In 1867 Swiss botanist Simon Schwendener was the first to recognize that lichens were symbiotic associations of fungi and algae (or, as subsequently discovered, cyanobacteria) (for more info, try this EOL podcast on lichen–“a tropical rainforest in miniature”).  Today about 13,500 species are described (lichens are named for fungal component), representing 18% of the 74,000 known fungi. It is remarkable that so few fungi have been named, given that estimated diversity is 1.5 million (Hawksworth, 2001). This presumably reflects difficulty of morphologic diagnosis of often microscopic, unculturable organisms with diverse life forms and highlights a need for molecular methods. Several recent epidemics causing serious animal and plant mortality have turned out to be newly recongized fungi [including Batrochochytrium dendrobatidis (chytridiomycosis in amphibians), Geomyces destructans (White-nose Syndrome in bats), Cryophonectria parasitica (Chestnut blight), and Ophiostoma spp. (Dutch elm disease)], hinting at the hidden diversity and importance of fungi.

Back to lichens–in March 2011 New Phytologist, researchers from Royal Botanic Garden (RBG) at Edinburgh and Kew report on DNA barcoding of lichenized fungi using internal transcribed spacer (ITS) region. ITS has been widely used in fungal taxonomy and has been proposed as a standard barcode region for this group (the standard barcode for animals, COI, has so far been difficult to reliably amplify from the diversity of fungi either due to variability at primer binding sites or introns). ITS refers to 2 regions in the nuclear ribosomal RNA gene complex (5′ external transcribed sequence—18s rRNA—ITS1–5.8s rRNA—ITS2—28s rRNA—3′ external transcribed sequence), which is present in several thousand copies in each cell. Advantages of ITS as a barcode region include availability of broad-range primers that bind to conserved regions in 18s and 28s rRNA; presence of multiple copies per cell, facilitating recovery from small or degraded samples; and the legacy of ITS fungal sequences in GenBank. Disadvantages of ITS as barcode locus are that is a non-coding region, making it more difficult to align and compare sequences; multiple copies per cell of which may differ from one another; and presence of misidentified sequences in the legacy data.

Kelly and colleagues sampled 112 freshly collected and herbarium specimens from one genus (Usnea) including 16 of the 19 species occurring in the British Isles and 248 specimens from native woodland habitats in Britain, comprising “94 species from 55, 28, and 8 genera, families and orders, respectively.” In the latter floristic set, 66.0% of species were represented by 3 or more samples and 77.7% by 2 or more samples. DNA was extracted using DNAeasy Plant Mini Kit and amplifications were performed with sets of standard primers that amplify the entire ITS segment (ITS1-5.8s rRNA-ITS); nested PCR was performed on “a small number of samples that failed to yield a single discrete product with standard PCR.” If these failed to generate a suitable product for sequencing, then a “thin slice of a single apothecium” was placed directly into the PCR mix and amplified as above or using primers for ITS2 only.  The full ITS region was obtained from 80.9% of combined 351 samples (75.9% of Usnea and 83.9% floristic). 22 (6.3%) of products showed heterogeneity on direct sequencing and required cloning to obtain suitable products for sequencing. The commonest regions for failure were no amplification [7.1% overall, largely with older (>3y) specimens; and amplification of non-target fungi (2.0% overall, only with field samples from floristic dataset)].

Is there a “barcode gap” (intraspecific<<interspecific distance) among fungal ITS sequences? In this study at least, usually yes. The RBG researchers defined clusters as nodes with ? 70 BP under BIONJ method or PP ? 0.95 under Bayesian inference. Under these criteria, species discrimination was 73.3% for Usnea dataset and 92.1% for floristic dataset. Simple BLAST analysis was also usually accurate–80% of Usnea species and 92.1 of floristic species were correctly assigned. This bodes well for cataloging the “dark matter” of fungal biodiversity using ITS DNA barcodes. So little is now known, it is exciting to contemplate what will be learned!

Infectious diseases may determine survival of individuals, entire species, and perhaps even large branches on the Tree of Life. Beginning in the late 1970’s, rapid declines in amphibian populations around the globe were noted and today about 40% of world’s 6,671 amphibian species are threatened with extinction (e.g. Stuart et al 2004). The major cause appears to global dissemination of a pathogenic chytrid fungus, Batrachochytrium dendrobatidis, first reported  in 1998 and formally described in 1999.

Although the global pattern is clear, many local population declines remain enigmatic due to absence of histologic data. In addition, the pattern of spread of the fungus and its timing in relation to mortality are not known. In April 2011 Proc Natl Acad Sci USA (open access), researchers from San Francisco State University and University of California, Berkeley, describe a non-invasive, DNA-based method for detecting B. dendrobatidis (Bd) in formalin-preserved specimens. Although exceptions are reported, DNA recovery after formalin treatment usually fails,  so these are remarkable results.

Cheng and colleagues analyzed formalin-preserved salamander and frog specimens collected in Mexico, Guatemala, and Costa Rica in areas where population declines had occurred. Specimens were rinsed in 70% ethanol, then, using a skin swab or dental brush, “stroked 30 times over the ventral surface…from neck to vent” [salamanders] or “on the ventral surface, including the inner thighs, abdomen, and between toes” [frogs]; the swab/brush was then stored in a microfuge tube at 4 ^oC until processing. DNA was extracted with a standard kit (Prepman Ultra or Qiagen DNeasy), and a 146-bp segment of Bd ITS-1 region was amplified, using 1/80th of recovered DNA for each amplification, run in triplicate using real-time PCR along with positive and negative controls.

Initial trials were done with 29 Bd-infected (as determined by histology) and 9 Bd-uninfected formalin-preserved Batrochoseps salamander specimens. Bd was detected in in 24 (84%) of infected specimens and none of uninfected  specimens. They suggest that their success with such unlikely specimens may reflect “(i) the very short length (146 bp) of the target sequence for Bd amplification, (ii) the presence of many copies per Bd cell of the ITS-1 region being targeted in our assay, and (iii) recovery of many cells  of Bd in our swabbing technique because Bd grows on the skin surface of the host.”

The researchers then applied this assay  to frogs and salamanders collected in Mexico (n=537), Costa Rica (n=74), and Guatemala (n=615) between 1964 and 2010. They found Bd as early as 1972, with a large increase (>50% prevalence) beginning in 1980, coincident with the observed population declines (see figure above). Combining their results with those of Lips et al 2006 indicated a steady southward movement of Bd from southern Mexico in 1972 to Panama in 2004. They interpret this remarkably slow expansion to mean that the pathogen is spread by the animals themselves, perhaps as they move between the tiny pools of water that collect in the crowns of bromeliads. The near coincident appearance of Bd around the world suggests additional modes of spread, possibly including human activities. I look forward to additional studies that will shed light on the global dissemination of Bd and point to interventions to limit this ongoing disaster for amphibians.

From the conference website:  www.dnabarcodes2011.org:

The Consortium for the Barcode of Life and the University of Adelaide invite you to join us in Adelaide, Australia from 28 November – 3 December 2011 for the Fourth International Barcode of Life Conference. Barcoding has seen extraordinary growth since the Mexico City Conference in November 2009 so join participants from around the world for the biggest barcoding event ever!

The organizers have developed this website to provide potential participants, co-sponsors, and other stakeholders with information about the conference. The conference organizers are also eager to have your feedback as we plan the conference so please share your ideas through Connect, the DNA Barcoding network. You can do this by using the links found throughout this website.