Osmunda pulchella sp. nov. from the Jurassic —reconciling molecular and fossil

Osmunda pulchella sp. nov. from the Jurassic —reconciling molecular and fossil
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
DOI 10.1186/s12862-015-0400-7
Open Access
Osmunda pulchella sp. nov. from the Jurassic
of Sweden—reconciling molecular and fossil
evidence in the phylogeny of modern royal
ferns (Osmundaceae)
Benjamin Bomfleur1*, Guido W. Grimm1,2 and Stephen McLoughlin1
Background: The classification of royal ferns (Osmundaceae) has long remained controversial. Recent molecular
phylogenies indicate that Osmunda is paraphyletic and needs to be separated into Osmundastrum and Osmunda
s.str. Here, however, we describe an exquisitely preserved Jurassic Osmunda rhizome (O. pulchella sp. nov.) that
combines diagnostic features of both Osmundastrum and Osmunda, calling molecular evidence for paraphyly into
question. We assembled a new morphological matrix based on rhizome anatomy, and used network analyses to
establish phylogenetic relationships between fossil and extant members of modern Osmundaceae. We re-analysed
the original molecular data to evaluate root-placement support. Finally, we integrated morphological and molecular
data-sets using the evolutionary placement algorithm.
Results: Osmunda pulchella and five additional Jurassic rhizome species show anatomical character suites
intermediate between Osmundastrum and Osmunda. Molecular evidence for paraphyly is ambiguous: a previously
unrecognized signal from spacer sequences favours an alternative root placement that would resolve Osmunda s.l.
as monophyletic. Our evolutionary placement analysis identifies fossil species as probable ancestral members of
modern genera and subgenera, which accords with recent evidence from Bayesian dating.
Conclusions: Osmunda pulchella is likely a precursor of the Osmundastrum lineage. The recently proposed root
placement in Osmundaceae—based solely on molecular data—stems from possibly misinformative outgroup
signals in rbcL and atpA genes. We conclude that the seemingly conflicting evidence from morphological,
anatomical, molecular, and palaeontological data can instead be elegantly reconciled under the assumption that
Osmunda is indeed monophyletic.
Keywords: Calcification, Evolutionary placement, Fern evolution, Organelle preservation, Osmundales,
Osmundastrum, Outgroup, Paraphyly, Permineralization, Phylogenetic networks
The royal ferns (Osmundales) comprise about 20 extant
species currently classified in four genera, i.e. Osmunda
L., Osmundastrum C.Presl, Leptopteris C.Presl, and
Todea Bernh. This small group of ferns is remarkable in
many respects and, consequently, has attracted considerable scholarly attention. Its members represent the most
* Correspondence: [email protected]
Department of Palaeobiology, Swedish Museum of Natural History,
Stockholm, Sweden
Full list of author information is available at the end of the article
primitive of all leptosporangiate ferns [1–4], with features that have been interpreted to be intermediate between Eusporangiatae and Leptosporangiatae [5–7].
Detailed investigations of their anatomy [8–11], cytology
and genetic structure [12–23], and evolution [24–34]
render the Osmundales one of the most intensively studied groups of ferns. Moreover, in contrast to their rather
limited modern diversity, Osmundales have a uniquely
rich and diverse fossil record [30, 35] currently considered to include more than 150 species, over 25 genera,
and at least three (sub) families. This extensive fossil
© 2015 Bomfleur et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution
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Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
record has been reviewed in several key works [7, 30, 36,
37] and, recently, been recruited for molecular dating
using three contrasting Bayesian approaches (traditional
node dating, total-evidence dating, and dating using the
fossilized-birth-death approach) [34].
The monophyly of Osmundales and their isolated position as the first diverging lineage within leptosporangiate
ferns are firmly established [1, 3, 38, 39]. However, the
resolution of systematic relationships within the group—
and especially the circumscription of Osmunda—continues to remain controversial. Linnaeus established Osmunda with three species: O. regalis L., O. claytoniana L.
and O. cinnamomea L. [40]. With subsequent descriptions
of additional species from East and Southeast Asia
[41–44], the genus was subdivided into several subgenera,
i.e. O. subgenus Osmunda, O. subgenus Plenasium
(C.Presl) J.Smith, O. subgenus Osmundastrum (C.Presl)
C.Presl, and O. subgenus Claytosmunda Y.Yatabe,
N.Murak. & K.Iwats. based on combinations of diagnostic
morphological and anatomical characters and—more
recently—molecular phylogenetic analyses [31, 32, 45].
However, independent lines of evidence based on morphology [11, 46, 47], anatomy [11, 29, 30], palynology [48],
hybridization experiments [49–52], and molecular and
genetic studies [31, 32, 53–55] have led to divergent opinions on the classification of these taxa. Most controversy
has arisen concerning the phylogenetic relationships and
taxonomic ranks of O. cinnamomea and O. claytoniana
(please refer to the nomenclatural remark in the methods
section for information on the use of taxon names herein).
Early molecular studies aiming to resolve specific relationships between O. regalis, O. claytoniana and O. cinnamomea produced remarkably incongruent results [53, 54].
Isozyme studies eventually demonstrated that O. claytoniana is probably more closely related to O. regalis than either is to O. cinnamomea [55], confirming previous
assumptions of early plant anatomists [8, 29, 30]. Subsequent nucleotide sequencing not only provided first robust
support for this relationship [31] but, unexpectedly, also
placed Todea and Leptopteris within Osmunda as traditionally defined. Consequently, the isolated O. cinnamomea at the base of the resulting tree was separated from
Osmunda s.str. and assigned to its own genus, sister to
Leptopteris plus Todea and the remaining Osmunda [32].
Here we describe a new Osmunda species based on an
exceptionally well-preserved rhizome from the Jurassic
of Sweden that combines features diagnostic of Osmunda and Osmundastrum. A phylogenetic analysis
based on a newly assembled morphological character
matrix places the new species intermediate between Osmunda and Osmundastrum, which is incompatible with
the recently established paraphyly and resulting classifications. Current notions in phylogenetic research
emphasize the significance of integrating morphological
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with molecular evidence for resolving evolutionary relationships (e.g. [56–58]), especially among ferns [59, 60].
Therefore, we re-analyse the molecular data and integrate morphological and molecular data-sets of fossil
and extant Osmundaceae to show that the recently
established paraphyly of Osmunda s.l. suffers from ambiguous outgroup signals; by contrast, we submit that all
evidence can instead be elegantly reconciled assuming
Osmunda s.l. is indeed monophyletic.
Systematic description of the fossil
Order Osmundales Link
Family Osmundaceae Bercht. & C.Presl
Genus Osmunda L.
Species Osmunda pulchella Bomfleur, G.Grimm &
McLoughlin sp. nov.
Rhizome creeping or semi-erect. Stem with ectophloicdictyoxylic siphonostele and two-layered cortex. Pith
entirely parenchymatous. Xylem cylinder about 8–12 tracheids (mostly ca 0.4 mm) thick, dissected by narrow,
complete, immediate leaf gaps, containing about twenty
xylem segments in a given transverse section. Phloem and
endodermis external only. Inner cortex ca 0.5–0.8 mm
thick, homogeneous, parenchymatous, containing about
ten leaf traces in a given transverse section; outer cortex
ca 1.5–2.5 mm thick, homogeneous, sclerenchymatous,
containing about 20 leaf traces in a given transverse section. Leaf traces in stem oblong, more or less reniform,
adaxially concave, endarch with a single protoxylem strand
at the point of emergence from stele, diverging at acute angles of ca 20–40°; protoxylem strand bifurcating only in
outermost cortex or upon departure from stem. Petiole
bases with adaxially concave vascular strand, one adaxial
sclerenchyma band in vascular-strand concavity, parenchymatic cortex, a heterogeneous sclerenchyma ring, and an
opposite pair of petiolar wings; adaxial sclerenchyma in
inner cortex of petiole appearing in the form of a single
patch or arch lining the vascular-bundle concavity with
homogeneous thickness, differentiating distally into two
thickened lateral masses connected by a thin strip, extending proximally only to base of petiole, not into
stem; sclerenchyma ring of petiole base thicker than
vascular bundle, heterogeneous, with a crescentic abaxial cap of thicker-walled fibres in the basal petiole portion differentiating distally into two lateral masses and
ultimately into two lateral and one abaxial mass; petiolar wings in distal portions containing an elongate
strip of thick-walled fibres. Roots diarch, usually arising
singly from one leaf trace, containing scattered sclerenchyma fibres.
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Type stratum and age
Mafic pyroclastic and epiclastic deposits informally
named the “Djupadal formation” [61]; Pliensbachian (late
Early Jurassic).
Type locality
Korsaröd lake (55°58’54.6”N, 013°37’44.9”E) near Höör,
central Skåne, southern Sweden.
Holotype (hic designatus)
A single specimen of permineralized rhizome, sectioned
and prepared into six blocks (specimens NRM S069649–
S069655) and three microscope slides, including two
transverse thin sections (slides NRM S069656 and
S069657) and one radial thin section (NRM S069658);
all material is curated in the collection of the Department of Palaeobiology, Swedish Museum of Natural History, Stockholm, Sweden.
The specific epithet pulchella (Latin diminutive of pulchra =‘beautiful’, ‘fair’) is chosen in reference to the exquisite preservation and aesthetic appeal of the holotype
The holotype is a calcified rhizome fragment about 6 cm
long and up to 4 cm in diameter (Fig. 1a–c). It consists
of a small central stem that is surrounded by a compact
mantle of helically arranged, persistent petiole bases and
interspersed rootlets (Fig. 1b, e). The rootlets extend
outwards through the mantle in a sinuous course almost
perpendicular to the axis, indicating low rhizomatous rather than arborescent growth; the asymmetrical distribution of roots in longitudinal sections of the rhizome
(Fig. 1d) points to a creeping habit.
The stem is ca 7.5 mm in diameter, and consists of an
ectophloic-dictyoxylic siphonostele surrounded by a
two-layered cortex (Figs. 1d, e, 2, 3, 7a). The pith is ca
1.5 mm in diameter and entirely parenchymatous (Fig. 2).
A thin region at the outermost periphery of the pith
consists of a few rows of parenchyma cells that are considerably more slender (ca 20–30 μm wide) than those
in the central portion of the pith (usually ≥ 50 μm wide;
Figs. 3, 4a, b). Furthermore, cell walls in some regions of
the pith periphery may be thicker and more clearly visible than in the centre (Figs. 3, 4b). However, there is no
evidence for the presence of an internal endodermis or
internal phloem. Given that endodermal layers are
recognizable in the stem and petiole cortices (e.g. Fig. 5f ),
we are certain that the absence of an internal endodermis is an original feature, and not the result of inadequate preservation. The xylem cylinder is ca 0.4 mm
and ca 8–12 tracheids thick, and dissected by narrow,
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mostly complete, immediate leaf gaps into about 20
xylem segments in a given transverse section. The
phloem forms an entire ring around the stele; it is most
easily recognizable opposite a leaf gap, where it forms a
narrow wedge-shaped patch of large, thin-walled cells
that projects slightly towards the gap in transverse section (Figs. 3, 4a).
The cortex of the stem is bi-layered (Figs. 1e, 2, 3, 7a).
The inner layer is ca 0.5–0.8 mm thick, consists entirely of
parenchyma, and contains about ten leaf traces in a given
transverse section (Fig. 2). The outer cortex is considerably thicker (ca 1.5–2.5 mm thick), and consists entirely of
homogeneous sclerenchymatic tissue (Figs. 1e, 2, 3).
Abundant leaf traces (about 20 in a given transverse section; e.g. Fig. 2) and rootlets traversing the outer cortex
(Figs. 1c, d 2) appear to have altered the original orientation of the sclereids, resulting in a somewhat patchy appearance of the outer cortical tissue (Fig. 2).
Phyllotaxy of the stem is helical with apparent contact
parastichies of 8 and 13 (Fig. 1b, e). Leaf-trace formation
begins with the appearance of a single protoxylem strand
in an eccentric position (about two-thirds to threequarters distance from the pith; Fig. 4a) in a stelar metaxylem segment. Distally, the protoxylem becomes associated with an increasing amount of parenchyma on its
adaxial side (making it effectively endarch for the rest of
its course), first occupying only the centre of the segment (resulting in an O-shaped xylem segment), then
connecting with the pith (resulting in a U-shaped xylem
segment), and ultimately forming the complete, narrow
leaf gap with the departure of the trace. Departing leaf
traces are oblong, only slightly curved adaxially, ca 300–
350 μm wide and two to four tracheids (ca 80–100 μm)
thick (Figs. 2, 4d); they diverge from the axis at angles of
ca 20–40° (Figs. 1a, 3).
In its course through the stem, a leaf-trace vascular
bundle becomes enveloped by increasing layers of tissue
through which it passes successively: first by phloem and
endodermis from the stele upon entering the inner cortex; by a sheath of parenchyma from the inner cortex as
it enters the outer cortex (Figs. 2, 3); and finally by a cylindrical sclerenchyma sheath from the outer cortex as it
departs from the stem (Fig. 1e). The initial bifurcation of
the leaf-trace protoxylem occurs in the outermost portion of the cortex or in the petiole base (Fig. 4e, f ).
In the inner cortex of the petiole, thick-walled fibres
appear in the form of a small irregular mass adaxial to
the vascular bundle (Fig. 5c, d). This mass develops distally into a thick band lining the bundle concavity
(Figs. 5e, 6a, b), and may further differentiate into two
lateral masses connected only by a rather thin strip
(Fig. 5f, g). Apart from the sclerenchyma inside the
vascular-bundle concavity, the inner cortex of the petiole
consists entirely of parenchyma.
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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Fig. 1 Osmunda pulchella sp. nov. from the Lower Jurassic of Skåne, southern Sweden. Holotype. a Reproduction of the only available print of
the original holotype material prior to preparation, showing the gross morphology of the rhizome. b, c Transverse sections through center
(B: NRM-S069656) and apex (C: NRM-S069657) of the rhizome. d Longitudinal section through the rhizome (NRM-S069658). (E) Detail of Fig. 1B.
Scale bars: (a–c) = 5 mm; (d, e) = 2 mm
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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Fig. 2 Osmunda pulchella sp. nov. from the Lower Jurassic of Skåne, southern Sweden. Transverse section through the stem (NRM-S069656).
Scale bar = 500 μm
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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Fig. 3 Osmunda pulchella sp. nov. from the Lower Jurassic of Skåne, southern Sweden. Radial longitudinal section through the stem (NRM-069658).
Scale bar = 500 μm
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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Fig. 4 Anatomical and cytological details of Osmunda pulchella sp. nov. from the Lower Jurassic of Skåne, southern Sweden (A–F, I: NRM-S069656;
G, H, J, K: NRM-S069658). a Detail showing pith parenchyma (bottom), stelar xylem cylinder dissected by complete leaf gaps, triangular section of
phloem projecting into leaf gap, and parenchymatous inner cortex (top); note mesarch leaf-trace protoxylem initiation in the stelar xylem
segment on the right. b Detail of (a) showing peripheral pith parenchyma and stem xylem. c Detail of stem xylem showing tracheid pitting. d
Endarch leaf trace emerging from the stele and associated with a single root. e Leaf trace in the inner cortex of the stem showing single, endarch
protoxylem cluster. f Leaf trace immediately distal to initial protoxylem bifurcation in the outermost cortex of the stem. g Root vascular bundle
showing well-preserved scalariform pitting of metaxylem tracheids. h Well-preserved pith parenchyma showing membrane-bound cytoplasm with
cytosol particles and interphase nuclei containing nucleoli. i, j Nuclei with conspicuous nucleoli (in interphase: I) or with distinct chromatid strands
(in prophase: J). k Transverse section through root showing diarch vascular bundle, parenchymatous inner cortex with isolated fibre strands, and
prominent fibrous outer cortex. Scale bars: (a) = 100 μm; (b, e, f, h) = 50 μm; (c, g) = 25 μm; (d) = 200 μm; (i, j) = 5 μm; (k) = 250 μm
The sclerenchyma cylinder of the petiole has an even
thickness that increases from about 300 μm near the
petiole base to ca 500 μm distally. Its composition is heterogeneous: near the petiole base, it contains a crescentic, abaxial arch of particularly thick-walled fibres
(Figs. 1e, 5, 6, 7b); distally, this arch begins to develop
two lateral masses (Figs. 5d–f, 7b) and ultimately two
lateral masses and one abaxial arch of thick-walled fibres whose lumina are more-or-less entirely occluded
(Figs. 5g, 7b).
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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Fig. 5 Basal to distal sections of petiole bases of Osmunda pulchella sp. nov. from the Lower Jurassic of Skåne, southern Sweden (NRM-S069657),
showing successive stages of petiole-base differentiation. a Parenchymatous inner cortex and petiolar wings. b–e Development of an abaxial arch
of thick-walled fibres in the sclerenchyma ring. c Appearance of a sclerenchyma patch in the bundle concavity. d Appearance of a sclerenchyma
mass in the petiolar wing. f Sclerenchyma ring with two prominent lateral masses of particularly thick-walled fibres. g Collapsed outermost petiole
(note rock matrix above) showing sclerenchyma ring with one abaxial and two lateral masses of particularly thick-walled fibres, and elongate
sclerenchyma strips (e.g. bottom, right) isolated from degraded stipular wings of adjacent petioles. Scale bars = 500 μm
The petiole bases are flanked by a pair of stipular
wings that consist initially of parenchyma only. As the
wings grow wider in more distal portions, they develop a
patch of thick-walled fibres (Figs. 5d, 7b) that forms an
entire, elongate strip (Figs. 5f, g 7b). The parenchymatic
ground tissue of the stipular wings is well-preserved only
in the innermost regions of the mantle (Fig. 1b, c, e);
outwards, it appears to be either increasingly degraded
or to have been removed by pervasive rootlet growth. In
the outermost portions of the mantle, all that remains of
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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The holotype of O. pulchella has a phenomenal preservational quality revealing cellular and subcellular detail
(Figs. 2, 3). Tracheids have exquisitely preserved wall
thickenings, which are scalariform in metaxylem (Figs. 4c
g, 6c) and annular to helical in protoxylem cells (Fig. 6c).
Most parenchyma cells contain preserved cellular contents
(Figs. 2, 3), including nuclei (Fig. 4h–j), membrane-bound
cytoplasm (Fig. 4h), and cytosol granules [62]. Some parenchyma cells, especially those adjacent to xylem bundles
in roots and leaf traces, contain varying amounts of
discrete, smooth-walled, spherical or oblate particles ca 1–
5 μm in diameter that have been interpreted as putative
amyloplasts [62]. Cell nuclei measure ca 10 μm in diameter, and contain nucleoli and, in a few cases, unravelled
chromosomes (Fig. 4i, j). Chromatid strands have a diameter of 0.3–0.4 μm (Fig. 4j).
Phylogenetic analyses
Phylogenetic relationships among fossil and modern members
of the Osmundaceae based on rhizome anatomy
Fig. 6 Details of petiole-base anatomy of Osmunda pulchella sp. nov.
from the Lower Jurassic of Skåne, southern Sweden, revealed via
scanning electron microscopy. a Distal cross-section through a
petiole. b Detail of (a) showing vascular strand with about eight
endarch protoxylem bundles and sclerenchyma mass lining the
vascular-strand concavity. c Detail showing helical wall thickening
of protoxylem strands (center) compared to multiseriate scalariform
wall thickenings of metaxylem tracheids in a petiole vascular bundle
(oriented with adaxial side facing upwards). Scale bars: (a) = 1 mm;
(b) = 100 μm; (c) = 50 μm
the stipular wings are usually just the isolated, elongate
strips of thick-walled fibres interspersed between petioles and rootlets (Fig. 5g).
Each leaf trace is usually associated with a single rootlet that diverges laterally at the point of departure from
the stele. The rootlets typically measure about 0.5 mm
in diameter, contain a diarch vascular bundle, parenchymatic ground tissue with interspersed sclerenchymatic
fibres, and a sclerenchymatic outer cortical layer.
The phylogenetic network based on pairwise distances
inferred from a matrix including 23 rhizome anatomical
characters resolved five major species groups: (1) extant
species of Leptopteris and Todea together with T. tidwellii
Jud, G.W.Rothwell & Stockey from the Lower Cretaceous
of North America; (2) all extant species of Osmunda subgenus Plenasium together with O. arnoldii C.N.Mill. and
O. dowkeri (Carruth.) M.Chandler from the Paleogene of
North America and Europe; (3) all species of subgenus
Osmunda sensu Miller, i.e. species of the extant subgenera Osmunda sensu Yatabe et al. and Claytosmunda
together with several Paleogene and Neogene species;
(4) all Jurassic rhizome species, including O. pulchella;
and (5) all extant and fossil members of Osmundastrum
(O. cinnamomea and O. precinnamomea) (Fig 8). Corresponding bipartitions, which would define clades in an accordingly rooted phylogram, were found in the bootstrap
replicate tree sample and the Bayesian sampled topologies
with varying frequency. Osmunda subgenus Plenasium
(BS = 47–80; PP = 0.76) and Osmundastrum (BS = 51–76;
PP = 0.95) received best support, whereas support values
for the other groups were generally low (BS ≤ 55; PP ≤
0.43). The Jurassic species bridge the morphological gap
between Osmundastrum and Osmunda subgenus Osmunda sensu Miller, with O. pulchella being the species
closest to Osmundastrum. A hypothetical clade comprising Osmunda subgenus Osmunda sensu Miller and
the Jurassic Osmunda species would receive BS up to
28 and PP of 0.28.
Especially remarkable is the diversification of subgenus
Osmundastrum as revealed by our independent coding
of the individual fossil records from Neogene [29, 63],
Paleogene [29], and Cretaceous deposits [64]; the individually coded fossil and extant representatives assigned
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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Fig. 7 Schematic drawings showing diagnostic anatomical characters of Osmunda pulchella sp. nov. from the Lower Jurassic of Skåne, southern
Sweden. a Stem cross section. b Successive cross sections of basal (bottom) to distal (top) petiole portions. Xylem in white; parenchyma in light-grey;
sclerenchyma in dark-grey; sclerenchyma with particularly thick-walled fibres in black; oc = outer cortex; ic = inner cortex; st = stele; p = pith; xs = stelar
xylem segment; lt/ic = leaf trace in inner cortex; lt/oc = leaf trace in outer cortex; rt = root trace; sr = sclerenchyma ring; aa = abaxial arch of thick-walled
fibres; sbc = sclerenchyma mass in bundle concavity; spw = sclerenchyma mass in petiole wing; lm = lateral masses of thick-walled fibres; am = abaxial
mass of thick-walled fibres
to O. cinnamomea show greater morphological disparity
than expressed between the separate species of any other
subgenus and genus.
Merging fossil and extant taxa into a molecular backbone
Of all taxa placed via the evolutionary placement algorithm (EPA; Fig. 9), Osmunda pulchella is the species
that is most incongruently placed between the different
weighting schemes: Using parsimony-based character
weights, the EPA places Osmunda pulchella at the root
of Claytosmunda, whereas it is placed either between
Osmundastrum and the remaining Osmunda s.str. or at
the root of the Plenasium clade using model-based character weights (Fig. 9). Single position swaps also occur in
most of the other Jurassic species [Ashicaulis (= Millerocaulis sensu Vera) plumites N.Tian & Y.D.Wang, A. (=
Millerocaulis sensu Vera) wangii N.Tian & Y.D.Wang,
Millerocaulis johnstonii Tidwell, Munzing & M.R.Banks,
M. liaoningensis Wu Zhang & Shao-Lin Zheng] and in
O. pluma C.N.Mill., O. iliaensis C.N.Mill., O. shimokawaensis M.Matsumoto & H.Nishida, and Todea tidwellii.
Except for Todea tidwellii (placed at the root of either
Leptopteris or Todea), all swaps occur within the Osmunda s.l. sub-tree. Swaps among the Jurassic species
mostly involve placements at the root of the Plenasium
sub-tree, the subgenus Osmunda sub-tree, and at the
branch between Osmundastrum and the remaining Osmunda. Osmunda shimokawaensis and O. iliaensis are
variably placed within the O. lancea Thunb.–O. japonica
Thunb. sub-tree.
By contrast, fixed placements congruent over all three
weighting schemes employed occur in: fossil members of
Osmundastrum (all at the O. cinnamomea branch); Ashicaulis (= Millerocaulis sensu Vera) claytoniites Y.M.Cheng
and O. wehrii C.N.Mill. (at the root of the Plenasium subtree); O. arnoldii, O. bromeliifolia (C.Presl) Copel., and O.
dowkeri (all at O. banksiifolia branch); O. oregonensis
(C.A.Arnold) C.N.Mill. (at the root of subgenus Osmunda),
and L. superba (at the branch of L. hymenophylloides).
Re-visitation of the outgroup-inferred Osmundaceae root
The gene jackknifing and single-gene analyses reveal ambiguity concerning the position of the Osmundaceae root
in the data of Metzgar et al. [32] (Fig. 10). As in the original analysis [32], support for backbone branches is effectively unambiguous based on the concatenated data,
and the outgroup-inferred root is placed between Osmundastrum and the remainder of the family, resolving the
traditional genus Osmunda (Osmunda s.l.) as a grade
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
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Fig. 8 Neighbour-net showing phylogenetic relationships among fossil and extant members of modern Osmundaceae inferred from a
morphological distance matrix based on rhizome anatomy. Edge (branch) support from bootstrapping (BS) and Bayesian inference (posterior
probability, PP) is annotated for modern genera and subgenera, and selected bipartitions. Further abbreviations: BSML/GTR = maximum likelihood
(ML) BS support using a general-time reversible transformation model; BSML/MK = BS support using Lewis’ one-parameter model [146]; BSP = parsimony
BS support; BSNJ = neighbour-joining BS support; see Additional file 1 [ESA]
(‘paraphyletic Osmunda scenario’). The signal for this root
placement stems from the two coding plastid gene regions
(atpA and rbcL). In the more (but not most) variable spacer regions (atpB-rbcL, rbcL-accD, and trnL-trnF to a
lesser degree), however, a competing signal is found resolving Osmunda s.l. as a clade (‘monophyletic Osmunda
scenario’). The most variable non-coding spacer regions
(trnG-trnR; rps4-trnS; and trnL-trnF to some degree)
provided only ambiguous signals including potential
outgroup-branch placements deep within the Leptopteris-Todea and Osmunda sub-trees or showed a preference for an Osmundastrum-Leptopteris-Todea clade
as sister to Osmunda s.str.
The gene-jackknifing results showed that the exclusion of
either one or both coding regions (atpA, rbcL)—which together account for 33 % of distinct alignment patterns in
the concatenated matrix—decreased support for the split
leading to an Osmunda grade with Osmundastrum
resolved as sister to the remainder of the family, whereas
the support for the alternative of an Osmunda clade or an
Osmundastrum-Leptopteris-Todea clade was increased. In
the case of O. (Claytosmunda) claytoniana, the genetic data
provided a coherent signal, with all plastid regions preferring a subgenus Osmunda sensu Yatabe et al.–Plenasium
clade over the alternatives of a subgenus Osmunda sensu
Miller or Claytosmunda-Plenasium clade. The gene-knifing
had no measurable effect (BSML = 98–100). The problem
concerning the placement of the root can also be illustrated
in the form of a neighbour-net splits graph based on genetic, uncorrected p-distances [see Additional file 2: Figure
S1 in Electronic Supplementary Archive (ESA)].
Placement of Osmunda pulchella within the two molecular
backbone topologies
Optimization of the anatomical characters on two specified backbone topologies inferred from the different
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Page 12 of 25
Fig. 9 Placement of fossil and extant members into the specified backbone topology of modern Osmundaceae inferred from molecular data of
Metzgar et al. [32] using the evolutionary placement algorithm [147] (see Additional file 1 [ESA]) and three different character-weighting schemes.
Dashed light-grey lines indicate weighting-scheme-dependent position swaps of taxa. Abbreviations: MLGTR = weighting scheme optimized
under a general-time reversible transformation model; MLMK, weighting scheme optimized under Lewis’ (2001) model; MP = weighting
scheme under parsimony
rooting scenarios (‘monophyletic Osmunda’ vs ‘paraphyletic Osmunda’ scenario) required 53 steps under parsimony (Fig. 11). Inserting Osmunda pulchella into the
‘paraphyletic Osmunda scenario’ tree, its most parsimonious
placement based on anatomical characters is alternatively
(1) at the most basal position as sister to all extant Osmundaceae, (2) as sister to O. cinnamomea, or (3) as sister to a
putative Leptopteris-Todea-Osmunda s.str. clade. In the
‘monophyletic Osmunda scenario’ tree, by contrast, the
most parsimonious placement of O. pulchella is as sister to
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Page 13 of 25
Fig. 10 Phylogenetic tree, optimized under maximum likelihood (ML), showing unambiguously resolved relationships among extant Osmundaceae
and the conflicting root-placement (outgroup-inferred) signals from individual gene regions. Based on the molecular matrix compiled and employed
by Metzgar et al. [32]. All backbone branches received full maximum-likelihood bootstrap support (BSML = 100) based on the concatenated data;
support for leaf-branches not shown (see Additional file 1 [ESA])
O. cinnamomea. In both trees, the least parsimonious positions of O. pulchella are within the Todea-Leptopteris clade
or at the root of or within the Plenasium sub-tree.
Many phylogenetic hypotheses have been proposed for
Osmundaceae over the past decades, each differing in
terms of the data matrices employed and the taxon relationships obtained [30–34, 37, 54, 55, 65–68]. The evolutionary history of the family is clearly difficult to resolve
based on the characters of the extant representatives
alone. Several researchers have, thus, urged the incorporation of fossil data to assist phylogenetic reconstructions of Osmundaceae [30, 34, 37, 65] and of ferns in
general [59, 60, 69, 70].
In the following sections, we (1) place the new fossil
species in the broader context of the Mesozoic–
Cenozoic fossil record of Osmundaceae; (2) explain the
rationale for the assignment of this and other fossil species to an (initially) extant genus; (3) examine the systematic relationships between Osmunda pulchella and
other fossil and extant species of modern Osmundaceae;
(4) provide a critical re-evaluation of the evidence for
generic separation of Osmundastrum and the paraphyly
of Osmunda s.l.; and (5) discuss the critical significance
of O. pulchella for the systematic classification and evolutionary history of modern Osmundaceae.
Osmundaceae in the regional fossil flora
Osmunda pulchella sp. nov. is among the earliest fossil
Osmunda rhizomes yet known, and the first such find
from the Mesozoic of Europe. Whole plants are rarely
fossilized, so identification of fossils depends on recognizing diagnostic characters in various dispersed organs.
Moreover, some isolated organs can only be identified to
taxa under special preservational states (e.g. where anatomical details are retained). Fossil evidence for Osmundaceae occurs in three main forms: (1) permineralized
axes with vascular, cortical and petiolar anatomy characteristic of the family; (2) compressions and impressions
of foliage (either fertile or sterile); and (3) dispersed
spores with sculptural characters typical of fertile macrofossil or extant representatives of the family.
Permineralized osmundaceous axes have a long-ranging
and geographically broad fossil record extending back to at
least the Permian of both hemispheres [36, 37, 71]. These
fossils are highly informative of the anatomical evolution of
the group since they preserve the three-dimensional architecture of axial tissues and the surrounding sheath of
petioles [30]. They provide further information on osmundacean ecology, since the excavations or coprolites of various invertebrates are commonly preserved within the
cortical tissues or petiole sheath [72]. However, occurrences of permineralized axes are generally restricted to
sedimentary rocks with a high proportion of volcanogenic
components. Free silica and, in some cases, carbonate ions
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Fig. 11 Diagram illustrating the most parsimonious phylogenetic
placement of Osmunda pulchella within the molecular-based topology
under both ingroup rooting scenarios. Left, outgroup-inferred
coding-gene-based root (Fig. 10 [31, 32]). Right, alternative
rooting (this study)
are liberated in particularly high concentrations from the
breakdown of glass and unstable calc-silicate minerals, especially in sediments derived from mafic to intermediate
volcanic terrains [73]. These ions preferentially link to free
hydrogen bonds of holocellulosic complexes in buried
plant matter, entombing the original cell walls in opaline
silica, quartz, or calcite. The exceptional circumstances of
such preservational conditions mean that permineralized
osmundaceous stems have a patchy record (see [36]
and [74] for summaries of occurrences). Although axes
are known from both older (Permian: [26]) and younger
(Cenozoic: [28, 75, 76]) rocks in the region, no osmundaceous rhizomes have thus far been reported from the
Mesozoic of Europe.
Compressions and impressions of foliage can only be
assigned to Osmundaceae with confidence where details of
the sori arrangement or sporangial annulus architecture
can be resolved [7]. Remains of such fertile fronds are variously assigned to Osmundopsis T.M.Harris, Todites Seward,
Anomopteris Brongn., Cacumen Cantrill & J.A.Webb, Cladotheca T.Halle, and Osmunda [7, 77–79] and possibly
Damudopteris D.D. Pant & P.K. Khare and Dichotomopteris
Maithy [80, 81]. Morphologically similar sterile fronds are
Page 14 of 25
typically assigned to Cladophlebis Brongn., although not all
forms referred to this fossil genus are necessarily osmundacean. Collectively, the record of fossil osmundacean foliage
matches that of the rhizomes, extending from the Permian
to Cenozoic and being distributed on all continents [30,
82–85]. Foliage referable to Todites or Cladophlebis is
widespread in the Mesozoic of Europe and is extensively
represented in Rhaetian to Early Jurassic strata of southern
Sweden [86–90].
Spores attributed to Osmundaceae found in situ within
fossil sporangia or dispersed within sediments are spherical to triangular and typically bear irregularly arranged
grana, bacula or pila of variable form and size. More
rarely, the spore surface is scabrate or laevigate. When
found dispersed, such spores are most commonly
assigned to Osmundacidites Couper, although some have
been attributed to Baculatisporites Pflug & P.W.Thomson, Cyclobaculisporites D.C.Bhardwaj, Todisporites Couper, Punctatisporites A.C.Ibrahim, Leiotriletes R.Potonié
& Kremp, or Triquitrites L.R.Wilson & E.A.Coe [78].
Such spores match the record of osmundaceous foliage
and permineralized axes in ranging from the Permian to
present, and occurring in considerable abundance during
the Mesozoic [78]. Osmundacidites wellmanii (Couper)
Danzé-Corsin & Laveine is one of the dominant spore
types recovered from sediments surrounding the fossil
rhizome studied herein [62] attesting to the strong representation of this family in the flora of the Korsaröd
area during the Pliensbachian. Moreover, Osmundacidites and Baculatisporites species are common elements
of palynofloras recovered from the uppermost Triassic
to Middle Jurassic strata throughout southern Sweden
[91–95], indicating that the family had an important role
in the ecology of the herbaceous stratum of the regional
mid-Mesozoic vegetation. Osmundaceae underwent a
notable decline in both relative diversity and abundance
accompanying the rise of the angiosperms in the Cretaceous [96, 97] and this trend appears to have persisted
through the Cenozoic resulting in the family’s low representation and, for some genera, relictual distribution
today [85].
Assignment to Osmunda
There is no standard rule in palaeontology deciding
whether fossil remains can (or should) be assigned to extant genera or species [79, 98, 99]. In each case, this decision must be taken individually after careful evaluation
of the completeness of preservation (i.e. the degree of
comparability with extant taxa) and of the diagnostic significance of the preserved morphological characters
available for comparison.
Historically, permineralized rhizomes similar to those
of extant Osmundaceae have been routinely placed in
fossil genera, such as Osmundites Unger [27, 28, 100].
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Based on a comparative study of fossil rhizomes and extant taxa, however, Chandler [75] concluded that Osmundites dowkeri Carruth. from the Paleocene of England can
be undoubtedly assigned to Osmunda subgenus Plenasium. Chandler’s rationale has since served as a precedence for subsequent authors to place other Paleogene,
Neogene, and—more recently—also Triassic to Cretaceous fossils of Osmundaceae in genera originally defined
for extant species [29, 30, 63, 65, 66, 101–104]. Finally,
well-preserved permineralized rhizomes from the Upper
Cretaceous of Canada that are strikingly similar to those
of modern Osmunda cinnamomea have led the authors to
even identify a particular extant species in the Mesozoic
fossil record [64]. These assignments and new combinations have been adopted in every subsequent systematic
treatment of fossil Osmundaceae [7, 36, 37]. Therefore,
the identification of extant genera and species of
Osmundaceae even in the Mesozoic fossil record is a
universally accepted practice, providing the fossils show
sufficient diagnostic detail to warrant affiliation with
their extant relatives. Fossils that have structural features unknown among modern taxa are, by contrast,
usually placed in more or less narrowly defined fossil
taxa, such as Palaeosmunda R.E.Gould, Osmundacaulis
C.N.Mill. emend. Tidwell, or Aurealcaulis Tidwell &
L.R.Parker [7, 36, 71]. The remaining osmundoid fossil
rhizomes that cannot be positively assigned to any of
these natural groups continue to be placed in the rather
broadly defined fossil taxon Millerocaulis Tidwell
emend. E.I.Vera (including the formerly separated Millerocaulis Tidwell emend. Tidwell and Ashicaulis Tidwell) [7, 36, 37, 105].
The calcified osmundaceous rhizome described here
contains all anatomical features diagnostic of Osmunda
[11, 30]: (1) ectophloic-dictyoxylic siphonostele with
complete leaf gaps; (2) thin parenchymatic inner cortex
and distinctly thicker, homogeneous, fibrous outer cortex;
(3) heterogeneous sclerenchyma cylinders in the petiole
bases; and (4) sclerenchyma fibres in the stipular wings of
the petiole. It shares an ample number of characters with
subgenera Osmundastrum and Osmunda sensu Miller, but
is markedly distinct from subgenus Plenasium [29, 30].
The rather high degree of stele dissection and the distant
point of initial bifurcation of leaf-trace protoxylem are typical of Osmundastrum and O. claytoniana [29, 30]; finally,
the presence of usually a single root per leaf trace together
with the development of (ultimately) one abaxial arch and
two lateral masses of thick-walled fibres in the petiole
sclerenchyma ring render the new species particularly
similar to subgenus Osmundastrum [29, 30]. Since the fossil differs from extant species merely in specific diagnostic
characters, we have no hesitation in assigning it to Osmunda in accordance with conventional practice [29, 30,
36, 63, 75, 101].
Page 15 of 25
By analogy, the same basic similarity also applies to at
least five of the >25 fossil species currently included in
Millerocaulis sensu Vera and Ashicaulis, which are all
characterized by having heterogeneous sclerenchyma rings
in the petioles: M. liaoningensis [106], A. claytoniites
[107], A. plumites [108], and A. wangii [109]—all from the
Jurassic of China—and M. johnstonii from Tasmania
[110], which we, therefore, included in our phylogenetic
analyses. The holotype of the last of these species was collected from a gravel pit; following Tidwell et al. [110], we
consider the age of this specimen to be likely concordant
with those of other Mesozoic permineralized fern stems
from eastern Tasmania, which have recently been dated as
Early Jurassic [111].
Systematic placement of fossil Osmunda rhizomes among
modern Osmundaceae
Phylogenetic network analysis
Relationships among extant species in the distance network based on our morphological matrix are congruent
with those of molecular phylogenetic analyses [31, 32],
confirming that the morphological matrix based on rhizome anatomy serves well in resolving systematic relationships among modern Osmundaceae. The only major
exception is expressed by O. claytoniana, which, together with extant species of subgenus Osmunda sensu
Yatabe et al. and Paleogene and Neogene fossils, forms
a group essentially consistent with subgenus Osmunda
sensu Miller.
The Jurassic taxa included in our analysis, including
O. pulchella, form a broad box-like structure that bridges the gap between the relatively derived Osmundastrum and the less derived Osmunda subgenus Osmunda
sensu Miller (Fig. 8). Their long terminal branches are
due to unique trait combinations intermediate between
their more derived fossil and extant relatives. Collectively, the Jurassic species probably represent ancestral
forms of Osmunda s.l., some being more similar to O.
cinnamomea (O. pulchella) and others to subgenus Osmunda sensu Miller (e.g. A. wangii).
Overall, the placement of the other fossil taxa accords
well with the basic assumption that they should be less
derived—and thus placed closer to the centre of the network—than their extant relatives. However, there is one
major exception: O. dowkeri from the Paleogene is the
furthest-divergent (i.e. most derived) of all fossil and extant species in the Plenasium group. This relates to its
unusually complex stele organization, which is highly
dissected and contains by far the largest number of
xylem segments of all species analysed (exceeding 30,
compared to less than 12 in all other Plenasium and less
than 20 in most other Osmunda).
Notably, a subdivision into two putatively monophyletic subgenera Osmunda sensu Yatabe et al. and
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Claytosmunda generates two taxa without discriminating anatomical and morphological features (potential
aut- or synapomorphies according to Hennig [112]).
Miller’s paraphyletic subgenus Osmunda accommodates
the fossil taxa, whereas the concept of Osmunda proposed by Yatabe et al. [31, 45] precludes infrageneric
classification of most fossil species (Fig. 8).
Compatibility with vegetative morphology
The systematic relationships revealed from our analysis
of anatomical characters of the rhizomes reflect the distribution of gross morphological and fertile features
within Osmundaceae very well. The isolated position
and tight clustering of subgenus Plenasium, for instance,
finds support through morphological data in the form of
its invariant, unique frond morphology: unlike any other
modern Osmundaceae, all extant Plenasium species are
characterized by having invariably simple-pinnate and
hemi-dimorphic fronds. The rather wide dispersion of
the (paraphyletic) subgenus Osmunda Miller is congruent with the variable frond morphology and dimorphism
in this group, ranging from pinnate–pinnatifid [e.g. O.
claytoniana (similar to O. cinnamomea)] to fully bipinnate and from fully to variably hemi-dimorphic.
The only major topology where anatomical data alone
probably fail to generate a realistic divergence distance occurs in the branch including Todea and Leptopteris. These
genera, having a rhizome structure broadly similar to that
of Osmunda and especially Osmundastrum [11] (but see
Fig. 8), are characterized by unique vegetative and fertile
characters (e.g. isomorphic fronds; tripinnate fronds, arborescent habit, and lack of stomata in Leptopteris) that
differentiate them very clearly from Osmunda s.l.
Integrating fossil species into the molecular backbone
Overall, the results of the EPA provide good support regarding the relationships between fossil and extant taxa
(compare Figs. 8 and 9). However, notable ‘position swaps’
occur between the placements obtained from different
weighting methods of several taxa, including Osmunda
pulchella. This incongruence is due to intermediate character combinations inherent to ancestral taxa, which we
interpret to result in ‘least conflicting’ placements at varying root positions; the EPA is designed to optimize the
position of a query taxon within a pre-defined backbone
topology. Because O. pulchella and other fossil taxa have
character combinations of genetically distant taxa, the
model-based weights in particular will down-weigh the
relevant characters. Maximum parsimony has a much
more naïve approach in this respect, which may help achieve
a more plausible placement of the fossils. Nevertheless, the
fact that this down-weighting results in a placement close to
the roots, but not in the tips of sub-trees, indicates that the
Page 16 of 25
remaining character suite is plesiomorphic in general, thus
supporting the interpretation of fossil taxa such as O. pulchella as ancestors of extant clades and possibly individual
species (Figs. 8 and 9).
Altogether, the results detailed above lead us to the following conclusions about the systematic and phylogenetic
placements of fossil species among modern Osmundaceae:
(1)The Jurassic Osmunda pulchella is an ancestral
member of Osmunda s.l. combining diagnostic
features both of Osmunda s.str. and of
(2)Other species reported from the Jurassic, together
with O. pluma (Paleogene) and O. wehrii (Neogene),
are representatives of the (paraphyletic) subgenus
Osmunda sensu Miller, including potential ancestors
of extant species of subgenus Osmunda and
(3)Osmunda oregonensis (Paleogene) is closely allied
with subgenus Osmunda sensu Yatabe et al. (see
(4)Osmunda arnoldii and O. dowkeri belong to
subgenus Plenasium and are closely similar to O.
banksiifolia; the highly derived O. dowkeri represents
the highest degree of specialization in the subgenus,
which is supposed to have reached its heyday in
distribution and diversity during the Paleogene [30].
(5)A close systematic relationship of extant and all fossil
Osmundastrum is unambiguous, despite their wide
stratigraphic age-span (Cretaceous, Paleogene, and
Neogene) and ‘trans-Pacific’ geographic distribution. It
is interesting to note, however, that the rhizomes of
O. cinnamomea show a far greater disparity in
anatomical characters than all other subgenera and
even genera of modern Osmundaceae, indicating
the existence of probably more than just a single
Osmundastrum species in the past (Fig. 8).
(6)Osmunda iliaensis and O. shimokawaensis are most
likely representatives of that species complex of
subgenus Osmunda that is today restricted to East
Asia (i.e. O. lancea and O. japonica); O.
shimokawaensis may be ancestral to O. japonica and
O. lancea.
(7)the Early Cretaceous Todea tidwellii may be as
related to modern Leptopteris as it is to Todea.
Re-evaluation of the Generic Status of Osmundastrum
The intermediate character combination and the resulting systematic placement of Osmunda pulchella and
other Jurassic species between Osmundastrum and subgenus Osmunda Miller challenges the current treatment
of Osmundastrum as a separate genus. In the following
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
section, therefore, we provide a detailed re-evaluation of
the sum of evidence that has been used to invoke generic separation of Osmundastrum. We begin with what
is perhaps considered the most novel and reliable body
of evidence—molecular data—and continue with additional evidence from morphological, anatomical, and
hybridization studies.
Molecular data
The comprehensive multi-locus phylogeny of Metzgar
et al. [32] has recently been interpreted to fully support
a separate generic status of Osmundastrum as suggested
earlier by Yatabe et al. [31]. Inter-generic and intersubgeneric ingroup-only relationships based on the molecular matrix employed by Metzgar et al. (reproduced
here in Fig. 10) indeed receive nearly unambiguous support from the concatenated gene matrix.
Our analysis of the root-placement stability, however,
revealed that the paraphyletic status of Osmunda s.l. inferred from the results of Metzgar et al. [32] is not unambiguously supported by all gene regions (Fig. 10).
Whereas this scenario indeed receives strong support
from the two coding regions (rbcL-gene, atpA-gene), the
molecular data matrix also yields a strong conflicting
signal from three relatively conserved spacer sequences
(i.e. atpB-rbcL, rbcL-accD, and trnL-trnF) that indicates
an alternative root placement between Leptopteris–
Todea and the remaining Osmunda s.l. This latter signal
offers an equally valid interpretation that would resolve
Osmunda s.l. as monophyletic.
The root-placement problem may be due in part to
the insufficiently comprehensive selection of out-group
taxa, which is limited to four samples of leptosporangiate ferns in the matrix of Metzgar et al. [32]: Matonia
pectinata R.Br. (Matoniaceae), Dipteris conjugata Reinw.
(Dipteridaceae) and Gleicheniella pectinata (Willd.)
Ching and Diplopterygium bancroftii (Hook.) A.R.Sm.
(Gleicheniaceae)—all members of Gleicheniales. Current
fern phylogenies indicate that Osmundaceae represent
the earliest-diverged group in the Polypodiopsida, which
include five other extant orders apart from Gleicheniales
(see e.g. [1, 2, 4]). We anticipate that a less ambiguous
molecular signal may be obtained by the selection of a
more comprehensive range of outgroup taxa, including
representatives from all major lineages within the Polypodiopsida (in particular Hymenophyllales and Schizaeales) and the sister clades of this class (Equisetopsida
and Marattiopsida). Comprehensive sampling of slowly
evolving nuclear genes (see e.g. [67, 68]) for the ingroup
and outgroup may help to identify outgroup-inflicted
branching artefacts in the current plastid-sequence-based
topology. Because representatives of Gleicheniales are relatively derived in comparison to Osmundales, they may inflict outgroup long-branch attraction with Osmundastrum
Page 17 of 25
[see Additional file 2: Figure. S1 (note the long terminal
edge bundles) and S2 in ESA].
Rhizomes of extant O. cinnamomea have several peculiar
and supposedly unique characters, including (1) the
common occurrence of an internal endodermis; (2) the
rare occurrence of a dissected, ectophloic to amphiphloic stele; (3) bifurcation of the protoxylem bundle only
as the leaf trace enters the petiole base; (4) the sclerenchyma ring of a petiole base containing one abaxial and
two lateral masses of thick-walled fibres; (5) usually single, rarely paired roots arising from the leaf traces; and
(6) a patch of sclerenchyma adaxial to each leaf trace in
the inner cortex (e.g., [11, 30]).
The first two characters occur inconsistently in extant
individuals, and are notably absent in fossil (Cretaceous
to Neogene) representatives of Osmundastrum [29, 30,
64], suggesting that these might represent recently acquired traits [30]. Moreover, dissected steles and dictyosteles, with either two endoderms or two phloem
layers connecting through a leaf gap, are conditions
only rarely and inconsistently developed below incipient rhizome bifurcations [8, 9, 11]. The significance of
both characters as diagnostic features of Osmundastrum is thus questionable.
The point of protoxylem bifurcation and the distribution
of patches of thick-walled fibres in the petiole sclerenchyma ring are consistent and arguably appropriate diagnostic characters of Osmundastrum. However, among the
remaining Osmunda s.l. species, these same characters are
regarded as diagnostic only at specific or subgeneric rank
[30]. Thus, it would seem inconsistent to afford greater
taxonomic weight to these characters in the delimitation
of Osmundastrum alone.
Roots typically arising singly is a useful character discriminating Osmundastrum and Osmunda pulchella
from the remaining Osmunda, although this feature is
inconsistent and may be difficult to observe [11, 30].
The occurrence of sclerenchyma patches adaxial to the
leaf traces in the inner stem cortex is the only invariant
and unique character of Osmundastrum that we consider might validate its separation beyond species level.
Apart from Osmundastrum, this feature occurs also in
Todea but not in its sister genus Leptopteris [30].
Morphological features commonly regarded as diagnostic of Osmundastrum include (1) generally complete
frond dimorphism; (2) pinnate–pinnatifid frond architecture; and (3) dense abaxial trichomes on pinna rachides
[32]. However, using frond architecture and dimorphism
as a strict diagnostic character has been shown to be
problematic (e.g. [11]). Pinnate fronds with deeply
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
pinnatifid segments occur in both O. (Osmundastrum)
cinnamomea and O. (Claytosmunda) claytoniana. Moreover, some common varieties and growth forms of O.
cinnamomea produce only hemi-dimorphic fronds
[113–116], some having apical fertile portions resembling those of O. regalis (see, e.g. [114, 117, 118]) and
others having intermittent fertile portions like those of
O. claytoniana (see, e.g. [114, 119]). Further, completely
dimorphic fronds are also predominant in O. lancea, common in O. japonica, and sporadic in O. regalis ([11, 120]).
Significantly, such ranges of variation are encountered
only in the species complex including Osmundastrum and
Osmunda subgenus Osmunda Miller (= subgenera Claytosmunda and Osmunda Yatabe et al.).
Finally, fronds of all Osmunda s.l. species emerge with a
more-or-less dense abaxial indumentum and differ merely
in the duration to which the trichome cover is retained in
the course of frond maturation [11]. In fully mature fronds
of all species considered, most of the hair cover is ultimately lost, with O. cinnamomea [especially O. cinnamomea
var. glandulosa Waters [121, 122] merely tending to retain
greater amounts of hairs than O. claytoniana, and those in
turn more than other species [11]. In summary, we follow
Hewitson [11] in arguing that none of these morphological
features provide consistent and reliable diagnostic characters for separating Osmundastrum from subgenus Osmunda Miller.
Metzgar et al. ([32] p. 34) suggested that the existence
of hybrids can be used to decide about the elevation of
subgenera to generic ranks. Numerous natural hybrids,
intra- and inter-subgeneric, are known to occur in Osmunda s.str.: O. × ruggii R.M.Tryon in eastern North
America (O. regalis × O. claytoniana; [49, 51]), O. × mildei C.Chr. in southern China (O. japonica × O. vachellii
Hook.; [123, 124]), O. × hybrida Tsutsumi, S.Matsumoto,
Y.Yatabe, Y.Hiray. & M.Kato in Southeast Asia (O. regalis × O. japonica; [68]), and O. × intermedia (Honda)
Sugim. (O. japonica × O. lancea) and O. × nipponica
Makino (O. japonica × ?O. claytoniana) in Japan [23, 67,
124]. The apparent absence of naturally occurring hybrids involving Osmundastrum has been interpreted to
result from its particularly isolated position within Osmunda s.l. [29, 30]. However, Klekowski [50] conducted
artificial breeding experiments and readily succeeded in
producing viable hybrid sporophytes from O. cinnamomea × O. claytoniana and O. cinnamomea × O. regalis,
with equal or even higher yields (1 out of 8 and 2 out of 9,
respectively) compared to O. claytoniana × O. regalis (1
out of 8). In addition, some authors suspect that there
may also be natural hybrids between O. cinnamomea and
Osmunda s. str. (see [67]). So far, there is no record of hybridisation between Leptopteris-Todea and Osmunda s.l.
Page 18 of 25
either ex situ or in situ (e.g., from southern Africa,
where the geographic ranges of Osmunda and Todea
overlap [125]).
We find that neither molecular, anatomical, morphological, nor hybridization studies have yet succeeded in
providing unequivocal evidence that would warrant separate generic status of O. cinnamomea, reject an (inclusive)
common origin of Osmundastrum and Osmunda s.str., or
else identify an (inclusive) common origin of LeptopterisTodea and Osmunda s.str. Rather we argue that the sum
of evidence for extant taxa detailed above allows for two
equally valid hypotheses: the ‘paraphyletic-Osmunda scenario’ [31, 32] and an alternative ‘monophyletic-Osmunda
scenario’ [30].
The impact of Osmunda pulchella on the classification of
modern Osmundaceae
The phylogenetic placement of Osmunda pulchella is critical to the systematic classification of modern Osmundaceae (Figs. 11, 12). In the specified topology of the
‘paraphyletic Osmunda scenario’, most parsimonious
placement of O. pulchella is at the base of the tree, at the
root of either Osmundastrum or of the remaining TodeaLeptopteris-Osmunda s.str. clade (Fig. 11). If this phylogenetic scenario is followed, and if only holophyletic
groups are considered valid taxonomic units (see, e.g.,
[126, 127] for critical discussion), then it follows that all
modern Osmundaceae need be included in one genus Osmunda, with Plenasium, Osmunda, Claytosmunda,
Osmundastrum, Todea, and Leptopteris being infrageneric
taxa (Fig. 12). Alternatively, the ‘four-genus classification’
proposed by Yatabe et al. and Metzgar et al. could of
course also be maintained under the ‘paraphyletic Osmunda scenario’ if fossil taxa were to be excluded from
systematic classification as a whole (Fig. 12). We expect,
however, that such practice would be broadly met with
criticism from palaeobiologists and neontologists (see e.g.
[59, 60, 69, 70]); in the present study, it would be particularly ignorant not to place the new fossil in a systematic
context given that it fully agrees with the circumscription
of an extant genus that is diagnosed by a considerable
number of informative anatomical characters.
If, by contrast, the specified topology of the ‘monophyletic Osmunda scenario’ is followed, in which the most
parsimonious placement of O. pulchella is as sister to O.
cinnamomea at the base of an Osmundastrum-Osmunda
s.str. clade (Fig. 11), then all fossil and extant species of
modern Osmundaceae can be resolved in three mutually
monophyletic genera: Todea, Leptopteris, and Osmunda,
the last of these including the subgenera Plenasium,
Osmunda, Claytosmunda, and Osmundastrum (Fig. 12).
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Page 19 of 25
molecular data set can be assembled that is immune to
outgroup long-branch attraction.
Evolutionary significance of fossil Osmunda rhizomes
Fig. 12 Diagram illustrating the critical significance of the placement
of Osmunda pulchella for a strictly cladistic-systematic classification of
modern Osmundaceae regarding the two alternative rooting
schemes (Figs. 10, 11). Genera names in bold; infrageneric taxa
names in regular font
In our opinion, this latter option integrates the apparently conflicting evidence from studies of the morphology, anatomy, molecular data, and fossil record of
Osmundaceae in a much more realistic and elegant way,
and offers a more practical taxonomic solution. We,
therefore, argue that Osmunda pulchella described here
exposes the recently established paraphyly of Osmunda
s.l. as a result of a sampling or reconstruction artefact in
the molecular matrix employed. A broader outgroup selection and more comprehensive gene sampling (e.g. including nuclear genes) may resolve the root of
Osmundaceae more reliably in the future, providing a
Grimm et al. [34] recently used the rhizome fossils and
molecular data studied herein together with an additional set of 17 frond fossils to infer divergence ages for
the major splits within modern Osmundaceae. Among
several tests, the authors employed a ‘fossilized-birthdeath’ (FBD) Bayesian dating approach in which only the
frond fossils were used for the calibration of agedistribution priors. The results of this test provide an independent temporal framework [34: supplement] that
can be used to assess the evolutionary significance of
fossil Osmunda rhizomes (Fig. 13).
Calibrated using only frond fossils, the FBD approach
dated the split between Osmundastrum and the
remaining Osmunda as being older than mid-Late Triassic. Consequently, Jurassic rhizomes with intermediate
or plesiomorphic anatomy represent either precursors or
extinct sister lineages of extant clades within Osmunda
s.l. With its Early Jurassic age and its intermediate anatomical character suite, O. pulchella emerges as an ideal
candidate for a true precursor of subgenus Osmundastrum (Fig. 13a), which became established in its present
form by the Late Cretaceous (Fig. 13g). The other Jurassic rhizomes have plesiomorphic character suites shared
with all remaining Osmunda, but lack the apomorphic
states that are characteristic of the highly specialized
subgenus Plenasium (Fig. 13b–e). This is in consonance
with the mid-Cretaceous root age inferred for Plenasium, which predates the occurrence of the oldest
known Plenasium rhizome fossils by at least 30 million
years (Fig. 13j, k). In most other cases, the estimated divergence ages also predate the earliest rhizome fossils
with lineage-specific characters, as would be expected.
One conflict occurs in the seemingly precocious appearance of O. shimokawaensis (Fig. 13q), which our analyses
identify as a precursor of the two East Asian Osmunda
species, in the late middle Miocene (12–14 million years
ago). According to the FBD dating calibrated via frond
fossils, the split between these two species (O. lancea
and O. japonica) and O. regalis occurred less than 10
million years ago. However, this conflict can be explained by the species-level molecular data used in the
dating, which mask the substantial intraspecific genetic
disparity between New-World and Old-World populations of O. regalis [68].
Finally, it needs to be pointed out that—following the
evidence gathered and presented here—Grimm et al.
[34] did not employ both rooting scenarios in their dating. However, during earlier stages of that study, preliminary dating analyses were performed for each of the two
different rooting scenarios, using a dirichlet probability
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Page 20 of 25
Fig. 13 Diagram illustrating the phylogenetic positions of fossil rhizomes within an independently obtained chronogram for modern
Osmundaceae that was calibrated using frond fossils only [34]. a = O. pulchella; b–d = M. liaoningensis, A. plumites, and A. wangii; e = A. claytoniites;
f = T. tidwellii; g = O. cinnamomea (Cretaceous, Canada [64]); h = O. precinnamomea [29]; i = O. pluma; j = O. dowkeri (Paleocene, UK [75]); k = O.
arnoldii; l = O. oregonensis; m = O. nathorstii; n = O. dowkeri (Eocene, USA; see [29]); o = O. cinnamomea (Neogene, USA [29]); p = O. wehrii; q = O.
shimokawaensis; r = O. cinnamomea (Miocene, Japan [63]); s = O. iliaensis
prior (DPP) model and including oldest fossils as minimum age constraints for the hypothetical most-recent
common ancestors of extant taxa (Additional file 3).
Dating using the DPP model shares the basic principles
of FBD dating, except that fossils are used in the traditional way as node-height constraints. The results
showed that the choice of the rooting scenario is
largely irrelevant to the estimated subsequent divergence ages. Thus, even if future studies should produce
more comprehensive and better-substantiated evidence
in favour of a paraphyletic rooting scenario over the
monophyletic scenario, O. pulchella would still remain
a likely member of the Osmundastrum lineage.
(i) Osmunda pulchella sp. nov. from the Early
Jurassic of Sweden is among the earliest
unequivocal records of fossil Osmunda rhizomes,
and a likely precursor of the extant O.
cinnamomea and its fossil relatives.
(ii)Intermediate anatomical character suites of O.
pulchella and other Jurassic osmundoid rhizomes
support re-inclusion of the recently separated,
monospecific Osmundastrum within Osmunda.
(iii)The sum of morphological, anatomical, molecular,
and fossil evidence supports modern Osmunda
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
(including Osmundastrum) and Todea-Leptopteris
being mutually monophyletic.
(iv)The recently established rooting of Osmundaceae
and the resulting paraphyly of Osmunda s.l., based
solely on molecular data, likely results from a
sampling or reconstruction artefact. More
comprehensive outgroup selection and broader gene
sampling may hold the potential to alleviate this
problem in future analyses.
(v)Our results accord remarkably well with
independently obtained divergence ages based on
molecular dating calibrated via frond fossils.
Fossil material
The studied specimen was collected from mafic volcaniclastic deposits (“Djupadal formation” [61]) near
Korsaröd lake (Höör municipality, central Skåne,
Sweden). The host strata are interpreted to be local
remnants of ash falls and lahar flows that spread from a
nearby volcanic centre, similar to other occurrences of
mafic volcaniclastic and epiclastic deposits associated
with basaltic necks in central Skåne [128]. Palynological
analyses indicate a late Pliensbachian (late Early
Jurassic) age [62], which agrees well with radiometric dating of associated basaltic necks that place the peak phase
of volcanism in central Skåne in the Pliensbachian to
Toarcian (ca 183 Ma [129]). Petrographic thin sections
(Figs. 1, 2, 3, 4, and 5) were studied and photographed
using an Olympus BX51 compound microscope with an
attached Olympus DP71 digital camera. Two sectioned
blocks of the holotype were selected for SEM analyses; the
sectioned surfaces of these blocks were polished, etched
with 5 % HCl for 5–10 s, mounted on aluminium stubs,
coated with gold for 90 s, and finally analysed using a
Hitachi S-4300 field emission scanning electron microscope at the Swedish Museum of Natural History (Fig. 6).
We applied conventional adjustments of brightness, contrast, and saturation to most of the digital images using
Adobe® Photoshop® CS5 Extended version 12.0; in some
cases, we performed manual image stitching and image
stacking [130] in order to obtain sufficiently sharp, large
composite images with optimal depth of field.
Phylogenetic analyses
In order to place the newly described fossil in a phylogenetic context, we assembled a morphological matrix
that is based on the phylogenetic assessment of Miller
[30], including all extant and fossil members of the extant genera plus those fossil rhizome species that agree
in all anatomical features with those of modern genera
[see Additional file 1; ESA].
Page 21 of 25
Network analysis
We rely exclusively on network methods as implemented
in SplitsTree v. 4.13.1 [131] to draw phylogenetic conclusions based on the morphological matrix (see [58,
132–135]): (1) a neighbour-net [136, 137] based on mean
inter-taxon distances, and (2) bipartition networks to
visualize support (Bayesian-inferred posterior probabilities, PP; non-parametric bootstrapping, BS) for alternative
phylogenetic relationships [58, 138, 139]. BS support was
established under three commonly used optimality criteria
using 10,000 bootstrap replicates: (1) Least-squares via the
BioNJ algorithm (BSNJ; [140]); (2) Maximum parsimony
(BSMP) using PAUP* [141, 142]; and (3) Maximum likelihood (BSML) via the fast bootstrapping implementation in
RAxML v. 7.4.2 [143, 144] using both available transition
models for categorical (multistate) data, i.e. (i) the general
time-reversible model (BSML/GTR) [145] and (ii) Lewis’
model (BSML/MK) [146]. For configuration details of
Bayesian inference, non-parametric bootstrapping, and
network-wise visualization refer to Additional file 1 [ESA].
Re-visiting the Osmundaceae root
We analysed the root placement in the phylogenetic tree
of Metzgar et al. [32] using the original molecular matrix.
First, a set of traditional phylogenetic analyses was run, including a gene jackknifing procedure. Trees and bootstrap
support were inferred using the concatenated data, each
gene partition separately, and matrices in which one partition was deleted. Second, the evolutionary placement algorithm (EPA [147, 148]) as implemented in RAxML was
used to determine the optimal position of the outgroup
taxa (i.e. the position of an outgroup-inferred root) within
an ingroup-only topology. The EPA has been originally
designed for placing fossils [147] or short-sequence
reads [148], but its metrics can also be used to generally test the position of one or many query sequences
—here: outgroup taxa—in a given topology—here: an
ingroup-only ML tree—in a ML framework (A. Stamatakis, pers. comm., 2014).
Character plotting and independent optimization of the
placement of fossils within a molecular framework of
modern taxa
Using the EPA we estimated a weight (probability) for
the placement of our fossil within the molecular backbone topology reproduced from the data matrix of
Metzgar et al. [32]. We also determined the most parsimonious placement of the newly described fossil within
the molecular tree of Metzgar et al. [32] implemented
into the morphological matrix using MESQUITE v. 2.75
[149]; this is done by simply moving the fossil within the
given topology and recording the incremental increase
in steps added to the resulting whole tree-length (see
[133, 150] for applications).
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Nomenclatural remark
In order to maintain consistent use of terminology, we
employ the following names: (1) Osmunda cinnamomea
instead of the currently used Osmundastrum cinnamomeum (L.) C.Presl; (2) ‘modern Osmundaceae’, referring
to those genera of Osmundaceae that are based on extant species, i.e. Osmunda (including Osmundastrum),
Todea, and Leptopteris; (3) ‘Osmunda s.l.’, referring to
the traditional generic concept that includes all extant
and several fossil species (e.g., Miller, 1971); and (4) ‘Osmunda s.str.’, referring to the recently proposed generic
concept of Osmunda that excludes O. cinnamomea and
O. precinnamomea C.N.Mill. (i.e., including only Osmunda subgenera Osmunda, Claytosmunda and Plenasium) [31]. Where necessary, we cite taxon authorities
to discriminate between formal subgeneric concepts
used by Miller [30] and Yatabe et al. [45].
For naming the additional Jurassic species included in
our analysis, we follow Vera’s [99] taxonomic revision of
Millerocaulis and Ashicaulis, in which a more broadly
defined generic concept of Millerocaulis was proposed
to include also those species that were previously
assigned to Ashicaulis [99]. Vera’s concept, however, has
so far not been universally adopted, and the genus Ashicaulis is still frequently used; new Ashicaulis species that
have been introduced since the revision appeared
[101–103] are provisionally listed here with their original
names and an additional remark; these include Ashicaulis (=Millerocaulis sensu Vera [99]) claytoniites [101], A.
(=Millerocaulis sensu Vera [99]) plumites [102], and A.
(=Millerocaulis sensu Vera [99]) wangii [103].
Availability of supporting data
An electronic supplementary data archive (ESA) containing all original data files and results, including the
employed matrices in NEXUS format is available for
anonymous download at www.palaeogrimm.org/data/
Bfr15_ESA.zip [please refer to the accompanying
index document (GuideToFiles.txt) for a detailed
Additional files
Additional file 1: Document detailing definition and coding of
characters in the morphological matrix, and the set-up for phylogenetic
analyses and the evolutionary placement algorithm.
Additional file 2: Neighbour net inferred from uncorrected pairwise
distances based on the concatenated data set of Metzgar et al.
(2008). Bootstrap support of (alternative) splits is annotated. Note the
occurrence of four genetically distinct lineages; the splits that place O.
cinnamomea as sister to all other Osmundaceae received strong support
only from atpA and rbcL partitions.
Additional file 3: Divergence ages and 95 % height posterior
densities obtained with fossilized birth-death dating [34] and the
DPP model using five minimum age constraints for calibration (P.
Kapli, G. Grimm, unpubl. data).
Page 22 of 25
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors contributed to the study design and manuscript preparation, and
read and approved the final manuscript. BB studied and documented the
fossil material. BB and GWG designed and assembled the morphological
character matrix. GWG conceived and performed all phylogenetic analyses
and carried out the re-analysis of molecular data.
We thank Ezequiel I. Vera (Buenos Aires), Wang Yongdong (Nanjing), and
Tian Ning (Shenyang) for helpful discussion and kind assistance in obtaining
literature; Alexandros Stamatakis (Heidelberg) for discussing methodological
approaches; Birgitta Bremer and Gunvor Larsson (Stockholm) for providing
live material of Osmunda for comparison; and Else Marie Friis and Stefan
Bengtson (Stockholm), Susanne S. Renner (Munich), and Ignacio H. Escapa
(Trelew) for discussion. We appreciate the comments of Mike Fay and Hassan
Rankou (Kew), Toshihiro Yamada (Kanazawa), Joselito Acosta and five
anonymous reviewers on earlier versions of this manuscript. Financial
support by the Swedish Research Council (VR grants 2014–5232 to B.
Bomfleur and 2014–5234 to S. McLoughlin) is gratefully acknowledged.
Author details
Department of Palaeobiology, Swedish Museum of Natural History,
Stockholm, Sweden. 2Department of Palaeontology, University of Vienna,
Vienna, Austria.
Received: 11 December 2014 Accepted: 1 June 2015
1. Pryer KM, Schuettpelz E, Wolf PG, Schneider H, Smith AR, Cranfill R.
Phylogeny and evolution of ferns (monilophytes) with a focus on the early
leptosporangiate divergences. Am J Bot. 2004;91:1582–98.
2. Smith AR, Pryer KM, Schuettpelz E, Korall P, Schneider H, Wolf PG. A
classification for extant ferns. Taxon. 2006;55:705–31.
3. Smith AR, Pryer KM, Schuettpelz E, Korall P, Schneider H, Wolf PG. Fern
classification. In: Ranker TH, Haufler CH, editors. Biology and Evolution of
Ferns and Lycophytes. Cambridge: University Press; 2008. p. 417–67.
4. Schuettpelz E, Pryer KM. Fern phylogeny inferred from 400 leptosporangiate
species and three plastid genes. Taxon. 2007;56:1037–50.
5. Bower FO. Is the eusporangiate or the leptosporangiate type the more
primitive in the ferns? Ann Bot. 1891;5:109–34.
6. Bower FO. The ferns (Filicales), treated comparatively with a view to their
natural classification. Vol. 2: The Eusporangiatae and other relatively
primitive ferns. Cambridge: University Press; 1926.
7. Tidwell WD, Ash SR. A review of selected Triassic to Early Cretaceous ferns. J
Plant Res. 1994;107:417–42.
8. Faull JH. The anatomy of the Osmundaceae. Bot Gaz. 1901;32:381–420.
9. Faull JH. The stele of Osmunda cinnamomea. Trans R Can Inst. 1909;8:515–
10. Seward AC, Ford SO. The anatomy of Todea, with notes on the geological
history and affinities of the Osmundaceæ. Trans Linn Soc Lond 2nd ser:
Botany. 1903;6:237–60.
11. Hewitson W. Comparative morphology of the Osmundaceae. Ann Mo Bot
Gard. 1962;49:57–93.
12. Strasburger E. Ueber Reductionstheilung, Spindelbildung, Centrosomen und
Cilienbildner im Pflanzenreich. Histologische Beiträge. 1900;6:1–224.
13. Yamanouchi S. Chromosomes in Osmunda. Bot Gaz. 1910;49:1–12.
14. Digby L. On the archesporial and meiotic mitoses of Osmunda. Ann Bot.
15. Sharp LW. Mitosis in Osmunda. Bot Gaz. 1920;69:88–91.
16. Manton I. Evidence on spiral structure and chromosome pairing in
Osmunda regalis L. Philos Trans R Soc Lond B. 1945;230:179–215.
17. Manton I. Chromosome length at the early meiotic prophases in Osmunda.
Ann Bot. 1945;9:155–78.
18. Manton I, Smiles J. Observations on the spiral structure of somatic
chromosomes in Osmunda with the aid of ultraviolet light. Ann Bot.
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
19. Tatuno S, Yoshida H. Kalyologische Untersuchungen über Osmundaceae I.
Chromosomen der Gattung Osmunda aus Japan. Bot Mag Tokyo.
20. Tatuno S, Yoshida H. Karyological studies on Osmundaceae II. Chromosome
of the genus Osmundastrum and Plenasium in Japan. Bot Mag Tokyo.
21. Klekowksi EJ. Populational and genetic studies of a homosporous fern–
Osmunda regalis. Am J Bot. 1970;57:1122–38.
22. Klekowksi EJ. Genetic load in Osmunda regalis populations. Am J Bot.
23. Yatabe Y, Tsutsumi C, Hirayama Y, Mori K, Murakami N, Kato M. Genetic
population structure of Osmunda japonica, rheophilous Osmunda lancea
and their hybrids. J Plant Res. 2009;122:585–95.
24. Kidston R, Gwynne-Vaughan DT. On the fossil Osmundaceae. Part I. Trans R
Soc Edinb. 1907;45:759–80.
25. Kidston R, Gwynne-Vaughan DT. On the fossil Osmundaceae. Part II. Trans R Soc
Edinb. 1908;46:213–32.
26. Kidston R, Gwynne-Vaughan DT. On the fossil Osmundaceae. Part III. Trans R Soc
Edinb. 1909;46:651–67.
27. Kidston R, Gwynne-Vaughan DT. On the fossil Osmundaceae. Part IV. Trans R Soc
Edinb. 1910;47:455–77.
28. Kidston R, Gwynne-Vaughan DT. On the fossil Osmundaceae. Part V. Trans R Soc
Edinb. 1914;50:469–80.
29. Miller CN. Evolution of the fern genus Osmunda. Contrib Mus Paleont.
30. Miller CN. Evolution of the fern family Osmundaceae based on anatomical
studies. Contrib Mus Paleont. 1971;23:105–69.
31. Yatabe Y, Nishida H, Murakami N. Phylogeny of Osmundaceae inferred from
rbcL nucleotide sequences and comparison to the fossil evidences. J Plant
Res. 1999;112:397–404.
32. Metzgar JS, Skog JE, Zimmer EA, Pryer KM. The paraphyly of Osmunda is
confirmed by phylogenetic analyses of seven plastid loci. Syst Bot. 2008;33:31–6.
33. Escapa IH, Cúneo NR. Fertile Osmundaceae from the Early Jurassic of
Patagonia. Int J Plant Sci. 2012;173:54–66.
34. Grimm GW, Kapli P, Bomfleur B, McLoughlin S, Renner SS. Using more than
the oldest fossils: Dating Osmundaceae by three Bayesian clock approaches.
Syst Biol. 2015;64:396–405.
35. Arnold CA. Mesozoic and Tertiary fern evolution and distribution. Mem
Torrey Bot Club. 1964;21:58–66.
36. Tian N, Wang Y-D, Jiang Z-K. Permineralized rhizomes of the Osmundaceae
(Filicales): Diversity and tempo-spatial distribution pattern. Palaeoworld.
37. Wang S-J, Hilton J, He X-Y, Seyfullah LJ, Shao L. The anatomically preserved
Zhongmingella gen. nov. from the Upper Permian of China: evaluating the
early evolution and phylogeny of the Osmundales. J Syst Palaeontol.
38. Hasebe M, Wolff PG, Pryer KM, Ueda K, Ito M, Sano R, et al. Fern phylogeny
based on rbcL nucleotide sequences. Am Fern J. 1995;85:134–81.
39. Schneider H, Schuettpelz E, Pryer KM, Cranfill R, Magallón S, Lupia R. Ferns
diversified in the shadow of angiosperms. Nature. 2004;428:553–7.
40. Linnaeus C. Species plantarum, exhibentes plantas rite cognitas, ad genera
relatas, cum differentiis specificis, nominibus trivialibus, synonymis selectis,
locis natalibus, secundum systema sexuale digestas. L Salvius: Stockholm;
41. Thunberg CE. Flora Japonica sistens plantas insularum Japonicarum:
Secundum systema sexuale emendatum redactas ad XX classes, ordines,
genera et species. Leipzig: Müller; 1784.
42. Presl CB. Reliquiae Haenkeanae, seu, Descriptiones et icones plantarum:
quas in America meridionali et boreali, in insulis Philippinis et Marianis
collegit Thaddaeus Haenke. JG Calve: Prague; 1825.
43. Blume CE. Enumeratio plantarum Javae et insularum adjacentium; minus
cognitarum vel novarum ex herbariis Reinwardtii, Kuhlii, Hasseltii et Blumii.
1828th ed. Leiden: van Leeuwen, J.W; 1828.
44. Hooker WJ. Icones plantarum; or figures, with brief descriptive characters
and remarks, of new or rare plants, selected from the author’s herbarium. T.
15. London: Longman, Rees, Orme, Brown, Green & Longman; 1837.
45. Yatabe Y, Murakami N, Iwatsuki K. Claytosmunda; a new subgenus of Osmunda
(Osmundaceae). Acta Phytotaxon Geobot. 2005;56:127–8.
46. Tagawa M. Osmundaceae of Formosa. J Jap Bot. 1941;17:692–703.
47. Bobrov AE. The family Osmundaceae (R. Br.) Kaulf. Its geography and
taxonomy. Botanicheskii Zhurnal. 1967;52:1600–10.
Page 23 of 25
48. Hanks SL, Fairbrothers DE. A palynological investigation of three species of
Osmunda. Bull Torrey Bot Club. 1981;108:1–6.
49. Tryon RM. An Osmunda hybrid. Am Fern J. 1940;30:65–6.
50. Klekowksi EJ. Ferns and genetics. Bioscience. 1971;21:317–22.
51. Wagner WH, Wagner FS, Miller CN, Wagner DH. New observations on the
royal fern hybrid Osmunda × ruggii. Rhodora. 1978;80:92–106.
52. Kawakami SM, Kondo K, Kawakami S. Reticulate evolution of the hybrid
produced artificially by crosses between Osmunda banksiifolia and Osmunda
lancea. J Plant Res. 2010;123:639–44.
53. Petersen RL, Fairbrothers DE. North American Osmunda species: a serologic and
disc electrophoretic analysis of spore proteins. Am Midl Nat. 1971;85:437–57.
54. Stein DB, Thompson WF, Belford HS. Studies on DNA sequences in the
Osmundaceae. J Mol Evol. 1979;13:215–32.
55. Li J, Haufler CH. Phylogeny, biogeography, and population biology of
Osmunda species: insights from isozymes. Am Fern J. 1994;84:105–14.
56. Wiens JJ. The role of morphological data in phylogeny reconstruction. Syst
Biol. 2004;53:653–61.
57. Mendes MM, Grimm GW, Pais J, Friis EM. Fossil Kajanthus juncaliensis gen. et
sp. nov. from Portugal: Floral evidence for Early Cretaceous Lardizabalaceae
(Ranunculales, basal eudicots). Grana. 2014;53:283–301.
58. Denk T, Grimm GW. The biogeographic history of beech trees. Rev
Palaeobot Palynol. 2009;158:83–100.
59. Schneider H. Plant morphology as the cornerstone to the integration of
fossils and extant taxa in phylogenetic analyses. Species, Phylogeny and
Evolution. 2007;1:65–71.
60. Schneider H, Smith AR, Pryer KM. Is morphology really at odds with
molecules in estimating fern phylogeny? Syst Bot. 2009;34:455–75.
61. Augustsson C. Lapilli tuff as evidence of Early Jurassic Strombolian-type volcanism
in Scania, southern Sweden. GFF. 2001;123:23–8.
62. Bomfleur B, McLoughlin S, Vajda V. Fossilized nuclei and chromosomes
reveal 180 million years of genomic stasis in royal ferns. Science. 2014;343:1376–7.
63. Matsumoto M, Nishida H. Osmunda shimokawaensis sp. nov. and Osmunda
cinnamomea L. based on permineralized rhizomes from the Middle
Miocene of Shimokawa, Hokkaido, Japan. Paleontol Res. 2003;7:153–65.
64. Serbet R, Rothwell GW. Osmunda cinnamomea (Osmundaceae) in the Upper
Cretaceous of Western North America: Additional evidence for exceptional
species longevity among filicalean ferns. Int J Plant Sci. 1999;160:425–33.
65. Jud NA, Rothwell GW, Stockey RA. Todea from the Lower Cretaceous of
western North America: implications for the phylogeny, systematics, and
evolution of modern Osmundaceae. Am J Bot. 2008;95:330–9.
66. Carvalho MR, Wilf P, Hermsen EJ, Gandolfo MA, Cúneo NR, Johnson KR. First
record of Todea (Osmundaceae) in South America, from the early Eocene
paleorainforests of Laguna del Hunco (Patagonia, Argentina). Am J Bot.
67. Tsutsumi C, Hirayama Y, Kato M, Yatabe-Kukagawa Y, Zhang S-Z. Molecular
evidence on the origin of Osmunda × mildei (Osmundaceae). Am Fern J.
68. Tsutsumi C, Matsumoto S, Yatabe-Kukagawa Y, Hirayama Y, Kato M. A new
allotetraploid species of Osmunda (Osmundaceae). Syst Bot. 2011;30:836–44.
69. Rothwell GW, Nixon K. How does the inclusion of fossil data change our
conclusions about the phylogenetic history of the euphyllophytes? Int J
Plant Sci. 2006;167:737–49.
70. Rothwell GW, Stockey RA. Phylogeny and evolution of ferns: a paleontological
perspective. In: Ranker TH, Haufler CH, editors. Biology and Evolution of Ferns
and Lycophytes. Cambridge: University Press; 2008. p. 332–66.
71. Gould RE. Palaeosmunda, a new genus of siphonostelic osmundaceous trunks
from the Upper Permian of Queensland. Palaeontology. 1970;13:10–28.
72. Tidwell WD, Clifford HT. Three new species of Millerocaulis (Osmundaceae)
from Queensland, Australia. Aust Syst Bot. 1995;8:667–85.
73. Jefferson TH. The preservation of conifer wood: examples from the Lower
Cretaceous of Antarctica. Palaeontology. 1987;30:233–49.
74. Tidwell WD. The Osmundaceae – a very ancient group of ferns. In:
Dernbach U, Tidwell WD, editors. Secrets of Petrified Plants. Heppenheim:
D’Oro Publishers; 2002.
75. Chandler MEJ. The generic position of Osmundites dowkeri Carruthers. Bull
Br Mus Nat Hist. 1965;10:139–62.
76. Kvaček Z, Manum SB. Ferns in the Spitsbergen Palaeogene.
Palaeontographica B. 1993;230:169–81.
77. Cantrill DJ, Webb JA. A reappraisal of Phyllopteroides Medwell
(Osmundaceae) and its stratigraphic significance in the Lower Cretaceous of
eastern Australia. Alcheringa. 1987;11:59–85.
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
78. Balme BE. Fossil in situ spores and pollen grains: an annotated catalogue.
Rev Palaeobot Palynol. 1995;87:81–323.
79. Taylor TN, Taylor EL, Krings M. Paleobotany – The Biology and Evolution of
Fossil Plants. Burlington: Academic; 2009.
80. Maithy PK. A revision of the Lower Gondwana Sphenopteris from India.
Palaeobotanist. 1974;21:70–80.
81. Maithy PK. Dichotomopteris, a new type of fern frond from the Lower
Gondwana of India. Palaeobotanist. 1974;21:365–7.
82. Herbst R. Palaeophytologia Kurtziana. III. 7. Revision de las especies
Argentinas del Genero Cladophlebis. Ameghiniana. 1971;8:265–81.
83. Anderson JM, Anderson HM. Palaeoflora of southern Africa. Prodromus of
South African megafloras: Devonian to Lower Cretaceous. AA Balkema:
Rotterdam; 1985.
84. Hill RS, Truswell EM, McLoughlin S, Dettmann ME. Evolution of the
Australian flora. In: Orchard AE, editor. Flora of Australia. 2nd ed.
Collingwood: ABRS/CSIRO Australia; 1999. p. 251–320. vol. 1.
85. Collinson ME. Cainozoic ferns and their distribution. Brittonia. 2001;53:173–235.
86. Nathorst AG. Bidrag till Sveriges fossil flora. II Floran vid Höganäs och
Helsingborg. K Sven Vetensk Akad Handl. 1878;16:1–53.
87. Antevs E. Die liassische Flora des Hörsandsteins. K Sven Vetensk Akad Handl.
88. Johansson N. Die rhätische Flora der Kohlengruben bei Stabbarp und
Skromberga in Schonen. K Sven Vetensk Akad Handl. 1922;63:1–78.
89. Lundblad AB. Studies in the Rhaeto–Liassic floras of Sweden. I. Pteridophyta,
Pteridospermae and Cycadophyta from the mining district of NW Scania. K Sven
Vetensk Akad Handl. 1950;1:1–82.
90. Pott C, McLoughlin S. The Rhaetian flora of Rögla, northern Scania, Sweden.
Palaeontology. 2011;54:1025–51.
91. Tralau H. En palynologisk åldersbestämning av vulkanisk aktivitet i Skåne.
Fauna och flora. 1973;68:121–5.
92. Lund JJ. Rhaetic to lower Liassic palynology of the on-shore south-eastern
North Sea Basin. Danmarks Geol Unders, 2 Rekke. 1977;109:1–129.
93. Guy-Ohlson D. Jurassic palynology of the Vilhelmsfält bore no. 1, Scania, Sweden.
Toarcian–Aalenian. Swedish Museum of Natural History: Stockholm; 1986.
94. Lindström S, Erlström M. The late Rhaetian transgression in southern
Sweden: Regional (and global) recognition and relation to the Triassic–
Jurassic boundary. Palaeogeogr Palaeoclimatol Palaeoecol. 2006;241:339–72.
95. Larsson LM. Palynostratigraphy of the Triassic–Jurassic transition in southern
Sweden. GFF. 2009;131:147–63.
96. Nagalingum N, Drinnan AN, McLoughlin S, Lupia R. Patterns of fern
diversification in the Cretaceous of Australia. Rev Palaeobot Palynol.
97. Coiffard C, Gomez B, Thevenard F. Early Cretaceous angiosperm invasion of
western Europe and major environmental changes. Ann Bot. 2007;100:545–53.
98. Escapa IH, Bomfleur B, Cúneo NR, Scasso R. A new marattiaceous fern from
the Lower Jurassic of Patagonia (Argentina): the renaissance of Marattiopsis.
J Syst Palaeont. 2015. in press.
99. Stewart WN, Rothwell GW. Paleobotany and the Evolution of Plants.
Cambridge: Cambridge University Press; 1993.
100. Unger F. Ein fossiles Farnkraut aus der Ordnung der Osmundaceen nebst
vergleichenden Skizzen über den Bau des Farnstammes. Denkschriften der
Kaiserlichen Akademie der Wissenschaften. Mathematischnaturwissenschaftliche Klasse. 1854;10:137–51.
101. Miller CN. Osmunda wehrii, a new species based on petrified rhizomes from
the Miocene of Washington. Am J Bot. 1982;69:116–21.
102. Phipps CJ, Taylor TN, Taylor EL, Cúneo NR, Boucher LD, Yao X. Osmunda
(Osmundaceae) from the Triassic of Antarctica: an example of evolutionary
stasis. Am J Bot. 1998;85:888–95.
103. Vavrek MJ, Stockey RA, Rothwell GW. Osmunda vancouverensis sp. nov.
(Osmundaceae), permineralized fertile frond segments from the Lower
Cretaceous of British Columbia, Canada. Int J Plant Sci. 2006;167:631–7.
104. Krassilov VA. Mesozoic lycopods and ferns from the Bureja Basin.
Palaeontographica B. 1978;166:16–29.
105. Vera EI. Proposal to emend the genus Millerocaulis Erasmus ex Tidwell 1986
to recombine the genera Ashicaulis Tidwell 1994 and Millerocaulis Tidwell
emend. Tidwell 1994. Ameghiniana. 2008;45:693–8.
106. Zhang W, Zheng S-L. A new species of osmundaceous rhizome from Middle
Jurassic of Liaoning, China. Acta Palaeontol Sin. 1991;30:714–27.
107. Cheng Y-M. A new species of Ashicaulis (Osmundaceae) from the Mesozoic
of China: a close relative of living Osmunda claystoniana. Rev Palaeobot
Palynol. 2011;165:96–102.
Page 24 of 25
108. Tian N, Wang Y-D, Philippe M, Zhang W, Jiang Z-K, Li L-Q. A specialized new
species of Ashicaulis (Osmundaceae, Filicales) from the Jurassic of Liaoning,
NE China. J Plant Res. 2014;127:209–19.
109. Tian N, Wang Y-D, Zhang W, Jiang Z-K. A new structurally preserved fern rhizome
of Osmundaceae (Filicales) Ashicaulis wangii sp. nov. from the Jurassic of western
Liaoning and its significances for palaeobiogeography and evolution. Sci China
Earth Sci. 2014;57:671–81.
110. Tidwell WD, Munzing GE, Banks MR. Millerocaulis species (Osmundaceae)
from Tasmania, Australia. Palaeontographica B. 1991;223:91–105.
111. Bromfield K, Burrett CF, Leslie RA, Meffre S. Jurassic volcaniclastic – basaltic
andesite – dolerite sequence in Tasmania: new age constraints for fossil
plants from Lune River. Aust J Earth Sci. 2007;54:965–74.
112. Hennig W. Grundzüge einer Theorie der phylogenetischen Systematik.
Berlin: Dt. Zentralverlag; 1950.
113. Steeves TA. An interpretation of two forms of Osmunda cinnamomea.
Rhodora. 1959;61:223–30.
114. Werth CR, Haskins ML, Hulburt A. Osmunda cinnamomea forma frondosa at
Mountain Lake, Virginia. Am Fern J. 1985;75:128–32.
115. Kittredge EM. Notes on cinnamon ferns. Am Fern J. 1925;15:93–8.
116. Torrey J. Catalogue of Plants. In: Annual Report. Vol. 4. Albany:
Geological Survey of the State of New York, Botanical Department; 1840.
p. 113–97.
117. Hollick A. Abnormal growth in ferns. Bull Torrey Bot Club. 1882;9:129.
118. Murrill WA. Note on abnormal fruiting in the cinnamon fern. Am Fern J.
119. Day EH. Osmunda cinnamomea. L var frondosa. Bull Torrey Bot Club.
120. Chrysler MA. Abnormalities in Botrychium and certain other ferns. Bull Torrey
Bot Club. 1926;53:279–88.
121. Waters CE. A new form of Osmunda cinnamomea. Fern Bull. 1902;10:21–2.
122. McAvoy WA. A new combination in the fern genus Osmundastrum
(Osmundaceae). Novon. 2011;21:354–6.
123. Zhang S-Z, He Z-C, Fan C-R, Yan B. A cytogenetic study of five species in
the genus Osmunda. J Syst Evol. 2008;46:490–8.
124. Kato M. Hybrids in the fern genus Osmunda. Bull Natl Mus Nat Sci Ser B Bot.
125. Kato M. Distribution of Osmundaceae. Bull Natl Mus Nat Sci Ser B Bot.
126. Hörandl E. Neglecting evolution is bad taxonomy. Taxon. 2007;56(1):1–5.
127. Hörandl E, Stuessy TF. Paraphyletic groups as natural units of biological
classification. Taxon. 2010;59:1641–53.
128. Ahlberg A, Sivhed U, Erlström M. The Jurassic of Skåne, southern Sweden.
Geol Surv Denmark Greenl Bull. 2003;1:527–41.
129. Bergelin I. Jurassic volcanism in Skåne, southern Sweden, and its relation to
coeval regional and global events. GFF. 2009;131:161–75.
130. Kerp H, Bomfleur B. Photography of plant fossils—new techniques, old
tricks. Rev Palaeobot Palynol. 2011;166:117–51.
131. Huson DH, Bryant D. Application of phylogenetic networks in evolutionary
studies. Mol Biol Evol. 2006;23(2):254–67.
132. Spencer M, Davidson EA, Barbrook AC, Howe CJ. Phylogenetics of artificial
manuscripts. J Theor Biol. 2004;227:503–11.
133. Friis EM, Pedersen KR, von Balthazar M, Grimm GW, Crane PR. Monetianthus
mirus gen. et sp. nov., a nymphaealean flower from the early Cretaceous of
Portugal. Int J Plant Sci. 2009;170:1086–101.
134. Schlee M, Göker M, Grimm GW, Hemleben V. Genetic patterns in the
Lathyrus pannonicus complex (Fabaceae) reflect ecological differentiation
rather than biogeography and traditional subspecific division. Bot J Linn
Soc. 2011;165:402–21.
135. Grímsson F, Zetter R, Halbritter H, Grimm GW. Aponogeton pollen from the
Cretaceous and Paleogene of North America and West Greenland:
Implications for the origin and palaeobiogeography of the genus. Rev
Palaeobot Palynol. 2014;200:161–87.
136. Bryant D, Moulton V. NeighborNet: an agglomerative method for the
construction of planar phylogenetic networks. In: Guigó R, Gusfield D,
editors. Second International Workshop vol. 2452, WABI. Rome, Italy:
Springer Verlag, Berlin, Heidelberg, New York; 2002. p. 375–91.
137. Bryant D, Moulton V. Neighbor-Net: An agglomerative method for the
construction of phylogenetic networks. Mol Biol Evol. 2004;21(2):255–65.
138. Holland B, Moulton V. Consensus networks: A method for visualising
incompatibilities in collections of trees. In: Benson G, Page R, editors.
Algorithms in Bioinformatics: Third International Workshop, WABI, Budapest,
Bomfleur et al. BMC Evolutionary Biology (2015) 15:126
Page 25 of 25
Hungary Proceedings vol. 2812. Berlin, Heidelberg, Stuttgart: Springer Verlag;
2003. p. 165–76.
Grimm GW, Renner SS, Stamatakis A, Hemleben V. A nuclear ribosomal DNA
phylogeny of Acer inferred with maximum likelihood, splits graphs, and
motif analyses of 606 sequences. Evol Bioinforma. 2006;2:279–94.
Gascuel O. BIONJ: An improved version of the NJ algorithm based on a
simple model of sequence data. Mol Biol Evol. 1997;14(7):685–95.
Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (and Other
Methods) 4.0 Beta. Sunderland, MA: Sinauer Associates; 2002.
Müller KF. The efficiency of different search strategies for estimating
parsimony, jackknife, bootstrap, and Bremer support. BMC Evol Biol.
Stamatakis A. RAxML-VI-HPC: Maximum-Likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics.
Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the
RAxML web servers. Syst Biol. 2008;57:758–71.
Rodriguez F, Oliver JL, Marin A, Medina JR. The general stochastic model of
nucleotide substitution. J Theor Biol. 1990;142:485–501.
Lewis PO. A likelihood approach to estimating phylogeny from discrete
morphological character data. Syst Biol. 2001;50(6):913–25.
Berger SA, Stamatakis A. Accuracy of morphology-based phylogenetic fossil
placement under Maximum Likelihood. In: IEEE/ACS International Conference on
Computer Systems and Applications (AICCSA). Hammamet: IEEE; 2010. p. 1–9.
Berger SA, Krompass D, Stamatakis A. Performance, accuracy, and web
server for evolutionary placement of short sequence reads under Maximum
Likelihood. Syst Biol. 2011;60:291–302.
Maddison WP, Maddison DR. Mesquite: a modular system for evolutionary
analysis. Version 2.75; 2011. http://www.mesquiteproject.org. Accessed 12
Feb 2015.
von Balthazar M, Crane PR, Pedersen KR, Friis EM. New flowers of Laurales
from the Early Cretaceous (early to middle Albian) of eastern North America.
In: Wanntorp L, De Craene LPR, editors. Flowers on the Tree of Life.
Cambridge: Cambridge University Press; 2011. p. 49–87.
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