Comparative Wood Anatomy in Pinaceae with Reference to Its Systematic Position

: The wood anatomy of 132 species of the genera Abies, Cathaya, Cedrus, Keteleeria, Larix, Nothotsuga, Picea, Pinus, Pseudolarix, Pseudotsuga and Tsuga was studied to determine the elements that characterise the xylem of each genus and discuss possible groupings by wood anatomy for compari-son with clades established by molecular phylogeny. The presence of resin canals and ray tracheids supports the family Pinaceae, although the absence of ray tracheids in Keteleeria and their occasional presence in Abies and Pseudolarix weakens it. Based on wood structure, Pinaceae clearly supports division into two groups, coinciding with molecular phylogeny: Pinoideae ( Cathaya-Larix-Picea-Pinus-Pseudotsuga ) and Abietoideae ( Abies-Cedrus-Keteleeria-Nothotsuga-Pseudolarix-Tsuga ). Although differences between genera are slight in Pinoideae, the Abietoideae group presents problems such as the presence of only axial resin canals in Keteleeria and Nothotsuga , absence of ray tracheids in Keteleeria and presence of traumatic radial resin canals in Cedrus. However, other features such as pitted horizontal walls and nodular end walls of ray parenchyma cells, indentures, scarce marginal axial parenchyma and presence of crystals in ray parenchyma strengthen the Abietoideae group.

Using the comparative anatomy of the canals in leaves, bark and wood, Wu and Hu [4] subsequently divided Pinaceae into three groups: Cathaya, Larix, Picea, Pinus and Pseudotsuga, with normal resin canals; Abies, Cedrus, Pseudolarix and Tsuga without normal resin canals; and Keteleeria, with or without normal resin canals. Lin et al. [5], after confirming the constant presence of axial resin canals in mature wood of Keteleeria and the arrangement of traumatic resin canals in this genus, proposed a classification of Pinaceae in three groups, more in line with the wood anatomy of the family: Cathaya, Larix, Picea, Pinus and Pseudotsuga with radial and axial resin canals; Keteleeria and Nothotsuga with only axial resin canals; and Abies, Cedrus, Pseudolarix and Tsuga, without resin canals.
The wood structure of Pinaceae has been widely studied both at the genus and species level (e.g., [5][6][7][8][9][10][11][12][13][14][15]). Comparative anatomy studies of the wood of this family have dealt with the presence/absence of resin canals (both normal and traumatic) and their arrangement, and elements of the rays (horizontal and end walls of ray parenchyma

Results and Discussion
Summary of features by genus of Pinaceae based on the species studied is given in Table 1. Appendix B Table A1 shows the 132 taxa analysed with their particular features.

Resin Canals
The presence of resin canals in conifers is restricted to Pinaceae. Cathaya, Larix, Picea, Pinus and Pseudotsuga have both axial and radial resin canals ( Figure 1A,B). Keteleeria, according to Lin et al. [5], has only axial resin canals, which generally have associated subsidiary tissue ( Figure 1C). Normal axial resin canals have been recorded in Nothotsuga by other authors [11], although none were observed in the sample studied, probably because the wood was juvenile and this type of wood has very few resin canals. Abies, Cedrus, Pseudolarix and Tsuga do not have normal resin canals ( Figure 1D).
Although in Pinus only thin-walled epithelial cells were observed ( Figure 1E), resin canals in Cathaya, Keteleeria, Larix, Picea and Pseudotsuga had thick-walled epithelial cells ( Figure 1F). Jin-Xing et al. [11] also reported thick epithelial in Nothotsuga. Moreover, axial resin canals in Pinus were usually larger and more numerous than in the other taxa.
Traumatic axial resin canals were observed in all genera studied except in the samples of Pseudolarix and Nothotsuga, although they were cited by Wu and Hu [4] as a response to wounding. In all cases, the traumatic resin canals were arranged in tangential rows and their diameter was greater than in normal resin canals.     Although in Pinus only thin-walled epithelial cells were observed ( Figure 1E), resin canals in Cathaya, Keteleeria, Larix, Picea and Pseudotsuga had thick-walled epithelial cells ( Figure 1F). Jin-Xing et al. [11] also reported thick epithelial in Nothotsuga. Moreover, axial resin canals in Pinus were usually larger and more numerous than in the other taxa. Cathaya, Larix, Picea, Pinus and Pseudotsuga had both axial and radial traumatic resin canals ( Figure 1G,H). In Keteleeria and Nothotsuga traumatic resin canals were only axial and appeared randomly and in tangential rows ( Figure 1I), differentiating this genus from the others, in which traumatic resin canals were always arranged in tangential rows ( Figure 1J). Jin-Xing et al. [11] suggested that Keteleeria and Nothotsuga have a high level of affinity due to the presence of axial resin canals in latewood and resin cavities in the bark. Cedrus follows a different pattern from the other genera without normal resin canals, as it is the only genus capable of forming both traumatic axial ( Figure 1K) and radial ( Figure 1L) resin canals. Molecular phylogenetic studies have shown that of the six Abietoideae genera, only Cedrus is difficult to include in the clade [22].
The presence of normal resin canals, therefore, allows three groups to be established: Cathaya-Larix-Picea-Pinus-Pseudotsuga, with axial and radial resin canals; Keteleeria-Nothotsuga, with only axial resin canals; and Abies-Cedrus-Pseudolarix-Tsuga, without resin canals. The groups established by the presence of traumatic axial resin canals are: Cathaya-Larix-Picea-Pinus-Pseudotsuga, with both normal and traumatic axial and radial resin canals; Keteleeria-Nothotsuga, in which traumatic resin canals are only axial, in conjunction with normal resin canals, similarly only axial; Abies-Pseudolarix-Tsuga, with only traumatic axial resin canals and no normal resin canals; and Cedrus, with traumatic axial and radial resin canals but without normal resin canals.
Some authors explained the presence of ray tracheids associated with wounding [7,28], as occurs with the presence of traumatic resin canals, although Holden [26] reported the presence of traumatic axial canals and the absence of ray tracheids as a response to wounding in Keteleeria. This absence of ray tracheids in Keteleeria disagrees with the clade proposed in the molecular phylogenetic study by Wang et al. [29], who concluded that the clade Abies-Keteleeria-Pseudolarix-Tsuga was well resolved.
In Cedrus, especially in marginal rows, irregularly shaped cells have been observed ( Figure 2J), described by Chrysler [30] as degenerated cells, although they are actually irregularly shaped ray parenchyma cells.
Taking into account the presence of ray tracheids and their characteristics, a single group can, therefore, be established of the genera in which ray tracheids are always present Cathaya-Cedrus-Larix-Nothotsuga-Pinus-Picea-Pseudotsuga-Tsuga, in addition to Abies-Pseudolarix, where they are occasionally present. Keteleeria is the only genus remaining outside this group and its inclusion is, therefore, unresolved.
Pitted horizontal ( Figure 2L) and distinctly nodular ray parenchyma end walls ( Figure 2M) are present in all genera of Pinaceae, although some species of Pinus do not conform (see Appendix B).
Indentures were also found in all genera, although they presented different degrees of development. In Abies ( Figure 2N), they were well developed but in Cedrus, Keteleeria, Nothotsuga and Pinus, they were only slightly developed.

Axial Tracheids
One of the most significant features of the axial tracheids of Pinaceae is the presence of helical thickenings: in Cathaya well developed in early and latewood; in some species of Larix only in latewood tracheids (L. decidua, L. griffithii, L. occidentalis); in Picea spinulosa well developed in early and latewood; in Picea abies, P. brachytyla, P. glauca, P. glehnii, P. jezoensis, P. koraiensis, P. mariana, P. sitchensis, P. torano only in latewood; in Pseudotsuga sinensis in early and latewood and in P. menziesii only in latewood ( Figure 3A). The other genera of Pinaceae do not have helical thickenings in axial tracheids. Therefore, this can Cedrus had scalloped tori ( Figure 3B), a feature not observed in other Pinaceae, present in all samples studied [7,25]. Although Willebrand [31] described intermediate forms of scalloped tori in Pseudolarix, they were not found in the present study. Therefore, this feature does not support grouping of genera and, as with the presence of traumatic axial and radial resin canals, Cedrus seems to differ from the other genera.
When biseriate tracheid pitting was present in earlywood, it was in opposite arrangement in all genera ( Figure 3C) and very rarely in alternate arrangement.
Organic deposits in tracheids were found only in A. pinsapo, agreeing with Esteban et al. [32], Abies guatemalensis and Pinus canariensis ( Figure 3D), but should not be regarded as characteristic of the genus, as they are in Agathis and Araucaria [33,34]. Organic deposits in tracheids have also been cited in Picea sitchensis [10].
All the genera of Abietoideae studied presented tracheid pitting in tangential walls ( Figure 3E) in the vicinity of the growth ring, generally in the first rows of latewood (Figure 3F). Tracheid pitting was very scarce in the other genera. A warty layer was observed only in Abies, Cedrus, Nothotsuga and Tsuga ( Figure 3G), although it has also been recorded in Pinus [25]. Cedrus had scalloped tori ( Figure 3B), a feature not observed in other Pinaceae, present in all samples studied [7,25]. Although Willebrand [31] described intermediate forms of scalloped tori in Pseudolarix, they were not found in the present study. Therefore, this feature does not support grouping of genera and, as with the presence of traumatic axial and radial resin canals, Cedrus seems to differ from the other genera.
When biseriate tracheid pitting was present in earlywood, it was in opposite arrangement in all genera ( Figure 3C) and very rarely in alternate arrangement.
Organic deposits in tracheids were found only in A. pinsapo, agreeing with Esteban et al. [32], Abies guatemalensis and Pinus canariensis ( Figure 3D), but should not be regarded as characteristic of the genus, as they are in Agathis and Araucaria [33,34]. Organic deposits in tracheids have also been cited in Picea sitchensis [10].
All the genera of Abietoideae studied presented tracheid pitting in tangential walls ( Figure 3E) in the vicinity of the growth ring, generally in the first rows of latewood ( Figure 3F). Tracheid pitting was very scarce in the other genera. A warty layer was  Figure 3G), although it has also been recorded in Pinus [25].

Axial Parenchyma
Although very scarce, axial parenchyma was observed in all genera of Pinaceae except Pinus. It was limited to the growth ring boundary, both in earlywood and latewood ( Figure 3H). Transverse end walls were nodular in all genera studied ( Figure 3I). The presence of this feature supports a single group for all genera except Pinus.

Crystals
The presence of crystals is a frequent feature in all genera of Abietoideae [35] and crystals also occur in some non-Abietoideae taxa. In this work, crystals were observed in many taxa of Abies (see Appendix B) and are a feature of this genus [18], all species of Cedrus and Keteleeria, Nothotsuga longibracteata, Pinus brutia, P. cembroides, P. contorta, P. halepensis, P. heldreichii, P. monophylla, Pseudolarix amabilis and all species of Tsuga except T. dumosa. They are usually located in marginal and submarginal rows of the ray parenchyma and in irregularly shaped marginal ray cells ( Figure 3J).
The presence of crystals has even been used to differentiate very close species in Pinus [36,37]. Crystal inclusions have also been recorded in subsidiary tissue of resin canals, e.g., in Abies pinsapo [32]. In this study, they were observed only in traumatic resin canals of Abies.
These considerations indicate that the presence of crystals must be interpreted as a feature allowing the formation of a single group in Pinaceae.

Anatomy and Phylogeny
Irrespective of their typology and arrangement, resin canals allow a well-resolved group to be formed in Pinaceae as they occur in all genera, either normal and traumatic or only traumatic. Although some non-Pinaceae genera, specifically Cupressaceae, can have traumatic canals, their presence is occasional, as reported by Bailey and Faull [38] in Sequoia sempervirens, Jeffrey [39] in Sequoiadendron, Benkova and Schweingruber [40] in Microbiota and Esteban et al. [41] in Tetraclinis articulata.
The presence of ray tracheids also supports the grouping of the family Pinaceae. Although this feature is also found in non-Pinaceae conifers (Cupressus L., Sequoia Endl. and Thujopsis Siebold & Zucc.) [7], it occurs in these species very rarely. However, the absence of ray tracheids in Keteleeria and their occasional presence in Abies and Pseudolarix weakens the group.
Other features such as pitted horizontal walls and nodular end walls in ray parenchyma cells, cross-field pitting and indentures do not support the grouping, as they occur in non-Pinaceae families. These considerations are supported by phylogeny studies by Gugerli et al. [42], who used the second intron in the mitochondrial gene nad1 to show that two clearly distinct clades can be established within the conifers: Pinaceae and non-Pinaceae.
Based on wood anatomy, other subgroups can be established in the family Pinaceae that differ from the subfamilies proposed by Frankis [2] and Farjon [3] but agree with the classification by Lin et al. [5]. However, this classification differs from molecular phylogenetic studies by other authors.
Wang et al. [29] established the existence of two distinct clades within Pinaceae: Cathaya-Larix-Picea-Pinus-Pseudotsuga and Abies-Keteleeria-Nothotsuga-Pseudolarix-Tsuga. Gernandt et al. [22] confirmed this division by phylogenetic analysis of chloroplast DNA (matK and rbcL) and concluded that Pinaceae supports two clearly distinct monophyletic groups: Abietoideae and Pinoideae, corresponding with the general structure of the family in the two subfamilies.
Moreover, Wang et al. [29], in their study of intergeneric relations of Pinaceae using sequences of the chloroplast matK, mitochondrial nad5 and nuclear 4CL genes, established a three-genome phylogeny consisting of the clade Larix-Picea-Pinus-Pseudotsuga similar to the phenogram established by Hart [43] based on cladistic analysis and to the clade established by Price et al. [44] through immunological comparison. The four genera of this clade have normal resin canals, both axial and radial, as does Cathaya.
Although some authors consider that the only species of the genus Cathaya (C. argyrophylla) belongs to the genus Pseudotsuga [45][46][47], using PCR-RFLP analysis of the chloroplast gene trnK, concluded that Cathaya is a separate genus and must be treated outside of Pseudotsuga. The presence of axial and radial resin canals with thick-walled epithelial cells supports the clade Cathaya-Larix-Picea-Pseudotsuga, but excludes the genus Pinus, which has thin-walled epithelial cells.
Despite the obvious differences in the anatomy of the secondary xylem between Keteleeria and Nothotsuga and the other Abietoideae genera (presence of axial resin canals, arrangement of traumatic resin canals and absence of ray tracheids in Keteleeria), the two genera appear to form a well-resolved clade with Abies-Pseudolarix-Tsuga [47]. Liston et al. [21] also confirmed that Nothotsuga-Pseudolarix-Tsuga form a sister clade to Abies-Keteleeria, with only Cedrus remaining unresolved.
However, molecular evidence supports the genus Cedrus as a sister group to the other Pinaceae and reinforces the idea that it should occupy a basic or root position in the family, to the extent that the subfamily Abietoideae can be considered paraphyletic [22]. This coincides with the peculiar behaviour observed in two features of Cedrus: it is the only genus with scalloped tori and has traumatic radial resin canals even though it has no normal resin canals. These features, particularly the latter, constitute different behaviour in Cedrus from the clade Abies-Keteleeria-Nothotsuga-Pseudolarix-Tsuga, as these genera do not have tori or traumatic radial resin canals.
Despite these distinctive features of Cedrus, the genera in Abietoideae form a homogeneous group in terms of wood structure, except for the resin canals in Keteleeria and Nothotsuga and the absence of ray tracheids in Keteleeria, which supports the clade Abies-Cedrus-Keteleeria-Nothotsuga-Pseudolarix-Tsuga, as all of these species have ray parenchyma with pitted horizontal walls and nodular end walls, indentures, scarce axial parenchyma on the growth ring boundary and crystals in ray parenchyma [35].

Conclusions
Wood anatomy and molecular phylogeny agree that Pinaceae is clearly differentiated from the other conifers by the presence of two features in its wood structure exclusive to the family-resin canals and ray tracheids-which are only very rarely present as exceptions in non-Pinaceae conifers.
Although division into two groups (Pinoideae and Abietoideae) is clearly supported by molecular phylogeny, the subgroups within Abietoideae differ from molecular phylogenetic studies that include Keteleeria and Nothotsuga with no exceptions and exclude Cedrus. However, the exclusion of Cedrus agrees with the differences in its wood structure compared to the anatomy of other genera.        [11]; 3 reported by Wu and Hu [4].