Contributions to the Classification of Capsicum annuum L . and Capsicum frutescens L . in West Africa Using Morphological Traits

In West Africa, the genus Capsicum is represented by two cultivated species namely C. frutescens and C. annuum with different varieties. However, the taxonomic identity of these two species has been unresolved because they are morphologically related. There has been considerable debate on their status as two separate species or just varieties of one species. The study was aimed at assessing the variability and relatedness among the four varieties of the two closely related Capsicum species in West Africa. The varieties used in this study are C. annuum var. abbreviatum, C. annuum var. acuminatum, C. annuum var. grossum and C. frutescens var. baccatum. The varieties were morphologically characterized using 80 quantitative and qualitative standard descriptors from seedling emergence to plant maturity. Data collected were subjected to statistical analysis using SPSS statistical package, descriptive statistics and cluster analysis. The results revealed overlapping similarities in the morphology of the four varieties. Classification analysis based on data collected showed two distinct clusters by grouping varieties sharing close phenotypic similarities into distinct cluster. The differences observed among the varieties majorly in flower and fruit traits do not provide enough evidence to conceive that the collections are two separate species. Our results therefore support the inclusion of C. frutescens var. baccatum as a variety of C. annuum. Additional comparative molecular profiling is needed to fully understand the phylogenetic relationship among the cultivated Capsicum species in this part of the world.


Introduction
The genus Capsicum commonly known as chilli or hot pepper belongs to the family Solanaceae.It originated from the Central and South America (Bosland and Votava, 2000).This genus consists of five domesticated and 22 wild species.The domesticated ones are C. annuum L., C. chinenses Jacqs., C. frutescens L., C. pubescens R. and C. baccatum L. (Bosland and Votava, 2000).
In West Africa, the genus is represented by two cultivated species namely C. frutescens and C. annuum with different varieties.However, the taxonomic identity of these two species has been unresolved because they are morphologically related.There has been considerable debate on their status as two separate species or just varieties of one species.Bosland and Votava (2000) defined species as a population or series of populations within which free gene flow occurs under natural conditions, with fertile and healthy progeny produced by interbreeding within the species whereas a botanical variety is a distinct morpho-logical subgroup within a species.On the basis of the form as annual or perennial, C. annuum and C. frutescens respectively were first distinguished as the only two species in the genus Capsicum by Linneaus in 1825.However, in the study of South American pepper, Bukasow (1930) rejected Linneaus' classification and considered the annual form to be nothing more than a variety of C. frutescens.Up till today, the distinction of these two plants as separate species or varieties of one species is still unresolved.
Identification and classification of botanical species correctly is a crucial step for managing germplasm collection efficiently.It is the basis for improving any plant species (Moura et al., 2013).The first step in the description and classification of germplasm is morphological characterization.Evaluation of morphological traits contributes valuable information for the assessment of genetic relatedness and variability of any plant species (Franco et al., 2005;Laurentin, 2009).This allows the description, identification and differentiation of species (Carvalho et al., 2014).Generally, the identification of Capsicum genus is carried out by morphological traits that are observed majorly in the Statistical analysis Data were subjected to Analysis of Variance (ANOVA) using SPSS statistical package version 16.0.Mean comparison was carried out using Duncan's multiple range test at 5% probability level.A dendrogram was generated using the Pair-Group Correlation Analysis (PGCA) clustering method to determine the relationship among the varieties.

Results
Variation in seedling/ vegetative qualitative traits Morphological characterization of the four varieties of the cultivated Capsicum species revealed little variation in qualitative agronomic traits.Among the agronomic seedling traits (juvenile traits), all the Capsicum varieties examined had white hypocotyls with no pubescence.The cotyledon leaf colour ranged from green to dark green, the shape of which was lanceolate, ovate and deltoid.Stem colour was green for all varieties; all stem are angular in shape and pubescence was sparse (Table 1).Of the 19 qualitative vegetative traits examined, a major variation was observed in the nodal anthocyanin pigmentation.Nodal pigmentation was purple in C. annuum var.abbreviatum, C. annuum var.acuminatum, and C. annuum var.grossum while the node color was green in C. frutescens var.baccatum till maturity (Fig. 1).

Variation in quantitative vegetative traits
According to analysis of variance (Table 2), the varieties of the cultivated Capsicum species differed significantly (p˂0.05) for all the quantitative vegetative traits examined.The plant height at maturity ranged from 17.57-59.9cm.flowers and fruits (Sudré et al., 2010).However, a combination of several diagnostic morphological traits is necessary to identify and differentiate the Capsicum species.
Cluster analysis has been used by several researchers to identify and distinguish accessions from each other and group accessions of plants based on their similarities (Sivaraj et al., 2012;Bibi et al., 2013;Tyagi et al., 2014;Dikshita and Sivarajb, 2015).On the basis of fruit form and size, four varieties of the cultivated species of Capsicum are recognized in West Africa.These are C. annuum var.abbreviatum, C. annuum var.acuminatum, C. annuum var.grossum and C. frutescens var.baccatum which are locally known as rodo, sombo, tatase and wewe respectively.The present study aimed to evaluate and characterize the commonly cultivated varieties of the Capsicum species using standard morphological descriptors and cluster analysis in order to assess their specific and varietal delineations.

Description of the study area
This study was carried out at the green house of University of Fort Hare, Alice, South Africa.University of Fort Hare lies between 32 47'1.23"S,26 51'9.85"E.

Collection of plant materials
Nigeria is a major producer and consumer of the cultivated varieties of Capsicum species in West Africa, therefore, mature fruits of the four varieties of C. annuum and C. frutescens were obtained from markets in major geographical zones in Nigeria.Seeds in the fruits were first removed, sun-dried and stored at room temperature of about 15-25 °C in paper bags and were later used for Planting.The voucher specimens of each variety was collected and deposited at the University of Ilorin's herbarium.

Growth of plant materials and experimental design
Planting was done in labelled plastic pots in the green house of the University of Fort Hare between September 2017 and February, 2018.The layout in the green house was a completely randomized design with 10 replications for each variety.The plastic pots were perforated to avoid water logging and to prevent fungal growth; they were filled with loamy soil and watered before sowing.Seeds of each variety were sown in their labelled plastic pots and covered with thinnest possible layer of soil.Watering was done based on climatic conditions with a fine watering can and weeding was done frequently as per the emergence of weeds.

Data collection
Data were collected from the four Capsicum varieties using 80 standard morphological traits as defined by the International Board for Plant Genetic Resources Descriptors for Capsicum (IPGRI, 1995).Genotype characteristics were recorded as quantitative or qualitative values as required.The methodology used to record qualitative values from seedling to harvest followed the descriptor for Capsicum.Variation in qualitative inflorescence traits Flower position was found to be the most useful diagnostic character that distinguished the two cultivated Capsicum species.Among the four varieties of the cultivated Capsicum species, the character erect flower position was present only in Capsicum frutescens var.baccatum while Capsicum annuum var.abbreviatum, Capsicum annuum var.acuminatum and Capsicum annuum var.grossum produced pendant flowers (Fig. 2).Other inflorescence characters varied considerably among the Capsicum varieties.Anther colour was predominantly purple as found in C. annuum var.abbreviatum, C. annuum var.acuminatum and C. annuum var.grossum, while the anther color in C. frutescens baccatum was green.Regarding the corolla colour, they all had white corolla the shape of which was rotate in all.Filament colour varied from white to purple, white being the predominant colour.The petals and sepals are five and six in number respectively in all the varieties.Flowers are borne singly on the node.Anular constriction in the calyx was present in all the pepper varieties with a dentate calyx margin (Table 3).

Variations in quantitative inflorescence traits
Morphological characterization of the four varieties of the cultivated Capsicum species revealed considerable variation in quantitative inflorescence traits.The earliest 138 number of days to 50% flowering was observed in C. annuum var.acuminatum (57 days), while the longest day to attain 50% flowering was recorded from C. annuum var.grossum (72 days) (Table 4).Number of flowers per plant significantly varied among the varieties (p˂0.05).The highest number of flowers per plant was recorded from C. annuum var.abbreviatum with a mean value of (37.Variation in qualitative fruits and seeds traits Variations were observed on fruit related traits such as shape, size and the position of fruit on the plant.Fruit shape was determined based on comparison with the shape proposed in the lists of descriptors of IPGRI.The four characteristic fruit shape recorded were campanulate in C. annuum var.abbreviatum, blocky type in C. annuum var.acuminatum, triangular in C. annuum var.grossum and the elongate fruit shape in C. frutescens var.baccatum (Fig. 3).All the cultivated Capsicum varieties produced shiny fruits.The fruit position was pendant in Capsicum annuum var.abbreviatum, Capsicum annuum var.acuminatum and in Capsicum annuum var.grossum while Capsicum frutescens var.baccatum produced erect fruits on the plant (Fig. 4).Fruit colour at intermediate maturation stage varied from light yellow to green, whereas at the mature stage, all the fruits turned red.Fruit shape at pedicel attachment varied from lobate to cordate and truncate and mature fruits on the pedicel were found to be persistent in all the Capsicum varieties studied (Table 5).Fruit outline was found to be slightly corrugated to corrugated and neck at the base of the fruit was either present or absent.The seeds of the Capsicum varieties were similar in shape and structure; they are all ellipsoid and oval in longitudinal section.The seed colour ranged from cream to light yellow and all had a wrinkled seed surface with intermediate seed size.

Variation in quantitative fruit and seed characters
Differences in mean performance of quantitative fruit and seed traits in the four varieties of cultivated Capsicum species are presented in Table 6.The shortest number of days to first fruiting was recorded in Capsicum annuum var.grossum while the longest days to first fruiting was recorded in Capsicum annuum var.abbreviatum.Analysis of variance indicated significant difference (p˂0.05)among the varieties on number of fruits per plant.C. annuum var.abbreviatum produced the highest number of fruits per plant while the least number of fruits per plant was recorded in C. annuum var.grossum (Table 6).With respect to the number of days to first fruit ripening, C. annuum var.acuminatum took the least number of days to ripen (96 days), followed by C. annuum var.abbreviatum and C. annuum var.grossum (98 days) and C. frutescens var.baccatum maturing latest (109 days).The highest number of seeds per fruit was recorded from C. annuum var.grossum while the least number of seeds was recorded from C. annuum var.abbreviatum.The maximum weight per 1000 seeds (7.09 g) was recorded from C. annuum var.acuminatum followed by C. annuum var.grossum (6.03g) and C. annuum var.abbreviatum (6.01 g).The least seed weight was registered from C. frutescens var.baccatum (4.10 g).

Cluster analysis
The dendogram separated the four Capsicum varieties studied into two distinct clusters by grouping varieties sharing close phenotypic similarities into distinct cluster.Consequently, C. frutescens var.baccatum and C. annuum var.abbreviatum were classified into the first cluster at 89% in the similarity matrix, while C. annuum var.grossum and C. annuum var.acuminatum were classified in the second cluster at 83% in the similarity matrix, indicating their close genetic affinities and phenotypic relationship (Fig. 5).

Discussion
Several researchers have stressed the importance of morphological characterization as a basic step towards the resolution of taxonomic conflicts in many plant species (Gerrano et al., 2017;Ranjit et al., 2013;Laurentin, 2009).According to Adebola and Morakinyo (2006), it is essential to use morphological descriptors first before the use of advanced biochemical and molecular techniques in resolving taxonomic problems because morphological characterization allows thorough investigation and provides basic information on existing genetic variability in the plant.The detailed morphological characterization of the four varieties of the cultivated Capsicum species in this study revealed that they are relatively uniform in gross morphology but considerable variations still exist.The four species showed overlapping similarities in qualitative and quantitative traits.In general, Capsicum annum and Capsicum frutescens could be distinguished by four diagnostic qualitative traits: nodal pigmentation, anther color, flower position and fruit position.All the three varieties of Capsicum annuum had purple nodal color and white anthers.Flowers and fruits were in pendant position on the plant in the three varieties of Capsicum annuum.C. frutescens var.baccatum had white nodal color, purple anthers and flowers and fruit stood erect on the plant.Similar findings was reported by Baral and Bosland, (2004) where pendant flower and fruit position separated accessions of C. frutescens from C. chinense in their study.The variations observed in nodal pigmentation, anther color, flower and fruit position are inherent as all the varieties examined were grown and characterized in the same controlled environment.Thus, these four qualitative traits may be of taxonomic importance and may be used to delimit these varieties.
The high overlapping similarities in most of the morphological traits examined is a reflection of their phylogenetic relationship.However, the significant differences in most quantitative characters among the four varieties of the cultivated Capsicum species suggest that there is a store of genetic variability in these species.Such variability could be harnessed for informed breeding and improvement programmes in these species (Saleh et al., 2016;Ince et al., 2009;Sudré et al., 2006;Guzmán et al., 2005).
The information gathered from cluster analysis are useful to identify genetic variability among plants.Clustering of genotypes signifies close genetic affinity between/among species and can be used in resolving taxonomic complexities (Maity et al., 2009).The result from the cluster analysis indicated that there was considerable variability among the varieties of the cultivated Capsicum species which allowed them to be separated into distinct group.
The cluster analysis from all the morphological traits examined did not separate Capsicum frutescens into a distinct cluster.Thus, the result does not support the distinction of Capsicum frutescens as a separate species.Based on the distance between species of different clusters, contrasting parents may be identified and used in hybridization programme for generating wider variability for selection and crop improvement.

Fig. 3 .
Fig. 3. Variation in size and shape of mature fruits of the Capsicum varieties

Fig. 5 .
Fig. 5. Dendogram constructed from quantitative morphological data using PGCA clustering method The highest plant height was recorded from C. frutescens var.baccatum while the lowest height at maturity was recorded in Capsicum annuum var.grossum.Highest total leaf length was recorded in C. frutescens var.baccatum with the least recorded in C. annuum var.grossum.However, C. annuum var.acuminatum and C. frutescens var.baccatum were found to be statistically the same.Furthermore, there was no statistical difference in C. annuum var.abbreviatum in comparism with C. annuum var.acuminatum and C. frutescens var.baccatum (Table 2).The widest leaf was obtained from C. annuum var.abbreviatum with C. frutescens var.baccatum recording the smallest leaf breadth.Highest stem girth was recorded in C. frutescens var.baccatum and the lowest was recorded in Capsicum annuum var.grossum (Table 2).With respect to the number of leaves, the highest number of leaves was recorded from C. frutescens var.baccatum with a mean value of 73.40.The least number of leaves was obtained from C. annuum var.grossum.The highest leaf area was found in C. annuum var.abbreviatum, followed by C. annuum var.acuminatum and C. frutescens var.baccatum, while the least was recorded in C. annuum var.grossum.However, C. annuum var.acuminatum and C. frutescens var.baccatum were found to be statistically the same (Table 2).

Table 2 .
Vegetative growth performance of the four varieties of Capsicum species.Data in mean ± standard error Means in the same row with the same superscript are not significantly different at p˂0.05.

Table 6 .
Fruit and seed growth performance of the four varieties of Capsicum species; data in mean ± standard error Traits C. annuum var.abbreviatum C. annuum var.acuminatum C. annuum var.grossum C. frutescens var.baccatum Means in the same row with different superscript are not significantly different at p˂0.05.