Poaceae pollen grains from southern Brazilian grasslands : Pollen grain size in species from dry and humid environments

Southern Brazil has a high diversity of grassland Poaceae species in the biomes of the region. However, few data on the morphology and the uniformity of Poaceae pollen grains cause taxonomic low resolution in pollen records. In this study, pollen samples of grassland Poaceae species from different regions of southern Brazil were analyzed. The pollen diversity of 70 grassland Poaceae species (59 genera, 15 tribes, and 6 subfamilies) of the Pampa and Atlantic Forest biomes is presented. Morphometric measurements indicated that pollen grains of humid grasslands species have a larger size than those of dry grasslands species. Though the average of measurements shows a small difference in pollen grain sizes between the Pampa and the Atlantic Forest, such differences cannot be applied to fossil records because non-significant differences occurred between samples. Regarding pollen size, non-significant differences also occurred for the C3 and C4 samples, probably because C3 species had high variance in size. These records provide relevant ecological inferences of Poaceae pollen grains, providing valuable information for reconstruction of vegetation. Furthermore, regarding taxonomic relationships, results suggest the trend of decrease of the size of Poaceae pollen grains towards derived tribes.


Introduction
Poaceae pollen grains are major pollen indicators used to analyze the grassland dynamics in South America during the Quaternary.Furthermore, they also indicate the modification of natural vegetation by the introduction of exotic species (corn and others cultivate) by the human (Behling et al., 2005;Macedo et al., 2009Macedo et al., , 2010)).
Poaceae pollen frequency was high during the late Pleistocene and early Holocene in southern South America.Poaceae pollen and pollen of Asteraceae and Fabaceae species shows a dominance of grassland vegetation (Campos) during these periods (Bauermann, 2003;Behling et al., 2004;Tonello and Prieto, 2010;Mourelle et al., 2015).However, the floristic diversity of Poaceae in South America cannot be observed in the pollen records due to the uniformity of the pollen grains of this family.The existence of few studies about the morphology of modern Poaceae in South America increases the difficulty to obtain better pollen taxonomic resolution (Heusser, 1971;Salgado-Labouriau, 1973;Markgraf and D'Antoni, 1978;Salgado-Labouriau and Rinaldi, 1990;Corrêa et al., 2005;Dórea, 2011;Bauermann et al., 2013;Radaeski et al., 2014).
In recent years, studies aiming to overcome the inaccessibility of ecological information of Poaceae pollen grains showed good results.Patterns in the size of the fossil Poaceae pollen grains could be obtained from different South American grassland ecosystems (Shüler and Behling, 2011).In relation to the photosynthetic metabolism of modern taxa, it was possible to observe trends in sizes of Poaceae pollen grains (Jan et al., 2014).Analyzing modern pollen grains was also possible to distinguish the pollen grains of grassland and forest Poaceae species of the southern Brazil (Radaeski, 2015;Radaeski et al., 2016).Even with these advances, interpretations about vegetation or climate change based on Poaceae pollen grains that predominated in the Quaternary fossil record of South America are hampered, especially in regions dominated by the grassland (Campos) vegetation as the Pampa biome.
About 400 species, 77 genera and 15 tribes of Poaceae are distributed in grasslands in southern Brazil (Boldrini et al., 2008;Boldrini and Longhi-Wagner, 2011).Poaceae, Asteraceae and Fabaceae are families that dominate natural grassland of the Rio Grande do Sul (Pillar et al., 2009;Pillar and Lange, 2015).This grassland vegetation distributed mainly in the Pampa biome, the southern half of Rio Grande do Sul, where these families colonized it when the climate was different from today (Bauermann, 2003;Behling et al., 2004;Bauermann et al., 2008).The permanence of the grassland for a long time in the south of Brazil allowed the distribution of floristic diversity of grassland in the region in response to different environ-mental and geological conditions in Rio Grande do Sul (Hasenack et al., 2010).However, the diversity of floristic grassland is not reflected in the pollen records due to the high concentration of different Poaceae species that may not have their pollen grains distinguished in palynological samples.To overcome these problems, more knowledge about the diversity of modern pollen grains of different grassland vegetation is required for better paleoecological inferences of fossil Poaceae pollen grains.Thus, more information about the pollen composition of fossil sediments based on the comparison of palynomorphs with their modern analogs will be available.
The aim of this study is to record the pollen diversity of Poaceae species of grasslands from Pampa and Atlantic Forest biomes in Rio Grande do Sul, determining the types of modern Poaceae pollen in southern Brazil.Furthermore, we will evaluate if there are differences between pollen grains from Atlantic Forest and Pampa biome species, as well as of humid and dry species and C 3 and C 4 species.

Study area
According to the Köppen climate classification, the state of Rio Grande do Sul (27º-34º S and 50º-58º W) has the predominant climate of the "Cfa" temperate type and annual precipitation with regular distribution (Nimer, 1989).The maximum annual average temperature is 28ºC recorded in the northwestern region of Rio Grande do Sul and the minimum annual average temperature is 10ºC located in the northeast region with high altitudes (Figure 1).The minimum annual rainfall of Rio Grande do Sul is 1100 mm located to the extreme west of the state and the maximum annual rainfall is 2000 mm in the north region (Rio Grande do Sul, 2012).The higher intensity of the winds in the Rio Grande do Sul is located in the east, in the coastal region, with winds that predominate in the northeast direction in the summer and south-west in winter (Atlas eólico: Rio Grande do Sul, 2014).
The high diversity of grassland species in Rio Grande do Sul is due to different environmental factors in the region, such as altitude, diversity of soils, rainfall and temperature (Boldrini and Longhi-Wagner, 2011).Altitude has a high influence to the distinction between the grassland of the Pampa and Atlantic Forest biomes of Rio Grande do Sul.The Campos de Altitude of the Atlantic Forest biome is located in the northeast of the state in the altitude above 800 m.The vegetation is a mosaic of grassland and Araucaria forest (Oliveira, 2009).The grassland of the Pampa biome are distributed in the southern half of Rio Grande do Sul at lower altitudes with gallery forests along the rivers in the region (Pillar et al., 2009;Boldrini and Longhi-Wagner, 2011).
Poaceae pollen grains from southern Brazilian grasslands: Pollen grain size in species from dry and humid environments

Data collection and analysis
Both fresh and herbarium material may be used for the analysis of pollen morphology (Erdtman, 1952;Salgado-Labouriau, 1973).The collection of pollen material was held in the herborized deposited material in the herbaria of HERULBRA of the Universidade Luterana do Brasil -ULBRA and ICN of Universidade Federal do Rio Grande do Sul -UFRGS (Figure 2, Table 1).The pollen material was chemically processed by the acetolysis technique proposed by Erdtman (1952) and slides with glycerin gelatin were deposited in the Laboratório de Palinologia of Universidade Luterana do Brasil.
Pollen morphological description followed the terminology proposed by Barth and Melhem (1988) and Punt et al. (2007).Species were organized in evolutionary order according to the classification of Soreng et al. (2015).Twenty-five pollen grains were measured for each species with optical microscopy under magnification of 1000x.In the equatorial view, the following measures were taken from pollen grains: polar diameters, equatorial diameters and thickness of the exine.Furthermore, the pore diameters and annulus diameters and thickness of the pollen grains were also measured.
The graphs showing the pollen grain size variation with correlation to the types of vegetation were generated BioEstat 5.0 and Statistix 8 softwares.Histograms were applied in order to know the distribution of grain sizes.An analysis of variance (ANOVA), followed by Tukey, was applied when the samples showed Gaussian distribution.

Results and discussion
Pollen diversity of 70 Poaceae species of Rio Grande do Sul grasslands distributed in Pampa and Atlantic Forest   biomes are presented (Tables 2 and 3, Figures 3 and 4).The analyzed species belonged to 59 genera representing tribes and six subfamilies.All species have monad pollen grain, with radial symmetry, heteropolar, circular amb, spherical shape, sexine and nexine of the same thickness.
The pollen grains of species varied from small (10-25 µm) to medium-sized (25-50 µm) with Gaussian distribution.The number of apertures in some species was only monoporate and other species showed monoporate and diporate apertures.The microechinate exine with thin thickness (1 to 1,24 µm) was observed in all species using a 1600x magnification.The microechinate ornamentation of some pollen grains can be often not observed in magnifications 400x or 1000x.Thus, this microechinate ornamentation is often described as psilate or scabrate (Radaeski et al., 2016).

Poaceae pollen grains of grasslands of the Pampa and Atlantic Forest biomes
The pollen sample of grasslands vegetation formations of Pampa and Atlantic Forest biomes were diverse in terms of pollen size (Figure 6A).The Poaceae species of the  5A) and monoporate aperture.On the other hand, the Poaceae species of the grassland of Pampa biome showed pollen grains with a smaller size (average size of 32 µm -Figure 5B), with monoporate aperture in some species and monoporate and diporate apertures in others species (Table 2).The variation in the average size of pollen grains of the species from the Atlantic Forest grassland was lower (33-42 µm) than the variation found in the grassland species of Pampa biome (22-45 µm).This difference may be related to high variation in the distribution of species and differences in temperature and volume of rainfall in the grasslands of the Pampa biome (Pillar et al., 2009;Boldrini and Longhi-Wagner, 2011;Rio Grande do Sul, 2012).Thus, the Pampa biome shows species occurring in different climatic conditions, whereas the climatic conditions in the Atlantic Forest biome are more stable (Rio Grande do Sul, 2012).These small differences in the grains sizes of Atlantic Forest and Pampa biome species cannot be applied to fossil records, because nonsignificant difference between the samples (ANOVA, p> 0,01; Table 4).Besides a significant difference between Poaceae pollen grains of forests and grasslands (Radaeski et al., 2016), differences of pollen size between grassland species (no forest species were considered) of Pampa and Atlantic Forest biomes are non-significant.

Humid and dry grasslands
Morphometric measurements (n=1750) of pollen grains from humid and dry grassland species in 70 Poaceae species of Rio Grande do Sul indicated that pollen grains of humid grassland species have a larger size than those of dry grassland species (Figure 6B, Table 4).The maximum pollen grain size was from species of humid grasslands, while the minimum size was from pollen grains of dry grassland species.The species of dry grasslands only presented the monoporate aperture, while pollen grains from humid grasslands had species either with monoporate aperture only or species with monoporate and diporate apertures.Thus, pollen size extreme ranges may indicate pollen grains of humid or dry grassland species on pollen records.The humid condition is determinant to the local occurrence of grassland species in Rio Grande do Sul.In this way, according to the humid conditions, Poaceae species of the humid grasslands, dry grasslands or species that can survive in both types of vegetation may occur in the region (Bond-Buckup, 2008).

C 3 and C 4 metabolism
Pollen grains of C 3 grassland species (24 taxa) show high variations of size accounting for the minimum (16 µm) and maximum (48 µm) sizes of the species studied (Figure 6B).The C 4 grassland species (46 taxa) presented pollen grains with a size between the minimum (18 µm) and maximum (46 µm) size of the pollen grains of C 3 species.C 4 species showed pollen grains with monoporate or diporate apertures, while that all C 3 species presented only monoporate aperture, except by Zizaniopsis bonariensis.Non-significant difference between the samples was showed for the C 3 and C 4 samples.
In general, species with C 4 metabolism are tropical and live in warmer and drier regions, while temperate species are C 3 and live in humid and cold conditions (Bond-Buckup, 2008;Boldrini, 2006).In Rio Grande do Sul, species with both types of metabolism may live together (Bond-Buckup, 2008;Boldrini, 2006).Thus, cold climate and high rainfall in the grassland of Atlantic Forest biome provide the predominance of C 3 species and the development of hibernal grasses.However, the megathermic species have a high frequency in the region because of the frequent use of fire by humans (Boldrini and Longhi-Wagner, 2011).In general, in the grassland of Pampa biome, the C 4 species with megathermic cycle have a high distribution, except for the region of the "campos de solos profundos" (south-west of state), that have a high presence of species with C 3 metabolism (Pillar et al., 2009).

Diporate apertures
Except by Zizaniopsis borariensis of the subfamily Oryzoideae, all species with a diporate aperture (five taxa including Zizaniopsis borariensis, Figure 7B) are of the   Southern Brazilian grassland species with diporate apertures showed larger size than the Forest herbaceous species Pharus lappulaceus with the same type of aperture (Radaeski, 2015;Radaeski et al., 2016).Pollen grains of grassland species have a size between 28 and 48 µm (Figure 7A), pollen grains of Pharus lappulaceus showed pollen grain size between 23 and 27 µm.These features allow the distinction between diporate pollen grains of the grassland and forest vegetation of southern Brazil.

Taxonomic information
Based on the recent phylogenetic classification of the Poaceae (Soreng et al., 2015) the results suggest a trend of decrease of the size of Poaceae pollen grains towards derived species (Figure 8).Radaeski et al. (2016) andSalgado-Labouriau et al. (1990) show that basal forest species (Bambuseae tribe) have larger pollen size (>45 µm) than species of other tribes.While species of Bambuseae tribe have larger pollen size, species of more derived tribes (Danthonieae, Eragrostideae, Zoysieae and Cynodonteae tribes) have smaller pollen size.

Conclusions
This work constitutes the greatest dataset of grassland Poaceae pollen diversity of South America.Although the taxonomic resolution at the species level was not obtained, the pollen database presented is important as they fill a gap about pollen morphology of the grassland of Pampa and Atlantic Forest biomes.This pollen database allows showing important information about the size of Poaceae pollen that can be used to identify species of dry and humid grasslands in Quaternary pollen samples.The pollen morphology of different groups of Poaceae species ana-   lyzed showed trends to decrease of pollen size towards more derived species.Thus, basal species have larger pollen grains while derived species have smaller pollen size.The records of diporate pollen grains are important to propose the identification of dry and humid grasslands in the two biomes of the region.Furthermore, pollen grains of humid grasslands of Atlantic Forest biome show a trend to have larger sizes than species of dry grasslands of Pampa biome.This pollen database of Poaceae species will serve as useful guide for studies of reconstruction of past vegetation in grasslands of southern Brazil.

Figure 1 .
Figure 1.Temperatures in Rio Grande do Sul, southern Brazil.(A) Maximum annual average temperatures in the region.(B) Minimum annual average temperatures in the region.(C) Average annual rainfall in the region.

Figure 2 .
Figure 2. Collection localities of Poaceae species from grassland vegetation of Rio Grande do Sul, southern Brazil (seeTable 1 for more details).

Figure 6 .
Figure 6.Pollen sizes of Poaceae species from grassland vegetation of Rio Grande do Sul, southern Brazil.(A) Variation of the average size between Pampa and Atlantic Forest biomes.(B) Variation of the average size between environments (dry and humid grasslands) and metabolism (C 3 and C 4 ).The horizontal line in bold within the box represents the median.The box shows 50% of the interquartile range and whiskers of the total variation.

Figure 8 .
Figure 8.Average size of pollen grains of Poaceae species from grassland and forest (species of Bambuseae) vegetation of Rio Grande do Sul, southern Brazil, considering their phylogenetic classification.The size of pollen grains of species of Bambuseae tribe is according to Radaeski et al. (2016).

Table 1 .
Poaceae species from grassland vegetation of Rio Grande do Sul, southern Brazil, examined in the present work.Note: (*) Numbers are the same used in Figure2.

Table 2 .
Average diameter, range diameter and type and number of aperture of the pollen grains of 70 Poaceae species from grassland vegetation of Rio Grande do Sul, southern Brazil.

Table 3 .
Measurements of the pores, annulus, annulus thickness and exine thickness of the pollen grains of 70 Poaceae species from grassland vegetation of Rio Grande do Sul, southern Brazil.

Table 4 .
Differences between the size of pollen grains of Atlantic Forest biome, Pampa biome, humid grassland, dry grassland, C 3 and C 4 species of Poaceae from grassland vegetation of Rio Grande do Sul, southern Brazil, obtained with ANOVA, followed by Tukey.Abbreviation: ns: not significant.