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Fauna FABIO STOCH Despite its inhospitable appearance and lack of any sign of life at first sight, groundwater is populated by large numbers of animal species of various taxa. These animals are generally very small, even tiny (between three-tenths of a millimetre to one centimetre). Only a few exceed one centimetre, and even fewer are quite big, like the underground Larva of stygoxene chironomid prawns of the genus Typhlocaris or the olm (Proteus anguinus). ● Stygoxenes. Not all organisms found in groundwater are exclusive to it: in fact, many of them are typical of surface environments and, through either active or passive dispersion, accidentally penetrate underground. They are therefore occasional guests in this habitat, to which they are generally carried by water percolating from the surface. This situation is very frequent in surface and underground karstic aquifers, with infiltration passages which are very efficient (sinkholes) or slower (like micro- and macro-fissures in limestone). The accidental guests occurring in groundwater are called stygoxenes. They do not have adaptations enabling them to survive in the harsh underground environment, where food supply is more restricted than in their habitat of origin. However, in particular conditions - for example if the aquifer is organically polluted - stygoxenes find optimal conditions for their survival and may even reproduce in hypogean (underground) habitats. Their populations may be quite large and compete with local ones, to the point of replacing them completely. They may also play important roles as prey or predators of underground species. ● Stygophiles. Stygophiles are organisms that exhibit some adaptation to life in groundwater environments, and may reproduce in both surface and underground waters. Stygophiles live within surface water-groundwater interfaces like springs, humid soil, wood litter and moss. These habitats share several characteristics with the underground environment, in particular darkness and limited living space. This is why stygophiles often have preMonolistra schottlaenderi (top) and Monolistra racovitzai (crustacean isopods, 7x) 41 42 adaptive features to life underground, which enable them to survive in transitional environments: they are often partially or totally depigmented and have reduced or totally absent visual organs. From the evolutionary viewpoint, since groundwater is a secondary colonisation environment, the species now found exclusively here are thought to have been stygophiles in the past. But the contrary cannot be ruled out - i.e., that all past stygophiles are now exclusive groundwater dwellers. Evolutionary destiny depends on the opportunities which single stygophilic species had of colonising groundwater, surviving and successfully reproducing. Stygophiles which regularly frequent the subterranean environment where they can reproduce, but which do not have marked adaptive characteristics, are called substygophiles. They include, for instance, some aquatic benthic insects found in watercourses, where they may spend the early phases of their development in the fluvial hyporheic environment. This is a winning adaptive strategy as regards avoiding predators, but their life-cycle is always completed in shallow waters, and adults often inhabit the subaerial habitat. This is true of many ephemeropterans, plecopterans and dipterans, mainly chironomids. Conversely, those stygophiles which show not only marked pre-adaptations but also an elective preference for the subterranean environment, where they are regular guests, are called eustygophiles. This is the case of many molluscs and crustaceans. ● Stygobionts. Animal species closely associated with underground environments, on which they depend for completing their life-cycle, are called stygobionts. They often have marked adaptations to underground life like depigmentation, anophthalmy (absence of eyes), well-developed sensory organs, and a reduced fecundity rate (described in the chapter on the ecological aspects of groundwater). These adaptations are partially shared by terrestrial troglobionts (cave-dwelling species) and by those living in soil (endogean species). Stygobionts may be ubiquitous and live in all types of aquifers, and sometimes in marginal waters (for instance, under dead leaves in humid forests), or they may be associated with specific habitats, like phreatobionts, which live exclusively in saturated alluvial aquifers, and karstic stygobionts, which are limited to aquifers in limestone and evaporites. A clear distinction between these ecological categories is difficult to make, and there are many intermediate levels. In some taxa, of which all species appear to be pre-adapted to life in humid soil and groundwater, like nematodes and some families of oligochaetes, it is impossible to divide them according to morphology, and their preference for underground habitats is evident from the frequency with which they are found there. This chapter on groundwater fauna describes exclusive dwellers, i.e., stygobionts. We will discover a fascinating multitude of dark-loving organisms, with sometimes bizarre morphological adaptations. Paracyclops imminutus (stygophyle) Niphargus steueri (stygobiont) 43 44 Methods for sampling and analysing groundwater fauna Groundwater habitats are difficult to reach, and researchers have had to come up with ingenious methods for sampling and analysing underground fauna which, according to the depth and accessibility of aquifers, are sometimes expensive and so complex that only specialised research institutes can carry them out. In karstic waters, traditional sampling methods include: ● continuous filtering of trickling water funnelled into containers with filters which are periodically emptied; ● collection of percolating water in gours and micro-gours, by means of rubber pumps or syringes; filtering of concretion water with plankton nets (60-100-µm mesh) which are emptied with rubber piping; ● direct filtering of water from large pools with plankton nets with handles; in streams and small watercourses: after coarse debris has been shaken out, water is filtered through plankton nets (with semi-circular openings 20-25 cm in diameter); ● direct collection of large organisms with aquarium nets and tweezers; ● in order to collect large predatory crustaceans (1), traps containing meat or tid-bits of food can be placed in suitable positions in open cans (to avoid the death of animals trapped inside, if the trap itself is lost); ● placing artificial substrates (twisted nylon netting compressed in tubes, or tubes filled with locally collected washed sediment) which are periodically emptied in order to analyse the colonisation of various types of substrates. Collecting specimens from wells or boreholes in alluvial soils may be carried out with: ● Fabio Stoch modified plankton nets (Cvetkov nets) with valves to prevent material from escaping when the nets are quickly lifted and replaced on the bottom of the well to agitate the sediments (2); ● various types of pumps (peristaltic, rotor, compressed-air), according to water-table depth (rotor pumps of greater power unfortunately easily destroy material). Lastly, in flooding watercourses where collection is concentrated in upwelling or outwelling stretches (see chapter on ecology), two methods are used: ● Karaman-Chappuis method: a hole is dug along the shore of a watercourse, and the water permeating from nearby sediments is collected and filtered through a plankton net; ● Bou-Rouch method: a hand-pump (3) is used to remove interstitial water from the bed of a watercourse, by means of a ● perforated tube inserted deeply into the sediments (for a detailed description of this method, see Teaching Suggestions). Research teams with the most recent equipment can use drills to place piezometers at varying depths, from which groundwater is extracted with pumps and filtered through plankton nets. Other, quite expensive methods, like freeze-coring, use liquid nitrogen to freeze sediment cores collected from boreholes and subsequently examined in the laboratory. More advanced research methods involve inserting transparent perspex piezometers with optical-fibre videocameras into the soil or sediment in the river bed. In this way, researchers can analyse large organisms in their natural environment without disturbing the underground community. 3 2 1 Filtering trickling water with a plankton net Equipment for sample collection 45 46 ■ Poriferans Among all stygobionts, sponges are certainly the least frequent and most primitive. These essentially marine organisms (there are few freshwater species belonging to the spongillid family) frequently colonise coastal caves shrouded in partial darkness. There is, however, at least one exception to this rule: Higginsia ciccaresei, a sponge which has recently been collected by scuba divers exploring the Zinzulusa Grotto in the Salento (Apulia). The species is endemic to the cave, and was found at a distance of 250 m from its entrance, at a depth of 12 m, in total darkness. The morphological characteristics of this species, like its depigmentation, have led researchers to consider it a stygobiont. ■ Platyhelminthes Flatworms are a primitive phylum of free-living or parasitic organisms. Stygobiont planarian worms are generally depigmented, eyeless, with slow reproductive cycles and high numbers of chromosomes. In Italy, although very little is known about these organisms living in phreatic environments, which are certainly inhabited by many minute Atrioplanaria morisii creatures (micro-turbellarians), some species living in karstic waters are well documented. Dendrocoelum collinii lives in pre-Alpine caves and in France, and D. italicum in Lombard caves, although the taxonomic position of Italian populations of both species requires revision. The genus Atrioplanaria is found in caves in Sardinia, central-southern Italy (A. racovitzai) and southern Piedmont (A. morisii); Polycelis benazzii lives in Ligurian caves. The taxonomy of these genera is uncertain, due to the fact that it requires examination of living specimens and the use of complex histological and karyological methods. This is why these animals are seldom included in lists of fauna. However, being predators, their trophic role in underground ecosystems may be of local importance. The taxon of temnocephalid flatworms is of uncertain position. These ectoparasitic species sometimes abound on the gills of crustaceans like stygobiont amphipods and decapods, whose haemolymph they suck. Very small (2 mm maximum), these animals have tentacles and adhesive discs with which they attach themselves to their hosts. In Italy, only three genera have been found so far (Bubalocerus, Scutariella, Troglocaridicola); they are parasitic on stygobiont shrimps of the genus Troglocaris which live in saturated karstic waters in the Karst areas of Trieste and Gorizia. ■ Molluscs All Italian stygobiont molluscs belong to the gastropod class, in particular to the hydrobioidean superfamily (spring snails). Although they are common in Italian groundwater, with about 70 species described, and are found in all types of habitats - except for the vadose zone of karstic aquifers - spring snails are still little known from the taxonomic viewpoint. This is because many species are only known by their Hadziella ephippiostoma shells, which are found in springs and hyporheic water, suggesting that the elective habitat of these populations is the deep, almost inaccessible underground environment. Many empty shells are found in river sediments, and therefore their unknown underground habitat may in fact be very far from where they were actually found. This is why their genera and species described in the past need to be revised. The shells of spring snails have various shapes. They are generally towershaped, conical and cylindrical, often truncated. Some genera have disc- and spiral-shaped shells. Stygobiont species of spring snails are usually very small (for example, the adults of the genus Hauffenia have a diameter of 2 mm and are only 0.7 mm high). The opening of the shell is generally large and round, closed with a thin, horny, egg-shaped lid, the operculum, which protects the soft tissues of these animals. They move by means of a complex set of muscles, the foot, which flattens ventrally and adheres to the substrate allowing the snails to crawl. Stygobiont spring snails feed on micro-particles of organic matter, encrusting micro-organisms and bacterial biofilms, which are scraped and ground by their radula, an organ bearing several rows of minute teeth. Except for a few species living in brackish water, most Italian spring snails live in freshwater and are crenobionts (spring-loving) or stygobionts. 47 Crenobiont species (genera Bythinella and Graziana) may enter groundwater bodies and burrow into interstices, probably in search of food, and therefore behave as stygophiles. A large number of endemic species of the genera Bythiospeum, Iglica, Istriana, Hadziella, Paladilhiopsis and Phreatica are strictly stygobiont, and live deep within karstic and alluvial underground networks in northern Italy. Some species, living in Alpine-Dinaric areas, are exclusive to the eastern Pre-Alps and are often strict endemics (like Paladilhiopsis robiciana, Phreatica bolei, Hauffenia tellinii and Belgrandia stochi). The area with the fewest species is the Piedmont Alps, with some endemic species of the genera Alzoniella, Iglica and Pseudavenionia. The Apennines host a small number of local endemics of the genera Pezzolia, Alzoniella, Pauluccinella, Orientalina, Fissuria and Islamia. Exclusive inhabitants of Sardinia are the genera Sardhoratia and Sardopaladilhia. Sicily hosts only one crenobiont species, Islamia cianensis. Thermal waters contain particular species of Bythinella and Belgrandia. 48 ■ Polychaetes Shells of gastropods Iglica vobarnensis, Paladilhiopsis virei and Hauffenia subpiscinalis (from top to bottom, ca. 30x) Polychaete worms are generally sea animals, and only a few species colonise anchialine coastal waters (land-locked bodies of water with a subterranean connection to the sea), and even fewer species are adapted to underground freshwater. Among them are two stygobiont species of great biogeographical interest. The nerillid Troglochaetus beranecki is an ancient thalassoid species (that is, one with marine affinities) originating from members of psammon in Tertiary epicontinental seas, from which it invaded underground freshwater. The characteristic of this species is its vast distribution area. In Europe, Troglochaetus beranecki is found in large rivers (Rhone, Garonne, Rhine, Weser, Danube, Oder, Elbe), in Finland and in Alpine streams. It has also been found in interstices in river beds of Colorado and Montana (up to 3050 m), although further molecular analyses are required to establish whether all these populations are really conspecific. Fewer numbers are found in caves of Switzerland, Germany, Poland, Hungary and Romania. In Italy, the species has recently been found in interstitial environments (Trentino) and in caves (Lessini Hills and Carnic Pre-Alps). This distribution is very wide, and includes ice-covered areas where underground fauna is minimal if not totally absent, and the rare stygobiont organisms have a remarkable capacity for adaptation, which enabled them to colonise these areas in post-glacial periods. 49 50 Alkaline waters in karstic caves in Gorizia and Trieste host the second Italian stygobiont polichaete, Marifugia cavatica. It belongs to the serpulids, which are marine polychaete worms living in limestone tubules. Marifugia cavatica, probably a micro-filterer, forms dense colonies that occasionally carpet extensive areas of walls of underground streams with tubules up to 1 cm long. The Marifugia formation is a little-known microhabitat rich in microfauna (protozoans, gastropods, oligochaetes, copepods, isopods and amphipods) that populates, like the colonies of marine serpulids, the complex mosaic of spaces between tubules. The species lives in the Dinaric region as far as Albania, together with other species of ancient marine origin, like isopods of the genus Sphaeromides and amphipods of the genus Hadzia, of probable Tertiary origin. Oligochaetes are freshwater and marine terrestrial annelids whose bodies are made up of segments (metameres) without appendages, with rows of transversal, movable bristles (setae). The shape and distribution of the setae play an important role in the taxonomy of this group. Earthworms are usually detrivorous and microphagous, and colonise microhabitats rich in organic matter. In Italian groundwater, the most common are the lumbriculid and tubificid families, together with the aquatic and semi-aquatic species of enchytraeids. The identity and similarity of groundwater oligochaetes have only recently been partially established, and research is beginning. The main problem in defining an oligochaete as stygobiont lies in the fact that many surface species (living in sediments of surface water and sometimes in humid soil) are pre-adapted to life in hypogean habitats (they are depigmented and anophthalmic). It is common practice to define as stygobiont those species which, as far as is known today, have exclusively been found in groundwater. The parvidrilid family is certainly the most interesting among the taxa recently found in Italy, from the Pre-Alps to Sardinia. So far, all records have been ascribed to the same species, Parvidrilus spelaeus. They are exclusive inhabitants of vadose zones in Italian caves, where they colonise the silty, muddy sediments on the bottom of concretions, pools of trickling water and interstices of hypogean streams. Presumably, the family has an ancient marine origin. Among the numerous species of tubificids found in Italian groundwater, there are the genera Haber (H. monfalconensis in springs of the Julian Pre-Alps and Trieste Karst, and H. zavreli in groundwater in Umbria and Emilia Romagna), two endemics of the genus Rhyacodrilus, recently described (R. gasparoi in Pre-Alpine caves and R. dolcei in small concretions of the Trieste Karst), Tubifex pescei, in phreatic waters of Umbria and Marches, Abyssidrilus cuspis, Troglochaetus beranecki (ca.50x) Cernosvitoviella sp. (ca. 40x) ■ Oligochaetes 51 52 collected in phreatic waters in Umbria and caves of Liguria and Friuli Venezia Giulia, Sketodrilus flabellisetosus in the Trieste Karst, and Aktedrilus ruffoi, recently described on specimens found in interstitial environments of the river Tione (Verona). Enchytraeids are less well-known, and several species of Cernosvitoviella, found in Pre-Alpine caves and considered to be stygobionts, are still being examined. Ostracods (from the Greek ostrakon, shell) are a diversified group of small crustaceans, whose body is enclosed by a bivalve carapace made of calcite, their unmistakable characteristic. Their carapace may be egg-, bean- or trapezoid-shaped, and is often knobbly or dimpled. The number and shape of their appendages is generally the same. Freshwater species have eight pairs of appendages, four of which are cephalic (antennules, antennae, mandibles, and maxillulae), three thoracic, and one caudal (uropod). They are generally small-sized (stygobionts seldom exceed 1 mm in length), and are found in all types of surface- and groundwater. Despite their large numbers in groundwater, both in caves and alluvial aquifers, stygobiont mussel shrimps are little known in Italy, and many species are still being studied. Among the most interesting is Cypria cavernae, thought to be endemic to alkaline karstic waters in Gorizia, Trieste and Slovenia. Other species are associated with anchialine habitats, like those in underground lakes of coastal caves in the Salento in Apulia (Trapezicandona stammeri, Pseudolimnocythere hypogea). Specimens of the genus Pseudolimnocythere were found in the brackish water of the Poiano springs, which issue from Triassic evaporites in the upper Val Secchia. A very interesting genus from the palaeogeographical viewpoint is Sphaeromicola. It is a commensal species living exclusively on stygobiont isopod crustaceans of the genera Monolistra (Sphaeromicola stammeri, in the Pre-Alps) and Sphaeromides (Sphaeromicola sphaeromidicola, in the Isonzo Karst). The same genus includes a commensal species on marine amphipod crustaceans, showing how both these ostracods and their hosts had marine ancestors. Mussel shrimps are very interesting in palaeogeographical research, because their shells are easily conserved in sediments, where they fossilise. The large numbers of fossil species, together with the great diversity of living ones, provide detailed information about the evolution of the animals of this class. Unfortunately, since stygobiont species are little known, have rarely been used to analyse the origin and evolution of stygobiont fauna. Cypria cavernae (ca. 100x) Pseudolimnocythere sp. aff. hypogea (ca. 100x) ■ Ostracods 53 54 ■ Copepods Copepods form the largest and most diversified group of crustaceans, with 13,000 species described, half of which are commensal or parasitic on other organisms. Copepods are divided into ten orders, four of which include freeliving species in groundwater (calanoid, cyclopoid, gelyelloid and harpacticoid copepods). Except for gelyelloid copepods, which live in the Jura mountains between Switzerland and France, the other three orders are found in Italy. Throughout their long evolution, copepods spread across continents, successfully colonising any aquatic environment. In freshwater, they live in standing waters (from lakes to transient pools), in benthic substrates of streams, and in all types of groundwater. They are also found in moss, forest litter soil and in wet meadows. Surface copepods spread easily thanks to their resting stages, which have been described in previous “Italian Habitats” volumes. These stages are also thought to exist in stygobiont species, although there is no evidence. This is why species exclusive to groundwater spread with difficulty, and are generally endemic. This characteristic is particularly evident in karstic systems, whereas species living in alluvial aquifers generally occupy larger areas, whose hyporheic habitats may easily be connected. Stygobiont copepods are very small (0.2-1 mm) and, with the sole exception of calanoids, have one main articulation between thoracic somites Speocyclops sp. aff. infernus (ca. 120x) (segments) 4 and 5, which divide their bodies into two distinct parts, the anterior prosome and the posterior urosome. The prosome includes the cephalothorax and four footed somites. The cephalothorax bears six pairs of cephalic appendages (antennules, antennae, mandibles, maxillulae, maxillae and maxillipeds), Antennule of a male harpacticoid copepod and each thoracic somite has a pair of (ca. 850x) oar-shaped limbs used for swimming (hence the name copepod, from the Greek, meaning “oar-shaped foot”). The urosome includes an anterior somite - bearing the fifth pair of thoracic appendages - and four appendage-free abdominal somites, the last of which, called anal somite, bears the two-branched furca, the unmistakable feature of copepods. Reproduction requires the participation of both sexes, and parthenogenesis is rare. Males are distinguished from females by one (in calanoids) or both (in cyclopoids and harpacticoids) antennules modified in the shape of claspers, used to hold the female during mating. The fertilised eggs are usually contained in one or two egg-sacs carried by the females. Groundwater species produce very few eggs, sometimes only one, large. Some stygobiont species do not have egg-sacs, and release their fertilised eggs directly on to the substrate. Among crustaceans, copepods exhibit the most complete metamorphosis. The eggs hatch into larvae called nauplii. There are six naupliar stages and, after the fifth moult, the nauplii turn into copepodids, which are segmented and similar to adults. Five copepodid stages follow, until the sexually mature adult stage is completed. Very little is known of the feeding requirements of stygobiont copepods. Calanoids, which are part of plankton, are filterers; larger cyclopoids (Acanthocyclops, Megacyclops) are predators and feed on other microorganisms. Most species are omnivorous, and the main source of food for small interstitial copepods (almost all harpacticoids and cyclopoids of the genera Speocyclops and Graeteriella) is particle-sized organic matter and its associated microbial biofilm. ● Calanoid copepods. The only Italian stygobiont species, Troglodiaptomus sketi, lives in caves in the Karst of Gorizia and Trieste, in Slovenia and Croatia. It is commonly planktonic in underground lakes. Very little is known of its ecology. 55 56 Cyclopoid copepods. So far, only about a hundred species are known to live in Italian freshwater, almost all of which belongs to the cyclopids. Few of them are planktonic in free waters (some Metacyclops in caves of Venezia Giulia, Apulia and Sardinia). Most are epibenthic or interstitial (e.g., genera Eucyclops, Acanthocyclops, Diacyclops, Graeteriella, and several Metacyclops and Speocyclops). Some species are exclusively associated with the vadose zone of karstic habitats, where they inhabit the network of microfissures and trickling pools (several species of Speocyclops), and others (such as Eucyclops, Acanthocyclops and Metacyclops) are exclusive to karstic phreatic waters. Niche segregation is therefore marked, although their habitat preferences may vary in different geographical areas. The most common and diversified cyclopoids in Italian groundwater belong to the group languidoides of the genus Diacyclops: this group of species, many of which are still being described, is found in all Italian regions. Other cyclopoid species live in restricted areas in the karstic waters of north-eastern Italy (Acanthocyclops troglophilus, A. gordani, Metacyclops gasparoi, M. postojnae, Diacyclops charon, Speocyclops infernus, to mention only a few), or are widely distributed (like Eucyclops graeteri along the Alpine chain, and Acanthocyclops kieferi, which colonises Pre-Alpine and Apennine areas). Noteworthy is Acanthocyclops agamus, an exceptional endemic species living in caves of the Alburni mountains (Salerno) and karstic areas in central Italy. It is an interesting example of progenetic paedomorphosis, a phenomenon described in the chapter on ecology. From the biogeographical viewpoint, other interesting species live in anchialine coastal groundwater, like the cyclopinid Muceddina multispinosa, recently described from caves in Sardinia, and many species of cyclopids of the genus Halicyclops. All groundwater species of cyclopids known so far probably derive from ancestors that used to inhabit surface freshwater, and the same applies to species living in brackish water, for which anchialine (and marine) environments are secondary habitats. Instead, cyclopinids probably have marine origins, although none of these species moves far from the coastline. ● Harpacticoid copepods. Except for species living in coastal marine groundwater, which host interesting biocoenoses, six families and 160 species of harpacticoids are known to live in continental Italian freshwater, half of which belong to the canthocamptids. The order includes numerous benthic and interstitial species, commonly found in all types of underground ecosystems. Stygobiont harpacticoid species have different origins, and include species with recent and ancient marine origin (like ameirids and ectinosomatids), as well as those deriving from surface freshwater ancestors, like most ● canthocamptids. There are several endemic species, many of which are limited to specific karstic areas (genera Nitocrella, Elaphoidella, Lessinocamptus, Moraria, Morariopsis, Paramorariopsis). Several species - only recently discovered or currently being described - populate microfissures in limestone or the tiny pools formed by trickling and percolating water in the vadose zone of caves. The nature of these environments and the isolation of limestone systems following karstification have presumably favoured speciation by vicariance, producing large numbers of endemics. Most endemics in karstic waters are known to live in caves in the Pre-Alps and on Sardinia. Recently, species of the genus Pseudectinosoma have been found in deep karstic systems of the Gran Sasso Massif, in the Alburni and Aurunci mountains. Before this extraordinary discovery, only two species were known in this genus, one marine species with amphi-Atlantic distribution and the other, stygobiont, known to live in French groundwater. The discovery of members of this puzzling genus of ectinosomatids in Italian groundwater has great biogeographical importance, as the genus is not known to inhabit the Mediterranean area, and Italian freshwater stygobionts may be relict species, the only survivors of an ancient fauna which became extinct in the marine environment during the salinity crisis that affected the Mediterranean in the Miocene. In addition, the surprising discovery of Pseudectinosoma galassiae in Australian groundwater confirms the extremely Elaphoidella pseudophreatica (ca. 50x) 57 ancient origin of the genus, which perhaps dates back to a period before the onset of continental drift in the Tertiary. Groundwater in alluvial aquifers also hosts endemic species like those of the genera Nitocrella, Parapseudoleptomesochra and Elaphoidella in much wider areas than in karstic environments. A particular feature of the genus Parastenocaris is that it fragmented into a myriad of species, most of which are known to inhabit only one or a few sites. Italy hosts 35 species - including those of the closely related genus Simplicaris - although many more species must still be described. Parastenocaris are the tiniest copepods (seldom longer than 3/10 of a mm), with worm-shaped bodies and small appendages, which enable them to wriggle through the minute fissures between sand and gravel grains in interstitial environments. 58 ■ Bathynellaceans SEM photos of harpacticoid copepods; from top to bottom: Pseudectinosoma reductum, Nitocrella pescei and Elaphoidella elaphoides (ca. 200x) Bathynellaceans are an order of totally stygobiont syncarid malacostracans of extremely ancient origin; some researchers believe they diversified as long ago as the Palaeozoic in littoral coastal waters, lagoons and estuaries, from which they colonised continental Bathynella skopljensis (ca. 15x) waters before the supercontinent Pangaea fragmented, causing them to spread into groundwater. Although only hypothetical, this fascinating scenario describes bathynellaceans as true living fossils, and shows how research on the taxonomy of these stygobionts is closely associated with the great palaeogeographical events which modelled the Earth’s surface. About 170 species of bathynellaceans are known, all of small size (between 0.5 and 3.5 mm), anophthalmic and diaphanous, with elongated, sometimes worm-like bodies. They do not have shells or brood pouches like isopods, amphipods and thermosbaenaceans, and their last abdominal segment (called telson) is free; these characteristics clearly identify these malacostracans. Italian bathynellaceans are still little known and studied: the first Italian representative of this group (Anthrobathynella stammeri) was discovered in 1954 in the interstitial environment of the river Adige in Verona, and since then only a few species have been described, belonging to the genera Bathynella, Hexabathynella, Hispanobathynella, Meridiobathynella and Sardobathynella. They include exclusively hyporheic interstitial species and 59 60 those associated with percolating water in caves. They are stenothermal animals, sometimes living in cold waters, as they have recently been found at high altitudes in the Alps. ■ Thermosbaenaceans Thermosbenaceans, like bathynellaceans, are an order of very ancient malacostracans, with about 30 stygobiont species living in fresh and slightly brackish water from the Caribbean sector to the circumMediterranean area, eastern Africa, Monodella stygicola (ca. 15x) Asia, and Australia. This group clearly differs from other malacostracans, due to the dorsal egg pouch formed by the carapace. The evolution of thermosbaenaceans is probably associated with the retreat of the sea following uplift caused by plate tectonics. The species often live in isolated locations and are of great biogeographical interest. Their name is misleading, as it refers to thermal water: in fact, it derives from the first species described, which was collected in an African thermal spring. Italy hosts four species - three of which are endemic to the country - living in saturated aquifers. Limnosbaena finki is found in karstic and alluvial water in north-eastern Italy (it is distributed as far as Bosnia); Monodella stygicola only lives in karstic habitats, occasionally in alluvial aquifers, in Apulia; Tethysbaena argentarii is an endemic species of anchialine waters in the Grotta di Punta degli Stretti (Argentario Promontory, Tyrrhenian), and Tethysbaena siracusae is endemic to the karstic area of Porto Palo in south-eastern Sicily. ■ Mysidaceans Spelaeomysis bottazzii (ca. 1x) Mysidaceans, or opossum shrimps, are malacostracans generally living in sea or brackish water. In Italy, there are two stygobiont genera, Stygiomysis and Spelaeomysis, in anchialine and freshwater in karstic areas in Apulia. The Mediterranean area hosts a third stygobiont species, Troglomysis, in the Dinaric karst. These detrivorous and saprophagous animals are 2-3 cm long thus, unusually large compared with other mysids - and are found in small lakes, seldom in flowing water. A euryhaline and eurythermal species, Spelaeomysis bottazzii, usually lives in anchialine habitats in south-eastern Italy, between the Gargano and Salento (Apulia), even in polluted water. Stygiomysis hydruntina is rare, and presumably lives further down, where the water-table recharges; so far, it has only been collected on the Ionian side of the province of Lecce. The two species may locally cohabit. Although electrophoretic analyses suggest that the species are of recent, perhaps Pliocene origin, similar species in Mexico, the Caribbean and eastern Africa imply a more ancient, Tethyan origin. ■ Isopods Woodlice are a very diversified order of malacostracan crustaceans, with more than 10,000 known species. They presumably colonised Italian groundwater from marine (cirolanids, microparasellids, microcerberids) and surface freshwater ancestors (asellotans and perhaps sphaeromatids). Each family is a microcosm in itself, Proasellus franciscoloi (ca. 6x) and their study reveals very interesting biogeographical aspects. Asellids. Almost all stygobiont species of this family are Italian endemics. Asellus cavernicolus lives in the river Timavo (Trieste Karst). Results from molecular studies reveal that it is a relict species deriving from pre-glacial colonisation of the Trieste Karst by an epigean species, Asellus aquaticus. In Italy, the genus Proasellus counts several surface as well as cavernicolous and interstitial species. The genus diversified into many phyletic lines, the taxonomy of which requires clarification: the group deminutus in north-eastern Italy; pavani in the central-eastern Pre-Alps; cavaticus in France, western Piedmont and Liguria, in karstic environments (P. cavaticus, P. franciscoloi), and the group patrizii, exclusive to Sardinian groundwater. In addition, there are many similar species: P. ligusticus, found from Liguria to the Apuan Alps, and P. acutianus, in Tuscany, Latium and the island of Elba are the most widely distributed 61 62 species. Other species are only known to live in restricted areas, like P. amiterninus, P. dianae, P. adriaticus and P. faesolanus. The similar genus Chthonasellus includes C. bodoni, endemic to karstic and interstitial waters in the Cuneo area (Piedmont), which is thought to be phylogenetically close to the French genus Gallasellus. Stenasellids. This family comprises exclusively stygobiont species of very ancient origin, found in Tuscany (Stenasellus gr. racovitzai) and Sardinia, in both caves and interstitial habitats. In Sardinia, techniques of molecular biology have identified many endemics, two of which have so far been attributed to S. racovitzai and are similar to French species, and two others (S. nuragicus, S. assorgiai) are similar to species found in eastern Europe. Two species collected in the area of Nuoro (Sardinia) are similar to Spanish species. Preliminary dating based on “molecular clocks”, suggests that the separation of the two phyletic lines dates back about 28 million years (Upper Miocene). This is one of the most fascinating biological pieces of evidence of the complex Tyrrhenid history. Microparasellids. This family of tiny isopods (a few mm long) deriving from ancient marine ancestors which colonised interstitial habitats during the marine regressive phases. Their distribution follows the ancient coastline of Tertiary seas. In Italy, six interstitial species are known, all belonging to the genus Microcharon (see drawing). Of these, only M. marinus is associated with transient groundwater along the Mediterranean coasts, and the geographical location of other species - e.g. M. novariensis, found in Piedmont springs reveals that they are relicts. Microcerberids. The family comprises mainly marine species, and only the relict Microcerberus ruffoi (see drawing) lives in Italian underground freshwater (water-table of the river Adige). Cirolanids. This family includes mainly marine species, and only two are Italian stygobionts. Sphaeromides virei inhabits alkaline water in the Gorizia Karst. A voracious predator, it is quite large (more than 3 cm long). Its typically Balkanic distribution and fragmentation into endemic subspecies suggest that it originally lived in groundwater in the Dinaric karst. Typhlocirolana aff. moraguesi is exclusive to the karstic system near Porto Palo (Siracusa, Sicily), and was distinguished from T. moraguesi (island of Majorca) with molecular biology techniques. Its origin is probably palaeoMediterranean. Sphaeromatids. Many species of the genus Monolistra, widely distributed in the Balkans, also live between the Italian-Slovenian border and the area of Como (north of Milan), in karstic groundwater in Pre-Alpine caves. Their absence north of the line that marked the southern boundary of the great Quaternary glaciers shows that they settled in groundwater during the Pliocene, perhaps deriving from surface freshwater ancestors which have become extinct. Ongoing molecular research will clarify their evolution. Each species and subspecies is endemic to a restricted karstic system. Monolistra schottlaenderi is exclusive to saturated aquifers of the Karst in the areas of Trieste and Isonzo, and is the only Italian member of the subgenus Microlistra, which also lives in Slovenia and Croatia. The species of this subgenus have knobby dorsal protrusions, and sometimes even long, robust spines that function as efficient defensive structures when the animal curls into a ball. Among other species, with smooth teguments, there is Monolistra julia, endemic to caves in the Julian Pre-Alps, where it lives in small streams of trickling water. It has two well-developed caudal appendages (uropods). Other species do not have uropods, which may be atrophic or barely visible. The furthest west (Monolistra pavani) is found in the underground stream of the Buco del Piombo (Como). Monolistra racovitzai (ca. 5x) 63 ■ Amphipods 64 Species of genus Niphargus; top: N. costozzae; centre: N. longicaudatus; left, bottom: N. pescei (top) and N. transitivus (bottom); right, bottom: N. bajuvaricus grandii (ca. 3x) This order of malacostracans includes several marine and freshwater species, sometimes sub-terrestrial, which colonised groundwater either directly from the sea, or from ancestors that once inhabited limnic surface water. In Italy, there are about 100 stygobiont species, almost all of which are endemic. Bogidiellids. Italian freshwaters host Bogidiella sp. (ca. 10x) seven stygobiont species, most of which are interstitial, occasionally euryhaline. Bogidiella albertimagni (in the Po Plain) and Bogidiella aprutina are the only continental species; the others are Tyrrhenian endemics, found in Sardinia and on the island of Elba. Gammarids. This family comprises exclusively surface species. Among those which only live in groundwater and are Italian endemics, two species of Tyrrhenogammarus live in karstic aquifers in south-eastern Sicily (Tyrrhenogammarus catacumbae) and Sardinia (T. sardous). One species of Longigammarus (L. planasiae) has recently been collected from a well on the limestone island of Pianosa (Tuscan archipelago), and a specialised species, Ilvanella inexpectata, is known to inhabit alluvial aquifers on the island of Elba and in Tuscany. Hadziids. The genus Hadzia - presumably a Tethyan relict - has four Italian species. Hadzia fragilis stochi, an endemic subspecies with delicate, elongated appendages, has been described in alkaline water in the karstic area of Trieste and river Isonzo. Hadzia minuta inhabits karstic waters in Salento, and H. adriatica has been collected from pools in Apulia. Another species, which is still under description, was recently found in southern Sardinia. Niphargids. The genus Niphargus (more than 250 known species, 70 of which live in Italy; size between 3 and 40 mm) have complex, controversial taxonomy, which is currently being revised by means of molecular biology techniques. Their distribution area includes most of Europe (except for the Iberian Peninsula and the upper northern areas) and stretches east towards Iran. This suggests that the genus colonised European surface freshwater from the basins of the Tertiary Paratethys, and later moved into groundwater. However, fossils similar to present species have recently been found in Baltic 65 66 amber, implying a perhaps more ancient origin. Almost all species are endemic and live in the Alps and Po Plain, in several distinct, although not welldefined phyletic lines (the main groups being stygius, kochianus, aquilex and bajuvaricus). The diversity of the genus decreases proceeding southwards down the Apennines, with species belonging to the groups speziae (northern and central Apennines), longicaudatus (throughout the Apennines, Sicily, Sardinia, and smaller islands), and orcinus. This last group includes species similar to Balkan ones (exclusively associated with karstic aquifers), which colonised Italy from the Julian karst in the north-east and, perhaps through trans-Adriatic pathways, the limestone systems of the central-southern Apennines and Apulia. In addition to these groups, there are several species of unknown affinity, like Niphargus stefanellii (found in caves in centralsouthern Italy), which seems related to Balkan species, and colonises even sulphureous waters. Niphargus species, which are greatly diversified in structure and size, colonise all types of underground habitats. In large alkaline karstic lakes, species are large (2-4 cm) with elongated antennae and other appendages, and large anterior claw-shaped limbs (gnathopods) for seizing prey (Niphargus steueri, N. tridentinus). Interstitial environments host small omnivorous species (3-10 mm) with globose (Niphargus pupetta, N. transitivus) or elongated, worm-like bodies (Niphargus bajuvaricus grandii, N. italicus). Other species colonise subsurface, non-karstic aquifers, and may even be found in moist soil. They have tapering bodies, like N. dolenianensis and various species of the group longicaudatus. Italy also hosts the only species of the related genus Carinurella (C. paradoxa), which has a globose body with stumpy appendages and lives in interstitial waters of Friuli Venezia Giulia. Salentinellids. This is possibly a palaeo-Mediteranean amphipod family that comprises only stygobionts deriving from marine ancestors, whose identity is still uncertain. Salentinella species are still undergoing revision, and the most common species, S. angelieri, is typically interstitial and lives in brackish water near the coastline. It is also found in caves of isolated karstic systems and in true continental groundwater, with other species like S. franciscoloi of Liguria. Salentinella gracillima is exclusive to groundwater in Apulia. Ingolfiellidae. This family includes many stygobionts with elongated bodies living in marine and freshwater interstitial environments. So far, only one species has been found in Italian fresh groundwaters: Ingolfiella (Tyrrhenidiella) cottarellii, from a cave on the island of Tavolara (off the northeastern coast of Sardinia). Metaingolfiellids. The family comprises the single species Metaingolfiella mirabilis, which is quite large (3 cm). Many specimens of this species were collected, on a single occasion, from water pumped out of a deep karstic well in Salento. Described in 1969, it has never been found since. It is perhaps one of the most ancient palaeo-endemics of Italian fauna. Its body structure and the shape of its gnathopods suggest that it is a predator. Pseudoniphargids. This family includes only stygobionts, and is particularly diversified in the Mediterranean area. In Italy, only a few species are known, living in interstitial environments and caves near the coastline, showing their marine origin. Two species (Pseudoniphargus africanus italicus, P. sodalis) live in Sicily, and one (P. planasiae) in the Tuscan archipelago. Pseudoniphargus adriaticus has been collected in wells near Bari, and is also known to inhabit the Pelagian Islands (between Sicily and Tunisia), although its taxonomic status is uncertain. Other specimens, collected in Sardinia, are still being examined. Metacrangonyctids. This family is distributed around the Atlantic, includes only stygobionts, and is very diversified in the Mediterranean area. Italy hosts only Metacrangonyx ilvanus, endemic to the island of Elba, where it was recently found only in one well in alluvial groundwaters. Salentinella angelieri (ca. 30x) 67 68 ■ Decapods Italian fauna has only two stygobiont decapod genera living in karstic waters: Troglocaris (Isonzo and Trieste Karst) and Typhlocaris (Apulia). Recent research has revealed that Italian caves actually host two species of shrimps of the genus Troglocaris of the anophthalmus group, morphologically difficult to distinguish, but easily identified by means of molecular Troglocaris anophthalmus biology techniques. They may belong to T. anophthalmus (Gorizia Karst) and T. planinensis (Trieste Karst), although their taxonomy still requires confirmations. Stygobiont species of the genus Troglocaris were thought to derive from marine ancestors. Very recent molecular biology analyses carried out at the University of Ljubljana (Slovenia) have dated the separation of the western anophthalmus group from the Dinaric-Caucasian one at between 6 and 11 million years ago, and the beginning of speciation within the Typhlocaris salentina anophthalmus group between 3.7 and 5.3 million years ago. Their marine origin is therefore very ancient, and populations colonised groundwater coming from surface freshwater. The third species of Italian stygobiont decapods, Typhlocaris salentina, is endemic to Apulian caves. It was discovered in the Grotta Zinzulusa at Castro Marina in 1922, and later collected from other caves in Salento, Murge and Gargano. This blind, depigmented prawn may reach exceptional sizes (up to 13 cm); a predator, it feeds on mysidaceans and stygoxene organisms. The genus Typhlocaris includes two stygobiont species living in groundwater in Israel and Libya, suggesting that it is an ancient relict of an otherwise extinct palaeo-Mediterranean pre-Pliocene surface fauna associated with a subtropical climate. Unfortunately, molecular data on this genus are not yet available. ■ Amphibians The olm (Proteus anguinus) is the only stygobiont amphibian of the Palaearctic fauna. The pétit dragon of the Postojna caves (Slovenia) - discovered by the Slovenian nobleman Valvasor in 1689 and briefly described by Laurenti in 1768 - is the best-known underground animal described so far and, in some ways, the most fascinating. It has a pinkish-white eel-shaped body, with atrophic eyes concealed under the skin and outer red gill plumes which it retains throughout its life. The olm is known for its neoteny, i.e., it reaches precocious reproductive maturity despite its larval appearance. Olms are predators feeding on other aquatic, even stygoxene animals; the females lay between 20 and 80 eggs, one at a time for over one month, and place them under rocks and stones. The greyish tadpoles have distinct eyes, which they retain until they are two months old. Until the age of three months, olms feed exclusively on yolk stored in the cells of their digestive tracts. In nature, reproduction seldom occurs before the tenth year of age. The origin of olms is debated. Fossils of proteids and iguanodonts, found at Bernissart in Belgium, date back to the Lower Cretaceous, when olms lived in surface water. Their colonisation of karstic groundwaters in the Dinaric area where they now live may have started in the Pliocene, when karstification began. In 1994, in the Slovenian Karst, a pigmented, eyed subspecies was Olms also live in the groundwaters of the Isonzo Karst 69 discovered (Proteus anguinus parkelj) genetically similar to the stygobiont populations of the same area, and thus suggesting that groundwater populations are more recent. Recent molecular analyses reveal that there may be more stygobiont olm species. In Italy, olms are known to inhabit only alkaline water in caves of the Trieste and Isonzo Karsts. An isolated population, introduced from Slovenia in 1850, still lives in Grotta Parolini at Oliero (Vicenza). Olms are the only Italian stygobionts listed as priority species in the Habitats Directive; they are also included in Annex IV and are therefore under strict protection. 70 71 Some olm specimens, coming from Slovenia, were introduced into the Oliero cave system (Veneto) in 1850 ■ Italian stygofaunal provinces As analysis of previously described taxonomic groups suggests, the present geographical distribution of stygobiont species in Italy is the result of a series of events which took place in ancient times (historical factors) and, to a lesser extent, of ecological factors, which occour in “real time”. The role played by both is described in the chapter on ecology. Since the evolution of many Italian taxonomic groups was similar over time, because they were affected by the same palaeogeographical events, Italy is divided into areas with similar fauna, particularly endemics. These areas are called stygofaunal provinces. Although these generalisations cannot tell us directly what the present fauna composition of a specific aquifer is, because this is also influenced by local events, they do describe the present situation of Italian stygobionts and explain where the most important endemic locations are found. Olm (Proteus anguinus) Dinaric province. This area includes only the easternmost portion of Italy the so-called “classic” Karst - an elliptical area of 200 km2, whose stygobionts are similar to those of the Karst in Slovenia, Istria and Dalmatia. The area was affected by karstification in the late Miocene, and hosts many palaeo-endemics. Exclusive to the karstic vadose zone are harpacticoid copepods of the genus Morariopsis, the bathynellacean Bathynella skopljensis and the amphipod Niphargus stygius. Exceptional fauna, whose western limit of distribution is the Karst, populates large cavities filled with alkaline karstic water. Among these, there are polychaetes (Marifugia cavatica), gastropods (Belgrandia stochi), ostracods (Cypria cavernae), calanoids (Troglodiaptomus sketi), and several cyclopoids and Sphaeromides virei (ca. 1x) harpacticoids (like Acanthocyclops troglophilus and Nitocrella stochi). This area hosts the only Italian stygobiont isopods of the genus Asellus, those of the subgenus Microlistra, and the large Sphaeromides, as well as amphipods, which are highly diversified, with many endemics (e.g., Niphargus stochi, Hadzia fragilis). Other remarkable inhabitants are decapods of the genus Troglocaris, and the most famous stygobiont species, the olm (Proteus anguinus). Aquifers in marl and sandstone also host very interesting fauna, with very different species from those found in adjacent karstic aquifers. Among the main biogeographical markers, there is the gastropod Istriana mirnae, and the large amphipods Niphargus spinulifemur and N. krameri. 72 Stygofaunal provinces in Italy Alpine province. The Alpine stygofaunal province includes a very complex area associated with Alpine orogenetic events. It is divided into a northern part (strictly Alpine), above the southern limit of the great Quaternary glaciations, and a southern Pre-Alpine one, below which is the recent alluvial area of the Po Plain. The Alpine area is populated by only a few stygobionts: cold-loving, stenothermal species which followed the retreat of Quaternary glaciers and colonised aquifers in carbonate and crystalline rocks in the Alps. In particular, a number of amphipods (Niphargus forelii, N. similis, N. strouhali) even live at high altitudes, above 2000 m, together with a few copepods and bathynellaceans. 73 74 In the Pre-Alps, the situation changes completely, as there are large numbers of endemics living in the numerous karstic systems. The main biogeographical component is of eastern origin and stretches westwards as far as the Pre-Alps near Como, with a few genera living on Mount Fenera (Piedmont) (hydrobioid gastropods of the genus Iglica, harpacticoids of the genus Paramorariopsis). Typical genera of this area are several hydrobioids (Bythiospeum, Hauffenia, Hadziella, Iglica, Paladilhiopsis), harpacticoids (Lessinocamptus, Paramorariopsis, and several species of the genus Elaphoidella), amphipod crustaceans (Niphargus of the stygius group) and isopods (Monolistra, Proasellus). The western and Ligurian Pre-Alps have fewer stygobionts, and their fauna is more similar to that in France (Proasellus of the cavaticus group) and the Apennine province (Alzoniella, Niphargus of the longicaudatus group). several cyclopoids of the groups languidus and languidoides of the genus Diacyclops, the bathynellacean Anthrobathynella stammeri, and the amphipods Bogidiella albertimagni and Niphargus bajuvaricus grandii. There are also more ancient organisms of pre-Quaternary marine origin, relict species survived to Pliocene events, perhaps also to Miocene sea retreat, like the recently discovered ectinosomatid harpacticoids and isopods of the genera Microcerberus and Microcharon. Padanian province. The alluvial areas of the Po Plain, which stretch into the Alpine and Apennine valleys, have many endemics. Here, as in the nearby Alpine province, there are many species from the east, such as the isopod Proasellus intermedius, and several endemic amphipods (Niphargus italicus, N. pupetta, N. transitivus, N. longidactylus, Carinurella paradoxa). Other species are associated with the fauna of the extensive plains of centraleastern Europe, and probably migrated towards Italy in more recent times, like Apennine province. Despite its extent, from the Colle di Cadibona (Savona) to the Madonie in Sicily (excluding Apulia), the fauna of this area is well characterised. There are areas rich in endemics (Ligurian Apennines, Alburni mountains, Gran Sasso Massif) that are widely distributed in the Apennines. The apparent monotony of this fauna may be related to historical events, as karstification here is a recent event, and the fragmented limestone outcrops in the Apennines were covered by little permeable soil in the interval between the Pliocene and Miocene, and were only uncovered in the Quaternary. These palaeogeographical events are very similar from Latium to Calabria, and the areas are inhabited by endemics which are found along the Apennines and do not have particular habitat preferences (cyclopoid copepods of the genus Diacyclops, harpacticoids like Attheyella paranaphtalica and Nitocrella stammeri, and the amphipod Niphargus longicaudatus, which live in recent tufa Diacyclops gr. languidus, females with egg-sacs (ca. 70x) Harpacticoids of the genera Lessinocamptus (left) and Paramorariopsis (right) (ca. 100x) 75 76 aquifers in the Sabini mountains and in karstic water in Cilento and evaporites in Calabria as well). These recent populations settled where palaeoendemics were already living and characterised Apennine areas. Thus, groundwater percolating in Triassic evaporites in Emilia hosts Niphargus poianoi; the karstic systems of the Aurunci, Gran Sasso and Alburni ranges are inhabited by the extraordinary copepod Acanthocyclops Stygiomysis hidruntina (ca. 2x) agamus and species of the genus Pseudectinosoma, which are perhaps Messinian relict. As regards gastropods, the genera Alzoniella, Avenionia and Fissuria live in the northern Apennines, and Arganiella, Orientalina and Pauluccinella in the central Apennines; the genus Islamia is divided into northern and central-southern species. Apulian province. Apulian groundwater is clearly different from that of the Apennines, and is rich in specialised endemics, especially in often brackish, saturated, karstic habitats. This is due to the extent and ancient origin of karstification, as well as to the geological history of Apulia, which palaeogeographers consider as part of a different tectonic micro-plate from those that shaped the Italian peninsula. These waters host exceptional specialised organisms of marine origin, like the sponge Higginsia ciccaresei, the gastropod Salenthydrobia ferrerii, the ostracods Trapezicandona stammeri and Pseudolimnocythere hypogea, the thermosbaenacean Monodella stygicola, the extraordinary metaingolfiellid amphipod Metaingolfiella mirabilis, the hadziid amphipod Hadzia minuta, the salentinellid Salentinella gracillima, mysids of the genera Spelaeomysis and Stygiomysis, and the large decapod Typhlocaris salentina. Tyrrhenian and Sardinian provinces. This province includes Sardinia, part of the Tuscan archipelago and isolated coastal areas in Italy deriving from the fragmentation of the Tyrrhenid, which started in the Oligocene. PalaeoTyrrhenian endemics are phylogenetically similar to evolutionary lines in the areas of Provence, the Pyrenees and Corsica. Among the several Sardinian endemics, the gastropod genera Sardhoratia and Sardopaladilhia, many species of cyclopoid and harpacticoid copepods, the bathynellacean Sardobathynella, the entire phyletic line of isopods related to Proasellus patrizii, and isopods of the genus Stenasellus, are also found along the Tuscan coast. Also presumably of palaeo-Tyrrhenian origin are the thermosbenacean Tethysbaena argentarii of Mount Argentario (Tuscany) and the amphipod Metacrangonyx ilvanus of the island of Elba. Other animals associated with local anchialine water are many amphipods of marine origin of the genera Bogidiella and Pseudoniphargus, and the subgenus Thyrrenidiella of genus Ingolfiella. More doubtful are the relationships between some gammarid genera, like Longigammarus, Tyrrhenogammarus, and the puzzling Ilvanella. Iblean province (Sicily). Sicilian stygobionts are biogeographically composite and little known, especially those living in the alluvial plains and karstic aquifers that sometimes develop in chalk. Clearly identified organisms are found in the Iblean area, especially in the water-table of the karstic system near Porto Palo. Although this small area is jeopardised by man-made alterations, exceptional, perhaps palaeo-Mediterranean endemics inhabit it, like the thermosbaenacean Tethysbaena syracusae, the cirolanid isopod Typhlocirolana aff. moraguesi, and the amphipods Tyrrhenogammarus catacumbae and Pseudoniphargus duplus. Examples of distribution of endemic stygobiont species; left: gastropods of the genus Paladilhiopsis (Eastern Pre-Alps, red circles) and Arganiella (Apennines, green circles); right: crustaceans of genera Stenasellus (Tyrrhenian, red circles), Spelaeomysis (Apulia, blue circles) and Tyrrhenogammarus (T. catacumbae, Iblean Mts, Sicily, green circles) 77 Ecology DIANA MARIA PAOLA GALASSI Although Stygobiology - the science that studies groundwater biology dates back to the 18th century, it was only in 1994 that the ecological importance of this environment was finally acknowledged and exhaustively described in the monograph Groundwater Ecology (see select bibliography). No matter how surprised we may be to learn that findings in groundwater ecology are recent, our surprise turns to shock on hearing that, although groundwater protection and management are of paramount importance for human survival, current legislation does not take into account the ecology of this particular Bottom of an old well for drinking-water environment. This is probably due to the unique characteristics of groundwater: invisible to most, not perceived as part of the territory, a totally dark world populated almost exclusively by tiny creatures. The biodiversity of this environment is truly “hidden”, as the subtitle of this volume suggests. This is perhaps the reason, albeit not a justifiable one, why man, in both law and culture, has always considered the exploitation of groundwater as more important than its ecology. It is a narrow-minded view, as we realise when reading the previous chapter on the complexity, diversity and scientific importance of stygofauna. It therefore comes as no surprise that, while there are only a few books to describe the geological and speleological aspects of these habitats, almost none has yet been published in Italy on underground ecology. This chapter is an attempt at overcoming the anthropocentric view of groundwater, at illustrating how the ecosystem works, and analysing the ecological factors that regulate the structure of groundwater communities and biodiversity. Waterfall produced by the karstic spring of Col del Sole (Friuli Venezia Giulia) 79 80 ■ The ecology of aquifers According to their hydrogeological and hydrological characteristics, aquifers are divided into three groups. Karstic aquifers are the most intensively studied from the biological viewpoint, perhaps because caves allow scientists easy access to them. These aquifers develop in large bedrock cavities, generally limestone, through a complex network of micro-fissures formed by the dissolution of carbonates. This group includes waters that penetrate chalk, and that flow into conglomerate, in which dissolution acts on evaporites and the cementing matrix of breccia and puddingstone. In the hydrological cycle, karstic aquifers undergo great variations in flow and, therefore, from the ecological viewpoint, they are less predictable systems. Unsaturated (vadose) karstic systems, which are more directly influenced by rainfall, are less stable than saturated ones. However, the stability and predictability of saturated karstic systems in turn depend on the age and depth of aquifers. Generally, ancient, deep aquifers are ecologically more stable than young, shallow ones. Porous and alluvial aquifers develop in unconsolidated sediments where interstices between sediment particles vary according to the particle size of the sediment itself. Porous aquifers may be divided into unsaturated or semi-saturated and saturated (water-tables). Medium-fine porous aquifers have greater physical inertia and, as water lasts in them for longer than in karstic aquifers, they are ecologically more stable and predictable. In non-karstic lithoid (stone) aquifers, water circulates in fractures (as in crystalline rock), between layers (marl-sandstone in flysch facies) and cavities of other origin (e.g., the lava flows of Mt. Etna). Water percolates in fissures whose size depends on the events that created them, and on the solubility and erodibility of rocks. Practically unknown from the ecological viewpoint, these aquifers are very similar to karstic ones if their fractures or cavities (as in lava tubes) are large, or behave like porous ones in the surface cortex, where soil and disintegrated rock host communities typical of porous systems. Lastly, a particular mountain environment is found in leaf litter with trickling water underneath, the so-called hypotelminorheic habitat. This does not represent a true aquifer, but is surface water flowing a few centimetres deep. Sketch showing the three main types of aquifers: fractured lithoid (A), porous (B), karstic (C) Hypotelminorheic habitat 81 82 Although the ecology of this unsaturated habitat is similar to that of porous surface aquifers, it is very unstable and unpredictable. Organisms from adjacent and underlying aquifers may migrate to these habitats to feed. The same aquifer often hosts more than one of the above categories: for instance, there are streams in cavities between flysch and limestone (in Friuli Venezia Giulia), between limestone and conglomerate (Veneto PreAlps), travertine and tuff (Latium), limestone and chalk (Grotte di Frasassi), limestone and lava (Lessini mountains), and limestone and granite (Sardinia). In addition, the water of karstic aquifers often drains to alluvial beds in valleys with complex hydrogeological and ecological relationships. The main ecological aspect deriving from the classification described above is that the various types of aquifers provide fauna with a complex availability of living space, with changes in structural complexity, food resources, stability of hydraulic conditions and water chemistry. Basically, different aquifers develop different habitats, and may host completely different animals. For instance, locally or extensively saturated karstic aquifers often have large hydric spaces (underground rivers or lakes), and therefore contain larger organisms which may reach a few centimetres in size, like large cirolanid isopods, mysids, decapods and the olm, the only Italian stygobiont vertebrate. Unsaturated karstic environments host smaller animals because the pools in caves - large pools or lakes of trickling water - are only transient habitats for fauna living in limestone micro-fissures above, adjacent to or underneath the pools. Medium-fine porous aquifers provide little living space, and only small animals - less than 1 cm long, often smaller than 1 mm, according to the size of particles - with particular adaptations have been able to colonise them. Fractured aquifers, in line with their nature, may offer different extension of living spaces, and therefore host relatively large animals (in flysch, amphipods of the genus Niphargus are up to 4 cm long) as well as microscopic organisms typical of porous systems. Unsaturated karstic habitat (micro-concretions) Porous habitat with (detail) a specimen of Niphargus in its environment ■ Groundwater habitats 83 84 The discovery of a new submerged world: anchialine environments Anchialine environments were first discovered in 1966, when the Austrian scientist Rupert Riedl described them as “marginal caves”. Since then, experts have debated the correct definition of anchialine systems. Today, they agree on defining them as caves or other underground aquatic habitats near the coastline of islands and continents, supplied by continental freshwater and with underground connections to the sea. Consequently, the water of anchialine pools is brackish, and light-weight freshwater generally floats on top of heavier seawater. The most typical feature of anchialine environments is the absence of a surface connection with the sea, which manages to reach far inland through deep infiltration passages in limestone and volcanic rocks. The most fascinating, extraordinary examples are the well-known Mexican cenotes, small bodies of crystalline brackish water, like blue eyes glittering in the tropical forests of Mexico and Belize, not far from the coast. In Italy, typical anchialine pools are found in the Grotta Zinzulusa, Abisso and Buco dei Diavoli (Salento), together with other water-tables in Apulia, groundwater in Porto Palo (Sicily), the Grotta Verde, Grotta di Nettuno and Grotta del Bue Marino (Sardinia) and the Grotta di Punta degli Stretti (Argentario, Tuscany). Anchialine pools are marked by few food resources, total darkness, and vertical gradients of salinity and oxygen concentrations. Although in the past anchialine ecosystems were believed to support themselves on allochthonous organic matter (deriving from the rock above and from seawater), today we know that part of the organic matter is locally synthesised by chemo-autotrophs. The most fascinating aspect that makes these environments true treasure troves of biodiversity is their exclusive fauna. Examples are remipedes, the most primitive class of living crustaceans, which have been found in anchialine caves on the Bahamas, in lava tubes on the island of Lanzarote and, more recently, in Australia. Remipedes, like many other animal groups found in these habitats, are true living fossils, whose distribution, enigmatically uneven in several areas of the world, dates back to the breakup of the ancient Tethys Sea. In addition to these unique organisms, there are also other extraordinary animals, some of which are Italian endemics with restricted distribution, like the sponge Higginsia ciccaresei, the thermosbaenacean Monodella stygicola, the mysid Stygiomysis hydruntina, and the decapod Typhlocaris salentina. It is worth noting that the copepod Muceddina multispinosa was recently discovered in the Grotta Verde (Capo Caccia, Alghero, Sardinia), a species with disjunct distribution also found in anchialine environments on the islands of Mallorca and Lanzarote (Spain). Generally speaking, anchialine habitats host heterogeneous assemblages which includes strictly marine animals and, to a lesser degree, freshwater organisms. Typically anchialine species are closely associated with the particular environment in which they live, and have never been found in other types of Diana Maria Paola Galassi underground habitats. They are also stygobionts, and have marked specialised features. Their origin dates back to various geological times, from the Tertiary to the more recent Pleistocene. Some researchers believe that their ancestors lived in the abysses of the sea; others trace their origin back to organisms living in shallow seawater on the continental shelf. Sea entrance to the Grotta Zinzulusa (Salento, Apulia) 85 87 86 Simplified sketch of a karstic aquifer ■ Ecology of karstic aquifers The Fontanon di Goriuda drains the karstic plateau of Mt Canin (Julian Pre-Alps, Friuli) The network of karstic conduits has two components that offer different living conditions to fauna: transmissive and capacitive components. The transmissive component is typical of highly karstified systems with great hydraulic conductivity and fast currents and, ecologically speaking, has little biodiversity. What kind of species could possibly survive in such hostile environments? Yet some actually can, and have even been incredibly successful in adapting to these harsh habitats. For example, some amphipod crustaceans of the genus Niphargus have evolved adaptations to exploit the advantages of fast currents in habitats where interspecies competition is very low. Research carried out in France reveals that these species, which perhaps live in fissures adjacent to the transmissive/drainage conduit, lay their eggs nearby, and exploit the speed of the current to disperse their young. Briefly, they synchronise their biological cycle to the discharge of the aquifer while developing growth strategies typical of changeable habitats: thus, they produce large numbers of fertilised eggs, a few of which will reach the adult stage, in ways which are typical of surface species rather than underground ones. Some species of isopod crustaceans of the genus Monolistra also exploit fast currents by curling up into balls and letting themselves be transported over great distances. In capacitive karstic systems, whose development varies according to the aquifer, water percolates in medium-sized and small fractures in branching 88 anastomoses, often adjacent to the main drainage system. Here, they form small pools of calm water which are connected to one another and flow into the main drainage conduit. This lateral network, which is generally more extensive and has greater volume than the drainage one, is called the capacitive annex system. Unlike the drainage system, which poses a severe threat to the survival of species, the situation is completely reversed in the capacitive system, where water flows slowly and there are large amounts of organic matter and inorganic sediments, creating habitats for many underground species. Biodiversity may even increase in capacitive systems with wide, diversified living environments, like underground lakes. Only in these habitats do we find plankton (calanoid copepods) together with benthic and interstitial organisms, large predators, like cirolanid isopods, large decapod crustaceans, and the olm. Unsaturated karstic systems also have a diversified network of storage microfractures, where living conditions are favoured by the three-dimensional complexity of the system. The diversity of substrates in small pools of water (pools and puddles with silt, clay, sand and organic material percolating from the surface, or complex calcium microstructures) give rise to diversified fauna, even if temporary water circulation prompts species to devise adaptations to withstand adverse conditions in small, locally saturated fractures. Sketch illustrating connection between transmissive system and capacitive annex subsystem during flood and drought periods ■ Ecology of alluvial aquifers The habitats of saturated porous aquifers are not very heterogeneous, and conditions are determined by the size of sediment grains in unconsolidated sediments. Food is restricted, because most of the organic matter coming from the surface is trapped in the subsurface layers of aquifers. The opposite is observed in surface unconsolidated sediments, generally unsaturated, where the surface aquatic and terrestrial environments are continuous or adjacent, giving rise to heterogeneous habitats due to the ecotonal nature of these environments and the greater availability of organic matter. Proximity to wet or dry surfaces produces greater concentrations of organic matter than in deeper saturated layers, thus contradicting the now out-dated idea that underground habitats lack niches and habitats. The hyporheic environment (where groundwater and surface water mix) is perhaps the most typical example of a subsurface alluvial aquifer and is, among underground habitats, certainly the best-known from the ecological viewpoint. It is an ecotone, that is, a transitional area between the surface habitat of running water (stream or river) and the saturated underground habitat (groundwater). Gravel bed of the Tagliamento (Friuli Venezia Giulia) 89 90 Literature defines this environment in myriad ways, each a nuance of the other, the only differences being the thickness attributed by each expert to the hyporheic layer. Aquatic ecotones are areas in which large-scale hydraulic exchanges occur and where biogeochemical activity - more intense than in adjacent habitats - affects the quality of water flowing through the interface. Hyporheic environments thus regulate water flow, and temporarily or permanently store organic, mineral and sometimes polluting matter. The microbial and animal components in them actively modify the timing and volume of nutrient and pollutant flows. From the structural viewpoint, the hyporheic zone is a matrix of permanently dark interstices saturated with water flowing slowly, with slight daily temperature variations and great bedrock stability. The hyporheic zone is an aphotic (without light), mechanical and biogeochemical filter between surface and underground water systems, ensuring their purification and maintenance. From this viewpoint, an important contribution came in 1993 from the American researchers Stanford and Ward with their concept of hyporheic corridor, which describes the connections and interactions between the hyporheic zone and the catchment basin. The role played by the hyporheic corridor in a catchment basin is essential for many ecological processes associated with nearby water sources: 1) the primary productivity in the stream above the hyporheic zone is strongly influenced by the distribution and frequency on the vertical scale of zones of upwelling (where the watertable supplies the stream), outwelling (areas where subsurface water supplies the stream laterally) and downwelling (areas where surface water supplies the water-table) of the stream, because the last two are usually richer in algal nutrients like nitrates and phosphates than surface water; 2) the temporal and spatial variability of processes of hydric exchange cause biodiversity in the hyporheic zone to increase more than in the adjacent water-table and surface habitats. Although Italian rivers are relatively well-known from the hydrological, biological and ecological viewpoints, an integrated overview of the Italian river ecosystem is still missing. Research emphasises a lateral dimension, i.e., the relationships between stream beds and surrounding floodplain and river areas in particular; a longitudinal dimension, i.e., the variations occurring along the length of rivers, from spring to outlet, although little is known about the vertical dimension, i.e., the relationships of rivers with their underlying aquifers. And the spatial scale is even less well-known in its temporal evolution. This three-dimensional spatial view, with the addition of a fourth temporal dimension, is the so-called four-dimensional nature of a river ecosystem (as defined by Ward). Three-dimensional (longitudinal, transversal, vertical) nature of a river system Spring at the base of a deposit of glacial origin (Pederù, South Tyrol) 91 92 The saline springs of Poiano: the importance of biological markers The Poiano Springs are the largest karstic springs in Emilia Romagna (mean discharge exceeding 400 l/s), and the main drainage system of groundwater flowing in the Triassic chalk of the Upper Val Secchia. Unlike other springs in the area, the Poiano Springs contain salt, with concentrations of dissolved sodium chloride between 5 and 7 g/l. Between autumn 2005 and spring 2007, the Trias Project (a research project by the Società Speleologica Italiana for the Ente Parco Nazionale dell’Appennino Tosco-Emiliano) was carried out on the Poiano aquifer, with automatic collection of the main environmental parameters (temperature, electric conductivity, pH, discharge) and continual collection of fauna by means of a net placed at the mouth of the spring. Geological and hydro-chemical tests showed that the chalk outcrops from which the springs gush are the top portion of a still active diapir, i.e., a chalk mass intruding vertically upwards because of its low density, bringing with it rock-salt, which makes the aquifer salty. Surface water, which infiltrates a few kilometres upstream through swallow holes, takes a few days to reach and mix with this salty water. This had led geologists to believe there was Nitocrella psammophila (left, 100 x), Niphargus poianoi (top, right, 6 x) and Pseudolimnocythere sp. (bottom, right, 100 x) Fabio Stoch · Mauro Chiesi a simple conduit system in the chalk bedrock, a typical feature of evaporites. The high content of sodium chloride is a limiting factor for biodiversity in the Poiano aquifer. Only three species of stygobiont crustaceans were collected from the springs: the harpacticoid Nitocrella psammophila and the ostracod Pseudolimnocythere sp., both of ancient marine origin, and the endemic amphipod Niphargus poianoi. However, contrary to previous ideas, biological research revealed that the conduit system does not carry to the springs the stygoxenic and stygobiotic organisms found in the streams flowing into swallow holes, none of which were collected in the Poiano Springs. Besides, the numbers of harpacticoids and ostracods coming from the springs are larger in periods of drought than in periods during which the aquifer is recharging, which suggested that the conduit system has a large groundwater basin. The integration of geological and biological research methods therefore enabled scientists to gain a better hydro-dynamic picture of the aquifer, and also revealed the unexpected presence of endemic species which had colonised the groundwater in the past and which are therefore now valuable bioindicators. Trends (measured during analysis) of discharge, precipitation and numbers of drifted stygobionts in the Poiano springs 93 ■ Ecology of springs 94 Springs are like windows opening on the underground environment Botosaneanu, a Romenian researcher, defined them as “the gates to the river Styx”. They are often the only means of analysing aquifers, because they are composed of surfacing groundwater which filters into recharge zones at different times, and reach spring points due to gravity. Springs may Sulphuric spring: the saline waters of Nirano therefore be studied from the “outside” (Emilia Romagna) to analyse surface organisms colonising the crenal zone (thus, crenobiology), or from the “inside”, to examine the fauna of the aquifers supplying them - the stygian zone (thus, stygobiology). Springs are particular physical environments which have constant temperature over time and sometimes undergo changes in the chemical composition of their waters, due to the nature of the aquifers supplying them. These parameters define extreme natural situations. For instance, according to their thermic regime, there are thermal springs (like those in the Euganean Hills (Veneto), which host an endemic species of gastropods of the genus Heleobia) and glacial ones (such as those in the Adamello-Brenta, between Lombardy and Trentino, which are colonised by endemic stygophilic harpacticoid copepods). Brackish springs are saline (like the Poiano springs in Emilia Romagna, whose exclusive guest is the stygobiotic amphipod crustacean Niphargus poianoi), and those with particular values of pH and hydrogen sulphide, i.e., sulphuric springs, which are found throughout Italy and its islands. ■ Groundwater inhabitants The perennial underground stream of the Pod Lanisce cave (Julian Pre-Alps, Friuli Venezia Giulia) The “darkness syndrome”. Contrary to ideas in the past, the underground environment can host great biodiversity. As described in the previous section, groundwater species may be divided into stygoxenes, stygophiles and stygobionts, according to their degree of dependency on this habitat for their survival. Stygobionts are species which are exclusive to groundwater and have developed special adaptations to life in this habitat. All their adaptations 95 96 Modified sensory setae on the antennule of a male harpacticoid (top: ca. 2000x; bottom: ca. 4000x, photo by SEM, scanning electron microscopy) define the so-called “darkness syndrome”, a condition made up of a series of morphological, physiological and behavioural changes that these species underwent during their evolution in the geological past, which brought their ancestors from surface waters to the underground ecokingdom. According to how species Aesthetasc on antennule of a harpacticoid (ca. react to the underground environment, 8000x, SEM photo) adaptations are distinguished from specialisations. Adaptations are strategies developed by species as responses to what are called the macrodescriptors of the underground environment, like constant darkness and scarce organic matter. Specialisation is the reaction to microdescriptors of the various types of habitats found in the hypogean environment in general. Groundwater organisms are depigmented (white, transparent or translucent), or sometimes pinkish (haematic pigments are visible through their semitransparent bodies), and their visual organs are generally small (microphthalmy) or totally absent (anophthalmy). Clearly, in totally dark environments, there is no advantage in having functioning visual organs or similarly, exhibiting gaudy colours. But it is more complex to understand what the disadvantages could be in maintaining these characteristics, since these same disadvantages often caused them to become extinct over time. Generally speaking, if an organism has a particular feature that is neither an advantage nor a disadvantage, a random neutral mutation may occur, causing that feature to disappear. In addition, if there is also an energy advantage, because during the ontogeny of these structures available energy can be used to develop compensatory sensory structures, then the loss of useless organs also has an adaptive logic. Presumably due to lack of resources, no stygobiont has developed the complex structures typical of animals living in sea abysses (like bio-luminescence). Moreover, pre-adaptive dynamics cannot be ruled out: in surface populations made up of both blind and sighted individuals, spatial segregation of blind phenotypes in groundwater and survival of sighted individuals in surface water may have given rise, over time, to two different, ecologically isolated genotypes. However, the evolutionary dynamics that led stygobionts to lose their eyes are still being discussed, as even within the same species eyes may show different evolutionary stages - for example, in some isopod, amphipod and decapod crustaceans. 97 98 The lack of eyes or their functional atrophy is generally accompanied by hypertrophic (excessively developed) alternative sensory organs for life in a dark world, where smelling or touching the surrounding space is the only way of sensing the approach of predators or potential prey, finding a partner, or making one’s way in three-dimensional space, large or small. The body surfaces of stygobionts are therefore covered with sensory organs, which have different shapes according to species. For instance, crustaceans have aesthetascs, setae with many chemoreceptors sensitive to chemical stimuli, and thigmoreceptors, end-organs which respond to touch and enable organisms to find their way around by feeling the lake bed or single sand particles in interstitial environments. Stygobiont crustaceans living in free water sometimes have longer antennules than their surface relatives. These modified cephalic appendages are used to sense at distance: it is better to know in advance if a predator is coming, before it is too late to escape! In stygobionts, the compensatory length of sensory appendages is contrasted by clearly rudimentary locomotory appendages, which are much smaller than those of their close relatives living on the surface, and generally with fewer segments, setae and spines. This adaptation is actually a type of specialisation, as it is typical, or even exclusive, to interstitial species. The interstitial environment of hyporheic zones of rivers, springs and karstic springs covered by alluvial sediments has one great disadvantage: living Body elongation favours movement in the interstitial habitat spaces are restricted. In these narrow habitats, movement is confined, and walking species, let alone swimmers, are rare. Most of their time is spent moving around single sand particles, feeding on the bacterial biofilm covering the surface. Long legs would only hinder movement. This morphological adaptation is accompanied by a reduction in body size, as stygobiont species are generally smaller than their surface relatives. It is no coincidence that shorter legs and smaller bodies are often associated. The most probable reason is that these adaptations are the result of developmental heterochrony. Heterochrony. This is a deviation from the typical developmental sequence of formation of organs and parts as a factor in evolution, both in animals with discontinuous development (with larval stages separated by moults) and those with continuous, gradual transformations into adults. Although there are various types of heterochrony, the most commonly found in underground environments are progenetic paedomorphosis (also known as progenesis) and neoteny. Why has heterochrony been so successful in the colonisation of groundwater? Progenesis is the process by which Evolutionary dynamics of stygobiont progenesis features of the sexually mature animal develop precociously. The result of this deviation is a reproductively mature adult which preserves the morphological and/or physiological characteristics typical of juvenile or larval stages. These small adults often look like larvae and, if they are metameric (segmented) animals or have segmented body appendages, they may have fewer metameres and segments than they would if they had developed normally. But what is the advantage of being small? Obviously, it enables these animals to creep and wriggle into tiny interstitial spaces. Although heterochrony is the result of modifications - genome alterations which are generally disadaptive - it actually provides free admission to interstitial environments. It may therefore be a pre-adaptation, i.e., nonadaptive, and at times a neutral character or set of characters acquired in their original habitats (i.e., surface water) which may become useful when 99 organisms find themselves in new environments. Many of the evolutionary lines in stygobiotic crustaceans (copepods, ostracods, bathynellaceans, thermosbenaceans, asellid isopods, ingolfiellid and bogidiellid amphipods) originated in this way. Another type of heterochrony is neoteny. The final result is the same: sexually mature adults that look like larvae. In this case, the dynamics are different, because larval development is so slow that individuals reach sexual maturity without having completed their larval development. In this case, animals may grow to the Elongation of the first antenna in the amphipod Hadzia fragilis stochi (ca. 8x) same size or larger than their nonheterochronic relatives. The classic example of neoteny in underground environments is provided by the olm. Stygobiont animals also have physiological adaptations to life underground, like low metabolic rates and slow body growth, longevity, reduced fecundity, larger eggs richer in yolk (enabling embryos to survive for longer periods), and less frequent reproduction, often independent of the season and occurring throughout the year. However, like all generalisations, this too has its exceptions. For instance, in open karstic environments, some Niphargus species depend on the seasons. In particular, in spring, when aquifers increase their discharge due to snowmelt, and surface water carries larger amounts of organic matter, the biological cycle of these animals is timed in order to produce their juvenile stages which exploit the greater amount of food in order to develop. 100 Theoretical evolutionary sequence which, starting from a large surface-living ancestor (1), through evolutionary steps (2-4), may have led to a progenetic interstitial species (5) Other adaptive strategies. Another characteristic of all interstitial organisms is body elongation, which makes even phylogenetically distant taxa acquire a common worm-like shape (turbellarians, annelids, crustaceans and mites). The advantage is that their bodies are capable of wriggling into tiny interstices. Only a few ethological strategies for survival in particular underground environments are known. In hyporheic habitats, mainly in the upper layers of river sediments, where current velocity may be considerable, animals without 101 102 adhesive organs to cling to the sediments may curl around single particles, to prevent being carried away by the current. This is the case of oligochaetes, harpacticoid copepods and amphipods with elongated bodies. Others may become globose (some isopods and amphipods), and drift with the current to colonise new habitats. Another strategy adopted by crustaceans is gender segregation in spatial niches, to avoid intraspecific competition. When sampling a particular underground environment, scientists often collect larger numbers of males or, vice versa, of females. In Harpacticoid female of Morariopsis aff. scotenophila with only one egg per sac addition to the sex ratio, which in (ca. 100x) nature is notably in favour of females, in interstitial environments, males and females may occupy microhabitats at different depths. Although little is known of the ethology of underground species, some male copepods occasionally re-clasp (mate with the same female) to ensure their paternity. Reproductive strategies. Until recently, the underground ecosystem was thought to be simple, stable and predictable. In fact, scientific research has proven this axiom invalid, at least for some habitats. Generally speaking, in physically predictable but ecologically unfavourable environments, species adopt a demographic strategy called A-selection (Adversity Selection), based on slow development and low fecundity in stressful environments with few resources and predictable changes. However, when habitats are still physically predictable and food resources are more abundant, species may turn to K-selection strategy, whereby populations grow to the carrying capacity of the environment. In this condition, intraspecies competition and predation may also become important for population dynamics. When environments are physically unpredictable but provide great food resources, there may be exponential population growth followed by plummeting figures when resources are no longer available: this is known as r-selection. Clearly, a single model cannot be applied to all underground species. ■ The food-chain A classic description of underground environments is generally made by comparing its physical and biological organisations with those of surface environments. For example, groundwater is permanently dark and has no nyctemeral (day-night) cycle, whereas surface water is illuminated by the sun, and therefore does have a day-night cycle. This condition influences the biological and, equally importantly, the energy characteristics of the ecosystem. Lack of light results in detritus food-chains because there are no photosynthetic organisms (green plants) and very few chemo-autotrophic organisms (ones feeding on oxidised inorganic chemical compounds). These are bacteria that exploit chemical energy instead of light energy, and synthesise organic compounds from carbon dioxide. One of the typical locations is the Movile Cave (Romania), where these organisms are primary producers and the entire food-chain relies on them. There are also examples in Italian groundwater, an underground stream in the Grotte di Frasassi (Marches) and sulphuric basins in the Grotta del Fiume Sotterraneo in the Lepini mountains (Latium), which are still being studied. Therefore, with very few exceptions, the entire groundwater ecosystem is not self-sufficient, but depends on inputs of organic matter from terrestrial Sulphur spring in the cave of Cala Fetente (Capo Palinuro, Campania) 103 104 Sketch showing degrading phases of particulate organic matter (POM) and its entry into the hyporheic environment (see text for abbrevations) and aquatic surface environments - defined by the French researcher Rouch as “epigean manna”. Due to the often total absence of primary producers, the aquatic underground environment is oligotrophic, i.e., deficient in nutrients and, as such, incapable of supporting long food-chains. There are only a few examples of dystrophic underground environments, which are periodically or occasionally filled with dissolved humic matter coming from the surface. Eutrophic conditions are associated with sudden increases in organic matter due to pollution. This happens in some saturated karstic habitats in periods of intense rainfall. Rain and surface organic matter percolate into aquifers through effective infiltration pathways, giving rise to large-scale although short-lived increases in organic matter available to biota. Similar events occur in unsaturated karstic environments, such as concretions and pools of trickling water in springs or hyporheic habitats, where the quantity of organic matter depends on the season. Clearly, the type of aquifer greatly affects the amounts of food available to underground communities, and may produce distinct community organisations. Most underground organisms are therefore detrivorous and feed on particles of organic matter produced by the decomposition of dead animal and plant organisms. This ingested matter is divided into two categories: Coarse Particulate Organic Matter (CPOM), with particles >1 mm, and Fine Particulate Organic Matter (FPOM), with particles <1 mm and >0.5 µm. Occasionally, some small organisms may feed on Dissolved Organic Matter (DOM) with particle sizes <0.5 µm, composed of proteins, aminoacids and simple sugars. Most interstitial organisms feed on bacterial biofilm made up of bacteria and fungi that provide proteins, fats and sugars, vitamins and oligo-elements. Bacterial biofilms develop on the surface of single sediment particles, exploiting the dissolved organic matter coming from the exterior; most bacteria are also heterotrophs (consumers). Several crustaceans and oligochaetes are limnivorous, because the inert matrix of silt - the finest soil fraction - traps the bacteria and organic matter on which these animals feed. Interstitial and karstic environments share the same predators, like copepods, cirolanid isopods, many amphipods, decapods, and olms. Cannibalism is not rare, and adults prey on their young - a process which may be used to keep demographic growth under control. In the surface water/groundwater ecotone and in underground environments rich in stygoxenes, there may be many hydrozoans, larvae of tanypodine chironomids and other insects that prey on copepods and amphipods, and microturbellarians, whose flat, diaphanous, transparent bodies show the prey (generally small copepods) that they have swallowed whole. Pools in Grotta dell’Acqua (Trieste Karst, Friuli Venezia Giulia) 105 Functional redundancy is a rare phenomenon in groundwater ecosystems, i.e., each trophic role is generally played by one or a few species, and there is little competition at each link of the food-chain. For the same reason, the ecosystem is certainly extremely vulnerable, as communities have low inertia (capacity for withstanding human disturbance unaltered). In fact, the extinction of one species of the community may bring the entire food-chain to a halt, with irreversible consequences for the whole ecosystem. Many underground species are therefore “key species”, as conservationist biologists call them. 106 107 Groundwater in a Tuscan cave ■ Stygodiversity: groundwater biodiversity Hierarchical approach to the study of underground ecosystems: from large regional to small microhabitat scales Several factors contribute towards the spatial and temporal distributions of the so-called stygodiversity, and they may be both historical (palaeoclimatic, palaeogeographical and palaeoecological events) and ecological. The structure and functioning of underground aquatic ecosystems are the result of complex processes that may act in different ways at different spatial - and temporal - levels. These factors are therefore studied at continental or regional scale (mega- or macro-scale), aquifer (meso-scale) and microhabitat level (micro- or fine-scale). These scales are a series of spatial configurations fitting one into the other, and each level integrates the processes occurring at lower levels and is associated with others at the same level. This hierarchical subdivision into continental, regional, and sometimes aquifer levels allows us to focus on the palaeogeographical and palaeoclimatic events that influenced the origin of biodiversity in groundwater. The origin of stygobionts in fresh groundwater is double. Some of them evolved from ancestors living in continental surface freshwater (lakes and rivers) - most hydrobiid gastropods, cyclopoid copepods and many canthocamptid harpacticoids, asellid isopods, niphargid amphipods and olms - and are called limnicoid stygobionts. Others - polychaetes, parvidrilid oligochaetes, amphipods of the genera Hadzia and Salentinella, microcerberid, 108 Stygodiversity in the Presciano springs: a pilot experiment The Presciano springs make up one of the three great sources giving rise to the river Tirino in Abruzzi, at the foot of the greatest karstic aquifer in the Apennines. They have proved to be excellent natural laboratories for scientific research on the environmental factors affecting the spatial distribution of groundwater species (the spring area does not exceed 2000 m2). This spring system is structurally complex, as a heterogeneous alluvial layer lies on strongly karstified bedrock, covering the underlying karstic aquifer. This situation has enabled researchers to examine how the different types of sediments affect the composition of underground communities. Detailed samplings have revealed that stygoxenic, stygophilic and stygobiont species are not at all homogenously distributed in the small spring area Presciano springs (Abruzzi) As depth increases, the relative importance of stygobionts increases to the detriment of other ecological categories, while stygophiles are generally found at intermediate depths (at 30 and 70 cm below the spring bed). This vertical distribution is a direct consequence of the ecological characteristics of stygobionts, which are morphologically and physiologically adapted to life in deep habitats, and of stygophiles, a transitional category that clearly prefers ecotonal areas. Stygoxenes are scattered, especially in subsurface sites, perhaps restricted by the low temperatures and oligotrophic conditions of the spring environment. Further analysis, carried out by dividing fauna living in karstic sites from that of alluvial sites, shows that stygophiles are almost exclusively found in the latter habitat. Diana Maria Paola Galassi · Barbara Fiasca In addition, alluvial sites characterized by heterogeneous grain-size composition, with clearly alternating temporal upwelling and downwelling phases, host greater biodiversity, as they provide more ecological niches than karstic sites, and contain greater quantities of organic matter, which is trapped in interstices. Copepod taxocoenoses, the most important group in the analysed springs, are found in alluvial sites, with 15 out of the 17 species collected in the entire spring system. Instead, karstic sites host more monotonous karstic fauna, with one harpacticoid copepod (Nitocrella pescei) making up 90% of the entire assemblage. However, it is precisely in karstic sites that we find the most biogeographically interesting species, Pseudectinosoma reductum, a relict of ancient marine origin. Nitocrella pescei Distribution of ecological categories (number of specimens) at various depths (in cm) 109 110 microparasellid and cirolanid isopods, ameirid and ectinosomatid harpacticoids and mysids - are related to taxa that are still living in marine environments and are called thalassoid stygobionts. The current distribution of stygobiont species is the result of an ever-changing mosaic of communities which have evolved on a geological time-scale and whose composition is still evolving. A particularly interesting aspect of the development of stygodiversity, which has long been debated by scientists, is the large number of relict species in underground communities. Some underground systems conserve species and even entire taxonomic groups related to now extinct ancient surface fauna (in one of his books, the French biologist René Jeannel spoke of cave-dwelling “living fossils”). Events like the Pleistocene glaciations, sea regressions and transgressions, and the salinity crisis of the Mediterranean have long been the focus of scientific debates on the origin of underground fauna. Some scientists believe stygobionts to be the “survivors” of surface populations which had taken refuge underground to escape harsh surface conditions (this “refugium” hypothesis goes back to the ideas of Charles Darwin). Others view colonisation as a continuous, ongoing process (the “active colonisation” hypothesis). Karstic spring (Friuli Venezia Giulia) According to the “refugium” theory, tropical regions, which were not affected by catastrophic events, would have scarce underground fauna; most importantly, surface populations, the ancestors of stygobionts, would generally have become extinct in geographical areas which underwent drastic climatic changes. Further knowledge has proved that both these theories were partly wrong, and suggested a new evolutionary scenario, according to which the present structure of stygodiversity has several causes. First, the extinction of surface populations is not a mandatory prerequisite for speciation in The endemic gastropod Bythiospeum calepii underground habitats. We already mentioned this when describing the coexistence of pigmented, eyed species of crustaceans with depigmented, anophthalmic or totally blind ones - many cases are known in Italy, especially for crustaceans of the genera Proasellus, Synurella and Gammarus. These are examples of ongoing active colonisation of groundwater not associated with dramatic changes in the surface environment. Stygobionts have also been found in all kinds of habitats in tropical areas - lava tubes on volcanic islands, cenotes in Mexico, caves in Somalia, anchialine systems in Australian deserts and in the caves of small islands in the Caribbean, and in Brazilian caves, inhabited by multitudes of blind fish - thus definitively contradicting the “refugium” hypothesis as the only explanation for the origin of underground communities. Although groundwater colonisation may occur independently of unfavourable conditions in the environments lying above, dramatic climatic and geological changes in surface habitats may in fact suddenly interrupt the genetic flow between hypogean and epigean populations. This explains the origin of “relicts”, both eco-geographical (separated from their closest surface relatives still living and sometimes well diversified) and phyletic (the last survivors of a now extinct surface evolutionary line). Phyletic relicts are very interesting from the scientific viewpoint. Entire taxonomic groups (like bathynellaceans and thermosbaenaceans) belong to this category, and their closest relatives can no longer be found among the 111 112 animal groups still living on the surface. However, since the chronology of colonisation and speciation is hard to establish, a solution has been found in the so-called “molecular clocks”, which synchronise DNA mutations in living organisms with palaeogeographical and palaeoclimatic events. An example is afforded by the examination of Sardinian, Provençal and Tuscan species of stenasellid isopods, living evidence of the ancient tectonic fragmentation of the Tyrrhenian plate and drifting of the Sardinian-Corsican continental plate and its fragments from what is now Provence towards present-day Italy. Although necessary for speciation, vicariance (i.e., the splitting of the area of the old species into two parts separated by boundaries) is not only associated with these ancient, wide-ranging events, and may occur on any scale, from small, isolated fractures in karstic systems, to continents. Vicariant events may be geoclimatic and ecological: obviously, the efficiency of these “boundaries” is closely related to the ecology of the species, especially their aptitude for dispersal. Many stygobiont species are not prone to dispersal, and show 113 Niphargus similis, found in relict sites in glacialised areas of the Alpine chain Affinities between Provençal, Sardinian-Corsican and coastal Tyrrhenian fauna are explained by their common palaeogeographic history limited geographic distribution (they are strict endemics). Furthermore, the low fecundity, benthic larval development and low dispersal potential of many interstitial crustaceans suggest that continuous and jump dispersal are quite rare in these groups. This is why stygobionts can be used as excellent historical (palaeogeographical) indicators, their descriptive capacity being similar to that of true fossils. Among the events that modelled present-day Italian stygofauna, the best known are certainly glaciations and sea regressions. The Quaternary glaciations depleted underground fauna in large areas of Italy, thus leading to the total absence of entire stygobiont genera of gastropods, copepods, isopods and amphipods north of the boundary of the Würmian glaciation. Sea regression, associated with glacial eustatism, trapped several taxa in coastal sediments, giving rise to stygobization. Very ancient regressive events led fauna of marine origin to become relicts, enigmatically confined and unevenly distributed in continental groundwater far from coastlines (amphi-Atlantic, Caribbean-Mediterranean, Caribbean-Mediterranean-Australian). In this case, the grandiose movements of plate tectonics were the main cause of relictualisation and, in the Mediterranean area, gave rise to the so-called Tethyan relicts, which date back to the disappearance of the ancient Tethys Sea in the Oligocene.