Deciphering the Neotropical Bignonia binata species complex (Bignoniaceae)

Bignonia is a genus of 28 species of Neotropical lianas. Most species are clearly characterized by morphological features, monophyletic and narrowly distributed. however Bignonia binata is polymorphic, polyphyletic and broadly distributed, from Mexico to Argentina. A detailed morphological survey of B. binata in the light of geographical and ecological data in its current circumscription recovered two clearly distinct morphological groups of plants. one group is found in Argentina, Paraguay and the Atlantic forest of Brazil, and characterized by non-winged stems, usually terminal inflorescences, usually non-glandular calyces, pantoaperturate pollen, narrowly elliptic fruits, and 1-winged seeds. the second group occurs in Central America and Amazonia and is characterized by winged young stems, usually axillary inflorescences, glandular calyces, inaperturate pollen, widely elliptic to circular fruits, and wingless seeds. the first group of plants corresponds to B. binata and the second group is recognized as another species, B. noterophila. Synonyms, morphology, distributions, and ecology are detailed for these newly circumscribed species. lectotypes are designated for B. noterophila and several synonyms: Adenocalymma ocositense, Arrabidaea schumanniana, and Petastoma laurifolium.


Introduction
Bignonia linnaeus (1753: 622) is a monophyletic lineage that was recently re-circumscribed (lohmann & taylor 2014) to now also include nine previously accepted genera: Clytostoma Miers ex Bureau (1868: 353), Cydista Miers (1863: 191), Macranthisiphon Bureau ex Schumann (1894: 219), Mussatia Bureau ex Baillon (1891[1888: 32), Osmhydrophora rodrigues (1891: 49), Phryganocydia Mart. ex Bureau (1872: 18), Potamoganos Sandwith (1937: 220), Roentgenia urban (1916: 747) and Saritaea dugand (1945: 262). under the new circumscription, Bignonia is characterized by eight phloem wedges in the stems (easily seen in cross-section), usually 2-foliolate leaves with simple tendrils, and opaque seed wings (lohmann & taylor 2014). While most species of Bignonia are morphologically well defined and narrowly distributed, B. binata thunberg (1821: 35) as it has been recognized is polymorphic and very broadly distributed, ranging from Mexico to northern Argentina (Gentry 2009). thunberg (1821) described B. binata based on a collection made by Freyreiss, from an unknown locality somewhere in southeastern Brazil. the identity of B. binata was not clear at that time and the name was seldom used. More than two decades later, de Candolle described Bignonia noterophila Mart. ex Candolle (1845: 148) based on a collection from Martius made in the flooded forests of Pará in northern Brazil. Bignonia noterophila was later transferred into Clytostoma by Bureau and Schumann (1896), who recognized a more widely distributed Clytostoma noterophilum (Mart. ex dC.) Bureau & Schumann (1896: 153) that ranged from French Guiana to rio Grande do Sul, in southern Brazil. their circumscription of C. noterophilum led to the recognition of a morphologically variable taxon that occupied many different habitats beyond just flooded forests. Sandwith (1937) later analyzed thunberg's original collection of B. binata and realized that this was an earlier name for the plants included in C. noterophilum. he therefore published the combination Clytostoma binatum (thunb.) Sandwith (1937: 231) andplaced B. noterophila, B. umbellulata Candolle (1845: 148), and B. purpurea lodd. ex hooker (1869: t. 5800) as synonyms. Following the broad circumscription of Sandwith (1937), additional species were later also included within the circumscription of B. binata by subsequent workers (e.g., Gentry 1973;Macbride 1961;Standley & Williams 1974). More recently, Clytostoma was synonymized under Bignonia and Bignonia binata became again the accepted name for this broadly circumscribed species (lohmann & taylor 2014). despite the wide geographic distribution and the morphological and ecological variation observed in B. binata, its broad circumscription has never been questioned. however this broadly circumscribed species, unusual in Bignonieae, has emerged as polyphyletic in a recent molecular phylogenetic study (Zuntini & lohmann, unpub. data). While hybridization, lineage sorting and other genetic events can lead to polyphyletic species (e.g., Majure et al. 2012;Soltis & Soltis 2009), molecular phylogenies have recently provided key insights into the identification of cryptic taxa (e.g., Govindarajulu et al. 2011). the molecular polyphyly of B. binata coupled with the wide morphological and ecological variation suggest that multiple taxa might currently be mixed under a single name and that this circumscription needs to be further investigated.
We conducted a detailed morphological analysis of the plants now included in Bignonia binata, also referred to as the Bignonia binata species complex, and analyzed these morphological data together with geographic distribution and ecological preferences. Because pollen morphology was shown to be variable among species of Bignonia (Gentry & tomb 1979), a palynological study was also conducted.

Methods
We examined specimens from the following herbaria: BA, BM, F, G, Gh, K, M, MBM, MG, MICh, Mo, NdG, Ny, P, Ph, S, SPF, uPS, uS (acronyms following thiers 2015). Morphological terms follow lohmann & taylor (2014) and radford et al (1974), with rare conditions presented within parentheses. the descriptions here are based on the genus description for Bignonia presented by lohmann & taylor (2014: 416) and do not repeat the genus-level characters. to look for useful palynological characters, flowering buds of selected specimens (Appendix 1) were dissected and the whole content of a single theca was spread in a stub. Stubs were sputter-coated with Au/Pd alloy on a denton desk V Cold Sputter Coater set at 35 mAmps for 120 seconds. Micrographs were obtained on a Jeol NeoScope JCM-5000 scanning electron microscope, using 10kV under high vacuum. the pollen terminology used here follows hesse et al (2009) and Gentry & tomb (1979). Selected images of pollen and representative specimens are available at troPICoS® (2015).

Results and discussion
the morphological analyses revealed many variable and overlapping characters, such as size and indument of leaves and corollas, which helps explain why B. binata has been treated as a single polymorphic species for more than a century. Nevertheless, two morphologically distinct groups are clearly distinguishable among the plants now classified in B. binata. the first group of plants is found in the Atlantic Forest from eastern Brazil through Paraguay to northern Argentina ( Fig. 1). Individuals from this morphological group can be recognized by their non-winged stems, usually terminal inflorescences, non-glandular calyces or calyces with just a couple of glands, narrowly elliptic fruits, and 1winged seeds (table 1, Fig. 2-A-e). the second morphological group occurs in Central America and Amazonia, and is characterized by winged young stems, usually axillary inflorescences, regularly glandular calyces, widely elliptic to circular fruits, and wingless seeds ( Fig. 2 the morphological features of this first group of plants match the characters of Bignonia binata. It is not completely clear where Georg W. Freyreiss collected the type specimen of B. binata, but it is known that he travelled to Bahia, espírito Santo, Minas Gerais and rio de Janeiro between 1813 and 1818 (urban 1906: 21), where the first group is distributed. on the other hand, the morphological features that characterize the second group of plants match the characters of B. noterophila, and its type was collected within the area where this second group of plants is found. Pollen morphology also supports the separation of these two groups, with pantoaperturate, coarsely reticulate or rarely medium-reticulate pollen grains found exclusively in the B. binata group (Fig 2-F) and inaperturate, medium-reticulate pollen grains found exclusively in the B. noterophila group (Fig 2-l).
Nomenclatural notes:-Candolle (1845) indicated in the protologue of B. noterophila that its type was deposited at M. however none of the three specimens located at M were annotated by Candolle. therefore, the best quality material (M0086398) is here designated as lectotype.
Kraenzlin's types were mostly deposited at B and were destroyed during World War II (Stafleu & Cowan 1981), so typification is required. In the case of Petastoma multiglandulosum, Gentry (1973: 826) cited a single specimen from K as the type despite the existence of additional specimens at MICh, Mo and Ny (not cited in the protologue), therefore lectotypification has been made. In the case of Petastoma laurifolium, however, Gentry (1973: 826) cited three specimens (B, K and MICh) but did not designate a lectotype. Among the three specimens listed by Gentry, one was desposited at B and is presumed to be destroyed, another is deposited at MICh (1115833), and the third at K. Given that Kraenzlin more likely saw the material designated at K, this specimen is here designated as lectotype. however, his designated type collection (Rusby 1144) is a mixed gathering of two species, as annotated by Sandwith (in sched.). three of these specimens correspond to Petastoma laurifolium and are cited as types of this name here. this same collection number was later cited as the type of Bignonia brevipes rusby (1900: 71), a species now synonymized with Stizophyllum riparium (Kunth 1819: 138) Sandwith (1938. the two duplicates that match rusby's description of B. brevipes are deposited at Ny (Ny00313136) and uS (1322428), and should not be considered as isolectotypes of P. laurifolium.
When Smith (1893) published Adenocalymma (?) ocositense, he only mentioned the collection number but did not designate a holotype. two sheets were located at uS, where Smith's material is deposited (Stafleu & Cowan 1985). Although one sheet (uS-47668) was probably mounted earlier than the other (uS-1322492), Smith was the collector of this material and clearly saw both specimens given that the description includes elements of both sheets. Both sheets are of equivalent quality and the sheet that was annotated by Smith (uS-1322492) is here designated as lectotype.
Arrabidaea schumanniana was described by huber (1906) based on his own unnumbered collection, deposited at MG. however, his type specimen includes mixed elements of two taxa: (1) the stems, leaves and flowers correspond to B. noterophila, and (2) the fruits and seeds are apparently those of a species of Fridericia Mart. Arrabidaea schumanniana has been considered a synonym of B. binata (e.g., Arbo & lohmann 2008;Brako & Zarucchi 1993), so we here designate the first part of huber's collection deposited at MG as the lectotype in order to maintain its current usage (rec. 9A.4, McNeill et al. 2012).