EDITORIAL Production Changes for Future Publication of the Journal of Cave and Karst Studies SCOTT ENGEL Production Editor The Journal of Cave and Karst Studies has experienced December 2011 issue, printed copies of the Journal will be budget shortfalls for the last several years for a multitude automatically distributed to paid subscribers, institutions, of reasons that include, but are not limited to, increased and only those NSS members with active Life and cost of paper, increased costs of shipping through the Sustaining level memberships. The remainder of the NSS United State Postal Service, increased submissions, and membership will be able to view the Journal electronically stagnant funding from the National Speleological Society online but will not automatically receive a printed copy. Full (NSS). The cost to produce the Journal has increased 5 to content of each issue of the Journal will be available for 20 percent per year for the last five years, yet the budget for viewing and downloading in PDF format at no cost from the the Journal has remained unchanged. To offset rising costs, Journal website www.caves.org/pub/journal. the Journal has implemented numerous changes over recent Anyone wishing to receive a printed copy of the Journal years to streamline the production and printing process. will be able to subscribe for an additional cost separate However, the increasing production costs, combined with from normal NSS dues. The cost and subscription process the increasing rate of good-quality submissions, has were still being determined at the time of this printing. resulted in the number of accepted manuscripts by the Once determined, the subscription information will be Journal growing faster than the acquisition of funding to posted on the Journal website. We anticipate that in the print those manuscripts. As a result, the Journal is future, subscription fees will be included as an optional experiencing an unprecedented backlog of manuscripts to payment along with annual NSS dues renewal or can be be published, resulting in long delays (up to one year) made separately online or through the NSS office. We hope between acceptance of a manuscript and its printing. In to reduce the financial burden on individuals by continuing order to clear our backlog and stay within budget, changes to allow free access online to all. have to be made. Journal staff and the NSS Board of While this is not an ideal solution for many current Governors have been evaluating numerous options for recipients of the Journal, it is consistent with the operation more than a year to find the best approach to maintain the and distribution of many scientific journals around the Journal as the premier peer-reviewed journal for karst world. The Journal has grown to become the most studies while keeping it within the budget constraints of the recognized karst science publication in the world and is modern economy. the cornerstone of science education outreach in the NSS. At the recommendation of the Journal Editor, the NSS We believe that the change to electronic publication will Board of Governors voted in July 2011 to begin moving to serve to preserve the Journal in a sustainable manner and an electronic publishing process to reduce production and will allow it to continue as a key resource for the Society’s printing costs (Act 81-854). Accordingly, beginning with the mission to advance the study and science of speleology. Journal of Cave and Karst Studies, August 2011 N 49 A. D. Asencio and M. Aboal – In situ nitrogen fixation by cyanobacteria at the Andragulla Cave, Spain. Journal of Cave and Karst Studies, v. 73, no. 2, p. 50–54. DOI: 10.4311/jcks2009lsc0129 IN SITU NITROGEN FIXATION BY CYANOBACTERIA AT THE ANDRAGULLA CAVE, SPAIN ANTONIA DOLORES ASENCIO1 AND MARINA ABOAL2 Abstract: Andragulla Cave is 2 m high, 6 m wide, and 2 m long (deep). Its lack of depth means that it is not isolated from external influences, and the microclimate is very similar to that experienced externally. The common stress factors on the growth of cyanobacterial communities on walls inside of Andragulla Cave include excess light, dryness, lack of nutrients, and cold temperatures. Nitrogen fixation, photon flux, relative humidity, and temperature in Andragulla Cave were measured hourly over 24 hours in winter. Nitrogen fixation by the reddish-brown mat formed by both cyanobacteria Scytonema mirabile and Gloeocapsa sanguinea in cave-like environments was measured in situ for the first time by acetylene reduction. The mat-specific rates (1.6 to 7.5 nmol C2H4 m22 s21) were lower compared to published values from laboratory measurements of rehydrated samples from different environments. Daytime fixation was slightly higher than nighttime fixation, where nitrogen fixation by Gloeocapsa sanguinea played an important role. The most influential parameters for environmental nitrogen fixation in Andragulla Cave were photon flux for daytime fixation, temperature for nighttime fixation, and relative humidity for both. Nitrogen fixation by cyanobacteria may be significant in these N-poor ecosystems. INTRODUCTION the in situ fixing behavior of organisms (Aranibar et al., 2003; Grimm and Petrone, 1997; Sivonen et al., 2007), and Atmospheric nitrogen fixation ability is limited exclu- such information in cave settings has been found only in sively to prokaryotes. Cyanobacteria are the only prokary- the study of Griffiths et al. (1987), for the genera Nostoc otes capable of both nitrogen fixation and oxygenic and Gloeothece. photosynthesis. The coexistence in a single organism of Because certain factors affecting nitrogen fixation are oxygenic photosynthesis and nitrogen fixation appears to sometimes irreproducible in the laboratory and there is be paradoxical, because nitrogen fixation is an intrinsically hardly any information available about nitrogen sources in anaerobic process. The key enzyme involved, nitrogenase, such adverse environments as cave settings, prompted us to is rapidly and irreversibly inactivated in vivo when exposed undertake this study. This work is the first to present in situ to even low oxygen partial pressures. nitrogen fixation data in the Scytonema mat communities In heterocysted cyanobacteria, a spatial separation growing in caves. takes place between the incompatible nitrogen fixation and oxygenic photosynthesis processes. Even though heterocysts retain photosystem activity I, they lack STUDY AREA photosystem II and activity of ribulose bisphosphate carboxylase, a fundamental enzyme in the Calvin cycle. Andragulla Cave (UTM 30SWH848264) is situated at an elevation of 1,200 m in the municipality of Moratalla, in Consequently, heterocysts are neither able to fix CO2 nor the Region of Murcia in southeastern Spain. The climate of able to produce O2. They also have a special cellular casing that restricts oxygen from entering (Walsby, 1985). For this this region is Mediterranean, with certain continental reason, heterocysts are practically anoxic inside and are characteristics and frequent frosts in winter. The mean nitrogen fixation sites (Fay, 1992). annual rainfall is 451 mm, and the average temperature is It is likely that all heterocysted cyanobacteria are able 16.3 uC. The area is made up of finely stratified limestone to fix molecular nitrogen (Gallon, 1980). When other with overlaying layers of differently colored loams. compounds of nitrogen are limited, using atmospheric The cave entrance (Fig. 1), faces northwest and nitrogen gives a competitive advantage over those organ- measures 2 m high, 6 m wide, and 2 m long (deep). Its isms lacking nitrogenase (Fogg et al., 1973). This capacity lack of depth means that it is not so isolated from external enables cyanobacteria to play a very important role in influences, and the microclimate in the cave is very similar hostile environments (Housman et al., 2006; Zielke et al., 2005). In general, studies into an organism’s atmospheric 1 Div. Bota´nica, Dpto. de Biologı´a Aplicada, Universidad Miguel Herna´ndez Avda, Universidad, s/n 03202 Elche, Alicante, Spain. [email protected] nitrogen fixation capacity have been done in a laboratory 2 Div. Bota´nica, Dpto. de Biologı´a Vegetal, Facultad de Biologı´a, Universidad de setting. Therefore, very little information is available about Murcia, Campus de Espinardo. 30100 Murcia, Spain. [email protected] 50 N Journal of Cave and Karst Studies, August 2011 A. D. ASENCIO AND M. ABOAL Figure 1. (a) Panoramic view of Andragulla cave in the Figure 2. Light micrographs [scale bar 10 mm]. (a) Scyto- Region of Murcia, southeastern Spain. (b) Walls of nema mirabile. (b) Gloeocapsa sanguinea. Andragulla Cave showing the cyanobacterial mats. to that experienced externally. Inside the cave there are were used. Electrodes were placed on the rock surface schematic-style cave paintings characterized by simple adjacent to the mats. figures dating from the end of the Neolithic to the Iron Nitrogen fixation in gaseous samples was quantified by Age (Beltra´n-Martı´nez, 1983). acetylene/ethylene reduction assays (Peterson and Burris, 1976) analyzed in a Shimadzu GC-14A gas chromato- MATERIAL AND METHODS graph. So as to not damage the cave paintings on surrounding RESULTS surfaces, great care was taken to collect material on the parts of the Andragulla cave wall where the Scytonema Nitrogen fixation was detected for the first time in a community grew (Asencio and Aboal, 2001). Samples were community formed by Scytonema mirabile (Fig. 2a) and not hydrated, but were incubated in situ in triplicate in Gloeocapsa sanguinea (Fig. 2b) growing as a brownish- transparent and opaque vials to reproduce light and dark reddish mat in Andragulla Cave (Asencio and Aboal, environments, respectively. Then 10% of the gas in these 1996). vials was replaced with acetylene using syringes inserted Nitrogenase activity during the day varied from 1.6 to 22 21 into the rubber stoppers (Hardy et al., 1973). Gas samples 7.5 nmol C2H4 m s (Fig. 3). The nitrogenase activity were taken at hourly intervals from sunrise to sunset on remained low and constant in the morning from 8:00 a.m.