Phalaris collecting summary, Czech Republic, 2010

During summer and fall of 2010, I completed an extensive collection of reed canarygrass (Phalaris arundinacea) along the six major rivers in the Czech Republic. This material will be assessed for genetic variation along with commercial cultivars. The specific research questions being asked are:
A) Does genetic variation differ along the CR rivers (Labe, Orlice, Vltava, Berounka, Lužnice, Dyje)?
B) Does genetic variation within populations differ along the river banks vs. inland from the river edges?

All of these rivers, with the exception of the Dyje, feed into the North Sea basin. The Dyje River runs into the Black Sea basin. There may be differences between these rivers that feed into the basins, although we do not have enough Black Sea basin rivers sampled to make this determination. Genetic variation assessment consisted of phenotypic observations of plants and populations and the use of inter-simple sequence repeats (ISSRs) to assess nuclear DNA genetic (amino acid sequence) variation (being done now).

Some of the river views, while collecting include:

The Dyje River which runs along the Czech Republic and Austrian borders:
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Who knows the parasitic species growing in the trees (right-hand side of the following picture)?

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Views along the Berounka River, near Praha-Radotinu:

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Scenes along the Labe River (which becomes the Elba River in Germany):

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Frosted Urtica (nettle) and Phalaris (reed canarygrass) leaves that morning:
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A scene from the Tame Orlice River in East Bohemia:

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And then, here's the Wild Orlice River, East Bohemia:

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Then, here's the scene below the confluence of the Wild and Tame Orlice Rivers:

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Pictures from a few collecting locations along the Vltava River:

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And who can identify the red-leafed plant growing along with the reed canarygrass??

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Initial results from our N. American reed canarygrass ISSR analysis, particularly that most of the genetic variation is within rather than among N. American populations, are being contrasted with Czech populations. Higher levels of variation within N. American populations caused us to expand from our initial proposal of collecting only 10 plants/population and 5 populations for each of the 5 major rivers. In a meeting with my hosts in July 2010 after my arrival, we expanded this to 6 rivers and sampling every 30 km along each river within the country, including above and below confluences. This was due to a new paper that had just been published that noted a distance of ~30 km separated river populations for different species.
At each sampling site, we collected from at least 5 locations along the river edges each of which were 100 m apart. At each site, as many as 15-18 samples were collected along the rivers (Table 1). In addition, we also included perpendicular transects within each site to determine whether the genetic variation along the rivers was the same or different from the populations extending back from the water's edge. At each site, if plants existed away from the river edges, we collected 1-3 transects with as many as 4-39 individuals for analyses (Table 1). Our total collections from the rivers, encompassed by ~13 000 km of driving around the country on multiple trips (July - November, 2010), totaled 30 sites within the six rivers and 155 locations at these sites for a total of 465 genotypes collected along all rivers (Table 1). At these locations, a total of 38 transects perpendicular to the rivers' edges were run and a total of 370 genotypes were collected (Table 1). Thus, our grand total of genotypes for Czech rivers is 835 genotypes--a very large sampling which will make our research papers and inferences very powerful.

Table 1.docx

In addition to these wild population collections in the Czech Republic, we collected large population transects in the wet meadows (two perpendicular and intersecting transects through the middle of each population) near some of the major historic fish ponds in the Trebon basin (Table 2). We also sampled along the Golden Canal (Zlata Stoka), using the same collection technique for the rivers (Table 1). The Golden Canal (Sites No. 12-13) feeds fresh water from the Lužnice River into all of these ponds. We hypothesized that the Golden Canal could be a major vector to spread reed canarygrass propagules (vegetative) through the fish pond system in Trebon. At Site No. 11, Rzmberk Pond, we collected two sets of perpendicular transects, one set each in the unmowed and mowed sections of Mokre Louky wet meadow. The unmowed wet meadow is a historic section of reed canarygrass that has been extensively studied by two of my hosts, Drs. Jan Květ and Keith Edwards; it is know to have a decreasing nutrient gradient across the site. The mowed wet meadow is harvested several times/year and used for biofuel production by Lane Aurora spa nearby. We theorized that the mowed and unmowed sections might be genetically divergent due to mowing. Genetic variation here may be important first steps in determining if there are differences between the mowed (cultivated) and unmowed (wild) populations and specific genotypes and whether the latter could be bred and selected for non-invasive reed canary grass cultivars for biofuel production. In 2011, I anticipate visiting the biofuel plants for Lazne Aurora as well as discussing future breeding directions with the Faculty of Agriculture, in particular Prof. Ing. Stanislav Kužel CSc.

Other collection sites for wild populations surfaced as we traveled around the country, including Site No. 10 (Rzmberk Pond; Table 2), which is where our University of Minnesota graduate student, Gina Quiram, sampled purple loosestrife populations, Lythrum salicaria, in 2008. It is also the site where Dr. Jan Květ has conducted numerous ecological studies for many decades. The historic salt marsh, the Nesyt Fish Pond, in South Moravia (near Lednice) has a unique population of reed canarygrass that are partially salt-tolerant and also grow in partial shade. Whether or not genetic changes have occurred in the population due to salt and shade conditions will be determined. The Nesyt Fish Pond has been studied extensively since 1968 by Drs. Jan Květ and Štěpán Husák (Institute of Botany). I visited this site with both of them in October 2010 so we could collect reed canarygrass samples (Site No. 32, Table 2). One biofuel cultivar, 'Chrastava', which was developed by the Czech Republic was also sourced from the breeders by Dr. Vladislav Čurn (Table 2). I also found 3 ornamental cultivars and have 4 genotypes of these 3 cultivars (Table 2). The genetic variation within cultivars will be compared with the wild Czech populations, as well as the N. American types (several of which were brought to the Czech Republic in July 2010 as tissue cultured specimens). Total germplasm collected or sourced were 4 cultivars, 4 canal or wet meadow sites, 7 transects (304 genotypes) within wet meadows, and 10 sets (30 genotypes) along the Zlata Stoka Table 2).

Table 2.docx

Genotypes were assessed for GPS coordinates (latitude, longitude, altitude) and the dominant plant community species were noted. Of particular interest was the unique correlation of reed canarygrass growing with nettles (Urtica) in all locations except one. What is unique about this finding is we have never observed this association in N. American populations. We also noted any morphological (phenotypic) abnormalities: tip burn, chlorosis, necrosis, insect damage (leaf borers vs. chewing insects), pathogens (Puccinia, Ustilago), potential vector(s) (recent flooding, Trumpeter swans) or other unusual characteristics. These were noted for each sample or population; should these be correlated or linked with any of the DNA markers in the future this information could be used to create linkage maps.

Leaf samples were collected (1 recently expanded leaf/plant), marked with site, location and plant-specific codes, kept on ice during collection and transport, and brought into the lab for descriptions. Leaf tips were cut into small (3-4 mm) pieces, placed in eppendorf tubes, labeled and frozen (-20•C) until extraction. The remainder of each leaf were placed in individual, labeled bags and frozen as back up samples in case they were needed. It took ~1-2 hours to cut, sample and freeze a single population of samples (15-40 genotypes). DNA extractions take 5 hours per 30 samples, so I have spent the bulk of time during November-December (when not teaching or preparing for lectures) doing extractions. We have now completed DNA extractions of all 465 samples collected along the rivers; the 370 perpendicular river transect samples are nearly completely extracted as well. I anticipate finishing extractions within the next week.

We ordered and have now received 3-5 ISSR primers from the University of British Columbia (UBC) that were used to score the N. American populations: UBC 810, 828, 853, 881, and 890. Once all of the DNA extractions are completed I will commence polymerase chain reactions (PCRs) and run gels to score the primers for differences between samples. Data analyses will follow. This will take several months to accomplish but I anticipate having this done by spring 2011.

At a later date, provided time allows, we will conduct similar genetic analysis of reed canarygrass from the European germplasm banks. I have obtained seed from the germplasm bank curators of ~150 sample populations from across the European Union. Seeds of related Phalaris species have also been sent and are being kept in the Ecosystems Biology seed vault. Also, if time allows, I will sample Phalaris herbarium specimens from the National Museum in Prague Herbarium. Dr. Otakar Sida is assembling all of the collections of reed canarygrass for this purpose during 2011.
We will be able to publish at least one if not 2-3 peer-reviewed publications from this research. It will add to the published body of knowledge on reed canarygrass and, more importantly, add new information about vegetative and seed spread of the species, whether rivers differ above and below confluences and if transects within populations differ from those by the water's edge. All of this will greatly influence future decision-making for use of Phalaris as a biofuel crop and risk assessment for potential future spread of this species within the Czech Republic.

Chrysanthemums in the Czech Republic

Of course, being a chrysanthemum breeder/geneticist, I'm always on the lookout for how this crop is used in Europe and the Czech Republic. Nowhere in the U.S. would you find a billboard devoted to just chrysanthemums, but here they are in prominence everywhere in Bohemia!!

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You might not be aware, however, that there are native species of chrysanthemums in the Czech Republic. Here, for instance, is Chrysanthemum serotinum, a species we've used at the University of Minnesota Chrysanthemum breeding program since the 1930s as a source of winter hardiness!

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This fine specimen is growing in the plant identification collection at the Institute of Botany in Trebon. It must be one genotype which is self-incompatible as there were no seeds in the composite inflorescences (I just had to check, of course!).

Ornamental reed canarygrass cultivars found!

I have been searching diligently for examples of the ornamental reed canarygrass, Phalaris arundinacea, here in the Czech Republic. Finally.....success! They were growing in places I didn't expect to encounter them.

Here, for instance, is the variegated type--either 'Variegata' or the variegated genotype of 'Feesey's Form'--found growing at the Vila Lanna in Prague where we stayed during the Fulbright Orientation. It was perfect placement, as several Fulbrighters asked what reed canarygrass looked like and I could easily show them!

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And then, when we stopped by an apple stand on the way back from Prague headed home to Ceske Budejovice....what should we discover as we bent over to look at the fall-blooming Colchicum autumnale (which contains the chemical, colchicum) but another variegated reed canary grass, this time most likely 'Strawberries and Cream'!

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We'll be able to identify exactly which cultivars each of these genotypes are when we compare the ISSR profiles with those of these cultivars already run at the University of Minnesota. Stay tuned!

Other ornamental species found growing near Phalaris

As a horticultural scientist, one cannot help but notice other species of ornamental or edible landscape value when we're out collecting.

Here are a couple of recent examples from this fall.

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Recognize it? It's Euonymous europaeus or burning bush!

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Also, at the edge of this farmer's field was a superb stand of:

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It's Rudbeckia laciniata, a N. American native plant that is invasive here in the Czech Republic. Isn't it interesting how we exchange invasive species across the continents!

Phalaris growing in shade and nearly dry conditions!

Since one of my Ph.D. students at the University of Minnesota is studying drought tolerance evolution in reed canarygrass, I'm always on the lookout for its occurrence here in the Czech Republic.

Here's a fine example of it growing in very dry conditions with low light. It's struggling due, most likely, to the lower light levels.

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We'll see whether the molecular markers differ significantly for this genotype when compared with other genotypes within this and other populations along the Luznice River.

A wet meadow on the other side of Rzmberk Pond & Zlatá stoka

This wet meadow (vlhké louky) is a very important site where faculty from the Academy of Sciences and the Institute of Botany have studied reed canarygrass since the 1970s! It is an ancient wet meadow, having been used for forage harvesting since the early days of fish pond establishment (~1200s!). The population in the unmowed segment has been intensely studied and highly published! We know that in the unmowed segment, the nutrient levels of Nitrogen vary in a gradient across the meadow.

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The mowed segment of the wet meadow is currently harvested for biofuel/biomass production. A bioreactor in Trebon uses it, along with corn stalks and pig manure, as a heating source for the famous Lazne Aurora Spa in Trebon. At the spa, one can get a peat moss bath (very healing and delightful) or swim in the therapeutic pools. Phalaris is helping out with promoting good health to everyone in South Bohemia!

Prior to 2002, the Phalaris was confined to the edges of the ditches transecting the wet meadow. The severe flood in 2002, however, brought a significant change to this meadow. Now the entire meadow, both the unmowed and mowed segments are virtually 100% reed canary grass. I wanted to sample both the unmowed and mowed meadows to determine whether these were derived from incoming seeds during the flood (highly likely) or from vegetative spread (less likely).

We sampled a transect across this nutrient gradient, as well as one perpendicular down the center of the gradient. It was interesting to observe that the above-ground biomass of the plants decreased visibily as one moved from the high nutrient levels to the lower.

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In the mowed section, we wanted to see if the variability changed from the unmowed meadow, as well as whether or not specific genotypes will be better in biomass production without being invasive (future studies!).

We found numerous examples of plant pathogens infecting reed canarygrass.

For instance, my host identified this as Puccinia phragmites:

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As the pathogen progresses, the leaf eventually senesces:

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We also encountered some type of smut infecting Phalaris. My host wasn't sure whether this was in the genus Ustilago or not. It had pustules of black spores exuding along the midveins which would leave a mark on your hand or clothing if you touched it!

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Particularly in the low nutrient areas the plant pathogens were common, as were herbivores (insects). Here's the common yellow grasshopper (Order Orthoptera) which likes reed canarygrass, leaving half-circle bites in the leaves!

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In late September and early October, we were out collecting reed canary grass samples for DNA analysis in two different wet meadows on opposite sides of the famous Rzmberk Pond just outside of Trebon.

In the first wet meadow, we ran two perpendicular transects through the large meadow which has not been mown for at least 50 years (pre-Soviet invasion, at least). This meadow has purple loosestrife around the edges (we saw this two years ago when Gina Quiram, an Ecology PhD student was here from the University of Minnesota -- we helped her collect samples from here). In the center of the large reed canary grass population is a stand of Spiraea; additional species include Glyceria, and Iris pseudoaccorus. We started a transect at the Golden Canal (Zlatá stoka), which runs through Trebon and is the source of fresh water for all of the fish ponds in this area--carrying water from the Luznice River to the ponds. Reed canarygrass also grows along the Golden Canal.

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Our sampling here will be informative in determining genetic variation within this well-established population since it is not documented whether most of the population is derived from seed or clonal (vegetative) source material. We shall see!

Since we've had a series of heavy rains, both the Vltava and Luznice Rivers have been flooded. The reed canarygrass growing along the river edges or in the floodplains were all laid flat (water can take a 2-3m plant and lay it down quite easily!). Once the waters subside, then new shoots emerge from the horizontal, existing stems....one adventitious bud at every node breaks forth into a new shoot. Soon it looks like newly mown reed canarygrass. In this picture, with shoots regrowing, the older plant stems (from earlier in the season) formed a mat approximately 0.5-1 m in depth that we had to walk on (more like bounce up and down on!) in order to get to the river's edge and collect.

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I pulled out one entire original stem so you can see the regrowth potential of the shoots!
You can see how, if a stem section breaks off, it could be carried down the river, lodge somewhere and start a new clonal plant! Thus, the easy spreading of clones in riparian habitats...

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Occasionally we note whether or not the samples collected have insect feeding damage (herbivory, in other words). Here you can see some damage inflicted on a sample. We remove the section that has been chewed on such that we don't have the potential DNA contamination from the insect.

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Additional invasive ornamental species that we run across while collecting reed canary grass include this gorgeous impatiens (Impatiens glandulifera). Sadly this has been used widely across the globe which has enhanced its spread. It grows right along with Phalaris arundinacea, Urtica (oh, those darn nettles!), and an occasional purple loosestrife (shown below; Lythrum salicaria).

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Lythrum salicaria....while it doesn't like growing water this deep (the Luznice River was flooded!), it can survive it for a short time.

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Isn't this picture from our collections along the Vltava River quite romantic? Here the drooping branches of weeping willow (Salix babylonica) reach downwards to the sward of reed canarygrass (Phalaris arundinacea). The fishing boats add an invitational to ride down the river!

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Just down the river from here, we noticed a bridge foundation on the west side of the river...a very tall concrete foundation (meaning that it was most likely from the 1900s). However, there was not a corollary foundation for the bridge on the other side of the river. I wondered whether or not this bridge had never been completed. As we trudged down the river collecting Phalaris samples, we ran across two fisherman on the banks of the river. We asked them about this structure and learned that this had been built by Hitler's army after the invasion but that it had, indeed, been abandoned before it was ever finished. This gave us a moment's pause of reflection on the history surrounding this site which I will never forget.

At one site along the Luznice River, we ran across a small portion of the population that was susceptible to the fungus, powdery mildew (order Erysiphales; there are too many species to know which one this was without an experienced plant pathologist along). The older leaves of all the plants were completely white!

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Here's how the collected leaf samples are processed once we get them into the lab. Each leaf is labelled with its sample number (population code, location code, replication number, etc.).

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A matching set of a zip bag and eppendorf tube are encoded with the sample information. Then the leaf tip is removed and put into the tube for grinding and DNA extraction while the remnant leaf sample goes into the -18oC freezer as a backup.

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More collections of Phalaris, Vltava River, 11 Aug 2010

At Population No. 2, on the Vltava River, which was near the site of the Golden Monastery (Zlatá Koruna) near Ceske Krumlov. See their website at:
http://www.czech.cz/en/66568-zlata-koruna-the-monastery
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We, of course, were needing to collect more reed canarygrass samples, so we didn't go to the Monastery (another time!). This site is ~30 km north of Site No. 1 (Rzmberk Castle area). We're collecting along each river at ~30 km intervals to prevent collecting populations that are in close proximity and unlikely to differ genetically since along the rivers the primary propagation method is generative (vegetative or asexual). Vegetative propagation would make many identical genotypes.

At each site, we're collecting every 100 m along the river's edge. At each spot, we collect three specimens (most recently matured leaf) that are 5 m apart from each other. When possible, we also collect along transects that run perpendicular to the water's edge (every 10 m apart, again with three plants collected at each spot that are 5 m apart from each other running parallel to the river's edge).

Here we are running a transect:
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Collecting samples:
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Here's the collection team that came with me that day: Jan, Albert, Bara and Dasa.
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