Phloem evolved gradually and asynchronously to xylem in early vascular plants

57 The Rhynie chert i s a 4 07 -million -y ear -ol d fo s s il s i t e 8 th at pr e s e r v e s a div ersi ty of p lant 58 speci e s sp anni ng th e orig in of the v a s c ul ar pla nt s 11, 12, 16, 17 , the sep ara tion o f th e l y cophy tes from t he 59 euphy llophy t e s a n d dive rsific a tion wi t h in the ly cophy t e s 10, 13, 17, 18, 19 . The s e spec ie s there fo r e provid e 60 an oppor t uni t y to inv e stiga te the o rigin a nd ev olution o f x ylem and phl oem at the ba s e of t he 61 va s c ular pla n t clad e . P reviou s w or k on th e w ater -conduc ting ti s s ue s o f th e Rhynie chert pla n ts ha s 62 doc umente d a clea r t raje ct ory fr om poly s po rang iophyt e s wi t h eith er no w at er - c o nduc t i ng ce lls 20 or 63 wa t e r-c onduct ing ce lls di stinc t fr om xyl em 16 , t o plant s with x ylem 11, 12, 13, 14, 17, 21. In contra st, t he 64 oc currence o f phlo em i n the Rhy nie c h ert l and pla n t s i s hig hly de ba ted . Phl o em is repor t e d in e arly 65 desc ription s o f th e Rh ynie c he rt land pl a nts, owing to th e po sition of t his ti ssue, i t s c ell ula r 66 organi s ati on 11, 12, 13, 14 , and pu t a t i ve subc e llular a n atomy 22 (critiqu ed in 23 ) . I t ha s b een sub sequ ently 67 deba te d if thi s ti ssue should b e con side r ed t ru e p hloem 9, 10, 16, 23, 24, 25 . Resolving t he identi ty of the 68 “p hloem-lik e” ti s sue o f th e Rhy nie c he rt plant s i s t he r e for e impera tive fo r u nde r standin g th e origi n s 69 and evolution o f va scu lar tissu e s. 70 To d eter m in e if phl oem wa s p r e sen t in the Rhynie che rt pla nts , the spo rophyt e s of f our 71 speci e s w ere inv e s tig ated : th e non -v as c u lar pol ysp or a ngiop hy t e A glao phy to n maj us ( w it h a c ent r a l 72 strand o f wate r-c on ducting cell s ( WC C s)) 12, 16 , and thr e e s p e cie s of v a s c ula r pl ant s w ith tr ue xy lem 73 Rhy nia gw ynne -v a ug hani i 11, 12 , and t w o l y cophy t e s p e cie s, th e zo ste r op hy ll Tric hophe r o p hyto n 74 teuc ha n sii 18 and t he lyc op s i d A s ter ox ylon m ack iei 13 . His tol og ica l and c ellu la r compa r i son s we r e t h en 75 made betwe e n th e s e f o ssils and fo ur spe c ies o f extan t l ycoph yte s . Thi s inve s tiga t i on wil l refe r to the 76 tis s u e of t h e Rhyni e che r t pl a nt s a s food - c onducting ce ll s ( F CC s), f o l lowing recen t l itera ture 25 , a nd 77 the t erm “ phloem” wil l only apply to tiss ue e xhibiting th e fe atu re s o f phloe m a s it is de fined in 78 ex t a nt va scul ar pla nt s. In ex tan t vascul a r plant s phlo em is h i s t olog ical ly dis tingui shed from 79 surroundi ng ti s s u e s by a peric yc le, at the c ellu lar lev el by elonga t e d cel l s w ith ve ry low a s pe ct r atio s, 80 and at th e subcel lula r lev el by s i eve po re s in t h e wall s o f siev e el e men ts 2 . Com pa r is on o f FC C s o f the 81 Rhyn ie ch ert p l ant s and the phl o em of ex tant lyc ophyt es we r e made on t h ree maj or lev el s of 82 organi s ati on t h a t de fine p rima r y phloem in va s c ular pla nts: hi s tologic al, cell ul ar a nd subc ell ula r. 83 The Rhynie chert plants la ck hi s tologic al distinction betwe en vascu lar and ground tiss ues 84 In e xtant va s c ula r plan ts , phloe m i s iden t ified hi stolo gica lly b y its po si t i on s ur roun ding or 85 periphe r a l t o xyl em, an d the s e ti s s ue s to gether co mp r i se th e vascul a r stra nd 2 . Th e vascul a r strand i s 86 deli mited from t he ground t i ss ue by a bo undi ng la yer, th e peric yc le 2, 26 , a nd in roo ts al s o an 87 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint endod e r mi s 27 or en dode r mo id 28 . The pe ric yc le is cl assic ally de fin ed a s a n irr egular la yer of on e or 88 more c ell s thick be twee n th e va s c ul atu re and t h e cor tex in s t e m s , a nd b etwe en th e v as c ula ture an d 89 endod e r mi s i n r oots 29, 30, 31 . The p ericyc le i s a wide spr ead fe a tur e ac ro s s vascula r plant s, th ough it i s 90 abs ent in the s t em o f s ome de rived grou ps 2, 31 . In the a n atomic al li ter atur e on ext ant ly cophy t e s , the 91 peric ycl e is o ft en di stingui sh ed i n figur e s (e.g ., 2, 26, 27 ), thoug h of ten la ck s a pr ec is e defini tion (e .g. , 26, 92 27, 32 ). In t h i s study, the pe ricy cle was ide ntifi e d in the e xtant ly cophy te s a s a laye r o f one to thr ee 93 ce lls t hick for mi ng a di s tinc t bounda ry be tween t h e phloem an d c or t ex . The cell s of th e pericy cle 94 lay er are l a r g er compa r ed to t h e adja cen t phl oem, bu t la ck air s pa ce s se en in th e cortex ( Fi g. 1 J ). 95 Given t h e wid e s p rea d occ ur renc e of a n a natomic ally di stinct p eric yc le in e xtan t va s c ular pl a nt 96 speci e s 2, 26. 27, 31.32 , evi dence for a co mpa r able l ayer w a s exami ned in t he Rhynie ch ert pla n ts. 97 The FCC s an d c orte x c ells wer e rea d ily d is ting ui s h e d in a ll f our o f the R hy nie c her t s pec ie s 98 ex amined. The F CC s c ompri se a ti s s ue o f clo s e -pa cki ng, el o ngat ed c ell s whi ch sur roun d the ce n tral 99 co r e of xy lem or W CC s 9, 10, 11, 12, 13 . By c ont ra st the c ortex is cha r a ct eri s e d by shor t er , roun der ce ll s 100 w it h m a n y in t r ac e l l ula r s p ac es ( Fig . S1 A- C ). Th e se t i s sue s have s im ila r cha r a ct eri stic s in t he ex tan t 101 ly cophy t e s. How ever , w herea s t h ere i s clea r dema rca t i o n betwe e n th e ti ssue s by t he pe ricy cle in the 102 ex t a nt s pe ci e s ( Fig . 1 I, J ) , a compa r a b le l a yer is a bs ent in the Rhynie ch ert pl ant s. Ins te ad, th e F C C s 103 grade out in to th e c ortex , and t h i s i s e sp ec ially pronounc e d in A. m ajus and R . g wy n ne - va ug han i i . 104 For the s e two s pe cie s, the g r a ding of th e FCC s int o the c o r tex ca n be se en i n tra n sve r s e s ec t i on . 105 Immed iat ely adjac ent to th e xy lem/WC C s t rand t her e is a ti ssue o f c lo se -pack ing c ells , a nd when 106 movi ng periphe r a lly, t he int r a cel lul ar spa ce s t hat c ha ract eri se t he c or t e x ap pea r i nitial ly small a nd 107 infre quen t, incre a sing in fr equenc y and size towa r d s the a xi s periph ery , with no sha r p t r an sition 108 betwe e n the s e ti ssue s ( Fig . 1 A- D ). I n l o ng it u di n a l s e ct ion , th e ti s s ue adjac ent t o t he xy lem/ WC C 109 strand i s o f clos e- pac king and e longa ted c ells, a nd whe n moving periphe ral ly the c ells app e ar 110 inc r e a s i ngly s ho rt er, and wit h more i ntra c ellula r s pa ce s, un til a ti ssu e of the t y pic a l rounded c o rt e x 111 ce lls with ma ny la r g e in trac e llular sp ac e s i s re ac hed ( Fi g. S 1 ). Thi s pa tte rn i s demo nst r a t e d 112 quan t i tativ ely in th e R. gw y n ne- va ug han ii ax is s h ow n in ( Fi g . S 2 ), whe re t h e cel l s i m mediately 113 adja cent t o the xyle m strand ( th e FC C s) a re elon ga ted , na r r ow and c lose -p ack ing, w ith an a verage 114 asp ec t r a tio ( A R) o f 0.20 (rang e 0 .31-0 .1 0 ). P eriph eral t o t h i s i s a r e gion of l oo ser - pac king but 115 elon gat ed c ell s, r ef erre d to he re a s t ran si t i onal ti s s u e (a t lea s t in pa rt c orr e sp ondi n g to the inn e r 116 co r tex in e. g. , 9 ). The se tra nsi tion al cell s ha ve a n ave r a ge AR of 0 . 30 (ra nge 0.58 -0 .14), indic a ting tha t 117 they are a ls o elo nga ted, thoug h on av e ra ge shor te r and w ider than th e FC C s ( Sup plementary T able 118 1 ). P e r i ph eral to th e tran si tional ti s sue i s a c las s ic al cor tical t i ssu e of sho rt e r , more r o unded c ell s 119 wi t h many i nter c ellul ar s pa ce s, and an av erage AR o f 0.62 (rang e 1 .22-0 .2 0) ( Fig. S 3). Thi s sh ows t hat 120 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint ther e is n ot a s har p tra nsi tion o f c ell mo rphol ogy b etween the cor tex a nd FC C s, b ut rat her a gra ding 121 of ti s s ue t y pe s, a s th e iden tifi c ation o f t h e tr a n s i tiona l ti s s ue d emon stra te s . 122 Inv estig ati on of the l ycoph yte s T . t e u chan s ii an d A. m ack iei i ndica t e th at t h e s e s p ec ie s hav e 123 a sha rper tran si tion from FC C s to c o r tex tissu e c ompar ed to A. m a j us and R . gwynn e- vau gh anii , 124 whi ch c an be s e e n in both tran sver s e sec tion ( Fi g . 1 E- H) an d longi tudi nal sec tion (F ig. S 1D - G) . 125 De spit e thi s, the r e i s still an ab s enc e o f a boundary layer i n t h e s e extinc t lyc ophyt es like the pe ric ycl e 126 of ex tan t lyc ophyte s. Th e ab sence o f an end ode rmal la yer in A. m a c ki e i r o ot i n g ax e s h as p r e vi o us l y 127 bee n pro po s e d 33 , b ut t he ob se r v atio n in t hi s s tudy of t he ab se nce of a peric ycl e in a ll A. mac kie i ax e s 128 is nove l . 129 In c oncl us i on , our hi s t ol ogic al inv e s tig a ti on into th e Rhynie cher t plan t s find s a di s tinc t FC C 130 tis s u e be tween the xyl em/ W CC an d co r t ex tissue s . Howev er , unlik e in e xtan t sp e cie s w her e th e 131 va s c ulat ure an d cort ex a r e s ep a r a te d by a cl ear pe ricy cle laye r , t h e Rhynie che r t p la nts la ck a 132 boundi ng la yer, an d exhibi t a gradi ng o f t is s ue cha rac t er i s tic s be t w een the c or t e x an d FC Cs . This 133 sugge st s tha t F CC s a re hi st ologic ally dis t i n ct from phlo em . 134 135 Cellul ar charac t e r i stics of the FCCs of th e Rhyn ie c he r t pla nt s are distinct fro m t hos e o f t he 136 phloem of extant lyc ophyt es 137 At th e c ellula r l evel, t h e cel l s of t h e phlo em, e s p ecia lly th e s p ec iali s ed s ug a r -c on d uctin g 138 sieve elemen t s, ar e na r r ow , clo se -pac kin g and e longat ed 34 . Overal l , ex cludin g so me outlyi ng 139 ang ios p er m group s , extant v a s c ular pl a n t s i e ve e leme nt s te nd to b e a r ou n d 10-2 0 μm in dia mete r , 140 and over 500 μm in l ength 35, 36 , produc in g a n aspec t ra tio ( AR) o f 0 .02 or l e ss. In li g ht of thi s, t h e 141 ce llular c har ac t e ris tic s of the F CC s o f t h e Rhynie che r t pl a nt s w er e exa mined a nd c o mpared t o tho s e 142 of th e ph loem o f ex tan t lyc ophyte s. 143 The phloem c ell s of the e xt ant ly cophy t e s ( si eve eleme nts and phlo em p ar enchy ma) 144 mea s u re be t w ee n 41 .4 μm and 2.2 μm i n diame t e r , with an aver ag e diame t e r o f 8 .9 μm a cro ss 145 speci e s a nd ti ssue ty pe s ( n = 994 7, 25 a x es ). I n c ontras t, t he F C C s o f t he Rhyni e chert pla nts mea s u re 146 betwe e n 231. 3 μm and 9.2 μm i n diame ter, w ith an ave rag e diame t e r o f 56.2 μm a cro ss sp ec ie s and 147 tis s u e t y pe s ( n = 290 7, 21 a xes ). Thi s indi cate s th ere i s a signi fic ant di f f e r e n ce be tw een the F CC a nd 148 phloe m cel l diam e t e rs (p ≤ 0.001 ), w ith t he FC C s o f t he Rhynie che rt plan t s bein g on av erage a round 149 six time s la r g e r in dia met er t han th e phl oem ce lls o f ext an t lyco phyte s ( Figure 2 ) . T his dif fe rence in 150 ce ll size betwe e n th e Rhyni e ch ert pl an t s a nd the ex ta nt lyc ophyte s r emain s signif ic ant (p ≤ 0 .001) 151 whe n the la r g er axis di a met er o f the R hyni e ch er t p l ant s i s tak en into a cc oun t ( Fig . S 4 ), a s we ll a s the 152 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint ove r a ll la r g e r cell s ize o f th e Rhynie cher t plant s ( Fig . S5, p ≤ 0 .00 1) ( see Suppleme nt ary D a ta Table 153 and Supplementary Data I m ages ). Thi s i nve s tig a tion o f c ellula r str u c ture of t he R hy nie c hert pla nt 154 FCC s sugge s t s tha t t h e s e cell s a r e marke dly diffe ren t in di ame ter from th e phloe m cel ls in ex t a n t 155 ly cophy t e s, a nd th at thi s i s inde pend e nt of oth er morp hologi cal and an a t o mical s i ze di ffe ren ce s . 156 157 Dis c overy of pu t ativ e s i eve pores in A. mackiei 158 The final w ay t ha t t he ph loem o f extan t v as c ular pl a nt s i s de fi ned i s a t the subc e ll ular sc a le. 159 The wall s o f p hloem siev e e leme n ts are perfora ted by s ieve por e s , whic h form fro m the enla r g emen t 160 of pla s mod e s ma ta l pi t s , and en able r api d long-di sta nc e s y mplas tic tra ns por t t hro ug h the phloe m 37 . 161 Si eve po r e s a re incre dibl y rare in the fo ss il rec or d, bu t have be en p r e s erved in f o s s il s w ith 162 ex ceptiona l p re se r v ation, d e mon s t rating t ha t t h e y c an be fos sili s ed 38 – 52 . T h e p r es en c e o f s ie ve p o r es 163 is o fte n take n a s the de fini ng fea tur e of ph loem in t he fo ssil rec o rd ( e.g., 7 ), owin g to their 164 dist inctne s s compa r ed to t h e ce ll wa ll s o f oth er par enc hymatou s ti s s ue s, wi t h onl y pl as m ode s m a tal 165 pits . Phl oem i s of t e n d esc ribed from de r i v ed ex tinct lin eage s in t he ab s ence o f s i e ve po r e s (e .g ., 53 ), 166 and t he r e ar e te nta tive de scrip tion s o f p hloem from o lder d epo sit s and ea r li er-di v erging groups, bu t 167 whi ch la ck t h e pre s e r v a tion qua li ty to pr es erve siev e po re s ( e.g., 54 ) . Owing t o th e importanc e of 168 the se struc t u re s, th e oc curr enc e o f si eve por e s in the F C Cs o f th e Rh ynie c he rt pla nts wa s 169 inv es t ig ate d. Fi rs tly, to ob ta in com par ati ve i mages o f ex tan t lyc ophyte siev e por e s and 170 pla s mod e smat al pit s , t h e extan t l ycoph yte s Lyc opodi um cl av at um a nd Hu perzi a se lago wer e i m a ge d 171 using S EM ( Fig . 3A and B a nd Fig . S6) . S ie ve por e s i n the l ate ral wall s of L yc opodi u m clav at um SE s 172 we r e fo und to b e 0 .15 µm i n diame ter o n averag e ( Fig . 3B), in the ra nge s previou sly r e po r t ed (0.2 to 173 0.86 µm 36, 55, 56 ). Plasmod e smat al pi ts in t he late ral w all s of phl oe m par enc hyma in Huperzi a s e l ago 174 we r e fo und to b e 0 .07 µm (70 nm) in di a meter o n av erage ( Fi g . S6 ) , s li g ht l y la r g e r t h a n r e po r te d fo r 175 pits in ly cophy te sho ot apica l meri st em s (2 6 to 41 nm 57 ). Th is show s th at t he siev e pore s o f 176 ly cophy t e s i e ve el eme nt s ar e at le a s t twi c e a s la r g e in d iame ter a s , and can be di s tinguish ed f rom, 177 the pl a s mod e s ma t a pi t s o f phl oem pa re n chym a. Us i ng th e se re s u l ts a s a sea r c h imag e, A. ma ck i ei 178 FCC s w ere inve s tiga t e d for t h e occ urre nc e of siev e po re s. 179 A. mac kie i alon e was foc u s ed on owi ng t o the i nte n s iv e imag ing requ ire d to d et e ct siev e 180 pore s, and a s thi s s peci e s i s a lyc opsi d, it is th e mos t like ly to ha ve trai t s in c ommon wi t h extant 181 ly cophy t e s. S ample s o f A. ma cki ei we r e subj ected t o S cann ing El ectr on Mic r o scop y (SEM) a nd 182 Airyscan Con foca l La se r Sc annin g Micro s copy (CLS M) imagin g to a sc ert ain if s i eve po r e s w er e found 183 in the wall s of the F CC s. Airysc an C LSM a nd SEM w ere u sed t ogeth er a s th e se t ec hnique s ar e 184 co mplimentary. W hi l s t S EM ha s a r e solut ion l imit (<1 nm 58 ) th at al low s s u bcell ul ar s tr uctur e s to be 185 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint cl early i maged, SEM pr epa r a t i on o f Rhyn i e che r t sampl e s requi r e s hy dr o flu or i c ac id etc hing, w hic h 186 co uld ca use the col l ap se o f ex po s e d cel l s. A i r y s c an CL SM i s a no n - d e structiv e tech ni que u sed on thi n 187 sec tion s, bu t doe s ha ve a re duced re s olv i ng pow er (th e re s o lu tion limi t o f Ai r y s c a n CL SM is 0.12 t o 188 0.2 µm fo r a 100 x objec tive len s with a 1. 4 Numeric al Ap ert ure 59-61 ). T he FC C s o f A . ma cki ei we r e 189 subjec t to S EM ima ging to iden ti fy ex pos ed wa lls o f FC C s oth erwi se suppo rt ed int ernall y wi t h int act 190 silic a, ther ef ore p reve n t i ng ce ll c ollap se i n s am ple p repa rati on (se e M ethods ) . This reve aled a n F CC 191 from a ro ot -be aring axi s pa r tia l ly e ncas e d in s il ica, bu t with e xpo sed r egion s of i n t a ct ex te r na l ce ll 192 wa ll ( Fig . 3C) . At two e xpos ed regi o ns of t hi s F C C (whit e a r row s i n Fig. 3C ), r egula r c ircula r 193 perf ora tion s o f the c el l w all w ere iden tif i e d ( Fig. 3D and E , black arrow s) , whic h mea s ure a r oun d 0.2 194 µm in diamet er. T hi s pr ov id ed the f i rs t te ntative e viden ce o f si ev e por e s in A. m a ckie i. Fu r th er 195 ev idenc e o f si eve pore s w a s s oug ht u s i ng Airyscan CLSM. A i r y s c an CL SM is ca p abl e o f re solv ing 196 submicron fe ature s in fo ssil sample s 60,62 , and is rea dily able to i mage th e 1-2 µm dia mete r xyl em pits 197 of A. ma ckie i ( Fig . S7) . T hin s ection s o f A. ma ckie i rooting a xe s with well- pre s erve d F CC s ( Fig . 3 F- H 198 and J- K ) w er e imag ed u sing Ai r y s c an CL S M with a x10 0 objec tive. Fi g . 3 I and L - M , showing regul ar 199 ci r c ular per for atio ns o f la ter al and en d w al ls , re spec t i vely , whic h are < 0 .5 µm i n di amete r. T her e fore , 200 under bo th S EM ( Fig . 3C -E ) and Airy s c an CL SM ( Fi g. 3F -I and J - M ) , t he F CC s o f A. mac kie i we r e fo und 201 to hav e per fo rati on s in the l a te r a l and e n d wa lls s imi lar in di ame ter to th e s ieve p ore s o f extant 202 ly cophy t e s a n d large r than t ho se o f pla s modesm ata pi t s, and a re th ere fo r e iden t ified a s s iev e po re s. 203 This rep re se nt s to o ur knowl edge th e ea r lies t r e co rd of si eve pore s in the fo s s il r e cord. 204 205 Dis c us sion 206 The Rhyni e ch ert pl an ts exhi b it a foo d -c onduc t i n g ti ssue d istinc t fr o m the p hl oe m of e xta nt 207 va s c ul ar pla nt s 208 The r e s ult s pr e s e n t e d he re show th a t th e Rhynie cher t pla nts ex hibi t a t i s sue in t he 209 ana t o mical po si t i on o f phlo e m ti s s ue an d with some a dapti on s fo r long di stanc e tran spo r t o f suga rs, 210 name ly el ongat ed, clo se -pac king cell s a n d, in the ca se o f A . m acki ei , si e ve p o res . H o we v er , t he F CCs 211 of th e Rhy nie c her t plan t s lac k othe r fe at ure s cha r a c teri stic of phl o em, name ly th ere i s no pe ricy cle 212 sep ara ting FC C s from t he cor tex, and t he F CC s grad e into t he cor tex. T he FC C s a re also s ig nif ica ntly 213 large r in diame t e r tha n phl oem cell s of e xtant lyc ophyte s, a nd are sho rt e r i n leng th t ha n th e typic al 214 SE s of l ycop hyt e s , f ern s a nd gy mno s pe rms. Thi s s ugge st s tha t th e FC C s of the Rh y nie c hert plan t s ar e 215 hist ologic ally and c ellul a rly dis tinc t f rom the phlo em of t he extan t v as c ul ar pla n t s , thoug h they d o 216 sha re subcel lul ar f eat ure s, the sieve po re s , implic a ted in a da pta tion t o a s ug a r con du cting fun cti on. 217 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint The FCCs o f th e Rhynie che r t pl an ts re v ea l the a s y nchron ou s an d ind ep e nde nt e v ol utio n ar y 218 his torie s o f phl oe m and x yle m 219 The identi fic ati on of a t i ssue be a r i ng som e, but no t a ll, o f th e k ey c harac te r i stic s o f the 220 phloe m in the R hyni e c her t plant s exa mi ned h ere s ug ge s ts a s c enari o fo r the ev olution o f phloem 221 tis s u e in whi ch F CC s i n poly spo rangi ophy te s ar e homol ogou s, phlo em is t he re fore a derive d f orm of 222 FCC an d ev olved gradual ly, a n d thi s occ u r red a s y nchron ou sly rel a tive to th e or i gi n and evolu t i on o f 223 xy lem ( Fi g . 4 ). I n thi s s c en ario, pl a s mod e s ma ta were p re sen t in t h e ance s t or o f all embryophy t e s 63 . 224 Fol lowing dive r g ence o f the poly s po rang i ophyte s, food -c onducti ng t i s s ue ev olved in the sporo phyte 225 gen era tion, b efo re w at er -conduc ti ng ti ssues . Ev idenc e suppor t i ng thi s come s fro m the e arly -226 div erging p olyspor angioph yt es t he e oph ytes 25 and H o rne o phyto n l igni eri 20 : in both taxa th ere i s a 227 ce ntr a l strand o f elo nga t e d cel l s de s c r i be d as F CC s or tran s fe r cell s, re sp ectiv ely, a nd the F C C o f 228 eophy te s exhibi t p its. T he se pit s from th e images in Edwa r d s et a l (2022 ) ar e e s ti mated he re a s bein g 229 ~0.2 2 µm in diame te r ( see Supple mentar y N ote ). Thi s i s large r than l ycop hyt e pla smode sma ta pi ts 230 but in t he r a nge o f t h e s i e ve pore s o f ex t ant lyc ophyte s and A. mack iei a s rep o r t e d he r e . From thi s, 231 we conc lude tha t elonga t e d F CC s with si ev e pore s wer e pre s ent e a r ly in the p olys porangiop hyt e s , 232 befo re th e or i gin o f w ate r- c o nduc t i ng ti s s ue s . The pre se nce i n th e non -va s c ular A . maj u s , and the 233 trache o phyte s R . gwy nne -v a ugh anii , T. t euc han sii a nd A. m ac kiei of a fo od-c o ndu cting tissu e w ith 234 some , but no t a ll, o f th e k ey fe a ture s o f e xtant t rache o phyte phl o em s ugges t s th at th e ass embly of 235 all phloem charac ter i stic s oc curred af ter the orig in o f x ylem. The FC C s of T. t e u c h a ns ii and A. m a c ki e i 236 also im ply tha t k ey fe a t u re s o f ex tan t phl oem t i s sue we re acqu ir e d fro m FCC s ind epe nd ently in the 237 euphy llophy t e s a n d the ex t a nt lyc op s i d s , through th e narr owing of s i eve el ement s and a cqui si tion o f 238 the pe ric yc le. F C C s a nd s i eve po r e s w ere also ac q uired in d epen dent ly and c onve r g ently in multiple 239 line age s o f bryoph yte 64-67 ( als o s e e Suppleme nt a r y N ote ) , in t h e game t o phyt e ge n era tion. Thi s 240 ev olutiona ry sce n ario sugge st s tha t s iev e pore s ar e not a s ol e de fining fe atu re o f t he ph loem, bu t 241 rath er one o f a suit e o f ada p t a t i on s to su ga r c onduc t i on . Furth ermor e, sieve po re s are like ly one o f 242 the ea r l ie r occ urr in g ada p t a t i on s to suga r c onduc t i on, owi ng to thei r ap pe aranc e in a n early-243 div erging p olyspor angioph yt e line age , a n d co nvergently in mul t ip le b ryophy te lin e a ges . Enla r g ing 244 pla s mod e smat a t o fo r m siev e por e s al so only require s co -op ting a dev elopm enta l programme 245 alrea dy inv olve d in pla s mode s ma ta pi t fo rmation 37 a n d t her e f or e is p ot en t ial l y m o r e r e ad i l y 246 origin at ed than mo re devel o pment ally compl ex trait s . Foll owing initial sp ec iali sat ion in sugar 247 co nduction t h roug h elonga t e d ce lls wit h sieve po re s like t h o se o f A. m ack iei, l ate r in trache op hyte 248 ev olution, a n d indep e nden tly i n the e up hy llophy t e and th e e xtan t ly cophy t e line a ges, fe atu re s aro se 249 as s o cia ted with o ptimi sa tion o f s ug a r co nduc tion, such a s n arrow er a nd lo nger c e lls. 250 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint The s c en ario ou tli ned a bov e i s underpi n ne d by i nter p re tati on of s i eve p ore s a s n ot the sol e 251 defini ng fea tur e of p hl oem ti s s ue , owing to the oc currenc e of s i milar s t ruc t u re s in suga r-c onduc ting 252 tis s u e s out sid e of the va scula r p la nt s , bu t r a th er f a vouring a mor e hol i s tic de fini ti on of t h e ti ssue , 253 inc orpor a t i ng hi stologic al , c ellu lar and s u bce llular a s p ec t s . How eve r, ev en i f si ev e pore s ar e tak e n a s 254 the sole d e fini ng fe a ture of phl oem, ou r interp r e t a t i on her e tha t eop hy te s exhib ite d s i eve p or e s a l so 255 impli es t ha t phl oem e volut ion w as async h r ono u s to tha t of the xyl em, as the s e pla nts la cked wat er -256 co nducting t i ssue s. H er e we do n ot t ake t hi s i n t e rpre ta tion, owing t o t h e hi sto log ic al and cel lular 257 diff er enc e s be tween the FC C s o f the Rhy nie c hert pl ant s and the p hlo em of ex tan t lyc ophyte s 258 sugge sting t ha t the se w e re sub s t antia lly di s t inc t t i ssu es. But r e gardl e s s of how F C Cs and phlo em a re 259 dist inguish ed p rec is ely, t hi s f urth er dem ons tr a t e s th at t he a ssembly of ph loe m a nd x ylem t rai ts 260 we r e indep en den t and a s yn chron ou s ac ros s t h e poly spora ngi ophyte phy logen y . 261 In c oncl us i on , th e iden tific a tion o f F C C s i n the Rhy nie c he rt plan t s with onl y s ome of the 262 fea tur es of t he phlo em of ex ta nt va scul a r plant s demon s tr a te s c lea rly t ha t , whils t xy lem an d phloem 263 are a lway s fou n d toge the r in e xtant va s c ular pla nts , they did no t origi na te simult aneou sly . In s t e a d, 264 also d raw ing on finding s in o t he r e arly p ol yspora ngiophy te s 20, 25 , we propo se a s c enario in whic h th e 265 fea tur es of phlo e m evol ved g r a dually fr o m ance stral F CC s a nd async hron ou s ly to that o f xy lem, w ith 266 some fea tur e s lik e si eve p ore s a r i sing be f ore th e origin o f xy lem, an d othe r a s s oc i ated fea t ur e s, s uch 267 as t he pe ric yc le and narro w and very l on g sieve el emen ts, ari sing l at er and in d ep enden tly in the 268 euphy llophy t e s a n d the ly cophy te s . 269 270 271 272 273 274 275 276 277 278 279 280 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 28 1 28 2 Figure 1 : The food condu cting c ells (FCCs) of the Rhynie ch ert plants grade into the c ort ex tis sue, 28 3 and there is no pericy cl e bet ween th e FCCs and the cortex. 28 4 (A- H) The a erial axe s o f th e R hynie c her t plant s s ho w a c ent ral stra nd of wa t e r- c o nducting ti ssue 28 5 (wate r c onducting cell s i n A glao phy to n maju s and xyle m in the re s t, shown in magenta fal se 28 6 colou r in g) s ur rounde d by an elonga te d, c lo se- p ac king tissu e (y ellow ) , r e f e r re d to a s t he fo od 28 7 conduc ting c ell s (F C Cs ), fol low ed by cort ex (bl ue) . 28 8 (I - J) I n extan t l ycoph yte s and euphy lloph y t e s, the phl oem ti s s u e (yell ow) i s sep ara ted f rom th e 28 9 cortex (bl u e) by a peric ycle (or a nge) . A c lear pe ric yc le is lac king in Rhy nie speci e s ( A - H) and in stea d 29 0 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint the F C C s g r a d e into t he cor tex ( show by a fuzzy bounda r y betwe e n FC C s and cor te x on B, D, F a nd 291 H). 292 Spe cie s abb rev iation s : A. m aj = Agl aop hyton m aj u s ; R. gwy = Rhyn ia gw ynne- v au g hani i ; T. te u = 293 Tric hophero phy to n te uc han sii ; A. m ac = As teroxyl on mac kiei ; L. cla = Ly copod ium c lavatu m. 294 Phy logeny ba sed on 17 , po siti on s of Ag l aop hy t o n maju s a nd Rhyn i a gwy nne -v a ug h anii fr o m 68 . 295 Spe cimen code s: A-B = M P E G 0 015, C = S COTT E U CM 13 61, D = MA N EMu 547 126, E - F = L YON 9 3.11, 296 G-H = GLAHM Kid 247 2 297 Sc ale bar s : 5 00 µm in ( A, E, G ), 200µm in (C, I), 1 00 µm in (F , H) ; 50µm in (B , D); 20 µm in (J ). 298 299 300 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 30 1 Figure 2: The F CCs of the Rhy nie c h ert pl ant s are signific antly (p≤0. 001) la rger in di am eter t ha n the 30 2 phloem c ells of extant lyc o phytes. Meas uremen ts of the diame t e r of p hlo em or f ood c onduc t i ng 30 3 cel ls i n ext ant l ycoph yte s compa red to e xtinc t s p ec ie s fro m the Rhyni e che rt. F ou r extant ly cophy te 30 4 sp e c i e s ( Hu perz i a goe be llii, H u p erz i a sel a go, Ly copo diu m c lav a tum , Sela ginel l a u n ci nata ) were 30 5 invest igate d cove r i ng both st em and r o o t ti s s ue s . F o ur Rhynie che rt speci e s c ove r ing axis ti ssue s 30 6 ( Agl ao phy t o n maju s, R hyn ia gw ynne - va u gha nii , T r i c ho ph e r o ph yt o n t eu c hans i i ) an d lea fy st e m , roo t 30 7 bearing axis , an d ro ot i ng ax is ( Aster oxy lon mac kie i ). The F C Cs o f th e Rhyni e che r t spec i e s are all 30 8 s ignific a ntly (p ≤0. 001 ) larg e r tha n t he phl oe m cel ls o f t h e extan t ly cophy te s , a s d e monstra ted by a t -30 9 te s t . 31 0 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint Spec ie s and orga n abbrev ia tion s: H. goe s te m = Hu pe r zia g o eb elli stem; H. s el s t em = Hu p e r zi a se l 31 1 st e m ; L. c la roo t = L yco podium clav at um root; L. c la st em = L yc opodium clav at um stem ; S. un c roo t = 31 2 Sela ginell a unc in at a roo t; S. un c stem = S elagin ella u n cinat a stem ; A. m a j axi s = A glao phy to n maju s 31 3 axi s ; R. gwy axis = R hy n i a g w y n ne- v a u ghan i i ; T. t e u ax i s = Trich oph erop hy t on t e uc ha n s ii axi s; A. ma c 31 4 leafy s hoo t = A s t eroxy lon ma cki ei lea fy s hoot; A. m ac ro o t -b e a ri n g a x i s = Asteroxylon mack iei root -31 5 bearing axis ; A. ma c rooting a xi s = A st ero xylo n mac kiei r ootin g ax is . 31 6 31 7 31 8 31 9 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint Fig ur e 3: Dis c overy of p ut ativ e s i eve pores in Asteroxylon mackiei like the siev e pores of extant 320 lyc ophytes. 321 (A, B ) SE M imagi ng of th e l at eral wa ll s o f Lycopo diu m c lava tum s ie ve el emen t s ( y e llow fal se 322 co louring) show s numero us si eve por e s ( bla ck arr ow s in B ). Th e r e gion w ithin the box on A i s shown 323 in B. 324 (C -E) S EM imagi ng of the r oot -be aring axi s o f Asteroxy lon m ac kie i show s lyc ophyt e- size d por es in the 325 later al wall of F CC s ; C, HF e tching expo se s va s c ul ar ti ssu e (ma gen ta = xyl em, ye llo w = FCC s) , whi lst 326 muc h of the F CC indic ate d in C i s s u rroun ded b y silic a, pa rt s o f th e exter na l s ur fac es o f th e lat eral 327 ce ll w alls ar e ex po s e d (arro ws); D sh ow s the e xpo sed c ell w all i n t he r egion indic a ted by t he d as hed 328 whi t e arro w in C , and E show s t he expo s ed c ell wall in the r egi on indic at ed by the solid w hit e arrow 329 in C , in both si ev e pore - s i zed struc ture s a re indic at ed by blac k a r row s. 330 (F- M) CL SM and A i r y s c an CL SM imag ing of r oo ting axe s of As teroxy lon mac kie i sh o ws lyc ophyte -331 siz e d pore s in the end and la te r a l wal l o f FCC s . 332 (F- I), F s h ow s a single plan e CLSM i mage of an A ste r ox ylon mack iei r oo ting axi s i n oblique s e cti on; G 333 max imum inten s i ty pr oj ection o f t h e regi on i n the solid w hit e box in F, and H sho ws the r egio n in th e 334 dash ed w hite box in f i mage d us ing the s ame method ; I s h ow s a max imum i nt e n s i ty pr o jection o f t h e 335 region in th e sol id b lack box in H, sieve p or e -sized st r uc ture s in t h e late ral cell wal l are indi cat ed b y 336 b l ac k ar r o ws . 337 (J -M), J s how s a si ngle p lane CL SM ima ge of a n A s ter ox ylon mack iei rooting axi s in cross secti on 338 obtain e d us ing an x40 o bjectiv e; K show s a maximu m intensity pr ojec tion o f an A i r y s c an CL SM z -339 sta ck image obtain ed u s ing a x 100 obje ctive of the regio n in th e solid white b ox in J; l and M bo th 340 show de t a il s o f K showing , re s p e ctive ly, t he reg ion in t h e da she d and in th e sol id b lack box in K, siev e 341 pore - siz ed s t ructu re s in th e e nd wal l ar e ind icated by bla ck arrow s. 342 Fa lse colou r o ve rlays : ma gent a = xyle m; yel low = phloem/ F C C s; bl ue = c or t e x. 343 Sc ale bar s = 50 µm in (C, F , J) ; 20 µm in ( G, H); 10µm i n ( A) ; 5 µm in (I , K); 1µm in (B, D , E, L , M). 344 Spe cimen code s = F -I = MPE G 00 62 , J-M = MPEG0043 345 346 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 34 7 Figure 4 : The c haracterist ic s o f FCCs evo lve d gradually across pol ys porang iophytes , out of s tep 34 8 w ith the evol ut ion of xylem. 34 9 P la smode sma ta w ere pre se nt in th e anc es t or o f a ll emb ry ophyte s. Th e pre se nc e of FCCs an d s ie ve 35 0 pore s in th e eoph yt e s tha t w e propo s e i mpli es t ha t s i ev e pore s o rigina te d in the c ommon a nces tor 35 1 of eop hy t e s + the re st o f the poly spo ran giophy te s , be fore th e evo lutio n of w at er conduc ting ti s s ue s. 35 2 The oc currenc e o f wa te r- c onducti ng cel ls (WCCs) in A gla op hyt on m aj us i mpli es th at thi s trai t wa s 35 3 pre s en t i n the c ommon anc e s to r of A gla ophyto n m aju s + tr a ch eop hyt es. Xylem arose from W CCs in 35 4 the com mon anc e stor o f ex tan t tra cheop hy te s . The pr e sence in A s t e r oxylon mack iei o f FCCs with 35 5 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint sieve po re s b u t without the na rrow and ve r y long cell s o f ph loem an d with out a p ericy cle betwe en 356 the F C C s a nd t he c ortex (a s indic a ted by t h e blur red c ortex / F CC b oundary in th e a x is s c hem atic s ) 357 impli es t ha t key fea ture s o f exta nt phl o e m tissue s we r e acqui re d f r om FC C s foll o wi ng the orig in of 358 xy lem a nd indepe nden tly in the eu ph yllo phy t e s a nd th e ex tan t lyc ops i d s. F C C s wit h sieve po re s wer e 359 also a cq uired c onve r g ently an d ind epe nd ently i n multiple lin eag e s o f bryophyt es , i n t he 360 ga metophyt e g ener atio n. Th i s sugg e st s t hat th e suga r and wat er c onducti ng ti s s u es o f 361 trache o phyte s followe d di s t i nct ev olu tio nary traje c torie s. 362 SP s = siev e por e s, Y = s i e ve po re s kn own; Axi s c . s . = c r o s s s ec tional sch ema tic of a xis ti ssue ; Cell s = 363 schema t i c of c ell d im en sion s ; * = pre s en ce of t r a it in onl y some o f th e c lade ; d as hed line on 364 cl adogram = unce rtai n t y regarding phylo g enetic plac em ent . 365 Tax on ab br e viatio ns = Bry o = Bryop hy t e s; E oph = E ophyte s ; Agl ao = A gla ophy to n maju s; Rhy nia = 366 Rhy nia gw ynne -v a ug hani i ; Euphy ll = Eup hyll ophyte s; As te r = Aster oxy lon mac kie i ; Exta nt lyc o = 367 Ex t a nt lyc ophyte s 368 Colou r k ey: b lu e = c or t e x, da s he d magen ta = W CC s , s ol i d mage nta = x ylem, yello w = ph loem or F C C s , 369 orange = p eric ycl e. 370 Phy logeny ba sed on 17 , po sitio ns o f A gla o phyto n ma ju s and R hy nia gw ynne -v aug hani i f r o m 68 , 371 posi tion o f eo phy te s f r om 25 . 372 373 374 375 Le ad conta c t 376 Furthe r inf ormati on and r eque s ts for re s ource s sh ould be di r e ct ed to , a nd wil l be fulfi ll ed by , th e 377 Le ad Con tac t, Alexa nde r J. H eth eringt on ( s andy. he the rington @e d.ac .uk ). 378 379 Ack nowledgme nt s 380 We w ould lik e to thank I. F e bbr a r i , Thin S ection s an d La pida ry F acil ity Mana ge r, U niversi ty of 381 Edin bur g h for the t hin s ection p r e pa rat io n of MPE G s lide s. We w ould li ke to than k Y. Ca ndel a fo r 382 as s i s ti ng us i n ac ce s s in g th e fo s s il s o f th e N ation al Mu seum s Sco t l a nd col lecti on , H . Kerp f or ac ce s s 383 to the c oll ec tion s a t th e Univer sity o f Mü nste r , L. L oughtm an fo r a cc e ss to mat eri al in the He rbarium 384 at th e Univ er s ity o f Manche ste r, and N. C lark f or ac ce s s t o t h e Kid sto n coll ectio n at th e Hunte r i an 385 Mus eum at th e Univ er s i ty of G l a s g ow. W e than k L . Pic h evin for a ssi stan ce w ith HF etch ing, a nd F. 386 La idlaw and A. Scho f i eld for a s s i s t anc e w ith SEM ima ging. W e th ank D. Ke lly a nd T . Mc H u gh at th e 387 Li ght Microsco py Cor e of t he W ell come D i sco very Re sea rc h Pla t form f or Hidden Ce ll Biol o gy, 388 Univer sity of E dinbu rgh fo r ac ce s s t o th e confoc al i maging faci li tie s . This work wa s suppo rt e d by 389 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint funding f or t h e Wellc ome Di scove r y Re s e arch Pl a tfo r m f o r Hidde n Cell Biol ogy (226791) and w e 390 grate full y ac know ledge s uppo rt from th e L ight Mic r osco py C ore . 391 392 Fundi ng 393 This w or k wa s suppo rt ed by U K Resea r c h and Innova t i o n Futur e L ead er s Fell ow shi p grant 394 MR/T018585 / 1 and MR/Y03399X / 1 (A. J. H.), Phil ip Le verhulm e Pr i ze gran t PLP -20 23-324 (A .J .H. ), 395 Eng ineering and P hy s ic al S cie nce s Re se ar ch Council grant E P/Y037 138/1 (A. J.H.) , a nd NERC E4 396 Docto ral T r a ining P ar t ne rship (L .M .C .) . 397 398 Author Cont ributions 399 A.J .H. con cep tuali z ed t h e s tudy . L.M .C . c olle cted an d anal y s e d da ta . A. J.H . an d L. M.C . wrote the 400 pape r. 401 402 Decla r a t ion of int e r e s t 403 The autho rs d e cla re no comp eti ng inter e st s. 404 405

s avail ability 406 Thin s e cti on s and mou nte d pe el s ex amined in thi s study ar e hou sed i n t he f ollow i ng c ollec tion s , 407 co llec t io n s a bb r e viati on s = Pb : For schun gs st e ll e fü r Pa l6iTD obot anik , In st i tu t für Geo log ie und 408 Pal 6iTDon tologie , U n iver s i t y o f Mün ste r , G e rman y. GLAHM Kid: K id s t on Co ll ection , The Hunte rian, 409 Univer sity of Gla sgow , UK. BHU T TA : Akhl aq Ahmed Bhu tta p e el co llec tio n, Un iver sit y o f Ca rdif f, UK . 410 NH MUK: N atur al Hi st o ry Museu m, Lond o n, UK. MA N : Ma nch e ste r Mu s eu m, Un i versi t y of 411 Manc he ster, UK. OXF U niv er s ity H e r ba r i a : Fie lding- Druc e H erb arium , U n iver si t y of Oxford, UK. LY ON: 412 Ly on Coll ection , U niv er s ity o f Ca rdi ff, UK . MPEG = M ole cula r P ala eo bo t a ny an d Evol ut i on Gr oup , 413 Univer sity of E dinbu rgh, UK. S C O TT = An drew Scott Coll ec tion, U ni ve r s ity of Edin burgh , U K . STA = St 414 Andrew s Coll ectio n, Univ er s i ty of St A ndr ews, UK. NMS = N atio na l Mu seum s Sco tl a nd, Edinb ur g h, 415 UK. 416 Acc es s io n number s f o r a ll figu r ed s p ec imens : MPE G0015 (Fig. 1A -B ) , S COTT EUCM 136 1 (Fig . 1C), 417 MAN EMu 5471 26 (Fig . 1 D), LY ON 93.11 (Fig. 1E-F ), GLAH M Kid 2472 (Fi g. 1G -H). MPEG0062 (F ig . 3F -418 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint I) , MPE G004 3 (Fig . 3J -M), NMS 1925.9. 7 (Fig. S1A -B) , NH M UK 156 33 (Fig . S1C) , STA355.7 6 (Fig . S1 D), 419 NH MUK 164 33 (Fig . S1E -G) , BHUTTA BL 2 9A.185 (F ig. S 2 ), STA 355.76 ( F ig . S 7A) , STA 3 55.61 (F ig. S 7B ), 420 MAN EMu 5474 08 (Fig . S7 C), NHMU K 16 433 (F ig. S 7D ) . 421 All ot h e r data are a vail a ble i n th e main te xt or su pplemen tary mat erial s . 422 423 Data and c ode av ailabil it y 424 • All da ta d e scribed in thi s pape r is inc lud e d w ithin the p ap er and suppl e men tary in forma tion. 425 • This pa pe r doe s no t rep ort o r i ginal code . 426 427

s 428 1. Lu cas, W .J ., Gro over, A., L icht en be r g er, R ., F ur u ta, K., Ya dav, S .R. , Hela r i utt a, Y., H e, X. Q., 429 Fuk uda, H., Kang, J ., Bra dy, S .M. and P a t r ic k, J.W. , 20 13. The plan t v as c ula r sy ste m: evol ution, 430 dev elopmen t a nd fu nction s f. J ourn al o f i ntegr ati ve pla nt bi olo gy , 55 (4), pp .2 94-3 88. 431 2. Esau, K . 1969. Th e Phl o em. Handbuc h d e r Pfl a nz en a nat omie, V, 2. G ebrüd er Bo rn tr a eg er. 432 3. Esau, K . , Che adl e , V. I. a n d Gi ffo r d, E.M ., 195 3. Compar ativ e struc t u re and po s s i bl e tren ds of 433 speci a lizati on of the p hl oem. A me r ic an J ourn al of B ot any , pp.9 -19. 434 4. Tay lor, E.L ., 199 0. P hloe m evol u t i on: an a ppr a i s a l ba s ed on t h e fo ssil r ec ord . In S ieve Eleme n t s : 435 co mpara tive s tr ucture, i nducti on a nd d e v elop me nt (pp. 28 5 -298) . Sp r in ger , Ber lin, Heid elbe rg. 436 5. Edwards, D., 2003. Xylem in early tracheophytes. Plant, Cell & Environment, 26(1), pp.57-72. 437 6. Edw ards , D . an d Davi e s , E. C. W., 197 6 . Ol dest rec o rded in sit u trach e id s . Nat ure, 26 3 (5577), 438 pp.49 4-495 . 439 7. Wight, D .C . a nd B ec k, C.B ., 1984. Si eve cel ls in phl oe m of a Middl e Devoni an 440 progy mnos p e r m. Sci ence , 225(46 69), pp . 146 9-1471. 441 8. Garwood , R.J. , O l ive r, H. a nd Spenc e r, A. R., 20 20. An in troduc t i on t o the Rhy ni e ch ert. G e o logic al 442 Maga zine , 157 (1), pp .4 7 -64 443 9. Kerp, H., 2 018. Organ s and ti ss ue s o f Rhynie chert pl an ts . Phi l o s op hical Tran sactio ns o f t he Royal 444 Soc iety B: Biolog ical Sci ence s , 373(1739 ), p.201604 95. 445 10. Edwards, D., 2003. Embryophytic sporophytes in the Rhynie and Windyfield cherts. Earth and 446 Environmental Science Transactions of The Royal Society of Edinburgh, 94(4), pp.397-410. 447 11. Kidsto n R, L ang W H. 19 17 On O l d Red S a ndstone pla nt s s ho wing struct ure , from t he Rhy nie 448 ch er t b e d, Abe rde en shire . P a r t I. Rhyni a gwyn ne-v au gha ni Ki dston & La ng. Tran s. R. Soc. 449 Edin bur g h 51, 761–784 . 450 12. Kidsto n R, L ang W H. 19 20a On Old Red S ands ton e plan ts showing s truc tur e, f rom the Rh ynie 451 ch er t b e d, Abe rde en shire . P a r t II. Additio nal note s o n Rh y n ia g w yn n e- va ugh an i Ki ds t on a nd 452 La ng; w ith desc riptio ns o f R hyn ia majo r , n .sp., and H orni a l ignieri , n .g., n . sp. Tran s . R . Soc. 453 Edin bur g h 52, 6 03–627 . 454 13. Kidsto n R, L ang W H. 19 20b On O l d Red S ands t on e plant s s h owing s truc tur e, f rom the Rhy nie 455 ch er t b e d, Abe rde en shire . P a r t III . As t e rox yl o n ma cki ei , Kid s ton a nd La ng . Tran s. R . S oc. 456 Edin bur g h 52, 6 43–680 . 457 14. Kidsto n R, L ang W H. 19 21a On Old Red S ands ton e plan ts showing s truc tur e, f rom the Rh ynie 458 ch er t b e d, Abe rde en shire . P a r t IV . Re s t or ation s o f the v a s c ula r c r y ptogam s, and di s c u ssion o f 459 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint their b ea r i ng on t he gene ral mor p h ology of the p te ridophyta and th e orig in of the orga nisa tion 460 of la nd -plan t s. Tran s . R. So c . Edi nburgh 5 2, 831–854 . 461 15. Kidsto n R, L ang W H. 19 21b On O l d Red S ands t on e plant s s h owing s truc tur e, f rom the Rhy nie 462 Cher t bed , A b erd een sh ir e . Pa rt V. The Th all ophyta oc cur ring i n the pe a t-be d ; th e succe ssion o f 463 the pl an t s thr oughou t a ver tic al sec tion o f t h e bed, a nd the c ondi tion s o f ac cumu la t i on and 464 pre serva tion o f th e depo si t. Tran s . R. So c . Edi nburgh 52, 855–902 . 465 16. Edw ards D.S . 1986. Aglaoph yt on major, a nonva s c ula r land -pl an t f r o m the Devon i an Rhyn ie 466 ch er t . B ot. J . Linn . Soc . 93, 17 3– 204 . 467 17. Kenric k, P. an d Cra ne , P.R ., 19 97. Or i gi n and e arly d iversi ficati o n o f la nd pl an ts . S miths onian 468 Ins titu t i on Pre ss . 469 18. Ly on, A.G. and Edwa r d s, D ., 19 91. The first z oste rophyl l fr om the Lo wer Devoni a n Rhyni e ch er t, 470 Aberd een shir e. E arth a nd En v iron men tal S cie nce Transaction s o f t he Royal S oci et y of 471 Edi nb urgh , 82 (4) , pp.324 -332 . 472 19. Pow ell, C. L., E dward s , D. a nd T r ew in, N .H ., 1999 . A new va scul ar plan t f rom the L o wer Dev onia n 473 Windyfi eld c h ert, Rhynie, N E Sco tla nd . E a r t h an d E nv ir o n m e nt a l Sc i e nc e Tr a ns a cti o ns o f t h e 474 Roy al Soc iety of Edinb ur g h , 90 (4) , pp. 331 -349. 475 20. Kenric k, P. an d L ong, E .J. , 2025. Tran s f e r c ells in H o rneoph y ton lign ieri illu min ate the orig in o f 476 va s c ular tis s u e s in l and pla n ts. N ew Phyt olo gi s t . 477 21. Kenric k, P. an d Cra ne , P.R ., 19 91. Wa te r- conduc ting c ell s in e arly f o ss il l and p lan ts: implic ation s 478 for t he ea r ly evol ution o f t rache o phyte s . Bota nic al G aze tte , 152(3 ), pp . 335-356 . 479 22. Sa t t ert hwa it and S chop f 19 72 480 23. Edw ards , D . , 1993. C ell s a nd ti s s ue s i n th e veg eta tive s p orophy te s of e arly la nd pl ant s. Ne w 481 Phy t o l ogi st , 125 (2), pp . 225-247 . 482 24. Rav en, J .A. , 20 18. Ev olution an d p alae o p hysiology of the v a s c ular sys tem and o t h e r m ean s o f 483 long -di s t anc e tr an sport. P hilo s ophic al Tr an sactio n s of the R oyal Soc iety B: Biologi c al 484 Sc ienc es , 373(17 39). 485 25. Edw ards , D . , Mor ri s , J.L. , Axe, L ., D uck et t, J. G ., Pre ssel , S . and Ken rick , P. , 20 22. Pi e cing t og eth er 486 the eo p hyte s– a new group o f a ncie n t p la nts con taini ng c r y ptos por es . N e w Phy t o lo g is t , 233 (3), 487 pp.14 40-1455 . 488 26. Ogura, Y. 1972. C ompar ative an atomy of vege t a t i ve o rga n s of t he Pte ridophyt e s. Handbuc h der 489 Pfl an z en a nat omie, VII , 3. G ebrüd er Bor n tr a eg er. 490 27. Ev er t, R.F ., 20 06. E sau' s pla n t a na tomy: me r i stem s, c ells , an d t i s sue s o f t he pla nt body: the ir 491 st r uc tur e , fu nctio n, an d d evel op men t. J o hn Wil ey & Son s . 492 28. Se ago Jr , J.L . , Moham ed, K.I ., L ea sure , B. and Bonac orsi , N.K. , 2 023. En igmatic Fea t ure s o f th e 493 Ly copod iace ae an d S ela ginel lac e ae —Lyc opodophy ta . I nter nati on al Jo urna l of Pl a nt 494 Sc ienc es , 184(1), pp .34-55 . 495 29. Van T ieghe m, P.H ., 188 2. Sur l e péricy cle . Répon s e à M. d ’ Ar ba umo nt. B ull. Soc. B ot . Fr ., 33: 152-496 153. 497 30. Van T ieghe m, P.H . and Doul iot , H., 1886 . Sur la polys téli e. A nn . Sc i. N at. B ot . Ser . 7 , 3: 275 - 3 22. 498 31. Bly t h, A. , 1958. O r ig in of p r im a r y ex t r a x y l a r y s t em f i b e rs i n d ic o t y l ed on s (pp. 145 -2 3 2). Be rkele y, 499 CA, US A: Univ ersi ty of C alif ornia P r e s s . 500 32. Sc houte, J. C., 1938 Ana t o my Manua l o f Pte ridolog y, ed . V er d oo r n, Ma rt i nu s Nijhof f, p 65-104. 501 33. Hethe rington , A. J. a nd Dolan , L., 20 18. St epw is e and i ndep ende n t or i gin s o f roo ts am ong la nd 502 pla nts. Natu re, 561 (7 722), pp .23 5-238 . 503 34. Behnk e, H .D . and Sjolund, R .D. ed s. (1990)). S iev e ele me nt s : com para tiv e st r uc t u re , ind uc tion 504 and d eve lo pme nt . S pringe r Sc ienc e & Bu s i ne ss M edia . 505 35. Li es c he , J. , Pace , M.R ., Xu, Q., L i, Y. an d C he n, S., 2017 . Heig ht-r ela te d s c alin g of p hloe m anat omy 506 and t he evol u t i on o f sieve el ement e nd wal l types in w oody plan t s . N ew Ph yt ol og i st , 214(1 ), 507 pp.24 5-256 . 508 36. Warmbrod t, R .D . and E vert , R.F. , 1974. S tr uc tu r e and d e velopme n t of t h e s i eve e l ement in the 509 ste m of Ly copodi um luc idulum. Ame r i ca n Jou rna l o f B ota ny, 61 (3), pp .2 67-277 . 510 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 37. Kal mbach and Hela r iu tt a 2 019 511 38. Rama nujam, C . G .K . and S te wart , W. N ., 1 969. T axodi ace ou s ba r k f rom th e Upper Cre tace o us of 512 Alber ta. Ame r ic an Jour nal o f Bo ta ny, 56 (1), pp.10 1 -107 513 39. Rama nujam, C . G .K . , 1970. A pe tr i f i ed b ar k o f Cupr e ssac ea e fr om the Up per Cre tac eou s of 514 Alber ta. Ca na dia n Jo urna l of B ot any , 48 (5 ) , p p . 85 5- 8 5 8 515 40. Eg gert, D. A. and Gaun t , D.D ., 1 973. Phl o em of S phen ophyl lµm. Americ an Jour nal o f 516 Bota ny , 60 (8), p p. 755 -770 517 41. Egg ert , D. A . a nd Ka nemot o, N .Y., 19 77. S tem phloem o f a middl e Penn sylv anian 518 Le pidodend ron. B ota nical Ga zet te , 13 8(1 ), pp.10 2 -111. 519 42. S moot, E.L . and T aylor , T .N., 1978. S ieve area s in fo s s i l phloem . Sc ienc e , 202(4 372 ), 520 43. Smoo t , E.L., 1979 . T he phlo em o f Etap ter is lec le rcqii and Bo tryopt eri s t r i den ta ta. Americ an 521 Jour nal o f Bo ta ny , 66 (5), 522 44. Pi gg, K.B. and Ro thw ell, G.W ., 198 3. Cha lo neria ge n . nov.; h et ero s po rou s lyc ophyte s f rom the 523 Penn syl vania n o f No r th Am e r ic a. Bot a ni ca l G a ze t te , 144 (1 ), pp.132 -14 7. 524 45. S moot, E.L ., 1984. Phlo em s tructu re in t he Car boni fero us fe r n P s a r oni u s ( Mara tti ale s ) . Ame r i can 525 journal o f b ota ny , 71 (8), p p.1 104 -1113 526 46. S moot, E.L ., 1984. Phlo em a natomy o f the Car boni fero us p te rido s p e r m Medu llo s a a nd 527 ev olutiona ry tren ds in g ymno sperm phl o e m. Bo tanic al Gaze t te , 145(4) , pp. 550 -56 4. 528 47. S moot, E.L ., 1985. Phlo em a natomy o f the Car boni fero us co e nop ter i d fe rn s Ana c horopt eri s and 529 Anky r op t e ri s. Am e r i ca n j o ur nal of b ot any , 72 (2), pp. 191 -208; 530 48. Smoo t , E.L. an d Wege , M. V., 1986 . Phlo e m anatomy in Staur opt eri s bi seri ata fro m the 531 Penn syl vania n o f No r th Am e r ic a. A m er ic a n jo u r na l of b ot an y , 73 (7), pp .1043-10 4 8 532 49. Erw in and S tocke y 199 1 533 50. Cordi, J . , 1993. A n a t o my and mor ph olog y of selec t D evo ni an pla nt s an d t he in fere nc e of 534 mac r o evol u t i onary pa tter n an d proce ss d uring t he r a d ia tion of e ar l y trache op hyte s . S tat e 535 Univer sity of New York at Bi ngham ton 536 51. Dega ni -Sc hmidt, I. a nd Guerra -S ommer , M., 20 16. C harc oali fi ed Agath oxy lon-typ e w ood w it h 537 pre serve d s eco ndary phlo em fr om the lo w er Permi an o f t he Bra z il ian Pa ra na Ba si n. Re view o f 538 Pala eob ot any and Pal ynol og y , 226 , p p . 20 - 2 9. 539 52. Franc o, M .J. , Bre a, M. and C e r de ñ o, E., 2 021. F irst Bign oniac eae li a na f rom the M i oc ene o f South 540 Americ a and i t s ev olution ary s ig ni fica nce . A m er ic a n J o ur n al of B ot an y . 541 53. Deco mbeix, A .L., G alti e r , J. and M ey er-B e r th aud, B. , 2014 . Sec ond ary phloem in Early 542 Carbo nif e r ou s se ed pl an ts: a na tomica l di v er si ty a nd ev olutio nary i mplica tio ns. In t ernati on al 543 Jour nal o f Pl an t Sc ienc es , 175(8) , pp.891 - 910. 544 54. Pf eil er, K.C . and Tome scu, A. M . , 2018 . An Early Dev onian pe rminer alized r hyni op s i d f r om t h e 545 Batt ery Poi nt Forma tion o f G as pé ( Ca n ad a). Bo ta n ic a l J our nal o f t h e Li nn e a n S o ci e t y , 18 7 (2), 546 pp.29 2-302 . 547 55. Burr, F .A. a n d Eve r t, R .F ., 197 3. S ome a s pect s of s i eve- el emen t st ruc t ur e a nd dev elopmen t in 548 Se lagine ll a krau s sia na . Prot opl a sma , 78 ( 1), pp.8 1 -97. 549 56. Kruatrach u e, M . and Evert , R.F . , 1974. St r u ctur e and d evelo pm ent o f sieve el eme nts in the l e af 550 of I so ete s muric ata . America n Jo urnal of Bota ny , 61 (3), p p.253 -266 551 57. Imai chi, R. and Hi rat suka , R. , 2007. Evolu tion of s h o ot apic a l meri ste m struc t u re s i n v ascular 552 pla nts wi t h r e s p ec t t o pla smode sma tal n e t w ork. A me r ic an jo urna l o f b ota ny , 94 (1 2), p p.1911 -553 1921 . 554 58. Vladá r , A.E . and Ara t , K.T ., 20 23. L imit s o f r e solu tion s in t h e s c anning elec tr on mic rosco pe. 555 Mic r osco py and Micro ana ly sis , Volum e 29, Issu e S upplem e nt_ 1, 1 Augu st 2023 , P age s 4 63–464 . 556 59. Huff , J. 2015 . The Airy sc an de tec tor f rom ZE ISS: confoc al ima ging with imp r ov ed si gnal - t o-n ois e 557 ratio an d s up e r-r es oluti on. N at M ethod s 12, i –ii 558 60. Voro nt s ov , D .D. , Kole sniko v, V.B ., V oron ezh skay a, E.E ., Perkov s k y, E.E ., B ert o, M. M., Mowe r y , J., 559 Ochoa, R . and Klimov, P.B ., 20 23. B ey ond the limi ts o f li ght: an a p plic ation of supe r-re s oluti on 560 co nfocal micro scopy ( sCL SM) to inv e s tig a te Eoc ene amb er micro fo s s il s. Li fe , 13 (4 ), p.865. 561 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 61. Davi dson, M. W . (2 025) Li mit of re sol uti o n of o pt i cal micro scope Acc e ss ed 16 / 03 / 2026 562 http s : / /www .microscopy u.c om/mic r o sc o py-ba sic s/re solu tion 563 62. Voro nt s ov , D . and Voron e z h skay a, E.E . , 2022. Pu shing the limi t s of op tical r e solu t i on in the study 564 of th e tini e st f os s i l a rth r opo d s. His torical Biology , 34 (12 ), p p.2415 -242 3. 565 63. Cook , M .E., G raham, L.E ., Bo tha, C.E. an d L avin, C.A. , 1997 . Compa ra tiv e ultr as t ru c t ure o f 566 pla s mod e smat a of Char a an d s e lec ted b ry ophyte s: towa rd a n eluc ida tion o f t h e e volutio nary 567 origin of plan t pl a s mo d es m a t a . America n Journ al o f Bo tan y, 84 (9 ), pp.1169 -11 78. 568 64. Hébant , C. , 1977. The c onducti ng tissu e of b r y ophyt e s. B r y o ph yt or u m B ib l i ot h ec a , 10 . 569 65. Sc heirer , D .C ., 19 90. Mo s s e s . In Siev e ele ment s : comp ara t iv e s tr uc ture , ind u ction an d 570 dev elo pm e nt (pp. 19 -33 ). Berlin , Heide l b erg: Sp r in ge r Berli n Heide l berg. 571 66. Li grone, R., D uc ket t, J .G . and Re nz a glia, K .S., 2000. Con duc ting t i ss ue s and phy le ti c rel ation shi ps 572 of bryoph yt e s . P hilo so phic al T r an s ac t i on s o f the R oy al Soc iety of Lon d on. Se r i e s B: Biolog ical 573 Sc ienc es , 355(13 98), pp .795 -813 . 574 67. Woudenb e r g , S. , Re n ema, J ., Tome scu, A .M., D e Rybel , B. and Wei jer s , D., 2022 . D eep origin a nd 575 gradua l ev oluti on of tran sp or ti ng ti s s ue s: per spectiv e s from a cro ss the la nd pl an ts . Pla nt 576 Phy s io l ogy , 190(1 ), pp.85 -99. 577 68. Ca sca le s-Miñ a na, B ., St eeman s, P. , S erva i s , T., L ep ot, K. a n d G e r rie nn e, P., 2 019. A n a lter n a tive 578 model fo r t he earli es t evo lu tion o f va s c ul ar pla nts . Le t ha i a , 52 (4), pp .44 5-453 . 579 580 581 582 583 584 585 586 587 588 589 590 591 592 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint Sup pleme ntar y Fig u res an d Caption s 59 3 59 4 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint Figure S1: Longitudinal sections of food conducting cells (FCCs) of the Rhynie chert species 595 shown in Figure 1. 596 (A) L ongit ud inal s ection of Aglaophyton majus ax is s h owi n g centr al W C C s and s u rroun ding 597 FCC s , transitional tiss u e and cor tex. 598 (B) D etai l of th e i m a ge in (A ) s h owi n g WC C s (left), FCCs and tr an s it i o nal t is s ue (middl e) and 599 cort e x (r ight). 600 (C) Longitu dinal section of Rhynia gwynne-vaughanii axis showin g c ent ral xy lem, 601 surro unding FC C s , t ransitional tis sue and cort i cal tis s ue. 602 (D ) CLSM of longitudinal sec t ion of Asteroxylon mackiei r ooting axis showin g c ent ral xyl em , 603 surro unding FC C s and cort ic al tis sue. 604 (E) Longitudinal section of Asteroxylon mackiei r oot- bear ing axi s with ro oti ng ax is meristem 605 showing cent ral FCC s and de ca yed co rtex tiss ue, F CC s measuring 194-405 µm . 606 (F) D etail o f the speci m en s hown in (E) showing tr ac heid s and elongated FC Cs . 607 (G) Det ai l of t he s p e cimen shown in (E) showin g a 40 7 µ m long F C C (arr ows ). 608 Specimen c o de s = A-B = NMS 1925.9. 7, C = NHM U K 1563 3, D = STA 355.76 , E-G = NHM U K 609 16433 610 Sca le bar s = 5 00 µm in (A ), 200 µm in (C, D ), 100 µm in (B, E) , 50 µ m in (F, G). 611 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 61 2 Figur e S 2 Rhynia gwynne-vaughanii (in cross section in Fig. 1 C- D) longitu d inal s ection 61 3 showing FCCs, tr ans it ional tiss u e an d c or tex cells . The F CC s are elongated and close 61 4 pac king, whil st t he c ells of the t ransi t i onal tiss ue are slightly s ho rter a n d l oos er -pac k ing, 61 5 with air s pace s bet ween c ells v isible. The c o rtex cell s are r ounded with many intr ac ellular 61 6 spa ce s . Spe cimen code = BHUTTA BL29A.185. S cale b a r = 500µm. 61 7 61 8 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 619 Figure S3 Aspect ratio (AR) of the parenchymatous cell types of the Rhynia gwynne-620 vaughanii axis in Figure S2 (c.s. of species in Figure 1 C-D). The FCC s have an average AR of 621 0.20 (r ange 0.3 1- 0.10), indicating that t hes e cells ar e nar row and elongated. A djac ent to the 622 FCC s is a tran s itional tissue of elongated cells (average AR o f 0.30, range 0. 58-0.14) with 623 some intercellular spaces, these cel ls have a significantly (p≤0.00 1, a s dem ons t rated by a t-624 test) lar g er AR th a n t he FCCs, a s the s e c ells are wider and short er than th e FC C s on average 625 (supplementary table 1). The c o rtex cel ls are mor e rounded t han the cells of t he t ransitional 626 tiss u e and t he FC C s ( a ver ag e A R of 0. 62, range 1. 22-0 .20 , si gnificantly larg er than t he 627 tr ans it i o nal tis sue). Mea surem ents from axis in Fig. S2. 628 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 62 9 Figur e S 4: Wh en d i f feren ces in axis d iameter ar e ac count ed for , the t rend shown in Fig ure 63 0 2, t hat th e F C Cs of t he Rhy nie cher t plant s are significantly (p ≤ 0.00 1) larger in diameter 63 1 t han the p hlo em cells of e x t ant lyc o phyte s, per s ists. Mea surement s of t he diamete r o f 63 2 phloem or food condu c t ing cells ex pr essed as a r a t io to the axis d i am eter a nd pr es ent ed as 63 3 a per c ent age in extant ly coph y t e s co mpar ed to ex tinct spec ie s from the R hy nie chert. Four 63 4 extant l y cophy t e specie s ( H uperzia g oebelli i, H uperzia s elago, Lyc opo di u m c lavatu m, 63 5 Selagine lla u nci nata ) were investigated covering both stem and r oot t is su es . Four Rhy nie 63 6 chert s pe cies covering axis t issue s ( Aglaop hyton majus , Rhynia gwynne-va ughan ii , 63 7 Tr ic hopher ophyton teuchans ii ), and r oot bearing ax is and roo ting ax is ( As ter oxylon mackiei). 63 8 The FC Cs of th e R hy nie cher t spe c ies are all significantly (p≤0.001) lar ge r than th e p hloe m 63 9 cells of the extant ly cophy tes , a s demon s tr ated by a t-t es t . 64 0 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint 64 1 Species and o r gan abbreviations: H. g oe stem = H u p e r z ia g o e b el l i stem; H. s e l s t e m = 64 2 H uperzia s el stem; L. c la r oot = Ly cop odium clavat um r oot; L. cl a s t e m = Ly copodium 64 3 clavatum stem; S. unc roo t = Sel agine lla unc inat a ro o t; S. unc stem = Sel agi nella u ncinat a 64 4 stem; A . maj ax is = Aglao phyton ma j us axi s ; R. gwy axis = Rhynia gwynne-v augha nii ; T. teu 64 5 axis = Tr i chopher ophyton teuch ans ii ax i s ; A. mac root -bearing axis = As ter oxylon mackiei 64 6 r oot-b e a r ing ax is; A. mac root ing ax is = As teroxylon mack iei ro oting ax is. 64 7 64 8 64 9 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint Figur e S 5: Wh en d i f feren ces in cell d iameter a r e ac cou nted fo r, the t rend shown in Fi gur e 65 0 2, t hat th e F C Cs of t he Rhy nie cher t plant s are significantly (p ≤ 0.00 1) larger in diameter 65 1 t han the p hlo em cells of e x t ant lyc o phyte s, per s ists. Mea surement s of t he diamete r o f 65 2 phloem or food condu c t ing cells ex pr essed as a rat io to the average cort ex cell diameter of 65 3 t he s ame axis in extant l y coph y tes co mpared t o ex t inc t s pecie s from t he R hy nie c h ert. Four 65 4 extant l y cophy t e specie s ( H uperzia g oebelli i, H uperzia s elago, Lyc opo di u m c lavatum , 65 5 Selagine lla u nc inata ) were investigated covering both stem and r oot t is su es . Four Rhy nie 65 6 chert s pe cies covering axis t issue s ( Aglaop hyton majus , Rhynia gwynne-va ughan ii , 65 7 Tr ic hopher ophyton teuchans ii ), and r oot bearing ax is and roo ting ax is ( As ter oxylon mackiei). 65 8 The FC Cs of th e R hy nie cher t spe c ies are all significantly (p≤0.001) lar ge r than th e p hloe m 65 9 cells of the extant ly cophy tes , a s demon s tr ated by a t-t es t . 66 0 Species and o r gan abbreviations: H. g oe stem = H u p e r z ia g o e b el l i stem; H. s e l s t e m = 66 1 H uperzia s el stem; L. c la r oot = Ly cop odium clavat um r oot; L. cl a s t e m = Ly copodium 66 2 clavatum stem; S. unc roo t = Sel agine lla unc inat a ro o t; S. unc stem = Sel agi nella u ncinat a 66 3 stem; A . maj ax is = Aglao phyton ma j us axi s ; R. gwy axis = Rhynia gwynne-v augha nii ; T. teu 66 4 axis = Tr i chopher ophyton teuch ans ii ax i s ; A. mac root -bearing axis = As ter oxylon mackiei 66 5 r oot-b e a r ing ax is; A. mac root ing ax is = As teroxylon mack iei ro oting ax is. 66 6 66 7 66 8 Figur e S 6: Plas mo d es mata pits in th e lat eral walls o f the ph l o em paren c h yma o f Huperzia 66 9 selago are d i stin guis hab le f rom siev e por es ( Figur e 3B). 67 0 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint A , SEM imaging o f longitudinally c ut Huper z ia s el ag o stem showing x y l em (magenta false 67 1 colour ing), phloem tiss u e ( y ellow), an d cor tex (blue); 67 2 B, higher magnifica t ion image of the regi o n enc losed b y the box in (A ) s howing phloem 67 3 par enchy m a l at eral c ell walls exhibiti ng mu l t iple p las mod es mat a p i t areas ( arrows, outlined 67 4 in pur ple); 67 5 C, higher magnifica t ion image s howin g th e p l asmodesmata pit ar eas in (B ); 67 6 D , higher magnification image of the c ent ral pit areas in (C) with ind ividual plasmodesmata 67 7 pit s clearly vi sible. 67 8 Scale bars = 2 0 µ m in (A), 10 µm in ( B ), 1 µm in (C) , 400nm in (D ). 67 9 68 0 68 1 68 2 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint Figure S7: Test imaging shows that xylem pits of Asteroxylon mackiei are readily imaged 683 using Airyscan CLSM, providing grounds for imaging of sieve pores in Asteroxylon mackiei 684 (Fig. 3C-M). 685 A, Xylem tr ac h ei d s in longitudinal sect ion 686 B, Xylem tr ac heids in oblique-t ransv er s e se c t ion 687 C, X ylem tr ac heids in oblique-longitudinal section 688 D, X ylem t racheids in longitud i n a l section 689 White arro w s indicate xy lem pit s . 690 Sca le bar s: 20 µm in ( A, C), 10 µm in (B , D). 691 Acce ssion code s : STA 355.76 ( A); S TA 355.61 (B) ; MAN EMu 54740 8 (C ); N H MUK 1643 3 (D ). 692 693 A verage cell length (µ m ) Cell length ra ng e (µ m ) Average cel l diameter (µm) Cell diameter range (µm) Average Aspect Ratio Aspect Ratio range FCC s 2 26.1 142.3- 346. 7 44.1 31.8-61.5 0.2 0.1-0.31 Transitional Tissue 1 89.6 93.9-2 82.1 53.7 3.1-72.6 0.3 0.14-0.58 Cor tex c ells 1 02.3 49.9-1 82.7 59.0 30.7-94.0 0.62 0.2-1.22 694 Supplementary Table 1: Cell length, diameter and Aspect Ratio (AR) summary for Rhynia 695 gwynne vaughanii parenchymatous tissue types. Measuremen ts f rom th e a xis s hown in 696 Fig. S2 , dat a sho wn in Fig. S3 . 697 698 699 700 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.23.713640doi: bioRxiv preprint