Reproductive and vegetative phenology of the micro endemic Stachytarpheta cassiae (Verbenaceae)

Abstract The flora of the Brazilian rupestrian grasslands represents a hotspot of species richness and endemisms. Stachytarpheta cassiae (Verbenaceae), is a micro endemic species, from which nothing is known. Here, we quantified the activity and intensity of vegetative and reproductive phenophases throughout 12 months and tested for their seasonality and their relationship with local climatic variables. Both vegetative and reproductive phenophases were continuous. No seasonality was observed in the vegetative phenophases and none of them was influenced by climatic variables. Only flower buds and mature fruits’ intensities showed seasonality in February (rainy season) and July (dry season), respectively. Accordingly, increased temperature and humidity combined explained increased production of flower buds whereas decreased rainfall explained increased mature fruits. Higher intensity in flower buds may respond to similar climatic conditions as other species in the community. However, S. cassiae is much different as it continues producing flowers continuously. Higher intensity of mature fruits in the dry season is expected as their seeds are abiotically dispersed. Due to constant flower and leaf production, S. cassia may be a key species for the conservation of many vertebrate and invertebrate species and for maintaining the biogeochemical functioning of the impoverished soils of the rupestrian grasslands.


Introduction
Plant species that comprise the flora of rupestrian grasslands are under risk of extinction as a result of the increasing anthropogenic environmental disturbances.Although they represent more than 5% of the Brazilian flora (Fernandes 2016;Fernandes et al. 2018), species from the rupestrian grasslands may undergo extinction as a consequence of synergistic disturbances among land-use and climate changes and the lack of management policies from public environmental organisms (Fernandes et al. 2014(Fernandes et al. , 2018(Fernandes et al. , 2020)).Another aspect that contributes to this worrying scenario is the lack of scientific knowledge about the natural history of many of these species (see Fernandes 2016).Endemic species are among the most threatened ones, in particular those with restricted distribution areas.Many endemic species from rupestrian grasslands have only a single known population such as Collaea cipoensis Fortunato (Fortunato 1995), Baccharis concinna G.M.Barroso (Marques et al. 2002) or Coccoloba cereifera Schwacke (Moreira et al. 2010).
In order to develop conservation and restoration strategies of threatened ecosystems, it is fundamental to understand the natural history of focal species, particularly about their phenology (e.g., Belo et al. 2013;Fernandes 2016).In this regard, phenological studies represent important tools for understanding the factors that influence reproduction and survival of plant species (Maderia & Fernandes 1999;Morellato et al. 2010).However, phenological studies in certain environments such as the rupestrian grasslands remain particularly scant, especially in endemic or threatened species (see Madeira & Fernandes 1999;Dutra et al. 2009;Miola et al. 2010).One plant genus of great relevance in the landscape of rupestrian grasslands is Stachytarpheta (S.Atkins) (Verbenaceae), as it interacts with a large number of animal species.Species from Stachytarpheta genus are generally shrubby or herbaceous with small colorful and showy flowers from intense purple to pale pink (Atkins et al. 1996;Barbola et al. 2006).Some species are considered ornamental or medicinal, such as S. jammaicensis (L.) and S. cayennensis (Rich.)Vahl (Rodríguez & Castro 1996).
Phenological studies with species of Stachytarpheta genus are still scarce, despite their high frequency in the rupestrian grasslands landscape.There are some studies about the floral biology of Stachytarpheta maximiliani Scham.(Barbola et al. 2006), and most of the studies in the genus are about pollination, floral visitors and their behavior, such as in S. cayennensis (Rich.)(Fonseca et al. 2006) and S. glabra Cham.(Antonini et al. 2005;Jacobi & Antonini 2008).The only phenological studies in the genus were also conducted in these two latter species (Jacobi & Antonini 2008;Fonseca et al. 2006).The focus of this research is the microendemic species Stachytarpheta cassiae (S.Atkins) from northern Minas Gerais, from which nothing is known about its natural history and phenology.
Our objective was to evaluate the periodicity of the phenophases of S. cassiae throughout the year in the rupestrian grasslands of the State Park Serra Nova in Minas Gerais, Brazil.We propose to i) analyze qualitatively and quantitatively the reproductive and vegetative phenologies of S. cassiae; ii) and to assess the relationship of phenological events with climatic variables.
The SPSN shows a striking transition between cerrado and caatinga domains, intermingled with rocky outcrops from the Espinhaço Setentrional mountains.The vegetation is characterized by physiognomies of the rupestrian grasslands, cerrado, campo cerrado, seasonal semideciduous forests (gallery forests) and seasonal deciduous forests (dry forest), with a clear stratigraphy of the vegetation in relation to the cliffs of the sierras.In the higher areas, the vegetation is typical of the rupestrian grasslands.Litholic soils and cambisols predominant in the area (IEF 2003).Drummond et al. (2005) consider the SPSN as a valuable region for scientific research given the high richness of endemic and threatened plant species.Phenological data were obtained in two sites within the SPSN, one located in the lowlands, known as "Escorregador" (15°36'56.1''S,42°44'00.8''W)at 818 m asl, with a population of 63 individuals of S. cassiae within an area of 0.06 ha.Another population was located in the higher lands of the SPSN at 1.059 m asl, known as "Miúdas" (15°36'24.9''S,42°45'00.4''W),with 153 individuals in an area of 0.8 ha.Individuals from these two populations of S. cassiae were adults of 0.4-2.5 m height, with stem circumference at the bottom of 4-15 cm.Flowers show an intense blue color and are displayed in terminal spike inflorescences (Fig. 1).No other population of S. cassiae was found within the region.

Phenological events
Twenty adult individuals of at least 1.2 m height were marked from the two known populations: 10 from Escorregador and 10 from Miúdas.These individuals were located at a minimum distance of 10 m from each other in order to assure a higher genetic variability and less degree of consanguinity among them (Kageyama & Gandara 1999), prioritizing sexually mature individuals.All individuals had a fully visible crown and were georeferenced and marked with aluminum tags.From these 20 individuals, five were newly tagged and georeferenced during the seventh expedition, as the five originally marked individuals had died in April 2015 and needed to be replaced.From these new five individuals, two were from Escorregador and three from Miúdas population.
Phenological monitoring was conducted monthly, initiating in September 2014 until August 2015, completing 12 expeditions throughout one year.The vegetative phenology accounted for the events of foliar changes, starting with foliar budding, young leaves, adult leaves and senescence (Fig. 2).Foliar budding implies the start of the development of foliar buds, which is defined by an intense light-green coloration and slight purple line in the edge of new leaves.This period lasts until these leaves attain 0.40 cm (Fig. 2a).Young leaves, which appear from the end of foliar budding until reaching 1.5 cm length, present an obovate aspect, are showy light-green colored with an intense purple in the edges (Fig. 2b).Adult leaves are fully developed leaves with an oval foliar limb of up to 5 cm long, they have a dark green color with purple edges of little intensity (Fig. 2c).Senescent leaves are similar in size to adult leaves but with different color and withered appearance (Fig. 2d).These senescent leaves have a characteristic yellowish color and they do not have the purplish edges like the young and adult ones.Soon after or together with senescence, leaf fall occurs and many leaves fall under or around the canopy.These fallen leaves show an intense brown color, giving the impression of decay and death of the plant (Guitman et al. 1991).
The reproductive phenophases involved the flowering and fruiting processes, initiating with the formation of flower buds (Fig. 3a) and followed by flowers at anthesis (Fig. 3b).Flower buds are 1.23-1.98cm long, have an intense purple color at the apex, extending from the lateral edges to the mid part of the flower bud.Flower buds are arranged in a terminal inflorescence with alternate organization within a spike.The flowers at anthesis are tubular, arranged in alternate shapes along the axis of the inflorescence and can reach up to 2.93 cm in length.Flowers have a purplish color and are very notorious at long distances, which is an important trait to attract pollinators.Flowers are 1.63-2.18cm long with a diameter of 0.72-1.1 cm.The fruiting phenophase implies immature and mature fruits.Immature fruits are developing fruits with a size from 0.90 to 1.1 cm long, with a visible persistent stylet in the fruit (Fig. 3c).These immature fruits have a characteristic light green color in the base and a light brown color at the apex, with a few light-purple spots.Mature fruits are dry, brownish, of 0.77-1.03cm long (Fig. 3d).

Analysis of phenological data
Phenological data were analyzed using activity and intensity indices.The activity index (percentage of individuals) is the simplest method, which verifies the presence or absence of a given phenophase in the individual.This method also estimates the synchronicity among the individuals of a population (e.g., Morellato et al. 1990).The greater the number of individuals manifesting the same phenophase at the same time, the greater the synchrony of the population.A phenological event is considered asynchronic when < 20% of individuals express the same phenophase; little synchronic when 20-60% of individuals express the same phenophase and highly synchronic when > 60% of the individuals in the population express the same phenophase (Bencke & Morellato 2002).
The intensity index was calculated using the method proposed by Fournier (1974).Values were obtained in the field through a semi-quantitative interval scale of five categories (0 to 4) and a 25% interval between each category, allowing the estimation of the intensity percentage of each phenophase in each individual as follows: 0 = absence of phenophase; 1 = intensity of 1 to 25%; 2 = intensity of 26 to 50%; 3 = intensity of 51 to 75% and 4 = intensity of 76 and 100%.At the end of each expedition, we summed the intensity values attributed to each individual, divided by the maximum intensity that can be reached by all individuals (N) in the sample (number of individuals multiplied by four).The value obtained, which corresponds to a proportion, was then multiplied by 100 to transform it into a percentage value, according to the formula: FI = (Σ grades) / (N*4) * 100.
Due to the cyclic nature of phenological data, we employed circular statistical analyses to calculate the existence of seasonality for each phenophase, following Morellato et al. (2010).The sampling months of each phenological event were converted into angles, where January corresponded to 0°, and December corresponded to 330°.We calculated the mean angle (and its corresponding month) of each phenophase, according to Morellato et al. (2010).The Rayleigh test (Zar 1996) was applied to test the significance of the mean vector angle (i.e., mean date of each phenophase) with a unimodal distribution.When the mean vector angle is significant, it corresponds to the average date of the year around which the phenological events are concentrated and the pattern is interpreted as significantly seasonal (Morellato et al. 2010).The vector (r) varies from 0 to 1 and indicates the concentration of phenological events (e.g., intensity) around the mean date or the degree of phenophases' seasonality (see Morellato et al. 2010).All circular analyses were done in the R environment (R Core Team 2022) using the "circular" package (Agostinelli & Lund 2017).The vegetative and reproductive phenologies of S. cassiae were classified according to Newstrom et al. (1994) as either continual, sub-annual or annual.
Finally, we assessed the influence of monthly mean temperature, humidity, rainfall and day length on the intensity of each reproductive and vegetative phenophase.Climatic variables were obtained from the Climatological Station 83395 -Janaúba, for the studied period (September 2014-August 2015; Fig. 4), with the exception of day lengths (hours), which were obtained from <http://www.solartopo.com/daylength.htm>,after providing the exact location of the study site and choosing the 15 th day of each month.We used stepwise multiple regression analyses to determine the combination of climatic predictor variables that best predict the dependent (phenophase intensity) variables.Before running the analyses, we tested for multicollinearity among predictor variables and found that day length was positively correlated with temperature (r = 0.85) and precipitation (r = 0.74; Tab.S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.22310752.v1>).For that reason, we decided not to include day length in the models.The other three climatic variables were uncorrelated amongst them (Tab.S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.22310752.v1>).For each of the stepwise multiple regression analysis we tested for the assumptions of normality of residual errors and homogeneity of variances.Stepwise multiple regression models (forward, backward, both) were selected using the "stepAIC" function from the MASS package, which selects the best model by exact AIC.These analyses were conducted in R environment (R Core Team 2022).

Results
All S. cassiae individuals assessed presented the four vegetative phenophases, foliar budding, young leaves, adult leaves and senescent leaves, across the twelve months (Fig. 5a), which imply a high synchronicity among the individuals of each population.Similarly, these vegetative phenophases can be classified as continual at both the individual and population levels.Some of these phenophases appeared to show mild increases in their intensity in different months: foliar budding in February and October, young leaves in May and August, adult leaves in May, and senescent leaves in April and October (Fig. 5a).Nevertheless, according to the circular analyses, the four vegetative phenophases did not show any trace of seasonality.The Rayleigh tests on the four vegetative phenophases resulted in lengths of mean vector (r) lower than 0.1 with non-significant p-values (Tab.1).The lower values of the length of mean vector (r) indicates an absence of temporal concentration of phenological activity.In other words, despite some apparent variation in intensities, the four vegetative phenophases in this species showed random or multi-modal pattern.Accordingly, the step-wise multiple regression models showed that none of the local climatic variables significantly explained variation in the intensity of any of the vegetative phenophases (Tab.S2, available on supplementary material <https:// doi.org/10.6084/m9.figshare.22310752.v1>).
All individuals of S. cassiae also presented the four reproductive phenophases (flower buds, flowers at anthesis, immature fruits and mature fruits) across the twelve months of observation (Fig. 5b), implying a high synchronicity among the individuals of each population.These reproductive phenology patterns can also be classified as continual at both the individual and population levels.All of these reproductive phenophases showed visual variations in their intensity across the studied year: flower buds in May, flowers at anthesis in November, immature fruits in March, and mature fruits in July (Fig. 5b).Based on the circular analyses for the reproductive phenophases, we found a significant pattern of seasonality in the flower buds and mature fruits phenophases (Tab.1).The mean vector angle corresponded to February for flower buds and to June for mature fruits (Tab.1).On the other hand, the phenophases of flower at anthesis and immature fruits did not show any signs of seasonality (Tab.1).Although the flower buds and mature fruits showed a significant seasonal pattern, the Rayleigh tests on the four reproductive phenophases resulted in lengths of mean vector (r) lower than 0.2 (Tab.1), indicating a weak or absent temporal concentration of reproductive phenological activity in S. cassiae.

Discussion
All the vegetative phenophases of Stachytarpheta cassiae were present concomitantly throughout the year, indicating that leaf renewal is constant and highly synchronic among individuals.Plants with this characteristic are extremely important for the rupestrian grassland environment, as constant leaf renewal along with their senescence provides a source of organic matter for the poor and shallow soil of this ecosystem.Such potential increased nutritional input to the soil can offer a more suitable environment for the development of other species, in addition to decreasing soil temperature by increased shading.
None of the vegetative phenophases showed seasonality across the year and none of them was influenced by climatic variables.Seasonal vegetative phenological strategies seem to be more frequent in cerrado and seasonal forest realms, where woody plants usually increase leaf budding at the onset of the rainy season and peak leaf senescence during the dry season (Lenza & Klink 2006; Santos de Oliveira et al. 2021).The continuous vegetative phenophases of S. cassiae coincides with other woody species from the rupestrian grasslands (Santos de Oliveira et al. 2021).The capability of constant leaf production in such impoverished soils implies the development of morphological and ecophysiological strategies (e.g., deep and efficient root system, the presence of reservoir organs such as xylopodia), that maximize the acquisition and maintenance of very limited resources (Belo et al. 2013;Dayrell et al. 2018).Thus, S. cassiae must have a greater tolerance to cope with water limitation, preventing increased leaf senescence during the dry season, which represents a nutrient conservation strategy in infertile environments, as observed in other species from the rupestrian grasslands (Dayrell et al. 2018;Santos de Oliveira et al. 2021).The constant production of leaves may also result in greater herbivory levels.However, growth defense tradeoff theory predicts that plants in low-resource habitats invest more energy in defense mechanisms against natural enemies than growth (Coley et al. 1985).Moreover, another defense strategy against herbivores involves the synchrony of leaf production at the population level (Aide 1993).This latter strategy can be effective if leaf biomass production exceeds the capacity of consumption by insects (Aide 1993;Lamarre et al. 2014).Thus, the high synchronicity of the vegetative phenophases along with other potential defense mechanisms may allow S. cassiae to cope with herbivory relatively well.
The production of flower buds showed a significant seasonality with a mean vector angle in February.Moreover, increased intensity of flower buds was explained by both increased temperature and humidity, which were highest between November and April, during the rainy period of warmer temperatures in the studied region.Such climatic conditions increase water availability, which along with constant leaf production would generate high resources to boost flower production (Batalha & Martins 2004;Belo et al. 2013) Species with continual flowering patterns such as S. cassiae are rather infrequent among angiosperms, which usually show annual seasonal flowering periods that last from a few days to few months at the most (e.g., Newstrom et al. 1994;Kang & Jang 2004;Le Stradic et al. 2018).However, such extended flowering period is reported for other Stachytarpheta species such as S. cayennensis (Rich.)J. Vahl in the Cerrado (Arruda et al. 2009) and S. glabra in rupestrian grasslands in southeastern Minas Gerais (Jacobi & Antonini 2008).On the other hand, Machado & Oliveira (2015) observed that the flowering of S. gesnerioides Cham occurred only from January to March in cerrado domains of central Brazil.Interestingly, extended flowering throughout the year were recorded in other species from the rupestrian grasslands, belonging to different botanical families such as Jatropha sp.(Euphorbiaceae), Prosopis rubriflora (Fabaceae) (De Freitas et al. 2013) and Hohenbergia ramageana (Bromeliaceae) (Santana & Machado 2010).The occurrence of these longflowering species represents key resources for pollinators and florivores throughout the year as they provide predictable resources over time (Loyola et al. 2007).Such extended flowering pattern increases the number of floral visitor and pollinator species seeking pollen and nectar, improving the reproductive success of these plant species (Antonini et al. 2005;Loyola et al. 2007).Finally, prolonged and synchronized flowering of plant individuals within and among populations in S. cassiae can increase the chances of crosspollination, enhancing the genetic diversity of the progeny (Kameyama & Kudo 2009).
The intensity of immature fruits showed no seasonality and was not affected by any climatic variable.This pattern indicates that fruit production is mostly dependent on the success of effective pollination.The intensity of mature fruits, however, showed a significant seasonality with a mean vector angle in June, during the dry season.Accordingly, rainfall explained the intensity of mature fruits, increasing the intensity as rainfall decreased.Such phenological pattern may be explained by the seed dispersal strategy of the species, as S. cassiae has schizocarp fruits that abiotically disperse their seeds once the mature fruits dry up and release their seeds.Thus, the higher probability of seed dispersal success is under low rainfall conditions during the dry season (e.g., Mantovani & Martins 1988;Oliveira & Moreira 1992).A similar phenological pattern of mature fruits was observed in S. cayennensis (Fonseca et al. 2006) and in other Cerrado species (Mantovani & Martins 1988).Almeida-Cortez (2004) explains that in areas with a seasonal climate, wind dispersal is more common during the dry season, as humidity makes it difficult to release the seeds.
Stachytarpheta cassiae presented all the reproductive and vegetative phenological phases during the studied 12 month-period.This micro-endemic species is of great relevance for maintaining the biogeochemical functioning of the impoverished soils of rupestrian grasslands as it provides continued input of organic matter and vegetation cover for the soil.Finally, the constant availability of resources for a wide range of floral visitors seeking for pollen and nectar, as well as for florivores and herbivores throughout the year, makes it a key species for the conservation of many vertebrate and invertebrate species in the rupestrian grasslands.

Figure 2
Figure 2 -a-d.Vegetative phenophases of Stachytarpheta cassiae (Verbenaceae) in an area of rupestrian grassland in State Park Serra Nova, MG -a.foliar budding; b. young leaves; c. adult leaves; d. senescent leaves.

Figure 3
Figure 3 -a-d.Reproductive phenophases of Stachytarpheta cassiae (Verbenaceae) in an area of rupestrian grasslands in the State Park Serra Nova, MG -a.flower bud; b. flowers at anthesis; c. immature fruits; d. mature fruits.

Figure 5
Figure 5 -a-b.Intensity index of phenophases in Stachytarpheta cassiae (Verbenaceae) in the rupestrian grasslands of the State Park Serra Nova, MG -a.vegetative; b. reproductive.

Table 1 -
. In Results of the circular analysis to determine the seasonality of the phenophases of Stachytarpheta cassiae (Verbenaceae) in the rupestrian grassland, State Park Serra Nova, MG.Mean vector length (r) and significance (p-value) according to Rayleigh uniformity test (α = 0.05).SD = standard deviation (degrees); NS = not significant.