Morphology of Juniperus Cone and Its Implications on Cone Evolution


 Background

The basic cone unit in Pinaceae is called bract-scale-seed complex (BSSC), in which the scale is supposed to be equivalent to an axillary shoot bearing ovules in Cordaitales. This correlation established by Florin provides a rational foundation on which an interpretation for the origin of cones in at least most Coniferales is built, and may be called Florin model for convenience. Cupressaceae is a family in Coniferales, in which the ovule-scale and its subtending bract are thought fully fused and hard to distinguish by external morphology.
Results

Different from Pinaceae and other typical conifers, Juniperus (Cupressaceae) appears not following Florin’s model closely. For example, the cone of Juniperus oxycedrus has only three rather than more BSSCs in a whorl, and its fleshy fructification appears more like a berry rather than a typical coniferalean cone. In this paper morphology and anatomy of Juniperus oxycedrus fructifications are documented using Micro CT. New observation demonstrates clearly that three seeds alternate the three surrounding bracts in J. oxycedrus.
Conclusions

Such spatial arrangement is quite different from that in typical BSSCs, in which the ovules should be aligned with their subtending bract. Together with other unexpected features in other cupressaceous cones, Juniperus my help to expand the avenue through which we can interpret the origin and homology of cones in Cupressaceae and other conifers or gymnosperms in general.

cone. In this paper morphology and anatomy of Juniperus oxycedrus fructi cations are documented using Micro CT. New observation demonstrates clearly that three seeds alternate the three surrounding bracts in J. oxycedrus.

Conclusions
Such spatial arrangement is quite different from that in typical BSSCs, in which the ovules should be aligned with their subtending bract. Together with other unexpected features in other cupressaceous cones, Juniperus my help to expand the avenue through which we can interpret the origin and homology of cones in Cupressaceae and other conifers or gymnosperms in general.

Background
Cones are reproductive organs characteristic of gymnosperms. They are frequently seen in Coniferales, Bennettitales, Pentoxyales, Gnetales, and Cycadales (Biswas and Johri 1997). Since 1930s the research of Florin (Florin 1939(Florin , 1951 and ensuing botanists (Schweitzer 1963, Rothwell 1982, Rothwell et al. 1997 appear to have resolved the homology of cones in Coniferales, in which a basic unit of a typical cone is called bract-scale-seed complexes (BSSC) and interpreted as derived from a secondary fertile shoot situated in the axil of its subtending bract. The Florin model appears to be very successful and can account for many evolutionary events related with Coniferales. However, persisting problems remain unsolved. For example, the spatial relationship between the assumed scale and bract is hard to decipher in some Cupressaceae, and the homology between coniferous cones and those in Cycadales, Pentoxyales, and Bennettitales remains obscure, leaving the evolutionary relationship among these taxa open to debate. At least some of the cones in Cupressaceae appear incompatible with the Florin model of cones although the latter appears to have resolved many problems of evolution in other Coniferales. It is interesting that, although cupressaceous cones are frequently investigated Tomlinson 1989, Zhang et al. 2000), the consistent inconsistency between these cones and Florin model is frequently down-played. Considering these all, investigating and thus understanding cupressaceous cones is necessary and crucial and has the potential to shed otherwise unavailable light on the evolution of coniferous cones.
Here we document the morphology and anatomy of the cones of Juniperus using Micro CT, a new technology available recently. Through the application of this technology, the morphology and organization of cupressaceous cone is more clearly visualized and demonstrated. The goal of this research is to call for decent attention to this non-Florin cone organization in Cupressaceae, discuss its potential for complementing the Florin model and prompt an integrated evolutionary map for gymnosperms based on cone morphology.

Materials And Methods
The material was collected from a tree of Juniperus oxycedrus macrocarpa (Silbth. & Sm.) in the Jardí Botànic de Sóller (IPEN ES-0-SOLLE-160019), Mallorca, Spain in June, 2016. The sample was photographed using a Sony ILCE-7 digital camera, scanned using a Bruker SkyScan1172 at the Institute of Botany, Chinese Academy of Sciences (CAS), Beijing, China. The machine-generated data was used to re-generate 3D image using a VG Studio and nal result was output as videos and pictures. All gures were organized as gures using a Photoshop 7.0 for publication.
It is frequently said that the scale and bract are fused each other in the Cupressaceae. We cannot con rm or deny this statement. In case of the material studied here, we would not use the word "scale" but only use "bract" in the description because 1) the scales, if present, should be closely related with seeds, 2) we saw nothing other than bracts closely associated with the seeds, 3) we cannot see any trace of scales distinguishable from the bracts anatomically and morphologically, 4) a scale, if present, should be aligned with the seed rather than a bract. The readers are welcome to interpret otherwise, but we do not think that this would in uence the validity of my following discussion and conclusion.

Results
The cones of Juniperus oxycedrus macrocarpa were collected when they were mature (Figs. 1a-c). The fructi cations appeared eshy and baccate due to the presence of three seeds inside the fructi cation (Figs. 1a-c). The fructi cations were about 19 mm long and 15 mm in diameter (Figs. 1a-c). Some longitudinal veins were seen in the eshy bracts that surround the seeds inside (Fig. 1b). The apices of the fructi cations were triangular in shape (Fig. 1c), suggestive of the tripartite organization of the cone. Three radiating sutures were seen on the cone apex (Fig. 1c), implying that positioning of former bracts.
The three sutures were also seen in Micro CT slices (

Discussions
According to the well-accepted Florin model of Cordaitales-Coniferales evolution (Florin 1949, 1951, Schweitzer 1963), BSSC in a typical cone should comprise a subtending bract and a secondary fertile shoot in its axil. This spatial relationship is easy to understand and accept considering axillary branching is almost ubiquitous in seed plants (except Cycadales), in which a branch is always in the axil of a subtending leaf (Eames 1961, Esau 1977 Part of the reason of the current academic situation is due to the shortcoming of traditional documenting technique, which cannot demonstrate the morphology and anatomy of cones to botanists and general public. Although para n section has contributed much to our understanding of plant anatomy in the past decades, its two dimensional presentation of three dimensional morphology and anatomy requires extra effort and education to correctly conceive the three dimensional relationship among different parts in an organ of interest. Newly developed technologies enable us to visualize and demonstrate the anatomy of plant organs in an easy-conceive way. Micro CT is one of such new technologies. Its application leaves no space for anyone to ignore the anatomical fact in cupressaceous cones. This is the reason we try to apply Micro CT to call for attention to the long-ignored fact about cupressaceous cones. According to the Florin model for coniferous cones, each basic unit of coniferous cone (BSSC) comprises a subtending bract and a secondary fertile shoot in its axil. This interpretation implies that a secondary shoot and its subtending bract are aligned in the same radius, namely, the secondary fertile shoot opposite the corresponding bract. This implication is well con rmed in most Coniferales, including Pinaceae, Araucariaceae, and Taxodiaceae. This explains the success and wide acceptance of the Florin model. If this implication were not con rmed in the reality, the fate of the Florin model would be quite different. It is interesting that the alignment between ovules and bracts expected by Florin model is not seen in at least above mentioned taxa in Cupressaceae. The existence of such consistent discrepancy between the Florin model and botanical observation undermines the validity of Florin model, or at least reduces the applicable scope of the model.
As documented here, the ovules consistently alternating the bracts in Juniperus are of importance in that they cast doubt over the validity of the Florin model in the Cupressaceae, and, more importantly, prompt new more widely applicable interpretations, at least including the Cupressaceae. Thus a new interpretation for cones valid in wider scope is needed in botany. It becomes more interesting, as you see below, that all cones in gymnosperms may be derived from a single common ancestor, and the Florin model may well be a specialization of a more general model applicable for more gymnosperms.
One of the important studies on plant organ evolution was performed about twenty years by Crane and Kenrick. After careful study of living and fossil plants, Crane and Kenrick came to a hypothesis that the variety of organs seen in living and fossil plants is a result of long time diverted development of reproductive organs throughout the geological history (Crane and Kenrick 1997). In their paper, their hypothesis is exempli ed by the provenance of microphylls in lycopsids and interseminal scales in Bennettitalean cones. Actually, the derivation of integument can also be taken as a result of diverted development, as suggested by Benson (Benson 1904) and favored by laters (Walton 1953, Andrews 1963, Taylor et al. 2009). The earliest reproductive organs in land plants are aggregates of sporangia, micro-or mega-, borne on shoot terminals, as seen in the earliest land plants (Taylor et al. 2009). It is conceivable that each of the ancestral female cones may comprise an axis and clusters of ovules (megasporoclads) helically arranged along its sides. This situation may be exempli ed by the lax cone of Cycas, in which clusters of ovules are helically arranged around the shoot apex. If each of the megasporoclads in Cycas is reduced into an ovule, a cone with helically arranged ovules/seeds around its axis, just as seen in Pentoxyales, may come into existence. The diverted development (sterilization) of these lateral appendages (ovules), as seen in Pentoxylalean cones, may produce interseminal scales, which surround and protect their fertile peers (ovules) in Bennettitales. The hybridization between axillary branching and the cones of Pentoxyales, turning sterilized ovules into subtending bracts, may give rise to cones seen in Cordaitales and typical Coniferales. Further modi cation of the secondary fertile shoot may make it a shoot terminating in an ovule with a micropylar tube, as seen in Gnetales. Lacking involvement of axillary branching, sterilization of some ovules into bracts in Pentoxylalean cones might produce the cone con guration documented here for Cupressaceae, namely, some of the former ovules may be sterilized and function as protecting bracts. When clusters of ovules (rather than single ovule) are retained, clusters of ovules dispersed between bracts as seen in Widdringtonia, Juniperus oxycedrus, J. communis, J. brevifolia, Libocedrus plumosa, L. bidwillii, Tetraclinis, Cupressus macnabiana, C. guadalupensis, and C. arizonica become something easy to conceive. When all except the terminal ovule(s) is retained and all others are sterilized into protecting surrounding and subtending bracts, the situation in Callitris rhomboidea (Takaso and Tomlinson 1989), Juniperus tibetica, J. squamata, J. satuaria, J. recurva, J. przewalskii, J. pingii, J. indica, J. angosturana (Farjon 2005), and Platycladus orientalis (Zhang et al. 2000) may come into existence. The signi cance of this interpretation lies in that it removes the former implicit requirement on spatial relationship between ovules and bracts, namely, the aligned arrangement of ovules and bracts in gymnospermous cones required by the Florin model becomes surplus and unnecessary. Ovules and bracts become independent each other and they have the freedom to combine and coalescence anyway in the new interpretation. Such a great freedom of combination among plant parts makes the great variety of gymnospermous cones easy to appreciate. Thus this lift of unnecessary restriction on spatial relationship between ovule and bract makes a common Bau-plan for gymnospermous cones within reach. If this is the case, botanists will not have to give ad hoc interpretations for various cones and will not have to play ostrich ignoring botanical facts any more. It may well be that the Florin model is a specialization of universal model that is applicable for all gymnospermous cones. If this is true, drawing a conceivable and rational evolutionary roadmap for gymnosperms will be a mission possible for botanists in the near future.

Conclusions
The most intriguing feature of Juniperus oxycedrus macrocarpa is the presence of angiospermy in it.

Competing interests
The authors declare that they have no competing interests. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript,

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