Wettable and unsinkable: the hydrodynamics of saccate pollen grains in relation to the pollination mechanism in the two New Zealand species of Prumnopitys Phil. (Podocarpaceae)

The pollination mechanism of most genera of the Podocarpaceae involves inverted ovules, a pollination drop and bisaccate pollen grains. Saccate grains have sometimes been referred to as 'non-wettable' due to their buoyant properties, while non-saccate pollen grains have been described as 'wettable'. The hydrodynamic properties of saccate pollen grains of seven podocarp species in five genera, Dacrydium Sol. ex G. Forst., Dacrycarpus (Endl.) de Laub., Manoao Molloy, Podocarpus L'Hér. ex Pers. and Prumnopitys Phil. have been tested in water, together with saccate and non-saccate pollen of four other conifer genera, Cedrus Trew (Pinaceae), Cephalotaxus Siebold & Zucc. ex Endl. (Cephalotaxaceae), Cupressus L. (Cupressaceae) and Phyllocladus Rich. ex Mirb. (Phyllocladaceae), and spores of three fern species and one lycopod species. All four spore types studied were non-wettable, whereas the bisaccate and trisaccate pollen types, like all other conifer pollen types, were wettable, enabling the grains to cross the surface tension barrier of water. Once past this barrier, grain behaviour was governed by presence or absence of sacci. Non-saccate and vestigially saccate grains sank, whereas saccate grains behaved like air bubbles, floating up to the highest point. In addition, the grains were observed to float in water with sacci uppermost, consistent with the suggestion that distally placed sacci serve to orientate the germinal furrow of the pollen grain towards the nucellus of an inverted ovule. Observations of pollen grains in the pollen chambers of naturally pollinated Prumnopitvs ovules confirmed this. The combination of buoyancy and wettability in saccate pollen has implications for the efficiency of the typical podocarp pollination mechanism.     

Comparative Biology of the Pollination Mechanisms in Acmopyle pancheri and Phyllocladus hypophyllus(Podocarpaceae s. l.)

The pollination mechanisms of Acmopyle pancheri(Brongn. &Gris) Pilg. andPhyllocladus hypophyllus Hook.f. were investigatedby conventional microscopical techniques and by nuclear magneticresonance (NMR) imaging. Dissimilarities include the orientationof the ovule and type of pollen;Phyllocladus has erect ovulesand wettable pollen with vestigial sacci, whereas Acmopyle hasmore-or-less erect ovules and non-wettable, functionally saccatepollen. Similarities include the mode of formation of the pollinationdrop and its response upon pollination. In both genera, pollinationtriggers pollination drop retraction and drop secretion ceases.Neither NMR imaging nor conventional histology of Phyllocladusovules revealed any specific tissue beneath the ovule whichcould be responsible for pollination drop retraction. It ismore likely, therefore, that the drop is channelled into thevascular supply or the apoplast. These findings invalidate thetaxonomic value of the pollination mechanism as a suite of characterstraditionally used to separate Phyllocladaceae from Podocarpaceae.Copyright 2000 Annals of Botany Company Acmopyle pancheri, gymnosperms, NMR imaging, nuclear magnetic resonance imaging, Phyllocladaceae,Phyllocladus hypophyllus , Podocarpaceae, pollination drop, pollination mechanism     

POLLINATION DROP IN RELATION TO CONE MORPHOLOGY IN PODOCARPACEAE: A NOVEL REPRODUCTIVE MECHANISM

Observation of ovulate cones at the time of pollination in the southern coniferous family Podocarpaceae demonstrates a distinctive method of pollen capture, involving an extended pollination drop. Ovules in all genera of the family are orthotropous and single within the axil of each fertile bract. In Microstrobus and Phyllocladus ovules are erect (i.e., the micropyle directed away from the cone axis) and are not associated with an ovule-supporting structure (epimatium). Pollen in these two genera must land directly on the pollination drop in the way usual for gymnosperms, as observed in Phyllocladus. In all other genera, the ovule is inverted (i.e., the micropyle is directed toward the cone axis) and supported by a specialized ovule-supporting structure (epimatium). In Saxegothaea there is no pollination drop and gametes are delivered to the ovule by pollen tube growth. Pollination drops were observed in seven of the remaining genera. In these genera the drop extends over the adjacent bract surface or cone axis and can retain pollen that has arrived prior to drop secretion (“pollen scavenging”). The pollen floats upward into the micropylar cavity. The configuration of the cone in other genera in which a pollination drop has not yet been observed directly suggests that pollen scavenging is general within the family and may increase pollination efficiency by extending pollination in space and time. Increased pollination efficiency may relate to the reduction of ovule number in each cone, often to one in many genera, a derived condition. A biological perspective suggests that animal dispersal of large seeds may be the ultimate adaptive driving force that has generated the need for greater pollination efficiency.     

Ovulate cone,pollination drop,and pollen capture in Sequoiadendron (Taxodiaceae)

In Sequoiadendron ovules are borne inside the ovulate cone, and pollination drops secreted from these ovules collect pollen. We examined: (1) the relation between ovular position and pollen capture; (2) pollen behavior when in contact with a pollination drop; and (3) ultrastructure of ovules during pollination drop secretion. During wet periods a water sheet forms on the surface of the cone due to bract shape and wettability. Pollination drops persist inside the wetted cone, and pollen capture resumes immediately after drying. Pollen landing on a pollination drop is taken inside the drop and carried into the micropyle when the drop contracts. Several notable ultrastructural features appear in the nucellus, integument, chalaza, and bract lamina during pollination-drop secretion. The abaxial surface of the lamina is covered by a membrane that may contribute to the wettable nature of the surface.     

Pollination of Picea orientalis (Pinaceae): saccus morphology governs pollen

Sacci of conifer pollen do not function primarily to increase the efficiency of wind pollination as is widely thought. Rather, they are bladders and cause pollen to float upwards in a liquid drop into the ovules. This observation is seemingly unsupported in the case of oriental spruce (Picea orientalis (L.) Link), which has saccate pollen. Ovulate cones are pendant at the time of pollination, which requires that pollen sink into the ovules. Pollen of oriental spruce floats at first but within 1-2 min sinks into the ovule. As sinking does not occur in saccate pollen of other Pinaceae, a variety of techniques was used to determine anatomical differences leading to this uncharacteristic tendency. Light, scanning electron, and confocal microscopy of the pollen surface yielded no significant appearing difference between pollen of oriental spruce and white spruce. However, transmission electron microscopy of freeze-fixed/freeze-substituted hydrated pollen revealed that the ektexine of oriental spruce pollen sacci is porous compared to that of white spruce. Confocal microscopy allowed examination of pollen hydration dynamics. Water enters pollen at the distal pole between sacci, and resulting rapid expansion of the tube cell forces air out of the saccate space. White spruce pollen remains buoyant because of enclosed air pockets in the saccus ektexine. Evolutionary change in pollen wall anatomy with resultant loss of saccus function is correlated with a change in ovulate strobilus orientation at pollination in oriental spruce. A suite of characters interact in the conifer pollination mechanism, and concerted change in these characters may lead to speciation.     

侧柏和北美香柏(柏科)的传粉机制(英)

观察了侧柏（Phaycladusorientalis（L．）Franco）和北美香柏（ThujaoccidentalisL．）散粉后花粉进入珠孔的过程。在散粉期,这两种植物的胚珠均分泌出传粉滴。当花粉落到传粉滴上后,引起传粉滴表面的形状发生改变或减弱胚珠的继续分泌,使得该传粉滴蒸腾加快,导致其比未授粉的传粉滴明显收缩。观察结果表明：不同植物的花粉导致侧柏传粉滴的收缩速率不尽相同。其中,与侧柏亲缘关系较近的植物花粉引起传粉滴的收缩速率和侧柏自身花粉引起的传粉滴收缩速率相似;反之,收缩速率变慢。侧柏传粉滴的收缩可能主要是由于花粉减弱胚珠分泌的结果。     

Pollination drop in Juniperus communis: response to deposited material

BACKGROUND AND AIMS: The pollination drop is a liquid secretion produced by the ovule and exposed outside the micropyle. In many gymnosperms, pollen lands on the surface of the pollination drop, rehydrates and enters the ovule as the drop retracts. The objective of this work was to study the formation of the pollination drop in Juniperus communis, its carbohydrate composition and the response to deposition of conspecific pollen, foreign pollen and other particulate material, in an attempt to clarify the mechanism of pollination drop retraction. METHODS: Branches with female cones close to pollination drop secretion were collected. On the first day of pollination drop exposure, an eyelash mounted on a wooden stick with paraffin was used to collect pollen or silica gel particles, which were then deposited by contact with the drop. Volume changes in pollination drops were measured by using a stereomicroscope with a micrometer eyepiece 3 h after deposition. The volume of non-pollinated control drops was also recorded. On the first day of secretion, drops were also collected for sugar analysis by high-performance liquid chromatography. KEY RESULTS: The pollination drop persisted for about 12 d if not pollinated, and formed again after removal for up to four consecutive days. After pollination with viable conspecific pollen, the drop retracted quickly and did not form again. Partial withdrawal occurred after deposition of other biological and non-biological material. Fructose was the dominant sugar; glucose was also present but at a much lower percentage. CONCLUSIONS: Sugar analysis confirmed the general trend of fructose dominance in gymnosperm pollination drops. Complete pollination drop withdrawal appears to be triggered by a biochemical mechanism resulting from interaction between pollen and drop constituents. The results of particle deposition suggest the existence of a non-specific, particle-size-dependent mechanism that induces partial pollination drop withdrawal. These results suggest that the non-specific response may decrease the probability of pollen landing on the drop, reducing pollination efficiency.     

Abiotic pollen and pollination: Ecological, functional, and evolutionary perspectives

The transport and capture of pollen in ~20% of all angiosperm families occurs in air and water. In other words, pollination is abiotic and occurs via the fluid media, not an animal vector. Whereas some early concepts considered abiotic pollination to be largely a stochastic phenomenon, there is sufficient evidence to indicate that wind pollination (i.e. anemophily) and water pollination (i.e. hydrophily) have deterministic features and are sophisticated fluid dynamic solutions to the problem of pollen release, dispersal, and capture.An abiotic pollination syndrome is defined in which there is spatial or temporal separation of carpellate and staminate flowers, which are drab, a reduction in perianth parts, stigmas and anthers are exposed to the fluid, and typically unclumped pollen may be produced in large amounts. Separate pollination syndromes are defined for anemophilous (i.e. wind-pollinated), ephydrophilous (i.e. surface-pollinated), and hydrophilous (i.e. submarine-pollinated) plants. Distinctions are based on habitat and physical conditions for pollination, pollen size, shape, and ultrastructure, morphology and ultrastructure of stigmas, and outcrossing rates. For example, anemophilous pollen are spherical and small, ephydrophilous pollen are spherical or reniform and large, while hydrophilous pollen are filiform (i.e. filamentous) or functionally filiform. The pollination mechanisms and mechanics associated with these syndromes reveals a strong evolutionary relationship between plant morphology and fluid dynamics.     

Orientation and withdrawal of pollination drops in Cupressaceae s. l. (Coniferales)

Pollination in the Cupressaceae is studied ex situ, focused on orientation and withdrawal of pollination drops. Orientation of pollination drops is a constant feature in most taxa studied and important for pollen capture. Conspecific pollen causes a withdrawal of pollination drops, varying in time among species from 8 to 24 min, but with little variation within species. Pollination drops of each tested Cupressaceae taxon are also withdrawn when pollinated with foreign, but Cupressaceous pollen. However, they remain unchanged and are not withdrawn immediately when pollinated with pollen of other seed plants. The results clearly indicate that the time for the total withdrawal of pollination drops is strongly influenced by the evolutionary distance of the taxa being involved in the pollination process. Among closely related taxa the withdrawal is much more rapid than in distantly related ones. This points to an effective recognition system regulating the withdrawal of pollination drops, probably controlled by the nucellus. This recognition system can be regarded as an important preadaption for the evolution of a self-incompatibility mechanism. The withdrawal of pollination drops is thus not exclusively a physically induced process as suggested in some earlier studies. Pollination drops of several ovules can fuse to form a large common one, perhaps increasing by this way successful pollen capture.     

Nectar and pollination drops: how different are they?

Background

Pollination drops and nectars (floral nectars) are secretions related to plant reproduction. The pollination drop is the landing site for the majority of gymnosperm pollen, whereas nectar of angiosperm flowers represents a common nutritional resource for a large variety of pollinators. Extrafloral nectars also are known from all vascular plants, although among the gymnosperms they are restricted to the Gnetales. Extrafloral nectars are not generally involved in reproduction but serve as ‘reward’ for ants defending plants against herbivores (indirect defence).

Scope

Although very different in their task, nectars and pollination drops share some features, e.g. basic chemical composition and eventual consumption by animals. This has led some authors to call these secretions collectively nectar. Modern techniques that permit chemical analysis and protein characterization have very recently added important information about these sugary secretions that appear to be much more than a ‘reward’ for pollinating (floral nectar) and defending animals (extrafloral nectar) or a landing site for pollen (pollination drop).

Conclusions

Nectar and pollination drops contain sugars as the main components, but the total concentration and the relative proportions are different. They also contain amino acids, of which proline is frequently the most abundant. Proteomic studies have revealed the presence of common functional classes of proteins such as invertases and defence-related proteins in nectar (floral and extrafloral) and pollination drops. Invertases allow for dynamic rearrangement of sugar composition following secretion. Defence-related proteins provide protection from invasion by fungi and bacteria. Currently, only few species have been studied in any depth. The chemical composition of the pollination drop must be investigated in a larger number of species if eventual phylogenetic relationships are to be revealed. Much more information can be provided from further proteomic studies of both nectar and pollination drop that will contribute to the study of plant reproduction and evolution.Key words: Nectar, pollination drop, ovular secretion, plant reproduction, proteins, sugars, gymnosperms, angiosperms, plant–animal interaction     

Anatomical observations on the nucellar apex of Wellwitschia mirabilis and the chemical composition of the micropylar drop

Summary In the young ovule of Welwitschia mirabilis the nucellar apex is dome shaped and starch begins to accumulate near the female gametophyte. With the degeneration of the cells of the nucellar apex, a pollen chamber is formed, which contains the micropylar fluid. Starch storage increases considerably in the upper part of the nucellus. Pollen drop emission is not a rhythmic process, and pollination does not produce the rapid withdrawal of droplets. The micropylar drop consists almost entirely of sugars, uronic acids and a very small amount of free amino acids and enzymes. The mechanism of micropylar drop secretion and its probable role in the process of pollination is discussed.This work was supported by a grant from MURST 40%     

GENE EXCHANGE IN LOBLOLLY PINE: THE RELATION BETWEEN POLLINATION MECHANISM,FEMALE RECEPTIVITY AND POLLEN AVAILABILITY

The pollination process in loblolly pine has been examined over several years, both in the field (seed orchards) and experimentally on greenhouse-grown material. Female strobili are receptive to pollination for periods of a wk or more. Initially, background pollen from outside the seed orchard is the main source of pollen but as peak receptivity approaches, pollen from the stand itself predominates especially in older orchards. Consequently, strobili can receive pollen both from outside the orchard as well as from within. The pollen lands on the micropylar horns where it is transferred through the micropyle onto the nucellus by either rainfall or the pollen drop, whichever comes first. Since the pollen drop does not occur until the latter part of the receptive period, rainfall is the most likely transfer agent and pollen flotation is vital if rain occurs. Early arriving pollen does not appear to have an advantage over later arriving pollen for uptake onto the nucellus, even if rain follows the first pollination immediately. Therefore, total pollination of the strobilus can result from both distant and nearby pollen sources.     

On the Systematic Position of Amentotaxus from Its Embryological Investigation

The present paper deals with the embryological study and the systematic position of Amentotaxus argotaenia (Hance) Pilger. The material used was collected during 1980-1981 from Jin-fo Shan, 1400-1600 m, Sichuan Province, China. The species is dioecious. The male cone sheds its pollen during the period from the end of May to the middle of June. The pollen at mature stage is 2-celled. Pollen chamber appears obvious at the end of the nucellus. When pollen grains are dispersed, megaspore mother cell, which is situated deep in the nucellus, is in meiosis. The megaspore divides mitotically after pollination and the free nuclei of female gametophyte divide for the last time at the end of June. The wall formation takes place at the stage of 256 free nuclei. The development of archegonia takes place at the beginning of July and the fertilization occurs about July 20-23. The fertilized egg divides successively four times and results in a 16-nucleate proembryo. The young embryo is developing in August. It is interesting to note that the development of the young embryo is very slow. When the seed reaches the mature stage from June to July in the following year, the multicellular masses of the young embryos resulted from simple polyembryony remain immature within the female gametophyte. No cleavage polyembryony has been found. The subsequent embryogeny takes place after the seed has shed. Keng (1975) considers that Amentotaxus links the Taxaceae with Cephalotaxaceae. Our embryological data support Keng’s conclusion since they share (1) compound microstrobilus, (2) 2-celled pollen grains at shedding stage and (3) the rather long life cycle. Keng (1975) also mentions that Podocarpaceae may connect with Taxaceae through Phyllocladus. According to Keng the Podocarpaceae is related to Taxaceae to certain degree. It is obvious that the primitive spike-like male strobilus like the one in Cordaitales is obviously retained in Podocarpus spicatus and P. andinus of Podocarpaceae and Amentotaxus of Taxaceae. In addition, like in Amentotaxus there are 16 nuclei before wall formation in the proembryo of Podocarpus nivalis. These facts may well indicate that at least the Podocarpaceae and the Taxaceae were derived from a common stock. As far as the Taxaceae is concerned the authors tend to support the view of Koidzumi (1932) that Amentotaxus and Austrotaxus should be put in the same tribe since both possess the spike-like strobilus, the long life cycle and the seed maturation in the following year. They are probably rather primitive genera in the Taxaceae. The proembryogeny of Torreya is more or less specialized. It may be placed in a rather advanced tribe and the tribe Taxeae (including Taxus and Pseudotaxus)may be between the above two tribes. In conclusion, the Taxaceae is related to the Coniferales in certain respects and, as Keng (1975), Harri (1976) and Wang et al. (1979) have pointed out recently, placing of the Taxaceae in Coniferales is rather justifiable.     

The behavior of pollination drop secretion in Ginkgo biloba L.

Pollination drop (PD) secretion plays a critical role in wind pollination in many gymnosperms. We conducted detailed investigations on PD secretion in Ginkgo biloba, and found that PDs could not form when the micropyle was removed, but were able to form after removal of the shoot, leaves, ovular stalk, or ovular collar. The duration and volume of the PD increased under high relative humidity, but addition of salt or sugar did not affect PD secretion, its size, or its duration. Morphological and anatomical observations showed that many secretion cells at the nucellus tip contributed to secreting the PD after the formation of pollen chamber. Under laboratory conditions, the PD persisted for approximately 10 d if not pollinated, and re-formed five times after it was removed, with the total volume of PDs reaching approximately 0.4 μL. These results suggested that PDs can be continuously secreted by the tip of the nucellus cells during the pollination stage to increase the chance of capturing pollen from the air. Importantly, PD secretion is an independent behavior of the ovule and PDs were produced apoplastically.     

The biomechanics of pollen release: new perspectives on the evolution of wind pollination in angiosperms

Evolutionary transitions from animal to wind pollination have occurred repeatedly during the history of the angiosperms, but the selective mechanisms remain elusive. Here, we propose that knowledge of pollen release biomechanics is critical for understanding the ecological and evolutionary processes underpinning this shift in pollination mode. Pollen release is the critical first stage of wind pollination (anemophily) and stamen properties are therefore likely to be under strong selection early in the transition. We describe current understanding of pollen release biomechanics to provide insights on the phenotypic and ecological drivers of wind pollination. Pollen release occurs when detachment forces dominate resistive forces retaining pollen within anthers. Detachment forces can be active or passive depending on whether they require energy input from the environment. Passive release is more widespread in anemophilous species and involves processes driven by steady or unsteady aerodynamic forces or turbulence-induced vibrations that shake pollen from anthers. We review empirical and theoretical studies suggesting that stamen vibration is likely to be a key mechanism of pollen release. The vibration response is governed by morphological and biomechanical properties of stamens, which may undergo divergent selection in the presence or absence of pollinators. Resistive forces have rarely been investigated for pollen within anthers, but are probably sensitive to environmental conditions and depend on flower age, varying systematically between animal- and wind-pollinated species. Animal and wind pollination are traditionally viewed as dichotomous alternatives because they are usually associated with strikingly different pollination syndromes. But this perspective has diverted attention from subtler, continuously varying traits which mediate the fluid dynamic process of pollen release. Reinterpreting the flower as a biomechanical entity that responds to fluctuating environmental forces may provide a promising way forward. We conclude by identifying several profitable areas for future research to obtain deeper insight into the evolution of wind pollination.     

Significance of exine shedding in Cupressaceae-type pollen

In conifers, which have non-saccate Cupressaceae-type pollen, the pollen must land on a pollination drop or be picked up by the pollination drop from the surface of the cone near the ovule before it can be taken into the ovule. After contact with the drop, the pollen intine absorbs moisture from the drop, expands and the exine is shed. In this study the significance of the shedding of the exine is interpreted from experiments in which simulated pollination drops and micropyles were used to determine the movement of pollen and other particles in suspension. The non-expanded pollen, which can be observed upon contact with the pollination drop, sheds the exine, which then functions as a non-elastic particle, while the pollen from which the exine was shed swells and functions as an elastic particle because it is enclosed by the flexible intine. Non-elastic particles are not easily transferred through narrow passages (the micropyle and micropylar canal) and tend to plug these passages. However, elastic particles, such as the swollen pollen, are easily transferred along narrow passages even when non-elastic particles are present. The simulated experiments demonstrate that exine shedding is an important feature in getting pollen through the narrow micropyle and micropylar canal to the nucellus of the ovule.     

Pollen and water limitation in Astragalus scaphoides, a plant that flowers in alternate years

Mast seeding is common in plant populations, but its causes have rarely been tested experimentally. We tested mechanisms of alternate-year flowering and fruit set in an iteroparous, bee-pollinated, herbaceous plant, Astragalus scaphoides, in semi-arid sagebrush steppe. Patterns of reproduction from 1986 to 1999 indicated that spring precipitation was a cue for synchronous flowering, and that increased pollination in high-flowering years was a fitness advantage of synchrony. We tested these patterns by adding supplemental water and pollen to plants in high- and low-flowering sites and years. Supplemental water had no effect on flowering or seed set, so water is not a proximate cue for reproduction, though it could be important over longer (>3 year) time scales. Supplemental pollination increased fruit set in low- but not high-flowering years, indicating that synchronous flowering increases pollination success. Many shorter-term studies also report increased fruit set after pollen supplementation, but not after resource addition. This pattern may reflect the fact that plants can store and reallocate resources, but not pollen, across multiple years. For animal-pollinated herbs such as these, uniting theories about pollination ecology and mast seeding may promote an understanding of the mechanisms that determine patterns of reproduction over time.     

Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis)

A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.     

茨藻目植物柱头特征与传粉系统的演化

茨藻目包含有风媒、水表水媒和水下传粉的多种传粉方式。在前人工作的基础上,补充了4科9 种植物的柱头特征和传粉机制的观察。该目花粉和柱头特征可分为两类,传粉系统表现出两种不同的柱头捕获花粉的机制。花粉为球形的种与其宽大的柱头相配;而那些花粉演化为丝状或花粉在水中具先期萌发能力的种,其花粉易被其丝状的柱头所捕获。此外,还推测了导致这个类群传粉系统分化的可能原因。     

Phylogeny of Podocarpaceae: comparison of evidence from morphology and 18S rDNA

Maximum parsimony analyses of the genera of Podocarpaceae were conducted using sequence data from 18S ribosomal DNA. Trees from sequence, morphological, and combined data differ in taxon arrangement, but are similar in that Podocarpus sensu lato and Dacrydium s.l. are unnatural, while Podocarpaceae (including Phyllocladus) are monophyletic. The clade Microcachrys + Microstrobos is recognized in all analyses, but its placement differs, i.e., nested among other scale-leaved taxa in the morphological analysis, but associated with Nageia and other tropical genera in the sequence analyses. Trees from combined data reflect this ambiguity. Podocarpus sensu stricto is paraphyletic according to most trees. Inferences of plesiomorphic character states within the family are largely consistent between analyses and support the view that prototypical podocarps had bifacial leaves, cones with several fertile cone scales, and large epimatia (cone scales) that covered the inverted ovules.