Abstract:
The origin of angiosperms has been a focus of botanical research.
Although macrofossils 1,2 and microfossils3,4 have suggested that angiosperms may have earlier
occurrence, there are some scholars reluctant to accept these fossil
records and their implications 5. To resolve this
conflict studying more fossils using new technology is helpful. Obvious
differences exist between pollen of seed plants and spore of ferns.
Applying the MFCFM technology, here we report a half pollen grain with a
mosaic combination of fluorescent spectral features of angiosperms and
ferns from the Jurassic of China. Although the mother plant is still
mysterious, the features of this fossil pollen grain apparently demands
a new thinking on pollen and its evolution.
Keyword: ferns, angiosperm, pollen, fluorescence, spectrum
Pollen grains of seed plants can be easily distinguished from the spores
of ferns in various aspects. Hitherto there is nothing intermediate
between these two categories of microspores in terms of wall
organization and fluorescence features, although such an intermediate,
if existed, would provide otherwise unavailable meaningful implications
for plant evolution. Here we report a half pollen grain from the
Jurassic of Inner Mongolia, China. The MFCFM (Multiple Frequency
Confocal Fluorescence Microscopy)6 reveals that 1)
fluorescence spectrum of this pollen grain has two peaks, one resembling
spore and the other resembling pollen; 2) its separated tectum and
supratectal spines resemble certain angiosperms. Such an inter-class
combination of features reveals a previously unknown aspect of plant
evolution.
The fossil was collected from the outcrop of the Jiulongshan Formation
(the Middle-Late Jurassic, >164 Ma) near Daohugou, Inner
Mongolia, China (Fig. S1). The sample was removed from an unidentified
fossil plant (Fig. S2a-c). Details of the pollen grain were observed and
photographed using a Leo 1530 VP SEM and a Tescan MAIA3 SEM at the
Nanjing Institute of Geology and Palaeontology. MFCFM observation was
performed using Olympus FV3000 laser scanning microscope at the Shanghai
Normal University. For comparison, Brassica juncea, Oryza sativa,
Actinidia sinensis, Pinus kwangtungensis and Cryptomeria
fortunei were selected as representatives of seed plants, andArachniodes, Ceratopteris and Lycopodium clavatum were
selected as representatives of spore plants. Lycopodium clavatumwas procured from Sigma (19108-100G-F). Other pollen and spores were
collected from the Botanical Garden of Shanghai Normal University for
MFCFM. We used fluorescence with wavelength of 405 nm for excitation,
and the bandwidth was set to 5 nm. Fluorescence of total 53 frequencies
ranging from 430 to 690 nm was used. To eliminate the noise from the
background, the signal of the background (sediment matrix) was
subtracted from that of the pollen grain. All photographs were saved in
TIFF format and organized together for publication using a Photoshop
7.0.
The pollen was embedded in siltstone of volcanic ashes (Figs. S2a-c). By
accident, the pollen grain was split in halves (Figs. 1a-b), and only
one half was used in this analysis and the wall organization was
observed using both SEM (Figs. 1a-d. g-h) and MFCFM (Figs. 1e-f). The
pollen grain is close to round in outline, 22 x 19 μm, uniaperturate.
The wall includes a foot layer, columella, and separated tectum (Figs.
1a-h). The foot layer is present in both aperturate and non-aperturate
regions, with smooth inner surface, slightly heterogeneous, 0.3-0.7 μm
thick (Figs. 1c-e,g). The columella is poorly developed only locally,
ranging from nil to 1.25 μm (Figs. 1b,g). The tectum is approximately
0.25 μm thick, separated from the foot layer, with its inner surface
visible in region where the neighboring foot layer is missing,
well-separated from the foot layer in the margin of aperture, with
supratectal spines (Figs. 1b-h). The foot layer and tectum are
well-separated in MFCFM image (Fig. 1e), similar to the pollen wall ofOryza sativa (Fig. 1i). The fluorescence spectrum of the pollen
grain has two peaks, namely at 535 nm and 495 nm, respectively,
different from both spore of ferns and pollen of seed plants (Figs.
2a-b).
The fluorescence spectrum of the present pollen grain combines the
features of both ferns’ spore and spermatophytes’ pollen. The
observation on extant plant taxa indicates that fluorescence spectra of
spores have their peaks in 510-545 nm, while those of pollen in 475-510
nm. As seen in Fig. 2, the fluorescence spectrum of the present pollen
grain has two peaks, namely 535 nm and 495 nm, respectively. One of two
peaks matches that of ferns’ spore and the other matches that of pollen
in seed plants. This unique spectral feature is perplexing if
consideration is restricted to extant plants. However, it becomes much
easy to accept and understand if fossil plants are included
consideration. There are fossil taxa termed as “seed ferns”, which
appear like ferns but are reproduced through seeds. Our new pollen seems
to be of another taxon that appears weird, chimeric, spanning formerly
isolated taxa in eyes of botanists who are more familiar to living taxa.
Wide morphological and molecular gap among living taxa is conceivable
because the intermediate ones have been deleted from the phylogeny tree.
We assumed our fossil pollen will help to narrows the otherwise
wide-remaining gap between ferns and spermatophytes.
Among the known spermatophytes, the present pollen grain appears more
similar to certain angiosperms in wall organization. Interestingly, the
well-separated tectum and foot layer seen in Fig. 1e are never seen in
any gymnosperms, while frequently seen in some Poaceae (Oryza
sativa ) (Figs. 1i, S3), suggestive of possible angiosperm affinity. The
supratectal spines on the tectum is another feature frequently seen in
some angiosperms. Taking all into consideration, pollen wall
organization of the present pollen grain favours that its mother plant
is most likely an angiosperm, which we think is an open question
requiring further investigation.
MFCFM oservation reveals that the Jurassic pollen grain reported here is
unique in its fluorescence spectrum resembling microspores of both ferns
and seed plants. The pollen wall organization demonstrates similarity to
that of some Poaceae. The implications of this new fossil material need
further exploration.
Acknowledgement We thank Mr. Yan Fang for his assistance with
the SEM. This research was supported by the Strategic Priority Research
Program (B) of Chinese Academy of Sciences (Grant No. XDB26000000), and
National Natural Science Foundation of China (41688103).