The Taxonomic Significance of ducts in the corolla lobes of Vernonia (Vernonieae: Asteraceae)

Abstract The multiple longitudinal ducts in the corolla lobes found in the closely related genera Vernonia, Vernonanthura and Trepadonia are microscopically studied and illustrated. The lack of such ducts in the two South American species that have until now been retained in Vernonia indicates that they should probably be excluded from the genus.


Introduction
Th e generic concept of the genus Vernonia was simple and uninsightful for over 150 years from the time of it's inception in Schreber (Gen. 2: 541. 1791, nom. cons.), with the eastern North American type Serratula noveboracensis L., type cons. Vernonia was the core genus of the tribe Vernonieae, having the combination of capillary pappus and non-liguliform fl orets that were common to all core genera of the Asteraceae during that time. Some segregates were named, such as Baccharoides Moench. and Gymnanthemum Cass., in Africa, Eremosis Gleason in Mesoamerica and Critoniopsis Sch.Bip. in South America, but these were not generally recognized at the generic level.
Th e concept of Vernonia began to change in the last 30 years as is partially summarized in the treatments by Robinson for the Paleotropical (1999a) and the American members (1999b) of the tribe. Th e altered concept reduced the genus Vernonia to a restricted group of species mostly directly related to the type species Vernonia noveboracensis (L.) Michx., a group with eleven species in eastern North America, ca. 5 species in the highlands of central Mexico, and one eastern North American species reaching the Bahamas. Until now, two species in South America have been retained in Vernonia. Th e genus consisted of species having a cymiform inforescence with seriate "scorpioid" cymose branches defl ected at the nodes, glanduliferous anthers, a somewhat rhizomiform rootstock and echinate weakly sublophate pollen. Closest relatives were considered to be members of the genus Vernonanthura H.Rob. with ca. 68 species in tropical America, and Trepadonia H.Rob. with two species on the eastern side of the Andes in Peru and western Brazil. Vernonanthura diff ered most notably by the more pyramidal infl orescence with cymiform branches and an often tuberous rootstock, and Trepadonia was scandent with widely spreading lateral branches in the infl orescence. Th e comparative close relationship between Vernonia and Vernonanthura was refl ected fully in the DNA sequence studies of Keeley et al. (2007).
A unifying anatomical structure of the three related genera in the typical element of the tribe Vernonieae is found in the study of the corolla lobes of the group. Th e corolla lobes of Vernonia are fi led with multiple parallel ducts containing resin or oils of some type ( Fig. 1a-d). Th ese ducts were fi rst noted in the related genus Trepadonia H.Rob., and they were illustrated crudely with a photomicrograph in the paper describing that genus (Robinson 1994). Subsequently, during casual observations during systematric work, the ducts were seen in typical Vernonia and in numerous species of the closely related Vernonanthura. In the latter genus the ducts were often observed in dried specimens under the dissecting microscope. Th e ducts were represented schematically in the plates of generic charactertistics in the study of American Vernonieae by Robinson (1999b).
All initial observations of the ducts were in material mounted in Hoyer's Solution, and no contents of the ducts were observed. It was when some material was observed microscopically in water mounts that resin or mucilage was seen in the ducts. Given the potential taxonomic importance of the ducts, a fi rst determined eff ort is made here to provide a proper illustration of the structure.

Methods
Material examined was from dried specimens in the US National Herbarium (US) plus fresh material of Vernonia noveboracensis (L.) Michx., the type species of the genus Vernonia Schreb., collected on Roosevelt Island and placed in FAA. Herbarium material studied included Vernonia noveboracensis, Vernonanthura brasiliana (L.) H.Rob., the type species of Vernonanthura, and the two species, Vernonia echioides Less., and V. incana Less., the latter two species that until the present have been the only South American species retained in the genus Vernonia. For purposes of the study, four additional species of Vernonia were examined from dried material, V. acaulis (Walt.) Gleason, V. baldwinii Torr., and V. blodgettii J.K.Small all from eastern North America, and V. karwinskiana DC. From Oaxaca, Mexico. A fi nal collection was of an unvouchered collection of Vernonia angustifolia Michx. from a garden in Arlington, Virginia, and placed directly on a microscope slide (See Appendix for citations).
For purposes of microtome sectioning, living material of Vernonia noveboracensis was fi eld collected and fi xed in formal-aceto-alcohol (FAA 1:1:18). Flower heads were subdivided, dehydrated with 2-2 dimethoxypropane (DMP) (Postek and Tucker 1976), then infi ltrated and embedded in paraffi n. Serial sections were made at 5 μm, stained with buff ered Toluidine blue O (Sakai 1973) and mounted in Lipshaw's synthetic mounting resin. Unopened buds were used to avoid the recurved condition of the lobes in opened fl orets. Placement of bud material in the mounting resin was uncertain since the fl orets had fi ve lobes that were themselves somewhat curved. Cross-sections were easily obtained. Longitudinal sections were by chance.
For views of unsectioned material, corolla lobes were taken from fi xed material and from dried herbarium specimens and one freshly collected specimen for whole mount observation.
Images (photomicrographs) were captured in bright fi eld or Nomarski (DIC) using a Zeiss Standard 16WL microscope, a Zeiss Universal Research microscope or a Zeiss Axiophot equipped with a Retiga 1300i digital camera (Q Imaging Corp.) and an image acquisition and processing system capable of live tiling and live EDF (extended depth of fi eld) by MediaCybernetics/ImageProPlus 7.0.
Th e material from dried herbarium specimens was remoistened in water and mounted on microscope slides in water to preserve any resin or mucilage that might be in the ducts.

Results
Th ere are two ways of showing the ducts in the corolla lobes of Vernonia. One is by sectioning the material, the other is by whole mounts of the corolla lobes placed under the microscope. Both methods have been used here, and both methods have unmistakably demonstrated the ducts.
Cross-sections of the corolla lobes of Vernonia noveboracensis clearly show the series of seven to twelve ducts in a lobe (Fig. 1A & B). Th e ducts are mostly surrounded by individual sheaths of cells, the same cells that form the partitions between the ducts that were obvious in the initial crude studies of the characteristic in Trepadonia. A longitudinal section (Fig. 1C) shows the ducts as elongate structures that lack cross-walls. Th e longitudinal sections of the corollas indicate that the ducts are not completely restricted to the corolla lobes, but that they begin below the lobes, especially along the main veins of the corolla. Both cross-and longitudinal sections show the ducts as empty; apparently Hoyer's Solution and preservative both completely remove all resin or mucilage contents of the ducts.
Th e second method of viewing the ducts is the one that showed the structure in the paper describing Trepadonia (Robinson 1994). However, that preparation was in Hoyer's Solution and retained no resin or mucilage in the ducts. Th e new views fi rst used material that had been placed in preservative, and the ducts were also empty. Mounts of corollas in water, however, showed the ducts clearly with their chemical contents, and the presence of material in the ducts made the ducts more obvious. Figures 1E & F show the lobes of Vernonia angustifolia and Vernonanthura brasiliana with resin or mucilage in the ducts. Th e duct contents show that multiple longitudinal ducts are present in both genera. Th e resin contents do not seem to appear in all the ducts of any lobe at the same time. Th e fresh material of Vernonia angustifolia obtained from garden plant shows lobe that has neither been dried nor treated with chemicals. Even here, the ducts seem to be only partially fi lled with resin. Observations of V. acaulis, V. baldwinii, V. blodgettii, and V. karwinskii were in water on slides simply to confi rm the presence of the ducts in a broader representation of the genus. While the ducts can be seen once the observer is familiar with their appearance, they are not quite obvious enough to be used as anything more than a supplementary key character in taxonomic treatments. Th e character could be useful in separating Vernonanthura from Critoniopsis Sch.Bip. in the Andes, and Vernonanthura from the adventive Gymnanthemum amygdalinum (Del.) Sch.Bip. ex Walp. in Brazil.
Th e survey of the whole tribe for presence of multiple ducts has not been rigorous, but certainly on a par with the observations that led to the initial discovery of the ducts in Trepadonia, Vernonanthura and Vernonia (Robinson 1994). It remains to be seen what diff erent kind of internal structure occurs in the corollas in other members of the Vernonieae. It can only be said that multiple longitudinal ducts have not been seen in any of the other genera. As an indication other possible specialization, at least one African species, still known as Vernonia potamophila Klatt, shows chambers of some kind in its lobes.
Th e nature of the chemical contents of the ducts remains unknown. Th e types of chemicals known from the Vernonieae include epoxy resins, but these have been found most notably in genera like Stokesia L'Hér. (Gunn and White 1974) and Centrapalus Cass. (Purdue et al. 1986(Purdue et al. , 1989, neither of which has the multiple longitudinal ducts in the corollas. Terpenoids are common in many Vernonieae (Herz 1977;Bohlmann and Jakupovic 1990), but their end products are not tolerated by Insects and Mammals or the primary tissues of the plants in which they are produced. For that reason, in the Asteraceae, the terpenoids are accumulated in special cells usually at the tips of glandular hairs such as those seen on the lobe of Vernonia echioides (Fig. 1G). Acetylenes are a stronger possibility, but they are not varied in Vernonieae where only pentaynenes have been reported (Bohlmann et al. 1973). In other tribes such as the Heliantheae, Acetylenes are highly varied and occur in ducts, and where they seem to have been credited with a mostly antifungal function that ceases with the death of the plant. Th ese are the types of secondary metabolites most widely known to occur in the tribe Vernonieae, and the identity of the duct contents remains unresolved.
It seemed particularly useful to examine the corolla lobes of the two South American species that had thus far been retained in Vernonia on the basis of the pollen and habit of the plants. Th eir position in Vernonia was always in question because of their remote geographical locations in Argentina, Brazil, Paraguay and Uruguay. Th e material was chosen from herbarium specimens that showed no indication of being preserved in alcohol or formalin. As shown in the fi gures (1G, H) no trace of multiple longitudinal ducts was seen. Th e character of the ducts is here regarded as a defi ning feature of the generic group of Vernonia, Vernonanthura and Trepadonia and the lack of the feature in the two South American species, combined with the remote geographical location is considered strong evidence that they do not belong in Vernonia. DNA of the two species has not yet been sequenced, and their proper disposition remains to be determined.