to characterize cellular development in the oldest root apices [ 3 , 4 , 5 , 6 , 7 ] , we inspected 139 thin sections made from carboniferous coal balls from britain ( see supplemental information ) . the first apex was the tip of a differentiated , non - growing root ( figure  1a ) . it was designated apex 76.1 and tentatively assigned to lyginopteris oldhamia on the basis of cellular organization ( figures 1a and 1b ; see supplemental information ) . the second apex ( figure  1c ) was larger , blunt , and represents an entirely new root apex type ; it was named radix carbonica ( see supplemental information for systematic paleobotany and comparisons with other fossil apices ) . the cellular organization of apex 76.1 and r.  carbonica can be compared with root meristems of extant species , because both thin sections are near median longitudinal in orientation . however , meristem organization of extant plants can be investigated only in actively growing roots , because meristem structure changes when root growth stops . it is therefore essential to establish if the root apices were fossilized during active growth . in roots that have stopped growing , differentiated tissues , including thickened xylem cells , are found very close to the promeristem as in the fossil meristems of apex 76.1 , lyginopteris , amyelon , and psaronius , ( figures 1a and 1b ) [ 3 , 4 , 6 , 7 , 9 ] , a feature not found in actively growing roots . by contrast , there were no distinguishable tissue types within the ground tissues or differentiated vascular cells found near the tip of r.  carbonica ( figure  1c ) . there is clear zonation of cell sizes in active root meristems of growing roots ; meristematic cells are relatively small and vary in size by 2-fold as dividing cells go through the cell cycle . consequently , there is a gradient from the smaller cells of the promeristem to the larger cells in the differentiating tissues . in contrast , there is no cell size gradient in inactive meristems where cell size abruptly increases from the small inactive initials , which abut much larger differentiated cells close to the apex ( figures 2a , 2b , and 2d).the distribution of cell areas in the differentiating ground tissues of r.  carbonica indicated that there was a gradual increase and a roughly 2-fold difference in cell area ( see heatmap in figures 2c and 2d ; there is a 2-fold difference in cell size between blue [ < 300  m ] and turquoise cells [ 300600  m ] throughout the majority of the body of the root ) , typical of actively growing root apices . these data indicate that r.  carbonica is the first and only example of a root fossilized during active growth , which has preserved the cellular organization of the meristem . comparison of the cellular organization of the different regions of the root apex indicates that the cellular dynamics in r.  carbonica conform to that observed in extant root meristems . root caps are typically tapered ( they are thinner in proximal positions than in distal positions ) because older cell layers are sloughed off as the root grows through the soil . r.  carbonica is covered by a protective root cap that tapers rapidly , indicating that cells were sloughing , typical of roots of extant species   ( figure  3a , yellow ) . the promeristem is the group of cells in a growing root that gives rise to all tissues , and it is identified in r.  carbonica as the region where the files of cells of fundamental tissues converge ( figure  3a , pink ) . the r.  carbonica promeristem is large , consisting of 138 cells arranged in 1015 tiers when imaged in the longitudinal plane of section ( figures 3a , pink , and 3b , pink and lilac ) . it comprises two morphologically distinct pools of initials ( figure  3b , pink and lilac ) ( see supplemental information ) , and initials give rise to many mature cell files , meaning that discrete initials for each cell layer do not exist . the r.  carbonica promeristem is different from all extant vascular plant root meristems ( see supplemental information ) , because of its large size and the spatial organization of cells that are arranged in more than ten tiers of initials . the distal promeristem ( figure  3b , lilac ) of r.  carbonica takes the form of a regular block of cells ; a similar organization of the promeristem is found in almost all extant gymnosperms [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ] . however , the structure of r.  carbonica differs from that of extant gymnosperm root meristems in two ways . the first feature that distinguishes r.  carbonica from extant gymnosperm root meristems is the discrete root cap that is not continuous with the distal promeristem in r.  carbonica ( figure  3b , lilac ) . in extant gymnosperms , it is not possible to distinguish a boundary between the promeristem and the root cap [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ] . however , in r.  carbonica , the promeristem is broad and not continuous with the root cap , which is discrete from other tissues . furthermore , within this broad promeristem there are large numbers of anticlinal cell divisions ( marked in yellow on figure  3c ) , which lead to the loss of the columnar organization of cell files between the promeristem and the cap . while some gymnosperm promeristems are columellar [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ] , and anticlinal division occurs in the promeristem of others [ 19 , 20 , 22 , 23 ] , numerous anticlinal divisions within a columellar promeristem have not been described in any species . no similar organization with broad promeristem and discrete root cap has been described in any root meristem to date ( see supplemental information ) . the second feature that marks r.  carbonica as distinct from the meristems of extant gymnosperms is the position of the krper - kappe boundary [ 1 , 13 , 24 , 25 ] ( see supplemental information for an extended description of the krper - kappe theory ) . the boundary between the krper and kappe complex is a highly conserved feature of all extant gymnosperms . in gymnosperms the krper complex contains the vascular tissue and , in some cases , a small number of layers of the ground tissues [ 13 , 14 , 15 , 16 ] ( figures 4a and 4c , red ) . the kappe complex , on the other hand , makes up the majority of the tissues ( remainder of the ground tissues , epidermis , and root cap ) of the root meristem [ 13 , 14 , 15 , 16 ] ( figures 4a and 4c , blue ) . the krper - kappe boundary is , therefore , located very close to the junction between the provascular tissues and the ground tissue ( figures 4a and 4c ) . the r.  carbonica krper complex constitutes the stele and almost all the ground tissue ( figure  3c , red ; figure  4d , red ) . the kappe complex ( figure  3c , blue shading ; figure  4d , blue ) comprises the root cap and the cell file abutting the root cap interpreted as the epidermis . therefore , the position of the krper - kappe boundary of r.  carbonica is structurally different from all extant gymnosperm root meristems ( figure  4 ) . r.  carbonica is the only root meristem that has been preserved in which the patterns of cell division in the active apex can be elucidated . this allowed us for the first time to compare the organization of cells in the promeristem of an extinct carboniferous root with the organization of cells in root meristems of extant plants . the organization of stem cells and differentiating cells suggests that the same general cellular dynamics in the self - renewing populations and their derivatives occurred in r. carbonica as in extant root meristems . however , the discrete root cap , zone of anticlinal cell divisions in the group of columella - like promeristem , and the position of the krper - kappe boundary mark r.  carbonica as structurally distinct from all other previously described meristems ( figure  4 ) . using the organization of cells in the promeristem and the meristem as criteria , schepp   identified nine classes of vascular plant root meristems . there were five classes of meristems in non - angiosperm tracheophytes ( lycophytes , monilophytes , and gymnosperms ) combined . the evolution of novel meristem types [ 13 , 28 , 29 ] in angiosperms has , therefore , been associated with their rise to dominance . the discovery of the organization of stem cells and their derivatives in r.  carbonica demonstrates that the diversity of developmentally distinct root meristem types that existed before the origin of angiosperms   ( figure  4e ) but are now extinct was greater than previously described . it also shows that extant root meristem organization represents a subset of the diversity that has existed since roots first evolved . the 139 thin sections of carboniferous coal balls from the oxford university herbaria and the university of oxford natural history museum were inspected for root meristems . the original l.  oldhamia root apex described by stopes and watson   and weiss   also was re - examined courtesy of the manchester museum , the university of manchester ( thin section r646 ) . meristems were imaged with an olympus bx50 microscope and quantitatively characterized using fiji . to quantitatively characterize the cell shape , cell area , and cell division pattern of r.  carbonica , a line drawing was made of the 988 cells that constitute the distal portion of the apex ( figure  2c ) and the 405 cells representing the development of the ground tissues ( figures 3b and 3c ) . line drawings also were made of the cells in the distal portion of apex 76.1 and l.oldhamia apices ( figures 2a and 2b ) .
summaryroots and shoots of plant bodies develop from meristems  cell populations that self - renew and produce cells that undergo differentiation  located at the apices of axes [ 1].the oldest preserved root apices in which cellular anatomy can be imaged are found in nodules of permineralized fossil soils called coal balls [ 2 ] , which formed in the carboniferous coal swamp forests over 300 million years ago [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ] . however , no fossil root apices described to date were actively growing at the time of preservation [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ] . 
 because the cellular organization of meristems changes when root growth stops , it has been impossible to compare cellular dynamics as stem cells transition to differentiated cells in extinct and extant taxa [ 11 ] . 
 we predicted that meristems of actively growing roots would be preserved in coal balls . here 
 we report the discovery of the first fossilized remains of an actively growing root meristem from permineralized carboniferous soil with detail of the stem cells and differentiating cells preserved . 
 the cellular organization of the meristem is unique . 
 the position of the krper - kappe boundary , discrete root cap , and presence of many anticlinal cell divisions within a broad promeristem distinguish it from all other known root meristems . 
 this discovery is important because it demonstrates that the same general cellular dynamics are conserved between the oldest extinct and extant root meristems . 
 however , its unique cellular organization demonstrates that extant root meristem organization and development represents only a subset of the diversity that has existed since roots first evolved .