Taxonomic updates in Amphitecna (Bignoniaceae): A new Mexican species and the re-establishment of the giant-leaved A. megalophylla

Abstract In this study, we analyzed the morphological affinities of the 24 species of Amphitecna based on detailed morphological studies and multivariate cluster analyses. Our results suggest that the genus Amphitecna includes six morphological groups that can be easily distinguished based on floral and fruits characteristics: A. donnell-smithii group, A. macrophylla group, A. megalophylla group, A. molinae group, A. spathicalyx group, and A. steyermarkii group. A new species from Mexico, Amphitecna fonceti, is described. This new species is clearly differentiated by the predominantly ramiflorous inflorescences bearing multiple flowers per shoot, buds rounded at the apex, large flowers with a transverse fold in the corolla throat, calyx surface pubescent and strongly costate, and fruits elliptic, apiculate at the apex. We discuss the characteristics of each morphological group and their geographical distribution, provide a detailed description of the new species including ethnobotany notes, and propose the re-establishment of the giant-leaved species A. megalophylla.


Materials and methods
To infer the morphological similarities among the 24 species of Amphitecna, we performed a hierarchical clustering analysis on a matrix that included 15 flower traits. The data were analyzed using the unweighted pair group method with arithmetic mean (UPGMA, Sokal and Michener 1958) and the Gower index (Gower 1971), allowing a simultaneous use of binary and continuous characters (Dunn and Everitt 1982;Yang et al. 2007;Zanella et al. 2011;Tuler et al. 2017; Svoboda and Ballard 2018; Wahlsteen Table 1. Species currently recognized in Amphitecna (Bignoniaceae) and their respective geographical distribution. The species are ordered by the morphological groups recovered in the clustering analysis. Amphitecna latifolia (from the A. molinae group, cluster "A") was excluded because it is the only broadly distributed species. NM = Northern Mesoamerica; SM = Southern Mesoamerica. and Tyler 2019). All morphological characters were obtained from original species descriptions (Gentry 1980;Gentry 1982b;Burger and Gentry 2000;Ortiz-Rodriguez et al. 2016), herbarium specimens deposited at MEXU (www.ibdata.ib.unam.mx), and type specimens available online (https://www.gbif.org/ and https://plants.jstor.org/). The UPGMA results were contrasted with those derived from other clustering algorithms, specifically Ward, single linkage, complete linkage, WPGMA, WPGMC, and UPGMC, implemented in the R-package stats, using the function hclust (R Core Team 2020: https://www.r-project.org/). We then determined the similarities and differences among the various clustering dendrograms by calculating the cophenetic correlation (a Pearson's measure) between each clustering result using the cor.dendlist and the corrplot functions from the corrplot R-package (Wei and Simko 2017). For each dendrogram, the agglomerative coefficient was calculated using the agnes function from the cluster R-package (Maechler et al. 2019). The agglomerative coefficient measures the amount of clustering structure, with values closer to 1 suggesting stronger clustering structure. Also, the Fowlkes-Mallows Index (from the dendextend R-package, Galili 2015) was used to compare the species composition within clusters (k = 3-8) obtained from the UPGMA analysis and other algorithms. The optimal number of morphological clusters in Amphitecna was determined based on the greater similarity between clustering algorithms (values closer to 1). We further performed an internal clustering validation (a cluster stability test) by calculating the average silhouette width (Si) for each cluster (k = 3-8) resulting from each of the algorithms used. While Si values greater than 0.71 suggest strong structure and cluster stability, values between 0.51 and 0.70 are interpreted as reasonable, values between 0.26 and 0.50 indicate weak structure, and values lower than or equal to 0.25 are not worth further discussion (Kaufman and Rousseeuw 2005). The graphical representation of the UPGMA dendrogram was carried out in the R software, using the function hclust implemented in the R-packages ape, and ggtree (Yu et al. 2017;Paradis and Schliep 2018).
The new species described was recognized by a unique combination of features (Donoghue 1985) identified through comparisons with morphologically similar taxa and literature review (Gentry 1982a, b, Burger andGentry 2000;Ortiz-Rodriguez et al. 2016). We assessed the conservation status by calculating the extent of occurrence (EOO) and the area of occupancy (AOO) using the GeoCAT tool (Bachman et al. 2011) and applying the IUCN Red List Categories and criteria (IUCN 2019).

results
The UPGMA dendrogram is shown in Figure 1. The results of this analysis are very similar to those obtained using other clustering algorithms (correlation values between 0.78 and 0.98, Suppl. material 1: Figure S1). The agglomerative coefficient value for the UP-GMA dendrogram was 0.67 (between 0.47 and 0.83 in analyses conducted with other approaches), suggesting a moderate to strong structure among species of Amphitecna. The Fowlkes-Mallows Index showed that six groups (k = 6) show significantly similar clusters when the UPGMA is compared to the other clustering algorithms (FM values between 0.71 and 1). Silhouette width values consistently showed the highest values (Si value 0.35 for all algorithms) when each dendrogram was divided into six clusters.
The UPGMA results indicate that the genus Amphitecna can be classified into six morphological groups ( Figure 1A) and three main clusters (A-C). The A. molinae, A. megalophylla, and A. spathicalyx groups are characterized by corollas with a transverse fold in the throat ( Figure 1B). In the UPGMA dendrogram, these species are nested within cluster "A" ( Figure 1A). The Amphitecna molinae group consists of 10 species with sessile leaves, 15-40 cm long, flower buds rounded at apex, and smooth fruit surface, rarely warty. The A. megalophylla group includes two species with short- petiolate leaves, long leaf blades (50-100 cm long), flower buds rounded at the apex, and fruit surface costate. The new species, Amphitecna fonceti, is part of the A. molinae group and is morphologically most similar to the A. latifolia and A. sessilifolia groups (Table 3). Amphitecna spathicalyx is also placed within cluster "A" (i.e., the A. spathicalyx group) and is distinguished from other species by the pointed flower buds and spathaceous calyces.
Cluster "B" is composed of the A. macrophylla and A.donnell-smithii groups (Figure 1A). The species from these groups are best recognized by their short-petiolate leaves, inflorescences born along the main trunk or leafless branches, mostly composed of 1-2 flowers per shoot, flowers without a transverse fold in the corolla throat, and calyces bilabiate or trilabiate ( Figure 1B). The A. macrophylla group contains five species with cauliflorous inflorescences and long and funnelform corollas (up to 70 mm long). The A. donnell-smithii group consists of two ramiflorous species with small flowers (less than 28 mm long) and broadly campanulate corollas. The only two species of Amphitecna with broadly campanulate flowers are included in the A. donnell-smithii group.
Cluster "C" consists of the Amphitecna steyermarkii group and is composed of three species that are characterized by their terminal inflorescences with several flowers per shoot, flower buds with a sharp acumen, flowers without a transverse fold in the corolla throat, and spathaceous calyces.
Key for the identification of species of Amphitecna (Bignoniaceae)

Morphological groups and their distribution
The results presented here show that Amphitecna consists of several morphological groups (Figure 1). These groups do not necessarily represent lineages and, according to internal clustering validation (Si value), their stability should be tested with additional data. Nonetheless, the resulting morphological grouping recovered provides new insights into the understanding of relationships among species of Amphitecna.
Although little is known about the reproductive ecology of Amphitecna, the flower and fruits differences among groups are likely linked to their pollinators and seed dispersers. Most species have exposed inflorescences (terminal and cauliflorous), consisting of one-to several flowers with a transverse fold in the corolla throat fitting the Crescentia-type pollination syndrome, which includes bat-pollinated flowers (Gentry 1980, Fleming et al. 2009). However, hummingbirds and other birds also visit flowers of some Amphitecna species, such as A. apiculata, A. latifolia, and A. sessilifolia (Richardson 1984). On the other hand, the fleshy and indehiscent fruits (mostly mammalian-dispersed) are only found in Amphitecna and close relatives (Gentry 1980), showing considerable variation in fruit shape and surface.
The distribution of species within the various morphological groups seems to follow a geographical pattern. Cluster "A" (species with a transverse fold in the corolla throat) includes taxa that are distributed throughout Mesoamerica (from Mexico to Colombia) (Table 1). On the other hand, sub-groups within cluster "A" show variable distribution patterns. For example, the A. molinae group from cluster "A" has members in both regions of Mesoamerica (i.e., Northern Mesoamerica and Southern Mesoamerica; Table 1), with the A. megalophylla group endemic to the northern portions of Mesoamerica (from Mexico and Guatemala), and the A. spathicalyx group endemic to the southern portions of Mesoamerica (found in Panama exclusively). On the other hand, cluster "B" includes members of the A. macrophylla and A. donnell-smithii groups, occurring predominantly in northern Mesoamerica. Except from A. parviflora that is endemic to Costa Rica, the remaining six species of cluster "B" are found in Mexico and Guatemala exclusively. Finally, two species placed in the Amphitecna steyermarkii group occur in northern Mesoamerica (Mexico, Guatemala, and Belize), with a single species endemic to Costa Rica. Based on the above, northern Mesoamerica is not only the center of diversity of Amphitecna, but also the most diverse region morphologically (Table 1).

Taxonomic implications
Amphitecna megalophylla was first treated as a synonym of A. macrophylla by Seibert (1940), which was subsequently followed by Standley and Williams (1974), Nelson (2008), and the iPlants Project (http://powo.science.kew.org/taxon/11655-2). Although Gentry (1980) highlighted the morphological features that characterize A. megalophylla, the species has continued to be treated as a synonym.
The results presented here show that Amphitecna megalophylla and A. macrophylla are clearly distinct and are best treated as separate taxa (Table 2). Amphitecna megalophylla is part of the A. megalophylla group together with A. costata, both of which are placed within cluster "A" based on its multi-flowered inflorescences, buds rounded at apex, and long pedicellate corollas with a transverse fold in the throat (Figure 1). In contrast, A. macrophylla is placed within the A. macrophylla group (cluster "B") along with two other giant-leaved species based on its inflorescences with 1 (rarely 2) short pedicellate flowers that lack a transverse fold in the corolla throat (radially symmetric). In addition, A. macrophylla is endemic to Veracruz (Mexico), while A. megalophylla is endemic to Guatemala (Gentry 1980). Results from our cluster analyses suggest that A. fonceti is part of the A. molinae group (cluster "A") along with A. apiculata, A. latifolia, and A. sessilifolia. Species within cluster "A" share multi-flowered inflorescences and flowers with a transverse fold in the throat, while showing several differences in their flower and fruit morphology (Table 3). Hence, A. fonceti is best treated as a separate taxon, which is described below and compared to other morphologically similar taxa.

Taxonomic treatment
Amphitecna fonceti Ortiz-Rodr. & Gómez-Domínguez, sp. nov. urn:lsid:ipni.org:names:77214647-1 Figures 2, 3 Type. Mexico. Chiapas, Municipio de La Concordia, Área de Protección de Recursos Naturales La Fraylesca, Rancho "Pacayal" a 3 kilómetros del ejido Solo Dios,1441 m, Diagnosis. Amphitecna fonceti is distinguishable from the other species of Amphitecna by its ramiflorous inflorescences that bear multiple flowers per shoot, buds rounded at apex, large flowers with a transverse fold in the corolla throat, calyx surface pubescent and strongly costate, and fruits elliptic, apiculate at the apex. Amphitecna fonceti is morphologically similar to A. apiculata and A. latifolia, both of which occur in Mexico. However, A. apiculata differs by the small and tubular corollas, and by the calyx with a smooth and glabrous surface. Amphitecna latifolia, on the other hand, differs by the smaller leaves, smooth and glabrous calyx surface, and globose fruits with a rounded apex. The three species show different climatic preferences (Table 3).
Description. Small to medium sized trees, 3-9 m alt., 6-25 cm DBH, the secondary branches terete. Leaves alternate-verticillate, clustered near the apex of branches, olive-green when dry, glabrous, coriaceous, 13-35 cm long × 6-13.2 cm wide, oblanceolate to obovate, short acuminate, acute to attenuate basis, midrib slightly raised on the upper surface, prominent on the lower surface; secondary veins 10-20 on each side, slightly raised above, prominent below; petiole shorter than 1 cm long, merging with attenuate leaf base, red wine in vivo. Inflorescences bearing three to six flowers (rarely with a single flower), borne on leafless portions of old branches, rarely terminal or along the main trunk (cauliflory), with a sour-odor; pedicels, outer side of buds, and calyces pubescent and densely covered with lenticel-like white dots. Flowers more or less erect, not pendant, pedicel 38-60 mm long; buds, rounded at apex; calyx campanulate, 25-32 mm long, coriaceous, evenly 2-3-labiate, strongly costate, with 6-10 longitudinal ridges per lobe; corolla funnelform, with a transverse fold on throat between 22-27 mm from the base, pale green, 38-46 mm long × 20-23 mm wide at the tube mouth, the basal portion of the corolla funnel-shaped, 9-13 mm long, lobes more or less fused into a frilly-margined rim; androecium with stamens 3 or 4, included, inserted 4-12 mm from base of the tube, anther thecae divergent, 5-6 mm long, filaments 12-29 mm long, staminodes shorter than 20 mm long when present, inserted 3-6 mm from base of the tube; gynoecium with ovary ca. 8 mm long × ca. 4 mm wide, broadly elliptic, glandular-papillose, style 25-29 mm long, stigma bifurcate; disc annular-pulvinate, ca. 11 mm in diameter. Fruits elliptic, 110-180 mm long × 70-105 mm wide, acute to short acuminate at apex, rounded to short acuminate at the base.
Habitat and ecology. This species is known only from the type locality in Chiapas, Mexico. The species inhabits areas with sedimentary soils, mostly formed by sandstones with a thin layer of organic matter, mostly within altered remnants of oak and pine-oak forest. The species with which it coexists are Quercus rugosa Née, Inga vera   the new species is distributed within a protected natural area, the oak, pine-oak forest at the type locality is seriously fragmented, with only small remnants persisting. Amphitecna fonceti is rare, with only 12 individuals being known to date.
Uses. The indigenous community where A. fonceti is found uses the fruits to treat respiratory diseases. The seeds of ripe fruits are extracted and soaked in a bottle of tequila for a week, after which a small glass is drunk in the morning to treat asthma. For whooping cough, two tablespoons of honey and almond oil are poured into the fruit after the removal of the fruit tip. The fruit is then cooked in water bath and its interior used as syrup. Its medicinal use likely helps the maintenance of this species within local coffee plantations.
Notes. In addition to A. apiculata and A. latifolia, A. fonceti can also be confused with A. sessilifolia, another species from the A. molinae group. However, A. sessilifolia (endemic to Costa Rica) shows terminal flowers, larger corollas, stamens inserted 13-18 mm from base of the corolla tube, larger pistils, smooth and glabrous calyces (Gentry 1980, Table 3). Amphitecna sessilifolia has been incorrectly reported to Mexico

Amphitecna megalophylla resurrected
Our results indicate that A. megalophylla is best treated as a separate taxon that can be identified by the following features: pachycaul trees, with leaves up to 1 m long, multiflowered inflorescences, cauliflorous and long-pedicellate flowers with a transverse fold in the corolla throat, and fruits with costate/angulate surfaces. The following species is thus treated as an accepted taxon here: