ISSR as a tool to support taxonomic decisions: a first approach for Chascolytrum species complexes (Poaceae)

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, Briza 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.


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 Universidade Federal do Rio Grande do Sul and latter at the Laboratory of Taxonomy of the Universidade Federal de Santa Maria.

Taxon Sampling
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.

Data analyses
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 relationships were portrayed by dendrograms built using the clustering method Unweighted Pair Group Method of Arithmetic Average (UPGMA).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 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 (total of 31 bands).The Jaccard similarity index ranged from 0.2260, between accession li201 and accession w10768, to 0.7442, between accessionHw5038 and accessionHw5056.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 fingerprints.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).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 bootstrap.
https://doi.org/10.20873/jbb.uft.cemaf.v5n2.essiIn 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 ISSR are one of the most polymorphic molecular markers; 2) Chascolytrum species are autogamous, mainly cleistogamous, so that a low intrapopulation variability and a high interpopulation 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 paperdata 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).The ISSR helped to solve some critical questions: 1) Concerning the acceptance of two varieties for Chascolytrum rufum: ISSR 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 remaining morphologically related accessions, thus supporting their acceptance as distinct species, in addition to the 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 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 some of the markers utilized (morphological, isoesterases) are more affected by environmental conditions than the ISSR markerswhich could be the main cause of the distinct phenotypes.Future works with ISSR should include a broader sampling of these three species and greenhouse essays.

CONCLUSIONS
Concluding, ISSR are useful as a tool to support taxonomic decisions in genus Chascolytrum, but it is important to keep in mind that ISSR are more effective as markers for polymorphism than for conservative features in this genus.

Figure 4 :
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.

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.https://doi.org/10.20873/jbb.uft.cemaf.v5n2.essi

Table 2 :
Primers code and sequences included in the analyses.