Background

Even if they are considered the quintessential “living fossils”, the fossil record of the extant genera of the Cycadales is quite poor, and only extends as far back as the Cenozoic. This lack of data represents a huge hindrance for the reconstruction of the recent history of this important group. Among extant genera,Bowenia (or cuticles resembling those of extantBowenia) has been recorded in sediments from the Late Cretaceous and the Eocene of Australia, but its phylogenetic placement and the inference from molecular dating still imply a long ghost lineage for this genus.

Results

We re-examine the fossil foliageAlmargemia incrassata from the Lower Cretaceous Anfiteatro de Ticó Formation in Patagonia, Argentina, in the light of a comparative cuticular analysis of extant Zamiaceae. We identify important differences with the other member of the genus, viz.A. dentata, and bring to light some interesting characters shared exclusively betweenA. incrassata and extantBowenia. We interpret our results to necessitate the erection of the new genusEobowenia to accommodate the fossil leaf earlier assigned asAlmargemia incrassata. We then perfom phylogenetic analyses, including the first combined morphological and molecular analysis of the Cycadales, that indicate that the newly erected genus could be related to extantBowenia.

Conclusion

Eobowenia incrassata could represent an important clue for the understanding of evolution and biogeography of the extant genusBowenia, as the presence ofEobowenia in Patagonia is yet another piece of the biogeographic puzzle that links southern South America with Australasia.

Electronic supplementary material

The online version of this article (doi:10.1186/s12862-017-0943-x) contains supplementary material, which is available to authorized users.

Keywords: Cycadales, stomata, cuticle, biogeography

Specimens investigated

The fossil specimens examined for this study were first described by Florin [42] and later by Archangelsky [43]. The specimens examined forAlmargemia incrassata are stored in the Natural History Museum (NHM), London, UK, to which they were donated as duplicates by Sergio Archangelsky in 1960, under accession numbers v52264 (macrofossil) and v52265 (cuticle slide). The specimens examined forA. dentata Florin are stored in the Swedish Museum of Natural History (NRM), Stockholm, Sweden, under accession numbers S085614–S085619. Two of the original specimens examined by Heer [44] and Florin [42] could be traced down at the Museu Geológico, Lisbon, Portugal (accession numbers 23,213 and 23,217). Sources of the samples from extant species are listed in Additional file1: Table S1. The slides produced from the latter are stored at the Department of Systematic and Evolutionary Botany of the University of Zurich, Zurich, Switzerland.

Extant material preparation

Whole leaves were fixed in 50% Ethanol. Sections of a leaf ofBowenia serrulata (W.Bull) Chamb. were stained and mounted according to Coiro and Truernit [45]. Cuticles were isolated by immersing leaf fragments in a 2:1 mixture of 30% hydrogen peroxide and 85% ethanol, warmed up to 60 °C until the leaf fragments turned transparent. Cuticles were then rinsed in distilled water and cleaned using a fine brush. Cuticles ofMacrozamia plurinervia (L.Johnson) D.L.Jones were isolated using overnight maceration in 10% Cr₂O₃. Cuticles were then stained in Auramine O (Sigma; 0.01%w/v in 0.05 M Tris/HCl, pH 7.2) for 10–15 min (see [46]). They were then rinsed with water and mounted in glycerol. All extant samples used for our comparative analyses are listed in Additional file1: Table S1.

Microscopy and image analysis

For light and epifluorescence microscopy, slides with fossil cuticles were observed using a Nikon Eclipse LV100ND microscope (Almargemia incrassata) or an Olympus BX-51 light microscope, which was modified for epifluorescence microscopy, and photographed with an Olympus DP-71 digital camera (A. dentata). Cuticles of extant cycads and whole-mount leaf samples were observed using a Zeiss Axioscope fitted with a Zeiss 38 HE fluorescence filter.

Confocal observations of fossil samples were made using a Nikon A1-Si laser-scanning confocal microscope, with two excitation lines: 488-nm line of 50-mW sapphire laser and 561-nm line of 50-mW sapphire laser (Coherent Inc., Santa Clara, California, USA). The autofluorescence signal was collected with two different photomultiplier detectors with the following wavelength emission windows: 500–550 nm for the 488-nm laser, 570–620 nm for the 561-nm laser. PS-PI stained samples and Auramine O stained cuticles ofBowenia spectabilis were observed using a Leica TCS SP8 microscope. Excitation was obtained using a 488 nm laser for the PI and a 405 nm diode laser for the Auramine O. Raw images were analysed and measured using the software FiJi [47]. Brightness and contrast were adjusted using the “auto” option in the software. Confocal stacks were combined using a Maximum Intensity projection. Scans of freshly cut leaves of extant cycads were taken at 1200 dpi using an Epson Perfection V600 Photo J252A scanner.

Phylogenetic analyses

To test the placement ofEobowenia in the phylogeny of the Cycadales, we codedEobowenia andAlmargemia by incorporating the new data from our investigation in a modified version of the morphological matrix from Martinez et al. [48]. We removed the taxa that had no character overlap with our foliage taxa from the matrix. We then changed some of the character states in the light of our comparative data. In detail, we codedBowenia andStangeria as having hypostomatic leaflets (character 49) and oblong stomata (character 51),Stangeria as having one accessory cell layer (character 53),Bowenia as having longitudinally-oriented stomata (character 52) and Bennettitales as having both flush and sunken stomata (character 50). We also added a character for the substomatal complex thickenings, coded as present inEobowenia, Encephalartos, Macrozamia andBowenia, and absent in the other extant Cycadales exceptLepidozamia. The state for such character in other fossil cycads as well as in the outgroups was coded as unknown.Eobowenia was coded conservatively regarding the architecture of the leaf, with leaf dissection coded as pinnate (character 24) and midrib coded as absent (character 32).

Two different sets of analyses were performed: first, we analyzed the morphological matrix separately, and secondly we combined the morphological matrix with the molecular matrix from Salas-Leiva et al. [7]. All analyses were conducted using both Maximum Parsimony (MP) as implemented in PAUP* ver 4.0b10 [49] and Bayesian Inference (BI) as implemented in MrBayes ver 3.2.6 [50]. Search for the MP trees was performed using heuristic search with 1000 addition replicates and random addition sequence of taxa, and the bootstrap analysis was conducted for 1000 replicates using 10 searches per replicate and keeping only one tree per replicate. We also ran an analysis with the relationships between the extant cycad genera from Salas-Leiva et al. [7] forced as a backbone constraint. The BI analyses were executed using two runs of four chains (one cold and three hot chains), with 1 million generations for the morphology-only analysis and 5 million generations for the combined morphological-molecular analysis. In the morphology-only analyses, we used the mk_inf model [51] with gamma-distributed rate variation. In the morphological-molecular analyses, we set one partition for each of the markers plus one morphological partition. For all molecular markers we used a GTR plus gamma model, and for the morphological partition we used a mk_inf plus gamma model. After discarding 25% of the trees as burn-in, trees were summarized as consensus trees including all compatible splits. Characters were then reconstructed on the trees using Maximum Parsimony as implement in Mesquite ver 3.03 [52] to identify synapomorphies.

We also used the modified morphological matrix to test the different placements ofEobowenia in relation to the extant genera of the Cycadales using Mesquite ver 3.03 [52]. We edited a tree of the extant genera according to the topology of Salas-Leiva et al. [7], and counted the length of the trees obtained by moving the placement ofEobowenia by hand.

Systematic Palaeontology

Order – Cycadales Pers. ex Bercht. et J.Presl.

Family – Zamiaceae Miq.

Subfamily – Bowenioideae Pilg. in H.G.A.Engler et K.A.E.Prantl.

Genus – Eobowenia M.Coiro et C.Pott gen. nov.

Type: Eobowenia incrassata (S.Archang.) M.Coiro et C.Pott comb. nov., from the Aptian (Lower Cretaceous) of Patagonia, Argentina.

Diagnosis: Emended from Archangelsky [43]. Leaves pinnate. Midrib delicate. Leaflets subopposite, insertion more acute towards the apex, oblong. Leaflet base broad. Leaflets with serrate margin. Veins parallel to the margin, converging at the base of the leaflet. Leaflets hypostomatic. Stomata with guard cell poles raised with respect to the aperture. Guard cells at same level with the epidermis, arranged longitudinally with respect to the leaflet axis. Stomatal complex monocyclic. Subsidiary cells with differentiated cuticle. Polar subsidiary cells sometimes differentiated from the lateral ones. Substomatal cells with thickened secondary cell walls. Epidermal cells elongated parallel to the leaflet axis, with darker-staining short cell distributed in rows of short cells. Anticlinal cell walls slightly wavy or concave.

Etymology: From Greek Ἕως, dawn, and the name of the extant cycadBowenia.

Remarks: Based on the new characters identified and a re-evaluation of the epidermal anatomy of extantBowenia and fossilAlmargemia dentata (Fig.1) andA. incrassata, we erect the new genusEobowenia. In our opinion, the leaflets ofA. incrassata share interesting characters withBowenia, but are distinct enough to deserve the institution of a new genus.Eobowenia is distinguished fromAlmargemia by the leaflets with serrate margin, the veins converging at the base of the leaflets, the guard cells at the same level of the epidermis arranged longitudinally with respect to the leaflet axis, and the monocyclic stomatal complexes. FromBowenia, it is distinguished by the presence of darker-staining cells arranged in rows, the broad attachment of the leaflets, and the smaller size of the leaflets.

Eobowenia incrassata (S.Archang.) M.Coiro et C.Pott comb. nov.

1966Almargemia incrassata – Archangelsky, p. 267; pl. I, Figs. 3, 4; pl. III, Figs. 13, 14; Text-Figs. 6–10, 13.

Diagnosis: As for the genus, with the following additions: Leaflet base with constricted acroscopic margin and decurrent basiscopic margin. Striations are visible in between the veins.

Holotype: LP6255, Museo de Ciencias Naturales, La Plata, Argentina.

Remark on types: Specimen LP6255, published by Archangelsky [43], automatically becomes the holotype of the new combination and the new genus. However, we chose specimen v52265 (a cuticle slide obtained from LP6255) as epitype; it serves as interpretative type because it perfectly presents the combination of characters necessitating the erection of the new genus.

Type locality: Estancia Bajo Grande, Santa Cruz Province, Argentina (not Bajo Tigre as erroneously reported by Archangeslky [43], see [53]).

Type unit and age: Baquero Group, Anfiteatro de Ticó Formation,Auracarites Bed. Lower Cretaceous (Aptian).

Description: Eobowenia incrassata (Fig.2) is represented by two fragmentary specimens [43]. The three (probably terminal) leaflets on specimen v52264 clearly show the serrate margin, the attachment of the leaflets, and the fine striations present between the veins on the leaflets (Fig.2 a). These characters were already identified as diagnostic for the species by Archangelsky [43]. The preserved portions of the leaflets are 7.4–9.3 mm long and up to 3.5 mm wide.

The cuticle fragments examined show that the leaflets are hypostomatic with epidermal pavement cells longitudinally elongated parallel to the leaflet axis (Fig.2 c, d). Ordinary epidermal cells are elongate and moderately cutinised. On the adaxial side, rows of cells with thicker cuticle than the ordinary pavement cells can be observed (darker staining; equivalent to the thin-walled cells of most Zamiaceae (see [54])), which seem to be arranged preferably in rows of short cells. The anticlinal walls of these dark-staining cells tend to be slightly concave. On the abaxial side, rows of darker staining cells as well as single darker staining cells are present. The stomata are confined to the abaxial side and are distributed uniformly in broad intercostal bands on the leaflet surface, with the guard cells oriented longitudinally (Fig.3a,c). Guard cells are in average 38.63 (35.53–42.30) μm long and 17.90 (16.86–19.71) μm wide, with an aperture that is 20.89 (14.78–23.98) μm long. The stomatal complexes are monocyclic, with four to six subsidiary cells that have a thicker, darker staining cuticle than the ordinary pavement cells. The cuticle of the guard cells presents a ventral thickening in the correspondence of the aperture as well as ridges that run parallel to the dorsal wall (Fig.2 d; Fig.3 c, e; Fig.4 a). In some stomatal complexes, is possible to observe differentially thickened or perforated cell walls, which are similar to the cell wall of the substomatal complex in extantBowenia (Fig.2 d; Fig.3 e).

Remarks: The characters that separate the fossils assigned toAlmargemia incrassata from those assigned toA. dentata and link the former withBowenia are depicted in Table1. The allocation ofA. incrassata to the new genusEobowenia retainsA. dentata as the only representative ofAlmargemia.

Phylogenetic analyses

The MP analysis of the modified morphological matrix of Martinez et al. [48] resulted in 242 equally parsimonious trees of 196 steps. In the strict consensus tree,Bowenia andEobowenia are in a polytomy with most of the other fossil taxa. This is due to the uncertainty in the placement ofStangeria andMesodescolea, which could be equally parsimoniously placed as sister toEobowenia plusBowenia, sister toBowenia alone withEobowenia as sister to this clade, or in a clade with other fossil taxa (Kurtziana,Pseudoctenis,Sueria,Mesosingeria). In the bootstrap analysis, a sister group includingEobowenia andBowenia does not receive support, being retrieved in only 50% of the bootstrap replicates. Forcing the molecular backbone constraint from Salas-Leiva et al. [7] on the modified morphological matrix of Martinez et al. [48] results in 594 trees of 222 steps. In the consensus tree, only a few relationships are resolved, butBowenia andEobowenia result sister taxa. In the Bayesian consensus tree of the morphology-only analysis, a clade includingEobowenia as sister toBowenia,Stangeria andMesodescolea receives a weak support (0.62 posterior probability).

The MP analysis of the combined matrix resulted in 368 trees of 2925 steps. The consensus tree is poorly resolved, butEobowenia results sister group to the twoBowenia species. In the bootstrap analysis, this relationship is weakly supported (55% of the bootstrap replicates). The BI analysis of the combined morphological-molecular matrix strongly supports a placement ofEobowenia as sister of the two species ofBowenia (0.91 posterior probability) (Fig.5 b). The presence of flush guard cells (char 50) and the absence of encircling cells (char 53) represent synapomorphies of theEobowenia andBowenia clade in this topology, whereas the presence of a thickened substomatal apparatus (char 89) is ambiguously resolved as either synapomorphic forEbowenia andBowenia or plesiomorphic for all Zamiaceae exceptDioon (Additional file2: Figure S1).

Using the topology from Salas-Leiva et al. [7] as a backbone and movingEobowenia by hand, the shortest tree is obtained withEobowenia as sister toBowenia (131 steps). A placement ofEobowenia as sister toBowenia plus Ceratozaminae and Encephalartinae, sister to Ceratozaminae plus Encephalartinae, or sister to either Encephalartinae or Ceratozaminae is one step longer. Placement as sister toDioon, Cycas, Zamiaceae orStangeria requires two more steps. Placement as sister toCeratozamia, sister toStangeria plusMicrocycas plusZamia plusChigua or sister toMicrocycas plusZamia plusChigua requires three more steps. Placement in any position in theMicrocycas-Zamia clade requires four more steps, and placement in any positions in theMacrozamia-Encephalartos-Lepidozamia clade requires six more steps (Fig.5 a).

Comparison of Almargemia dentataand Eobowenia incrassata

The leaves from the Lower Cretaceous of Portugal later referred to by Florin [42] asAlmargemia dentata were first described by Heer [44] asCtenidium dentatum Heer andC. integerrimum Heer. In the generic diagnosis, Heer [44] distinguishedCtenidium fromPtilophyllum andPtilozamites by the decurrent leaf bases and fromCtenis by the absence of vein anastomoses. Florin [42] investigated the epidermal anatomy of the specimens described by Heer [44] in detail and, as a consequence, transferred both species in the new combinationAlmargemia dentata, correctly recognizing that the genus name selected by Heer [44] was pre-occupied by a genus of extant mosses. The main diagnostic epidermal characters ofAlmargemia according to Florin [42] were the predominantly incompletely amphicyclic haplocheilic stomata, arranged irregularly in stomatal bands running between the veins on the abaxial surface of the leaflets, the sunken guard cells and the presence of both weakly and strongly cutinised pavement cells. Macromorphologically, the diagnostic characters included slightly contracted leaflet bases, parallel (rarely dichotomizing) venation and the presence of lobe-like teeth (Fig.1 a, b, e).

When Archangelsky [43] describedEobowenia incrassata (asAlmargemia incrassata), he decided to assign such specimens toAlmargemia on the base of the serrate margin of the leaves (erroneously identified as ‘dentate’ by Archangelsky [43]) and the differently thickened cutinization of the epidermal cells. However, most of the other diagnostic characters ofA. dentata are absent inE. incrassata (Table1). The stomatal characters are strikingly different (Fig.4), withE. incrassata having guard cells at the same level of the epidermal cells, monocyclic stomatal complexes and longitudinally oriented guard cells, whileAlmargemia dentata has stomata sunken below the epidermal level, as in most extant Zamiaceae, incompletely amphicyclic stomatal complexes and randomly oriented guard cells. To use only the differentially thickened cutinisation of the epidermal cells is, in our opinion, too weak a character to assign the fossils in question (viz.Eobowenia incrassata) toAlmargemia, because of their common presence in most members of extant Zamiaceae [34,41,54]. Moreover, the dentation of the margin in the two species is quite different, withE. incrassata having relatively small, acute teeth andA. dentata having larger, lobe-like teeth. For these reasons, we reconsider the allocation made by Archangelsky [43] by erecting a new genus because a new generic definition is needed for this fossil taxon.

Comparison of Eobowenia incrassatawith other fossil cycadophytes

The leaves ofEobowenia incrassata are easily distinguishable from all other cycadalean leaf taxa described from the Baquero Group (i.e.Ticoa,Mesosingeria,Mesodescolea,Sueria; [40,43]) by their leaf shape and epidermal anatomy (see [40,43]). Among other Mesozoic cycadophyte leaves with parallel venation,E. incrassata differs fromPseudoctenis [55] by its basally converging veins, the serrate margin and by epidermal characters (i.e. guard cells at the same level as the epidermal cells, darker-staining pavement cells, longitudinally elongated pavement cells), and fromCtenis [55] by the absence of vein anastomoses as well as the very different cuticle. It differs from segmentedNilssonia leaves [55] by the lateral attachment of the leaflets and the anatomy of the cuticle and fromEncephalartites by the leaf base that is contracted only on the acroscopic side, and by the oblong leaflets.Eobowenia incrassata is distinguished from any segmented bennettitalean leaf by the haplocheilic architecture of the stomata in contrast to the syndetocheilic architecture characterising bennettitalean leaves [19,14].

A similar combination of differentially thickened epidermal cells, monocyclic stomatal complexes and guard cells at the same level with the aperture is present in some species assigned to the tentative pteridosperm genusStenopteris. Monocyclic stomatal complexes with differentiated subsidiaries are present inS. nana T.M.Harris from the Bajocian of Yorkshire [55], but the overall morphology of the leaf easily distinguishes this species fromEobowenia incrassata. Another interesting species isS. cyclostoma K.Saiki, T.Kimura et J.Horiuchi, from the Lower Cretaceous Choshi Group of Japan [56]. The cuticle of this species presents many similarities withE. incrassata including the rows of dark staining cells [56], but presents a very dissimilar morphology of the leaf. However, there are differences even at the cuticular level, withS. cyclostoma being clearly amphistomatic and having an external vestibulum. Moreover, we were not able to identify the peculiar perforations of the substomatal complexes in the illustrations of Saiki et al. [56]. The cycad-like characters ofS. cyclostoma are definitely interesting, but a more thorough discussion would include a revision of the morphology of the entire genus, and falls outside the scope of the present investigation.

Comparison of Eoboweniaand Bowenia

Our reinvestigation pinpoints numerous similarities betweenEobowenia incrassata and the extant cycad genusBowenia (Table1). Among the most interesting characters are the flush guard cells, which clearly separateEobowenia fromAlmargemia dentata as well as from all Zamiaceae and Cycadaceae sensu Stevenson [6] (Fig4, Additional file3: Figure S2; Additional file4: Figure S3; Additional file5: Figure S4; Additional file6: Figure S5; Additional file7: Figure S6). The cuticle of the guard cells also presents cuticular thickenings both on the dorsal and ventral surfaces, and single cuticular ridges running parallel to the dorsal wall of the guard cells (Fig.3 c, d; Fig.4 a, b), the monocyclic stomatal complexes (Fig.3 a, c), and the presence of substomatal cell complexes with secondarily thickened walls (Fig.3 e). The first set of characters is present among extant cycads inBowenia andStangeria, with some differences between the two genera [34]. Monocyclic stomatal complexes with stomata at the same level with the epidermis are restricted in extant Zamiaceae toBowenia [41,57,58] (Additional file6: Fig. S4). The perforations associated with some of the stomatal complexes inEobowenia incrassata presents some striking similarities to the substomatal complex inBowenia, which present secondarily thickened cell walls. This structure was interpreted by Greguss [34] as a perforation of the subsidiary cells, not dissimilar to the condition present inCycas [54], where all epidermal cells present perforations of the inner periclinal wall. The structures inEobowenia incrassata more closely resemble the structures inBowenia (which also occur but are less developed in some species ofEncephalartos andMacrozamia; see Additional file7: Figure S6) in being mostly restricted to the substomatal complexes (Fig.3 e, f). The main difference between the epidermis/cuticles ofEobowenia incrassata andBowenia is the presence of files of short cells with thickened cuticle in the former. This character has been compared to the state present inCeratozamia [57] by Kvaček [28], where files of short, dark-staining cells are present on both surfaces of the leaflets. However, the slightly concave and sometimes wavy anticlinal cell walls of the dark-staining cells is closer to the cuticle of the cell files present inDioon (Additional file3: Figure S2 C, D) [41]. Darker staining cells are present inBowenia, but they are organised as single or small groups of cells, commonly of the same length as the other epidermal cells (Fig.3 b, d).

Eobowenia andBowenia not only share significant and interesting characters in epidermal and cuticular anatomy, but also share commonalities at the macromorphological level, one being the serrate leaflet margin, which occurs in many extant cycads, such as some species ofZamia andStangeria (Fig.6). Marginal teeth are also present in a few species ofEncephalartos (Fig.6 b). InBowenia, a serrate margin is present in bothB. serrulata (Fig.6 a) and individuals ofB. spectabilis Hook. ex Hook.f. growing in more open environments [59], as well as in the fossilB. eocenica R.S.Hill [29] and other fossil members of the genus that have not yet been formally described [33]. The thickened, almost glandular-like aspect of the teeth inEobowenia is compatible with the situation present in extant (Fig.6) as well as fossil cycads (i.e.Restrepophyllum, [38]). In extant cycads, the thickened aspect of the tooth is given by a concentration of marginal fibres. The teeth inAlmargemia dentata, on the other hand (Fig.1) remind more closely of the lobe-like teeth present in some species ofEncephalartos (Fig.6 e). The basally converging veins in the leaflets are another character shared betweenEobowenia incrassata andBowenia. This character is also present in members ofZamia, butZamia has articulated leaflets in contrast to the decurrent insertion of the leaflets inBowenia andEobowenia. The striations on the leaflets ofEobowenia remind of similar striations present in fossil representatives ofBowenia (described by [29] as “veinlets”), which correspond to interspersed fibres in the leaflets of extantBowenia.

However,Bowenia andEobowenia also differ in details that are mainly restricted to the morphology of their leaflets. All extant and extinct species ofBowenia are characterised by dichotomous venation, with veins ending at the margin. In the species with serrate leaflet margin, the veins commonly end in the teeth. InEobowenia, the details of the venation are not clear from the material available, even if some dichotomies are potentially present on the specimen (Fig.2 a, b).

Another striking difference betweenEobowenia andBowenia lies in the size of the leaflets. The two extant species ofBowenia have leaflets with length varying from 9 to 14 cm [59], which are markedly larger than the 0.6–1.0 cm long leaflets ofEobowenia. However, fossil leaves assigned toBowenia commonly have rather short leaflets (e.g.B. eocenica andB. papillosa R.S.Hill: 3–4 cm; [33]). ExtantBowenia is characterised by bipinnate leaves, which are an autapomorphy of the genus, while the fragmentary nature of the leaflets ofE. incrassata does not allow us to evaluate the character in this taxon.

Despite the striking similarities presented betweenEobowenia incrassata andBowenia, we refrain from assigning the specimens toBowenia, mainly in the light of the differences outlined above, and considering the institution of the new genusEobowenia to represent the best solution for the accommodation of this fossil taxon.

On the other hand, the differences and uncertainties in macromorphological characters do not preclude a relationship between the two genera. Regarding, for example, leaflet size, size variation is not uncommon among extant and fossil Cycadales. For example, in extantZamia, leaflet length can vary from 1 to 8 cm inZ. pygmaea Sims [60] to 30–60 cm inZ. wallisii A.Braun. In the fossil genusCtenis, leaflet length can vary from 1.5–3.5 cm inC. nathorstii Moeller [42] to 15–20 cm inC. kaneharai Yokoyama [55].

Phylogenetic evidence for the placement of Eoboweniaand Almargemia

Our investigation is not the first to hypothesise a link betweenEobowenia (Almargemia) incrassata andBowenia. In their phylogenetic analysis of extant and fossil cycads, Martinez et al. [48] retrieved a maximum parsimony tree withAlmargemia (predominantly coded afterA. incrassata) as sister toBowenia plusStangeria andMesodescolea. However, such relationship does not receive any support from the bootstrap analysis, and it is not retrieved in other analyses of morphology, which considerAlmargemia predominantly coded forA. incrassata [61,62].

Using the topology from Salas-Leiva et al. [7] and the modified matrix from Martinez et al. [48] as a backbone, the best placement forEobowenia is as sister toBowenia (Fig.5 a). However, alternative placements are possible at the cost of only one or two steps more. This could be due to the low number of characters coded forEobowenia (21 characters out of 89), and the few informative epidermal characters linking the different clades of the Zamiaceae. If we consider the placements which are only one step longer, these placements imply that the unique characters of the stomatal complex ofEobowenia (guard cells at level with epidermis and monocyclic stomatal complexes) either evolved independently in this taxon and inBowenia (ifEobowenia is placed as sister to the Ceratozaminae or the Encephalartinae), or represent a potentially plesiomorphic status of all Zamiaceae exceptDioon. This would imply that all the similarities of the stomatal complexes of the Encephalartinae andDioon could represent parallel evolution of sunken, protected guard cells.

Our phylogenetic analyses based on the Martinez et al. [48] matrix retrieve a relationship betweenEobowenia andBowenia in both the MP and BI analyses of the morphological data, withEobowenia being sister to the Stangeriaceae sensu Stevenson [6] but such relationships only receive low support in the BI analysis. In the MP analysis this is partially due to the uncertainties surrounding the relationships betweenBowenia andStangeria and many other fossil taxa with peculiar character combinations, such asKurtziana, Mesosingeria, Sueria andPseudoctenis. When information from the molecular analysis of Salas-Leiva et al. [7] is added, resulting in the breakup of the Stangeriaceae,Eobowenia is preferentially retrieved as sister toBowenia instead ofStangeria. The characters linkingEobowenia andBowenia in these topologies regard the unique structure of the stomatal apparatus, which combines the flush guard cells with the lack of encircling cells. The combined analysis using a Bayesian framework retrieves the strongest support for the sister relationship of the two genera. This is in our knowledge the first attempt of integrating morphology and molecular data in a matrix that includes fossil taxa in the Cycadales, and shows the potential of this practice to resolve some of the uncertainties in the relationships between extant and fossil cycads.

The placement ofAlmargemia, on the other hand, is much more uncertain, with no clear placement in any of the analyses. However, a sister relationship betweenEobowenia andAlmargemia is never retrieved.

Our phylogenetic analyses show that the link betweenEobowenia andBowenia is the best hypothesis to explain the relationship between the fossil taxon and the diversity of the Cycadales, even when adopting a conservative approach to its macromorphological character coding. Such phylogenetic evidence, which is lacking for many fossil cycads that have been linked with extant groups, such asRestrepophyllum [38] andAustrozamia [25], as well as for the many fossil leaves assigned to extant genera [21–24,26–28], makeEobowenia a reliably placed cycad fossil foliage.

Such a placement is also compatible with at least some of the inferred age for the divergence ofBowenia based on molecular dating. The age of the deposition of the Anfiteatro de Ticó Formation, whereEobowenia is found, is very well constrained to 118.23 ± 0.09 Ma [63] or 116.85 ± 0.26 Ma [64] representing an Aptian (Lower Cretaceous) age, which is compatible with the ages inferred for the stem ofBowenia by Nagalingum et al. [17] using a relaxed log-normal clock and by Condamine et al. [16] using the favoured birth-death prior with both the calibration implemented, but is older than the dates retrieved by Salas-Leiva et al. [7] (Table2). This probable early divergence of the genusBowenia is, however, compatible with the phylogenetic placement retrieved by the multilocus analysis of Salas-Leiva et al. [7], which seesBowenia as sister to all the other Zamiaceae apart fromDioon. A Cretaceous stem history ofBowenia/Eobowenia is also compatible with the presence of cuticle indistinguishable from modernBowenia in the Upper Cretaceous of Central Australia [32].

Eoboweniaand the biogeography of Bowenia

The occurrence of a potential sister ofBowenia in the Early Cretaceous of Patagonia helps to strengthen some of the hypotheses around the biogeography ofBowenia. Until now, the phylogenetic isolation ofBowenia, as well as the presence of fossil records limited to Australia, had complicated the resolution of the biogeography of the genus. Indeed, Salas-Leiva et al. [7] retrieved two different results in their analysis: using S-DIVA, they retrieved an ancestral area including Australia, Africa and Mexico for the stem ofBowenia, while their DEC analysis hypothesises a model of stasis in Australia. The presence ofEobowenia in Patagonia during a period of connectivity between southern America and Australasia supports the hypothesis of a Gondwanan distribution for the stem of the group, with subsequent extinction shaping the current Australian endemic distribution.Bowenia would indeed represent yet another case of eastern survival [65]. Even if we know that some cycads persisted in southern South America until the Palaeocene [25], the identification of the precise timing of the extinction ofEobowenia in South America is hindered by the potential rarity of this fossil leaf type in the record. However, it is clear that this fossil represents another important clue to the biogeography of Gondwana coming from Patagonia [20].

Funding

The visits of MC to NRM and NHM were funded by the European Union’s Seventh Framework Programme (FP7/2007–2013) under grant agreement n 226,506 (SYNTHESYS projects SE-TAF-5774 and GB-TAF-5763). CP acknowledges funding from the Swedish Research Council (Vetenskapsrådet), Stockholm, Sweden, under grant number 621-2012-4375.

Availability of data and materials

All data generated or analysed during this study are included in this published article, its Additional files1,2,3,4,5,6,7,8.

Authors’ contributions

MC designed the study. MC and CP collected data and analysed the specimens and the data, and MC wrote the manuscript with inputs from CP. Both authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

Data Availability Statement

All data generated or analysed during this study are included in this published article, its Additional files1,2,3,4,5,6,7,8.