Filling in the gaps: evidence of leaf endodermis and vein sheath in gymnosperms

James L Seago; Kamal I Mohamed; Kristen R Haynes

doi:10.1093/aob/mcaf165

. 2025 Jul 19;136(7):1497–1514. doi: 10.1093/aob/mcaf165

Filling in the gaps: evidence of leaf endodermis and vein sheath in gymnosperms

James L Seago ^1,^✉,^b, Kamal I Mohamed ^2,³, Kristen R Haynes ⁴

PMCID: PMC12718014 PMID: 40682443

Abstract

Background and Aims

Botanical literature is filled with studies which tried to demonstrate that leaves of many gymnosperms have an endodermis with Casparian bands or a sheath of sclerified cells around leaf veins. Direct photographic evidence of an endodermis with Casparian bands (strips) is lacking for the leaves of many gymnosperms. Our goal was to confirm with direct evidence an endodermis with Casparian bands or a vein sheath in leaves of representative gymnosperms via histochemical staining and epifluorescence microscopy, while extending previous work by examining understudied petioles and leaf bases.

Methods

We sectioned leaves fresh with razor blades and viewed them unstained or stained, usually with berberine hemisulfate, counterstained with gentian violet, and phloroglucinol HCl. Bright-field, epifluorescence and/or laser confocal microscopies on a Zeiss LSM700 were used for viewing and imaging.

Key Results

Most members of Pinaceae of Pinophyta (13 species of genera Cedrus, Pseudotsuga, Larix, Picea and Pinus) had endodermis with distinct Casparian bands in needles. We identified endodermis in the distal petiolar regions of nine of these same species. Sclerified vein sheaths or partial vein sheaths were observed in 10 species of 41 studied among Cycadophyta (Cycas), Ginkgophyta (Ginkgo) and the Pinophyta Podocarpaceae (Dacrycarpus, Dacrydium), Cupressaceae (Metasequoia), Taxaceae (Amentotaxus) and Pinaceae (Keteleeria, Abies, Tsuga).

Conclusions

Endodermis with Casparian bands is only characteristic of most species of Pinaceae; vein sheaths are found in three genera of Pinaceae and six genera of the other families of gymnosperms, but most gymnospermous leaves lack endodermis and vein sheath, particularly in petiolar and leaf base regions. The presence of endodermis with Casparian bands could have contributed to the adaptation of the Pinaceae to extreme environments; members of genera such as Picea and Pinus are the typical treeline species in many mountain ranges across the world.

Keywords: Endodermis, Casparian bands or strips, conifers, gymnosperms, needles, phylloclades, vein sheath

INTRODUCTION

The leaves of gymnosperms have been variously described as linear with abaxially/adaxially oriented veins, broad with petioles, needles with petioles, cataphylls or scale leaves, cladodes with obliquely oriented veins, or phylloclades with perpendicularly orientated veins and stem-like concentrically arranged vascular bundles (Napp-Zinn, 1966; Bierhorst, 1971; Gifford and Foster, 1989; Dörken and Stützel, 2011; Kaplan, 2022). Within these leaves, structures surrounding the vascular tissues, or veins, have been described and designated in many different ways, but usually structures termed vascular bundle sheaths (vein sheaths) or endodermis have been assigned to them; both of these are historically known.

From Chamberlain (1935) to Napp-Zinn (1966), Esau (1977), Gifford and Foster (1989), Biswas and Johri (1997) and Kaplan (2022), texts and major books have long extolled the characteristics of gymnosperm leaves, including the occurrence of endodermis, especially in leaves of pines and related Pinaceae. In his review less than 30 years ago, however, Lersten (1997) noted that there was no real proof of Casparian bands in an endodermis in gymnosperm leaves, and earlier, in his major tome on gymnosperm leaves, Napp-Zinn (1966, citing mostly much earlier researchers) did not present any photographic evidence of leaf endodermis or of sclerified vein or vascular bundle sheath (or ‘Leitbündelscheiden’, pp. 156–157, 226–227). More recently, the Dörken group has studied many of the families, genera and species of gymnosperms (e.g. Dörken et al., 2010, 2019, 2021, 2023a, b; Dörken and Stützel, 2011, 2012; Dörken, 2012, 2013a, b, 2014; Dörken and Parsons, 2016; Dörken and Lepetit, 2018; Dörken and Nimsch, 2018, 2021), but their techniques did not usually allow direct Casparian band evidence.

Endodermis and vein sheaths are different anatomical structures, although both form the innermost layer of root and shoot cortex or leaf mesophyll, and will be illustrated initially in the first figure of the Results section. The endodermis in vascular plants is a specific structure that has been most characterized in roots. In its simplest form, as described and discussed variously by Peterson and colleagues (Seago et al., 1999; Enstone et al., 2003; Ma and Peterson, 2003), Schreiber and Franke (2011) and Soukup and Tylová (2018), it comprises a ring or layer of cells of the innermost cortex with mostly lignified Casparian bands impregnated in its radial and transverse cell walls: a State I endodermis has Casparian bands only. Endodermis can also form additional chemical structures; a State II endodermis has Casparian bands with associated suberin lamellae within the limits of the primary cell wall and outside the protoplast. Further, endodermis can form a State III endodermis with secondarily lignified wall lining the inside of the suberin lamellae, often with its thickness greatest on the inner tangential wall in root endodermis (and outer tangential wall of an innermost layer of an exodermis, which may form the outermost layer of cortex). An endodermis in stems has also been well documented (Seago, 2020), as has endodermis of some gymnosperm roots (e.g. Wilcox, 1968 for Pinus resinosa; Bonacorsi and Seago, 2016 for Ginkgo biloba; Song et al., 2019 for Cunninghamia lanceolata; Brundrett and Tedersoo, 2020 for various gymnosperms with mycorrhizae).

Importantly, as noted by Enstone et al. (2003), Geldner (2013), Soukup and Tylová (2018) and others, the endodermis regulates the movement of water, dissolved minerals and carbohydrates into and out of the vascular cylinder of roots; further, the endodermis filters and arrests unwanted harmful molecules from being translocated in the xylem strand up to the shoot while at the same time allowing necessary inorganic molecules essential for plant growth. While water and dissolved minerals can pass apoplastically from cell wall to cell wall, symplastically or even transcellularly through the protoplasts of cortex cells, all movement of water and minerals passes through the endodermis only symplastically or transcellularly due to the presence of Casparian bands in the radial/transverse walls of the endodermis. It is known that in the needles of conifers, endodermis also plays an important ecological role in the survival of pines in extreme cold habitats; for example, Stegner et al. (2023) suggested that the lignification of the endodermis blocks the penetration of ice from the frozen vascular tissue into the mesophyll, allowing needles to remain active during extended periods of extreme cold temperatures. This, in turn, may be associated with oxygen and carbon dioxide pathways between vein and mesophyll (Roden et al., 2015; Sage and Khoshravesh, 2016) as well as movement of nutrients like nitrogen (Gao et al., 2023; Valderrama-Martin et al., 2024) and carbohydrate (Liesche et al., 2011) and structural support (e.g. Leegood, 2007; Meicenheimer et al., 2008).

An endodermis with Casparian bands has been well demonstrated in leaf blades or needles in relatively few species, with the most striking examples being in Pinus nigra and P. resinosa of the Pinaceae by Meicenheimer et al. (2008), where P. nigra had prominent Casparian bands and P. resinosa had prominent outer tangential, secondarily lignified walls in its endodermis. Other authors (e.g. Campbell, 1972; Delian and Săvulescu, 2022) have presented photographs of P. nigra clearly showing endodermis, sometimes with well-demonstrated Casparian bands. Often published illustrations are specimens in which Casparian bands do not show, but the cells have a characteristic endodermal shape and pattern, as in Esau (1977) for Larix and Pinus and Gifford and Foster (1989) for Pinus. Using a variety of techniques, Soar (1922) had drawings of leaf endodermis in Pinus, Picea, Abies, Larix, Pseudotsuga, Cedrus and Tsuga. Others (e.g. Hu and Wang, 1984; Eguchi et al., 2004; Bercu et al., 2010; Ghimire et al., 2015a) demonstrated images with endodermis patterns in a variety of Pinaceae, but Casparian bands were not visible. Vein sheath was labelled in Abies (Dörken, 2014; Dörken and Lepetit, 2018); these studies were not suggestive of an endodermis with Casparian bands.

Evidence seems to show that endodermis in gymnosperm leaves may be a more recently derived trait and of interest evolutionarily. Fossil cycads, for example, do not have endodermis in cataphylls or scale leaves (Hermsen et al., 2006), and they may be among the oldest of gymnosperms (e.g. Taylor et al., 2009; Crisp and Cook, 2011; Lu et al., 2014; Lubna et al., 2021). Fossils from basal gymnosperms like Podocarpaceae (Andruchow-Colombo et al., 2023) do not show any evidence of endodermis in leaves. On the other hand, fossil pines, like Pinus hokkaidoensis (Stockey and Ueda, 1986) and P. arnoldii (Klymiuk et al., 2011), are recent in evolutionary appearance and have been shown to have a distinct endodermis (Taylor et al., 2009; Gernandt et al., 2016).

A vein sheath or its modifications consists of cortex sclerenchyma cells surrounding the vascular bundles that are sclerified or lignified without Casparian bands. The occurrence and nature of vein sheaths in gymnosperm leaves have been little addressed in major tomes (e.g. Bierhorst, 1971; Gifford and Foster, 1989; Kaplan, 2022), but they have been variously described as a ‘layer of lignified cells’ in Ginkgo by Dörken (2013b, p. 90), a ‘parenchyma sheath’ or ‘unstratified endodermis with thin-walled large cells’ by Cargo et al. (2000, p. 38), or a zone of ‘scattered thin-walled lignified fibers’ in Cycas by Griffith et al. (2014, p. 241). Vein sheaths are clearly irregular in pattern and structure, but without Casparian bands. In addition to facilitating the movement of water, nutrients, photosynthates and gases between vascular tissues and mesophyll, the vein sheath reinforces and supports the vascular tissues and even the inner mesophyll (Esau, 1977; Leegood, 2007), potentially serving a similar function as endodermis.

Among other gymnospermous families, evidence of endodermis with Casparian bands or vein sheath is spotty and sometimes lacking. Sethi et al. (2020) reported a sclerenchyma sheath in Cycas. Cargo et al. (2000, p. 38) stated ‘an unistratified endodermis with thin-walled large cells’ in Ginkgo biloba, but it was not so described by Duarte et al. (2013); Dörken’s (2013a) bright-field micrographs demonstrated a vein sheath in its lamina. Liesche et al. (2011, p. 184) reported a ‘suberized bundle sheath’ and ‘endodermis-like bundle sheath’ for the same lamina image in Ginkgo, whereas Klimko et al. (2015, p. 185) noted a ‘parenchymatous sheath’ around its veins. Bundle sheaths appear to be present in some members of the Podocarpaceae (Andruchow-Colombo et al., 2024) and not others (Lee, 1952; Griffith, 1957). Endodermis is claimed but not demonstrated in leaves of Cupressaceae by Yousif and Shahbaz (2023); presumed endodermis was also noted in Juniperus (Bercu et al., 2010; Güvenc et al., 2011). In their broad survey of leaf characteristics in conifers (7 families, 37 genera, 111 species), Yao and Hu (1982) reported the presence or absence of endodermis or vein sheath, but their drawings did not demonstrate endodermis with Casparian bands or vein sheaths. The scale leaves or cataphylls reported by many (e.g. Soar, 1922; Sacher, 1955; Kaplan, 2022) have reduced vascular tissue and no apparent endodermis.

Thus, the evidence for endodermis with Casparian bands in gymnosperm leaves is incomplete and uncertain. The situation in petioles or leaf bases has been far less documented, even though Napp-Zinn (1966), for example, devoted much to leaf origin – without a single depiction of a mature leaf with petiole. Lee (1952, p. 395) illustrated by drawing a petiole and its leaf vascular bundle without an ‘endodermal sheath’ in the leaf of Dacrydium taxoides of the Podocarpaceae. An illustration by Cross (1940) showed a vein sheath in the petiolar or proximal region of a Cunninghamia lanceolata leaf. Doak (1935) had only drawings of the fascicle bases in pine and leaf bases in Araucariaceae. Soar’s (1922) drawings of the origin of needles in fascicles of Pinus sylvestris showed no endodermis in lower or proximal levels of the developing needles and an incomplete endodermis in Picea excelsa and in Abies pectinata. Needles of Pinus mugo and P. monophylla were photographed by Bierhorst (1971) with endodermis around the two vascular bundles of a needle; his photograph of a P. mugo fascicle without endodermis is one of the few examples of photographic evidence of endodermis presence or absence in petiolar regions within a fascicle of gymnosperm leaves. Bright-field images of Picea mariana needles by Way and Sage (2008) show a typical endodermis but without manifest Casparian bands. Hu and Yao (1981) and Hu and Wang (1984) reported an endodermis in the needle and stem of Cathaya argyrophylla; an endodermis is apparent in its leaves without visible Casparian bands and in a petiole in a ring around the vein, but endodermis or vein sheath was not mentioned in its petiole. Duarte et al. (2013) had micrographs of petioles in Ginkgo biloba without noting vein sheath or endodermis, but Dörken (2013a) clearly illustrated one vein sheath around the two vascular bundles in its petioles. Yet Klimko et al. (2015) labelled an endodermis in Ginkgo petioles. The dearth of photographic evidence and conflicting reports indicate that more investigations are merited for petioles in gymnosperms.

Given the paucity of photographs adequately demonstrating an endodermis with Casparian bands or vein sheaths in gymnosperm leaves, we examined leaves (the Dörken group; Napp-Zinn, 1966; Kaplan, 2022) and other gymnospermous leafy appendages like cladodes (Dörken and Stützel, 2011), phylloclades (Dörken et al., 2021; Kaplan, 2022), cataphylls (Kaplan, 2022) and their associated leaf petioles or petiolar regions (or leaf bases where no petiole was apparent), which have been rarely included in analyses of leaves. We demonstrate the presence or absence of endodermis with Casparian bands along radial (and sometimes transverse) cell walls, mostly by epifluorescence of magnified images after selective staining and microscopy. We concentrate on the endodermis and the xylem of the vascular tissue because xylem is much easier to demonstrate in relation to Casparian bands than is phloem tissue. For many species, we use higher-magnification close-ups of veins to show more clearly the presence or absence of endodermis Casparian bands. We will note other leaf features, like transfusion tissues, which may be appropriate to anatomical characterizations and functions in veins. It is sometimes very difficult to determine a vein sheath with lignified cell walls but no Casparian bands. These observations should help researchers studying the associations among plant structure and function in various ecological/environmental conditions. We have at least one image for all species studied so that we can demonstrate the situations in many species and genera, but we emphasize, with more detail, select species among 11 families, 30 genera and 41 species.

MATERIALS AND METHODS

For two centuries, Oswego, NY, USA, has been a small but major port on Lake Ontario, easternmost of the North American Great Lakes that flow into the St Lawrence River and then the Atlantic Ocean. During that time, ships have brought seeds or plants into the Oswego area from many places around the world; for example, on the SUNY Oswego campus there are non-native species of gymnosperms, including dawn redwood or Metasequoia glyptostroboides, which is believed to have been planted on campus in the 1950s, not many years after it was reported extant in China, and Chamaecyparis pisifera, located within 50 m of Lake Ontario shoreline. In the city of Oswego, well-established trees of Ginkgo biloba, Cryptomeria japonica, and Picea abies occur within 200 m of Lake Ontario and the Port of Oswego, and in the surrounding towns of Minetto, Oswego and Scriba many other non-native as well as native gymnosperms are found.

With that background, we were long interested in these gymnosperms. Leaves were obtained from plants growing in the city of Oswego, NY, USA, the towns of Minetto, Oswego and Scriba, NY, USA, the campus and greenhouse of SUNY at Oswego, and a few botanical gardens and growers (examples are named in the Acknowledgements section). Multiple leaves of deciduous species like Ginkgo biloba and Larix occidentalis were sampled during early to late summers, June into October. Evergreen species like Chamaecyparis pisifera and Pinus strobus were sampled during their first year from early summer into winter, and we examined leaves in summer and winter (we note, however, that summer to winter first-year leaves were similar in structure). Leaves and stems were fresh-sectioned with Wilkinson sword razor blades (split in half to make single blades; sometimes the halves were held together to section through a leaf, a technique taught to J.L.S. by Chaodong Yang). Specimens were then run through stain procedures or mounted directly in dH₂0 or 80 % glycerine.

Following methods in Seago et al. (1999), Soukup and Tylová (2018) and Seago (2020), staining procedures of freshly cut specimens included berberine hemisulfate for 1–2 h to reveal leaf cell walls under epifluorescence, berberine hemisulfate counterstained briefly in 1 % gentian violet or in 0.5 % toluidine blue O for 30–60 s to reveal lignins in endodermis Casparian bands and xylem under epifluorescence, fluorol yellow for 1 h to reveal suberin under epifluorescence, phloroglucinol HCl for 5–10 min to stain lignin in cell walls in bright-field, Sudan red 7B or Sudan IV for 1 h to reveal suberin in bright-field, and toluidine blue O for 30–60 s as a metachromatic stain to colour different wall chemicals of various tissues in bright-field. These procedures allowed us to visualize endodermis with Casparian bands, vein sheaths, xylem tracheids and transfusion tissues (e.g. Hu and Yao, 1981). We do not include images of all of the staining methods.

We used a Zeiss LSM700 with a camera at SUNY Oswego for the vast majority of images under epifluorescence at 509 nm, but a few at 419 nm. Many Zeiss LSM700 images were taken in bright-field and in laser confocal microscopy at 488 nm, and an Amscope bright-field scope with a Samsung Galaxy A42 camera was used for some images.

RESULTS

After first presenting illustrations of specimens with endodermis, vein sheaths or no layers conspicuously surrounding veins (Fig. 1), the remainder of the Results will begin with the Cycadophyta, Ginkgophyta and Gnetophyta – gymnosperms not usually classified with the Pinophyta; these will then be followed by the Pinophyta from Araucariaceae through Pinaceae. These descriptions will include references to specific, appropriate literature.

Structures surrounding leaf veins

Figure 1A is a transverse section of a fresh-cut, unstained bright-field image of a white pine (Pinus strobus) needle in which there is a clear ring of cells, i.e. endodermis, surrounding the central vein but no easily visible Casparian bands. Epifluorescence images reveal endodermis around a vein in Fig. 1B, C with the radially oriented Casparian bands showing clearly around the xylem tracheids and vein transfusion tissue. As seen in Fig. 1D, E, there is a vein sheath or ring or zone of lignified sclerenchyma cells around large veins in a small section of a broad maiden hair/ginkgo (Ginkgo biloba) leaf. Staining for Casparian bands in a small vein reveals that veins and vein sheath cells do not have Casparian bands (Fig. 1F). An example of a flattened gymnospermous needle in a coastal redwood (Sequoia sempervirens) without endodermis or vein sheath is shown in epifluorescence in Fig. 1G, H, where there are xylem tracheids and transfusion tissue visible in a vein, but no endodermal Casparian bands or sclerified cells.

Cycadophyta, Cycadophyceae

Cycas revoluta

The compound leaf of Cycas revoluta has leaflets (Fig. 2A) and a petiolar region (Fig. 2B) with a singular vein of little xylem surrounded by a vein sheath (Fig. 2B) but no endodermis. This is similar to the drawings in Lamb (1923) and to the photographs in Jeffrey (1917), Gifford and Foster (1989) and Stevenson et al. (1996). Griffith et al. (2014, p. 244) demonstrated a thick, U-shaped ‘limiting layer of vein’ with phloroglucinol stain, but no endodermis. The related Zamia leaves have many veins with only vein sheaths (Beck, 2010). Vovides et al. (2020) and Glos et al. (2022) show vein sheath very clearly in standard microscopic preparations in another cycad, Ceratozamia, and in Zamia, respectively, as well as Coiro et al. (2020) in various microscopies for many Zamiaceae.

Ginkgophyta, Ginkgophyceae

Ginkgo biloba

The deciduous broad leaf lamina (Fig. 1D, E) and the petiole (Fig. 2C, D) of G. biloba long shoots and spur shoots (Gifford and Foster, 1989) are characterized by adaxial/abaxial vein orientation in the lamina with a vein sheath (Fig. 1D, E). There is no endodermis with Casparian bands around the leaf lamina or petiole veins as shown by berberine gentian violet staining in a small peripheral lamina vein (Fig. 1F). Along most of their length, petioles also have a vein sheath, extending even between the two veins of the petiole (Fig. 2C). Nearer petiole bases, xylem may be united and surrounded by a less distinct lignified vein sheath (Fig. 2D); there is no endodermis. This corroborates images by Dörken (2013b) and by Leigh et al. (2011) but contradicts the claims of an endodermis by Cargo et al. (2000) and Klimko et al. (2015, for petiole), neither of whom used techniques to demonstrate Casparian bands. Our Ginkgo leaves contrast with roots of G. biloba, which have been well documented (Bonacorsi and Seago, 2016) to have an endodermis and exodermis with Casparian bands and suberin lamellae, and even its first stem internode has such exodermis, which was revealed using similar techniques to the present study (from the same batch of seeds used to produce one of the plants in the present study).

Gnetophyta, Gnetaceae

Gnetum gnemon

The broad leaf lamina (Fig. 2E) and narrower petiole (Fig. 2F) of G. gnemon are multi-veined with no endodermis (Fig. 2F) or vein sheath around the veins (Fig. 2E). Tomlinson and Fisher (2005) noted no endodermis but an ‘indistinct’ vein sheath in Gnetum, while Ajuru (2018) noted an endodermis, which does not appear evident.

Ephedraceae

Ephedra tweedieana

Appressed to the stem, scale leaves (Fig. 2G) of E. tweedieana have no endodermis or vein sheath; these can also lack xylem in very small-scale leaves. Dörken (2012) showed no endodermis or vein sheath.

Pinophyta, Araucariaceae

Araucaria araucana

The relatively wide leaf of A. araucana has several veins but no endodermis or vein sheath (Fig. 2H). Leaf tips reduce to one vein and leaf bases have a few veins with no endodermis (Fig. 2I). Thomson’s (1913) early photographs do not reveal a possible endodermis. Mastroberti and Mairath (2003) label endodermis in their images, but there is no clear ring of cells nor Casparian bands. The bright-field images of A. araucana by Rivera et al. (2022) or of Wollemia by Burrows and Bullock (1999) and Vasheka et al. (2019) do not mention or show endodermis or vein sheath. Multi-veined leaf and petiole images of Agathis do not reveal endodermis or vein sheath in Arbicheva and Pautov (2018).

Podocarpaceae

Members of the Podocarpaceae have not been shown by most to have an endodermis around their vasculature structures in phylloclades, cataphylls or needles (Lee, 1952; Griffith, 1957; Kausik, 1975; Tomlinson et al., 1989; Dörken and Parsons, 2016; Dörken et al., 2021; Andruchow-Colombo et al., 2024). The species that we examined have no endodermis around their leaf veins or concentric vascular bundles in phylloclades or leaves.

Phyllocladus alpinus

A phylloclade (Dörken et al., 2021) is illustrated at mid-phylloclade level in Fig. 2J. Viewed in berberine (Fig. 2J) and berberine gentian violet (Fig. 2K), some of the vascular tissues are stem-like and arranged in a concentric fashion, hence concentric vascular bundles, with very few xylem elements and no endodermis (Fig. 2K); there may be one stem-like stele (concentric vascular bundles) or a few stem-like vascular bundles. Lateral veins generally have a few xylem elements without endodermis and are oriented perpendicular to the epidermis, not adaxially/abaxially (Fig. 2K). At the phylloclade base (Fig. 2L) there is a ring of vascular bundles, i.e. a stem stele, sometimes with sclerified cells and no endodermis. All veins and concentric vascular bundles are without vein sheath or endodermis (Dörken et al., 2021).

Dacrycarpus dacrydioides

For the needle-like ends of the cataphylls and their bases, there is no endodermis, but vein sheath cells occur (Fig. 2M). Dörken and Parsons (2016) presented no evidence of endodermis or vein sheath in their standard histochemical preparations.

Dacrydium elatum

In D. elatum, needle-like ends of the cataphylls (Fig. 2N) do not have an endodermis (Fig. 2O), but there are vein sheath cells in the cataphylls and their bases (Fig. 2N). In one of the few studies to look at petioles, Lee (1952, p. 396) similarly noted that there was no ‘endodermal sheath’ in the leaf or petiole. Dörken and Parsons (2016) also showed no evidence of endodermis or a vein sheath in Dacrydium cupressinum; however, there was some evidence of a vein sheath in the bright-field micrograph of a leaf in Sahni and Mitra (1927).

Podocarpus macrophyllus

Like Griffith (1957) or Kausik (1975), under berberine (Fig. 2P) and berberine gentian violet (Fig. 2Q) staining our specimens of P. macrophyllus had no endodermis or vein sheath in the needle or petiole (Fig. 2R). We did observe transfusion tissue under berberine but not under berberine gentian violet staining (Fig. 2P; see Griffith, 1957).

Sciadopityaceae

Sciadopitys verticillata

The shoot structures on our plants were cladodes (Dörken and Stützel, 2011) in which two well-separated veins have an oblique orientation seen under berberine epifluorescence (Fig. 2S); there is no endodermis (Fig. 2T, berberine gentian violet) even at their base and no obvious vein sheath. Scale leaves have a very small vein with little to no xylem and no endodermis or vein sheath of lignified cells (Fig. 2U). The Dörken and Stützel (2011) bright-field images show that there is no evident ring of endodermis, but there is a vein sheath (or vein sheet, per Dörken and Stützel, 2011). The image of a fossil Sciadopitys (Stockey et al., 2005) shows two veins but no endodermal or sheath region.

Cupressaceae

The leaves of members of the Cupressaceae have such a seeming variety of morphologies from phylloclades, needles and flattened laminae to cataphylls (Gifford and Foster, 1989; Kaplan, 2022) that we were not surprised to find that many researchers reported a variety of situations for their vascular tissues. For example, Cross (1940) showed no evidence of endodermis but did report a vein sheath in Cryptomeria, whereas Ivănescu et al. (2007) and Yousif and Shahbaz (2023) noted but did not demonstrate an endodermis in various species not covered herein; in addition, some of the vascular tissues photographed and referred to were stem vascular bundles, not leaf or phylloclade tissues. Fossil Cupressaceae with phylloclade-like morphologies have a wider central vein or probable concentric vascular bundles much like typical, extant phylloclade-bearing plants (Stockey et al., 2005; Dörken et al., 2023a, b; the current study for Phyllocladus of the Podocarpaceae and Fokienia of the Cupressaceae).

Cunninghamia lanceolata

Neither needles (Fig. 3A) nor petioles (Fig. 3B) have endodermis or a distinctive, lignified vein sheath in C. lanceolata. Cross (1940) showed no endodermis but noted a vein sheath without good evidence (even referring to a wrong photograph).

Athrotaxis cupressoides

The tightly positioned, scale-like cataphylls of A. cupressoides lack an endodermis and vein sheath (Fig. 3C). Only a faint vein sheath was reported by Napp-Zinn (1966, noting a 19th-entury report).

Metasequoia glyptostroboides

The needle (Fig. 3D) and petiolar end of the needle (Fig. 3E) in M. glyptostroboides have no endodermis, but they do have a vein sheath in needles and petiolar regions. Sterling (1949, p. 468) stated that ‘No distinct endodermal layer is present’. Standard preparations and micrographs in Ng and Smith (2020) reveal a possible vein sheath, but no endodermis, including in fossil Metasequoia. In Metasequoia roots, however, endodermis and exodermis have been clearly demonstrated (Yang et al., 2019).

Sequoiadendron giganteum

We found no endodermis or vein sheath in the needle-like end of the cataphylls (Fig. 3F, G, with transfusion tissue) or at the bases of cataphylls of S. giganteum. Chin and Sillett (2016) showed no endodermis, but they did demonstrate fibres around the vein of a cataphyll.

Sequoia sempervirens

There is no endodermis or vein sheath in needles (Fig. 1G) or petioles (Fig. 1H) of S. sempervirens. After Napp-Zinn (1966, noting far earlier researchers) noted only faint vein sheath in Sequoia, photographs in Oldham et al. (2010) and Chin et al. (2022) did not reveal any evidence of endodermis, although a vein sheath or fibres may have been present.

Cryptomeria japonica

Our sections of C. japonica did not reveal an endodermis or vein sheath with lignified cell walls in either the needle (Fig. 3H) or the petiolar base, both with xylem and transfusion tissues (3I). Cross (1940, p. 577) stated that an ‘incipient endodermal sheath may be present’. Azuma et al. (2015) labelled a vein sheath in needle leaves of C. japonica, but they did not reveal sclerification.

Thuja occidentalis

Where cataphylls arise from stems and partially surround the stem (Fig. 3J), they have no endodermis or vein sheath. Dörken (2013a) also did not find endodermis or vein sheath in Thuja plicata.

Chamaecyparis pisifera

Chamaecyparis has cataphylls that terminate in a needle-like tip; neither their needle-like portions (Fig. 3K) nor bases have an endodermis or vein sheath, although there is transfusion tissue in the vein (Shiraki et al., 2016). Ivănescu et al. (2007) noted, without demonstrating, that its leaves had endodermis.

Fokienia hodginsii

The photosynthetic organs of F. hodginsii are phylloclades (Fig. 3L). The central vascular tissue comprises stem-like concentric vascular bundles (Fig. 3L, M; also see Dörken et al., 2021), and the two lateral vascular strands are leaf veins (Fig. 3L, N), each with a single bundle of xylem; these are arranged with xylem lateral to phloem rather than in an adaxial/abaxial pattern without endodermis or vein sheath (Fig. 3N). Near phylloclade bases, the veins have their perpendicular-to-epidermis orientation, but the stem vasculature is opposite that found in the phylloclade (Fig. 3O); phylloclades lack endodermal Casparian bands or vein sheaths (Fig. 3L–O). The phylloclade pattern was shown long ago by Saxton (1930) for Fokienia, with a variation of the position of the two lateral veins in Libocedrus. While Fokienia has been found to be very closely related to Chamaecyparis and Thuja of the Cupressaceae (Lu et al., 2014; Zang et al., 2019), its leaves are clearly more similar structurally to Phyllocladus of the Podocarpaceae (Zang et al., 2019; Dörken et al., 2021); phylloclades were termed branchlets by Saxton (1930), and Dörken et al. (2021) clarified their nature and structures. These phylloclades in Fokienia are not like the leaves or cataphylls of Chamaecyparis or Thuja. Yousif and Shahbaz (2023) had bright-field micrographs of cupressaceous plants with stem-like vasculature in the leaves of Platycladus; they also stated that an endodermis was present in these genera, including around secretory canals, but an endodermis was not revealed in their micrographs.

Juniperus

We did not find an endodermis or vein sheath in the needle-like or scale leaves and petiolar regions of J. chinensis (Fig. 3P) or J. horizontalis (Fig. 3Q). Güvenc et al. (2011) labelled endodermis in standard preparations of Juniperus species, but there was no evidence of Casparian bands.

Cephalotaxaceae

Cephalotaxus harringtonia

Neither the needle (Fig. 3R) nor the petiole (Fig. 3S) has an endodermis or vein sheath in C. harringtonia. Similarly, Biswas and Johri (1997) stated that the lone vein does not have a vein sheath in C. fortunei, nor did Hu (1984) reveal endodermis in Cephalotaxus, although transfusion tissue was present. However, the autofluorescence photograph in Elpe et al. (2017) has an apparent vein sheath.

Taxaceae

Ghimire et al. (2014, p. 379) found an endodermis ‘scarcely evident’ or ‘parenchymatous’ in the Taxaceae, yet they labelled an endodermis in Taxus. Photographs in Elpe et al. (2017) reveal a possible vein sheath in Taxus, Pseudotaxus and Torreya.

Amentotaxus formosana

The leaf lamina has no endodermis, but there are vein sheath cells in the leaf lamina (Fig. 3T) and petiole (Fig. 3U); the extension of sheath fibres/sclerenchyma throughout the lamina from the single vein ending is striking (Fig. 3T). For A. formosana, Chin and Lin (2022, p. 4) also identified a vein sheath around the singular vein, as well as a vascular cambium in the vein and the extension of transfusion cells with ‘elongated sclerenchyma’ throughout the mesophyll (see also Ghimire et al., 2014). Elpe et al. (2017) labelled some cells as vascular sclereids in Amentotaxus argotaenia and Torreya californica, but did not demonstrate endodermis or vein sheath.

Taxus cuspidata

In Taxus, the needle (Fig. 3V, W) and petiole (Fig. 3X) have no endodermis or vein sheath (Roden et al., 2015), but transfusion tissue is readily apparent (Fig. 3V). Gostin and Predan (2022) found no endodermis in bright-field images, but Ghimire et al. (2014) labelled an endodermis in bright-field sections, but no Casparian bands were visible. Elpe et al. (2017) showed a possible vein sheath in T. baccata and Pseudotaxus chienii in fluorescence microscopy.

Pinaceae

Dörken and Stützel (2012) and Dörken et al. (2010) have demonstrated with standard preparation techniques and bright-field microscopy an endodermis in many members of the pines. Interestingly, Du et al. (2020, p. 1) categorized the differences between the ‘flattened leaves’ of Abies, Larix, Pseudolarix and Pseudotsuga and the ‘needlelike leaves’ of Cedrus, Picea and Pinus in regard to photosynthetic mesophyll capacity and adaxial/abaxial tissue polarity, but endodermis presence does not fully follow their categories; our findings below will show that only certain species of Pinus have oblique vein orientations. A bright-field image of Cathaya leaf shows an apparent endodermis (Hu and Wang, 1984); Pant and Basu (1977) show Casparian bands only in drawings. Fossilized needles of Pinus foisyi (Miller, 1992) demonstrate endodermis.

Cedrus atlantica

Cedrus atlantica has a clearly demonstrable endodermis with Casparian bands in its needles (Fig. 4A, B), but in petioles there are no Casparian bands or vein sheaths (Fig. 4C); veins are oriented adaxially/abaxially. In bright-field images of needles, Fahn (1990) clearly demonstrated endodermal Casparian bands in C. deodara, and Bercu et al. (2010) and Bakkali and Amraoul (2022) illustrated endodermis with Casparian bands in C. atlantica.

Keteleeria davidiana

In our specimens of K. davidiana, there is a vein sheath in needles (Fig. 4D) and petioles (Fig. 4F), but no demonstrable endodermis with Casparian bands in needles (Fig. 4E) or petioles (Fig. 4F, G). Pant and Basu (1977) showed only drawings of the ring of endodermis in Keteleeria. Pant and Basu (1977) noted that there was an endodermis with Casparian bands, suberin and lignified secondary walls, but these were not apparent in their photographs of the closely related Cathaya, only in drawings with a ring of endodermis. Hu and Wang (1984) noted Casparian bands without demonstrating them in Cathaya needles, but not in petioles; a ring of cells in the position of endodermis was visible in both leaf parts. Han (1984) showed only an incomplete ring of endodermis without identifiable Casparian bands in Keteleeria, but no such ring of endodermis was in Cathaya.

Abies

Around the two veins of needles, A. balsamea has a vein sheath with some lignified cell walls (Fig. 4H) even on radial walls in the position of Casparian bands (Fig. 4I, arrow), but no distinct endodermis. In petioles, there is a vein sheath without endodermis (Fig. 4J). Abies concolor has a vein sheath around the two veined needles without endodermis (Fig. 4K) and in petioles (Fig. 4M); Casparian bands via berberine gentian violet epifluorescence are not revealed in either needles (Fig. 4L) or petioles. Dörken (2014) and Dörken and Lepetit (2018) illustrated a vein sheath in the related A. alba, although others (e.g. Bercu et al., 2010; Ghimire et al., 2015a) illustrated endodermis, but Casparian bands were not evident. Endoh et al. (2012) had a ring of endodermis under bright-field, but Casparian bands were not visible in cryo-SEM images.

Tsuga canadensis needles (Fig. 4N) and petioles (Fig. 4P, Q) have a vein sheath without endodermis (Fig. 4O). There is usually just one vein in needles, although it may appear somewhat divided, whereas petioles have greater division between two units of the xylem. Soar (1922) showed lignified and suberized endodermal cells in a drawing, and Gambles and Dengler (1974) clearly demonstrated a partial vein sheath in T. canadensis.

Pseudotsuga menziesii

Needles (Fig. 4R, S) and petioles (Fig. 4T) have distinct endodermis Casparian bands (Fig. 4S); they also have lignified outer tangential secondary walls without suberin lamellae (Fig. 4T). Soar (1922), Pădure et al. (2008), Chin and Sillett (2019) and Apple et al. (2002) also demonstrated the Casparian bands in this species.

Larix

In L. laricina, endodermis with Casparian bands is present in needles (Fig. 5A, B) and leaf bases or petioles (Fig. 5C) with some evidence of lignified tangential walls in needles in berberine staining (Fig. 5A, low magnification). In L. kaempferi (Fig. 5D, E) and L. occidentalis (Fig. 5F, G) endodermis is present in needles and leaf bases (petioles); in L. kaempferi outer tangential endodermal cell walls were thickened under low magnification in berberine staining (Fig. 5D) and in L. occidentalis at higher magnification under phloroglucinol staining (Fig. 5G). Endodermis in Larix has been shown by Eguchi et al. (2004) and Bercu and Popoviciu (2013) without demonstrable Casparian bands.

Picea abies, P. glauca and P. pungens In the three species of Picea studied here, P. abies has endodermis in the needles (Fig. 5H, I) and distal petioles (Fig. 5J, K), but not in proximal petioles; the outer tangential walls fluoresce lightly in berberine (Fig. 5H). Picea glauca has distinctive endodermis in needles (Fig. 5L, M), but no endodermis or vein sheath in petioles (Fig. 5N), and P. pungens also has endodermis with Casparian bands in needles (Fig. 5O, P), but no endodermis or vein sheath in petioles (Fig. 5Q). Endodermis was demonstrated in P. abies by Soukupová et al. (2000), in P. glauca by Hacke et al. (2015) and in P. pungens by Soukupová et al. (2001) and Qin et al. (2014). Endodermis was also shown in needles of Picea species by Ghimire et al. (2015a) without evident Casparian bands.

Pinus

Pinus strobus (Fig. 1A–C) has single vein needles with a distinctive endodermis with Casparian bands (Figs 1B, C and 6A) and some lignification on tangential cell walls (Fig. 6A) but no suberin lamellae (Fig. 6B); the xylem of the vein is often in two slightly separated units (Fig. 1C). In the needle bases within the middle-lower fascicle or petiolar region, there is no endodermis or vein sheath (Fig. 6C); there is endodermis in the most distal petiolar regions (not shown). Pinus mugo needles have an endodermis with Casparian bands around the two veins (Fig. 6D); the fascicle base has two petiolar needles without endodermis or vein sheath around the veins (not shown). In P. sylvestris, the two-veined needles have a distinctive endodermis with lignified Casparian bands (Fig. 6E, F) but no suberin lamellae (Fig. 6G); the outer tangent wall is thickened but stained unevenly for lignin (Fig. 6F). Casparian bands are found in distal fascicled needles (Fig. 6H, I) but not in proximal fascicled needles (Fig. 6J), indicating that endodermis has developed before needles become separate in fascicles. Scale leaves have no endodermis and often show no xylem (Fig. 6K).

In P. nigra leaves, needles have endodermis with Casparian bands around the two veins (Fig. 6L); Casparian bands stain for lignin (Fig. 6M, on either side of arrow or e), but outer tangential cell walls do not stain for lignin via phloroglucinol HCl in bright-field or berberine gentian violet in epifluorescence, although they appear thicker (Fig. 6M at arrow). There is no evidence of suberin lamellae (Fig. 6N). In unstained specimens viewed in epifluorescence (Fig. 6O, difficult to see) distal and partially separated needles within a fascicle develop an endodermis with Casparian bands (Fig. 6P). In proximal, unseparated needles within the fascicle there is no endodermis in the two needles (Fig. 6Q). Pinus resinosa has mature needles with two veins surrounded by an endodermis (Fig. 6R) with Casparian bands (Fig. 6R, S) and distinctly secondarily thickened outer tangential cell walls of lignin (Fig. 6R, S); they do not have suberin lamellae (Fig. 6T). Distal, slightly separated needles within a fascicle have Casparian bands in endodermis around each two-veined needle (Fig. 6U, V), whereas proximal or petiolar needles in fascicles do not have endodermis (Fig. 6W). Scale leaves have little to no xylem and no endodermis (Fig. 6X). In three species of Pinus with two veins within an endodermis we find varying obliquely oriented veins (Fig. 6E, L, R; cf. Du et al., 2020).

Chamberlain (1935), Keng and Little (1961), the Dörken group (e.g. Dörken and Stützel, 2012) and Kaplan (2022), have had illustrations of pine needles with an endodermis. Kuusk (2024) has recently shown bright-field images of needles of P. nigra that seem to have more lignin in their endodermal cell walls after 2 years than in their first-year needles after phloroglucinol HCl treatment. Endodermis in P. sylvestris showed clearly in images via laser confocal images in Lin et al. (2001) and in bright-field by Dörken and Stützel (2012). No cataphyll endodermis was revealed in bright-field images in P. lambertiana by Sacher (1955). Carde (1978) questioned the occurrence of Casparian bands in P. pinaster needles, but Gao et al. (2023) clearly demonstrated Casparian bands. Like Meicenheimer et al. (2008) for P. resinosa, Donaldson and Williams (2018) demonstrated unequivocally that there were secondarily lignified outer tangential cell walls in P. radiata, but their fluorescence photographs also showed clearly Casparian bands without suberin lamellae in the endodermis using techniques like ours. The bright-field images of various Abies, Picea (Ghimire et al., 2015a) and Pinus species (e.g. P. sylvestris, P. parviflora, P. densiflora, P. taeda; Ghimire et al., 2015b) also showed thickened outer tangential endodermal cell walls. Miller (1992) also demonstrated clearly thickened outer tangential cell walls in the fossilized endodermis of P. foisyi. In P. nigra, Meicenheimer et al. (2008) had bright-field and fluorescent sections of P. nigra with no secondary cell walls, but Lux et al. (2017) and many others (e.g. Ghimire et al., 2015a, 2015b; Donaldson and Williams, 2018 (P. radiata)) had bright-field photographs with distinct outer tangential cell walls that were thickened and lignified. Soar (1922) illustrated (but did not present photographs of) the lack of endodermis in the proximal regions of the fascicled base of pines and its presence developing prior to emergence of the needles between the scale leaves of fascicles. Few other researchers have determined the presence or absence of endodermis or vein sheaths in petioles or scale leaves in pines.

DISCUSSION

As shown in Table 1, an endodermis with Casparian bands is found around leaf veins only in the Pinophyta family Pinaceae, where it is found in most members (Cedrus atlantica; Pseudotsuga menziesii; Larix laricina, L. kaempferi and L. occidentalis; Picea abies, P. glauca and P. pungens; Pinus mugo, P. nigra and P. resinosa with lignified secondary outer tangential walls, P. strobus and P. sylvestris). Petioles, in contrast, almost always lack endodermis; the notable exceptions with Casparian bands in petiolar regions are Pseudotsuga menziesii, Larix laricina, Picea abies, and distal petiolar regions within fascicles of Pinus nigra, P. resinosa, P. strobus and P. sylvestris. Within the 11 genera of Pinaceae and the 8 we studied, only needles of Keteleeria, Abies and Tsuga lack distinctive Casparian bands in an endodermis-like ring around veins, and only these same three Pinaceae genera have vein sheaths. Both Keteleeria and Abies are interesting because their needles and petioles showed indications of cell wall modifications that might represent incipient endodermis. Thus, in detail, with our study and with other examples in the literature, we have resolved the issues of Napp-Zinn (1966) and Lersten (1997) that endodermis with Casparian bands around veins had not been positively identified in needles of many members of the Pinaceae.

Table 1.

The presence of endodermis and vein sheath in representative gymnosperms.

Phylum and family	Species	Endodermis with Casparian bands		Vein sheath
Phylum and family	Species	Leaf	Petiole	Leaf	Petiole
Cycadophyta, Cycadaceae	Cycas revoluta			✓	✓
Ginkgophyta, Ginkgoaceae	Ginkgo biloba			✓	✓
Pinophyta, Araucariaceae	Araucaria araucana
Pinophyta, Podocarpaceae	Phyllocladus alpinus
	Dacrycarpus dacrydioides			^*	^*
	Dacrydium elatum			^*	^*
	Podocarpus macrophyllus
Pinophyta, Sciadopityaceae	Sciadopitys verticillata
Pinophyta, Cupressaceae	Cunninghamia lanceolata
	Athrotaxis cupressoides
	Metasequoia glyptostroboides			✓	✓
	Sequoia sempervirens
	Sequoiadendron giganteum
	Cryptomeria japonica
	Thuja occidentalis
	Chamaecyparis pisifera
	Fokienia hodginsii
	Juniperus horizontalis
	Juniperus chinensis
Pinophyta, Cephalotaxaceae	Cephalotaxus harringtonia
Pinophyta, Taxaceae	Amentotaxus formosana			^*	^*
	Taxus cuspidata
Pinophyta, Pinaceae	Cedrus atlantica	✓
	Keteleeria davidiana			✓	✓
	Abies balsamea			✓	✓
	Abies concolor			✓	✓
	Tsuga canadensis			✓	✓
	Pseudotsuga menziesii	✓	✓
	Larix occidentalis	✓	✓
	Larix kaempferi	✓	✓
	Larix laricina	✓	✓
	Picea abies	✓	✓
	Picea pungens	✓
	Picea glauca	✓
	Pinus nigra	✓	✓
	Pinus resinosa	✓	✓
	Pinus strobus	✓	✓
	Pinus sylvestris	✓	✓
	Pinus mugo	✓
Gnetophyta, Gnetaceae	Gnetum gnemon
Gnetophyta, Ephedraceae	Ephedra tweedieana

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Taxa are listed according to Yang et al. (2022).

^*Partial vein sheath.

In contrast to endodermis, vein sheaths are prominent in the most early evolved gymnosperms, namely Cycadophyta and Ginkgophyta, but they are present in relatively few members of the Pinophyta and are absent in the Gnetophyta. Viewed in current phylogenies (Gernandt et al., 2016; Yang et al., 2022), the distribution of this trait (Table 1) suggests a loss in several lineages or several independent origins. A few gymnosperms in the Pinophyta families of Podocarpaceae (Dacrycarpus dacrydioides and Dacrydium elatum), Cupressaceae (Metasequoia glyptostroboides) and Taxaceae (Amentotaxus formosana) have vein sheaths or sclerified cells in positions of possible vein sheaths. The derived lineage Gnetophyta (Gnetum gnemon and Ephedra tweedieana) has no endodermis or vein sheath in leaves.

If we consider the fossil record (Taylor et al., 2009), for example, Robison (1977) had micrographs of fossil Pinus fascicles to show that an endodermis, without visible Casparian bands, was present in distal needles bases, but not proximal bases. He also claimed that there was only a thick-walled sheath in an earlier fossil, Prepinus (Robison, 1974). Then, Stockey and Ueda (1986) produced light micrographs of five-needled fossil Pinus hokkaidoensis that clearly reveal a ring of endodermal cells without visible Casparian bands around the vein. Taylor et al. (2009, in a photograph by R. A. Stockey) had a fossil five-needle pine, P. hokkaidoensis, like P. strobus, with a quite distinct endodermis ring around the vein in each needle with distinctly thickened radial and outer tangential walls. The absence of an endodermis with Casparian bands in the Abietoideae of the Pinaceae fossil records supports the idea that it is a more recently evolved structure and that it may have evolved multiple times. It would help to have more anatomical images of fossil members of the Abietoideae and Pinoideae of the Pinaceae to track the evolution of endodermis with Casparian bands. The fossil record of other families like the Cupressaceae seems particularly difficult for obtaining a clear picture of internal structure with vein sheaths or no vein-encircling structures (e.g. Taylor et al., 2009; Herrera et al., 2017), complicating the task of tracking the evolution of vein sheath across gymnosperms.

Soar (1922) was among the first to discuss any physiological and ecological roles of Pinaceae leaves in enabling them to withstand temperatures in alpine and subalpine regions. She described the leaves as xerophytic in nature, with defined endodermis, placing them in contrast with angiosperm leaves where there is no endodermis. The development and structure of endodermis with Casparian bands in needles/leaves of the members of Pinaceae could have contributed to their ability to evolve in, colonize and radiate into extreme environments, especially in cold, high-elevation environments where other gymnosperms are not found. Indeed, members of Pinaceae such as Picea and Pinus are the typical treeline species in many mountain ranges around the world (e.g. Kaku, 1971; Skärby et al., 1998; Miller, 1999; Wu et al., 2005; Taylor et al., 2009; Roden et al., 2009, 2015; Leslie et al., 2018; Stegner et al., 2023) where blowing ice and rime ice, consequent desiccation and extreme low temperatures limit other species (e.g. Kaku, 1971; Skärby et al., 1998; Wu et al., 2005; Roden et al., 2009, 2015; Taylor et al., 2009; Stegner et al., 2023). As Wu et al. (2005) have importantly shown, endodermis with Casparian bands in needles of Pinus bungeana is more permeable than endodermis with Casparian bands in roots; a salient reason could be the lack of suberin lamellae as we have shown for other species of Pinus. In Picea mariana, Way and Sage (2008) showed temperatures had a pronounced effect upon vein and leaf size, but it was not apparent in their photographs if there were any effects upon the obvious endodermis which we have shown in other Picea species. Endoh et al. (2012) showed in a species of Abies that changes in needles were structurally visible during freezing events. Freeze tolerance gained through an endodermis with Casparian bands is a logical driver of its evolution and persistence in Pinaceae.

The lack of a clear, distinct endodermis in Keteleeria and Abies of Abietoideae could relate to their subtropical habitat and origin (Zhang et al., 2023) – and a possible lack of selection pressure for endodermis, potentially explaining why this anatomical feature is absent in these lineages but present in Cedrus of Abietoideae and members of Pinoideae. The possible incipient endodermis seen in these genera could suggest an intermediate evolutionary step between vein sheaths and endodermis. Selection pressure for the development of endodermis is understandable for species like Abies balsamea, which is a treeline species subject to extreme cold temperatures.

This relationship between leaf structures and the plant environments of the gymnosperm has also been studied with regard to levels of CO₂ (Sage and Khoshravesh, 2016), as well as nutrients and other compounds. In terms of the control and facilitation of movements of nutrients, phosphates and gases through needle veins, endodermis and mesophyll, Gao et al. (2023) have demonstrated the importance of endodermis with Casparian bands (see also Uchida et al., 2012). Other researchers have shown how photosynthetic and respiratory processes with CO₂ and O₂ movements could be salient in considering how the endodermis functions in conifer leaves (e.g. Sutinen et al., 2000; Eguchi et al., 2004; Roden et al., 2015; Sage and Khoshravesh, 2016). These interactions and relationships between leaf structural and environmental effects need to be examined far more thoroughly. Indeed, this goes beyond normal leaf functions to the effects of air pollution on these gymnosperm leaves in which there is endodermis – very evident in bright-field images of Pinus nigra by Delian and Săvulescu (2022).

As noted in the Results, members of the Pinaceae generally have a State I endodermis with Casparian bands only, i.e. there are no suberin lamellae in the endodermis cells, as per Seago et al. (1999), Enstone et al. (2003), Soukup and Tylová (2018) and Yang et al. (2019). The occurrence of a secondarily lignified outer tangential wall might seem to make these cells more like an exodermis, except that they are lacking suberin lamellae. However, a typical exodermis has Casparian bands and suberin produced concomitantly in plant roots (Pazourek and Votrubová, 1997; Seago et al., 1999; Seago, 2002; Enstone et al., 2003; Schreiber and Franke, 2011; Bonacorsi and Seago, 2016; Soukup and Tylová, 2018) or in plant stems (Seago and Fernando, 2013; Bonacorsi and Seago, 2016). Where produced, secondary cell wall lignification between suberin lamellae and protoplasm in these cells is normally produced in the innermost tangential cell wall of endodermis and the outermost cell wall in exodermis (e.g. Meyer et al., 2009), as found in gymnosperm roots (e.g. Bonacorsi and Seago, 2016; Brundrett and Tedersoo, 2020).

Therefore, the development and structure of endodermis in conifer leaves are both similar to and different from conifer roots. Conifer leaf endodermis is typically in State I, Casparian bands only. Where lignified, secondary cell wall thickening occurs, as in Pinus resinosa needles and Pseudotsuga petioles, it occurs only in the outer tangential cell wall and without suberin lamella formation. However, our findings also show that some outer tangential cell wall thickening in needles seen under epifluorescence occurs in these outer tangential walls in Cedrus, Metasequoia, Larix (deciduous species all), Picea, Pseudotsuga and most Pinus species, but these do not stain for lignin by the phloroglucinol test as they do in Pinus resinosa (Meicenheimer et al., 2008). However, other Pinus species, e.g. P. radiata (Donaldson and Williams, 2018) and P. laricio (Coulter and Chamberlain, 1901; Popham, 1966; Clegg and Cox, 1978), have what is like an innermost State III exodermis, except that there are no suberin lamellae and no U-shaped lignified walls. Interestingly, Griffith et al. (2014) showed a U-shaped wall in Cycas hainanensis leaf via phloroglucinol staining, but no suberin lamellae were demonstrated.

Age of needles must be very important to these differences (Soar, 1922; Cross, 1940; Ewers and Schmid, 1981; Sutinen et al., 2000; Wu et al., 2005; Sack and Scoffoni, 2013; Vasheka et al., 2019; Bozkurt et al., 2023; Huusk, 2024). In her recent thesis Huusk (2024) had bright-field images of phloroglucinol-stained Pinus nigra, P. halapensis and P. pinea in which needles older than first year in P. nigra appeared to reveal more heavily lignified cell walls under low magnification; yet, needles may remain on some pines like P. sylvestris for 3–4 years (Pensa and Jalkanen, 1999) and many Picea species retain needles for years. There would seem to be major physiological and ecological ramifications for leaves and the plants if leaf endodermis develops more lignified cell walls, even if only in outer tangential walls, over time as leaves survive and remain active, especially through seasons of warmth and then freezing cold, etc. Lastly, larger questions are: do Pinaceae represent earlier or later evolved gymnosperms (cf. Taylor et al., 2009; Lu et al., 2014; Gernandt et al., 2016; Yang et al., 2022) and thus when did endodermis evolve in gymnosperms and Pinaceae?

ACKNOWLEDGEMENTS

We are extremely grateful to the following persons for aiding this project by encouragement, providing access to plants, providing background information or helping to obtain some supplies: Marilyn A. Seago, Melodie Seago, Zenab Mohamed, Samouel Beguin, Yulia Artemenko, Ashley Mears, Beverly Jones, Cathy Van Patten, Chaodong Yang, Aleš Soukup, Edita Tylová, Olga Votrubová, Stan Kollar at Kollar Nursery, Silas Zoeller at Far Reaches Botanical Conservancy, Ontario Orchards, Wilson Bros Gardens, Juniper Level Botanic Garden at Plant Delights Nursery, Conifer Kingdom, Woodies Garden Goods and the AoB reviewers and editors.

Contributor Information

James L Seago, Department of Biological Sciences, SUNY Oswego, Oswego, NY 13126, USA.

Kamal I Mohamed, Department of Biological Sciences, SUNY Oswego, Oswego, NY 13126, USA; Rice Creek Field Station, SUNY Oswego, Oswego, NY 13126, USA.

Kristen R Haynes, Rice Creek Field Station, SUNY Oswego, Oswego, NY 13126, USA.

FUNDING

We received no funding except that we had the use of the facilities and Zeiss LSM700 microscope at SUNY Oswego.

AUTHOR CONTRIBUTIONS

The study was conceived and begun by the senior author, but all three authors participated in the preparation and examination of specimens, as well as the microscopy and photography. The senior author began and wrote the early version of the manuscript, but all three authors contributed to the writing.

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PERMALINK

Filling in the gaps: evidence of leaf endodermis and vein sheath in gymnosperms

James L Seago

Kamal I Mohamed

Kristen R Haynes

Abstract

Background and Aims

Methods

Key Results

Conclusions

INTRODUCTION

MATERIALS AND METHODS

RESULTS

Fig. 1.

Structures surrounding leaf veins

Cycadophyta, Cycadophyceae

Cycas revoluta

Fig. 2.

Ginkgophyta, Ginkgophyceae

Ginkgo biloba

Gnetophyta, Gnetaceae

Gnetum gnemon

Ephedraceae

Ephedra tweedieana

Pinophyta, Araucariaceae

Araucaria araucana

Podocarpaceae

Phyllocladus alpinus

Dacrycarpus dacrydioides

Dacrydium elatum

Podocarpus macrophyllus

Sciadopityaceae

Sciadopitys verticillata

Cupressaceae

Cunninghamia lanceolata

Fig. 3.

Athrotaxis cupressoides

Metasequoia glyptostroboides

Sequoiadendron giganteum

Sequoia sempervirens

Cryptomeria japonica

Thuja occidentalis

Chamaecyparis pisifera

Fokienia hodginsii

Juniperus

Cephalotaxaceae

Cephalotaxus harringtonia

Taxaceae

Amentotaxus formosana

Taxus cuspidata

Pinaceae

Cedrus atlantica

Fig. 4.

Keteleeria davidiana

Abies

Pseudotsuga menziesii

Larix

Fig. 5.

Pinus

Fig. 6.

DISCUSSION

Table 1.

ACKNOWLEDGEMENTS

Contributor Information

FUNDING

AUTHOR CONTRIBUTIONS

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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