Networks of highly branched stigmarian rootlets developed on the first giant trees
Edited by Peter R. Crane, Yale School of Forestry and Environmental Studies, New Haven, CT, and approved February 29, 2016 (received for review July 22, 2015)
Significance
Coal swamps were the carbon burial factories of the Carboniferous period, forming huge coal deposits and driving climate cooling. The Carboniferous forests were also home to the first giant (>50 m) trees to grow on the planet. These trees were anchored by a unique structure termed a stigmarian system, which is hypothesized to represent a leafy shoot modified to function as a root. Here, we report the discovery of the complex, highly branched rootlet structure of these trees. Our findings demonstrate that rootlet architecture is conserved from the giant extinct trees of the Carboniferous to the small extant herbs of today’s flora.
Abstract
Lycophyte trees, up to 50 m in height, were the tallest in the Carboniferous coal swamp forests. The similarity in their shoot and root morphology led to the hypothesis that their rooting (stigmarian) systems were modified leafy shoot systems, distinct from the roots of all other plants. Each consists of a branching main axis covered on all sides by lateral structures in a phyllotactic arrangement; unbranched microphylls developed from shoot axes, and largely unbranched stigmarian rootlets developed from rhizomorphs axes. Here, we reexamined the morphology of extinct stigmarian systems preserved as compression fossils and in coal balls from the Carboniferous period. Contrary to the long-standing view of stigmarian systems, where shoot-like rhizomorph axes developed largely unbranched, root-hairless rootlets, here we report that stigmarian rootlets were highly branched, developed at a density of ∼25,600 terminal rootlets per meter of rhizomorph, and were covered in root hairs. Furthermore, we show that this architecture is conserved among their only extant relatives, herbaceous plants in the Isoetes genus. Therefore, despite the difference in stature and the time that has elapsed, we conclude that both extant and extinct rhizomorphic lycopsids have the same rootlet system architecture.
Sign up for PNAS alerts.
Get alerts for new articles, or get an alert when an article is cited.
The spread of the first wetland forests with tall trees during the Carboniferous period (359–300 million years ago) had a dramatic impact on the carbon cycle by burying large amounts of organic carbon in the form of peat in coal swamps (1, 2). Lycophyte trees up to 50 m in height (3, 4) were dominant components of coal swamp forests (5, 6). They were key components of coal-forming environments throughout the Carboniferous period but dominated in the lower–middle Pennsylvanian (Namurian–Wetsphalian) where they typically contribute between 60% and 95% of the biomass in buried peat (7–13). The preserved remains of lycophyte trees form some of the most extensive fossil plant deposits of any geological period. This is in part because of their size and ecological dominance but also the result of the high probability of preservation in the waterlogged conditions in which these trees grew (4). Detailed descriptions of the morphology of these plants on a range of scales—from entire in situ tree lycophyte forests (14, 15) to cellular descriptions of developing spores (16)—have made these trees some of the best understood fossil plants of the Carboniferous coal swamps.
The rooting system of the arborescent lycopsids—stigmarian systems—consist of large shoot-like axes (rhizomorphs) that develop lateral organs called rootlets (4, 17–20). Rootlets, which have been described as largely unbranched and root hairless (4, 5, 17–24), are arranged in a characteristic pattern or rhizotaxy on the rhizomorph (25). It is the arrangement of these largely unbranched leaf-like rootlets on a shoot-like axis that first led to the theory that stigmarian systems were modified leafy shoots (26–28). The modified shoot hypothesis got more support toward the end of the 20th century. The discovery of fossilized embryos showing that the shoot and root axes were derived from a branching event during embryogenesis (16, 29), the documentation that rootlet abscission resembled foliar abscission (17, 18), observations on well-preserved fossil rhizomorph apices (30–32), and their interpretation within a phylogenetic context (31, 33) led to a complete revival of Schimper’s (27) modified shoot hypothesis.
The only living relatives of these Carboniferous giant trees are small herbaceous plants in the genus Isoetes (24, 33–35). The rooting system of Isoetes also consists of a rhizomorph meristem that develops rootlets in a regular rhizotaxy (31, 36, 37). Aside from the reduction and modification of the rhizomorph, the rooting systems of Isoetes and the tree lycopsids are morphologically similar (4, 19, 21, 31, 38–40). However, current models suggest that rootlet architecture is different in extant Isoetes and extinct stigmarian rootlets. Isoetes rootlets form dense, highly branched networks of rootlets covered in root hairs (41, 42), whereas stigmarian rootlets are thought to be largely unbranched and root hairless (4, 5, 17–24). This difference is even more puzzling because the cellular anatomy of stigmarian rootlets and Isoetes rootlets is almost identical (21, 38).
Given that the architecture of the stigmarian rootlet systems differs markedly from rooting systems of their extant relatives, we hypothesized that the rootlet architecture of the stigmarian rooting system may have previously been misinterpreted. Here, we report the discovery of the complex structure of stigmarian rootlet systems from quantitative analysis of rootlet branching and multiple lines of geological evidence. The proposed model reveals that the highly branched rootlet architecture has been conserved over the past 300 million years and is found in the closest living relatives of arborescent lycophytes.
Results
To compare rootlet architecture of the stigmarian systems with their extant relatives, we first defined quantitatively rootlet branching in Isoetes echinospora Durieu and Isoetes histrix Bory (Fig. S1). Isoetes rootlets branch dichotomously along their length, and rootlet diameter decreases by ∼25% at each dichotomy (Fig. 1 A, B, D, E, and H); the average diameter of the rootlet that develops from the rhizomorph is 0.73 mm (SD, ±0.21 mm; SE, ±0.02 mm), and the average rootlet diameter of the fifth-order branch is 0.21 mm (SD, ±0.04 mm; SE, ±0.002 mm) after four rounds of dichotomous branching (X4 in Fig. 1E). Rootlet diameter does not decrease between branch points, i.e., the branches do not taper (Fig. 1 G and H). These data indicate that Isoetes rootlets are highly branched—there are up to five orders of branching on each rootlet—and decrease in diameter by ∼25% at each dichotomy but do not taper.
Fig. 1.
Fig. S1.
To test the hypothesis that stigmarian rootlets formed branched networks like Isoetes, we characterized the branching morphology of rootlets preserved as compression fossils in Carboniferous sediments (Supporting Information and Fig. S2). We found rootlets with up to four orders of branching (Fig. 1C). Furthermore, rootlet diameter decreased by ∼25% with each order of branching (Fig. 1 D, F, and I) with no evidence of tapering (Fig. 1 G and I). Together, these data from rootlets preserved as compression fossils demonstrate that stigmarian rootlets were branched and rootlet diameter decreased by ∼25% at each branch point and did not taper. This indicates that the pattern of rootlet branching is similar in extinct stigmarian and extant Isoetes rootlets.
Fig. S2.
To test independently the hypothesis that stigmarian rootlets were highly branched, we modeled the predicted frequency of rootlet diameters sampled from thin sections of coal balls (Fig. S3). Coal balls are permineralized peat from the coal swamps in which the anatomical and cellular detail of growing stigmarian rootlets are preserved in situ (4, 43–45). If rootlets branched dichotomously (Fig. 2A), as we observed in the compression fossils described above (Fig. 1C), we hypothesized that there would be more thin rootlets than thick rootlets in coal ball-preserved stigmarian systems (Fig. 2C, red). This is because of the geometric increase in the number of progressively smaller terminal rootlets in the dichotomously branching stigmarian rootlet system. However, if rootlets were relatively unbranched, as the long-standing model suggests, and therefore decreased in diameter by tapering (Fig. 2B), we would expect to observe equal numbers of small- and large-diameter rootlets in a sample of roots preserved in coal balls (Fig. 2C, blue). Our model therefore allows us to determine whether stigmarian rootlets preserved in coal balls were unbranched or branched.
Fig. 2.
Fig. S3.
We measured the diameter of 785 stigmarian rootlets preserved in 94 coal ball thin sections (Supporting Information) (Fig. S4). Rootlet diameter was then calculated, and the frequency of rootlets in each 0.5-mm-diameter class was calculated (Fig. 2D). These data demonstrate that there are many more small-diameter rootlets than large-diameter rootlets, which supports the dichotomous branching rootlet model. To ensure that the distribution of rootlet diameter is not due to variation in local growth conditions we plotted separately the data from 16 individual thin sections with more than 15 stigmarian rootlets and thin sections collected from a variety of collection sites in Central Britain [Yorkshire and Lancashire coal fields (46)] (Figs. S5 and S6). In all cases, the same frequency distribution was observed—there were more (Figs. S5 and S6) small-diameter rootlets than large-diameter rootlets. This demonstrates that the relationship between rootlet diameter and frequency does not vary from place to place or in different samples. This is consistent with the model in which the stigmarian rootlets are highly branched, and this branching pattern did not vary from site to site or from sample to sample.
Fig. S4.
Fig. S5.
Fig. S6.
To determine whether young, developing rootlets near the rhizomorphs apex could have contributed to the large numbers of thin rootlets in our sample, we measured the diameters of the earliest stages of rootlet development on two preserved apices (Fig. S7 A and C). Mean rootlet scar diameter within the first 10 cm from the rhizomorph apex was 3.48 mm (SD, ±0.95). The measurements of the rootlet scars on these apices are similar to those observed on other stigmarian apices (30, 32). The diameter of these young rootlets were more than twice the diameter of the most abundant, small-diameter rootlets that we observed in coal balls (Fig. 2D). This demonstrates that the youngest developing rootlets near the apex of the rhizomorph could not account for the large numbers of thin rootlets observed in coal balls. Instead, it supports the hypothesis that the thin rootlets were formed through progressive rounds of dichotomous branching of large rootlets.
Fig. S7.
To verify independently that stigmarian rootlets branched, we searched the coal ball thin sections for rootlets in which there were two vascular strands. Stigmarian rootlets have a single vascular strand (4, 17, 21, 22) (Fig. 3E, white arrowhead); however, above a point of branching, the vascular strand locally bifurcates (Fig. 2F, white arrowheads). Finding rootlets in coal balls containing two vascular strands therefore indicates that rootlets in coal balls branched (Fig. 2G, white arrowheads). Of the 785 samples observed, twin vascular strands were found in 51 rootlets (Fig. 2G, white arrowheads), demonstrating that these sections were made just above a branch point (Fig. S8). Of these, the circumference of 42 rootlets could be measured. Twin vascular strands were observed in rootlets with diameters ranging from 1.1 to 12.8 mm, indicating that rootlets of all size classes branched (Fig. S8). The frequency distribution of the diameters from the 42 rootlets further supported the branched-rootlet model (Fig. S8). The observed peak frequency diameter of branching rootlets was 2–2.5 mm (Fig. S8), indicating that the smallest and most frequent rootlets undergoing dichotomous branching were in the 2- to 2.5-mm-diameter range. We previously showed that daughter rootlet diameter is ∼73% the diameter of the rootlet from which they formed. According to this measure, the peak diameter that we would expect to see produced from the branching of 2- to 2.5-mm-diameter rootlets would be in the 1.5- to 1.8-mm-diameter range. The peak frequency rootlet diameter found in all rootlets examined being between 1 and 2 mm (Fig. 2D). Taken together, diameter frequency distributions of rootlets preserved in permineralized coal balls demonstrate that stigmarian rootlets were highly branched.
Fig. 3.
Fig. S8.
Root hairs have not previously been found on stigmarian rootlets and their absence led to the suggestion that root hairs did not develop in these plants (5, 20, 22, 23). However, because root hairs develop on Isoetes rootlets (41, 42, 47) (Fig. 3 A–C, black arrows), we hypothesized that root hairs would have formed on stigmarian rootlets. A total of 21 root hairs were discovered on nine stigmarian rootlets on seven individual thin sections made from different coal balls (Fig. 3 D and E; Supporting Information and Fig. S9). Mean stigmarian root hair diameter was 14.3 µm (SD, ±2.6 µm; SE, ±0.56 µm), and the root hair highlighted with an arrow in Fig. 3E was 13.9 µm in diameter. Root hair diameter of the two Isoetes root hairs shown in Fig. 3C were 9.2 and 10.7 µm. These data indicate that root hairs developed on stigmarian rootlets and that they were morphologically similar to the root hairs that develop on extant Isoetes species.
Fig. S9.
The lycopsid trees of the British Carboniferous wetland forests comprised both sigillarian and nonsigillarian species (7, 33, 46). To determine whether the rootlet branching pattern was the same in each, we scored the presence of rootlets with twin vascular strands and determined the distribution of rootlet diameters in both sigillarian and nonsigillarian rootlets. There is a “connective” of cortical tissue between the vascular trace and the outer cortex in sigillarian rootlets (4, 17, 18, 21, 48) (Fig. S10A). By contrast, there is no connective in the central cavity of the nonsigillarian rootlet and the central vascular trace is free within the rootlet cavity (4, 17, 21) (Fig. S10B). First, we identified twin vascular stands in both sigillarian and nonsigillarian rootlets, which indicates both sigillarian and nonsigillarian rootlets in the coal balls sampled were branching (Supporting Information). Second, the frequency distribution of rootlet diameters is similar for sigillarian and nonsigillarian rootlets (Fig. S10C) (Supporting Information). These data indicate that both sigillarian and nonsigillarian rootlets branched three to four times (Supporting Information). Furthermore, root hairs are present on both sigillarian and nonsigillarian rootlet types (Supporting Information). We conclude that both sigillarian and nonsigillarian rootlets formed similar bifurcating rootlets systems to those found in Isoetes today.
Fig. S10.
Using the quantitative data from this analysis, we constructed a model for the stigmarian system (Fig. 4). Because rootlets developed at densities of ∼1,600 rootlets per m of rhizomorph (25) (this study) and we assumed that each rootlet branched at least four times (this study), we calculated a density of 25,600 terminal rootlets per m of rhizomorph with a surface area 5.5 times larger than unbranched rootlet systems (assuming that living root hairs are present only on the terminal two orders of branching) (Methods). This model shows a stigmarian system with a densely packed cylinder of interwoven rootlets around the rhizomorph axes (Fig. 4).
Fig. 4.
Discussion
We demonstrate that the rootlets of stigmarian systems were highly branched—branching dichotomously up to five times—and were covered in root hairs. We verified the highly branched architecture through quantitative analysis of the numbers and diameters of stigmarian rootlets preserved in coal balls. Analysis of the size distribution of stigmarian rootlets in coal balls provided us with the unique opportunity to investigate the entire population of stigmarian rootlets growing in situ regardless of either the diameter of the rootlet or the proximity of the rootlet to the rhizomorph axis. This analysis was possible because stigmarian rootlets are ubiquitous in coal balls (49, 50), and can be readily identified because of their unique cellular anatomy composed of three zones of cortex, the middle of which rapidly disintegrates, leading to the formation of a large air space containing the inner cortex and central vascular strand (17, 21). This anatomical detail allows stigmarian rootlets to be easily distinguished from the rooting structures of other plants that grew in the coal swamps (Fig. S4). Furthermore, the exquisite cellular preservation of these in situ fossils allowed the visualization of root hairs developing from the rootlet epidermal surface for the first time (to our knowledge). Such an extensive branched system would have formed a subterranean network with a large surface area available for nutrient uptake and tethering these giant trees in place.
We suggest that the previous model for stigmarian rooting systems was incomplete because it was based on compression fossils in which the full extent of the rootlet network was obscured. Furthermore, isolated stigmarian rootlets preserved in compression fossils have few features distinguishing them at this coarse level of preservation, making them difficult to identify (51). Therefore, isolated branched rootlets have not contributed to the construction of the long-standing model of stigmarian rootlet architecture. This means that previous interpretations of stigmarian systems were biased; reconstructions were based on the proximal portions of the rootlets where they attach to the rhizomorphs and could be identified unequivocally. However, because rootlets can extend for over 90 cm from the rhizomorph surface (52–55), this bias means that the morphology of the distal branched regions of the rootlets remained undescribed. Through quantitative analysis of rootlet architecture in both compression and in situ-preserved permineralized fossils, we have been able to demonstrate that stigmarian rootlets were highly branched.
Highly branched rootlets would have contributed to the anchoring of these giant trees. Branched root structures are between twice and seven times more resistant to pull-out compared with unbranched structures (56–58) and the discovery of root hairs would not only have increased the surface area but would have further contributed to anchorage (59). The tree lycophytes would have formed large root plates as individual rhizomorph axes could extend for over 12 m (19) from the trunks of large trees. Given that tree lycopsids have additionally been reported to grow at high densities [up to 1,769 stems per ha (15)] in coal swamp forests (14, 60), root plates would have also interlocked with neighboring stigmarian systems. Highly branched rootlets would have further consolidated these extensive root plates (Fig. 4). It is the ability of root plates to resist movement when the aerial parts of the tree are subjected to lateral force that provides structural support to tall trees (61). We predict that highly branched stigmarian rootlets would have contributed to the anchorage of these giant trees.
The first giant wetland trees to grow on Earth with their unique stigmarian rooting systems have attracted the attention of scientists for well over 150 y (4, 17–19, 21, 31, 52, 62). Recent studies have built on this foundation of knowledge and have shed fresh light on physiological mechanism controlling their development, structure, and interaction with other organisms (63–66). The discovery that stigmarian rootlets were highly branched, developed root hairs and share the same branching architecture as extant Isoetes rootlets reveals a remarkable conservatism in rootlet architecture between the first giant trees and their only living herbaceous relatives.
Methods
Isoetes Collection and Plant Growth.
Isoetes histrix was collected in March 2014 on the Lizard Peninsula (Cornwall, UK) with the permission of the National Trust and Natural England. Isoetes echinospora was collected in September 2013 and 2014 from North West Sutherland (Scotland, UK) with the permission of the John Muir Trust and the Scourie Estate. Isoetes histrix plants were grown in Levington M2 compost. Isoetes echinospora were grown submerged in aquaria in Levington M2 compost topped with coarse gravel. Both were grown at 20 °C with a 16-h photoperiod.
Quantifying Isoetes and Stigmarian Rootlet Architecture.
Isoetes rootlets were imaged with a Leica M165 FC (Fig. 1B and Fig. S1 A–I). Isoetes rootlet diameter was measured using Fiji (67) (Fig. 1H and Fig. S1 A′–I′). SD and SE were calculated using Microsoft Excel 2013. Graphs were plotted using Microsoft Excel 2013. Box plots were made in RStudio (2013) (68). To establish whether rootlets taper, the diameter of 167 rootlet segments (only including segments covered by five or more diameter measurements) were plotted against distance along their respective rootlets. A linear trend line was then applied to each data series allowing the gradient of each trend line to be calculated (Microsoft Excel 2013). An average gradient of y′ = −0.0011x (Fig. 1G) was calculated from the 167 rootlet segments. Decrease in rootlet diameter at each branching point was calculated by comparing the average diameter of 227 daughter rootlets with the average diameter of their parent rootlet. Daughter rootlets had an average diameter of 74% of their parent rootlet. Stigmarian rootlet architecture was quantified using the same method described above for Isoetes rootlets (Fig. 1C and Fig. S2 A–D). The same method was used to investigate whether stigmarian rootlets tapered; this time, the average rootlet gradient was calculated from 40 rootlet segments giving an average rootlet gradient of y = 0.0067x (Fig. 1G). Average decrease at each branching point was calculated in a similar fashion to Isoetes using the measurements of 36 daughter rootlet diameters compared with their parent rootlets. Daughter rootlets had an average diameter of 73% of their parent rootlet.
Modeling the Predicted Frequency of Rootlet Diameters in Coal Balls.
The model is based on the principle that the length of a rootlet segment is equal to the frequency of finding that segment in a random rootlet sample from coal balls.
The branched rootlet (Fig. 2A and Fig. S3A).
The branched rootlet model undergoes four rounds of dichotomous branching, resulting in 16 terminal rootlets. At each dichotomy, the diameter of the daughter rootlet is 0.73 that of the parent rootlet (based on the measurements made in this study). After a bifurcation point, the daughter rootlet segment is 0.92 the length of the previous segment. This value is based on measurements of 96 Isoetes daughter rootlet segments compared with their parent rootlet segments (only using rootlet segments that started and terminated with a branching point to avoid the bias of using rootlets that had not finished growing or had been broken off).
The tapered rootlet (Fig. 2B and Fig. S3B).
The tapered rootlet is made up of five segments of equal length. Each segment is 0.73 the diameter of the previous segment, such that the size decrease (tapering) between the branched rootlet and the tapered rootlet is the same.
The model (Fig. S3C).
To compare between the two types of rootlets an initial starting diameter (D) and a combined length of the five segments (L) was assumed for both rootlets. From this, it is possible to calculate the length (frequency) of finding a particular diameter (D) of rootlet segment. To determine a realistic value for diameter (D), a starting diameter of 6 mm (4, 12, 17, 20) was used (Fig. 2C). After four orders of decreasing in diameter by 0.74, this results in a terminal rootlet diameter of 1.7 mm, a value similar to the terminal rootlets of the compression fossil (Fig. 1C) and approaching the smallest sizes of isolated stigmarian rootlets previously reported from coal balls (48) interpreted as coming from distal portions of stigmarian systems (18).
Measuring the Diameter of Stigmarian Rootlets from Coal Balls.
Thin sections prepared from Carboniferous coal balls held in the Oxford University Herbaria (97 slides) and Oxford University Museum of Natural History (42 slides) were inspected, and stigmarian rootlets were identified. All of the available slides were used rather than only those that were made to display stigmarian systems, to take an unbiased approach. Images were captured of 785 rootlets from 94 thin sections with a Leica M165 FC stereo microscope. The circumference of 785 rootlets was measured using Fiji (67). The rootlets were grouped into 0.5-mm size bins and plotted on a histogram (Fig. 3C) (Microsoft Excel 2013).
Measurement of Rootlet Scar Diameter on Rhizomorph Apices.
Two rhizomorph apices were photographed by A.J.H. in the collections of The University of Manchester, Manchester Museum. The diameter of 36 rootlet scars were measured from the well-preserved apex (Collection No. LL. 15952.470; Fig. S7A), and 12 rootlet scars we measured form the poorly preserved apex (Collection No. LL. 15952.471; Fig. S7C) using Fiji (67) (Fig. S8 A and B). Average rootlet scar diameter was plotted for each 2-cm interval from the apex (Fig. S7 A′ and B′) (Microsoft Excel 2013).
Isoetes and Stigmarian Root Hairs.
The distal portion of a single Isoetes echinospora rootlet was imaged using a Leica M165 FC stereo microscope (Fig. 3A). Additional rootlets were embedded in paraffin, sectioned, and stained with toluidine blue. Slides were imaged with an Olympus BX50 compound microscope using bright field (Fig. 3 B and C). Stigmarian root hairs were imaged with an Olympus BX50, and root hair diameter was measured with Fiji (67).
Estimating Surface Area Increase.
To estimate the increase in surface area due to branching, we again used the simplified rootlet models (branched and unbranched; Fig. 3 A and B, and Fig. S3 A–C). Rootlets were assumed to be cylindrical and the surface area of each segment of rootlet was calculated with SA = πdh. The presence of branching results in the branched-rootlet model having a surface area 3.9 times larger than the tapered model. Next, we included an estimate of the increased surface area provided by root hairs. Dittmer (69) estimated that the surface area of the rye (Secale cerale L.) root systems was 6,875.4 ft−2, with root axes contributing 2,554.09 ft−2 and the root hairs contributing 4,321.31 ft−2. We therefore assumed that an axis with root hairs has a surface area 1.7 times that of the same axis lacking root hairs. The Dittmer (69) estimate is applicable for the stigmarian system as 50% of the epidermal cells in Isoetes form root hairs (70) as they do in rye (71). As a conservative estimate, we did not take into account root hairs on the top three branching orders (where they may have been sloughed off in the soil) but estimated that root hairs would contribute an additional 1.7 times the surface area over the final two orders of branching. This resulted in our branched-root model having a surface area 5.5 times that of the tapered model (4, 17, 20–22).
Stigmarian Rootlets
Eight branched stigmarian rootlets were identified from five compression fossils. Four branched stigmarian rootlets were found in the photographic collections of the British Geological Survey and reproduced with the permission of the British Geological Survey (CP15/032):
Two branched rootlets were identified in Asset No. P685925 (Fig. S2A).
Caption: Stigmaria ficoides—dichotomous rootlets. Monckton Main Coll, Barnsley. (Fossil plant).
Description: Stigmaria ficoides—dichotomous rootlets. Kidston negative number: Kidston 50. Thin section no. Kidston 2600. Magnification, 100×. Half plate. Box 1.
Date taken: 01/01/1899.
Photographer: Kidston, R.
One branched rootlet was identified in Asset No. P687428 (Fig. S2B).
Caption: Stigmaria ficoides (Sternberg). Maltby Bore, Rotherham. (Fossil plant).
Description: Stigmaria ficoides (Sternberg). Kidston negative number: Kidston 1648. Magnification, 100×. Quarter plate. Box 3.
Date taken: 01/01/1911.
Photographer: Kidston, R.
One branched rootlet was identified in Asset No. 687585 (Fig. 1C).
Caption: Stigmaria/dichotomously divided rootlet. Shipley Clay Pit, Ilkeston. (Fossil plant).
Description: Stigmaria/dichotomously divided rootlet. Kidston negative number: Kidston 1836. Dr. Moysey's specimen. Magnification, 100×. Quarter plate. Box 4.
Date taken: 01/01/1912.
Photographer: Kidston, R.
One branched rootlet was photographed by A.J.H. in the collections of the London Natural History Museum.
Collection No. V.24929 (Fig. S2C).
Description: Stigmarian rootlet (branching).
Formation: Coal Measures.
Locality: Hensies Boring, nr, MONS. Pit No. 1. 786 m.
Three branched rootlets were photographed by A.J.H. in the collections of The University of Manchester, Manchester Museum.
Collection No. W.1896 (Fig. S2D).
Description: Stigmaria (dichotomizing branches).
Location: Barnsley, 625 yd deep.
Coal Balls Thin Sections
All Carboniferous coal ball thin sections from the Oxford University Herbaria (97 slides) and Oxford University Museum of Natural History (42 slides) were examined for the presence of stigmarian rootlets. All of the available slides were used rather than only those that were made to display stigmarian systems, to take an unbiased approach. All slides examined were believed to come from individual coal balls because there was no evidence of sequential sections on consecutive slides. Most slides lack maker’s marks, but 38 are marked as being produced by James Lomax (72). Individual coal ball slides thus provide us with individual time points during the swamp ecosystem. The coal balls used were also collected from a variety of collection sites in the Lancashire and Yorkshire coal fields (46). The Carboniferous plants from the Lancashire and Yorkshire coal deposits were the principal material used for the early anatomical characterizations of the stigmarian system (19, 48–50, 52, 73), thus making these slides ideal for a reinvestigation.
Rhizomorph Apices
Two rhizomorph apices were photographed by A.J.H. in the collections of The University of Manchester, Manchester Museum.
Collection No. LL. 15952.470.
Description: Stigmaria ficoides, Brong. (root contracting to point). Coal Measures. E Coll Williamson.
Collection No. LL. 15952.471.
Description: Stigmaria ficoides, Brong. (root contracting to point). Coal Measures. E Coll Williamson.
Stigmarian Root Hairs
The same slides used to investigate stigmarian rootlet diameter were investigated for the presence of root hairs as well as additional collections of acetate peels (74) from the teaching collection in the School of Earth and Ocean Sciences, Cardiff University. Nine rootlets were found with stigmarian root hairs (Fig. 3 D and E, and Fig. S9 A–J). The collection localities of the coal balls containing root hairs are as follows: Oxford University Herbaria 78, 94, and 95 were from individual coal balls from Dulesgate (46); Manchester Museum LL15952, unknown locality. The acetate peels from the teaching collection in the School of Earth and Ocean Sciences, Cardiff University, are of unknown locality but are believed to be have been made for Prof. A. G. Lyon by his technician from coal balls collected at the Sutcliffe (quarry/mine) (Burnley, UK).
Sigillarian Rootlets Branched
To test the hypothesis that sigillarian rootlets were highly branched, we scored each of the 785 rootlets for the presence of a connective characteristic of sigillarian rootlets. There was a connective in 122 and no connective in 464, and 199 rootlets could not be scored equivocally. This indicates that ∼20% of the rootlets that could be scored for the connective were sigillarian. The frequency of sigillarian and nonsigillarian rootlet diameters was plotted (Fig. S10C). The distribution of diameter frequencies of sigillarian and nonsigillarian rootlets was similar: there were few large-diameter rootlets and many small-diameter rootlets. The observation that there are many more small-diameter rootlets than large-diameter rootlets in both sigillarian and nonsigillarian rootlets demonstrates that both were highly branched and supports the branched-rootlet model reported in Fig. 2C.
To independently verify that sigillarian rootlets branched, we scored for the presence of a connective in the 51 rootlets identified with twin vascular strands. There was a connective in seven rootlets with twin vascular strands, indicating that they were sigillarian. Twenty-two rootlets with twin vascular strands lacked a connective, indicating that they were nonsigillarian rootlets. The connective could not be scored in the remaining 22 sectioned rootlets. This presence of twin vascular strands in rootlets with and without connectives indicates that both sigillarian and nonsigillarian rootlets branched.
The maximum diameter of a sigillarian rootlet found in our analysis was 4.1 mm. If a typical sigillarian rootlet starts with an initial diameter of 3–4 mm, which is consistent with our observed results (Fig. S10C), it would require three to four orders of branching for the rootlet diameter to decrease to the peak rootlet size of 1–1.5 mm. With the removal of sigillarian rootlets, the peak rootlet frequency of nonsigillarian rootlets is 1.5–2 mm in diameter. If a typical nonsigillarian rootlet starting diameter is 5–7 mm (4, 17, 20–22), it would decrease in diameter to this peak frequency through three to four orders of branching (four orders for any rootlet starting with a diameter of >5.2 mm). We conclude that the majority of rootlets—sigillarian and nonsigillarian—underwent approximately four orders of branching.
To determine whether root hairs developed on both sigillarian and nonsigillarian rootlets, we determined whether connectives were present in rootlet sections on which root hairs had been identified. Both sigillarian (four rootlets; Fig. 3D and Fig. S9 A–E) and nonsigillarian rootlets (two rootlets; Fig. S9 F and J) developed root hairs. Taken together, these data indicate that both sigillarian and nonsigillarian rootlets were highly branched and developed root hairs similar to the rootlets of extant Isoetes.
Acknowledgments
We are grateful to Dr. K. Bacon, Prof. P. Kenrick, Prof. J. Langdale, Prof. H. Dickinson, Prof. A. M. Hetherington, and Prof. K. Niklas for helpful discussions. L.D. is grateful to Ms. Iris Marston and Prof. Richard Bateman for valuable insights at the beginning of this project. We are grateful to Bill DiMichele and an anonymous reviewer for insightful comments on our manuscript. We are grateful to the John Muir Trust, the National Trust, Natural England, the Scourie Estate, Oxford University Museum of Natural History, Oxford University Herbaria, London Natural History Museum, British Geological Survey, and University of Manchester, Manchester Museum; Dr. N. J. Hetherington, Miss J. N. Shaw, Mr. J. D. Hetherington, and Mrs. R. Wise (Oxford University; for drawing the reconstruction of the stigmarian system and for the drawing of the rhizomorph apex); and Mr. J. Baker (Oxford University; for photographic assistance). This research was supported by a Biotechnology and Biological Sciences Research Council (Grant BB/J014427/1) Doctoral Training Partnership Scholarship (to A.J.H.) and European Research Council Advanced Grant EVO500 (to L.D.).
Supporting Information
Supporting Information (PDF)
Supporting Information
- Download
- 4.55 MB
References
1
T Algeo, S Scheckler, Terrestrial-marine teleconnections in the Devonian: Links between the evolution of land plants, weathering processes, and marine anoxic events. Philos Trans R Soc Lond B Biol Sci 353, 113–130 (1998).
2
R Berner, The carbon cycle and CO2 over Phanerozoic time: The role of land plants. Philos Trans R Soc Lond B Biol Sci 353, 75–82 (1998).
3
BA Thomas, J Watson, A rediscovered 114-foot Lepidodendron from Bolton, Lancashire. Geol J 11, 15–20 (1976).
4
W Stewart, GW Rothwell Paleobotany and the Evolution of Plants (Cambridge Univ Press, 2nd Ed, Cambridge, UK, 1993).
5
TL Phillips, WA DiMichele, Comparative ecology and life-history biology of arborescent lycopsids in Late Carboniferous swamps of Euramerica. Ann Mo Bot Gard 79, 560–588 (1992).
6
WA DiMichele, TL Phillips, Paleobotanical and paleoecological constraints on models of peat formation in the Late Carboniferous of Euramerica. Palaeogeogr Palaeoclimatol Palaeoecol 106, 39–90 (1994).
7
TL Phillips, RA Peppers, WA DiMichele, Stratigraphic and interregional changes in Pennsylvanian coal-swamp vegetation: Environmental inferences. Int J Coal Geol 5, 43–109 (1985).
8
ME Collinson, AC Scott, Implications of vegetational change through the geological record on models for coal-forming environments. Geol Soc Lond Spec Publ 32, 67–85 (1987).
9
AT Cross, TL Phillips, Coal-forming through time in North America. Int J Coal Geol 16, 1–46 (1990).
10
AV Lapo, IN Drozdova, Phyterals of humic coals in the U.S.S.R. Int J Coal Geol 12, 477–510 (1989).
11
ME Collinson, PF Van Bergen, AC Scott, JW De Leeuw, The oil-generating potential of plants from coal and coal-bearing strata through time: A review with new evidence from Carboniferous plants. Geol Soc Lond Spec Publ 77, 31–70 (1994).
12
TL Phillips, WA DiMichele, From plants to coal—peat taphonomy of Upper Carboniferous coals. Int J Coal Geol 16, 151–156 (1990).
13
GM Rex, AC Scott, The sedimentology, palaeoecology and preservation of the Lower Carboniferous plant deposits at Pettycur, Fife, Scotland. Geol Mag 124, 43–66 (1987).
14
M MacGregor, J Walton The Story of the Fossil Grove of Glasgow Public Parks and Botanical Gardens, Glasgow (Glasgow Public Parks and Botanic Gardens Department, Glasgow, UK, 1948).
15
WA DiMichele, PJ Demaris, Structure and dynamics of a Pennsylvanian-Age Lepidodendron forest: In the colonizers of a disturbed swamp habitat Herrin (No. 6) Coal of Illinois. Palaios 2, 146–157 (1987).
16
TL Phillips, Reproduction of heterosporous arborescent lycopods in the Mississippian–Pennsylvanian of Euramerica. Rev Palaeobot Palynol 27, 239–289 (1979).
17
JM Frankenberg, DA Eggert, Petrified Stigmaria from North America. I. Stigmaria ficoides, the underground portions of Lepidodendraceae. Palaeontogr B 128, 1–47 (1969).
18
DA Eggert, Petrified Stigmaria of sigillarian origin from North America. Rev Palaeobot Palynol 14, 85–99 (1972).
19
W Williamson, A monograph on the morphology and histology of Stigmaria ficoides. Palaeontogr Soc 40, 1–62 (1887).
20
EL Taylor, TN Taylor, M Krings Paleobotany: The Biology and Evolution of Fossil Plants (Academic, 2nd Ed, Burlington, MA, 2009).
21
W Stewart, A comparative study of stigmarian appendages and Isoetes roots. Am J Bot 34, 315–324 (1947).
22
Y Lemoigne, Les appendices radiculaires des Stigmaria des lycopodiales arborescentes du Paleozoique. Ann des Sci Nat Bot Paris 12, 751–774 (1963).
23
JC Schoute Manual of Pteridology, ed F Verdoorn (Martinus Nijhoff, The Hague, The Netherlands, 1938).
24
WA DiMichele, RM Bateman, The rhizomorphic lycopsids: A case-study in paleobotanical classification. Syst Bot 21, 535–552 (1996).
25
WA Charlton, J Watson, Patterns of arrangement of lateral appendages on axes of Stigmaria ficoides (Sternberg) Brongniart. Bot J Linn Soc 84, 209–221 (1982).
26
M Renault, Ètude sur les Stigmaria rhizomes et raciness de sigillaires. Ann des Sci Géologiques 12, 1–48 (1882).
27
WP Schimper, Traité de Paléontologie Végétale ou la Flore du Monde Primitive dans ses Rapports avec les Formations Géologiques et la Flore du Monde Actuel, Tome 2 (J. B. Baillière et Fils, Paris). (1872).
28
HG Solms-Laubach Fossil Botany Being an Introduction to Palaeophytology from the Standpoint of the Botanist. Translated into English by Garnsey, H. E and Balfour, I. B (Clarendon, Oxford, UK, 1891).
29
SP Stubblefield, GW Rothwell, Embryogeny and reproductive biology of Bothrodendrostrobus mundus (Lycopsida). Am J Bot 68, 625–634 (1981).
30
GW Rothwell, The apex of Stigmaria (Lycopsida), rooting organ of lepidodendrales. Am J Bot 71, 1031–1034 (1984).
31
GW Rothwell, D Erwin, The rhizomorph apex of Paurodendron: Implications for homologies among the origins of Lycopsida. Am J Bot 72, 86–98 (1985).
32
GW Rothwell, JS Pryor, Developmental dynamics of arborescent Lycophytes—apical and lateral growth in Stigmaria ficoides. Am J Bot 78(12):1740–1745. (1991).
33
R Bateman, W DiMichele, D Willard, Experimental cladistic analysis of anatomically preserved arborescent Lycopsids from the Carboniferous or Euramerica: An essay on paleobotanical phylogenetics. Ann Mo Bot Gard 79, 500–559 (1992).
34
P Kenrick, PR Crane, The Origin and Early Diversification of Land Plants: A Cladistic Study. Smithsonian Series in Comparative Evolutionary Biology. (Smithsonian Institute Press, Washington, DC). (1997).
35
J Xue, Phylogeny of Devonian lycopsids inferred from Bayesian phylogenetic analyses. Acta Geol Sin (English Ed) 85(3):569–580. (2011).
36
DJJ Paolillo The Developmental Anatomy of Isoetes (University of Illinois Press, Urbana, IL, 1963).
37
DJ Paolillo, Meristems and evolution: Developmental correspondence among the rhizomorphs of the lycopsids. Am J Bot 69, 1032–1042 (1982).
38
EE Karrfalt, A further comparison of Isoetes roots and stigmarian appendages. Can J Bot 58, 2318–2322 (1980).
39
WH Lang, Studies in the morphology of Isoetes. I. The general morphology of the stock of Isoetes lacustris. Mem Proc Manchester Lit Philos Soc 59:1–28. (1915).
40
WH Lang, On the apparently endogenous insertion of the roots of stigmaria. Mem Proc Manchester Lit Philos Soc 67:101–107. (1923).
41
D Scott, T Hill, The structure of Isoetes hystrix. Ann Bot (Lond) 14, 413–454 (1900).
42
C West, H Takeda, X. On Isoetes japonica, A. Br. Trans Linn Soc Lond 2nd Ser Bot 8(8):333–376. (1915).
43
MC Stopes, DMS Watson, On the present distribution and origin of the calcareous concretions in coal seams, known as “Coal Balls.”. Philos Trans R Soc Lond B Biol Sci 200, 167–218 (1908).
44
AC Scott, DP Mattey, R Howard, New data on the formation of Carboniferous coal balls. Rev Palaeobot Palynol 93, 317–331 (1996).
45
AC Scott, G Rex, The formation and significance of Carboniferous coal balls. 311(1148):123–137. (1985).
46
J Galtier, Coal-ball floras of the Namurian-Westphalian of Europe. Rev Palaeobot Palynol 95, 51–72 (1997).
47
RG Leavitt, Trichomes of the root in vascular cryptogams and angiosperms. Proc Bost Soc Nat Hist 31, 273–313 (1904).
48
S Leclercq, A monograph of Stigmaria bacupensis, Scott et Lang. Ann Bot (Lond) 44, 31–54 (1930).
49
FE Weiss, A re-examination of the stigmarian problem. Proc Linn Soc Lond 144(1):151–166. (1933).
50
DH Scott Studies in Fossil Botany (Adam and Charles Black, London, 1900).
51
EW Binney, JW Kirkby, On the upper beds of the Fifeshire Coal-measures. Q J Geol Soc 38, 245–256 (1882).
52
EW Binney, R Harkness, XXXV. An account of the fossil trees found at St. Helen’s. London, Edinburgh Dublin Philos Mag J Sci 27, 241–252 (1845).
53
J Lindley, W Hutton The Fossil Flora of Great Britain: Or, Figures and Descriptions of the Vegetable Remains Found in a Fossil State in This Country (J. Ridgway and Sons, London) Vol 1–3 (1831).
54
WE Logan, XXIX.—On the characters of the beds of clay immediately below the coal-seams of South Wales, and on the occurrence of boulders of coal in the Pennant Grit of that district. Trans Geol Soc Lond Ser 2, 491–497 (1842).
55
EW Binney, XXVII. On the remarkable fossil trees lately discovered near St. Helen’s. London, Edinburgh, Dublin Philos Mag J Sci 24, 165–173 (1844).
56
L Dupuy, T Fourcaud, A Stokes, A numerical investigation into factors affecting the anchorage of roots in tension. Eur J Soil Sci 56, 319–327 (2005).
57
SB Mickovski, et al., Material stiffness, branching pattern and soil matric potential affect the pullout resistance of model root systems. Eur J Soil Sci 58, 1471–1481 (2007).
58
F Giadrossich, M Schwarz, D Cohen, F Preti, D Or, Mechanical interactions between neighbouring roots during pullout tests. Plant Soil 367, 391–406 (2013).
59
AR Ennos, The mechanics of anchorage in seedlings of sunflower, Helianthis annuus L. New Phytol 113, 185–192 (1989).
60
WA DiMichele, CF Eble, DS Chaney, A drowned lycopsid forest above the Mahoning coal (Conemaugh Group, Upper Pennsylvanian) in eastern Ohio, U.S.A. Int J Coal Geol 31, 249–276 (1996).
61
KJ Niklas, The influence of gravity and wind on land plant evolution. Rev Palaeobot Palynol 102, 1–14 (1998).
62
H Steinhauer, On fossil reliquia of unknown vegetables in the coal strata. Trans Am Philos Soc 1, 265–297 (1818).
63
CK Boyce, WA DiMichele, Arborescent lycopsid productivity and lifespan: Constraining the possibilities. Rev Palaeobot Palynol 227, 97–110 (2016).
64
H Sanders, GW Rothwell, SE Wyatt, Parallel evolution of auxin regulation in rooting systems. Plant Syst Evol 291, 221–225 (2011).
65
GW Rothwell, SE Wyatt, AMF Tomescu, Plant evolution at the interface of paleontology and developmental biology: An organism-centered paradigm. Am J Bot 101, 899–913 (2014).
66
M Krings, TN Taylor, EL Taylor, N Dotzler, C Walker, Arbuscular mycorrhizal-like fungi in Carboniferous arborescent lycopsids. New Phytol 191, 311–314 (2011).
67
J Schindelin, et al., Fiji: An open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012).
68
RStudio Team (2015) RStudio: Integrated Development for R (RStudio Inc, Boston). Available at www.rstudio.com. Accessed February 12, 2014.
69
HJ Dittmer, A quantitative study of the roots and root hairs of a winter rye plant (Secale cereale). Am J Bot 24, 417–420 (1937).
70
L Dolan, Pattern in the root epidermis: An interplay of diffusible signals and cellular geometry. Ann Bot (Lond) 77, 547–553 (1996).
71
FAL Clowes, Pattern in root meristem development in angiosperms. New Phytol 146, 83–94 (2000).
72
AC Howell, James Lomax (1857–1934): Palaeobotanical catalyst or hindrance? Geol Soc Lond Spec Publ 241, 137–152 (2005).
73
EW Binney, On Sigillaria and its roots. Trans Manchester Geol Soc 3, 110–122 (1862).
74
KW Joy, AJ Willis, WS Lacey, A rapid cellulose peel technique in palaeobotany. Ann Bot 20(80):635–637. (1956).
Information & Authors
Information
Published in
Classifications
Copyright
Freely available online through the PNAS open access option.
Submission history
Published online: May 25, 2016
Published in issue: June 14, 2016
Keywords
Acknowledgments
We are grateful to Dr. K. Bacon, Prof. P. Kenrick, Prof. J. Langdale, Prof. H. Dickinson, Prof. A. M. Hetherington, and Prof. K. Niklas for helpful discussions. L.D. is grateful to Ms. Iris Marston and Prof. Richard Bateman for valuable insights at the beginning of this project. We are grateful to Bill DiMichele and an anonymous reviewer for insightful comments on our manuscript. We are grateful to the John Muir Trust, the National Trust, Natural England, the Scourie Estate, Oxford University Museum of Natural History, Oxford University Herbaria, London Natural History Museum, British Geological Survey, and University of Manchester, Manchester Museum; Dr. N. J. Hetherington, Miss J. N. Shaw, Mr. J. D. Hetherington, and Mrs. R. Wise (Oxford University; for drawing the reconstruction of the stigmarian system and for the drawing of the rhizomorph apex); and Mr. J. Baker (Oxford University; for photographic assistance). This research was supported by a Biotechnology and Biological Sciences Research Council (Grant BB/J014427/1) Doctoral Training Partnership Scholarship (to A.J.H.) and European Research Council Advanced Grant EVO500 (to L.D.).
Notes
This article is a PNAS Direct Submission.
Authors
Competing Interests
The authors declare no conflict of interest.
Metrics & Citations
Metrics
Altmetrics
Citations
Cite this article
Networks of highly branched stigmarian rootlets developed on the first giant trees, Proc. Natl. Acad. Sci. U.S.A.
113 (24) 6695-6700,
https://doi.org/10.1073/pnas.1514427113
(2016).
Copied!
Copying failed.
Export the article citation data by selecting a format from the list below and clicking Export.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.