Chapter 3 - Herbarium Methods and Exchanges

Glime, J. M. and Wagner, D. H. 2017. Herbarium Methods and Exchanges. Chapt. 3-1. In: Glime, J. M. Bryophyte Ecology.
3-1-1
Volume 3. Methods. Ebook sponsored by Michigan Technological University and the International Association of Bryologists. Ebook last
updated 1 August 2020 and available at <http://digitalcommons.mtu.edu/bryophyte-ecology/>.
CHAPTER 3-1
HERBARIUM METHODS AND EXCHANGES
Janice M. Glime and David H. Wagner
TABLE OF CONTENTS
Folding Packets ....................................................................................................................................................................... 3-1-2

Packet Machine ............................................................................................................................................................... 3-1-4
Followers ........................................................................................................................................................................ 3-1-5
Herbarium Sheets ............................................................................................................................................................ 3-1-6
Herbarium Labels ................................................................................................................................................................... 3-1-6
Multiple Species.............................................................................................................................................................. 3-1-6
Annotations ..................................................................................................................................................................... 3-1-8
Multiple Access .............................................................................................................................................................. 3-1-8
Storage .................................................................................................................................................................................... 3-1-9
Cabinets .......................................................................................................................................................................... 3-1-9
Packet storage ................................................................................................................................................................. 3-1-9
Type Specimens ............................................................................................................................................................ 3-1-10
Storage Containers ........................................................................................................................................................ 3-1-11
Palm Folders ................................................................................................................................................................. 3-1-11
Storage Boxes from Genus Covers ............................................................................................................................... 3-1-12
Specially Made Storage Boxes ...................................................................................................................................... 3-1-13
Preservation .................................................................................................................................................................. 3-1-14
Cool Preservation .................................................................................................................................................. 3-1-15
Minute Species and Special Structures ......................................................................................................................... 3-1-15
Herbarium Arrangement ....................................................................................................................................................... 3-1-15
Guide Cards .................................................................................................................................................................. 3-1-15
Herbarium Care..................................................................................................................................................................... 3-1-15
Pest Control................................................................................................................................................................... 3-1-15
Agral 600 .............................................................................................................................................................. 3-1-16
Moth Balls (Naphthalene) ..................................................................................................................................... 3-1-16
Microwave Oven ................................................................................................................................................... 3-1-16
Bromomethane (Methyl Bromide) ........................................................................................................................ 3-1-16
Freezing ................................................................................................................................................................ 3-1-16
Insect Traps ........................................................................................................................................................... 3-1-17
Drowning .............................................................................................................................................................. 3-1-18
Steam Sterilization ................................................................................................................................................ 3-1-18
Moisture Control ........................................................................................................................................................... 3-1-18
Dehumidifier ......................................................................................................................................................... 3-1-18
Silica Gel............................................................................................................................................................... 3-1-18
Herbarium Cabinet Materials ................................................................................................................................................ 3-1-19
Sending Specimens for Identification ................................................................................................................................... 3-1-19
References............................................................................................................................................................................. 3-1-20
Current Names .............................................................................................................................................................. 3-1-20
Indexing ........................................................................................................................................................................ 3-1-20
Herbarium Programs ............................................................................................................................................................. 3-1-22
Shipping Live Bryophytes .................................................................................................................................................... 3-1-22
Sharing Images ..................................................................................................................................................................... 3-1-22
Herbaria ................................................................................................................................................................................ 3-1-22
Herbarium Specimen Mapping ............................................................................................................................................. 3-1-23
Live Collections .................................................................................................................................................................... 3-1-23
Cryopreservation ................................................................................................................................................................... 3-1-23
When You Depart – Willing Your Herbarium ...................................................................................................................... 3-1-26
Exchange Programs .............................................................................................................................................................. 3-1-26
Borrowing Specimens ........................................................................................................................................................... 3-1-27
Type Specimens ............................................................................................................................................................ 3-1-27
Summary .............................................................................................................................................................................. 3-1-27
Acknowledgments ................................................................................................................................................................ 3-1-27
Literature Cited ..................................................................................................................................................................... 3-1-27
3-1-2 Chapter 3-1: Herbarium Methods and Exchanges
CHAPTER 3-1
HERBARIUM METHODS
AND EXCHANGES
Figure 1. Benito Tan and herbarium cabinets for bryophytes at the Hattori Botanical Laboratory in Nichinan, Japan. Photo by
Janice Glime.
Folding Packets
The standard for bryophyte storage is to put them in At CAS, curators use Strathmore ultimate white 100%
packets. These are made from a sheet of white paper, pure cotton 24 lb watermarked paper, available in reams of
preferably acid-free, 100% cotton to reduce decomposition 500 sheets, CODE 318003 (US standard size 8.5 x 11 in)
of brittle paper. Some herbaria use brown packets made (Jim Shevock, Bryonet 8 April 2015). CAS also uses
from shelf liners or grocery bags (e.g. Kraft paper), and use Strathmore 25 percent cotton fiber paper for printing of
of these is somewhat common in the field. Those are not herbarium labels and to process additional labels for
quite as easy to read, but they do last well. Wagner uses 3" specimen exchange. Both are acid free and of archival
margins for the packets, but Glime finds that 1-1.5" quality and print well on photocopiers. Karen Golinski
margins work well. The size depends in part on the size of (Bryonet 8 April 2015) similarly uses 100% cotton, acid
the herbarium box or drawer used to hold the packets. free, Avon Brilliant White, wove finish, 24 basis
Having an exact size isn't critical, so after a little practice it <http://www.neenahpaper.com/finepaper/morebrands/cotto
probably won't be necessary to measure. If the housing for npapers/classiccottonpapers/productdetail?color=Avon+Bri
the packets permits larger sizes, larger packets may be lliant+White&finish=Wove>. Mary Zimmerman (Bryonet
desirable for some large taxa. Note that the outside (last) 8 April 2015) uses Byron Weston Linen Record Ledger
fold should be a little shorter than the others (Schofield Paper from Talas: 100% cotton ('linen' is just the slight
1985). This permits more space for the bryophyte and texture on the paper) and it has the year of manufacture
makes it clear which side is to be opened. watermarked into the sheets
Chapter 3-1: Herbarium Methods and Exchanges 3-1-3
<http://apps.bnt.com/ecom/catalog/product_specific.cfm?C into the box or drawer than those where sides are folded
lientID=15&ProductID=24235> last. And it is the only folding system that works well
Bryophyte specimens should be placed into the when the packet is glued to a herbarium sheet. The typical
packets. An 8 1/2 x 11" (21.6 x 28 cm) sheet of paper, or resulting packet is 4x6" (10x15 cm), a convenient size for
size close to that such as the standard European size, should storage in shoe boxes. These packets may be stored in
be folded in thirds like a business letter (Figure 2). After boxes as packets or glued to a herbarium sheet, with the
the first fold, the two open ends are folded inward. It is an packet glued across the middle section of the back so the
important consideration that the first fold is up, then the opening flap faces you like the flap of a pocket. See
sides are folded in before the top is folded down. This storage below.
folding is less likely to lose specimens and fits more neatly
Left to right: 1. Mark 3" (7.6 cm) in from top of 8.5x11" (21.6x25.4 cm) sheet. 2. Mark 3" in from other side at top. 3.
Mark 3" from top using 3" card template. 4. Fold bottom up to mark 3" down side. [Change 3" on sides to 1.5" 3.8 cm) if you
desire.]
Left to right: 5. Fold left edge to mark 3" (7.6 cm) from left. 6. Fold right edge to mark on right. 7. Fold top flap
down.
Left to right: 8. Crease well. Packet is complete. 9. Packet with preprinted label data.
Figure 2. Steps for folding herbarium packets. Colors were used to make it easier to see the folds in these images. Photos by
David Wagner.
Packet Machine
Miller (1988) offers an alternative way to expedite
making packets. He uses a file folder to make a packet
machine. We have modified it here to make the same type
of packet as the one shown in Figure 2 and to maintain
packet size close to 4x6" (10x15 cm) with maximum space
on the flap for the label [3.5" (8.9 cm)] (Figure 3-Figure
13) (Schofield 1985).
Figure 6. Staple or tape the ends so that it forms a pocket.

Once stapled, this packet machine is ready to prepare packets.
Photo by Janice Glime.
Figure 3. Cut the tabs from the folder to leave all edges
straight and square. Then carefully measure 3.75" (9.5 cm) wide
on one end of the opened folder, parallel to the folder fold. Score
this line with a ball point pen and ruler to make it easy to fold.
Photo by Janice Glime.
Figure 7. The machine is now complete with staples. For

the first fold, 2-3 sheets can be folded together. Place one end of
the 8.5" (21.6 cm) wide paper in the 3.75" (8.9 cm) pocket and
fold it over the pocket. An old stainless steel spoon under the
thumb or just the thumbnail helps to get a good crease on the
packet. Photo by Janice Glime.
Figure 4. On the opposite end prepare a similar pocket;

measure 1.25" (3.2 cm) from that end, score, and fold both ends to
make pockets. Photo by Janice Glime.
Figure 8. Separate the sheets and place one side that is

Figure 5. Line up the pocket creases carefully and press perpendicular to the fold into the 1.25" (3.2 cm) pocket. Photo by
them with a spoon or your fingernail. Photo by Janice Glime. Janice Glime.
Figure 12. Place the bottom folded edge of the packet into
Figure 9. Fold the packet over the pocket and crease. Photo the 3.75" (9.5 cm) pocket of the folder and fold the exposed part
by Janice Glime. of the sheet over the pocket just above the pocket top edge so that
when folded the dimensions are 3.75x6" (9.5x15 cm) with the last
flap being 3.5" (8.9 cm). You won't be able to fold along the edge
of the folder pocket this time, but must fold just above it. Photo
by Janice Glime.
Figure 13. Now it is ready to use. The label should be

placed on the top flap. Photo by Janice Glime.
Followers
Figure 10. Repeat the operation on the other side of the
David Wagner (pers. comm. 2009) has found a way to
keep folded packets neatly stacked, in order, under constant
but light pressure. This also works for sorting, since
specimens can be added anywhere in the row with ease and
it will expand readily to fit. The trick is to use a cylinder
(can of beans in this case) in a tray that is propped up to
provide an incline for the can to roll against the packets
(Figure 14).
Figure 11. After you fold this side of the packet, you have an
envelope and only the top flap needs to be folded down. Photo by Figure 14. Packets held in place with food can in inclined
Janice Glime. box. Photo by David Wagner.
Card files (4x6" card size ≈ 10x15 cm) have a movable Label data should include name of the species (if
back on the drawers that can be adjusted to hold the packets known), the author of the scientific name, altitude,
upright. Shoe boxes can be packed with wadded paper in habitat, substrate, date of collection (with month
back to keep packets upright. written out), and location (country, state, county, distance
to nearest town), GPS coordinates, name of collector,
Herbarium Sheets collection number, determiner (name of person
Jerry Jenkins (Bryonet 31 January 3013) reports identifying or verifying identification). Persons adding
getting good herbarium paper from Herbarium Supply identifications or verifications to specimens often precede
<http://www.herbariumsupply.com/nu_listCategoriesAndP their names with an exclamation mark (!) to indicate
roducts.asp?idCategory=31> in Bozeman, Montana. determined by. Additional information may include name
of associated species, color, height of plant, abundance or
Herbarium Labels other information not evident from the pressed specimen.
Rob Gradstein (pers. comm. 26 July 2012) states that For liverworts, it should include descriptions of the oil
"labels should be a little smaller than herbarium packets bodies because these will disappear upon drying.
and glued on the outer surface (top, not bottom!) of the The family name is less commonly used for bryophyte
packet." But we agree with Schuster (1966) that the label labels because the family concept is less stable than in
should be printed directly on the front flap of the packet. flowering plants and there are fewer families and genera.
This saves time, and glued-on labels have a tendency to Specimens are usually stored alphabetically by genus (see
come loose from packets after time in storage. This can Herbarium Arrangement below).
result in loss of data, or worse, incorrect information when The label will usually also include the name of the
the label is matched to the wrong specimen. (Glime herbarium and the accession number for that herbarium.
inherited a herbarium where loose and lost labels were a The herbarium name aids in getting loans back to the
serious problem.) If the specimen needs to be put in a new rightful owner.
packet, the label can always be cut from the original packet A sample herbarium label is shown in Figure 15. Note
and glued to it or stored inside if a new label is printed on that the date is written out in full to avoid confusion
the packet. In either case, the label should be on the among different country annotations, and a detailed
opening face of the packet. collection location is included.
CRYPTOGAMIC HERBARIUM OF
MICHIGAN TECHNOLOGICAL UNIVERSITY
FAMILY: Fontinalaceae
SPECIES: Fontinalis duriaei Schimp.
DATE: 1 May 1969
LOCATION: USA, New Hampshire, Grafton Co., 1 km. north of Plymouth in woods on left of
Texas Hill Rd.
45°8'N, 71°40'W, R21, T15, sec 6 ELEV. 300 m
HABITAT: on granite rock in mountain stream in Tsuga canadensis woods
NOTES: few dark capsules with ends abraded away; plants dark green with little algal growth
COLLECTED BY: Janice Glime COLLECTION NUMBER: 281
DETERMINED BY: Janice Glime ! Winona Welch
ACCESSION NUMBER: 12896
Figure 15. Sample herbarium label from Michigan Technological University. Designed by Janice Glime.
Multiple Species
Bryophytes often grow intermixed (Figure 16). Here
need of ecologists and taxonomists/systematists differ. For
ecological studies, the associations contribute important
information. I (Glime) am reminded of a letter I received
from Sin Hattori, along with his careful notes on the
species in a set of collections of Frullania. Most of the
collections contained multiple species. He encouraged me
to "do something" with the information of the mixes – so I
did (Li et al. 1989; Glime et al. 1990).
On the other hand, when Niels Klazenga (Bryonet 15
Figure 16. At least 3 species are tucked in between lobes of
July 2013) collected bryophytes in Borneo in 1997, he Conocephalum conicum. When mixes like these are in
grabbed what he could – as told to by his PhD supervisor, collections it is best to make a minipacket if the mixed-in species
hence including many mixed collections. Curation officers are important. In any case, they should be noted on the label.
at the museum spent ten years cleaning up the mess. Photo by Janice Glime.
Jean Faubert (Bryonet 15 July 2013) disagrees. parent packet should retain the letter a, alerting the
Rather, he admonishes not to try to make single species researcher that there are other packets. If the other taxa
collections. Grab the lot, put it in the bag and DO identify have been identified, they should be listed in the notes
everything you see in it when back in the lab. Sure enough, along with their collection number and letters.
that is when the goodies show up. He declares that most of David Wagner suggests a way to have all the
his lifetime big finds were made that way. Philip E. Hyatt specimens catalogued in the herbarium: Make duplicate
(Bryonet 15 July 2013) agrees and adds that since most labels and file the duplicates for the subordinate species in
info is slowly going on line, someone who is desperate to their appropriate places, but indicate the specimen label
find a specimen won't have too much problem running where the actual specimens are located. This does cause
it down in the future like we might have had to do with problems when the systematics are updated, but can be
mixed collections in the past. In 1990, if you were not in a helpful in locating the smaller associates.
herbarium you probably didn't know the specimen existed. Alas, in large herbaria, as noted by Ambroise Baker
Life changes. (Bryonet 16 July 2013), 1 specimen =1 species at 1
In habitats like the Sahara Desert or epiphylls, species location at 1 collection date. This is also true for higher
are often not separable. Tamás Pócs (Bryonet 14 plants, but it is easier to do for them. As stated by Niels
December 2015) makes a packet with one good specimen Klazenga (Bryonet 15 July 2013), "mixed collections are
of the mix, then makes as many copies of the label as the not okay." But Jon Shaw (Bryonet 15 July 2013) disagrees.
number of species present. Packets with just labels (no My (Glime) own solution to the mixed collection is to
specimens) or just labels are filed by each species, with the make minipackets in which a bit of each minor species is
appropriate species for that location underlined. placed. A sample of the dominant species can also be
As Philip Hyatt suggested, the herbarium in placed in a minipacket to assure the right specimen/species
Trondheim is databasing every species (present in the same is examined. If the community is important, only samples
packet) as a separate record. They have a way to track of each species are removed, but if the specimen is
which of the species is the 'main' species in the packet, i.e. important for taxonomic purposes, I might attempt to
in what cabinet the specimen is stored. This is more remove all the minor associates. Blanka Shaw, herbarium
practical for the data users (and less practical for the curator at Duke, likewise treats the plants that are mixed
herbarium curators), and curation of synonyms is easier this together and a separation is not an option by making small
way, too. fragment packets with a few plants of each named species
The practices and reasons are varied, as demonstrated separated from the rest. "If you spend the extra time to
by a Bryonet discussion in mid July 2013. There are name more than one taxon in the packet, definitely do make
certainly pros and cons for both approaches. Separating the a fragment packet. There is nothing more frustrating than
species is likely to lose the growth habit. Parts may be having a specimen with a rare liverwort in it, that is present
broken and underground structures lost. One loses the in few stems only, and there's no way to find it out among
information gained by determining which species form all the other dried species that look identical in the
associations and how reliable those associations are. dissecting scope." Blanka Shaw further distinguishes
Baranabas Malombe (Bryonet 15 July 2013) also considers between plants associated in the same microhabitat in the
it important to collect and retain all the species in the field and those associated in the collection/packer.
collections to demonstrate the diversity of the site. Blanka Shaw (Bryonet 15 July 2013) does issue a
If it is desirable to have archival specimens of more warning about only listing the species on the same packet
than one species, then removal to a separate packet is as associated species. These species might not be
necessary. If only one species is of interest, it is safest to searchable in some databases. The bryophyte portal
make minipackets to represent the accompanying species <http://bryophyteportal.org/portal/index.php> currently
and to include their names in the notes on the packet label, doesn't enable one to search the associatedTaxa field.
or at least indicate that it is mixed with other species. Keep However, the field is available there for this purpose, and
in mind that beginners may use this collection to learn you can get at the data by downloading the result of your
species. Rod Seppelt (Bryonet 16 July 2013) agrees. search. In the Duke database, there are about 5,000
"While it may be desirable to have monospecific specimens with the associatedTaxa field filled in (out of ca.
collections, in practice it is rarely possible." For example, 160,000 records). But this information is rarely used – she
Seppelt states "I have been looking at Fissidens (collected has never considered it when preparing species lists. When
by the late Ilma Stone); the label clearly indicates that the a species name is updated, the name(s) in the
packet also contains fruiting material of two, sometimes associatedTaxa field does not get updated automatically
three, additional Fissidens. It would be impossible to (=you have to search for every synonym). So, it is not very
separate these into separate collections and still have a practical for the data users. Hence, David Wagner's
meaningful herbarium voucher." As Seppelt points out, "if method of making a separate label to be filed as if it were a
all threads/plants of a particularly species are removed herbarium packet would put it into the database and enter it
from a mixed collection, what can be important information in both the search and nomenclatural updates.
about associated taxa is lost." Dorothy J. Allard (Bryonet 15 July 2013) suggests the
While it is desirable to separate species into separate following from the perspective of a bryophyte collector and
packets, exemplars of intermixed species can be housed in curator:
minipackets within the herbarium packet. When
accompanying species are removed to separate packets, the • If you have enough material, split all of it into separate
collection numbers should be retained, but individualized packets and establish one collection for each species.
by adding a letter at the end of the collection number. The Then in an "associated taxa" field, indicate which other
species are present. Separating material can sometimes label for name changes, verifications, or other notes.
be difficult and destructive. However, this slip of paper can easily come loose, so
• If it is easy and non-destructive you can separate the several options are used. One is to glue the packet to a
material into individual packets. Information on the larger card and attaching the paper, fully glued, to that.
associated taxa is still useful to express on the label. This seems to defeat some of the advantages of the packets
• If you don't have enough material, label the specimen and can create storage problems, unless the packets are in
with a single species and include information about the palm folders, but packets could get tangled with each other,
other species in the packet in an associated species field. causing glue to come loose. Another alternative is to place
If possible include one smaller packet inside for each of the annotations in a waxed envelope and to place that
the associated species with its own label. inside the packet. (Putting it in without protection could
result in smudging or mold.) The disadvantage is that one
It is not unusual for one of the minor species to be the must open the packet to know that something has been
one of interest. Dorothy J. Allard (Bryonet 15 July 2013) added. If the addition is extensive, one could place a note
reports that sometimes she collects a specimen because of a on the outside label instructing one to see inside.
small and interesting liverwort, for example, embedded At the University of Colorado Museum, William
within a clump of Brachythecium. In this case she labels Weber reports that annotations are placed on the back of
the specimen with the name of the liverwort and indicates the packet (with packets stored in boxes or palm packets,
that it is within a matrix of the Brachythecium in a habitat not on herbarium sheets).
field, but she also lists the Brachythecium in an associated NEVER DISCARD THE ORIGINAL LABEL.
taxa field. In essence she treats the Brachythecium as the Handwritten and even typed labels must be interpreted, and
liverwort's substrate. sometimes that interpretation is in error. Keeping the
Ken Kellman (Bryonet 15 December 2015) separates a original label permits researchers to check for possible
collection out when the identification is made. Each will alternative interpretations. And there is always the
become a separate collection with a suffix like a,b,c etc. possibility of transcription error.
On the front of the label, you can then put “growing with
Tortula muralis, Didymodon fallax...) and this information
is included on all the separated packets. But he only does Multiple Access
this if there is some special reason to separate the Guido van Reenen (Bryonet 15 December 2015) has
collection: 1) the separated plant needs to be documented refined his relational database to overcome the problems
for a study; 2) the separated plant is rare or disjunctive etc. mentioned above. In his database structure the specimen
It would take so much time to separate out all collections information is basically stored in two tables, an
that you have to prioritize. In any event, he tries to put all 'Observation' table and a 'Collection' table. In the
species found in a collection on the label so future workers 'Observation' table the information of the specimen is
can read what he was seeing. stored (taxon name, determiner, date, substrate, phenology,
Genevieve Lewis-Gentry (Bryonet 14 December 2015) if there is a microscope slide and/or a photo, etc.). If the
and coworkers use three different options for a mixed specimen is also collected (that is not necessarily the case)
collection: a link is established to the 'Collection' table. In database
1. Put a separate barcode for each different biological jargon: the 'Observation' table has a many-to-one relation
organism on the single packet. Use this to track to the 'Collection' table. In other words a collection can
name changes/annotations for each. contain one or more specimen.
2. Use a single barcode that corresponds to the packet. In the case of multiple specimen in a collection, van
Add many biological organisms to this one identifier. Reenen defines one specimen as the 'main' specimen.
3. Only use a single barcode, pretend it is a simple Mostly this is the most abundant specimen in the
specimen, and note all the other species in a remarks collection. Under this specimen the collection is stored in
field. (This is exclusively what was done with older the herbarium. The herbarium label also lists the names of
systems, but this is never done now.) the other specimens in the collection. He also uses the
method of placing cards in the herbarium to reference to
Their database and workflow centers around barcodes. the physical collection, but that takes too much time and
Everything has to have at least 1 to go into their computer. requires a lot of discipline, especially after a name change.
Their system is flexible so that depending on the situation And it is not necessary anymore because all the information
they can choose to either put many barcodes on a single is now in the database. He does most of the queries in the
packet/sheet/slide/etc. or to put a single barcode with the 'Observations' table, because all other tables in the database
packet etc. and tell the computer that this preparation has are linked to this table and it gives him all the information I
multiple organisms they would like to track separately. need, including herbarium information.
As Claudio Delgadillo-Moya (Bryonet 15 July 2013) Van Reenen also maintains a 'Taxa' table with
summed it up, "What and how you collect mosses and information on the name, if it is a synonym, if it contains
other small plants depends on where you live, the purpose synonyms, place in the taxonomic hierarchy, etc., a
of your research, or what you want the herbarium for." 'Location' table, a 'Persons' table and a 'Relevé' table, to
name the most important. The last one gives some
Annotations
headaches as well, because when collecting in a 400 m2
Sometimes labels are filled with information and little relevé, often one species was collected more than once.
room remains for further annotation. A common practice is And a percentage cover should only be attached to every
to glue one end of a slip of paper to the edge of the packet unique species in the relevé.
The printing of the herbarium labels is done from the rather than glued so that it can be removed without
'Collections' table (Figure 17). The collection is stored in destroying the packet.
the herbarium under Fissidens ornatus.
Figure 17. Sample herbarium label for a mixed collection

from Colombia. Courtesy of Guido van Reenen.
Storage
Cabinets
Herbarium cabinets are the standard method for
storage of preserved plant material. For most tracheophyte
specimens, pest control is essential and it is important that
the cabinets be sealed or nearly so to keep specimens dry
and to discourage pests. Bryophytes, on the other hand, are
usually not bothered by pests, so in less humid climates,
less expensive storage cabinets are acceptable.
But cabinets require lots of space, so many larger
herbaria with larger budgets have converted to compactors
(Figure 18) that are used for both bryophytes and other
plants. Although these can be a nuisance at times, they are
Figure 18. Herbarium compactor at Missouri Botanical
great space savers and also make it somewhat easier to Garden showing cabinet with open door. Labels on the ends
control humidity and pests because access is reduced. denote the contents of that section. Photo by Paul J. Morris
through Flickr Creative Commons.
Packet Storage
Nearly everyone stores bryophytes in packets, but Cargill (Canberra) reports that one can prevent
some herbaria glue the packet to a standard size herbarium specimens from falling to the bottom of the packets by
sheet. This has the advantage that the herbarium can use storing the specimens in polypropylene archival bags. In
the same storage method for the bryophytes as they use for some cases they are also wrapped in Kimwipes® before
tracheophytes. But the packets take much more room this placing them in the bag.
way, and a herbarium sheet is difficult or impossible to put Kerry Barringer (Brooklyn Botanic Garden) reported
under the microscope for closer inspection. It also makes that they were changing their method from packets on
your working space more crowded. My biggest concern is sheets to packets in cardboard boxes (51 x 16.5 x 6.3 cm).
that the large format forces me to remove the specimen to The boxes are open and two will fit lengthwise on a
observe it under the microscope, and when comparing standard herbarium cabinet shelf. They made new packets
several specimens, it is easy to mix them up, returning and photocopied disintegrating old ones to store inside the
specimens to the wrong packet. Single packets can be packet.
placed under the microscope without removing the Those who disliked the placement of packets onto
bryophyte from the packet. herbarium sheets cited concerns such as glue yellowing the
Dale Kruse conducted a survey of bryonet members in packet, glue coming lose, packets getting caught and being
2008 and got a mixed response. Susana Rams Sánchez has torn off, glue catching dirt, difficulty in removing packets
worked with specimens at MA, MUB, BM, E, S, MO and from the sheet (resulting in loss of specimens), greater cost
others. She finds the method at MO (Missouri Botanical for sending loans, more storage space required. To this list,
Garden) to work the best, i.e., packets. Others using one must consider where the packet is to be placed on the
packets included Noris Salazar Allen (Herbarium, sheet. If it is placed in the lower right corner, where a label
University of Panama), Chris Cargill (Canberra), Stephen would normally go, then the stack becomes very lopsided.
Rae (MUSCI Natural Resource Assessment). Rudolf If packets are arranged at random on different sheets, then
Schuster (1966) considered packets in shoe boxes, trays, or it makes sorting through the sheets to find a particular
drawers to be "much better" than pasting the packets to specimen a more difficult job. Placement of more than one
herbarium sheets. He also recommended that if the packet packet on a sheet brings its own problems – renaming
must be affixed to a herbarium sheet, it should be stapled some, but not all, specimens; shipping for loans or
verification of identification, and still has the problem of

locating the labels when sorting through to find something.
David Long (Royal Botanic Garden, Edinburgh), a
proponent of herbarium sheets, cited advantages of gluing
packets to herbarium sheets: being able to use standard
herbarium cabinets, species covers, and genus covers;
specimens do not get lost as easily as those in loose
packets; it is easier to flick through sheets to find individual
specimens (if packets are in a standard position and only
one per sheet); hunting for specimens requires less
handling and thus less chance for damage; specimens are
kept horizontal so that soil does not collect at the bottom of
the packet and damage specimens; specimens are better
protected when sent on loan; there is greater ease to arrange
packets geographically by sheets (this could also be
accomplished in a palm folder); types can have the
traditional red folder and be easier to spot; useful literature
can be placed in the folder with them (Bryonet July 2008). Figure 20. Type specimen packet (red top) among other
packets. The red top is made by a red felt pen. Photo by Janice
Bill Buck (New York Botanical Garden) further
Glime.
supports the use of packets glued onto herbarium sheets.
The greater protection of the specimen seems to be a
primary concern for supporters of this method, including
problems with settling in vertical packets and provision for
extra padding without tight packing. The herbarium sheet
also will accommodate large packets for such taxa as
Spiridens (Figure 19) and Polytrichum; when just packets
are used, large specimens must either be cut into sections or
stored elsewhere. And packets, due to their small size, are
more easily lost, especially when sent out on loan. Figure 21. Close view of type specimen packet among other
Catherine La Farge England (Bryonet 18 July 2008) reports packets. The red top is made by a red felt pen. Photo by Janice
the same reasoning for the University of Alberta Glime.
Herbarium, an approach established by Dale Vitt.
When a palm folder is used, a felt pen can be used to
Type Specimens make a colored dot on the folder to indicate the presence of
Colored folders are traditionally used for tracheophytes a type specimen (Figure 25).
to indicate special collections. Red is standard for type
specimens, whereas blue or other color may be used to
indicate a particular geographic area. The same system can
be used if bryophytes are stored on herbarium sheets and
provides one of the arguments in favor of this method. A
red felt pen run across the top of a packet will serve the
same purpose (Figure 20-Figure 21), or a red herbarium
folder can be cut to fit around the packet (Figure 22).
Figure 22. Sample type specimen folder for bryophyte

When slides are made of the type specimen, it is

important to retain the slide with its specimen (Singh Deo
& Majumdar 2020). These can be mounted in a permanent
medium, or allowed to dry on the slide. For the latter, in
particular, the slide should be placed in a small packet to
Figure 19. Spiridens flagellosus, a large epiphytic moss. prevent loss of the specimen. These latter ones can be
Photo by John Game through Flickr Creative Commons. rewet to position the specimen as needed.
Storage Containers Palm folders can be stacked so that 6-7 will fit on one
standard herbarium shelf (Figure 25). The folders will
For those using 4x6 (10x15 cm) packets, a 4x6 card allow specimens up to 27 mm thick. This permits a
file cabinet can be used to hold the packets. It has a pull- collection of various sizes to remain together. Jim Shevock
out drawer that can be removed and a movable back that points out that a further advantage is that the 27 mm
can hold the packets up even when the drawer is not full. thickness permits labelling the end of the folder (Figure
Brian Eversham uses plastic boxes that can hold a 25), making it easier to find the right folder.
double row if the packets are folded small enough (Figure
23). I use shoe boxes because they are free at the local
shoe stores and keep the packets covered, avoiding excess
dust. It is easy to attach a label to the end of the box to
indicate the part of the alphabet contained therein. I try to
leave enough room for half as many more packets to be
added, i.e., 2/3 full.
Figure 23. Herbarium drawer with packets. Photo by Brian

Eversham.
Packets on herbarium sheets can be stored in a

standard herbarium cabinet, and that seems to be the main
asset for those who prefer them. The boxes or drawers,
however, can also be stored in a herbarium cabinet and
require much less space than a packet plus herbarium sheet.
Bryophytes are seldom eaten by pests in a herbarium,
unlike tracheophytes, so most bryologists store them
without mothballs or other deterrents.
Palm Folders
Palm folders were originally constructed to handle Figure 24. Herbarium palm folders showing arrangement of
large or thick tracheophyte specimens like palms, hence the packets with a variety of labels, some as part of the packet, others
name. Palm folders can hold 10-20 packets, or even more, glued on. Note the map on one of the labels indicating its location
depending on the size and thickness of the packets. Those in the state of Nevada. Photo by Jim Shevock.
using packets placed in palm folders (Bryonet July 2012)
included Jaakko Hyvönen (Plant Biology, Helsinki), Dan
Norris (Berkeley University Herbarium), Xiaolan He-
Nygren (Helsinki), and Jim Shevock (California Academy
of Sciences). This method permits the packets to lie flat,
overcoming the crushing problem and the problem of
having specimens collect at the bottom of the packet in a
pile of soil.
Dan Norris (Bryonet July 2012) cites the flexibility
Susana Rams Sánchez warns against making species
offered by palm folders for having different sizes of
packets to accommodate large specimens. The folders are sheets with more than one packet glued to them. These
30.5 mm x 56 mm and have additional flaps on each side, will soon be a problem as identifications change with
top, and bottom (Figure 24). The large size of the folder, revisions. And when specimens are sent for loan, all the
like the large herbarium sheet, can accommodate large packets must be shipped, making them unavailable at the
specimens like Spiridens (Figure 19) or Dawsonia. home herbarium and increasing shipping costs.
Figure 26. To make boxes, use scissors, razor knife, or paper

Figure 25. Herbarium cabinet with palm folders. Photo by cutter to remove 4.2 cm from long side of a 43x61 cm genus
Jim Shevock. cover. Save trim as template for trimming additional genus
covers. Score with hard metal edge such as door key and
fold/unfold along indicated lines. Scoring controls exact line of
fold. Rub smooth, hard object down folded edge to make creases
sharp. To save measuring scoring lines, create template strips
from cardboard or genus covers to guide scoring tool.
Storage Boxes from Genus Covers
If you are familiar with large herbaria, you are familiar

with the heavy poster board or Manila folder quality of
genus covers used for storing tracheophytes. Davison
(2002) suggests using these for making storage boxes for
bryophyte packets. These are similar to the palm folders,
but the ones Davison has designed are the width of a
"standard" packet and are not covered. The following
instructions (Figure 26-Figure 28) are only slightly
modified from his:
The finished box occupies the full length of a standard
herbarium cabinet. Two boxes fit side by side on the shelf.
The boxes can hold 40-130 upright specimens, depending Figure 27. To form corners of box, push slightly inward at
arrows and align edge a with edge b. Hold edges a and b firmly
on the size of the specimens. Be sure to measure the shelf together and crease from inside. The corner crease will find itself
size of your cabinet before making the boxes because the as you align, meet, and hold edge a to b. Press firmly while
cabinet sizes can vary somewhat. creasing. Fold/unfold all four corners.
Size 3 (4 3/4 x 3 3/8 x 7/8): ~pricing range $0.72 to $0.88

Size 4 (7 5/16 x 4 15/16 x 7/8): ~pricing range $0.93 to
$1.19
Purchase is direct from:
Jay Cordeiro
Northeast Natural History & Supply Co.
Distributor: HH Elements, Inc.
24 North Grove Street
Middleboro, MA 02346 USA
<unionid@comcast.net>
Figure 28. Once all folds have been made, shape the box and
adhere each end with tape, glue, or staples. Davison uses clear 2"
(5 cm) wide commercial-grade box packing tape and cover the
entire outer face of each end. The tape provides a tear-resistant
surface for taping and removing labels that identify the box
contents. If handled carefully, as specimens should be, the boxes
are adequately sturdy. Placing cardboard inside the upright ends
strengthens the boxes but is not required. Figure 29. Wooden tray. Photo by Jay Cordeiro, Northeast
Natural History Supply Co.
Specially Made Storage Boxes

Jay Cordeiro of the Northeast Natural History &
Supply Co. supplies herbarium drawers and trays to your
specifications (Figure 29-Figure 34). Specimen drawers
and unit trays are designed for curation, storage, rehousing,
and display. They can be used for shells, minerals, skeletal
material, feathers, eggs, skins, anthropological objects,
fossils, glass vials, and memorabilia, as well as bryophytes.
Archival trays are custom manufactured to any dimension;
they are rigid, unbuffered, and acid-free with neutral pH.
Trays are constructed of white corrugated cardboard, come
free-assembled (not flat and self-folding), are overwrapped,
and nested for maximum storage efficiency. Archival Figure 30. Wooden tray. Photo by Jay Cordeiro, Northeast
drawers are available in standard sizes to fit typical Natural History Supply Co.
Cornell, California Academy, and National Museum of
Natural History style storage cabinets. Trays can be lined
with plastazote or ethafoam, unbleached cotton, or
polyester batting for use with delicate specimens. Lids are
optionally available for better protection from ambient
environmental damage and for use in layered storage. The
trays are sturdy and affordable.
The company does not have an online catalog because
their product is custom designed. The trays and drawers
can be purchased in sets of 100 or more. Sample sizes and
prices include:
Size 2 (2 3/8 x 3 5/16 x 7/8): ~pricing range $0.62 to Figure 31. Wood pull of wooden tray. Photo by Jay
$0.76 Cordeiro, Northeast Natural History Supply Co.
Glime once stored Splachnum ampullaceum in a plastic

film can.
Preservatives should be avoided so the material can be
used later for DNA or chemical analysis. If preservation is
necessary for maintaining the morphology of a liverwort,
maintain some of the specimens in preservative and others
dried, and in some cases (flat thallose species), like
Conocephalum (Figure 35), pressed. Rob Gradstein (pers.
comm. 26 July 2012) suggests using FAA (fenyl-acetic-
alcohol). This preservative served Barbara Crandall-Stotler
for her morphological work and Rudy Schuster for making
the drawings used in his liverwort volumes.
Figure 32. Box opener of wooden tray. Photo by Jay

Cordeiro, Northeast Natural History Supply Co.
Figure 35. Conocephalum conicum, a mostly flat thallose

liverwort that preserves better if it is pressed. Photo by Robert
Figure 33. Herbarium drawer with boxes. Photo by Jay Klips.
Cordeiro, Northeast Natural History Supply Co.
Gradstein (pers. comm. 26 July 2012) also suggests
that dry, shrunken herbarium material of thallose liverworts
can be rehydrated and stained with methylene blue (see
Rico 2011), a method that works well for him in studying
Riccardia (Figure 41). Rico developed this method of
rehydrating the moss in a solution of sodium hypochlorite
(commercial bleach) diluted to 20% in distilled water. This
restores the form of the liverwort and the structure of the
cells. The cells are cleared, making observation easier.
Species like Riccia fluitans (Figure 36) can be teased
apart and floated onto a 3x5 (7.6x12.7 cm) card. The algae
on these aquatic plants will serve as a glue to make them
adhere to the card. Once affixed, they will retain their
shape and remain flat.
Figure 34. Trays. Photo by Jay Cordeiro, Northeast Natural

History Supply Co.
Preservation
Most specimens will keep well in packets if they are
not packed together too tightly. If a specimen has fragile
parts sticking out, it helps to pack crumpled tissue paper
around the specimen. An alternative is to cut out space for
the specimen in a piece of styrofoam or corrugated
cardboard of appropriate thickness and dimensions.
However, if the specimen falls out of the styrofoam, it may
suffer even greater damage than with no packing, so it Figure 36. Floating form of Riccia fluitans, a species that
might be necessary to staple or tape a minipacket in the cut can be floated on a card before putting it in a packet. Algae help
out space. Small jewelry boxes can sometimes be useful. to glue it to the card. Photo by Janice Glime.
Since leafy liverworts will lose their oil bodies upon The disadvantage of systematic arrangements is that
drying, one should preserve them by a photograph that our knowledge of bryophyte systematics is constantly
shows details of the oil bodies. In addition, describe the oil changing. The publication of Shaw and Goffinet (2000)
bodies in detail. moved a lot of genera to other families and split some
families. Because of the instability of our understanding of
Cool Preservation the systematics, the cabinets would require an updated list
Bryophytes prosper in cool temperatures, so it is not of the locations of each genus and family. Flora North
surprising that cooling them during drying can improve the America is making further changes. As we gather more
quality of the specimen over air-drying. Victor Ardiles H. molecular information we keep moving things. Hence, this
(Bryonet 25 September 2013) reports that both arrangement can be expensive because it would require
gametophytes and sporophytes look better when fresh constant monitoring and rearrangement whenever a taxon
samples, still in their paper bags, are placed in a freezer for has been moved or redefined.
15 days at 7°C, 37% Relative humidity. The method The alphabetical arrangement is more practical. In
discourages fungal growth and retains colors, leaf details, some cases, the packets are arranged in families with an
structure of thallose and leafy liverworts, bottle liverworts, alphabetical arrangement of families. In other herbaria, the
and hornworts. The low temperature slows the dehydration genera are arranged alphabetically with no family
of the tissues, a more natural approach. groupings. The latter arrangement is the most stable
arrangement.
Minute Species and Special Structures Jim Shevock reports that the University of California
The really tiny species can present special problems herbarium files their bryophytes alphabetically by genus
because they are easily lost among the soil in the packet. (Figure 25). My own experience is that most bryological
Several methods can help to make these locatable in the herbaria use that method because it is easier and less
future. One popular method is to remove some of the expensive to maintain. Missouri Botanical Garden uses
specimens from the soil and place them in a minipacket family groupings. At the California Academy of Sciences
(Rothero & Blackstock 2005) or small envelope without the genera are filed by family, but the genera and species
the soil. Another possibility, suggested by Richard Zander are filed alphabetically within the family, and the families
(pers. comm. 1 August 2012) is to glue the plants (without are arranged alphabetically.
soil) to a white card with a polyvinyl-alcohol-based glue. I like the advice of Jaakko Hyvönen (Bryonet July
That glue is water soluble, so the specimens can be soaked 2008) regarding phylogenetic vs alphabetical: we are...still
loose. too far away from the classification that would enable
In some cases, only a few plants may have capsules, arrangement accordingly. Alphabets have been pretty
antheridia, archegonia, or propagules. To help avoid loss stable for quite some time and this makes it easy for ALL
of these important structures, make a small packet or use a people (most of whom are NOT bryologists) to locate
small envelope to store these within the species packet specimens in collections. On the long run one would be
(Rothero & Blackstock 2005). Microscope slides can also able to save a LOT of precious volunteer, student etc.
be put in a small packet and stored within the species herbarium time by adopting this simple system. At the
packet. They may survive better in a waxed paper same time, need for rearrangement is minimized.
envelope because the slide can be sticky and the paper may
stick to the slide. The waxy surface can reduce this but Guide Cards
won't necessarily eliminate it. A practical way to help the user is to provide guide
cards. William Weber (University of Colorado Museum)
Herbarium Arrangement uses blue cards for Colorado material and yellow for other
areas. A salmon guide card indicates the genus, yellow the
There are two choices in widespread use in the species. Alternatively, one could color code the top of the
arrangement of bryophyte herbaria – systematically or packets with a felt pen. Note that red is reserved for type
alphabetically. They each have their advantages and specimens.
disadvantages, so one needs to choose based on resources When a herbarium is rearranged or names change,
and needs. The majority of those who commented on this guide cards can be placed where the alternative name
to Dale Kruse in his survey preferred a strictly alphabetical would occur, directing the user to the location of that
system. group. This can be useful if staff lack the time to rearrange
The systematic arrangement provides groupings that the collection. A guide card can be placed where the new
make it easier for someone making a systematic study. All name should be, directing users to the name on the packets.
members of a family would be grouped together. This
method is further divided into choices – systematic or Herbarium Care
alphabetical arrangement of genera. Richard Zander
(Bryonet 13 November 2008) considers this family Pest Control
grouping with alphabetical arrangement of genera to be "a Pests can be a problem in a herbarium, and methods to
nice compromise." Rod Seppelt (Australian Antarctic eliminate or minimize them can be detrimental to future
Division) practices a further compromise to group genera studies that rely on untreated material for historical
into the family, but to arrange the families alphabetically. pollution studies or DNA testing. In November of 2010
This solves the problem of trying to linearize the non-linear there was a discussion on bryonet-L regarding means of
systematics of families. One could also arrange the species eliminating pests without compromising future studies.
systematically, but that does not seem to be a common Historically, most bryophyte herbaria have not treated
practice. for pests with the same care as that used for vascular plants.
For example, beetles can be real pests among tracheophytes prevent the introduction of agricultural pests, Rod Seppelt
and some algae, but are usually not rampant among (Bryonet 13 April 2016) warns that it should not be used on
bryophytes. The popular belief that nothing eats them let specimens to be used for molecular work. Rather, he
of a somewhat false confidence in storing the with no recommends freezing.
pesticide treatments. However, if you have ever tried to
import them into a country, you know that the border Freezing
quarantine agents are concerned about pests in the soil, and
this alone should suggest that the bryophytes may introduce It appears that the safest and most common method in
pests into the herbarium. Scattered publications, and current use is freezing. And this is standard practice in
especially more recent ones, as cited in the interactions many herbaria (Figure 37). In this method, one
volume on this website, demonstrate that our assumption recommendation is to freeze the packets for 24-48 hours;
that nothing eats bryophytes was incorrect. the process should be repeated annually to maintain the
pest-free environment (Denis Oliver, Bryonet 26 November
Agral 600 2010). Rod Seppelt (Bryonet 26 November 2010)
As mentioned in the Laboratory Techniques recommended three days at -18ºC for material collected in
subchapter on Slide Preparation and Stains, Tom the region or -18ºC for seven days if it has come from a
Thekathyil (Bryonet 12 May 1210) submerses the different biogeographic region or outside the country. He
bryophytes in Agral 600 (horticultural wetting agent). It later modified this (Bryonet 6 February 2012), based on a
kills the animal life that often accompanies the bryophytes response from the herbarium in Auckland, New Zealand.
but does not seem to affect the plants. They found that a more effective treatment was to use
cycles of room temperature to -15ºC over a few days. It is
Moth Balls (Naphthalene) kinder to the bryophytes and more effective against insects.
For tracheophytes, the standard treatment has been to At Christchurch (CHR), freezing is for 7 days at -20°C
put moth balls in the cabinets. These have contained such (Allan Fife, Bryonet 15 August 2002).
compounds as naphthalene (highly flammable and At the University of Alberta Herbarium (ALTA)
carcinogenic), 1,4-dichlorobenzene, or camphor. These specimens are frozen at -20°C (Catherine La Farge
all have strong odors that are very offensive to some England, Bryonet 15 August 2002). The specimens are
people, especially when they work for many hours in that stacked as single sheets or only a few sheets overnight;
environment. larger stacks are stored at that temperature for four days to
Rod Seppelt (Bryonet 26 November 2010) reports be sure the center gets cold enough. The specimens are
using fumigation with Pyrethrum in a spray. The plant that sealed in poly freezer bags in the freezer and kept in them
produces the Pyrethrintn, however, is known to cause until they reach room temperature afterwards, for up to a
human health problems among long-term growers of the day for larger stacks. A similar procedure is followed at
plant. the New York Botanical Garden (NYBG) and Missouri
Botanical Garden (Marshall Crosby, Bryonet 15 August
Microwave Oven 2002), where freezing is for 3-4 days (Barbara Thiers,
A more recent method for killing bryophyte Bryonet 15 August 2002).
inhabitants has been to put them in the microwave oven,
but such treatment renders the bryophytes unusable for
future DNA studies due to the ability of the gamma rays to
alter the DNA. Lars Hedenäs (Bryonet 30 November
2010) reports that the Swedish Museum of Natural History
would never send material to another herbarium if there is
the danger that the material on loan would be subjected to
microwaves. The risk of destroying DNA would "seriously
reduce its value for future research."
Wagner finds that the microwave is not effective,
largely because of the uneven distribution of microwaves
inside the oven. The oven has the further problem of being
too small unless you purchase a commercial grade oven.
Wagner had a friend who trapped a fly inside his otherwise
empty microwave, turned it on for 60 seconds, and when he
opened it the fly flew out. It had survived by cowering in a
safe corner. Some herbarium material absorbed
microwaves and overheated. Wagner has even had charred Figure 37. New specimens are being placed into the freezer
herbarium specimens, and blackened paper under them, at Beaty Biodiversity Museum in Vancouver, BC, Canada. Photo
that resulted from too long a treatment. by Derek Tan, copyright Beaty Biodiversity Museum
Bromomethane (Methyl Bromide) At the Helsinki Herbarium (H), all loans are frozen for
at least a week before putting them in herbarium cabinets
Juan Larraín (Bryonet 13 April 2016) raised concerns (Johannes Enroth, Bryonet 15 August 2002). The same
that Chile requires that all specimens with soil, including procedure is followed at the British Museum (Brian
bryophytes, must be treated with bromomethane before O'Shea, Bryonet 15 August 2002). At the Chicago Field
entering the country. While this may be a good means to Museum (Matt von Konrat, Bryonet 15 August 2002), new
specimens and loans are frozen unless they are to be used particularly in the tropics. An alternative in temperate
for DNA analysis or study of oil bodies in liverworts. The climates is an air-conditioning system that chills the air
concern is more for the protection of other plants and fungi before heating it, thus dehumidifying it. Keeping
in the herbarium since bryophytes are seldom eaten by vulnerable specimens, especially fungi, away from the
herbarium pests. Fungi are particularly vulnerable. bryophytes solves a lot of the problems, especially if low
Lloyd Stark (Bryonet 15 August 2002) warns that humidity can be maintained.
freezing as described above may be too effective, killing The downside to all this pest control is that the
the bryophytes as well as the pests. In regeneration tests on specimens are no longer suitable for longevity tests on
specimens from UC, MO, and Cal Acad, mosses were not spores or plant tissues and might not be usable for DNA
able to regenerate, but members of Pottiaceae that had not testing. The specimen label should indicate treatments
been frozen were able to regenerate several years later. At such as these to protect against faulty conclusions by
the University of Nevada Herbarium (UNLV), the dry people using the specimens for physiological purposes or
climate makes freezing unnecessary. David Wagner agrees DNA analysis.
that low humidity is almost as effective as low temperature Eva Krab (Bryonet 3 February 2012) found that a
for controlling typical herbarium pests. number of approaches did not work. After a number of
Rod Seppelt (Bryonet 16 August 2002) reports the failed attempts, she took the approach of flushing the moss
additional precaution of freezing specimens that have been cores [Sphagnum fuscum (Figure 38) and Hylocomium
taken out of the herbarium cabinets for more than a few splendens (Figure 39)] in a gas-closed chamber with 100%
hours. If the specimens are kept in the herbarium facility, CO2 for 12 hours, then leaving the cores at room
overnight freezing is usually adequate. If they reside temperature for 24 hrs (so that eggs would hatch) before
anywhere else while outside the cabinets, they are frozen freezing them at -20°C. But even after 3 rounds of all
for several days. those treatments – and still no success – the springtails
At the Provincial Museum of Alberta (PMAE), the were still active! (It worked a lot better in the Hylocomium
procedure is even more extreme. They do a quick freeze to cores than in the Sphagnum cores.) The mosses actually
-70°C for small accessions (fewer than 50 specimens). For survived these treatments surprisingly well. These were
larger collections they fumigate. Roxanne Hastings subarctic springtails, so maybe temperate springtails might
(Bryonet 16 August 2002) reports that creatures are killed be more sensitive to the freezing part of the cycle.
within 24 hours at the very low temperature and have no I like the suggestion from Javier Martínez-Abaigar
chance to acclimate to it. (Bryonet 3 February 2012). He suggested using a Berlese
Herbarium personnel have done some experimenting, funnel (Figure 40) to chase the springtails out of the moss,
although it may not appear in the literature. John Braggins then returning them to their natural habitat.
reported to Rod Seppelt (Bryonet 26 November 2010) that
multiple freezing events were more effective than a single
event. He found at AK that silverfish could be killed with a
number of cycles, from room temperature down to -6ºC or -
10ºC and back to room temperature. That procedure was Insect Traps
more effective than just one cycle to -10ºC. Freezing Some passive means include insect traps, apparently
overnight is most likely useless. After all, these organisms somewhat standard procedure in large herbaria, but these
survive such cycles in nature in many parts of the world. are ineffective against eggs that may be dormant for long
Rod Seppelt (Bryonet 6 February 2012) also reported that periods, causing new outbreaks when new material is
he had greater success with several low temperature (- introduced. What traps adults may not work for larvae that
1°C)/warm temperature cycles for several days. The sit and chew on bryophytes and packets for weeks or
multiple freezing event treatment seems to be gaining months.
popularity, and many of the herbaria cited above may
already be using it.
Domestic freezers vary in their temperatures, but
generally only go down to about -15ºC, and depending on
their arrangement may have zones that are warmer or
slightly colder.
Jeff Duckett (Bryonet 26 November 2010) points out
that one advantage to freezing the bryophytes is that it does
not always kill the bryophytes, despite killing their
inhabitants. These are plants that can spend the winter,
often for three months, under snow, or in many cases
exposed with no snow ab below freezing temperatures. In
the polar regions they survive in areas that may be snow-
free for some time at very low temperatures. Yet these
species survive. Such is probably not the case for tropical
bryophytes.
Adequate freezing facilities are not available in many
herbarium locations. David Wagner (Bryonet 16 August Figure 38. Sphagnum fuscum, a hummock moss that
2002) suggests that baking or poisoning, coupled with survives cryopreservation with a pretreatment in 100% CO2 to
closely contained quarantining may be necessary instead, eliminate pests. Photo by Michael Lüth.
Figure 39. Hylocomium splendens, a moss that survives

cryopreservation with a pretreatment in 100% CO2 to eliminate
pests, but invertebrates do not survive as well as those on
Sphagnum. Photo by Janice Glime.
Drowning
Eleanor Edye (Bryonet 2 February 2012) found that
washing the collections with a surfactant before drying
them increases the effectiveness in killing them. She
reports that springtails usually have a very hydrophobic
cuticle and thus tend to float. Forced immersion in water
will reduce their populations. If bryophytes are the only
concern, some of the predatory mites will eat the springtails
but not the bryophytes.
Steam Sterilization
Soil can be sterilized with steam. While this will most
likely kill the pests, it will likewise kill the bryophytes.
Rod Seppelt (Bryonet 16 December 2009) reported that
Alison Downing found that some bryophyte spores, such as
the thick-walled spores of Riccia species, survive standard
autoclaving of soil.
UV radiation can be used to sterilize the air and even
for a short distance (a few cm) into water (Javier Martinez-
Abaigar, Bryonet 16 December 2009). However, soil
shields it, so it is not an effective tool for sterilizing soil Figure 40. Berlese funnel showing its parts. A = liquid for
adhering to bryophytes, and most likely will not kill trapping insects (not needed if you want to keep them alive). B =
invertebrates hiding among the bryophytes. air space above liquid in jar or bottle. C = lid with hole for funnel
end. D = open funnel area with screen or wider mesh on top. E =
area of funnel where sample is placed. F = space above funnel to
Moisture Control avoid scorching bryophyte sample. G = light/heat source that
causes invertebrate inhabitants to go downward to escape.
Moisture is another challenge in some herbaria, Drawing from Creative Commons.
especially in the tropics. Fungi may appear as tiny hairs
projecting upward or as a mass of hairs forming a mat. In
worse cases they may form spores that spread easily to
other specimens and that are not healthy to breathe. Roxy
Hastings (Bryonet 26 November 2010) found that fungi
could be a problem at relative humidities above 40%. Silica Gel
Modest problems can be controlled with silica gel
packs, available from herbarium suppliers (Roxy Hastings,
Dehumidifier
Bryonet 26 November 2010). They are available from
Use of a dehumidifier may be sufficient in some cases "Herbarium Supplies" to maintain various humidity levels
to prevent the growth of fungi and bacteria, but it adds to in the range of 25-40%. These packs can be "recharged"
the operating expenses and may be insufficient in large by putting them in an oven to dry and usually provide a
herbaria in very humid climates. color indicator of their state of moisture.
Herbarium Cabinet Materials import regulations and the name of the country can
be a good start.
If you choose to make your own cabinet for herbarium 8. Remove as much soil as possible.
specimens, be aware that particle board can contain 9. Be sure the specimen is dry and in paper, not plastic,
formaldehyde in the glue, presenting a long-term health to avoid mold.
risk (Rod Seppelt, Bryonet 26 November 2010). 10. Get permission from the recipient before sending the
Herbarium cabinets are usually made of metal with a specimen. Otherwise, you might never see your
somewhat spongy material around the door to seal it. A specimen or any identification again.
good cabinet will not allow pests to gain entry unless they 11. Provide a clear address and email address for
travel with the herbarium specimen. providing you with the names of bryophytes
identified.
12. If you have a target deadline, be sure you discuss that
with the person identifying before you send the
Sending Specimens for Identification specimens.
13. Try not to send more than three specimens at a time
Understanding accepted courtesy and rules for sending
so the task will not seem so daunting to the recipient
bryophyte specimens can make it easier for one to get
(Zander 1993).
much needed help. These guidelines should keep you out
14. Don't include more than one species in a packet if you
of trouble and avoid misunderstandings:
can avoid it. If not, tease out the individual species
1. Assign each of your collections a unique collection
and put some branches of them in small packets
number. Many bryologists pre-number collection
within the larger one, giving each the same collection
bags and keep a life list of numbers to avoid
number but a unique letter to distinguish it (and keep
ambiguity.
duplicates of the individuals). It is important to
2. When mailing a specimen for identification, keep part
maintain the growth form to help in identification.
of the sample yourself and be sure it has the same
collection number on both yours and the identifier's 15. If you are borrowing samples for DNA analysis or
packets. This will permit correspondence with the other destructive purpose, be sure the
least ambiguity. person/institution loaning them understands that, and
3. Be prepared to donate the collection to the person be sure that at least some material is left for
doing the identification (Loeske 1925; Raup 1926; verification by anyone later.
Zander 1993). This is a courtesy for the time that 16. Include in your packet a carefully prepared slide with
person spends helping you. Be aware that some a semi-permanent or permanent mount of the
recipients will assume that the specimen is now specimen of interest, including stem leaves, branch
theirs. leaves from the middle of the branch, a short branch
4. Make it clear who will be considered author(s) of any from which the middle leaves have been removed,
scientific publications resulting from the and if available, a peristome (Holzinger 1900). It is
identification. If possible, offer to make the also very helpful to provide a permanent mount slide
determiner a co-author. If there is a reason you can't of leaf cross sections. These inclusions will save
do this, explain why you must be the only author. considerable time for the identifier and make it more
Establishing this at the onset can avoid awkward likely that you will get your identifications in a timely
misunderstandings. manner. These should be protected in a small
5. Include details of name of collector, collection envelope within the packet.
number, date, substrate, habitat, and location on the 17. Karen Golinski (Bryonet 12 July 2012) suggests
packets, including latitude, longitude, and more providing a spreadsheet with the collection numbers
precise coordinates, including GPS if possible. and collection information with space for adding the
6. Include on the packet label any notes that might be name. This makes it easy for the identifier to provide
important. Information included in an accompanying you the names and makes it easier for that person (or
letter will usually not be added to the label by others. you) to add the information to a herbarium database.
7. Check and follow the import/export laws regarding 18. Make an attempt to identify your specimens before
herbarium specimens in both yours and the receiving you send them to experts. Not only will you learn
countries. Usually it is sufficient to label a package more this way, but it makes the task less daunting for
as "herbarium specimens, no commercial value," but those helping you. And some bryologists will take
some countries have very rigorous import standards the time to tell you where you went wrong in those
to protect against introducing soil organisms and that are identified incorrectly. David Wagner
disease, and more recently, against collections of rare (Bryonet 12 July 2012) states "First, for anybody
or endangered species. For example, specimens sending specimens to an expert you have not had
entering Australia and New Zealand require communication with before, send only one or two
paperwork in advance and treatment protocols (Rod specimens with your best guess as to identification.
Seppelt, Bryonet 12 July 2012). The sender or This is far more likely to get a prompt response. As
recipient may have to pay inspection and/or soon as somebody sends me a box with a dozen or
fumigation costs, the alternative being destruction of more specimens, it gets put on a shelf as a 'when I get
the specimens. And in some countries you could get time to do it' task. This shelf has accumulated
the recipient in trouble because the necessary specimens for many years with most boxes advancing
paperwork is lacking. A Google search for plant only very slowly to the top of priority tasks. Send me
one specimen and I'll look at it and respond within 24

hours. Offering to pay helps to advance priority
ranking, of course." Wagner also points out that your
identifications will give the expert some idea of your
level of expertise.
19. Make use of the internet to see if your identification
matches the images there. But of course be aware
that some ID's shown there may be wrong.
20. If you took pictures of the bryophytes and their
habitats, send the best of these to the person doing
identification (Figure 41-Figure 43). Field growth
habit can help in the identification, and if the pictures
are good, they are an additional way of saying thank
you to the one helping you. Photographs of
microscopic characters are even better.
Figure 43. Riccardia cf. elata microscopic view posted to

Bryonet by Zhang Li for further help in identification. This view
enabled Bryonetters to be certain the species was not a fern, but
rather one of the liverwort Riccardia. Note the oil bodies. The
suggestions were narrowed to Riccardia elata or R. prehensilis.
Photo by Zhang Li.
Current Names
The choice of references depends on your geographic
location, so it would most likely be of little help for us to
make suggestions. Old references can be useful if you
check the names in TROPICOS
<http://www.tropicos.org/> for nomenclatural changes. If
Figure 41. Riccardia cf. elata, posted to Bryonet by Zhang you can't locate them there, Google might help, or The
Li for help in identification. With only this view, Bryonetters Plant List <http://www.theplantlist.org/tpl/record/tro-
suggested the fern Hymenophyllum. A view showing its habitat 35156923>. Be careful of your spelling – these lists don't
and growth habit might have helped. Photo by Zhang Li.
find similar spellings. They don't care about capitalization.
Bryologists will always disagree among themselves
References about generic placement using the Linnean naming system.
This generates healthy discussion about relationships, but
Most bryologists identify their specimens at a bench creates problems for a herbarium and the ability to relocate
surrounded by two microscopes, dissection tools, a specimen. It is best for a herbarium to choose a published
herbarium specimens, and an assortment of references. classification system and be consistent in its use. If a
These references need to be both broad (floristic treatments different system is chosen, then the entire collection should
with keys and descriptions) and specific ones for the family be updated. This might be at the generic or family level,
or genus (often as monographs or journal papers). A good not necessarily at the level of the entire herbarium. But it
glossary such as that by Malcolm and Malcolm (2006) is should not be store partly by phylogeny and partly by
also helpful, especially for the beginner. alphabet at the same hierarchical level.
Two easy sources for names, authors, synonyms, and
currently accepted legitimate names are TROPICOS
<http://www.tropicos.org/>, a service of the Missouri
Botanical Garden, and The Plant List
<http://www.theplantlist.org/>, a collaboration between the
Royal Botanic Gardens, Kew, and the Missouri Botanical
Garden. I (Glime) don't like the higher level classification
used by the Missouri Botanical Garden (it puts the
bryophytes in the class Equisetopsida to use classification
levels considered commensurate with those of animals). At
least I don't have to look at them in the Kew list!
Indexing
Wagner recommends indexing your taxonomic
Figure 42. Riccardia cf. elata, posted later to Bryonet by reference books. He found the books more inviting when
Zhang Li for help in identification. With the addition of this they were easier to use, and found that the accuracy of his
view, Bryonetters could be more certain the species was one of work definitely increased as a result. With bryophytes he
Riccardia. Photo by Zhang Li. indexes the major genera. Figure 44 shows an indexed
copy of Paton's "The Liverwort Flora of the British Isles." The second type of index Wagner uses is an invention
Figure 45 shows the thumb tabs in greater detail and Figure that began with an address book. The index is printed on
46 shows the method for cutting them. An index card only one side of the paper and stapled on the right side.
serves as template for the area to be cut out. A cutting mat This might seem counterintuitive because most booklets
is placed ON TOP of the page to be indexed. Wagner uses have the binding on the left side when it is face up.
a #11 scalpel blade to cut through forty pages (twenty However, because our writing is from left to right, it means
sheets of paper). The important part is to plan which pages the words to be indexed will appear lined up on the left side
will be indexed. It is easy to want to do too many and run of a sheet. (This might need to be reversed for some Asian
out of space on the outer margin of the book. countries.) By staggering the sheets and trimming on the
left, any item is quickly found. There are two examples
here. The first (Figure 47) shows an index to the five most
common references Wagner uses for liverworts, directing
one to the pages for species of liverworts found in Oregon.
The species are designated by six letter codes. This index
is kept with the appropriate books on the workbench or
book shelf.
Figure 44. Index tab indentations in The Liverwort Flora of

the British Isles. Photo by David Wagner from Bryological Times
#136 (2012).
Figure 47. Excel file printout of six-letter liverwort codes,

showing reference where it can be found and page. Photo by
David Wagner from Bryological Times #136 (2012).
The second example of this kind of index is an older

one, made in 1998 when Wagner was doing extensive
cryptogam inventories in southwestern Oregon. It has all
the mosses and liverworts known from the entire state,
almost 700 names (Figure 48). It is useful to check spelling
Figure 45. Close view of index tab indentations in The or authority of a name when typing memos, labels, or
Liverwort Flora of the British Isles. Photo by David Wagner from annotating. Again, the names are designated by six letter
Bryological Times #136 (2012). codes manufactured for rapid data entry both in the field
and when databasing. By using small type all 678 names
fit onto 14 pages.
Figure 48. List of all bryophytes found by David Wagner in

Figure 46. Tools for cutting indentations in page edges. Oregon, used to check spelling and supply authors. Photo by
Photo by David Wagner from Bryological Times #136 (2012). David Wagner from Bryological Times #136 (2012).
Herbarium Programs Sharing Images

There are several programs on the internet, e.g. Many herbaria have web pages where they provide
<http://www.flmnh.ufl.edu/herbarium/pl/>, to help make it images of bryophytes. If you choose to set up your own
easier for you to produce labels and make a herbarium web page, a few guidelines will make it more useful.
database. If you can use Access, UC Davis has a free Make it clear what you consider fair use. If you prefer
Herbarium Management System to download restricted use or permission, provide contact information
<http://herbarium.ucdavis.edu/database.html> that allows for those seeking permission and make clear what
you to print up labels from Access data (Stephen Lodder, information you will need to give that permission (e.g.,
Bryonet 12 July 2012). intended use, size and resolution, whether it will be
Specify 6 is another free herbarium program modified, attribution, web address). When I (Glime)
<http://specifysoftware.org/>, funded by the U.S. National request images for this book, I state that the image is for an
Science Foundation. This program can handle specimen online book on Bryophyte Ecology
data for computerizing collections, tracking specimen and <www.bryoecol.mtu.edu>, sponsored with no financial
tissue management transactions, and moving species data support by the International Association of Bryologists and
to the internet. It runs on Windows, Mac OS X, and Linux the Department of Biological Sciences of Michigan
operating systems. Technological University. I clearly state that I will give
One consideration for computer programs is that their credit for the image and ask if there is additional attribution
database is compatible with other programs in widespread they would like included besides the name of the
use. Current efforts to create a national database of US photographer.
holdings are underway, supported by government funding. Sending large images by email can really slow down
Part of this effort includes converting the many individual the system at both ends, so you might want to share images
databases into a single one that is accessible online. This with specific individuals through a free downloadable
will permit researchers to find the location of needed program called DropBox <www.dropbox.com>. There are
specimens and to determine the contact person(s) for loans. also a number of websites where you can post images that
are available to everyone, or by becoming a "friend" for
that group, much like FaceBook. If you give full
permission for use, provide the attribution information you
would like the user to include.
Shipping Live Bryophytes BE SURE OF IDENTIFICATION! It is okay to post
species where your identification is doubtful, but be clear
Bryophytes don't like to be wet and hot at the same that it is doubtful, or ask for help when you post the
time, and this can be exacerbated by also being dark. Such picture.
conditions are ideal for fungi to grow, and once a fungus Don't post pictures taken by anyone else without
attacks the bryophytes, they most likely won't recover. getting their permission and all the information discussed
Hence, shipping live bryophytes can be a major challenge. above.
To reduce these ideal fungal conditions, whenever possible Some posters restrict the resolution and size of the
pack some of those frozen picnic cooler gels with your images they post to avoid having them used commercially
bryophytes to keep them cool and use an insulated for profit as posters, calendars, or advertisements. Many
container, or insulate one with something like crumpled posters give permission for educational use, but not for
paper or styrofoam peanuts. If the bryophyte is drought other purposes. If you have no plans of publishing your
tolerant, send it dry. If you are shipping aquatic pictures, or using them for profit, why not give permission
bryophytes, seal them with clean water in plastic bags and for all but commercial use? This book is built on the
keep them cool. Use a rapid shipping method to ensure the willingness of people to share. And the less time one must
best results. Don't provide nutrients as they will encourage spend hunting for a contact person to gain permission, the
growth of algae, fungi, and bacteria on the surface. I have more time can be spent on creating and sharing the final
had some success packing aquatic mosses with wet paper product.
towels or newspaper, but heat will quickly spoil all your
efforts. Herbaria
Your first concern may be to keep the bryophytes
alive, but getting them across the border might be even There are numerous herbaria around the world, and
more challenging. Even within the same country, it might many of them are able to loan specimens to other herbaria.
be necessary to have a nursery license to ship plants across When requesting specimens, it is important to state the use
state borders. For example, in North Carolina, the you will make of them and anticipated return date. If you
Department of Agriculture and Consumer Services requires need them for DNA or chemical analysis, or any other
a nursery license for shipping within the US (Annie Martin, destructive sampling, be sure the loaning herbarium
Bryonet 8 December 2010). All live plants and bryophytes understands that. NEVER use type specimens for
need to be inspected for nematodes, insects, or diseases in destructive sampling. And likewise, avoid using voucher
advance. A certificate documenting certification must be specimens unless the destruction is necessary to verify
included in any shipment of live plants (bryophytes). For identification or compare then and now. Try not to use the
international shipments, at least from the USA, a local entire specimen.
inspector must examine each and every shipment that Index Herbariorum
leaves the country. Shipping overseas is a laborious <http://www.nybg.org/bsci/ih/ih.html> provides a list of
process and shipping is costly. the registered herbaria of the world. The index lists 1610
herbaria in 117 countries. The site permits searching by right nutrients must be present for development. Hence,
institution, city, state, acronym, staff member, caution should be used in using cultured bryophytes for
correspondent, and research specialty. taxonomic identifications. Nevertheless, live cultures are
one way to maintain rare species on the verge of extinction.
Herbarium Specimen Mapping An alternative to living, growing cultures, is
cryopreservation. Michael Christianson (Bryonet 10 June
Some herbaria include a dot map on the herbarium 1999) reported that he had taken over the culture collection
label (Figure 49). Phytogeographers need to understand established by Malcolm Sargent and that he had begun
plant distributions, and floras typically include the using cryopreservation of the species, including successful
distributions of the species. The size of the map depends cryopreservation of liverworts.
on the level of detail needed for that herbarium or project.
For instance, specimens collected for the BBS vice county
records will have a dot in the county of collection. Cryopreservation
Before we have scratched the surface of the
complexity of evolution and biogeographic pathways,
many plants and animals are disappearing from the planet
forever. We have struggled with our fossil record to make
sense of the small samplings we have through time and we
do not want to compound our struggle for understanding by
losing the species we have today. Nature does not preserve
species as fossils on a regular basis, so to ensure these
disappearing taxa remain available for study, we as
scientists must help out.
We knave known for a very long time that most
bryophytes have the ability to survive being frozen (Gubin
et al. 2003), so our knowledge about cryopreservation for
this group of animals already has a sound scientific basis.
Some of the early studies on cryopreservation for scientific
purposes have included bryophytes (Sugawara et al. 1980).
But several bryologists led the way toward building a
collection of cryopreserved endangered and rare bryophyte
species (Burch & Wilkinson 2002; Burch 2003; Burch &
Ramsay 2003).
Developing such a collection requires considerable
testing to be assured that most of the cultures will survive
and begin growth again. However, this method for
conservation has advantages over the traditional live
Figure 49. Dot map for Michigan, USA, indicating location culture methods. It requires much less maintenance time
of a specimen in one county. From Voss 1996. once the species has been cryopreserved, and it is less
likely to get contaminated while frozen. Furthermore,
Computers have brought us mapping programs that cultured bryophytes tend to lose vigor over time and both
greatly facilitate these tasks. Brent D. Mishler (Bryonet 13 their physical and physiological characters may change in
July 2008) has alerted us about the free program the unnatural conditions of culture, making them look like
BerkeleyMapper <http://berkeleymapper.berkeley.edu/>. a different species (Christianson 1998).
This program uses Google maps and places GIS-based
As in standard culture, it is desirable to obtain a pure
points on the maps.
culture free of algal and fungal contaminants. Burch and
The best way to look at a map is to run the query first at
Ramsay (2003) and Christianson (1998) suggest
<http://ucjeps.berkeley.edu/bryolab/UC_bryophytes.html>.
eliminating algae by growing protonemata in (not on) a
For example:
medium where they will grow toward the light. The
1. Search for Scientific name Mnium (or any other)
photosynthetic ends of these protonemata will emerge from
2. Submit query
the medium free of algae.
3. Select on the return page the link: "Map the results
using BerkeleyMapper (192 records with coordinates Dehydration prior to freezing will minimize the
[those with a light green checkbox])" formation of ice crystals that damage cells. Desiccation-
tolerant species are able to survive the prolonged
Live Collections dehydration that makes this successful, but desiccation-
intolerant species may not (Burch 2003). Survival of these
Maintenance of live collections requires a solid intolerant species is more likely to be successful if the
background in the ecological and physiological needs of growth medium is supplemented with abscisic acid (ABA)
the species to be cultured. These details will be covered and sucrose (see volume 1 for a discussion of desiccation
elsewhere in this volume. In the present chapter, we wish tolerance in bryophytes) (Christianson 1998; Burch 2003;
to caution you that cultured species may not look like the Burch & Ramsay 2003). Exact levels needed will require
same species in the field. For genes to be expressed, the experimentation, with needs differing by species.
Preparation of the bryophytes can be important to their In a study of a desiccation-tolerant, a non-desiccation

survival, and as you might expect, the ones from wet tolerant, and an intermediate-tolerant bryophyte, Burch
habitats lack desiccation tolerance, making them more verified this expectation (Figure 53Figure 56). Burch
difficult to preserve through cryopreservation (Burch tested a protocol in which the moss protonemata were
2003). Christianson (1998) found that only 3-4 days in a cultured on sucrose-free 1/2 strength MS medium
medium supplemented with 10-5 M ABA and 100 mM (Murashige & Skoog 1962), pH 5.8, solidified with 3.5 g
proline prepared the mosses Ceratodon purpureus (Figure L-1 Gelrite®. These were cultured in 5 cm Petri plates
50), Funaria hygrometrica (Figure 51), Physcomitrella sealed with Micropore® tape and maintained at 20±2°C
patens (Figure 52), and two species of Sphagnum (Figure with 16:8 hour light:dark cycle. Light was provided by
38) to survive at least one year in cryopreservation at - Growlux® and cool white fluorescent tubes (22-29 µmol
80°C. m-2 s-1). After sufficient cultured material developed, the
protonemata were air dried for 18 days with half the
cultures encapsulated and half not. The encapsulation
process started with a double thickness sterile filter paper
cut into 0.5x1.5 cm strips placed into sucrose-free 1/2 MS,
3% sodium alginate (from Sigma) encapsulation medium.
This medium was solidified using 100 mM calcium
chloride solution. The two pieces of filter paper were
separated so that one side was coated in alginate. 2-4 mm
diameter circles of protonemata were embedded in the
alginate, re-immersed in 3% sodium alginate, and set again
using 100 mM calcium chloride solution (Wilkinson et al.
1998). Each strip of filter paper had only one sample
protonemata, and each strip was placed separately in a 5 cm
Petri plate. An equal number of samples was cultured the
same way, but without the encapsulation procedure. When
Figure 50. Ceratodon purpureus, a widespread moss that these were transferred onto fresh control media, and little
survives when treated with ABA and proline prior to difference was visible between the cultures. After 18 days
cryopreservation. Photo by Michael Lüth. of dehydration in empty Petri plates sealed with
Micropore® tape, they were again tested for viability. The
three species exhibited 100% survival of the desiccation-
tolerant species, 40% for the intermediate species, and 0%
survival for the desiccation-intolerant species. After 18
days, one strip was placed in each cryovial and immersed
directly into liquid nitrogen, cooling rapidly to -196°C.
After 20 hours of cryopreservation, the protonemata were
warmed rapidly by immersing the vials in a 40°C water
bath for two minutes. The thawed samples were
transferred once again to 12 MS medium and returned to
the original cultural conditions. This procedure indicated
that encapsulation did little to affect the survival of
cryopreservation in these species. Hence, Burch concluded
Figure 51. Funaria hygrometrica, a disturbed habitat soil that for desiccation-tolerant species, pretreatment may be
moss that survives when treated with ABA and proline prior to
unnecessary.
cryopreservation. Photo by Janice Glime.
Figure 52. Physcomitrella patens, a disturbed habitat soil Figure 53. Bryum rubens, a desiccation-tolerant bryophyte
moss that survives when treated with ABA and proline prior to that survives dehydration and cryopreservation. Photo by Michael
cryopreservation. Photo by Janice Glime. Lüth.
Pence (1998) developed a protocol similar to that of

Burch (2003), testing three liverworts and one moss. The
thallose aquatic Riccia fluitans (Figure 36) was sensitive to
desiccation and required either abscisic acid (ABA)
pretreatment or encapsulation in alginate beads with 0.75
M sucrose to achieve 100% survival of drying. ABA had
little effect on the leafy liverwort Plagiochila sp. (Figure
57); it survived with simply drying, encapsulation, and
liquid N exposure. The thallose liverwort Marchantia
polymorpha (Figure 58) required both ABA and
encapsulation. Hence, ABA was needed as pretreatment
for both thallose species to avoid total mortality upon
drying. Rowntree and Ramsay (2009) reported that
pretreatment methods, including ABA and encapsulation,
were successful for 22 species of bryophytes having a
Figure 54. Ditrichum cornubicum, a bryophyte with broad range of moisture and other ecological requirements.
intermediate desiccation tolerance that has partial survival Some species had 100% survival, and overall regeneration
following dehydration and freezing. Photo by Des Callaghan. rates were more than 68% for all species tested.
Figure 57. Plagiochila asplenioides, member of a genus for

which ABA had little effect on survival of cryopreservation.
Photo by Dick Haaksma.
Figure 55. Cyclodictyon laetevirens, a bryophyte that lacks
desiccation tolerance and that has no survival following Duckett et al. (2004) suggest ways of streamlining the
dehydration and freezing. Photo by Sean Edwards. cryogenic process. Spores, gemmae, and vegetative
fragments can be surface sterilized and grown in Petri
plates on media with inorganic salts. Phytogel or Gelrite
are preferable to most traditional agars because these are
often toxic due to impurities. And some bryophytes benefit
from dilution of nutrients. Spore availability can be
extended by storing ripe capsules at 4°C. Temperatures
above 25°C can cause excess respiration and reduce the
health of the propagule/culture; light intensity should be
much lower than that in nature to prolong the culture
viability.
Figure 56. Comparison the effects of encapsulation in

alginate on survival in a desiccation-tolerant (Bryum rubens), an
intermediate-tolerant (Ditrichum cornubicum), and an intolerant
(Cyclodictyon laetevirens) bryophyte species. Bars with the same Figure 58. Marchantia polymorpha, a thallose liverwort
letter are not significantly different from each other (α = 0.05). species that requires both ABA and encapsulation before
Redrawn from Burch 2003. cryopreservation. Photo by Jan-Peter Frahm.
Bryophytes such as Ditrichum plumbicola that

produce specialized propagules may be easier to preserve
through desiccation and cryopreservation (Rowntree et al.
2007). Some species cultured in preparation for
cryopreservation will produce protonemal gemmae hitherto
unknown in nature (Ditrichum cornubicum, Saelania
glaucescens, Seligeria camiolica, and Zygodon gracilis)
(Duckett et al. 2004). Protonemal gemmae suspensions are
an ideal way to re-introduce these species to the natural
environment.
Ditrichum plumbicola protonemata exhibited
unexpectedly low survival of cryopreservation (Rowntree
et al. 2007). Rowntree and coworkers (2007) found that
pretreatment of Ditrichum plumbicola protonemata with
ABA and sucrose caused protonemal growth to be arrested
and propagules were induced. Most protonemal cells died,
but those that survived were char by thick, deeply
pigmented walls, numerous small vacuoles, and lipid
droplets in their cytoplasm. The protonemal propagules
were highly desiccation- and cryopreservation-tolerant,
behaving like the desiccation tolerant rhizoids in the natural
environment where they are induced by extreme
conditions. Figure 60. Four ecotypes of Physcomitrella patens in
Not all mosses need to be cultured as protonemata to culture in Ralf Reski's IMSC lab <http://www.moss-stock-
preserve well. Schulte and Reski (2004) used fresh plants center.org/>. Photo by Ralf Reski.
to preserve 140,000 mutants by cryopreservation (Figure
59-Figure 60). They used a combination of several of the
pre-treatment techniques described above, but with some When You Depart – Willing Your
additions. They used a complete Knop medium (Egener et
al. 2002), amended with 920 mg L-1 ammonium tartrate, 87
Herbarium
g L-1 mannitol (Grimsley & Withers 1983), 10 µM ABA Your personal herbarium is valuable, but non-botanists
dissolved in DMSO (dimethyl sulfoxide), and 100 mM might not recognize its value (Miller 1988). Therefore, it is
proline (Christianson 1998). The liquid medium was filter wise to be sure you have either included it in your will or
sterilized; the solid medium was supplemented with 1.2$ your heirs understand its value and where it should go.
(w/v) agar. Macro- and microelements, FeSO4 x 7H2O, Since herbaria will not always be willing to accept
glucose, and mannitol were autoclaved. The other collections, it is wise to make arrangements with the
supplements were filter-sterilized with a 0.22 µm millipore receiving herbarium so you know they will accept your
filter and added to the medium after it was autoclaved. The specimens. It is the responsibility of the receiving
medium pH was adjusted to 5.8 before autoclaving and herbarium to let the bryological community know that they
before filter sterilizing. have received your herbarium. If your herbarium is a
personal herbarium and is duplicated elsewhere, consider
giving it to an exchange program or to a struggling
bryologist where the herbarium is inadequate. And be sure
provisions are made for return of any specimens you might
have on loan.
Exchange Programs
Several of the bryological societies sponsor bryophyte
exchange programs. For example, the ABLS (American
Bryological and Lichenological Society) program has
separate liverwort and moss exchanges. To join the
program, one needs to send several species with five
duplicates to the current appropriate director of exchange.
For each specimen you send, you can select a species from
the next exchange list. Hence, if you send six species with
five specimens of each, you are eligible to receive 30
specimens from among the forthcoming lists. Specimens
contributed must be of adequate size, typically palm size,
but this depends on the abundance and size of the species.
Figure 59. Cryopreservation equipment in Ralf Reski's Sending rare species for exchange should be avoided. The
IMSC lab <http://www.moss-stock-center.org/>. Photo by Ralf packets must have complete label information, as discussed
Reski. earlier in this chapter.
Borrowing Specimens methods of storing and labelling depend on the purpose

of the collection. Storage cabinets need to protect from
Funk (2007), US National Herbarium, has provided a pests but usually do not require moth balls. Keeping
good introduction into the many uses of a herbarium specimens dry is most important.
<http://www.virtualherbarium.org/vh/100UsesASPT.html> Herbaria have preferences for specimen storage,
. Top among these uses for ecologists is to compare your including boxes, drawers, folders, and herbarium
specimens with those of others to verify your identification. sheets. Cool preservation works best, but is expensive.
If you are not near with a large herbarium, it may be Minute species and special structures may require liquid
necessary to borrow specimens to verify your preservation or minipackets. Arrangement in the
identifications. There is an etiquette for borrowing and herbarium may be alphabetical (for ease of filing) or
asking in the right way is more likely to get you the phylogenetic (useful for systematic studies). Type
specimens you need. Visit the New York Botanical Garden specimens are usually indicated by red folders, but
website for instructions on how to borrow specimens other marks of red can be used.
<http://sciweb.nybg.org/science2/herb/tips.asp.html>. Killing inhabitants and soil pathogens is necessary
for new collections, whether fresh from the field or
Type Specimens
obtained from another herbarium. This can be
You should only borrow type specimens when non- accomplished by Agral 600, moth balls, microwave,
types will not do. This would include revisions of a genus freezing, steam, insect traps, moisture control, or
or species when you must verify the original description. drowning.
Type specimens must be handled with utmost care and A herbarium should be equipped with both
returned to the loaning herbarium quickly. This method of dissecting and compound microscopes and equipment
verification may change somewhat as our use of molecular named in Chapter 2-1. Its workspace should include
identification becomes more common and a larger database good taxonomic references, and it helps to add indexing
is available. tabs. A computer station is useful for entering data,
The first problem in borrowing a type specimen is to using online keys, updating nomenclature, making dot
locate it. Generally there are multiple paratypes placed in maps, and finding images, as well as making herbarium
multiple herbaria, but there is only one holotype. The labels.
location of the holotype can be determined by checking the When shipping specimens to other countries, be
Index Herbariorum sure you know and comply with pertaining laws. Most
<http://sciweb.nybg.org/science2/hcol/bryotypes/index.asp. prohibit soil. Be sure the recipient knows they are
html>. Index Herbariorum provides the physical location coming, and whenever possible, ship to a herbarium
of a herbarium, its web address, holdings (number and type where the recipient can receive them. There are many
of specimens), history, staff, areas of expertise of acts of courtesy that can help when you ask others to
associated staff, and contact information. Only permanent identify your specimens. Posting pictures online to ask
collections with active management and accessibility to for identification help should include as much
scientists are included. information as possible, show habit, plant, leaf, and
When using Index Herbariorum, you can locate cross sections, and be kept small so as not to clog
herbarium personnel by entering the person's name on the inboxes or be slow in loading. Be sure you have
Text Search page. For example, when I entered "Deguchi," permission to post pictures that are not yours.
it provided me Person: Hironori Deguchi; Herbarium Living culture can maintain rare species and permit
Acronym: HIRO; Institution: Hiroshima University; testing without decimating the extant populations.
Location: Japan, Hiroshima; Research Pursuits: Cryopreservation can also maintain the genome for later
Taxonomy; morphology; and ecology of bryophytes. study and cultivation.
To locate a herbarium where a type specimen is Exchange programs are available through some of
housed, one can use the Virtual Herbarium for Bryophytes the societies, e.g. the American Bryological Society,
and visit the Type Specimen Catalog. where members of the program can exchange
specimens with others in the group to build the diversity
in a herbarium.
Herbaria can borrow specimens from each other,
Summary but loans to individuals might be refused. Type
specimens are more likely to be carefully protected, so
Most bryophytes are stored in packets folded in you might have to travel to the host herbarium.
thirds of a standard sheet of 100% rag paper. It is easy
to make your own packet folding machine. Labels can
be designed on a word processor or produced by a
herbarium label program. Labels need to include name Acknowledgments
of species, author of the scientific name, altitude,
habitat, substrate, date of collection, location Dale Kruse kindly sent me a printout of all the emails
(country, state, county, distance to nearest town), GPS he received on Bryonet and offline regarding herbarium
coordinates, name of collector, collection number, and practices.
determiner (name of person identifying or verifying
identification). Once they are placed into the herbarium Literature Cited
collection, an accession number should be added. Burch, J. 2003. Some mosses survive cryopreservation without
Packets with multiple species should indicate so; prior pretreatment. Bryologist 106: 270-277.
Burch, J. and Ramsay, M. 2003. The ex situ conservation of Murashige, T. and Skoog, F. 1962. A revised medium for rapid
bryophytes. Bryol. Times 108: 7-8. growth and bioassays with tobacco tissue cultures. Physiol.
Burch, J. and Wilkinson, T. 2002. Cryopreservation of Plant. 15: 473-497.
protonemata of Ditrichum cornubicum (Paton) (sic) Pence, V. C. 1998. Cryopreservation of bryophytes: The effects
comparing the effectiveness of four cryoprotectant of abscisic acid and encapsulation dehydration. Bryologist
pretreatments. Cryo-letters 23(3): 197-208. 101: 278-281.
Christianson, M. L. 1998. A simple protocol for Raup, L. C. 1926. Hunting for and collecting mosses. Bryologist
cryopreservation of mosses. Bryologist 101: 32-35. 29: 73-74.
Davison, P. G. Date: 2002. Making storage boxes for bryophyte Rico G., R. R. 2011. Methods for cell clearing and rehydration in
packets. Evansia 19: 125-127. thalloid liverworts. Trop. Bryol. 33: 12-18.
Duckett, J. G., Burch, J., Fletcher, P. W., Matcham, H. W., Read, Rothero, G. and Blackstock, T. 2005. Recording and
D. J., Russell, A. J., and Pressel, S. 2004. In vitro conservation. Submitting new vice-county records. Field
cultivation of bryophytes: A review of practicalities, Bryol. 85: 41-43.
problems, progress and promise. J. Bryol. 26: 3-20. Rowntree, J. K. and Ramsay, M. M. 2009. How bryophytes
Egener, T., Granado, J., Guitton, M. C., Hohe, A., Holtorf, H., came out of the cold: Successful cryopreservation of
Lucht, J. M., Rensing, S., Schlink, K., Schulte, J., Schween, threatened species. Biodiv. Conserv. 18: 1413-1420.
G., Zimmermann, S., Rak, B., and Reski, R. 2002. High Rowntree, J., Duckett, J. G., Mortimer, C. L., Ramsay, M. M., and
frequency of phenotypic deviations in Physcomitrella patens Pressel, S. 2007. Formation of specialized propagules
plants transformed with a gene-disruption library. BMC resistant to desiccation and cryopreservation in the
Plant Biology 2: 6. threatened moss Ditrichum plumbicola (Ditrichales,
Funk, V. 2007. 100 Uses for a Herbarium. US National Bryopsida). Ann. Bot. 100: 483-496.
Herbarium. Accessed 28 February 2013 at Schofield, W. B. 1985. Introduction to Bryology. Macmillan
<http://www.virtualherbarium.org/vh/100UsesASPT.html>. Publishing Company, New York, 431 pp.
Glime, J. M., Hudy, P. S., and Hattori, S. 1990. Diversity and Schulte, J. and Reski, R. 2004. High throughput cryopreservation
altitudinal niche width characteristics for 35 taxa of the of 140,000 Physcomitrella patens mutants. Plant Biol. 6:
Papua New Guinea Frullania flora with consideration of 119-127.
sibling pairs. Trop. Bryol. 2: 103-117.
Schuster, R. M. 1966. The Hepaticae and Anthocerotae of North
Grimsley, N. H. and Withers, L. A. 1983. Cryopreservation of America, East of the Hundredth Meridian. Vol. I. Columbia
cultures of the moss Physcomitrella patens. Cryo-Letters 4: University Press, New York, 802 pp.
251-258.
Shaw, A. J. and Goffinet, B. (eds.). 2000. Bryophyte Biology.
Gubin, S. V., Maksimovich, S. V., Davydov, S. P., Gilichinskii, Cambridge University Press, Cambridge, UK, 476 pp.
D. A., Shatilovich, A. V., Spirina, E. V., and Iashina, S. G.
2003. O vozmozhnosti uchastiia pozdnepleistotsenovoi bioty Singh Deo, S. and Majumdar, S. 2020. Recommendation for
v formirovanii bioraznoobraziia sovremennoi kriolitozony. preserving dissected parts of holotype specimens of names of
[The possible contribution of late Pleistocene biota to bryophytes. Taxon 69: 625.
biodiversity in present permafrost zone.]. Zhurn. Obshchei Sugawara, Y., Matsushima, H., and Takeuchi, M. 1980.
Biol. 64(2): 160-165. Cryopreservation of plant protoplasts and their
Holzinger, J. M. 1900. Suggestion for glycerine jelly mounts. differentiation after thawing. Cryobiology 17: 623.
Bryologist 3: 42. Voss, E. G. 1996. Michigan Flora: Part III: Dicots Concluded.
Li, X., Glime, J. M., and Hattori, S. 1989. Ecological gradient The Regents of the University of Michigan, Ann Arbor, MI.
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Loeske, L. 1925. Wie man Moose sucht und sammelt. Sonder- Wilkinson, T., Wetten, A., and Fay, M. 1998. Cryopreservation
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Chapter 3 - Herbarium Methods and Exchanges

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Glime, J. M. and Wagner, D. H. 2017. Herbarium Methods and Exchanges. Chapt. 3-1. In: Glime, J. M. Bryophyte Ecology.

Folding Packets ....................................................................................................................................................................... 3-1-2

Figure 6. Staple or tape the ends so that it forms a pocket.

Figure 7. The machine is now complete with staples. For

Figure 4. On the opposite end prepare a similar pocket;

Figure 8. Separate the sheets and place one side that is

Figure 13. Now it is ready to use. The label should be

Figure 17. Sample herbarium label for a mixed collection

verification of identification, and still has the problem of

Figure 22. Sample type specimen folder for bryophyte

When slides are made of the type specimen, it is

Figure 23. Herbarium drawer with packets. Photo by Brian

Packets on herbarium sheets can be stored in a

Figure 26. To make boxes, use scissors, razor knife, or paper

Storage Boxes from Genus Covers

If you are familiar with large herbaria, you are familiar

Size 3 (4 3/4 x 3 3/8 x 7/8): ~pricing range $0.72 to $0.88

Specially Made Storage Boxes

Glime once stored Splachnum ampullaceum in a plastic

Figure 32. Box opener of wooden tray. Photo by Jay

Figure 35. Conocephalum conicum, a mostly flat thallose

Figure 34. Trays. Photo by Jay Cordeiro, Northeast Natural

Figure 39. Hylocomium splendens, a moss that survives

one specimen and I'll look at it and respond within 24

Figure 43. Riccardia cf. elata microscopic view posted to

Figure 44. Index tab indentations in The Liverwort Flora of

Figure 47. Excel file printout of six-letter liverwort codes,

The second example of this kind of index is an older

Figure 48. List of all bryophytes found by David Wagner in

Herbarium Programs Sharing Images

Preparation of the bryophytes can be important to their In a study of a desiccation-tolerant, a non-desiccation

Pence (1998) developed a protocol similar to that of

Figure 57. Plagiochila asplenioides, member of a genus for

Figure 56. Comparison the effects of encapsulation in

Bryophytes such as Ditrichum plumbicola that

Borrowing Specimens methods of storing and labelling depend on the purpose

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