BACKGROUND
unreduced gametes, or gametes with the somatic chromosome number , are thought to play an important role in the evolution of polyploid species  <cit> .
if two unreduced gametes unite, a fertile polyploid hybrid may form-either autopolyploid  or allopolyploid .
most plant species are now thought to be of recent or ancestral polyploid origin  <cit> .
however, little is known about the frequency of unreduced gamete formation and the genetic and environmental factors which affect unreduced gamete production in most genera  <cit> .
in solanum tuberosum and trifolium pratense, unreduced gamete production appears to be initiated by a monogenic recessive allele with other genes affecting the frequency of production   <cit> ).
unreduced gamete-producing mutants linked to defects in the meiotic cell cycle machinery have also been recently identified in model plant arabidopsis thaliana, leading to greater understanding of the mechanisms behind unreduced gamete formation  <cit> .
however, little is known about the genetic or environmental factors that influence the production of unreduced gametes within most species, or in interspecific hybrid plants.
interspecific hybrids tend to produce greater frequencies of unreduced gametes than their parents, as suggested by ramsey and schemske   <cit> .
unreduced gametes may be important in polyploid evolution via a triploid bridge  <cit> .
a triploid bridge results from the union of an unreduced gamete  with a reduced gamete .
the triploid plant  may then produce unreduced gametes in a backcross with bb pollen to produce a new polyploid species .
the triploid bridge hypothesis builds on the possibility that unbalanced interspecific hybrid plants produce more unreduced gametes than the parental species, but this has never been quantitatively tested under controlled experimental conditions  <cit> .
the triploid bridge hypothesis may provide a more likely scenario for polyploid evolution than alternative hypotheses which require two unreduced gametes to unite by chance in an interspecific hybridization event , or which require chromosome doubling to occur in somatic tissue of a seed-derived hybrid   <cit> .
unreduced gamete production may be stimulated by stressful environmental conditions  <cit> .
cold spells in the field, cool glasshouse conditions and temperature cycling in growth chambers have all been implicated in increased unreduced gamete production   <cit>  and briefly by felber   <cit> ).
in rosa, high temperatures induced spindle abnormalities causing an increase in unreduced pollen grain formation  <cit> .
however, the interaction of temperature  and genotype on unreduced gamete production in interspecific hybrids has not been evaluated  <cit> .
the brassica "u's triangle"  <cit>  species have valuable attributes for investigating the role of genotype and temperature on production of unreduced gametes in interspecific hybrids.
u's triangle includes three diploid species with genome complements aa, bb and cc  and three allotetraploid species aabb, aacc and bbcc .
interspecific trigenomic hybrids between the allotetraploid species  may easily be created  <cit> , and the hybrids often flower and produce viable gametes.
the presence of one diploid genome  in these unbalanced hybrids provides a moderate level of fertility  <cit> , which is useful for assessing the production of unreduced gametes.
unreduced gametes have been observed in a number of brassica interspecific hybrid types  <cit>  including hybrids of the brassica allotetraploids  <cit> , although the frequency of unreduced gametes in parents and hybrids has never been quantified.
no genetic or environmental factors influencing unreduced gamete production have been reported in brassica species or their interspecific hybrids.
most experiments on production of unreduced gametes have targeted male gametes  <cit> , which are more easily assessed than female gametes.
in dicotyledonous species, a structure known as a sporad is formed after meiosis in microspore mother cells, and this normally contains four daughter cells within an outer membrane and is known as a tetrad .
sporads that contain unreduced gametes are of two types.
the first type is a dyad, which contains two unreduced cells bound together within an outer cell membrane  <cit>  .
the second is a giant tetrad, which contains four unreduced gametes  <cit> .
unreduced gametes are also expressed as "giant" pollen in some species   <cit> ) including brassica  <cit> , which is useful for assessment of the frequency of unreduced gametes and their viability.
in this study, we investigated genetic and temperature effects on male unreduced gamete production in interspecific hybrids between allotetraploid species in the brassica triangle of u  <cit> .
these species are ideal for this purpose since they produce hybrid plants that flower and many hybrids produce some viable male gametes.
we evaluated male unreduced gamete production in five b. napus, two b. carinata and one b. juncea parental genotypes, and thirteen interspecific hybrid combinations among these parents.
the effect of temperature during floral development on male unreduced gamete production was investigated in a subset of five parental genotypes and five interspecific hybrid combinations.
based on previous work  <cit> , we hypothesized that the hybrids would have elevated frequencies of unreduced male gametes compared to their respective parents, and that this frequency would be influenced by genetic factors and by temperature.
RESULTS
characterization of putative interspecific hybrid plants
seed set in the  <dig> possible brassica interspecific cross combinations varied widely, and in  <dig> successful crosses there was an average of  <dig>  seeds per pollinated bud .
all three species were successful as male parents, but b. carinata was the least successful as a female parent .
the  <dig> putative hybrid plants from  <dig> combinations were assessed by genome-specific polymorphic simple sequence repeat markers, some of which were dosage-sensitive   <cit>  and mason et al.
<cit>  for details), and characterized for morphological attributes .
of these,  <dig> plants were true hybrids resulting from a reduced  gamete from both parents.
dosage-sensitive markers revealed four plants which were derived from an unreduced gamete from b. napus and a reduced gamete from b. juncea , and one plant which was derived from an aneuploid gamete from b. carinata and a reduced gamete from b. juncea .
the remaining six plants were matromorphs  .
another group of  <dig> putative hybrid plants were grown for the temperature experiment, and were all interspecific hybrids derived from a normal reduced gamete from both parents.
b. napus genotypes: n <dig>  n <dig>  n <dig>  n <dig> and n <dig>  b. carinata genotypes: c <dig> and c <dig> and b. juncea genotype: j <dig>  data are given as seeds per bud pollinated.
within-species combinations and b. carinata ♀ × b. napus n <dig> ♂ crosses were not performed .
a missing some marker loci from b. carinata parent, presumed aneuploid gamete
b two copies of alleles from female parent to one copy of alleles from male parent, presumed unreduced female gamete.
hybridity was confirmed using molecular marker analysis and phenotyping.
true hybrids from molecular marker results which had abnormal phenotypes were further characterized using ten additional dosage sensitive molecular markers.
b. juncea genotype "j1", b. napus genotypes "n1", "n2", "n3", "n4" and "n5" and b. carinata genotypes "c1" and "c2" were crossed to produce the experimental hybrid population, and a subset of the seeds produced sown out.
estimates of male unreduced gamete production through sporad observations
sporads were classified according to the number of daughter cells present within the structure: monads, dyads, triads, tetrads, pentads, hexads and heptads.
in addition, "giant sporads" were observed in some hybrids.
these tetrads were disproportionately larger than other tetrads from the same anther.
in order to estimate unreduced gamete formation from sporad observations, dyads were assumed to form two unreduced gametes, and giant sporads were assumed to produce four unreduced gametes  <cit> .
tetrads of normal size were assumed to produce four normal, reduced gametes.
in order to estimate abnormal sporad production, monads, dyads, triads, pentads, hexads and heptads were assumed to form one, two, three, five, six and seven abnormal nuclei respectively.
all eight b. juncea, b. napus and b. carinata parent genotypes produced extremely low levels of unreduced gametes based on sporad observations .
four dyads were observed out of more than  <dig>  <dig> sporads in parent genotypes, equating to an overall unreduced gamete frequency of  <dig> %.
dyads were only observed in 3/ <dig> parent genotypes: b. napus n <dig> and n <dig> and b. juncea j <dig> .
in contrast, dyads were observed in all interspecific hybrid combinations , and a few giant sporads were also observed in hybrid combinations b. juncea × b. carinata j1c <dig>  b. juncea × b. napus j1n <dig> and b. napus × b. carinata n1c <dig> .
average male unreduced gamete production in hybrids was estimated by sporad production at  <dig> % .
* 2n male gamete production was estimated by the formula /* <dig>
both dyads and giant sporads were assumed to produce unreduced  male gametes, whereas monads, dyads, triads, pentads, hexads and heptads were assumed to produce abnormal male gametes.
** significant differences between genotypes
***significant differences between genotypes
† both dyads and giant sporads were assumed to produce unreduced  male gametes, and monads, dyads, triads, pentads, hexads and heptads and giant sporads were assumed to produce abnormal male gametes.
hybrids were produced between five doubled-haploid derived genotypes of b. napus , two doubled-haploid derived genotypes of b. carinata  and one near-homozygous inbred genotype of b. juncea .
interspecific hybrid combinations are given as a combination of parent codes.
hybrid combinations with different maternal parent but the same parent genotypes were pooled after the model unreduced gametes ~ genotype + maternal parent revealed no significant effect of maternal parent on unreduced gamete production.
hybrid combinations varied in the frequency of total abnormal sporads, and the derived estimate of unreduced gamete production at the sporad stage ranged from  <dig> % in b. juncea × b. carinata j1c <dig> to  <dig> % in b. juncea × b. napus j1n <dig> .
there was no significant effect of maternal parent  on unreduced gamete production as estimated by sporad observations, based on linear mixed models.
overall, interspecific hybrid combinations produced more unreduced gametes  as estimated from sporad observations than their parent cultivars  .
the effect of temperature on unreduced gametes observed at the sporad stage
parental genotypes j <dig>  n <dig>  c <dig> and c <dig> and b. juncea × b. carinata j1c <dig> averaged less than  <dig> % unreduced male gametes across all temperature treatments, as estimated from sporad observations .
the average unreduced gamete production across temperature treatments of b. juncea × b. napus j1n <dig> and j1n <dig>  was much larger than in the parent genotypes  but there was no apparent effect of temperature on these hybrids .
however, b. napus × b. carinata n1c <dig> and n2c <dig> produced 23% and 9% unreduced gametes respectively in the cold temperature treatment , which was more than two orders of magnitude greater than in the parent species.
giant viable pollen was visibly prevalent in these hybrid genotypes under cold temperatures .
viable pollen in hybrids and parents
viable pollen in hybrids was on average larger  than viable pollen in parent species , with a greater size range   and more spherical shape.
there were small but significant differences in average pollen diameter between genotypes.
b. napus and b. carinata genotypes averaged  <dig>  to  <dig>  μm, and the b. juncea genotype averaged  <dig>  μm diameter.
giant pollen grains were observed very infrequently in the parents .
a maximum of two giant viable pollen grains were observed per parent genotype across  <dig> plants .
"giant" pollen grains were defined as viable pollen grains with a minimum diameter greater than  <dig>  times the genotype mean in the parent genotypes, and in interspecific hybrid combinations as  <dig>  times the reduced  pollen mid-parent mean diameter of the two parent genotypes of that hybrid.
this represents approximately double the volume of reduced gametes.
viable giant pollen was observed in all nine interspecific hybrid combinations which produced viable pollen .
the frequency of giant pollen production varied significantly between interspecific hybrid genotypes .
brassica juncea × b. carinata hybrids produced significantly less giant pollen  than other hybrid types .
b. juncea × b. napus j1n <dig> and b. napus × b. carinata n1c <dig> produced the most giant pollen as a fraction of viable pollen , while the remaining genotypes fell in between the two extremes .
overall, interspecific hybrids produced significantly more giant pollen than their parents .
a pollen grain was determined to be "giant" if the minimum diameter of the pollen grain exceeded  <dig>  × the mean pollen diameter observed in pollen production by that plant.
no significant differences in giant pollen production were observed between genotypes.
*** significant differences between genotypes
Ɨ numbers in the same column followed by the same letters are not significantly different .
hybrids were produced between five genotypes of b. napus , two genotypes of b. carinata  and one genotype of b. juncea .
hypothetical "giant" pollen size in the hybrids was estimated under the assumptions that a) doubling dna content would double pollen grain volume, and b) that reduced pollen in hybrids would have a maximum dna content of  <dig>  times parent  dna content.
hybrid combinations with different maternal parent but the same two parent genotypes were pooled after the model unreduced gametes ~ hybrid genotype + maternal parent revealed no significant effect of maternal parent on unreduced gamete production.
estimation of unreduced gametes derived from sporads and viable pollen
the frequency of unreduced gametes in hybrids, as estimated from the proportion of viable giant pollen compared with total viable pollen  was much higher than estimates based on observations of sporads  in interspecific hybrids .
however, there was a high proportion of pollen grains in hybrids that were not viable.
consequently, giant pollen as a fraction of total pollen production  was  <dig> %, which was similar to estimates of unreduced nuclei at the sporad stage.
there was no difference between these two measures of male unreduced gamete frequency in the b. juncea, b. napus and b. carinata parent genotypes.
evidence of meiotic abnormalities
abnormal sporads  were also observed, including monads, triads, pentads, hexads and heptads.
these were assumed to contain gametes with abnormal chromosome numbers.
abnormal sporad production in all  <dig> b. juncea, b. napus and b. carinata plants at 18°c/13°c day/night temperature was extremely low, ranging from 0% to  <dig> % .
hybrid plants produced abnormal sporads with a frequency ranging from  <dig> % to  <dig> % .
triads, pentads and hexads had nuclei with variable size: almost all pentads and hexads showed four large nuclei and one and two extra small nuclei respectively.
pentad and hexad frequencies were highly positively correlated , and triad and dyad frequencies were also positively correlated across hybrid plants , but there was no significant relationship among other sporad types.
some chromosomes were observed to be excluded from nucleus formation at telophase ii, and multiple chromosomes were often observed as laggards at anaphase ii .
parallel spindles  were also observed in some hybrid genotypes .
hybrid genotype b. napus × b. carinata n1c <dig> produced significantly more sporads with more than four nuclei  in the hot temperature treatment  than in the warm , cool  and cold  temperature treatments.
brassica napus n <dig> also produced more sporads with more than four nuclei  in the hot temperature treatment compared to the other temperature treatments .
the synchronous timing of meiosis was also deregulated in b. carinata c <dig>  b. napus n <dig> and b. juncea × b. napus j1n <dig> in response to the hot temperature treatment, with many stages of meiosis from prophase i to sporads often present in the same anther .
brassica juncea × b. napus j1n <dig> also exhibited asynchronous meiotic divisions in the warm temperature treatment.
the effects of genotype and temperature on pollen viability
hybrid combinations varied significantly in pollen viability and seed set .
all b. juncea × b. napus  and b. juncea × b. carinata  genotypes produced some viable pollen .
however, all six b. napus × b. carinata  hybrid genotypes had < 2% viable pollen, and four of these were male-sterile .
brassica juncea × b. napus  hybrids produced the most viable pollen , but b. juncea × b. carinata  hybrids produced the most self-pollinated seed .
most interspecific hybrids produced at least some flowers with developed anthers and viable pollen in all  temperature treatments.
however, b. juncea × b. carinata j1c <dig> hybrids produced entirely male-sterile flowers in the cold temperature treatment  , and the majority of flowers produced by both b. napus × b. carinata genotypes in the hot temperature treatment were also male-sterile .
some male-sterile flowers were also produced by b. napus × b. carinata genotypes under the warm and cool temperature treatments, and by b. carinata c <dig>  b. napus n <dig> and b. juncea × b. napus hybrids j1n <dig> and j1n <dig> under the hot temperature treatment.
pollen viability in the parent genotypes was not significantly affected by temperature treatment, with two exceptions: b. juncea j <dig> pollen viability was lower in the cold treatment , and b. carinata c <dig> pollen viability was lower in the hot treatment .
brassica juncea × b. carinata j1c <dig>  b. juncea × b. napus j1n <dig> and b. napus × b. carinata n2c <dig> pollen viability was also affected by temperature .
flowering time in most interspecific hybrids was intermediate between their maternal and paternal parent varieties across all temperature treatments in the temperature experiment .
the cold temperature treatment delayed flowering by  <dig> days on average within the temperature experiment .
discussion
the frequency of unreduced gametes produced by some brassica interspecific hybrids exceeded the frequency in parental genotypes by more than one order of magnitude , and there was significant variation among genotypes .
at cold temperatures, some genotypes produced unreduced male gametes at two orders of magnitude higher level than in the parents .
the frequency of viable giant pollen from unreduced gametes, as a proportion of total viable pollen, was high in hybrids due to the low viability of reduced pollen in hybrids.
under these conditions, viable unreduced gametes would be readily available for polyploid species evolution via brassica interspecific hybrids, as required by the triploid bridge hypothesis of allopolyploid evolution  <cit> .
high temperature did not stimulate formation of unreduced gametes in any parental or hybrid genotypes.
the parental genotypes produced very low frequencies of unreduced gametes , as expected from established species  with diploidized meiosis  <cit> .
the interspecific hybrid genotypes had unbalanced genome complements  most likely with univalent chromosomes at meiosis  <cit> , which may be associated with the increased formation of unreduced male gametes in these hybrid types.
the relatively low level of unreduced gametes observed in b. juncea × b. carinata  hybrids  and b. napus × b. carinata  types;  <cit> ) supports this hypothesis.
however, different genotypes of b. napus × b. juncea  and b. napus × b. carinata  hybrids produced a wide range of frequencies of unreduced gametes under the same conditions , which indicates that genetic factors inherited from parent species mediate the production of unreduced gametes.
the triploid bridge hypothesis of allopolyploid evolution has recently gained support  <cit> .
the triploid bridge hypothesis suggests that unreduced gamete yy from a diploid species with genome complement yy unites with reduced gamete z from a diploid species with genome complement zz to give triploid hybrid yy+z = yyz  <cit> .
this triploid hybrid then produces unreduced gamete yyz which unites with reduced gamete z from parent species zz to give new balanced polyploid yyz + z = yyzz.
a key factor in the triploid bridge hypothesis of allopolyploid evolution is the production of unreduced gametes by the interspecific hybrid  <cit> .
our results show that unreduced gamete production by brassica interspecific hybrids is higher than in their parent genotypes, which will promote polyploid evolution via a triploid bridge.
the hybrid pollen size distribution, expected to be distributed around a predicted 2x mean pollen size, was biased to the right  in our experiment .
this suggests that loss of univalent chromosomes conferred a viability penalty for gametes produced by the interspecific hybrids.
unreduced gametes were also more viable during pollen development than reduced gametes produced by the interspecific hybrids in our experiment, as the fraction of unreduced gametes estimated in the viable pollen fraction was much greater  than the fraction of unreduced gametes estimated in the sporad population .
this supports a similar finding of high viability of male unreduced gametes in arabidopsis  <cit> .
we also observed selection of unreduced gametes in the initial crossing event to produce four "triploid" hybrids with a diploid genome from b. napus and a haploid genome from b. juncea .
this suggests that unreduced gametes may be more viable in all interspecific crosses irrespective of ploidy level.
mechanisms of polyploidization and speciation  are expected to be conserved with increasing ploidy  <cit> , as evidenced by the multiple rounds of polyploidy found in most species  <cit> .
hence, unreduced gamete production by interspecific hybrids among brassica allotetraploids may be expected to mimic processes of unreduced gamete production in diploid brassica interspecific hybrids.
interestingly, palmer et al.
<cit>  predicted from chloroplast dna analysis that back-crossing of a novel hybrid to the paternal parent population must have occurred several times during the evolution of b. napus from progenitor species b. rapa and b. oleracea, supporting the triploid bridge mechanism of polyploid formation in this genus.
abnormal sporad production is predicted to be the result of three mechanistic processes from our study: laggard chromosomes, abnormal spindle formation and pre-meiotic doubling.
firstly, pentad and hexad production were highly positively correlated , and most sporads of this form appeared to have four larger nuclei and one or two small nuclei.
these extra nuclei are probably formed by laggard chromosomes at meiosis   <cit> ), which form micronuclei visible at the sporad stage .
the correlation between dyad and triad frequency observed in our experiment may be due to a shared meiotic mechanism.
the most likely meiotic mechanism that accounts for both dyads and triads is abnormal spindle formation.
several major gene mutations in brassica relative arabidopsis result in high frequencies of dyads and triads through the same mechanism of parallel spindles at meiosis ii   <cit> .
a single gene is thought to be responsible in solanum for fused, parallel and tripolar spindles  <cit> , which may give rise to dyads, dyads and triads respectively.
if a single gene is also responsible for abnormal spindle orientation in brassica, this may explain the correlation between dyads and triads observed in our experiment.
finally, the occasional observation of "giant" sporads in our study   <cit> ) suggests that somatic doubling of some pollen mother cells may occur prior to meiosis, although possible causes of this effect are not known.
temperature had two different effects on meiotic behavior as assessed by meiotic products at the sporad stage in our study.
firstly, the cold temperature treatment stimulated unreduced gamete production in b. napus × b. carinata interspecific hybrid combinations n1c <dig> and n2c <dig> .
secondly, the hot temperature treatment appeared to stimulate abnormal meiosis in b. napus genotype n <dig> and in b. napus × b. carinata n1c <dig>  meiosis was poorly synchronized within each anther and frequently resulted in additional nuclei or micronuclei, probably as a result of chromosome laggards or spindle abnormalities.
chromosome synapsis in meiosis has long been known to be influenced by temperature  <cit> .
recent studies in arabidopsis and yeast have implicated chromatin remodeling in response to cool temperatures, resulting in physical blocks to gene transcription  <cit> .
dna methylation has also been implicated in the cool temperature vernalization response for a number of plant species  <cit> .
as the heat and cold treatments used in this study  could potentially be reached in normal growing conditions worldwide for brassica, this highlights the need for further investigation of the role of meiotic response to temperature in polyploid fertility, speciation and establishment.
CONCLUSIONS
unreduced gametes were produced at an order of magnitude higher on average in some interspecific hybrids compared to their parent genotypes.
unreduced gametes were also more viable than reduced gametes in interspecific hybrids.
genotypic variation was present among hybrid combinations in the production of unreduced gametes in brassica interspecific hybrids, and some hybrid genotypes were stimulated by cold temperatures to produce high levels of unreduced gametes.
these results demonstrate that a source of unreduced gametes, required for the triploid bridge hypothesis of allopolyploid species formation, is readily available in brassica interspecific hybrids especially if cold temperatures are present during flowering.

 unreduced gametes, or gametes with the somatic chromosome number, are thought to play an important role in the evolution of polyploid species. if two unreduced gametes unite, a fertile polyploid hybrid may form-either autopolyploid or allopolyploid. most plant species are now thought to be of recent or ancestral polyploid origin. however, little is known about the frequency of unreduced gamete formation and the genetic and environmental factors which affect unreduced gamete production in most genera. in solanum tuberosum and trifolium pratense, unreduced gamete production appears to be initiated by a monogenic recessive allele with other genes affecting the frequency of production ). unreduced gamete-producing mutants linked to defects in the meiotic cell cycle machinery have also been recently identified in model plant arabidopsis thaliana, leading to greater understanding of the mechanisms behind unreduced gamete formation. however, little is known about the genetic or environmental factors that influence the production of unreduced gametes within most species, or in interspecific hybrid plants. interspecific hybrids tend to produce greater frequencies of unreduced gametes than their parents, as suggested by ramsey and schemske. unreduced gametes may be important in polyploid evolution via a triploid bridge. a triploid bridge results from the union of an unreduced gamete with a reduced gamete. the triploid plant may then produce unreduced gametes in a backcross with bb pollen to produce a new polyploid species. the triploid bridge hypothesis builds on the possibility that unbalanced interspecific hybrid plants produce more unreduced gametes than the parental species, but this has never been quantitatively tested under controlled experimental conditions. the triploid bridge hypothesis may provide a more likely scenario for polyploid evolution than alternative hypotheses which require two unreduced gametes to unite by chance in an interspecific hybridization event, or which require chromosome doubling to occur in somatic tissue of a seed-derived hybrid. unreduced gamete production may be stimulated by stressful environmental conditions. cold spells in the field, cool glasshouse conditions and temperature cycling in growth chambers have all been implicated in increased unreduced gamete production and briefly by felber ). in rosa, high temperatures induced spindle abnormalities causing an increase in unreduced pollen grain formation. however, the interaction of temperature and genotype on unreduced gamete production in interspecific hybrids has not been evaluated. the brassica "u's triangle" species have valuable attributes for investigating the role of genotype and temperature on production of unreduced gametes in interspecific hybrids. u's triangle includes three diploid species with genome complements aa, bb and cc and three allotetraploid species aabb, aacc and bbcc. interspecific trigenomic hybrids between the allotetraploid species may easily be created, and the hybrids often flower and produce viable gametes. the presence of one diploid genome in these unbalanced hybrids provides a moderate level of fertility, which is useful for assessing the production of unreduced gametes. unreduced gametes have been observed in a number of brassica interspecific hybrid types including hybrids of the brassica allotetraploids, although the frequency of unreduced gametes in parents and hybrids has never been quantified. no genetic or environmental factors influencing unreduced gamete production have been reported in brassica species or their interspecific hybrids. most experiments on production of unreduced gametes have targeted male gametes, which are more easily assessed than female gametes. in dicotyledonous species, a structure known as a sporad is formed after meiosis in microspore mother cells, and this normally contains four daughter cells within an outer membrane and is known as a tetrad. sporads that contain unreduced gametes are of two types. the first type is a dyad, which contains two unreduced cells bound together within an outer cell membrane. the second is a giant tetrad, which contains four unreduced gametes. unreduced gametes are also expressed as "giant" pollen in some species ) including brassica, which is useful for assessment of the frequency of unreduced gametes and their viability. in this study, we investigated genetic and temperature effects on male unreduced gamete production in interspecific hybrids between allotetraploid species in the brassica triangle of u. these species are ideal for this purpose since they produce hybrid plants that flower and many hybrids produce some viable male gametes. we evaluated male unreduced gamete production in five b. napus, two b. carinata and one b. juncea parental genotypes, and thirteen interspecific hybrid combinations among these parents. the effect of temperature during floral development on male unreduced gamete production was investigated in a subset of five parental genotypes and five interspecific hybrid combinations. based on previous work, we hypothesized that the hybrids would have elevated frequencies of unreduced male gametes compared to their respective parents, and that this frequency would be influenced by genetic factors and by temperature.  characterization of putative interspecific hybrid plants seed set in the possible brassica interspecific cross combinations varied widely, and in successful crosses there was an average of seeds per pollinated bud. all three species were successful as male parents, but b. carinata was the least successful as a female parent. the putative hybrid plants from combinations were assessed by genome-specific polymorphic simple sequence repeat markers, some of which were dosage-sensitive and mason et al. for details), and characterized for morphological attributes. of these, plants were true hybrids resulting from a reduced gamete from both parents. dosage-sensitive markers revealed four plants which were derived from an unreduced gamete from b. napus and a reduced gamete from b. juncea, and one plant which was derived from an aneuploid gamete from b. carinata and a reduced gamete from b. juncea. the remaining six plants were matromorphs. another group of putative hybrid plants were grown for the temperature experiment, and were all interspecific hybrids derived from a normal reduced gamete from both parents. b. napus genotypes: n n n n and n b. carinata genotypes: c and c and b. juncea genotype: j data are given as seeds per bud pollinated. within-species combinations and b. carinata ♀ × b. napus n ♂ crosses were not performed. a missing some marker loci from b. carinata parent, presumed aneuploid gamete b two copies of alleles from female parent to one copy of alleles from male parent, presumed unreduced female gamete. hybridity was confirmed using molecular marker analysis and phenotyping. true hybrids from molecular marker results which had abnormal phenotypes were further characterized using ten additional dosage sensitive molecular markers. b. juncea genotype "j1", b. napus genotypes "n1", "n2", "n3", "n4" and "n5" and b. carinata genotypes "c1" and "c2" were crossed to produce the experimental hybrid population, and a subset of the seeds produced sown out. estimates of male unreduced gamete production through sporad observations sporads were classified according to the number of daughter cells present within the structure: monads, dyads, triads, tetrads, pentads, hexads and heptads. in addition, "giant sporads" were observed in some hybrids. these tetrads were disproportionately larger than other tetrads from the same anther. in order to estimate unreduced gamete formation from sporad observations, dyads were assumed to form two unreduced gametes, and giant sporads were assumed to produce four unreduced gametes. tetrads of normal size were assumed to produce four normal, reduced gametes. in order to estimate abnormal sporad production, monads, dyads, triads, pentads, hexads and heptads were assumed to form one, two, three, five, six and seven abnormal nuclei respectively. all eight b. juncea, b. napus and b. carinata parent genotypes produced extremely low levels of unreduced gametes based on sporad observations. four dyads were observed out of more than sporads in parent genotypes, equating to an overall unreduced gamete frequency of %. dyads were only observed in 3/ parent genotypes: b. napus n and n and b. juncea j. in contrast, dyads were observed in all interspecific hybrid combinations, and a few giant sporads were also observed in hybrid combinations b. juncea × b. carinata j1c b. juncea × b. napus j1n and b. napus × b. carinata n1c. average male unreduced gamete production in hybrids was estimated by sporad production at %. * 2n male gamete production was estimated by the formula /* both dyads and giant sporads were assumed to produce unreduced male gametes, whereas monads, dyads, triads, pentads, hexads and heptads were assumed to produce abnormal male gametes. ** significant differences between genotypes ***significant differences between genotypes † both dyads and giant sporads were assumed to produce unreduced male gametes, and monads, dyads, triads, pentads, hexads and heptads and giant sporads were assumed to produce abnormal male gametes. hybrids were produced between five doubled-haploid derived genotypes of b. napus, two doubled-haploid derived genotypes of b. carinata and one near-homozygous inbred genotype of b. juncea. interspecific hybrid combinations are given as a combination of parent codes. hybrid combinations with different maternal parent but the same parent genotypes were pooled after the model unreduced gametes ~ genotype + maternal parent revealed no significant effect of maternal parent on unreduced gamete production. hybrid combinations varied in the frequency of total abnormal sporads, and the derived estimate of unreduced gamete production at the sporad stage ranged from % in b. juncea × b. carinata j1c to % in b. juncea × b. napus j1n. there was no significant effect of maternal parent on unreduced gamete production as estimated by sporad observations, based on linear mixed models. overall, interspecific hybrid combinations produced more unreduced gametes as estimated from sporad observations than their parent cultivars. the effect of temperature on unreduced gametes observed at the sporad stage parental genotypes j n c and c and b. juncea × b. carinata j1c averaged less than % unreduced male gametes across all temperature treatments, as estimated from sporad observations. the average unreduced gamete production across temperature treatments of b. juncea × b. napus j1n and j1n was much larger than in the parent genotypes but there was no apparent effect of temperature on these hybrids. however, b. napus × b. carinata n1c and n2c produced 23% and 9% unreduced gametes respectively in the cold temperature treatment, which was more than two orders of magnitude greater than in the parent species. giant viable pollen was visibly prevalent in these hybrid genotypes under cold temperatures. viable pollen in hybrids and parents viable pollen in hybrids was on average larger than viable pollen in parent species, with a greater size range and more spherical shape. there were small but significant differences in average pollen diameter between genotypes. b. napus and b. carinata genotypes averaged to μm, and the b. juncea genotype averaged μm diameter. giant pollen grains were observed very infrequently in the parents. a maximum of two giant viable pollen grains were observed per parent genotype across plants. "giant" pollen grains were defined as viable pollen grains with a minimum diameter greater than times the genotype mean in the parent genotypes, and in interspecific hybrid combinations as times the reduced pollen mid-parent mean diameter of the two parent genotypes of that hybrid. this represents approximately double the volume of reduced gametes. viable giant pollen was observed in all nine interspecific hybrid combinations which produced viable pollen. the frequency of giant pollen production varied significantly between interspecific hybrid genotypes. brassica juncea × b. carinata hybrids produced significantly less giant pollen than other hybrid types. b. juncea × b. napus j1n and b. napus × b. carinata n1c produced the most giant pollen as a fraction of viable pollen, while the remaining genotypes fell in between the two extremes. overall, interspecific hybrids produced significantly more giant pollen than their parents. a pollen grain was determined to be "giant" if the minimum diameter of the pollen grain exceeded × the mean pollen diameter observed in pollen production by that plant. no significant differences in giant pollen production were observed between genotypes. *** significant differences between genotypes Ɨ numbers in the same column followed by the same letters are not significantly different. hybrids were produced between five genotypes of b. napus, two genotypes of b. carinata and one genotype of b. juncea. hypothetical "giant" pollen size in the hybrids was estimated under the assumptions that a) doubling dna content would double pollen grain volume, and b) that reduced pollen in hybrids would have a maximum dna content of times parent dna content. hybrid combinations with different maternal parent but the same two parent genotypes were pooled after the model unreduced gametes ~ hybrid genotype + maternal parent revealed no significant effect of maternal parent on unreduced gamete production. estimation of unreduced gametes derived from sporads and viable pollen the frequency of unreduced gametes in hybrids, as estimated from the proportion of viable giant pollen compared with total viable pollen was much higher than estimates based on observations of sporads in interspecific hybrids. however, there was a high proportion of pollen grains in hybrids that were not viable. consequently, giant pollen as a fraction of total pollen production was %, which was similar to estimates of unreduced nuclei at the sporad stage. there was no difference between these two measures of male unreduced gamete frequency in the b. juncea, b. napus and b. carinata parent genotypes. evidence of meiotic abnormalities abnormal sporads were also observed, including monads, triads, pentads, hexads and heptads. these were assumed to contain gametes with abnormal chromosome numbers. abnormal sporad production in all b. juncea, b. napus and b. carinata plants at 18°c/13°c day/night temperature was extremely low, ranging from 0% to %. hybrid plants produced abnormal sporads with a frequency ranging from % to %. triads, pentads and hexads had nuclei with variable size: almost all pentads and hexads showed four large nuclei and one and two extra small nuclei respectively. pentad and hexad frequencies were highly positively correlated, and triad and dyad frequencies were also positively correlated across hybrid plants, but there was no significant relationship among other sporad types. some chromosomes were observed to be excluded from nucleus formation at telophase ii, and multiple chromosomes were often observed as laggards at anaphase ii. parallel spindles were also observed in some hybrid genotypes. hybrid genotype b. napus × b. carinata n1c produced significantly more sporads with more than four nuclei in the hot temperature treatment than in the warm, cool and cold temperature treatments. brassica napus n also produced more sporads with more than four nuclei in the hot temperature treatment compared to the other temperature treatments. the synchronous timing of meiosis was also deregulated in b. carinata c b. napus n and b. juncea × b. napus j1n in response to the hot temperature treatment, with many stages of meiosis from prophase i to sporads often present in the same anther. brassica juncea × b. napus j1n also exhibited asynchronous meiotic divisions in the warm temperature treatment. the effects of genotype and temperature on pollen viability hybrid combinations varied significantly in pollen viability and seed set. all b. juncea × b. napus and b. juncea × b. carinata genotypes produced some viable pollen. however, all six b. napus × b. carinata hybrid genotypes had < 2% viable pollen, and four of these were male-sterile. brassica juncea × b. napus hybrids produced the most viable pollen, but b. juncea × b. carinata hybrids produced the most self-pollinated seed. most interspecific hybrids produced at least some flowers with developed anthers and viable pollen in all temperature treatments. however, b. juncea × b. carinata j1c hybrids produced entirely male-sterile flowers in the cold temperature treatment, and the majority of flowers produced by both b. napus × b. carinata genotypes in the hot temperature treatment were also male-sterile. some male-sterile flowers were also produced by b. napus × b. carinata genotypes under the warm and cool temperature treatments, and by b. carinata c b. napus n and b. juncea × b. napus hybrids j1n and j1n under the hot temperature treatment. pollen viability in the parent genotypes was not significantly affected by temperature treatment, with two exceptions: b. juncea j pollen viability was lower in the cold treatment, and b. carinata c pollen viability was lower in the hot treatment. brassica juncea × b. carinata j1c b. juncea × b. napus j1n and b. napus × b. carinata n2c pollen viability was also affected by temperature. flowering time in most interspecific hybrids was intermediate between their maternal and paternal parent varieties across all temperature treatments in the temperature experiment. the cold temperature treatment delayed flowering by days on average within the temperature experiment. discussion the frequency of unreduced gametes produced by some brassica interspecific hybrids exceeded the frequency in parental genotypes by more than one order of magnitude, and there was significant variation among genotypes. at cold temperatures, some genotypes produced unreduced male gametes at two orders of magnitude higher level than in the parents. the frequency of viable giant pollen from unreduced gametes, as a proportion of total viable pollen, was high in hybrids due to the low viability of reduced pollen in hybrids. under these conditions, viable unreduced gametes would be readily available for polyploid species evolution via brassica interspecific hybrids, as required by the triploid bridge hypothesis of allopolyploid evolution. high temperature did not stimulate formation of unreduced gametes in any parental or hybrid genotypes. the parental genotypes produced very low frequencies of unreduced gametes, as expected from established species with diploidized meiosis. the interspecific hybrid genotypes had unbalanced genome complements most likely with univalent chromosomes at meiosis, which may be associated with the increased formation of unreduced male gametes in these hybrid types. the relatively low level of unreduced gametes observed in b. juncea × b. carinata hybrids and b. napus × b. carinata types; ) supports this hypothesis. however, different genotypes of b. napus × b. juncea and b. napus × b. carinata hybrids produced a wide range of frequencies of unreduced gametes under the same conditions, which indicates that genetic factors inherited from parent species mediate the production of unreduced gametes. the triploid bridge hypothesis of allopolyploid evolution has recently gained support. the triploid bridge hypothesis suggests that unreduced gamete yy from a diploid species with genome complement yy unites with reduced gamete z from a diploid species with genome complement zz to give triploid hybrid yy+z = yyz. this triploid hybrid then produces unreduced gamete yyz which unites with reduced gamete z from parent species zz to give new balanced polyploid yyz + z = yyzz. a key factor in the triploid bridge hypothesis of allopolyploid evolution is the production of unreduced gametes by the interspecific hybrid. our results show that unreduced gamete production by brassica interspecific hybrids is higher than in their parent genotypes, which will promote polyploid evolution via a triploid bridge. the hybrid pollen size distribution, expected to be distributed around a predicted 2x mean pollen size, was biased to the right in our experiment. this suggests that loss of univalent chromosomes conferred a viability penalty for gametes produced by the interspecific hybrids. unreduced gametes were also more viable during pollen development than reduced gametes produced by the interspecific hybrids in our experiment, as the fraction of unreduced gametes estimated in the viable pollen fraction was much greater than the fraction of unreduced gametes estimated in the sporad population. this supports a similar finding of high viability of male unreduced gametes in arabidopsis. we also observed selection of unreduced gametes in the initial crossing event to produce four "triploid" hybrids with a diploid genome from b. napus and a haploid genome from b. juncea. this suggests that unreduced gametes may be more viable in all interspecific crosses irrespective of ploidy level. mechanisms of polyploidization and speciation are expected to be conserved with increasing ploidy, as evidenced by the multiple rounds of polyploidy found in most species. hence, unreduced gamete production by interspecific hybrids among brassica allotetraploids may be expected to mimic processes of unreduced gamete production in diploid brassica interspecific hybrids. interestingly, palmer et al. predicted from chloroplast dna analysis that back-crossing of a novel hybrid to the paternal parent population must have occurred several times during the evolution of b. napus from progenitor species b. rapa and b. oleracea, supporting the triploid bridge mechanism of polyploid formation in this genus. abnormal sporad production is predicted to be the result of three mechanistic processes from our study: laggard chromosomes, abnormal spindle formation and pre-meiotic doubling. firstly, pentad and hexad production were highly positively correlated, and most sporads of this form appeared to have four larger nuclei and one or two small nuclei. these extra nuclei are probably formed by laggard chromosomes at meiosis ), which form micronuclei visible at the sporad stage. the correlation between dyad and triad frequency observed in our experiment may be due to a shared meiotic mechanism. the most likely meiotic mechanism that accounts for both dyads and triads is abnormal spindle formation. several major gene mutations in brassica relative arabidopsis result in high frequencies of dyads and triads through the same mechanism of parallel spindles at meiosis ii. a single gene is thought to be responsible in solanum for fused, parallel and tripolar spindles, which may give rise to dyads, dyads and triads respectively. if a single gene is also responsible for abnormal spindle orientation in brassica, this may explain the correlation between dyads and triads observed in our experiment. finally, the occasional observation of "giant" sporads in our study ) suggests that somatic doubling of some pollen mother cells may occur prior to meiosis, although possible causes of this effect are not known. temperature had two different effects on meiotic behavior as assessed by meiotic products at the sporad stage in our study. firstly, the cold temperature treatment stimulated unreduced gamete production in b. napus × b. carinata interspecific hybrid combinations n1c and n2c. secondly, the hot temperature treatment appeared to stimulate abnormal meiosis in b. napus genotype n and in b. napus × b. carinata n1c meiosis was poorly synchronized within each anther and frequently resulted in additional nuclei or micronuclei, probably as a result of chromosome laggards or spindle abnormalities. chromosome synapsis in meiosis has long been known to be influenced by temperature. recent studies in arabidopsis and yeast have implicated chromatin remodeling in response to cool temperatures, resulting in physical blocks to gene transcription. dna methylation has also been implicated in the cool temperature vernalization response for a number of plant species. as the heat and cold treatments used in this study could potentially be reached in normal growing conditions worldwide for brassica, this highlights the need for further investigation of the role of meiotic response to temperature in polyploid fertility, speciation and establishment.  unreduced gametes were produced at an order of magnitude higher on average in some interspecific hybrids compared to their parent genotypes. unreduced gametes were also more viable than reduced gametes in interspecific hybrids. genotypic variation was present among hybrid combinations in the production of unreduced gametes in brassica interspecific hybrids, and some hybrid genotypes were stimulated by cold temperatures to produce high levels of unreduced gametes. these results demonstrate that a source of unreduced gametes, required for the triploid bridge hypothesis of allopolyploid species formation, is readily available in brassica interspecific hybrids especially if cold temperatures are present during flowering. 
we evaluated the effect of genotype and temperature on male unreduced gamete formation in brassica allotetraploids and their interspecific hybrids. based on estimates at the sporad stage, some interspecific hybrid genotypes produced unreduced gametes at more than an order of magnitude higher frequency than in the parents. these results demonstrate that sources of unreduced gametes, required for the triploid bridge hypothesis of allopolyploid evolution, are readily available in some brassica interspecific hybrid genotypes, especially at cold temperatures. the frequency of unreduced gametes post-meiosis was estimated in sporads from the frequency of dyads or giant tetrads, and in pollen from the frequency of viable giant pollen compared with viable normal pollen. in nine hybrids that produced viable mature pollen, the frequency of viable giant pollen was much greater than in the parents. two b. napus × b. carinata hybrids produced 9% and 23% unreduced gametes based on post-meiotic sporad observations in the cold temperature treatment, which was more than two orders of magnitude higher than in the parents. the effect of genotype was assessed in five b. napus, two b. carinata and one b. juncea parents and in interspecific hybrid combinations. giant pollen was defined as pollen with more than × normal diameter, under the assumption that the doubling of dna content in unreduced gametes would approximately double the pollen cell volume. giant pollen, most likely formed from unreduced gametes, was more viable than normal pollen in hybrids.
 unreduced gametes may provide a pathway for evolutionary speciation via allopolyploid formation. we evaluated the effect of genotype and temperature on male unreduced gamete formation in brassica allotetraploids and their interspecific hybrids. the frequency of unreduced gametes post-meiosis was estimated in sporads from the frequency of dyads or giant tetrads, and in pollen from the frequency of viable giant pollen compared with viable normal pollen. giant tetrads were twice the volume of normal tetrads, and presumably resulted from pre-meiotic doubling of chromosome number. giant pollen was defined as pollen with more than × normal diameter, under the assumption that the doubling of dna content in unreduced gametes would approximately double the pollen cell volume. the effect of genotype was assessed in five b. napus, two b. carinata and one b. juncea parents and in interspecific hybrid combinations. the effect of temperature was assessed in a subset of genotypes in hot, warm, cool and cold treatments.  based on estimates at the sporad stage, some interspecific hybrid genotypes produced unreduced gametes at more than an order of magnitude higher frequency than in the parents. in nine hybrids that produced viable mature pollen, the frequency of viable giant pollen was much greater than in the parents. giant pollen, most likely formed from unreduced gametes, was more viable than normal pollen in hybrids. two b. napus × b. carinata hybrids produced 9% and 23% unreduced gametes based on post-meiotic sporad observations in the cold temperature treatment, which was more than two orders of magnitude higher than in the parents.  these results demonstrate that sources of unreduced gametes, required for the triploid bridge hypothesis of allopolyploid evolution, are readily available in some brassica interspecific hybrid genotypes, especially at cold temperatures. 