Dugan & al. • Vouchering with identification through morphology and DNA TAXON 56 (4) • November 2007: 1238–1244 METHODS AND TECHNIQUES A large scale plant survey: efficient vouchering with identification through morphology and DNA analysis Laura E. Dugan1,3, Martin F. Wojciechowski2 & Leslie R. Landrum2 1 Global Institute of Sustainability, Arizona State University, Tempe, Arizona 85287, U.S.A. 2 School of Life Sciences, Arizona State University, Tempe, Arizona 85287, U.S.A. [email protected] (author for correspondence) 3 Current address: Integrative Biology, University of Texas at Austin, 1 University Station C0930, Austin, Texas 78712, U.S.A. A large-scale survey of the Central Arizona-Phoenix Long-Term Ecological Research study area was conducted in which many kinds of samples were collected (e.g., plant, insect, soil). The plant samples were of a smaller size than typical herbarium accessions due to limitations in personnel, storage space, available plant materials, and time. The vouchers were identified in the field if possible. The vast majority of the rest were identified in the herbarium morphologically after the survey; less than 5% of the vouchers remained unidentified. Of these, a subset were subjected to molecular analysis using sequences of the nuclear ribosomal ITS region and to a BLAST search of sequences in GenBank to find closely related taxa. The close relatives along with the unknown specimens were subjected to maximum parsimony analysis to identify the potential phylogenetic relationships of the unknown specimens. Through this analysis nine unknown plant accessions were identified to family, tribe, genus, and species. Once this level of identification was established, some specimens were re- examined morphologically and compared with potentially related taxa in the herbarium to confirm or improve identifications. We outline an efficient method for surveying and collecting plant vouchers for a large sampling area. We also demonstrate that one can combine morphological and molecular data in the identification process to produce more complete datasets. KEYWORDS: Arizona, nrDNA ITS sequences, plant identification, survey, voucher specimens known to exist in the county before. It is highly likely INTRODUCTION that many had been present in the county for years, but Voucher specimens are fundamental in the biological had not been documented. Voucher specimens help to sciences (Boom, 1996). Documenting what organisms create a measure of a particular area’s biodiversity and are being studied, whether it is for molecular systemat- vegetation structure at a given point in time and provide ics, taxonomic monographs, ethnobotanical studies or documented proof that a plant did, in fact, exist in a given ecological studies, links names of organisms to specific place at a particular time. In the Sonoran Desert about specimens that can be re-examined by future workers. half of the species of plants are ephemerals that respond The identifications can be verified or corrected, greatly to seasonal rainfall and do not appear every year. Based enhancing the value of the study when voucher specimens on records provided by vouchers, we know that some are obtained. Studies without voucher specimens are plants have disappeared from the Phoenix area as wild sometimes without value, while incorrect determinations plants (e.g., Cephalanthus occidentalis L., Prosopis have a negative effect by providing false information that pubescens Benth.) and that others have only appeared may be perpetuated in subsequent publications. in the last few years (e.g., Oncosiphon piluliferum (L. Collecting voucher specimens is crucial to obtain- f.) Källersjö). Thus, the visible flora changes according ing accurate identifications of plants present in a study to rainfall patterns, and the total flora changes because area. Morgan & Overholt (2005) document a situation of local extinctions and introductions. in St. Lucie County, Florida in which very poor records The use of DNA sequence analysis using molecular had been maintained of the vegetation in the county, phylogenetic methods can further enhance our knowledge and the result is a gap in the knowledge of the com- of the flora of an area by aiding in the identification of munity structure of plants in that area. In one survey unknown specimens and biodiversity (e.g., Kress & al., they found an additional 18 category one invasive plant 2005; Cowan & al., 2006). Specimens may be unidenti- species (community-altering species) that had not been fiable morphologically because they do not have mature 1238 TAXON 56 (4) • November 2007: 1238–1244 Dugan & al. • Vouchering with identification through morphology and DNA vegetative or reproductive structures, or do not have an Method of collection. — Instead of standard-sized essential identifying structure present (e.g., leaf, flower, herbarium sheets (30.5 × 43.0 cm) specimens were col- fruit). A specimen may remain unknown because the spe- lected in the field into herbarium packets 10 cm by 10.5 cies has not been known to exist in an area previously, or cm in size. The design of the ASU packet is such that they is extremely rare in an area, and thus there is little or no do not easily open (see ASU website for instructions at previous knowledge of it existing in a particular locality. http://lifesciences.asu.edu/herbarium/packet.html). The Also, as in the survey described here, many plants were packets each had a pre-printed label that could easily be left unidentified (“unknown”) because they were rarely filled-in in the field, with information about the partic- used or are non-native horticultural plants (found at urban ular survey point locality, field identification if any, and sites). By analyzing the sequence of a variable, nuclear a collection number for that point. Each collection was spacer region that is commonly used for phylogenetic given a unique identification code that is a combination studies in plants, we were able to determine the identity of the collection point and a separate specimen number. of some otherwise unidentifiable plants collected during The “collector” is CAP LTER survey crew for 2005. Suc- the course of this biodiversity survey. culent plants that could not be easily collected or plants in urban settings where sampling would have disrupted a landscape design were photographed only. The packets were placed into a standard plant press and placed in a METHODS drier within two days. When the survey was completed The CAP LTER 200 Point Survey. — From mid- the identified specimens were organized alphabetically February to early June of 2005 a large-scale survey of by family, genus, and species. Five thousand two hundred the Phoenix, Arizona, metropolitan area and outlying and twenty-five voucher specimens collected were stored agricultural and desert areas was conducted as part of in 28 CD storage boxes. The boxes required ca. one-half the interdisciplinary Central Arizona-Phoenix Long-Term of one herbarium cabinet for storage. Their label data are Ecological Research (CAP LTER) study at Arizona State available at http://seinet.asu.edu/collections/selection2.jsp, University (ASU) funded by the National Science Founda- and the collection is stored in the ASU herbarium. tion. This survey is performed once every five years and Taking photographs of every plant collected was part the 2005 survey was the second such survey conducted. of the plan at the beginning of the survey, but after one One of the purposes of this survey is to create a “snapshot” week photography was discarded in most cases because of the biodiversity present in the study region at that point of the amount of time it required. Field crews usually con- in time, which can then be analyzed alone and can also sisted of three or four members. Sites contained up to 60 be compared to the “snapshots” of the area in other years plant species, and it was quickly realized that photograph- (e.g., the 2000 survey as well as those surveys yet to be ing every individual species was not practical for the field conducted). crew available. An additional member whose job it was to An area of 6,387 km 2 was surveyed by collecting data photograph specimens would have been desirable and a from 204 randomly selected sites, each measuring 30 × 30 more complete record of each site could have been made. m, over a range of land-use types from heavily urbanized Plants were collected, when possible, that were either commercial and residential sites within the city, to agri- in fruit or flower, and as much of the plant was collected cultural land on the outskirts of the city, to undeveloped as (1) could easily be removed from the ground and (2) desert surrounding the city. Several values were measured fit into a collection packet. When the plants were neither and many kinds of specimens collected (e.g., soil, plant, flowering nor fruiting, as much of the plant (e.g., basal and insect). We anticipated that a few thousand plants would stem leaves, stems showing branching, roots if possible) be encountered in this survey. Vouchering each plant on a was collected as feasible. For every plant that was not standard herbarium sheet at each site seemed impractical identifiable in the field, notes were generally taken on for various reasons: (1) adequate material for a full-sized the collection packet regarding architecture of the plant, sheet was often not available; (2) sufficient personnel were microhabitat, and other pertinent characteristics to aid in not available to collect complete, full-sized specimens; (3) later identification. storage of the anticipated number of specimens would have The large majority of the specimens collected in the required about five full-sized herbarium cabinets. An al- 2005 survey were identified in the field and only required ternative method that produced vouchers but that required verification by comparison to existing specimens in the less time and expense was devised.