Significance of Fruit and Seed Coat Morphology in Taxonomy and Identification for Some Species of Brassicaceae

Fruit, seed morphology and seed coat sculpturing were recorded for 10 species belongs to 9 genera and five tribes of Brassicaceae in eastern region of Saudi Arabia by using light microscope (LM) and scanning electron microscope (SEM). Four seed patterns were recognized based on surface sculpturing pattern; Reticulate, with four subtypes, undulate, verrucate and ocellate. The results of numerical analysis showed that species were grouped into two major clusters and each cluster divided into two groups. The cluster analysis indicates that the characters of fruit and seed are very important characters for identification and taxonomical classification of the tribes. The results indicate that the most tribes of Brassicaceae are polyphyletic, and some are related to each other’s as Lepidieae, Alyssieae and Sisymbrieae.


Introduction
The Brassicaceae (Cruciferae), or mustard family, is one of the largest angiosperm families, can be recognized easily by its floral and fruiting characters.It is cosmopolitan but chiefly distributed in the temperate and Mediterranean region, it represented by 338 genera and 3709 species [1].In Saudi Arabia, it is represented by 53 genera and 74 species [2].In eastern region of Saudi Arabia, [3]  sub tribe, then [6] put the members of the family in 10 tribes by using nectar glands and myrosin cells characters.[7] divided the family into three groups according to fruit characters into Siliquosae, Siliqulosae and Lomentaeae, each composed of number of tribes.[8] classifies the genera of the family in 19 tribes.[9] reduced the total number of Schulz's tribes from 19 to 15 tribes depending on very large numbers of features of flowers and fruit.[10] recognized 6 tribes under the family Cruciferae according to fruit characters, but [11] divided the family into 6 groups and 10 tribes.[12] [13] reduced Schulz's tribes to three tribes: The lypodieae, Brassiceae and Sisymbrieae.[14] classified the family Brassicaceae of the southeastern United States into 7 tribes; then [15] recognized 25 tribes for the family Brassicaceae.Recently there are some researches based on molecular and phylogenetic studies for the family as [16]- [29].
The morphology of seed coat structure is considered to be stable characters and is little influenced by external environmental conditions whilst the seeds develop and ripen within the fruit [30] [31] [32] [33].The seed coat characters have been used successfully to solve systematic and phylogenetic problems between different taxa [34] [35].In the Brassicaceae, several studies have been carried out on the seed coat and its taxonomic significance.[36] [37] [38] [39] studied the external morphology and anatomy of seeds of 90 genera and 200 species of the Cruciferae.[40] [41] [42] investigated the seed coats and development of epidermal slime bodies of Malcolmia.[43] studied the seed coat of Lepidium in East tropical Africa.[44] investigated the structure of seed coat and development in certain species of Brassicaceae.[45] [46] studied some taxa of Brassica seeds by using scanning electron microscope (SEM).[47] used SEM on the seed surface to distinguish Matthiola, Morettia, and Diceratella.[48] studied the seed coat morphology, particularly the epidermal slime structure of Morettia.[49] studied the seed coat of genus Farsetia by using SEM to distinguish between species.[50] investigated the seed coat sculpture in species of the tribe Brassiceae in Egypt by using SEM.[51] investigated the seed morphology and taxonomy with SEM in the tribe Lepidieae in Egypt.More recently, [52] gave detailed descriptions of seed morphology in 44 species of the subtribes Brassicinae, Raphaninae and Moricandiinae, and elucidated the phylogenetic relationships between taxa.
[53] study seed morphology of 45 taxa belonging to 23 genera from tribes Arabideae, Euclidieae, Hesperideae, Lunarieae, Matthioleae, and Sisymbrieae, of Brassicaceae were examined using light and scanning electron microscopy.[54] studied the macro and micromorphological characters of approximately 22 genera, 30 species of Brassicaceae.[55] study seed coat sculpture of 93 taxa belonging to 45 genera of the family Brassicaceae from Egypt by using light and scanning electron microscope.[56] investigated seed morphology and seed coat sculpturing of 32 Taxa of Family Brassicaceae by using light microscope and scanning electron microscope.[57]

Material and Methods
Ten species belong to nine genera were collected fresh in spring season from different area in eastern region of Saudi Arabia (Table 1).The materials studied were identified according to plant key of [2] [3].Foliar details for fruit and seed were examined with the aid of binocular stereo microscope (Olympus Bx53).
The details of seed morphology were investigated in electron scanning microscope (SEM) with the use of FEI, ISPECT S50 (Czech Republic).SEM was operated at 20 kV with working distance 10 mm.Long and high magnification was performed to capture the recognized features of the specimen.The seeds were The terminology of [32] [61] [62] was adopted to describe the SEM aspects of the seed coat.The morphological characters of the fruit and seed for the studied species were collected and creating data matrix used for numerical analysis.The relationships between the studied species have been demonstrated as dendrograms Figure 4 by using PRIMER 6, version 6.1.6analyses using agglomeration of Schedule measure Euclidean distance, using complete linkage between groups.

Results
The fruit and seed morphological characters for ten species of the family Brassicaceae are summarized in Tables 2-6 and the image by and SEM are represented in Figures 1-3.

Fruit Pedicel
The texture of pedicel is glabrous in most studied species except in Coronopus didymus and Farsetia burtonae are hairy.The pedicel length has greeted variation in the studied species; it ranged from short, long and very long pedicel.

Fruit Types
The family Brassicaceae is characterized by two types of fruit; siliqua and silicula, in the present study the silicula fruit noted in Savignya parviflora and Coronopus didymus.The reminder studied species have the type of siliqua.

Fruit Segmentation
Most studied species are non-segmented, segmented fruit are present in Raphanus sativus, Cakile Arabica and Farsetia burtonae.

Fruit Shape
The shape of fruit showed great variation.In the studied species the fruit shape recorded three main types; linear or terete in Brassica juncea, Brassica tournefortii, Farsetia burtonae, Cardamine hirsute and Sisymbrium irio.Oblong in Eruca sativa, Raphanus sativus, Cakile arabica and Savignya parviflora.Subreniform in Coronopus didymus.

Fruit Direction and Opening
The most species of family Brassicaceae are characterized by erect and dehiscent fruit.In the studied species four spices [Eruca sativa, Savignya parviflora, Cardamine hirsute and Sisymbrium irio] are erect and dehiscent, while erect and indehiscent in Cakile arabica and Coronopus didymus.Erect to slightly curved and dehiscent in Brassica juncea, Brassica tournefortii and Farsetia burtonae.Curved and indehiscent in Raphanus sativus only.

Fruit Size
The size of fruit in the studied species are different from very long (more than 3 cm.),long (1 -3 cm.) and short (less than 1 cm.) in length or narrow (less than 2 mm.) or wide (more than 2 mm.) in width.Very long and narrow in Brassica tournefortii and Sisymbrium irio; long and narrow in Brassica juncea, Farsetia burtonae and Cardamine hirsute while long and wide in Eruca sativa, Raphanus sativus and Cakile arabica.Short and wide in Savignya parviflora and Coronopus didymus.

Beak Characters
The size of the beak ranged from 0.2 mm -16 mm in length.The smallest size 0.2 mm present in Coronopus didymus, while the longest 16 mm present in Raphanus sativus.The beak is seedless in all studied species except in Brassica tournefortii have one or two seed.The shape of the beak in the studied species recorded three types, conical in Brassica juncea, Brassica tournefortii, Eruca sativa and Raphanus sativus, flat in Cakile Arabica and Savignya parviflora, cylindrical in the reminder.

Seed Color
The color of seeds is highly important for distinguish between different species.
The seed color in studied species varied from yellow in Cardamine hirsute and Sisymbrium irio; yellowish-brown in Cakile arabica, Savignya parviflora, Coronopus didymus and Farsetia burtonae; brownish-yellow in Eruca sativa and brown in the remainder species.

Seed Shape
Seed shape of the studied species recorded four types; globose in Brassica juncea and Brassica tournefortii; sub-globose in Eruca sativa; kidney shape in Coronopus didymus; oblong ovate in Savignya parviflora, Farsetia burtonae and Cardamine hirsute; oblong in the reminder.

Seed Hilum
The hilum of seed in most studied species is terminal except in Cakile arabica, Savignya parviflora, Farsetia burtonae, and Sisymbrium irio are sub terminal.

Seed Size
Seed length and width varied among the studied species; it ranged from 0.

Seed Surface
The seed surface of the studied species recorded four types; reticulate in Brassica juncea and Raphanus sativus; warty in Brassica tournefortii; glabrous in the reminder.

Seed Margin
The seed are winged in two species only [Savignya parviflora and Farsetia burtonae], and not winged in the rest studied species.

Shape of Outer Epidermal Cell
Outer epidermal cells can be of considerable important value for systematics.
The outer epidermal cells varied from polygonal to elongated cell in Brassica juncea, Cakile arabica, Coronopus didymus and Farsetia burtonae; irregular poly gonal cell in Brassica tournefortii and Savignya parviflora; isodiametric polygonal cells in Eruca sativa; polygonal cell in the reminder species.

Anticlinal Wall
The characters of anticlinal wall can be used for identification and differentiate the different species.The shape of anticlinal wall recorded three types in the studied species; undulate in Brassica tournefortii, Savignya parviflora, Cardamine hirsute and Sisymbrium irio; straight in Brassica juncea only and straight to sinuous in the reminder species.The main types for the texture of the anticlinal wall is smooth, but it smooth to fold in Brassica tournefortii; smooth to warty in Eruca sativa and smooth to striated in Cakile arabica.The texture is reticulate in Brassica juncea and striated in Raphanus sativus.The anticinal wall varied from thin to thick, it is thick in Brassica tournefortii, Eruca sativa, Raphanus sativus and Coronopus didymus; while thin in the reminder species.All the studied D. G. Gabr DOI: 10.4236/ajps.2018.93030394 American Journal of Plant Sciences species with raised anticlinal wall except in Savignya parviflora and Sisymbrium irio are grooved.

Periclinal Wall
The characters of the outer periclinal cell wall can be good diagnostic characters.
The present study recorded five types for the shape of Periclinal wall; flat in Brassica juncea and Cakile arabica; flate to concave in Farsetia burtonae; flat and convex in center in Cardamine hirsute; convex in Savignya parviflora and Sisymbrium irio; concave in the rest of species.The texture of Periclinal wall have five types; micro-reticulate in Brassica juncea and Raphanus sativus; fine folded in Brassica tournefortii; striate in Cakile arabica and Savignya parviflora; smooth to fine striate in Farsetia burtonae; smooth in the rest studied species.

Seed Wing
There are two seeds from the studied species are winged, the coat pattern sculpture for these seeds are tuberculate in Savignya parviflora and scalariform in Farsetia burtonae.The outer epidermal cell shape is isodiametric to irregular polygonal in Savignya parviflora while elongated in Farsetia burtonae.The shape of anticinal wall is straight to undulate in Savignya parviflora while straight to sinuous in Farsetia burtonae.The texture of anticinal wall is striate in Savignya parviflora and smooth in Farsetia burtonae.The thickening and level of anticinal wall for the two speces are thin and grooved.The shape of Periclinal wall is convex in the two winged species, but the texture is striate in Savignya parviflora while smooth to folded in Farsetia burtonae.

Numerical Analysis
The fruit and seed morphology characters of the studied species obtained by light and scanning electron microscope 34 characters (14 characters for fruit and 20 characters for seed) were used for numerical analysis.
The cluster analysis obtained from fruit characters only (Figure 4(a)) showed that species were grouped into two major clusters and each cluster divided into two groups, the first cluster I consisted of five species: Coronopus didymus and Farsetia burtonae recorded in group I while Cardamine hirsute, Cakile arabica and Sisymbrium irio present in group II.The cluster II comprised five species: Raphanus sativus (group I) and Brassica juncea, Eruca sativa, Brassica tournefortii and Savignya parviflora (group II).
The results obtained from seed characters only (Figure 4

Discussion
The tribal classification of Brassicaceae has long been problematic and not well understood phylogenetically because of the traditionally used few characters as cotyledons in the embryo, fruit characters, number of rows of seeds in each locule, trichome type, and features of the nectarines etc. to delimit different tribes of the family Brassicaceae [23] [63].In the present study, the species are recorded in five tribes (Alysseae, Lepideae, Sisymbrieae, Cardamineae, Brassiceae) [15].
The tribe has recently been studied phylogenetically as [65] [66].The position of Alysseae relative to the other tribes varied in different studies; [20] put the tribe with tribe Brassiceae in lineage II, while [63] say the tribe appeared either related to Erysimum or unrelated to any tribe.[67] found the tribe related to the Noccaeeae, while [68] it grouped together with the tribes Chorisporeae, Hesperideae, and Malcolmieae.In the present study tribe Alysseae (Farsetia burtonae) are placed with tribe Lepideae (Coronopus didymus) in the cluster result from fruit characters only, this differ from the traditional classification which separate them into different tribes or different groups, but it agree with recent molecular studies as [27] which put the two tribes in the same lineage.While in the clusters results from seed or seed and fruit characters the tribe Alysseae (Farsetia burtonae) are placed with species of tribe Brassiceae (Savignya parviflora), this agree with some molecular studies as [27] [69].
The tribe Lepideae consists of three to five genera and over 240 species, [15] [70].It is polyphyletic.Some genetic studies as done on the tribes, [27] [69] [71] [72] [73].In the present study the tribe Lepideae (Coronopus didymus) are placed with some species of tribe Brassiceae, this agree with some traditional studies as [5] which put them in the same tribe but in different sub-tribes, or placed with species of tribe Alysseae (Farsetia burtonae) in the same group.
The tribe Sisymbrieae was recognized with 70 genera and 400 species according to [8] [74] but molecular data as, [18]  Brassieae are placed together but some species present with different tribes.This results support the Brassiceae to be heterogenous.

Conclusion
The fruit and seed coat characters are very important characters that can be used for identification and differentiate between the different tribes and also between species belong to the same genera.Most tribes of Brassicaceae are polyphyletic, and some are related to each other's as Lepidieae, Alyssieae and Sisymbrieae.
The results indicate the tribe Brassiceae to be heterogenous.
(b)) also divided the studied species into two main clusters and two groups.Cluster I contained Savignya parviflora and Farsetia burtonae.While cluster II consisted of eight species present in two groups; Cardamine hirsute, Raphanus sativus, Coronopus didymus, Eruca sativa and Cakile arabica in group I and Brassica juncea, Brassica tournefortii and Sisymbrium irio in group II.The results obtained from combined of fruit and seed characters (Figure 4(c)) divided the species into two main clusters and two groups.Cluster I contained D. G. Gabr DOI: 10.4236/ajps.2018.93030395 American Journal of Plant Sciences

Figure 4 .
Figure 4. Dendrograms showing the interrelationships between ten species of Brassicaceae based on 34 characters, (I) cluster one, (II) cluster two, (G1) group one, (G2) group two, (a) fruit characters, (b) seed characters, (c) combined fruit and seed characters by using PRIMER Program.
[75] [76][77], supported the removal of many of its component genera to other tribes.The tribe is monophyletic and is sister to the Schizopetaleae[20] or the Brassiceae[18] [75].In the present study the tribe Sisymbrieae (Sisymbrium irio) found together with species from tribe Brassiceae, this agrees with[5] [18] [27] [69] [75].[9] presented a phylogenetic network and indicated close relationships of D. G. Gabr DOI: 10.4236/ajps.2018.93030397 American Journal of Plant Sciences Sisymbrieae with Arabideae and Lepidieae.[78] [79] have shown that the boundaries between the tribes Lepidieae, Lunarieae, Sisymbrieae, Euclidieae, and Alysseae are highly artificial.The results from previous studies of phylogenetic relationships [71] [72] [73] demonstrated a close relationship among the tribes Sisymbrieae, Lepidieae and Arabideae.[70] indicated that the tribes Arabideae, Lepidieae and Sisymbrieae are polyphyletic.The tribe Cardamineae DC is monophyletic.The tribe has been subjected to several molecular studies as; [80] [81] [82] [83] [84] that support its monophyly.The position of Cardamineae relative to the other tribes is done by some studies as, [27] [69].The study put the tribe Cardamineae with tribe Lepidieae and tribe Alysseae in the same lineage.In the present study the tribe Cardamineae (Cardamine hirsute) placed together in the same group with some species of tribe Brassiceae and tribe Sisymbrieae in all clusters, this agree with [5].The tribe Brassiceae DC is consider monophyletic according to some molecular studies as [19] [85] [86].This tribe was subjected to extensive molecular studies, [20] [63] [67] [69] [87] [88].In the present study the most species of tribe [59]study the seed exomorphic characteristics of 14 taxa of Lepidium L. in Brassicaceae from Turkey by light microscope and scanning electron microscope (SEM).[59]studymacro and micromorphological characters of 9 species and 30 accessions seeds in Ricotia L. by light (LM) and scanning elec- study the relation between 26 taxa of family Brassicaceae from Egypt by using morphological characters including fruit and D. G. Gabr DOI: 10.4236/ajps.2018.93030382 American Journal of Plant Sciences seed characters, [

Table 1 .
Collection data of the studied species and its tribal classification.All species from eastern region of Saudi Arabia.
ES, UK) to avoid charging and capture high quality images.

Table 2 .
Fruit morphological characters of the studied species.

Table 3 .
Macro morphological characters of seed of studied species.

Table 4 .
Seed coat micromorphology of the studied species.

Table 5 .
Characters list for the numerical analysis of the studied species of Brassicaceae.

Table 6 .
Data matrix of morphological characters listed in Table5.

Table 4 &
Figure 3Examination by scanning electron microscope on the studied species shows four different types of seed coat pattern; the reticulate is the main types in which b) Reticulate-foveolate in Eruca sativa.c) Irregular reticulate present in Cakile arabica.d) Reticulatewith globular central body in Cardamine hirsute.2) Undulate in Brassica tournefortii.3) Verrucate in Savignya parviflora.4) Ocellate in Sisymbrium irio.
Savignya parviflora and Farsetia burtonae.While cluster II consisted of eight species present in two groups; Cardamine hirsute, Raphanus sativus, Cakile arabica, Eruca sativa and Sisymbrium irio in group I and Brassica juncea, Brassica tournefortii and Coronopus didymus in group II.