1THEVETIA PLANT ECONOMIC POTENTIAL:CHEMISTRY’S KEY POSITIONCOURTESIESThe Vice Chancellor,Deputy Vice-Chancellors (Academic & Administration),Other Principal Officers of the University,Provost, College of Health Sciences,Deans of Faculties, Postgraduate School and Student Affairs,Professors and other members of Senate,My Academic and Professional Colleagues,Non-Teaching Staff of the University,My Lords, Spiritual and Temporal,Great Unilorin Students,Distinguished Invited Guests,Gentlemen of the Print and Electronic Media,Ladies and Gentlemen.INTRODUCTIONI give honour, adoration and glory to God Almighty forthe unique opportunity to serve this great University as a pioneermember of academic staff and and for seeing me through todayto give this inaugural lecture. I am doubly grateful to God andthe University authorities for honouring me to give this lecturetoday, during a historic event, the convocation ceremony.Ordinarily, the inaugural lecture should have been shifted, butbecause the University Management and I believe the governingCouncil wish to honour me for whatever reasons they may haveadduced. I remain particularly very grateful for this honour.One common characteristic of plants and animals is life,similarly both plants and animals feed for survival. In mostcases, animals feed on plants, this simple mode of feeding interrelationshipis referred to as herbivorous.God at creation provided a model for the study ofscience and the perfect food chain model. God created the very2basic essentials for life first i.e. air, water and light. Thereafter,He created plants and finally animals. Man was created as theking of animals and given the authority to take control over allHe had created. Man exercises the control in progression as hisknowledge of the environments, particularly plants, increases. Asthe population increased, survival challenges increased,adaptation and utilization of the plants for his good alsoincreased. These conditions for survival called for betterknowledge i.e. study of the plant, became more and morechallenging. There arose a steady need to explore plants aroundhim to his economic advantage.Farming, in the simple concept, is the multiplication of aplant to meet the need of man, consequently increase inpopulation demands increase in scope of farmland and farmingtechnology. Farming also calls for selection of crop with

preferred qualities, this accounts for man’s activities whereby acrop native to a location readily gets migrated by man to aforeign land. The colonial explorers and traders who migrated inthe early history contributed very largely for easy spread ofplants from one region of the world to others.Virtually all plants cultivated in plantations today wereonce plants that grew in the wild, in their natural environment.We are all familiar with fruit trees, e.g. mango and orange, palmtrees, and other economic tress, how man has cultured each tomaximize their plantations. Plantations today have developedbeyond just large scale farming; care is given to selecting bestvariety for propagation.Thevetia PlantThevetia plant is a tropical shrub which grows in thewild and remains ornamental, despite the abundance of the plantaround our homes, schools and other buildings. The plant isgrown as hedges and kept for its bright and attractive flowers.Thevetia plant is recorded to be more than 2000 years in itsnative countries – West Indies, Brazil and Mexico. It was takento Europe about three hundred years ago, and today it hasnaturalized in virtually all countries in the tropics. Thevetia plant3thrives very well in all the climatic and vegetation belts ofNigeria, it is readily found in Port Harcourt and in Maiduguri orSokoto. To date, thevetia plant remains a plant of no economicvalue whereas it has a lot of potentials. This is the focus of thislecture.Before I go into serious business of the lecture, Mr. ViceChancellor sir, please allow me to introduce you to the plant ofthe lecture.Thevetia Plant MorphologyThevetia plant is a dicotyledon which belongs to theAponaceae family. It is a composite, evergreen shrub, which isfound to have a milky sap. It is native to West Indies, Mexicoand Brazil. It is known as yellow oleander (nerium), gum bush,bush milk, exile tree in India, cabalonga in Puerto Rico, ahanaiin Guyana, olomi ojo by Yorubas in Nigeria. The plant is ashrub, reaching a height of 3 to 3.9metres. The plant is perennial;the leaves are linear, narrow, sword-like and green. The flower isyellow flute which develops to a fruit which has a pair of4follicles or drupes, it has one to four compartments, eachcontaining a seed. The fruit when unripe is hard and green butgradually turns black as it ripens. The fruit has varying masses(2-6.1g) which are dispersed by man and propagated by seed orstem. The plant fruits virtually ten out of the twelve months ofthe year. The seed contains about 60 – 64% oil on dry matterbasis. The plant produces white latex (sap) that is highlypoisonous. The seed also is highly poisonous. This attributeaccounts solely for the lack of interest in the development of the

plant. In spite of the toxicity of the plant, it has found usefulapplications in several spheres of life. Its latex is used as ananalgesic for toothache when the stem part is chewed in Juccata;and as an insecticide, the latex or extract of the stem as vesicantand the bark as a febrifuge and effective abortificacent. Thewood is used as axe handle.5THE CHEMIST IN ACTION ON THE POTENTIALITYOF A PLANTCuriosity is one dominant factor a natural productchemist possesses to explore his environment, particularly theplants. The plant everybody does not pay any attention to couldreadily engage the research interest of the natural productchemist. It is an in-built trait for a chemist to notice a peculiarityof a plant for his research and kick- start the study of the plant.On the other hand, a plant scientist of any discipline say,herbalist/pharmacist, agronomists/plant breeder, a nutritionist etcmay, by questions in his area of interest on a certain plantmaterial, stimulate interest of a chemist to pay attention on theplant. Even in the event of the latter, the chemist will only acceptthe challenge and do something meaningful if he is curious andzealous about the problem.Every plant is a stored-up treasure (nature) God hasprovided for the exploration of man. It is like the inorganicmineral and crude oil deposits. The deposit remains a thing ofpotentiality until man ventures to explore it and analyze it for itsconstituent and adapt these to the advantage of man. Thepetroleum deposit in Nigeria did not get there after ourindependence. Indeed had the British government a goodknowledge of the deposit and had explored this, ourindependence would have been delayed. All the same theexploration and development did not start until some peopleshowed interest, invested money and energy. The Obasanjoadministration had shown interest in the solid mineralexploration, this is the initial impetus; there remains a need forgood implementation by all concerned. For now, most solidminerals in Nigeria still remain potential as far as theircontribution to the economy of the country is concerned.Like the solid minerals and crude petroleum deposits,every plant is a treasure that must be explored to give manmaximum benefit of the plant. The plant, as we have studied atthe elementary school, is made up of the leaves, stem, root and inmost common plants, flowers and fruits. Many of the plants6around us are used in their natural forms, deriving only basic andminimium benefits. Plants that have been explored by allscientific input have been developed at varying degree forvarying level of benefits to man. It is common knowledge that afruit tree produces fruits at its season, so its product is availablefor a short period even when the fruit provides valuable nutrients

we need throughout the year. Unless the fruit is processed, theservices derivable from the fruit shall be limited.Plants influence the good life of man in various spheressuch as in medicine, food and industry (agro allied industry). Theplant however shall remain potential or partly developed untilthe chemist makes his contribution on the constituents of theplant. For instance, malaria was a deadly disease until thediscovery of cinchona plant which contains quinine. The planteven after man’s initial knowledge of its herbal proficiencycertainly required the input of the chemist to identify the activeingredient and eventually work to supply and meet demand ofthe drug, quinine.Scurvy was a disease that was ravaging among sailorsand treated by taking fresh fruits. With the input of the chemists,vitamin C (ascorbic acid) was mass produced and put on hold thedisease henceforth.The role of the chemist goes beyond identification of theactive ingredient in a plant; he is also saddled with theresponsibility to synthesize the compound as a true replicate ofthat which the plant biosynthesizes. By this singular role, he cansupply the populace beyond what the plant can, even ifplantations are developed. Ability to synthesize helps to provideimproved variety and new drugs and other chemicals in otherapplied fields.Plants Position in the World EconomyPlants in various forms provide the raw materials forevery agro—based industry. This sector accounts for a highpercentage of the economy of every nation. Countries notendowed with petroleum deposit or mineral deposits have nooption other than develop its agricultural sector to supply raw7materials for the agro-based industries. Nigeria was in thisposition until the 70s when crude petroleum accounted for theforeign exchange of the nation.Plants may be classified to include oil seed plants, whichtoday contribute tremendously to the economy of all nationseven the developed countries. Malaysia and Indonesia investedin the development of palm tree in the mid 50s, today this singlecrop/plant gives the nation a high percentage per capital andforeign exchange earnings.Oils and fats are products of plants which produce fruitsand majority of plant fruits/seeds contain oils/fats in varyingquantities. A plant is referred to as an oil/fat seed plant when itsoil/fat is worthy of extraction for one use or another.There are about twenty major oil seed plants recognizedworldwide today as oil seed plants. Nigeria is ratedunderdeveloped judged by the state of the development of its oilseed plants. Nigeria NIFOR has it on record to have provided thefirst set of palm oil seedling to Malaysia in the 50s. Nigeria wasonce rated as a world leading palm oil producing country up till

the 80s. Since the discovery of the crude petroleum, Nigeria hadabandoned the palm tree plantation and turned to import palm oilfor its industries requiring vegetable oils. Nigeria climate andvegetations are best suited to develop almost all the followingmajor oil seed plants - palm tree, coconut, groundnut, cotton,castor, sunflower, jathropha, melon, soybean, beniseed and sheabutter. It is sad to note that Nigeria is not noted in the productionof any of these among the first twenty leading producingcountries of the world.Basic requirements to the emergence of a plant as aneconomic plantEvery plant in the first instance grows in the wild. Whenman discovers one use for the crop or product of the plant, heinvests energy, time and his saving to explore the potentialitiesof the crop. To achieve early dividend in the exploration, acombined efforts of each of the following agents must becoordinated: the research team (universities and research8institutions), crop farmers association, government agents,industries and bankers. The emergence of a few selected oil seedcrop plants from wild to economic status is highlighted in thissection. Each plant is selected for at least one peculiar propertyworthy of development for an existing application. Whereas inthe distant past, the emergence of a plant as an economic plantwas basically dependent on the quantity of the oil, today oil yieldis secondary, whereas individual characteristics now becomeprominent, as will be seen in the following four oilseed plants.Jojoba1

Jojoba is a slow-growing perennial plant, native to theSonora Desert of Arizona; California and Mexico. Its oilscomposed of liquid esters with major components 40 and 42carbon wax esters. The plant was first commercially harvested inthe US in 1982. Its plantations are being established in India,Australia, South Africa and the Middle East. Jojoba oil isspecialty oil for use in cosmetic, this prospect excited researchersto reach higher heights for the promotion of the plant; its oil andoleochemicals. There are multi-tract approaches on theproduction of jojoba seed and oil. Productivity average in 1991was 226 pounds per acre and it is projected to quadruple by2010, going by the investment on the plant production in 2004.Lesquerella1

The plant belongs to the mustard family and is native toNorth America. It is a potentially valuable source of hydroxyfatty acid, supplementing castor seed oil. It is seen as a potentialdrought – tolerant oilseed crop that could produce fatty acids forlubricants, plastics, protective coatings, surfactants andpharmaceuticals. One of its fatty acids is lesquerolic acids (14 –hydroxy cis – 11- eicoseneoic acid) a hydroxy fatty acid, similarto the castor oil derived recinoleic acid but two carbon atomslonger.

Meadow foam1

Meadow foam is a slow growing herbaceous winter,annual, wild flower, native to the Pacific North West. It is still atexperimental and developmental stage as a good alternate seed9oil. Its emergence success as a world oil-seed is hampered by thecompetition its oil faces in the world market. The high price ofthe oil and the need to increase yield is a major deterent. Goodfunding to farmers and research will facilitate early marketimpact.Cuphea1

Cuphea belongs to the family lyathraceae, a wild plantwhich still must be domesticated to gain commercial value. Itsoil is rich in medium chain fatty acids. It competes favourably inthe market with coconut and palm kernel oils. The latter two aretropical plants. The oils are well adapted to the manufacture ofsoaps, detergents, surfactants, lubricants and related products. Itsoil is a good source of capric acid which is currently obtainedprincipally from petroleum source.Mr. Vice Chancellor Sir, I wish to mention in passing atthis point that for any plant to transform from wild to economic,concerted, collaborated efforts of various bodies must besupported by government policies and adequate funding.Contributions of oils and fats in the world economyEach oil seed crop contributes it quota into the worldpool of oils and fats. Invariably oils and fats have uses first in thedomestic sector as part of food, and then conversion to otherproducts now called– oleochemicals – in the industrial sector.Soybean is one major oil in the oleochemical industry and hasone of the largest adaptations and conversion products. Scheme 1gives at a glance major new uses of soyabean in theoleochemical sector.10Scheme 1 New uses of soybean oilNew usesProducts Soy ink Soy toners Plasticisers Soy protein Biolubricant soy oil soy polyolsCompetitizers adhesivesProducts Printing copiers PVC, Plastic wood transports cosmetic plasticsMarkets Inks & laser adhesiveprintersConsumptions(M. Bush/Y) 100 20 100 150 100 1 100

Source: INFORM (2005) 16 (10) 66011Oils chemists and oleochemicals manufacturers withhigher concern are ever becoming more and more anxious overrates of production vis-a-viz consumption and demand for oiland oleochemicals. The concern and anxiety comes from theever increasing demand by the oleochemical sector for oils thatprimarily were once for food. If prices of food containingvegetable oils will not fly out of reach of the common masses,there must be matching new sources of oils that shall specifically

be oleochemical concern only. Towards this general concern,Frank Gunstone2 in his lecture at the Stephen Chough Award byAOCS in May, 2006 attempted to answer the question “will oiland fat supply meet oil and fat demand in 2007?” He usedavailable figures for the 15 years (1990 to 2005 to consider whatsupply and demand will be in the next 15years i.e 2005 – 2020.He classified oils and fats uses into three categories. These threeclassifications have oils and fats distribution as 80:14:6. Theratio is changing rapidly, principally due to high demand forbiodiesel alone. Table 1 presents the supply and uses of oils andfat in 1990 and 2005 based on 4 animal fats and 13 vegetableoils.Table 1: Oils and fats: supply and usage (MMT)Supply Food Oleochemicals Others199020058013564108111958Source: INFORM (2006) 17 (18) 541.World supply of vegetable oils and fats shall ever be onthe increase, and with widening gap between supply anddemand, because of the environmental advantages ofoleochemicals over petro-chemicals. Furthermore, there is thethreat that petroleum resource decline with daily pumping ofcrude pteroleum whereas there is ever encouraging increase interms of man input in acreage available for oil seed cropsproduction. Oleochemicals have a bright future, because of thebelief in many quarters that after a while, quantities of12oleochemicals will be cheaper than the petrochemicals.Furthermore, oleochemicals are environmentally friendly.The greatest challenge for oils and fats in theoleochemicals demand remains biodiesel. It is Gunstone’sforecast that the world demand for production of biodiesel in2020 may be 40-50MMT; this in turn will demand from theworld vegetable and animal oils and fats. Gunstone is hislecture also considered the world production and use of oils andfats in terms of population and quantities of oils and fats, heviewed the world as made up of three categories. The developednations with a strong oleochemical industry but use of their oilsand fats in food are less than the global average. New Zealandwas taken to represent developed countries with strongagricultural activity but probably with no oleochemicalsindustry. Nigeria represents developing nations with populations

greater than 100million. In this third category, there is growingdemand for food fats and oils. Table 2 presents per caput use ofoils and fats (kg/yr) for all purposes in selected countries in 2005(the world average is 21 MMT for 6,454 million persons.Table 2: Per caput use of oils and fats (kg/yr)Country Population(Million)Kg Country Population kgEU-25 456 50.8 China 1,299 19.6USA 380 49.0 India 1,097 11.7New Zealand 4 38.2 Indonesia 225 18.2Russia 141 22.2 Brazil 184 25.1Mexico 106 25.9 Pakistan 161 19.4Nigeria 130 13 Bangladesh 152 7.5Source: INFORM (2006) 17 (18) 54-2Biodiesel: a major consumer of world oils and fatsBiodiesel is methyl ester of fatty acid. It may beproduced by batch or continuous process represented in the twoequations for reactions involved.13(a)(i) Fat/oil + NaOH RCOONa + glycerolsoap(ii) RCOONa + MeOH RCOOMe + NaOHbiodiesel(b) Fat/oil + MeOH RCOOMe + glycerolBiodieselBiodiesel production at the present day volume isrelatively recent, but that not withstanding, it is experiencingvery dramatic expansion in the developed countries forclassification of national development, it is almost an index. Theneed for the commodity, which is preferred to diesel, serves as agreat driver for success in the sector. The drive is greatlysupported by high price arising from artificial scarcity and fearfor future real scarcity of petroleum diesel.According to the US National Biodiesel Board, thenumber of active and proposed biodiesel plants grew by morethan 67% in six months in 20052. Projected production capacityfor 2005 was 545 million gallons per year. As the capacity ofbiodiesel production increases, there shall be a correspondingincrease in demand for oils and fats. In USA, soybean is thefavorite oil, because it is easily available and the ease ofprocessing it into biodiesel. Even in the developed nations, thereis an aggressive drive for alternate seed oil feedstock forbiodiesel, in particular. This is in anticipation of a major need ofbiodiesel by internal combustion engines; a justification forsource in unorthodox new oil seed crops world over.Palm oil and palm kernel oil: the plant oil wealth neglectedby NigeriaMalaysia purchased its first palm oil seedling in mid 50sfrom NIFOR. In less than 50 years after, Malaysia andIndonesia led the world in the production of palm oil and palm

kernel oil. In 2002, Malaysia produced 37MMT crude palm oiland 11.9 MMT of crude palm kernel oil3. Today Malaysia has14the largest oleochemicals capacity of any country in the worldand her capacity represents 25% of the world capacity in 2002.16 oleochemical companies were in operation in Malaysia with atotal production capacity of 1.756 MMT. About 1.4MMT ofthese oils were processed in to oleochemicals and 1.27 MMT ofthese products (89%) were exported. Major oleochemicalsexported were fatty acids, fatty esters, fatty alcohols andglycerine. Oleochemicals from Malaysia have been exported toover 100 countries, including north America, European Unioncountries, Japan and China. It is worthy to note at this point thatthe volume of trade to Nigeria on the oleochemicals and crudevegetable oils is negligible, not worthy of mention, yet Nigeriaimports a lot of its oleochemicals for the few oleochemicalindustries in the country possibly mainly from Malaysia andIndonesia. Furthermore, Mr. Vice Chancellor Sir, it is pertinentto remark at this point that Nigeria has greater potentialities tohave been producers of oleochemicals which now competeeffectively with petrochemicals. We have potentials to produce,but painfully we have neglected this ability. In no distant future,we shall have to import biodiesel to supplement if not replaceour petroleum diesel then at a high price and a major drain on theeconomy of the nation, what a pity this will be to us then as anation.THE JOURNEY IN THE STUDY OF THEVETIA PLANTThe plant grows with widespread in Kwara state. Theabundance attracted my attention to consider what could bemade of the plant; so literature review was conducted to obtaininformation on quantity of studies done so far on the plant. Inall, literature report was very scanty, which means very limitedwork has been reported on the plant; even then, the little reportsavailable were on the toxicity of the latex, and the seeds.The first experiment4 was organized to study the impactof fertilizers on the seedlings, this involved pots experiments oneffect of NPK, calcium, potassium, and phosphorus. The resultof the studies revealed that application of NPK, single15superphosphate, calcium nitrate, and murate of potash had effecton the uptake of nitrogen, potassium and calcium, particularlyafter seven weeks of application. Whereas the fertilizers rich incalcium tended to depress uptake of potassium and nitrogen,application of CAN, however, tended generally to enhance, moresignificantly, uptake of calcium.The plant was thereafter studied for the properties of theseeds which the plant produces abundantly yearly. The seed is60-65% oil and 40-45% protein. These two parametersstimulated us to embark on aggressive studies particularly onintroducing it as a good substitute to orthodox supply of oils

used by the commercial soap making industries.The oil physical properties, particular its saponification value,120-124, and unsaponifiable matters, 0.24-0.41% prompted us tostudy it in soap making. Tables 3a - c present some informationon the oil compared with other orthodox commercial oils.Table 3a: Sterol content of a few selected seed oils (%)Fat/Oil Sterol content (%)Coconut 0.06 – 0.08Cotton 0.20 – 0.31Linseed 0.37 – 0.42Palm 0.23 – 0.31Palm kernel 0.06 - 0.12Groundnut 0.19 - 0.25Rapeseed 0.35 - 0.50Soybean 0.09 - 0.11Wheat gem 1.3 - 1.7*T. Peruviana 0.44 -1.40 (plus phospholipids)* Fadipe V.O., 1992 MSc. Thesis16Table 3b: Oil seed mean oil content of a few selected seeds.Type\source AOCS Ac – 44 Exhaustive extraction AOCS 2-93Soybean 19.35 21.98Rape seed 43.74 43.5Cotton seen 18.17 20.61Sun flower 12.71 46.10Safflower 37.99 38.0* Groundnut 21.24* Lasquerella 25* Varnonia 40- T. peruviana 60-64Source: INFORM (1997) 8 (10), 1048; Ibid (1998) 9(8) 749-835;(1999) 2 (5) 686 – 691.- Result in the departmentTable 3c: Oil composition of selected seedsType/Source Sat Mono unsat Poly unsatSoybean 15 24 67Cotton seed 24 26 50Palm 52 38 10Palm kernel 86 12 2Sun flower 11 20 69Coconut 92 6 2Ground nut 18 49 33.5* T. peruviana 30-45 46-51 1-3Source: INFORM (1990) 11 (4) 250* Ibiyemi et al5.The first and oldest6 oleochemical is soap. This is aproduct of saponification of vegetable oils and fats. It isbelieved to have been produced by the ancient Egyptians throughPhoenician into the Rhone valley in France as early as 600BC.In 23-79 AD, Plinus, described soap as hair pomade which wasmade from a combination of fats and ashes. At the early uses,soap was not a cleansing agent. About 130-200 AD, Galenos, a

Roman doctor, discovered the cleansing property of soap. In 17th

17Century, the general use of soap for cleansing and bathing firststarted when Justus V.Liebig (1803-1873) gave a citation that“soap is an index of the prosperity and culture of any nation”.This simply means that soap in the days was used to comparetwo nations with the same population and to ascertain which oneis richer, more prosperous and better cultured.The first type of soap in Nigeria is the black soap6 whichwas milder, creamer and foam better than some imported soapsas at the time under review.The basic principle and process of soap making remainspractically unchanged for the past 1000 years. This involvessaponification of oils and fats with alkali and salting out of thesoap.OCH2-O-CR CH2OHOCH-O-CR + 3NaOH CHOH + 3RCOONaOCH2-O-CR CH2OHOil/fat glycerol SoapGuided by the high level of the oil in the seed coupledwith the abundance of the plant with its widespread in thecountry, the team including Mr. S.A. Akanji decided to study theoil of the seed starting with saponification reaction. Towardsthis goal, several kilograms of the fruit/seeds were processed toobtain some gallons of the oil. In the first instance, samples ofthe oils were made available for undergraduate students at theirpracticals to conduct saponification reaction to obtain soap. Theperformance of the oil at this level prompted us to provide theProduction Manager of Lever Brothers Nig. Ltd, Apapa (nowUnilevers Nig. PLC) one gallon of the oil for soap trialproduction in 1981. The Production Manager was veryenthusiastic in accepting the oil and indeed, in a very short timereported back to us his findings. The oil was very good inproduction of soap that was suitable for bar and toilet soap.18There was no need to bleach the oil before use, thereby savingprocessing cost, and the lathering property was appropriate. Weconsidered the finding as good grounds for the department’sbreakthrough in its interaction with an industry.In 1978, December 4-7, a symposium sponsored byUNESCO was held in Toronto, Canada, and a team of twelvechemists led by Prof. Ekong DEU, attended. The theme wasuniversity – industry interaction in chemistry in Africa. Theuseful thrust of the symposium is for members of the universitychemistry departments to forge a close relationship withchemical-based industries in Africa. So the finding by theproduction manager was to have signaled an opportunity to

record success of the aims and objectives of the Torontosymposium. But low and behold this was not the case as therelationship broke down with the Production Manager questions,“what quantity (kilogram or gallons) of the oil we expected willbe produced from one hectre of the crop plantation”? We havenever thought of this because we saw ourselves concerned withthe chemistry of the oil not the agriculture and economics. Thatnot withstanding, however, to keep the relationship alive, wesuggested that we needed one thousand naira to engage theservices of labourers to keep one acre of farm to be planted at thebeginning of the next rains. The production manager was frankand quick to let us know that such request is not common andwould require management decision. All other follow-up provedabortive and so terminated the much cherished and anticipatedcelebration of break-through with an industry.The disappointment was taken as a challenge and wesought alternative means to determine the yield of oil per acre, ifthe plant was grown on a semi large scale. The team of chemistsdid not see itself handling this assignment effectively, so talkedwith our colleagues in the crop production department, Facultyof Agriculture to accept to place final year project students onthe scheme. All our efforts yielded no response. So we venturedinto acquiring a plot in the Faculty demonstration farm for theproject, there was no success recorded.19In 1992/93 I was away on sabbatical leave in ABU,Zaria, and I enjoyed the favour and cooperation of Prof DanielSarror, the Vice Chancellor, who was good to grant me a specialfund for the project. The farm management, under thechairmanship of Prof Duro Olarewaju, made a plot available formy project. At the beginning of the rains, 1000 seedlings wereplanted and kept until the expiration of my sabbatical leave inSeptember 1993. There was no staff collaborating and the plantswere to flower in 1994, it was not convenient for me to maintainthe project. Thereafter I moved to Ilorin consequently andpainfully the farm was lost to weeds.During the period I was in Zaria, I organized an MScstudent and an academic staff, Mr. J.O. Ojokuku, together withMr. V.O. Fadipe, an MSc student of University Ilorin to do acomparative study of the plant seed oil obtained from seedscollected in Ilorin, Edidi, Zaria and Enugu. I was briefly inEnugu on WAEC assignment and I noted a variety of the plantwith purple flower rather than yellow. This peculiarity attractedmy attention for comparative studies of its oils with others withyellow flowers. The results of analysis of the seeds on thevarietals based on the number of kernel per seed andgeographical location are presented in table 5.Table 4a: Summary results of analysis of the four varietiesof seedsAnalysis Data Oneseed

TwoseedsThreeseedFourseed% oil yield 56-61.1 59-63.1 54.58.8 54-59.3Saponification value 121-140 124-186 120.181 120-196Unsaponifiablematters (%)0.27 0.21 0.42 0.38Free fatty acids 0.488 0.434 0.564 0.513Acid value 0.97 0.86 1.12 0.02Peroxide value 16.46 14.47 19.97 19-41Iodine value 76-. 81.2 80.2 79.2Refractive index 1.461 1.462 1.462 1.462Specific gravity 0.926 0.913 0.927 0.937Viscosity (cent) 19.00 21.36 15.80 17.04Source: Ibiyemi et al520Table 4b: Common fatty acids present in oils of the varieties of thevetia seeds based on kernel number andgeographical locations.Source: Ibiyemi et al5LocationVarietyC14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 Sat UnsatEnugu 12340.310.370.480.4317.719.318.420.90.100.350.180.305.26.026.317.0046.140.3839.9436.74-15.5713.128.470.70.70.9-23.3525.72

25.1028.2946.8657.9856.1679.57Edidi 12340.560.170.720.3114.220.616.019.810.30.30.200.275.017.755.486.5634.0640.8534.1236.5813.858.8510.767.951.350.120.161.3419.7828.2222.6576.0551.5850.1145.2146.15Ilorin 12340.310.170.170.3220.4218.3421.0015.970.250.210.91

0.217.355.287.205.8045.0630.4443.3933.7612.5814.0514.0816.970.441.330.450.7128.9023.7928.2722.0958.3346.0358.7157.65Zaria 12340.140.160.220.3820.8123.7020.1220.210.240.230.350.288.018.977.397.6345.7940.9647.0040.0016.8112.6514.7716.000.360.600.470.4928.9634.9327.7328.22

56.7854.4462.4962.71

21Enugu has the highest rainfall over 10 months, Zariawith lowest rainfall, while Edidi and Ilorin lie in-between inamount of rainfall, and period of rains. Seeds from Ilorin andEdidi record highest oil content (62-64%), Zaria 58% and Enugu61%. In terms of quality of the oil, Zaria seeds contain highestunsaturation (59.3%) and Enugu lowest value (51.1%).Basic research, in general, in most purposes and intent,provides data that would guide the applications of the materialbeing investigated. This principle guided our plan of actions onthe analysis of thevetia seed oil, i.e. to accumulate as much dataon its physical and chemical properties, peradventure, some daythis will adapt the oil for an industrial use. Towards this goal,the oil was studied for its thermal stability7. The result obtainedshowed characteristic changes in physicochemical propertieswhen heated at 180oC, 200oC, and 220oC, over a period of 15hours. There was no significant difference in the five parameters(iodine value, acid value, peroxy values, saponification valueand amount of polar compounds) studied for the three workingtemperatures. The study proved that the seed oil would be stableto heating, therefore could be good for cooking, if adopted foruse in frying food. This property also shows justifications forthe oil to be used or converted for use in the making of lubricantsand greases.Synthetic Lubricants: Thevetia seed oil and its chance.Synthetic lubricants came into use during the World WarII in Germany, primarily due to lack of petroleum. However,their use today is based on specific applications whereconventional petroleum base-lubricants fall short of the desiredrequirements, primarily in the area of low and high temperatureperformances. Table 5 presents some compound classes that fallinto the category of lubricants from vegetable oils and fats.22Table 5: Types of synthetic lubricantsThe combination of chemically compactable thickeningagents with either petroleum oil or synthetic fluids results ingreases. There are greases which are soaps and non-soap types.The soap-types contain the metal salt of organic acids, while thefatty acids (non-soap) greases are usually palmitic acid, andother saturated fatty acids.Thevetia seed oil was therefore investigated with a viewto establish its suitability in preparing a lubricant or grease sinceit has good thermal stability properties. Several project studentswere placed on the modifications of the seeds oil. Like any otherseed oils, thevetia seed oil responded favourably to severalchemical reactions but analysis of the products to establish theproducts was truncated by lack of facilities even as elementary as

separation techniques beyond plate and column chromatography.At this point it may be mentioned that the university hadpurchased an equipment vital for our studies of vegetable oils,GLC, but the equipment is not adequately equipped to perform,for lack of standards. All efforts made since 2004 to acquire thestandards directly from SIGMA in UK, and then through ZAYO,the sole agent in Nigeria, based in Jos, is yet to yield fruit,despite payment for the standards in pound sterling and naira.Thevetia seed oil, like any organic liquid, could beconsidered worthy to be tried for production of emulsion.Emulsion could be cosmetic or lotions, depending on variousfactors that may include the presence of more than one phase,stability, or the phenomena of surface activity. Thevetia seed oilwas investigated peradventure it will make good varnishingcreams, an essentially oil-in-water emulsion with its fatty acidClass Typical compoundFatty acid esters EthylstearateDibasic acid esters Dibutylphythalate, di (-2-ethyl hexyltricresy phosphate)Silicone esters Tetraethylsilicate, hexa(-2-ethyl butoxyl disiloxanePolyglycols Poly(propylene glycol)Poly alkyl and aryl ethers Poly phenyl ethers23salts as the emulsifying agent. Stable emulsions of the oil wereprepared but the qualities other than stability could not beestablished.Thevetia biodiesel: Its chances as a seed oilThe world is daily seeking to substitute petrochemicalsin general, but most specifically biodesel and other enginecombustion fuels. The first use of vegetable oils8 in internalcombustion engines dates back to about 1900 when RudolfDiesel (1858-1913) experimented with groundnut oil and FujioMagao achieved operation with pine oil in 1948. The two oilpricesharp increases (1973) sparked world-wide desire indeveloping energy sources as alternate to petroleum in internalcombustion engines, boilers and other combustors. Effort sincethen, has stimulated investigations in favour of use of ethanol inBrazil, sunflower oil in South Africa9, rape seed oil in Europe,and currently, and on a large scale spent cooking oils andvegetable oil esters are gaining acceptance and use at asurprising scale. For almost ten years, Malaysia and Indonesiatend to lead the world by the nose by virtue of its success story inbiodiesel investments and production. Malaysia in 2005announced a national biodiesel policy to stimulate thedevelopment of the biofuel industry by four-prong strategy thatencompases the production of a biofuel blend of 5% processedpalm oil and 95% petroleum diesel (B5), encouraging the use ofB5 among the public and establishing biodesel plants inMalaysia for the export market10.Our research team which kept changing as the researchstudents graduated made coordinated planning very weak and

inefficient. This not withstanding, thevetia seed oil has beenstudied to provide data on its conversion to biodiesel.Production of its biodiesel involved both batch and continuousprocesses. All available analytical techniques have been used toestablish a level of success in the esterification reactions. Themost reliable confirmation of the success should have come fromNNPC; however we have lately approached LUBCON for this.Samples of our biodiesel were made available to NNPC Kaduna24in 1993 for analysis. The “no response” was taken as negativeresults, but at the same time we needed their response on what todo to achieve success. Of recent, our effort, whereby weinvestigated the effects of catalysts on our production ofbiodiesel has taken us to LUBCON to determine our level ofsuccess. Samples have been presented to LUBCON; the resultof analysis is being awaited. Since LUBCON is close and morecordial, there will be close interaction and we hope to havesuccess through this in the near future.Thevetia seed oil in livestock feed formulationThe oil, if freed of the toxins of the plant, and theplantation developed, and the oil is not used as cooking oils, itmay serve as oil component in the livestock feed mealformulation. The team led by Prof. J.O. Atteh12 had the first trialon the replacement of palm oil by thevetia oil in broiler chickdiets. In the experiment, the effects of replacing 0, 25, 50 or100% of the dietary palm oil (5%) with oil of Thevetia peruvianaseed was investigated using broiler chicks 0-3 weeks old.Increasing the content of thevetia oil in the diet reduced averagefeed in-take and weight gain (P<0.05) and decreased protein andfibre retention (P<0.05) and fat retention (P<0.01). However,there was no significant effect on feed: gain ratio or mortalityrate. It was concluded that there was a need for furtherprocessing of the seed oil before it can be used effectively as aningredient in broiler feed.On our part, we have established that the oil indeedrequires refining. Charcoal treatment removes all taint colourand recently we have confirmed the presence of the plant toxinsin the oil. A repeat of the trial feed experiment with the refinedseed oil is desirable, depending on the cooperation of ourcolleagues in the animal science department.Thevetia seed oil in the paint industryThe utility value of the seed oil is unlimited, so the seedoil was considered possible alternate oil for the paint industry.D.N. Meyer, sometimes in 2004, invited the departmentto a round-table discussion on what the department could do to25source a local alternative alkyd, the major ingredient in the paintformulation; an item if replaced by local sources would reduceproduction cost. The alkyd accounts for about 50% of allingredients compounded to make gloss paints.

Table 6: Typical recipe for gloss paint formulationIngredients % WeightAlkyd resins 49.6Talcum powder 21.9Turpentine 15Titanium dioxide 17Cobalt naphthenate 6.6Source: Turner13

Soybean oil accounts for one of the major ten seed-oilscurrently in use by major paints manufacturing companiesworld-wide. Nigeria, to-date, imports every bit of its alkyds, avegetable oil polyester. The chemical reactions basic to thepreparation of alkyds are presented in the following equations.1. Esterifications:R’COOH + ROH R’COOR + H2O2. Etherification of polyols (a possibility):ROH + R’OH ROR’ + ROH3. Transesterification:RCOOR1 + R2COOR3 R2COOR1 + RCOOR3

The alkyd is a resin, synthesized from a dicarboxylicacid such as phthalic anhydride or its acid and a polybasicalkanol e.g. glycerol, pentaerythritol etc in the presence of asuitable catalyst at about 220-240oC. The reaction is acondensation reaction and produces polymers. It is believed thatthe properties such as rapid drying, good flow, and excellentweathering. They have almost completely outstripped dryingoils e.g. linseed oil, as binder for paints e.g. enamel, varnishes,wood primers and lacquers.14

The percentage of oil can be varied giving rise to shortalkyd resins when the oil content is low and long alkyd resins26when oil content is high. Typical alkyd resins’ ingredientscontents are presented in table 7.Table 7: Classification of alkyds based on the amount of oilcontentParameter Long alkyd Medium alkyd Short alkyd(%) oil >70 50-70 <50phthalicanhydride (%)20-30 25-35 35-60Viscosity (Sec) 90-120 120-150 150-700Acid value 12 max 30 max 32 maxSource: Martens C.R. (2003) Alkyd Resins. Reinhold PublishingCorp. N.Y. Pp 108.The impact of the properties of the oil and performanceof the paint is presented in tables 8 (a) & (b)Table 8a: Effect of oil on alkyd propertiesSource: Kirk–Othmer (1963) Encyclop of Chem Techn.2nd Ed-Intersci. Publ. JohnWiley & Sons Inc. NY Pp851-882.Oil Type Coating PropertiesIodine

valueSpeed drying Colour retention Gloss retentionLinseed 180Tuna 170Dehydrated castor 155SafflowerSegregated140Conton seed 130-140Cotton 110Tall oil 125Soybean 135Ground nut 108Castor 85Olive 85Coconut 9027Table 8b: Division of each drying and semi drying oils intoshort, medium and long oil resins categoriesSource: Deffar D and Soucek MD (2001) Journal Coat Techn 73(919) 95Thevetia seed oil has properties comparable with thecommon oils being used in the preparation of alkyds; wetherefore embarked on the preparation of the alkyds, if only inresponse to D.N. Meyer’s challenge. There are three teams ofmembers of staff in the department who have accepted thechallenge and have studied at least four seed oils in alkyd resinpreparations and characterization of the alkyds. Alkyds havebeen prepared from thevetia seed oil, jathropha seed oil,dehydrated castor oil, tobacco seed oil and parinary seed oil.Tables 9a-c presents findings on the alkyds of thevetia andjathropha seed oils.Drying oilresinsshort oil resins Cure at elevated temp, give very hard, glossyfinishesUsed in finishes for appliances, signs and toys.Drying oilresinsMedium oil resins May be air dried or heated, give durable glossyfinishes.Used for farm hardwares and metal furnitureDrying oilresinsLong oil resins Have good brushing characteristics, dry rapidlyin air, reasonably durable, glossy filmUsed in house-hold paints.Semi-dryingresinsNo division based onoil lengthGive film with improved resistance to yellowingon ageing.Used particularly for high gloss white finishesNon-dryingresins

Short oil resins Used mainly in conjunction with amino resins.Give improved adhesion and flexibility.Used in storing finishes for appliances.Non-dryingresinsMedium oil resins Used mainly as plasticizers for cellulose nitratefor furniture finishes.

28Table 9a: Solidification time of thevetia seed oil andjathropha seed oilAlkyds solidification time (sec)(i) (ii) (iii) (i) (ii) (iii)JAK-1 T-AKD-1 P-AKD-1 150 132 109JAK-2 T-AKD-2 186 180 -JAK-3 T-AKD-3 294 207 -JAK-4 T-AKD-4 300 240 -JAK– Jathropha alkyds; T-AKD - Thevetia alkyds; P-AKD –Parinary alkyd.Table 9b: Viscosity of Alkyds(i) (ii) (iii) (i) (ii) (iii)JAK-1 T-AKD-1 P-AKD-1 140 210 190JAK-2 T-AKD-2 97 100 -JAK-3 T-AKD-3 92 105 -JAK-4 T-AKD-4 86 85 -Table 9c: Drying characteristics of alkyd resins containingno drierOut-doorDFTIndoorDFTOutdoorSTTTIndoorSTTTJAK-1 T-AKD-15.3* 16.8/28+

JAK-1T-AKD-111.6* 20/31+

JAK-1 T-AKD-151.8 4.8/15+

JAK-1 T-AKD- 170.1 8.7/19+

JAK-2 T-AKD- 210* 21/38+

JAK-2 T-AKD-216.5* 28/45+

JAK-1 T-AKD-199 10/24+

JAK-1 T-AKD- 1125 14/27+

JAK-3 T-AKD-319.8* 39/50+

JAK-3 T-AKD-325.3* 44/55+

JAK-3 T-AKD-

113 12/23+

JAK-3 T-AKD-149 16/34+

JAK-4 T-AKD-426.5* 41/55+

JAK4 T-AKD-431.7* 48/65+

JAK-4 T-AKD-416.1 18/37+

JAK-1 T-AKD-117.5 19/42+

P-AKD19.2P-AKD24P-AKD5.67P-AKD8.5DFT = Dust-free Time, STTT = Set-to-touch-timeSource: Akinwatimi (2006). B.Sc.Project.Seeds processed all over the years would have producedtesta, if allowed to accumulate, would have amounted to severalkilograms, which if it is not properly disposed could readily be29an environmental nuisance of the type similar to saw dust insawmills and palm kernel shell in palm kernel processingindustry. On careful consideration, we thought that the testacould be considered for board-particle production. Samplestherefore were sent to the Africa Timber and Plywood, Sapele in1987. The product made out of the material was very hard andbrittle. The industry indicated no further interest in theexperiment, otherwise we would have wanted to have a blend ofthe hard material with a soft material, particularly, baobao fruittesta. (See appendix for the letter from the MD).Thevetia seed: providing protein in animal feedsThe seed on the basis of its protein content (40-45%)should be preferred to most orthodox protein sources in theformulation of animal feeds. Brain et al15 and Bisset16 wereamong the first few to report on the seeds for its toxins, cause ofdeath as recorded for two children, horses and other animals.Compounds I to IV below serve as representatives of thetoxins.OHCH3

CHOO HCH2

OHOHOHHHHHH

OO

OOCH2OHHOHOHHOHHHH

OOCH3

OMe HOHHHH H

O

Thevetin A30OCO2ROHCH2OH O- ß - D - glucose

(Theveside: R = H; Theveridoside: R = Me)OHOOCH3

HCH3

OOHCH3

OHHHHHOMeOH

NeriifolinHOOHOODigitoxigenin (Thevetigenin)31The seed is shown to contain between 3.6 and 4%thevetin17, the major glycoside of the seed, and the most lethaltoxin. It is cardiotonic. Other compounds that have beenidentified are cerberin, ruvoside, perusitin and neriifolin18. Paperchromatography reveals that fresh seed of the plant containsfifteen compounds. Some of the glycosides have been subjectedto clinical trials, especially in the treatment of congestive heartfailure19 and cardiac insufficiency. However the margin betweentoxic and the therapeutic doses have been found to be too smallfor many of the glycosides, especially thevetin, to be usefultherapeutically until further research is done in this regard. Someof the compounds have been commercialized by ALDRICHchemical company. This already paves way for possible use ofthe extract when the seed will eventually be processed in largequantities.

In favour of the plant emerging as a possible commercialplant is the fact that other useful compounds have been isolatedfrom the plant, these include flavonones and flavonolsglycosides, extracted from the leaves of T.peruvianaThe presence of anti-nutrients in oil seeds is notsufficient reasons to neglect a plant with prospect as the casewith thevetia plant. There are not many seed that are free of antinutrientsor toxins of any one type. The quantity and lethal levelof the agent and ease of removal matter. Soybean, cotton seedand castor seed in their raw forms are all intolerable to majorityof animals, particularly monogastric animals. Processing eachunder specified conditions have been adopted in thedetoxification of such seeds. Irradiation is well established andproperly utilized to effect genetic re-engineering, therebyproducing improved variety of plant and animal types. Thiscould lead to variations of contents of seeds and could beeffective detoxification technique.In the first set of detoxification treatment, dilute strongalkaline solutions and dilute hydrochloric acid were usedseparately and detoxification monitored by the level of bitternessof the cake. The cake with minimum bitterness was used to32compound broilers meals containing 0,5, 10 15% thevetia cake.20

Inclusion of thevetia cake in broiler diets, irrespective of level ofinclusion, drastically reduced feed intake and weight gain(P<0.01) at both the starter and finisher stages. The resultsshowed that both methods of detoxification are not efficient andsufficient. In pursuant of effective detoxification of the seed,other methods adopted include acid leaching using solutions atpH 6-9, organic solvents extraction, followed by aqueous ethanolextraction. Another method employed activated charcoal andboiling at varying periods ranging from 1 to 5hrs. Table 10presents cardiac glycoside contents of the raw cake and cakesafter various treatments.Table 10: Cardiac glycosides content of thevetia cakesSample Total cardiac glycosideRaw seed cakeAcid treated cakeEthanol treated cakeCharcoal treated cake4.27 ±0.44% or 4.27g/kg0.22 ±0.71% or 2.25kg/kg0.08 ± 0.25% or 0.83g/kg0.24 ± 0.22% or 2.4g/kgSource: Oluwaniyi et al21.Charcoal treatment and ethanol extraction have beenfound to be more efficient and effective in thedetoxification/debiterisation of the seed cakes. Acid hydrolysisprior to ethanol extraction also proved to be efficient but residualacid tended to leave a sharp taste that may not be desirable. Thework of Finnigan and Lewis22 using acid hydrolysis followed by

ethanolysis to remove glucosinolates as food component in therapeseed provides support that our results are as reliable andefficient methods of detoxification process21. The detoxificationeffectiveness was further established by a measure of theremains of the cake by monitoring the quality and quantity ofprotein in the cake, peradventure, protein may also have beenextracted, along with the glycosides. Loss of protein wouldnegate the primary objective of securing a good alternate proteinsource.Variations in the time of extraction, volume of ethanoland quantity of cake were investigated and products obtained33analyzed for the protein content23. Results obtained arepresented in tables 11a & b.Table 11a: Glycoside (%) extracted using varying ethanol:cake ratioTime (hrs) 10:1Ethanol: Cake15:1Ethanol: Cake20:1Ethanol: Cake0.00 5.44 5.44 5.440.75 0.61 0.48 0.5824 0.56 0.48 0.4848 0.52 0.45 0.2472 0.46 0.32 0.24Table 11b: Moisture and Protein content of cake afterextraction.Time (hrs) 10:1Ethanol: Cake15:1Ethanol: Cake20:1Ethanol: CakeMoistureProteinMoistureProteinMoistureProtein0.75 17.24 64.92 12.10 69.09 15.80 65.9224 16.95 65.76 11.13 68.91 15.63 66.0548 16.09 63.45 12.80 68.91 15.80 66.3472 18.00 65.77 12.89 68.58 15.46 65.91Source: Oluwaniyi & Ibiyemi23

Table 12c: Effect of varying water in ethanol on the qualityof cake using 15:1 ethanol: cake for 72 hrs% Ethanol inwaterMoisture ProteinContent Content(%) (%)

GlycosideContent(%)50 25.89 62.13 5.4460 22.82 65.35 0.4970 15.35 67.98 0.4580 13.39 68.95 0.3290 14.44 65.50 0.32100 15.89 62.35 0.32Source: Oluwaniyi22

Results in graphic form in Fig. 1(a) and (b)3401234560.00hrs 0.75hrs 24hrs 48hrs 72hrsTimePercentage glycoside10: 1 solvent: meal ratio15: 1 solvent: meal ratio20: 1 solvent: meal ratio

Fig 1a: Extraction of cardiac glycosides from thevetia seedmeal by 80% aqueous ethanol/methanolmixture.0123456Crude 50 60 70 80 90 100% (v/v) Aqueous alcoholic solutionPercentage glycoside

Fig 1b: Extraction of thevetia glycosides using varying concentrations ofaqueous alcohol at 15: 1 solvent to meal ratio for 72 hrs.

35The best adjudged treated cake was formulated as feedmeals and fed to chicks to establish the efficacy of thedetoxification technique. The feed experiments results arepresented in figures 2a & b.020406080100120140Feed Intake /Bird/Week Weight Gained /Bird/Week Gain: Feed Ratio (FeedEfficiency Ratio)Mortality (%)ControlT1AT1BT1C

Fig 2a: Performance Pattern of birds fed acid detoxified TSM020406080100120140160Feed Intake /Bird/Week Weight Gained /Bird/Week Gain: Feed Ratio (Feed Efficiency Ratio)ControlT2AT2BT2C

Fig 2b: Performance pattern of alcohol detoxified TSM36Result obtained from detailed analysis of the bloodsamples for birds fed each of the meals corroborated the resultpresented above from chemical analysis of the mealperformance.The qualities of the detoxified seed cake were further

established by the analysis of the seed cake for the proteincontent and amino acids profile.Table 13 presents the amino acids profile of the rawcake, and each of the two treated cakes. The efficiency of thisethanolysis by all standards is confirmed even by this parameterto be more efficient and effective in the detoxification exercise.Table 13: Amino acids profile of the seed cakesSeed Cak eAmino AcidRaw Acid treated Ethanol treatedAlanineArginineAspartic acidCystineGlutamic acidGlycineHtistidineIsoleucineLeucineLysineMethioninePhenylalanineProlineSerineThreomine4.494.4819.851.6914.213.631.622.945.494.470.883.384.243.932.613.044.2521.861.0520.102.241.392.094.883.970.643.223.853.12

2.044.565.1920.341.6915.673.701.652.975.595.650.903.704.494.002.67Source: Oluwaniyi22

The result agrees with the report by El-Adawy and El-Kadousy24 that ethanolysis led to an increase in the total,essential, basic, and aromatic amino acids of the seed cakedetoxified.37Irradiation has been put into divergent uses in variousaspect of life including agriculture; it has been widely adoptedfor genetic re-engineering to produce improved varieties in plantand animals. This technique is imagined could influence geneticvariation that may effect the reduction and possibly eliminationof the glycosides-toxins of thevetia plant.Thevetia seeds and kernels were subjected to 100 rads,300 rads and 500 rads treatment (100 rad=I Gray when (GY) isequal to IJK/ kg. Rad = radiation absorbed dose). Some wereanalyzed, some planted and plants produced monitored withinthe limit of chemists abilities for effect of the irradiation. Resultobtained on chemical analyzed of the seeds is presented in Table14.Table 14: Monitoring effects of Co-60 gamma rays on the oilcontent of four sets of seeds based on kernel numbervariety.Oil yield (%) of the types based on number of kernel/seeSeed Type dOne-seed Two-seed Three seed Four seed AverageUntreated /rawkernel41.00 43.30 58.80 54.20 54.32100 rad kernel 68.40 61.54 61.30 63.63 63.71300 rad kernel 50.80 50.00 50.80 48.80 50.50500 rad kernel 40.00 59.60 46.80 41.87 60.00Source: Akinduro (1999) BSc. thesisThis result seems to suggest that seeds treated at 300 radcontain lowest oil content, an undesirable result, if the plantwould be selected and nurtured after irradiation. The study ofthe plant morphology for seeds irradiated at the three doseshowever, suggests contrary performances. Table 15 presents

results of the seeds harvest from plants grown from seeds witheach of the three doses. Furthermore, the canopy size of theplants right from the 6th month till the 24th month providedconvincing evidence in favour of 300 rad treatment. Plants from500 rad seeds were dwarf, majority grew to about half the size of38others and such were never able to flower and fruit, even in thefourth year.Table 15: Summary results of the profile of fruits and seedsfrom thevetia plants (treated and untreated seeds).Seed-type (i) unirradiated (ii)300rad treated(iii)500rad treated (using 20-30 plants per plot)Average(i) (ii) (iii)Number fruits /plant 610 876 300Weight of total fruit/plant (g)230.5 204.3 198.69Weight of kernel 5.72 7.25 4.86Weight of fruit 11.45 14.00 12.31Seed populationdistribution based onkernel/seedType1Type2Type 3Type4131617412151641514156oil yield (Pet etherextract of seed cake (%) 54.46 62.40 47.00Chloroform extract ofSeed cake (%) 2.10 1.70 2.00Ethanol extractof seed cake (%) 1.57 1.30 1.49Source: Alabi (2004) B Sc thesis.The apparent contradiction in the results obtained byworkers in 1999 and 2004 lends credence to the team convictionthat as chemists we should limit our concerns with analysis ofproducts provided by plant scientists and / or agronomists.Furthermore, irradiation effects on plant may not manifest andstabilize in the first or even second filial. i.e the changes39

anticipated may emerge in latter generation. The desiredmodifications may therefore manifest slowly.Another monitoring conducted on the effect of theirradiation of the seed with 300 rads was to analyse cakesprepared from seeds harvested as first filial generation of plantstreated and untreated seeds. The results obtained for the aminoacids analysis are presented in tables 16a & bTable 16a: Amino acid analysis of thevetia seed cakesAmino acid T1 T2 T3 T4 T5AlanineArginineAspartic acidGlysteineGlutamic acidGlycineHitistidineIsoleucineLeucineMethiominePhenylalanineProlineThreomineTyrosineValine4.494.4819.851.6914.213.631.622.945.490.883.384.742.612.494.013.044.2521.501.0520.102.241.392.094.880.643.223.852.041.943.57

4.565.1920.341.6915.673.701.652.975.570.903.704.492.672.494.012.902.99144.881.3716.772.001.031.783.840.502.282.951.911.662.484.394.8719.171.6115.013.591.693.005.70.793.304.302.502.494.01Table 16b: Distribution of amino acids on basis of groupAnalysis T1 T2 T3 T4 T5Total Amino acids 84.41 83.35 89.27 65.54 84.88% Difference - - 0.66% + 5.76% -22.36% + 0.56%Essential amino acids (%) 35.4 31.07 36.22 32.00 35.65Acid amino acids (%) 40.35 50.04 40.34 48.29 40.27Basic amino acids (%) 17.55 16.05 19.02 15.91 19.34Sulphur amino acids (%) 3.04 2.73 2.90 2.85 2.83Aromatic amino acids (%) 5.87 5.16 6.19 4.64 5.79Source: O. O. Oluwaniyi (2007) Ph.D thesis (in progress)

40T1 = crude (untreated thevetia seed cake)T2 = acid detoxified seed cakeT3 = ethanol detoxified seed cakeT4 = Charcoal detoxified seedcakeT5 = 300 rad treated seed cakeTable 16(b) reveals that detoxification processes byethanolysis and radiation are worthy of further and detailedstudies particularly irradiation effect on toxin level. Amino acidsmake up proteins. There are different members of proteindepending on the sequence of the amino acids. Majorclassifications are globulin, albumin, prolamin and to lesserextent and of limited occurrence is gluten, majorly sourced fromwheat. Solubility properties of the protein members arepresented in Table 17.Table 17: Protein classification based on solubility.Proteins of the seed cakes have been analyzed for thevarious protein types; globulin and albumin have beenestablished in various proportions. There are indications, subjectto confirmation, that gluten may be present in an appreciablequantity that may justify intense research of the seed for itsprotein content, for gluten in particular.Table 18a: albumin and globulin content of thevetia seedsCakeHCl treatedCakeNaOH treatedCakeCa(OH)2 treatedCakeNaCl treatedExtractant: protein(%)Alb GlbExtractant: protein(%)Alb GlbExtractant: protein (%)Alb GlbExtractant: protein (%)Alb Glb0.5M 0.6 0.50 0.2M 0.26 0.21 0.5M 2.63 2.61 0.5M 0.98 0.76Protein Soluble mediumAlbumin WaterGlobumin Salt solutionProlamin 70% ethanolGluten Alkaline solution41Table 18b: Composition of individual member proteins inseed cakeType/member Content %Untreated Irradiated Detoxified Irradiated & detoxifiedCrude totalprotein53.3 53.11 54.25 53.14Albumin 11.70; 10.41* 12.89 - 13.88; 16.06* 10.24

Globulin 1.07; 7.65* 5.98 8.84; 5.66* 2.06Gluten 15.62; 13.94* 14.38 12.54; 10.28* 15.84Prolamin 4.56; 2.81* 2.38 0.99; 0.93* 0.85* Result from two different proceduresIn the event that gluten is unequivocally confirmed to bepresent as indicated in table 18b and in a quantity comparable toits presence in wheat, this singular parameter may be sufficientto achieve the vision for the emergence of thevetia plant as aneconomic plant. Table 18a provides good information on theprotein isolates of thevetia seed. This prompted us for furtherstudy of the protein fractions.CONCLUSIONWe have studied Thevetia peruviana plant seed from asmany points of view, believing we have accumulated as manypieces of information and data that should encourage scientistsof related disciplines, particularly agronomists to show sufficientinterest and work to bring out the potentialities of the plant. Theplant deserves to be studied to establish it valuable prospectsother than ornamental.Mr. Vice Chancellor sir, I wish to inform this augustgathering that I have presented a poster paper at the annualconference of American Oil Chemists Society held in Quebec,Canada, May 12-15th 2007 with the purpose to stimulate interestsof chemists worldwide on the study of the seed for its oil and theseed cake. The plant, if given the right quantum of research andfunding, could by 2015 be on field trials and its oil and cakeavailable in commercial quantities as a major feedstock for anyof the oleochemical and livestock feed meal industries.42RECOMMENDATIONSMr. Vice Chancellor sir, I wish to let you know that Ihave specifically sent special invitation letters to persons whoshould be in good knowledge of the research efforts on Thevetiaperuviana plant for one reason or another. Invitees include thepresidency, Raw Material Development Council, a few selectedpaints industries, Soap /detergent industries, cosmetic industries,flour mills; the Permanent Secretary of Federal Ministry ofEducation, Secretary of NUC and MD of NNPC and LUBCON.These people I have specially invited with belief that each, ifsufficiently informed and convinced of my vision for the plant,are strategically placed to promote:(i) research with focus on the plant,(ii) promote the right relationship for healthy interactionbetween industries and university research on vegetableoils and fats in general and the plant in particular,(iii) fund research in a satisfactory quantum,(iv) appreciate the need for good and guided policies,political will, and drive in Nigeria to invest NOWspecifically in the study of thevetia plant and in generalin the production of vegetable oils and fats in Nigerian tomake Nigeria contribute its quota effectively in the

world struggle for production of oleochemicals.To achieve the stated objectives, Nigeria research needsgood and well articulated polices of the Federal Government andNUC shall steer the ship of research to ensure the right drive forgood production of vegetable oils and fats in Nigeria. If thefederal government shall popularize and provide necessaryincentives to the farmers as has been done for cassava in the lastfive years, the country doors to foreign investors are certainlywider and more attractive in the production of oleochemicalsthan we got on cassava. A very major drive in processing anycrop is production cost of the crop within a short period. Nigeriais well suited to produce vegetable oil to storm the world marketwithin five years if it can be focused on the well established43annual oil crops- e.g. soybean, sun flower, melon in addition tothevetia seed. If Malaysia and Indonesia have excelled as worldleaders on two major oils- palm oil and palm kernel oil, within20years, Nigeria with the same oil plants and at least ten othershas better potentials that can readily make it be the leading notjust one of the leading oleochemicals producers by 2020, aperiod oleochemicals may take over from petrochemicals, whenthe latter may be less relevant.Mr Vice Chancellor, Sir, please allow me to suggest thefollowing specific recommendations that I belief will put theeconomy of Nigeria on a sound footing and rank Nigeriaeconomy at par with what obtains in the developed nations.1. Government should provide policies and legislations forthe creation of:(a) Oil farmers association to ensure production of a targetvolume of the commodity in the short, medium and longterms,(b) A governing council to work for the establishment of aviable oleochemicals production in Nigerian with atarget time for its product to be available in the Nigeriaand subsequently the world markets,(c) A fund that derives its source directly from the crudepetroleum sale to foster the activities set up in (a) and (b)above2. National Universities Council should:(i) Create and enforce compliance of a new policy oninterdisciplinary research in preference over and aboveindividual research in the universities. It should also provideguidelines whereby a minimum of say 10% of fund allocatedto Universities shall be devoted to research studies,(ii) Constitute a monitoring unit to ensure compliance in theinvestment of research grants in every university,(iii) Work discretely through a committee of NUC/ industries topromote university research and industries interactionthrough healthy collaborations between the two bodies andwith adequate funding by the industries,

44(iv) Fill the gap between Raw Material Development Council,and the universities through a committee of the GoverningCouncil for the RMDRC and NUC. The committee shouldgive premium to collaborative interdiscipline research in theuniversities with sharper focus on direct and immediaterelevance on the life and developmental efforts of the nation.3. The universities: Universities in Nigeria should createa special central account specifically designated for equipmentthat shall function and be properly maintained thereafter torender useful services. In addition:i. every University shall have a virile consultancy division tofoster a healthy and productive University-Industrialrelationship and attract necessary funding for research in theuniversity,ii. every university senate shall constitute its committee to seekand develop appropriate links with universities outsideNigeria for exchange of research information andcollaboration on uses of facilities that make up for short fallsin Nigerian universities. The committee shall mandatorilygive reports, at least once, every academic session to thesenate on its progress.In my final submission sir, Mr. Vice Chancellor, theprimary objective of the University is to provide unbiasedintellectual leadership for the development of the nation.Thevetia plant is a challenge for the University of Ilorin to placeNigeria in the world map as a pacesetter in the studies of thevetiaplant so that by 2015 it shall be an economic plant. It is anachievable target and I sincerely plead with the University ofIlorin management and Governing Council to accept thechallenge to act positively. Towards this goal, sir, distinguishedladies and gentlemen and particularly my special invitees,captains of industries, please accept this very specialrecommendation as worthy of immediate implementation. Thatthe University of Ilorin Governing Council should establishTHEVETIA PLANT SPECIAL RESEARCH FUND into whichevery stakeholder in Nigeria shall subvent generously. The fund45shall ensure success of thevetia plant research studies such thatthe University of Ilorin shall be “World Centre of Excellence onThevetia Plant Development”. It is an achievable feat worthy ofcareful consideration.Thank you and God bless you for your attention.ACKNOWLEDGEMENTTo God be glory, honour and adoration for Hisgoodness, mercies and for making today a reality.God has made so many people to work to make today areality. I am sincerely grateful to all the people that God hasdirected to contribute in any one form no matter how small.I am particularly grateful to all undergraduate students

who have done their project studies with me. My sincerelygratitude also goes to all my M.Sc. and Ph.D students. I say Godbless you all. Your own day of honour will not elude you.I register my sincerely appreciation to the Universityauthorities who have given me employment, peace of heart andthe facilities and challenge of “publish or perish” that havestimulated the research studies I have the opportunity to giveaccount of today.Relations, social associates, Christian friendsparticularly and many Muslim friends have been supportive invery many ways.Members of my family beginning with my wife haveendured so much, I thank you for your understanding andendurance when you have missed me physically and when I havebeen tight financially because I must succeed in my studies. Thehonour and joy of today belong to all us.Thanks to all who have come today in response to myspecial invitation. You are great and the benefits that will accruefrom the challenges of the lecture certainly remain your greatreward.God bless you ALL.46REFERENCES1. Features (1991); INFORM 2 (8) 678-692.2. Frank Gumstone (2006); INFORM 17 (8) 541-543.3. Salmiah Ahmad (2003); Malaysia: the hub for plantbasedoleochemicals. INFORM 14 (10) 604-606.4. Ibiyemi S.A. and T. Faloye (1988); Potassium, Nitrogenand Calcium uptake by T. peruviana seedlings asaffected by various nutrient sources. Nig. J. Agronomy.3 (2) 68-73.5. Ibiyemi S.A., Fadipe V.O., Akinremi O.O. and BakoS.S., (2002); Variation in Oil Composition of Thevetiaperuviana juss (Yellow Oleander) Fruits Seeds. J. Appl.Sci. Environ Mgt. 6 (2) 61-65.6. Fagbule, M.O. & O.A. Sosanwo (1983); Nig. J. Sci. &Tech. 1 (11) 31-36.7. Ibiyemi S.A., S.S. Bako, G.O. Ojokuku, & V.O. Fadipe(1995); J. Am. Oil Chem. Soc. 72 (6) 745-747.8. Thomas Mielke (2003); The world outlook for majoroilseeds; INFORM 14 (12) 712-713.9. Maruyama T. (1994); INFORM 5 (10) 1338-1140.10. INFORM staff reporters (2006); Ten years of oilseed, oiland meal forecast. INFORM 17 (5), 290.11. Ibiyemi, S.A and Oluwaniyi, O.O. (2003); Efficacy ofcatalysts in the batch esterification of the fatty acids ofT. Peruviana seed oil. J. Appl. Sci. Environ Mgt. 7 (1)15-17.12. Oluwaniyi, O.O. (2007). Ph.D. thesis (in progress)13. Turner G.P. (1988); Introduction to paint chemistry and

principles of paint chemistry and paint technology. 3rd

ed. Chapman and Hall, pp 108-169.14. Algers, S. M. (2001); Polymer Science dictionary;Elsevier Science Publ. Co. N.Y. 140 – 145.15. Brain Author, A Fox, Altan G. Cameron, a textbook onFood Science and Health, 5th ed. pp 172.4716. Bisset, N. G. (1963); Cardiac glycosides. IV,Apocynaceae: a preliminary paper chromatographicstudies of the glycosides from T. peruviana. Ann. Bogor4(2) 145-152 (Chemical Abstract 58:14438h).17. Sun N.C. and N.I. Libizor (1965). The glycosides of T.Peruviana. Chemical Abstract 6: 20496.18. (a) Perez – Amador, M., E.A. Bratoeff and S.B.Hernandez (1994); Thevetoxide and digitoxigenin,cardenolides from two species of Thevetia(Apocynaceae). Chemical Abstract 120: 319441t.(b) Lang, H.Y. and N.C. Sun; (1964); The cardiacglycosides of T. Peruviana II. Isolation andidentification of cerberin, ruvoside and a new cardiacglycoside, perusitin. Yao Hsueh Hsueh Pao 11(7) 464-472 (Ch.) (Chem. Abstr. 62: 9465a, 1965).(c) Huang, C.C., K.H. Hung and S.H. Lo (1965);Pharmacology of the glycosides of T. Peruviana I.Thevetin. Yao Hsueh Hsueh Pao 12(2) 824-826 (Ch.)(Chem. Abstr. 64: 18275d, 1966).19. Arora, R.B., J.N. Sharma, and M.C. Bhatia, (1967);Pharmacological evaluation of peruvoside, a new cardiacglycoside from T.neriifolia with a note on its clinicaltrials in patients with congestive heart failure; Indian J.Exp. Biol. 5(1) 31-36 (Chem. Abstr. 67: 20362f, 1967).20. Atteh, J.O., S.A. Ibiyemi, and A.O. Ojo (1995);Response of broilers to dietary levels of thevetia cake. J.Agric. Sci Cambridge. 125, 307 – 310.21. Oluwaniyi, O.O., S.A. Ibiyemi and A.L. Usman, (2007).Effect of detoxification on the nutrient content of T.Peruviana seed cake. Res. J. of Applied Sci. 2 (2) 188-191.22. Atteh, J.O., S.A. Ibiyemi, F.O. Onadepo andO.O.Ugboma, (1990); J. Agric. Sci. Cambridge. 115,141-145.4823. Oluwaniyi, O.O. and S.A. Ibiyemi (2007); A study of theextractability of thevetia glycosides with alcoholmixture. African Journal of Biotechnology (Accepted).24. El-Adawy, T.A. and El-Kadousy, S.A. (1995); Changesin chemical composition, nutritional quality, physicochemicaland functional properties of peach kernel mealduring detoxification. Food Chemistry 52: 143 – 148.

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Morphology and Embryology of Holarrhena antidysenterica Wall.

C. S. Lattoo

Botanical Gazette, Vol. 135, No. 3 (Sep., 1974), pp. 173-180 (article consists of 8 pages)

Published by: The University of Chicago Press

Stable URL: http://www.jstor.org/stable/2474236

Morphology and Embryology of Holarrhena antidysenterica Wall., by C. S. Lattoo © 1974 The University of Chicago Press.

Abstract

The wall of the microsporangium in Holarrhena antidysenterica Wall. consists of an epidermis, endothecium, one or two middle layers, and a secretory tapetum. The endothecial cells develop fibrous thickenings in later stages. Division of microspore mother cells is successive, and cytokinesis takes place by cell-plate formation. Isobilateral, decussate, T-shaped, and linear tetrads of microspores are formed; the pollen grains are two-celled at anthesis. The ovule is hemianatropous and tenuinucellate. The development of the embryo sac conforms to the Polygonum type. Synergids are long with prominent hooks, and antipodals are ephemeral. Endosperm development is of the nuclear type and ultimately becomes cellular. Embryo development follows the Caryophyllad type (Johansen 1950). The seed coat formed by the integument consists of three regions: an outermost epidermis, a thick-walled middle region, and thin-walled inner cells. The epidermal cells contain starch and tannin.

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1Current Address: Bridgewater College, Box 1568, Bridgewater, Virginia 22812, U.S.A.J. Bot. Res. Inst. Texas 2(1): 489 – 493. 2008

CHROMOSOME NUMBER OF THEVETIA AHOUAI(APOCYNACEAE: RAUVOLFOIDAE: PLUMERIEAE) WITH DISCUSSIONON THE GENERIC BOUNDARIES OF THEVETIAJustin K. Williams and Julia K. Stutzman1Department of Biological SciencesSam Houston State UniversityHuntsville, Texas 77341-2116, U.S.A.abstractThe mitotic chromosome count (2n = 20) for Thevetia ahouai is the first reported chromosome count for Thevetia sect. Ahouai. The counttogether with a previous count in Thevetia sect. Thevetia (also 2n = 20) provides an additional synapomorphy that further supportsthe monophyly of Thevetia as traditionally recognized. A discussion on the proposal to recognize Thevetia sect. Thevetia as the genusCascabela is provided.Key Words: Thevetia, Apocynaceae, chromosome number, Cascabela, Cerbera, PlumerieaeresumenEl recuento cromosomático mitótico (2n = 20) de Thevetia ahouai es el primero para Thevetia sect. Ahouai. Este recuento junto con otroprevio en Thevetia sect. Thevetia (también 2n = 20) aporta una sinapomorfía adicional que apoya la monofilia de Thevetia como se hareconocido tradicionalmente. Se aporta una discusión de la propuesta de reconocer Thevetia sect. Thevetia como el género Cascabela.Thevetia L. belongs to the Apocynaceae subfamily Rauvolfioideae tribe Plumerieae and comprises eight speciesof shrubs occurring from Central Mexico to northern South America (Gensel 1969; Williams 1996; Allorge1998; Endress et al. 2007). According to various specialists in the Apocynaceae, the generic boundaries ofThevetia vary. In the concept of Thevetia sensu K. Schum. (Schumann 1895; Gensel 1969; Williams 1996;Allorge 1998; Alvarado-Cardenas 2004), eight species are sub-divided between two sections: sect. AhouaiK. Schum. with three species—T. ahouai (L.) A. DC., T. amazonica Ducke, and T. bicornuta Mull. Arg.—andsect. Thevetia K. Schum. with five species—T. gaumeri Hemsl., T. ovata (Cav.) A. DC., T. peruviana (Pers.) K.Schum., T. pinifolia (Standl. & Steyerm.) J.K. Williams, and T. thevetiodes (H.B.K.) K. Schum. In the conceptof Thevetia sensu Lippold (Lippold 1980; Alvarado-Cardenas & Ochoterena 2007) the three species ofsect. Ahouai are retained in Thevetia; the other five species of sect. Thevetia are segregated into the genusCascablea Raf. Although Lippold (1980) and Alvarado-Cardenas and Ochoterena (2007) segregate Thevetiasensu K. Schum. into the two genera Thevetia and Cascablea, at no point do they argue against the genera’s“close morphological relationship” (Alvarado-Cardenas & Ochoterena 2007). In fact, a recent morphologicalcladistic analysis (Fig. 1, Alvarado-Cardenas & Ochoterena 2007) nests all eight species of Thevetia sensu K.Schum. in a clade supported by six synapomorphies. In short, Thevetia sensu K. Schum. is clearly shown to bemonophyletic. Nevertheless, Alvarado-Cardenas and Ochoterena (2007) argue for its paraphyly and chooseinstead to recognize the two sub-clades of the clade (Fig. 1) as distinct genera: Thevetia and Cascabela.Chromosome numbers have proven useful in resolving generic relationships in the Apocynaceae (Vander Laan & Arends 1985; Williams 2007). To date, 73 of the 179 genera of the Apocynaceae s.str. (subfamiliesRauvolfioideae and Apocynoidoideae) have been counted (Van der Laan & Arends 1985; Goldblatt & Johnson

2003; Williams 2007). Although previous chromosome counts for sect. Thevetia (T. peruviana; 2n = 20) exist,none have been reported for sect. Ahouai. The present paper provides the first reported chromosome countfor a species of sect. Ahouai and discusses the utility of chromosome numbers in interpreting the systematicrelationship of Thevetia and Cascabela.490 Journal of the Botanical Research Institute of Texas 2(1)Fig. 1. Morphologically constructed dendogram of “Plumerieae” clade (from Alvarado-Cardenas & Ochoterena 2007). a = Thevetia sensu K. Schum. and/or “Cascabela-Thevetia” clade of Alvarado-Cardenas and Ochoterena 2007. This clade is supported by seven synapomorphies. b = Thevetia sect. Ahouaiand/or Thevetia sensu Lippold. This clade is supported by two synapomorphies. c = Thevetia sect. Thevetia and/or Cascablea Lippold. This clade is supportedby four synapomorphies.Williams and Stutzman, Chromosome number of Thevetia ahouai 491materials and methodsRoots tips and voucher specimens were collected from a greenhouse specimen of Thevetia ahouai housedat the greenhouse of the Department of Biological Sciences, Sam Houston State University (Table 1). Theroot tips were fixed and analyzed for chromosome number using standard procedures (Raffauf 1964; Vander Laan & Arends 1985). A voucher specimen of the greenhouse plant was made and is preserved in theWarner Herbarium (SHST).resultsThe format used for reporting chromosome numbers in this article follows that established by Strother andNesom (1997). A mitotic chromosome number of 2n = 20 was recorded for Thevetia ahouai. Van der Laanand Arends (1985) reported chromosome lengths in the Apocynaceae to be between 0.5–4.0 μm, with theaverage chromosome length between 1–2 μm. The length of the chromosomes in T. ahouai varied between1–3 μm, consistent with most other chromosomes in the Apocynaceae. The base chromosome number (x =10) for Thevetia ahouai is consistent with previous reports of x = 10 for Thevetia (T. peruviana 2n = 20; Gadella1977; Ugborogho 1983; Van der Laan & Arends 1985; Santhosh & Omanakumari 1997).discussionVan der Laan and Arends (1985) postulated a base chromosome number of x = 11 for the Apocynaceae s.str. based on its prevalence in the family and on the observation that many of the plesiomorphic taxa possessa base number of x = 11. A base chromosome number of x = 10 is found in four genera representingthree of the 11 tribes recognized in the Rauvolfioideae (Endress et al. 2007): Hunterieae (Gonioma E. Mey.),Plumerieae (Cerbera L., Thevetia), and Vinceae (Ochrosia Juss.). Based on tribal circumscription (Endresset al. 2007) along with molecular evidence (Simóes et al. 2007), x = 10 is reconstructed as having evolvedindependently at least three times in the Rauvolfioideae.A cursory review of chromosome counts for the Apocynaceae (Van der Laan & Arends 1985; Goldblatt& Johnson 2003) reveals that at present the only chromosome counts for genera in the Plumerieae, and thusrelatives to Thevetia (2n = 20), are for Allamanda L. (n = 9; 2n = 18), Cerbera (2n = 40), Himatanthus Willd. exSchult. (2n = 18), Mortoniella Woodson (2n = 32), and Plumeria L. (2n = 36). At present there are no chromosome

counts for the remaining four genera in the Plumerieae: Anechites Griseb., Cameraria L., CerberiopsisVieill. ex Pancher & Sébert, and Skytanthus Meyen., and it is suggested here that effort be made to obtaincount of these taxa. Figures 1 and 2 present cladograms of the Plumerieae constructed from morphological(Alvarado-Cardenas & Ochoterena 2007) and molecular evidence (Simóes et al. 2007), respectively. Diploidcounts for the respective genera included in the phylogenies are presented for both Figures 1 and 2.Alvarado-Cardenas and Ochoterena (2007) presented six synapomorphies that described the Thevetiasensu K. Schum. clade (Fig. 1, branch a). The diploid count of 2n = 20 presented here adds a seventh synapomorphy.When interpreting their data Alvarado-Cardenas and Ochoterena (2007) state that “(t)here isstill no consensus regarding the question of whether one should recognize one genus with two (sections)or two distinct genera (Lippold 1980), given that Cascabela and Thevetia are sister taxa.” We would arguethat the consensus in evolutionary systematics is to assign generic boundaries that reflect both monophylyand shared ancestry. The decision to divide a well supported clade into two separate genera may supportmonophyly; however, it excludes shared ancestry. Without prior knowledge, most botanists would be unawarethat Thevetia and Cascabela are sister taxa that share seven synapomorphies. Instead, an evolutionarilymore meaningful interpretation of the clade would be that Thevetia sensu K. Schum. is monophyletic andsupport is provided for the recognition of two sections as defined by Schumann (1895). In order to maintainsystematic consistency as pertains to current trends in phylogenetic nomenclature we recognize Thevetiasensu K. Schum. and regard Cascabela and all taxa pertaining to the genus as synonyms of Thevetia.492 Journal of the Botanical Research Institute of Texas 2(1)Table 1. Voucher specimen for the chromosome number of Thevetia ahouai.Taxon Voucher specimen Chromosomenumber (2n)Thevetia ahouai TEXAS: Sam Houston State University 20Department of Biological SciencesGreenhouse specimen, 22 Jan 2008Williams 2008-1 (SHST).Fig. 2. Molecularly constructed dendogram of “Plumerieae” clade (from Simóes 2007).acknowledgmentsWe thank Tami Cook for providing us with access to her digital light microscope, and Bob Rhodes formixing the Carnoy’s solution and aceto-orcein. Sibyl Buceli and an anonymous reviewer provided valuableeditorial comments.referencesAllorge, L. 1998. Les Thevetia, compagnons des succulentes. Succulentes 21(1):23–32.Alvarado-Cardenas, L.O. 2004. Apocynaceae. Flora del Valle de Tehuacan-Cuicatlan 38:1–57.Alvarado-Cardenas, L.O. and H. Och oterena. 2007. A phylogenetic analysis of the Cascabela-Thevetia species complex(Plumerieae, Apocynaceae) based on morphology. Ann. Missouri Bot. Gard. 94:298–323. 2007.Endress , M.E. and P. Bruyns. 2000. A revised classification of the Apocynaceae s.l. Bot. Rev. 66:1–56.Endress , M.E., S. Liede-Sch umann, and U. Meve. 2007. Advances in Apocynaceae: The enlightenment, an introduction.Ann. Missouri Bot. Gard. 94:259–267.Gadella, T.W.J. 1977. IOPB chromosome number reports LVI. Taxon 26:257–274.

Gensel, W.H. 1969. A revision of the genus Thevetia (Apocynaceae). Masters Thesis University of Connecticut.Goldblatt, P. and D.E. Johnson. 2003. Index to plant chromosome numbers 1998–2000. Monogr. Syst. Bot. MissouriBot. Gard. 94.Lipp old, H. 1980. Die Gattungen Thevetia L., Cerbera L. und Cascabela Raf. (Apocynaceae). Feddes Repert.91:45–55.Raff auf, R.F. 1964. Some chemotaxonomic considerations in the Apocynaceae. Lloydia 27:288–298.Santhosh , B. and N. Omanakumari. 1997. Karyomorphological studies on two varieties of Thevetia peruviana. J.Cytol. Gene. 32:95–98.Williams and Stutzman, Chromosome number of Thevetia ahouai 493Sch umann, K. 1895. Apocynaceae. In: A. Engler and K.A. Prantl, Die Natürlichen Pflanzenfamilien. Wilhelm Engelmann,Leipzig. 4(2):109–189.Si móes, A.O., T. Livsh ultz, E. Conti, and M.E. Endress . 2007. Phylogeny and systematic of the Rauvolfioideae (Apocynaceae)based on molecular and morphological evidence. Ann. Missouri Bot. Gard. 94:268–297.Strother, J.L. and G.L. Nesom. 1997 Conventions for reporting plant chromosome numbers. Sida 17:829–831.Ugborogho, R.E. 1983. IOPB chromosome number reports LXXIX. Taxon 32:321.Van der Laan, F.N. and J.C. Arends. 1985. Cytotaxonomy of the Apocynaceae. Genetica 68:3–35.Williams , J.K. 1996. A new combination in Thevetia (Apocynaceae). Sida 17:185–190.Williams , J.K. 2007. Documented chromosome numbers 2007: Chromosome number of Laubertia contorta(Apocynaceae: Apocynoideae) and its phylogenetic importance. J. Bot. Res. Inst. Texas 1:431–435. 2007.

Biological Journal of the Linnean Society (1998), 63: 553–577. With 6 figures

The timing of insect/plant diversification:might Tetraopes (Coleoptera: Cerambycidae) andAsclepias (Asclepiadaceae) have co-evolved?B. D. FARRELL*Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138, U.S.A.C. MITTERDepartment of Entomology, University of Maryland, College Park, MD 20742, U.S.A.Received February 1997; accepted for publication 24 October 1997

Ehrlich and Raven’s essay on coevolution has stimulated voluminous work on the mechanismsof insect/plant interaction, but few explicit tests of their model’s prediction that theevolutionary success of entire insect and plant clades is governed by their putative reciprocaladaptations. This paper begins an inquiry into possible coevolutionary diversification forNorth American milkweeds of the genus Asclepias and one of their few major herbivores, thelonghorn beetle genus Tetraopes, focusing first on the historical duration and continuity of theinteraction. A phylogeny for Tetraopes and relatives, estimated from morphology and allozymes,shows evident similarity to a morphology based hostplant cladogram synthesized from theliterature, though the significance of the correspondence under heuristic statistical testsdepends on the treatment of one beetle species reported (without certainty) from multiple hostspecies. Fossils and biogeography support the interpretation that cladogram correspondencereflects synchronous diversification of these two clades, hence opportunity for coevolution,rather than beetle ‘host-tracking’ of previously-diversified plants. Cladogram correspondenceis more evident at higher than at lower levels, as expected under Ehrlich and Raven’s model.An apparent phylogenetic progression in the potency and location of milkweed cardenolides,seemingly related to species diversity of both Asclepias and Tetraopes subclades, provides furthersuggestive evidence for that model. The phylogeography of the Tetraopes/Asclepias assemblagesuggests that extant species evolved largely in their current, often quite localized biomes,facilitating potential experimental tests for hypotheses of adaptation and counteradaptationand their importance to diversification.Ó 1998 The Linnean Society of LondonADDITIONAL KEY WORDS—coevolution – herbivores – plant–animal interactions –phylogeny – cardenolides – escalation.CONTENTSIntroduction . . . . . . . . . . . . . . . . . . . . . . . 554*Correspondence to: B. D. Farrell. Email:bfarrell@oeb.harvard.edu.5530024–4066/98/040553+25 $25.00/0/bj970207 Ó 1998 The Linnean Society of LondonB. D. FARRELL 554 AND C. MITTERNatural history of Tetraopes and allies . . . . . . . . . . . . . 555Relationships among hostplants . . . . . . . . . . . . . . . 559Material and methods . . . . . . . . . . . . . . . . . . . 560Morphological analysis . . . . . . . . . . . . . . . . . . 560Allozyme electrophoresis . . . . . . . . . . . . . . . . . 561Phylogenetic analyses . . . . . . . . . . . . . . . . . . 563Comparisons of phylogeny estimates . . . . . . . . . . . . . 563Results . . . . . . . . . . . . . . . . . . . . . . . . 564Allozyme data . . . . . . . . . . . . . . . . . . . . 564Phylogenetic analyses . . . . . . . . . . . . . . . . . . 564Concordance of insect and plant cladograms . . . . . . . . . . 566Discussion . . . . . . . . . . . . . . . . . . . . . . . 567Ages of associated beetles and hosts . . . . . . . . . . . . . 568Coevolution? . . . . . . . . . . . . . . . . . . . . . 569Concluding observations . . . . . . . . . . . . . . . . . 572Acknowledgements . . . . . . . . . . . . . . . . . . . . 573References . . . . . . . . . . . . . . . . . . . . . . . 573Appendix . . . . . . . . . . . . . . . . . . . . . . . . 577INTRODUCTION

Among explanations offered for the great diversity of phytophagous insects andtheir hostplants, none has been more influential than Ehrlich & Raven’s (1964)theory of coevolution. For three decades this proposal has stimulated work on thephysiological, genetic and ecological mechanisms of insect/plant interactions, focused

especially on the role of plant secondary chemistry (Futuyma & Keese, 1992). Onlyrecently, however, has there begun comparably rigorous investigation of the model’smacroevolutionary implication, namely that these interactions have promoted diversificationof associated insect and plant groups.This study opens an inquiry into the possibility of coevolutionary diversificationfor the North American longhorn beetle genus Tetraopes and its primary hosts, themilkweed genus Asclepias. We first present a phylogenetic analysis of Tetraopes, basedon morphology and allozymes. In conjunction with published information on thephylogeny, biogeography and secondary chemistry of Asclepias, we then use theseresults to examine several questions raised by Ehrlich & Raven’s theory.The main focus of this paper is on how long and continuously these beetle andplant species, and their ancestors, have interacted: what has been the opportunityfor coevolution? One expectation, if insect and plant lineages have diversified inassociation, is that the phylogenetic order of divergence among extant host taxashould correspond in some way to that among their associated herbivores. Forexample, if the insects never switched hosts, the association could be continuousover time, yielding an essentially perfect match of speciation sequences. In contrast,the ‘escape and radiation’ process envisioned by Ehrlich and Raven should producean only imperfect correlation of speciation sequences, but a closer match of insectphylogeny to the evolutionary succession of plant defences.The few explicit studies so far suggest great heterogeneity in the correspondencebetween insect and hostplant phylogenies. In a review of 14 assemblages for whichat least partial cladogram comparison can be made (Mitter & Farrell, 1991; Farrell,Mitter & Futuyma, 1992), the mean fraction of groupings on which insects andhostplants agreed was about 50%, suggesting that some degree of parallel diversificationmight be common. However, in only about a quarter of the comparisonsTIMING OF INSECT/PLANT DIVERSIFICATION 555

was the match significant or nearly so under heuristic statistical tests, and in aboutthe same number there was no correspondence at all.A biological pattern to this variation was suggested by a study of Phyllobrotica leafbeetles, which show the most detailed match to host phylogeny of any herbivorousinsect group yet reported (Farrell & Mitter, 1990). These beetles exhibit unusuallyintimate dependence on their hosts, mostly herbaceous mints in the genus Scutellaria.Each beetle species is restricted to a single host species; the larvae are internalfeeders, in the roots, and the adults both feed and mate on the larval host.The adults, moreover, appear aposematically coloured, suggesting that like otherherbivores of plants that contain iridoid glycosides (Bowers, 1988), they might relyon host-derived toxins for protection from their predators. All these traits have beenargued to enforce long-term host fidelity (Feeny, 1987), and may make paralleldiversification especially likely.Tetraopes and its hosts provide a test of this conjecture, because the life history ofthese beetles is strikingly similar to that of Phyllobrotica. We will present evidencethat phylogenetic relationships within Tetraopes are also strongly concordant withthose of their hosts. Phylogeny concordance by itself, however, is not sufficient todemonstrate parallel phylogenesis: the entire insect clade could instead be youngerthan any of the host species, but have undergone colonization and speciation in asequence dictated by features that mirror host phylogeny. For example, derivedplant species might often bear unusual secondary chemistries, causing them to becolonized later than their less distinctive, more primitive relatives.We will present initial evidence consistent with such ‘escape and radiation’:Asclepias shows a phylogenetic progression in the potency and location of cardenolides,suggesting escalation of defence, which appears related to species diversity of bothmilkweeds and beetles.Natural history of Tetraopes and alliesTetraopes and its apparent nearest relatives, the genus Phaea, belong to themonophyletic tribe Tetraopini, subfamily Lamiinae, of the Cerambycidae. The tribes

Tetraopini, Hemilophini and Phytoecini together form the apparently monophyletic‘Phytoeciides vrais’ of Chapuis (1872), distinguished by bifid tarsal claws, andconstitute nearly 25% of the 50 000 described cerambycid species. Larvae of thesetribes invariably bore in stems or roots of their hostplants, which are typicallyherbaceous, while the adults consume the foliage and flowers. A preliminarymorphological phylogenetic study treating all 40 genera of Tetraopini plus outgroupsconfirmed that Tetraopes and Phaea together form an entirely New World clade,defined by appendiculate tarsal claws. This clade in turn appears most closely relatedto the Philippine genus Eustathes, with which it shares a pronotal umbone. Tetraopesis distributed from Guatemala to Canada, and is most diverse in the Sonoranregion (Chemsak, 1963; Chemsak & Linsley, 1979). Phaea, currently under revision(Chemsak, 1977; J. Chemsak, pers. comm.), appears to contain about 30 speciesdistributed from northern South America (Colombia and Venezuela) to the centralUnited States, and is most diverse in Central America.The following account of life histories in Tetraopes is based largely on the revisionB. D. FARRELL 556 AND C. MITTERTABLE 1. Distributions and host affiliations of Tetraopes species, plus the subset of species in the sistergenus Phaea included in this study. An asterisk follows the name of each species sampled for allozymesTaxa Hosts DistributionPhaea jucunda Ipomoea pandurata Southeastern U.S.P. canescens I. leptophylla Midwestern U.S.P. mirabilis* Thevetia ovata S. MexicoP. maryannae* Stemmadenia palmeri S. Mexico NicaraguaP. biplagiata Stemmadenia palmeri S. Mexico to GuatemalaTetraopes mankinsi Honduras, GuatemalaT. melanurus* Asclepias tuberosa Eastern U.S.T. cleroides Central MexicoT. paracomes Matelea quirosii Guatemala to Costa RicaT. comes S. Mexico to Costa RicaT. elegans Baja CaliforniaT. ineditus Marsdenia lanata Western MexicoT. batesi Western MexicoT. umbonatus* A. glaucescens S. Mexico to NicaraguaT. linsleyi A. linaria AZ, TX, NMT. discoideus* A. subverticillata AZ, TX, NMA. curassavica Mexico to El SalvadorT. annulatus* A. sullivantii (MO) TX, NM, AZ, North to CanadaA. subverticillata (AZ)A. speciosa (UT)T. pilosus* A. arenaria TX, KS, NE, OKT. tetraophthalmus* A. syriaca Northeastern U.S.T. varicornis* A. notha S. MexicoT. texanus* TX, OKT. mandibularis* A. latifolia TX, OKT. quinquemaculatus* A. amplexicaulis Midwestern U.S., to S. OntarioT. subfasciatus Central MexicoT. thoreyi Central Mexico

T. sublaevis* A. erosa S. CaliforniaT. thermophilus SE TX, S to El SalvadorT. basalis* A. eriocarpa CA, S.OregonT. femoratus* A. speciosa Central and western North America

by Chemsak (1963), supplemented by observations from fieldwork by the seniorauthor across North and Central America (Tables 1, 2).Tetraopes larvae attack roots, while the adults, whose emergence coincides withhostplant flowering, feed on upper foliage and flowers. The adult females of Tetraopestetrophthalmus (Forster) oviposit 8–20 eggs inside grass stems within a few centimetersof a milkweed plant, gaining access via a hole chewed for the purpose. About 10days later, the hatchling larvae emerge from the oviposition access, fall to the groundand immediately burrow downward, in presumed search for host roots. Hartman(1977) documented extensive damage to the roots of Asclepias syriaca by larvae ofTetraopes tetrophthalmus, while Chemsak (1963) presented evidence of similar damageto the roots of A. erosa by its herbivore, Tetraopes sublaevis Casey; to A. eriocarpa by T.basalis LeConte; and to A. speciosa by T. femoratus LeConte. Similar damage is causedby other species of Tetraopes. Tetraopes larvae feed both inside and outside of the rootsystem and there seems to be a correlation of adult body size with the size of thelarval resource (Price & Wilson, 1976; Hespenheide, 1973). Larval root feeding isunique to Tetraopes in the subfamily Lamiinae; Phaea and other genera mine stems(Linsley, 1961).With few confirmed exceptions, individual Tetraopes species or their subspecies areTIMING OF INSECT/PLANT DIVERSIFICATION 557TABLE 2. Localities from which population samples of Tetraopes and Phaea species were obtained forprotein electrophoresisTaxa Localities sampledPhaea mirabilis MEXICO: Estado Morelos, Zumpango del RioP. biplagiata MEXICO: Estado Guerrero, IgualaP. maryannae MEXICO: Estado Guerrero, IgualaTetraopes discoideus (1) AZ: Portal MEXICO: (2) Taxco (3) Puebla, AtlixcoT. umbonatus MEXICO: (1) Guerrero, Xochichalca Ruins (2) Oculixtlhuacan (3) Iguala (4)Puebla, AtlixcoT. melanurus MD: Prince Georges Co., Patuxent Wildlife Research CenterT. quinquemaculatus MO: (1) Clay Co., Flemington; (2) Le Petite Grande Prairie; (3) Neiwathe Prairie;(4) KS: Reno Co., HutchinsonT. texanus (1) MO: Clay Co., Flemington (2) TX: Cooke Co., GainesvilleT. annulatus NM: (1) McKinley Co. Mesita [16] (2) Gallup [19] (3) AZ: Apache Co., HouckT. pilosus (1) KS: Reno Co., Sandhill State Park, Hutchinson (2) TX: Ward Co., MonahansT. tetropthalmus MO: Clay Co.: (1) Independence; (2) Flemington; (3) VT: Chittenden Co.:

Colchester; (4) MD: Prince Georges Co. Patuxent Wildlife Research CenterT. mandibularis TX: Dickens Co., DickensT. varicornis MEXICO: Puebla, Rio FrioT. femoratus (1) NV: Reno (2) CA: GraegeleT. basalis CA: Plumas Co.; QuincyT. sublaevis CA: Riverside Co., Blythe

affiliated with single, differing species of milkweeds in the subgenus Asclepias (Asclepias).The following brief account is summarized in Table 1. Tetraopes umbonatus LeConteis affiliated with A. glaucescens throughout southern Mexico (Chemsak, 1963; Farrell,1991). Tetraopes paracomes Chemsak and Tetraopes ineditus Chemsak are associated withMarsdenia and Matelea, respectively, vining milkweeds in the lowland forests of CentralAmerica and Mexico (B. Farrell, unpublished). Tetraopes discoideus LeConte is theonly species confirmed to use two hostplant species (Chemsak, 1963; Farrell, 1991).It ranges from southern Mexico, where it feeds on A. curassavica (Chemsak, 1963;Farrell, 1991), to the southwestern United States, where it attacks A. subverticillata(Chemsak, 1963; Farrell, 1991), and is absent from the area north of Mexico Cityto approximately the U.S. border. Both of these hostplant species are in Woodson’s(1954) series Incarnatae and their cardenolide profiles are similar (Roeske et al.,1976). Tetraopes melanurus Schonherr attacks A. tuberosa throughout the eastern UnitedStates (Chemsak, 1963; Farrell, 1991). Tetraopes tetrophthalmus feeds on Asclepias syriacathroughout its range (Farrell, 1991; Hartman, 1977), though an isolated populationin a disturbed site in Illinois was reported on A. verticillata, where the adults maysuffer reduced fitness (Price & Willson, 1976). Tetraopes femoratus is affiliated with A.speciosa throughout the western United States (Chemsak, 1963; Farrell, 1991), buthas been reported in very low numbers from A. syriaca at the eastern edge of itsrange (Lawrence, 1982). Tetraopes pilosus Chemsak and its host, A. arenaria, arerestricted to the Quaternary sandhills of the midwestern U.S. (Farrell, 1991). Bothbeetle and host are clothed in white pubescence, possible adaptations against moistureloss and overheating in their xeric dune habitats. Tetraopes mandibularis Chemsak is

affiliated with Asclepias latifolia in the Llano Estacado region of northwestern Texasand adjacent Oklahoma (Rice, Turnbow & Hovore, 1985; Farrell, 1991). Tetraopesvaricornis Castelnau uses A. notha in southern Mexico (Farrell, 1991). Tetraopes sublaevisand its sole host A. erosa are confined to the lower Colorado Desert (Chemsak, 1963;Farrell, 1991).B. D. FARRELL 558 AND C. MITTER

Two additional species of Tetraopes have been reported only from single hostAsclepias species, and we have no direct observations that contradict these associationsthough we cannot confirm them first hand. Tetraopes quinquemaculatus Haldeman wasreported to attack A. amplexicaulis in the midwestern U.S. (Price & Wilson, 1979)and we have collected several specimens of this species in the vicinity of A. amplexicaulisbut have not observed adult or larval feeding. Tetraopes linsleyi Chemsak reportedlyuses A. linaria, the only milkweed to occur in its dry Chiricahuan canyon habitats(Hovore, 1983; pers. comm.). We provisionally accept these associations pendingconfirmation. In contrast, while Tetraopes elegans Horn was inferred to use A. subulataby Chemsak (1963) on the grounds that this is one of very few milkweeds that occursthroughout its range in Baja California, the distribution of Asclepias albicans is alsovery similar (Woodson, 1954) to that of T. elegans and two other Asclepias species,very closely-related to A. albicans, also occur in Baja. Therefore, we regard the hostof T. elegans as unknown.The hosts of T. annulatus LeConte are uncertain, but this may be among the veryfew Tetraopes species to use more than one host. While adults of this species havebeen found on A. subverticillata in Arizona, they have also been collected from A.speciosa in Utah, and from the closely-related A. sullivanti in Missouri (M. Rice, pers.comm.). The remarkable range in body size of this species (Chemsak, 1963; Farrell,1991) also suggests larval feeding on different hostplants, although this remainsunconfirmed.

Like other herbivore groups popular with amateur collectors, Tetraopes are sometimesrecorded from plant species other than those supporting growth and reproduction.For example, while Tetraopes tetrophthalmus adults most commonly feedon and oviposit near Asclepias syriaca, we have also seen this beetle feeding on flowersof A. incarnata (swamp milkweed), which often occurs in the wet margins of fieldsoccupied by A. syriaca and flowers slightly later. However, we have never observedoviposition near A. incarnata, and it is very unlikely that the beetle larvae couldsurvive the combination of very wet soil and the very shallow and fine root systemoffered by this milkweed species (Hartman, 1977). We have also occasionally observedadults of several other species on non-host milkweeds, at the edge of the beetles’range, and after peak adult emergence. Thus, at least some accidental records maybe attributable to post-reproductive dispersal of adults, which sometimes undertakelong-distance flights (Davis, 1980a,b, 1984; D. McCauley, pers. comm.).With one exception, adult oviposition and larval habits of Phaea species have beenheretofore undescribed. The genus appears to consist of two morphological subgroupswhich are affiliated respectively with Convolvulaceae and Apocynaceae, membersof related orders in the subclass Asteridae (Olmstead et al., 1992) which share byconvergence the presence of latex canals. Phaea jucunda has long been known to borein the stems of the convolvulaceous vine Ipomoea pandurata (Craighead, 1923; Riceet al., 1985), and other morphologically similar species also attack Ipomoea species.For example, P. canescens, the only other North American species of Phaea, attacksthe shrubby Ipomoea leptophylla (M. Rice, pers. comm.), while the Central AmericanP. acromela is affiliated with an as yet unidentified species of arborescent Ipomoea (F.Hovore, pers. comm.). The remaining Central American species in this subgroupseem likewise affiliated with woody Ipomoea species ( J. Chemsak, F. Hovore, E.Giesbert, pers. comm.). In southern Mexico, at least some of the species in the other

Phaea subgroup oviposit in stems of arborescent Apocynaceae. For example, Phaeamaryannae and P. biplagiata both attack Stemmadenia palmeri, while P. mirabilis ovipositsTIMING OF INSECT/PLANT DIVERSIFICATION 559

in Thevetia ovata (Farrell, 1991). Verbal descriptions of the hosts of other Phaea speciesindicate that these are also latex-bearing trees with tubular flowers, strongly suggestingeither Apocynaceae or Convolvulaceae, but these await identification.Adults of species of Phaea and Tetraopes bear apparently aposematic, bright orangeor red markings of varying extent. Aposematism is unusually widespread amongherbivores of the Apocynaceae and Asclepiadaceae, and many of these specializedinsects, including Tetraopes, sequester toxins from their hostplants for defense againstpredators (Brower & Brower,1964; Scudder & Duffy, 1972; Rothschild, 1973; Isman,Duffy & Scudder, 1977; Marsh et al., 1977; Nishio, Blum & Takahashi, 1983; Broweret al., 1984a,b; Berenbaum & Miliczsky, 1984; Ackery & Vane-Wright, 1984).Variation in adult coloration and flight among Tetraopes species may be correlatedwith host chemistry. The more primitive beetle species, affiliated with apparentlyless toxic hostplants (see below), have much less surface area brightly-colored(Chemsak, 1963), are smaller in body size on average, and are also more difficultto capture.Relationships among hostplantsThe following review is summarized in Figure 1. The two main hostplant familiesof Tetraopes and relatives, Asclepiadaceae and Apocynaceae, form a group sometimestermed the Apocynales. Its monophyly is supported by, among other derivedcharacters, the possession of latex canals and cardiac glycosides (Cronquist, 1981).A recent molecular phylogenetic study (Sennblad & Bremer, 1996) has shown theApocynaceae to be paraphyletic. Of the two apocynaceous hosts of Phaea, Thevetiaand Stemmadenia, Thevetia is closest to the monophyletic Asclepiadaceae, while thegroup that includes Tabernaemontana, the closest relative of Stemmadenia (not includedin the study), is more basal (Sennblad & Bremer, 1996). Among the three hostgenera

of Tetraopes, all in Asclepiadaceae, Marsdenia is closer to Asclepias than Matelea(Woodson, 1941).Asclepias is a North American genus of about 120 species, most recently revisedby Woodson (1954). Most of the known Tetraopes hosts are in the nominate subgenus,the largest of nine subgenera recognized by Woodson. Woodson grouped the 72species of this subgenus into eight ‘series’, among which he postulated the phylogeneticrelationships shown in Figure 1.Striking, independent support for Woodson’s arrangement comes from subsequentstudies of the distribution of cardenolide types among milkweed species which arehost to the monarch butterfly. These have included 20 species representing all butone of Woodson’s series (Nelson, Seiber & Brower, 1981). Series 5–8 are unique inproducing cardenolides of the labriniformin type, whereas series 1–4 producecardenolides of only the calotropogenin type, which are widespread across Asclepiadaceaeand presumably primitive (Brower et al., 1984a,b). Within the chemicallyadvanced group of series, the levels, locations and identities of characteristic cardenolidescan be further arranged into transformation series consistent with Woodson’sarrangement (Fig. 1). As detailed in the Discussion, these features, in additionto supporting Woodson’s phylogeny, also suggest a sequence of increasingly effectivedefenses. In summary, while Woodson’s phylogeny needs re-examination, it providesa credible first estimate to which the independent estimate for Tetraopes can becompared.B. D. FARRELL 560 AND C. MITTERFigure 1. Taxonomic arrangement of hostplants used by Tetraopes and Phaea following Woodson (1954,1941) within Asclepias; and Sennblad & Bremer (1996) and Leeuwenberg (1994) outside of Asclepias.MATERIAL AND METHODS

Morphological analysisAdult morphological characters were scored for all species of Tetraopes; sevenspecies of Phaea, including all those with known hostplants; and the Philippine genusEustathes, nearest relative of Tetraopes+Phaea. Morphological features were examinedunder a Wild dissecting microscope. Dissections were performed with sharpened

insect pins and stored in alcohol for further examination. Soft tissues were clearedfrom genitalic dissections in 10% KOH. The 26 features showing essentially fixedTIMING OF INSECT/PLANT DIVERSIFICATION 561TABLE 3. Morphological characters in Tetraopes and Phaea scored for phylogenetic analyses. Numbersfollowing descriptions are ordered character state codingsHEAD1. First antennomere longer than scape 0; shorter than scape 12. Mandible sexually monomorphic 0; dimorphic 13. Labial palpi long, narrow 0; subovoid 1; ovoid 24. Antennal annulation absent 0; present 15. Scape lateroapical carinae absent 0; present 1THORAX6. Pronotal umbone absent 0; present, weakly developed 1; strongly developed 2; strongly developed with lateralridge 37. Umbonal lateral sulci absent 0; present at frontal 1/2 1; sulci along length of umbone 2; sulci continuousaround umbone 38. Lateral macula absent 0; present 19. Umbonal maculae absent 0; present 110. Procoxal cavities widely separated by prosternum 0; cavities contiguous or nearly so 111. Lateral umbone absent 0; present, weakly developed 1; strongly developed 2ELYTRA12. Elytral disc coarsely punctate 0; smooth 113. Discal chevron absent 0; present 114. Apical chevron absent 0; present 115. Humeral maculae absent 0; present 116. Discal maculae absent 0; present 117. Apical maculae absent 0; present 118. Elytral form mesally constricted, subparallel, slender 0; stout, parallel 119. Lateral macula absent 0; present 1APPENDAGES20. Claws appendiculate 0; bifid 121. Male metatrochanter spur absent 0; present 1ABDOMEN22. Female sternal sulcus present 0; absent 123. Aedeagus dorsal piece subtending ventral 0; overlapping ventral 124. Aedeagal apex lanceolate 0; explanate 125. Aedeagus with dorsal piece lateral explanation absent 0; present 1OVERALL HABITUS26. Exoskeleton with scattered, sparse golden pubescence 0; with dense, white pubescence 1

differences among species were coded as discrete characters for phylogenetic analysis(Tables 3,4).Allozyme electrophoresisFresh material of three species of Phaea and 13 species of Tetraopes, including allthose with confirmed host affiliations, was fresh-frozen in liquid nitrogen in the field,and stored at -85°C. Whenever possible, electrophoretic analyses for each speciesincluded samples from several geographic regions (Table 2).Starch gel electrophoresis was performed using methods modified from Hillis &Moritz (1990). Frozen individual beetles were homogenized in 7ml of ice-coldhomogenization buffer (200mM Tris-HCL, pH 8.0, 26mM sodium metabisulfite,10mM MgCl2 1.5mM EDTA, 5% w/v PVP-40, 0.05% 2-mercaptoethanol; modifiedfrom Futuyma & McCafferty, 1990), and spun for 5 minutes in a refrigerated

microfuge. Half the supernatant was frozen for subsequent runs. The rest was loadedB. D. FARRELL 562 AND C. MITTERTABLE 4. Matrix of morphological and allozyme characters states for all species of Tetraopes plus a subset of Phaea species, following their respective orderings inTable 3 and the AppendixP. canescens 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?P. jucunda 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?P. biplagiata 0 0 0 0 1 2 2 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3 7 1 ? 3 ? 2 3 5 5 2 2 5 2 3 2 ? 2 4 2 2 ? 6P. maryannae 0 0 0 0 1 2 2 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 4 2 1 5 2 3 2 4 6 5 3 5 7 3 1 3 2 6 6 2 2 ? 4P. mirabilis 0 0 0 0 1 2 2 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 7 2 6 1 ? 2 6 6 5 2 7 2 3 2 1 2 1 3 2 2 8 5T. mankinsi 0 0 0 0 0 2 2 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. paracomes 0 0 0 0 0 3 2 0 0 1 0 0 1 0 1 1 0 1 1 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. cleroides 0 0 0 0 0 3 2 0 0 0 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. comes 0 0 0 0 0 3 2 0 0 1 0 0 1 0 1 1 0 1 1 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. ineditus 0 0 0 0 0 3 2 0 0 1 0 0 1 0 1 1 0 1 1 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. elegans 1 0 1 0 0 3 2 0 0 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. batesi 1 ? 0 0 0 3 2 0 ? 0 0 0 1 1 0 0 1 1 1 0 1 1 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. discoideus 0 0 0 0 0 2 2 0 1 0 1 0 1 0 1 0 1 1 0 0 0 1 0 0 0 0 4 2 1 7 1 3 3 3 6 1 1 4 8 2 3 8 9 5 4 1 1 7 6T. umbonatus 0 1 1 0 0 2 2 0 1 0 1 0 1 1 0 0 1 1 0 0 0 1 0 0 0 0 4 2 1 7 1 4 1 2 3 1 1 4 8 2 3 ? 5 5 1 1 1 7 6T. melanurus 0 1 2 0 0 2 2 0 1 0 1 0 1 1 0 0 1 1 0 1 0 1 1 1 1 0 3 4 1 1 1 1 1 1 1 2 1 4 7 4 3 ? ? 5 1 1 1 ? 1T. texanus 1 1 1 0 0 1 1 0 1 0 2 0 0 0 1 1 1 1 0 1 1 1 0 0 0 0 3 4 1 1 1 2 3 5 4 2 1 ? 6 4 3 3 ? 6 1 1 1 5 1T. linsleyi 1 0 2 0 0 2 2 0 1 1 0 0 0 1 1 0 1 1 1 0 1 ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. thermophilus 1 1 2 1 0 2 2 0 1 1 0 1 0 0 1 ? 1 1 1 0 1 1 1 ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. quinquemaculatus 1 1 2 0 0 2 2 0 1 1 1 0 0 0 1 0 1 1 0 1 0 1 1 1 1 0 3 4 1 3 1 2 3 5 4 2 1 6 8 4 2 2 ? 7 1 1 1 5 2T. annulatus 1 1 1 1 0 1 1 0 1 1 2 1 0 0 1 1 1 1 0 1 0 1 1 1 0 1 3 ? 1 1 1 1 1 1 1 2 1 4 4 4 4 2 4 7 1 1 1 5 1T. pilosus 1 1 1 1 0 1 1 0 1 0 2 1 0 0 1 1 1 1 0 1 0 1 1 1 0 1 5 4 1 1 1 1 1 1 1 2 1 4 4 4 4 7 4 7 1 1 1 5 1T. tetropthalmus 1 1 1 1 0 2 2 1 1 1 2 1 0 0 1 1 1 1 1 1 0 1 1 1 0 0 3 3 1 ? 1 2 1 5 4 2 1 6 3 4 3 7 5 4 1 1 1 4 1T. mandibularis 1 1 1 1 0 1 2 1 1 1 2 1 0 0 1 1 1 1 1 1 0 1 1 1 1 0 3 3 1 1 1 1 1 5 4 3 1 6 3 4 3 7 5 4 1 1 1 4 1T. thoreyi 1 1 1 1 0 3 2 1 1 1 2 1 0 0 1 1 1 1 1 1 1 1 1 1 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. subfaciatus 1 1 2 1 0 3 2 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?T. varicornis 1 1 1 1 0 3 2 1 1 1 2 1 0 0 1 1 1 1 1 1 1 1 1 1 0 0 5 3 1 4 1 1 3 5 4 2 1 6 3 4 2 7 5 7 5 1 1 6 3T. femoratus 1 1 2 1 0 2 2 1 1 1 2 1 0 0 1 1 1 1 0 1 1 1 1 1 0 0 5 3 1 ? 1 1 3 5 4 2 1 6 3 4 2 5 5 7 5 1 1 6 1T. basalis 1 1 1 1 0 2 2 1 1 1 2 1 0 0 1 0 1 1 0 1 1 1 1 1 0 0 2 3 1 1 1 2 3 5 4 2 1 6 3 4 2 5 7 7 3 1 1 6 1T. sublaevis 1 1 1 1 0 2 2 1 1 1 2 1 0 0 1 1 1 1 0 1 1 1 1 1 0 0 5 3 1 4 1 1 3 5 4 2 1 6 3 4 2 5 7 7 3 1 1 6 1TIMING OF INSECT/PLANT DIVERSIFICATION 563

into cold gels (11% starch from Connaught Laboratories) using 5mm square wicksof Whatman #2 filter paper. All enzymes were run for 14–15 hours on small (200 mltotal volume ) or large (450 ml total volume) gels, in one of seven buffer systemsdetermined by an initial survey to be optimal for allele separation and resolution(Appendix). Trays of ice were placed on top of the gels, which were run in arefrigerator at 0–4°C. Gels were sliced two or three times and stained followingrecipes in Hillis & Moritz (1990). Electromorph differences and identities wereconfirmed through multiple, side-by-side comparisons.Phylogenetic analysesMorphological characters with more than two states (5 of 26 characters) werecoded to reflect transformation series inferred from morphological similarity (Table3). For the allozyme data, each locus was treated as a character, with alleles as theunordered states (Table 4). Polymorphism was treated as ambiguous.

Morphological and allelic data were first analysed separately for the subset of 16taxa on which both were obtained, which includes nearly all species with knownhosts. As these analyses yielded largely concordant groupings, a third phylogenyestimate was obtained for these species by combining the two data sets. This treewas then compared to that for all 29 species based on both data sets, as a test forthe possible effects of missing taxa on the estimate for the reduced set. Finally, atree was calculated for all taxa with known hosts, using both character sets, andcompared to the host phylogeny.Most parsimonious trees were generated in each analysis using the heuristic searchroutines in PAUP 3.1.1 (Swofford, 1993), with 100 random addition sequences andsuccessive weighting using mean retention indices. Decay analyses used AutoDecay1.1 (courtesy of N. Erikkson), with 10 random addition sequences for each reverseconstraint tree analysis. Similarity clustering was also performed on the allozymeallele frequencies, using the Manhattan, Nei’s and Roger’s distance coefficients andthe UPGMA routine in NTSYS-pc (Rohlf, 1990).Phaea mirabilis was deleted from the data matrix for calculations based on distancesbecause it showed 100% divergence from the most derived Tetraopes species, renderingNei’s distance infinite.Comparisons of phylogeny estimatesThe problem of measuring correspondence between host and parasite phylogenyestimates has been recently reviewed by Page (1995). Our earlier work (Farrell &Mitter, 1990) applied quantitative methods developed for similar problems inbiogeography (Page, 1990) and in evaluation of cladograms generated by differentdata sets for the same taxa (Shao & Sokal, 1986). Recent conceptual advancespermit more realistic evaluation of phylogeny correspondence when host-parasiteassociations may result from several underlying processes (Page, 1995). We thereforeapplied cospeciation analysis as implemented in the TreeMap program package of

Page (1995). In this approach, one maps the number of cospeciation events thatexplain the distribution of the observed insect species across hostplant species entirelythrough parallel phylogenesis, minimizing the total number of host-shifts, sortingB. D. FARRELL 564 AND C. MITTER

and duplication events. Statistical significance is evaluated by comparing the observedcospeciation count to the distribution of values for randomly generated parasiteand/or host phylogenies (Page, 1995). TreeMap permits generation of trees undereither Markov or proportional-to-distinguishable models (Page, 1995). We selectedthe more conservative Markov model of tree generation, which consistently producedhigher (i.e. of lower significance) P values. We also chose to randomize the beetlephylogeny estimate as this contains fewer taxa for the purposes of this study. Whencomparing trees of unequal size, randomization of the smaller tree will producehigher P values for any given number of cospeciation events, and thus is moreconservative.Using this approach, the one species apparently associated with multiple Asclepiashosts, Tetraopes annulatus, was scored for association with each host separately, andscored for association with all three hosts. The strikingly allopatric Arizonan andMexican populations of Tetraopes discoideus were treated as separate entities, eachassociated with a different host, as these populations are also divergent in morphologyand allozymes (see Results). We did not include the Tetraopes species for which therewere no direct observations of host-use.RESULTS

Allozyme dataTwenty-three apparent loci were resolved (Appendix), all of which showedinterspecific variation . These include all the loci analysed in Tetraopes tetrophthalmusby McCauley & Eanes (1987), except that we did not assay leucine amino peptidaseor hexokinase. Intra-population polymorphism was uncommon, and there were fewinstances where more than one allele was shared with other species. Thus, very

little phylogenetic information was lost in the discrete coding. In no instance ofpolymorphism was there significant departure from Hardy–Weinberg genotypeproportions.Nei’s distance between the populations of T. femoratus, T. annulatus and T. tetropthalmusranged between 0.007 and 0.044, suggesting moderate levels of divergence accompanyinggeographic isolation. For each of these species, mean distances wereused in the phenetic analysis reported below. The small samples of T. discoideus fromsouthern Mexico and Arizona differed more strongly. Chemsak (1963) also notedconsistent (though not invariant) differences in head color between these populations.Phylogenetic analysesFor the 16 species scored for both morphology and allozymes, and for whichhostplants are known, the morphological data alone yield four most parsimonioustrees of which the strict consensus is shown in Figure 2a. The allozyme data aloneTIMING OF INSECT/PLANT DIVERSIFICATION 565Figure 2. (a) Strict consensus tree of the four most parsimonious trees based on morphology alone,for the subset of Tetraopes species for which allozyme data are also available. Morphological charactersdo not resolve relationships among T. femoratus, T. sublaevis, and T. basalis, and are equivocal aboutplacements of T. melanurus and T. quinquemaculatus. Length is 42 steps, rescaled consistency index=0.5.(b) Strict consensus tree of two most parsimonious trees for subset of Tetraopes and Phaea species basedon allozyme data, coded as in Table 4. Length is 90 steps, rescaled consistency index=0.7. Allozymesresolve all relationships but are equivocal about the placements of Phaea biplagiata and P. maryannae,which are almost completely divergent from Tetraopes species. (c) Single most parsimonious tree forTetraopes and Phaea species for which allozyme data are available, based on Table 4 (all data). Lengthis 140 steps, rescaled consistency index=0.6.resulted in two most parsimonious trees, whose strict consensus tree is shown inFigure 2b. UPGMA clustering on allele-frequency distances specifies an almostidentical tree (Fig. 3). The allozyme-based estimate is slightly more resolved withinB. D. FARRELL 566 AND C. MITTERFigure 3. UPGMA phenogram of Nei’s distances for Tetraopes and Phaea species specifies nearlyidentical relationships to cladistic analysis of same data. UPGMA clustering under Manhattan, Rogers’and Prevosti’ distances specifies same tree.

Tetraopes than that based on morphology, but less resolved for the outgroup Phaeaspecies. The two disagree only by minor re-positioning among nearby taxa, namelyT. quinquemaculatus, T. mandibularis, and the pair T. annulatus/T. pilosus. The two datasets analysed in combination yield six most parsimonious trees. Following successiveweighting by the mean retention indices of these, a single most parsimonious treeresults (Fig. 2c) which is completely resolved, with at least one disagreement resolvedin favor of each data set. The relationships among these taxa are little changed inthe most parsimonious trees resulting when the 13 species scored only for morphologyare added to the combined data set (Fig. 4). This tree confirms the monophyly ofTetraopes and suggests that Phaea is paraphyletic, with the Apocynaceae-feeding groupmost closely allied to Tetraopes.Concordance of insect and plant cladogramsThe phylogeny for the beetle species with recorded hosts, extracted from thephylogeny for all species based on the combined data sets (Fig. 4), is compared tothe literature-synthesized estimate of host relationships (Fig. 1) in Figure 6. Thereare several points of disagreement, of which the most striking involve T. pilosus andT. mandibularis, both of which appear to represent colonization of the advanced hostseries Roseae from ancestors affiliated with more primitive milkweeds. Overall,however, the phylogenies appear to match fairly well, an impression supported byheuristic statistical analyses. Under cospeciation analysis as implemented in theTreeMap package of Page (1995), 13 cospeciation events explain the distribution ofbeetle across host species when Tetraopes annulatus is scored for association with eitherTIMING OF INSECT/PLANT DIVERSIFICATION 567Figure 4. One of the six most parsimonious trees for all species of Tetraopes plus a subset of Phaeaspecies, based on all available data. Trees were obtained under heuristic search routine and 100random addition sequences in PAUP (vers. 3.1.1:courtesy of D. Swofford). Length is 173 steps,consistency index=0.62, retention index is 0.79. Character changes were optimized on branches underDELTRAN routine, which favors parallelisms over reversals. The numbers adjacent to internal nodes

are the numbers of unambiguous changes followed by the maximum number of changes for eachgrouping, with the decay index in parentheses.Asclepias sullivantii or A. speciosa (P=0.01). Ten cospeciation events result in theanalysis when Tetraopes annulatus is scored for association with A. subverticillata or allthree plant species (P=0.07). When Tetraopes annulatus is omitted from the analysis,on the grounds that its affiliations are ambiguous, the estimate is 12 cospeciationevents (P=0.038). If Phaea is also removed, confining the analysis to herbivores ofAsclepiadaceae, the estimate is 10 cospeciation events. Ten or more cospeciationevents are significant when the host or both host and beetle phylogeny estimatesare randomized (P<0.05).DISCUSSION

The agreement between morphological and allozyme analyses suggests that theTetraopes phylogeny estimate is reasonably robust, but addition of other molecularB. D. FARRELL 568 AND C. MITTER

character sets, now in progress, is needed to settle several weakly-resolved regionsof the tree. The need for modern re-examination of Asclepias phylogeny, alsounderway, is even more evident, and dictates caution about any conclusion to bedrawn here.Nonetheless, the correspondence between the beetle and plant phylogenies appearsstronger than expected by chance, and seems more likely to increase than decreaseas those independent estimates are improved (i.e. unless error in the current estimatesproduced this pattern, correspondence should become more clear as error decreases).The match is inexact, suggesting that pairs of associated species have typically arisenmost immediately by beetle colonizations from a related host rather than parallelspeciation, but it is consistent with broadly synchronous diversification betweenlineages, providing the opportunity for long-term coevolution.Ages of associated beetles and hostsThe earliest fossils of Apocynaceae are Paleocene (Muller, 1984), with extantgenera appearing in the Eocene. The divergence between Apocynaceae+Asclepiadaceae and Convolvulaceae, which are in different orders, was probably

considerably earlier. The earliest fossils of Asclepiadaceae appear in the Oligoceneand Miocene (Muller, 1984); as these already represent extant genera, includingAsclepias, the family is likely to be older. Fossil datings of subgroups within Asclepiasare not available.Tetraopini are represented by a single, Oligocene fossil, which cannot be furtherplaced (Statz, 1938). Biogeography suggests that the Phaea+Tetraopes clade itself isthis old or older: the disjunction between this New World lineage and its SoutheastAsian sister group Eustathes mirrors the distributions of many pantropical groups forwhich the fossil record suggests Late Eocene to Early Oligocene origins (Tiffney,1985; 35–>45 Mya), before northerly dispersal routes were cut off by Oligocenecooling.Mapping of species distributions on the phylogeny estimate (Fig. 5) suggests atropical lowland origin for Tetraopes, followed by more recent occupation of temperateupland savannah and most recently, Sonoran desert and midwestern Sandhills. Asimilar biogeographic history is suggested for Asclepias by Woodson’s arrangement.Most species in the relatively primitive series 1–4 occupy mesic tropical habitatsand are distributed from Mexico south, while most species in the derived series 5–8occupy grassy temperate savannah habitats. Species within the highly-derived Roseae(series 8) have invaded the youngest, most severe habitats, the Sonoran Desert andcentral Sandhills of the United States.These sequences of habitat occupation parallel the order of appearance of thevarious habitats in the plant fossil record (Axelrod, 1979; Wolfe, 1978, 1985; Tidwell& Nambudiri, 1989), as expected if endemic beetle and milkweed lineages and theirhabitats differentiated synchronously. However, they also parallel the order ofseverity of these habitats with respect to moisture. It is conceivable that both beetleand milkweed colonization would have been constrained to follow a similar sequence,

even if they occurred entirely after differentiation of the habitats, by the need forsuccessive pre-adaptations to successively harsher environments. This alternativeseems less parsimonious to us, but we cannot entirely rule it out. If we provisionallyaccept the time scale implied by our habitat datings, the origin of association withTIMING OF INSECT/PLANT DIVERSIFICATION 569Figure 5. Biogeographic distributions optimized on tree from Figure 4 imply tropical origin followedby colonization of more temperate latitudes, and and at least one secondary invasion of the tropics(by the ancestor of T. thermophilus, T. subfasciatus, T. thoreyi, and T. varicornis).Asclepiadaceae, corresponding to the split between Tetraopes and Phaea, is dated to40–47 Mya; association with subgenus Asclepias at more than 20 Mya (pre-datingdivergence of T. discoideus); and exclusive association with the advanced series (5–8)of Asclepias (T. mandibularis through T. basalis in Figure 6) at >7 Mya. Theseprojections, like the sparse fossil evidence, are at least consistent with the hypothesisthat Tetraopes and its subgroups have diversified in approximate synchrony with theirasclepiadaceous hosts.In contrast, the divergence between the Phaea groups feeding on Convolvulaceaeversus Apocynaceae is probably not as old as that between their more distantlyrelated host groups. Transfers between these families are relatively common, andmay reflect both shared placement in the subclass Asteridae and the convergentpossession of latex canals, which together account for a majority of the insect faunalconnections between Apocynaceae/Asclepiadaceae and other plant families (Farrell& Mitter, 1993).Coevolution?The phylogenetic and temporal evidence adduced above suggests that Tetraopesand Asclepias, particularly subgenus Asclepias, have been associated during much oftheir respective histories, providing abundant opportunity for evolution in responseB. D. FARRELL 570 AND C. MITTERFigure 6. Comparison of most parsimonious tree, based on all data, for Tetraopes and Phaea specieswith known hosts, to hostplant phylogeny of Figure 1. There are 10–13 cospeciation events, dependingon which reported host of T. annulatus is scored. Correspondence is significant under 2/3 scorings

(P<0.01), and if T. annulatus is omitted on the grounds that its host affiliations are ambiguous (P=0.038: see text for details). *T. annulatus is here depicted opposite its host A. sullivantii, though adultsof this species have also been collected from A. subverticillata and A. speciosa (see Introduction).to their interaction. Direct evidence for coevolution has not been sought, but severalsuggestive observations point to directions for future study.Reciprocal adaptation between particular pairs of plant and phytophagous insectsis considered rare (Futuyma & Keese, 1992; Farrell & Mitter, 1993), and has beenreported in just a few cases involving long-standing, highly specific interactions suchas exclusive plant/pollinator associations (Thompson, 1994). However, such pairwisecoevolution has rarely been directly looked for in antagonistic interactions. ForTetraopes and Asclepias, pairwise coevolution should most profitably be sought inpaired endemics of distinctive, extreme habitats, such the affiliation of Tetraopes pilosusand Asclepias arenaria in the Central Sandhills. Such species seem especially likely tohave differentiated together, and the harshness of their environment may bothrestrict the number of other herbivores with which the plant must contend, andplace a premium on effective defense (Coley, Bryant & Chapin, 1987). Tetraopes isclearly capable of impairing milkweed fitness (see earlier references), and its combinationof larval root feeding and adult feeding on reproductive parts may imposeespecially severe selection on hostplants (Brown, 1990).Diffuse coevolution as embodied in Ehrlich & Raven’s model (1964)—evolutionof plant lineages in simultaneous response to suites of herbivore species and viceversa—has been accorded a wider influence on the structure and diversity of insect/plant communities (Futuyma & Keese, 1992). However, there are as yet fewplausible instances, let alone convincing demonstrations, of such coevolution effects.Berenbaum (1983) spelled out the predictions of Ehrlich & Raven’s (1964) model,and built a persuasive case for stepwise elaboration of coumarin compounds, inApiaceae and other plant families, in response to counter-adapting herbivores.

However, the phylogenies needed to secure this inference are not yet availableTIMING OF INSECT/PLANT DIVERSIFICATION 571

(Miller, 1987). Coevolutionary ‘escape’ may also explain the supplementation orreplacement of glucosinolates by other, very different secondary compounds in somelineages of crucifers (Feeny, 1977). Perhaps the strongest case for Ehrlich & Raven’sscenario is provided by secretory canals containing latex or resin. These structuresare effective defenses against a spectrum of enemies, and no other hypothesizedfunction for them seems plausible (Dussourd & Eisner, 1987). The multiple lineagesin which they have independently arisen show consistently elevated diversificationrates (Farrell, Dussourd & Mitter, 1991). They have in turn evoked characteristiccounter-adaptations by some insect herbivores, some of which circumvent the canalsby severing them before feeding (Dussourd, 1993).The subgenus Asclepias presents a prima facie case for coevolution sensu Ehrlichand Raven with enemies including Tetraopes, that parallels on a smaller scale theexamples just cited. There is, first, a phylogenetic progression in the types and tissuedistribution of cardenolides, alluded to earlier (Fig. 1), which can be plausibly readas a stepwise escalation of defense (Nelson et al., 1981). Cardenolides in the moreprimitive of Woodson’s series (1–4), like those in other subgenera and genera ofasclepiads, are of the simpler, presumably primitive calotropogenin type. Series 5–8are unique in producing cardenolides of the structurally complex labriniforminfamily. These compounds are among the most emetic and toxic cardenolides known(Detweiler, 1967; Brower et al., 1982, 1984a,b). In series 5 (Syriacae, host to T.tetropthalmus and T. linsleyi ), the labriformin-type cardenolides are present in onlytrace amounts; they increase in successive series to a maximum in series 8 (Roseae,host to T. sublaevis, T. pilosus, T. basalis and T. mandibularis). In Roseae, moreover,cardenolides are confined principally to the latex, where their deterrent effects on

herbivores should be maximal (Nelson et al., 1981). Species in this series have thelargest laticifers in the subgenus (Nelson et al., 1981), and the highest concentrationof cardenolides known in any milkweed. Perhaps the ability of these plants to invadeharshly xeric habitats unoccupied by other milkweeds derives in part from enhanceddefences.While this phylogenetic pattern suggests escalation of defence, demonstrating thatsuch plant traits evolved as defences at all, let alone as a response to any particularenemy, has proven notoriously complex (review in Futuyma & Keese, 1992). Thereis some evidence that cardenolides are toxic or repellent to insects and vertebratesthat do not feed on plants containing them (Detweiler, 1967; Cohen, 1983), butthere has been little systematic attempt to compare the defensive effectiveness ofthe varying cardenolide profiles and deployments within subgenus Asclepias. However,preliminary field and lab observations suggest that milkweeds in series Roseae arefree from the assemblages of oligophagous ctenuchine arctiid moths and chrysomelidbeetles that attack chemically and phylogenetically more primitive congeners. Indeed,Tetraopes and the monarch butterfly are the only known folivores of Roseae. Thus,enhanced defences may have allowed these plants to escape former enemies, includingmost insects. Support for this inference, for example, would come from demonstrationof negative effects of advanced cardenolides on the fitness of primitive beetles.Extending this scenario to the limit, one could further interpret Woodson’sphylogeny as supporting Ehrlich and Raven’s conjecture that origin of novel defensespromotes diversification. That is, the three nested Asclepias subgroups characterizedby the successive defence innovations postulated above are each more species-richthan their apparent sister groups (Fig. 1). In turn, the Tetraopes clade associated withthe chemically advanced Asclepias series (circumscribed by T. linsleyi and T. basalis inB. D. FARRELL 572 AND C. MITTER

Fig. 5) is likewise more diverse than its sister group, suggestive of beetle radiationfollowing colonization of a newly-diverse host clade.Tests of this scenario for Asclepias faces the difficulty that each of its componentadaptations has evolved only once. This makes unavailable the criterion of repeatabilityacross independent lineages, a powerful form of evidence on both theadaptive value of traits (Williams, 1992), and their consequences for diversification(Heard & Hauser, 1995). On the other hand, the most derived, and apparentlymost toxic, series Roseae has been colonized by beetles three times (T. pilosus, T.mandibularis and the common ancestor of T. sublaevis and T. basalis), thus providingopportunity for comparative study of the necessary adaptations and possible advantagesinvolved in use of these plants. Moreover, the phylogeography of theTetraopes/Asclepias assemblage suggests that extant species and their adaptationsevolved largely in the habitats, often quite restricted, that they currently occupy.The ecological circumstances under which putative reciprocal adaptations havearisen in these relatively young, still-localized lineages may be better preserved thanthose surrounding the origin of older, now widespread traits such as the possessionof cardenolides per se. This should permit correspondingly more compelling experimentaltests of hypotheses about both the adaptive origins of traits, and themechanisms whereby improved adaptation translates into increased diversification(Sanderson & Donoghue, 1994). Experimental studies of examples such as Roseaeare needed for rigorous evaluation of Ehrlich and Raven’s model, now thatbroad statistical approaches have provided initial, presumptive evidence for diffusecoevolution between insects and plants.Concluding observationsHostplant use in Tetraopes is unusually conservative: our evidence suggests thatthese beetles’ exclusive association with Asclepiadaceae dates to the mid Tertiary,perhaps to as long as 40 Mya. Moreover, unlike that of most insect groups whichhave been similarly examined, the phylogeny of the Tetraopes/Phaea lineage showssignificant concordance with that of its hosts. This concordance is plausibly ascribed

to approximately synchronous diversification, at least between Tetraopes and Asclepias.These findings parallel results of an earlier analysis of Phyllobrotica leaf beetles (Farrell& Mitter, 1990). Thus, they support the conjecture that parallel phylogenesis withhostplants, with its attendant opportunity for long-term coevolution, is promoted byshared features of these two assemblages which probably reinforce specificity andconservatism of their habits. These traits include larval endophagy, adult feedingand mating on the larval host, and ‘toxic’ host chemistry on which the apparentlyaposematic adult beetles may be dependent for defence.The hostplants of Tetraopes and Phyllobrotica, Asclepiadaceae and Lamiaceae, likethe coumarin-rich Apiaceae (Berenbaum, 1983), belong to the derived angiospermsubclass Asteridae s.l. (Olmstead et al., 1992), which is characterized by an exceptionalprevalence and diversity of toxic and repellent compounds (Cronquist, 1981). Allthree families are derived relatively recently (mid-Tertiary) from woody tropicalancestors, and consist partly (milkweeds) to mostly of temperate herbs that havepresumably diversified with the spread of open, seasonal habitats since the Eocene( Judd, Sanders & Donoghue, 1994). For reasons advanced earlier, such plantTIMING OF INSECT/PLANT DIVERSIFICATION 573

lineages should provide test cases for the prevalence and detectability of escape andradiation coevolution, rigorous study of which has barely begun.ACKNOWLEDGEMENTS

For help with obtaining beetles and collecting locales for populations of Tetraopesand Phaea species we thank Marlin Rice, John Chemsak, James Farrell, Ed Wappes,Ed Riley, Ed Giesbert and Frank Hovore. We also thank the Patuxent WildlifeResearch Center for permitting access to field populations of Tetraopes and Asclepias,and Felipe Noguerra for aid in locating beetles at the Chamela Field Station ofUNAM. Special thanks to Irina Ferreras and Eva Silverfine for providing essentialand enthusiastic help with protein electrophoresis, and to Doug Futuyma and DaveMcCauley for providing recipes for allozymes. For loan of specimens used inmorphological studies, we thank the Museum of Comparative Zoology (HarvardUniversity), the Bishop Museum, The Field Museum of Natural History, The

American Museum of Natural History, the Essig Museum (U.C. Berkeley), theCalifornia Academy of Sciences, the Sacramento Dept. of Food and Agriculture,and the National Museum of Natural History.REFERENCESAckery PR, Vane-Wright RI. 1984. Milkweed Butterflies: Their Cladistics and Biology. New York: CornellUniversity Press..Axelrod DI. 1979. Age and origin of Sonoran Desert Vegetation. Occasional Papers of the CaliforniaAcademy of Sciences 132: 1–74.Berenbaum MR. 1983. Coumarins and caterpillars: A case for coevolution. Evolution 37: 163–179.Berenbaum MR, Miliczky E. 1984. Mantids and milkweed bugs: efficacy of aposematic colorationagainst invertebrate predators. American Midland Naturalist 105: 64–68.Bowers MD. 1988. Chemistry and coevolution: Iridoid glycosides, plants and herbivorous insects.In: Spencer KC, ed. Chemical Mediation of Coevolution. San Diego: Academic Press, 133–166.Brower LP, Brower JVZ. 1964. Birds, butterflies and plant poisons in ecological chemistry. Zoologica49: 137Brower LP, Seiber JN, Nelson CJ, Lynch SP, Tuskes PM. 1982. Plant-determined variation inthe cardenolide content, thin-layer chromatography profiles, and emetic potency of monarchbutterflies, Danaus plexippus, reared on the milkweed Asclepias eriocarpa in California. Journal of ChemicalEcology 8: 579-633.Brower LP, Seiber JN, Nelson CJ, Lynch SP, Hoggard MP, Cohen JA. 1984a. Plant-determinedvariation in cardenolide content and thin layer chromatography profiles of monarch butterflies,Danaus plexippus, reared on milkweed plants in California. 3. Asclepias californica. Journal of ChemicalEcology 10: 1823–1857.Brower LP, Seiber JN, Nelson CJ, Lynch SP, Holland MM. 1984b. Plant-determined variationin the cardenolide content, thin-layer chromatography profiles, and emetic potency of monarchbutterflies, Danaus plexippus L. reared on milkweed plants in California: 2. Asclepias speciosa. Journalof Chemical Ecology 10: 601–639.Brown VK. 1990. Insect herbivory and its effect on plant succession. In: Burdon JJ, Leather SR, eds.Pests, Pathogens, and Plant Communities. Oxford: Blackwell Publishers, 275–288.Chapuis F. 1872. Famille du Longicornes. In: Lacordaire T. Genera des coleopteres. 9(2): 411–930.Chemsak JA. 1963. Taxonomy and bionomics of the genus Tetraopes (Cerambycidae: Coleoptera).University of California Publications in Entomology 30. Berkeley and Los Angeles: University of CaliforniaPress.B. D. FARRELL 574 AND C. MITTERChemsak JA. 1977. Records and descriptions of some Mexican species of the genus Phaea Newman(Coleoptera: Cerambycidae). Pan-Pacific Entomologist. 53: 269-276.Chemsak JA, Linsley EG. 1979. New Cerambycidae from Honduras (Coleoptera). Pan-PacificEntomologist 55: 267–272.Cohen JA. 1983. Chemical interactions among milkweed plants (Asclepiadaceae) and lepidopteranherbivores. PhD. Dissertation. University of Florida.Coley PD, Bryant JP, Chapin FS. 1987. Resource availability and plant antiherbivore defense.

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Nelson CJ, Seiber JN, Brower LP. 1981. Seasonal and intraplant variation of cardenolide contentin the California milkweed Asclepias eriocarpa, and implications for plant defense. Journal of ChemicalEcology 7: 981–1010.Nishio S, Blum MS, Takahashi S. 1983. Intraplant distribution of cardenolides in Asclepias humistrata(Asclepiadaceae), with additional notes on their fates in Tetraopes melanurus (Coleoptera: Cerambycidae)and Rhyssomatus lineaticollis (Coleoptera: Curculionidae). Memoirs of the College of Agriculture, KyotoUniversity 122: 43–52.Olmstead RG, Michaels HJ, Scott KM, Palmer JD. 1992. Monophyly of the Asteridae andidentification of its major lineages inferred from rbcL sequences. Annals of the Missouri BotanicalGarden 80: 700–722.Page RDM. 1990. Temporal congruence and cladistic analysis of biogeography and cospeciation.Systematic Zoology 39: 205–226.Page RDM. 1995. Parallel phylogenies: Reconstructing the history of host-parasite assemblages.Cladistics 10: 155–173.Price PW, Willson MF. 1976. Some consequences for a parasitic herbivore, the milkweed longhornbeetle, Tetraopes tetrophthalmus, of a host-plant shift from Asclepias syriaca to A. verticillata. Oecologia 25:331–340.Price PW, Willson MF. 1979. Abundance of herbivores on six milkweed species in Illinois. AmericanMidland Naturalist 101: 76–86.Rice ME, Turnbow RH, Hovore RT. 1985. Biological and distributional observations on Cerambycidaefrom the southwestern United States (Coleoptera). Coleopterists Bulletin 39: 18–24.Richardson BJ, Baverstock PR, Adams M. 1986. Allozyme Electrophoresis. A Handbook for AnimalSystematics and Population Structure. Sydney: Academic Press.Roeske CN, Seiber JN, Brower LP, Moffitt CM. 1976. Milkweed cardenolides and theircomparative processing by Monarch butterflies (Danaus plexippus). Recent Advances in Phytochemistry 10:93–167.Rohlf PJ. 1990. NTSYS-pc, version 1.60. Stony Brook: Applied Biostatistics, Inc.Rothschild M. 1973. Secondary plant substances and warning colouration in insects. In: van EmdenHF, ed. Insect Plant Relationships. London: Blackwell Scientific Publishers, 59–83.Sanderson MJ, Donoghue MJ. 1994. Shifts in diversification rate with the origin of angiosperms.Science 264: 1590–1593.Scudder GGE, Duffey SS. 1972. Cardiac glycosides in the Lygaeinae (Hemiptera: Lygaeidae).Canadian Journal of Zoology 50: 35–42.B. D. FARRELL 576 AND C. MITTERSennblad B, Bremer B. 1996. The familial and subfamilial relationships of Apocynaceae andAsclepiadaceae evaluated with rbcL data. Plant Systematics and Evolution 202: 153–175.Shao K, Sokal RR. 1986. Significance tests of consensus indices. Systematic Zoology 35: 582–590.Statz G. 1938. Funf neue fossile Cerambyciden-Arten aus den mitteloligocanen Ablegerungen vanRott am Siebenbirge. Entomologische Blatter 34: 173–179.Swofford DL. 1993. PAUP: Phylogenetic analysis using parsimony, Version 3.1.1. Laboratory of MolecularSystematics, Smithsonian Institution.

Thompson J. 1994. The Coevolutionary Process. Chicago: University of Chicago Press.Tidwell WD, Nambudiri EMV. 1989. Tomlinsonia thomassonii, gen. et specie novo, a permineralizedgrass from the upper Miocene Ricardo Formation, California (USA). Review of Paleobotany andPalynology 60: 165–178.Tiffney B. 1985. Perspectives on the origin of the floristic similarity between eastern Asia and easternNorth America. Journal of the Arnold Arboretum 66: 73–94.Werth CR. 1985. Implementing an isozyme laboratory at a field station. Virginia Journal of Science 36:53–76.Wolfe JA. 1978. A paleobotanical interpretation of Tertiary climates in the Northern Hemisphere.American Scientist 66: 694–703.Wolfe JA. 1985. Distribution of major vegetational types during the Tertiary. In: Sundquist ET,Broecker WS, eds. The Carbon Cycle and Atmospheric CO2: Natural Variations Archean to Present. Washington,DC: American Geophysists Union Monograph 32.Williams GC. 1992. Natural Selection. Oxford: Oxford University Press.Woodson RE. 1941. The North American Asclepiadaceae I. Perspective of the genera. Annals of theMissouri Botanical Gardens 28: 193–210.Woodson RE. 1954. The North American species of Asclepias L. Annals of the Missouri Botanical Gardens41: 1–211.TIMING OF INSECT/PLANT DIVERSIFICATION 577APPENDIXEnzyme loci scored for phylogenetic analyses. The suffix 2 indicates a cathodal locus. 2. TBE: Trisborate-EDTA; TM: Tris-maleate; LiOH: discontinuous Lithium hydroxide; TC7, 8: Tris citrate; TVB;Salb: Salamander B (Hillis & Mortiz, 1990; Richardson et al., 1986; Werth, 1985). The matrix of allelefrequencies will be supplied on request by the senior author.Enzyme E. C. # Locus1 Buffer2

1. Aconitase 4.2.1.3 Aco-1 1/2 LIOH2. Aconitase 4.2.1.3 Aco-2 TVB3. Aldolase 4.1.2.13 Ald SalB4. Catalase 1.11.1.6 Cat SalB5. Fructose-1,6 diphosphate 3.1.3.11 Fdp TBE6. Glyceraldehyde-3-phosphate dehydrogenase 1.2.1.12 G3p TC77. Glucose-6-phosphate dehydrogenase 1.1.1.49 G6p TC8dil8. Aspartate aminotransferase 2.6.1.1 Got1 TC8dil9. Aspartate aminotransferase 2.6.1.1 Got2 TBE10. Hydroxybutyrate dehydrogenase 1.1.1.30 Hbd TC8dil11. Alpha-glycero-phosphate dehydrogenase 1.1.1.8 Agpd TC8dil12. Isocitrate dehydrogenase 1.1.1.42 Idh TC713. Malate dehydrogenase 1.1.1.37 Mdh-1 TC714. Malate dehydrogenase 1.1.1.37 Mdh-2 TC715. Malic enzyme 1.1.1.40 Me TC716. Mannose-6-phosphate isomerase 5.3.1.8 Mpi TC8dil17. Phosphoglucomutase 5.4.2.2 Pgm TM18. Glucose phosphate isomerase 5.3.1.9 Phi 1/2 LIOH19. 6-phosphogluconate dehydrogenase 1.1.1.44 Gp6 TC8dil20. Sorbitol dehydrogenase 1.1.1.14 Sdh TBE21. Superoxide dismutase 1.15.1.1. Tox TBE22. Xanthine dehydrogenase 1.1.1.204 Xdh LIOH23. Adenylate kinase 2.7.4.3 Ak LIOH

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Performing your original search, chemical test of thevetia, within the ACS Publications collection will retrieve 9 results. Go to resultsPrev. Article Next Article Table of Contents NoteCardenolide Glycosides of Thevetia peruviana and Triterpenoid Saponins of Sapindus emarginatus as TRAIL Resistance-Overcoming CompoundsAbstract

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Takashi Miyagawa†, Takashi Ohtsuki†, Takashi Koyano‡, Thaworn Kowithayakorn§ and Masami Ishibashi* † Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan, Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164-0012, Japan, and Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, ThailandJ. Nat. Prod., 2009, 72 (8), pp 1507–1511DOI: 10.1021/np900202nPublication Date (Web): July 13, 2009Copyright © 2009 The American Chemical Society and American Society of Pharmacognosy* To whom correspondence should be addressed. Tel: +81-43-290-2913. Fax: +81-43-290-2913. E-mail: mish@p.chiba-u.ac.jp., † Chiba University., ‡ Temko Corporation., § Khon Kaen University.Abstract

A screening study for TRAIL resistance-overcoming activity was carried out, and activity-guided fractionations of Thevetia peruviana and Sapindus emarginatus led to the isolation of four cardenolide glycosides (1−4) and four triterpenoid saponins (5−8), respectively. In particular, cardenolide glycosides (1 and 2) from T. peruviana were shown to have a significant reversal effect on TRAIL resistance in human gastric adenocarcinoma cells, and real-time PCR showed that thevefolin (2) enhanced mRNA expression of death receptor 4 (DR4) and DR5. In addition, 1H and 13C NMR characterizations are shown for thevefolin (2) for the first time.View: Full Text HTML | Hi-Res PDF | PDF w/ Links

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__African Journal of Pharmacy and Pharmacology Vol. 4(2). pp. 087-089, February, 2010Available online http://www.academicjournals.org/ajppISSN 1996-0816 © 2010 Academic Journals

Short Communication

Anti-termite and antimicrobial properties of paint madefrom Thevetia peruviana (Pers.) Schum. oil extractP. G. Kareru1*, J. M. Keriko1, G. M. Kenji2 and A. N. Gachanja1

1Chemistry Department, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya.

2Food Science and Technology, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya.Accepted 5 January, 2010Thevetia peruviana (Pers.) K. Schum. seed oil was used to make a surface coating with antifungal,antibacterial and anti-termite properties. The paint exhibited inhibitory activity against Escherichia coli,Staphylococcus aureus, Bacillus subtilis and Candida albicans in a concentration dependent manner.The antibacterial activities were statistically significant (p = 0.05). The repellent action of paint againstsubterranean termites (Microtermes spp.) was significant (p = 0.03). From these results, it wasconcluded that the Thevetia peruviana-based oil paint was self-preserving against microbes andsubstantially protected wood from subterranean termite attack.Key words: Thevetia peruviana, anti-termite, antifungal.INTRODUCTIONThevetia peruviana (Pers.) K. Schum. (commonly knownas Yellow oleander) is an ornamental plant which growsin Kenya and other parts of the World, such as tropicalAmerica, Western Asia, Southern Europe, India andtropical Africa. Extracts from T. peruviana plant speciescontain glycosides, whose toxicity against snails, slugs(Panigrahi and Raut, 1994), bacteria (Obasi andIgboechi, 1991), insects (McLaughlin et al., 1980) andhumans (Langford and Boor, 1966) has been documented.T. peruviana plant extracts have also been reportedto have antifungal properties against Cladosporiumcucumerinum (Gata et al., 2003).Toxicity and repellent effects of medicinal plant extractson subterranean termites (Isoptera: Rhinotermitidae)have also been demonstrated (Verena-Ulrike and Horst,2001). The presence of unsaturated linoleic acid inYellow oleander oil (Obasi et al., 1990), which has dryingproperties (Cecilia et al., 2005), makes Yellow oleanderoil suitable for making a surface coating such as paint.The aim of this study was to formulate an oil-based paintusing crude Yellow oleander oil and to determine itsinsecticidal and antimicrobial properties.*Corresponding author. Email: pgkareru@yahoo.com,patgkareru@gmail.com. Tel: +254-6752-223. Fax: +254-6752-446.MATERIALS AND METHODSCollection of Yellow oleander kernel seeds and extraction of oilYellow oleander kernel seeds were collected from Jomo KenyattaUniversity of Agriculture and Technology campus. After removingthe kernel, seeds were macerated using a blender. Oil was extractedwith methanol. Filtered crude oil was stored in a refrigerator at4°C till used.Paint formulationCommercial grade long oil (alkyd resin), titanium dioxide, anti-skinagents, white spirit and paint dryers were purchased from a localchemical supplier, Industrial area, Nairobi. Five kilogrammes (5 kg)of paint was made by mixing appropriate ingredients. Yellowoleander oil extract was used to make paint batches whose oilconcentration ranged from 0.0 to 80.0%.Antibacterial and antifungal assays

The procedure followed was as described by Cheesbrough (1984).Inhibition zone diameters for paints were determined against E. coli,S. aureus, B. subtilis and C. albicans. The results were presented inFigure 1.Anti-termite activityLabeled dry plywood plates (6 x 6 inches) were painted on bothsides, (in triplicate) with the formulated paints. One set of control

__088 Afr. J. Pharm. Pharmacol.Figure 1. Inhibition zone diameters (mm) of oleander paint.Figure 2. Repellent activity of Oleander paint towards Microtermes spp.plates was painted with neat oleander oil, while the other waspainted with a paint in which Yellow oleander oil was not added.After drying to constant weight in the laboratory environment, eachplate weight was determined. The wooden plates were then placedside by side and covered with foliage under a termite (Microtermesspp) nest and left for a period of one month. Moisture wasconstantly maintained by pouring water on the foliage within theexposure period, so as to maintain appropriate environmentalconditions favourable to termites. After the exposure period, thewooden plates were washed with clean water to remove soil anddebris, and dried in the oven at 50°C to a constant weight. Themass of each plate was then determined and the average weightloss calculated.RESULTS AND DISCUSSIONThe Oleander paint inhibited the tested microbes in aconcentration dependent manner. The control paint(containing 0.0% oil) did not inhibit the test bacteria andfungus. From these results, it was concluded thatoleander paint was self-preserving against bacterial andfungal attack. Antibacterial and antifungal activity of T.peruviana plant extracts had been earlier established(Obasi and Igboechi, 1991; Gata et al., 2003) andcollaborates with the present findings. From Figure 2 it__was evident that the oleander paint repelled Microtermesspp. The repellent action was highest when pureoleander oil was used. However, no termite deaths werereported in this study. Insecticidal and toxicity of Yellowoleander oil has been reported (McLaughlin et al., 1980;Panigrahi and Raut, 1994; Langford and Boor, 1966).Also anti-termite activity of medicinal plant extracts hasbeen documented (Verena-Ulrike and Boor, 2001). Thepresent findings demonstrate that paint made from T.peruviana plant oil extract could substantially protecttimber from termite attack.ConclusionYellow oleander paint possesses antimicrobial and antitermiteactivities. T. peruviana oil extract would serve asan environmentally friendly bactericide and fungicide foroil based paints.ACKNOWLEDGEMENTSThe authors wish to thank Jomo Kenyatta University ofAgriculture and Technology for funding this study. Thanksgoes to Prof. Rosabella Maranga for identification of thetermite species used in this investigation.

Kareru et al. 089REFERENCESCecilia S, Martin S, Mats J (2005). A study of the drying of linseed oilswith different fatty acid patterns using RTIR-Spectroscopy andChemiluminescence (CL), Ind. Crops Prod. 21(2): 263-272.Cheesbrough M (1984). Medical Laboratory Practice in TropicalCountries, Part II. Cambridge University Press, pp. 401-402.Gata GL, Nogueira JMF, Bruno de Sousa OMR (2003). Photoactiveextracts from Thevetia peruviana with antifungal properties againstCladosporium cucumerinium, J. Photochem. Photobiol. Biol. 70: 51-54.Langford S D, Boor PJ (1966). Oleander Toxicity: An examination ofhuman and animal toxic exposures. Toxicology, (Medline). 109: 1-13.McLaughlin J L, Freedman B, Powel R G, Smith C R, (1980). Neriifolinand 2’acetylneriifolin. Insecticidal and cytotoxic agents of Thevetiatheveotides seeds. J. Econ. Entomol. 73: 398-402.Obasi NBB, Igbochi AC (1991). Seed-soil distillates of ThevetiaperuvianaSynonym Thevetia-neerifolia: Analysis and antibacterialactivity. Fitoterapia 62(2): 159-162.Obasi NBB, Igboechi AC, Bejamin TV (1990). Seasonal variations in theseed oil of Thevetia peruviana (Pers.) K. Schum. J. Am. Oil Chem.Soc. 67(10): 624-625.Panigrahi A, Raut SK (1994). Thevetia peruviana (Family:Apocynaceae) in the control of slug and snail pests. Mem. Inst.Oswaldo Cruz, Rio de Janeiro, 89(2): 247-250.Verena-Ulrike B, Horst H (2001). Repellent and Toxic effects of plantextracts on subterranean termites (Isoptera: Rhinotermitidae). J.Econ. Entomol. 94(5): 1200-1208.

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