Sep 082016
 

One of the main problems with sampling largely from herbarium specimens, rather than from material that has been specifically collected for DNA work (rapidly dried in silica gel then maintained at low humidity), is that the quality of the DNA is unpredictable and usually rather poor. Therefore, despite starting out with 169 accessions and about 20 species of Marchantia, the actual successes, where we were able to get good quality DNA sequence data, were substantially lower. What we currently have is a slightly unbalanced data matrix, with 82 Marchantia accessions for rbcL, and 78 Marchantia accessions for psbA-trnH.

Reboulia hemisphaerica thallus, photographed by David Long (Long 34254)

Reboulia hemisphaerica thallus, photographed by David Long (Long 34254)

We also sequenced both rbcL and psbA-trnH from material of two accessions that we thought were Marchantia but where the sequences turned out to be Reboulia (from Texas) and Wiesnerella (from Bhutan). A quick check of the herbarium voucher specimens for both of these showed that they represented mixed collections of more than one complex thalloid species, for which the “wrong” plant parts had ended up in our silica dried tissue collection. Taking fortune from misfortune, both Reboulia and Wiesnerella form quite adequate outgroups for the phylogeny!

Wiesnerella denuda, photographed by David Long

Wiesnerella denuda thallus, photographed by David Long (Long 36267)

Out of the 20 species that we HAD hoped to sample, we ended up with only 12 named Marchantia species for rbcL (Marchantia polymorpha, M. paleacea, M. linearis, M. papillata, M. inflexa, M. emarginata, M. pinna, M. chenopoda, M. debilis, M. hartlessiana, M. quadrata and M. romanica), and 15 for psbA-trnH (Marchantia polymorpha, M. paleacea, M. linearis, M. papillata, M. inflexa, M. emarginata, M. pinna, M. chenopoda, M. debilis, M. globosa, M. pappeana, M. hartlessiana, M. subintegra, M. quadrata and M. romanica); we also had three Marchantia polymorpha subspecies (polymorpha, ruderalis and montivagans) and two Marchantia paleacea subspecies (paleacea and diptera).

That’s a little disappointing, representing, as it does, fewer than half of the 38 currently recognised species in the genus. However, we did also sequence a number of Marchantia accessions that had not been determined to species, and although many of them were good DNA matches to species that we had sampled, several are clearly different to everything else that we have included: one distinct lineage in Yunnan, China, another that occurs in Yunnan and Nepal, and a third in Indonesia and Malaysia. That’s balanced again by taxa that may not have been identified correctly; the psbA-trnH sequences from African material of M. debilis, M. globosa, M. pappeana and M. polymorpha, for example, are identical.

Intriguingly, in the “Preissia” clade, as well as M. romanica, there appear to be two lineages of Marchantia quadrata, one consisting of accessions from Denmark, Sweden and Sichuan, China, and the other with accessions from Svalbard, Norway and Utah, USA. These may tie in with subspecies quadrata (for the first lineage) and subspecies hyperborea (for the material from Svalbard and Utah), but the degree of genetic divergence is far higher than that found between many of the recognised species in Marchantia. It is a bit disconcerting, however, to notice that we have managed to overlook any Marchantia quadrata material from Scotland in our sampling!

The next step in the project, before it’s time to reveal any of the phylogenetic trees I’ve alluded to, is a phase of reciprocal illumination where we reconcile morphological information from the herbarium specimens with the information derived from the molecular sequence data. In other words, it’s time to double check our plant identifications, a part of the project that’s now in the capable hands of Dr David Long; the pile of Marchantia specimens is already on his desk!

 

 

Relevant posts

A rapid phylogeny of Marchantia, from the RBGE collections. I. Sampling

A rapid phylogeny of Marchantia, from the RBGE collections. II. Illuminating our sampling

Jun 302016
 

The genus Aitchisoniella contains a single species, A. himalayensis, which was described by Pakistani botanist Professor Shiv Ram Kashyap from plants that he collected in Mussoorie, Uttarakhand, India (1914). Subsequently (1929) he also found the species in Shimla and Kullu in Himachal Pradesh. Although reports of new locations followed (Kanwal, 1977, Pant et al., 1992, Bischler et al., 1994), near Nainital in Uttarakhand, the species was only known from the north-west Himalayas of India until 2010, when its range was extended by RBGE bryologist Dr David Long, on the ‘Kunming/Edinburgh Expedition to Sichuan’. David found Aitchisoniella at two localities in China, in Litang and Daocheng counties of south-west Sichuan Province.

Athalamia pinguis, Sichuan, photographed by David Long (Long # 40305)

Fig. 1. Athalamia pinguis in Sichuan, showing stalked sexual branches (carpocephala), photographed by David Long (Long # 40305)

Aitchisoniella looks quite different to other complex thalloid liverworts: it doesn’t have the stalked sexual branches (carpocephala) that are present in most complex thalloid species like Marchantia, or Athalamia (as can be seen in the photograph, Fig. 1). Instead, the female sex organs (archegonia), and therefore the sporophytes, grow on the lower (ventral) side of a short receptacle (as can be seen in the photograph, Fig. 2). The receptacle is part of the main thallus, with air chambers and air pores, and a groove on the underside of the thallus from which characteristicly complex-thalloid pegged rhizoids grow.

 Aitchisoniella himalayensis in Sichuan showing terminal sporophyte-bearing receptacles, from Long 39886

Fig. 2. Aitchisoniella himalayensis in Sichuan, showing terminal sporophyte-bearing receptacles, photographed by David Long (Long # 39886)

Originally, Aitchisoniella was thought to belong to the Exormothecaceae family of complex thalloid liverworts, along with Exormotheca and Stephensoniella. Earlier this year, we transferred all the plants in Exormothecaeae into another family, Corsiniaceae, where they joined Corsinia and Cronisia (Long et al. 2016a).

Complex thalloid phylogeny reconstructed by Villarreal, J.C., B.J. Crandall-Stotler, M.L. Hart, D.G. Long, L.L. Forrest. 2015. Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate. New Phytologist. DOI: 10.1111/nph.13716

Fig. 3. The phylogenetic position of Aitchisoniella, from the complex thalloid phylogeny reconstructed by Villarreal et al. (2015)

However, having fresh material of the plant meant that we were able to extract DNA from it, and add it into our molecular phylogeny for the group (Villarreal et al. 2015). The results were unexpected, with Aitchisoniella grouping with species from a different complex thalloid family, Cleveaceae (Fig. 3). The growth forms are quite different, as all the species in Cleveaceae have got stalked carpocephala. However, once we started to think more about the evolution of these plants, and look more objectively at differences and similarities, one thing struck us: the spores of Aitchisoniella (see Fig. 4) look more like the spores of plants in Cleveaceae (e.g. Athalamia, Fig. 5) than they do like spores of plants in Exormothecaceae (e.g. Fig. 6).

Spores of Aitchisoniella himalayensis. (A) Distal view; (B) proximal view; (C) lateral view; (D) detail of distal view. (A and C) from Long 39886. (B and D) from Long 40020. Scale bars: (A–C) = 5 μm, (D) = 2 μm.

Fig. 4. Spores of Aitchisoniella himalayensis from Long et al. 2016: (A) Distal view; (B) proximal view; (C) lateral view; (D) detail of distal view. (A & C) from Long 39886. (B & D) from Long 40020. Scale bars: (A–C) = 5 μm, (D) = 2 μm.

Athalamia hyalina spore images from M. P. Steinkamp and W. T. Doyle American Journal of Botany Vol. 68, No. 3 (Mar., 1981), pp. 395-401

Fig. 5. Spores of Athalamia hyalina (Cleveaceae) from Steinkamp & Doyle (1981): 1. distal view (x 1,100); 2. proximal view (x 1,100); 3. equatorial view (x 1,100); 4. close-up of pore (x 6,000); 5. distal face (x 3,000).

 

Exormotheca spores: E. bulbigena - A, distal view, B, proximal view. E. holstii - C, distal view, D, proximal view, E, distal view, F, proximal view. From Bornefeld et al., 1996.

Fig. 6. Spores of Exormotheca from Bornefeld et al. (1996): E. bulbigena – A, distal view, B, proximal view. E. holstii – C, distal view, D, proximal view, E, distal view, F, proximal view.

Characters like spore shape used to be considered to be quite neutral in bryophyte evolution, features that were not really acted on by natural selection. Following this theory, spore characters were thought to be indicative of true and ancient relationships, changing very little across huge amounts of time. We have since moved away from this view, with, for example, small changes in the shape and size of spores known to have drastic effects on their aerodynamics. However, within the complex thalloids, it does seem that characters like the presence or absence of carpocephalum branches are quite variable within families, while spore morphology can be indicative of deeper relationships.

As a result of this work, based on both molecular and morphological evidence, we have transferred the genus Aitchisoniella to the family Cleveaceae (Long et al. 2016b), where it joins the four genera accepted by Rubasinghe et al. (2011): Athalamia, Clevea, Peltolepis and Sauteria.

 

REFERENCES

Bischler, H., Boisselier-Dubayle, M.C. & Pant, G. 1994. On Aitchisoniella Kash. (Marchantiales). Cryptogamie. Bryologie-Lichénologie, 15: 103–10.

Borenfeld, T., O.H. Volk & R. Wolf. 1996. Exormotheca bulbigena sp. nov. (Hepaticae, Marchantiales) and its relation to E. holstii in southern Africa. Bothalia 26,2: 159–165.

Kanwal, H.S. 1977. Marchantiales of district Naini Tal (Kumaun Hills) U.P., India. Revue Bryologique et Lichénologique, 43: 327–38.

Kashyap, S.R. 1914. Morphological and biological notes on new and little known West-Himalayan Liverworts. I. New Phytologist, 13: 206–26. doi: 10.1111/j.1469-8137.1914.tb05751.x.

Kashyap, S.R. 1929. Liverworts of the Western Himalayas and the Panjab Plain. Part 1. Lahore: The University of the Panjab.

Long, D.G., L.L. Forrest, J.C. Villarreal & B.J. Crandall-Stotler. 2016a. Taxonomic changes in Marchantiaceae, Corsiniaceae and Cleveaceae (Marchantiidae, Marchantiophyta). Phytotaxa, 252: 77–80.

Long, D.G., L.L. Forrest, J.C. Villarreal & B.J. Crandall-Stotler. 2016b. The genus Aitchisoniella Kashyap (Marchantiopsida, Cleveaceae) new to China, and its taxonomic placement. Journal of Bryology.

Pant, G., S.D. Tewari & S. Joshi. 1992. An assessment of vanishing rare bryophytes in Kumaun Himalaya – thalloid liverworts. Bryological Times, 68/69: 8–10.

Rubasinghe, S.C.K., D.G. Long, R. Milne & L.L. Forrest. 2011. Realignment of the genera of Cleveaceae (Marchantiopsida, Marchantiidae). The Bryologist 114: 116-127. http://dx.doi.org/10.1639/0007-2745-114.1.116.

Steinkamp, M.P. & W.J. Doyle. 1981. Spore wall ultrastucture in the liverwort Athalamia hyalina. American Journal of Botany 68: 395-401.

 

Villarreal, J.C., B.J. Crandall-Stotler, M.L. Hart, D.G. Long & L.L. Forrest. 2015. Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate. New Phytologist. 209: 1734–46, doi: 10.1111/nph.13716.

 

Long, D.G., L.L. Forrest, J.C. Villarreal, B.J. Crandall-Stotler. 2016. The genus Aitchisoniella Kashyap (Marchantiopsida, Cleveaceae) new to China, and its taxonomic placement. Journal of Bryology.

Sep 182015
 

Sadly, although not surprisingly, I was not able to amplify the regions of Monocarpus DNA needed to compare it to other complex thalloid liverworts from a 1950s collection that we had been sent by our Australian colleagues.

Monocarpus specimen collected by Carr, photographed by Chris Cargill

Monocarpus specimen collected by Carr, photographed by Chris Cargill

However, this was not the end of the story. Just over a year later, in September 2009, we received another email from Australian National Herbarium’s Chris Cargill, who had just attended the Australasian Bryophyte Workshop in Western Australia.

There, Chris met up with Pina Milne and Helen Jolley, who had travelled to WA the previous week before to do some collecting north of Perth and had actually found living Monocarpus! Even better for us, they were happy to send us some of the recent collection for DNA sequencing. 

Complex thalloid liverwort Monocarpus sphaerocarpos, photographed by Pina Milne

Complex thalloid liverwort Monocarpus sphaerocarpos, photographed by Pina Milne

The specimens were sent back from PERTH herbarium at the end of October, and reached Edinburgh in time for the DNA to be extracted before the year end. Successful sequence data was generated for several of the regions that we tried to amplify, allowing us to finally place Monocarpus sphaerocarpus in the liverwort tree of life. (To be continued…)

Monocarpus photographed by Pina Milne

Complex thalloid liverwort Monocarpus sphaerocarpus, photographed by Pina Milne

 

Monocarpus heading

 

 

On Monocarpus – http://stories.rbge.org.uk/archives/17112

Finding Monocarpus, in the Herbarium – http://stories.rbge.org.uk/archives/17146

Finding Monocarpus, in the field – http://stories.rbge.org.uk/archives/17272

Lost before found: Was there more than one species in Monocarpus? – http://stories.rbge.org.uk/archives/17904