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Vol. 5, n.2: pp. 202-210, May, 201 4 ISSN: 2179- 4804
Journal of Biotechnology and Biodiversity
Liliana Essi1, Hilda Maria Longhi Wagner2, Tatiana Teixeira de Souza Chies 2
ISSR as a tool to support taxonomic decisions: a first
approach for Chascolytrum species complexes
(Poaceae)
ABSTRACT
Chascolytrum Desv. is a South American grass genus, with extension up to Central America, which presents several taxonomic controversies concerning genera and species circumscriptions. Morphological studies were not able to provide robust elements for taxonomic decisions in some species complexes. It was the case of Chascolytrum subaristatum and morphological allies, and of Chascolytrum rufum plus the acceptance (or rejection) of two varieties. In order to provide additional elements for taxonomic decisions in these species complexes, it was performed a preliminary survey using Inter Simple Sequence Repeat (ISSR) markers. Nine primers were used to build similarity dendrograms, and 25 collections were included, analysed in two separated complexes. ISSR were able to separate the two varieties of Chascolytrum rufum, supporting their acceptance. Two species recently described could be clearly separated from their morphologically related taxa, but the species Briza erecta, B riza macrostachya and Chascolytrum subaristatum, as well as Briza subaristata var. interrupta, could not be separated, adding elements to the synonymization of these three taxa under Chascolytrum subaristatum . The ISSR contributed to solving some controversies on genus Chascolytrum, but its use as an exclusive species-marker is limited in this genus due to high band polymorphism.
Keywords: Briza, molecular marker, Poeae, systematics.
ISSR como uma ferramenta para apoiar decisões taxonômicas: uma abordagem preliminar para complexos de espécies em Chascolytrum (Poaceae)
RESUMO
Chascolytrum Desv. é um gênero sul-americano de gramíneas, com extensão da distribuição até a América Central, o qual apresenta diversas controvérsias taxonômicas com relação à circunscrição de gêneros e espécies. Estudos morfológicos não foram capazes de fornecer elementos robustos para a tomada de decisões taxonômicas em relação a alguns complexos de espécies. Esse foi o caso de Chascolytrum subaristatum e espécies morfologicamente afins, e de Chascolytrum rufum com relação à aceitação (ou rejeição) de duas variedades. Para fornecer elementos adicionais para decisões taxonômicas nesses complexos de espécies, foi realizado um estudo preliminar utilizando marcadores do tipo Inter Simple Sequence Repeat (ISSR). Foram utilizados nove primers para construir dendrogramas de similaridade, e 25 coletas foram incluídas, analisadas em dois complexos distintos. Os marcadores ISSR foram capazes de separar as duas variedades de Chascolytrum rufum, apoiando a aceitação das mesmas. Duas espécies recentemente descritas puderam ser claramente separadas de seus táxons morfologicamente relacionados, porém as espécies Briza erecta, Briza macrostachya e Chascolytrum subaristatum, bem como Briza subaristata var. interrupta, não foram discriminadas nos dendrogramas, o que adiciona elementos para justificar a sinonimização desses três táxons sob Chascolytrum subaristatum. Os ISSR contribuíram para a resolução de algumas controvérsias taxonômicas no gênero Chascolytrum, mas seu uso como um marcador para espécies é limitado no gênero devido ao alto polimorfismo de bandas.
Palavras-chave: Briza, marcador molecular, Poeae, sistemática .
*Autor para correspondência
1Universidade Federal de Santa Maria
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INTRODUCTION
Chascolytrum Desv. lato sensu (Poaceae) is
differences can be a result of environmental variation, species polymorphism or even of
a morphologically diverse and evolutionary divergence processes, such as
taxonomically controversial grass genus from subtropical and highland areas in South America, some species presenting potential to use as forage, and also there are endangered species. It is represented by
genetic isolation or speciation. The presence or absence of trichomes on certain structures, for instance, may be taxonomically negligible for some groups, or be a stable and reliable morphologic
perennial species, caespitose or marker for other taxa.
rhizomatous, with extravaginal basal innovations, spikelets many-flowered and panicles open to contracted, erect or pendulous. The spikelets are very variable inside the genus, and this is one of the reasons that made authors propose several different circumscriptions for this alliance of species. These species can be placed into several other genera, like Briza L. l ato sensu, Poidium Nees or Lombardochloa
Roseng. & B. R. Arrill., according to each author. Its evolutionary history was recently analysed (Essi, Longhi- Wagner and Souza-Chies, 2008), and a proposal of a lato sensu circumscription was done, excluding only the euroasiatic species ( Essi, Longhi-Wagner andSouza-Chies, 2011) . Some taxa presents delimita tion controversies, forming species complexes not clarified with morphological studies. It is not clear, for instance, whether species like Briza erecta Lam. and Brizamacrostachya (J. Presl) Steud. should be treated as separate taxa, as done by Longhi-Wagner (1987), or whether they represent only intra-specific variation, and should be synonymised, as proposed by Matthei (1975). The occurrence of several intermediates further complicates this case: morphological links can group these two species with the broadly distributed B riza subaristata Lam. (here treated as Chascolytrum subaristatum (Lam.) Desv.).There is also no consensus whether varieties distinguishable only byspikelet measures, like B. subaristata var. interrupta (Hack. ex Stuck.) Roseng.,Arrill. &Izag.orbased on single discrete characters , like B. rufa var. sparsipilosa Roseng., Arrill. & Izag., should be accepted, or whether their variation is merely intra - specific polymorphism or environment plasticity.
It is often difficult to interpret morphological divergences among closely related taxa. Minor morphological
Decisions concerning accepting or not certain features such as taxon markers or like establishing a given taxonomic level for a morphologically distinct group may provide to systematics a certain level of subjectivity. In such cases, the addition of extra approaches can bring light and objectivity to the studies.
Inter Simple Sequence Repeat (ISSR) PCR is a fast, cheap approach to genetic characterization and is widely used for ecological (Wu et al., 2004; Poulin, Weller and Sakai, 2005), population (Esselman et al., 1999; Alexander, Liston and Popovich, 2004; Song et al., 2006; Zhang et al., 2013) or phylogenetic studies (Joshi et al., 2000; Bussell, Waycott and Chappill, 2005), as well as in crop cultivar or native plant fingerprinting (Mattioni et al., 2002; Souza et al., 2005). It has been also applied to explore the hybrid origin of species (Ayres and Strong, 2001) and for systematic purposes (Blair, Panaud and McCouch, 1999; Vanderpoorten, Hedenäs and Jacquemart, 2003; Dinelli, 2004; Vergara et al., 2008).
ISSRs have a few advantages over other molecular markers. Their primers annealto SSR (Simple Sequence Repeat) that are abundant throughout the eukaryotic genome and evolve rapidly, and hence may reveal a high level of polymorphism (Zietkiewicz , Rafalski and Labuda, 1994; Li and Ge, 2001). In addition, ISSR may produce more reliable and reproducible bands than RAPD (Random Amplified Polymorphic DNA), due to the higher annealing temperature and longer primer sequences (Qian, Ge and Hong, 2001).
The aim of this study was to test whether ISSR can provide markers to help at intra - specific or species-level decisions for taxa belonging to genus Chascolytrum lato sensu (with some taxa referred in the text as Briza, according to the existence or absence of a combination under Chascolytrum) ,
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whenever the study of morphological characters alone is insufficient.
The results obtained by ISSR markers are compared to previous approaches on the genus, and suggestions for applications for ISSR in the group are provided.
MATERIAL AND METHODS
The study was carried out at the Laboratory of Plant Genetics of the Universidade Federal do Rio Grande do Sul, following the procedures described in the Molecular Protocols, between the years 2006 and 2007. Plants were collected previously during field trips (from 2003 to 2006), or obtained from herbarium specimens, as described in the Taxon Sampling. Data analysis was carried out at the Laboratory of Plant Genetics of the U niversidade Federal do Rio Grande do Sul and latter at
the Laboratory of Taxonomy of the Universidade Federal de Santa Maria.
Taxon S ampling
Taxon sampling included representatives of two species complexes, which were analysed separately, according to morphological similarities (Table 1). The first complex (Group 1) included three species (Figures 1 and 2), and the second (Group 2) included one species (Figure 2). Twenty-five accessions were analysed , including representatives of major morphological variation found in each complex. Characters such as spikelet colour and size, lemma width, presence of trichomes, or habitat, were most often considered for sampling. Geographical information was collected, although it was not considered for sampling purposes.
Figure 1: Comparison of specimens showing part of the morphological variability of Chascolytrum subaristatum- Size and shape variation in spikelets and panicles. A. “Briza macrostachya-like” spikelet (Voucher Information: L. Essi 13, São Pedro do Sul, RS, Brazil, 12/10/2003; ICN herbarium); B. Cylindrical type spikelets (Voucher Information: T. M. Petersen, La Yela, Dep. Empedrado, Prov. Corrientes, Argentina, 29/10/1969; S herbarium); C. Comparison between two panicles, with small spikelets (left) and big spikelets (right) – Location: São Pedro do Sul, RS, Brazil, 2003 (no voucher associated to the picture).Squares [A-B]: 1mm2 .
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Figure 2: Species included in the analysis. A. Lema of Chascolytrum altimontanum (Group 1) – Voucher Information: Wood 10768, Oropeza, Chuquisaca, Bolivia, 25/02/1996, LPB herbarium. The arrow shows the complanate trichomes at the base of the lema, typical of the species. Squares: 1mm2; B. Panicle of Chascolytrum latifolium (Group 1)– Picture location and date: Urubici, Santa Catarina, Brazil, 07/12/2006; Vouchers Information: Longhi-Wagner et al. 10228 – 10230, ICN herbarium; C. Panicle of Chascolytrumrufum (Group 2) – Picture location and date: Guarapuava, PR, Brazil, 16/01/2001 (no voucher associated to the picture).
Table 1: Accessions included in this study. All the taxa included are considered belonging to genus Chascolytrum; Some taxa were referred as genus Briza, when there is not yet a published combination inside Chascolytrum [one new combination submitted]. Collector’s abbreviations: Hl: H. M. Longhi Wagner; Li: L. Essi; W: W. J. R. Wood.
Species [morphological or habitat type] Collector number
Vou cher
Geographical origin of the sample (Country – State – Locality)
Group 1
C. subaristatum (Lam.) Desv. [Brizaerecta Lam.]
Hl5056
ICN
Uruguay, Carrasco
C. subaristatum [intermediate C. subaristatum- B. erecta ]
Li332 ICN 149.322 Brazil, Santa Catarina, Florianópolis
C. subaristatum Li7 ICN 132.506 Brazil, Rio Grande do Sul, Porto Alegre
C. subaristatum Li8 ICN 134.887 Brazil, Rio Grande do Sul, Porto Alegre
C. subaristatum Li10 ICN 132.509 Brazil, Rio Grande do Sul, Porto Alegre
C. subaristatum Li11 ICN 132.510 Brazil, Rio Grande do Sul, Porto Alegre
C. subaristatum Li12 ICN 132.511 Brazil, Rio Grande do Sul, Porto Alegre
C. subaristatum Li202 ICN 132.542 Brazil, Santa Catarina, Urubici
C. subaristatum Hl5029 ICN Brazil
C. subaristatum
Hl5040 ICN 131.398 Brazil, Rio Grande do Sul, Bagé - Minas de Camaquã
C. subaristatum[B. subaristata var. interrupta (Hack. exStuck.) Roseng., Arrill. &Izag. ]
Li19 ICN 132.518 Brazil, Rio Grande do Sul, São Pedro do Sul
C. subaristatum[Briza macrostachya (J. Presl) Steud.]
Li13 ICN 132.512 Brazil, Rio Grande do Sul, São Pedro do Sul
C. subaristatum[Briza macrostachya] Li18 ICN 132.517 Brazil, Rio Grande do Sul, São Pedro
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do Sul
C. subaristatum[Brizamacrostachya] Li54 ICN 132.553 Brazil, Rio Grande do Sul, Piratini
C. subaristatum[Brizamacrostachya] Li57 ICN 132.556 Brazil, Rio Grande do Sul, Piratini
C. subaristatum[Brizamacrostachya ]
Hl5038 ICN 131.396 Brazil, Rio Grande do Sul, Bagé - Minas de Camaquã
ChascolytrumlatifoliumEssi, Souza - Chies &
Longhi- Wagner
Li201 ICN 132.700
Brazil, Santa Catarina, Urubici
Chascolytrum altimontanum Essi, Souza- Chies
& Longhi- Wagner
W10768
K, LPB
Bolivia, Chuquisaca, Oropeza
Group 2
C. rufum J. Presl var. rufum[80 cm tall plant]
Li278 ICN 134.875 Brazil, Rio Grande do Sul, Canguçu
C. rufum var. rufum Li279 ICN 134.876 Brazil, Rio Grande do Sul, Canguçu
C. rufum var. rufum Li280 ICN 134.877 Brazil, Rio Grande do Sul, Canguçu
C. rufumvar. rufum[broad leaves] Li372 ICN 132.586 Brazil, Rio Grande do Sul, Porto Alegre
C. rufum var. rufum [broad leaves] Li373 ICN 132.587 Brazil, Rio Grande do Sul, Porto Alegre
C. rufum [Brizarufa var. sparsipilosaRoseng., Arrill. &Izag., open panicle type]
Li281 ICN 134.878 Brazil, Rio Grande do Sul, Canguçu
C. rufum [Briza rufa var. sparsipilosa] Hl8061 ICN 135.198
Brazil, Rio Grande do Sul, São Francisco de Paula
Molecular P rotocols
DNA was extracted from fresh, silica dried or herbarium leaves, following a CTAB method (Doyle and Doyle, 1987) adapted for micro-centrifuge tubes. Total DNA was quantified by agarose gel visualization. Thirteen primers described by Joshi et al. (2000), Martins, Tenreiro and Oliveira, (2003) and Lin et al. (2005) were tested for ISSR amplification, and nine of them were selected (Table 2). PCR reactions were carried out in an Applied Biosystems 2400 termocycler, in a total volume of 25 µl containing 12 µl sterile Milli-Q purified water, 0.2 µl Taq DNA Polymerase (5U/ul), 2.3 µl MgCl2 (25mM), 2.5 µl 10× buffer, 1
µl primer 10 pmol, 1 µl of 40 mM dNTP mixture (10mM each dNTP), 1 µl DMSO (2%), and 5 µl DNA (total 30-50 ng). PCR amplifications included 40 cycles of 1 min at 94°C, 45 sec at 50°C and 2 min at 72°C, preceded by a period of 5 min at 92°C and completed by a final extension of 5 min at 72°C, for all the primers, except primer F11, whose annealing temperature wa s 48°C. The ISSR amplification products were stained by ethidium bromide, run until the complete separation of the ladder (100 and 50 bp, PB-L Produtos Bio- Lógicos, Universidad Nacional de Quilmes), approximately 2h30min, at 100V on 1.8% agarose gel, and visualized by UV.
Table 2: Primers code and sequences included in the analyses.
Primer code: Primer sequence: Described by:
P1 (AC)8T Lin et al., 2005 (as 25)
P2 (GA)8T Joshi et al., 2000 (as 810)
P3 (CTC)4RC Poulin, Weller and Sakai, 2005 (as n. 15)
P4 (CT)8G Joshi et al., 2000 (as 815)
F3 (AG)8YC Joshi et al., 2000 (as 835)
F4 (GA)8YC Joshi et al., 2000 (as 841)
F7 (GT)8A Joshi et al., 2000 (as 819)
F11 (GACA)4 Lin et al., 2005 (as 73)
F12 (GTGC)4 Martins, Tenreiro and Oliveira, 2003 (as IS06)
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Data analyse s
Polymorph bands were scored for presence/absence. Analyses were performed using the NTSYS-pc version 2.10 software (Rohlf, 2000). For each of the two groups, the genetic similarity among individuals was calculated using Jaccard’s Similarity Coefficient (J), which takes only shared presence into account. The similarity
polymorphic. For Group 2, the average was 8.6 DNA bands, 85.89% of which were polymorphic. The group with the highest number of monomorphic bands was group 2, with 11 bands (14.11% of the matrix). The primer with the smallest number of bands, considering both complexes, was F7 (total of 7 bands), and the highest number of bands was obtained with the primer P4
relationships were portrayed by (total of 31 bands).
dendrograms built using the clustering method Unweighted Pair Group Method of
The Jaccard similarity index ranged from 0.2260, between accession li201 and
Arithmetic Average (UPGMA). accession w10768, to 0.7442, between
Bootstraping analyses, with 2,000 replicates each, were performed by the Winboot software (Yap and Nelson, 1996), to access the robustness of nodes in the dendrograms, as proposed by Felsenstein (1985).
RESULTS AND DISCUSSION
For Group 1, an average of 12.66 DNA bands was produced using nine primers, 98.24% of which were found to be
accessionHw5038 and accession Hw5056. Low levels of similarity were found in both groups. The absence of a similarity index of 1.0 indicates that no clone or repeated germplasm was included, and all accessions represent plants with distinct f ingerprints. The limits for the acceptance of the clusters were established by the similarity average inside the group (see values of the averages for each group in Figures 3 and 4 ).
Figure 3: UPGMA dendrogram of individuals from group 1. The cluster is based on the Jaccard similarity index. Vertical line marks the average of similarity indexes.*1= C. subaristatum [B. subaristata var. interrupta (Hack. ex Stuck.) Roseng., Arrill. & Izag.]; *2=C. subaristatum [intermediate C. subaristatum- B. erecta] Numbers below branches correspond to bootstrap values >50.
In Group 1, the two recently described species (accessions li201 and w10768) appeared very distinct from the remaining taxa, being grouped with a very low similarity index. The two are distinct taxa, and grouped together probably due to the absence of other more closely related accessions. All other accessions appeared
clustered. Although the grouping between Cluster A and Cluster B is under the similarity average for the group, it is supported by bootstrap. The high similarity among accessions of Briza macrostachya and the accession of B. erecta is also supported by bo otstrap.
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Figure 4: UPGMA dendrogram of individuals from group 2. The cluster is based on the Jaccard similarity index. Vertical line marks the average of similarity indexes. Numbers below branches correspond to bootstrap values >50.
In Group 2, the two varieties could be separated, being variety sparsipilosa grouped with the highest similarity level (Cluster B), in spite of its different geographical origins. The main clusters (A and B) represent the two varieties, and the secondary clusters follow a geographical order (AI: Canguçu; AII: Porto Alegre). Low similarity levels were already expected, due to two main factors: 1) The
data supported the recognition of two genetically distinct groups, which correspond to the two varieties sampled. This is also in agreement with other approaches based on flavonoid variation, pollen and satellite chromosomes position (Winge et al., 1984).
2) Concerning the robustness of recently described species: The accessions sampled were absolutely distinct from the
ISSR are one of the most polymorphic remaining morphologically related
molecular markers; 2) Chascolytrum accessions, thus supporting their acceptance
species are autogamous, mainly as distinct species, in addition to the
cleistogamous, so that a low intra - population variability and a high inter - population variability were expected. Although a high level of polymorphism was not a surprise, the use of ISSR as a species marker was not possible: only one band was exclusive of a particular analysed species (however not exclusive when comparing to other Chascolytrum species, not included in this paper – data not show), and the similarity indexes were lower than those usually published for species- complexes. Their use as phylogenetic markers for Chascolytrum should be avoided, since a minimum monomorphism (20%) across all taxa is required to consider the markers potentially homologous to such studies (Bussell, Waycott and Chappill, 2005). T he ISSR helped to solve some critical questions:
1) Concerning the acceptance of two varieties for Chascolytrum rufum: ISSR
morphological findings.
3) Concerning the circumscription for the Briza macrostachya- B. erecta - C. subaristatum complex: The obtained data place B. erecta asgenetically very close to B. macrostachya, a relation supported by bootstrap. This is in agreement with the opinion of Matthei (1975), who considered these two species synonymous. But the different accessions of C. subaristatum are widespread over the clusters, producing the same impact caused by morphological analysis –not only B. macrostachya and B. erecta should be synonymised, but all three, B. erecta and B. macrostachya considered an intra-specific polymorphism. This idea may appear not convincing morphologically considering the analysis of the Uruguayan material of B. erecta, but it is perfectly acceptable when the Brazilian material is studied. Different spikelet sizes (mature spikelets) are commonly found in the same
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plant. And the material usually identified as B. erecta is collected mostly in sandy soils. In the case of this complex, a study observing the morphology under different environmental conditions and through different plant generations would be useful to check the influence of the environment on the colour and size of the spikelets , which are the main characters used to distinguish species from this complex. If the three species are accepted as distinct taxonomic entities, it is necessary to consider the hypothesis of hybridization among the species, due to intermediates types. The results obtained for this complex contradict the results of Winge et al. (1984), which allowed the differentiation of the three species, and influenced the circumscription published by Longhi - Wagner (1987). Although their sampling was broader (average of ten individuals per species), it is important to emphasize that
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Recebido: 20/02/2014 Received: 02/20/2014
Aprovado: 10/04/2014 Approved: 04/10/2014
J. Biotec. Biodivers. v.5, n.2: pp. 202-210 May 201 4
https://doi.org/10.20873/jbb.uft.cemaf.v5n2.essi