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Insights into the evolution of Iberian Calendula species depicted from genome size and
chromosome numbers
Sofia Nora1, Ana Carla Gonçalves3, Sílvia Castro2, João Loureiro2, Helena Oliveira3, Mariana Castro2, Conceição Santos3, Paulo Silveira3*
1Department of Plant Biology and Ecology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain, 2CFE - Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, 3001 - 455 Coimbra, Portugal and 3Department of Biology & CESAM, University
of Aveiro, 3810 - 193 Aveiro, Portugal
Introduction
Calendula L. is mostly distributed throughout the Mediterranean basin and is ataxonomic and cytological
complex genus mainly due to hybridization events and large morphological variability, that results in the
appearance of a wide range of intermediate forms, recognized by various names under different taxonomic
categories (Heyn and Joel 1983).
References
Heyn CC, Joel A (1983). Pl Syst Evol 143:311-329
Loureiro J, et al. (2007). Ann Bot 100:875-888
Nora S. et al. Pl. Syst. Evol. In press
NorlindhT (1946). Feddes Repert 85:245-283
Ohle H (1975 a). Feddes Repert 86:1-17
Ohle H (1975 b). Feddes Repert 86:525-541
Acknowlegements
Authors are very grateful to the ICNB for allowing the collection of samples on several nature reserves, to Miguel Sequeira for the samples
of Calendula incana ssp. maderensis,to Abelardo Aparicio for methodological help in the chromosome counts. Several herbaria (AVE, BC,
BCN, BM, BR, C, COI, ELVE, G, GAT, GDA (GDAC), HAL, K, LD, MA, MAF, MGC, O, P, PO, RAB, SALA, SANT, SEV, TFMC) allowed the study
and loaned specimens of Calendula. Thanks are also due to Portuguese Foundation for Science and Technology and European Social Fund
for financing the work of SC with the scholarship FCT/BPD/41200/2007, the work of HO with the scholarship SFRH/BPD/48853/2008 and
the work of AG with the scholarship SFRH/BD/51464/2011.
Material & Methods
The study includes 11 Iberian taxa and two additional taxa from Morocco (34 field locations).
Chromosome counts were made using the squash technique in root tips and
flower buds: pre-treatment for 12hin cold water, fixation for 24hin
ethanol/glacial acetic acid (3:1) and staining with alcoholic hydro-cloridric acid-
carmine.
Nuclear DNA contents were assessed using propidium iodide flow cytometry
following Loureiro et al. (2007).
Nuclear DNA
content
Nº of nuclei
Aims
This study aimed to assess the role of genome size:
1) to decipher the relationships and evolution of Calendula species;
2) to support taxonomic decisions for a revision of this genus.
Results & Discussion
Nuclear DNA content and chromosome number were positively correlated. Still, C. tripterocarpa and C. stellata had higher values of genome size than expected, suggesting a distinct evolutionary line. Results from Calendula
officinalis also get aside from the pattern, presenting a decrease in genome size that might be due to cultivation processes.
Hybridization and polyploidization events were proposed by Heyn and Joel (1983), as the main mechanisms of evolution between Calendula species. C. maroccana and C. stellata belong to the region considered as the primary
centre of evolution of Calendula (Norlindh 1946). These species played a central role in the origin of several taxa. Hybridization and polyploidization events were proposed as the main mechanisms giving rise to new entities.
Conclusions
This study contributes to the knowledge of:
genome size and chromosome numbers in Calendula species
taxonomic and evolutionary relationships, reviewing the hypotheses proposed by Heyn & Joel (1983)
intraspecific variation in genome size
new insights into the evolutionary history of Calendula
background information for the taxonomic revision of this genus
Hypothesis for the speciation process, adapted from Heyn and Joel (1983)
Hyp 1 Origin of C. suffrutiosa, C. incana and C. officinalis
Chromosome losses by C. maroccana originated an entity with 2n=16, than
suffered a genome duplication event, originating an entity with 2n=32
Hyp 2 Origin of C. suffrutiosa, C. incana and C. officinalis
Hybridization between taxa with different chromosome numbers, such as
2n=14 and 2n=18, followed by chromosome doubling of the hybrid with 2n =
16 (Ohle 1974, 1975a, b).
Hyp 3 Origin of C. stellata
Proposed to have originated from C. maroccana after chromosome reductions.
Hyp 4 Origin of C. tripterocarpa
Suggested to have originated after hybridization between C. stellata and: 1) C.
maroccana xC. stellata hybrid or C. maroccana after the loss of 2
chromosomes.
Hyp 5 Origin of C. arvensis
Hypothetically originated from a cross between C. stellata and C.
tripterocarpa, and subsequent duplication of the genome.
GENOME SIZE DO NOT SUPPORT
HYP 1 AND HYP2
Considering a descending dysploidy event from C. maroccana,
resulting in a 2n=16 entity with the same genome size of the origin
species with subsequent genome duplication, a theoretical genome size
of 3.5 pg/2C would have been obtained.
GENOME SIZE DO NOT SUPPORT HYP 3 The genome size of C. stellata is higher than that of C. maroccana. C.
stellata may have been originated by another path.
Dysploidy should be considered. Genome duplication of an individual
originated from the hybridization between C. stellata and a 2n=16 entity
with the same genome size of C. maroccana through dysploidy would
have a genome size with the values obtained for C. tripterocarpa.
GENOME SIZE DO NOT SUPPORT HYP 4
GENOME SIZE SUPPORT HYP 5
(for more details see Nora S. et al. Pl. Syst. Evol. In press).
C. arvensis. A) flowering capitula.
B) fruiting capitula.
C. tripterocarpa. A) flowering capitulum.
B) fruiting capitulum.
C. stellata.
C. incana subsp. incana. A) flowering capitulum.
B) fruiting capitulum.
C. suffruticosa subsp. greuterii. A) flowering
capitulum. B) fruiting capitulum.
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Article
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After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high-quality suspensions of intact nuclei suitable for DNA flow cytometry.
  • C C Heyn
  • A Joel
Heyn CC, Joel A (1983). Pl Syst Evol 143:311-329
  • S Nora
Nora S. et al. Pl. Syst. Evol. In press Norlindh T (1946). Feddes Repert 85:245-283
  • H Ohle
Ohle H (1975 a). Feddes Repert 86:1-17