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. 2022 Nov 28;8(12):e11895. doi: 10.1016/j.heliyon.2022.e11895

Phenotypic diversity of rosemary (Salvia rosmarinus Schleid.) accessions for qualitative characters

Zewdinesh Damtew Zigene a,∗, Bizuayehu Tesfaye Asfaw b, Tesfaye Disasa Bitima c
PMCID: PMC9718970  PMID: 36471846

Abstract

Rosemary is one of the industrially significant crops traded over the world. Its cultivation in many countries depends on locally adapted populations rather than on the use of improved varieties. Collection and characterization of the local populations could contribute to their conservation and selection of desirable traits. Therefore, this study was undertaken to estimate the diversity of 45 rosemary accessions collected from different parts of Ethiopia using qualitative morphological traits. The frequency distribution of characters reflected the polymorphism of the studied characters, and a total of 39 phenotypic classes were observed. Shannon-weaver diversity indices estimated across accessions ranged from 0.43 to 0.99 with a mean value of 0.79, demonstrating the existence of adequate phenotypic diversity among the accessions. Estimates of diversity indices within and among growing regions revealed that intra-region diversity (0.57) exceeds inter-region diversity (0.43). Cluster analysis classified the accessions into six major clusters regardless of the accessions' geographical origin. This was consistent with the estimated within and among growing regions diversity indices. The results clearly showed the presence of considerable levels of phenotypic diversity that could be exploited as a source of a valuable gene pool for future breeding programs.

Keywords: Accessions, Qualitative characters, Salvia rosmarinus, Shannon diversity

Accessions; Qualitative characters; Salvia rosmarinus; Shannon diversity.

1. Introduction

Analysis of genetic variability and estimation of relationships among accessions are the first basic steps in a meaningful breeding program (Aremu, 2012; Birhanu et al., 2017). The presence of genetic diversity in crops is essential and provides an option for the development of improved varieties (Govindaraj et al., 2015). Diversity in crops has been assessed using molecular markers, morphological, agronomical, chemical and nutritional traits (Suresh et al., 2014; Animasaun et al., 2015; Temesgen et al., 2015; Shimeles et al., 2016; Satyal et al., 2017; Solomon et al., 2019; Lee et al., 2020; Prasad et al., 2020; Zewdinesh et al., 2021). However, the use of morphological traits stands out as the first and the main instrument for the quantification of genetic diversity and identification of plant populations (Gerrano et al., 2017; Lazaridi et al., 2017; Carneiro et al., 2019).

Collected germplasm needs to be evaluated both for quantitative and qualitative morphological traits, and these traits are the targets of selection in crop improvement programs (Bonny et al., 2019). Qualitative morphological traits are essential to estimate genetic diversity among germplasm because: they are governed by allelic differences, have high heritability, can be easily identified by naked eyes, and their expressions are less influenced by environmental factors (Briggs and Knowles, 1967; van Hintum et al., 2000). They appeared to be expressed in all environments and the classification of plants using these qualitative traits could be applied to any year, location, or environment, and hence they are considered more important for characterizing plants’ genetic resources (Berg et al., 1993). Qualitative traits are also the basis of selection both in breeding and utilization of accessions by farmers.

Several studies have been conducted to estimate the genetic diversity of crops using qualitative traits (Ayan and Bekele, 1998; Hadish, 2013; Beemnet, 2018; Shimeles, 2018; Bonny et al., 2019). So far, morphological, molecular, and chemical characterization of rosemary genotypes has been conducted in different countries, and the studies showed the presence of high variability among genotypes (Satyal et al., 2017; Nunziata et al., 2019; Carrubba et al., 2020). Characterizations of rosemary genotypes for morphological traits denoted the presence of variability in terms of growth habit, leaf morphology, flower color, calyx and style characteristics, and flowering habit (Mulas et al., 2002; Cervelli and Masselli, 2013; Mateu-Andres et al., 2013; Carrubba et al., 2020). Furthermore, characterization of the essential oils and extracts for their flavonoid content, antioxidant, anticancer, antimicrobial, and cholinesterase inhibitory activity has been studied in rosemary and related species and showed higher variability (Zaouali et al., 2010; Sytar et al., 2015; Karakaya et al., 2020; Leporini et al., 2020).

In Ethiopia, limited study has been conducted on morphological, molecular, and chemical traits variability (Beemnet et al., 2013; Bekri et al., 2018, Zewdinesh et al., 2021). However, no research effort has been done in the investigation of qualitative morphological traits diversity. But analyzing qualitative morphological traits and knowing the extent of variability among accessions and populations is crucial for effective management, improvement, and utilization of the crop. Given the scarcity of studies regarding the characterization of the available rosemary germplasms and the significance of the study towards its use in the collection, conservation, and breeding programs; this experiment was designed to determine the phenotypic diversity of Ethiopian rosemary accessions using standard qualitative morphological descriptors.

2. Materials and methods

2.1. Study area

The experiment was conducted in the southern region of Ethiopia from 2018 to 2019 at Wondo Genet Agricultural Research Center, Wondo Genet, Ethiopia. The site is located at 7°19′N latitude and 38° 38′E longitudes with an altitude of 1780 masl. The annual mean rainfall of the area in 2018 and 2019 were 1441.3 mm and 1317.5 mm, respectively. The annual average minimum temperature varies from 11.8 to 15.1 °C, whereas the annual mean maximum temperature is between 25.1 and 29.7 °C. The soil of the experimental area is fertile, well drained and sandy loam with a pH of 6.4 (Abayneh et al., 2006).

2.2. Plant materials and experimental procedures

A total of 45 rosemary accessions were used for this activity (Table 1). The forty three rosemary accessions were originally collected from farmers’ fields at different agro-ecology of Ethiopia (Figure 1), and two accessions were obtained from commercial farms, which were introduced by private investment sectors in Ethiopia. Seedlings were prepared from soft stem cuttings of all accessions and raised in the nursery. After three months, well performing and uniformly grown seedlings were transplanted to the experimental field using Randomized Complete Block Design (RCBD) with three replications. Plants, plots and blocks were spaced by 0.6 m, 1 m, and 1.5 m, respectively. Watering the experiment was carried out once a week during dry seasons, and the experimental field was managed properly and kept weed-free throughout the experimentation.

Table 1.

Rosemary accessions used for the study and their area of collections.

No Accessions code Geographic origin
1 Ros02 Wolaita
2 Ros03 Wolaita
3 Ros05 Wolaita
4 Ros14 Wolaita
5 Ros35 Wolaita
6 Ros36 Wolaita
7 Ros01 Hadiya
8 Ros04 Hadiya
9 Ros15 Hadiya
10 Ros37 Hadiya
11 Ros16 Hadiya
12 Ros08 Gurage
13 Ros30 Gurage
14 Ros31 Gurage
15 Ros33 Gurage
16 Ros38 Gurage
17 Ros39 Gurage
18 Ros32 Gurage
19 Ros13 Sidama
20 Ros42 Sidama
21 Ros43 Sidama
22 Ros44 Sidama
23 Ros45 Sidama
24 Ros40 Arssi
25 Ros41 Arssi
26 Ros26 Arssi
27 Ros27 Arssi
28 Ros12 Arssi
29 Ros20 North Shewa
30 Ros21 North Shewa
31 Ros22 North Shewa
32 Ros23 North Shewa
33 Ros24 North Shewa
34 Ros25 North Shewa
35 Ros06 Gonder Zuria
36 Ros07 Gonder Zuria
37 Ros09 Harari
38 Ros10 Harari
39 Ros11 Harari
40 Ros34 Harari
41 Ros17 Harari
42 Ros18 Harari
43 Ros19 Harari
44 Ros28 Commercial farm
45 Ros29 Commercial farm
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Figure 1.

Figure 1

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Map of Ethiopia depicting the collection areas of rosemary accessions used for the study. N. Shewa, North Shewa; N. Gonder, North Gonder.

2.3. Data collection

Data were recorded for 15 qualitative morphological traits using International Union for the Protection of New Varieties of Plants descriptors given for rosemary (UPOV, 2007). All flower traits related data were recorded for accessions that produced flowers. Ten representative samples per plot were selected and data were recorded as described in Table 2.

Table 2.

Codes and descriptions of the characters used for the study.

Characters Code Description
Growth habit 1. Erect
2. Semi-erect
3. prostrate
Flower arrangement 1. Opposite
2. Whorl
Position of long side branches 1. Mainly basal
2. Mainly upper
3. Along whole stem
Stem pubescence 1. Absent or weak
2. Moderate
3. Strong
Leaf variegation 1. Absent
9. Present
Leaf green color 3. Light
5. Medium
7. Dark
Curvature of longitudinal axis 1. Incurved
2. Straight
3. Re-curved
Re-curving of margin 3. Weak
5. Medium
7. Strong
Intensity of flower blue color 3. Light
5. Medium
7. Dark
Calyx shape 1. Funnel-shape
2. Campanulate
Calyx anthocyanin coloration 1. Absent
9. Present
Calyx pubescence 3. Weak
5. Medium
7. Strong
Style length in relation to stamen 1 Equal
2 Longer
Flowering habit 1 Not flowering
2 Seasonal flowering
Time of beginning of flowering 3 Early
5 Medium
7 Late
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2.4. Data analysis

For data analysis, the accessions were grouped into nine classes based on their area of collections (Wolaita, Hadiya, Gurage, Sidama, Arssi, North Shewa, Gonder, Harari and Commercial farm). Phenotypic frequency distributions of the qualitative traits were worked out for all of the accessions and collection regions. A chi-square test of independence was conducted to determine whether or not the characters and growing areas were dependent, while, a chi-square test of homogeneity was applied to test the homogeneity of the populations from the different growing areas. The calculation was performed using the formula given by Bolboaca et al. (2011). Calculated chi-square values were tested against tabulated value for its significance at df = (# rows–1) × (# columns–1) for independence test and at df = # rows–1 for homogeneity test.

Where, # rows and # columns are the number of collection regions and the number of phenotypic classes of each character, respectively.

The diversity index (H′) of Shannon and Weaver (1949) was used to measure the phenotypic diversity of the entire sample and the samples group for each growing regions. The Shannon-Weaver diversity index (H′) as described by Hennink and Zeven (1991) is given as:

H′=−∑i=1npi∗ln(pi)

where: H′, Shannon-Weaver Diversity Index; pi, the proportion of genotypes in the ith class of an n-class character; n, the number of phenotypic classes of a character. To normalize and keep the value between 0 and 1, each diversity index value was divided by Ln (n).

The partitioning of the phenotypic diversity into within (Hcr/Hsp) and between regions of collections ((Hsp − Hcr)/Hcr) was executed following the methods given by Wachira et al. (1995).

Where: Hcr and Hsp, are Shannon-Weaver diversity index across collection regions and across accessions, respectively.

From the qualitative data of the entire accessions cluster analyses were performed using MINITAB ver.17 software after standardizing the data to mean zero and unity variance (van den Berg et al., 2006). The average linkage method based on Euclidean distances was carried out to generate the dendrogram.

3. Result and discussion

3.1. Characterization of the entire collection

The entire rosemary collections were characterized by the presence of 39 varied phenotypic classes (Table 3). For the whole accessions, three categories of growth habits were observed (Figure 2). The predominant type was semi-erect growth habit (53.3%) followed by erect growth habits (40%). Only 6.7% of the accessions showed prostrate growth habit. Our result was in line with Mulas et al. (2002) who found upright, intermediate, and prostrate growth habit, with predominance of intermediate growth habit for rosemary genotypes in Sardinia. Besides, our observation partially agrees with Cervelli and Masselli (2013) who categorized the growth of Italian rosemary cultivars into three distinct growth habit (erect, semi-erect, and prostrate type), but with the predominant of erect growth type. Consistent result was also reported by Carrubba et al. (2020) for Sicilian rosemary genotypes. Growth habit of the crop could be used as an important trait for selection purpose. Accessions with intermediate and prostrate growth habit are suitable for leaf production, whereas accessions representing upright/erect growth habit producing lesser leaves and essential oils; and might be suitable for ornamental as well as hedge purposes (Mulas et al., 2002; Beemnet et al., 2013).

Table 3.

Proportion of phenotypic classes for 15 qualitative traits in rosemary accessions.

Characters Code Character classes Frequency Percentage
Growth habit 1 Erect 18 40
2 Semi-erect 24 53.3
3 prostrate 3 6.7
Flower arrangement 1 Opposite 19 46.3
2 Whorl 22 53.7
Position of long side branches 1 Mainly basal 11 24.4
2 Mainly upper 10 22.2
3 Along whole stem 24 53.3
Stem pubescence 1 Absent or weak 20 44.4
2 Moderate 17 37.8
3 Strong 8 17.8
Leaf variegation 1 Absent 38 84.4
9 Present 7 15.6
Leaf green color 3 Light 4 8.89
5 Medium 37 82.22
7 Dark 4 8.89
Curvature of longitudinal axis 1 Incurved 20 44.4
2 Straight 10 22.3
3 Re-curved 15 33.3
Re-curving of margin 3 Weak 12 26.7
5 Medium 14 31.1
7 Strong 19 42.2
Intensity of flower main blue color 3 Light 34 83
5 Medium 3 7.3
7 Dark 4 9.7
Calyx shape 1 Funnel-shape 30 73.2
2 Campanulate 11 26.8
Calyx anthocyanin coloration 1 Absent 32 78
9 Present 9 22
Calyx pubescence 3 Weak 25 61
5 Medium 8 19.5
7 Strong 8 19.5
Style length in relation to stamen 1 Equal 7 17
2 Longer 34 83
Flowering habit 1 Not flowering 4 8.89
2 Seasonal 41 91.11
Time of beginning of flowering 3 Early 10 24.4
5 Medium 11 26.8
7 Late 20 48.8
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Figure 2.

Figure 2

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Diversity in growth habit and leaf variegation of rosemary accessions. Erect growth habit (A), semi-erect growth habit (B), prostrate growth habit (C) and leaves with variegation (D).

The majority of the studied accessions had long side branches along their whole stem (53.3%), while the respective proportion of accessions with long side branches mainly basal and mainly upper were 24.4% and 22.2%. It was found that most of the accessions had no or weak pubescence on their stem (44.4%) however, 37.8% and 17.8% of the accessions were characterized by their moderate and strong stem pubescence, respectively. Regarding flower arrangement, two phenotypic variances were observed with higher frequencies of whorl arrangement (53.7%) followed by opposite arrangement (46.3%).

The studied accession also showed phenotypic variation in respect to leaf characteristics such as leaf variegation (Figure 2), leaf green color, leaf longitudinal curvature, and re-curving of leaf margin (Table 3, Figure 3). Out of the analyzed accessions, only 15.6% had variegation on their leaves, whereas leaf variegation was absent in all of the remaining accessions (84.4%). A higher frequency of leaf color was observed for medium green (82.22%), while light green (8.89%) and dark green (8.89%) leaf colors were less frequent and equally distributed among the rest accessions. The most frequent leaf longitudinal axis was incurved (44.4%) followed by re-curving (33.3%) and straight (22.3%) leaf longitudinal axis. Furthermore, accessions varied in their re-curving of leaf margin, and the majority of the accessions displayed strong re-curving (42.2%) followed by medium (31.1%) and weak (26.7%) re-curving of the leaf margin. Majority of the accessions demonstrated strong leaf margin curving, which is in agreement with leaf characteristics of most rosemary species (Lorenzi and Matos, 2006; Begum et al., 2013; Ribeiro-Santos et al., 2015).

Figure 3.

Figure 3

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Variety in style length in relation to stamen and leaf morphology of rosemary. Equal style length in relation to stamen (A), longer style length in relation to stamen (B), incurved, straight and re-curved longitudinal axis of leaves (C), weak, medium and strong re-curving of leaf margin (D).

The variation in leaf characteristics could have practical value for selecting the desired type for conservation and breeding activities. In this study, accessions that demonstrated medium green leaf color were performed best for leaf and essential oil yields, while the majority of the accessions with light green leaf color were yielded higher essential oil content (Zewdinesh, 2021). Our data also showed that accessions with variegated dark green leaves produced lower leaves and essential oil contents, but were preferred by growers for pot planting due to their attractive ornamental features (Cervelli and Masselli, 2013). Thus the observed variation in leaf phenotype could be used as an important marker for selection in the development of improved cultivars for different purposes.

Rosemary species displayed a wide variability regarding flower color, calyx, and style characteristics (Lorenzi and Matos, 2006; Zaouali et al., 2010; Cervelli and Masselli, 2013; Nunziata et al., 2018). Three different phenotypic classes were observed for flower color (Figure 4). Among them, light blue flower color was recorded for the majority of the accessions (83%). Accessions with medium and dark blue flower colors were less frequent by 7.3% and 9.7%, respectively. Regarding calyx shape, funnel and campanulate shape were noted (Figure 4), and most of the accessions demonstrated funnel-shaped (73.2%) followed by campanulate shape (26.8%). Anthocyanin coloration on the calyx was absent for the majority of the accessions (78), but 22% of the accessions had anthocyanin coloration (Figure 4). The presence of anthocyanin coloration in some accessions might indicate high levels of rutin (flavonoids) in extracts of rosemary accessions. This can be supported by the finding of Sytar et al. (2014), who reported a direct correlation of anthocyanin's contents with rutin content in vegetative organs of buckwheat. Rosemary leaves extract contains up to 3.22% flavonoids (expressed as rutin), and it is one of the contents responsible for its anti-oxidant property (Gird et al., 2017). Therefore, accessions with anthocyanin coloration might be selected for their flavonoid content, and this qualitative character would help as a marker for selection purposes.

Figure 4.

Figure 4

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Variety in flower color, calyx shape and calyx anthocyanin coloration of rosemary accessions. Dark blue flower color (A), medium blue flower color (B), light blue flower color (C), campanulate shaped calyx with anthocynin coloration (D1), funnel shaped calyx without anthocyanin coloration (D2).

Furthermore, accessions were classified into three distinct phenotypic classes based on the intensity of calyx pubescence. Based on this, 61% of the accessions exhibited week calyx pubescence, while accessions with medium and strong calyx pubescence were equally frequent with 19.5% each. Two character classes were observed for style length in relation to stamen (Figure 3), and the majority of the studied accessions showed longer style length in relation to stamen (83%). Whereas accessions with equal style and stamen length were less frequent (17%).

The wider flower traits variabilities noted in the studied accessions showing the divergence of the accessions and could have practical value in selection and improvement activities. Previous studies have also shown the presence of large flower characters variability that has been used as an important trait in distinguishing rosemary accessions (Ulbricht et al., 2010; Mattia et al., 2011; Begum et al., 2013; Carrubba et al., 2020).

The studied accessions also displayed variation in flowering habit and time of flowering. Two phenotypic classes of flowering habit (not flowering and seasonal flowering) were recognized in the present study. Except some accessions which did not produce flowers (8.9%), most of the accessions demonstrated seasonal flowering habit (91.1%). Moreover, flower beginning time marked three character classes, and most of the evaluated accessions were late flowering (≥10 months, 48.8%) followed by medium (8–10 months, 26.8%) and early flowering habit (6–8 months, 24.4%). Noticeable variation in flowering habit and flowering time of rosemary cultivars has also been reported by different authors (Cervelli and de Lucia, 2004; Cervelli and Masselli, 2013; Banjaw et al., 2016).

The flowering time of rosemary depends on cultivar types and most cultivars bloom in full sun (Leporini et al., 2020). But environmental conditions such as day length and soil rich in nitrogen nutrients may prevent the flowering of some rosemary cultivars (Garden report, 2021). From the studied accessions, four samples (Ros01, Ros04, Ros26, and Ros37) did not produce flowers. The presence of non-flowering rosemary genotypes in Ethiopian conditions has also been reported before (Beemnet et al., 2013; Banjaw et al., 2016). Since rosemary is mainly produced in Ethiopia for its leaf and essential oil obtained from leaves, the non-flowering types might be preferred by growers due to their year-round evergreen leaf production.

The variability observed in the studied phenotypic characters showed the presence of genetically diverse accessions. High variability within cultivated crops could result from human or natural selection, genetic drift, and natural variations (Joshi and Baniya, 2006). These facts could be attributed to the observed variability in the evaluated rosemary accessions. The wide variability of rosemary germplasm regarding growth habit, leaf morphology, and flower color has also been reported elsewhere (Carrubba et al., 2020). Overall, the qualitative traits investigated were found variable and can serve as a marker for future collection, selection, and hybridization activities.

3.2. Distribution of characters in collection regions

Frequency distribution and chi-square test of independent and homogeneity of the 15 qualitative characters in collection regions were evaluated (Table 4). Out of the fifteen qualitative characters studied, only six characters (leaf green color, re-curving of leaf margin, intensity of flower blue color, calyx anthocyanin coloration, calyx pubescence, and flowering habit) with 13 phenotypic classes showed a dependent distribution of the collection regions with significant chi-square test (X2gk > 15.51 for df = 8 and X2gk > 26.3 for df = 16) at the level α = 0.05. This demonstrated that these characters did not distribute consistently across the collection regions, and the regions differed in reference to phenotypic class of these traits. The homogeneity test also corroborated this and verified that character classes of these traits, namely light green leaf color, strong re-curving of leaf margin, medium blue flower color, strong calyx pubescence and non-flower habit have showed a heterogeneous distribution across the collection regions (X2g > 15.51 for df = 8).

Table 4.

Frequency distribution and chi-square test of qualitative traits for rosemary accessions grouped into nine collection regions.

Characters
 Growth habit
 Flower arrangement
 Position of long side branches
 Stem pubescence
 Leaf variegation
Character class 1 2 3 1 2 1 2 3 1 2 3 1 9
Regions
Wolaita 3 3 0 2 4 1 1 4 3 2 1 5 1
Hadiya 2 1 2 0 2 1 1 3 2 2 1 5 0
Gurage 2 5 0 3 4 1 1 5 3 2 2 7 0
Sidama 3 2 0 2 3 1 1 3 2 2 1 5 0
Arssi 2 2 1 2 2 1 2 2 3 2 0 5 0
N.Shewa 3 3 0 3 3 1 2 3 3 3 0 3 3
Gonder 0 2 0 1 1 0 1 1 1 1 0 2 0
Harari 3 4 0 4 3 3 1 3 3 3 1 4 3
Commercial 0 2 0 2 0 2 0 0 0 0 2 2 0
Xg2 3 5 12 2.8 2.3 6.8 2.7 2.2 1.3 1.3 10.5 2.2 12
Xgk2 20 (26.3)ns 5.1 (15.51)ns 11.74 (26.3)ns 13.1 (26.3)ns 14.2 (15.51)ns
Characters
 Leaf green color
 Curvature of longitudinal axis
 Re-curving of margin
 Intensity of main blue color
 Calyx shape
Character class 3 5 7 1 2 3 3 5 7 3 5 7 1 2
Regions
Wolaita 0 5 1 3 1 2 1 1 4 5 0 1 5 1
Hadiya 3 2 0 3 0 2 1 2 2 2 0 0 2 0
Gurage 0 7 0 5 1 1 1 2 4 7 0 0 5 2
Sidama 0 5 0 2 1 2 1 2 2 5 0 0 4 1
Arssi 1 4 0 1 1 3 1 2 2 4 0 0 3 1
N.Shewa 0 6 0 2 3 1 3 1 2 3 3 0 3 3
Gonder 0 2 0 1 0 1 1 1 0 2 0 0 2 0
Harari 0 4 3 1 3 3 3 1 3 4 0 3 4 3
Commercial 0 2 0 2 0 0 0 2 0 2 0 0 2 0
Xg2 18.5 2.5 12.3 5.2 5.7 3.5 3.7 5.1 19 2 17.5 11 1.4 3.8
Xgk2 33.3 (26.3)∗ 14.4 (26.3)ns 27.8 (26.3)∗ 30.5 (26.3)∗ 5.2 (15.51)ns
Characters
 Calyx anthocyanin coloration
 Calyx pubescence
 Style length in relation to stamen
 Flowering habit
 Time of beginning of flowering
Character classes 1 9 3 5 7 1 2 1 2 3 5 7
Regions
Wolaita 5 1 5 1 0 1 5 0 6 2 1 3
Hadiya 2 0 2 0 0 0 2 3 2 0 0 2
Gurage 7 0 5 1 1 0 7 0 7 0 3 4
Sidama 5 0 3 1 1 0 5 0 5 0 2 3
Arsi 4 0 3 1 0 0 4 1 4 0 1 3
N.Shewa 3 3 2 1 3 3 3 0 6 3 1 2
Gonder 2 0 2 0 0 0 2 0 2 0 1 1
Harari 4 3 3 3 1 3 4 0 7 3 2 2
Commercial 0 2 0 0 2 0 2 0 2 2 0 0
Xg2 3.8 13.6 4.1 3.3 27.6 10.3 2.1 18.5 1.8 12.4 2.9 3.7
Xgk2 17.4 (15.51)∗ 35 (26.3)∗ 12.4 (15.51)ns 20.3 (15.51)∗ 19 (26.3)ns
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Xg2 > χ2 α = 15.51 (df = 8) is significant at the level α = 0.05 and indicates that collection regions frequencies are significantly different for a particular phenotypic class; Xgk2 > χ2 α = 15.51 (df = 8) or Xgk2 > χ2 α = 26.3 (df = 16) is significant at the level α = 0.05 and indicates that collection region frequencies are significantly independent for a particular trait (characters are region dependent).

The remaining nine qualitative traits, which together expressed 26 phenotypic classes, exhibited independent distribution of the collection regions (X2gk < 15.51 for df = 8 and X2gk < 26.3 for df = 16), and revealed the presence of high similarity among the collection regions concerning phenotypic character classes of these traits. The homogeneity test also confirmed the presence of similarity among collection regions in relation to the 26 phenotypic classes (X2g < 15.51 for df = 8).

For all materials, a wide range of variation was observed across the growing regions (Table 4). Most growing regions were represented by plants with erect and semi-erect growth habits. The prostrate growth habit was observed only in Hadiya and Arssi samples. Leaf variegation and green color showed monomorphic distribution across the collection regions, and the non-variegated medium green leaf color was the predominant in majority of collection regions. Similar trends were also observed for flower color, calyx anthocyanin coloration, style length in relation to stamen, calyx pubescence and flowering habit. Collection regions are less polymorphic in regard to these characters and the characters were predominated in specific locations. The remaining qualitative traits showed polymorphic distributions across all regions.

The commercial accessions showed monomorphic distribution for all of the studied qualitative characters. These commercial accessions were introduced from abroad (Israel) by private investors for large-scale production purposes. The two farms might introduce the same cultivars, and this could be the reason for the complete uniformity displayed for all of the studied qualitative traits.

The frequency distribution of each character class and chi-square test showed the existence of resemblance among the collection regions for the majority of the studied qualitative traits, though there were some growing area dependent characters. The similarity shared among the growing areas indicated lack of distinct rosemary populations related to collection areas. This could be resulted from the common ancestor of the accessions, presence of gene flow due to plant material exchange among different growing areas, and or due to the presence of similar evolutionary factors in growing regions.

3.3. Estimates of phenotypic diversity

Shannon-Weaver diversity index (H′) estimated across all accession, across collection regions and within collection regions for all the examined qualitative traits were presented in Table 5. The value of H′ across accessions varied from 0.43 (for flowering habit) to 0.99 (for flower arrangement) with an overall mean of 0.79. The entire studied qualitative traits contributed to the phenotypic diversity at various levels, with the highest contribution of flower arrangement, re-curving of leaf margin, curvature of leaf longitudinal axis, time of flowering, stem pubescence, and position of long side branches.

Table 5.

Shannon-Weaver diversity index across accessions, within and among collection regions.

H′ of local collections
Characters H′sp H′cr H′cr/Hsp (H′sp − H′cr)/H′sp H′cr/Hsp (H′sp − H′cr)/H′sp
Growth habit 0.80 0.55 0.69 0.31 0.77 0.23
Flower arrangement 0.99 0.76 0.77 0.23 0.87 0.13
Position of long side branches 0.92 0.74 0.80 0.20 0.9 0.10
Stem pubescence 0.94 0.73 0.78 0.22 0.88 0.12
Leaf variegation 0.62 0.29 0.47 0.53 0.53 0.47
leaf green color 0.54 0.23 0.43 0.57 0.49 0.51
Curvature of longitudinal axis 0.97 0.66 0.69 0.31 0.77 0.23
Re-curving of margin 0.98 0.70 0.72 0.28 0.81 0.19
Intensity of main blue color 0.52 0.18 0.35 0.65 0.4 0.60
Calyx shape 0.84 0.56 0.66 0.34 0.74 0.26
Calyx anthocyanin coloration 0.76 0.29 0.39 0.61 0.43 0.57
Calyx pubescence 0.86 0.39 0.46 0.54 0.52 0.48
Style length in r. to stamen 0.66 0.29 0.44 0.56 0.50 0.50
Flowering habit 0.43 0.19 0.43 0.57 0.49 0.51
Time of beginning of flowering 0.95 0.51 0.54 0.46 0.60 0.40
Mean 0.79 0.47 0.57 0.43 0.65 0.35
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H′sp, diversity index for each character calculated from the entire data set; H'′cr, average diversity index of each character pooled over the nine collection regions; H′cr/H′sp, proportion of diversity within collection regions; (H′sp − H′cr)/H′sp, proportion of diversity between collection regions.

The average diversity index (H′) of individual character across collection regions ranged from 0.18 (for intensity of flower blue color) to 0.76 (for flower arrangement) with an overall mean value of 0.47 (Table 5). Three stem characteristics viz. flower arrangement, position of long side branches, stem pubescence, and one leaf character, namely re-curving of leaf margins, were found to be the most diverse traits across all collection regions with H′’ ≥ 0.7. However, flowering habit, the intensity of flower main blue color, style length in relation to stamen, calyx anthocyanin coloration, leaf green color, and leaf variegation were less diverse across all regions with H′ value <0.3, and indicated the existence of unbalanced frequency distribution of these character classes across collection regions. Therefore, the collection regions lack diversity in regard to these flower and leaf characteristics.

Partitioning of the phenotypic diversity into within (Hcr/Hsp) and between collection regions (Hsp − Hcr/Hsp) demonstrated the presence of more intra-region diversity (0.57) than inter-region diversity (0.43), indicating the presence of gene flow among growing regions. Position of long side branches, stem pubescence, flower arrangement, re-curving of leaf margin, curvature of longitudinal axis, growth habit and calyx shape were among the studied qualitative traits that mainly contributed for within collection region heterogeneity with H′ value >0.65.

Even though more phenotypic diversity was observed within collection regions (0.57), a considerable amount of diversity also existed among regions (0.43) (Table 5). The intensity of flower blue color (0.65), calyx anthocyanin coloration (0.61), flowering habit (0.57), leaf green color (0.57), and style length in relation to stamen (0.56) contributed largely to among region diversity, and agreed with the chi-square test of independence and our field observation. Therefore, these characters could be useful in discriminating accessions of different collection regions for future selection and characterization work.

Since an estimate of phenotypic diversity within the collection region was made across all collection regions, the monomorphic nature of commercial farm accessions somehow masked the available level of variability within local populations. Considering only the local collections, 65% of the total phenotypic variation was found within the collection regions and 35% differentiation was existed among collection regions (Table 5). Hence, it is important to identify the actual level of variability existing among local accession to exploit it for future conservation and improvement programs.

A higher level of within collection regions diversity for different qualitative traits was also reported for other crops in Ethiopia and other countries (Ayan and Bekele, 1998; Nsabiyera et al., 2013; Birhanu et al., 2017; Shimeles, 2018). Due to the presence of a higher level of within region diversity, all growing regions could serve as a source of important traits and equal weight should be given to all regions for further collection, characterization, and conservation activities of the rosemary crop.

3.4. Cluster analysis

Cluster analysis of qualitative traits using average linkage criterion grouped the 45 rosemary accessions into six major clusters at different similarity coefficient cutting ages (Table 6; Figure 5). The pattern of accessions clustering did not follow the basis of their geographic origin, except the only clue in cluster V, where three accessions from North Shewa formed a distinct group. Among all, clusters II contained the larger number of accessions (27) from all collection areas except commercial farms at 67.3% similarity coefficient. Accessions in this group were similar in their flower color, flowering habit, anthocyanin coloration of calyx and style length in relation to stamen. Cluster I consisted of four accessions (3 from Hadiya and 1 from Arssi) at 68.3% similarity coefficient. Classification of accessions in this cluster was mainly due to their light green leaf color, absence of leaf variegation, along whole stem branching habit and absence of flower. Light green leaf color and non-flowering habit were the distinct characteristics of this group.

Table 6.

Distribution of the 45 rosemary accessions into six different clusters based on 15 qualitative morphological traits.

Clusters Number of accessions Accessions included Similarity coefficients
I 4 (8.89%) Ros01, Ros04, Ros26, Ros37 68.30%
II 27 (60%) Ros02, Ros03, Ros06, Ros07, Ros08, Ros10, Ros11, Ros12, Ros13, Ros15, Ros16, Ros19, Ros20, Ros21, Ros25, Ros27, Ros30, Ros31, Ros32, Ros33, Ros35, Ros36, Ros40, Ros41, Ros42, Ros43, Ros45 67.30%
III 4 (8.89) Ros05, Ros17, Ros18,Ros34 88.2%.
IV 5 (11.1%) Ros09, Ros14, Ros38, Ros39, Ros44 68.68%
V 3 (6.67%) Ros22, Ros23, Ros24 100%
VI 2 (4.44%) Ros28, Ros29 100%
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Figure 5.

Figure 5

Open in a new tab

Dendrogram generated by average linkage cluster analysis method showing the interrelationships among 45 rosemary accessions using 15 qualitative traits.

Thee accessions from Harari and one from Wolaita formed the third cluster with a similarity coefficient of 88.2%. Accessions in this cluster were differed only in one out of the 15 qualitative traits, and had distinctively dark green leaf and dark blue flower color. Cluster IV has five accessions from four different growing regions (Gurage, Wolaita, Sidama and Harari) at 68.68% similarity coefficient. The absence of leaf variegation and calyx anthocyanin coloration, medium green leaf and light blue flower color, strong re-curving of leaf margin, funnel-shaped calyx, longer style length in relation to stamen, and late-flowering habit were the common characteristics of accession in this cluster. Cluster V contained three accessions from North Shewa which had all characters in common (100% similarity coefficient), with uniquely medium blue flower color. Similarly, accession from commercial farms formed a separate cluster; cluster VI, at a 100% similarity coefficient.

The distribution of the accessions into six different clusters suggests the existence of genetic variation among the accessions. The result of cluster analysis also revealed that the grouping of accessions was not influenced by the area of collections, and most of the accessions clustered regardless of their area of collections. Anyone of the growing region formed a distinct cluster signifying the presence of relatedness among accessions from different collection regions. While discrete clustering of accessions from similar collection areas implied the presence of within collection region variation, and consistent with frequency distribution and Shannon-Weaver diversity index analyses, which showed higher within region diversity than among collection region diversity. It is also consistent with our finding at the molecular level (Zewdinesh et al., 2021). Similar results were also stated in rosemary and other spice and medicinal crops (Roy et al., 2011; Santos et al., 2012; Ballina-Gómez et al., 2013; Nsabiyera et al., 2013; Edoardo et al., 2015; Birhanu et al., 2017; Beemnet, 2018; Bonny et al., 2019). The observed clustering pattern might be resulted from the presence of planting material exchange among growing communities which result in increased within region diversity, but lowered among growing regions diversity.

4. Conclusions

Characterization of 45 rosemary accessions using 15 qualitative morphological traits was performed. Most of the qualitative traits were found variable, and the overall higher phenotypic diversity observed across accessions (H’ = 0.79) demonstrated the presence of genetically diverse accessions. The frequency distribution of characters among collection regions showed the occurrence of related accessions, and collection regions shared similarities for the majority of the traits. The within collection region diversity (0.57) exceeds the among region diversity (0.43). This could also be seen from the clustering pattern, where most of the accessions collected from different growing regions clustered together, while accessions of similar growing areas showed dispersed clustering.

Even though there was similarity for the majority of the studied characters among collection regions, some phenotypic traits, viz intensity of flower blue color, calyx anthocyanin coloration, flowering habit, leaf green color, style length in relation to stamen, calyx pubescence, and leaf variegation were found variable and contributed for regional differentiation. Therefore, these characters could be used as key markers to differentiate accessions of different growing regions. Due to the existence of higher variability among accessions within the growing areas, all regions could serve as a source of desirable genes and future collection, conservation, and improvement activities should be based on actual diversity, and give equal weight to the growing areas.

Based on the above result it may be possible to state that, in the farming system of rosemary, there may be a practice of extensive planting material exchange among communities irrespective of their geographic distance and a culture of maintaining variable types of landraces for medicinal and spice purpose. Overall, this study was conducted for the first time in Ethiopia and provides initial results to have a better insight into the genetic resource of Ethiopian rosemary. The study also pointed out the need of giving more focus to the local accessions for further collection, characterization, and breeding studies to identify useful germplasm with desirable characteristics.

Declarations

Author contribution statement

Zewdinesh Damtew Zigene, PhD: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Bizuayehu Tesfaye Asfaw, PhD: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Tesfaye Disasa Bitima, PhD: Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This work was supported by Ethiopian Institute of Agricultural Research and Agricultural Growth program II.

Data availability statement

Data will be made available on request.

Declaration of interest's statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Acknowledgements

The authors would like to duly acknowledge Ethiopian Institute of Agricultural Research and Agricultural Growth program II for finding the experiment. We would also like to acknowledge the immense contribution made by Wondo Genet Agricultural Research Center for providing and facilitating the necessary facilities for the experiment.

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