CYTOTAXONOMICSTUDIES IN THE EUPHORBIACEAE, SUBTRIBE PHYLLANTHINAE' GRADY L. WEBSTER and J.R. ELLIS Departmentof Biological Sciences, Purdue University,Lafayette, Indiana and Galton Laboratory,University College, London, England

ABSTRACT WEBSTER, GRADY L. (Purdue U., Lafayette,Ind.), and J. R. ELLIS, Cytotaxonomicstudies in the Euphorbiaceae, subtribePhyllanthinae. Amer. Jour. Bot. 49: (1): 1X18. Illus. 1962.-Chromosome numbersare reportedfor 18 of mostlyWest Indian Euphorbiaceae, subtribe Phyllanthinae, 13 of these for the first time (including the firstpublished count for the Margaritaria). For 4 species, a number differentfrom previous determinationshas been recorded. The base chromosomenumber in Breynia, Fluggea, Margaritaria,and most species of Phyllanthusappears to be 13. However, in subg. Isocladus haploid numbers of 8 and 18 were observed.One species, Phyllanthuspulcher, is a sterile hexaploid (n - 39) of presumablyhybrid origin. The cytological data do not support Perry's suggestionthat annual taxa are primitivein the Euphorbiaceae.

THE 7,000 species of the famllyEuphorbiaceae regarded as unequivocallyconfirmed. The few presentsuch a vegetativeand floraldiversity that workerswho have studiedtaxa of the subtribein- systematictreatment of the grouphas alwaysbeen clude Perry (1943), Raghavan (1957), Raghavan controversial.Not only have there been many and Arora (1958), JanakiAmmal and Raghavan schools of thoughtin delimltingtaxa withinthe (1958), and Thombre(1959). family,but its recognitionas a naturalgroup has In the presentcontribution we wish to report been seriouslyquestioned on the basis that it is the resultsof cytologicalobservations on 18 spe- polyphyleticin origin.Hutchinson (1959) suggests cies belongingto 4 generaof subtribe Phyllanthinae, derivationof the familyfrom at least 4 different most of which are eithernative to or cultivated orders(Blixales, Tiliales, Malvales, and Celastrales). in the West Indies. In view of the paucityof data Erdtman (1952), on the basis of palynolog,ical availableup untilnow, we believethat these counts studies,has suggestedpossible relationshipswith are moresignificant than a similarnumber of mis- theBuxaceae and Thymelaeaceae.Hurusawa (1954) cellaneousreports in moreextensively studied taxa. proposes a division of the Euphorbiaceaeinto 4 However,the tentativeand exploratorynature of families. our conclusionsmust be recognized,since even In contrastto such familiesas the Gramineae, withthe addition of therecords in thispaper, chro- Leguminosae,and Compositae,the Euphorbiaceae mosomenumbers are knownfor only about 3% have been relativelylittle studied cytologically. Of of thespecies of Phyllanthus. the 7,000 species, fewerthan 250 have been re. MATERIALS AND PROCEDUREs-Studies weremade portedon, and the bulk of the countsare in the ofplants growing in thefield in theWest Indies dur- singlegenus Euphorbia. The subfamilyPhyllanthoi. ing thesummer of 1959,and werelater supplemented deae has been especially under-represented.as by observationson plantsgrown in the greenhouses heretoforecounts have been publishedon less than at Purdue. Observationsof mitoticchromosomes 2%o of the 1500 species. In the subtribePhyllan. were made fromroot-tip squashes. Root-tipswere thinae,which includes about 1000 species (700 of pre-treatedfor 2-3 hr in a saturatedsolution of thesein Phyllanthus),counts have been published paradichlorobenzene,fixed overnightin acetic al- for only 8 species, and only I of these may be cohol,hydrolyzed in 1 N HCI at 60 C for 18 min stainedin Feulgen 1 Receivedfor publication May 12, 1961. for2 hr and squashedin aceto- carmine.For meiotic Field workfor this studywas carriedout in the West observations,buds were col- Indies during the summersof 1959 and 1960. The work was lectedin thefield and fixeddirectly in aceticalcohol supported by the National Science Foundation (Grant in whichthe acetic acid componenthad been sat- G-7148). uratedwith ferricacetate; the antherswere then The authorsare indebted to Mr. Kim Miller for valuable dissectedfrom the male flowers assistance in collecting and preparing material. Acknowl- and squashed in edgementfor assistance is also due to the directorsand staffs aceto-carmine.Voucher herbarium specimens of the of cooperatinginstitutions in the West Indies, includingthe plantsstudied have been depositedin the Kriebel Instituteof Jamaica (Kingston), U. S. AgriculturalExperi- Herbarium,Purdue University(PUL). ment Station (Mayaguiez), Universityof Puerto Rico Agri- RESULTS AND DISCUSSION-In 1 cultural ExperimentStation (Rio Piedras), Institutede la Table are pre- Recherche Agronomique (Basse-Terre), Imperial College sentedcounts made by us for 18 speciesbelonging of Tropical Agriculture (Port-of-Spain), and the Dept. of to 4 generaof the subtribePhyllanthinae, of which Agricultureand Marine Products (Nassau). 17 representeither different determinations ciom- 14

This content downloaded from 169.237.8.35 on Mon, 16 Sep 2013 14:22:41 PM All use subject to JSTOR Terms and Conditions January,1962] WEBSTER AND ELLIS-EUPHORBIACEAE 15 pared with previous reportsor counts recorded Margaritariahas not been reportedon cytologi- for the firsttime. For convenienceof comparison, callybefore, possibly because the commonest species publishedobservations by other workersare in- M. nobilisis disguisedin manybooks as a species cluded in the table,but are set in italic type.The of Phyllanthus.Cytologically, it agreesclosely with circumscriptionof the Phyllanthinaeand the Fluggeain havinga diploidcomplement of 26 chro- arrangementof subgenerictaxa withinPhyllanthus mosomes(Fig. 1). Margaritariaand Fluggea are, followsthe treatment of Webster(1956-58), which therefore,diploid genera, so faras we know,where- differsin a numberof respectsfrom the classifica- as Breyniais tetraploid.This relationshipis notun- tionof Pax and Hoffmann(1931). expected,since the formergenera have been con- It is interestingthat in the3 othergenera studied sideredprimitive on the basis of floraland vegeta- (besides Phyllanthus)x = 13. Perry'srecord of tivemorphology, whereas Breynia is obviouslymore 2n = 56 in Breynianivosa is questionable,inas- specialized. muchas we have found2n = 52 in meioticprepara- The 16 species of Phyllanthusreported on here, tionsof B. disticha(the correctname forthe same togetherwith 1 species (P. emblica) studied by ). Likewise,his reportof 2n 16 in Fluggea Indian workers,represent 13 sectionsbelonging to obovatais probablyerroneous, for in our plantF. 8 subgenera.Although these species constituteless virosa the diploid number is clearly 26. Since than 3% of those in the genus,they are sufficient Fluggea obovata and F. virosa are synonymous fora preliminarysurvey of cytologicalrelationships namesfor the same plant,this large discrepancyin at the supraspecificlevel. reportedchromosome number suggests that perhaps One of the most strikingresults of our studies Perrv's determinationwas for some completely is the demonstrationthat the base numberin most differentplant. sectionsof Phyllanthusis 13, as in the 3 genera

W.~ ~ ~ ~~~~~~~~~~~~I,

....S ...... I

Fig. 1-5. Chromosomesof Margaritaria and2800..... PI&yllantl&us.-Fig. 1. Margantaria nobilis, mitotic 2n 26 X diakm Fig. 2-3 Phyllathuspolgonoide, diakiesis, anphase I 2n = 1, X 1900 Fig. 4.PI&yllathuscarltnzenss sis, 2n =36, x 1900.-Fig. 5. Phyllantl&us2300~~~~~~~~~~~...... pulcl&er, anaphase I, x

This content downloaded from 169.237.8.35 on Mon, 16 Sep 2013 14:22:41 PM All use subject to JSTOR Terms and Conditions 16 ANIERICAN JOURNAL OF BOTANY [Vol.49 mentionedabove, but that in 2 sectionsit is dis- Isocladus,which consists of specieswith unspecial- cordant.In P. polygonoidesof sect. Isocladus (not ized branching.On the otherhand, all taxa with West Indian but includedhere because of its in- n - 13 have phyllanthoidbranching (except for terest)n 8 (Fig. 2, 3), whilein P. caroliniensissect. Botryanthus).This would appear to suggest of sect.Loxopodium, n - 18 (Fig. 4). Perry'sre- thatthe taxa in subgenusIsocladus may be phylo- portof 2n - 28 in the latterspecies appears very geneticallyisolated fromthe other groups within dubious,even though he may have studieda differ- Phyllanthus.However, Arora (accordingto Janaki ent subspecies.It is noteworthythat sectionsIso- Ammaland Raghavan,1958) has reporteda chro- cladus and Loxopodiumboth belong to subgenus mosomecount of 2n - 52 forP. maderaspatensis,

TABLE 1. Chromosomenumbers in the Phyllanthinae

Taxon Chromosomenumber gam. spor. Reference or voucher collection Forst. 26 Jamaica; Websteret al. 8069 56 Perry,1943a Fluggea 'obovata' 16 Perry,1943b Fluggea virosa (Willd.) Baill. 13 Jamaica; Proctor8131 Margaritarianobilis L. f. 26 Guadeloupe; Webster et al. 9157 Phyllanthus Subgenus ISOCLADUS Section ISOCLADUS P. polygonoidesNutt. ex Spr. 16 Texas; Johnston3547 P. maderaspatensisL. 52 Arora (cited by Janaki Ammal and Raghavan, 1958) Section LOXOPODIUM P. caroliniensisWalt. ssp. caroliniensis 28 Perry,1943C ssp. guianensis (Kl.) Webster 36 Martinique; Webster et al. 9200 Subgenus & section KIRGANELIA P. reticulatusPoir. 13 Webster and Wilson 5237 Subgenus & section CICCA P. acidus (L.) Skeels 26 Jamaica, cult. 14 Thombre,1959 Subgenus PHYLLANTHIUS Section EMBLICA P. emblica L. 28 Perry,1943 98 Raghavan, 1957 98-104 Janaki Ammal and Raghavan, 1958 Section URINARIA P. urinaria L. 26 Jamaica; Webster et al. 8177 52 Raghavan and Arora, 1958 Section PHYLLANTHUS P. amarus Schum. & Thonn. 26 Jamaica; Websteret al. 8025A P. 'niruri' 26 Raghavan, 1957 P. pentaphyllusWright ex Griseb. 52 Florida; Webster et al. 9983 Subgenus & section CONAMI P. acuminatusVahl 26 Puerto Rico; Webster et al. 8851 P. subglomeratusPoir. 26 Guadeloupe; Websteret al. 8985 Subgenus & section ERiococcus P. pulcher Wall. ex Muell. Arg. c.39 Trinidad; Websteret al. 9969 Subgenus & section BOTRYANTHUS P. nutans Sw. ssp. nutans 26 Jamaica; Webster et al. 8019 Subgenus XYLOPHYLLA Section ASTERANDRA P. juglandifoliusWilld. ssp. juglandifolius c.156 Puerto Rico; Webster et al. 8594B Section EPISTYLIIJM P. axillaris Sw. 26 Jamaica; Webster et al. 8472 Section HEMIPHYLLANTHUS P. mimosoidesSw. 26 Guadeloupe; Webster et al. 8987 P. ovatus Poir. 26 Martinique; Webster et al. 9172 a Determinedby Perryas Breynianivosa; this is a synonymof B. disticha. b Determinedby Perry as Fluggea obovata; if the determinationis correct,the plant is the same as F. virosa. Perry's plant came froma nurseryin Virginia,so presumablyit is the subspecies native there.

This content downloaded from 169.237.8.35 on Mon, 16 Sep 2013 14:22:41 PM All use subject to JSTOR Terms and Conditions January,1962] WEBSTER AND ELLIS-EUPHORBIACEAE 17 a specieswhich certainly belongs in sect. Isocladus suspicion,since in the other 2 species (Breynia along withP. polygonoides.We thinkthat this re- distichaand Phyllanthuscaroliniensis) in whichhe portshould be reinvestigated,as 1 pair of chromo- reportedx = 7, his countscould not be confirmed. somes in P. polygonoideshas very large satellites At any rate, furthercytological investigation of (not apparentin Fig. 2, 3 due to higherdegrees of this variable and economicallyimportant species constrictionin meiotic material), which during appears warranted. prophaseand earlymetaphase could easilybe mis- Consideringthe great diversity of the indigenous takenfor small chromosomes. If in P. maderaspaten-West Indian species belonging, to subgeneraConami, sis x -8 also, it may well be a hexaploidin which Botryanthus,and Xylophylla,it was interestingto 2n 48. find that all species investigatedare tetraploids Whateverthe diploid number of P. maderas- (2n 52), with1 exception.In P. juglandifolius patensismay prove to be, it is clear that sub- of sect. Asterandra,the somaticnumber proves to genus Isocladusdoes not fit into the cytological be ca. 156, so thatthe plant must be a dodecaploid; patternestablished for the Phyllanthinae.However, thisis thehighest chromosome number yet recorded untiladditional taxa in subgenusIsiocladus can be in the subfamilyPhvllanthoideae. Of course,since studied,it wouldbe unwiseto drawany phylogenetic only 7 of the 44 West Indian membersof the 3 conclusionsor to make any formaltaxonomic ad- subgenerahave been investigated,it is quite pos- justments.It is quitepossible, for instance, that still sible thatother species may proveto have chromo- otherbase numbersmay be foundwithin this rather some numbersat intermediateploidy levels. The diversegroup. highchromosome number of P. juglandifoliusdoes Turning,to the othersubgenera of Phyllanthus,not seemto be correlatedwith any greatdegree of in whichx - 13, we finda more understandable morphologicaladvancement, since the plant is actu- correlationbetween chromosome number and taxo- ally rather unspecializedwithin subgenus Xylo. nomicstatus. Here mostof the speciesstudied are phylla.More significantis the factthat the other6 polyploid,and since the diploid species belong to species, belongingto 4 sectionsin 3 subgenera, the smaller sections,it is clear that by far the have a commonnumber of 2n - 52 despitetheir greaterpart of the speciesin the genuswill prove manysuperficial differences. This adds supportto to be polyploidwhen studied.Only in subgenera the conservativephilosophy of retainingall these Kirganeliaand Cicca did we find diploids,with taxa withinPhyllanthus rather than segregating 2n - 26. Since these 2 subgeneraare the only Conami and Asterandraas distinctgenera (as diploid taxa with phyllanthoidbranching, it fol- treatedby Brittonand Wilson,1924). lows that they occupy a strategicposition in the The native herbaceousWest Indian species of phylogeneticsense. In thisconnection, it is interest- Phyllanthuswith phyllanthoid branching, as wellas ing that Janaki Ammal and Raghavan (1958) theirOld Worldrelatives, have been placed in sub- foundvery high chromosomenumbers, 2n - 98- genus Phyllanthus.Our studiesshow thatmost of 104, in P. emblica,which has oftenbeen placed in the species investigatedare also tetraploids,with the Ciccagroup because of its drupaceousfruit. If 2n = 52 (Fig. 8). However,Rag,havan (1957) has x - 13 in P. emblica,as seems highlyprobable, reported2n 26 forPhyllanthus 'niruri' collected then the species should be an octoploidwith the in India. Since trueP. niruriis an Americanspecies primarynumber of 2n 104. Perry's (1943) not knownto be immigrantin India, the identity accountof 2n - 28 in thisspecies is again open to of Raghavan'splant is doubtful;it mayrnot even

~ ~ ~ *z-**Zj **'* W

0~~~~~

40

4b. 8

Fig. 6-8. Chromosomesof Phyllanthus.-Fig.6. Phyllanthuspulcher, anaphase I, showing12 dividingunivalents, X 2200.-Fig. 7. Phyllanthuspulcher, anaphase I, showing16 dividingunivalents and 2n 78, X 2200.-Fig. 8. Phyl- lanthuspentaphyllus, mitotic, 2n = 52,X 2000.

This content downloaded from 169.237.8.35 on Mon, 16 Sep 2013 14:22:41 PM All use subject to JSTOR Terms and Conditions 18 AMERICAN JOURNAL OF BOTANY [Vol. 49 belongto subgenusPhyllanthus. For thetime being, Finally,it may be notedthat the data here col- therefore,there is no unequivocal record of an lecteddo not supportPerry's thesis that perennial herbaceousdiploid species except for the ones with taxa have evolved from annual ones in the Eu- unspecializedbranching in subgenusIsocladus. phorbiaceae. The most primitivegenera in the A quiteexceptional case is providedby P. pulcher Phyllanthinae,Fluggea and Margaritaria,are either (subgenus Eriococcus), a hexaploid plant with treesor shrubsand are diploid.With the exception 2n 78. The speciesas observedby us in Trinidad of subgenusIsocladus, the more primitivegroups flowersfreely but neverproduces seed. Cytological withinPhyllanthus are woody and diploid,while observations(Fig. 5-7) showed very irregular theherbaceous species so farinvestigated are tetra- meioticdivisions, due to a variable number(10- ploid. From other lines of evidence it is clear 16) of chromosomeswhich failed to pair. It that these herbaceousspecies are not ancestralto appears,therefore, to be a sterilehybrid between 2 the tetraploidwoody species. In the evolutionof distinctspecies of sect.Eriococcus, but its parentage thePhyllanthinae there has been a tendencytoward is still unknown,nor is it knownwhether other higher chromosomenumbers and a trend from species of the sectionare also hexaploid.We com- the woody to the herbaceouscondition, but these mend this interestinggroup to the attentionof trendsare not strictlycorrelated in all groups. cytotaxonomistsin easternAsia. LITERATURE CITED CONCLUSIONS-It is now clear that in most taxa of the subtribePhyllanthinae the original base BRITTON,N. L., AND P. WILSON. 1924. Sci. Surv. Puerto Rico and Virgin Islands; Bot. Puerto Rico and Virgin numberis 13, and that the taxa whichwould be I. 5: 472-476. regardedas most primitiveon the basis of gross ERDTMAN,G. 1952. Pollen morphologyand plant . morphologyare diploids.However, the majorityof Angiosperms.Almquist & Wiksells,Uppsala. 539 p. bothherbaceous and woodyspecies of Phyllanthus HURUSAWA,I. 1954. Eine nochmalige Durchsicht des her- are at thetetraploid level or higher.Janaki Ammal k'ommlichenSystems der Euphorbiaceen im weiteren and Raghavan (1958) suggestthat the herbaceous Sinne. Jour. Fac. Sci. Tokyo III. Bot. 6(6): 209-341. speciesof Phyllanthus,with x -_ 13, have a differ- HUTCHINSON,J. 1959. The families of floweringplants. I. entbase numberfrom the woody ones, with x - 7. Dicotyledons.Oxford UniversityPress, London. 510 p. Presumablytheir suggestion is influencedby Perry's JANAKI AMMAL,E. K., AND R. S. RAGHAVAN.1958. Poly- (1943) reportof 2n - 28 in P. emblica (and per- ploidy and vitamin C in Emblica officinalisGaertn. haps also by his record of 2n 56 in Breynia Proc. Indian Acad. Sci., B, 47: 312-314. MANGENOT,S., AND G. MANGENOT.1957. disticha) whichwould suggestx - 7. Thombre's Nombres chro- - mosomiques nouveaux chez diverses dicotyledoneset (1959) reportof n 14 in Phyllanthusacidus monocotyledonesd'Afrique occidentale. Bull. Jard. Bot. mayreflect the same influence.However, we believe Bruxelles 27: 639-654. that Perry's observationsare incorrectand that , AND . 1958. Deuxiieme liste de nombres x - 13 in the woody as well as the herbaceous chromosomiquesnouveau chez diverses dicotyledones species. et monocotyledonesd'Afrique occidentale. Bull. Jard. The demonstrationof x - 13 in most of the Bot. Bruxelles 28: 315-329. Phyllanthinaeis especiallyinteresting in viewof the PAx, F., AND K. HOFFMANN. 1931. Euphorbiaceae. In A. factthat Mangenot and Mangenot(195-8) have re- Engler and K. Prantl. Naturl. Pflanzenfam.2nd ed. portedn 13 in Antidesma,Bridelia, Hymeno- 19c: 11-251. PERRY,B. A. cardia, ,and Uapaca. 1943. Chromosomenumber and phylogenetic , relationshipsin the Euphorbiaceae. Amer. Jour. Bot. Since thesegenera represent subtribes Antidesminae 30: 527-543. and Uapacinae of the ,as wellas tribe RAGHAVAN,R. S. 1957. Chromosomenumbers in Indian Bridelieae,it is apparentthat 13 is a very wide- medicinal . Proc. Indian Acad. Sci., B, 45: 294- spread base numberin the Phyllanthoideae.How- 298. ever,it is not necessarilythe originalnumber for , AND C. M. ARORA.1958. Chromosomenumbers in the subfamily,for Reese (1957) has reportedn = Indian medicinal plants. II. Proc. Indian Acad. Sci., B, 12 in ,which is more primitivethan 47: 352-358. mostof thegenera mentioned above, and Mangenot REESE,G. 1957. UYberdie Polyploidiespecktrenin der nord. and Mangenot (1957) have found n 20 in saharischen Wiistenflora.Flora 144: 598-634. Drypetes,which is a primitivetaxon in many re- THOMBRE, M. V. 1959. Chromosomenumbers in some com- mon floweringplants. Sci. and Cult. 25: 208. spects. Sampling of genera in the still unstudied WEBSTER, G. L. 1956-58. A monographicstudy of the West subtribeswill be necessarybefore a clear picture Indian species of Phyllanthus.Jour. Arnold Arb. 37: of karyotypeevolution in the Phyllanthoideaecan 91-122, 217-268, 340-359. 1956. 38: 51-80, 170-198, be constructed. 295-373.1957. 39: 49-100,111-212. 1958.

This content downloaded from 169.237.8.35 on Mon, 16 Sep 2013 14:22:41 PM All use subject to JSTOR Terms and Conditions