was pointed out that A. kaynakiae has a close morphological affinity with A. niveum and A. harputicum . There are currently no sufficient detailed anatomical studies on A. niveum , but the anatomy of A. harputicum was thoroughly by Kürşat et al. (2008) . The anatomical comparison of A. kaynakiae with A. harputicum is summarised in Table 1 . This anatomical comparison emphasises the differences between the two species in root and stem structures. Regarding the root structure, while A. harputicum has a secondary structure ( Kürşat et al. 2008 ), A. kaynakiae has a primary structure as the most significant distinction. Supplementarily, the stem structure of A. harputicum contains both a wood-like and herbaceous stem, but the stem structure of A. kaynakiae is only herbaceous. These findings suggest that anatomical characteristics could be helpful for the distinction of plants whose phylogenetic relationships are yet unknown. Consistent with this idea, a study that comprised taxa in tribe Alysseae by Karaismailoğlu (2020) proposed that anatomical characters can be helpful in the further delimitation of plants that necessitate new arrangements. FIGURE 5. A. Root cross-section (×100), B. Root cross-section (×400), C. Stem cross-section (×100), D. Stem cross-section (×400), E. Leaves (×100), F. Leaves (×400); e: epidermis, cl: collenchyma, phl: phloem, xy: xylem, co: cortex, sp: spongy parenchyma, pp: palisade parenchyma, bs: bundle sheat, v: vascular bundle (11/17 BULU). In this study, the properties of pollen morphology of A. kaynakiae were determined by using a Light microscope and a Scanning electron microscope. The results show that the pollen shape of A. kaynakiae is prolate. In addition, pollen grains have tricolpate apertures and reticulate ornamentation. Some Alyssum species’ pollen morphology was also reported earlier ( Bulbul et al. 2019 , Başer et al. 2018 , Kürşat et al. 2008 ). As was noted by these studies, the pollen grains of the Alyssum species are sub-prolate and prolate, and their ornamentation is reticulate and has tricolpate apertures. The pollen structure of A. kaynakiae complies with this information. Erdtman (1966) described the Brassicaceae as a stenopalynous family. According to Erdtman’s (1966) study, pollen grains of Brassicaceae are usually tricolpate, and exine ornamentation is usually reticulate. Khalik et al. (2002) indicated that exine ornamentation plays a significant role within the tribe, family, and between species in the same genus of Brassicaceae . The authors also reported minor differences in pollen size, shape, and aperture but significant variation in exine ornamentation. There are three different types of exine ornamentation (microreticulate, reticulate, and coarsely reticulate) based on variation in diameter of the lumina in the studied species of the family Brassicaceae ( Khalik et al. 2002 ) . Gabr (2018) reported two types of exine ornamentation (reticulate and coarsely reticulate) in ten species represented in 9 genera and 5 tribes of Brassicaceae . Besides exine ornamentation, exine thickness is also an important character to distinguish species of the family Brassicaceae ( Amina et al. 2020 ) . It was reported that morphological properties of exine are significant to explain the phylogenetic relationship of taxa ( Walker 1974 , Kuprianova 1967 ). Some studies showed that exine ornamentation and thickness are affected by enhanced UV irradiation ( Koti et al. 2004 , Yeloff et al. 2008 ). Yeloff et al. (2008) reported that the exine thickness of pollen grains increased with the UV treatments. Besides the UV irradiance, habitat and soil conditions, including nutrient and water availability, may also influence pollen morphology. According to the study by Mutlu and Erik (2012) , habitat properties and altitudes are significant for the exine thickness of some species of the family Brassicaceae . When the exine thickness of A. kaynakiae is compared to other species ( Bulbul et al. 2019 , Başer et al. 2018 , Kürşat et al. 2008 ), A. kaynakiae has been found to have the highest exine thickness, as shown in Table 2 . This quality of A. kaynakiae can be attributed to its arid and open habitat. This information is consistent with the explanation mentioned above regarding the habitat effect on exine thickness. TABLE 1. Comparing anatomical properties of A. kaynakiae (present study) and A. harputicum ( Kürşat et al. 2008 ) .

Kürşat et al. (2008)	Present study
Alyssum harputicum	Alyssum kaynakiae
Structure	Secondary structure	Primary structure
Root Anatomy	Collenchyma	Inside the cortex layer	Under the epidermis layer
Sclerenchyma	Inside the lignified xylem area	–
Structure	Wood-like stem Herbaceous stem Secondary structure –	Primary structure
Epidermis	– Single-layered	Single-layered
Collenchyma	Inside the cortex layer –	Under the cortex layer
Stem Anatomy	Sclerenchyma	Inside the lignified Inside the cortex layer xylem area	–
Number of vascular bundles	– 12–15	9–11
Pith region	Composed Composed of parenchyma cells of parenchyma cells	Composed of parenchyma cells
Type	Isolateral	Isolateral
Trichomes	Lepidote hairs	Lepidote hairs
Leaf Anatomy	Epidermis	Single layered	Single layered
Stomata	On both surfaces	On both surfaces
Mesophyll	2–3 layered palisade parenchyma cells	2–3 layered palisade parenchyma cells

Uysal et al. (2017) revealed that the chromosomes generally give data relevant to the taxonomy of plants. The Brassicaceae family is well known for the considerable variation in chromosome numbers and the common occurrence of polyploids ( Marhold and Lihová, 2006 ). Polyploidy has a significant role in the taxa of the Brassicaceae family in terms of conducting to speciation and evolution ( Španiel et al. 2015 ). Chromosome and ploidy variation is essential also in the tribe Alysseae and the genus Alyssum ( Španiel et al. 2015 ) . According to the AlyBase database, the genus Alyssum includes diploid, polyploid, or diploid-polyploid taxa ( Španiel et al. 2015 ). Diploid taxa with 2n = 16 are most common in this genus, and the most frequent base chromosome number is x = 8; on the other hand, x = 7 ( Alyssum umbellatum Desv. ) was also reported to a lesser extent ( Španiel et al. 2015 , Cetlová et al. 2021 ). In line with these data, the counted 16 chromosomes in metaphase cells of Alyssum kaynakiae demonstrate that it is diploid. This study brought the first chromosome number record for this species.