THE VIOLETS OF THE BRAZILIAN SAVANNA: A REVISION OF THE POMBALIA LANATA COMPLEX (VIOLACEAE), WITH DESCRIPTIONS OF TWO NEW SPECIES

Pombalia Vand. (Violaceae) is represented in the Cerrado by a well-supported monophyletic group of species known as the Pombalia lanata complex, characterised by some unique vegetative and reproductive features within the genus. This group includes six species – Pombalia cristalina , P. insignis , P. lanata , P. poaya , P. strigoides and P. velutina – the first two of which are new to science. Pombalia lanata shows the widest geographical distribution throughout the Central Brazilian Plateau and the Espinhaço Range in Minas Gerais state, with a disjunction in Misiones Province, Argentina. Because most species in this group are narrow endemics, we evaluated the conservation status of all taxa. An identification key for the species, descriptions, illustrations, maps of geographical distribution and nomenclatural notes are also provided.


Introduction
The genus Pombalia Vand. (Violaceae) comprises 42 species distributed throughout the Americas Paula-Souza & Souza, 2015). Half of these species are found in Brazil, and in this country the Cerrado (the Brazilian savanna), with 13 taxa, is the richest phytogeographical domain in terms of number of species of the genus (Paula-Souza, 2018).
The savannas of the Central Brazilian Plateau are also home to a morphologically well-defined group of species called the Pombalia lanata complex, which takes its name from its most common and widely distributed species. The complex is characterised by two unique features within the genus: anthers ornamented with reduced, hyaline connective appendages (except Pombalia insignis Paula-Souza; see discussion below) and a well-developed, bud-bearing underground root-like system. Preliminary phylogenetic studies have resolved a clade of six species corresponding to the Pombalia lanata complex (Paula-Souza, 2009). Five of these species occur in the Brazilian Cerrado [P. cristalina Paula-Souza, P. insignis, 4 A revision of the Pombalia lanata complex (rarely white in the latter), yet all bearing the same basal yellow or white spot ( Figure 2E and Figure 9A-D, respectively).
Investigations of reproductive biology in tropical taxa of the Violaceae are scarce, but authors of the few studies carried out with species of Hybanthus sensu lato (Augspurger, 1980, andRosero-Lasprilla, 1997, with Pombalia;Munzinger & Pauly, 2003, with Afrohybanthus) reported similar pollination mechanisms to those observed in the widely studied Viola (Beattie, 1969(Beattie, , 1971Freitas & Sazima, 2003), possibly constituting a pattern throughout the strongly zygomorphic-flowered taxa, given their overall similar floral architecture. In these groups (and the weakly zygomorphic-flowered Rinorea sensu lato), the androecium shows an elaborate morphology that prevents the possibility of self-pollination in self-compatible species; this mostly involves subsessile, introrse anthers that are longitudinally fused around the ovary through numerous papillae, and the anthers' connective apically furnished with large membranous projections that are imbricate to form a cone around the style ( Figure 6G).
The tubular structure formed by the imbricate membranous appendages efficiently encloses the released pollen grains; therefore, although the species are generally self-compatible, an external agent is required for pollination to be accomplished. Changes in the relative positions of the stigmatic opening and the connective appendages, in response to the visitors' movements, are essential for pollination success, and in their attempts to access pollen grains, the insects sometimes engage in specialised behaviours such as buzz pollination (Roubik, 1992;Freitas & Sazima, 2003).
The species of the Pombalia lanata complex, however, exhibit two significant modifications in this general morphological pattern that suggest loss of the mechanism that prevents the easy release of pollen, thus allowing not only self-pollination (although compatibility is entirely unknown in this group), but also, more importantly, pollen access to generalist visitors. First is elevation of the anthers on slender and long filaments, thus exposing the base of ovary where the released pollen grains accumulate, a feature that is also found in a few species of Pombalia, including their hybridising sister groups P. arenaria and P. calceolaria. Second, and exclusive to the Pombalia lanata complex (except P. insignis and the unrelated P. graminifolia), is reduction of the anthers' apical connective appendages, such that the typical conical structure around the style is not formed ( Figure 6F).
Bees and wasps are the most frequent pollinators reported for the strongly zygomorphicflowered genera of Violaceae, particularly small, solitary bees and the eusocial stingless bees (Apidae: Meliponini). The latter are of special interest in our context, because they are the most generalist in their use of floral resources in the Neotropics (Pacheco Filho et al., 2015) and the Brazilian savanna is a hotspot for this group of bees (Pioker-Hara et al., 2014). The same aspects that make the Meliponini such a relevant group in maintaining genetic variability in remnant fragments of the Cerrado flora, namely large colonies of polylect insects consisting of numerous workers gathering resources (Michener, 1979), combined with the drastic morphological shifts of the androecium observed in this group, might favour the formation of hybrids within the Pombalia lanata complex. In a broader context, this association might have contributed to the occupation of open habitats by members of this clade, from elements typical of forested areas [e.g. Pombalia brevicaulis (Mart.) Paula-Souza and P. heterosepala (Eichler) Paula-Souza] or the Caatinga seasonally dry woodlands (P. arenaria), to the widespread P. calceolaria or those showing adaptations to the climatic and edaphic conditions of the Cerrado (discussed below) that are unique in the group.

Underground root-like system
Fire damage to and information on the labels accompanying herbarium specimens from species of the Pombalia lanata complex suggest that collections were mostly made soon after a wildfire, sometimes as recently as a month after the disturbance. Formation of a well-developed, bud-bearing underground root-like system is a common plant survival strategy; in the Pombalia lanata species complex, it has presumably evolved in response to the recurrent fires in the Cerrado and as an adaptation to the strongly seasonal climate and edaphic conditions of deep, acidic, well-drained soils with high aluminum content (Filgueiras, 2002;Alonso & Machado, 2007;Appezzato-da-Glória & Cury, 2011;February et al., 2020). A bud-bearing underground system ensures regrowth after fire events and enhances water absorption in deeper soil layers during the dry months.
Ongoing anatomical studies will allow better characterisation of the underground root-like systems in the Pombalia lanata complex. Preliminary examination of several herbarium specimens that were collected with the upper portion of subterranean tissues show a thickened and prolifically bud-bearing structure, just below ground level, from which the shoots originate after fire ( Figure 8A). This thickening is followed by a cylindrical axial root of 5-10 mm in diameter, and field observations have shown that this primary root in Pombalia lanata has orthotropic growth and is flexible, reaching more than 50 cm in depth. It is noteworthy that the underground parts of Pombalia velutina ( Figure 9E) are not as thick and developed as in the remaining species that occur in the core area of the Cerrado, a difference that might be associated with the species' distribution in peripheral areas (southern São Paulo state in Brazil to Paraguay) that are not subject to fire regimes and edaphic restrictions like those of the Central Brazilian savanna.
The only specialised root system in Violaceae besides that of the Pombalia lanata complex was reported for Paypayrola arenacea, a dwarf species of an unrelated woody group of the family growing in scrubby savannas of Venezuela (Aymard et al., 2014). The Amazonian oligotrophic white sand savannas of northern South America are floristically and ecologically distinct from the Central Brazilian Cerrado (Eiten, 1972;Lenthall et al., 1999), and no species of the Pombalia lanata complex have been recorded in this phytogeographical zone of the South American savanna.

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A revision of the Pombalia lanata complex

Indument
The indument, when associated with androecium morphology, is a useful feature with which to distinguish the species within the Pombalia lanata complex. Despite the considerable amount of variation in this character, it is possible to recognise three patterns of indument and their associated trichome types.
• Spreading-villous. Exclusive to Pombalia poaya, this indument is readily recognised by its simple, long, soft and spreading trichomes covering the entire plant body ( Figure 5E). • Strigose. This indument is observed in Pombalia cristalina and P. strigoides and is composed of simple and stiff trichomes that are adpressed to the organs ( Figures 1C,  9D). The indument is overall densely distributed throughout the plant body, except on the leaves of Pombalia cristalina, in which the trichomes are restricted to the basal parts, becoming increasingly sparse upwards along the midrib (Figure 2A,B,D). • Tomentose-velutinous with stellate trichomes. This is the pattern for Pombalia insignis, P. lanata and P. velutina, and in its typical form is displayed as a dense indument of 5-9 simple, tufted trichomes (Figures 1A,E, 5C, 9G), the grouped hairs thus forming a stellate trichome (sensu Webster et al., 1996;Inamdar & Gangadhara, 2008). Some populations display a sparser indument, and in such cases the hairs on the leaves are so scattered as to not form the typical stellate structures, or they are grouped in poorly elaborated units ( Figure 1B; additional illustrations in . Although the stellate trichomes on the leaves can vary in number of radii within a single individual, and very frequently are all distributed as ungrouped, single hairs, the typical stellate structure in a dense indument is always found in floral parts (e.g. sepals and ovary), possibly as a strategy to protect against predation or heat loss.
Plant species in alpine tropical systems often have indument, which provides protection against frosts (Hedberg, 1964), ultraviolet radiation (Karabourniotis & Bornman, 2002), and the loss of water (Ehleringer & Björkman, 1978;Ehleringer, 1982) and heat (higher floral temperatures are associated with significantly increased seed production; Miller, 1986). In this context, the development of a dense indument in the Pombalia lanata complex could be related to the occupation of colder habitats, as has been hypothesised for the lianescent lineage of Violaceae extending to the Andean highlands (Paula-Souza & Pirani, 2014). The stellate trichomes in this family are found exclusively in the Pombalia lanata complex and its sister group, P. arenaria. Although the indument is equally variable across the wide distribution of Pombalia arenaria in northeastern Brazil, it has been observed that its typical morphotypes -densely velutinous with multibranched stellate hairs -are found mostly in the highlands of the Chapada Diamantina. The morphotypes of Pombalia arenaria at lower elevations tend to display a much sparser indument of single or few-branched stellate hairs, and these are areas where its simple-haired sister lineage P. calceolaria becomes more abundant than in the highlands, also occurring in sand dunes at sea level, where the former species is absent. Unfortunately, the only available (yet still unpublished) phylogeny including the Pombalia lanata complex (Paula-Souza, 2009) lacks sufficient resolution to provide a definitive picture of the evolution of indument among its species. However, the same mechanism that favoured the occupation of new habitats in the lianescent Violaceae and Pombalia arenaria might also be inferred for the Cerrado group in areas of lower temperatures where a denser indument might confer some adaptive advantage. The broad, fimbriate sepals and denser coating on the congested inflorescences of Pombalia velutina ( Figure 9F,G) might additionally play an important role in maintaining warmer temperatures inside the flowers (Miller, 1986;Yang et al., 2008), thus contributing to the occurrence of this species in more southern areas of the group's distribution, which are prone to colder temperatures.
Etymology. The epithet refers to the municipality of Cristalina, where the new species is found. In addition to being well known for the production of gemstones and crystals, Cristalina is also recognised here for inhabiting 'precious' plants.
Distribution and habitat. Pombalia cristalina is known only from open savannas and campos rupestres around the municipality of Cristalina, Goiás state, central Brazil ( Figure 3).
Phenology. The species has been collected with flowers and fruits in September and October, with a single flowering record for July.
Conservation status. The municipality of Cristalina is situated at the junction of Brazil's two major highways, which link the largest cities of the country to its capital city, Brasília. This has contributed to much of the growth and urbanisation of the area at the expense of its natural ecosystems. This region is one of the most important centres of production of crystals and gemstones in Brazil (hence the name), and potential environmental effects of this industry are an intense threat to its natural habitats. Personal observations have further indicated that agriculture is a relevant threat; at least two known populations have been drastically reduced and are struggling in cultivated fields of heavily mechanised crops. There is a single occurrence of this species from a small conservation unit. Therefore, according to IUCN (2012) criteria B1a,b(i,iii), Pombalia cristalina is considered Critically Endangered (CR) based on its very limited distribution (EOO, 13,413 km 2 ) and the continuing decline in the quality of its habitat. The habit of this new species resembles that of Pombalia strigoides, but it has slightly larger leaves. In respect to floral features, a striking difference between the two species is in the connective scales of the anthers (see Figure 10E,F), which are much larger in Pombalia strigoides. Additional differences are listed in the    Etymology. The epithet is the Latin word for 'remarkable, outstanding', in reference to the new species' large and showy flowers compared with the other species of this group.
Distribution and habitat. Pombalia insignis has a restricted distribution, known only from a few populations in savannas of Emas National Park, Goiás state, Brazil (see Figure 3).
Phenology. Flowering and fruiting specimens were recorded in October.
Distribution and habitat. Pombalia lanata occurs in savannas, grasslands and campos rupestres of the Brazilian Plateau from Tocantins to Mato Grosso do Sul, also reaching Espinhaço Range in Minas Gerais, with a disjunction in Misiones province, Argentina (Figure 7; see also Paula-Souza et al., 2011). The collection Gardner 2397 at K registered for Ceará state is probably a case of mixed labels, because there is a gathering of Pombalia

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A revision of the Pombalia lanata complex arenaria by Gardner with the same number from the same location ("Cachoeira"), which is situated in a typical xerophytic, deciduous Caatinga vegetation quite distant from the known geographical limits of P. lanata.

Vernacular names. Poaia, poaia-do-campo.
Etymology. The epithet is the vernacular name of the species, which was widely used in traditional medicine in Saint-Hilaire's time.

Pombalia strigoides
Phenology. The species has been collected with flowers and fruits from September to November, with a single flowering record in August.
Etymology. The epithet refers to the dense, villous indument of the species.
Distribution and habitat. Pombalia velutina is found in grasslands and savannas from southern São Paulo to Paraná states in Brazil and adjacent areas of Paraguay, showing the southernmost distribution of this group, isolated from the other species that are typical of the core area of the Brazilian Cerrado (see Figure 3; for a more detailed discussion on its geographical distribution, see Paula-Souza et al., 2011).
Phenology. Flowering and fruiting specimens of Pombalia velutina have been collected from September to November, with a single record in December. This species is readily recognised by its broad and fimbriate sepals, unique among species of the Pombalia lanata complex, and the flowers usually forming congested inflorescences.

Conclusions
The Cerrado of the Brazilian Plateau is home to a strongly supported clade of Pombalia species that show specialisations in their root systems to cope with recurring disturbances such as fire and limiting edaphic conditions. The Pombalia lanata complex takes its name from its most common and widely distributed species, and comprises six taxa that frequently show overlapping morphological characteristics and distributions. Our analyses have shown that half the species of this complex, including the two new species described here, are Critically Endangered according to IUCN criteria (IUCN, 2012). Although the taxonomic boundaries of species in the Pombalia lanata complex are now better understood, multidisciplinary studies focusing on the reproductive mechanisms, ecology, and evolutionary relationships are urgently needed to guide conservation strategies.