Stojković et al. 2023, Biologica Nyssana 14(2) 14 (2) December 2023: 97-106 DOI: 10.5281/zenodo.10209805 Morphological variability and differentiation of selected Achillea species (sections Achillea sensu lato and Anthemoideae) from Serbia Original Article Jelena P. Stojković Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia jelenapstojanovic93@gmail.com (corresponding author) Zorica S. Mitić Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia Bojan K. Zlatković Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia Received: September 26, 2023 Revised: October 22, 2023 Accepted: October 27, 2023 Abstract: In the present study, morphological variability and differentiation of 13 Achillea species, including 11 from the Achillea section Achillea sensu lato (A. chrysocoma, A. clypeolata, A. coarctata, A. collina, A. crithmifolia, A. distans, A. grandifolia, A. millefolium, A. nobilis, A. pseudopectinata and A. vandasii) and two species from the Achillea section Anthemoideae (A. ageratifolia and A. lingulata) from Serbia were analyzed. Morphometric analysis included a total of 9 quantitative characteristics from the vegetative and reproductive plant parts. Multivariate statistical analyses reflected almost clear differentiation between species from two studied sections whereby ligulate floret length and stem leaf segment length had the greatest influence for their distinction. Thus, the section Achillea sensu lato was characterized by significantly shorter ligulate florets and longer segments of stem leaf compared to the section Anthemoideae. Furthermore, A. grandifolia featured the longest segments of stem leaf and showed considerable separation from all other species within the section Achillea sensu lato. It could be concluded that the selected set of morphological characteristics in the present study mainly positioned the studied species in accordance with the current infrageneric classification of Achillea. Key words: Achillea, morphometry, multivariate statistical analysis, Balkan Peninsula Apstrakt: Morfološka varijabilnost i diferencijacija odabranih vrsta roda Achillea (sekcija Achillea sensu lato i Anthemoideae) iz Srbije U ovom istraživanju analizirana je morfološka varijabilnost i diferencijacija 13 vrsta roda Achillea, uključujući 11 iz sekcije Achillea sensu lato (A. chrysocoma, A. clypeolata, A. coarctata, A. collina, A. crithmifolia, A. distans, A. grandifolia, A. millefolium, A. nobilis, A. pseudopectinata i A. vandasii) i dve vrste iz sekcije Anthemoideae (A. ageratifolia i A. lingulata) iz Srbije. Morfometrijskom analizom obuhvaćeno je ukupno 9 kvantitativnih karaktera iz vegetativnih i reproduktivnih delova biljaka. Multivarijantne statističke analize pokazale su gotovo jasnu diferencijaciju izmedju vrsta iz dve istraživane sekcije, pri čemu su dužina jezičastog cveta i dužina režnja lista stabla imale najveći uticaj na njihovo razlikovanje. Tako se sekcija Achillea sensu lato odlikovala znatno kraćim jezičastim cvetovima i dužim režnjevima lista stabla u odnosu na sekciju Anthemoideae. Osim toga, A. grandifolia je imala najduže režnjeve lista stabla i pokazala značajno odvajanje od svih ostalih vrsta u okviru pomenute sekcije. Može se zaključiti da je odabrani skup morfoloških karaktera u ovoj studiji pozicionirao istraživane vrste uglavnom u skladu sa aktuelnom infrageneričkom klasifikacijom roda Achillea. Ključne reči: Achillea, morfometrija, multivarijantne statističke analize, Balkansko polu- ostrvo Introduction Genus Achillea L. (yarrows) is one of the most polymorphic and taxonomically complicated genera in the family Asteraceae, including more than 100 species, distributed in Europe, temperate climate Asia and few species in North America (Saukel et al., 2003; Ehrendorfer & Guo, 2006; Guo et al., 2006). In the flora of Serbia, 27 Achillea species have been recognized (Euro+Med, 2006). © 2023 Stojković et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 97 The genus is characterized by the ability to adapt in different ecological amplitudes and ranges in extreme environmental conditions, such as deserts to water-logged habitats and from sea coastal areas to the high mountains (Ehrendorfer & Guo, 2006). Consequently, several widespread Achillea species show extremely high morphological variability and complexity. In addition, the presence of spontaneous hybrids, allo- and autopolyploids, aneuploids and phenocopies results in a great cytological, morphological and phytochemical variability both at inter- and at intraspecific level. Therefore, taxonomic evaluation, identification of species and classification has been a difficult task for decades (Inotai et al., 2016). Taxonomical grouping within the genus has been rearranged several times in the past and intensively researched even today (Kidlovits & Nemeth, 2012). Based on previous morpho-anatomical and ecogeographical analyses, many classification schemes of the genus Achillea have been proposed (Afanasyev & Bochantsev, 1961; Huber-Morath, 1975). However, the most recent multidisciplinary studies recognized five sections within this genus: Achillea sensu lato, Anthemoideae (DC.) Heimerl, Babounya (DC.) O. Hoffm., Ptarmica (Mill.) W. D. J. Koch and Otanthus (Hoffmanns. & Link) Ehrend. & Y. P. Guo (Saukel et al., 2003; Guo et al., 2004; Ehrendorfer & Guo, 2005). Certain morphological characteristics, including characteristics of leaves, florets and pollen grains may be useful for morphological characterization and classification of Achillea taxa (Saukel & Länger, 1992; Valant-Vetschera & Kastner, 2000; Rauchensteiner et al., 2002; Nedelcheva, 2008; Azani et al., 2009; Akyalcin et al., 2011). The main aims of the present study were: 1) determination of highly variable morphological characteristics as well as those of low variability in two sections, Achillea sensu lato and Anthemoideae, and 2) assessment of the degree of differentiation of the studied species based on selected set of morphological characteristics. Materials and Methods Plant material Plant material (whole plants) of 13 Achillea species, including 11 from the section Achillea sensu lato (A. chrysocoma Friv., A. clypeolata Sm., A. coarctata Poir., A. collina (Becker ex Rchb. f.) Heimerl, A. crithmifolia Waldst. & Kit., A. distans Waldst. & Kit. ex Willd., A. grandifolia Friv., A. millefolium L., A. nobilis L., A. pseudopectinata Janka and A. vandasii Velen.) and two species from the section Anthemoideae (A. ageratifolia (Sm.) Benth. & Hook. f. and A. lingulata Waldst. & Kit.) were collected in their natural habitats in Serbia. Additionally, plant material of one population of A. pseudopectinata was collected in North Macedonia. The voucher specimens of each population were deposited in the „Herbarium Moesiacum Niš” (HMN) of the Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš. The map of the studied area, with locations of the selected populations, is shown in Fig. 1. Furthermore, the original data on position, habitat and voucher information for each population are listed in Tab. 1. Plant material was identified by Dr. B. K. Zlatković, Dr. Z. S. Mitić and M.Sc. J. P. Stojković. 98 BIOLOGICA NYSSANA ● 14 (2) December 2023: 97-106 Stojković et al. ● Morphological variability and differentiation of selected Achillea species (sections Achillea sensu lato and Anthemoideae) from Serbia Fig. 1. Geographical position of studied populations. For the description of the location and habitat conditions of the populations, cf. Tab. 1 Morphometric analysis A total number of 384 individuals from 26 populations and 13 species of Achillea, from two different sections, were compared at the level of 9 quantitative morphological characteristics. Quantitative traits such as rosette leaf segment length (RLSL) / rosette leaf tooth length (RLTL), stem leaf segment length (SLSL) / stem leaf tooth length (SLTL), ligulate floret length (LFL), tubular floret length (TFL) and involucral bract length (IBL) of each examined individual were mounted as permanent microscope slides and photographed after preparation with a Leica MZ-16A binocular magnifier (Leica Microsystems©, Wetzlar, Germany). As a background, graph paper was used in the photography process. Each characteristic was measured with three consecutive measurement repetitions. All measurements of the mentioned properties were performed using Digimizer Image Analysis Software (MedCalcSoftware©, Belgium), BIOLOGICA NYSSANA ● 14 (2) December 2023: 97-106 Stojković et al. ● Morphological variability and differentiation of selected Achillea species (sections Achillea sensu lato and Anthemoideae) from Serbia 99 Ta bl e 1. P os iti on a nd h ab ita t d es cr ip tio n of s tu di ed p op ul at io ns o f A ch ill ea s pe ci es Se ct io n Sp ec ie s L oc al ity L at itu de (N ) L on gi tu de (E ) A lti tu de (m a. s.l .) Su bs tr at um N um be r of in di vi du al s an al yz ed D at e of co lle ct io n N um be r of va uc he r (H M N ) Ac hi lle a se ns u la to A. c hr ys oc om a Se rb ia : M t. B es na K ob ila 42 °3 1' 05 " 22 °1 3' 05 " 19 23 Si lic at e 15 Ju ly 2 02 0 14 30 0 A. c ly pe ol at a Se rb ia : S ić ev ač ka go rg e 43 °1 8' 36 " 22 °1 0' 49 " 27 6 Li m es to ne 15 M ay 2 01 9 13 95 7 A. c ly pe ol at a Se rb ia : V lk ov ija 43 °0 5' 02 " 22 °5 4' 24 " 10 20 Li m es to ne 15 Ju ne 2 02 1 14 31 0 A. c oa rc ta ta Se rb ia : P či nj a riv er va lle y, B ud ov ija 42 °2 0' 23 " 21 °5 3' 26 " 44 0 Si lic at e 15 Ju ne 2 01 8 13 68 1 A. c oa rc ta ta Se rb ia : S ić ev ač ka go rg e 43 °3 3' 42 " 22 °0 6' 95 " 36 5 Li m es to ne 15 Ju ne 2 01 8 13 68 5 A. c oa rc ta ta Se rb ia : T em sk a 43 °1 7' 06 " 22 °3 5' 06 " 44 0 Si lic at e 15 Ju ly 2 02 0 14 30 3 A. c ol lin a Se rb ia : P či nj a riv er va lle y, B ud ov ija 42 °2 0' 23 " 21 °5 3' 26 " 68 0 Si lic at e 15 Ju ne 2 01 9 13 95 2 A. c ri th m ifo lia Se rb ia : P či nj a riv er va lle y, B ud ov ija 42 °2 0' 23 " 21 °5 3' 26 " 44 0 Si lic at e 22 Ju ne 2 01 8 13 68 4 A. c ri th m ifo lia Se rb ia : K un ov ic a 43 °1 7' 30 " 22 °0 6' 34 " 46 7 Li m es to ne 15 Ju ne 2 01 8 13 68 0 A. d is ta ns Se rb ia : M t. St ar a pl an in a, B ab in z ub 43 °2 3' 04 " 22 °3 5' 35 " 16 00 Si lic at e 15 Ju ly 2 02 0 14 30 5 A. d is ta ns Se rb ia : K op ao ni k 43 °1 8' 51 " 20 °5 1' 01 " 13 70 Si lic at e 15 Ju ly 2 02 0 14 30 6 A. g ra nd ifo lia Se rb ia : J er m a go rg e 42 °5 8' 18 " 22 °3 7' 23 " 54 8 Li m es to ne 15 Ju ne 2 02 0 14 30 2 A. g ra nd ifo lia Se rb ia : T em sk a 43 °1 6' 56 " 22 °3 5' 04 " 44 0 Si lic at e 5 Ju ly 2 02 0 14 61 4 A. m ill ef ol iu m Se rb ia : S ić ev ač ka go rg e 43 °2 0' 04 " 22 °0 4' 09 " 24 0 Li m es to ne 15 Ju ne 2 01 8 13 68 2 A. m ill ef ol iu m Se rb ia : N iš 43 °1 8' 19 " 21 °5 3' 50 " 26 0 N eo ge ne se di m en ts 15 Ju ne 2 01 9 13 95 5 A. m ill ef ol iu m Se rb ia : L al in ač ka sa lt m ar sh 43 °2 0' 42 " 21 °4 4' 45 " 20 0 N eo ge ne se di m en ts 15 M ay 2 01 9 14 40 0 A. m ill ef ol iu m Se rb ia : P či nj a riv er va lle y, B rn ja re 42 °2 3' 30 " 21 °5 5′ 01 " 50 7 N eo ge ne se di m en ts 15 Ju ne 2 01 9 13 96 1 100 BIOLOGICA NYSSANA ● 14 (2) December 2023: 97-106 Stojković et al. ● Morphological variability and differentiation of selected Achillea species (sections Achillea sensu lato and Anthemoideae) from Serbia Ac hi lle a se ns u la to A. n ob ili s Se rb ia : S ić ev ač ka go rg e 43 °2 0' 04 " 22 °0 4' 09 " 36 5 Li m es to ne 15 Ju ne 2 01 8 13 68 3 A. n ob ili s Se rb ia : L al in ač ka sa lt m ar sh 43 °2 0' 42 " 21 °4 4' 45 " 20 0 N eo ge ne se di m en ts 15 Ju ne 2 02 0 14 29 8 A. p se ud op ec tin at a N or th M ac ed on ia : K aj m ak ča la n 40 °5 9' 12 " 21 °4 0' 06 " 71 8 Si lic at e 12 Ju ne 2 02 0 14 30 9 A. p se ud op ec tin at a Se rb ia : O bl ač in a 43 °1 8' 35 " 21 °4 0' 15 " 33 3 Si lic at e 15 Ju ne 2 02 0 14 30 1 A. v an da si i Se rb ia : V ra nd ol 43 °1 6' 41 " 22 °1 5' 31 " 42 6 C ar bo na te 15 Ju ne 2 02 0 14 30 4 An th em oi de ae A. a ge ra tif ol ia Se rb ia : B or , M t. V el ik i K rš 44 °1 0' 47 " 22 °0 4' 30 " 99 5 C ar bo na te 15 Ju ne 2 01 9 13 95 4 A. a ge ra tif ol ia Se rb ia : J el aš ni čk a go rg e 43 °1 6' 37 " 22 °0 4' 09 " 36 0 Li m es to ne 15 M ay 2 02 1 14 54 2 A. li ng ul at a Se rb ia : M t. St ar a pl an in a, B ab in z ub 43 °2 2' 06 " 22 °3 5' 42 " 16 00 Si lic at e 15 Ju ne 2 01 9 13 95 3 A. li ng ul at a Se rb ia : M t. B es na K ob ila 42 °3 1' 45 " 22 °1 3' 40 " 18 50 Si lic at e 15 Ju ly 2 02 0 14 29 9 on previously taken photographs. Characteristics of large plant parts, such as rosette leaf length (RLL), and stem leaf length (SLL), were determined using a ruler. In contrast, rounded structures, capitulum length (CL) and capitulum width (CW), were measured with a high-precision digital meter (Mahr Federal 410710716U, Esslingen, Germany). Statistical analysis The statistical analysis of the obtained data was performed using the STATISTICA 8 software (StatSoft, Inc., Tulsa, OK, USA). Statistical data processing included descriptive statistics and multivariate statistical analyses: canonical discriminant analysis (CDA), and agglomerative hierarchical clustering (AHC). Unweighted pair group average (UPGMA) was used as a criterion for the clusters development, and Euclidean distances as diversity assessment criteria in AHC analysis. Results and discussion Variability of morphological characteristics of the studied Achillea species Results of descriptive statistics for 9 quantitative morphological characteristics of 13 Achillea species were presented in Tab. 2. According to the obtained results, most of the studied characteristics were within a moderate degree of variability (10