1. Introduction
Infrafamiliar classification of Burseraceae historically has been defined primarily by fruit characters, especially the degree of connation of the pyrenes (a pyrene is a seed with bony endocarp) in the compound drupe. Engler’s two tribal classifications of Burseraceae [
1,
2] used the configuration of the pyrenes as the only diagnostic character, and until recently, this has been the traditional classification of Burseraceae [
3]. Although recent molecular phylogenetic reconstructions are challenging the recognized infrafamiliar classification of the family [
4,
5], there is not yet a consensus from DNA data or additional evidence regarding synapomorphies from anatomy or morphology. Thus, current Burseraceae tribes are these: Protieae, with fruits dehiscent and pyrenes free or connivent but separable, the mesocarp and pericarp usually carnose or sometimes dry (some
Protium); Boswellieae (=the autonym Bursereae), with fruits dehiscent consisting of pyrenes more or less connate but usually detachable from each other or separating when mature, the mesocarp and pericarp dry and dehiscent; and Canarieae, with fruits indehiscent and pyrenes more or less and often entirely connate, always forming a plurilocular putamen, pericarp carnose, rarely more or less dry.
Despite the importance of fruit structure in Burseraceae, there are few references in the literature regarding fruit anatomy. In general, literature on Burseraceae anatomy is scant [
3]. In the tribe Canarieae, the most important of those references is Lam’s description of a three-winged bony axial “intrusion” in the fruit of
Canarium [
6], a characteristic Lam considered important enough to divide Canarieae into two subtribes based on the presence or absence of the intrusion. Another contribution to fruit structure in Canarieae was Cuatrecasas’s work on Neotropical
Dacryodes, where he described “carpelar sutures” in the fruit [
7] and an endocarp with “only one seminiferous cavity and one tiny, appressed sterile cell” [
8]. The sterile cell has been used as part of the description of a New World species of
Dacryodes as an articulated plate [
9].
The present study documents fruit anatomy in tribe Canarieae, with the goal of a better understanding of what has been vaguely defined for a long time as a “degree of connation of the pyrenes”, which traditionally has formed the basis for Burseraceae infrafamilial classification. Understanding Burseraceae fruit anatomy will help elucidate the generic limits and phylogenetic relations in this tribe, as well as relations among Burseraceae tribes. To accomplish this, first it is important to review some definitions.
Burseraceae fruits have been considered as drupes [
8,
10], “more or less drupaceous” [
11], or “variations on the drupe” [
3]. According to Spjut [
12], the Burseraceae have two fruit types, drupes and nuculania; in his system, a drupe is composed of a fleshy pericarp, with generally one to five stones (a stone is defined as a hard shell that encloses one or more seeds) whereas a nuculanium is a simple fruit with a dry pericarp and a hard endocarp, of one or more stones, the external layer(s) crustaceous or fibrous or coriaceous, indehiscent (
Garuga) or dehiscent (
Boswellia). To this last category belong the fruits of the subtribe Boswelliinae Daly, related to Canarieae in molecular studies [
5,
13]. These definitions correspond to those of Roth [
14], in which drupes comprise a highly developed fruit type characterized as an indehiscent sarco-sclerocarpium, “where the pericarp reaches its classical subdivision into exo-, meso- and endocarp”. For this study, tissues were distinguished and classified based on the previous definitions of Spjut and Roth: “The exocarp is formed by the outer epidermis, but subepidermal layers may also be included; the mesocarp, generally composed of parenchyma, consists of large cells which may possibly be elongated radially. The endocarp is hard and consists either of sclereids or of fibers or of a mixture of both”.
3. Discussion
The availability of material on this study permits the description of the tissues observed, their relative position, and particular characteristics, but not the tissue origin and/or the developmental biology of the fruit components.
Fruits of Paleotropical
Canarium are large and ellipsoid, oblong or ovoid; the pericarp is carnose and very resinous [
6], with an endocarp generally described as thick and bony [
15] or woody [
16]. The endocarp was described by Lam [
6] as composed of pyrenes separated by a three-winged axial intrusion and covered by mesocarpal lids that open when the seeds are germinating. However, Ng reported that the endocarp of
Canarium is fused to part of the stony inner layer of the mesocarp to form a thick-walled pyrene [
17]. Similarly, Hill described the germination of
C. schweinfurthii Engl., in which the ‘lid’ of a locule is present with a window-like fenestra that the embryo pushes and opens when germinating [
18]. This last structure is more commonly named an operculum, and occurs in Burseraceae’s sister family, the Anacardiaceae, especially in the tribe Spondioideae [
19].
Trattinnickia is a Neotropical group from humid forests of Central and South America, transferred from Protieae to Canarieae by Daly [
20]. This genus is characterized by its hard bony endocarp and recent molecular studies reaffirm
Trattinninckia as forming a strong supported clade with Neotropical
Dacryodes [
21].
Dacryodes is a genus that occurs in the Caribbean, Central and South America, Southeast Asia, and Africa. The presence of sutures along the carpels of
Dacryodes has not been investigated anatomically but it has been mentioned in the literature as carpellar sutures or appressed sterile cells by Cuatrecasas [
7,
8] or as an articulated plate [
9]. The later definitions refer to the structure composed of the carpels whose locules are aborted and become compressed and reduced.
The fruit of the rare monotypic genus from Central Tropical Africa
Pseudodacryodes leonardiana has two locules, of which one develops a seed. The aborted locule, however, is not reduced as dramatically as
Dacryodes and most of the other genera of Canarieae. The fruit of
Pseudodacryodes is described as thin, not bony, with resinous pericarp and an eccentric intrusion separating the two locules [
22].
As currently circumscribed,
Santiria is a Paleotropical genus from the Western Malaysian region, the Philippines, the Moluccas, New Guinea, and Africa.
Santiria fruits are similar in structure to those of
Dacryodes: there are two sutures that evidence the presence of an articulated plate (which develops from the two undeveloped carpels), but the difference is that in
Santiria, the articulated plate is oriented towards the base of the fruit, the result being that the stigma is distinctly excentric in the fruit [
6].
Rosselia bracteata Forman is a recently described monotypic genus from New Guinea. Rosselia fruits have a coriaceous pericarp, with fibers running longitudinally, and the pyrene is similar to those of Dacryodes and Santiria, due to the presence of articulated plates; the ones in Rosselia are similar in shape and position to those of Santiria, but they are less fused to the developed locule, and the apices of the carpels remain free.
Haplolobus is a Paleotropical genus from Eastern Malaysia. The fruit of
Haplolobus has been considered dry by some authors [
23] or more or less dry [
6] because of its membranaceous pericarp and really thin mecocarp. However, in
Haplolobus, the articulated plate differs from that present in
Dacryodes,
Santiria, and
Rosselia in that the former is not detachable from the fruit, and the two sutures are somewhat imbricate while in the latter genera, the articulated plate is detachable by hand and does not overlap.
Scutinanthe is a small genus with two species in Malaysia. It is the only genus in Canarieae with pentamerous flowers. The fruits have a fleshy pericarp, and the mesocarp has long radially arranged palisade cells; the pyrene is hard and bony, usually with two locules, one strongly reduced [
10]. However, in
Haplolobus, the articulated plate differs from that present in
Dacryodes,
Santiria, and
Rosselia in that the former is not detachable from the fruit, and the two sutures are somewhat imbricate while in the latter genera, the articulated plate is detachable by hand and does not overlap.
Garuga is a small genus with four species in India and Southeast Asia. It currently remains in an unnamed subtribe in the Bursereae [
24]. The fruit has one to five seeds, a carnose pericarp, and one to five small pyrenes that are gibbous, bony, thick, and resinous (Lam 1932a).
The endocarp is the most distinct part of the drupe [
14] and it is indeed the most variable anatomical structure in Canarieae fruits. A study of Anacardiaceae fruits by Wannan and Quinn describes the Spondias-type, “which is composed of a mass of usually strongly lignified and irregularly oriented sclerenchyma” [
19]. The authors also conclude that “the occurrence of valves or opercula that are dislodged at germination, appears to be restricted to this type of endocarp” but recognize this same fruit type in Canarium, suggesting this character is plesiomorphic.
Observations of the exterior of the pyrenes and cross-sections in this study show two different fruit morphologies (
Table 1). The first group, composed of
Canarium and
Trattinnickia, has a very thick and bony endocarp, and the sterile locules are asymmetric, but the carpels are not compressed (that is, the abortion of locules does not affect the symmetry of the pyrene).
Canarium differs from
Trattinnickia by the presence of an intercarpellar tissue (Lam’s axial intrusion). Overall, there is a close congruence of fruit characters between
Trattinnickia and
Canarium, including the presence of very hard endocarps and a similar distribution of epicarp and mesocarp. Additionally, some species of
Canarium and all
Trattinnickia (but also
Garuga) have a corrugate endocarp. The second fruit type is represented by
Santiria,
Dacryodes,
Rosselia, and
Haplolobus, sharing the presence of an articulated plate, compressed and displaced abortive carpels, and a smooth endocarp.
Scutinanthe exhibits an intermediate fruit type, with fused bony pyrenes, no axial intrusion, and no evidence of a separable articulated plate, although the aborted locules are compressed and displaced. Finally, the placement of
Pseudodacryodes is intriguing as well, being the only fruit with a cartilaginous endocarp. Germination of indehiscent fruits or with delayed indehiscence is explained by Roth: “many of the so-called indehiscent fruits open “passively” by the withering of the pericarp or by the pressure developed from the germinating embryo” [
14].
The tissues of the fruits within the Canarieae tend to repeat certain patterns, with some modifications in the thickness of the different layers and their degree of lignification. The epidermis is conspicuously lignified in Dacryodes and Haplolobus but without evident lignification in the other genera. There is a very evident cuticle in Pseudodacryodes, Rosselia, Santiria, and Trattinnickia. The epidermis of all genera studied except Scutinanthe is accompanied by a resinous hypodermal layer, composed of smaller and densely distributed parenchyma cells with a resin-like content.
The mesocarp was defined in this study as the parenchymatic layer with vascular bundles, located between the epidermis and the endocarp. All taxa studied (except Scutinanthe) have parenchymatous cells with a resin-like content, and vascular bundles associated with secretory canals distributed through the mesocarp. In general, the mesocarp is the thickest layer in the fruit, the reduced mesocarp of Haplolobus being the only exception.
The distribution of endocarp tissues in Canarieae and
Garuga is similar and consists of a parenchymatous layer outside a lignified layer (no evidence of lignification was found in
Pseudodacryodes). This parenchymatous layer stained differentially in
Dacryodes, and it was very rich in cells with a resin-like content in
Pseudodacryodes. An extra innermost layer of thin parenchyma, staining black, was observed in
Pseudodacryodes and
Santiria, corresponding to what some authors interpreted as an inner epidermis [
14]. Additionally, the descriptions presented here agreed with those made by Wannan and Quinn for the fruit of
Canarium oleosum (Lam.) Engl. [
19].
This study, based on herbarium specimens, revealed some promising characters for the understanding of fruit evolution in this group, but fresh material is needed to perform more complete analyses, including histological series with a broader representation of species for each genus, in order to document the developmental biology of the endocarp.
This study used fruit samples from herbarium specimens with available material for all eight genera of traditionally defined Canarieae (
Canarium,
Dacryodes,
Haplolobus,
Pseudodacryodes,
Rosselia,
Santiria,
Scutinanthe, and
Trattinnickia).
Garuga, indehiscent but with free pyrenes, was originally placed in Protieae but was informally placed in Bursereae [
15]; this has been supported by molecular studies [
13,
24]. For each genus, few mature fruits were available except for Neotropical
Dacryodes and
Trattinnickia.