cytogenetic analysis of lathyrus japonicus willd

INTRODUCTION

The genus Lathyrus comprises 150 species,which are distributed mainly throughout the tem-perate zones of the northern hemisphere, howev-er, some species reach tropical East Africa andSouth America. In general, scarce species of thisgenus grow in the Pacific coast of Asia, and veryfew in Japan (GAGNEPAIN 1916; OHWI 1953).Among them, the most conspicuous and distrib-uted species is the beach pea L. japonicus. Thisspecies belongs to section Orobus, which is con-sidered the most ancestral of the genus (KUPICHA

1983). Approximately, half of the species of thissection are restricted to North America and theother half live in Eurasia (HITCHCOCK 1952;BASSLER 1973). However, L. japonicus and L.

palustris grow along the shorelines of artic andsubartic regions of the old and new world, fromGreenland to Siberia and Japan (SENN 1938,BASSLER 1973). L. japonicus was cited for Chile,but according to BURKART (1935), it has been in-troduced there. This species is a perennial herb,with decumbent to climbering stems. The racemesare composed by 2-8 conspicuous red or purplishto light blue flowers loosely arranged, bloomingfrom spring to summer (HITCHCOCK 1952).

As L. japonicus belongs to the most ancestralsection of the genus (KUPICHA 1983) and it is al-so widely distributed, chromosome data may givehints to understand the evolutive tendencies fol-lowed by species of different sections of thegenus. Karyological studies in this species arescarce and contradictory, and detailed meioticanalysis is lacking (SENN 1938; SAKAI 1952; LA-VANIA and SHARMA 1980). Therefore, the presentresearch was conducted in order to provide da-ta for futures phylogenetic analysis by perform-

CARYOLOGIA Vol. 54, no. 2: 173-179, 2001

Cytogenetic analysis of Lathyrus japonicus Willd.(Leguminosae)GUILLERMO SEIJO 1, * and AVELIANO FERNANDEZ 2

1, 2 Instituto de Botánica del Nordeste (UNNE-CONICET), Casilla de Correo 209, 3400 Corrientes, Argentina. 2 Facultad deCiencias Exactas Químicas y Naturales y Agrimensura, Universidad Nacional del Nordeste, Corrientes, Argentina.

Abstract – L. japonicus belongs to section Orobus and grows in most of theseashores of the Northern Hemisphere and as adventitious in Chile. In this re-port a detailed analysis of karyotype and meiotic behaviour was carried out in or-der to provide data for phylogenetic analysis. This species presented a symmet-rical karyotype with 2n=14, 6m+8sm chromosomes. The longest and the short-est chromosomes of the complement were metacentrics while the sm were verysimilar and medium in size. NORs, determined by argentic staining, were lo-calised in secondary constriction of the long arm of the longest m chromosome.The symmetrical karyotype is in accordance with the ancestral morphologicalcharacters showed by L. japonicus. Meiotic behaviour was regular with a chias-ma frequency of 2.5 per bivalent at metaphase I. This fact agrees with the 98.2%of pollen stainability estimation. However, in ana-telophase I a bridge and frag-ment (<1%) were observed, while in interphase only fragments were detected(1%). In addition, out of plate chromosomes appeared in metaphase I and II(2%). These chromosomes segregated independently, formed micronuclei intelophase II and they became micromicrospores at the tetrad stage. These mi-cromicrospores correspond to the micro pollen grains observed in anthesis.

Key words: Lathyrus japonicus, meiosis, NORs, phylogenetic analysis.

* Corresponding author: fax ++54 3783 27131; e-mail:[email protected]

ing a detailed characterization of the karyotype,a complete analysis of the meiotic behavior andan estimation of pollen viability.

MATERIAL AND METHODS

The studied material was collected in Kashima Is-land, Shikoku, Japan, (Seijo 2353, CTES), were sev-eral populations were identified. Plants were also ob-tained from seeds in the experimental farm of the Col-lege of Agriculture, Ehime University at Matsuyamacity.

Somatic chromosome number and karyotype weremade on meristematic cells of root tips obtained fromgerminated seeds. Roots were pre-treated at 0ºC for24 h, then fixed in 5:1 ethanol:lactic acid (FERNÁNDEZ

1973) and kept in 70% aqueous ethanol. Staining wasperformed following the Feulgen’s technique and themeristems were macerated in a drop of 3% acetic or-cein before squash. Permanent slides were preparedusing Euparal.

The karyotypes were described according to chro-mosome morphology determined by the centromericindex (CI=short arm x 100/total chromosome length)as suggested by LEVAN et al. (1964). Chromosomes wereclassified in two categories: metacentrics (m): CI=50-37.5 and submetacentrics (sm): CI=37.5-25. Idiogramsand chromosome measurements were done from draw-ings using a Wild camera lucida attached on a Zeiss mi-croscope. Karyotype asymmetry has been determinedusing the ROMERO ZARCO’S (1986) intrachromosomal(A1) and interchromosomal (A2) indices, as well as bythe classification proposed by STEBBINS (1971).

Meiotic studies were performed in floral buds ofplants from natural populations. They were fixed inethanol:lactic acid (5:1) for 24h, then transferred to70% aqueous ethanol and kept in refrigerator untiluse. Pollen mother cells were stained with acetic or-cein and slides were made permanent as describedabove for mitotic analysis.

Pollen viability was estimated by the carmine glyc-erine technique and by the fluorescein diacetate–phenosaphranine technique (WIDHOLM 1972).

RESULTS

All the plants analysed have in their somaticcells 2n=14 chromosomes (Fig. 1A). Chromo-some morphology and measurements for eachpair are given in Table 1. The karyotype formula,karyotype length, mean chromosome length (LT),mean centromeric index (CI), chromosome lenghrange and asymmetry indices are detailed in Table2. Idiogram is illustrated in Fig. 2, where chro-

mosomes were arranged by morphology (m, sm)and among each category by size. The longest andthe shortest chromosomes of the complement aremetacentrics, while the submetacentric chromo-somes are very similar and of medium size. Thelongest metacentric chromosome bears in thelong arm a macrosatellite, which length is equiv-alent to the 30% of the arm. In prometaphasesand early metaphases, these satellites appeared farfrom the proximal segment of the arm, resem-bling small acentric chromosomes. Silver stainingconfirms the localization of the nucleolar orga-nizing regions (NORs) in the secondary constric-tion present in the long arm of the longest meta-centric pair (Fig. 1B). The maximum number ofnucleoli (2) observed in interphase nuclei is in ac-cordance with the number of NORs found inmetaphasic chromosomes.

According to the asymmetry indices of RO-MERO ZARCO’S (1986), the karyotype of L. japon-icus is symmetrical. Considering the classificationof STEBBINS (1971), this karyotype lay in the 2Acategory because the ratio between the longestand the shortest chromosomes is lower than 2:1and because the proportion of chromosomes witharm ratio bigger than 2:1 is lower than 0.5. Thiscategory also represents symmetrical karyotypes.

Meiotic analysis showed normal prophases. Inmetaphase I, seven bivalents were formed witha mean chiasma frequency per bivalent of 2.5,and a mean chiasma frequency per pollen moth-er cell of 17.25. Table 3 shows meiotic configura-tion of bivalents in metaphase I. Most of the chi-asmata were terminal, some interstitial while theproximal ones were very rarely observed.

Bridges and fragments were observed in near-ly 1% of anaphases I (Fig. 1C), while only frag-ments were detected in interphase (1%) and inthe second division (Fig. 1D), though with lowerfrequency.

In anaphase I, asynchrony in bivalent segre-gation occurred. One bivalent with precocioussegregation was observed (Fig. 1E) in less than1% of the cells. On the other hand, with ap-proximately the same frequency, one bivalent re-mained in the equator without segregation by thetime the other chromosomes reach the spindlepoles.

Out of plate bivalents were observed in 2% ofmetaphases I (Fig. 1F). These bivalents can segre-gate partially or completely (Fig. 1G), but almostalways the chromosomes remained as laggards anddo not reach the poles to integrate the nuclei. Out

174 SEIJO and FERNANDEZ

CYTOGENETICS IN LATHYRUS JAPONICUS 175

Fig. 1 – A, mitotic metaphase stained with Feulgen´s technique. B, Silver stained metaphase, arrows indicate NORs. In thispicture it can be also seen most of the kinetochores stained. C, Anaphase I with a bridge and fragment. D, Telophase II witha fragment. E, Asynchrony of bivalents division, this case a precocious segregation. F, Out of plate bivalent, this picture wastaken using phase contrast. G, Prophase II with four chromosomes that remained out of the nuclei. H, Prometaphases II withtwo out of plate chromosomes. I, Telophase II with two sister chromatids out of the poles. J, Phase contrast picture of a tetradwith one micromicrospore. Bar=5 µm.

of plate chromosomes were also observed inmetaphase II with a frequency of 1% of the cells(Fig. 1H). If these chromosomes were still com-posed by two chromatids, equational segregationcould occur, though not always this phenomenonhappened. In both cases, those chromosomes re-mained out of the telophase II nuclei as micronu-clei, no matter they were composed by one or twosister chromatids. Micronuclei formed by com-plete chromatids became micromicrospores. Af-ter the cytokinesis, besides the four expected mi-crospores, in 2.3% of the tetrads one or two mi-cromicrospores were observed (Fig. 1J). The esti-mation of pollen viability was high (98.2%), how-ever empty microspores (1.4%) and micromi-crospores (0.4%) were found (Table 4).

DISCUSSION

Under the name of L. japonicus Willd., sever-al species were synonimysed by FERNALD (1932),and one of those is L. maritimus (L.) Bigel. How-ever, the name L. maritimus continued being usedin some cytological publications where idiogramswere represented (SAKAI 1951; LAVANIA andSHARMA 1980). For a better discussion of the re-sults the synonymy will be considered, thereforein this report, the name L. japonicus will be theonly used.

The somatic chromosome number 2n=14 iscoincident with the number published in previ-ous reports (SENN 1938; SAKAI 1952). However,the karyotype formula found here differs from


Table 1 – Mean chromosome measurements by pairs and Levan´s types. S: short arm, L: long arm, TL: Total length and CI: cen-tromeric index.

Chromosome length (µm)

Pair S L TL CI Type

1 3.10 ± 0.17 3. 72 ± 0.49 6.82 ± 0.65 45.63 ± 2.34 m, SAT2 1.85 ± 0.22 2.90 ± 0.48 4.75 ± 0.69 39.15 ± 1.53 m3 1.73 ± 0.19 1.99 ± 0.20 3.73 ± 0.37 46.48 ± 1.70 m4 1.71 ± 0.24 3.53 ± 0.37 5.24 ± 0.45 32.68 ± 3.64 sm5 1.50 ± 0.22 3.40 ± 0.41 4.90 ± 0.50 30.63 ± 3.54 sm6 1.42 ± 0.14 3.20 ± 0.39 4.62 ± 0.47 30.75 ± 2.45 sm7 1.47 ± 0.12 2.88 ± 0.21 4.35 ± 0.33 33.72 ± 0.83 sm

Table 2 – Total chromosome length in µm (TCL), mean chromosome length in µm (CL), chromosome length range (Range), meancentromeric index (XCI) and intra (A1) and interchromosomal (A2) asymmetry indices in Lathyrus japonicus.

Karyotype formula TCL XCL Range XCI A1 A2

6 m+8 sm 34.40 4.91 37.003.73–6.82 0.40 0.20

± 3.12 ± 0.96 ± 1.03

Table 3 – Frequency of metaphase I configurations.

Configuration 7 ring 6 ring + 1 rod 5 ring + 2 rod Total

Cells 12 9 2 23Frequency 0.52 0.39 0.09 1

Table 4 – Pollen viability estimated by carmine-glycerine. Both non-stained and micrograins were considered sterile.

Pollen viability %

Stained Non-stained Micrograin

Mean 98.2 1.4 0.4Range 96.2-100 0-1.6 0-3.2

the ones previously reported. LAVANIA and SHAR-MA (1980) showed an idiogram where no refer-ence to chromosomes morphology was made,however, our measurements from that idiogramshow that the karyotype formula consist of3m+3sm+1st. This formula is almost coincidentwith the formula found in the present report.One difference is the presence of 1 st chromo-some in LAVANIA and SHARMA (1980), which isnot present in the individuals studied here. Theother difference resides in the position of thesatellite. In LAVANIA and SHARMA (1980) the sec-ondary constriction is located in the short arm ofthe longest metacentric chromosome, while in theplants studied here, it is in the long arm of thesame chromosome.

These differences may be attributed to thepre-treatment used for chromosome preparation,since in the present report 0ºC was used to de-polymerised the microtubules while paradi-clorobenzene was used in LAVANIA and SHARMA’s(1980) work. This fact may cause different chro-mosome condensation and slightly change inchromosome morphology. The total chromosomelength found here is around 30% shorter thanthat published by Lavania and Sharma (1980),while the mean centromeric index is a little high-er. This is in accordance with what may be ex-pected when the karyotype became shorter dueto pretreatmet, i.e.- the centromeric index in-crease, especially when we are dealing with meta-centric or submetacentric chromosomes. Becauseof the above mentioned reasons, the karyotypeformulae cannot be considered different, and foraccurate comparisons pre-treatment conditionsshould be standardized.

The other idiogram reported for L. japonicus(under L. maritimus) is the one of SAKAI (1952).The karyotype formula we obtained from mea-surements of the chromosomes drawn by this au-thor, is coincident with the one found here, al-though, they differ in type and position of satel-lite. SAKAI (1952) showed a microsatellite in theshortest chromosome of the complement, whilethe individuals analysed here have a macrosatel-lite in the long arm of the longest m chromosome.

The karyotype of L. japonicus is symmetricalin both the ROMERO ZARCO’s (1986) and STEB-BINS (1971) classifications. In many plant groups(e.g. some genera of Asteraceae and Ranuncu-laceae) high symmetrical karyotypes are associat-ed with ancestral characters, and it was postulat-ed that in flowering plants there is a general trend

toward the increase of asymmetry by means ofpericentric inversions and occasional transloca-tions (STEBBINS 1971). Reversible trends, howev-er, were cited for several other groups (STEBBINS

1971) like in Lycoris (DARLINGTON 1963) andsome genera of Commelinaceae (JONES 1970).According to these postulates, the symmetricalkaryotype of L. japonicus is associated with manymorphological characters considered ancestralfor the genus. Moreover, when the karyotype ofL. japonicus (section Orobus) is compared withthose reported for some species of the derivedsection Notholathyrus (BATTISTÍN and FERNÁN-DEZ 1994; KLAMPT 1997), it is evident that thekaryotype of the former is more symmetric thanthose of the laters. This increment of karyotypeasymmetry is determined by the fact that Notho-lathyrus species have only one or two m chromo-somes and in many cases also one st (subtelocen-tric). KUPICHA (1983) postulated that southamer-ican species (section Notholathyrus) are derivedcompared with the ones of section Orobus, con-sidered as one of the most ancestral sections. Ac-cording to the primary tendency postulated bySTEBBINS (1971), from symmetrical toward asym-metrical karyotypes, chromosome data supportKupicha´s hypothesis, i.e. ancestral position of L.japonicus and the derived state of southamericanspecies. However, much more data are necessaryto generalize this tendency for the genus.

Silver staining confirmed that active NORswhere only located in the conspicuous secondaryconstrictions of the longest m chromosome, whichnumber coincides with the maximum nucleolinumber in interphase nuclei. In prometaphase,the satellites are generally far from the chromo-some arms to which they belong. However, when


Fig. 2 – Idiogram of L. japonicus. : satellite.

µm

silver staining was used, the long secondary con-striction appeared as a long stained filament con-necting both elements of the chromosome arm. Atearlier stages, instead of these filaments, appearedthe nucleoli. This phenomenon was also observedin several plants species, like in Arachis (FERNÁN-DEZ and KRAPOVICKAS 1994) and in Vicia (NARAN-JO et al. 1998), being the later, the Lathyrus clos-est genus.

The regular meiotic behaviour was confirmedby the high percentage of pollen stainability. Thepresence of bridges and fragments may corre-spond to the occurrence of a chiasma in a loopformed by heterocigote chromosome region fora paracentric inversion. Although, it may also cor-respond to a U-type interchange (BRANDHAM

1969). However, as the frequency of bridge andfragment occurrence is very low, statistical analy-sis of fragment size could not be done, and there-fore we are not able to attribute the bridge andfragment to any of these phenomena. If they cor-respond to a paracentric inversion the invertedsegment should probably be so small that loopsoccurred only rarely and, therefore, dicentric andacentric chromosomes appeared in low frequen-cy.

The asynchrony of bivalent segregation is inrelation to chiasmata distribution; bivalents withonly one and terminal chiasma segregate very ear-ly, while ring bivalents with proximal chiasmatasegregate later in the anaphase. This phenome-non was observed in some other plants and asummary is given by DARLINGTON (1965). How-ever, it seems that this asynchrony has no effecton bivalent segregation. When chromosomeswere counted in telophase I, the nuclei analysedshowed seven chromosomes confirming that bi-valent segregation was regular in spite of the asyn-chrony.

Out of plate chromosomes are the cause ofmicromicrospore formation, which are evident attetrads stage. The constitution of the micromi-crospores depend on the segregation of the outof plate bivalent; they can be formed by one ortwo chromatids and this fact determines theirsize. In case that micromicrospores were com-posed by two chromatids, they always are sisterchromatids, which remained together because thechromosome did not suffer equational segrega-tion. However, when there is complete segrega-tion, all the micromicrospores formed contain on-ly one chromatid. The formation of these mi-cromicrospores reduced pollen viability, not on-

ly by their formation but also because a numberof microspores (depending on the segregation ofthe out of plate chromosomes) with normal sizewill be deficient for one chromosome. Eventhough the consequences of out of plate chro-mosomes occurrence is now known, the cause oftheir origin is poorly understood and still have tobe revealed.

Acknowledgements – This work has been sup-ported by grants of Ministry of Education of Japan(MOMBUSHO), Consejo Nacional de Investiga-ciones Científicas y Técnicas (CONICET) and Sec-retaría de Ciencia y Técnica Universidad Nacionaldel Nordeste, Argentina, which are greatly ac-knowledged. This report is part of a Doctoral the-sis that will be submitted to the Facultad de Cien-cias Exactas, Físicas y Naturales of the UniversidadNacional de Córdoba (Argentina).

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Received February 7, 2001; accepted March 12, 2001


cytogenetic analysis of lathyrus japonicus willd

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