5- analysis of reproductive biology poa annua...

5- ANALYSIS OF REPRODUCTIVE BIOLOGY OF POA ANNUA

5.1. OBJECTIVE

To determine selfing and outcrossing potentials in Poa

annua by observation of morphology, anthesis, pollen tube

development, apomixis, and seed yield from self, sib, and

cross pollination.

5.2. OBSERVATIONS

Anthesis in Poa annua has been reported to proceed from

the uppermost florets of the uppermost spikelets down the

panicle (Lynch, 1903; Hackel, 1904; Hovin, 1957; and Tutin,

1957). Deviations from this pattern were noted on biotypes

in the University of Minnesota collection.

5.2.1.1. Materials and Methods

Observations of anthesis behavior of individual Poa

annua culms were made on selected greenhouse and floral pic

isolation samples. The opening of the first spikelet to

expose stigmas, pollen dehiscence, the site of anthesis,

and/or direction of anthesis on a panicle were recorded.

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5.2.1.2. Results and Discussion

Anthesis of individual culms varied both within and

among genotypes. Most genotypes underwent anthesis as cited

in section 5.2.1. Dichogamy has been observed in the

investigated selections of Poa annua, with respect to

individual inflorescences. Some samples had entire

inflorescences exhibiting protogyny (Plate 3) , although

partial dichogamy was most common (CAN321, FRA15105, NY2061,

NY4093, NY84253215, NY84253220, and OH84073), with anthesis

progressing from the distal end toward the proximal end of

the inflorescence. Stigmas of male sterile florets in the

uppermost spikelets of a panicle were exposed from less than

one hour (NY1605) to more than one day (NY6, NY118, NY11804,

and NY11805) prior to dehiscence. In some instances, an

entire panicle had stigmas from male sterile florets of each

spikelet exerted prior to any dehiscence from the same culm.

A gradation of exposed stigmas of male sterile florets

occurred between these extremes. Pollen shed typically

occurred next from perfect florets below the male sterile

florets in each spikelet.

Individual culms of some genotypes deviated from typical

anthesis patterns. Dehiscence was usually basipetal, though

progress from the middle of the panicle either basipetally

or both acropetally and basipetally was observed in some

genotypes (ITA11699, SWE01, IL85Q004, IL850006r NY10,

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NY3024, NY5041, and NY5081). This phenomenon occurs in corn

where pollen matures and dehisces from the middle of the

inflorescence outward in both directions. One inflorescence

each of OH84074, NY1861, NY4062, and NY5033 even began

anther dehiscence from the bottom spikelets, with anthesis

progressing upward. Protandry, with pollen shed prior to

stigma exertion, was rare, being observed once on NY1861.

A completely male sterile inflorescence was occasionally

observed on Poa annua (CAN321). The same plants also had

panicles with both male sterile and perfect florets. Thus,

the use of male sterility as a breeding option was not

likely with any genotypes observed. However, variation for

male sterility in Poa annua suggests that selection for

complete male sterility or maximizing male sterility

expression by manipulating environmental conditions may be

possible. Based on these observations, physical outcrossing

mechanisms exist within Poa annua.

5.2.2. Pollen Tube Growth

Pollen tube development may be examined to evaluate

compatibility of specific selfs and crosses as early as one

day after pollination. This makes it possible to visualize

compatibility and incompatibility responses for rapid

identification of successful crosses.

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Pollen tube growth was studied using staining techniques

described by Kho and Baer (1966). These rapid methods could

be used to observe pollen tube growth into styles and ovary.

The procedure was based on secondary fluorescence after

selective uptake of fluorochromes by cells. Only fixed

material was used, although fresh material may also be

examined. A careful maceration in NaOH was not necessary

with this species. The stain was 0.1 percent aniline w.s.

dissolved in 0.1N K3P04. The ultraviolet wavelength source

was 350 to 400 millimicrons.

For the observations reported here, inflorescences were

emasculated prior to anthesis, as described in 4.2.1, and

placed in isolation for self, sib or outcross pollination.

After incubation times of 2 to 48 hours after pollination,

inflorescences were fixed in CRAF for a minimum of 2 hours.

Each flower was dissected to expose the pollinated pistil.

Excised pistils were placed on a glass slide to which 1 to

2 drops of aniline blue stain was added. A cover slip was

placed over the pistils, which were then gently squashed.

Pollen germination and pollen tube growth observations were

completed using both light and fluorescent microscopy.

Callose is found in walls of pollen tubes, but not

usually in stylar tissue. Pollen tubes are outlined by

callose and callose plugs formed at irregular distances in

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the tube. The callose takes up aniline blue and fluoresces

a bright yellow green when illuminated by blue or

ultraviolet light.


Pollen tube growth was observed extending through the

stigma toward the ovary in both self and cross pollination.

Pollen grains which did not germinate, or which had pollen

tubes that stopped growing immediately upon germination,

were observed for both self and cross pollination. Self

incompatibility, as expressed by differential pollen tube

growth, was not apparent.

5.2.3. Apomixis

Apomixis has been reported for several polyploid Poa

species, such as Poa pratensis (Beard, 1973), and has been

questioned for Poa annua (Koshy, 1969,- Ellis et al 1973).

This phenomenon may be exemplified by uniform progenies of

open-pollinated parents (Hanna and Bashaw, 1987).


Seed set on selfed panicles isolated alone versus in

pairs and selfed versus outcrossed panicles were compared

using paired t-tests. Selected progeny were also observed

for morphological uniformity.

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Among genotypes examined, apomixis was not apparent.

As noted in section 4.3., single, selfed inflorescences in

isolation, set less seed per inflorescence than selfed

inflorescences paired with another culm from the same

genotype. If apomixis was occurring, seed set would be

expected to be similar between the two kinds of selfed

inflorescences. No seed was set on several isolated culms

(genotypes NY3091, NY3074, NY4045, OH84074, CAN311, and

CAN313) with exposed stigmas, but no pollen present.

Other evidence for the lack of apomixis within Poa annua

included the significant differences in seed set between

self and outcross pollination (Section 5.3.2.). Seed

production on apomictic species should be unaffected by the

genotype of the external pollen source. In addition,

progeny from open pollination of selected genotypes varied

for germination rate, leaf width, vigor, and/or color.

Apomictic populations would have identical phenotypes

because of clonal propagation. Koshy (1969) and Ellis, et

al (1973) also found none of the meiotic irregularities

associated with apomixis.

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5.3. COMPARISONS OF SELF, SIB, AND OUTCROSS SEED SET

Based on observations of the current collection of

diverse genotypes and conflicting published reports,

reproductive biology of Poa annua may not be easily

classified into either self or outcross fertilization.

Further clarification was pursued by seed yield analyses of

selected isolations.

5.3.1. Materials and Methods

Twenty-five hundred isolations included 1 to 74

inflorescences each of 180 genotypes from 67 families of Poa

annua collected from the United States, Canada, and Europe.

Appendices Al, A2, and A3 contain descriptions of accessions

and progeny. Collection sites and morphological

characterizations are included. Poa annua self, sib, and

outcrosses were made in diallel, when possible, using the

floral pic isolation method described in 4.2.1. Seed was

collected from each inflorescence at maturity and counted.

Seed counts from selected self, sib, and outcross

pollination were analyzed using t-tests. Factors related

to reproductive biology, which could affect seed set,

included potential incongruity as related to geographic

distance between origins of paired accessions (MILE); days

elapsed between panicle excision and anthesis (BPOL);

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asynchrony of anthesis between paired culms (ASYN); and

number of open spikelets per inflorescence (OSPK). Effects

of these factors were evaluated by t-tests, histogram

analyses, and multiple linear regression analyses.

Data used for regression analyses included

inflorescences least influenced by extraneous variables,

such as differing sucrose and/or 8 HQC concentrations. The

master data set, consisting of seed counts from 868 self,

sib, and outcross pollination, was randomly split in half

to form 2 subsets with 4 34 cases each. One to 17

inflorescences from 109 genotypes were unequally represented

among the 3 pollination types in one subset. The other

group included 1 to 18 inflorescences from 103 genotypes,

also unequally distributed among pollination groups. These

subsets were again subdivided into self and cross

pollination (sib pollination were not considered in these

analyses).

Several subsets were randomly and non-randoraly generated

for regression comparisons between and within pollination

types. One subset produced Files 7 and 8, while the other

subset consisted of files 9 and 10. File 5 was generated

from the combined Files 7 and 8. File 6 was similarly

produced from combined files 9 and 10. Files 11, 12, 13,

and 14 were subsampled from files 7, 8, 9, and 10,

respectively. Files 3 and 4 were stratified to have equal

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representation of self and outcross pollination,

respectively from 3 6 genotypes (1 to 16 panicles per

genotype). Files 1 and 2, generated from Files 3 and 4,

respectively, contained one pollination randomly selected

from each genotype. Self- and cross-pollinated subset

derivations are described in more detail in Table 3.

Model building initially used a step-up format.

Variables were added as long as the t-test for the last

added variable was significant at p < 0.05. Other models

tested adding variables of interest (e.g. MILE in subsets

using outcross pollination only) without regard to step-up

procedure. Square root, Freeman-Tukey, log10, inverse, and

squared transformations were tested for potential

improvement of normality and residuals. No data were

deleted as potential outliers (studentized residuals > 2.5)

since results were already screened to eliminate suspect

data.

5.3.2. Results and Discussion

5.3.2.1. Grand Means for Self, Sib and Outcross Pairings

There were no significant differences between self and

outcross seed set in a sample averaged across genotypes

(Table 4). The similarity of seed set between culms paired

with culms from itself, sib, and/or unrelated genotypes

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seemingly affirmed the previously reported self

compatibility. since genotypes could influence potential

seed production, self and outcross samples were paired by

genotype for t-test analysis which was significant to p <

0.001 (Table 5). These results suggest that Poa annua is

not an exclusively self-compatible species.

5.3.2.2. Examples of Specific Genotypes Illustrating

Outcrossing

Two plant families (NY842532## and NY16###) were fairly

consistent in producing greater numbers of seed in sib

pairings than in outcross or self pairings (Table 6). sib

seed set was as much as two times greater than some self

pollination. A finding of self seed set lower than sib or

outcross seed set may be an indication of self

incompatibility occurring within Poa annua.

The percent decrease in self seed set compared to

outcross seed production (l-[mean self/mean outcross seed

set] X 100), was calculated (Table 7). CAN320 and MN66 had

significantly reduced self seed set of 42 and 55 percent,

respectively. The relatively high percent of self

fertilization could be the result of one or more factors,

including pseudo-self compatibility (Ascher, 1976) .

A genetic basis for variation in seed number was

observed by the similar performance of full sibs (Table 6).

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In some genotypes, with data expressed as seed set perinflorescence or per open spikelet, self seed numbers weregreater than or equal to outcross seed set, with sib seedset either greater or less than self or outcross seed set(NYII0002 and NYII0005). For other genotypes (NY84253205and NY84253206), outcross seed set was greater than selfseed set with sib seed set variable in relation to selfs,but less than outcrosses. This demonstrated the range ofpossible responses (from selfing to outcrossing) amonggenotypes for seed set.

In other pairings, outcross seed set was significantlyless than self seed set (Table 8). Data included only seedset on inflorescences where anthesis asynchrony betweenpaired culms was 2 days or less to assure pollenavailability from both culms of each pair during themajority of the anthesis period. since self pollination waspossible for these culms, reduction of seed set inoutcrosses suggests a post-fertilization phenomenon, or

incongruity, which reduces seed yield.Reduction in seed yield in these pairings, then,

reflects the degree of outcrossing. To better visualizethis outcrossing phenomenon, percent outcrossing wascalculated from the formula, l-(mean seed set of outcrosspollinated inflorescences/mean seed set of self pollinatedinflorescences) X 100. For genotypes with significant t-

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tests (p < 0.05), outcross pollination seed set for NY10,

MN21, and AZ1716 was 37, 58, and 83 percent less,

respectively, than self seed set (Table 8). Other

genotypes, without statistically significant t-tests, had

varying levels of potential outcrossing from 2 percent

(AZ1719) to 53 percent (NY604).

Some accessions, such as AZ1716 and IL850006, yielded

nearly equal seeds per open spikelet for self and outcross

pollination, indicating that these genotypes could be self

compatible (Table 9) . When each accession was compared

individually to each different genotype with which it was

paired, however, variable results were observed. Outcross

seed set was greater in some cases and less in other cases.

An interesting note was that male biotypes collected nearest

to the Arizona or Illinois accessions produced the greatest

seed set.

Reproductive barriers may be active prior to

pollination, between pollination and fertilization, and

after fertilization (Levin, 1971). One such barrier may be

a by-product of divergent evolution, resulting from

incongruities in reproductive formats of isolated

populations. The longer genotypes are isolated, (e.g. by

geographic distance) , the longer they can evolve separately,

which could result in stabilization of genetic barriers.

This phenomenon of evolutionary divergence is termed

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incongruity (Hogenboom, 1975). Possible evidence of this

type of barrier in Poa annua may be found in genotypes from

Minnesota (MN44), Ohio (OH84074), and New York (NY10)

(8/16/87 date in Table 10).

Since given female genotypes, with apparently equal

outcross and self seed set, varied in outcross seed set when

different males were used, the data were separated for each

male genotype. Results of seven partial or complete

diallels appear in Table 10. Self seed set was greater than

outcross seed set in many cases, indicating the likelihood

of male interference when unrelated inflorescences were

matched for mutual anthesis. In other examples, self seed

set was less than outcross seed set, which could result from

some level of self incompatibility.

Differences between self- and outcross-pollinated seed

set may represent reduction in self seed set due to

reproductive failure of incongruous outcross fertilizations.

It may also indicate the percent of potential outcrossing

if there was no incongruity. If any seed resulted from

outcross pollination, the outcrossing percentage would be

even higher.

Genotype variation in response to selfing and

outcrossing may contribute to an explanation for the

worldwide adaptability of Poa annua. One implication for

a breeding program is that each genotype must be evaluated

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for selfing and/or outcrossing, rather than assuming that

the response of one biotype is typical for the species.

5.3.2.3. Effects of Days between Panicle Excision and

Anthesis

The last inflorescences paired on a particular date were

most likely to have unequal or uneven pollen shed. An

increasing number of days elapsed between excision and

pollination negatively affected seed production (Table 11} .

Both self and outcross seed set decreased as days between

panicle excision and anthesis increased. To avoid potential

confounding by genotypic differences in response to outcross

pollination, only self seed production was compared.

Overall, seed set was not significantly affected when 4 or

fewer days elapsed between stem excision and pollen shed.

Genotype CAN320 had seed production drop off with only 2

days elapsing between excision and anthesis, while genotypes

AZ1716 and AZ1719 were not affected by 5 days (Table 12).

Seed set decreased for most genotypes when 3 or 4 days

elapsed between excision and anthesis (Table 12).

The mean and standard error for seed production per

inflorescence decreased as days from pollen shed increased

(Table 11) . The square root of seed set and square root of

open spikelets (Tables 11, 12) stabilized the variances best

of all transformations previously examined with regression

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analysis. Results indicated the potential to either account

for fluctuations in anthesis timing with regression models,

to discard isolations when anthesis is too severely delayed,

or to be extremely critical in selecting inflorescences for

isolation. Although the latter method is preferable, it

would also limit the number of inflorescences suitable for

collection from a single plant on a given day. Clonal plant

increases could provide additional flowering culms of

similar maturity at the same time.

5.3.2.4. Effects of Anthesis Asynchrony between Paired Culms

Although every attempt was made to match culms of

similar anthesis dates, asynchrony occurred in several

paired Poa annua pic isolations. A similar situation

probably exists for bagged inflorescences and could have

profound effects on resulting seed set. The occurrence of

asynchronous anthesis, while not intentional, provided

useful information on reproductive biology.

Start of anthesis was recorded at first observation of

anther dehiscence. First stigmas usually exerted no more

than 24 hours before dehiscence. Effects of anthesis

asynchrony between paired panicles on seed set were

significant (p < .05), with highest seed yields resulting

when anthesis of paired culms occurred within 2 days of each

other (Tables 13, 14? Figures 1-4).

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Self seed set was enhanced on culms opening 1 or 2 days

prior to their matched culm (Tables 13, 14; Figures 1, 3).

More stigmas would have been receptive to pollination on the

earlier opening culm when the latter culm began dehiscing,

while stigmas from latter culms would not have been exerted

during initial dehiscence from the early-opening culm. The

first culms to open received more pollen from both culms,

compared to later-opening culms, and yielded more seed,

suggesting a pollen load effect.

For outcross pollination pairings (Tables 13, 14;

Figures 2, 4), culms which opened prior to culms with which

they were matched (culms with positive anthesis asynchrony

values), often produced greater seed than when pollen was

immediately available from the unrelated matched

inflorescence (culms with negative anthesis asynchrony

values) . This may be interpreted as an indication of

incongruity in the form of male interference with self seed

production, resulting from a post-fertilization barrier

developed through divergent evolution. These differences

were observable whether self versus outcross comparisons

were matched by genotypes (Table 14,* Figures 3, 4) or were

pooled across several genotypes unequally represented in

each pollination type (Table 13; Figures 1, 2).

Higher outcross-pollinated seed set on culms opening

before their partners (positive asynchronous anthesis

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values) may reflect self fertilization prior to potential

male interference from the unrelated mated panicle. If

there was no incongruity, this higher seed yield may also

reflect selfing plus outcrossing, since stigmas from culms

with positive asynchronous anthesis values would be exposed

to both self and outcross pollination for longer periods

than later opening culms.

Simultaneous pollen shed ensures maximal pollen load

containing a mixture of pollen from each culm (i.e. self and

outcross pollen would be present with respect to any

inflorescence). In the case of partially overlapping

anthesis, some stigmas would be exposed only to self pollen

(culms with negative anthesis asynchrony), while others

would be exposed to both self and outcross pollen (culms

with positive anthesis asynchrony). Each culm would only

be exposed to self pollen during discontinuous anthesis,

with pollen load being only a fraction of the maximum

potential (e.g. 1/2 if two culms are paired) . If male

interference occurred between self and outcross pollen,

simultaneous anthesis of paired culms would result in

reduced seed set compared to culms with negative anthesis

asynchrony.

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5.3.2.5. Effects of Number of Open Spikelets per

Inflorescence

Open spikelet numbers per inflorescence on Poa annua

biotypes were influenced genetically and environmentally for

total potential spikelets which could open and for the

number which actually opened, As stock plants matured,

panicle size decreased due to increasing vegetative

competition and clipping. Increased days elapsed between

culm excision and anthesis also seemed to reduce the number

of open spikelets per inflorescence. These open spikelets

represented total potential seed yield.

After observing variability on the first 100 isolations,

open spikelets were counted on subsequent samples. Open

spikelet numbers fluctuated among and within genotypes

(Table 15) . Computations of seeds per open spikelet

minimized effects of variability in number of open spikelets

per inflorescence. Similar diversity in floret number has

been reported between species and between and within plants

of a particular species (Mulford, 1937,* Boyle and Stimart,

1986).

5.3.2.6. Multiple Regression Analyses

Multiple regression analyses estimated the partial

influences of genetic isolation as measured by geographic

distance between origins of paired accessions (MILE) , number

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of open spikelets per inflorescence (OSPK), days elapsing

from excision to anthesis (BPOL), and anthesis asynchrony

between mated culms (ASYN) on seed set, using actual or

transformed data. Transformations which improved normality

of data included square roots of seed set per inflorescence

(SSDS) and number of open spikelets per inflorescence

(SSPK).

Data from self and outcross pollination were originally

combined because the original hypothesis was that BPOL,

OSPK, and ASYN would interact similarly with seed set

regardless of pollination type. The geographic separation

factor (MILE) was initially the only element considered to

be testable for evidence of incongruity in Poa annua.

When variability, due to incongruity based on geographic

distance between accession origins (MILE), was not

significant in many models, subsets were derived containing

either self or outcross pollination data (Tables 3, 16).

The rationale was that data sets combining self and outcross

data may have resulted in masked incongruity expression of

outcross pollination by greater representation of self

pollinations. If Poa annua is fully self compatible, models

constructed from the two subset types would have similar

included variables and coefficients.

For all subsets using self-pollination data only, the

best models contained SSDS regressed on SSPK, BPOL with an

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R2 from 0.35 to 0.39 (Table 16, Files 3, 1, 9). Similar

coefficients of the largest files (7 and 9), with no data

in common, indicated good model fit for approximately one-

third of the variability in self seed production in Poa

annua. The model for the smaller subset 3 contained the

same variables as larger sets, but different coefficients.

The smaller file with fewer samples and genotypes, reflects

a more restrictive predictive model. The remaining 65

percent variability in seed yield not explained by these

models may represent genotypic and/or self incompatibility

interactions, for example.

Data subsets using only outcross pollinations also

resulted in models containing SSDS regressed on SSPK and

BPOL (Table 16, files 4, 8 10). They differed, however, in

that MILE could also be included at a significant level (p

< 0.05) for some files (e.g. Table 16, File 10). In

addition to examples in Table 16, models were also

constructed to test whether variables which could be

incongruity indicators (MILE, ASYN, OSPK) were

interchangeable based on the order they were added.

Files 3 and 4 (self and outcross pollinated seed set,

respectively, and represented by the same genotypes)

resulted in similar models with different coefficients. No

difference in coefficients or R2 would be expected if Poa

annua were self compatible. Incongruity is one possible

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explanation for the twelve percent more variability of

outcross-produced data compared to selfed results.

Comparing self- and outcross-pollinated groups, with

files containing the same variables, 10 is more similar to

7 and 9 than 8 is. File 10 is also the only example listed

which included MILE at a significant level after the

variables SSPK and BPOL. Four to 5 percent variability may

be explained by MILE, a potential indicator of incongruity

in outcross fertilization. One explanation for not fitting

MILE into the File 8 model may be that variability

attributable to MILE in File 8 is already partly included

in the model as contributed by SSPK and/or BPOL, such that

MILE is no longer significant when added after the first two

variables.

Considering the R2 of Files 8 and 10, 4 to 12 percent

more variability is attributable to SSPK and BPOL than for

Files 7 and 9. All or part of this may be attributable to

incongruity between divergent genotypes. The low percentage

indicated here does not necessarily conflict with examples

in Table 7, since incongruity expression here may be

confounded with factors other than MILE (e.g. by genotypes

which are cross compatible, or by divergence resulting from

separations other than geographic distance). Asynchronous

anthesis (ASYN) , which may also be an indicator for

incongruity, did not fit outcross data files unless either

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SSPK or BPOL were omitted. There may have been confounding

of variability assignable to ASYN with SSPK or BPOL as was

suggested for MILE. Another reason may be low variation in

ASYN in the smaller subsets generated, since ASYN did fit

in models of larger data sets with self- and outcross-

pollinated seed set.

5.3.2.6.1. Days between Excision and Anthesis

The days elapsed between culm excision and anthesis

contributed significantly to most regression models,

regardless of pollination type. Of the 4 factors included

in the multiple regression modeling, this factor was the

only one inversely correlated with seed set. No

transformations of this variable improved models from any

data sets.

5.3.2.6.2. Asynchronous Anthesis between Paired Culms

Regression models generally were not improved by

including the ASYN variable in data subsets containing self

pollinations only, possibly due to minimal asynchronous

anthesis between selfed culms. Since these culms opened

similarly, they were often much easier to pair for

simultaneous anthesis, Asynchronous anthesis may also be

less critical to selfed seed production compared to

outcrossed seed production. In the latter case, incongruity

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may be expressed when asynchrony occurs and may affect

apparent pollen load.

Using data from outcross pollinations, the asynchronous

anthesis element was added to some models at a significant

level before and/or after number of open spikeiets or days

between excision and anthesis, partially contributing to an

explanation of seed set variability. Interestingly, either

anthesis asynchrony or geographic distance could be added

to models based on data from outcross pollinations, but not

both. This result would be logical if both variables are

at least partially attributable to the same phenomenon, e.g.

incongruity. The low contribution to R2 (0,01 or less for

some models) when these variables were added after number

of open spikeiets could be due to some of the same

incongruity factored into number of open spikeiets.

Subsequent isolations were chosen to minimize anthesis

asynchrony between matched culms and days elapsing between

excision and anthesis. A model, using 2 00 self and cross

pollinations generated from these isolations, resulted in

a low amount of variability attributable to these factors

(R2 < 0.20), indicating success in decreasing undesirable

seed set variation.

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5.3.2.6.3. Number of Open Spikelets per Inflorescence

For most data sets, open spikelet numbers, or their

square roots, were most highly correlated to seed set.

There were reservations, however, about using number of open

spikelets per inflorescence in the regression modeling. If

number of open spikelets affected total possible seed set,

then use of this factor would be appropriate in the modeling

process. Alternatively, if an inflorescence had a maximum

seed set confounded by one or more factors, such as source-

sink capacity or incongruity, then factoring of numbers of

open spikelets could erroneously separate equal seed set or

result in overestimation of predicted seed set. Any

incongruity expressed through an interaction with open

spikelet number and seed set per inflorescence could also

affect identification of incongruity with other variables,

such as asynchronous anthesis and geographic separation.

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5- analysis of reproductive biology poa annua...

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