2002 Final Report Summaries
Discover Life in America ATBI Grant Program

I. Promised Products

In my revised proposal I indicated that the following activities would be carried out during the 2002-2003 grant cycle.

1. Complete sampling at standard sites in the GSMNP.
2. Begin collecting at specialized sites such as seeps and caves.
3. Isolate and identify backlogged samples.
4. Submit data to ATBI database.
5. Begin statistical analysis of ecological predictors of tardigrade diversity and species effort curves.    

II. Completed Products  

We made good progress on all of the goals, but due to major illness of the taxonomist for the project, Diane Nelson, we did not get as far as predicted. Still, over 450 samples have been collected and over 1600 specimens have been identified. We have brought the number of species records in the park from 3 to 42, and we believe 3 of these are new to science (see attached species list).

1. Sampling Standard Sites We have now sampled from 14 of the 19 terrestrial ATBI plots. Most of the remaining plots are balds, which have few lichens and mosses, and therefore few tardigrades. We have also now collected in 12 streams from 5 watersheds in the park, leaving only a single day of collection left to complete the aquatic component of our sampling.  

2. Sampling Specialized Sites Collections have been made in the dolomitic caves around Cades Cover, and our first collections of seeps have been made. Specimens have been identified from the cave samples, including one species which is a new record for North America (Doryphoribius sp.). The seep samples have yet to be processed.  

3. Processing of Backlogged Samples Good progress has been made toward processing samples. Students are cranking out microscope slides, as I write. The bottleneck has shifted somewhat from samples needing to be processed to microscope slides needing to be identified.  

4. Submission of Data to ATBI Database A database of 1590 specimens has been sent to Michael Kunze. This is almost double the size of last year’s submission.  

5. Ecological Analysis The useable database for ecological analysis includes 1300 specimens from 60 samples. This is adequate to let us judge our progress by calculating species effort curves. It also gives us the ability to begin looking for ecological variables associated with tardigrade diversity. A WWC student is currently working on a project in his GIS class, attempting the first distribution models for tardigrades.  

Species Richness Estimate curves were calculated using EstimateS 6 software by Rob Colwell of the University of Connecticut. These curves are attached below (Figs. 1-4). They indicate that there are somewhere between 47 and 76 species of tardigrades in the park (Fig. 5), more or less evenly distributed in soil, lichens, moss, and streams. Habitat associations are beginning to be found for tardigrade species (Fig. 6), and it is clear that height above ground level is an important predictor of diversity for species living on tree moss (Fig. 7).    

In-Kind Budget   Considering the personnel time spent on this project beyond that funded by the grant, the following are reasonable estimates for in-kind contributions from partner organizations.  

In Kind Support Estimated Value Faculty research $5000 (200 hrs @ $25/hr) Lab tech research $1500 (150 hrs @$10/hr) Student research $1250 (250 hrs @$5/hr) GSMNP & DLIA personnel $ 600 (60 hrs @ $10/hr) WWC lab facilities $6000 (30 days @ $200/day) ETSU lab facilities $3000 (15 days @ $200/day) GSMNP facilities $ 600 (6 days @ $100/day) WWC transportation for student groups $ 156 (520 miles @ $0.30/mi) TOTAL $18,086    

Publications/Presentations   Bartels, P.J. & Nelson, D.R. (2001). Tardigrade inventory status report. Annual ATBI Conference, Gatlinburg, TN.

Bartels, P.J. & Nelson, D.R. (2002). Tardigrade inventory status report. Annual ATBI Conference, Gatlinburg, TN.

Bartels, P.J. (2003). The ATBI and the Phylum Tardigrada. Natural Science Seminar, Warren Wilson College, Asheville, NC.

Bartels, P.J. & Nelson, D.R. (ms in prep). A large-scale, multi-habitat inventory of the Phylum Tardigrada in the Great Smoky Mountains National Park. 9 th International Tardigrade Symposium, St. Petersburg, FL.

General accomplishments and failures

The most significant accomplishment under this grant has been, in the judgment of the Principal Investigator, to get him trained in pseudoscorpions well enough that he can now begin to make critical identifications of the thousands of specimens collected in the ATBI endeavor. The wisdom of the reviewers of the proposal was greater than that of the investigator—the proposal promised to do too much with too little. It is solely the fault of the investigator that most of the promised training did not occur.

The primary objective of ATBI—that of inventory—will still be accomplished by the investigators. The schedule will be stretched for years beyond what was proposed and what was desirable.

Status of approved research objectives

1. To assemble records of occurrences of pseudoscorpions in GSMNP and the region and to assemble supporting literature.

The records of occurrence have been assembled, primarily by Dr. William Muchmore and Mr. James Cokendolpher, and I have assembled approximately 185 books, monographs, and published papers from the international literature that are pertinent to studies of the taxonomy, systematics, and ecology of regional pseudoscorpions. In addition, the pictured key to Smokies pseudoscorpions which was developed by Muchmore and Cokendolpher for the DLIA web site has been tested and revised by Cokendolpher and Clebsch and is now available on the web site.

2. To collect forest floor samples monthly from April through October from several distinctive plant communities along an altitudinal gradient from low elevation old growth deciduous forest to high elevation spruce-fir or fir forests. Specimens will be identified to the lowest taxonomic level possible and will be sent to world experts for verification or further identification.

This objective was not accomplished in work supported by this grant, but was accomplished in part in the following ways. As the litter samples from Dr. Fred Coyle’s study of the spiders of the Smokies were sorted, the pseudoscorpions from those samples were turned over to me for study. They are still in my possession, and the identification of specimens has begun. Dr. Coyle has provided me with descriptions of his study sites and the plant communities sampled, and that information will be related to fuanistics of each site as identifications are completed.

The eleven ATBI intensive sample sites are scattered across many elevations and plant communities in the park. As the litter samples (and some funnel trap samples containing flies with phoretic pseudoscoprions) from the 11 intensive ATBI sites were sorted, Ian Stocks turned the pseudoscorpions over to me. Those samples are in my possession, and will be studied. The pseudoscorpions from the pitfall traps at the intensive ATBI sites have now been sorted and have also been turned over to me for study.

My promise to send specimens to authorities in other parts of the world was made based on experience in botanical systematics and naivete about the nature of pseudoscorpion systematists. As far as I can determine there are very few pseudoscorpion systematists that are specialists in only one or a few families or genera. As a consequence, this task has not been completed. Collaboration with international workers in pseudoscorpion systematics remains a personal goal.

3. Sample forest floor layers quantitatively once per month at two ATBI plots through the year. This objective was abandoned after it became clear that the time and effort required to accomplish it were beyond the scope of available personnel and equipment. A few quantitative spot samples were taken and the pseudoscorpions were extracted in loaned Tullgren funnels. The animals have not yet been identified.

4. Collect extensively on living tree bark, under bark of dead tress, and under stones and logs, and develop experiments to make collecting more efficient. Once again, my inexperience in the group of organisms and unrealistic projections of the labor required have resulted in few measurable accomplishments. Experience has taught me that the most efficient collecting (in terms of numbers of organisms) is done with Tullgren funnel extractions. Careful searching of habitats and sieving of substrates are inefficient in terms of numbers of animals captured but yield good diversity of species.

5. Develop a gallery of photographs of living pseudoscorpions, to be posted on the DLIA web site. No progress. I now have laboratory space in a residence which I own, and have equipment and supplies that will allow me to begin to maintain animals in captivity. I have purchased a digital camera (with personal funds) which is capable of micro-photography. When I am able to keep pseudoscorpions in culture, I will seek help in photographic documentation from the several volunteer photographers associated with DLIA/ATBI.

6. Test the null hypothesis that the pseudoscorpion fauna does not differ in the rotting wood from hollow trees of different species. No progress. Still a viable hypothesis, but worth the effort of a master’s thesis.

7. Test the null hypothesis that the pseudoscorpion fauna does not differ among forest litter samples that are derived predominantly from different single plant species. No progress. It is still a viable hypothesis, but the time and effort required to gather the data have proven to be beyond the scope of this study. Also worth a master’s thesis.

Status of approved training objectives

1. Get the lead investigator and students trained in field and laboratory techniques with the help of James Cokendolpher. I spent a week with James in the Smokies, searching, collecting, and identifying pseudoscorpions, scorpions, and opiliones (the latter two his specialties). Efforts to involve students failed, largely because of scheduling problems. The training was invaluable for the lead investigator. Cokendolpher took his collections back to the Museum at Texas Tech University, and I have not received reports of his identifications. His determinations will either be sent directly to DLIA or will be forwarded through me.

2. To work intensively with three or four middle school or high school students from Anderson County, the students to be selected by and largely supported by Mr. John Byrd.

John selected bright, eager students. An initial, exciting field exercise produced pseudoscorpions. The follow-up tedious lab work of dissection and slide preparation dulled their excitement. The tedium, coupled with poor organization and planning on the part of the lead investigator, resulted in loss of interest on the part of three of the students. The fourth persisted without much guidance and has now turned to other interests. Further field collections with the whole group did produce pseudoscorpions.

3. Photograph pseudoscorpions with the flex-cam equipment at Clinton High School. The flex-cam apparatus was limited in the magnifications it could produce to the point that it was essentially useless for photographing pseudoscorpions. The lead investigator now has a digital camera with microscope adapter and software for processing images, and that setup has the potential of yielding useful images of preserved and mounted whole specimens or dissected parts.

Inventory data

I have attempted to install the free database program provided by Michael Kunze, and have been unsuccessful in both attempts. As a consequence, I have no geo-referenced data to provide at present. I intend to contact Chuck Parker soon to seek help. As specimens are identified to species they and their occurrence data will be entered into the evolving database being developed by Michael and Chuck.

Expenditures of grant funds

Cokendolpher air fair, fee, and subsistence $ 1758
Mileage (approximate) @ $0.35/mile in personal vehicles 600
Field subsistence for Clebsch 110
Supplies and minor equipment (Winkler bag, slides, fluids, etc.) 985
TOTAL funds expended to date $ 3453

Please note: The Principal Investigator did not utilize any of the grant funds for personal salary.

Leveraged funds and “in-kind” contributions

Personal funds were expended for microscopes and microscope accessories, including a digital camera and lens, in the amount of $ 3328.28. The equipment will, of course, be used initially on this project but will also be used on other studies as well.

Volunteer time:
Clebsch professional time 156 hrs.
Dr. William Muchmore professional time 16
Mr. James Cokendolpher professional volunteer time 40
Technician time from Ian and Stephanie Stocks (estimated)* 100 hrs.
Sorters* 200 hrs.
Students 20 hrs.

* The contributed time of these individuals may be accounted for under other accounts and may thus not be appropriate for entering here.

Publication

The only publication to date was a short general paper on the scorpion and pseudoscorpions of Great Smoky Mountains National Park, co-authored by Clebsch and Cokendolpher and printed in the ATBI Quarterly. It has been approved for reprinting in the Southeastern Naturalist.

Dept. Entomology

Products Described in Proposal Submitted 18 February 2002

In last year's proposal I stated that we would provide the following products or initiate the following activities during this reporting period:
1. Continue the sorting, processing, and distribution activities at the Louisiana State Arthropod Museum.
2. Enhance communication and cooperation among TWIG cooperators through the use of the TWIG website that was established during year one of this project.
3. Continue field research designed to survey beetle habitats in GSMNP that may not be covered by the park's established collecting protocols.
4. Prepare two publications describing previously undescribed species from GSMNP.

Products and Activities Delivered
(numbers correspond to list above)

1. Space and procedures have been established at the LSAM to sort beetles. We have devoted most of our 400 square foot sorting and alcohol storage laboratory to this project. Early in 2002 we hired a Master's Degree candidate to perform family sorting in preparation for distribution to the cooperating specialists described above. She was with us for only a short time, but we were able to complete processing of beetles collected during the 2001 Beetle Blitz. New records obtained during the Blitz were included on the checklist and those data were presented at the Annual Conference. Priority taxa that were delivered to specialists included Lampyridae to Kathrin Stanger-Hall and Chrysomelidae to Edward Riley. Riley sent us an updated list that included many additions to the checklist reporting the results of his and Shawn Clark’s chrysomelid research. Two genera of carabids, Anillinus and Trechus, were sorted and detailed taxonomic work was performed by myself and Igor Sokolov, resulting in the discovery of five species new to science (discussed in more detail below). A large specimen loan of the leiodid beetle genus Agathidium was made to Cornell postdoctoral researcher Kelly Miller.

During early 2003 we hired museum intern and prospective graduate student Stephanie Gil to coordinate sample and specimen processing. She expects to continue with the project through at least fall 2003.

In summary, 190 species new to the park were logged into our list during the 2002-2003 funding period. Five species new to science were added, bringing the total number of new taxa discovered during the past two years to eight new species and one new genus. Assuming a starting baseline of 700 species on the original two lists, we have added almost 600 species new to the park during that period. Total beetle species documented for GSMNP stands at approximately 1300.

2. Our network of cooperators has now grown to 30, and we are excited about the addition of Dr. James S. Ashe from the University of Kansas, a specialist of one of the most neglected and diverse groups on our list, the staphylinid subfamily Aleocharinae. Also, we have added Sergey Kazantsev of Chicago, who will have responsibility of the superfamily Cantharoidea, and Robert Ward, of Bolivar, TN, who studies the carabid beetles and their allies.

3. Fieldwork associated with this project was headquartered at the Appalachian Highlands Science Learning Center at Purchase Knob. During 15-21 July LSAM Curator Victoria Bayless, undergraduates Stephanie Gil and Lisa Peri, and myself set up flight intercept traps at several high altitude sites near the center and on Purchase Knob. Following their departure I remained in residence through 15 August conducting fieldwork and sorting samples (n=55) from the park’s structured collecting initiative. I also had the pleasure of interacting with large numbers of middle and high school students during regularly scheduled educational field trips, other groups (e.g., the Asheville Mushroom Club) and was filmed briefly during the production of Heron Production’s instructional feature“Biodiversity All Over the Place.”

In addition to the four flight intercept traps set up during the first week, fieldwork centered around collecting forest litter samples (n=25) from the east, southeast, and southern areas of the park. A series of 10 fruit baited traps were run in the vacinity of Purchase Knob, and valuable observational data were collected on various aspects of beetle behavior and life history.

Significant research findings that directly resulted from this fieldwork included the discovery (finally) of new specimens and additional locality records of the previous identified undescribed genus of staphylinid, opening the door for its formal description. Other discoveries included additional records of undescribed species of Anillinus ground beetles, including a new species not previously identified.

During my extended visit at Purchase Knob I had the opportunity to observe the insect phenology of the large stinkhorn mushroom Dictyophora duplicata thanks to information provided by members of the Asheville Mushroom Club. In addition to the usual flies and carrion beetles (Nicrophila americana), I was able to observe up to a dozen larvae of the unusual stinkhorn feeding beetle Psilopyga nigripennis during approximately one week of laboratory study. The larvae grew amazingly fast, from 5 mm to approximately 15 mm in only four days. They spent all of their time completely immersed in decomposing stinkhorn slime with only the tips of the spiracles exposed for gas exchange. Dr. Ernie Bernard ( University of Tennessee ) was kind enough to set up his video camera and record live shots of the larvae near the end of the observation period. The larva of this species has never been fully described, so a publication is planned that will accomplish that and report the life history findings.

4. Project activities and results continued to be reported on the LSAM's website: http://www.agctr.lsu.edu/arthropodmuseum/index.htm. In addition to the updates to the list and cooperators already described, I have added two research briefs describing the discovery of the new carabid species and the life history information on Psilopyga.

Additional publication activity relevant to this objective is reported in the summary that follows.

Summary of Presentations, Publications, and Educational Activities

Presentations
1. Carlton, C. E. Progress Report on the Coleoptera Twig. Discover Life in America Annual Meeting, Gatlinburg TN, Dec. 2002.
2. Carlton, C. E. Update on Coleoptera Twig to Coleopterists Society Executive Council. Entomological Society of America National Meeting, Ft. Lauderdale, FL, Nov. 2002.
Publications (published and accepted).
1. Konstantinov, A., and A. Tishechkin. 2003. The first Nearctic leaf litter flea beetle (Coleoptera: Chrysomelidae) from the Great Smoky Mountains National Park. Coleopterists Bulletin 57: in press (publication anticipated Dec. 2003).
2. Sokolov, I., and C. Carlton. 2003. Review of Anillinus with Descriptions of 13 New Species and a Key to Soil and Litter Species (Carabidae: Trechinae: Bembidiini). Coleopterists Bulletin 57: in press (publication anticipated Dec. 2003).
Educational Activities
1. Taxonomic training for three graduate students (Alexey Tishechkin, Andrew Cline, and Erin Watson) and two undergraduate students (Lisa Peri and Stephanie Gil) in LSAM laboratory. The two undergraduates also participated in fieldwork described above.
3. Extensive interaction with school groups during residency at Purchase Knob, including instructional presentations and insect field walks.
4. P.I. featured on “Biodiversity All Over the Place’ instructional video.

In-kind Support Estimates

Commitment of Principal Investigator (Assoc. Professor) to project
10% salary + fringe --$7700
Commitment of Curator/Research Associate to project
5% salary + fringe --$2000
Unrecovered indirect cost
43% of MTDC --$2100
Total --$11,800

Additional undocumented support was provided by the LSU Agricultural Center through the use of facilities and equipment, including vehicles, microscopes, computers, and specimen storage.

Information Management

Specimens derived from research activities conducted by LSAM staff and students are processed in-house and data capture at the specimen level is accomplished using Biota software (R. Colwell, Sinauer Assoc.). Relevant fields for all GSMNP specimens were selected and exported to flatfile and imported to Excel. This file has been sent to Michael Kunze at Keith Langdon's office at Twin Creeks, GSMNP.

All specimens derived from our research carry unique specimen codes consisting of our four letter acronym, LSAM, and a seven digit numerical code. Thus, subsequent information, such as updated taxonomic information, can be precisely matched using the unique specimen codes, but this adds an additional upstream step in the data consolidation process.

Specimens sorted from the structured GSMNP sampling protocols that have been forwarded to taxonomic specialists in alcohol were accompanied by explicit instructions that specimen data capture is required. Individual specialists have the option of accomplishing the data capture themselves or returning properly mounted and labeled specimens to us for incorporation into our own databasing system for submission during the next reporting period.

BACKGROUND

Native bees play a vital role as pollinators in most terrestrial ecosystems. It is estimated that 70% of flowering plants are dependent on these services (Axelrod 1960). Most rare plants appear to be included in this dependence. Studies of the reproductive and pollination biology of numerous species of rare plants by the USDA-ARS Bee Biology & Systematics Lab found that almost all were dependent on insects, primarily bees, for their reproductive success (Tepedino unpub.). The relationships of bees to plants are still little known. Some, such as the exotic honey bee are broad generalists, visiting many kinds of flowers for pollen and nectar. Others have specialized pollen foraging habits (Linsley 1958, Wcislo & Cane 1996) but these are poorly documented.

Several comprehensive studies of local bee faunas have been conducted in the western United States (Griswold et al. 1998) but only four east of the Mississippi River, in Illinois (Marlin & LaBerge 2001), Ithaca, New York (Ascher unpub.), southern Mississippi (Michener 1947) and Everglades National Park (Pascarella et al. 1999). There has been no systematic study of bees in the Great Smoky Mountains National Park (GSMNP). The only published information is fragmentary records scattered in revisionary studies of specific bee genera. Because of the diversity of habitats in GSMNP, the presence of undisturbed and only moderately disturbed habitats, and the rich array of flowering plants, a diverse bee fauna is expected. At a state level 194 species are recorded from Tennessee, while the better known North Carolina fauna stands at 509 (Mitchell 1966, 1968; updated Griswold unpub.). A park fauna of over 250 species is anticipated.

A preliminary inventory of GSMNP bees was conducted in 2002 to obtain initial information on the bee fauna of the park, their distributions and floral preferences augmented by existing records in the literature and the U. S. National Pollinating Insects Collection in Logan, Utah. Limited additional sampling was conducted in 2003. The study was a four-way collaboration between the two science centers (Michelle Prysby, Tremont Institute and Paul Super, Purchase Knob), Pat Lincoln, Coker College and Terry Griswold, USDA-ARS Bee Biology & Systematics Laboratory.

METHODS

Both traditional and structured collections were made by a combination of net collections on flowering plants and passive pan traps. Net collecting was focused on collecting at plants in flower. Pan trapping consisted of placing 30 small bowls (10 each of three colors: fluorescent blue, fluorescent yellow, white) in a transect 5m apart. Water with a biodegradable surfactant was placed in the bowls. Emphasis in sampling was placed on providing opportunity for volunteers and interns at the two GSMNP science centers and undergraduate students of Coker College to be involved to the greatest degree possible. Training was conducted by Griswold and Lincoln with continued supervision by Prysby and Super. Emphasis was placed on obtaining floral associations. The goal was to cover as much of the flowering season as possible. Locality (with GPS derived coordinates), date, and floral relationship were recorded for each specimen. Specimens were mounted and labeled at the science centers, then sent to the USDA-ARS Bee Lab, where they were barcoded, identified and entered into a specimen-level relational database. Specimens are housed in the Bee Lab’s U.S. National Pollinating Insects Collection, and GSMNP. In an attempt to capture as much data as possible on bee distributions in the park material previously collected at Tremont Institute and in 2003 at Purchase Knob as well as records in the database of the U. S. National Pollinating Insects Collection were identified and included in the analysis.

Most specimens are deposited in the U. S. National Pollinating Insects Collection and the collection of Great Smoky Mountains National Park with vouchers returned to Tremont Institute and Purchase Knob.

RESULTS

More than sixty sites were collected at least once from April to October with sampling concentrated in seven areas (Fig. 1). Limitations in funding prevent even spatial coverage of collecting sites. The 4 most collected sites, Cataloochee, Purchase Knob, Tremont, and Cades Cove, had an average of 40 collector-days (range, 18 to 85 collector-days). These sites represented 74%, 70%, 59%, and 37% (respectively) of the total genera collected throughout the park and 39%, 56%, 27% and 18% of the total species. This study yielded a total of 1774 specimens for analysis. Collections were nearly evenly divided between pan trapping and net collecting; 45% of the records have floral relationships.

Abstract : 41 species and 10 genera of lichens are reported new to the park. New to science are Biatora pontica Printzen & Tønsberg (in print), Biatora sp. A, Lepraria lanata Tønsberg ined., L. placodiolica Tønsberg ined., Lepraria salazinica Tønsberg ined., Lepraria sp. A. P. May & Tønsberg ined., Rinodina sp. A, and Vainionora americana Kalb, Tønsberg & Elix in prep. ”Catillaria” croatica is new to North America. New to eastern North America are the genus Leioderma, and the species Fellhaneropsis cf. vezdae, Gyalideopsis anastomosans, G. (Microlychnus) epicorticis,Gyalideopsis sp. A, and Mycoblastus caesius. The park appears to be a diversity center for the genus Lepraria. Substratum is indicated for taxa new to the park. A list of the localities visited is provided. All specimens are georeferenced.

The aim of my ATBI project in the Great Smoky Mountain Nat. Park in 2002 was to add as many names as possible to the park lichen checklist with emphasis on sterile, corticolous crusts. Field work was carried out from June 12 to June 22, 2002. The lichen herbarium (DUKE) at Duke University, North Carolina, was visited (as part of the project) June 23-24.

According to their growth form, lichens are either folious (leaf-like and with an upper side and a lower side separable from the substratum), fruticose (more or less shrub-like) or crustose (crust-like and not separable from the substratum). Many of the crustose species are so small that a hand-lens is necessary to recognize them in the field (examples are Gyalideopsis anastomosans and Opegrapha corticola), whereas other species may cover extensive patches on tree bark (e.g., Biatora printzenii) and rock (e.g., Psilolechia lucida). Of the species listed below 4 species, Heterodermia japonica, Leioderma sp., Leptogium phyllocarpum, and Phaeophyscia squarrosa are foliose, whereas the other 36 are crustose.

My project focuses on sterile, crustose lichens. Sterile crusts are adapted to dispersal by vegetative diaspores (such as isidia and soredia). Lichens are classified according to their sexual structures (such as, e.g., apothecia). Sexual stuctures are usually necessary to elucidate generic affinity (when a molecular study is not possible). When apothecia are present a lichen is said to be fertile. Lepraria species are always sterile, i.e., apothecia are not known. Fortunately, most Lepraria species can be recognized on morphology and chemistry. Many crusts that are usually sterile may occasionally develop apothecia. However, among the park material are several species that I never found with sexual structures. These species have been sorted out by morphology and chemistry, but a search in the field for specimens with sexual structures is necessary before such specimens can be identified. Many of them probably represent new species.

Species are regarded as new to the park when they they are not listed in the (unpublished) park checklist.

Material and methods.
This report is based on material collected by the author in June 2002. Most specimens, including all sterile ones, have been subjected to thin-layer chromatography (TLC) according to the methods of Culberson (1972) and Culberson & Kristinsson (1970), and later modifications. Map datum is WGS84.

Before new cyanobacterial species can be described one should demonstrate not only that a cyanobacterium with similar morphology and ecophysiology has not been previously described, but that the new species can be phylogenetically placed based on the DNA sequence of its 16S rRNA gene. Chlorophyte and eustigmatophyte species generally require observation of the flagellated stages of their life histories. Both of these endeavors require extensive manipulation of cultures, as well as additional bench work.

For our last Discover Life in America Award, we requested funds to examine existing cultures of putative new algal species and provide the additional characterization that could lead to publication of the new species we have observed. We made an additional trip to collecting sites to obtain fresh material so that we could isolate some of the new taxa. Sites in Big Creek, Little River Gorge, Hen Wallow Falls, and Cataloochee were sampled in early May, 2002 along with other selected sites. We developed special low-nutrient media based on the water chemistry of these sites to isolate these unique and unusual strains. We also proposed to begin determination of sequences of 16S rRNA genes for cyanobacterial strains currently in culture and those isolated from the fresh samples.

We proposed to examine and photograph algae using Nomarski DIC photomicroscopes, and to write descriptions of the new taxa. As part of analysis, we promised to scan images to produce J-peg images for web-site construction. We stated our intention to publish a floristic study of our aerial habitat study in which the new taxa were to be described.

We have accomplished a great deal on the project this year, although not everything we had hoped to accomplish. The major achievements are 1) the floristic study on the algae of epilithic aerial algae of the Great Smoky Mountains National Park was published, 2) the species record list for new algal species records for the Great Smoky Mountains National Park has finally gone to print, and will be out in Algoglogical Studies, volume 111, in February 2004, 3) a paper describing three new Leptolyngbya species has been submitted to Hydrobiologia, 4) a paper describing a new species of Capsosira (Nostocales, Cyanobacteria) and a new genus of Nostocales (Rexia) was also submitted to Hydrobiologia, 5) we successfully used these seed projects to complement an NSF proposal (written with Rex Lowe, Bowling Green State University) that was funded in August 2003. This last grant ensures that our research in the park can continue, and will grant us some independence from the DLIA funds that have helped us in our work thus far.

Results

The floristic paper by Johansen et al. (2004) has been in the hands of the journal for some time, and will finally appear in two months time. It forms the basis from which our more recent work which was supported by a DLIA award can be evaluated. In that work, we reported that we had found a total of 173 new species records, of which 65 were distinct but not identified to species. These 65 taxa needed further work, and were potentially new species. The total number of algal records for the park was 584 species: 108 cyanobacteria, 97 chlorophytes, 12 tribophytes, 1 chrysophyte, 2 synurophytes, 3 eustigmatophytes, 353 diatoms, 4 dinoflagellates, 3 rhodophytes, and 1 euglenoid. A copy of the galley proofs for this project is attached.

During the period of DLIA support, we prepared a floristic report of the epilithic aerial algae based on the thesis work of Shannon Gomez (Gomez et al. 2003). The taxa in this paper are included in Johansen et al. (2004), so new records did not appear in this work that do not appear in the larger work. However, Gomez et al. (2003) give distributional information for 118 algal taxa occurring in several aerial habitats in the park. The work clearly demonstrates the fact that we have undersampled the park, and are likely to find many more species with further collections. A reprint of this paper is attached to this report.

The molecular characterization of new cyanobacterial species has been slower than we thought it would be. Isolation of many of the cyanobacteria has been very difficult, and so subsequent molecular analysis has not been done. However, we did manage to grow some very unusual strains in culture. One is a new Capsosira species that grows as the phycobiont in the rare aquatic lichen Hydrothyria venosa found growing in Hen Wallow Falls. This species is the second species of Capsosira to be described, and is thus quite a discovery. It has been named Capsosira lowei Casamatta, Gomez & Johansen, and will appear in a paper that is part of a festschrift volume for Rex Lowe (in Hydrobiologia). In the same paper, we describe a new genus in the Microchaetaceae, Nostocales, Cyanobacteria. It is a very unusual alga from aerial habitats that will be named Rexia erecta Casamatta, Gomez & Johansen in the same publication. Both of these taxa had their 16S rRNA gene and associated ITS region sequenced, and so they are very solidly published under both the botanical and bacteriological codes of nomenclature. A copy of the manuscript that was recently submitted (Casamatta et al., in review) is attached to this report.

We were not able to isolate the Leptolyngbya species we knew were new to science. We elected to describe three of these taxa under the botanical code only rather than not name them. One in particular is very distinct (Leptolyngbya badia sp. nov.), with a dark sooty brown sheath. We have only recently succeeded isolating this taxon, and so will likely sequence it as part of other studies of cyanobacteria from the park. The paper describing these Leptolyngbya species, and illustrating 7 taxa in the genus, has been recently submitted (Olsen et al., in review). It is attached for reference.

Olsen is currently writing her master's thesis, and will have many new records. So far, we know that her thesis will add at least 21 cyanobacterial taxa to the 108 cyanobacteria currently on record. She will likely add some diatom and green algal taxa as well.

Cost Share

I was able to use the DLIA award to leverage the following funding from my university.

Summer support for Shannon Gomez: $1,860
Overhead on salary for Gomez: 865
Lab supplies: 1,200
Sequencing expenses 400
Publication costs for papers 390 (with anticipation of more in future)
Total direct support from JCU $4,715

I worked on this project in collection, student mentoring, and manuscript preparation. My commitment in the proposal was for two weeks, but I have easily spent 6 solid weeks (spread out over the course of 18 months) of my time. The university paid my salary, fringe benefits, and overhead for this time, but I consider it an in kind contribution. The value of this in kind contribution is $14,966 (overhead and fringes added to salary).

Thus, the total value leveraged directly by this grant is $19,681.

Summary

I feel that substantial progress has been made this year in the algal twig. The preparation of four separate manuscripts (2 having completed the peer review process) as well as a funded NSF proposal seems to us to be an excellent outcome from the generous but limited DLIA award. I had actually hoped to be further along in describing the new species in the park, but this is a more arduous task than initially anticipated. However, I intend to continue to work on the flora and publish the new species we find (and have found). A website for the algal twig has been started by Rex Lowe, and I intend to install our species images on that website.

References

Casamatta, D.A., S.R. Gomez, & J.R. Johansen. In review. Capsosira lowei sp. nov. and Rexia erecta gen. et sp. nov., two newly described cyanobacterial taxa from the Great Smoky Mountains National Park (USA).

Gomez, S.R., J.R. Johansen & R.L. Lowe. 2003. Epilithic aerial algae of Great Smoky Montains National Park. Biologia, Bratislava 58:603-615.

Johansen, J.R., R. Lowe, S.R. Gomez, J.P. Kociolek and S.A. Makosky. 2004. New algal species records for the Great Smoky Mountains National Park, U.S.A., with an annotated checklist of all reported algal species for the park. Algological Studies 111:17-44.

Olsen, C.E., J.R. Johansen & R.L. Lowe. In review. New epilithic cyanobacteria records from Great Smoky Mountains National Park with descriptions of three Leptolyngbya species new to science.

A survey of the insect fauna associated with eastern hemlock was conducted from March 20 to November 20, 2002, at four sites (two representing mature growth and two representing new growth) in the Great Smoky Mountains National park, to provide information on species common on this host tree. The objectives of this project were: 1) develop a species list of insects associated with eastern hemlock, and 2) compare the insect fauna in relationship to their location on the host plant within the Great Smoky Mountain National Park. Insects were sampled using direct observations/sweep-nets, malaise, and pitfall traps. Two sites with mature hemlocks and two sites with new growth hemlocks were selected representing high and low elevation gradients within the park. Insects were collected from two trees per site using the following methodology:

1. Visual Observations/Direct Sampling of Insects : were obtained from the branches and foliage every 14 days (30-40 min/tree) within each study site by netting and handpicking. Sweep-net (a canvas net bag ca. 38 cm in diameter and 82 cm deep) samples were collected, placed in a vial/bag, provided a field label that included date, site number, and host identification, and taken to the lab for processing and identification.

2. Malaise/pan traps : consisted of a 60 cm 3 cube frame constructed with 1.9 cm diameter PVC pipe. The frame was covered with No-see-um ® fabric (73 perforations per 645 mm 2) on the two parallel sides, and in a pyramidal shaped top. In the middle of the cube, a separate piece of the fabric was used to form a flat surface, positioned perpendicular to the ground that would act as a barrier to flying insects. Some insects strike the barrier and fly upwards where the fabric is attached to the pyramid- shaped top (also constructed of No-see-um ® fabric) funneling the insects into the collecting head (ca. 60 mm wide x 65 mm deep; 120 ml vol) containing 30-60 ml of a 50% mixture of propylene glycol and water. Traps were suspended by nylon rope on two trees at each of the four sites. A pan that also containing 50% propylene glycol and water mixture was attached to the base of the malaise trap. Specimens were collected every 14 days in each of two mature and two new growth trees at each site and returned to the lab where they were processed and identified.

3. Pitfall Traps (24 cm diam.): were placed into shallow holes dug into the ground at each site to collect ground-inhabiting insects. Traps consisted of two 120 ml plastic cups (60 mm wide x 65 mm deep/120 ml vol) - one cup placed inside the other aided in sample collection and reduced impact of flooding. The inner cups were filled with a 50% mixture of propylene glycol and water. A plexiglas cover equipped with 90˚ directional fans were placed on the ground surface above the cups - the directional fans will guide the insects into the pitfall traps. Samples were retrieved every 14 days/site and taken to the lab for processing and identification. On each sampling date, the collection cup with preservative was removed, capped, and taken back to the lab, and a new cup was added.

Processing and Identification of Specimens : From 36 trips to the study sites, insects were collected representing18 sampling periods from March through November resulting in 16,987 specimens obtained from the three collecting methods. Specimens have beensorted and pinned, labeled (order, family, names, date, locality, host, collection method, number of specimens, collector, determiner), mounted on slides, or placed into alcohol vials. Processed specimens were separated into similar taxonomic taxa for identification using a Leitz stereoscope.

Insects collected are in the process of being identified to genus and species using standard keys; the more difficult species either have been or in the process of being sent to approved specialists (USNM, USDA, University specialists, etc.) for identification or verification. Currently, 114 species representing 46 families in 8 orders have been identified (Table 1). Voucher specimens of each species identified are being systematically arranged into Cornell drawers for future incorporation into the National Park and University of Tennessee insect museums.

Upon completion of the identification of the specimens within the next two months, we will compile a species list, analyze the specimens obtained from all collection methods at all sites, and assess any site differences in regard to species. Data consisting of species, family, and Order name, site, number of specimens, collection date and collection method are being entered into computer databases (Access, Excel, and Biota) for analysis. Determinations of any significant differences in the abundance of insect specimens, families, and species will be made using the Chi-square formula (SAS Institute 1989). Differences in the mean numbers of insect specimens and species captured at each location over the sampling period will be determined by using the Tukey’s test. Diversity and evenness will be assessed for each individual sampling method and taxa using the Shannon index. Species diversity and richness will constitute the total number of species in an area. Species evenness, essentially a percentage of the diversity, will be presented as a measurement on a scale of 0 to 1, where one represents the most even value for a community.

Successful Aspects : This study has been successful in achieving the objectives as outlined in the proposal. We estimate that we have completed about 80% of the project components at this time.

Insects were consistently obtained from each collection method during the 18 sampling periods, 114 of the projected 200+ species associated with eastern hemlock have been identified, we developed and presented posters on the project at the DLIA/ATBI conferences, the annual meeting of the Tennessee Entomological Society, and at the National Meeting of the Entomological Society, our work has generated positive newspaper articles on the adelgid project that serves eastern Tennessee, and this project has provided the opportunity for a graduate student to obtain experience in biodiversity and taxonomy, while engaged in a project that will be beneficial to the objectives of the ATBI.

Introduction

During my first year at Western Carolina University, I submitted a proposal to Discover Life in America (DLIA) in order to begin a bacterial and archaeal survey within Great Smoky Mountains National Park (GSMNP). I was awarded approximately 54% ($4063 of $7500) of requested funding for the 2002 DLIA grant proposal that I submitted and have been successful in obtaining monies in support of the DLIA grant as well as other ATBI-related endeavors. In my proposal, I suggested the following general deliverables: 1) data to be gathered for publication, 2) DNA sequences to be generated and added to online databases, 3) information obtained from GSMNP studies to be accessible on my WCU webpage (including a linking of microbial diversity to a map of he Park), 4) work on a pamphlet or booklet for the general public on the microbial ecology of the Park, and 5) storage of microbial cultures and DNA for future studies. I have made significant progress in all of these areas with goals 1, 2, and 5 being the most developed. With new DLIA funding this year and a reduced teaching load next year, I hope to continue progress on all five goals, with goals 3 and 4 receiving more attention. Below are specific details about each goal while scientific results are included in the next section.

Goal 1: I have had three undergraduates (Kim Lowery, Gina Parise, and Kristina Reid) and one graduate student (Henry Angelopulos) working with me over the last year and a half on projects in GSMNP. Along with independent work on my part, we have produced nine talks and 12 posters for seven local, regional, national and international meetings. We have gathered data not only on the identities of organisms that live in the Park but also relating to their ecology and distributions. Seven species of Archaea (six or seven new to science) and 52 species of Bacteria (37 new to science) have been thus far detected in our work and we have a backlog of microorganisms to identify (~170). The three undergraduate projects are publishable and a rough draft of each is being undertaken by each of the students with planned submissions over the summer and into fall. One ATBI Quarterly article was published and my article from last year’s ATBI Quarterly was reprinted (see Publications/Presentations section for more detail).

Goal 2: Seventy-two DNA sequences have been generated to help identify the archaeal and bacterial species we are examining from the Park. These will be added to two databases this summer (GenBank and Ribosomal Database Project II) and assigned “GSMNP.A-X” and “GSMNP.B-Y” to designate them as being from the Park and “A-X” or “B.Y” further designating them as archaeal sequences or bacterial sequences, respectively. The use of DNA sequences in microbial ecology is widespread and critical. This sharing of data with other researchers and linking novel species to the Park will give our work and the ATBI some additional attention.

Goal 3: Poster presentations from this year’s American Society for Microbiology Annual Meeting in Washington, DC will be made available online for interested persons. I am also working on enhanced webpages related to my courses taught that utilize the ATBI as a teaching tool in my classes. I hope to have these things completed this summer and revised in the coming years as new projects are generated.

Goal 4: With the generation of substantial information about the prokaryotes of GSMNP, we have the beginnings of a decent story to tell about the microbial ecology of the Smokies. I plan to add this summer to the number of ATBI sites sampled (increasing from three to twelve ATBI plots) and work over the next year to two on a visitor’s guide pamphlet to the Park. Even if the creatures I study are “sub-visible”, I believe some of the general public would be interested in the microbes that make the world go round.

Goal 5: For organisms that can be cultivated in the laboratory, we are freezing samples away for future studies (i.e., cultures can be revived and grown again). Such studies will include the biochemical testing of bacterial organisms for their metabolic capabilities – tools that can help differentiate between species. For those organisms that resist cultivation (e.g., all Archaea detected thus far) we are archiving DNA samples for future studies as well. It is hoped that we can begin to cultivate Archaea and have whole organisms to study in the future – indeed this would be a scientific breakthrough.

This report details the work completed to-date under the project entitled “A Web-based Identification Guide to the Leaf Beetles (Coleoptera: Chrysomelidae) of Great Smoky Mountains National Park.” This project proposal received partial funding (53%) from DLIA during spring 2002.

The partial funding received was allocated entirely to the purchase of a single software package essential to the production of high-quality digital images of small insects. All of the original objectives of the proposal could not be met with partial funding; none-the-less, substantial progress has been made.

Accomplishments Toward Original Objectives

Objective 1

The software package Auto-Montage (Syncroscopy, Synoptics Ltd.) was purchased and integrated with existing computer, digital camera, and microscope to establish a digital imaging station in the TAMU Insect Collection. The senior PI learned the system sufficiently to produce images of leaf beetles of the mid- to large-size range (2.5 to 10 mm) and for species with varying degrees of surface reflection. Lighting techniques for the smaller specimens were tested but need further refinement. Dorsal habitus images were completed for 26 leaf beetle species (18% of the species confirmed for GSMNP). An image of each species is included as part of the “species page” for each species in the Identification Guide (see objective 3).

Step-by-step methods were established for the production of stock images of leaf beetles. These procedures are broken down as follows:

Pre-imaging:
1) selection of a specimen as model for image,
2) manipulation of specimen to position legs and antennae,
3) re-mounting specimen on new point to hide original point/pin,
4) entering specimen name, label data, etc. into imaging log book.

Imaging:
1) set lighting, magnification, and camera settings particular to species and record in these details in log book,
2) generate a series of images at different focal planes (usually 7 to 12 separate images),
3) execute Auto-Montage program to produce a single in-focus image.

Post-imaging:
1) a copy of the Auto-Montage image is further refined using Adobe Photoshop to complete a “stock” image.

With the above procedures established and implemented, stock images can be completed at a rate of approximately 1 image/hour. The pre-image specimen manipulation has proven to be more time-consuming than originally estimated.

Objective 2

Not attempted under present award.

Objective 3

An identification guide to the leaf beetle species confirmed or expected to occur in GSMNP has been initiated (see http://insects.tamu.edu/re sea rch/collec tion/chrysomelidae/) The guide has three components:

1) Identification Key (working draft completed)
2) Help Boxes (ca. 22 required, none completed)
3) Species Pages (ca. 330 required, 26 completed)

Identification Key : The identification key serves as the backbone of the identification guide. In its present format, it is a simple html document with hyperlinks to help boxes and species pages. Additional “internal” links allow for forward or backward jumps within the key.

The key is modeled in part after a traditional dichotomous key except that the end result (or identification) may be a single species or a group of similar species. When the end result is a group of similar species, a nested set of further divisions within the numbered couplet may be given (for example, see couplet 21b). Further tracing among these divisions may result in the identification of a single species or a group of similar species. For discrimination of species within groups of similar species, the user should view the linked species pages where images and diagnostic remarks are found. This key structure was adopted for three reasons: 1) to keep the key short and simple, 2) to cluster similar species closely in the key, and 3) to allow for easier editing as the key is further refined.

Where possible, the key employs less rigorous taxonomic characters and uses terms likely to be understood by laypersons with minimal training. None-the-less, some technical terminology must be used. For many such cases, “help buttons” have been placed at the appropriate places in the key (indicated by the text “[HELP]”). Much of the higher classification of leaf beetles is preserved in the structure of the key, i. e., most leaf beetle subfamilies and major tribes key-out as blocks.

Help Boxes : Help boxes are pages linked to “help buttons” placed in the identification key. Help boxes contain explanations (and will ultimately include line illustrations) that explain and depict taxonomic characters which may be difficult for the layperson to interpret correctly. The primary purpose of help boxes is to keep the identification key simple. This is accomplished by removing lengthy explanations and illustrations, which will not be needed by many users, yet are essential for first-time users and non-specialists. Text and illustrations for the help boxes are not presently prepared.

Species Pages : Species pages will likely be the most useful component of the identification guide (See any of the 26 species pages currently linked from the Identification Guide (see http://insects.tamu.edu/rese arch/colle ction/chrysomelidae/). Each page follows a template that includes a dorsal habitus image (produced under objective 1), and blocks of text under the following seven headings:

1) Synonymy: The species synonymy is presented here. This text is taken from the recently published leaf beetle catalog of Riley, et al. (2003). The completion of that work was supported in part by the Small Grants Program of DLIA, 2001 funding cycle. Synonymies are complete for all confirmed and potential leaf beetles species of GSMNP.

2) Classification: The formal classification levels between superfamily and genus are listed here for the respective species.

3) Diagnostic Remarks: Distinguishing characters or other remarks appropriate for species recognition is given here. These remarks are prepared on a species by species basis and often include detail specific for GSMNP and surrounding region.

4) Distribution: The Canadian provinces and states of the United States are listed here using their two-letter postal codes. Dubious province/state records are given in lowercase and enclosed in brackets. These data are taken from the recently published leaf beetle catalog of Riley, et al. (2003). The completion of that work was supported in part by the Small Grants Program of DLIA, 2001 funding cycle. These data are presently compiled for all confirmed and potential leaf beetles species of GSMNP.

5) Food Plants: Leaf beetles are often highly specific in their choice of food plants. Tracing beetles to plants or plants to beetles is probably the most important way to foster an interest in leaf beetles and the watching of micro-wildlife among persons with a general interest in field biology. The data under this section includes the names of plants that are known or likely food plants. These text blocks are condensed versions of the text to be published soon in a catalog of food plants for North American leaf beetles ( Clark, et al., in prep.). The plants listed may or may not occur in GSMNP, none-the-less, all food plants are listed since they provide a clue which may lead to the discovery of food plants that do occur in the park.

6) Comments: These remarks are prepared on a species by species basis and will sometimes include detail specific for GSMNP and surrounding region.

7) References: Included here are the reference for the original description of the leaf beetle species and any references cited in the text blocks (other than original descriptions of the junior synonyms). In additional, important references on taxonomy and biology are also be included if they exist and are deemed appropriate.

Each species page pops up as a separate web-browser window that can be re-sized by the user. More than one window can be opened and re-sized at anytime, so that images of similar species can be compared side-by-side on the screen (for example, open and re-size both Neolema species, couplet 10a).

Objective 4

Not attempted under present award.

The Future

Simple html text pages are not the optimal way to present web-based identification keys. We are currently exploring options for producing multiple-entry keys such as the software package Lucid (CSIRO Publishing), and considering the further development of a key-generating program originally developed at TAMU by the junior PI (the original objective 4).

Future effort on this project should be focused primarily in two areas: 1) additional field collecting, and 2) the production and linkage of additional species pages. Further field work is needed to refine the list of leaf beetles confirmed and expected in the park and to generate within-park distribution data that can ultimately be linked to each species page. Additional fieldwork will also provide plant association observations that are specific for GSMNP and surrounding areas.

For GSMNP, the current number of confirmed leaf beetle species is 139, the current number of additional possible species is at ca.190, although this latter figure is probably an overestimate.

Literature Cited

Clark, S. M., D. G. LeDoux, T. N. Seeno, E. G. Riley, A. J. Gilbert, and J. M. Sullivan. (in prep., expected in 2004). Host plants of leaf beetle species occurring in the United States and Canada (Coleoptera: Orsodacnidae, Megalopodidae, Chrysomelidae exclusive of Bruchinae).

Riley, E. G., S. M. Clark, and T. N. Seeno. 2003. Catalog of the leaf beetles of America north of Mexico (Coleoptera: Megalopodidae, Orsodacnidae and Chrysomelidae, excluding Bruchinae). Coleopterists Society Special Publication no. 1, 290 pp.

The DLIA grant made it possible for me to visit some of the collecting plots currently used in the All Taxa Biodiversity Inventory and from which material earlier had been sent to me for identification (e.g. Road Prong, Clingmans Dome, Newfound Gap, Greenbrier Cove, Twin Creek, Ravensford). Valuable information as to soil types and vegetation could be documented for use in the future description of the Pauropoda fauna of the GSMNP. The result of this collecting is hundreds of specimens of 38 species, 7 or 8 of them new to science, indicating an unusual high diversity.

In the previous ATBI collecting the soil fauna had been extracted by funnels (type Tullgren). Now I got opportunities to collect manually (from the underside of stones and logs and under bark of dead trees) and to extract specimens by water flotation. Thanks to several cooperating biologists, who were clever collectors of the very small pauropods (Ernest C. Bernhard, Charles Parker, Ian and Stephanie Stocks), the manual collecting was prosperous. In spite of the dry soils we could find pauropods at about 30 different sites from the lowest levels in the Cades Cove area to the highest near Clingmans Dome. The collecting results from the lower western part was unfortunately meagre, probably because the pauropods, as a result of the dry weather, had migrated to deeper soil levels. Collection there in winter or spring should be of interest. When our collecting was directed on the northern and eastern parts of the forest it became obvious that pauropods were often met with. It was really astonishing to find that almost everywhere we searched for them we also found. That shows that pauropods form a common constituent of many moist soil types in almost the whole GSMNP, at least where the soil cover is not too thin and the humus content not too small. The picture from the time before the start of the All Taxa Biodiversity Inventory, 5 species (20 specimens, 12 of them from Cherokee), gives a completely wrong description of their occurrence in the park..

The time consuming taxonomical study of both the manually collected specimens and the flotation material will begin in early 2003. Then a first manuscript with descriptions of new species will be prepared and forwarded for publication.

During my visit I also held a lecture on pauropods (morphology, systematic, reproduction, ecology, diversity) at the University of Tennessee, with Professor Dr. Ernest C. Bernhard, Entomology and Plant Pathology.