Heliyon 8 (2022) e11895

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

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

qualitative characters
Zewdinesh Damtew Zigene a, *, Bizuayehu Tesfaye Asfaw b, Tesfaye Disasa Bitima c
a
Ethiopian Institute of Agricultural Research, Wondo Genet Agricultural Research Center, Ethiopia
b
Hawassa University College of Agriculture, P.O. Box 05, Hawassa, Ethiopia
c
Ethiopian Institute of Agricultural Research, National Agricultural Biotechnology Research Center, Ethiopia

A R T I C L E I N F O A B S T R A C T

Keywords: Rosemary is one of the industrially significant crops traded over the world. Its cultivation in many countries
Accessions depends on locally adapted populations rather than on the use of improved varieties. Collection and character-
Qualitative characters ization of the local populations could contribute to their conservation and selection of desirable traits. Therefore,
Salvia rosmarinus
this study was undertaken to estimate the diversity of 45 rosemary accessions collected from different parts of
Shannon diversity
Ethiopia using qualitative morphological traits. The frequency distribution of characters reflected the poly-
morphism 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 ex-
istence 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.

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 identifi-
cation 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 se-
lection 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 envi-
ronments 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 re-
sources (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, morpho-
logical, 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; Nun-
ziata 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

* Corresponding author.
E-mail address: damtewzewdinesh@gmail.com (Z.D. Zigene).

https://doi.org/10.1016/j.heliyon.2022.e11895
Received 14 April 2022; Received in revised form 12 May 2022; Accepted 17 November 2022
2405-8440/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Z.D. Zigene et al. Heliyon 8 (2022) e11895

Masselli, 2013; Mateu-Andres et al., 2013; Carrubba et al., 2020).

Furthermore, characterization of the essential oils and extracts for their Table 1. Rosemary accessions used for the study and their area of collections.
flavonoid content, antioxidant, anticancer, antimicrobial, and cholines- No Accessions code Geographic origin
terase inhibitory activity has been studied in rosemary and related spe- 1 Ros02 Wolaita
cies and showed higher variability (Zaouali et al., 2010; Sytar et al., 2 Ros03 Wolaita
2015; Karakaya et al., 2020; Leporini et al., 2020). 3 Ros05 Wolaita
In Ethiopia, limited study has been conducted on morphological,
4 Ros14 Wolaita
molecular, and chemical traits variability (Beemnet et al., 2013; Bekri
5 Ros35 Wolaita
et al., 2018, Zewdinesh et al., 2021). However, no research effort has
6 Ros36 Wolaita
been done in the investigation of qualitative morphological traits di-
7 Ros01 Hadiya
versity. But analyzing qualitative morphological traits and knowing the
8 Ros04 Hadiya
extent of variability among accessions and populations is crucial for
9 Ros15 Hadiya
effective management, improvement, and utilization of the crop. Given
10 Ros37 Hadiya
the scarcity of studies regarding the characterization of the available
rosemary germplasms and the significance of the study towards its use in 11 Ros16 Hadiya

the collection, conservation, and breeding programs; this experiment was 12 Ros08 Gurage

designed to determine the phenotypic diversity of Ethiopian rosemary 13 Ros30 Gurage

accessions using standard qualitative morphological descriptors. 14 Ros31 Gurage
15 Ros33 Gurage
2. Materials and methods 16 Ros38 Gurage
17 Ros39 Gurage
2.1. Study area 18 Ros32 Gurage
19 Ros13 Sidama
The experiment was conducted in the southern region of Ethiopia 20 Ros42 Sidama
from 2018 to 2019 at Wondo Genet Agricultural Research Center, Wondo 21 Ros43 Sidama
Genet, Ethiopia. The site is located at 7
190 N latitude and 38
380 E lon- 22 Ros44 Sidama
gitudes with an altitude of 1780 masl. The annual mean rainfall of the 23 Ros45 Sidama
area in 2018 and 2019 were 1441.3 mm and 1317.5 mm, respectively. 24 Ros40 Arssi
The annual average minimum temperature varies from 11.8 to 15.1
C, 25 Ros41 Arssi
whereas the annual mean maximum temperature is between 25.1 and
26 Ros26 Arssi
29.7
C. The soil of the experimental area is fertile, well drained and
27 Ros27 Arssi
sandy loam with a pH of 6.4 (Abayneh et al., 2006).
28 Ros12 Arssi
29 Ros20 North Shewa
2.2. Plant materials and experimental procedures
30 Ros21 North Shewa
31 Ros22 North Shewa
A total of 45 rosemary accessions were used for this activity (Table 1).
32 Ros23 North Shewa
The forty three rosemary accessions were originally collected from
33 Ros24 North Shewa
farmers’ fields at different agro-ecology of Ethiopia (Figure 1), and two
accessions were obtained from commercial farms, which were intro- 34 Ros25 North Shewa

duced by private investment sectors in Ethiopia. Seedlings were prepared 35 Ros06 Gonder Zuria
from soft stem cuttings of all accessions and raised in the nursery. After 36 Ros07 Gonder Zuria
three months, well performing and uniformly grown seedlings were 37 Ros09 Harari
transplanted to the experimental field using Randomized Complete Block 38 Ros10 Harari
Design (RCBD) with three replications. Plants, plots and blocks were 39 Ros11 Harari
spaced by 0.6 m, 1 m, and 1.5 m, respectively. Watering the experiment 40 Ros34 Harari
was carried out once a week during dry seasons, and the experimental 41 Ros17 Harari
field was managed properly and kept weed-free throughout the 42 Ros18 Harari
experimentation. 43 Ros19 Harari
44 Ros28 Commercial farm
2.3. Data collection 45 Ros29 Commercial farm

Data were recorded for 15 qualitative morphological traits using In-

ternational Union for the Protection of New Varieties of Plants de-
scriptors 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.
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 fre-
quency 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 ¼ (# rows–1)  (# columns–1)
for independence test and at df ¼ # 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 (H0 ) 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
(H0 ) as described by Hennink and Zeven (1991) is given as:

2
Z.D. Zigene et al.
Figure 1. Map of Ethiopia depicting the collection areas of rosemary accessions used for the study. N. Shewa, North Shewa; N. Gonder, North Gonder.
0
X
n
H ¼ pi*lnðpiÞ
i¼1
where: H0 , Shannon-Weaver Diversity Index; pi, the proportion of geno-
types 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 inter-
mediate 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
3
Heliyon 8 (2022) e11895
growth habit (erect, semi-erect, and prostrate type), but with the pre-
dominant 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. Ac-
cessions 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).
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 pubes-
cence 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 lon-
gitudinal 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
Z.D. Zigene et al. Heliyon 8 (2022) e11895

Table 2. Codes and descriptions of the characters used for the study. Table 3. Proportion of phenotypic classes for 15 qualitative traits in rosemary
accessions.
Characters Code Description
Characters Code Character classes Frequency Percentage
Growth habit 1. Erect
Growth habit 1 Erect 18 40
2. Semi-erect
2 Semi-erect 24 53.3
3. prostrate
3 prostrate 3 6.7
Flower arrangement 1. Opposite
Flower arrangement 1 Opposite 19 46.3
2. Whorl
2 Whorl 22 53.7
Position of long side branches 1. Mainly basal
Position of long side branches 1 Mainly basal 11 24.4
2. Mainly upper
2 Mainly upper 10 22.2
3. Along whole stem
3 Along whole stem 24 53.3
Stem pubescence 1. Absent or weak
Stem pubescence 1 Absent or weak 20 44.4
2. Moderate
2 Moderate 17 37.8
3. Strong
3 Strong 8 17.8
Leaf variegation 1. Absent
Leaf variegation 1 Absent 38 84.4
9. Present
9 Present 7 15.6
Leaf green color 3. Light
Leaf green color 3 Light 4 8.89
5. Medium
5 Medium 37 82.22
7. Dark
7 Dark 4 8.89
Curvature of longitudinal axis 1. Incurved
Curvature of longitudinal axis 1 Incurved 20 44.4
2. Straight
2 Straight 10 22.3
3. Re-curved
3 Re-curved 15 33.3
Re-curving of margin 3. Weak
Re-curving of margin 3 Weak 12 26.7
5. Medium
5 Medium 14 31.1
7. Strong
7 Strong 19 42.2
Intensity of flower blue color 3. Light
Intensity of flower main blue 3 Light 34 83
5. Medium
color
7. Dark
5 Medium 3 7.3
Calyx shape 1. Funnel-shape
7 Dark 4 9.7
2. Campanulate
Calyx shape 1 Funnel-shape 30 73.2
Calyx anthocyanin coloration 1. Absent
2 Campanulate 11 26.8
9. Present
Calyx anthocyanin coloration 1 Absent 32 78
Calyx pubescence 3. Weak
9 Present 9 22
5. Medium
Calyx pubescence 3 Weak 25 61
7. Strong
5 Medium 8 19.5
Style length in relation to stamen 1 Equal
7 Strong 8 19.5
2 Longer
Style length in relation to stamen 1 Equal 7 17
Flowering habit 1 Not flowering
2 Longer 34 83
2 Seasonal flowering
Flowering habit 1 Not flowering 4 8.89
Time of beginning of flowering 3 Early
2 Seasonal 41 91.11
5 Medium
Time of beginning of flowering 3 Early 10 24.4
7 Late
5 Medium 11 26.8
7 Late 20 48.8
margin curving, which is in agreement with leaf characteristics of most
rosemary species (Lorenzi and Matos, 2006; Begum et al., 2013; Ribeir-
o-Santos et al., 2015). accessions (83%). Accessions with medium and dark blue flower colors
The variation in leaf characteristics could have practical value for were less frequent by 7.3% and 9.7%, respectively. Regarding calyx
selecting the desired type for conservation and breeding activities. In this shape, funnel and campanulate shape were noted (Figure 4), and most of
study, accessions that demonstrated medium green leaf color were per- the accessions demonstrated funnel-shaped (73.2%) followed by
formed best for leaf and essential oil yields, while the majority of the campanulate shape (26.8%). Anthocyanin coloration on the calyx was
accessions with light green leaf color were yielded higher essential oil absent for the majority of the accessions (78), but 22% of the accessions
content (Zewdinesh, 2021). Our data also showed that accessions with had anthocyanin coloration (Figure 4). The presence of anthocyanin
variegated dark green leaves produced lower leaves and essential oil coloration in some accessions might indicate high levels of rutin (flavo-
contents, but were preferred by growers for pot planting due to their noids) in extracts of rosemary accessions. This can be supported by the
attractive ornamental features (Cervelli and Masselli, 2013). Thus the finding of Sytar et al. (2014), who reported a direct correlation of an-
observed variation in leaf phenotype could be used as an important thocyanin's contents with rutin content in vegetative organs of buck-
marker for selection in the development of improved cultivars for wheat. Rosemary leaves extract contains up to 3.22% flavonoids
different purposes. (expressed as rutin), and it is one of the contents responsible for its
Rosemary species displayed a wide variability regarding flower color, anti-oxidant property (Gird et al., 2017). Therefore, accessions with
calyx, and style characteristics (Lorenzi and Matos, 2006; Zaouali et al., anthocyanin coloration might be selected for their flavonoid content, and
2010; Cervelli and Masselli, 2013; Nunziata et al., 2018). Three different this qualitative character would help as a marker for selection purposes.
phenotypic classes were observed for flower color (Figure 4). Among Furthermore, accessions were classified into three distinct phenotypic
them, light blue flower color was recorded for the majority of the classes based on the intensity of calyx pubescence. Based on this, 61% of

4
Z.D. Zigene et al. Heliyon 8 (2022) e11895

Figure 2. 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).

Figure 3. 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 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).

5
Z.D. Zigene et al.
Figure 4. 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).
The studied accessions also displayed variation in flowering habit and
time of flowering. Two phenotypic classes of flowering habit (not flow-
ering and seasonal flowering) were recognized in the present study. Except
some accessions which did not produce flowers (8.9%), most of the ac-
cessions 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 con-
ditions 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 inthe studied phenotypic characters showedthe
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 vari-
ability 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 homoge-
neity of the 15 qualitative characters in collection regions were evaluated
(Table 4). Out of the fifteen qualitative characters studied, only six characters
6
Heliyon 8 (2022) e11895
(leaf green color, re-curving of leaf margin, intensity of flower blue color,
calyxanthocyanincoloration,calyxpubescence,andfloweringhabit)with13
phenotypic classes showed a dependent distribution of the collection regions
withsignificantchi-squaretest(X2gk >15.51fordf¼8andX2gk >26.3fordf¼
16) at the level α ¼ 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 hetero-
geneous distribution across the collection regions (X2g > 15.51 for df ¼ 8).
The remaining nine qualitative traits, which together expressed 26
phenotypic classes, exhibited independent distribution of the collection
regions (X2gk < 15.51 for df ¼ 8 and X2gk < 26.3 for df ¼ 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 ¼ 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 pre-
dominant 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 pro-
duction 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.
Z.D. Zigene et al. Heliyon 8 (2022) e11895

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
X2g 3 5 12 2.8 2.3 6.8 2.7 2.2 1.3 1.3 10.5 2.2 12
X gk 2 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

Xg2 > χ2 α ¼ 15.51 (df ¼ 8) is significant at the level α ¼ 0.05 and indicates that collection regions frequencies are significantly different for a particular phenotypic
class; Xgk2 > χ2 α ¼ 15.51 (df ¼ 8) or Xgk2 > χ2 α ¼ 26.3 (df ¼ 16) is significant at the level α ¼ 0.05 and indicates that collection region frequencies are significantly
independent for a particular trait (characters are region dependent).

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 evolu-
tionary factors in growing regions.
3.3. Estimates of phenotypic diversity
Shannon-Weaver diversity index (H0 ) 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 H0
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 pu-
bescence, and position of long side branches.
The average diversity index (H0 ) 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 H0 ’  0.7. However, flowering habit, the intensity

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Z.D. Zigene et al. Heliyon 8 (2022) e11895

A higher level of within collection regions diversity for different

Table 5. Shannon-Weaver diversity index across accessions, within and among qualitative traits was also reported for other crops in Ethiopia and other
collection regions.
countries (Ayan and Bekele, 1998; Nsabiyera et al., 2013; Birhanu et al.,
H0 of local collections 2017; Shimeles, 2018). Due to the presence of a higher level of within
Characters 0
H sp 0
H cr 0
H cr/ 0
(H sp  H0 cr/ (H0 sp  region diversity, all growing regions could serve as a source of important
Hsp H0 cr)/H0 sp Hsp H0 cr)/H0 sp traits and equal weight should be given to all regions for further collec-
Growth habit 0.80 0.55 0.69 0.31 0.77 0.23 tion, characterization, and conservation activities of the rosemary crop.
Flower arrangement 0.99 0.76 0.77 0.23 0.87 0.13
Position of long side 0.92 0.74 0.80 0.20 0.9 0.10 3.4. Cluster analysis
branches
Stem pubescence 0.94 0.73 0.78 0.22 0.88 0.12
Cluster analysis of qualitative traits using average linkage criterion
Leaf variegation 0.62 0.29 0.47 0.53 0.53 0.47
grouped the 45 rosemary accessions into six major clusters at different
leaf green color 0.54 0.23 0.43 0.57 0.49 0.51
similarity coefficient cutting ages (Table 6; Figure 5). The pattern of
Curvature of 0.97 0.66 0.69 0.31 0.77 0.23 accessions clustering did not follow the basis of their geographic origin,
longitudinal axis
except the only clue in cluster V, where three accessions from North
Re-curving of 0.98 0.70 0.72 0.28 0.81 0.19
Shewa formed a distinct group. Among all, clusters II contained the larger
margin
number of accessions (27) from all collection areas except commercial
Intensity of main 0.52 0.18 0.35 0.65 0.4 0.60
blue color farms at 67.3% similarity coefficient. Accessions in this group were
Calyx shape 0.84 0.56 0.66 0.34 0.74 0.26
similar in their flower color, flowering habit, anthocyanin coloration of
Calyx anthocyanin 0.76 0.29 0.39 0.61 0.43 0.57
calyx and style length in relation to stamen. Cluster I consisted of four
coloration accessions (3 from Hadiya and 1 from Arssi) at 68.3% similarity coeffi-
Calyx pubescence 0.86 0.39 0.46 0.54 0.52 0.48 cient. Classification of accessions in this cluster was mainly due to their
Style length in r. to 0.66 0.29 0.44 0.56 0.50 0.50 light green leaf color, absence of leaf variegation, along whole stem
stamen branching habit and absence of flower. Light green leaf color and non-
Flowering habit 0.43 0.19 0.43 0.57 0.49 0.51 flowering habit were the distinct characteristics of this group.
Time of beginning 0.95 0.51 0.54 0.46 0.60 0.40 Thee accessions from Harari and one from Wolaita formed the third
of flowering cluster with a similarity coefficient of 88.2%. Accessions in this cluster
Mean 0.79 0.47 0.57 0.43 0.65 0.35 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
H sp, diversity index for each character calculated from the entire data set; H'0 cr,
0

average diversity index of each character pooled over the nine collection regions; five accessions from four different growing regions (Gurage, Wolaita,
H0 cr/H0 sp, proportion of diversity within collection regions; (H0 sp  H0 cr)/H0 sp, Sidama and Harari) at 68.68% similarity coefficient. The absence of leaf
proportion of diversity between collection regions. 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
of flower main blue color, style length in relation to stamen, calyx habit were the common characteristics of accession in this cluster.
anthocyanin coloration, leaf green color, and leaf variegation were less Cluster V contained three accessions from North Shewa which had all
diverse across all regions with H0 value <0.3, and indicated the existence characters in common (100% similarity coefficient), with uniquely me-
of unbalanced frequency distribution of these character classes across dium blue flower color. Similarly, accession from commercial farms
collection regions. Therefore, the collection regions lack diversity in re- formed a separate cluster; cluster VI, at a 100% similarity coefficient.
gard to these flower and leaf characteristics. The distribution of the accessions into six different clusters suggests
Partitioning of the phenotypic diversity into within (Hcr/Hsp) and the existence of genetic variation among the accessions. The result of
between collection regions (Hsp  Hcr/Hsp) demonstrated the presence of cluster analysis also revealed that the grouping of accessions was not
more intra-region diversity (0.57) than inter-region diversity (0.43), influenced by the area of collections, and most of the accessions clustered
indicating the presence of gene flow among growing regions. Position of regardless of their area of collections. Anyone of the growing region
long side branches, stem pubescence, flower arrangement, re-curving of formed a distinct cluster signifying the presence of relatedness among
leaf margin, curvature of longitudinal axis, growth habit and calyx shape accessions from different collection regions. While discrete clustering of
were among the studied qualitative traits that mainly contributed for accessions from similar collection areas implied the presence of within
within collection region heterogeneity with H0 value >0.65. collection region variation, and consistent with frequency distribution
Even though more phenotypic diversity was observed within collec- and Shannon-Weaver diversity index analyses, which showed higher
tion 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 Table 6. Distribution of the 45 rosemary accessions into six different clusters
color (0.57), and style length in relation to stamen (0.56) contributed based on 15 qualitative morphological traits.
largely to among region diversity, and agreed with the chi-square test of Clusters Number of Accessions included Similarity
independence and our field observation. Therefore, these characters accessions coefficients
could be useful in discriminating accessions of different collection re- I 4 (8.89%) Ros01, Ros04, Ros26, Ros37 68.30%
gions for future selection and characterization work. II 27 (60%) Ros02, Ros03, Ros06, Ros07, Ros08, 67.30%
Since an estimate of phenotypic diversity within the collection region Ros10, Ros11, Ros12, Ros13, Ros15,
was made across all collection regions, the monomorphic nature of Ros16, Ros19, Ros20, Ros21, Ros25,
commercial farm accessions somehow masked the available level of Ros27, Ros30, Ros31, Ros32, Ros33,
Ros35, Ros36, Ros40, Ros41, Ros42,
variability within local populations. Considering only the local collec-
Ros43, Ros45
tions, 65% of the total phenotypic variation was found within the
III 4 (8.89) Ros05, Ros17, Ros18,Ros34 88.2%.
collection regions and 35% differentiation was existed among collection
IV 5 (11.1%) Ros09, Ros14, Ros38, Ros39, Ros44 68.68%
regions (Table 5). Hence, it is important to identify the actual level of
V 3 (6.67%) Ros22, Ros23, Ros24 100%
variability existing among local accession to exploit it for future con-
VI 2 (4.44%) Ros28, Ros29 100%
servation and improvement programs.

8
Z.D. Zigene et al. Heliyon 8 (2022) e11895

Figure 5. Dendrogram generated by average linkage cluster analysis method showing the interrelationships among 45 rosemary accessions using 15 qualitative traits.

within region diversity than among collection region diversity. It is also out the need of giving more focus to the local accessions for further
consistent with our finding at the molecular level (Zewdinesh et al., collection, characterization, and breeding studies to identify useful
2021). Similar results were also stated in rosemary and other spice and germplasm with desirable characteristics.
medicinal crops (Roy et al., 2011; Santos et al., 2012; Ballina-G
omez
et al., 2013; Nsabiyera et al., 2013; Edoardo et al., 2015; Birhanu et al., Declarations
2017; Beemnet, 2018; Bonny et al., 2019). The observed clustering
pattern might be resulted from the presence of planting material ex- Author contribution statement
change among growing communities which result in increased within
region diversity, but lowered among growing regions diversity. Zewdinesh Damtew Zigene, PhD: Conceived and designed the ex-
periments; Performed the experiments; Analyzed and interpreted the
4. Conclusions data; Wrote the paper.
Bizuayehu Tesfaye Asfaw, PhD: Conceived and designed the experi-
Characterization of 45 rosemary accessions using 15 qualitative ments; Analyzed and interpreted the data; Wrote the paper.
morphological traits was performed. Most of the qualitative traits were Tesfaye Disasa Bitima, PhD: Contributed reagents, materials, analysis
found variable, and the overall higher phenotypic diversity observed tools or data; Wrote the paper.
across accessions (H’ ¼ 0.79) demonstrated the presence of genetically
diverse accessions. The frequency distribution of characters among
Funding statement
collection regions showed the occurrence of related accessions, and
collection regions shared similarities for the majority of the traits. The
This work was supported by Ethiopian Institute of Agricultural
within collection region diversity (0.57) exceeds the among region di-
Research and Agricultural Growth program II.
versity (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 Data availability statement
clustering.
Even though there was similarity for the majority of the studied Data will be made available on request.
characters among collection regions, some phenotypic traits, viz intensity
of flower blue color, calyx anthocyanin coloration, flowering habit, leaf
Declaration of interest's statement
green color, style length in relation to stamen, calyx pubescence, and leaf
variegation were found variable and contributed for regional differenti-
The authors declare no conflict of interest.
ation. 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 re- Additional information
gions could serve as a source of desirable genes and future collection,
conservation, and improvement activities should be based on actual di- No additional information is available for this paper.
versity, and give equal weight to the growing areas.
Based on the above result it may be possible to state that, in the Acknowledgements
farming system of rosemary, there may be a practice of extensive planting
material exchange among communities irrespective of their geographic The authors would like to duly acknowledge Ethiopian Institute of
distance and a culture of maintaining variable types of landraces for Agricultural Research and Agricultural Growth program II for finding the
medicinal and spice purpose. Overall, this study was conducted for the experiment. We would also like to acknowledge the immense contribu-
first time in Ethiopia and provides initial results to have a better insight tion made by Wondo Genet Agricultural Research Center for providing
into the genetic resource of Ethiopian rosemary. The study also pointed and facilitating the necessary facilities for the experiment.

9
Z.D. Zigene et al. Heliyon 8 (2022) e11895

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