Notes on the sexual condition of Myriophyllum aquaticum, Haloragaceae Notas sobre la condición sexual de Myriophyllum aquaticum, Haloragaceae

Resumen. Myriophyllum aquaticum (Vell.) Verdc. es nativa de Sudamérica con distribución pantropical. Esta especie ha sido citada como dioica, monoica y polígama. El objetivo de este trabajo es contribuir a la discusión sobre su condición sexual, basada en material de herbario, y apoyada por observaciones de campo. Se examinó material de herbario de Argentina. En la Reserva Natural de Punta Lara (Buenos Aires, Argentina), se muestrearon periódicamente veinte ramas para registrar el sexo de las flores presentes en cada verticilo durante el período de floración de 2002. Tanto en material de herbario, como en el campo, observamos especímenes con ramas con flores femeninas, especímenes con ramas con flores masculinas y especímenes con flores femeninas y masculinas en la misma rama. Algunos de estos materiales presentan frutos. Nuestras observaciones apoyan la idea que M. aquaticum no es una especie estrictamente dioica, al menos en Argentina.

Myriophyllum aquaticum is a submerged and emergent herb with stem branches up to 1 m long and emerging apexes, perpetuating growth after anthesis.The leaves are in whorls of four to six, 1-3 cm long, oblong in outline, pinnate, with thread-like segments.The flowers are unisexual, solitary, axillar, pedicellate and 4-merous.Male flowers have ovate-deltoid sepals, yellow petals and eight stamens.Female flowers have deltoid sepals, no petals, a pyriform ovary and a clavate style with four white densely fimbriate stigmas.Fruits are ovoid with four mericarps (Meijden & Caspers, 1971;Orchard, 1981;Lahitte et al., 1997;Tur et al., 2009).
This species is commonly found in fresh-water bodies, forming patches in quiet, shallow water, usually associated with other free-emergent floating species (Orchard, 1981;Negritto & Anton, 1996).It is frequently found in an emergent form producing dense populations (the immersed leaves often deteriorate when plants grow in standing water).This species, which has been sold commercially as an ornamental aquatic plant, is invasive on most continents (Moody & Les, 2010).
On the sexual issue, there are some disagreements that became worse because of the nomenclatural problem (Tur et al., 2009).Enydria aquatica Vellozo (1825), basionym of M. aquaticum, is described as a likely monoecious species.Myriopyllum brasiliense Cambess., a nomenclatural synonym of M. aquaticum, is cited as monoecious (Kanitz, 1882), mostly monoecious or more-or-less dioecious (Meijden & Caspers, 1971), and polygamous or dioecious (Ayres Fevereiro, 1975).Meijden & Caspers (1971) mentioned that all naturalized specimens are sterile or female but do not produce fruits (in Malaysia, male specimens are absent), and even fruits rarely occur in South America.In contrast to these authors, Ayres Fevereiro (1975) described male, female and bisexual flowers, and observed that this species flowers and fruits throughout the year in Brazil.On the other hand, M. proserpinacoides Gill.ex Hook.& Arn., another nomenclatural synonym of M. aquaticum, was cited as a subdioecious (Gillies, 1833;Schindler, 1905) or dioecious plant species (Kanitz, 1882;Reiche, 1898).Orchard (1979Orchard ( , 1981) ) reported that the lectotype of M. proserpinacoides is a monoecious specimen (Dr.Gillies s.n., K).Finally, M. aquaticum was cited as monoe-cious or mostly dioecious by Boutique & Verdcourt (1973) or as dioecious by Orchard (1979Orchard ( , 1981)), Li & Hsieh (1996), Negritto & Anton (1996) and Moody & Les (2010).In revisions of the genus for South America and Australia, Orchard (1979Orchard ( , 1981) ) only found female plants of M. aquaticum outside South America, and concluded that reproduction is entirely vegetative.According to Meijden & Caspers (1971), male flowers and fruits are rare even in South America.Li & Hsieh (1996) cited entirely vegetative reproduction, with male plants absent, in Taiwan.Moody & Les (2010) studied the phylogenetic relationships in the genus Myriophyllum.They concluded that all the specimens of M. aquaticum collected in North America were exclusively female plants with identical genotypes.
The objective of this paper was to contribute to the discussion of the sexual condition of M. aquaticum, based on herbarium material and field observations.

MATERIALS AND METHODS
Herbarium material.Herbarium acronyms follow Holmgren et al. (1990).Herbarium material deposited at BA, LP and SI was examined to check the sex of the specimens, and to record the localities where the taxon had been collected.The study herbarium material is cited in three groups within Results: (1) specimens with only female flowers, (2) specimens with only male flowers, and (3) specimens with male and female flowers on the same branch.The presence of fruits had also been registered (fr).Some of these specimens are whole plants, whereas others are constituted only for branches or branch fragments.
Field observations.The study area was the Punta Lara Nature Reserve (34° 47´ 22´´ S, 58° 00´ 21´´ W), Buenos Aires province, Argentina, located in the Pampean biogeographical province (Cabrera, 1976).The climatic conditions are warm temperate, with frosts in winter and early spring; rainfall occurs throughout the year, but it is more intense in spring and autumn, scarce in winter and insufficient in summer.It ranges from 600 mm to 1100 mm annually (Cabrera, 1976).
In the field, M. aquaticum grows in dense patches with only female or male flowers at sight, which have a light difference in colour.We call them "female patches" or "male patches", in spite of the sex of all the flowers present in all branches.Twenty branches of M. aquaticum were selected from patches growing in the interior of the water bodies, and the last fertile whorl was marked on each branch.Of these whorls, ten of them came from a "male patch", and the other ten from a "female patch".We registered the sex of the flowers in the fertile whorls previous to its marking (before 6 October 2002).On each marked whorl, the number of new whorls and the sex of the flowers present on it were recorded during the flowering period of 2002(18 October 2002, 1 November 2002and 15 November 2002).

RESULTS
Herbarium material.The flowering period of Myriophyllum aquaticum ranges from August to December in Argentina; it rarely starts in June.Specimens with branches bearing either female or male, or female and male flowers on the same branch were recorded (Table 1).Some of the study material also had fruits (Table 1).Field observations.At the time of marking, nine out of 10 branches marked as male presented two or more whorls of female flowers at the base (Table 2, branches 1 to 9).All branches marked as female later developed whorls of male flowers (Table 2, branches 11 to 20).

Specimens with only
Four out of the 20 study branches presented alternate whorls of female and male flowers (Table 2,branches 3,6,8 and 20).At the same time, five of them presented both female and male flowers on the same whorl (Table 2, branches 1, 3, 8, 18 and 19).Table 1.Number of herbarium specimens examined with female flowers (f), female flowers and fruits (f+fr), male flowers (m), male flowers and fruits (m+fr), male and female flowers (m+f), and male and female flowers and fruits (m+f+fr) in different provinces of Argentina.
Tabla 1. Número de especímes de herbario examinados con flores femeninas (f), flores femeninas y frutos (f+fr), flores masculinas (m), flores masculinas y frutos (m+fr), flores masculinas y femeninas (m+f), y flores masculinas, femeninas y frutos (m+f+fr) en diferentes provincias de Argentina.Table 2. Sex of the flowers present on each whorl of 20 branches (10 in a male patch and 10 in a female patch at sight) registered at the field during the flowering period of 2002.The highlighted row shows the whorl used to mark the branches in the patch identified at sight as either male or female.Abbreviations: m: whorl with male flowers; f: whorl with female flowers; mf: whorl with male and female flowers; -: sterile whorl.

Sexual condition of Myriophyllum aquaticum
All branches developed whorls of male flowers at the end of the flowering period, and all presented fruits on the submerged parts.The branches that survived drought and herbivory followed their flowering period developing vegetative whorls.

Is Myriophyllum aquaticum dioecious or monoecious?
By analyzing herbarium material we noted the presence of monoecious specimens in a broad range of distribution of M. aquaticum in Argentina.This indicates that M. aquaticum is not strictly dioecious, at least in this part of the world.This conclusion was supported by field observations.
Our observations demonstrated that there were herbarium specimens and field grown plants that showed male flowers and/ or fruits (Table 1; Table 2; Fig. 1).These results disagree with findings of Meijden (1969), Meijden & Caspers (1971) and Orchard (1979Orchard ( , 1981)), who reported that male flowers and fruits were rare in South America.Furthermore, it was verified that this species developed abundant fruits, although they are not easily seen because they promptly become submerged.These findings agree with those of Ayres Fevereiro (1975), who pointed out that M. aquaticum flowers and fruits during almost the whole year.
The disagreement about the presence or absence of male flowers and fruits may be because of the timing of collection.In agreement with Ayres Fevereiro (1975), our results demonstrated that branches with female flowers at the base later developed male flowers in the upper part (Table 2).Depending on the timing of sampling, it is possible to find specimens with only female flowers, female and male flowers, or male flowers and submerged fruits.More fertile whorls at the moment of marking were found on branches marked as male than female (Table 2).We can conclude that "male patches" began to flower earlier than "female patches".Thus, a "female patch" will be seen later than a "male patch".
In his work, Orchard (1981) mentioned that "all specimens examined of M. aquaticum, with one exception, are strictly dioecious, bearing either male or female flowers", recognizing only the specimen Gillies s.n.(K) as monoecious.Among the other five specimens from Argentina that he cited as dioecious, two were cited as monoecious in this study (Pedersen 3977 and Venturi 629).We observed the original material deposited in LP, and Orchard studied its duplicates deposited at F, MO, NY, P, and US.It is likely that the branches present in the duplicates had only female flowers, whereas the original material that we observed had both types.
The absence of male flowers in the region where M. aquaticum is adventitious, and the conclusion that its reproduction is sexual in its natural distribution area, and vegetative in the region  Torres Robles SS et al., FYTON 80 (2011) where it is adventitious, has previously been reported (Meijden, 1969;Meijden & Caspers, 1971;Orchard, 1979Orchard, , 1981;;Li & Hsieh, 1996;Moody & Les, 2010).This conclusion is reinforced by the results of Moody & Les (2010); these authors remarked that all their collected plants had the same genotype.Moreover, there is some evidence of different sexual strategies on aquatic plants depending on competition or environmental conditions (Dorken & Barrett, 2004).Aquatic plants display a remarkable range of reproductive strategies, including diverse sexual systems and clonal propagation (reviewed by Barrett et al., 1993).Variation in reproductive traits will influence the ability of populations to colonize and persist in different types of habitats (Dorken & Barrett, 2004).These authors suggested that monoecy represents a flexible reproductive strategy to adjust allocation to female vs. male sex function in response to environmental heterogeneity, i.e. reproduction might be sexual in competitive habitats, whereas vegetative reproduction might be common under favorable environmental conditions.
Nomenclatural issue?Our results demonstrated that Myriophyllum aquaticum is monoecious in Argentina, whereas other authors (Orchard, 1979(Orchard, , 1981;;Li & Hsieh, 1996;Negritto & Anton, 1996;Moody & Les, 2010) affirmed that the species is dioecious.Only the type specimen of M. proserpinacoides (nomenclatural synonym of M. aquaticum) is accepted as monoecious by Orchard (1981).Considering the list of monoecious specimens cited here, and that this material was collected in Argentina, another question arises: Might monoecy vs. dioecy define in fact the existence of two species?Reiche (1898) alluded in the Flora of Chile that M. proserpinacoides is identical to M. verticillatum var.limosum (in spite of the dioecy ocurring in the former).Orchard (1981) mentioned that M. verticillatum was listed in Chile, but the specimens examined by him were in fact M. aquaticum or M. quitense.Then, he excluded M. verticillatum from the flora of South America.Very likely, there has been confusion among these species most of the times.
These examples suggest that the disagreements on the sexual issue could be taxonomic in nature.A much broader analysis should be made to examine the distribution of monoecious and dioecious populations, in combination with general morphological studies of monoecious and dioecious plants in the context of a nomenclatural revision.

Fig. 1 .
Fig. 1.Flowers and fruits in Myriophyllum aquaticum.A: Branch with male and female flowers in different whorls.B: branch showing whorl with mature fruits.C: detail of a whorl with fruits.Abbreviations: m: male flower; f: female flower; fr: fruit.B, C, from Tur 1840 (LP).