Phenology of Sclerocarya birrea ( A . Rich . ) Hochst . Provenances

Phenology study was conducted to assess 22 genotypes of Sclerocarya birrea (A. Rich.) Hochst, collected from West, Eastern and Southern Africa. Assessments were done on time for bud onset, flower opening, leaf flush, fruit set, fruit maturity period and fruit production. Highly significant (P< 0.001) variations between provenances were obtained in all the phenological traits assessed including variations between sexes in time from flower bud set to anthesis. There was flowering overlaps and synchrony between provenances and sexes with males flowering earlier than females. So far two subpopulations have emerged within the trial referred to as early and late flowering genotypes. The early flowering included provenances from Mozambique, and Swaziland while the late flowering encompassed provenances from Malawi, Zimbabwe, Namibia, Tanzania and Zambia. Fruit maturity period ranged from 76±2 to 192±15days. The early flowering genotypes flowered, fruited and matured between August and January while the late genotypes flowered and fruited from September to May. There were highly significant (P≤0.001) variations in fruit yield of S. birrea provenances between 2016/2017 and 2017/2018 seasons with the former being more productive than the later confirming that S. birrea fruit yield is not constant across seasons due to seed mating effects. There were very strong positive relationship ranging r=0.81 to r=0.78 between leaf flush, bud set, flower opening and fruit set significant at (P<0.001). Some trees classified as females in the first year as based on flowers were found have male flowers which calls for more detailed investigations into this sex change behaviour.


1-Introduction
Sclerocarya birrea (A.Rich.)Hochst is an important multipurpose indigenous fruit tree of high social, cultural and commercial value in Africa [1,2].The species is common and widespread throughout the semi-arid, deciduous savannas in sub-Saharan Africa [3].In Malawi, the species occurs mainly in hot dry areas between 500 and 1,000m above sea level with mean annual rainfall of 900-1,000mm and mean annual temperature of 22 -23°C [4].Ecological distribution, biological description and uses of S. birrea are well explained by Mkwezalamba et al. [5] and Nyoka et al. [6].Briefly, the tree of S. birrea is famously known because of its fruits which are used to make products of economic importance such as Amalura beer and the oil which are sold worldwide.
In order to sustain the supply of Marula fruit production, domestication initiatives started in the 1990s in SADC region [7] and in Israel [8].An international provenances trial of 22 genotypes of Sclerocarya birrea was established in Mangochi in 1999 to evaluate growth, tolerance to pest and diseases, adaptability (survival), fruit productivity and quality in order to select genotypes with superior traits and well adapted to local conditions for the domestication.The purpose of this study was to examine the phenology (inflorescence development) of the 22 provenances of S. birrea.Phenology reveals the transformational mechanisms of plant life, timing of recurrent life cycle stages (phenophases).The timing of flowering phenology within and between populations is of fundamental biological importance because of its influence on total seed production, dispersal, pollinator abundance and fitness [9].More importantly the synchronization of flowering phenology between sexes is crucial in ensuring gene mixture through increased outcrossing and effective fertilization [10].
Since the establishment of the International Provenance trial of S. birrea, published papers on survival percentage [11], vegetative growth and fruit production [6,12,13], morphometric fruit traits [5], have been written on the trial.But no study has been conducted on the trial to address issues of phenology.The information on the phenology of S. birrea genotypes is pertinent for successful selection for domestication and breeding program [14].It is also important in assessing barriers to seed and fruit set [15].
In order to study the phenology of S. birrea we aimed to answer the following questions: (1) Do all the geographic populations have the same flowering time and length of flowering?(2) Are there variations between populations in time for bud set, flower opening, leaf flush and fruit set? (3) Do variations exist between sexes from flower bud set to anthesis?(4) Is there flowering synchrony between male and female trees?(5) Does provenance vary in the duration for fruit maturation?(6) Are there variations in fruit productivity between seasons?

2-1-Study Site and Experimental Material
The study site and experimental material has been well described by Mkwezalamba et al. [5] and Nyoka et al. [6] Briefly, the trial was established in February 1999 in the Palm Forest Research, Mangochi (14°28'S, 35°14'E, and m above sea level) (Figure 1) with twenty populations of S. birrea ssp.caffra and one population of S. birrea ssp.birrea (Table 1).The trial was laid out as a randomized complete block design replicated four times.The plot size was a 20tree row plot.The spacing was 5 m between row plots and 4 m between trees within a plot.There were, therefore, trees per population in the test.Weather during the study period are given in Figure 2.

2-2-Data Collection
At age 18 of S. birrea, the trial was assessed for bud onset, flower opening and leaf flush for both male and female individual trees except for the bulked provenance from Tanzania.Assessments on fruit set, fruit maturity period and productivity were done on fruiting female trees.Fruit productivity assessment was done in two consecutive fruiting seasons (2016/2017 and 2017/2018).The assessment was done using the procedure described by Khanduri et al. [16].

2-3-Statistical Analysis
The data on time for leaf flush, bud formation to anthesis, fruit onset to maturity and productivity were tested for normality and homogeneity.After the two criteria were met the data were subjected to analysis of variance (ANOVA) using MINITAB 17.0 with provenances and block as fixed factors.Differences between treatments means were separated using Fischer's least significant difference (LSD) at the 0.05 level.Pearson correlation coefficients were calculated to determine the relationship between leaf flush, bud set, flower opening and fruit set.A summary of the research methodology is presented in Figure 3.

3-1-Variations among Provenances of S. Birrea in bud Set, Bud Opening, Leaf Flush, Fruit Set, Fruit Maturity Period and Production
The results on the parameters studied (bud set, bud opening, leaf flush, fruit set, fruit maturity period and fruit production) are presented in Tables 2 and 3 and Figures 4 and 5.The results indicate that there were significant (P<0.001)differences among provenances on all the parameters studied.The first provenances to set buds included Marracuene, Magunde, Moamba, Kalanga and Missira within a range of 242±3.7 and 256±1.9days.

3-2-Variations between Sex in Days from Bud to Anthesis
Variations between males and females in number of days from bud set to flower opening were highly significant (P<0.001)(Figure 6).The males reached anthesis within the range of 5.0±0.4 and 8.0±0.8 days while females reached anthesis within 4.0±0.3 and 7.0±2.0days.The males that took the longest period (8 days) to reach anthesis included Ohangwena and Chikwawa provenances while Missira had the shortest period of about 5 days.Amongst the female trees, Mudzi, Biriwiri and Kalanga had the longest period of 7 days between bud set and flower opening while Magamba, Mangochi and Siavonga took the shortest period within a range of (4.0±0.0 to 5.0±1.00)days.

3-3-Relationship between Leaf Flush and Phenology Characteristics
There were significant (P<0.001)positive correlation between leaf flush and phenology characteristics (bud set, flower opening and fruit set).The correlation between leaf flush and phenology characteristics (bud set, flower opening, and fruit set) were 0.80, 0.81 and 0.78 respectively.

4-1-Variations in Time for Bud Set to Flower Opening (Anthesis)
The results of this study have shown wide variability between provenances of S. birrea in bud set to anthesis with an extended flowering period of about 57 days starting from August and ending October.The time for flower opening corresponded with time of bud set implying that provenances that were first to set buds also reached anthesis earlier but with a lot of tree to tree variations.The flowering initiation time of August is consistent with what [12]  age three to four years on the same provenances trial.The long flowering period among different genotypes of S. birrea enhanced overlapping between different genotypes and flowering synchrony between males and females which will permit cross pollination if so desired without the need to collect, process and store pollen in a breeding programme.
From the results five provenances comprising of Marracuene, Moamba, Magunde, Kalanga and Missira emerged as early flowering while the other 15 provenances including, Chikwawa, Mangochi, Ohangwena, Kalimbeza, Magamba, Siavonga, Ngundu, Biriwiri, Mudzi, Matebeleland North and Matebeleland South were classified as late flowering genotypes.Two distinctive groupings were evident basing on flowering time although there was slight overlap in September among genotype.The existence of two distinct groups based on flowering time can be used to create two breeding populations from selections of the superior trees in the trial.However, among the early flowering provenances, selections from Missira cannot be combined with the selections from the other early flowering provenances as this genotype belongs to the subspecies birrea while Marracuene, Moamba, Magunde and Kalanga to caffra.
The results suggest that variations in flower bud set to anthesis may either be due to origin of S. birrea provenances or genetic because the genotypes are sharing same environmental conditions.Site had no influence on the early or late flowering of provenances possibly they were influenced by locality of origin such as latitude and altitude for example Marracuene, Magunde, Moamba and Kalanga (Table 1).The late flowering trait in different genotypes doesn't seem to correspond with latitudinal and altitudinal influence across provenances probably this might be evident at country level (Table1).Despite that Chikwawa and Mangochi (Malawi) synchronized with Mudzi, Matebeleland North, Matebeleland South, Magamba, Ngundu (Zimbabwe), Siavonga (Zambia) and Kalimbeza (Namibia) genotypes but they are from different ecological zones in terms of latitude and altitude, this therefore may signify genetic influence [12,13].This is in agreement with reports of Munthali et al., [17] where similarities in phenotypic and genetic attributes between geographically different populations of Adansonia digitata were attributed to genetic closeness.Consequently, the overlapping in flowering between populations, provide opportunities for hybridizing selections made in the populations without the need for storing pollen and conducting control pollinations which is expensive.The overlapping of flowering give confidence that the geographic populations may not necessarily be maintained through isolation based on origin.
The trait for early flowering in S. birrea is good in selection process for domestication as it corresponds with early fruiting and maturity which may be preferred by farmers [18].Early fruit maturity is advantageous as it coincides with lean period (December and January) and may fetch higher prices on the market.In addition, variations at provenance and individual level are more specific and useful during selection for domestication and breeding programs while flowering overlaps and synchrony enriches the genetic composition of the progenies due to increased outcrossing [19].According to Giménez-Benavides et al., [20] early flowering plants flower for longer periods resulting into high fruit yield however current findings have shown that the early flowering genotypes of S. birrea flowered the least number of days with no significant variations in fruit yield.On the contrary Rios et al., [21] in the study of Chamaedorea pinnatifrons (Arecaceae) found out that the late flowering females with lower synchrony presented the highest reproductive success but S. birrea fruit yield showed no significant variations among the early and late flowering genotypes in the two reproductive seasons although this may require continued monitoring for subsequent seasons because of 'seed masting' effect [22].
We concluded that the variations observed between populations of S. birrea are largely influence by genotype and origin.This is consistent with published reports on the same trial [5,12,13], which attributed variations in vegetative growth, fruit traits and productivity of S. birrea to genetic influence.

4-2-Differences between Males and Females in Days from Bud Set to Anthesis
Generally, the development of male flower buds and females started in the same months (August to October).The results have shown that in all populations the female flowers were developing in tandem with male flowers.This shows that the flowering ensures availability of pollen to influence effective fertilization.The results also revealed variations in period of flower bud initiating and opening between provenances.The earlier flowering in males of S. birrea is associated with the competition among individuals of the population for greater dispersal of gametes while delayed flowering in females enabled them to synchronize with males at the peak of their flowering intensity for increased pollination [10,23,24].The longer period from bud to anthesis in males suggest that males requires longer period for pollen development for increased pollination capacity while females need shorter period for pollen reception to ensure seed set [24].This agrees with the report of Fonseca et al., [25] on dioecious species of Baccharis playtypoda where distinctive variations between sex in flowering initiation and duration existed and Forrest [26] in a Protandry study where most male dioecious plant species reached flowering peak before females.In a related scenario, Williams and Adam [27] in the study of Guioa semiglauca (Sapindaceae) species reported males reaching anthesis one to three weeks earlier before the opening of female flowers.Consequently, in cases of isolated populations, phenological shifts may lead to reduced chance of pollination between female's plants and pollinator populations that could potentially result in the extinction of both populations [28] or limited number of successful pollinators.Therefore, reproductive synchronization between and within populations of S. birrea and extended flowering period in males is an important trait as it provides opportunities for pollination without resorting to pollen storage if and when pollination between selected trees in different provenances is envisaged.Hence differences in flowering time between different provenances, future design of fruit orchards should group those genotypes that synchronize in flowering including males and females and plant them at same place to ease management and further selection for future breeding programmes.

4-3-Leaf Flush
The present results have revealed extensive variation in time for leaf flush within and between provenance levels denoting some provenances as early while others as late leaf flushers.The early leaf flushers included Moamba, Marracuene, Magunde and Kalanga while the late flushers comprised of Missira, Magamba, Ngundu, Biriwiri, Mudzi, Matebeleland North and South, Kalimbeza, Ohangwena, Mangochi, Chikwawa, and Siavonga.Early flushing in Moamba, Marracuene, Magunde and Kalanga is associated with latitude in the locality of origin (Table 1).According to Murali and Sukumar [29] early flushers are more advantageous than the late ones as the early flushers produce new leaves before the emergence of herbivorous insects.This is in consistent with the findings of the current study where late flushers such as Siavonga, Chikwawa and Mangochi provenances of S. birrea that flushed late in October to November had all their leaves defoliated while the early flushing genotypes were not affected.Consequently, the losing out of leaves is crucial as it affects photosynthetic activities that eventually affects physiological processes within the tree resulting in stunted growth and poor productivity [30].
Latitudinal gradient and temperatures are reported to influence leaf flushing phenology in many tree species [31,32].However, Elliott et al., [33] and Rivera et al. [34], attributed leaf flushing phenology among species around the world to water storage in deep soils and photoperiodic induction of leafing.In the current study leaf flushing coincided with increasing temperatures (Figure 1) and displayed variations that showed a pattern of originality mainly basing on latitude, elevation and temperatures [12].The results support the findings of Kaszkurewicz and Fogg [35] reported in Friedman et al., [32] where latitudinal difference between 34.9 and 47.6°N resulted in delayed date of leaf flush of Populus deltoides by 44 days.Likewise, latitude differences in place of origin of S. birrea genotypes had an influence in time of leaf flush for example latitudinal variations between Marracuene (25 o 58'S) and Mangochi (14 o 02'S) brought about 30days difference in leaf flush between provenances [13].On the same note Chikwawa (Malawi) and Siavonga (Zambia) provenances with a minimal latitudinal difference between 16 o 46'S and 16 o 30'S respectively flushed new leaves at the same period.The differences in time of leaf flush were largely influenced by latitude of origin.The provenances also displayed individual tree to tree variations signifying genetic variability within provenances.Individual variations are mostly valued and used in selection for domestication or breeding programs [19].The trait for early flushing among others is important in selection for domestication as it enhances tree growth, reproduction and protection from herbivorous insects.
Missira sub species birrea a provenance from North West Africa (Table 1) was among the early fruiting and maturing genotypes however it differed with other provenances at fruit maturity and in time for leaf flushing.The fruits of Missira provenance ripened whilst in the tree like mangoes and dislodges there after unlike other provenances of subspecies caffra where fruits drop from the canopy while green and ripens on the ground after a few days depending on the prevailing temperatures [36].A second observed difference was in leaf flush where Missira put up new leaves about days after fruit set while the rest of the provenances of subspecies caffra flushed new leaves within a range of 2 -9 days after or before fruit set (Table 2).The long time lag between fruit set and leaf flush in Missira provenance suggest that the genotype is capable of storing adequate food resources to sustain the plant during the reproductive phase [37].The phenology behaviour of Missira appears to be largely influenced by genotype and locality of origin.
S. birrea provenances also displayed high tree to tree variation in period of fruit maturation.The observed variation is important for fruit tree like Marula since the maturity is well spread for a period of seven months.This is ideal for price stabilization and also farmers will have sufficient time to make Marula products without the fruit spoiling or farmers resorting to storage.Late maturing trait can also be economically valuable as fruits mature off peak when demand and price are likely to be high.Furthermore, accurate determination of fruit maturity plays an important role in its timely planning for harvesting, packaging, transportation and the guarantee of commodity quality [38].

4-5-Fruit Productivity of S. Birrea in 2016/2017 and 2017/2018 Seasons
The results indicated significant variations between the two seasons with higher fruit production in 2016/2017 season as compared to 2017/2018 season.Some individual trees that had high fruit yield during the first season performed poorly in the second while other families stayed dormant without flowering (Table 3).The 2016/2017 fruiting season was characterized by cooler temperatures as compared to 2017/2018 which had cooler nights but very high day temperatures (Figure 1).Furthermore, in 2017/2018 season by October, the site had received more rainfall than the 2016/2017 season during same period of time suggesting that poor fruit yield in 2017/2018 season was not primarily influenced by rainfall rather by high temperatures during the flowering phase [39].Furthermore, reports from previous studies [5,13] plus the current results have indicated that at age 18 some provenances/ families have not yet started fruiting suggesting long precocity period for example the Rumphi genotype.This behaviour was also reported in the wild populations of Macadamia and Dacryodes edulis where some trees started producing fruits at age 20 and 22 respectively [40].Hence continued monitoring is required on those provenances the have not yet started flowering.
Periodicity in fruit production among S. birrea provenances was also reported by other researchers [12,14,[40][41][42] indicating that many individual trees of S. birrea become reproductively inactive in a season following a high fruit reproductive season and referred the phenomenon as 'mast seeding' behaviour.Nevertheless, mast seeding behaviour is a demerit to domestication because famers require satisfactory fruit yield yearly.Premature fruit shedding and sex change were also observed among some populations.According to Diallo et al., [43] fruit abortion is an adaptation that permit plants to match fruit and seed number with the available resources across a range of environmental circumstances.Tree to tree variations in fruit production are of paramount importance and commonly used by geneticists during selection for domestication and tree breeding.It offers a wider chance for a combined selection to be carried out both at provenance, family and individual tree level to attain high genetic gains [17,44].Therefore, continued monitoring for fruit production and sex change among genotypes is required for several consecutive seasons before effecting selection of those trees that do not exhibit mast seeding.

4-6-The Relationships between Leaf Flush and Flowering Phenology Traits
The strong association between leaf flush, bud set, flower opening and fruit set corresponded to locality of origin basing on latitude and altitude (Table 1) in the early flowering provenances (Moamba, Magunde and Marracuene and Kalanga) while in the late flowering there was a lot of diversity both in locality of origin basing on latitude and elevation at provenance level.However strong association basing on latitude could be noted at country level.The strong relationship between the reproductive phase and leaf flush suggest the need for food production to sustain fruit development and seed maturation in females and vegetative growth in males although the new leaves do not photosynthesize immediately after flush [45].However, the strong association is important because as soon as the new leaves attain the green colouring matter would start manufacturing food using solar energy [30].The manufactured food would then replenish the used up food reserves during leaf flush and flowering phases as well as for continued plant growth, maintenance and sustenance of fruit development up to maturation.

5-Conclusion
The study has revealed substantial variation between and within provenances in time of leaf flush, bud initiation to flower opening (Anthesis), fruit set to maturity with increased flowering synchrony between male and female trees as well as overlaps between populations.The variations in fruit yield and maturity period between and within provenances are adequate to warrant selection for domestication.This study has also generated information of prime importance to tree breeders for instance variations in genetic traits between and within provenances including early flowering, maturity period and productivity.Therefore, the existence of variations in different traits offers a wider chance for combined selection to be carried out both at provenance and individual family level to attain high genetic gains from tree breeding programs.Basing on differences in flowering time between different provenances future design of fruit orchards should group and plant those genotypes that flower at the same time to ease management and further selection for breeding programs.Future studies should investigate change of sex among genotypes, premature flower bud and fruit shading as well as continued study in fruiting behaviour of different provenances.

Figure 1 .
Figure 1.Map of Malawi showing the location of the study site

Figure 2 .
Figure 2. Weather from June 2016 to June 2018 for the study area

Figure 3 .
Figure 3. Research Methodology Flow Chart

Table 1 . Geographic details of the Marula germplasm tested in Malawi
* Locality name not given.