Genetic distances and phylogeography of selected disjunct moss populations in Europe

Disjunct ranges are a common phenomenon in bryophytes. To test, whether these disjunctions are the result of the former continuous ranges (and therefore relicts) or the result of long-distance dispersal (and therefore result of spreading), five moss species were chosen for a case study: Campylopus oerstedianus, Hilpertia velenovskyi, Isothecium holtii, Rhytidium rugosum and Dichelyma capillaceum. Also, the problem of genetic diversity in space and time within the species morphologically not very variable has been studied. For the determination of genetic distances between the populations, the variation of the nuclear ribosomal non coding DNA region of the Internal Transcribed Spacer (ITS) and the trnL-F region of the chloroplast DNA was used. Campylopus oerstedianus has a scattered circum tethyan distribution ranging from Costa Rica to Georgia, the Pyrenees, the southern Alps and northern Greece. It is worldwide sterile and can propagate only vegetatively. In addition it is found in the Massif Central and the Vosges, in France. The molecular results showed that the populations in the Pyrenees, southern Alps and Greece have no genetic exchange and are possibly separated for long time. The disjunct populations in France are clustering with the population in the Pyrenees and are therefore possibly derived from the latter. The Greek population is highly isolated and genetically unrelated to the other European populations of C. oerstedianus, which counts for a very long isolation. Hilpertia velenovskyi is mainly a species of cold loess steppes in inner Asia. It is also found in Europe on loess cliffs from southern Poland to northern Serbia. It was recently discovered in Germany (Saxony and Rhinehessia). The molecular tree shows that Saxonian population is related to the Czech and both cluster with all other European populations except that from Rhinehessia. The latter is totally isolated also from a Chinese one which means that it has spread from somewhere else, probably from some populations which were not included in the study, or it has to be interpreted as an result of long lasting isolation since the last Ice Age. The continuity of the habitat (loess cliff), however, support this hypothesis. It would be the first molecular proof of a glacial relict among bryophytes. Isothecium holtii has been regarded either as good species, dubious species, or as a variety of either I. alopecuroides or I. myosuroides. Its distribution range includes the British Isles, western France and western Norway, with disjunct occurrences in Central Europe and Turkey. A molecular study was designed to clarify the taxonomic position of this taxon as well as to assess whether the disjunct populations in Germany are a result of recent migration or whether they are relicts of a wider distribution in more humid climatic periods. The analysis of nuclear ribosomal DNA sequences (ITS) revealed that Isothecium holtii is a good species, and that it is closer related to I. myosuroides than to I. alopecuroides. The disjunct populations in Germany are heterogenous. Two from the Harz Mts. cluster with different populations in Western Europe, indicating independent dispersal events, whereas another from the Eifel Mts. is genetically isolated, indicating a long separation and a relict. The populations in Brittany and the Massif Central in France are genetically related but rooted with the populations from Britain and Ireland and indicate that they are relicts of a former continuous range. The boreal moss Rhytidium rugosum, today widely but scattered spread over Europe, is considered to be a glacial relict of its wide but scattered holarctic range. Due to molecular data, it survived glaciation in southern European refugia, but also in some regions of central and western Europe. According to haplotypes distinguished in Europe, the species range is a result of migrations, range extension, colonization and spreading events. However, some relic haplotypes remain within some regions of Europe. The species extremely rarely produces spores and spreads mainly vegetatively. The geographical regions of higher genetic diversity among populations can be the regions where some relic haplotypes survived and afterwards were settled by some other haplotypes during migrations, as well. South-western and western Germany is determined as one of these region in Europe. According to molecular data sampled from selected European, American and Asian populations, a high genetic diversity of this species is present although it is mostly sterile. Dichelyma capillaceum is a rare and protected pleurocarpous moss from riparian habitats with scattered amphi-atlantic distribution. The European center of its distribution is concentrated within Scandinavia with 85 populations mostly in Sweden. Outside Scandinavia two further population are still present in Europe (in W. Germany and NW France). It is sterile although there are a few rare old records with sex organs or sporophyte from herbaria. The German population of D. capillaceum is genetically long time separated but originated from some ancestor from Scandinavia. According to the genetical data obtained it is rather result of relatively recent long distance dispersal event than of relict origin. Due to bio-geographical and molecular data, it can be assumed that the Scandinavian populations derived from North American. This can be proved by the fact that genetic variation among European populations studied is lower compared to all studied populations, which means a longer existence of American populations. It is furthermore is supported by the fact that atlantic North America is centre of species diversity of the genus Dichelyma. From the molecular data obtained, from the dispersal strategies of the species and from the geographical interpretations, the following conclusions can be drawn: 1. Both, long distance dispersal and relicts are responsible for the present distribution of the species studied. 2. The basal position of the southern and western European populations in species like Campylopus oerstedianus and Isothecium holtii supports the refugial hypothesis during the last Ice Age, from which the species established populations in Central Europe by long distance dispersal. The site of Dichelyma capillaceum in Germany has originated from the Scandinavian population by long distance dispersl, too. 3. In case of Hilpertia velenovskyi and Rhytidium rugosum, persistence during and since the last Ice Age in Central Europe can be postulated. 4. Long-distance dispersal is possible in the species studied, although they are sterile at present and lack specialized brood bodies. It can only be explained by the fact that fragments of plants can serve even for long distance dispersal

Back to Hungary: a story on reintroduction of a European Habitat Directive moss species, Hamatocaulis vernicosus

Hamatocaulis vernicosus is rare and threatened species Europe-wide, and it is a member of many national red lists. The main reason for this can be regarded as habitat quality changes. The last records from Hungary comes from 1968, and in years after despite of intensive search it was not found in any historically known habitats. Thus, it was considered as extinct in Hungary. An attempt on getting back this species to Hungary has been made. Less than one year old herbarium specimens from neighboring countries have been used to establish new population but with limited success. The same material that could be revived has been used to start axenix in vitro culture. Once the culture has been established, the optimization and propagation started. The lab originated material was grown under controlled condition but in xenic medium originated from Hungary. Finally, after two years the moss plants were reintroduced to two national parks in Hungary and two populations were established. This is just the first report but there are continuous attempts to stabilize the populations. The problems, achievements and solutions will be discussed. A Hamatocaulis vernicosus, egy ritka és veszélyeztetett faj Európában és számos nemzeti vörös listán is szerepel. A fő veszélyeztető tényező az élőhelyeinek, a lápréteknek, a visszaszorulása, illetve leromlása. Az utolsó magyarországi record 1968-ból származik és bár az utóbbi években a közösségi jelentőségű fajok monitorozása intenzíven folyik, a Hamatocaulis vernicosus nem került elő egyetlen korábban ismert élőhelyéről sem. Így Magyarországról kipusztultnak tekinthető. Ennek a fajnak a visszatelepítésére tettünk kísérletet. Szomszédos országokból származó, egy éven belül gyűjtött herbáriumi példányokat próbáltunk meg felhasználni új populáció létrehozására, de ez csak korlátozott eredménnyel járt. Az egyik ilyen herbáriumi anyagból azonban sikerült in vitro kultúrát létrehozni és felszaporítani. A laborban kontrollált körülmények között tartott kultúrát magyarországi lápokról származó médiumon neveltük. Végül, két év után visszatelepítést végeztünk két nemzeti parkban és így két lápon sikerült egy-egy populációt létrehozni. A populációk stabilizálásán folyamatosan dolgozunk, nyomonkövetjük a változásokat. Bemutatásra kerülnek az elért eredmények, a felmerült problémák és lehetséges megoldások

Gibberellin influence on the morphogenesis of the moss Bryum argenteum Hedw. in in vitro conditions

The moss Bryum argenteum Hedw. was treated with gibberellins as well as some inhibitors of gibberellin biosynthesis in order to investigate their influence on B. argenteum morphogenesis. Generally, gibberellins have not been chemically identified in bryophytes, while other groups of classical phytohormones (auxins, cytokinins, abscisic acid and ethylene) have been chemically identified in these plants. The in vitro culture of the moss Bryum argenteum was established from sterilized spores. The apical shoots of untreated gametophytes grown in vitro were used to investigate the influence of different substances on secondary protonema and on the growth and multiplication of the gametophytes. B. argenteum reacts differently to the growth regulators applied. Both gibberellins applied in vitro (GA3 and GA7) have a positive effect on B. argenteum morphogenesis. Shoot multiplication was negatively affected by three tested growth retardants (ancymidol, BX-112 and chlorocholine chloride), while these substances did not have such strong effects on the moss protonema development.Projekat ministarstva br. 143031 i 14301

In vitro culture and apogamy: Alternative pathway in the life cycle of the moss Amblystegium serpens (Amblystegiaceae)

In vitro culture of the moss Amblystegium serpens (Amblystegiaceae) was established on hormone-free Murashige and Skoog (MS) medium that contained a half amount of MS micro- and macro- mineral salts and vitamins, 100 mg/l myoinositol, 30 g/l sucrose, and 0.70% (w/v) agar. Spores were germinated and primary protonema developed on the above medium at 16 h day/8 h night 25±2ºC, 60-70% air humidity, and irradiance of 47 μmol/m2s. Three months after development of primary protonema, seven sporophytes appeared directly from primary protonema without generation alternation. The phenomenon of apogamous sporophyte formation is very rare, both in nature and under in vitro conditions. This is the first report of apogamy induced by Amblystegium serpens

Effects of selected bryophyte species extracts on microorganisms

Two dozen of bryophyte species was collected from native habitat types across Europe to test if the ethanol extract can affect the growth of various microorganisms. After start-up test the ethanol extract did not show any influence on growth and development to Gram-negative bacteria and fungi Candida albicans (ATCC10231). Thus, the further tests focused on the Gram-positive bactreia. The ethanol extract of 5g bryophyte material was macerated in liquid nitrogen, and then extract was evaporated till dry. The dry residue was dissolved in 5ml of methanol. This was used in further analyses by MIC and MBC techniques. The result obtained showed the most effective extracts were those made from Pellia endiviifolia and Bazzania trilobata. Phytopathogenic bacteria and fungi C. albicans showed resistance to extracts of any bryophyte species tested. Gram-positive bacterias, namely Staphylococcus aureus (ATCC25923), Listeria monocytogenes (ATCC19111) and Bacilus subtilis (ATCC6633) were intolerant to extracts of liverworts P. endiviifolia and B. trilobata. MIC value for two above mentioned liverworts extract tested were 7-12mm, while referent antibiotic (rifampicin) inhibitory zone were 15-35mm. The most resistant to any bryophyte extracts was Escherichia coli (ATCC25922), while the most sensitive was B. subtilis (ATCC6633) with MIC values obtained 0.01-0.19 mg/ml. The results obtained show clear antimicrobial potential of P. endiviifolia and B. trilobata, however further research are needed

Effects of different cytokinins on chlorophyll retention in the moss Bryum argenteum (Bryaceae)

Background and Purpose: Cytokinins are a group of plant hormones that have an important role in plant growth and developmental processes. Chlorophyll content is an extremely important parameter in estimating the plant production level. Since bryophytes do not have such economical importance as vascular plants and their production in many ecosystems is small, they remain uninteresting for studying their chlorophyll level. The aim of this study was to compare the effect of different cytokinins on chlorophyll retention in moss B. argeteum gametophyte shoots grown in natural conditions with those grown in in vitro culture. Material and Methods: The effect of different cytokinins: kinetin (KIN), 6-benzylaminopurine (BAP) and thidiazuron (TDZ) on chlorophyll retention of the moss Bryum argenteum Hedw. (Bryaceae) derived from in vitro culture or grown in nature was tested. Plants derived from in vitro culture were grown on Murashige and Skoog (MS) medium at 25±2°C. Gametophyte shoots were used in experiments where influence of different concentrations (0.001–10 μM) of three cytokinins was used to investigate their effect on chlorophyll-a, -b and total chlorophyll retention. Results and Conclusions: Cytokinins had a positive but unequal influence on chlorophyll retention in both plant groups – plants derived from in vitro culture and plants grown in the nature. Kinetin proved to be the most effective cytokinin in chlorophyll retention. Exogenous application of kinetin increased chlorophyll content with concentration (0–10μM). BAP had similar trends in in vitro and native mosses, increasing chlorophyll content up to 1 μM and then significantly decreasing, although the chlorophyll content was greater in in vitro grown plants. TDZ showed significantly better effect in in vitro cultured moss shoots, but when applied in concentrations higher than 0.1 μM, total chlorophyll content decreased