diversity Article Ojo Guareña: A Hotspot of Subterranean Biodiversity in Spain Ana Isabel Camacho 1, * and Carlos Puch 2   Citation: Camacho, A.I.; Puch, C. Ojo Guareña: A Hotspot of Subterranean Biodiversity in Spain. Diversity 2021, 13, 199. https:// doi.org/10.3390/d13050199 Academic Editors: Tanja Pipan, David C. Culver and Louis Deharveng Received: 31 March 2021 Accepted: 6 May 2021 Published: 8 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Dpto. Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales (CSIC), José Gutiérrez Abascal 2, 28006 Madrid, Spain 2 Grupo Espeleológico Edelweiss, Club Cántabro de Exploraciones Subterráneas, Víctor de la Serna 26, 28016 Madrid, Spain; carlospuch@gmail.com * Correspondence: mcnac22@mncn.csic.es Abstract: Ojo Guareña Natural Monument in Burgos (Spain) is an important and large karstic system. It consists of more than 110 km of surveyed galleries, and it has rich sources of organic material from the surface and permanent water circulation. It is the fourth largest cave system in the Iberian Peninsula, and one of the 10 largest in Europe. Ojo Guareña also ranks 23rd among the world’s largest caves. To date, only volcanic caves in the Canary Islands, in which between 28 and 38 subterranean species occur, are considered subterranean diversity hotspots in Spain. Here, we provide the first list of subterranean taxa present in Ojo Guareñ, which is comprised of 54 taxa that includes 46 stygobiotic and eight troglobiotic species (some still unidentified at the species level), revealing Ojo Guareña as the largest known subterranean biodiversity hotspot in Spain and Portugal. In addition, we provide a list of an additional 48 taxa, 34 stygophiles and 14 troglophiles, found in the system, whose ecological status could change with detailed biological studies, which may change the number of strictly subterranean species present in the system. Indeed, at present, these numbers are provisional as they correspond to a small part of this sizeable cave system. The biodiversity of large areas of the system remains unknown as these areas have yet to be explored from the biological point of view. In addition, a large number of samples of both terrestrial and aquatic fauna are still under study by specialists. Furthermore, evidence of cryptic species within Bathynellacea (Crustacea) indicates an underestimation of biodiversity in the karstic system. Despite these limitations, the data available reveal the typical uneven distribution of subterranean aquatic fauna, and suggest that the great heterogeneity of the microhabitats in this wide and highly connected karstic extension led to the great richness of aquatic subterranean species. Keywords: hotspot caves; Ojo Guareña natural monument; stygobionts; troglobionts; subter- ranean biodiversity 1. Introduction Iberian Peninsula, together with Balearic Islands, is one of the regions in Europe with the greatest development of karst areas. Numerous caves, many of them large (more than 3 km of development and/or more than 300 m in depth), have been surveyed in the country [1]. Despite this, very little data on the subterranean fauna within these caves are available. Potential biospeleologists are discouraged by the difficulties of access and progression in the caves (e.g., large vertical shafts, meanders, narrow passages, etc.), the in- accessibility to man of much of the underground environment (flooded galleries, mesovoid shallow substratum-MSS-, hyporheic sites), and the low density of specimens in accessible populations (epikarst, especially). Thus, most samplings of these ecosystems have been limited and sporadic, and mainly focused on the study of specific taxonomic groups using capture techniques suited for them. Due to the great sampling deficiencies and the scarce biospeleological tradition in our country, there are very few inventories of the region’s underground fauna beyond some lists of a few terrestrial or aquatic taxa in the more well-known caves. The only caves considered in the literature as subterranean diversity Diversity 2021, 13, 199. https://doi.org/10.3390/d13050199 https://www.mdpi.com/journal/diversity