Strategies for reintroduction and conservation of Gymnopogon doellii, an endemic grass at risk of extinction

Abstract

The Brazilian savanna, regionally known as Cerrado, is characterized by a great diversity of physiognomies and holds the highest species diversity and level of endemism among world savannas. However, due mainly to agribusiness, this vegetation is among the most threatened ones, currently presenting alarming rates of extinction. Among the species present in the “red list” we find the endemic Gymnopogon doellii, a grass whose distribution is restricted to a few sites. In the Federal District and surroundings, for example, only one population, with scattered subpopulations, is known, within the limits of the Brasilia National Park. By this study we raised information related to the biology of G. doellii, as population size and distribution, genetic variability and germination characteristics. Besides, we produced seedlings in greenhouse, planted in different physiognomies of the Cerrado and followed them for almost four years to check for their survival, growth and fruiting under natural conditions. Seedlings transplanted to the field presented high rates of recruitment (> 25%), growth patterns similar to wild plants and produced viable caryopses. We recommend transplanting of individuals of G. doellii for both in situ conservation as well as for the revegetation of degraded areas of the Cerrado.

Keywords:
conservation; endangered species; genetic variability; recruitment; reintroduction; savanna

Resumo

A savana brasileira, também conhecida como Cerrado, é caracterizada por sua grande diversidade de fitofisionomias e detém a maior diversidade de espécies e nível de endemismo entre as savanas mundiais. Contudo, devido principalmente às atividades agropecuárias essa vasta vegetação está entre as mais degradadas, apresentando elevadas taxas de extinção. Entre as espécies atualmente presentes na “lista vermelha” encontra-se a endêmica Gymnopogon doellii, uma gramínea cuja distribuição é extremamente restrita a algumas poucas áreas. No Distrito Federal e arredores apenas uma população é conhecida, localizada dentro dos limites do Parque Nacional de Brasília. Neste estudo levantamos informações relevantes relacionadas a biologia dessa população de G. doellii, como sua área de distribuição, variabilidade genética e características de germinação. Mudas a partir de suas cariopses foram produzidas em casa de vegetação, plantadas em diferentes fisionomias do Cerrado, incluindo áreas degradadas, e suas taxas de sobrevivência e desempenho fisiológico acompanhados por quase quatro anos. A população apresenta baixa variabilidade genética. As mudas apresentaram altas taxas de sobrevivência (> 25%), padrões de crescimento similar aos indivíduos selvagens e produziram cariopses viáveis. Recomendamos o plantio de indivíduos de G. doellii tanto para sua conservação in situ como para recuperação de áreas degradadas do Cerrado.

Palavras-chave:
conservação; espécie ameaçada; variabilidade genética; recrutamento; reintrodução; cerrado

Introduction

It is well established that Brazil is home of the world’s greatest biodiversity but, at the same time, among the most threatened countries in respect to species conservation ( Amaral et al. 2017 ; Françoso et al. 2020 ). In respect to grasses, around 1,410 species occur within its territory, of which some 115 are at risk of extinction and 67 are currently on the Red List of Threatened Species. Gymnopogon doellii Boechat & Valls is among the grasses at risk of extinction, classified as “CR” - in critical danger - by the Red Book of Brazilian Flora ( Filgueiras et al. 2013 ).

The genus Gymnopogon belongs to the family Poaceae Barnhart, subfamily Chloridoideae Kunth ex Beilschm., tribe Cynodonteae Dumort., subtribe Hubbardochloinae ( Soreng et al. 2017 ), and occurs in South America, Central America, North America and Southeast Asia. It is composed of 14 described species ( Cialdella & Zuloaga 2011), seven of which are found in Brazil ( Boechat & Valls 1990a, b). The species are megathermic ( Burkart 1975), preferring sandy and dry soils, though some species, e.g., G. burchellii (Munro ex Döll) Ekman and G. fastigiatus Nees, can occur in humid areas, such as natural fields, marshes and plains. Gymnopogon doellii is exclusive to the Brazilian flora, and its occurrence is restricted to the states of Minas Gerais (MG), Goiás (GO) and the Federal District (DF) ( Boechat & Valls. 1990a, b; Filgueiras 1992; Cialdella & Zuloaga 2011; Vinícius-Silva et al. 2020 ). Filgueiras ( 1992) classified G. doellii as a species of rare occurrence within the Federal District. The great devastation of the Cerrado by agribusiness ( Lahsen et al. 2016 ) most likely contributed to the reduction of its distribution over native areas. Besides, the invasion of natural areas by exotic plants has become a particular threat to the conservation of the native flora ( Sühs et al. 2020 ), and alien species represent the second most common threat associated with species extinction since AD 1500 ( Bellard et al. 2020 ). At the Brasilia National Park, for example, 28 species of alien grasses were registered, which represent 19% of the total number of grasses (147) in this protected area. For the effective preservation of the native vegetation, a monitoring and control/eradication program should be elaborated including all alien species in this protected area ( Martins et al. 2007 ).

The Brasília National Park has the largest known protected population of G. doellii within a Conservation Unit. To date, the extent of its area of occurrence and population size, physiological attributes, genetic variability of the remaining plants, germination capability and recruitment potential over native areas remain unknown.

Previous studies revealed a low production of, and a high level of dormancy among viable caryopses collected from natural populations of G. doellii ( Martins et al. 1997 ), suggesting that these attributes may have contributed to its low occurrence and distribution over natural areas. As far as we know, there is no other study on germination characteristics, initial plant growth and development of this endangered species neither conducted in the laboratory nor under field conditions.

Within this context, the present study aimed at contributing for a better knowledge of the endangered native grass G. doelli by mapping its occurrence areas, determining its seed germination characteristics, analyzing the genetic variability of these remaining populations and evaluating growth parameters and establishment rates of individuals introduced in different savanna physiognomies occurring within the Brasília National Park. By this study we also intend to contribute to unraveling limitations for its reproductive capacity and determining efficient management strategies for its reintroduction in natural.

Material and Methods

Occurrence of G. doellii in the Brasília National Park

The survey of G. doellii populations at the Brasília National Park was carried out from February 2014 to June 2017, totaling 287 individuals sampled. Fieldwork was combined with a Global Positioning System (GPS) and laboratory signal receiver, with the support of Geoprocessing techniques. The areas of occurrence of G. doellii were georeferenced and, subsequently, they were spatialized using the ArcGIS SIG program. Areas of influence with a radius of 50 m were generated from each point of occurrence.

Seed germination experiments

Our field observations at the Brasilia National Park revealed that individuals of G. doellii reach the reproductive period between late April and May. The experiments of germination were conducted with mature caryopses collected between May- June of 2015 at the Brasilia National Park. Around 40 inflorescences from as many individuals as possible were randomly harvested from natural areas of open savanna as well as from dense grasslands, stored in paper bags and dried under natural conditions (~23 ºC) in the Laboratório de Termobiologia of the University of Brasilia, DF. The spikelets were manually separated to remove the dispersal units (caryopses), which were stored in Gerbox at lab temperature (~23 ºC) in a dark room for up to one year. Full spikelets (i.e., those containing embryos) could be easily differentiated from the empty ones by their color and size; while embryo-bearing spikelets are bigger and reddish, the empty ones are smaller and yellowish ( Carmona et al. 1997 ).

Germination tests started in September 2015, four months after the dispersal units were collected. The caryopses were placed in transparent polystyrene (6 cm) plates lined with three sheets of qualitative filter paper moistened with distilled water. Tests were performed in a germination chamber set for alternating temperature of 22 ºC/28 ºC (12/12 hours) under a photoperiod of 12 hours (white, fluorescent light). This temperature regime was set according to the average minimum and maximum temperatures recorded during the wet season, which represents the growing season for most savanna species in the Cerrado, including grasses ( Ramos et al. 2017 ). Tests in the dark were carried out at the same temperature regime, however the dark treatment was applied by wrapping the plates with two sheets of aluminum foil. Five replicates of twenty embryo-bearing caryopses were used for each light and dark treatments. Germination was monitored daily and distilled water was added as needed. For experiments conducted in the dark, observations were made under a green safelight (490–560 nm) ( Labouriau 1983). The germination experiments lasted 83–95 days, according to the treatment, and ended when no germination was detected for 15 consecutive days. Germinability (%) and its standard deviation were calculated according to Labouriau ( 1983).

DNA extraction and genetic analyses Genetic variability of G. doellii populations was assessed using RAPD markers.

Samples were collected from 96 individuals of G. doellii in the following areas: open savanna, dense grassland, and disturbed area. Total genomic DNA was extracted from freeze-dried leaf tissue using a modified CTAB protocol ( Grattapaglia & Sederoff 1994). DNA was quantified comparing the fluorescence intensities of the samples to those of lambda DNA standards in ethidium bromide- stained 1% agarose gels under UV light.

RAPD reactions were performed in a 13 μl solution, containing 10X PCR buffer, 1 μg/μl purified BSA (New England Biolabs), 200 μM of each dNTP, 0.4 μM 10-base primer (Operon Technologies Inc.), 7.5 ng of genomic DNA and 1 unit of Taq DNA polymerase. Amplification reactions were performed in a Veriti Thermal Cycler (Applied Biosystems) programmed for 40 cycles of 1 min at 92 ºC, 1 min at 35 ºC and 2 min at 72 ºC, and a final DNA extension cycle at 72 oC for 7 min. RAPD products were analyzed by electrophoresis in 1.5% agarose gels in 1X TBE and 1.5 μg/μl ethidium bromide.

PCR amplification products of the 96 samples were scored as presence (1) or absence (0) of bands. The data matrix was used to calculate Jaccard’s similarity coefficient. Dendrograms were constructed using the unweighted pair-group method analysis (UPGMA). These analyses were performed using NTSYS-pc software, version 2.21 ( Rohlf 2009).

Management strategies for reintroduction into the National Park of Brasilia

Seedlings of G. doellii were produced in a greenhouse covered by a shade cloth blocking 50% of solar radiation of the state company NOVACAP/ Federal District, located near the Brasília National Park. In the greenhouse, plastic trays (20 × 30 × 10 cm) were filled with a substrate consisting of a mixture of 25 kg of Bioplant fertilizer (agricultural substrate with coconut fibers) plus 2 kg of the NPK Osmocote fertilizer (18–5–9). In the first week of June 2015, seeds were sown in the trays and covered with a thin layer of the substrate (~1 mm). The trays were watered frequently and around 30 days after sowing the growing seedlings were transplanted into 50cm3 tubules (conical tube of rigid plastic) filled with the same substrate as the germination trays.

The transplanting of G. doellii seedlings into natural areas started on November 2015 in the following areas: a) open savanna; b) dense grassland; and c) disturbed area. Seedlings were planted one per each corner and one in the center of four plots of 16 m2 (4 × 4 m) previously demarcated, so resulting in 20 seedlings transplanted in each studied area.

The survival and grow of the transplanted G. doellii seedlings were followed during the next four years. In December 2015, May 2017 and May 2019, the number of living individuals, the number of tillers per individual and length of the longest tiller (as a proxy for shoot height) per individual were accessed. In order to compare the performance of the transplanted seedlings with the performance of those naturally occurring in the Park, in May 2019, the same growth parameters were measured on 40 adult individuals randomly selected from native subpopulations of G. doellii.

Statistical analyses

Analysis of Variance (ANOVA) followed by Tukey HSD was performed to detect differences in the germination percentages of freshly harvested or one-year stored caryopses of G. doellii, incubated under both light and dark conditions. Two-tailed Kruskal-Wallis - Bonferroni posteriori test was performed to compare the number of tillers and the shoot height of individuals measured in December 2015, May 2017 and May 2019 in the three areas under study ( Rahardja 2017).

Results and Discussion

The area of potential occurrence of G. doellii in the Brasília National Park was estimated in 104.87 ha (Fig. 1). Our exhaustive field surveys revealed that the spatial distribution of individuals of G. doellii was generally clumped, and subpopulations were detected in the Cerrado physiognomies of open savanna, dense grassland, and grassland ( sensu Ribeiro & Walter 2008). Individuals of G. doellii were also detected in an area formerly subjected to mining (disturbed area), but now going through spontaneous vegetation recovery. The population mapping shows that this native species can occur in a wide range of physiognomies of the Cerrado, as well as in disturbed areas (Fig. 2). The identification of areas of spontaneous, natural occurrence of G. doellii is critical to most effectively allocate efforts in reintroduction of native grasses for restoration purposes ( Schmidt et al. 2019 ).

Figure 1 –
Populations of Gymnopogon doellii recorded (black dots) and area of occurrence estimated (circled dark grey area) at the Brasília National Park (BNP), Federal District.

Figure 2 –
Individuals of Gymnopogon doellii of spontaneous occurrence at the Brasília National Park, Federal District.

Freshly harvested caryopses of G. doellii were shown to present a considerable level of dormancy and absolute light requirement for germination (Tab. 1), as already reported by Carmona et al. ( 1997). However, after one year of storage, the germination percentage increased significantly, and the light-dependence for germination virtually disappeared (Tab. 1). Moreover, the results indicate that the caryopses can maintain a high level of viability up to one year of storage.

Table 1 –
Germination percentage and standard deviation of freshly harvested (Sept 2015) and one year stored (Sept 2016) caryopses of Gymnopogon doellii at alternating temperature of 22 ºC/28 ºC under light or dark conditions.

Treatment	Sept 2015		Sept 2016
	Germination (%)	Standard deviation	Germination (%)	Standard deviation
Light	57 *, **	21.1	81 *, ns	11.4
Darkness	0 **, **	0	88 **, ns	7.58

First symbol represents comparison within rows, second symbol represents comparison within columns. Shapiro-Wilk test did not reject data normality (W = 0.93908, p-value = 0.2304), and Levene’s Test did not reject variance homogeneity (F-value = 2.5106, p-value = 0.0956). ANOVA two-way test followed by Tukey HSD test: * = significance level of 0.05; ** = significance level of 0.01; ns = no significant difference among treatments).

Dormancy among caryopses of native grass species has been previously reported ( Ramos et al. 2017 , 2019; Saraiva et al. 2020 ) and different cues, as smoke and heat shock ( Ramos et al. 2019 ) as well as KNO3 and alternating temperatures ( Carmona et al. 1998 ; Saraiva et al. 2020 ) were reported to improve their germination. Caryopses of G. doellii are dormant at harvest, but alternating temperature treatments, coupled or not with KNO3 and light were shown to considerably improve germination ( Carmona et al. 1997 ).

The level of dormancy and considerable longevity of G. doellii caryopses seem to represent a recruitment strategy to reduce the probability of failed germination during the dry season and, at the same time, to keep a seed bank until the following rain season, as already reported among other grass species of the Cerrado ( Ramos et al. 2017 ). In fact, previous studies have reported a higher level of dormancy among grass caryopses dispersed early in comparison to those dispersed late in the dry season ( Ramos et al. 2017 ). Due to the presence of a positive photoblastism, it is also expected that caryopses of G. doellii germinate primarily on or near the soil surface, so reducing their probability of germination either buried or in shaded environments.

A total of 60 RAPD primers were screened for polymorphism using eight samples. Of these, only 10 revealed at least one polymorphic fragment and were used to analyze the 96 individuals. The 10 primers amplified 51 fragments, which were used for the genetic variability analysis. All primers produced identical RAPD patterns for the selected fragments during the primer-screening step and in the final analysis.

An UPGMA dendrogram based on Jaccard’s similarity index was constructed for the 96 individuals (Fig. S1, available on supplementary material < https://doi.org/10.6084/m9.figshare.21349773.v1>). Some groups were evident, but they were not associated neither with the area nor with the vegetation type in which the samples were collected. The average genetic similarity indices between the 96 plants were very high, estimated at 0.92. These results revealed a very low genetic variability of G. doellii within the National Park of Brasilia, proving the urgency of finding new populations, due to the high risk of extinction of this species.

Well planned reintroduction of new individuals has been viewed as a promising strategy for conserving rare plant species worldwide ( Maschinski & Albrecht 2017), and conservation units have emerged as relevant areas for in situ conservation of threatened ones ( Costa et al. 2018 ). One month after transplanting the seedlings to the selected areas, all individuals of G. doellii were still alive. This high survival rate is very probably associated to the fact that they were transplanted in November, a period when rains are relatively regular and frequent in the region. After 18 months, survival was still high among individuals planted in the open savanna physiognomy (90%) but dropped to 50% and 65% in dense grassland and disturbed areas, respectively (Tab. 2). After 42 months of transplanting, the survival dropped to 40%, 30% and 25% in the open savanna, dense grassland and disturbed area, respectively. The reduction in survival rates among transplanted individuals was very likely related to the periods of water shortage which plants face every year during the dry season, when the grass-herb component of the vegetation usually dries out in the open physiognomies of the Cerrado ( Silva et al. 2008 ). The establishment rates reached in our transplanting approach are within the range observed in similar studies; for example, in a study of reintroduction of Artemisia tridentata seedling in communities subjected to fire, Davies et al. ( 2020) reported survival rates between 46% and 7%, depending on microsite type, after four growing seasons of planting. A survival rate above 25% as reached by our study is very promising, since at least one out of four individuals survive in disturbed area, revealing that this strategy of seedling transplanting may be effective both for reintroduction efforts of endangered grasses as well as for revegetation of open and disturbed areas with native species.

Table 2 –
Survival (in %) of individuals of Gymnopogon doellii after three years of transplanting into different types of vegetation at the Brasilia National Park, Federal District. Six-month old individuals produced under greenhouse conditions were transplanted to the areas in November 2015.

Physiognomy	Survival (%)
	December 2015	May 2017	May 2019
open savanna	100	90	40
dense grassland	100	50	30
disturbed area	100	65	25

Differently from scleromorphic forests and even savanna physiognomies, which are likely to regenerate naturally following low- or medium- intensity land use due to extensive resprouting of woody plants, the restoration of grassland physiognomies may require reintroduction of grass and forb species that do not tolerate soil disturbance and exotic grass competition ( Schmidt et al. 2019 ). Although this approach may be unsuccessful for forest tree species ( Souza et al. 2020 ), direct seeding could work for open physiognomies. In fact, direct seeding effectively established many native neotropical savanna species of different life forms (including grasses) in tropical savanna- grassland mosaics ( Sampaio et al. 2019 ) and may reach up to 30% of soil cover for grass species ( Pellizzaro et al. 2017 ). On the other hand, the establishment success of our seedling transplanting approach (> 25%) was very close to those studies and shows that the use of grass seedlings for revegetation of open areas and reintroduction of endangered species may be effective.

It is worth mentioning that, over the first year after transplanting, four out of 20 seedlings originally transplanted reached their reproductive stage in the open savanna physiognomy. Laboratory tests conducted at the same experimental conditions described above with caryopses collected from these individuals revealed that 55% were fertile (bearing viable embryos) and reached a germination of 43% under the experimental conditions described above. In the second year of monitoring, we observed that six, eight and four individuals reached their reproductive stage in the open savanna, dense grassland and disturbed areas, respectively, representing 30%, 40% and 20% of the total individuals transplanted. These results revealed that, within its first years of life, this species presents a great potential to recruit and reproduce over different physiognomies of the Cerrado, what is promising in terms of recovery of natural grasslands ( Overbeck et al. 2013 ).

Besides survival and reproduction, growth parameters can also provide evidence of successful recruitment and establishment of a species at new sites ( Monks et al. 2012 ). The monitoring of the transplanted seedlings revealed a decline in the number of tillers per individual over the experimental period, however, the average shoot height increased in all experimental areas, in particular between the first and second years of monitoring (Tab. S1, available on supplementary material < https://doi.org/10.6084/m9.figshare.21349773.v1>). Taking together, these results indicate that the growth of the seedlings started with a higher investment in the number of tillers, later supplanted by an increase in the average shoot height, in particular among individuals transplanted in the open savanna and dense grassland areas (Tab. S1, available on supplementary material < https://doi.org/10.6084/m9.figshare.21349773.v1>). After four years of monitoring the number of tillers and average shoot height was the lowest among individuals transplanted into the disturbed area. We have not intended to check for effects of spatial variability in our planting responses ( Davies et al. 2020 ), but the establishment and grow performance of seedlings cleared differed across the areas of study (Tab. S1, available on supplementary material < https://doi.org/10.6084/m9.figshare.21349773.v1>). The low growth performance of seedlings observed in the disturbed area may be related to edaphic factors: the soil in this anthropized area is much more compacted than in the undisturbed, natural areas (C.R. Martins 2018, personal communication).

A field survey conducted over native subpopulations of G. doellii revealed an average number of four tillers per clump, and an average shoot height of 70.4 cm (± 19.8 cm) among adult individuals (n = 40). These observations allowed us to conclude that the individuals of G. doellii transplanted to the open savanna and dense grassland areas showed a physiological performance comparable to individuals growing spontaneously in these physiognomies.

Our study revealed and characterized a remnant population of G. doellii thriving in the Brasília National Park. This population spread over physiognomies of grassland and open savanna, coexisting with a large number of herbs and grasses. The considerably high survival rates after transplanting, comparable physiological performance and capability to produce viable caryopses show that the production and transplanting of G. doellii seedlings into natural areas may represent a viable alternative both for restoration purposes as for the in-situ conservation of this endangered grass in the Cerrado.

The analyses using molecular markers revealed that the genetic variability among subpopulations of G. doellii occurring spontaneously at the Brasília National Park is low, which highlights the urgency of finding new areas of occurrence of this species. After 149 years since its first collection in Minas Gerais State (Lagoa Santa locality), new occurrences of G. doellii were recorded in that state, some 650 km far from the Brasília National Park ( Vinícius-Silva et al. 2020 ). This discovery is extremely relevant for the conservation of G. doellii and renews the hope that new populations be found in areas where this species was formerly registered.

Acknowledgments

Fabian Borghetti and José Francisco Montenegro Valls gratefully acknowledge the Conselho Nacional de Desenvolvinento Científico e Tecnológico - CNPq, Brazil, for their fellow grants (PQ number 312152/2018-3 and 310026/2018-0, respectively). Studies conducted at the Laboratório de Termobiologia were partially supported by a research grant from the Fundação de Apoio à Pesquisa do Distrito Federal - FAP-DF (process number 193.000.922/2015). We thank the staff of the Parque Nacional de Brasilia who provided research facilities for the field work.

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Edited by

Area Editor:

Dr. Pedro Viana

Publication Dates

• Publication in this collection
  21 Nov 2022
• Date of issue
  2022

History

• Received
  01 June 2021
• Accepted
  25 Feb 2022