Megaceros is a genus of hornwort found within the moister areas of Australia and neighbouring regions. Five species were recognised for Australia by 1916, but few taxonomic studies have since been performed to verify this finding. In this study, modern techniques, including scanning electron microscopy of the spores and DNA sequencing of genetic markers were used to investigate the diversity of Megaceros within Australia. Four distinct species were identified from spore morphological data, including one previously unlisted for Australia.

The hornworts (Division Anthocerophyta) are amongst the oldest extant land plant taxa. Distinguished from the other bryophyte groups by their horn-like sporophyte, the hornworts are a relatively small group, with only 100 to 150 species worldwide. Classification has been controversial both at the species and generic levels, with anywhere between 5 and 12 genera recognised. Clarification of the taxonomic boundaries and clear species definitions are required before the biogeography, ecology and phylogeny of this unique group can be fully understood.

Megacerosis a hornwort genus distinguished from other taxa by a number of features including, its green spores, pseudoelaters with spiral thickenings, sporophyte capsules that lack stomata and multiple chloroplasts per cell that lack pyrenoids. Typically found growing on rocks and soil banks in streams, Megaceros populations are spread throughout the moister areas of Australia, with an eastern distribution ranging from northern Queensland down to Tasmania. As a genus, Megaceros was first described by Campbell in 1907 and by 1916, Stephani had recognised 43 species worldwide, including five present within Australia. These included four species unique to Australia (M. gracilis, M. longispirus, M. carnosus and M. crassus) and one species originally named for New Zealand, M. denticulatus. However, recent authors have emphasised the need for revision of this genus in Australia, (Scott, 1985) following studies investigating the taxonomy of Megaceros in nearby regions (Hasegawa, 1983). Thus, the aim of this project was to determine which species of Megaceros are represented in Australian populations. Both a molecular and morphological approach was taken for this investigation.

Mature air-dried spores were removed from the capsules of Megaceros herbarium specimens originating from a variety of Australian populations and type specimens from Australia and the surrounding regions. The spores were mounted on aluminium plates using double-sided sticky tape and gold coated before viewing under a scanning electron microscope. Micrographs of both the proximal and distal spore surfaces were recorded and later analysed. Spore preparations were also viewed under a light microscope to record colour information and measure spore diameters.

DNA was extracted from the growing tips of gametophyte tissue or sporophyte capsules of both live plants and herbarium specimens less then two years old. Universal primers were used in a polymerase chain reaction to amplify the following regions of the chloroplast genome; the trnL intron; trnL-trnF intergenic spacer, trnT-trnL intergenic spacer, rpL16 intron, psbA-trnH intergenic spacer and the atpB-rbcL intergenic spacer. If amplification was successful, DNA sequences were obtained from the resulting PCR product.

Morphologically, analysis of spore ornamentation patterns has been a powerful tool used for differentiating taxa within the Anthocerophyta. Scanning electron micrographs of spores from Australian Megaceros populations revealed the presence of four distinct morphological types, each representing a different species. The distal spore surface often contained the most informative characters for differentiating between taxa at the species level, whilst relatively little variation was present on the proximal surface. The dominance of each species changes from the northern to the southern parts of Australia, with some overlap in their geographic distributions.

Found in the tropics of northern Queensland, M. flagellaris represents the most northerly Australian Megaceros species. Previously unrecorded for Australia, M. flagellaris was originally described for Fiji and has since been found in other areas of the Pacific and South-east Asia, including Japan (Hasegawa, 1983). M. flagellaris is characterised by its unique reticulate patterning around the equatorial region of the distal surface and the small size of its spores, which range in diameter from 20-25 m m. It is here recognised for the first time and recorded for Australia. Also of taxonomic significance is the large, round papillae on the proximal surface, which are clustered towards the centre of each triradiate face.

Spread over a relatively large geographic area, ranging from the mountainous areas of Queensland, through to NSW and Victoria, Megaceros populations with relatively simple spore ornamentations were present. The distal surface of such spores either displayed a ridge-like pattern or irregular globules across the equatorial region. Papillae present on the proximal face are relatively irregular in shape and are smaller and more spread out then those in M. flagellaris. The spores of this species are quite large, ranging from 26-29 m m in diameter. No type specimen with matching spore ornamentation could be found, implying that this could either represent an as yet unnamed species, or M. gracilis, the only Australian species for which a type specimen was unavailable for comparison.

With a relatively limited Australian distribution, M. denticulatus populations were found only within the south-eastern corner of Victoria. Characterised by the large tubercules that are evenly distributed around the distal spore surface, M. denticulatus has a distinct morphology that separates it from the two species located further north. The size of M. denticulatus spores are also slightly bigger then that of the aforementioned species, ranging from 27-32 m m in diameter. Although previously listed for Australia, M. denticulatus was originally named for New Zealand and appears to be restricted to the cooler, southerly regions.

Ranging from New South Wales and Victoria, down to Tasmania, M. longispirus represents the southernmost Megaceros species in Australia. Superficially, this species is similar to M. denticulatus in that the distal surface is ornamented with large, globose tubercules. However, in M. longispirus, these tubercules are restricted to the equatorial region and central mamilla, and do not extend into the intervening papillate area. The spores of M. longispirus range from 27-33m m in diameter, continuing the trend of increasing spore size with species located further south. Originally named for Tasmania, M. longispirus spores bear a similar ornamentation pattern to those of two species previously named for southern Australia, M. carnosus and M. crassus. Spores removed form the type specimens of these species are also of similar size to M. longispirus, ranging from 24-29 m m and 26-30m m in diameter for M. carnosus and M. crassus, respectively. The high similarity in spore ornamentation, size and distribution of these species suggest that these are conspecific and may be synonymised to M. longispirus.

Of the six regions within the chloroplast genome that were targeted for amplification, PCR products could only be obtained for the trnL intron and trnL-trnF intergenic spacer. However, DNA sequence analysis of these regions revealed less then 1% divergence between taxa and thus this data could not be used for analysing differences at the species level. The limited success in amplification of the four remaining regions may be due to the use of universal primers during the PCR, which were not specifically designed for hornworts, despite successful amplification of the corresponding regions in the liverwort, Marchantia polymorphia.

Through analysis of spore morphology at the SEM level, this study has shown that at least four species of Megaceros are present within Australian populations, M. flagellaris, M. denticulatus, M. longispirus and an as yet unidentified species. Close scrutiny of the spore ornamentation of M. carnosus and M. crassus type specimens suggest that these species are synonymous to M. longispirus. Molecular data obtained in future studies is likely to provide better resolution of species level differences.

Much gratitude is expressed towards Christine Cargill (supervisor), for all the encouragement, enthusiasm and support provided during this project. Many thanks also to all the staff at the Australian National Botanic Gardens, CSIRO Microscopy Centre and Australian National Herbarium and in particular the Molecular systematics group for all their technical input. Thanks to the Centre for Plant Biodiversity Research for the opportunity to be a part of the summer scholarship program.

Campbell, D. H. (1907) Studies on some Javanese Anthocerotaceae. Annals of Botany. 21(84): 467-491.

Hasegawa, J. (1983) Taxonomic studies on Asian Anthocerotae III. Asian species of Megaceros. Journ. Hattori. Lab. 54: 227-240.

Kugita, M; Kaneko, A; Yamamoto, Y; Takeya, Y, Matsumoto, T; Yoshinaga, K. (2003) The complete nucleotide sequence of the hornwort (Anthoceros formosae) chloroplast genome: insight into the earliest land plants. Nucleic Acids Research. 31(2): 716-721.

Scott, G. A. M. (1985) Southern Australian Liverworts. Australian Government Publishing Services, Canberra. pp 34-35.