Abstract: The macro- and micromorphological characters of five selected species of Myrtaceae was examined representing five genera, two tribes and two sub-families to see if it could throw light on the general morpho-anatomical features. A general description of macro- and micro morphological characters of vegetative and floral parts is given using LM and SEM and the importance of them for the identification and distinctness between taxa is assessed by means of statistical analysis utilizing criteria from morphology and anatomy of stem, petiole, lamina, flower and seeds. The floral anatomical criteria provide data which support the appendicular nature of the inferior ovary in all the species under investigation. The dendrogram produced from the cluster analysis of all combined characters indicates that there is high degree of homoplasy amongst both morphology and anatomy of the studied taxa of tribe Myrteae with the Eucalyptus camaldulensis from tribe Leptospermeae.
INTRODUCTION
Family Myrtaceae is spread over 80 genera and 3000 species. Cronquist[1] and Willis[2] regard 100 genera and 3000 species. The plants are distributed in tropical and sub-tropical regions with centers of distribution in Australia and America. The family plays a significant role in characterizing the vegetation of Brazilian floras. The genus Eugenia L., with 14 species being the most numerous representative in the ecosystem of Rio de Janeiro[3]. Barroso et al.[4] pointed out that the American species of Myrtaceae are very similar morphologically and their identification being therefore difficult.
Myrtaceae attract the attention of many authors to do with it, some of the recent literature in this subject are the work of Andrew et al.[5], McDonald et al.[6], Michael et al.[7], Pound et al.[8], Rachel et al.[9] and Stephen and David [10].
Myrtaceae includes trees or shrubs bearing essential oils. Helophytic to xerophytic. Heterophyllous sometimes markedly so, e.g. Eucalyptus spp. or not heterophyllous. There are gland dotted aromatic edgewise to the stem and leaves commonly in Callistemon and Eucalyptus[11].
Among studies in comparative anatomy, Bailey and Howard[12] studied the wood characteristics and their potential to separate groups within the family and to define lines of phylogenetic specialization. Howard[13], Johansen[14] and Khatijah et al.[15] when commenting on the taxonomic significance of the anatomical characteristics, asserted that they sometimes show affinity between species.
Cuticular and epidermal studies on Myrtaceae have been carried out by many workers as characters to separate up to the level of genera and species[16-26].
The nature of the inferior ovary has always received a great attention on telling evolutionary pathways; and all the floral phenomena that of the inferior ovary has doubtless been extensively discussed. Since the nineteenth century two theories about its nature have received strong support and, simply presented, these are the appendicular theory[27] and the axial theory[28,29].
Not easy as above either theory can be accepted since the whole situation is further complicated by the so-called hypanthium[10]. Eames[29] stated that the inferior ovary had developed in two morphologically different ways, by adnation of the floral appendages and by hollowing the axis tip. In monocots, however, the case is not as easy as in dicots. This is because, as Stebbins[30] states, epigyny in monocots is derived by routes which differ from those of the dicots.
More recently, SEM and embryological development in certain taxa are helping considerably in solving some of the taxonomic problems[31-35].
The ultimate goal of the present study is threefold: (1) to gain further insight into morpho-anatomy of selected taxa of Myrtaceae by using LM and SEM, (2) to further our understanding of floral anatomy in general and specifically the nature of the inferior ovary and (3) to clarify the interrelationships among the present members of Myrtaceae.
MATERIALS AND METHODS
The five investigated taxa, the sources of collection and classification cited after Willis[2] are listed in Table (1). These selected taxa represent the famous ornamental and cultivated myrtaceous plants in Egypt. The voucher specimens are kept at the Herbarium of Botany Department, Faculty of Science, Ain Shams University. The identification of the studied taxa was done according to Bailey[36].
Macromorphological characters were studied from the fresh specimens as well as relevant literature[37, 2, 38].
The anatomical investigation was achieved through transverse sections of stem, petiole and lamina by a hand microtome at 15-20 μm and stained with safranine and light green. Also, stomata and epidermal cells were examined in epidermal peels stained with toludine blue. In addition to some microphotographs of some specific structures.
For floral anatomy, the flower buds were fixed in F.A.A. and then stored in alcohol[39]. These buds were dehydrated and embedded in paraffin wax of melting point 56-58°. Transverse sections were cut from pedicel upward at 10-18 ìm thick on a Rotary microtome and double stained with crystalviolet-erythrosine[40]. These sections were examined under light microscope. In addition to some microphotographs of some specific structures.
For SEM examination, the seeds were cleaned and washed carefully then mounted on brass stubs and coated with gold palladium in sputter coating unit. The scanning was carried out by a Joel JSM 100 SEM at accelerating voltage 15 Kv. The terminology used here to describe the seed surface sculpture was adopted after Stearn[41], Barthlott[20] and Munson[42]. The comparative evaluation were made at 600, 1500 and 2000 magnification according to the seed size.
The taxa under investigation were collected from Botanic Garden, Department of Botany, Faculty of Science, Ain Shams University, Abassia, Cairo, Egypt.
For the data analysis, the data obtained from the total number of the recorded characters in each taxon were subjected to the numerical analysis. The presence or absence of each of all characters was treated as a binary character in a data matrix. For computation the SPSS, version 10 was used.
| Table 1: | Collected data and their classification after Willis[2] |
SPSS (Statistical Package for the Social Science), version 10 is a data management and analysis product. It can perform a variety of data analysis and presentation functions, including statistical analysis and graphical presentation of data.
The OTU’s (Operational Taxonomic Units), produced from the analysis of present characters of the taxa under investigation and resulted in a dendrogram that was compared with the current taxonomic treatments of Myrtaceae.
RESULTS AND DISCUSSION
The morphological and anatomical characters of the vegetative and floral organs of the studied taxa provide 312 indicative characters illustrated in Table 2 using LM and SEM.
By comparing the present data with the former results of Willis[2], it was observed that the macro-morphological characters of the vegetative and floral organs are more or less consistent among the studied species of tribe Myrteae and Leptospermeae Table 2 (specimens 01, 02 and 04) for more details. The characters of Eugenia uniflora and Psidium guajava provide data to support the inclusion of them in one series and Callistemon citrinus, Eucalyptus camaldulensis and Melaleuca leucadendra in another series. The morphological features of the seed surface sculpture as revealed by SEM show no clear boundaries between the species of the same tribe except minor variations in the aspect of the anticlinal and periclinal walls (Table 2 and Plate II). The former results supported by the dendrogram produced from the cluster analysis of 152 macromorphological characters of vegetative and floral organs (Fig. 1) in which the species have highest taxonomic distance of 25. The dendrogram shows two series, Series I includes Eugenia uniflora and Psidium guajava while series II includes Callistemon citrinus, Eucalyptus camaldulensis, Melaleuca leucadendra. This is in accordance with Willis classification (Table 1).
The stem anatomical characters of the species under investigation recorded in Table 2 show great homogeneity amongst Callistemon citrinus, Eucalyptus camaldulensis, Melaleuca leucadendra and Eugenia uniflora (Plate I). These observations was supported by the phenogram produced from the cluster analysis of 54 microanatomical characters (Fig. 2) in which the studied species show highest average of taxonomic distance of 25. Two series are recorded one with Psidium guajava and the second with the remaning studied taxa.
Johansen[39] when commenting on the taxonomic significance of anatomical characteristics asserted that they sometimes show affinity between species and define the position of given species.
| Table 2: | Macro- and micromorphological criteria of the selected studied taxa of Myrtaceae. 01: Callistemon citrinus; 02: Eucalyptus camaldulensis; 03: Eugenia uniflora; 04: Melaleuca leucadendra; 05: Psidium guajava; 0: Absent; 1: Present |
Also, in the available bibliography of Myrtaceae there is an emphasis on the anatomy of the leaf blade, but the anatomy of the petiole can also provide data for the taxonomists[13,15,32]. Khatijah et al.[15] and Paula[43] recommended that the leaf anatomy play an important role in the systematics of certain families.
In agreement with those the petiole and lamina anatomical characteristics in the current study provide data which enclose the studied species of tribe Myrteae with Eucalyptus camaldulensis from Leptospermeae in one series (Table 2 and Plate I). This is supported by the dendrograms produced from the cluster analysis of 29 petiole anatomical characters and 42 lamina characters (Fig. 3 and 4).
The pathway of the vascular traces to the different floral organs in all investigated species is the same. The vascular supply at the receptacular region in all the studied species show continuous siphonostelic structure.
| Fig. 1: | Morphology |
| Fig. 2: | Stem anatomy |
| Fig. 3: | Petiole anatomy |
Few micrometers above the central stele diverges into group of protrusions ranging from eight as in Eugenia uniflora and ten as in the rest of the studied taxa. These protrusions represent vascular complexes to the sepals, petals and stamens. Four outer and four inner as in Eugenia uniflora and five outer and five inner as in the rest studied taxa. After the departure of these complexes, the remaining central stele is differentiated into group of separate vascular bundles. Four as in Eugenia uniflora, six as in Callistemon citrinus and Melaleuca leucadendra, eight as in Eucalyptus camaldulensis and eight to ten as in Psidium guajava. These bundles representing two ventrals and two dorsals carpellary bundles as in Eugenia uniflora.
| Fig. 4: | Lamina anatomy |
| Fig. 5: | Floral anatomy |
| Fig. 6: | All combined characters |
Three ventrals and three dorsals as in Callistemon citrinus and Melaleuca leucadendra. Four ventrals and four dorsals as in Eucalyptus camaldulensis. Four to five ventral and the same dorsal as in Psidium guajava. The septal bundles are numerous and derived from the ventral bundles whereas the wall carpellary bundles are derived from the dorsals. At higher level and beneath the level of locules differentiation the outer complexes migrate through the receptacular tissue to the sepals, petals with numerous ramifications to the sepals and petals margins and to the numerous filaments. From the former data it was observed that the behavior of the traces is consistent amongst the taxa of tribe Myrteae and Leptospermeae. The only difference is the number of the traces to each whorl (Plate III).
| Plate I: | Fig. 1-13: Microphotographs |
| Palte II: | |
| Plate III: | Fig. 1-7: Floral microphotographs of Eugenia uniflora from pedicel upwards showing the appendicular nature of the inferior ovary. Scale bar (x =x 3.2x5.3) |
The phenogram produced from the cluster analysis of 35 floral micro-characters encourage the separation of Eugenia uniflora from the rest of studied species in one series (Fig. 5) which consider the more advanced in its floral characters.
In view of the above data in the Table 2, it is of primary importance to draw attention towards what is meant by epigyny.. The phenomenon of epigyny is the reversed topographical position of the ovary relative to the other floral organs.
The interpretation of data obtained from the vasculature of the flower of the selected studied taxa clarified the state of epigyny as follow, the ovary in all the flowers is wholly appendicular, where the vascular traces to all the floral organs arise free from the early beginning as totally independent traces which run distinct at a level lower than that of the locules and remain as such. This indicates that the flower in Myrtaceae is false epigyny. This is in agreement with the work of Barabe[44] on the pistillate flower of Begonia handellii. His results showed that the inferior ovary is formed by the union of appendicular organs. Awasthi and Kumar[45] studied the floral histogensis in Amaryllis belladonna L. and A. vittata Ait.(Amaryllidaceae) and suggested that the stylar part of gynoecium is formed by zonal growth below the carpel primordia. On the basis of ontogeny the inferior ovary is interpreted as appendicular, thus supporting the observation of earlier workers based on floral anatomy. Also the criteria here is compatible with the work of Stephen and David[10] on the development of hypanyhium of Acmena smithii and Syzgium australe (Acmena alliance).
When employing all available morphological and anatomical characters of the vegetative and floral organs including the phenogram produced from the cluster analysis of 312 characters Fig. 6 in which the species have highest average taxonomic distance of about 25. The phenogram based on the combination of all characters shows two main series; Series I includes Eucalyptus camaldulensis in one sub-series of one cluster and Eugenia uniflora and Psidium guajava in another sub-series. Series II includes Callistemon citrinus and Melaleuca leucadendra in one cluster as closely related taxa with great affinity.
The combination of all morphological and anatomical characters of both vegetative and floral organs of the studied taxa for computation and illustrated phenogram (Fig. 6) support the position of all taxa under the specific tribes as cited previously by Willis[2] except Eucalyptus camaldulensis which shares important morpho-anatomical features with the taxa studied under the tribe Myrteae. Finally we recommend to do extensive morphological and molecular studies to obtain more criteria to interpret the relationships and affinity between the studied taxa.