Introduction to the collection of plant samples during field work

with emphasis on the production of herbarium specimens by Alexander N. Schmidt-Lebuhn (schmidtleb@yahoo.de)

CONTENTS 1. Introduction 2. Herbarium specimens 2.a. What and how to collect? What needs to be written down? 2.b. Collection numbers 2.c. Drying and pressing 2.d. Mounting 2.e. Labelling 2.f. Storage and conservation 2.g. Some additional information on professional collections 3. Tissue samples for molecular analysis 3.a. What and how to collect? 3.b. Drying and storage 3.c. Use in the laboratory 4. Dried nectar samples 4.a. What and how to collect? 4.b. Drying and storage 4.c. Solubilising 13 2 4

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1. INTRODUCTION
The value of the ability to collect and conserve usable samples is, unfortunately, increasingly underestimated. Today, a fair number of scientists only sees the analysis in the laboratory and on the computer as important, but tends to forget that it is by no means sufficient to extract DNA from a few plants to conduct research according to good scientific practice. Regrettably, in the last few years this has lead to a situation where an increasing number of students start their masters thesis or even their dissertation without ever having been introduced to basic collecting and preservation methods. The present reader is intended to close this gap as far as is possible without a practical approach. The focus is consciously placed on the production of herbarium specimens. Not only are they often considered as particularly old-fashioned and unnecessary, but they are, as we will soon see, in a sense the basis of several types of botanical studies, regardless of with what other kinds of samples these studies also work. To show why herbarium specimens are in no way superfluous in the age of molecular biology, and why it is not irrelevant how samples are conserved, we will begin with a short overview of the uses of this kind of sample. What purpose do they serve, then? Types: the most important samples in taxonomy. --- Taxonomy is the science of the naming of organisms. The most important taxon is the species, and the question what exactly a species is has been a source of dissent for ages and probably will be forever. Nevertheless, it is important that any species has only one accepted (Latin) name, and that it has to be clear which species exactly is meant by that particular name. For this latter purpose, the concept of types was introduced. They are those samples in botany mostly herbarium sheets, in zoology stuffed animals, skulls or needle-mounted insects that a name is attached to. The taxonomist who is describing a species as new to sciences is required to designate a (holo-)type for the species to be deposited at an established herbarium, so that colleagues or later generations of researchers will be able to look up what precisely he or she meant with the given name. This helps to solve subsequently arising problems, for example, if the species description was not accurate enough to distinguish it from closely related species only discovered later. When what had been considered to be one species in the past is now separated into two distinct species, the type has to be examined to decide which of the two segregates will have the old common name attached to it and which of them will have to receive a new name. There are various kinds of types, all with their particular relevance and definition. Holotype: The one single herbarium sheet that was designated as a reference by the describer of the new taxon. Isotype: A duplicate of the holotype, i.e., another part of the same collection by the same collector from the same day, but mounted on a different herbarium sheet and usually sent to a different herbarium. For larger plants this should preferably be a part of the same plant, for smaller herbs at least nearby conspecific plants of the same population. Paratypes: All other herbarium specimens the describer of the new taxon examined for the description and that he or she cited as belonging to the same species. Lectotype: Originally an isotypus, but selected to replace a holotype that has become lost (e.g., because of an accident or acts of war). Neotype: A replacement of the original type selected after the loss of all holo- and isotype material. This should preferably be a paratype, if there is still one available. There are some other, but less important kinds of types.

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Part of a list of specimens examined for a taxonomic study

Specimens examined: the basis of morphological, floristic and taxonomic studies. --Herbarium specimens, usually loaned from important herbaria, sometimes collected by the researcher himself, are the basis of morphological, floristic and taxonomic studies. They may, for example, serve the description of new species, the synonymisation of species names in the case of repeated descriptions, or the updating of our knowledge of distribution, ecological adaptations or relationships of a certain group of plants. An indispensable part of the publication of taxonomic or floristic studies is consequently a list of the examined specimens (see example above). It shows, in a formulaic and internationally relatively consistent way, all or a representative selection of the herbarium sheets that were available to the researcher of the present publication. This allows colleagues to see, even after decades, what material the study was based on, and to understand the logic used in it, and it is thus a prerequisite for the studys reproducibility and comparability. At the same time, the list allows the reader to get an instant impression of the distribution of the group in question. Voucher specimens: did they identify the samples correctly? --- This introduction began with a comment on the increasing importance of laboratory-based analyses in biological research. Many systematic and ecological studies are, as we know, today conducted partly or entirely with molecular techniques: DNA sequencing with subsequent cladistic analysis in systematics, genetic fingerprint methods in population biology, analysis of genetic diversity for the assessment of the consequences of habitat fragmentation, etc.. For these kinds of studies it is often not sufficient to simply pick some leaves from the plants and extract their DNA. Serious scientific journals will normally only publish the results of those studies where a so-called voucher specimen has been deposited in an established herbarium for every genetic sample or at least for every population that was examined. This enables other researchers, if necessary fifty years afterwards, to re-examine the results of a study, and this possibility is an important aspect of good scientific practice. What good is, for example, a spectacular phylogenetic publication leading to a new circumscription of genera if about half of the samples used in the study were misidentified? What use is an examination of the variability of pharmacologically interesting chemical components of plants whose identity nobody will be able to verify afterwards? And even if, at first sight, the identity of the study species seem unequivocal, it may be that the next scientist working on the plant group simply prefers a different species concept and would like to know how the names in the results section of our publication would be according to his or her own concept.

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As we can see, molecular samples can by no means simply substitute for herbarium specimens, but both have to supplement each other. Other kinds of plant samples. --- There are, of course, countless other kinds of samples that could be collected for research on plants, like phloem juice or root tips, to name just two. Nevertheless, this reader will cover only two others apart from herbarium specimens: tissue samples for molecular analyses and nectar samples. The quality of the first is important for certain types of analyses, and the second are dried and stored in a similar manner, so that they lend themselves to be treated together.

2. HERBARIUM SPECIMENS
2.A. WHAT AND HOW TO COLLECT? WHAT NEEDS TO BE WRITTEN DOWN? It depends on the taxonomic, and sometimes also the ecological group of plants, what parts of them should be collected. Some taxa are nearly undeterminable without fruits, while the elsewhere indispensable flowers are nearly irrelevant (e.g., Valerianella, Papaver), others are difficult to determine without rhizomes (e.g., tropical ferns). It is therefore a good idea to have as many characters available as possible. Often, this is obviously impossible because a certain organ is not present at the time of collection, or undesirable because the rootstocks of rare species should not be unearthed. In these cases it is all the more important to carefully note all possible observations directly in the field that could be useful for identification. Generally a collection should comprise the following: Large flowering plants. --- At least one well developed inflorescence, if possible partly flowering, partly fruiting; below that a branch with several leaves, so that the leaf arrangement can be observed; if the leaves closer to the inflorescence are different from lower, purely vegetative leaves, both types have to be collected; in the case of herbs, the leaves of the basal rosette are often conspicuously different from those on the stems, so at least one of them must be included. Ranunculus is an example for a genus that is nearly undeterminable without basal leaves. It is a matter of common sense to decide if roots or rhizomes are needed: for the private herbarium of a student they can be regarded as superfluous, but their presence would be desirable in the collection of a possibly unknown geophyte. For some herbs, the rhizome, bulb or tuber is an important character; for woody plants, it is uncustomary and usually unnecessary to collect roots. Small, short-lived flowering plants. --- Small annuals like Erophila verna are usually collected whole (with roots), and several individuals are collected for one herbarium sheet. Not only does one plant appear a bit lost on the average sheet, it also does not give an impression of the populations variability, which can be especially high in short-lived organisms. These plants generally grow in large groups, so that only rarity or their protection status should stand against this procedure. Again, it depends on the taxon whether fruits or flowers are more important preferably both should be present. Ferns. --- For European species, the collection of rhizomes is normally superfluous, at least if the collector notes in the field whether the species has a creeping rhizome with distant fronds or whether the fronds are arranged in a cluster. In areas with higher species numbers or for morphologically complicated fern groups, the rhizome should be collected. Important for the identification of ferns are fertile fronds, and preferably those where the shape of the indusia is already or still recognizable. Very large fronds are partitioned, and in these cases the collection should

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comprise at least the tip, one middle pinna (leaflet), one basal pinna and the petiole. The scales of the petiole are often an indispensable character. For tree ferns, stem characters should be noted, most importantly colour, scales or hairs, and whether the petioles of dead fronds are shed or not. Mosses and liverworts. --- Bryophytes can often easily be collected in sufficient quantities to give a good sample for identification, but some can hardly or never be identified without sporogoniums. To collect plants that are hard to reach, especially epiphytes, a variety of tools has been developed. The crown of smaller trees can be browsed with poles that have a kind of clipper or pincer attached to their end. For taller trees, climbing equipment is employed. Rarely this is comprised simply by special boots with hooks and a safety belt; more commonly, however, botanists today use professional climbing gear with a rope that is first attached to a weight and thrown over a high branch or, in extreme cases, shot into the trees crown by means of a crossbow. If the woody plants themselves are sampled, it is a good idea to pack a sturdy outdoor knife or a machete. To avoid wilting of collected samples during ongoing field work, they are generally kept in plastic bags, and then transferred to the plant press (see below) once one has returned to camp, research station, hotel or home. This usually has to be done the same evening. If only a small number of samples is collected, it may be easier to directly put them into a handy plant press, but they are often too much of a nuisance to carry around during work. As important as the collection of the sample itself is the writing down of certain observations directly at the place and time of collection. They will be discussed in more detail in section 2.e., but here the most important characters should already be mentioned. They include especially those that are usually lost in the process of pressing or unavailable in partial collections of large plants, like flower or bract colours or growth form. For bryophytes, it is a great help for identification to know the substrate (bark of living tree, dead wood, rocks, earth). 2.B. COLLECTION NUMBERS Collection numbers can be regarded as optional for private herbaria, but they are indispensable if large numbers of samples are collected from the same species and must be related to other data afterwards, or if duplicates are collected for each sample and then distributed to different herbaria. The latter procedure is a prerequisite for obtaining a collecting permit in other countries, where a local herbarium expects to receive at least one duplicate of every collection by a foreign visitor. Another likely case would be that a researcher wants to keep one duplicate of every sample for himself, but send another to an internationally recognized specialist as a so-called gift for determination: send me the names, and you can keep the duplicates. In both cases, only collection numbers allow us to relate individual samples from two different herbaria, that is, they allow us to know which sheets represent the same collection. Every scientifically relevant herbarium specimen should be unmistakably identifiable from the combination of its collector name and collection number, e.g., Krmer 244. Specimens that have, for some reason or another, never received a number, are in publications cited with the abbreviation for sin numero, like Kuntze s.n.. There are different ways to count sample numbers, but the best and most commonly accepted international practice is the following: every collector starts exactly once with the number one and then consecutively uses increasing numbers until his or her death. No

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number should ever be used twice. If it is realized too late that an error has occurred, it is customary to put letters behind the number (Mller 394a and Mller 394b). When a group of researchers is collecting together, all can appear on the label with their names to acknowledge the work of ones collaborators, but the numbers should be counted only after the collector mentioned in the first place (Schulz 1003, Schulz & Meyerhofer 1004, Schulz 1005). To avoid confusing later users of your specimens, it is obviously essential to avoid starting repeatedly with one or using a different numbering approach when collecting together with colleagues. On the other hand, it should never be blindly assumed that all collectors whose specimens you are examining have followed these conventions in every detail. In the field, collection numbers can be attached to the specimen in various ways. The most common are to strap small numbered paper labels to the samples before pressing and writing the number on the newspaper that contains the sample during drying. 2.C. DRYING AND PRESSING While mosses, liverworts and lichens are simply stuffed into labelled paper bags and then air-dried, higher plants must not only be dry but also flat for mounting on a herbarium sheet. To this end, plant presses are employed. They consist of two plates of wood or, more rarely, metal grids that are bound together with strapping belts as can be obtained from home improvement or outdoor shops. Between the two plates are newspaper sheets, alternating between folded simple sheets containing the plant samples and thicker layers of sheets that are regularly exchanged to remove moisture from the press (diapers). When putting a plant into the press, care has to be taken that no parts of the plant sample lay on top of each other. If that is unavoidable, additional sheets of paper can be put between branches or leaves that would otherwise cover each other. Cable harnesses or paper slings can be employed to keep uncooperative shoots in the desired position. The layers between those containing the samples take up moisture from the plants and are regularly exchanged for fresh, dry sheets, while the samples stay in the same sheets all the time, so that the latter can also be labelled. Many circumstances influence the frequency with which sheets have to be changed, like ambient temperature, air moisture and the nature of the plants themselves. For starters, they should usually be changed daily. Binding of the plant press should not be so strong as to squash fragile organs, but not so loose that small parts can crumple up in drying. Several variants of this procedure have been developed over the years: Pressing at room temperature. --- This approach is taken by most recreational collectors and is usually sufficient for the temperate climate zone and normal plants. In the tropics it also often the only way, but then due to lack of superior equipment. The sheets are changed often at the start, less often later, and are dried for their next turn in the press by spreading them on the floor, preferably in the sun and weighed down with stones or similar to protect them from wind. After two to four weeks, the samples are hopefully dry and flat. Problems occur with longer periods of rain, tropical and wet ambient climate, or succulent plants. In these cases, the plant samples turn dark and finally begin to rot. Another drawback of this method is the need to carry around large quantities of newspaper. Drying ovens. --- Professional herbaria in the tropics often have hot drying ovens to dramatically accelerate the drying process, sometimes to only two nights, and to avoid the problems of the previous approach. They are often simply wooden boxes with heat radiators below; the presses are placed vertically on top, so that the heat can enter them from the side. The presses are generally

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augmented with corrugated iron for heat transmission and additional layers of soft material. The obvious drawbacks of this method: even though this is regularly done by some researchers, not everybody wants to carry a bulky radiator to field work, if electricity is available at all. In addition, these constructs have a tendency to go up in flames if left unguarded, and sometimes they take half a research station with them. Preservation in alcohol. --- This method was developed by the U.S. American ethnobotanist Richard Evans Schultes when he was faced with the problem of conserving herbarium specimens during several month-long journeys in tropical rainforests while not having a field oven at his disposal. The plants are first placed in newspaper sheets as if they were to be put into the plant press, but then they are only bound together to form a thick and densely packed bundle. Several of these bundles are placed into a watertight (!), durable plastic bag. An amount of ca. 70% alcohol is poured into the bag to infuse the bundles thoroughly, but not so much as to drown them. The samples can now be transported and stored for long time periods without fear of rotting. Once one has returned to the research station or university, the samples are transferred to a plant press and dried and pressed in a drying oven as usual. Drawbacks of this method: the need to carry inflammable alcohol, the samples often loose their colors and become rather brittle, and the resulting herbarium specimens are unsuitable for later DNA extraction. Specific problems with drying and pressing arise in some ecological or systematic plant groups: Asteraceae/Compositae tend to develop ripe fruits in the plant press. This, however, is not as bad as it seems, as fruit characters are highly relevant for determination in this family. Succulent plants like Sedum have large reserves and die very slowly, so that they often shed all their leaves and produce long, etiolated shoots in the plant press. To avoid this, the plants could be poisoned (alcohol) or, perhaps even less aesthetic, the shoots could be squashed before drying. Rhinanthoideae (the hemi- or fully parasitic species that were previously placed in Scrophulariaceae, now Orobanchaceae) and a few other groups nearly always turn black while drying. There are even some species with tell-tale Latin names like nigrescens. Needle-leaved Conifers and Ericaceae nearly always shed their leaves in the plant press. It is possible that collection in alcohol helps against that, otherwise the only solution is to put the leaves into a paper bag and glue it to the herbarium sheet.

Left: a wooden plant press; right: alternating layers of newspapers for storing the plant sample (red arrow; opened only for demonstration purposes) and for taking up the moisture (white arrows).

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An oven for drying plant samples in the Bolivian National Herbarium, currently containing two different plant presses, one of them rather thick. They are placed on their sides so that the heat can enter from below and the moisture can escape from the top.

2.D. MOUNTING Dried mosses are stored in paper envelopes folded from a sheet of standard office paper. Higher plants are mounted on cardboard sheets; their size usually corresponds to standard office paper sheets for private herbaria (i.e., German DIN A4), but is twice as large in professional herbaria (DIN A3). Paper strength should not be below 160 g/m2, as otherwise handling of the sheets becomes difficult, and the mounted samples tend to break more easily. For mounting on the sheet, special gummed paper strips are placed strategically over parts of the plant sample. Obviously, parts that are especially relevant for determination, like fruits and flowers, should not disappear under a strip, so that leaf tips and internodes are preferred. Very thick and heavy objects, especially woody plants, are sometimes sewed to the sheet in professional herbaria. Admittedly, some herbaria have, faced with shortages of staff, time and money, turned to using other mounting methods, like hot glue pistols. These deceptively easy and quick approaches all have the drawback that specimens cannot be separated from their sheets without damage when this becomes necessary for examination. Many glues, especially the Scotch tape/Sellotape used by some beginners, also dissolve after only few years and may then even leave a smear over the sample. Herbarium specimens should be mounted in an approximately natural position (e.g., roots at the bottom, flowers at the top) and in a way that allows different leaf sides to be seen; if only one leaf is available, like in large fern fronds, a part of it is usually folded over. It is also important to leave some space for the label. Long, narrow samples like grasses are often folded up or, in extreme cases, cut to pieces to make them fit onto the sheet. If parts of a sample have broken off, they are stored in a paper pocket glued to the same sheet.

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Paper envelope for the storage of moss, liverwort and lichen specimens. It can be folded from a standard sheet of office paper.

Left: a roll of tape, cut glue strips, pincer and other equipment for mounting higher plant specimens; right: the usual size and placement of glue strips on the sheet.

One possible variant of paper pockets for storing loose sample fragments on a herbarium sheet.

2.E. LABELLING Every herbarium specimen is identified and described by a label which is glued or stapled to the paper envelope in the case of mosses, liverworts or lichens and glued to one corner (frequently bottom right) of the sheet in the case of higher plants. The following data should be available from the label: Collectors name and, for professional specimens, the collection number: J. Doe 311. Scientific name including taxonomic authority: Bellis perennis L..

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Scientific name of plant family: Asteraceae. Place of collection: Country, relevant political subdivisions below country level, name of closest town, exact locality. The description should be given in a way that allows others to find the place again, like Bolivia, Departamento Beni, near Santa Rosa, 3 km north of the town on the road to Riberalta. In contrast, Bolivia, Santa Rosa alone is nearly useless, as there are numerous towns of this name in the country, and the population of a rare plant is hard to locate without the more precise directions omitted in the second case. Of course, less details are necessary for private herbaria, especially if one never collects outside of ones home country. GPS coordinates, if available, but this is also unnecessary for private herbaria. Elevation; in Germany usually unnecessary outside of the alps. Habitat, i.e., ecological parameters. It is not sufficient to simply write forest, grassland or even roadside, a typical beginners mistake. Other researchers subsequent users of your specimen are interested in light conditions, soil type, intensity of disturbance, degree of moisture, etc., and thus the following examples are much more useful: dry open grassland on sandy soil, wet meadow, cattle pasture, mixed forest of oak and hornbeam on limestone soil, commercial spruce forest on red sandstone soil, high alpine cushion plant community. Ethnobotanical notes like common names of the plant, its medicinal or culinary uses, etc., as far as relevant to the collector and as far as available. Characters that will most certainly not be seen on the dried and mounted specimen, like colours of corolla, calyx and bracts, growth form, growth height, scent, etc. Date of collection. The following notes are also often found on labels, but they are optional from a scientific perspective: Name of the herbarium whose researcher has collected the plants and/or distributed the duplicates to other herbaria. Name of the expedition or research project in whose framework the collections were conducted, and sometimes the name of an institution that has financed the project.

Label of a herbarium sheet from a professional collection. It was printed with most of the data, but the species identification was added at a later time by a specialist. He has also included his name and the date, so that the reader knows on whose expertise and on what state of knowledge the identification was based.

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2.F. STORAGE AND CONSERVATION Paper envelopes containing mosses, liverworts or lichens are typically sorted one after the other into cardboard boxes. Herbarium sheets of higher plants are either stacked one above the other in half-open cardboard boxes or placed one above the other between two stronger cardboard sheets and then strapped together to form a fascicle. Boxes or fascicles are stored in boards, mostly ordered alphabetically first after family name, then after genus, finally after specific epithet. Often, however, some plant groups are stored separately, like pteridophytes or monocotyledons. Old herbaria may also have collections of historical interest in separate rooms, or there may be especially protected boards for type specimens. The sheets should be stored under dry conditions to avoid mould. Even then they must be checked from time to time for pest infestations. The most frequent pests in herbaria are tiny booklice, which over time consume especially the anthers and petals, and the hairy carpet beetle larvae or related species, which destroy various kinds of material and can even chew tunnels through several layers of cardboard sheets. They are combated by storing the samples for several days in freezers at 20C or lower, or less commonly in heat chambers. In the first case, fascicles have to be wrapped in plastic bags to keep them from taking up moisture from the freezer. A more problematic but, at least in the moist tropics, still popular alternative is the use of poison to protect the specimens. Unfortunately, it makes handling them and working in the herbarium less pleasant and less healthy. For the work with fascicles it is important to note that the individual sheets should never be turned like pages. They are treated very carefully, always held with the mounted plant up, and sorted by restacking. If a part of a specimen breaks off or has to be separated from the rest for study, it must subsequently be deposited in a paper bag on the sheet, otherwise the sample will get smaller and smaller over the years. Beginners sometimes think that breakage can be avoided and handling of specimens facilitated by storing them in transparent folders. Regrettably, these damage the plant samples through electrostatic charges, so that they are never used by professional herbaria.

Unbound fascicles in the herbarium of the University of Halle, Germany.

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2.G. SOME ADDITIONAL INFORMATION ON PROFESSIONAL COLLECTIONS Finally, we will consider some general aspects of scientific herbaria that may be of interest. Botanical collections including herbaria are usually found in museums of natural history, in botanical gardens or at universities. They are overseen by a scientific collections manager or curator, but they also sometimes employ technical staff or offer internships. A silent agreement exists between all major herbaria with sufficient financial capabilities to loan specimens to each other on request, usually for one year, although it should be unproblematic to extend this to the duration of an average Ph.D. project. A formal request is written on the lines of we are conducting a study on genus xyz, and would like to ask for a loan of. In exchange, the respective herbaria expect to be mentioned in the acknowledgements section of resulting scientific publications, to receive a reprint of the same, and the willingness to lend them specimens when they next need some from your institution. Shipping is first paid by the herbarium sending out the specimens, then the return shipment is paid for by the researcher who borrowed them. Smaller herbaria or those in economically less prosperous countries can, obviously, not afford this, and so it frequently becomes necessary to visit them personally to view their collections. These visits should, of course, be announced and discussed in advance, so that the respective collections manager is not taken by surprise, and to make sure that the herbarium is not closed at the relevant time. On the other hand, many herbaria appreciate visits by experts on specific taxonomic groups who are then expected not only to obtain information for their own use, but also to leave small labels with an up-to-date identification on the specimens they have examined, e.g., Sanchezia oblonga Ruiz & Pav., Acanthaceae, det. Jane Doe 7/2005. Older specimens can wear several of these labels reflecting historically changing state of knowledge. Even large, comparatively wealthy herbaria hesitate to send out historically important collections or old types, so that in these cases a personal visit may also become necessary. On the other hand, an increasing number of specimens is being digitised, so that some simpler tasks can today be completed by consulting online specimen databases. Often, DNA for sequencing can be extracted from herbarium specimens, at least if they were not collected alcohol or dried too slowly. Some herbaria, however, forbid damaging their precious samples in this way. Information about herbaria worldwide can be found in the Index Herbariorum, available as a book or online at http://sweetgum.nybg.org/ih/. Besides addresses, staff and important collections, the index also lists the acronyms that unmistakeably identify every herbarium and are used as their abbreviations in scientific publications. The herbarium of the German University of Halle, for example, has the acronym HAL, B identifies the Botanical Museum Berlin-Dahlem, K the herbarium of the Royal Botanic Gardens in Kew, United Kingdom, etc.. It is commonly assumed that either Kew or Paris (P) is the largest herbarium in the world. German Berlin-Dahlem would presumably be a member of the same league if not for the fact that much of it was destroyed in WWII. The fastest growing and a candidate for the largest herbarium of the future is said to be that of the Missouri Botanical Garden (MO). Other important herbaria in Europe and the U.S.A. are, for example, Aarhus (AAU), the Field Museum in Chicago (F), New York Botanical Garden (NY), Stockholm (S), University of California/Berkeley (UC) and Smithsonian

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Institution/U.S. National Herbarium (US). Next to Berlin, the most important German herbarium is probably the Botanische Staatssammlung Mnchen (M).

3. TISSUE SAMPLES FOR MOLECULAR ANALYSIS

As mentioned above, DNA can often be extracted in sufficient quantities from herbarium specimens. Nevertheless, these extracts are, in many cases, unsuitable, because the specimens are too old, they were collected in a manner that degraded the DNA or they were dried too slowly, or because high molecular DNA is needed for certain methods, e.g., restriction analyses or AFLP. It is also possible that there simply does not exist a sufficient number of available samples, especially in the case of studies of population genetics. In all these cases, fresh samples are collected specifically for use in the laboratory. 3.A. WHAT AND HOW TO COLLECT? For molecular analyses, it is best to concentrate on young leaf material, as it contains more DNA than the stems, which largely consist of vascular tissue. It is self-evident that contaminations by lichen growths, fungi or plant pests must be avoided; the first are usually a problem on older leaves only, but aphids suck preferably on soft, young shoots that would otherwise be ideal for collection. A limited number of leaves is generally sufficient for molecular study. It is often better to limit oneself a bit rather than collect cubic centimetres of plant material for each sample and then run out of drying material and fail to get the samples well dried at all, so that they might be of limited use. On the other hand, one should always collect enough material for repeated attempts at extraction, just in case the first one fails. 3.B. DRYING AND STORAGE The plant material is placed in a paper bag labelled with the sample number. This bag should preferably be designed so as to keep fragments of the sample from falling out. A slightly complicated but very well-sealed design is shown in the figure below; alternatively, tea-bags could be used. Several such sample bags are stored together in an air-tight Ziploc plastic bags as can be obtained from the supermarket. To quickly and entirely remove the moisture from the samples, silica pearls are poured between the paper bags, and the plastic bag is closed tightly. Silica gel is a reusable, hygroscopic drying material with a colour indicator: if dry, it is blue or red, if spent and full of water, it is transparent. To finish the drying process, transparent pearls are instantly switched against fresh pearls, and then heated on or in an oven to prepare them for their next shift. This has to be done gently, for they char above ca. 130C. They are also stored under airtight conditions, otherwise they take up water from the air and must be heated again. Silica gel, especially the older, blue version, is poisonous and should not be swallowed. Once tissue samples are dried, they are stored over longer periods together with a smaller amount of silica. Some botanists even put the bags into a freezer for long time storage, but that should not be necessary for well-dried samples.

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Example for a sample bag folded from a quarter of a sheet of standard office paper.

Left: Ziploc-bag with one tissue sample on a large amount of silica gel (this amount would be sufficient to dry a considerably higher number of samples); right: long-time storage of several samples in a bag with a lower amount of silica.

3.C. USE IN THE LABORATORY For DNA extraction, about finger nail-sized leaf fragments are carefully removed and then submitted to the respective extraction protocol. It is obviously important to avoid mixing up parts of several samples, as sometimes very small amounts of tissue are sufficient for obtaining a PCR product.

4. DRIED NECTAR SAMPLES

For HPLC-based analyses of nectar sugar concentration and composition, nectar samples were previously often collected in liquid preservation fluids to keep the sugars from degrading. It is,

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however, very cumbersome having to carry around samples in numerous small tubes. In the following chapter, a method for the easy collection and dry storage of nectar samples is described. 4.A. WHAT AND HOW TO COLLECT? Nectars degrade in the course of few hours, and consequently it is important to collect them when they are reasonably fresh, before the flower is getting old. The liquid, which is often found at the base of the flower, sometimes in special nectar pouches or spurs, is collected with calibrated glass capillaries of 1 or 10 l capacity as can be obtained from sellers of laboratory supplies. The liquid enters them by capillary force, so that no sucking mechanism is needed. For sympetalous plants, especially those with a slender corolla tube, it is often easier to pluck a corolla from its calyx and collect the nectar from below. The amount of nectar collected is carefully noted by estimating the portion of the capillary that it has filled. Afterwards, a special plastic pipette bulb with a perforation (also available from lab suppliers) is put onto the capillary; the perforation prevents that the nectar is already pressed out by this manoeuvre. The capillary is held onto a small, triangular piece of filter paper, and the nectar is pressed out by carefully squeezing the bulb while covering the perforation. The filter paper takes up the nectar, and a small pencil mark is made slightly above the moist area to indicate how much of the paper holds the sample, as this can hardly be seen after drying. It also helps to know where the piece of paper can be touched without contaminating the sample. This sample-free area can also be labelled with sample number and amount of nectar collected. Each capillary is used only once.

Left: nectar is transferred to a capillary from the base of a corolla of Justicia tenuistachys (Acanthaceae). The capillary is filled for about two thirds, so it contains ca. 6-7 l. Right: Transfer of the nectar onto a piece of filter paper.

4.B. DRYING AND STORAGE The paper should air-dry until it no longer sticks to other paper. Several samples are then placed in folded paper sheets or sample bags and stored on silica gel in airtight plastic bags (see 3.b.). The silica should not touch the filter paper directly, and individual samples should not touch each other at least while there is still some moisture left. Dried and stored on silica, nectar samples can be safely kept for at least several months without showing signs of degradation.

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4.C. SOLUBILISING Before analysis, a piece of filter paper carrying a sample is simply bathed in a defined amount of demineralised water to resolubilise the nectar sugars. With the help of the notes on the original volume of the nectar sample, the original sugar concentration can be calculated from the total amount of sugars that are measured in the analysis.