CARYOLOGIA Vol. 63, no. 3: 304-313, 2010 *Corresponding author: e-mail: [email protected]Microsporogenesis in three wild species of the genus Antirrhinum L. (A. litigiosum Pau, A. pulverulenthum Lazaro and A. subbaeti- cum Güemes, Sánchez and Mateu) Nikolova Vesselina 1, * and Isabel Mateu-Andrés 2 1, *Maritsa Vegetable Crops Research Institute, 32, Brezovsko shose Str. 4003 Plovdiv, Bulgaria, phone: + 359 32 991 227; fax: + 359 32 650 177. 2 Instituto Cavanilles de Biodiversidad y Biología Evolutiva y Departamento de Biología vegetal, Universidad de Valencia, C / Dr. Moliner 50, E-46100- Burjassot, Valencia; e-mail: [email protected]Abstract — The diploid chromosome number (2n = 2x = 16) in three wild species A. litigiosum Pau, A. pul- verulenthum Lazaro and A. subbaeticum Güemes, Sánchez & Mateu, belonging to the genus Antirrhinum was cytologically observed. Genetic stability of the simultaneous meiotic division type was found in these natural growing snapdragons. The synchronicity at the initial meiotic stages and at tetrad and pollen creation was high and disappeared with the diakinesis initiation. Reflecting the systematic position of the studied species into Antirrhinum, the performed investigation mani- fested similarity: in the presence of univalents and quadrivalent configurations parallel with bivalents at the early meiotic stages; in the type of disturbances at metaphase and anaphase stages; in the tetrad formation and pollen characteristics; in the particularity of the tapetal cell division. There were dissimilarities in the bivalent chiasma formation and localisation and in the level of the meiotic dis- orders. Seven different chromosome associations were observed at diakinesis and metaphase one in A. pulveru- lenthum PMCs, while in the other two Antirrhinum species they ranged between 12 and 13. The highest value of ring bivalents, total and mean chiasma number per bivalent, was found in A. pulverulenthum PMCs, while the lowest value of these characteristics was evaluated in the A. litigiosum cells. The chromosome behaviour during anaphase stages showed more changes in A. subbaeticum PMCs and the most regularity in the A. pul- verulenthum cells, which were reflected in the value of the pollen fertility (83.7% and 98.4% respectively). The established specify and differences demonstrated genetic diversity between A. litigiosum, A. pulverulenthum and A. subbaeticum genomes. Key words: A. litigiosum, A. pulverulenthum, A. subbaeticum, chiasma, chromosome behaviour, meiosis. INTRODUCTION The genus Antirrhinum (2n = 2x = 16) has some 25 species, all of them perennial diploid plants, distributed in the Mediterranean area (SUTTON 1988). The Iberian Peninsula is their centre of diversification, as 23 species are nat- ural from this area. The origin of the genus is considered as recent (GÜBITZ et al. 2003), most of whose species have diverged within the last 1myr (GÜBITZ et al. 2003). As a model system, the ornamental species A. majus L. has been genetically and molecularly well investigated (SCHMIDT and KUDLA 1966; STUBBE 1966; LAI et al. 2002; SCHWARZ-SOMMER et al. 2003a, 2003b). Some cytological (SPARROW et al. 1942; STEIN 1942; BERGER et al. 1951; STUBBE 1996) and molecular cytogenetic investigations (ZHANG et al. 2004; XUE et al. 2009) have been performed to reveal the origin and the nature of A. majus. According to the publications the domesticated snapdragon showed meiotic in- stability and variability. SPARROW et al. (1942) described simultaneous type of the microsporo-
Microsporogenesis in three wild species of the genus Antirrhinum L. (A. litigiosum Pau, A. pulverulenthum Lazaro and A. subbaeti-cum Güemes, Sánchez and Mateu)
Nikolova Vesselina1,* and Isabel Mateu-Andrés2
1,*Maritsa Vegetable Crops Research Institute, 32, Brezovsko shose Str. 4003 Plovdiv, Bulgaria, phone: + 359 32 991 227; fax: + 359 32 650 177.2Instituto Cavanilles de Biodiversidad y Biología Evolutiva y Departamento de Biología vegetal, Universidad de Valencia, C / Dr. Moliner 50, E-46100- Burjassot, Valencia; e-mail: [email protected]
Abstract — The diploid chromosome number (2n = 2x = 16) in three wild species A. litigiosum Pau, A. pul-verulenthum Lazaro and A. subbaeticum Güemes, Sánchez & Mateu, belonging to the genus Antirrhinum was cytologically observed. Genetic stability of the simultaneous meiotic division type was found in these natural growing snapdragons. The synchronicity at the initial meiotic stages and at tetrad and pollen creation was high and disappeared with the diakinesis initiation.Refl ecting the systematic position of the studied species into Antirrhinum, the performed investigation mani-fested similarity: in the presence of univalents and quadrivalent confi gurations parallel with bivalents at the early meiotic stages; in the type of disturbances at metaphase and anaphase stages; in the tetrad formation and pollen characteristics; in the particularity of the tapetal cell division. There were dissimilarities in the bivalent chiasma formation and localisation and in the level of the meiotic dis-orders. Seven different chromosome associations were observed at diakinesis and metaphase one in A. pulveru-lenthum PMCs, while in the other two Antirrhinum species they ranged between 12 and 13. The highest value of ring bivalents, total and mean chiasma number per bivalent, was found in A. pulverulenthum PMCs, while the lowest value of these characteristics was evaluated in the A. litigiosum cells. The chromosome behaviour during anaphase stages showed more changes in A. subbaeticum PMCs and the most regularity in the A. pul-verulenthum cells, which were refl ected in the value of the pollen fertility (83.7% and 98.4% respectively). The established specify and differences demonstrated genetic diversity between A. litigiosum, A. pulverulenthum and A. subbaeticum genomes.
Key words: A. litigiosum, A. pulverulenthum, A. subbaeticum, chiasma, chromosome behaviour, meiosis.
INTRODUCTION
The genus Antirrhinum (2n = 2x = 16) has some 25 species, all of them perennial diploid plants, distributed in the Mediterranean area (SUTTON 1988). The Iberian Peninsula is their centre of diversifi cation, as 23 species are nat-ural from this area. The origin of the genus is considered as recent (GÜBITZ et al. 2003), most
of whose species have diverged within the last 1myr (GÜBITZ et al. 2003).
As a model system, the ornamental species A. majus L. has been genetically and molecularly well investigated (SCHMIDT and KUDLA 1966; STUBBE 1966; LAI et al. 2002; SCHWARZ-SOMMER et al. 2003a, 2003b). Some cytological (SPARROW et al. 1942; STEIN 1942; BERGER et al. 1951; STUBBE 1996) and molecular cytogenetic investigations (ZHANG et al. 2004; XUE et al. 2009) have been performed to reveal the origin and the nature of A. majus. According to the publications the domesticated snapdragon showed meiotic in-stability and variability. SPARROW et al. (1942) described simultaneous type of the microsporo-
MICROSPOROGENESIS IN ANTIRRHINUM 305
genesis in intra and inter – varietal A. majus tetraploids. ERNST (1938) (in STUBBE 1966), de-scribed cytokinesis following the telophase one, producing dyad formation in A. majus meio-cytes. Reduction of the meiosis, in some cases to a single division, was observed by BERGER (1951) in the ornamental Antirrhinum, as the dyads had the diploid chromosome number. This process was similar to an endomitotic division. ZHANG et al. (2004) investigated centrometric sequences, chromosome identifi cation and recombination in the A. majus genome. According to XUE et al. (2009) molecular genetic analyses showed that an extra telocentric chromosome contain-ing AhSLF-S1 is responsible for self-compatible expression in A. majus and A. hispanicum hybrid progeny.
The cross ability between the domesticated and wild species in the genus Antirrhinum and between some representatives of natural grow-ing snapdragons are also well known. There is very limited information in the literature about the wild species chromosome morphology and meiotic behaviour of their chromosomes.
Three species were selected, A. litigiosum Pau, A. pulverulenthum Lazaro and A. subbae-ticum Güemes, Sánchez & Mateu, each species belonging to different subgroups of the genus Antirrhinum (ROTHMALER 1956). A. pulveru-lenthum and A. subbaeticum are closer to each other, than to A. litigiosum, as the former species are included into separate series of subsection Kickxiella, while the last one belongs to subsec-tion Antirrhinum.
The aim of the present study was to evaluate particularity of the microsporogenesis in wild Antirrhinum species and to gain insights about the behaviour of chromosomes on the cytology of the genus. That knowledge would allow us to know if a different level of similarity exists in the bivalent confi gurations and in meiotic chro-mosomes behavior, refl ecting A. litigiosum, A. pulverulenthum and A. subbaeticum systematic relationships.
MATERIALS AND METHODS
Microsporogenesis and some bivalent charac-teristics were investigated in pollen mother cells of three Antirrhinum species – A. litigiosum, A. pulverulenthum and A. subbaeticum in the labo-ratory of the Department of Botany – Valencia University, Spain in 2009. The A. litigiosum plants were grown in the fi eld and in greenhouse
conditions during 2008/2009 respectively, while A. pulverulenthum and A. subbaeticum plants were cultivated in a greenhouse (2009). For the evaluation of the meiotic chromosome behav-iour, fl ower buds of different size were fi xed in 3:1 ethanol:glacial acetic acid fi xative during the blossoming period. The slides were obtained by temporary squash preparations and stained with 4% acetocarmine solution.
The bivalent characteristics were described at pachytene, diplotene and early diakinesis stages. The chromosome associations and frequencies of meiotic confi gurations were recorded at di-akinesis and metaphase one. Some disturbances of the chromosome behavior were noted during metaphase, anaphase and telophase stages.
Pollen fertility and some particularity of the pollen germination were evaluated by acetocar-mine staining and the unstained pollen grains were recognized as sterile.
RESULTS
Cytological study confi rmed the diploid chro-mosome number (2n = 2x = 16) in investigated wild Antirrhinum species – A. pulverulenthum and A. subbaeticum (GÜEMES 2009). We observed the same chromosome number in A. litigiosum pollen mother cells (PMCs), which relates to the postulate, that the Antirrhinum species are pe-rennial diploid plants, possessing n = x = 8.
In the performed research a simultaneous type of the microsporogenesis was found in the PMCs of the studied A. litigiosum, A. pulveru-lenthum and A. subbaeticum plants.
The level of synchronicity of the initial meiot-ic stages (leptotene, pachytene and diplotene) in the wild species was high and it was possible to observe the same phase in the four anthers of the fl oral bud. The synchronicity disappeared with diakinesis initiation, as different stages, from diakinesis to tetrad formation could be seen in the anthers of the fl ower bud, including, in a few cases the microspore stage also. Most frequently, tetrad formation and subsequent phases of the microgametogenesis were signifi cant synchronic processes.
Formation of two nucleoli in the meiocyte nucleus often occurred in plants from the inves-tigated species.
Chromosome associations and bivalent confi g-urations - The data from Tables 1 and 2 demon-strates different chromosome associations and the average frequency of chromosome confi gura-
VESSELINA and MATEU-ANDRÉS306
tions at diakinesis and metaphase one (MI). The homologous chromosomes paired and formed 8 bivalents (II): in about 70-73% (2008/2009) of the pollen mother cells in A. litigiosum (7.47 and 7.59II, respectively per cell) (Fig. 1a, d); in 82.1% of the A. pulverulenthum PMCs (7.69II per cell) (Fig. 2d); and in 84.1% of the A. sub-baeticum cells (7.70II per cell) (Fig. 3a). The data from Table 3 shows that the greenhouse condi-tions did not infl uence the chromosome pairing in A. litigiosum meocytes and that were no clear differences between A. litigiosum plants devel-oped in the fi eld and these grown in greenhouse, at least concerning the PMC percentages with various chromosome associations. Chromosome conjugation demonstrated higher variability among the plants cultivated in greenhouse than
in those growing in the fi eld. The morphology of the bivalent confi gura-
tions did not differ signifi cantly in these three Antirrhinum species (Fig. 4, Fig. 5 and Fig. 6). Formation of X shaped bivalent, with single proximal chiasma localization was described in all species, as this bivalent visually was the short-est. A signifi cant part of the heterochromatin was located in the telomere region of the A. li-tigiosum homologous chromosomes in this pair. The presence of a butterfl y type bivalent and a bivalent with specifi c shape (Fig. 4; Fig. 6 a), both of them looking the longest, with three chi-asmata formation, were observed in the PMCs of the investigated snapdragons. The other fi ve pairs presented two chiasmata, positioned in dif-ferent chromosome regions.
TABLE 1 — Chromosome associations at diakinesis and metaphase one in pollen mother cells (PMCs) of A. litigiosum, A. subbaeticum and A. pulverulenthum plants.
TABLE 2 — Average frequency of chromosome confi gurations at diakinesis and methaphase one per pollen mother cell (PMC) of A. litigiosum, A. subbaeticum and A. pulverulenthum plants.
Species Chromosome confi gurations per a pollen mother cell
II I III IV fragments chromatidsA. litigiosum - 2008 7.47 1.00 0.0 0.015 0.0 0.0 - 2009 7.59 0.77 0.0 0.014 0.002 0.0
A. pulverulenthum 7.69 0.54 0.0 0.020 0.0 0.0
A. subbaeticum 7.70 0.52 0.005 0.009 0.0 0.06
MICROSPOROGENESIS IN ANTIRRHINUM 307
The data regarding to the bivalent type and total chiasmata in the part of the cells at diaki-nesis and MI is shown in Table 4. The highest
values of ring bivalents (5.78), total chiasmata (14.04) and mean chiasma number per bivalent (1.76) were found in A. pulverulenthum PMCs,
Fig. 1 — Microsporogenesis and pollen grain in A. litigio sum plants. (a) Diakinesis with 8II; (b) diakinesis with 5II+6I; (c) diakinesis with 6II+1IV; (d) MI with 8II; (e,f) irregural MI; (g) normal AI; (h,i,j,k) al with disturbances; (l,m) two types of tetrads; (n) polyad formation; (o) germinated pollen grain.
Fig. 2 — Microsporogenesis, tapetal cells and pollen grain in A. pulverulenthum plants. (a,b) Diakinesis with bivalents and quadrivalents; (c) diakinesis with 7II+2I; (d) MI with 8II; (e) normal AI with 8-8 chromosomes; (f,g,h,i) AI, TI, MII, AII with disturbances; (j,k,l) two types of tetrad formation, polyad and triad; (m) binuclear tapetum (n) 4 nuclei, result of mitotic division after endomitotical cycles in tapetal cell; (o) germinated pollen grain.
VESSELINA and MATEU-ANDRÉS308
while the lowest value of these characteristics was established in the A. litigiosum cells (4.17, 12.11 and respectively 1.51).
Abnormalities of chromosome pairing - In case of incomplete synapsis or of early chiasma termi-nalisation, between 2 and 12 univalents (I) were formed at diakinesis and MI (average frequency 1.0 and 0.77I per cell, 2008/2009 respectively) in A. litigiosum plants (Fig. 1b) (Table 1 and Table 2). During the diakinesis in this species, the per-centage of the cells with univalents was higher (34.8 and 28.2 - 2008/2009) compared to the one established at MI (11.9 and 15.9). Univalents (from 2 to 10) were observed in 13.8 and 16.4% of the PMCs in the A. subbaeticum (Fig. 3b, c, d, f) and respectively in A. pulverulenthum (Fig. 2c) plants (0.52 and 0.54I per cell). The percentage of the PMCs with univalents and the number of the univalents in the cells of these two species logically increased in MI. Formation of a qua-drivalent confi guration or a trivalent + univalent
was established in a few cells of the plants from the investigated species (Fig. 1c; Fig. 2a, b; Fig. 3d). Early orientation of some univalents (up to 4) toward the spindle poles (Fig.1e; Fig. 3e) and 1 or 2II outside of the metaphases plate (Fig. 1f) were described also in MI. Two additional small fragments to the 8II in A. litigiosum PMCs, early division of the univalents to the chromatids and loss of the chromosome orientation to the spin-dle poles in A. subbaeticum cells are among the other disturbances during the above mentioned stages.
Disturbances at anaphase stages - The specifi c greenhouse conditions infl uenced unfavorably the running of anaphase one (AI) in A. litigio-sum plants (Fig. 1h, i, j, k), as the major PMCs of the buds collected from the fi eld presented normal chromosome distribution toward the spindle poles (Fig. 1g). The large number of the PMCs was with equal 8-8 chromosome orien-tation at AI in A. pulverulenthum and A. sub-
TABLE 4 — Some characteristics of the bivalents in A. litigiosum, A. subbaeticum and A. pulverulenthum PMCs.
Studied Ring Rod Total Chiasmata Univalents
Species PMC bivalents bivalents chiasmata per bivalent number mean number mean number mean number mean number
TABLE 3 — Chromosome associations at diakinesis and metaphase one in pollen mother cells (PMCs) of A. litigiosum plans growing in different conditions.
PMC total number Chromosome associations in the fi eld – 2008 PMCs in the greenhouse – 2009 PMCs
baeticum cells (Table 5) (Fig. 2e; Fig. 3g). Some abnormalities (non separated bivalent, up to 6 lagging chromosomes, unbalanced number of the chromosomes in the spindle poles, bridge formation, early division of the univalents to the chromatids) were defi ned in 8.1% of A. pulveru-lenthum PMCs (Fig. 2f, g) and in 15.3% of A. subbaeticum cells (Fig. 3h, i) (Table 5).
The meiotic chromosome behaviour at sec-ond metaphase and anaphase (MII and AII) changed the most in A. subbaeticum PMCs (Fig. 3j, l, m), as in the AII, parallel with the afore-mentioned irregularities, three-pole orientation of the chromosomes and division of the chroma-tides to chromonems were seen in 20% of the
cells. Less irregularities were noted in the A. pul-verulenthum cells (Fig. 2h, i) at these stages.
Microspore formation and pollen characteristic - In all three wild species, the microsporogenesis fi nished with cytokinesis after second telophase (TII) and tetrad formation in about 91-99% of the cells (Table 6). Two types of the tetrads - iso-bilateral or tetraederic (Fig. 1l, m; Fig. 2j; Fig. 3n) were established together in the PMCs of the studied Anthirinun plants. Meiotic abnor-malities originated dyad, triad and polyad for-mation (Fig. 1n) in 15.3% of the PMCs in some A. litigiosum plants (No 1-2, 1-4) growing in the greenhouse. Those abnormalities resulted in the reduction of the pollen fertility (87.6%). Similar
Fig. 3 — Mikrosporogenesis and pollen grain in A. subbaeticum plants. (a) Diakinesis with 8II and two nucleoli; (b) diakinesis with 6II+4I; (c) diakinesis with 6II+1III+1I; (d) MI with 5II+1IV+2I; (e,f) irregular MI; (g) normal AI with equal distribution of chromosomes; (h,i) AI with disturbances; (j) irregural MII; (k) normal AII; (l,m) AII with irregularities; (n) two types of tetrads; (o) pollen grain with generative cell.
Fig. 4 — Bivalents of A. litigio sum pollen mother cells.
VESSELINA and MATEU-ANDRÉS310
type of sporad creation was observed very rarely in the other two species (Fig. 2k, l).
Irregularities of the meiotic chromosome be-havior were refl ected in the lowest pollen fertility (83.7%) in the A. subbaeticum plants (Table 6). A relation between the lesser amount of meiotic disturbances and the highest value of the pollen fertility (98.4%) was found in the A. pulveru-lenthum.
The pollen grains of the studied Antirrhinum species were tricolporate with small generative cell, while two sperm formation occurred in the pollen tube (Fig. 1o; Fig. 2o; Fig. 3o). The pres-ence of two sperms in the pollen grains was ob-served very rarely.
Tapetal cells division - The fi rst tapetal divi-sion, appearing as normal mitotic one (Fig. 7a, b, c) in all three species, resulted in the forma-tion of binucleate cells (Fig. 2m). After several endomitotic cycles, tetraploidal (Fig. 7d, e) and octoploidal (Fig. 7f) chromosome number in the
poles of the tapetal cells was established. During the microsporogenesis, some tapetal cells had four nuclei (Fig. 2n). Very frequently there were more than two nucleoli in the interphase tapetal nuclei (Fig. 2m, n).
DISCUSSION
In the performed study, a simultaneous type of the microsporogenesis was found in the PMCs of the studied A. litigiosum, A. pulverulenthum and A. subbaeticum plants, growing naturally or under greenhouse conditions. Cytokinesis oc-curred after second telophase, producing tet-rad formation. The results demonstrate genetic stability of the meiotic division type in the three wild Antirrhinum species. According to BERGER (1951) and ERNST (1938) (in STUBBE 1966), the domesticated snapdragon showed instability and variability of the meiotic division type, which al-
TABLE 6 — Microspore stage and pollen fertility in A. litigiosum, A. subbaeticum and A. pulverulenthum plants.
TABLE 5 — Chromosome behaviour at anaphase one and second anaphase in pollen mother cells (PMCs) of A. litigio-sum, A. subbaeticum and A. pulverulenthum plants.
Meiotic stages
A. litigiosum PMC A. pulverulenthum PMC A. subbaeticum PMC number % number % number % A n a p h a s e o n ewithout disturbances 428 94.1 136 91.9 183 84.7with: - bridge formation 6 1.3 3 2.0 6 2.8- 1 non separated bivalent 5 1.1 1 0.7 2 0.9- from 1 to 6 lagging chromosomes 12 2.6 2 1.4 22 10.2- unbalanced orientation of chromos. 3 0.7 2 1.4 0 0.0- early division to chromatides 1 0.2 4 2.7 3 1.4 S e c o n d a n a p h a s ewithout disturbances 167 94.9 91 98.9 86 79.6with: - bridge formation 1 0.6 1 1.1 4 3.7- from 1 to 3 lagging chromosomes 7 4.0 0 0.0 4 3.7- unbalanced orientation of chromos. 1 0.6 0 0.0 6 5.6- three poles orientation of chromos. 0 0.0 0 0.0 6 5.6- polyad orientation of chromosomes 0 0.0 0 0.0 1 0.9- division of chromatid to chromonem. 0 0.0 0 0.0 1 0.9
MICROSPOROGENESIS IN ANTIRRHINUM 311
Fig. 5 — (a) Pachytene; (b) diplotene; (c) diakinesis and bivalents of A. subbaeticum pollen mother cells.
Fig. 6 — Schematic imagine of (a) A. Litigiosum, (b) A. pulverulenthum, (c) A. subbaeticum bivalents.
lows to hypothesize a spontaneous mutagenic origin of A. majus. The simultaneous and succes-sive type of the microsporogenesis in the PMCs of Phaseolus vulgaris mutant, induced by gamma ray Co60 irradiation was observed by NIKOLOVA and PORJAZOV (1991).
Crossover is cytologically observable as chi-asmata (JANSSENS 1924). The chiasmata also are responsible for: the protection of the chromatid connection until the metaphase stage; correct bipolar orientation of the bivalents at MI; equal segregation of the univalents toward the spindle poles at AI. According to DARLINGTON (1931), localisation of chiasmata occurred in particu-lar regions along a bivalent, as the frequency of chiasma formation depends on chromosome length. JOHN and LEWIS (1965) concluded that the chiasmata decrease or are completely absent in the heterochromatin chromosome regions. The three wild Antirrhinum species showed an X shaped bivalent with one proximally chiasma localisation (Fig. 4, Fig. 5 and Fig. 6). It is pos-sible that the short length of the chromosomes and the signifi cant concentration of the hetero-chromatin observed in their telomeric regions did not allow distal chiasma formation in this bivalent. It is also possible that the DARLINGTON (1931) suggestion explains three chiasma forma-tions in the observed two longest bivalents. The other fi ve bivalents had two chiasmata situated in different chromosome regions, and according to which, some of the pairs of these wild species did not look similar. Some bivalents were asym-
metric, having one chiasma occurs in the termi-nal part of the two homologous and the second one between distal and respectively centromeric regions. There were genome differences be-
VESSELINA and MATEU-ANDRÉS312
tween the studied wild Antirrhinum species in the shape of some bivalents, as high similarity was seen in the other chromosome pairs. In con-trast to the pachytene, the analysed PMCs did not show high variability in the bivalent charac-ter at diplotene phase and the genetic factor was probably involved in the regulation of chiasma creation and localisation. The genetic determi-nation of the chiasma formation has been sup-posed by DARLINGTON (1937), REES (1955), REES and THOMPSON (1956), ROSEVEIR and REES (1962) and REES and JONES (1977).
The cytological study generally manifested similarity of the meiotic chromosome behav-iour in the studied snapdragons, refl ecting their systematic belonging to the genus Antirrhinum. There were some particularity and dissimilari-ties between these species, which showed their genome diversity.
Data on the performed investigation dem-onstrated the highest average value of ring bi-valents (5.78), total chiasmata (14.04) and mean chiasma number per bivalent (1.76) in A. pul-verulenthum PMCs. In this species, at diakinesis and metaphase one, seven different chromosome associations were observed, while in the other two Antirrhinum species they ranged between 12 and 13.
Early chiasma terminalisation produced the lowest average number of ring bivalents, total and mean number of chiasmata per bivalent (4.17, 12.11 and 1.51 respectively) in the A. liti-giosum cells. As a result, the highest percentage of PMCs with univalents and the highest univalent frequency (around 1 per PMC) were observed (Fig. 1b). Part of the homologous chromosomes in this species at diakinesis looked decondensed, without connection, but probably some of them were not real univalents and the chiasmata were not completely missing.
The data from tables 1 and 2 presented more similarity in the chromosome associations of the A. pulverulenthum and A. subbaeticum plants, refl ecting their systematic relationship.
Quadrivalent formation in about 1.4-1.6% of the cells was described in the observed three snapdragons (Fig. 1c; Fig. 2a, b; Fig. 3d) and ac-cording to their origin, it is possible to suppose either the presence of heterozygous transloca-tion, probably concerning very small regions of non-homologous chromosomes, or some homol-ogy between small terminal segments.
Early division of the univalents to chromatids was found in a few cells in initial meiotic phases, as only in A. subbaeticum cells, in some cases
the chromatids divided to the chromonems. The telophase nuclei included chromatids or chromonems parallel with the chromosomes, as this genetic instability produced unviable gam-ete formation.
The meiotic chromosome behaviour during the anaphase stages changed the most in A. sub-baeticum PMCs and was most regular in the A. pulverulenthum cells. The specifi c greenhouse conditions unfavorably defi ned the running of the anaphase one in A. litigiosum plants. A high-er percentage of cells with disturbances (10.7) and a larger specter of chromosome abnormali-ties (non-separated bivalent, up to 6 lagging chromosomes, unbalanced number of chromo-somes in the spindle poles, bridge formation, early division of the univalents to the chroma-tids) (Fig. 1h, i, j, k) was observed, compared to those established in the buds collected from the fi eld (only individual meiocytes appeared with a bridge or with one non-separated bivalent).
Similarities between the three studied Antir-rhinum species were found in the tetrad forma-tion and in the pollen germination. Microsporo-genesis in these species fi nished with two types of tetrad creation - isobilateral or tetraederic (Fig. 1l, m; Fig. 2j; Fig. 3n), according to the second metaphase plate position (parallel or perpendicular). The pollen grains were tricol-porate, presenting a small generative cell with large nucleus. Two sperm formation occurred in the pollen tube, which is specifi c for these wild species. MILOCANI et al. (2006) observed a small generative cell of Tillandsia seleriana Mez pollen grain, as a large vegetative cell occupied the re-maining volume of the grain. The generative cell possessed a large nucleus and did not show any plastids in cytoplasm.
The described abnormalities in the chromo-some behaviour during microsporogenesis in the observed species related with the pollen fertility values – the lowest in A. subbaeticum (83.7%) and the highest in A. pulverulenthum plants (98.4%).
The fi rst tapetal division (Fig. 7a, b, c) in all three species resulted in formation of binucleate cells. BERGER (1951) suggested about the fi rst ta-petal division of A. majus, either normal mitosis or endomitosis, similar to that described by GEI-TLER (1939), STEIN (1942) and WITKUS (1945). Simultaneously, these two nuclei went through several endomitosis, as the chromosome in the later endomitosis visually looked shorter than those in the earlier mitotic and endomitotic di-visions. A few tapetal cells had four nuclei. Ac-
MICROSPOROGENESIS IN ANTIRRHINUM 313
cording to PAPINI et al. 1999, the dissolving of the tapetal cell wall and shrinkage of the whole cell and nuclei generally produce degeneration and mortality of the tapetum in Tillandsia albida Mez et Purpus and Lobivia rauschii Zecher. It is possible that either the dissolving of the cell walls may cause the presence of more then two nuclei in the tapetal cell, or after several endomi-tosis the mitotic division may occur without cy-tokines in some cells.
The cytological characterization performed on the species A. litigiosum, A. pulverulenthum and A. subbaeticum is the fi rst step and will initi-ate future investigation, concerning the meiotic chromosome behavior in their remote hybrids. That will allow us to obtain more information about the genome similarity or differences be-tween these three wild snapdragons, refl ecting their systematic relationship.
REFERENCES
BERGER C.A., WITKUS E.R. and JOSEPH T.C., 1951 — Tapetal cell and meiotic divisions in Antirrhinum majus L. and Linaria vulgaris Hill. Caryologia, IV, 1: 110-115.
DARLINGTON C.D., 1931 — Chiasma formation and chromosome pairing in Fritillaria. Proc. Interna-tional Botany Congress, 189, 1.
DARLINGTON C.D., 1937 — Recent advances in cytol-ogy. London Chrurchill.
GEITLER L., 1939 — Die Enstehung der polyploiden Somakerne der Heleropteren durch Chromosomen-teilung ohne Kerneteilung. Chromosma, 1: 1-22.
GÜBITZ T., A. CALDWELL and A. HUDSON, 2003 — Rapid molecular evolution of Cycloidea-like genes in Antirrhinum and its relatives. Molecular Biol-ogy and Evolution, 20: 1537-1544.
GÜEMES J., 2009 — Antirrhinum. In Castroviejo et al. (eds.) Flora Ibérica 13, www.rjb.csic.es/fl oraiber-ica
JANSSENS F.A., 1924 — La chiasmatypie dans les in-sects. Cellule, 34: 135-359.
JONH B. and K.R. LEWIS, 1956 — The meiotic system. Protosplasmalogia, 6, Wien.
LAI Z., W. MA, B. HAN, L. LIANG and Y. ZHANG et al., 2002 — An F-box gene linked to the self-in-compatibility (S) locus of Antirrhinum is expressed specifi cally in pollen and tapetum. Plant Molecular Biology, 50: 29-42.
MILOCANI E., A. PAPINI and L. BRGHIGNA, 2006 — Ultrastructural studies on bicellular pollen grains of Tillandsia seleriana Mez (Bromeliaceae), a neo-tropical epiphyte. Caryologia, 59(1): 88-97.
NIKOLOVA V. and I. PORJAZOV, 1991 — Pollen mother cell meiosis in mutant forms of garden bean (Pha-
seolus vulgaris L.) resistant to Xanthomonas camp-estris var. phaseoli. Genetics and Plant Breeding, 24, 6: 422-425 (Bulgaria).
PAPINI A., S. MOSTI and L. BRGHIGNA, 1999 — Pro-grammed-cell-death events during tapetum develop-ment of angiosperms. Protoplasma 207: 213-221
REES H., 1955 — Genotypic control of chromosome behaviour in rye. I. Inbred lines. Heredity, 9: 93.
REES H. and R. JONES, 1977 — Chromosome Genetics. Edward Arnold, London.
REES H. and J. THOMPSON, 1956 — Genotypic control of chromosome behaviour in rye. III. Chiasma fre-quency in homozygotes and heterozygotes. Hered-ity, 10: 409-424.
ROSEVEIR J. and H. REES, 1962 — Fertility and chro-mosome pairing in autotetraploid rye. Nature, 195: 203.
ROTHMALER W., 1956 — Taxonomische Monographie der Gattung Antirrhinum. Feddes Repert.spec.nov.regni.vegetabilis. Beiheft 136, Akademie-Ver-lag Berlin.
SCHMIDT T. and J. KUDLA, 1966 — The molecular structure, chromosomal organization and interspe-cies distribution of a family of tandemly repeated DNA sequences of Antirrhinum majus L. Genome, 39: 243-248.
SCHWARZ-SOMMER Z., B. DAVIES and A. HUDSON, 2003a — An everlasting pioneer: the story of Antirrhinum research. Nat. Rev. Genetics, 4: 657-666.
SCHWARZ-SOMMER Z., E. ANDRADE SILVA, R. BERNDT-GEN, W.E. LONNING and A. MULLER et al., 2003b — A linkage map of an F2 hybrid population of Antirrhinum majus and A. molle. Genetics, 163: 699-710.
SPARROW A.H., M.L. RUTTLE and B.R. NEBLE, 1942 — Comparative cytology of sterile intra - and fertile inter-varietal tetraploids of Antirrhinum majus L. American Journal of Botany, 29: 711-715.
STEIN E., 1942 — Cytologischen untersuchungen an Antirrhinum majus mutant canrroidea. Endomi-tosen-Entwicklung. Chromosoma, 2: 308-333.
STUBBE H., 1966 — Genetik und Zytologie von Antir-rhinum L. sect. Antirrhinum. Veb Gustav Fischer Verlag Jena.
SUTTON D.A., 1988 — A revision of the tribe Antir-rhineae. Oxford University Press, London.
WITKUS E.R., 1945 — Endomitotic tapetal cell divi-sions in Spinacia. American Journal of Botany, 32: 326-330.
XUE Y., Y. ZHANG and Q. YANG, 2009 — Genetic fea-tures of a pollen-part mutation suggest an inhibitory role for the Antirrhinum pollen self-incompatibility determinant. Plant Mol Biol., 70: 499-509
ZHANG D., Q. YANG, W. BAO, Y. ZHANG, B. HAN, Y. XUE and Z. CHENG, 2004 — Molecular cytogenetic characterization of the Antirrhinum majus genome. Genetics, 169: 325-335.
Received February 19th 2010; accepted May 31th 2010
