Ichnanthus robustus (Renvoize) R.P. Oliveira, A.C. Mota & C. Silva, 2017

Oliveira, Reyjane Patricia, Junior, Luiz Antonio De Jesus, Mota, Aline Costa Da & Silva, Christian, 2017, The end of a mystery: transferring Streptostachys robusta to Ichnanthus (Poaceae, Paspaleae) based on DNA sequences, morphology and leaf anatomy, Phytotaxa 326 (3), pp. 159-174 : 162-171

Ichnanthus robustus (Renvoize) R.P. Oliveira, A.C. Mota & C. Silva , comb. nov. ( Figs. 1−7 ).

Basionym:— Streptostachys robusta Renvoize (1984: 182) .

Type:— BRAZIL, Bahia: Ilhéus, 16 km SW of Olivença on road to Vila Brasil (ex Ruinha), 40 m, 21 May 1976, Calderón, Santos & Oliveira 2443 (holotype CEPEC 28766!, isotypes K 000309352!, SP 349344!).

Epitype (here designated):— BRAZIL, Bahia: Barro Preto, Serra da Pedra Lascada, floresta ombrófila densa montana, 14°46’27.5”S, 39°32’08.3”W, 834 m, 14 December 2013 (fl, fr), Silva, Daneu & Bezerra 1049 ( HUEFS!, isoepitypes CEN!, CEPEC!, ICN!, K!, MG!, MO!, P!, RB!, US!).

Perennial plants, herbaceous, cespitose, with short rhizomes, 0.8−2.2 m tall. Culms erect or decumbent, sometimes scandent, rooting at the lower nodes, adventitious roots rigid, 3.5−6.8 mm in diameter at base, green to vinaceous; internodes glabrous; nodes covered by leaf sheaths. Leaves distributed along the culms; leaf sheaths pilose at the base and along the margins with trichomes 1.7−3.8 mm long, or glabrous, sometimes with small auricles 0.7−1.8 mm long; collar glabrous, sometimes with very short trichomes ca. 0.15 mm long; leaf blades 8.5−44.5 × 1.3−5.3 cm, lanceolate, shiny green on the adaxial surface, pale green with or without vinaceous spots on the abaxial surface, apex acuminate, base attenuate to rounded, glabrous on both surfaces, sometimes sparsely pilose with trichomes 0.8−2.1 mm long, margins pilose basally with trichomes 2−5 mm long; midrib conspicuous on both surfaces and more pronounced abaxially, transverse cross-veins (tessellation) slightly conspicuous or inconspicuous abaxially, all ribs scabrous, the marginal ones more sclerified; pseudopetioles present, 1.3−2.8(−6.1) mm long; ligules membranous, 0.4−1.1 mm long. Inflorescences paniculate, open, pyramidal, terminal; peduncle 8.2−32.5 cm long, entirely included in the terminal leaf sheath to 16 cm exserted, glabrous, smooth; main axis 22−55.5 cm long, glabrous, scabrous towards the apex; primary branches 1.9−29.8 cm long, alternate, sometimes opposite or verticillate, scabrous toward the apex, with a small pulvinus at the base; secondary branches 2−16.7 cm long, only present on the lower primary branches, sometimes absent, scabrous, with a small pulvinus at the base; spikelets in pairs, sometimes single, pedicels of the lower ones 1−2 mm long, and pedicels of the apical ones 2.6−5.1 mm long, both scabrous. Spikelets 5.9−7.1 × 1.8−3.4 mm, ellipticlanceolate, sparsely distributed along the branches, green to vinaceous; internode between glumes conspicuous, ca. 0.5 mm long; glumes unequal; lower glume 4−5.8 mm long, 5−7-nerved, margins hyaline toward the base and pilose toward the apex (trichomes 0.4−2.3 mm long), midrib scabrous, apex acute to acuminate, base embracing the upper glume; upper glume 5.4−6.6 mm long, 5−7-nerved, margins hyaline toward the base, apex navicular; lower anthecium staminate; lower lemma ca. 0.5 mm longer than the upper glume, 5−7-nerved, margins hyaline toward the base, apex navicular; lower palea as long as the lemma, membranous, stramineous, apex acute; stamens 3, anthers 2.3−2.8 mm long, light black; upper anthecium 4.6−5.2 × 1.2−1.5 mm, ovate-lanceolate to lanceolate, pale to dark brown at maturity, glabrous, smooth; upper lemma with 2 rows of stomata at the apex; upper palea base rounded, sulcate to flat at maturity; rachilla appendages 0.75−1 mm long (0.2−0.3 mm free from the lemma), winged, fleshy and translucent, up to 1/5 the length of the anthecium, apex rounded, pale to dark brown at maturity, emerging from a short stipe at the base of the upper lemma, 0.15−0.3 mm long; stamens 3, anthers 2.3−2.8 mm long, light black; stigmas 2, strongly vinaceous. Caryopsis not seen.

Morphological comments: —This robust perennial forest grass resembles other species of Ichnanthus in some aspects. Vegetatively, the cespitose habit, erect culms, and large and shiny lanceolate leaves distributed along the culms ( Figs. 1A , 2B ) resemble I. leiocarpus ( Sprengel 1820: 243) Kunth (1831: 507) . However, I. leiocarpus is a smaller plant (up to 1.2 m tall) with densely branched and flowered panicles, spikelets 4−6 mm long, and upper anthecia 3−5.1 mm long with large appendages, ca. 1/3 of the length of the anthecia (see Silva et al. 2015a, and fig. 5D 12 in Silva et al. 2015b), whereas plants of I. robustus are larger, up to 2.2 m tall ( Figs. 1A , 2B ), with few-flowered panicles usually lacking secondary or tertiary branches ( Figs. 1A , 2H ; secondary branches sometimes present on the proximal branches), spikelets 5.9−7.1 mm long ( Figs. 1E− G , 2I− J ), and upper anthecia 4.6−5.2 mm long with very short appendages, ca. 1/5 of the length of the anthecia ( Figs. 1H− M , 3A− B ).

The few-branched panicles with spikelets loosely distributed along the branches associated with lanceolate leaves ( Figs. 1A , 2H ) of Ichnanthus robustus also resemble those of I. longhi-wagnerae Mota & Oliveira (2012: 117) , a species known only from the type collection. It differs from I. robustus mainly by the upper anthecium with large appendages, ca. 1/3 its length (see fig. 2C− E in Mota & Oliveira 2012). Lastly, the spikelets and upper anthecia with short appendages of I. robustus ( Figs. 1E− M , 2I− J , 3A− B ) are very similar to those of I. bambusiflorus ( Fig. 3I, J ; see also Silva et al. 2015a and fig. 5D2, D 3 in Silva et al. 2015b). Nevertheless, I. bambusiflorus is usually a scandent plant, with leaf blades coriaceous and cordate to subcordate at the base (see Silva et al. 2015a), whereas I. robustus is a cespitose plant ( Fig. 2B ), with leaf blades chartaceous and attenuate to rounded at the base ( Fig. 2D ).

Phylogenetic relationships:— The ndhF Bayesian and ML trees are better resolved than the ITS Bayesian and ML trees (trees partly shown in Fig. 4A− D ). The only strongly supported incongruences between plastid and nuclear trees are the placement of the accession ‘ Ichnanthus panicoides ’, the relationships among the accessions ‘ Hildaea pallens ’, ‘ H. sp. PA’, and ‘ H. tenuis ’, and the relationships among the three accessions of ‘ I. calvescens ’ (BA, CE, and GO) . Nevertheless, we analyzed the data sets in combination since none of these relationships affect the conclusions of this study. Results of the individual analyses are presented in Fig. 4A− D . The trees from individual analyses were limited to the Ichnanthus clade for clarity. The full tree resulting from the combined Bayesian analysis is presented ( Fig. 5 ) because it is more resolved and better supported than the tree from the ML combined analysis. Major clades are in agreement with the results of the multilocus analyses presented by Silva et al. (2015b).

The two accessions of Ichnanthus robustus formed a strongly supported clade (1 PP/100 BS) in the combined analyses, and this clade is moderately to strongly supported as sister (0.99 PP/67 BS) to the clade including all accessions of I. bambusiflorus (1 PP/100 BS) ( Fig 5 ). The clade I. robustus – I. bambusiflorus is strongly supported (1 PP/100 BS) as sister to the remaining species of Ichnanthus ( Fig. 5 ). These relationships were also recovered in the ITS analyses ( Fig. 4C, D ), but there is a basal polytomy in the ndhF Bayesian and ML trees ( Fig. 4A, B ). The lower resolution of the ndhF trees is probably due to a reduced number of informative characters of this marker in relation to ITS, as observed by Silva et al. (2015b).

The sister relationship of Ichnanthus robustus and I. bambusiflorus is interesting because both species have the smallest winged appendages found in the genus, whereas the remaining species have well developed winged appendages (see fig. 5 in Silva et al. 2015b). The morphology of the spikelets and the upper anthecium corroborates the relationship between I. robustus and I. bambusiflorus ( Figs. 1−3 ), but the differences in habitat, distribution ( Fig. 7), and vegetative morphology are considerable. Although we have not performed dating analysis, the distinctive morphology suggests these lineages have been evolving separately for a long time. Ichnanthus bambusiflorus is more widely distributed, possibly because it is adapted to dry, sunny, and hot habitats whereas I. robustus is restricted to Southern Bahia, possibly because it is adapted to the cooler, shaded, and humid environments of the ombrophilous montane forests ( Fig. 2A ).

Scanning electron microscopy: — Ichnanthus robustus and I. bambusiflorus have short winged appendages at the base of the upper anthecium ( Fig. 3C− E, K ), which arise from the rachilla ( Fig. 3F, L ). The winged appendages do not have macrohairs. As we have observed for other species of Ichnanthus ( Mota & Oliveira 2012, Silva et al. 2013, 2016), the winged appendages change in shape after fertilization in I. robustus ( Fig. 3C− D ) and in I. bambusiflorus ( Fig. 3I− K ). A change in color also occurs ( Fig. 3A− B, I− J ), suggesting there may be an accumulation of oils in the appendages, as found in other panicoid taxa with rachilla appendages ( Berg 1985, Silva et al. 2017). If confirmed, the presence of oils in the appendages may be indicative of myrmecochorous dispersal since the presence of elaiosomes is a sign of adaptation for myrmecochory ( Lengyel et al. 2009).

The surface of the upper lemma and palea is smooth, without macrohairs, papillae or prickles in both species ( Fig. 3C− D, K ), and there are few silica bodies at the apex of the lemma ( Fig. 3H, M ). The palea base is V-shaped in Ichnanthus robustus ( Fig. 3C− D ) and rounded in I. bambusiflorus ( Fig. 3K ). The germination lid is acute in I. robustus ( Fig. 3F ) and obtuse in I. bambusiflorus ( Fig. 3L ). Stomata are present in two rows on each side of the lemma and palea apices in both species ( Fig. 3G− H, M ).

Leaf anatomy: — Ichnanthus robustus has leaf blade surfaces straight to slightly undulate between vascular bundles, a thin cuticle on both surfaces, and vascular bundles associated with small and narrow sclerenchyma girders ( Fig. 6A− C ), whereas I. bambusiflorus has both leaf surfaces with medium furrows between vascular bundles, a thick cuticle on both surfaces, and vascular bundles associated with well-developed anchor-shaped sclerenchyma girders ( Fig. 6E, F ). These set of characters may be correlated with habitat preferences, since the former species inhabits the forest understory of humid forests ( Fig. 2A ), in medium to high elevations, which is a more stable environment, and the latter usually inhabits the edge of dry/gallery forests or open areas, exposed to drought stress and seasonal variations.

The epidermis is uniseriate in both species ( Figs. 6A− C, E, F ), with bulliform cells on both surfaces in Ichnanthus robustus , composed of numerous cells that occupy the space between vascular bundles, the cells larger in the center ( Fig. 6B, C ). In I. bambusiflorus , bulliform cells are composed of few large cells confined to the center of the furrows, present only on the adaxial surface ( Fig. 6E, F ). No macrohairs or papillae were observed in I. robustus and I. bambusiflorus ; however, trichomes may occur on the leaf blades of both species ( Fig. 2F ). Prickle hairs were observed along the costal zones of both species, being uniseriate and present on both surfaces in I. robustus ( Fig. 6B ), and pluriseriate and present only on the abaxial surface in I. bambusiflorus ( Fig. 6E, F ).

The midrib is quite distinct in relation to the remaining regions of the leaf blade in Ichnanthus robustus ( Fig. 6A ), whereas in I. bambusiflorus there is only a slight differentiation ( Fig. 6E ). This region includes a single first-order vascular bundle in both species ( Fig. 6A, E ), being prominent abaxially in I. robustus ( Fig. 6A ) due to the presence of sclerenchyma and colorless parenchyma, and equidistant from both surfaces in I. bambusiflorus ( Fig. 6E ). Primary vascular bundles are surrounded by a double sheath in both species ( Fig. 6C, D, G, H ): the parenchyma sheath (outer) and the mestome sheath (inner). The parenchyma sheath is colorless and has inflated cells in both species ( Fig. 6C, D, G, H ),

characteristic of a C 3 photosynthetic pathway, corroborating the findings of Morrone & Zuloaga (1991) for I. robustus (under Streptostachys robusta ).

The mesophyll cells have a lax distribution in Ichnanthus robustus ( Fig. 6B, C ), and are more compact in I.

bambusiflorus ( Fig. 6E, F ). Substomatic chambers were observed throughout the mesophyll in both species, always associated with vascular bundles in I. robustus ( Fig. 6C ), and less evident in I. bambusiflorus ( Fig. 6F ). The mesophyll vascularization consists of first and third orders vascular bundles ( Fig. 6B− H ) in both species, located at the same level along the mesophyll, as well as in the midrib, equidistant from both epidermal surfaces ( Fig. 6B, C, E, F ). The first order vascular bundles are circular or elliptical in both species ( Fig. 6A, C, E, G ), occasionally varying in the same leaf blade. In the third order vascular bundles, which are elliptical in shape, the xylem and phloem are indistinguishable and the parenchyma sheath cells are bigger than the vessels cells ( Fig. 6D, H ).

Geographic distribution, habitat preferences, and conservation status: — Ichnanthus robustus is endemic to Southern Bahia, Brazil ( Fig. 7). It was described based on a specimen collected in the middle 1970s between the municipalities of Ilhéus and Olivença ( Renvoize 1984). The type locality and other areas where I. robustus occurs are within the Atlantic Forest domain. The type locality comprises lowland forests similar to those found in the vicinity of Una; however, most records of I. robustus are from montane forests (e.g., Fig. 2A ), which occur above 700 m (700−1100 m: low-montane forests; up to 1100 m: high montane forests; see Oliveira-Filho & Fontes 2000 and Amorim et al. 2009). The climate in these montane areas is hot and humid, but the temperature is lower and the humidity is higher than in the lowland areas ( Amorim et al. 2009), promoting a more stable environment.

In our collections made in that region since the 1990s, we have not identified populations of this species in lowland areas. Like Ichnanthus robustus , other taxa typical of montane forests also have been recorded in the surroundings of Una, such as Podocarpus sellowii Klotzsch ex Endlicher (1847: 209) ( Marinho et al. 2016). According to A. Amorim (pers. comm.), a taxonomist specialized in the flora of that region, the particular environmental conditions of these lowland areas are similar to those found in montane forests, allowing such species to inhabit that region. Ichnanthus bambusiflorus , the closest relative of I. robustus , is more widely distributed, occurring in Central, Northeastern, Southeastern, and Southern regions of Brazil, in the Cerrado, Caatinga, and Atlantic Forest domains ( Fig. 7; see also Silva et al. 2015a).

Based on all known records for Ichnanthus robustus , the Extent of Occurrence and Area of Occupancy calculated using the GeoCAT tool (Bachman et al. 2001) were 3,848.695 km ² and 20,000 km ², respectively. According to these values, I. robustus fits the IUCN (2012) Red List Criteria B 1ab(i,ii)+2ab(i,ii), being assessed as Endangered ( EN). Southern Bahia is the main cacao production region of Brazil. It is characterized by an agroforest system regionally called cabruca, in which cacao trees are planted under thinned-out native forests after removing the understory and some of the canopy trees (see Sambuichi et al. 2012). The montane forests where I. robustus occurs are surrounded by cabrucas, threatening the conservation of these areas.

Additional specimens examined: — BRAZIL. Bahia: Almadina, Serra do Corcovado, fazenda São José , floresta ombrófila densa montana, 14°42’21”S, 39°36’12”W, 800 m, 19 April 2007 (fl), Lopes et al. 1283 ( CEPEC!) ; ibid., 12 August 2007 (fl), Perdiz et al. 90 ( CEPEC!, HUEFS!) ; ibid., 03 September 2011, Coelho et al. 368 ( CEPEC!, UESC) ; ibid., 17 December 2006 (fl, fr), Borges et al. 444 ( CEPEC!, HUEFS!) ; ibid., 15 December 2013 (fl, fr), Silva & Daneu 1057 ( CEPEC!, CEN!, HUEFS!, ICN!, K!, MG!, MO!, RB!). Arataca, RPPN do IESB, Serra do Peito de Moça, Rodovia Arataca / Una , trilha da serra, entrada ca. 1.5 km NE da sede, floresta ombrófila densa montana, 15°09’43”S, 39°20’37”W, 700−900 m, 02 July 2008 (fl), Jardim et al. 5324 ( CEPEC!, HUEFS!). Barro Preto, Serra da Pedra Lascada , fazenda São Miguel, floresta ombrófila densa montana, 14°46’22”S, 39°32’16”W, 960 m, 18 August 2003 (fl, fr), Fiaschi et al. 1557 ( CEPEC!, SPF) ; ibid., 02 November 2003 (fl), Fiaschi et al. 1798 ( CEPEC!) ; ibid., 16 January 2007 (fl), Borges et al. 505 ( CEPEC!) ; ibid., 11 February 2011 (fl), Mota & Carvalho-Sobrinho 393 ( HUEFS!, CEPEC!) ; ibid., 13 March 2007 (fl), Oliveira et al. 1216 ( CEPEC!, HUEFS!). Una, Reserva Biológica do Mico-leão ( IBAMA), entrada no km 46 da rodovia BA-001 Ilhéus-Una , região da mata higrófila Sul Baiana , 15°09’S, 39°05’W, 24 September 1992 (fl, fr), Amorim et al. 780 ( CEPEC!, MO, NY, K!) ; margem da rodovia Una- Olivença , mata costeira, 01 June 1966 (fl), Belém & Pinheiro 2381 ( CEPEC!, UB!) ; rodovia de Una a Olivença, mata, 06 September 1971 (fl), Pinheiro 1591 ( CEPEC!, MO). Wenceslau Guimarães , Estação Ecológica , trilha atrás do alojamento, passando a captação, floresta ombrófila densa, 13°36’00”S, 39°43’06”W, 702 m, 16 October 2012 (fl), Milliken et al. 5095 ( HURB!, K!) .

Figures

FIGURE 1. Ichnanthus robustus. A. Habit (including inflorescence); B. Culm, leaf sheath base (node region), and leaf blade base; C. Pulvini at the base of the primary branches; D. Segment of an inflorescence branch with pairs of spikelets; E. Pair of spikelets, lateral view; F. Spikelet, dorsal view; G. Spikelet, ventral view; H–J. Young upper anthecium; H. ventral view; I. lateral view; J. dorsal view; K–M. Mature upper anthecium; K. ventral view; L. lateral view; M. dorsal view. (based on C. Silva et al. 1049)

FIGURE 2. Ichnanthus robustus.A. Habitat,montane forestin SouthernBahia; B. Habit; C. Baseof the plant,including culms,adventitious roots, and rhizome; D. Culm, leaf sheath base (node region), and leaf blade base; E. Node region glabrous; F. Trichomes in the adaxial surface of the leaf blade; G. Vinaceous spots in the abaxial surface of the leaf blade; H. Inflorescence; I. Pair of spikelets, dorsal (left) and lateral (right) views; J. Spikelet, dorsal view. (Photos by C. Silva)

FIGURE 3. Stereomicroscope (SM) and scanning electron microscope (SEM) images of the upper anthecium. A–H. Ichnanthus robustus [SM: Amorim et al. 780 (CEPEC) and Silva & Daneu 1057 (HUEFS); SEM: Amorim et al. 780 (CEPEC), Borges et al. 444 (HUEFS), and Silva & Daneu 1057 (HUEFS)]. A. Young anthecium, ventral view; B. Mature anthecium, ventral view; C. Young anthecium, ventral view; D. Mature anthecium, ventral view; E. Young anthecium, base, lateral view; F. Mature anthecium, base, dorsal view; G. Mature anthecium, apex, ventral view; H. Mature anthecium, apex, lateral view. I–M. Ichnanthus bambusiflorus [SM: Ribeiro-Filho 221 (HUEFS); SEM: Silva et al. 295 (HUEFS)]. I. Young anthecium, ventral view; J. Mature anthecium, ventral view; K–M. Partially mature anthecium; K. Ventral view; L. Base, dorsal view; M. Apex, lateral view. [Scale bars A, B, I, J = 1 mm; C–H, K–M = 500 μm. GL = germination lid; L = lemma; P = palea; PB = palea base; R = rachilla; SB = silica bodies; ST = stomata; W = winged appendages]

FIGURE 4. Part of the trees resulting from the Bayesian and Maximum Likelihood (ML) individual analyses representing the Ichnanthus clade. Bayesian posterior probabilities> 0.5 and ML bootstrap support values> 50% are reported below branches. A. ndhF Bayesian consensus tree; B. ndhF ML tree; C. ITS Bayesian consensus tree; D. ITS ML tree. Ichnanthus robustus is highlighted in bold, followed by a bracket. The accession followed by an asterisk was identified as Ichnanthus sp. BA in Silva et al. (2015b; voucher: Oliveira et al. 1216).

FIGURE 5. Bayesian consensus tree resulting from the combined (ndhF and ITS) analysis. Bayesian posterior probabilities> 0.5 and Maximum Likelihood bootstrap support values> 50% are reported above and below branches, respectively. Ichnanthus robustus is highlighted in bold, followed by a bracket.

FIGURE 6. Cross-sections of the leaf blades showing the midrib and the mesophyll of Ichnanthus robustus [Calderón et al.2443 (CEPEC), Silva et al. 1049 (HUEFS), and Silva & Daneu 1057 (HUEFS)] and I. bambusiflorus [Silva et al. 1025 (HUEFS)]. A–D. I. robustus. A. Midrib overview; B. Mesophyll overview; C. First order vascular bundle; D. Third order vascular bundle. E–H. I. bambusiflorus. E. Midrib and mesophyll overview; F. Mesophyll, third order vascular bundles; G. First order vascular bundle; H. Third order vascular bundle. [Scale bars A–C, E = 100 μm; D, G–H = 20 μm; F = 50 μm]