Chromosome numbers in the genus Lippia (Verbenaceae)

L. F. Viccini1, P. M. O. Pierre

1, M. M. Praca

1, D. C. Souza da Costa

1, E. da Costa Romanel

1,

S. M. de Sousa1, P. H. Pereira Peixoto

2, and F. R. Goncalves Salimena

2

1Departamento de Biologia, Universidade Federal de Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil2Departamento de Botanica, Universidade Federal de Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil

Received October 25, 2004; accepted April 30, 2005Published online: October 20, 2005� Springer-Verlag 2005

Abstract. The genus Lippia (Verbenaceae)comprises about 200 taxa mainly distributed inBrazil, Mexico, Central America, Africa, Argentinaand Paraguay. Some problems involving the numberand delimitation of species have been reported. Inorder to contribute to the solving of these problems,the chromosome numbers of 14 Lippia species aredocumented. The following species were collected atEspinhaco Range, Southeast Brazil: Section Zapa-nia (L. corymbosa, L. diamantinensis, L. hermannio-ides, L. lacunosa, L. rotundifolia, L. rubella), sectionRhodolippia (L. florida,L. lupulina,L. pseudothea,L.rosella), section Goniostlachyum (L. glandulosa, L.pohliana, L. sidoides) and sectionDioicolippia (L. fil-ifolia). Immature inflorescences were collected andthe ideal size for chromosome observation wasdetermined. The majority of species have a haploidchromosome number from 10 to 14. Few specieshave a higher chromosome number, which suggeststhe occurrence of polyploidy. The relationshipsbetween chromosome numbers and the taxonomicsections are also discussed.

Key words: Lippia, Verbenaceae, chromosomenumber, meiosis.

Introduction

Verbenaceae, subfamily Verbenoideae, com-prises about 36 genera and more than 1035

species, widely distributed from tropical totemperate regions (Judd et al. 1999). Thisconstitutes one of the five most importantEudicotyledonous families of Campos Rupes-tres (Giulietti et al. 1987). In recent years,some genera have been recognized for theirmedicinal proprieties. Among them, Lantana,Stachytarpheta and mainly Lippia are the mostimportant ones (Salimena-Pires 1991, Salimena-Pires and Giulietti 1998).

The genus Lippia comprises nearly 200taxa (Salimena 2000) and 160 species distrib-uted mainly in Brazil, Mexico, CentralAmerica, Africa, Paraguay and Argentinawith few endemic African species (Troncoso1974). Brazil, one of the largest centers ofdiversity, has approximately 70–75% of theknown species. Most of them are endemicand concentrated at Espinhaco Range, MinasGerais and Goias State, where the occurrenceof rocks is common and many Lippia speciesgrow on them. Due to this particular char-acteristic, human actions in this region suchas mining, have put at risk some endemicspecies.

A taxonomic problem in Lippia is thedefinition of species, given disagreement con-cerning the segregation of some genera, such as

Pl. Syst. Evol. 256: 171–178 (2006)DOI 10.1007/s00606-005-0351-3

Phyla and Aloysia, from Lippia (Nash and Nee1984). In addition, some problems of speciesdelimitation are due to the absence of goodtaxonomic characters. Cytogenetic analysishas been extensively recognized as an auxiliarytool, since chromosome numbers are notinfluenced by environmental factors and con-stitute an important trait, not only to help thecharacterization of species, but also to estab-lish evolutionary trends (Stebbins 1971, Shanet al. 2003).

Despite its medicinal potential, endemism,extinction risk and innumerous taxonomicproblems, there are few cytogenetic studies ofthe genus Lippia (Bose and Choudhury 1960,Smith 1966, Navaneethan 1982, Coleman1982, Filippa 1984, Kumar and Dutt 1989).This paper shows, for the first time, thechromosome number of 14 Lippia species fromthe Espinhaco Range in Southeast Brazil. Thebasic chromosome number is also discussed.

Material and methods

Plant material. The immature inflorescences of 14species of Lippia (Verbenaceae) were collected atEspinhaco Range in Southeast Brazil, according tothe bloom season of each species. Inflorescenceswere fixed in fresh cold methanol : acetic acidsolution 3:1 for at least 24 hours. Vouchers aredeposited at the Herbarium CESJ of UniversidadeFederal de Juiz de Fora. Locality information, theseason and register numbers are described inTable 1.

Pre-selection of the inflorescences. In order toobtain cell suspensions with meiotic stage cellsappropriate to chromosome counts, the immatureinflorescences were divided into arbitrary sizes andthe cells of each one were observed. Cell suspen-sions for each species were made based on thepreviously defined size.

Cell suspension. Considering the small size ofthe flower buds of this group (1–4 mm), the cellsuspension was prepared according to Caixeta andCarvalho (2001) with some modifications. Approx-imately 40 anthers were excised from 12 flowerbuds and placed in a special microtube (0.5 ml).This microtube was cut at the bottom and a nylonscreen attached (60 lm mesh). The material was

washed three times in distilled water to removefixative. The adapted tube containing the antherswas immersed in another normal microtube(1.5 ml) with concentrated enzymatic solution(Pectinex NOVO FERMENTTM). The materialwas placed in an incubator at 34 �C for 20 min.After enzymatic maceration, the material waswashed again in order to remove the excess ofenzymatic solution. The adapted microtube wasthen placed in a new common microtube (1.5 ml)containing distilled water. The anthers weremechanically fragmented with an adapted pin inorder to remove the pollen mother cells (PMCs),which fall into a normal microtube with distilledwater. The cellular suspension obtained was thencentrifuged three times at 2000 rpm for 12 min. Ineach step the supernatant was removed and thevolume of the microtube was maintained withdistilled water. After the last centrifugation, thevolume of the microtube was replenished with freshfix. The cellular suspension was carefully resus-pended. The final suspension was stored at )20 �C.

Slide preparation. For slide preparation, aboutsix drops of the suspension were dropped on to aclean slide. Slides were air-dried and stained withGiemsa solution for 3 min.

Results and discussion

Due to the great difficulty in obtainingmeristematic roots because these species ofLippia produce few seeds with low percentagegermination and do not root in hydroponics,or sand systems (data not shown), we chooseto work with reproductive cells.

With the objective to obtain good cellularsuspensions, the season they were collectedwas recorded and the best size of flower buddetermined (Table 2). Ideal flower bud sizevaried from 1 to 4 mm. The small size of budshinders the use of conventional techniques andconsequently the observation of meiotic cells.In order to solve this problem, we adapted amethodology that can be useful to otherspecies with small flowers. Using this method-ology it was possible to observe manypreserved cells with litlle background staining.

In general, few abnormalities were ob-served in all the species studied, which suggests

172 L. F. Viccini et al.: Chromosome numbers in Lippia (Verbenaceae)

that these plants are stable in their meioticprocess. On the other hand, most of themproduce few seeds with a small percentage ofgermination (data not shown). According toour data, this is not a consequence of unbal-anced gametes and may be a consequence of apost-meiotic event or due to an altered phys-iological process. During three years of study,in our experimental area, we observed newindividuals originating naturally by seed ger-mination only for L. sidoides, L. diamantinensisand L. rotundifolia. The reason for theirinability to reproduce from seeds is not clearbut may explain in part the narrow geographicdistribution of some species.

Chromosome numbers varied from n=10to n=28 (Figs. 1, 2; Table 3). The majority ofthe species show chromosome haploidnumbers between 10 and 14. Comparatively,three species have high chromosome numbers:L. diamantinensis, L. rotundifolia and L. lacun-osa. Although we have few studies for thesespecies, the small number of species with highchromosome number suggests that they couldbe a result of auto- or allopolyploidy.

Although there are few cytogenetic publi-cations for Lippia, previous reports showsimilar chromosome numbers to those ob-served in the present paper. The chromosomenumber has been described for L. juncea(2n=30) and L. nodiflora (2n=36) (Junell1934), L. alba (2n=30) (Bose and Choudhury1960), L. lanceolata (2n=32) (Smith 1966),L. citriodora (2n=36) (Navaneethan 1982),L. lycioides (2n=36) and L. salviaefolia(2n=24) (Coleman 1982), L. turbinata(2n=30) (Filippa 1984) and L. geminata(2n=32) (Kumar and Dutt 1989). Of thesespecies, only L. alba, L. geminata, L. turbinataand L. salviaefolia are still classified in thegenus Lippia. The others are now classified inAloysia, Phyla or Ghinia. Among the speciesthat belong to Lippia, L. alba (n=15),L. geminata (n=16) and L. turbinata (n=15)belongs to section Zapania which is, accordingto our data, the most complex section. In thissection we observed the largest variation inchromosome number (n=10 to n=28). On the

other hand, L. salviaefolia, which belongs tosection Goniostachyum has the same chromo-some number as the other species in thepresent paper (n=12). Apparently, this sectionis more uniform than Zapania. For Rhodolip-pia we observed n=12, 13 and 14. Althoughwe did not observe the same number for all thespecies studied, the numbers are similar andsupport the idea that this is a uniform group.In section Dioicolippia, only one species(n=12) was studied and it was not possibleto extend our analysis.

Morphology, a particular inflorescencecharacter indicates that Rhodolippia andGoniostachyum are consistent sections. Rho-dolippia was considered by Salimena (2000)as a monophyletic group. On the other hand,Zapania is a confusing section and hasdifferent patterns of inflorescences. We ob-served various chromosome numbers in thissection, which is in accordance with thediversity of morphology– Zapania is the onlysection that has subsections. Our data sug-gest that a new revision of Zapania isdesirable. In this section, considering thekind of inflorescences, it is possible to recog-nize two groups: Group I (L. corymbosa,L. diamantinensis, L. lacunosa and L. rotun-difolia) and Group II (L. alba, L. turbinata,L. geminata, L. rubella and L. hermannio-ides). For group I, L. corymbosa has a smallchromosome number (n=14) – which isexactly the half number observed forL. rotundifolia and L. lacunosa (n=28). Forgroup II we observed n=10 (L. rubella) andn=13 (L. hermannioides). This reinforces theneed for revision of this section.

According to Sanders (2001), the chromo-some numbers of the Verbenaceae family arediverse, suggesting aneuploidy both in basenumbers among genera and in genomes amongspecies within genera. Depending on theimportance attached to ascending vs. descend-ing aneuploidy, any number below x=10could be the ancestral base number. The sameauthor reports that the most reasonablehypothesis is that x=5 gave rise to x=6 and7 by ascending aneuploidy and to x=10 by

L. F. Viccini et al.: Chromosome numbers in Lippia (Verbenaceae) 173

Fig. 1. Chromosome complements of some Lippia species. a. L. corymbosa; b. L. diamantinensis; c. L. filifolia;d. L. florida; e. L. glandulosa; f. L. hermannioides; g. L. lacunosa; h. L. lupulina. Bars = 10 lm

174 L. F. Viccini et al.: Chromosome numbers in Lippia (Verbenaceae)

polyploidy, which in turn gave rise to x=8 and9 by descending aneuploidy. In some genera,the presumed ancestral base number of five hasbeen retained whereas in others only basenumbers derived from multiples of five areknown. The genus Duranta, for example, ishighly aneuploid but presumably has basenumbers of 9 and 8, derived from x=10.Petrea is probably derived from x=10 due toloss resulting in x=9, followed by doubling to18, and further loss to produce x=17.

If we consider other genera of Verbena-ceae (sensu lato) it is possible to find x=7

for Verbena (Schnack 1964, Sanders 2001),x=5 for Glandularia (Schnack 1964, Poggioet al. 1993), Bouchea, Diostea and Junellia(Sanders 2001), x = 12, 13, 15, 23, 24, 25,26 for Clerodendrum (Choudhary and Roy1983, Poggio et al. 1993), x=8 or 9 forAloysia (Corazza-Nunes et al. 1995, Sanders2001), x=18 for Holmskioldia (Choudharyand Roy 1983), x=11 or 12 for Lantana(Sanders 1987, Ojha and Dayal 1993, Sinhaet. al. 1995), x=8 for Vitex (Dijkgraaf et al.1995), and Neospartum (Sanders 2001), x=9for Stachytarpheta (Sanders 2001), x=6 for

Fig. 2. Chromosome complements of some Lippia species. a. L. pohliana; b. L. pseudothea; c. L. rosella;d. L. rotundifolia; e. L. rubella; f. L. sidoides. Bars = 10 lm

L. F. Viccini et al.: Chromosome numbers in Lippia (Verbenaceae) 175

Priva and Pitraea (Sanders 2001). Somegenera probably have low base chromosomenumbers but are known only from specieswith chromosome complements at high poly-ploid levels.

In the genus Lippia, although none of thespecies with 2n=10 has been described, ourdata support the hypothesis that x=5 (Sanders2001) is the ancestral base chromosome num-ber. Other derived base chromosome numbers

Table 1. Collected data from Lippia species originated from Southeast Brazil

Species Herbariumregisternumber

Place Altitude Latitude Longitude

Lippia corymbosa Cham. 34.741 Diamantina 1262 m 18�1404800 S 43�360600 WLippia diamantinensis Glaz. 34.734Lippia filifolia Mart. & Schauerex Schauer.

34.740

Lippia glandulosa Schauer 34.744Lippia lacunosa Mart. & Schauer 42.706Lippia lupulina Cham. 34.742Lippia pohliana Schauer 39.360Lippia pseudothea (St. Hil.) Schauer 34.340Lippia rosella Moldenke 34.746Lippia rotundifolia Cham. 34.338Lippia rubella Moldenke 42.706Lippia florida Cham. 34.738 Serra do Cipo 794 m 19�2704700 S 43�3301000 WLippia hermannioides Cham. 34.739Lippia sidoides Cham. 34.737

Table 2. Collection season, ideal size of immature inflorescences and flower bud observed for chromosomenumber of some Lippia species

Species Section Collectionseason

Inflorescencesize (mm)

Flowerbud size (mm)

Lippia corymbosa Cham. Zapania Schauer Rainy 10 3Lippia diamantinensis Glaz. Rainy 10 2Lippia hermannioides Cham. Rainy 2 1Lippia lacunosa Mart. & Schauer Dry 20 2–3Lippia rotundifolia Cham. Dry 20 2–3Lippia rubella Moldenke Dry 3 1Lippia florida Cham. Rhodolippia Schauer Dry 10 3–4Lippia lupulina Cham. Rainy 8 3Lippia pseudothea (St. Hil.) Schauer Dry 10 3Lippia rosella Moldenke Dry 5 2–3Lippia glandulosa Schauer Goniostachyum Shauer Rainy 3 1Lippia pohliana Schauer Dry 3 1Lippia sidoides Cham. Rainy 4 2Lippia filifolia Mart. & Schauerex Schauer.

Dioicolippia Troncoso Rainy 7 3

176 L. F. Viccini et al.: Chromosome numbers in Lippia (Verbenaceae)

could be 6, 7, 8 and 9, originated by ascendinganeuploidy or by polyploidy followed bydescending aneuploidy. It could be a case ofa genus with low base number deduced fromspecies with chromosome complements athigher polyploidy level. In addition, somechromosome combinations can be a conse-quence of natural hybridization followed bychromosome duplication. Other possibilitiesinclude the occurrence of a second cycle ofpolyploidy (Stebbins 1971). However, it isimportant to consider that the low number ofstudied species and lack of phylogenetic anal-ysis hinders the precise determination of thebasic chromosome number. It is difficult todetermine if chromosome counts obtainedrepresent polyploidy in the individual species,or a polyploidy base number retained in thesample species.

Although we present here, for the first time,chromosome numbers of 14 species of Lippia,additional studies with other species areneeded to better understand the chromosomebase number and the phylogeny of this inter-esting and not so thouroughly studied group.

The authors thank Fundacao de Amaparo aPesquisa do Estado de Minas Gerais (FAPEMIG),Conselho Nacional de Desenvolvimento Cientıfico

e Tecnologico (CNPq) and Financiadora de Estu-dos e Projetos (FINEP) for financial support.

References

Bose R. B., Choudhury J. K. (1960) Cytologicalstudies in L. alba (Mill) N. E. Br. Bull. Bot. Soc.Bengal. 14: 71–72.

Caixeta E.T., Carvalho C.R. (2001) An improvedcytogenetic method for maize pachytene chro-mosomes. Cytologia 66: 173–176.

Choudhary S. S., Roy R. P. (1983) Karyologicalstudies and trend of speciation in some membersof Verbenaceae. Cytologia 48: 735–740.

Coleman J. R. (1982) Chromosome numbers ofangiosperms collected in the state of Sao Paulo.Rev. Bras. Genet. 3: 533–549.

Corazza-Nunes M. J., Pagliarini M. S., Silva N.,Silva I. (1995) Hexaploidy and fertility interre-lationships in Aloysia lycioides (Verbenaceae).Arq. Biol. Tecnol. 38: 627–630.

Dijkgraaf A. C., Lewis G. D., Mitchell N. D.(1995) Chromosome number of the NewZealand puriri, Vitex lucens Kirk. New ZealandJ. Bot. 33: 426–426.

Filippa E. M. (1984) El numero cromossomico deL. turbinata (Verbenaceae). Kurtziana 17:169–170.

Giulietti A. M., Menezes N. L., Pirani J. R.,Meguro M., Wanderley M. G. (1987) Flora daSerra do Cipo, Minas Gerais: caracterizacao e

Table 3. Chromosome numbers of some Lippia species according to each section

Species Subgroup Section Chromosomenumber (n)

Lippia corymbosa Cham. I Zapania Schauer 14Lippia diamantinensis Glaz. I 26Lippia hermannioides Cham. II 13Lippia lacunosa Mart. & Shauer I 28Lippia rotundifolia Cham. I 28Lippia rubella Moldenke II 10Lippia florida Cham. Rhodolippia Schauer 12Lippia lupulina Cham. 14Lippia pseudothea (St. Hil.) Schauer 13Lippia rosella Moldenke 14Lippia glandulosa Schauer Goniostachyum Schauer 12Lippia pohliana Schauer 12Lippia sidoides Cham. 12Lippia filifolia Mart. & Schauer ex Schauer. Dioicolippia Troncoso 12

L. F. Viccini et al.: Chromosome numbers in Lippia (Verbenaceae) 177

lista das especies. Bol. Bot. Univ. Sao Paulo 9:1–52.

Judd W. S., Campbell C. S., Kellogg E. A., StevensP. F. (1999) Plant systematics. A phylogeneticapproach. Sinauer Associates, Inc. Publishers,Suderland, Massachusetts.

Junell S. (1934) Zur Gynaceummorphologie undSystematik der Verbenaeen und Labiaten. Sym.Bot. Upsal. 4: 1–219.

Kumar P., Dutt B. (1989) Cytogenetic basis ofbreeding system in some verbenaceous species.Cytologia 54: 347–353.

Nash D. L., Nee M. (1984) Flora de Veracruz,Mexico, Verbenaceae. Inst. Nac. de Investigaci-ones sobre Recursos Bioticos, Xalapa, Veracruz.Mexico.

Navaneethan N. (1982) Karyomorphological stud-ies in two species of Cryptolepis R. Br. Proc.Indian Sci. Congr. Assoc. 69 (3VI): 231–232.

Ojha B. M., Dayal N. (1993) Cytological investi-gations in the genus Lantana in India. Cytologia57: 9–13.

Poggio L., Botta S. M., Greizerstein E. J., FerrariM. R. (1993) Natural hybridization in Glandu-laria (Verbenaceae). I. Evolutionary implicationsof chromosome pairing. Darwiniana 32: 77–90.

Salimena F. R. G. (2000) Revisao taxonomica deLippia L. sect. Rhodolippia Schauer (Verbena-ceae). D.Sc. Thesis. Universidade de Sao Paulo.

Salimena-Pires F. R. (1991) Verbenaceae da Serrado Cipo, Minas Gerais, Brasil. M.Sc. Thesis.Universidade de Sao Paulo.

Salimena-Pires F. R., Giulietti A. M. (1998) Florada Serra do Cipo, Minas Gerais: Verbenaceae.Bol. Bot. Univ. Sao Paulo. 17: 155–180.

Sanders R. S. (1987) Taxonomic significance ofchromosomeobservations inCaribbean species ofLantana (Verbenaceae). Am. J. Bot. 74: 914–920.

Sanders R. W. (2001) The genera of Verbenaceae inthe southeastern United States. Harv. Pap. Bot.5: 303–358.

Schnack B. (1964) Bases naturales de la separaciongenerica de Verbena y Glandularia (Verbenace-as). Comision de Investigacion Cientifica Notas.2: 1–13.

Shan F., Yan G., Plummer J. A. (2003) Karyotypeevolution in the genus Boronia (Rutaceae). Bot.J. Linn. Soc. 142: 309–320.

Sinha S., Sinha B., Sharma A. (1995) Chromosomecomposition of Lantana camara L. karyotype,basic number and DNA diversity. Nucleus 38:16–22.

Smith E. B. (1966) Cytogenetics and phylogeny ofHaplopappus section Isopappus (Compositae).Can. J. Gen. Cytol. 8: 14–36.

Stebbins G. L. (1971) Chromosomal evolution inhigher plants. Addison-Wesley Publishing Com-pany, Great Britain.

Troncoso N. S. (1974) Los generos de Verbenaceasde Sudamerica extra-tropical (Argentina, Chile,Bolıvia, Paraguay, Uruguay y sur de Brasil).Darwiniana 18: 295–412.

Addresses of the authors: Lyderson FacioViccini (corresponding author) (E-mail: lviccini@icb.ufjf.br, lyderson.viccini@ufjf.edu.br), Patrıcia MariaOliveira Pierre, Milene Miranda Praca, DeboraCristine Souza da Costa, Elisson da Costa Romanel,Saulo Marcal de Sousa, Departamento de Biologia,Universidade Federal de Juiz de Fora, Juiz de Fora,Minas Gerais, 36036-330, Brazil. Paulo HenriquePereira Peixoto, Fatima Regina Goncalves Salime-na, Departamento de Botanica, Universidade Fed-eral de Juiz de Fora, Juiz de Fora, Minas Gerais,36036-330, Brazil.

178 L. F. Viccini et al.: Chromosome numbers in Lippia (Verbenaceae)