Evolution of secondary heads in Nassauviinae (Asteraceae, Mutisieae)

KATINAS, L.; CRISCI, J. V.; SCHMIDT JABAILY, R.; WILLIAMS, C.; WALKER, J.; DREW, B.; BONIFACINO, J. M.; SYTSMA, K. J.

São Paulo, Brasil

Congreso; 58° Congresso Nacional de Botânica; 2007

Sociedade Botanica do Brasil

The aggregations of two or more capitula into inflorescences of a second order in Asteraceae are termed secondary heads, syncephalia or pseudocephalia (Troll 1928; Claßen-Bockhoff 1996). Stebbins (1967) suggested that the union of few-flowered heads into a secondary head provides a more efficient mechanism to increase the size of the functional inflorescence relative to the acquisition of new flowers into a reduced capitulum.  Stebbins (1967) hypothesized a scenario for the formation of secondary heads involving three steps: 1) the capitula aggregate closely without losing their individual identities; 2) the capitula aggregate into a functional head, the individual capitula being still recognizable, but without the formation of a pseudoinvolucre (the series of bracts that surrounds the secondary head); and 3) the complete loss of identity of individual capitula and the acquisition of a pseudoinvolucre.  Secondary heads occur in approximately 70 genera (4.4 % of all genera in Asteraceae) and in 11 tribes (Crisci 1974a). One of these tribes is the Mutisieae, an early-diverging lineage in the family, which is constituted by two subtribes, the monophyletic Nassauviinae and the non-monophyletic Mutisiinae (Crisci 1974b; Cabrera 1977). The focus of our investigation is the Nassauviinae (25 genera, about 300 species), a Neotropical group with the greatest concentration of genera and species in the southern Andes.             Four genera of Nassauviinae, Moscharia (two species), Nassauvia (38 species), Polyachyrus (seven species), and Triptilion (seven species), display secondary heads with differing degrees of capitula condensation. In Nassauvia and Triptilion all stages are found between a cymose, lax conflorescence and the aggregation of few-flowered capitula into condensed, glomerulose conflorescences still containing the identities of the individual capitula. Each capitulum has one to five flowers surrounded by two series of bracts or phyllaries. The sister genera Moscharia and Polyachyrus (Crisci 1974b; Katinas & Crisci 2000) have syncephalia bearing and not bearing a pseudoinvolucre, respectively. In Polyachyrus each capitulum is two-flowered with an involucre of four phyllaries, the outermost being keeled and more or less surrounding the whole capitulum; both flowers are separated by a palea. Moscharia has a central one- or two-flowered capitulum with an involucre of 4-7 phyllaries, that is surrounded by marginal, two-flowered capitula. As in Polyachyrus, each marginal capitulum is enclosed by a keeled bract and both flowers are separated by a palea, but the total number of phyllaries are reduced to only the keeled phyllary.             Some authors have interpreted Moscharia as having a simple capitulum instead of a secondary head. Burtt (1977) and  Hellwig (1985) stated that Moscharia has a much greater resemblance to some species of Leucheria, a genus of Nassauviinae with simple capitula. Recent molecular phylogenetic analyses (e.g., Funk et al. 2005) show Leucheria as sister to Jungia, another member of the Nassauviinae, with Nassauvia and Triptilion also sister taxa but independent of the pair Leucheria-Jungia. The genera Moscharia and Polyachyrus were not sampled in these studies.            The goal of this study is to use molecular data to answer the following questions: Does the molecular approach support the sister relationship between Moscharia and Polyachyrus? What are the phylogenetic positions of Leucheria, Nassauvia, and Triptilion with respect to Moscharia and Polyachyrus? How can the evolution of secondary heads in Nassauviinae be interpreted with new molecular evidence in context of structure and ontogeny? MATERIAL AND METHODS Forty-six taxa representing 20 genera of Asteraceae from the two subtribes of Mutisieae, Nassauviinae and Mutisiinae, were sampled. Three outgroups were selected from the monophyletic subfamily Barnadesioideae: Chuquiraga, Doniophyton, and Duseniella.  Total genomic DNA was extracted from silica-dried leaves. Both the nuclear ITS region and chloroplast trnL-trnF region were amplified by polymerase chain reaction (PCR) using trnL-trnF primers following Taberlet (1991) and ITS primers ITS leu1 and ITS 4 (White et al. 1990). The nuclear ITS and plastid trnL-trnF regions were analyzed separately and together using maximum parsimony and maximum likelihood. Congruence between the two datasets under parsimony was assessed with the Incongruence Length (ILD) test (Farris et al. 1994) using a heuristic search and simple taxon addition for 100 random partitions of data in PAUP 4.0b10 (Swofford 2002). All characters were unweighted and treated as unordered. Bootstrap replicates (Felsenstein 1985) were performed for each analysis to assess character support. Consistency indices (CI) and retention indices (RI) (Farris 1989) were calculated to evaluate the amount of homoplasy in the data.  Bremer support (Bremer 1988) for all clades was calculated. Parameters for maximum likelihood (ML) analysis of the combined datasets excluding gaps was obtained with ModelTest v. 3.6 (Posada & Crandall 1998).  ML analysis was run with 100 replicates, TBR branch swapping and random taxon addition.                RESULTS MP analysis of trnL-trnF and indels found 2932 most parsimonious trees of 178 steps and a CI of 0.775, and RI of 0.913. MP analysis of the ITS data yielded 284 trees of length 1284 with a CI of 0.431 and RI of 0.744. The ITS and trnL-trnF data sets were highly congruent (P=0.9). Combining the ITS  and trnL-trnF datasets yielded 24 trees of length 1650 with a CI of 0.471 and RI of 0.795. The strict consensus of the 24 MP trees from the combined data analysis is well resolved. The bootstrap support of two groups is 100%: first, Leucheria embedded in the Polyachyrus and Moscharia clade (and 99% for the clade of Leucheria + Polyachyrus); and second, the two other genera with syncephalia, Nassauvia and Triptilion, sister to Perezia and Panphalea. ML analysis of the combined datasets excluding gaps using the TIM+I+G model selected by ModelTest generates a tree very similar to those obtained with MP. Nassauvia with four species sampled is monophyletic relative to Triptilion. Only the genus Leucheria still remains non-monophyletic, and appears paraphyletic in the ML tree although the branch length placing L. salina as sister to L. rosea + Polyachyrus is extremely small.          The most striking finding of the MP and ML analyses is the sister relationship of Polyachyrus with Leucheria, both constituting a monophyletic group sister to Moscharia. This clade is independent of the pair Nassauvia-Triptilion, the other group with secondary heads.             DISCUSSION Our molecular analysis strongly supports Leucheria sister to Polyachyrus, and both sister to Moscharia. This clade of three genera is not sister to a clade of Nassauvia and Triptilion, indicating that the secondary heads have evolved twice independently in the subtribe in Nassauviinae.             It is widely accepted that Polyachyrus has secondary heads and that Leucheria has single capitula. However, whether the two species of Moscharia have capitula or secondary heads is still debatable with good arguments in favor of both views. In the context of this debate, close relationships were postulated between Moscharia and Polyachyrus (e.g., Crisci 1980; Freire et al. 1993; Katinas & Crisci 2000) and between Leucheria and Moscharia (e.g., Burtt 1977; Helwig 1985). The results presented here differ fundamentally from these traditionally held views. The analysis supports, with 99% of bootstrap replicates, a sister relationship between Leucheria and Polyachyrus.             A genus, with single capitula like Leucheria, nested between two genera with secondary heads is not implausible if we analyze a very unusual feature in the capitula of species of Leucheria. The genus has species with and without phyllaries placed among the marginal flowers. These phyllaries, interpreted sometimes as paleae, change their spatial orientation in the different species (Crisci 1976). Some species (e.g., Leucheria gilliesii, L. glandulosa) have the paleae enclosing florets with the open part facing the center of the capitulum. In other species (e.g., Leucheria amoena, L. rosea) the paleae enclosing florets face the outer part of the capitula.             A new hypothesis of secondary head evolution should now be considered for these three genera. This hypothesis is based on the strong molecular phylogenetic relationships presented here, the clear homology in the order of floral maturation of all three genera (centripetal sequence) and between the reproductive structures in the syncephalia of Moscharia and Polyachyrus, and the presence and absence of paleae and the change of the paleae orientation in Leucheria.  It is most parsimonious to view the ancestor of the group as possessing morphologically complex secondary heads with subsequent descendants exhibiting re-arrangements and reductions. A plausible reconstruction of the evolution in floral and head arrangement for this group would involve the following events: (A) a common ancestor with syncephalia of many central capitula and many marginal capitula with  keeled outer phyllaries, and pseudoinvolucre; (B) Polyachyrus-like ancestor with central capitula but with re-arrangement of their phyllaries to the marginal capitula; (C) loss of central capitula (truncation) and pseudoinvolucre in Polyachyrus; (D) loss of marginal capitula in Leucheria; the remnants of the phyllaries re-arrangement in the ancestor is evidenced in the phyllaries position seen among different extant species; and (E) planation to a common receptacle in Moscharia, which is very similar to the hypothetical ancestor except that the former has only one central capitulum. This hypothesis of syncephalia evolution thus explains two interesting features of this group: the different palea orientation in Leucheria and the centripetal sequence in the flowering of Moscharia and Polyachyrus.             Previous hypotheses postulated the evolution from simple to more complex secondary heads within this group. The emerging hypothesis established here suggests the opposite, the ancestor of Moscharia, Polyachyrus, and Leucheria had a more complex type of secondary head than the extant genera. The reduction in complexity of the syncephalia within the group involved principally loss of capitula and re-arrangement of phyllaries and flowers. Acknowledgements. Field work was supported by the National Geographical Society (grant # 7646-04) and the Facultad de Agronomía, Montevideo. Laboratory work was supported by NSF grant EF-0431233 (KJS). 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