The bracts are green and extend beyond the tepals. Notably, pistillate flowers possess bracts that are comparatively longer than those of staminate flowers . Pistillate flowers have five tepals, which are rounded or blunt with an apical notch . Each pistillate flower has one superior gynoecium. Fruit are single seeded utricles and become wrinkled when dry . Seeds are dark reddish-brown to black, lens-shaped, and 1.0 to 1.3 mm long. Staminate flowers have five stamens and five acute tepals with a dark green midrib . Based on these observations, the floral diagrams and formulae for Amaranthus palmeri pistillate and staminate flowers are shown in Fig. 4. In summary, a staminate flower has one to three bracts, five tepals in a quincuncial sequence, and five stamens. A pistillate flower has one to three bracts, five tepals in a quincuncial sequence and one superior gynoecium made of a single ovary containing one ovule.In the axils of the young leaves, inflorescence primordia appear soon after the reproductive transition. The initiation of tepal primordia is the first indication of floral organogenesis . The diameter of the central meristem above the tepal primordia is about 30 µm. The meristem broadens, and five stamen primordia are initiated as hemispherical mounds in a spiral pattern along the outer rim of the floral meristem . A protuberance forms at the center of the meristem shortly thereafter . This protuberance has similarmorphology and position to the early stages of carpel initiation in pistillate flowers. Stamen primordia soon become elliptical, and anthers have elongated sufficiently to arch inward and cover the floral apex . Differentiation into anther and filament is obvious when the stamen primordia broaden, commercial grow racks which is accompanied by a change of shape . The lobes will later develop into the pollen sacs. The filament remains short and starts to elongate just prior to anthesis. The diameter of A. palmeri pollen is about 31 µm and the number of apertures per grain is about 25.

The centrally-located putative carpel primordium shows little change in size, with the approximate diameter ranging between 45 µm and 70 µm, and the approximate height between 30 µm and 50 µm . This structure remains undeveloped and surrounded by the bases of the filaments rather than progressing to form a fertile mature gynoecium .With the transition to flowering, five tepal primordia are initiated first, in a weakly spiral pattern on the flower apical meristem . At the center of the flower meristem, the gynoecium arises, consisting of one carpel primordium. The primary carpel primordium is differentiated into an annular ovary wall primordium around a central single ovule primordium . The ovary wall grows up from the ring primordium, forming two style primordia . Each style primordium elongates and forms one long style. The gynoecium elongates by intercalary growth and forms the ovary that later closes post-genitally at the top . Styles elongate and a stigmatic region differentiates along the adaxial surface. The stigmatic branches are unifacial and papillate. After fertilization and seed maturation, stigmas and styles desiccate . The single ovule of A. palmeri is anatropous or campylotropous . We also found somemature pistillate flowers with three styles . We speculate that the protuberance located near the inwardly elongating two styles may develop and become the third style. Further research is needed to confirm this.This is the first study of floral development in Palmer amaranth and documents the developmental differences between staminate and pistillate flowers. Flower and plant sex in A. palmeri can be distinguished morphologically by sharpness of bracts and shape of tepals prior to anthesis. Bracts are sharper in female plants than those in male plants. Tepals are of obtuse shape in pistillate plants while tepals are acutely pointed in staminate plants. Based on the progression of organogenesis in flower development, we also classified floral development of A. palmeri into ten stages. The distinction between the two flower types only became apparent at stage 4 with the formation of stamen primordia .

Pistillate flowers only develop female reproductive structures whereas staminate flowers develop both female and male reproductive organs initially. This finding places A. palmeri in the type I group of unisexual species.Unisexual species are believed to have evolved from hermaphroditic ancestors through a variety of evolutionary processes . Both androecial and gynoecial primordia are often initiated in the unisexual flower of dioecious plants. The developmental halt of the organs of the opposing sex, which occurs at different phases of development in different species, gives rise to unisexual flowers . In Silene latifolia , the development of male and female organs is identical until stage 5 when stamen development is terminated after anther differentiation in pistillate flowers, but in staminate flowers rudimentary gynoecia continue to grow throughout flower development . In Celtis iguanaea, termination of gynoecium development in staminate flowers happens earlier than the arrest of androecium development in pistillate flowers . Results of A. palmeri flower development show staminate flowers initially develop both androecium and gynoecium, but eventually become functionally male with a central bulge instead of a fertile gynoecium whereas pistillate flowers do not go through a hermaphroditic stage. This implies that the sex of pistillate flowers is determined earlier than staminate flowers. Sex determination of pistillate flowers may occur before flower initiation or even before floral evocation because pistillate flowers only develop a fertile gynoecium with no anther primordia at any stage. Differences in the timing of residual organ termination suggests that the developmental program that suppresses gynoecium development in staminate flowers is probably independent from the program that stops stamen growth in pistillate flowers . This observation implies the existence of different mechanisms involving different genes, which does not corroborate the hypothesis that stage of organ abortion in male and female flowers is temporally correlated within species .

Similarly, Grant et al. found that genetic lesions in the Y-linked genes which prevent gynoecium growth have no effect on stamen development in S. latifolia.It is interesting that S. latifolia with heteromorphic sex chromosomes exhibits a similar pattern of late floral sex differentiation as A. palmeri which lacks visually distinct sex chromosomes. Sex in A. palmeri is controlled by a male specific genome region perhaps with an XY system without dimorphism between X and Y . However, sex-determination loci have not been identified in these taxa. Early cytogenetic studies in S. latifolia show that there are three regions identified on the Y chromosome related to sex expression: a gynoecium suppression region and two promotion regions of stamen development . The speculation was made that the male Y chromosome linked genes with gynoecium-suppressing functions are expressed in staminate flowers before the first sex-specific difference appears . Miller and Kesseli suggests the Y chromosome in S. latifolia remains quite similar to the X chromosome, probably with the main differences in the primary sex determination regions. The slow differentiation of staminate flowers in A. palmeri may relate to the allocation of resources to male compared with female reproductive functions . According to Bateman’s Principle , female fitness tends to be limited by resources needed to fill seeds and fruits, dry racks for weed whereas male fitness is more likely to be limited by mating opportunities. Reproduction is more costly to females than males; sex allocation theory therefore predicts that the environmental conditions favorable for plant growth should induce femaleness whereas resource-poor environments induce maleness . This strategy is very important for A. palmeri to establish a population after colonizing a new habitat. In expressing an initial stage of hermaphroditism, the A. palmeri pattern seems to differ from a few other species studied in this family. For example, in dioecious Spinacia oleracea L. flowers appear to be unisexual from inception and only initiate development of either stamens or pistils . In Amaranthus hybridus, a monoecious species, flowers bear only either a primary gynoecium primordium or stamen primordia at early stages. Our result suggests the evolution of A. palmeri from a cosexual ancestral state to dioecy is at an early or intermediate stage, which is in line with the findings from the whole-genome sequencing analysis . By identifying a potential Y chromosome in the A. palmeri draft genome sequence, Neves et al. suggested that dioecy in A. palmeri is at an intermediate evolutionary state with a young Y chromosome. Our study is the only detailed floral developmental study of a dioecious member of the Amaranth genus and may offer unique insights into the evolution of sex determination in plants and into the development of novel control strategies to control dioecious weeds such as changing the sex through manipulation of the environment . Amaranthus palmeri is of major interest because it represents one of the most important agronomic weeds in North America. The species can evolve resistance rapidly and repeatedly to herbicides . Understanding the reproductive biology of weeds can aid in the development of agronomic strategies and to reduce herbicide resistance and weed populations. Mesgaran et al. found water stress induced a female sex expression of Palmer amaranth resulting in female to male ratio of 1.78:1, which was significantly different from 1:1 sex ratio.

This finding is consistent with our observation that males produce rudimentary gynoecia and can be potentially bipotent . If there are more females than expected due to water stress, they could have been produced from males since males initially produce female organs. Further, water stress reduced the synchrony in anthesis between the two sexes of A. palmeri mainly through a delay in male anthesis whilst females were almost unaffected . To better decipher the above observations, future research should investigate the floral development in male and female plants under water stress conditions.Comparing floral organogenesis of both sexual types, we found that Amaranthus palmeri staminate flowers initially develop both androecium and gynoecium, but eventually become functionally male with a central bulge instead of a fertile gynoecium, whereas pistillate flowers only develop a fertile gynoecium with no anther primordia at any stage. Timing of residual organ termination varies across the two sexes in A. palmeri. Sex determination in pistillate flowers probably occurs before flower initiation or even before floral evocation, which is much earlier than staminate flowers. The results of our floral development study suggests that the evolution of A. palmeri from a cosexual ancestral to complete dioecy is still in progress since males exhibited transient hermaphroditism whilst females produced strictly pistillate flowers. It is very important to understand the reproductive biology of this species as it can help develop weed control methods and reduce herbicide resistance. Techniques such as skewing the sex ratio and reducing the flowering overlap between the two sexes have been offered to reduce seed output in A. palmeri and perhaps other dioecious weeds for which a better understanding of weed reproductive biology is required.Current agriculture systems rely heavily on the use of herbicides and tillage for weed management but both have negative impacts on the environment and farm productivity in longterm use while herbicide-resistance is increasing . An integrated approach to weed management which incorporates ecological principles and involves using multiple tactics that vary in timing and type of control is needed to reduce the probability of rapid weed adaptation to management practices . Moreover, weed management decisions should aim to prevent soil seedbank inputs rather than just minimize current yield loss for agricultural profitability . While there have been many studies focused on weed seed biology and seedbank management , research focused on reducing weed seed production by manipulating flowering and seed set is lacking. At anthesis, pollen grains landing on a compatible stigma may germinate and produce pollen tubes which grow through the style to fertilize the ovules. Later pollen tubes are unable to enter fertilized ovules as the first pollen tube’s sperm cell delivery causes an immediate block to further fertilizations . During its journey inside the pollen tube, the generative cell of a pollen grain divides into two male gametes. One gamete fuses with the egg cell nucleus and the other fuses with the pair of central cell nuclei. Together, these two fertilization processes are referred to as double fertilization , which is unique to angiosperms . The fertilized egg cell will give rise to an embryo while the fertilized central cell will give rise to the endosperm .