Hymenaea L. is a genus of caesalpinioid legumes with 14 species (Lee and Langenheim 1975). The genus is distributed throughout tropical America, from Mexico to Paraguay, with one species in coastal East Africa (Mackinder 2005). Hymenaea is included in the Resin-producing Clade of the tribe Detarieae, and is most closely related to the genera Guibourtia Benn. and Peltogyne Vogel (Bruneau et al. 2001, 2008; Fougère-Danezan et al. 2007), all presenting similar leaf morphologies, with two asymmetrical pellucid-punctate leaflets. Hymenaea can be differentiated from these related genera by its rather larger and more massive bat-pollinated flowers with a robust hypanthium, and indehiscent, pulpy and woody pods with very large seeds.

The current taxonomy of the genus is largely based on Lee and Langenheim’s (1975) revision. In addition to recognizing the 14 currently accepted species, they reduced several species to varietal rank. These polytypic species were viewed by Lee and Langenheim (1975) as showing complex relationships with other species of Hymenaea. They hypothesized, for example, that Hymenaea oblongifolia Huber var. oblongifolia is more closely related to Hymenaea aurea Lee and Lang. and Hymenaea eriogyne Benth., while Hymenaea oblongifolia var. davisii (Sandwith) Lee and Lang. is probably more closely related to Hymenaea parvifolia Huber, Hymenaea rubriflora Ducke, and Hymenaea reticulata Ducke. According to their concepts, Hymenaea oblongifolia should be regarded as a polyphyletic species. Another example of a putative polyphyletic species, according to Lee and Langenheim’s (1975) conceptual framework, is Hymenaea courbaril, with var. villosa Lee and Andrade-Lima hypothesized as being more closely related to Hymenaea martiana Hayne, and var. longifolia (Benth.) Lee and Andrade-Lima to Hymenaea velutina Ducke and Hymenaea stigonocarpa Mart. ex Hayne.

Hymenaea courbaril is the most widely distributed species of the genus, almost matching the geographic range of Hymenaea in the New World. It also has the greatest economic importance in the genus, due to the high quality of its wood and its resin, the latter being used by native populations as incense, cement, in the manufacture of varnishes, and for medicinal purposes. Its nutritive fruits are sought after by mammals and birds (Rizzini 1971; Langenheim 1967; Lee and Langenheim 1975). Hymenaea courbaril is the most taxonomically complex species, with six varieties: var. altissima (Ducke) Lee and Lang., var. courbaril, var. longifolia, var. stilbocarpa (Hayne) Lee and Lang., var. subsessilis Ducke, and var. villosa. These varieties are differentiated by their leaflet sizes, shapes, and indumentation, calyx indumentation, petal shapes, ovary stipe sizes, and pod sizes and shapes. These variations in several diagnostic features make the boundaries of putatively related taxa rather imprecise. The widely circumscribed Hymenaea courbaril, as defined by Lee and Langenheim (1975), is hereafter referred to as the Hymenaea courbaril complex.

The species and varietal limits of Hymenaea courbaril, Hymenaea stigonocarpa, and Hymenaea martiana were investigated by Pestana (2010). This author did not employ objective analytical methods and used the same classical taxonomic approach as Lee and Langenheim (1975), thus coming to similar conclusions as the latter authors in keeping Hymenaea courbaril as a polytypic species with six varieties.

Delineating precise species boundaries is a key task in plant taxonomy. This process has direct impacts on society, as there is a growing demand for credible taxonomic information that allows us to conserve, manage, and understand natural biodiversity (Wheeler et al. 2004). However, questions of species recognition can be affected by several theoretical, methodological, and practical issues. De Queiroz (2005, 2007) defined species as separately evolving metapopulation lineages (the Unified Species Concept–USC), and proposed that all other previously considered properties of species should be reinterpreted as contingent rather than critical. These additional contingent properties, such as phenetic distinctiveness, reciprocal monophyly, genetic coalescence, or ecological distinctiveness, are acquired during speciation and should be considered as different lines of evidence relevant to assessing lineage separation. The USC, by treating species conceptualization and species delimitation as clearly separate issues, allows the use of properties formerly treated as secondary criteria in species delimitations. More importantly, it allows for currently accepted species limits to be considered as hypotheses to be tested using the presence of any one of those secondary properties as evidence for the existence of a species.

Analyses of morphometric data can be useful in objectively demonstrating species limits, especially when combined with molecular markers (Andrés-Sánchez et al. 2009; Newmaster and Ragupathy 2009). Additionally, the use of morphological information represents the fastest and least expensive manner of assessing taxonomic complexes–and has been used to solve problems of species limits in many different plant groups, especially when molecular data was not easily available (Handerson 2006; Estrella et al. 2009; Pedersen 2010; Ceolin and Miotto 2012; Rahman and Rahman 2012; Castello and Galetto 2013; Scrivanti et al. 2013), as was the case of the group studied here.

The problem of defining species limits in the polytypic Hymenaea courbaril complex is revisited here under the USC conceptual framework by exploring morphometric, geographical, and ecological patterns as lines of evidence for the existence of separate metapopulation lineages. Specifically, we sought to test Lee and Langenheim’s (1975) hypothesis that Hymenaea courbaril should be treated as a polytypic species with six varieties.

A total of 96 specimens of the Hymenaea courbaril complex were examined in this study (vouchers listed in Appendix 1). All analyzed materials were sheets from the following herbaria: B, CEN, CEPEC, HRB, HUEFS, IBGE, IPA, LAGU, M, MBM, NY, RB, SP, SPF, U, UB, and UC. We selected specimens that displayed branch ends with fully-developed (mature) leaves. This criterion avoided considering young leaves from the tips of the branches or leaves at the bases of the branches that are often much larger. Each specimen analyzed was considered an individual, and identifications strictly followed Lee and Langenheim (1975), although these names were only used as nomenclatural references in this study.

The leaves in the group studied here are bifoliolate; the leaflets range from oblong to ovate or obovate, with rounded, acute or obtuse apices; the main vein is displaced towards the inner margin resulting in an asymmetrical base, the outer portion being wider than the inner and extending beyond the attachment to the petiolule (Figure 1). Fourteen quantitative (continuous) characters were examined (Table 1; Figure 1). Only leaf traits were measured and quantified, as most herbarium sheets lacked flowers and/or fruits. In any case, flower morphology is much conserved in the species studied and the herbarium material examined usually contained only incomplete or damaged flowers. Measurements were taken of two fully developed leaves per dried herbarium sheet, using a graduated ruler (precision 1 mm).

All multivariate analyses were carried out using Past software (Hammer et al. 2001). Cluster analyses used the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based on Bray-Curtis dissimilarity matrices. Principal Component Analyses (PCA) were carried out based on the same morphometric matrix. Two sets of Analyses of Similarity (ANOSIM; Warwick, Clarke and Suharsono 1990) and Non Parametric Multivariate Analysis of Variance (NPMANOVA; Anderson 2001) were carried out to evaluate statistical support for: (1) the major groups recovered in the UPGMA and PCA; and (2) the varieties as defined by Lee and Langenheim (1975). Hymenaea courbaril var. villosa was not included in the second analysis because it is known from only two specimens (Lee and Langenheim 1975) and only one was located during this study. Both ANOSIM and NPMANOVA used Bonferroni corrections, 10, 000 permutations, and Bray-Curtis distances. As Hymenaea courbaril var. villosa was represented by only a single specimen, we carried out a second set of UPGMA, PCA, and similarity analyses for the major groups without including this variety in order to test its influence on the results.

Distribution maps of the specimens studied were prepared using DIVA-GIS software (Hijmanns et al. 2005), based on the geographic coordinates recorded on the herbarium sheet labels. For material lacking original coordinates, a central coordinate for the municipality was used as provided by the Species Link website (available at http://specieslink.org.br ).

Species limits were tested following the USC framework (de Queiroz 2005, 2007). Species ranks were ascribed to groups that showed morphological and habitat distinctiveness and geographical consistency. We considered as morphologically distinct groups those that were recovered in UPGMA and PCA and that exhibited statistical significance in both ANOSIM and NPMANOVA tests. Habitat distinctiveness was assessed from the vegetation type where the taxon occurs, following the UNESCO (1973) classification. Species diagnoses were prepared based on vegetative and reproductive characters.

Both UPGMA and PCA recovered three major groups (Figure 2): Group 1 included all specimens of var. altissima; Group 2 all specimens of varieties courbaril, stilbocarpa, subsessilis, and the single specimen of villosa; and Group 3 all specimens of var. longifolia. Within Group 2, individuals of the different varieties did not cluster together and appeared intermixed in UPGMA, or formed highly overlapping groups in two first axes of PCA. The first PCA axis accumulated 88.3% of the total variance, with the two first axes summing 93% of the observed variation. Leaflet length was the trait that explained most of the variation found in first axis, and the three major groups were sorted mostly by leaflet size. These results indicate that Group 3 includes specimens with largest leaflets, and Group 1 the smallest leaflets (Figure 3).

The results of the UPGMA and PCA were consistent with both ANOSIM and NPMANOVA. Comparisons of the varieties of Hymenaea courbaril showed significant differences (p < 0.05) between the varieties altissima and longifolia and all other varieties, but no significant differences between the varieties courbaril, silbocarpa, and subsessilis that clustered in Group 2 (Table 2). The same analyses comparing the three groups recovered in UPGMA and PCA found significant morphological differences between them (Table 3).

The complex morphological variations found in Group 2 appear to reflect its wide geographic range and large genetic variability (Ramos et al. 2009). Phylogeographic studies in part of the geographic range of Hymenaea courbaril var. stilbocarpa clearly demonstrated the wide genetic base and geographic structure of this genetic variation (Ramos et al. 2009). The morphological distinctions among the varieties clustered in Group 2 are made by rather continuous characters, such as the color of the calyx lobes (ochraceous to golden or rusty brown), leaflet shape (broadly to narrowly falcate), and the shapes of the outer sides of the leaflet base (straight to nearly straight or rounded). The var. subsessilis presents a short ovary stipe (c. 2 mm long) that grades to a medium-sized stipe in var. stilbocarpa (3–4 mm) and then large in var. courbaril (4–6 mm).

Leaflet indument distinguishes var. villosa from the remaining varieties of Group 2 (Lee and Andrade-Lima 1974, 1975). The ovary is also described as slightly pilose on one side, a condition not observed in other varieties of Hymenaea courbaril. The exclusion of var. villosa from the similarity analyses did not alter the results obtained with its inclusion (Table 3). The var. villosa is known from only two specimens from the eastern coastal areas of the states of Paraíba and Pernambuco (northeastern Brazil) where the range of var. stilbocarpa overlaps with that of Hymenaea martiana. This species (Hymenaea martiana) has tomentose leaflets and an ovary with a tuft of trichomes near its base. The rarity of Hymenaea courbaril var. villosa, as well as the transitional nature of the leaflets and ovary indumentation in relation to Hymenaea martiana and the other varieties of the Hymenaea courbaril complex clustered in Group 2, all suggest that var. villosa could represent a hybrid between Hymenaea martiana and Hymenaea courbaril.

The habitats of the taxa of Group 2 include mostly tropical ombrophilous alluvial (gallery) forests in areas subject to seasonally dry climates from Mexico and the Caribbean islands to central Brazil, but not the Amazonian region (var. courbaril and stilbocarpa), tropical ombrophilous lowland forests in central and eastern Amazon basin (var. subsessilis), or tropical ombrophilous submontane forests in the northeastern Brazilian state of Paraíba (var. villosa) (Figure 2).

The var. altissima (Group 1) constitutes a morphologically, geographically and ecologically consistent taxon. It is distinguished from the other varieties of Hymenaea courbaril complex by having smaller, falcate and acuminate leaflets, not exceeding 6.5 cm long on the flowering branches (Table 4). The leaflets of this taxon have a thinner texture than the remaining varieties, allowing its tertiary venation to appear as raised and reticulate veins. The flowers of var. altissima are also the smallest within the Hymenaea courbaril complex, measuring less than 15 mm long (Lee and Langenheim 1975, Pestana 2010). This variety is distributed in tropical ombrophilous lowland and submontane forests of the Atlantic Forest phytogeographical domain along the eastern coast of Brazil (Figure 2). It was cited for the southeastern Brazilian states of São Paulo and Rio de Janeiro (Mattos 1968, Lee and Langenheim 1975, Rizzini 1978, Pestana 2010), but we found specimens occurring northwards into Espírito Santo and Bahia states. It was originally described as Hymenaea altissima Ducke (Ducke 1935). Lee and Langenheim (1974: 448) considered that the foliar characters and relative flower size “do not appear sufficiently significant to warrant specific status for this taxon” and considered it as a variety of Hymenaea courbaril.

Var. longifolia (Group 3) comprises the morphs with largest leaflets. This variety also differs from the remaining groups by having oblong leaflets with an obtuse apex, clawed petals, and fruits compressed and enlarged toward the apex (Table 4). It occupies a very particular habitat in submontane tropical drought-deciduous thorny forests in the Caatinga and Cerrado phytogeographical domains that extend from western Bahia and Piauí states to the Araripe mountain range in the border area between the states of Ceará and Pernambuco (Figure 2). It was first proposed as a variety of Hymenaea splendida Vogel. Lee and Langenheim (1974, 1975) considered Hymenaea splendida var. splendida to be synonymous with Hymenaea courbaril var. stilbocarpa and segregated Hymenaea splendida var. longifolia as another variety of Hymenaea courbaril.

In addition to morphological distinctions, ecological and geographical information can be used for interpreting the three groups recovered by UPGMA and PCA as separately evolving lineages that would be considered different species under the USC framework (see Taxonomy section). With respect to the variation in Group 2, studies of wider samplings, including especially the collections made after Lee and Langenheim’s (1975) work, shows that the diagnostic features used for defining the varieties included in Group 2 have more complex variations. Indumented leaflets, for example, the diagnostic character for var. villosa, can be found to varying degrees in var. stilbocarpa. A short stipe was used as a diagnostic feature of var. subsessilis, but this trait is variable and grades into var. courbaril. Finally, the distinctions between vars. courbaril and stilbocarpa rely on the types and colors of the calyx lobe indumentation, traits that tend to change during the duration of the flower. Thus, a more parsimonious way to treat the taxonomy of Group 2 is to consider it a variable species, without recognizing varieties.