Zostera Marina Seeds Research Articles

Microbial communities around seeds promote Zostera marina germination

Eelgrass meadows are vital not only for sustaining marine biodiversity as marine ecosystems but also for carbon fixation as blue carbon. However, the national decline in eelgrass meadows in Japan, which was initiated by economic growth in the 1980s, prompted the implementation of conservation measures. This study explored the fundamental techniques of the sediment environment to enhance Zostera marina seed germination rates. We investigated the microbial origin of Zostera marina seeds under anaerobic conditions and assessed their germination efficiency. Anaerobic sediments in the eelgrass meadows were found to increase eelgrass germination rate by 6% more than aerobic sediments. And interestingly, the sulfate-reducing bacteria Desulfovibrionaceae and Sulfurospirillaceae were detected mainly on eelgrass seeds. Our result clearly indicated that the sulfate-reducing bacteria around Zostera marina seeds create anaerobic conditions that promote germination through their sulfate-reducing function. Therefore, when developing new eelgrass meadows, it can be expected to improve germination rates by preparing the sediment environment to facilitate the increase of SRB coexisting with seeds. However, the difference in bottom sediment conditions varies by region, so it is necessary to be careful when adding or removing something. In addition, germination and growth of eelgrass must be considered separately, and factors that promote growth are not described in this work. The result of this study was an important finding for the implementation of Blue Carbon activities to improve sediment quality prior to the creation of eelgrass meadows and can also support eelgrass meadows maintenance after establishment.

Insights into the regulation of energy metabolism during the seed-to-seedling transition in marine angiosperm Zostera marina L.: Integrated metabolomic and transcriptomic analysis.

Seed development is a crucial phase in the life cycle of seed-propagated plants. As the only group of angiosperms that evolved from terrestrial plants to complete their life cycle submerged in marine environments, the mechanisms underlying seed development in seagrasses are still largely unknown. In the present study, we attempted to combine transcriptomic, metabolomic, and physiological data to comprehensively analyze the molecular mechanism that regulates energy metabolism in Zostera marina seeds at the four major developmental stages. Our results demonstrated that seed metabolism was reprogrammed with significant alteration of starch and sucrose metabolism, glycolysis, the tricarboxylic acid cycle (TCA cycle), and the pentose phosphate pathway during the transition from seed formation to seedling establishment. The interconversion of starch and sugar provided energy storage substances in mature seeds and further acted as energy sources to support seed germination and seedling growth. The glycolysis pathway was active during Z. marina germination and seedling establishment, which provided pyruvate for TCA cycle by decomposing soluble sugar. Notably, the biological processes of glycolysis were severely inhibited during Z. marina seed maturation may have a positive effect on seed germination, maintaining a low level of metabolic activity during seed maturation to preserve seed viability. Increased acetyl-CoA and ATP contents were accompanied with the higher TCA cycle activity during seed germination and seedling establishment, indicating that the accumulations of precursor and intermediates metabolite that can strengthen the TCA cycle and facilitate energy supply for Z. marina seed germination and seedling growth. The large amount of oxidatively generated sugar phosphate promotes fructose 1,6-bisphosphate synthesis to feed back to glycolysis during seed germination, indicating that the pentose phosphate pathway not only provides energy for germination, but also complements the glycolytic pathway. Collectively, our findings suggest these energy metabolism pathways cooperate with each other in the process of seed transformation from maturity to seedling establishment, transforming seed from storage tissue to highly active metabolic tissue to meet the energy requirement seed development. These findings provide insights into the roles of the energy metabolism pathway in the complete developmental process of Z. marina seeds from different perspectives, which could facilitate habitat restoration of Z. marina meadows via seeds.

Effect of Manila clam Ruditapes philippinarum on burial depth of eelgrass Zostera marina seeds and sediment porosity

Eelgrass Zostera marina is widely distributed in coastal areas of the Northern Hemisphere; however, its continuous population decline is increasingly serious. Besides exogenous stresses, there are endopathic reasons for the decline, e.g., its seedling establishment rate, which is affected by burial depth of seeds and sediment types, is extremely low. Studies have shown that eelgrass seedlings in bivalve distribution areas are easier to establish. Moreover, large-scale bottom sowing culture for bivalves affects the bulk density of sediments which affects seed germination and seedling establishment of eelgrass. This study sought to examine the relationship between sediment modification induced by Manila clams with different body size and stocking density and the burial depth of eelgrass seeds caused by clams. The porosity of surface sediments varied with Manila clam biomass and was mainly affected by clam body size. Clams with a weight of 2.56 ± 0.12 g created more porosity. Clams with a weight of 3.71 ± 0.10 g caused more porosity than clam with a weight of 7.47 ± 0.22 g did. The sand diving of Manila clams affected the distribution of eelgrass seeds in sediments at different depths. The moderately abundant and large Manila clams promoted the burial depth of eelgrass seeds in sediments, and this effect was more obvious at 1–3 cm near the burial depth of 2 cm which is conducive to germination of eelgrass seeds and seedlings establishment. Thus, based on the bioturbation of Manila clams, the efficiency of sowing methods to restore the eelgrass population can potentially be improved.

An examination of seed germination and seedling growth of Zostera marina for planting-time selection in Rongcheng Bay, Shandong Peninsula, China

This study firstly quantified the responses of seeds of Zostera marina to different planting times (22 September, 5 October, 23 October, 7 November and 20 November in 2015) through a field seed-planting experiment over a two year period. The suitable seed planting time required by the seeds of Z. marina was evaluated. The seedling establishment rate of Z. marina subjected to different planting times ranged from 7% to 55%, with the higher values attained on the treatments of 22 September and 5 October. New plant patches from seed were fully developed and well maintained on the planting time of 22 September, 5 October and 23 October after 2 years following planting. The shoot density under the three treatments ranged from 62 shoots per replicate to 72 shoots per replicate with an average of 67 shoots per replicate in September 2017. According to the propagation assessment and growth analysis, we found that the planting time from mid-September to mid-October may be the optimal time to plant seeds of Z. marina in our experimental site. Our results demonstrate that seed planting time has an important effect on the effectiveness of eelgrass restoration and provide data that could prove helpful in the development of successful eelgrass restoration.

Reciprocal Field Transplant Experiment and Comparative Transcriptome Analysis Provide Insights Into Differences in Seed Germination Time of Two Populations From Different Geographic Regions of Zostera marina L.

Seagrasses are the only submerged marine higher plants, which can colonize the sea through sexual (via seeds) reproduction. The transition between seed dormancy and germination is an important ecological trait and a key stage in the life cycle of higher plants. According to our observations, the seeds of Zostera marina L. (eelgrass) in Swan Lake (SL) and Qingdao Bay (QB) in northern China have the same maturation time (summer) but different germination time. To investigate this phenomenon, we further carried out reciprocal transplantation experiment and transcriptome analysis. Results revealed that differences in the seed germination time between the two sites do exist and are determined by internal molecular mechanisms as opposed to environmental factors. Furthermore, we conducted comparative transcriptome analysis of seeds at the mature and early germination stages in both locations. The results that the number of genes related to energy, hormone and cell changes was higher in SL than in QB, could account for that the dormancy depth of seeds in SL was deeper than that in QB; consequently, the seeds in SL needed to mobilize more related genes to break dormancy and start germination. The results could have important practical implications for seagrass meadow restoration via seeds and provide in-depth and comprehensive data for understanding the molecular mechanisms related to seagrass seed germination.

Canopy-Mediated Hydrodynamics Contributes to Greater Allelic Richness in Seeds Produced Higher in Meadows of the Coastal Eelgrass Zostera marina

Seagrass meadows, which mediate ocean acidity and turbidity, sequester carbon, and increase biodiversity by providing shelter for larvae and small fish, are among the fastest disappearing ecosystems worldwide. Seagrasses are ecosystem engineers, creating distinct regions of enhanced and diminished flow and turbulent mixing, dependent upon canopy physical parameters, such as canopy density and blade morphology, which in turn impact the transport of pollen, sediment, and nutrients. The health and resilience of seagrass meadows increase with intraspecies genetic diversity, which depends on successful sexual reproduction and the transport of pollen particles between reproductive shoots, which in turn depends on the hydrodynamic conditions created by the meadow. This paper explored the transport of pollen grains in seagrass meadows using a random walk model. The model was parameterized with profiles of mean velocity and eddy diffusivity derived as functions of shoot density, canopy height, canopy shear velocity, canopy drag coefficient, and blade width, and validated with experimental measurements of a tracer plume evolving in a submerged model canopy. Model results showed that release at the top of the canopy led to significantly greater dispersal than release within the canopy, which was consistent with observed patterns of genetic diversity in Zostera marina seeds collected from coastal Massachusetts meadows. Specifically, seeds produced from upper inflorescences had greater allelic richness than seeds from lower inflorescences on the same reproductive shoot, and were the product of a greater number of fathers, reflecting the greater in-canopy pollen movement farther from the bed. Pollen grains modeled with a realistic elongated shape experienced significantly higher rates of capture by the canopy relative to spherical grains of the same volume. The effect of submergence depth (the ratio of water depth to canopy height) on pollen dispersal depended on the nature of the surface boundary: when pollen reflected off the water surface, the mean travel distance before pollen capture decreased with decreasing submergence depth. In contrast, when pollen accumulated at the water surface, surface transport increased pollen dispersal distances, especially at low submergence depths.

Zostera marina seed burial can be enhanced by Manila clam Ruditapes philippinarum: A microcosm study

Seagrass seed bank plays a key role in the regeneration of new vegetation when seagrasses are removed by the natural or man-made disaster. Various factors may affect the development of sediment seed bank. We conducted a microcosm experiment to test the effects of burrowing and feeding activities of Manila clam, Ruditapes philippinarum on the burial of Zostera marina seeds in sediments. The effects of lasting time (3-hour, 1-day, 3-day, 7-day, 14-day and 28-day), clam density (0, 2, 4 and 8 clams with shell length of 3 cm in each microcosm) and clam size (shell length of 2, 3 and 4 cm at 4-clam density) on seed burial were examined in plastic microcosm cores (30 cm high × 10 in inner diameter) in a 28-day period. Results showed that the seed burial depth significantly increased with time, the density and the size of clams. No seeds were buried in the sediment in the cores without clams during the whole experiment period. For the 3-cm clams, about 91.61% of the seeds were buried in the sediment at the end of the experiment in the high-density treatment (8 clams at each core); while in the medium and low-density treatments (4 and 2 clams in each core, respectively), about 76.93% and 60.61% of the seeds were buried in the sediment, respectively. For the size treatments, large (4 cm) clams buried 89.56% of the seeds at the end of the experiment, much more than those of medium (3 cm, 76.93%) and small (2 cm, 61.50%) size clams. During the whole experiment period, nearly all of the buried seeds were at a depth of from 0 cm to 5 cm. These results suggested that Manila clam Ruditapes philippinarum may play an important positive role in seagrass seed bank dynamics in the field.

Copper treatment during storage reduces Phytophthora and Halophytophthora infection of Zostera marina seeds used for restoration

Restoration is increasingly considered an essential tool to halt and reverse the rapid decline of vital coastal ecosystems dominated by habitat-forming foundation species such as seagrasses. However, two recently discovered pathogens of marine plants, Phytophthora gemini and Halophytophthora sp. Zostera, can seriously hamper restoration efforts by dramatically reducing seed germination. Here, we report on a novel method that strongly reduces Phytophthora and Halophytophthora infection of eelgrass (Zostera marina) seeds. Seeds were stored in seawater with three different copper sulphate concentrations (0.0, 0.2, 2.0 ppm) crossed with three salinities (0.5, 10.0, 25.0 ppt). Next to reducing seed germination, infection significantly affected cotyledon colour: 90% of the germinated infected seeds displayed a brown cotyledon upon germination that did not continue development into the seedling stage, in contrast to only 13% of the germinated non-infected seeds. Copper successfully reduced infection up to 86% and the 0.2 ppm copper sulphate treatment was just as successful as the 2.0 ppm treatment. Infection was completely eliminated at low salinities, but green seed germination was also dramatically lowered by 10 times. We conclude that copper sulphate treatment is a suitable treatment for disinfecting Phytophthora or Halophytophthora infected eelgrass seeds, thereby potentially enhancing seed-based restoration success.

Persistence of Zostera marina L. (eelgrass) seeds in the sediment seed bank

Two separate field experiments in the Newport River/Back Sound, North Carolina (NC) and the lower Chesapeake Bay (CB), Virginia were conducted in 2007 and 2008 to quantify the effects of time (6, 12, 15months), seed source (mixed-annual, perennial NC; perennial CB), site (local environmental factors), and sediment type (fine, coarse) on the persistence of Zostera marina seeds in the sediment seed bank. It is here, at the southern limit of the species distribution along the western Atlantic, that the probability of population loss may be high and the importance of a seed bank in the resilience and recovery of these populations great. Experimental results indicate that viability of both NC and CB seeds decreased significantly after just 6months in the sediment following the seasonal period of maximum germination and continued to decline over time with no seeds viable remaining in CB cores and <5% of seeds remaining viable after 15months in NC treatments. In these experiments time was the overriding factor affecting the persistence Z. marina seed banks for all treatments in both NC and CB and viability was not significantly affected by seed source, site, or sediment type. Based on the results of the in situ experiments, mixed-annual and perennial Zostera marina populations in North Carolina and perennial populations in Virginia produce transient seed banks (seeds viable <12months). The lack of a persistent seed bank may reduce the resilience of Z. marina at the limits of the species distribution to repeated stress events. As a result these populations may be particularly susceptible to disturbance with only a limited capacity for recovery if sexual reproduction is impaired.

Burial of Zostera marina seeds in sediment inhabited by three polychaetes: Laboratory and field studies

The large number of seeds produced by eelgrass, Zostera marina, provides this plant with a potential to disperse widely and colonise new areas. After dispersal, seeds must be buried into sediment for assuring long-term survival, successful germination and safe seedling development. Seeds may be buried passively by sedimentation or actively through sediment reworking by benthic fauna. We evaluated the effect of three polychaetes on the burial rate and depth of eelgrass seeds. Burial was first measured in controlled laboratory experiments using different densities of Nereis (Hediste) diversicolor (400–3200indm−2), Arenicola marina (20–80indm−2), and the invasive Marenzelleria viridis (400–1600indm−2). The obtained results were subsequently compared with burial rates of seed mimics in experimental field plots (1m2) dominated by the respective polychaetes. High recovery of seeds in the laboratory (97–100%) suggested that none of these polychaetes species feed on eelgrass seeds. N. diversicolor transported seeds rapidly (<1day) into its burrow, where they remained buried at a median depth of 0.5cm. A. marina and M. viridis buried seeds by depositing their faeces on top of the sediment. At their highest abundance, A. marina and M. viridis buried seeds to a median depth of 6.7cm and 0.5cm, respectively, after a month. The burial efficiency and depth of these species were, in contrast to N. diversicolor, dependent on animal abundance. Only 2% of seed mimics casted in the field plots were recovered, suggesting that physical dispersion by waves and currents was considerably important for horizontal distribution. However, polychaete affected significantly the vertical distribution of seeds. Overall the effects of these three polychaetes indicate that benthic macroinvertebrates may significantly impact eelgrass seed bank at the ecosystem scale. Some species have a positive effect by burying seeds to shallow depths and thereby reducing seed predation and facilitating seed germination, while other species bury seeds too deep for successful seed germination and seedling development.

The carbohydrate catabolism of Zostera marina influenced by lower salinity during the pre-germination stage

The distribution of starch granules in ungerminated seeds of Zostera marina was examined by electron microscopy and histochemistry. Quantitative changes during germination in starch and sugars [glucose/maltosaccharides (MS), sucrose, and fructose] were examined in the seeds using biochemical methods. At natural seawater salinity (30.2 psu), the number of starch granules and the amount of starch markedly decreased, resulting in no increase in the sugar content in the embryos during the early germination stage. A substantial increase in sugars in the shoots occurred after the development of true leaves, probably due to photosynthetic activity in the green true leaves. At natural seawater salinity, the amount of starch did not significantly change during the initial 14 days of germination. In contrast, at lower salinities, starch levels decreased significantly from day 0 to day 6, resulting in higher levels of glucose/MS and fructose in the basal hypocotyls. These results suggest that compared with the salinity of natural seawater, lower salinity may interfere with starch catabolism in seeds, thereby causing the seed coat to break open earlier.

The structural and histochemical analyses and chemical characters of the cuticle and epidermal walls of cotyledon in ungerminated seeds of Zostera marina L.

How Zostera marina L. adapts to environmental stresses is of major interest to researchers wanting to obtain a better understanding of how to preserve this ecologically important seagrass. We have examined the structure and chemical properties of the cuticle and epidermal walls of Z. marina cotyledons at the pre-germination stage by microscopy, histochemistry, and chemical analyses. The epidermal cells, which have a smooth plasma membrane and slight wall-ingrowth, are surrounded by thickened outer tangential walls consisting of cellulose and pectic substances. The thick cuticle layer covers the outer surface of the outer tangential walls; the former were observed here to be much thicker than has been reported earlier for the leaves and sheaths of Z. marina. Chemical analysis of the isolated cuticle by attenuated total reflectance–infrared Fourier transform spectrometry and gas chromatography–mass spectrometry detected C16 and C18 fatty acids, ω-hydroxy fatty acids and β-sitosterol as components of the wax and cutin. The outermost cuticle layer was also observed to be covered with a slimy layer consisting of some polysaccharides. These results suggest that the extremely thick cuticle and slimy layer could play a significant role in protecting the cotyledons from environmental stresses at the pre-emergence stage and just after emergence.

Effects of the Loess Coating on Seed Germination and Seedling Growths of the Eelgrass, Zostera marina

Seagrass bed is an important component in coastal and estuarine ecosystems, providing food and habitats to a wide variety of marine organisms. Recently, seagrass coverage has declined significantly due to anthropogenic impacts such as cultural eutrophication and reclamation, and thus efforts are under way to prevent further losses and restore disturbed seagrass habitats worldwide. Seagrass transplantation techniques for habitat restoration include vegetative and seed-based methods. Seagrass seeds can be collected easily, and sowing seeds is an economically effective method for large-scale restoration. However, large numbers of seed can be lost by seed predation and physical disturbance in the planting areas. In the present study, Zostera marina seeds were coated with loess to reduce seed loss by predation and sweeping away by the water currents, and germination rates of coated seeds and seedling growth were examined to assess the feasibility of the seed-coating method for large-scale restoration. Germination rate of the coated seeds with loess was significantly higher than that of the uncoated seeds. Additionally, seedling growths were not significantly different between the coated and the uncoated seeds. These results suggest that coating of eelgrass seeds with loess enhances success of seed germintion with no harmful effects on seedling growth. Therefore, the seed coating method using loess may be an effective and applicable seedbased transplanting technique for large-scale restoration.

Growth characteristics of Zostera marina seedlings under anaerobic conditions

Seeds of Zostera marina L. are capable of germinating in anaerobic (nitrogen-gassed) seawater (10%) and developing into seedlings with unusual morphology. The axial hypocotyl of seedlings grown in light and dark was characteristically elongated (mean in dark = 32 mm), but the pumule and adventitious roots remained rudimentary and embryonic in appearance. Aerobic seedlings by contrast exhibited little hypocotyl elongation (mean in dark = 5 mm), and plumule nad roots were well developed. The stalk-like blade of the cotyledon elongated in both aerobic and anaerobic cultures, but growth of the cotyledonary sheath surrounding the plumule was most marked in the presence of oxygen. When seedlings were grown anaerobically in the dark for 16 days and then transferred to aerobic-light conditions, elongation of the axial hypocotyl ceased instantly, but plumule and cotyledonary sheath growth was evident within 32 h, and by 96 h after transfer there was pronounced greening of the plumule leaves and swelling of the root primordia. Hypocotyl elongation of seedlings from seeds planted in sediment varied directly with planting depth. Elongation of the hypocotyl pushed the cotyledonary node with rudimentary plumule and root primordia up through the sediment to a position below the sediment surface where hypocotyl elongation stopped and plumule and root growth commenced. The cotyledonary blade which grew concomitantly with the axial hypocotyl continued to elongate after protruding from the sediment into the overlying water. Results from experiments in which the distal part of a seedling cotyledon was exposed to aerated water while the rest of the seedling was maintained in N 2-gassed water suggested that the cotyledon may function as a conduit for oxygen diffusion to the plumular region of the seedling.

Nitrogen content of seeds of Zostera capricorni and Zostera marina

The nitrogen (N) content of mature seeds of Zostera capricorni Aschers, was found by Kjeldahl analysis to be 1.70% of dry matter. The larger seeds of Zostera marina L. were analysed individually by the more sensitive Heraeus procedure. The mean seed N content of these seeds was 1.37% of dry matter (range 1.20–1.56%, standard deviation (SD) 0.13). Seed water contents were similar for both species, 34.7% and 32.3% of seed mass, respectively. The ecological, physiological and evolutionary significance of these observations is discussed.

Phenology and reproductive biology of eelgrass ( Zostera marina L.) at Bahia Kino, sea of Cortez, Mexico

At Bahia Kino, Mexico, seeds of Zostera marina L. (eelgrass) germinate in October and early November. By January seedlings are widespread throughout the area. A very rapid vegetative growth ensues. By March all eelgrass at the depth limit (7 m) is flowering; and most is flowering in shallow water (low tide to 4 m depth). In April all plants are in flower; a few plants display mature seeds. Windrows of detached plants occur on the beaches. In May all reproductive plants have mature seeds, and few attached plants remain. In July all eelgrass in the area has disappeared. All reproductive activities and plant detachment are completed before the lethal upper limit for the species is reached (30°C). Thus, eelgrass at Bahia Kino is a true annual and represents an ultimate response to high water temperatures.

Field studies on seed germination and seedling development in Zostera marina L.

Seeds of Zostera marina L. were collected in July 1978 and July 1979 and planted within two weeks in sediment-filled containers in shallow water at Northwest Creek, New York. The containers were retrieved at periodic intervals over the next 12 months and the seeds and seedlings examined. Between 76 and 93% of the recovered seeds germinated; primarily during October–December, 3–4 months after release. Seedling growth and development occurred during the autumn and spring, but not in winter. A progressive loss of seedlings was observed during the winter and spring, however, and none survived past May. Three stages of seedling development were distinguished. Stage 1 represented germination including emergence and straightening of the cotyledon. Stage 2 seedlings exhibited continued growth of the cotyledon and variable, but often extensive, elongation (2–50 mm) of the axial hypocotyl. The plumule remained undeveloped (length ⩽ 3 mm), and there were no hairs on the basal hypocotyl. Stage 3 seedlings showed the same range in axial hypocotyl length, but they were distinguished by the onset of plumule growth and hypocotyl hair formation.

Transplantation of seagrasses, with special emphasis on eelgrass, Zostera marina L.

A background, listing the history of transplanting seagrasses is given. There is a need for seagrass field planting methods owing to the extremely high productivity of seagrasses and the perturbations they are subjected to. I have planted vegetative stocks and seeds of Zostera marina L. (eelgrass) across tidal zones, across latitudinal zones (from Alaska into Puget Sound), and into water deeper than indigenous growth. Data from these plantings indicate: (1) varietal distinctions based on leaf dimensions are invalid; (2) the possibility of local physiological race distinction in eelgrass; (3) the depth limit of eelgrass is due to a lack of suitable light deeper than a certain minimum depth; and (4) eelgrass seedlings require a higher light intensity than is present deeper than a certain minimum depth. Thus, transplanting experiments yield much more data concerning seagrass biology than just planting method information.

STUDIES OF THE DEVELOPMENT OF ZOSTERA MARINA L.: II. GERMINATION AND SEEDLING DEVELOPMENT

Seeds of Zostera marina L. are shed in Prince Edward Island waters during mid to late summer and germinate the following spring. Occasionally seeds germinate in nature before winter; they can be germinated during autumn and winter in the laboratory. Elongation of cells of the cotyledon and doubled-back axial hypocotyl pushes the embryo out of the seed coat. Cell increase plays no part in this or subsequent elongation of cotyledon and hypocotyl. Leaves emerge through the slit-like opening at the top of the cotyledonary sheath. Hairs develop from cells of the convex surface of the basal swollen hypocotyl; two adventitious roots grow from the cotyledonary node, develop root hairs, and anchor the seedling. Stelar tissues of the hypocotyl and cotyledon differentiate at germination. No characteristically thickened tracheary elements differentiate in hypocotyl or cotyledon. In the hypocotyl, phloem differentiates around the central xylem parenchyma and abuts externally on endodermis which develops Casparian strips. Xylem parenchyma differentiates adaxially against the phloem in the single cotyledonary strand. Differentiation of procambium and vascular tissues in the leaf primordia follows as these expand. Vascularization of the stem nodes is complicated by differentiation of strands to the roots and leaves. All internodes except the first have one pair of cortical bundles. The pair of lateral veins of the first true leaf differentiate in connection with the stele at that node; the lateral bundles from each later-formed leaf pass through one internode as cortical bundles and unite with the stele in the node below.