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الملخص

تمت دراسة الصفات المورفولوجية والجزيئية لعدد خمس وعشرون وحدة تصنيفية تابعة لتحت الفصيلة البقمية (الفصيلة القرنية). وقد مثلت العينات المدروسة أربعة قبائل ، خمسة عشر جنسا وخمسة وعشرون نوعا كلها عينات محلية منزرعة فى عدد من الحدائق النباتية فى مصر.

الهدف من الدراسة:

اجريت الدراسة بهدف استنباط الصفات الشكلية الظاهرية للأعضاء الخضرية للنبات، النورة، الزهرة، الثمرة و البذرة. كذلك سطح الورقة لتتبع الشبكة المعمارية لسطح النصل ، الثغور، الشعيرات و البلورات باستخدام الميكروسكوب الضوئى والالكترونى الماسح. كما تهدف الدراسة الى استنباط البصمة الوراثية البيوكميائية والجزيئية ودرجات التشابه بين الوحدات التصنيفية قيد الدراسة باستخدام التباين فى التضاعف العشوائى لأجزاء من الدنا فى جهاز سلسلة تفاعلات إنزيم البلمرة (RAPD-PCR) ومشابهات الأنزيمات. كذلك ايجاد وتفسير العلاقات البينية بين الانواع المدروسة من خلال تحويل النتائج الى شفرة وادخالها الى البرنامج الاحصائى المشار اليه فى الرسالة والحصول على الشجرة التصنيفية مع مقارنة هذا الشكل مع بعض انظمة التصنيف التى تتبعت تصنيف تحت الفصيلة البقمية.

وقد خلصت الرسالة الى مجموعة كبيرة ومتنوعة من الشواهد والدلالات والأستنتاجات نوجزها فيما يلى:



اولا: الشواهد المورفولوجية للنبات

1. نمط حياة النبات : تنوع مابين اشجار وشجيرات، طول النبات : تنوع ما بين الطويل ومتوسط الطول، الملمس : تنوع ما بين الخشن ، الشوكي ،الشعيرى أوالأملس، تركيب الورقة: اما بسيطة أو ريشية احادية التضعيف، ثنائية الطرف أو ثنائية التضعيف، شكل النصل : قلبى ، بيضاوي ، مستطيل، مستطيل بيضاوي، إهليلجي بيضي ، مستطيل بيضي ، بيضى مقلوب، أو مسحوب، قمة النصل : غائرة، مذنبة ، حادة ، دائرية ، مشقوقة ، مقرنة حادة أو مستدقة، قاعدة النصل : قلبية ، رمحية أو دائرية، حافة النصل: مسننة وهذة الصفة كانت متفردة في Gleditsia caspica أو كاملة فى باقى العينات قيد الدراسة، الاذينات: غائبة، صوانية ، قلبية ، شوكية، بيضاوية ، رمحية أو شريطية، النورة : تنوعت ما بين سنبلية ، مشطية أو عنقودية. كما ان عدد الازهارفى النورة تراوح بين العدد القليل أو الكثير. الطول : قصيرة أو طويلة، المكان : مقابلة للورقة ، ابطية وطرفية ، إبطية أو طرفية، القنابة: غائبة او ممثلة فى شكل قنابة مثلثة ، قلبية، حرشفية، بيضية، رمحية، بيضية، بيضية مقلوبة أو مستطيلة، الكأس: تراوح بين الأملس ،الشعيرى أو المخملي. أحمر، وردي ، قرمزي ، بني ، أخضر أو أصفر، الكاس اربعة او خمسة سبلات ما بين الملتحم والسائب. اما رمحية ، بيضية رمحية، بيضية ،بيضية مقلوبة أو مستطيلة، التويج : اربعة أو خمسة بتلات سائبة، تتراوح ما بين الأملس و الشعرى. أبيض، أخضر، أصفر، أحمر، وردي أو قرمزي. والبتلات اما بيضية مقلوبة، بيضية ، بيضية مقلوبة الى مستطيلة ، إهليلجي بيضي ، مستطيل ، شبه دائري ، مخروطية أو ملعقية، الطلع : تراوح ما بين ثلاثة ، خمسة ، سبعة ، ثمانية ، تسعة، عشرة أو اثنى عشركلها خصيبة أو عشرة اسدية( سبعة خصيبة وثلاث عقيمة ، أوستة خصيبة وأربعة عقيمة أو خمسة خصيبة وخمسة عقيمة). الطول :مساوية للبتلات، اطول من البتلات، أقصر من البتلات. ملمس الخيط : شعيرى، غدي أو املس. الشكل : مخرز أو خيطي. وهذا الأخير قد يكون على شكل ملتوى أو به انتفاخ. حجم المتوك : غير متكافئة ، شبه متكافئة أو على قدم المساواة. أتصال المتك بالخيط: ظهرى ، قاعدى أو مدلى، المبيض : أملس ، شعيرى أو زغبى. الشكل: مبطط ، مستطيل أو اسطوانى . وضع المبيض : جالس ، شبه جالس أو محمول. طول القلم: قصير أو طويل. الشكل: مبطط ، اسطوانى أو خيطي. الانحناء: منحنى، منحنى لأعلى او مستقيم. شكل الميسم : هامي، شبه هامى، مقعر ، مثلثى ، درعى الشكل أو بسيط، القرن : زغبى أو املس. أصفر أو بنى. شريطى ، شريطى مستطيل ، مستطيل ، اسطوانى أو اهليجى. متفتح أو غير متفتح. ذو قمة حادة، مقرنة، شوكية، مستدقة أو دائرية. بدون منقار أو بمنقار طويل أو قصير. النوع : غشائي ، قشرى، شبه خشبي أو خشبي. البذرة : برتقالية ، سوداء ، سوداء مخضرة، خضراء أو بنية. اسطوانية، شبه اسطوانية، مضغوطة أو مبططة. بيضية، بيضية مقلوبة ، دائرية ، شبه قلبية، كمثرية أو مستطيلة.

2. أنماط الشبكة المعمارية للنصل (Lamina Architecture): الابتدائي المنوال : campylodromous أو ريشي.

الثانوية المنوال : cladodromous ، brochidodromous أو festooned brochidodromous. عدد العروق السفلية: تراوحت ما بين 1 الى 7 و 13 او 14 الفراغات بين العروق الثانوية: متجانسة أو غير نظامية. الثلاثية المنوال : قد تكون غير متميزة، متفرعة ، alternate percurrent أو عشوائي شبكي. مسار العروق الثلاثية المنوال : متعرج ، مستقيم ، متشعبة جهة العرق الوسطى أو متشعبة بعيدا عن العرق الوسطى. زاوية العروق الثلاثية المنوال: منفرجة أو حادة. رباعي المنوال : غير متميز، alternate percurrent ، ثنائى التفرع ، أو مضلع منتظم شبكي. الجزر بين العروق: غائبة ، ضعيفة التميز ، متوسطة التميز أو عالية التميز. نهايات الأوردة : غائبة ، غير متفرعة ، متفرعة مرة أو اثنتين أو أكثر. هامشية العروق : غائبة ، fimbrial أو مفصصة.

3. خصائص سطح البشرة العلوى والسفلى للنصل (باستخدام الميكروسكوب الضوئى والالكترونى الماسح): شكل الخلية تراوح ما بين رباعي الزوايا ، المضلعة ، خماسية ، سداسية أو غير منتظمة. الجدر القطرية : تباينت ما بين المستقيمة ، المتعرجة ، المنحنية. نوع الشعيرات : غائبة ، وحيدة الخلية أوثنائية الخلية. نوع الثغور : paracytic ، isotricytic & tetracytic ، cyclocytic ، anomocytic أو paracytic. والأصناف قيد الدراسة تتفاوت اوراقها بين hypostomatic أو amphistomatic. الشكل : دائري أو بيضاوي. البلورات : غائبة ، غير منتظمة ، نجمية و/ أو مفردة. النحت أو زركشة سطح البشرة تراوح بين colliculate ، ruminate، reticulate ، pusticulate أو tuberculate. عرض الجدر القطرية: واسعة أو ضيقة. الارتفاع : مرتفعة او منخفضة. الملمس : أملس أو حبيبي. أرتفاع الجدر المماسية: مرتفعة او منخفضة. الملمس : أملس أو حبيبي.

ثانيا : شواهد البيولوجيا الجزيئية

1. التضاعف العشوائى لأجزاء من الدنا فى جهاز تفاعلات البلمرة المتسلسل ((RAPD-PCR

تم التحصل على عينات من الحمض النووي باستخدام عشر بادءات عشوائية في تضخيم قطع من الحمض النووي وهى على التوالى:ٍ ( SC10-5, SC10-14, SC10-17, SC10-18, SC10-22, SC10-23, SC10-25, SC10-59, SC10-64 and SC11-30 ).وقد دللت البصمات الناتجة عن بعض الحزم المميزة لكل الأنواع . وقد تم التحصل على عدد اجمالى من الحزم متعددة الأشكال (323 حزمة) من بينها 91 حزمة فريدة، استخدمت للتمييز بين الأنواع التي شملتها الدراسة.

2. مشابهات الانزيمات

تم تحديد البصمات الكيميائية الحيوية بواسطة خمسة أنظمة من مشابهات الانزيمات وهى (acid phosphatase (Acph) ,alcohol dehydrogenase (Adh), aldehyde oxidase (Alo), and esterase (Est). حيث كشفت النتائج المتحصل عليها فى هذة الدراسة عن وجود مستوى عال من تعدد الأشكال بين الوحدات التصنيفية المدروسة والتى سهلت التعرف على كل من هذة الوحدات التصنيفية مما يدل على أن الأخذ بالبصمات الجينية البيوكيميائية هو أسلوب موثوق فيه الى حد كبير جدا فى الفصل بين الانواع المدروسة.

ثالثا : التحليل العددي

أظهرت نتائج التحليل العددي الذي أعتمد علي الصفات المورفولوجيه (الشكل الظاهري للنبات والصفات الجزيئية حيث أخضعت البيانات التي تم التحصل عليها وعددها (679حالة صفة) وكذلك الشجرة التصنيفية ((dendrogram انا الوحدات قيد الدراسة قد تم فصلها الى عدد اثنان مسلسل ، ثلاثة خصل و ثمانية مجموعات شملت المسلسلة الاولى على خصلة واحدة ومجموعتين تضمنت خمسة وحدات تصنيفية بينما شملت المسلسلة الثانية على خصلتان وستة مجموعات تضمنت عشرون وحدة تصنيفية. وقد برزت أهم الاستدلالات على العلاقات البينية بين الانواع كالتالى:

 توافقت المعاملة التصنيفية الحالية الى حد بعيد مع بعض الأنظمة التصنيفية لعديد من العلماء ومنهم:

(Bentham & Hooker, 1862, Post, 1932, Emberger, 1960, Engler, 1964, Willis, 1966, Brenan, 1967, Hutchinson, 1967, Pettigrew & Watson 1977, Smith, 1977, Polhill & Raven, 1983, Watson & Dalwitz, 1983 and Lewis et al., 2005).

حيث ان معظم الوحدات المدروسة رتبت تحت القبائل الخاصة بها والتى اكدت من ذى قبل.

 تجمعت الأنواع المدروسة من جنس Bauhinia في مجموعة واحدة واتصلت اتصالا وثيقا بالنوع Cercis chinensis ، وهذا يؤيد تقسيم قبيلة Cercideae (Bauhinieae) الى تحت قبيلتين وهما Cercidinae وBauhiniinae من قبل Wunderline et al. (1981 & 1987).

 توافق انعزال Haematoxylum campecianum بعيدا عن بقية الانواع المدروسة ذات الصلة وقد توافق هذا مع ما ذكره.Pettigrew & Watson, 1977).)

 على الرغم من ان جنسى الكاسيا و السينا فى جميع التصنيفات القديمة كانت تندرج تحت جنس الكاسيا بمفهومه الواسع فان الدلالات المستخلصة من التصنيف العددى اظهرت ان جنسى الكاسيا والسنا وحدتين تصنيفيتين منفصلتين وذلك يرجع الى ان الوحدات المدروسة تشتت فى عدد من المجموعات مما يدل على انها مجموعة غير متجانسة . وعليه فان الدلالات المستخلصة تؤيد اقتراح فصل جنسا الكاسيا إلى ثلاثة تحت اجناس Fistula, Senna and Lasiorhegma (Bentham, 1871 & Taubert, 1891) or Cassia, Senna and Absus (Randell, 1976).

 أكدت الدراسة ان مجموعة الكاسيا (كاسيا و سننا) قد تم توزيعها على عدد من المجموعات على الرغم من ان لها منشأ واحد وعليه تعتبر مجموعة الكاسيا

( (paraphyleticوهذا ما توافق مع اراء بعض العلماء من ان مجموعة الكاسيا ليست (monophyletic)



 كما لوحظ من مقارنة الشجرة التصنيفية الناتجة عن الصفات المورفولوجية وعددها 353 حالة صفة بمثيلتها الناتجة عن مجموع الصفات المورفولوجية والجزيئية وعددها 679 حالة صفة ان ترتيب الانواع قيد الدراسة متشابة الى حد كبير جدا فى الحالتين مما يدلل على ان الصفات المورفولوجية أساسية ولا يمكن الاستغناء عنها حيث انها حجر الزاوية فى اى دراسة تصنيفية.



وعليه فان الدراسة القائمة قد خلصت الى ان الصفات المورفولوجية والجزيئية المستخلصة من الانواع قيد الدراسة قد ساهمت الى حد كبير فى الفصل بين الانواع وتفسير العلاقات البينية بينها وكذلك استتباب الوضع التصنيفى لمعظم الانواع والاجناس تحت قبائلها الرئيسة كما جاء فى الانسقة التصنيفية المعنية بدراسة تحت الفصيلة البقمية. الا ان هذا يتطلب فى الدراسات المستقبلية عدد أكبر من الانواع النباتية واستنباط صفات من اكثر من مصدر باستخدام التقنيات العلمية الحديثة.

Summary

The morphological characters of the whole plant, Leaf architecture and lamina epidermal characteristics (LM & SEM) as well as Molecular data (RAPD-PCR & Isozymes) of 25 taxa of the Caesalpinioideae were investigated. These taxa represent four tribes, 15 genera, 25 species.

The morphological and molecular criteria and their states (679 attributes) were numerically analyzed. The observation obtained led to various conclusions of taxonomic importance, which could be summarized in the following:



Section A: Morphological Characters

1. Whole Plant

Habit: tree or shrubs. Length: varied between tall or medium.Texture: rough, prickly, spiny, Pubescent or glabrous. Leaf Composition: Simple, Bipinnate (paripinnate), oncepinnate (paripinnate) oncepinnate & bipinnate (paripinnate).

Shape of Blade: Suborbicular, elliptic-oblong, oblong, oblong-elliptic, ovate-lanceolate, ovate-elliptic, oblong-ovate, obovate in, cordate, oblanceolate or oblong-lanceolate. Apex of Blade: emarginated, caudate, acute, obtuse, cleft, mucronate, acute-mucronate, retuse or acuminate. Base of Blade: cordate, cuneate or obtuse. Margin of Blade: Serrate in Gleditsia caspica or entire in the rest. Stipules: exstipulate in Cassia fistula, C. grandis & C. marginata, cresentiform, auricle, cordate, spiniform, ovate, lanceolate or linear. Number of Flowers / Inflorescence: Few or many. Length: short or long. Position: leaf opposed, axillary & terminal, axillary or terminal. Type: spike-like, corymbose or raceme. Bracts: ebracteate (Cassia fistula), deltoid, suborbicular, subulate, scaly, ovate, ovate-lanceolate, ovate-oblong or obovate. Calyx Texture: glabrous, tomentose, velvety, pubescent. Colour: red, pink, crimson, scarlet, brown , green or yellow. Sepal Shape: gamosepalous or polysepalous. The sepals may be lanceolate, ovat-lanceolate, ovate, obovate, oblong, oblong-ovate, oblong-obovate or ovate-oblong. Number: four or five. Corolla Texture: glabrous or pubescent. Colour: whitish green, white, yellow, red, lavender, pink or scarlet. Petal Shape: obovate, obovate-elliptic, obovate-oblong, ovate-elliptic, oblong, sub-orbicular, obconical, spathulate or ovate. Number: in all the taxa under investigation the number of petals are five except Gleditsia caspica (four petals) and Ceratonia siliqua & Saraca indica (apetalous). Androecium Fertility: ten fertile stamens, three, five, seven, eight, nine or 12, seven fertile & three sterile, six fertile and four sterile or five fertile & five sterile. Length: as long as petals, shortly exerted, longly exerted or included. Filament Texture: pubescent, hairy, pilose, glandular or glabrous. Form: awl-shaped or Filiform. The latter may be sigmoid or nodulated. Anthers Size: unequal, subequal or equal. Attachement: dorsifixed, basifixed or versatile. Ovary Texture: glabrous, pilose or pubescent. Form: flattened, oblong or terete. Setting: sessile, subsessile or stipitate. Style Length: short or long. Form: flattened, teret or filiform. Curvature: upcurved, straight or incurved. Stigma Form: capitate, sub-capitate, concave, truncate, peltate or simple. Pod Texture: pubescent or glabous. Colour: yellow or Brown. Form: linear, linear-oblong, oblong, terete or elliptic. Dehiscence: dehiscent or indehiscent. Apex: acut, mucronate, cuspidate, tapering or obtuse. Peak: peakless, long or short. Type: membranous, crustaceous, sub-woody or woody. Seed Colour: orange, black, greenish black, green or brown. Form: teret, sub-terete, compressed or flattened. Shape: ovate, obovate, orbicular, suborbicular, elliptic, pear–shaped or oblong.

2. Lamina Architecture

Primary Vein Category: campylodromous or pinnate. Secondary Vein Category: poorly developed in Parkinsonia aculeata, cladodromous, brochidodromous or festooned brochidodromous. Basal Vein Number: one, two, three, four, five, six, seven, 13 or 14. Secondary Vein Spacing: uniform or irregular. Tertiary Vein Category: poorly developed, dichotomizing, alternate percurrent or random reticulate. Tertiary Vein Course: sinuous, straight, admedially ramified or exmedially ramified. Tertiary Vein Angle: obtuse or acute. Quaternary Vein Category: alternate percurrent, dichotomizing, poorly developed or regular polygonal reticulate. Areolation: absent, poorly developed, moderately developed or highly developed. Freely Ending Veins: absent, unbranched, once branching or two or more branching. Marginal Ultimate Venation: absent, fimbrial or looped.

3. Lamina Epidermal Characteristics (LM)

Cell Shape: quadrangular, polygonal, pentagonal, hexagonal or irregular. Anticlinal Wall: straight, sinuous, slightly curved or curved. Trichome Type: wanting, unicellular or bicellular. Stomata Type: paracytic, isotricytic & tetracytic, cyclocytic, anomocytic or paracytic. The taxa under investigation vary between hypostomatic lamina or amphistomatic one. Shape: circular or elliptic. Crystals: wanting, irregular, druses or/and prisms.

4. Lamina Epidermal Characteristics (SEM)

Sculpture: colliculate, ruminate, reticulate, pusticulate or tuberculate. Anticinal Wall Width: broad or narrow Elevation: raised or depressed. Texture: smooth or granular. Periclinal Wall Elevation: depressed or raised. Texture: smooth or granular.

Section B: Molecular Characters

1. RAPD Diversity Aspects

The present investigation was conducted to develop biochemical and molecular genetic fingerprints for the studied taxa and to elucidate relationships among these samples.

Extracted DNA from fresh leaf samples was used to identify the molecular fingerprints. Ten 10-mer arbitrary oligonucleotide primers were used to establish their randomly amplified polymorphic DNA based on polymerase chain reaction (RAPD-PCR) fingerprints. The ten primers were successfully generated reproducible polymorphic products. The primers are SC10-5, SC10-14, SC10-17, SC10-18, SC10-22, SC10-23, SC10-25, SC10-59, SC10-64 and SC11-30. The fingerprints generated by these primers revealed characteristic profiles for each taxa, in terms of number and position of RAPD bands. The results revealed that both the number and size of the amplified products varied considerably with the different primers.

A sum of 323 polymorphic bands was generated by these primers in the taxa under study. In the present study, a total of 91 unique bands were identified out of the polymorphic ones. These unique bands were used to discriminate among the studied taxa. Most samples of the studied taxa were discriminated by one or more unique bands.

2. Isozyme Analysis

Fresh and young leaves were used separately to identify the fingerprint based on polymorphism in isozymes banding patterns.

Polyacrylamide gel electrophoresis was employed to identify the biochemical fingerprint based on five isozyme systems, alcohol dehydrogenase (Adh), aldehyde oxidase (Alo), and esterase (Est) and acid phosphatase (Acph) profiles. Isozymes results revealed a high level of polymorphism among the studied taxa and have proved to be effective in identifying the each of the studied taxa by a unique class pattern, which indicates that biochemical genetic fingerprinting, is a reliable technique to discriminate among these taxa.

Section C: Numerical Analysis

The data obtained from the morphological and molecular attributes (679) of the investigated taxa were subjected to the numerical analysis to produce the dendrogram and comparing it with the current systems of taxonomic classification. The results illustrated that the taxa investigated were split into eight groups, three clusters and two series.

The most important of several conclusions and predictions are obtained as the following:

The suggested current taxonomic treatments agree in far extent with the current systems of classification (Bentham & Hooker, 1862, Post, 1932, Emberger, 1960, Engler, 1964, Willis, 1966, Brenan, 1967, Hutchinson, 1967, Pettigrew & Watson 1977, Smith, 1977, Polhill & Raven, 1983, Watson & Dalwitz, 1983 and Lewis et al., 2005).

Haematoxylum was isolated away from the rest of the related taxa viz. Ceasalpinia, Delonix, Parkinsonia and Peltophorum. And this is confined with Pettigrew & Watson, 1977.

The studied Bauhinia species were nested together in one group and closely related to Cercis, this supported the division of tribe Cercideae into two subtribes, Cercidinae, the subtribe containing Cercis and Bauhiniinae, the subtribe containing Bauhinia by (Wunderlin et al., 1981, 1987).

Although Cassia and Senna group were heterogenous, in far extent the suggested treatment supports the segregation of genus Cassia L. into three subgenera viz. Fistula, Senna and Lasiorhegma by Bentham, (1871) & Taubert, (1891) or subgenera viz. Cassia, Senna and Absus by Randell, (1976).

Discussion Numerical Analysis

Section C: Numerical Analysis


The taxonomic treatment based on 326 morphological attributes, 353 molecular attributes, sum of 679 used for computation and produced dendrogram revealed classification of the studied taxa of Caesalpinioideae which compared with the crrent system treatments as mentioned in Table (1). The resulted dendrogram showed that the taxa under investigation at each level of morphological, molecular or sum of 679 attributes were split into two seiries , thee clusters and a number of groups varied between six groups (at morphological level), five groups (molecular) and eight groups (at the sum level).

The application of such treatment could be discussed as in the following































Table (25): The Proposed Treatment of 326 Morphological Attributes, Based on Numerical Analysis of the Taxa Under Investigation of Caesalpinioideae.





Series Cluster Group Taxa under investigation

I A 1 Bauhinia alba, Bauhinia hookeri, Bauhinia variegata

II B 2 Brownea grandiceps, Saraca indica, Schotia brachypetala, Tamarindus indica, Haematoxylum campecianum

3 Caesalpinia ferrea, C. gilliesii, Peltophorum africanum, Delonix regia, Parkinsonia aculeata

4 Cassia fistula, C. nodosa, C. grandis, C. javanica, C. marginata, Ceratonia siliqua

5 Senna alata, S. didymobotrya, S. sophera, S. surattensis

C 6 Cercis chinensis, Gleditsia caspica



Table (26): The Proposed Treatment of 353 Molecular Attributes (RAPD & Isozymes), Based on Numerical Analysis of the Taxa Under Investigation of Caesalpinioideae.



Series Cluster Group Taxa under investigation

I A 1 Bauhinia alba, Bauhinia hookeri, Bauhinia variegate, Cassia javanica, Cercis chinenceis, Parkinsonia aculeata

2 Brownea grandiceps, Peltophorum africanum, Senna sophera, Cassia grandis, Gleditsia caspica, Cassia nodosa, Ceratonia siliqua, Senna surattensis

B 3 Caesalpinia gilliesii, Haematoxylum campecianum, Schotia brachypetala, Tamarindus indica, Delonix regia, Senna alta, S. didymobotrya

4 Cassia fistula, C. marginata, Saraca indica

II C 5 Caesalpinia ferrea





Table (27): The Proposed Treatment of 679 Morphological and Molecular Attributes, Based on Numerical Analysis of the Taxa Under Investigation of Caesalpinioideae.



Series Cluster Group Taxa under investigation

I A 1 Bauhinia alba, Bauhinia hookeri, Bauhinia variegata

2 Cercis chinensis, Gleditsia caspica

II B 3 Brownea grandiceps, Senna sophera, Peltophorum africanum

4 Cassia grandis, C. javanica, Ceratonia siliqua, Senna surattensis

5 Senna alata, S. didymobotrya

6 Cassia fistula, C. marginata, C. nodosa

C 7 Caesalpinia ferrea, C. gilliesii, delonix regia, Parkinsonia aculeata

8 Haematoxylum campecianum, Schotia brachipetala, Tamarindus indica, Saraca indica.







1. Phenetic Analysis of Morphological Attributes



The data obtained from the macromorphological attributes of the studied taxa of Caesalpinioidea were subjected to numerical analysis using the NTSYS-pc version 2.02 (Rolf, 1989).

The resulted dendrogram clarifies that the taxa under investigation divided into two main series. Series I includes only one cluster (A) and one group (1) with three studied species and series II includes two clusters (B & C) with five groups; (from 2 to 6) cluster B and (from 5 to 6 ) cluster C. The former cluster includes 16 studied species while the latter cluster includes six studied taxa. The interrelationships between these taxa are discussed.



2. Phenetic Analysis of Molecular Attributes



The resulted dendrogram clarifies that the taxa under investigation divided into two main series. Series I includes two clusters (A & B) and four groups (from 1 to 4). Cluster A with group 1 and 2 includes 14 studied species while cluster B with group 3 and 4 with ten studied species. Series II includes only one cluster (C) with one group (5) and one species. The interrelationships between these taxa are discussed.



3. Phenetic Analysis of both Morphological and Molecular Attributes



The generated dendrogram clarifies that the taxa under investigation divided into two main series (I and II) at taxonomic distance 1.3. Series I includes one cluster (A) with two groups (1 and 2). Cluster A with group 1 and 2 includes five studied taxa. Series II includes two clusters (B andC); cluster B with four groups (from 3 to 6) including 12 studied taxa while cluster C with two groups (7 and 8) including eight taxa. The interrelationships between these taxa are summarized as follows.

Series I

Group 1: includes Bauhinia alba, B. variegata and B. hookeri which separated at the taxonomic distance of 1.09.

Group 2: includes Cercis chinensis & Gleditsia caspica which separated at taxonomic distance 1.2.

The grouping of studied Bauhina sp. and Cercis chinensis in one cluster and two closely related groups (cluster A; Group 1 & 2) is comparable with current system of treatment of Caesalpinioideae as mentioined by Bentham & Hooker (1862), Post (1932), Emberger (1960), Engler (1964), Willis (1966), Brenan (1967), Hutchinson (1967), Pettigrew & Watson (1977), Smith (1977), Polhill & Raven (1983), Watson & Dalwitz (1983) and Lewis et al. (2005) Who classified Bauhinia and Cercis under Tribe Bauhinieae or Cercideae. Wunderlin et al. (1981; 1987) suggested the division of tribe Cercideae or Bauhinieae into two subtribes, Cercidinae and Bauhiniinae. The data extracted from cluster A; group 1 & 2 encourage this suggestion (studied Bauhinia species are classified under subtribe Bauhinineae while Cercis under subtribe Cercineae).

According to Watson & Dallwitz (1983), Gleditsia caspica (tribe Caesalpinieae), separated away from Caesalpinia, Delonix, Parkinsonia and Peltophorum in a separate subgroup.

Gleditsia caspica in the present study and on the bases of morphological and molecular criteria was separated away from tribe Caesalpinieae and grouped with Cercis chinensis (group 2 at 1.2 taxonomic value). It was suggested that data extracted enhance the grouping of this taxa with Cercis chinensis under subtribe Cercidineae.







Series II

Group 2: includes Brownea grandiceps, Senna sophera & Peltophorum africanum at a taxonomic value 1.16.

According to the different authors as mentioned in Table 1 & 2 in the present study, Brownea, Tamarindus, Saraca and Schotia were grouped under tribe Amhestieae. Hutchinson (1967) and Watson & Dallwitz (1983) separated Brownea away from the related taxa viz. Tamarindus, Schotia & saraca in a separate subgroup.

In the present study the morphological and molecular data was supported the suggestion of Hutchinson (1967) and Watson & Dallwitz (1983).



Group 4: included Cassia grandis, C. javanica, Ceratonia siliqua & Senna surattensis separated at 1.18. the two former Cassia sp. are more closely related than Ceratonia and Senna. In this connection Irwin and Barneby (1981) divided Cassieae into five subtribes viz. Ceratoniinae (Ceratonia), Dialiinae, Duparquetiinae, Cassiinae (Cassia), and Labicheinae and this is in accordance with the proposed treatment in the present study.

Group 5: includes Senna alata & S. didymobotrya at 1.11 taxonomic value.

Group 6: includes Cassia fistula, C. marginata & C. nodosa at taxonomic value 1.23. In the present study the morphological and molecular data supported the separation of studied Cassia and Senna species from each other and this is in agreement with Bentham (1871) & Taubert (1891) in which genus Cassia L. is segregated into three subgenera viz. Fistula, Senna & Lasiorhegma and into Cassia, Senna & Absus (Randell, 1976).



Group 7: includes Caesalpinia ferrea, C. gilleisii, Delonix regia & Parkinsonia aculeata at 1.25 taxonomic value. These taxa belong to tribe Caesalpinieae or Eucaesalpinieae in most of the current taxonomic treatments of classification (Bentham & Hooker, 1862; Post, 1932; Emberger, 1960; Engler, 1964; Willis, 1966; Brenan, 1967; Hutchinson, 1967; Pettigrew & Watson 1977; Smith, 1977; Polhill & Raven, 1983; Watson & Dalwitz, 1983 and Lewis et al., 2005).

Group 8: includes Haematoxylum campecianum, Schotia brachypetala, Tamarindus indica & Saraca indica at taxonomic level 1.25. The taxa under this group represent tribe Detarieae or Amhersiteae (except Haematoxylum, tribe Caesalpinieae or Eucaesalpinieae) as mentioned by Bentham & Hooker (1862), Post (1932), Emberger (1960), Engler (1964), Willis (1966) Brenan (1967), Hutchinson (1967), Pettigrew & Watson (1977), Smith (1977), Polhill & Raven (1983), Watson & Dalwitz (1983) and Lewis et al. (2005). Schotia brachypetala and Tamarindus indica are closely related at taxonomic value 0.94 and this is in contradiction with Pettigrew & Watson (1977) where Schotia and Brownea were placed together in a single subgroup, Saraca in subgroup and Tamarindus in subgroup. In this respect Haematoxylum was delimited by Pettigrew & Watson (1977) and Watson & Dallwitz (1983), this is in accordance with the data extracted in the present study. From the proposed treatment (Table 26 and Fig. 14) the following subcequent points revealed a taxonomic meaning:

The majority of studied taxa are arranged under the specific tribes based on morphological and molecular attributes.

The studied taxa of Cassieae (Cassia, Senna & Ceratonia) are considered paraphyletic (one ancestor; Cassia s.l. segregated away from the remainig descendants). This is supported by Irwin & Barneby (1981), Doyle et al. (1997), Kajita et al. (2001), Herendeen et al. (2003) and Wojciechowski et al. (2004) who concluded that Cassieae is not monophyletic based on analysis of molecular sequence data.

The proposed treatment and dendrogram resulted from morphological and molecular attributes supported the separation of Cassia and Senna as two taxonomic entities.

Discussion Molecular Stidies

Section B: Molecular Characters




Characterization and quantification of genetic diversity have long been a major goal in species, sub-species and cultivar discrimination as well as plant breeding. Isozymes and random amplification of polymorphic DNA (RAPD) markers are powerful techniques for determining intra- and interspecific variations and allow direct comparison of plant variation at both biochemical and molecular levels (Williams et al., 1990; Welsh & McClelland, 1990; and Lu et al., 2009).



1. RAPD Diversity Aspects (Tabble 23)

In this study of genetic diversity in Caesalpinioid taxa with RAPD molecular technique, the level of DNA polymorphism detected with ten primers was very high and allowed the distinction of all taxa analysed. The high discriminatory power of the primers used indicates that the RAPD technique provides an effective tool for germplasm analysis in Caesalpinioideae.

All primers produced 323 bands and showed no monomorphic bands, meaning that the polymorphism investigated by these primers reached 100%.

Primer SC10-5 produced 17 polymorphic bands (14 common & three unique); SC10-14 showed 32 polymorphic bands, (eight unique); SC10-17 generated 24 polymorphic bands (seven unique); SC10-18 generated 46 polymorphic bands (ten unique); SC10-22 produced 22 polymorphic bands (12 bands unique); SC10-23 produced 47 polymorphic bands (nine unique); SC10-25 generated 32 polymorphic bands (11 unique); SC10-59 produced 31 polymorphic bands (13 unique); SC10-64 generated 39 polymorphic bands (nine unique) and SC11-30 showed 33 polymorphic bands (nine unique).

The data extracted from RAPD-PCR analysis for the studied taxa were amalgmated with the data from morphological and isozymes analysis then subjected to numerical analysis to interprete and discuss the interrelatioship between the taxa under investigation at generic and infra-specific level. Also, comparison of the schematic presentation with some of current systems of classification.

2. Isozyme Analysis (Table 24)

The data obtained from isozymes anlysis revealed highest degree of genetic diversity between the studied taxa of Caesalpinioideae. Acid phosphatase generated seven bands; alcohol dehydrogenase generated four bands; esterases generated 15 bands and aldehyde oxidase produced four bands.

All the enzyme systems analyzed were polymorphic where the inter-specific polymorphism reached 100%.

The data extracted from isozyme analysis for the studied taxa were amalgmated with the data from morphological and RAPD-PCR analysis then subjected to numerical analysis to interprete and discuss the interrelatioship between the taxa under investigation at generic and infra-specific level. Also, comparison of the schematic presentation with some of current systems of classification.

Discussion Epidermal Studies SEM

7. Lamina Epidermal Characteristics (Table 22 & Plate XXIX; SEM)




i. Sculpture: Abaxial epidermis; colliculate in 11 studied taxa; ruminate in seven studied taxa; reticulate in Cassia fistula, Cercis chinensis, Gleditsia caspica & Senna didymobotrya; pusticulate in Cassia nodosa & Schotia brachypetala or tuberculate in Senna alata. Adaxial epidermis; colliculate in ten studied taxa; ruminate in seven studied taxa; reticulate in Cassia fistula, C. nodosa, Cercis chinensis, Gleditsia caspica & Senna didymobotrya; pusticulate in Ceratonia siliqua & Schotia brachypetala or tuberculate in Senna alata.

ii. Anticinal Wall Width: Abaxial epidermis; broad in six studied taxa or narrow in the rest of the taxa under investigation. Adaxial epidermis; broad in eight studied taxa or narrow in the rest of the studied taxa.

iii. Elevation: Abaxial epidermis; raised in. Cassia fistula, Cercis chinensis, Gleditsia caspica & Senna didymobotrya or depressed in the remaining. Adaxial epidermis; raised in five taxa viz. Cassia fistula, Cercis chinensis, Gleditsia caspica & Senna didymobotrya or depressed in the remaining.



iv. Texture: Abaxial epidermis; smooth in 17 out of the studied taxa or granular in the rest (eight taxa). Adaxial epidermis; smooth in 16 studied taxa or granular in the rest of the studied taxa.

v. Periclinal Wall Elevation: Abaxial epidermis; depressed in four taxa viz. Cassia fistula, C. nodosa, Cercis chinensis, Gleditsia caspica & Senna didymobotrya or raised in the rest. Adaxial epidermis; depressed in five taxa viz. Cassia fistula, C. nodosa, Cercis chinensis, Gleditsia caspica & Senna didymobotrya and raised in the remaining.

vi. Texture: Abaxial epidermis; smooth in eight out of the studied taxa or granular in the rest. Adaxial epidermis; smooth in 13 studied taxa or granular in the rest.

Discussion Epidermal Studies

6. Lamina Epidermal Characteristics (Table 21 & Plate XXVII; XXVIII; LM)




i. Cell Shape: Abaxial epidermis; quadrangular, polygonal, pentagonal, hexagonal in ten studied taxa or irregular in 15 taxa. Adaxial epidermis; quadrangular, polygonal, pentagonal, hexagonal in 18 studied taxa or irregular in seven taxa.

ii. Anticlinal Wall: Abaxial epidermis; straight in eight studied taxa; sinuous in Caesalpinia gilliesii, Brownea grandiceps & Saraca indica; slightly curved in seven studied taxa or curved in the remaining taxa. Adaxial epidermis; Sinuous in Brownea grandiceps, Caesalpinia gilliesii, Cassia fistula & Saraca indica; curved in Parkinsonia aculeate, & S. sophera or slightly curved in Caesalpinia ferrea, Cassia marginata, C. nodosa & Senna alata or straight in the remainder.

iii. Trichome Type: Abaxial epidermis; wanting in 11 taxa; unicellular in 13 taxa or bicellular in Peltophorum africanum. Adaxial epidermis; wanting in 13 out of the studied taxa or unicellular in the rest.



iv. Stomata Type: Paracytic, isotricytic & tetracytic in Bauhinia alba, B. hookeri & B. variegata; cyclocytic in Gleditsia caspica & Schotia brachypetala; anomocytic in Caesalpinia ferrea & C. gilliesii or paracytic in the remaining studied taxa. The taxa under investigation vary between hypostomatic lamina (15 taxa) or amphistomatic lamina (ten taxa). In this respect this character shared in the presnt study in separation of Cassia species (hypostomatic) away from Senna species (amphistomatic). This is in accordance with Bentham (1871), Taubert (1891) and Randell (1976) who segregated genus Cassia into three subgenera viz. Fistula, Senna and Lasiorhegma or subgenera viz. Cassia, Senna and respectively.

v. Shape: Abaxial epidermis; circular in four out of the studied taxa viz. Parkinsonia aculeate, Schotia brachypetala, Senna surattensis & Tamarindus indica; oblong-elliptic in Haematoxylum campecianum or elliptic in the remaining studied taxa. Adaxial epidermis; circular in Parkinsonia aculeata, Peltophorum africanum & Senna surattensis and elliptic in the remainders.

vi. Crystals: Abaxial epidermis; wanting in ten studied taxa; irregular in Ceratonia siliqua, Cercis chinensis & Schotia brachypetala; druses or/and prisms in the remaining studied taxa. Adaxial epidermis; wanting in ten studied taxa; irregular in Ceratonia siliqua, Cercis chinensis, Schotia brachypetala & Tamarindus indica; druses or/and prisms in the remaining studied taxa.

Discussion Architecture

5. Lamina Architecture (Table 20, Plate XXVI)




i. Primary Vein Category: Campylodromous type in four out of the studied taxa viz. Bauhinia alba, B. hookeri, B. variegata & Cercis chinensis or pinnate type in the remaining of the studied taxa. The former type is considered unique character for Bauhina & Cercis. This is in accordance with the conclusion reached before by Bentham & Hooker (1862), Post (1932), Emberger (1960), Engler (1964), Willis (1966), Brenan (1967), Hutchinson (1967), Pettigrew & Watson (1977), Smith (1977), Polhill & Raven (1983), Watson & Dalwitz (1983) and Lewis et al. (2005) where the studied Bauhinia sp. and Cercis chinensis are grouped under Bauhinieae or Cercidieae.

ii. Secondary Vein Category: Poorly developed in Parkinsonia aculeata; cladodromous in Haematoxylum campecianum & Gleditsia caspica; brochidodromous in Bauhinia alba, B. hookeri & B. variegata or festooned brochidodromous in the rest of the studied taxa. The festooned brochidodromous type segregates Cercis chinensis away from the studied Bauhinia sp. and supported the division of tribe Cercideae or Bauhinieae into two subtribes, Cercidinae and Bauhiniinae by Wunderlin et al. (1981; 1987).

iii. Basal Vein Number: One in Parkinsonia aculeata; two in Caesalpinia ferrea, C. gilliessii & Delonix regia; three in 12 taxa; four in Peltophorum africanum & Senna alata; five in Cassia grandis; six in Senna surattensis; seven in Cercis chinensis; 11 in Haematoxylum campecianum; 13 in Bauhinia alba & B. variegata or 14 in B. hookeri.

iv. Secondary Vein Spacing: Uniform in eight out of the studied taxa or irregular in the rest.

v. Tertiary Vein Category: Poorly developed in Delonix regia & Parkinsonia aculeata; dichotomizing in Caesalpinia ferrea; alternate percurrent in Bauhinia alba, B. variegata, Saraca indica & Senna alata or random reticulate in the remainder.

vi. Tertiary Vein Course: Sinuous in Saraca indica; straight in Bauhinia alba & B. variegata; admedially ramified in Caesalpinia ferrea, C. gilliessii & Senna alata or exmedially ramified in the rest.

vii. Tertiary Vein Angle: Obtuse in 13 studied taxa or acute in the rest.

viii. Quaternary Vein Category: Alternate percurrent in Bauhinia alba & B. variegata; dichotomizing in B. hookeri, Cassia marginata, C. nodosa, Senna surattensis & Tamarindus indica; poorly developed in Caesalpinia ferrea, C. gilliessii, Delonix regia & Parkinsonia aculeata or regular polygonal reticulate in the rest.

ix. Areolation: Absent in Caesalpinia ferrea, Delonix regia & Parkinsonia aculeata; poorly developed in Cassia marginata & C. nodosa; moderately developed in Caesalpinia gilliesii or highly developed in the remaining studied taxa.

x. Freely Ending Veins: Absent in 11 taxa; unbranched in Bauhinia hookeri & Brownea grandiceps; once branching in Cassia fistula & Haematoxylum campecianum or two or more branching in the rest of the taxa studied.

xi. Marginal Ultimate Venation: Absent in Delonix regia & Parkinsonia aculeata; fimbrial in Bauhinia alba & B. variegata or looped in the rest.

Discussion Whole Plant

Section A: Morphological Characters



In the present part, the different macromorphological characters of the studied caesalpinioid taxa are presented in cumulative tables in order to facilitate deducing the most important diagnostic characters.



1. Whole Plant (Table 19)



i. Habit: The taxa under investigation may be tree in 16 taxa or shrubs the remainder studied nine taxa. In this respect Rendle (1925) stated that Caesalpinioideae are trees or shrubs, more rarely herbs.

ii. Length: The studied taxa varied between tall plants in 11 taxa, medium in nine taxa or small five taxa.

iii. Texture: Rough (Tamarindus indica), prickly (Haematoxylum campecianum), spiny (Gleditsia caspica & Parkinsonia aculeata), Pubescent (16 taxa) or glabrous (Caesalpinia ferrea, C. gilliesii, Saraca indica & Senna alata).



2. Leaf (Table 19)



i. Composition: Simple in Bauhinia alba, B. hookeri, B. variegata & Cercis chinensis, Bipinnate (paripinnate) in Caesalpinia ferrea, C. gilliesii, Delonix regia, Parkinsonia aculeata & Peltophorum africanum, oncepinnate (paripinnate) in 14 taxa or oncepinnate & bipinnate (paripinnate) in Gleditsia caspica & Haematoxylum campecianum.

The variations in leaf composition among the studied taxa are in accordance with the current taxonomic systems of classification, to cite but a few one can refer to Bentham & Hooker (1862), Post (1932), Emberger (1960), Engler (1964), Willis (1966), Brenan (1967), Hutchinson (1967), Pettigrew & Watson (1977), Smith (1977), Polhill & Raven (1983), Watson & Dalwitz (1983) and Lewis et al. (2005). Where Bauhinia and Cercis (simple leaf) separated in tribe Bauhinieae or Cercideae. Pettigrew & Watson (1977) segregated Haematoxylum away from the rest of related taxa viz. Ceasalpinia, Delonix, Parkinsonia and Peltophorum. The data in the present study support the segregation of Haematoxylum and Gleditsia (oncepinnate & bipinnate; paripinnate) away from the related taxa. Hutchinson (1973) pointed out that the variations in leaf composition of Caesalpiniaceae may indicate the general trend of evolution within the family vs simple-pinnate (imparipinnate)-compound.

ii. Shape of Blade: Suborbicular in Bauhinia alba, B. hookeri, B. variegata, elliptic-oblong Caesalpinia ferrea, oblong in six taxa, oblong-elliptic in Cassia grandis, C. javanica & Senna alata, ovate-lanceolate in Cassia fistula, Gleditsia caspica, Senna sophera, ovate-elliptic in Cassia nodosa & Senna surattensis, oblong-ovate in Senna didymobotrya, obovate in Brownea grandiceps, Ceratonia siliqua & Haematoxylum campecianum, cordate in Cercis chinensis, oblanceolate in Parkinsonia aculeata or oblong-lanceolate in Saraca indica. In the current study the shape of the blade distinguishes the Bauhinia sp. Under investigation away from Cercis chinencis. In the former the blade is suborbicular and cordate in the latter. This conclusion is comaparable with the work of Wunderlin et al. (1981, 1987) "tribe Cercideae or Bauhinieae is divided into two subtribes viz. Cercidinae (Cercis) and Bauhiniinae (Bauhinia)".

iii. Apex of Blade: Emarginated in Bauhinia alba, B. hookeri, B. variegata; caudate in Brownea grandis; acute in Caesalpinia ferrea, Cassia fistula, Senna sophera; obtuse in six taxa, cleft in Cassia grandis & C. javanica; mucronate in Cassia nodosa, Peltophorum africanum, Saraca indica; acute-mucronate in Gleditsia caspica; retuse in five taxa or acuminate in Cercis chinensis. The variations in blade apex support the segregation of the studied Bauhinia sp. away from Cercis chinensis. This is in accordance with the work of Wunderlin et al. (1981, 1987).

iv. Base of Blade: Cordate in Bauhinia alba, B. hookeri, B. variegata & Cercis chinensis; cuneate in. Caesalpinia ferrea, Gleditsia caspica, Haematoxylum campechianum, Parkinsonia aculeata & Saraca indica or obtuse in the remaining studied taxa (16 taxa).

The data extracted here are in accordance with some of the current taxonomic systems of classification viz. to cite but a few one can refer to Bentham & Hooker (1862), Post (1932), Emberger (1960), Engler (1964), Willis (1966), Brenan (1967), Hutchinson (1967), Pettigrew & Watson (1977), Smith (1977), Polhill & Raven (1983), Watson & Dalwitz (1983) and Lewis et al. (2005) where the studied Bauhinia sp. and Cercis chinensis are grouped under Bauhinieae or Cercidieae.







v. Margin of Blade: Serrate in Gleditsia caspica or entire in the remainder studied taxa.

vi. Stipules: Exstipulate in Cassia fistula, C. grandis & C. marginata; cresentiform in Cassia javanica, auricle in Cassia nodosa, cordate in Senna didymobotrya, spiniform in Haematoxylum campechianum & Parkinsonia aculeata; ovate in seven taxa, lanceolate in five taxa or linear in five taxa.



3. Inflorescence (Table 19)

i. Number of Flowers / Inflorescence: Few in five taxa viz. Bauhinia alba, B. hookeri, B. variegata, Cercis chinensis & Senna sophera and many in the rest of the studied taxa.

i. Length: Short in 11 taxa and long in the remaining taxa studied.

ii. Position: Leaf opposed in Bauhinia hookeri; axillary & terminal in Bauhinia alba, B. variegata & Cercis chinensis; axillary in 14 taxa or terminal in the remaining studied seven taxa.

iii. Type: Spike-like in Gleditsia caspica; corymbose in Saraca indica or raceme in the remaining studied taxa.



iv. Bracts: Ebracteate (Cassia fistula); deltoid (Bauhinia alba & B. variegata); suborbicular (Brownea grandiceps); subulate (Peltophorum africanum); scaly (Ceratonia siliqua, Cercis chinensis & Gleditsia caspica); ovate (ten taxa), ovate-lanceolate (Bauhinia hookeri, Cassia javanica & C. nodosa); ovate-oblong (Caesalpinia gilliesii, Schotia brachypetala & Tamarindus indica) or obovate (Senna alata).



4. Flower (Table 19)

i. Calyx Texture: Glabrous (ten taxa); tomentose (Cassia grandis & C. nodosa); velvety (Cassia javanica & C. marginata); pubescent in the remaining studied taxa.

ii. Colour: Red, pink, crimson or scarlet in ten taxa; brown in five taxa; green in eight taxa or yellow in S. alata & Tamarindus indica.

iii. Sepal Shape: Gamosepalous in Bauhinia alba, B. variegata & Cercis chinensis or polysepalous in the remaining studied taxa. The sepals may be lanceolate in Bauhinia hookeri, Delonix regia & Gleditsia caspica or ovat-lanceolate in Schotia brachypetala & Tamarindus indica; ovate in five taxa; obovate in four taxa; oblong Brownea grandiceps & Caesalpinia gilliesii; oblong-ovate in Cassia fistula; oblong-obovate in Senna alata or ovate-oblong in four taxa. The data in the oresnt study about the union of sepals support the conculsion reached before by Rendle (1925) "Bauhinieae have a gamosepalous calyx" except Bauhinia hookeri (polysepalous calyx).

iv. Number: For sepals in Brownea grandiceps, Gleditsia caspica, Saraca indica, Schotia brachypetala & Tamarindus indica or Five in the remaining taxa under investigation.

v. Corolla Texture: Apetalous in Ceratonia siliqua & Saraca indica; glabrous in 13 taxa; pubescent in the remaining taxa under investigation..

vi. Colour: Whitish green (Gleditisia caspica); white (Bauhinia alba & B. hookeri); yellow (11 taxa); red (Brownea grandiceps, Cassia marginata & Schotia brachypetala), lavender (Bauhinia variegata); pink (Cassia grandis, C. javanica & C. marginata) or scarlet (Delonix regia).

vii. Petal Shape: Obovate in nine taxa; obovate-elliptic in Cassia grandis, C. javanica; obovate-oblong in Haematoxylum campechianum, Schotia brachypetala & Tamarindus indica; ovate-elliptic in Cassia marginata & Parkinsonia aculeata; oblong in Brownea grandiceps & Gleditsia caspica; sub-orbicular in Bauhinia hookeri & Delonix regia; obconical in Bauhinia alba spathulate Caesalpinia gilliessii or ovate in Cassia nodosa.

viii. Number: In all the taxa under investigation the number of petals are five except Gleditsia caspica (four petals) and Ceratonia siliqua & Saraca indica (apetalous).

ix. Androecium Fertility: Ten fertile stamens in 12 taxa; three in Tamarindus indica, five in Ceratonia siliqua, seven in Saraca indica, eight in Gleditsia caspica, nine in Cassia marginata or 12 in Brownea grandiceps; seven fertile & three sterile stamens in Cassia fistula, C. nodosa, Senna alata & S. didymobotrya; six fertile and four sterile in S. sophera or five fertile & five sterile in Bauhinia alba & B. variegata.

x. Length: As long as petals in Haematoxylum campecianum, Schotia brachypetala, Senna sophera & Tamarindus indica; shortly exerted in Caesalpinia ferrea, Cassia fistula, C. nodosa, Ceratonia siliqua & Gleditsia caspica; longly exerted in Brownea grandiceps, Caesalpinia gilliesii, Cassia marginata & Saraca indica or included (not exerted) in all the remaining studied taxa.

xi. Filament Texture: Pubescent (Bauhinia hookeri, Haematoxylum campechianum & Tamarindus indica); hairy (Caesalpinia gilliesii, Cercis chinensis, Delonix regia, Parkinsonia aculeata, Peltophorum africanum); pilose (Gleditsia caspica); glandular (Caesalpinia ferrea) or glabrous in the rest of the studied taxa.

xii. Form: Awl-shaped in Peltophorum africanum or Filiform in the rest of the studied taxa. The latter may be sigmoid in Cassia grandis, C. javanica & C. marginata, nodulated in C. nodosa. The filament form (sigmoid & noduated) in taxa under investigation is comparable to those mentioned by Bentham (1871), Taubert (1891) and Randell (1976) who suggested that the filament form help in segregation of genus Cassia L. into three subgenera viz. Fistula, Senna and Lasiorhegma, or subgenera viz. Cassia, Senna and Absus respectively.

xiii. Anthers Size: Unequal in Cassia marginata, Senna alata & S. didymobotrya; subequal in Cassia fistula, C. grandis, C. javanica, C. nodosa, Senna sophera & S. surattensis or equal in the remaining studied taxa.

xiv. Attachement: Dorsifixed in Cassia grandis, C. javanica, C. marginata & Schotia brachypetala; basifixed in seven taxa or versatile in the rest of the studied taxa. In this respect Rendle (1925) distinguished tribe Cassieae by having more or less basifixed anthers.

xv. Ovary Texture: Glabrous in four out of the studied taxa viz. Caesalpinia ferrea, Cercis chinensis, Gleditsia caspica & Haematoxylum campechianum; the others with pilose in three taxa viz. Saraca indica, Schotia brachypetala & Tamarindus indica or pubescent in the rest (18 taxa).

xvi. Form: Flattened in nine taxa; oblong in 11 taxa or terete in the rest (five). Bentham (1871), Taubert (1891) and Randell (1976) concluded that the flattened and terete ovary enhance the separation of Cassia and Senna. This conclusion is in agreement with the data extracted in the present study particularly in that point.

xvii. Setting: Sessile in Caesalpinia gilliessii & Delonix regia; subsessile in seven taxa or stipitate in the remaining studied taxa.

xviii. Style Length: Short in eleven out of the studied taxa or long in the remainders.

xix. Form: Flattened in five out of the studied taxa viz. Bauhinia alba, B. hookeri, B. variegata, Haematoxylum campecianum & Senna surattensis; teret in ten taxa or filiform in the remainder.

xx. Curvature: Upcurved in Cercis chinensis; straight in seven taxa or incurved in the rest.

xxi. Stigma Form: Capitate (Bauhinia alba, B. hookeri, B. variegata, Brownea grandiceps); sub-capitate (Cercis chinensis & Tamarindus indica); concave (Caesalpinia ferrea & Haematoxylum campecianum); truncate (Caesalpinia gilliessii & Delonix regia); peltate (Ceratonia siliqua & Peltophrum africanum) or simple in the remainder.

xxii. Pod Texture: The pod not available in Brownea grandiceps; pubescent in Cassia grandis, Senna didymobotrya & S. surattensis and glabous in the remainder.

xxiii. Colour: Yellow in Haematoxylum campecianum or Brown in the rest.

xxiv. Form: Linear in six taxa; linear-oblong in Cercis chinensis; oblong in 12 taxa; terete in four viz. Cassia fistula, C. javanica, C. marginata & C. nodosa and elliptic in Haematoxylum campecianum.

xxv. Dehiscence: The pod dehiscent in 13 taxa or indehiscent in 11 taxa.

xxvi. Apex: Acut in Senna alata; mucronate in Haematoxylum campecianum; cuspidate in Bauhinia hookeri & Senna didymobotrya; tapering in Cercis chinencis, Parkinsonia aculeata & Peltophorum africanum or obtuse in the remaining studied taxa.

xxvii. Peak: Peakless in six out of the studied taxa; long in Bauhinia alba, Bauhinia variegata & Delonix regia or short in the remainders.

xxviii. Type: Membranous in Haematoxylum campecianum; crustaceous in Tamarindus indica; sub-woody in Caesalpinia ferrea, Gleditsia caspica & Saraca indica or woody in eight taxa or coriaceous in the remainder.

xxix. Seed Colour: Not available in case of Brownea grandiceps & Cassia nodosa; orange in Cassia fistula; black or greenish black in Cercis chinensis, Saraca indica & Senna alata; green in Senna didymobotrya and brown in the remaining taxa.

xxx. Form: Teret in Parkinsonia aculeate; sub-terete in Delonix regia, & Saraca indica; compressed in 12 taxa or flattened in the remaining taxa.

xxxi. Shape: Ovate in Caesalpinia ferrea, C. gilliesii, Cassia fistula, C. marginata & Schotia brachypetala; obovate in Ceratonia siliqua & Senna surattensis; orbicular Bauhinia alba, B. variegata, Cassia javanica & Tamarindus indica or suborbicular in Bauhinia hookeri; elliptic in Cassia grandis & Gleditsia caspica; pear–shaped in Senna alata & S. sophera or oblong in the remainder.

Methods

II. Methods


1. Morphological Investigations

i. Whole Plant

Macromorphological attributes of the whole plant, inflorescence, flowers, fruits and seeds were described from the investigated specimens or compiled from text books such as Bailey (1949) and others. Macrophotographs for some of the studied taxa were made from living representatives and others were derived from specific sites.*

ii. Lamina Vein Architecture

 Leaves were cleared in 5–20% KOH, rinsed in water, bleached in 25% NaHClO 2 , and placed in 250% chloral hydrate (CCl 3 CH(OH) 2 ) for 24 h.

 Cleared and bleached leaves were then rinsed in water, dehydrated through an ethanol series, stained with 1% safranin, then placed onto glass slides and investigated by a microscope, photographs were presented.

 Leaf architectural terminology generally follows Hickey (1973, 1979) and LAWG** (1999).



iii. Lamina Epidermal Morphology

 Lamina epidermal characteristics were studied using fresh materials.

 Leaf fragments of 5–10 mm2 were sampled from the median portion of each leaf.

 The leaf fragments were then soaked in concentrated acetic acid and 9% hydrogen peroxide in Petri dishes for periods ranging from 1 to 3 days. The appearance of air bubbles on the surface of the leaf fragments indicated their suitability for separation.

 They were transferred into water in a Petri dish with a pair of forceps.

 Both epidermal surfaces were carefully separated by tearing up a corner of the leaf and pulling back the upper epidermis.

 The epidermal surfaces were cleaned with a camel hair brush, rinsed several times in distilled water and later transferred into 50% alcohol to harden for about 2 min.

 The epidermal srips were stained in safranine for about 3–5 minutes and excess stain washed off in water.

 The ab- and adaxial epidermises were mounted in glycerine on a slide with the edges of the cover slip ringed with nail varnish to prevent dehydration.

 The photomicrographs were taken using a Reichert Microstar IV microscope at the Plant Taxonomy Research Laboratory, Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt.

 Descriptive terminology of epidermal characteristics based on Matcalfe & Chalk (1950), LAWG (1999) and Prabhakar (2004).

 The stomatal index (SI) was calculated using the formula of Stace (1965): S/S+E×100, where "S" denotes the number of stomata per unit area and "E" the number of epidermal cells in the same unit area.

 For SEM small pieces (7 mm2) of the leaf material were fixed on SEM stubs with double-sided tape, coated with gold in SPI-Module sputter coater, examined and photographed in Jeol JSM 5200 at different magnifications.









2. Molecular Investigations

i. RAPD-PCR

Genomic DNA extraction was performed as suggested by DNA extraction kit’s manufacturer Jena Biosciences, Plant DNA Preparation Kit, Genomic DNA purification from plant tissue.

a) Sample Collection: Fresh lamina tissue finely grounded with a mortar and pestle in liquid nitrogen prior to DNA isolation, tissue kept cold to minimize DNase activity.

b) Cell Lysis: The finely grounded tissue (10-30 mg) transferred to a 1.5 ml microtube, 300 µl Cell Lysis Solution added to the tissue, then incubated at 65°C for 60 min, the tube inverted occasionally during the incubation.

c) RNase Treatment: The RNase A Solution 1.5. µl added to the cell lysate, the sample mixed by inverting the tube 25 times and incubated at 37°C for 15-60 min.

d) Protein Precipitation: The sample cooled to room temperature and 100 µl of Protein Precipitation Solution added to the cell lysate, the solution mixed well by vortexing, centrifuged at 15,000 g for 3 min. (The precipitant form a tight, green pellet.



e) DNA Precipitation: DNA containing supernatant transferred to a clean 1.5 ml microtube containing 300 µl of Isopropanol >99%, The sample mixed by inverting gently 50 times, then Centrifuged at 15,000 g for 1 min, the supernatant discarded and tube drained briefly on clean absorbent paper, 300 µl Ethanol 80% added and tube inverted several times to wash the DNA Pellet, centrifuged at 15,000 g for 1 min, the ethanol discarded carefully, finally air dried at room temperature for 10-15 min.

f) DNA Hydration: DNA Hydration Solution 50-100 µl added to the dried DNA pellet, the DNA hydrated by incubating sample at 65°C for 60 minutes.

g) PCR analysis (Table 4): Polymerase chain reactions (PCR) were carried out in 50 µl volumes containing around 100 ng of sample DNA, nucleotides at 200 µM and 10-base oligonucleotide primers at 200 nM . each reaction containing 0.8units of Taq polymerase and 5 µl of Taq polymerase buffer was overlaid with 30 µl of light mineral oil. Reactions were carried out in a Techne PHC1 DRI block. The reaction parameters were 92C for 1 min; 35C for 1.5 min; 72C for 1.5 min for 45 cycles with a final extension step of 72C for 5 min. samples were then stored at room temperature.

Products from the PCR reaction were separated by agarose gel electrophoresis and visualized under UV light following ethidium bromide staining.



Table (4): Primers Used in RAPD Analysis

No. Primer Sequence No. Primer Sequence

1 SC10-5 TCGGAGTGGC 6 SC10-14 TCCCGACCTC

2 SC10-17 GTTAGCGGCG 7 SC10-18 GCCCTACGCG

3 SC10-22 CTAGGCGTCG 8 SC10-23 GGCTCGTACC

4 SC10-25 CGGAGAGTAC 9 SC10-30 CCGAAGCCCT

5 SC10-59 GCATGGAGCT 10 SC10-64 CCAGGCGCAA



ii. Isozyme Technique

 Native-polyacrylamide gel electrophoresis (Native-PAGE) was conducted to identify isozyme variation among studied Caesalpinioideae using five isozyme systems.

 Fresh and young leaf samples for each species were used separately for isozymes extraction.

 The utilized isozymes are - and -esterase (- and - Est), acid phosphatase (Acph), alcohol dehydrogenase (Adh), and aldehyde oxidase (Alo).

 These isozymes were separated in 10 % polyacrylamide gel electrophoresis according to Stegemann et al. (1985).

a) Stock Solutions

 Extraction Buffer: 1M Tris HCl (pH 8.8): Solution was prepared by dissolving 12.11 g Tris in about 50 ml distilled water and pH was adjusted to 8.8 by conc. HCl, then the volume was completed to 100 ml with distilled water then kept at 4oC.

 Acrylamide Stock (30%): The solution was prepared by dissolving 30 g acrylamide and 0.8 g N, N, methylene bis–acrylamide in about 70 ml distilled water, then the volume was completed to 100 ml by distilled water. The stock solution was kept at 4oC.

 Electrode Buffer (pH 8.65): Electrode buffer was prepared by dissolving 18.55 g boric acid and 2.5 g sodium hydroxide in 500 ml distilled water and mixed well with magnetic stirrer, then pH was adjusted into 8.6 by distilled water then kept at 4oC.

 Gel Buffers: a. Separating gel buffer (1.5 M Tris – HCl, pH 8.8) (Tris 18.15 g, distilled water up to 100 ml). b. Stacking gel buffer (0.5 M Tris – HCl pH 6.8) (Tris 6.05 g, distilled water up to 100 ml). c. Ammonium persulfate solution (APS 10 %) (Amm. persulfate 0.1 g, distilled water up to 100 ml).

b) Extraction of Isozymes: Isozymes extraction from the different samples was performed separately for each taxa by homogenizing 0.5 g fresh leaves samples in 2 ml extraction buffer using a mortar and pestle. The extract was then transferred into clean eppendorf tubes and centrifuged at 10,000 rpm for 5 minutes. The supernatant was transferred to new clean eppendorf tubes and kept at –20 oC until use for electrophoretic analysis.

c) Gel preparation (Table 5): Vertical slab gel electrophoresis apparatus was used. All glass plates were washed with tap and distilled water, then surface sterilized with ethanol. Spacers of 1.5 mm were used. Separating gel was prepared by mixing the chemical components. The prepared gel solution was poured immediately between the two glass plates and overlaid with isopropanol and left to polymerize for at least one hour. After polymerization, isopropanol was removed. Stacking gel was similarly, prepared by mixing the chemical ingredients listed in the table and then poured over the separating gel. The comp was placed immediately. The gel was left to polymerize.









Table (5): Composition of Separating and Stacking Gels (- = absence)

Stock Solution 10% Separating Gel 4% Stacking Gel

Acrlyamide 8.3 ml 1.7 ml

Separating gel buffer 6.3 ml -

Stacking gel buffer - 1.25 ml

Distilled water 9.9 ml 6.8 ml

10 % Ammonium persulfate 250 μl 100 μl

TEMED 10 μl 10 μl



d) Application of Samples: A volume of 40 μl extract of each sample was mixed with 10 μl sucrose and bromophenol blue, then a volume of 50 μl from this mixture was applied to each well.

e) Electrophoresis Conditions: The gel glasses were fixed to both lower and upper tanks of the electrophoresis apparatus. The run (electrode) buffer was added to both lower and upper tanks. The apparatus was connected to the power supply. The run was performed at 100 volt until the bromophenol blue dye has reached the separating gel and then the voltage was increased to 125 volt. Electrophoresis apparatus was placed inside a refrigerator during running duration.

f) Enzymes Assay: After electrophoresis, the gels were stained according to their enzyme system with the appropriate substrate and chemical solutions then incubated at room temperature in dark for complete staining. In most cases incubation for about 1 to 2 hours is enough.

 Acid Phosphatase (Acph): After electrophoresis, the gel was soaked in 100 ml of 50 mM Na-acetate buffer pH 5.0 containing 100 mg Fast blue BB salt, 100 mg -naphthyl phosphate, 100 mg MgCl2 and 100 mg MnCl2 (Wendel & Weeden, 1989). 50 mM Na-acetate buffer pH 5.0 was prepared by adding 5.15 ml glacial acetic acid and 2.85 g sodium hydroxide to 500 ml distilled water).

 Alcohol Dehydrogenase (Adh): After electrophoresis, the gel was soaked in a solution of 4 ml isopropanol, 25 mg NAD, 20 mg NBT and 5 mg PMS in 100 ml of 0.05 M Tris HCl pH 8.5 (Jonathan & Wendel, 1990).

 - and - Esterase Enzyme (- and - Est): After electrophoresis, the gel was soaked in 0.5 M borate buffer (pH 4.1) for 90 minutes at 4 oC. This procedure lowers the pH of the gel from 8.8 to about 7 at which the reaction proceeds readily. The low temperature minimizes diffusion of the protein within the gel. The gel then was rinsed rapidly in two changes of double distilled water. The gel was stained for esterase activity by incubation at 37 oC in a solution of 100 mg -naphthyl acetate or - naphthyl acetate (as a substrate) and 100 mg fast blue RR salt in 200 ml of 0.1 M phosphate buffer pH 6.5 (Scandalios, 1964).

 Aldehyde Oxidase (Ao): After electrophoresis, the gel was soaked in 100 ml of aldehyde oxidase staining buffer pH 8.6 containing 20 mg NBT, 10 mg EDTA, 25 mg NAD, 100 mg KCl, 10 ml benzaldehyde and 5 mg PMS ((Jonathan & Wendel, 1990). Aldehyde oxidase staining buffer was prepared by dissolving 5.44 g Tris, 1.55 Boric, 0.37 g EDTA in 50 ml distilled water. After dissolving, the solution was completed to 100 ml by distilled water.

g) Gel Fixation: After the appearance of the enzyme bands, the reaction was stopped by washing the gel two or three times with tap water. This was followed by adding the fixative solution, which consists of ethanol and 20 % glacial acetic acid (9 : 11 v/v). The gel was kept in the fixative solution for 24 hours and then was photographed.

3. Numerical Analysis

a) Coding of Characters: Prior to analysis, the presence of character states (morphological and molecular) should be indicated as numerical values in order to make the comparison during similarity estimation feasible.



b) Standardization: The average data for each character was standardized using standardization program to get the linear transformation of the variables in the data matrix.

c) Similarity or Dissimilarity Assessment: This requires comparative recording of a set of characters for all studied taxa and is made by the use of a certain similarity coefficient. Then, average taxonomic distance was generated by using the similarity of interval data program.

d) Estimation the States of Characters: UPGMA (Unweighted Pair-Group Method using Arithmetic Averages) was used to estimate states of characters variation among the species, each taxa was considered as operational taxonomic unit (OTU) and states of characters analysed as binary characteristics.

e) Clustering: The formation of groups depending on the values of similarity. The sorting strategies are numerous and variable, but in erecting taxonomic relations it is usually hierarchical agglomerative i.e. forming groups or grouping the most similar OTU in groups, then these groups are linked in more larger groups and so on to the end of the analysis at which all OTU’s are in one group. Each step of the clustering is based on values of similarities between the groups. SAHN (Sequential, Agglomerative, Hierarchial and Nested clustering method) program was used as defined by Sneath & Sokal (1973) and Dunn & Everitt (1982).

f) Plotting the Tree: Phenogram was obtained by use of tree display graphics program.

All computations were carried out by the aid of the NTSYS-PC version 2.02 (Numerical Taxonomy and Multivariate Analysis System) software program (Rohlf, 1989).



Terminology

The terms used for lamina archirecture study.

Term Meaning

Admedially ramified Branching orinted toward the primary or midline.

Alternate percurrent Veins cross between secondaries wit an offset.

Brochidodromous Secondaries joined together in a series of prominent arches.

Campylodromous Several primary veins or their branches, originating at or near a single point and running in strongly recurved arches that converge apically.

Cladodromous Secondaries freely branching toward the margin.

Dichotomizing Veins branch freely.

Exmedially ramified Branching orinted toward the leaf margin.

Festooned brochidodromous Having one or more additional sets of loops outside of the main brochidodromous loop.

Fimbrial vein Higher vein orders fused into a vein running just inside the margin.

Opposite percurrent Veins cross between adjacent secondaries in parallel paths witout branching.

Pinnate Single primary vein

Random reticulate Tertiaries anastomose (rejoin) withother 3° veins or 2° veins at random angles.

Regular polygonal reticulate Veins anastomose with other 3° veins to form polygons of similar size and shape.

Semicraspedodromous Secondary veins branching just within the margin, one of the branches terminating at the margin and the other joining the superadjacent secondary.



The terms used for stomatographic study (LM).

Term Meaning

Anomocytic Five or more cells enclosing the guard cells, cells adjacent to the guard cells not differentiated in any way from the normal epidermal cells.

Cyclocytic Single ring of five or more small cells enclosing the guard cells.

Isotricytic Stomata completely surrounded by three subsidiaries, more or less equal in size.

Paracytic Two cells enclosing the guard cells with their long axis parallel to the long axis of the guard cells.

Tetracytic Stomata surrounded by only four subsidiaries of variable size and shape.







Terms used for Leaf Surface Sculpture (SEM)



Term Meaning

Colliculate With rounded broad elevations closely spaced.

Pusticulate With small broad slight elevations not so high or abundant as on colliculate and not having as abrubt elevations as a minutely tuberculate surface.

Reticulate With a rasied network of narrow and sharply angled line frequently presenting a geometric appearance, each area or depression outlined by the reticulum being an interspace.

Ruminate Penetrated by irregular channels giving an eroded appearance and running in different directions.

Tuberculate With small smooth rounded projections or knobs.