Subterranean Biology 2: 7-14, 2004

Trophic sources partition and population genetic structure of the isopod Androniscus dentiger from a chemoautotrophy based underground ecosystem
Gabriele GENTILE,(1,2), Serban M. SARBU(3)
Department of Biology, Tor Vergata University, Viale della Ricerca Scientifica, 00133 Roma Italy, e-mail: gabriele.gentile@un iroma2.it 2 Department of Ecology and Evolutionary Biology, Yale University, OML 165 Prospect St. New Haven, CT 06520-USA 3 Department of Biological Sciences, University of Cincinnati, 821 A. Rieveschl Hall PO Box 210006, Cincinnati, OH 45211USA
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ABSTRACT In this paper we present data not published, but yet cited in a previous work concerning with the subterranean community from the Grotta del Fiume, in Central Italy (Sarbu et al 2000). In particular, we discuss the role of food abundance and distribution as a possible determinant of the population genetic structure of Androniscus dentiger, a cave-dweller terrestrial isopod inhabiting that cave, where guano and chemoautotrophic bacteria are trophic sources, spottily distributed in different areas. Key words: Chemoautotrophic underground ecosystems, population genetic structure, cave ecology, isopods, stable isotope ratio.

INTRODUCTION Because of their trophic structure and energy cycles, underground habitats such as caves are usually considered very simple environments, which mostly depend on energy-inputs from surface. Predation and the processing of organic debris represent the main pathways through which energy is transferred between cave dwellers. An exception to this paradigm is those caves where, because of the presence of particular environmental conditions, chemoautotrophic bacterial communities can develop, providing organic matter through chemosynthetic processes. In the last decade, a number of communities, which are supported at various levels by a chemoautotrophic energy base, have been described. Chemoautotrophic communities have been found in marine, freshwater, and, very recently, cave habitats (Conway et al 1994; Stevens and McKinley 1995; Sarbu et al 1996). Only very recently, for the first time, studies carried out on the Rumanian Movile Cave system showed that, under particular conditions, chemoautotrophic bacteria can produce a great deal of organic matter, sufficient to support a taxonomically differentiated, and trophically well structured cave community. In fact, the Movile Cave can be considered an almost isolated system, with the energy inputs from surface being negligible if compared to the amount of organic matter produced in situ (Sarbu et al 1996). Thus, although the study of the Movile underground system answered the question whether chemoautotrophic processes can satisfy the energy requirements of a whole cave community, it also raised new issues regarding the

stability of environments such as the Movile Cave. In particular, we wondered if a cave community could develop and maintain a high degree of differentiation when the condition of geographic isolation is not met. The second question arose from the relationship, well known to bio-speleologists, between pattern of food distribution and cave-dwellers dispersion. In fact, often times cave organisms show patterns of dispersion that overlap those of sources of food. Based on this observation, we wondered whether and to what extent, abundance and distribution of food can influence the genetic structure of cave-organisms and their actual dispersal. The Grotta del Fiume, a cave located in Central Italy (Fig. 1), proved to be a very suitable case study to address those issues. The Grotta del Fiume, naturally accessible to man, is part of a more vast and complex underground system (Grotta del Fiume-Grotta del Vento), which is connected with the surface at many sites. A continuum in the habitat makes routes available for the dispersal of cave-dwellers. This cave is inhabited by a number of species that have access to abundant trophic resources. Organic debris or bat guano is spottily available in different areas of the cave. An important source of food, available only in a few areas of the cave is chemoautotrophic bacteria. These bacteria are present in the deep section of the cave where, after a preliminary sampling survey, we also found 15 species of invertebrates, seven of which are endemic to the karst system where the Grotta del Fiume develops. Five of these species are new to science (Sarbu et al 2000). Plus, the Stable Isotope Ratio Analysis (SIRA), that already proved very useful when investigating the food web structure of cave communities (Pohlman et al 1987; Sarbu et al

G. Gentile, S.M. Sarbu

Figure 1- 1. Grotta di Frasassi, 2. Grotta del Mezzogiorno, 3. Grotta del Vento, 4. Grotta del Fiume. The systems 1-2 and 3-4 are separated by the Sentino River. a) Guano room in Grotta di Frasassi (MEZ); b) Lago Verde in Grotta del Fiume (LAV); c) Guano room in Grotta del Fiume (GUA); d) Ramo Sulfureo in Grotta del Fiume (RSO). The asterisks indicate the sampling sites.

occurred as aggregations of large number of individuals, in correspondence of spottily distributed food sources. A. dentiger samples were collected in two sulfidic and in one non-sulfidic habitat within the Grotta del Fiume (Fig. 1): the Ramo Sulfureo section (RSO), the Lago Verde room (LAV), and the Guano room (GUA). The first two habitat are characterized by the occurrence of Beggiatoacea (unpublished data) and two species of sulfur-oxidizing bacteria (Thiobacillus thiooxidans and T. cfr. ferrooxidans) (Vlasceanu et al 2000), whereas the GUA site is a room which contains a small pile of guano. An additional non-sulfidic habitat was also investigated for comparison. It consisted in a large cave passage in the Grotta di Frasassi (MEZ), which is part of the Grotta di Frasassi - Grotta del Mezzogiorno system. The Grotta di Frasassi is a good candidate for a comparison to the Grotta del Fiume because it is (1) geographically close the Grotta del Fiume (approx. 1Km), being separated from the sites in the Grotta del Fiume.by the Sentino River. (2) Food in the Grotta di Frasassi is not spottily distributed as in the Grotta del Fiume, but is represented by guano and organic debris scarcely distributed throughout the whole cave. Allozyme electrophoresis In a previous study based on allozymes, A. dentiger revealed a high level of genetic variation (Gentile and Sbordoni, 1998). For this reason we decided to investigate the genetic structure of A. dentiger within the Grotta del Fiume, using allozyme electrophoresis as a first, preliminary approach. Twenty-five individuals were sampled from each site. Each individual was scored for all 18 presumptive allozyme loci. Gene loci scored, and protocols used are reported in Table 1. A replica sample was collected from each site seven months after the first sampling (i.e. after at least one generation). Statistical analysis Chi square tests were performed in order to detect temporal change in genotype distributions. The exception to this was the LAV site, for which no original sample was available. A linkage disequilibrium test was carried out between pairs of polymorphic loci. The algorithm used is by Black and Krafsur (1985) as implemented in LINKDIS. Heterozigosity, Neis (1978) genetic distance, F-statistics, and gene flow were estimated by using GENETIX ver. 4.0.1. Gene flow was estimated from FST (Wright 1951). Theta () was used as an unbiased estimator of FST (Weir and Cockerham 1984). Two tests of selective neutrality were performed: the Ewens-Watterson homozygosity test (Ewens 1972; Stewart 1977; Watterson, 1978) and Chakrabortys test of population amalgamation (Chakraborty 1990). The

1996; Southward et al 1996), showed that invertebrate communities of Grotta de Fiume rely on different autochthonous sources of food (Sarbu et al 2000). Among the various species inhabiting the Grotta del Fiume, we also found the oniscidean isopod Androniscus dentiger, which is the most abundant species throughout the different habitats of this cave. The occurrence of A. dentiger is very important because the evolutionary dynamics and gene flow of this species have been studied extensively over a large geographic scale (Gentile and Sbordoni, 1998; Gentile 1998; Gentile and Allegrucci 1999) including the area where the Grotta del Fiume is located. Thus, the data on the population genetics of A. dentiger provides a good framework for a study at a micro-geographic scale aimed at addressing the issue regarding the influence of food distribution on the genetic structure of the organisms inhabiting this cave. This paper reports the results of an allozyme survey aimed at catching a glimpse at the population genetic structure of A. dentiger from this cave. Although preliminary, data presented in this paper show a remarkable genetic structure and suggest a possible role of the spatial distribution of food as one of the determinants of the genetic structure of A. dentiger from Grotta del Fiume. MATERIALS AND METHODS Sample sites Although A. dentiger is locally abundant in the Grotta del Fiume, it does not occur throughout the entire cave. Despite an accurate exploration of the cave, this species could be found only in few sites, where it

Trophic sources and population genetics structure of Androniscus dentiger Table 1 - Proteins, buffers and stain. Protein Adenosine deaminase Aldolase Arginine phosphokinase Carbonate anidrase Diaphorase Alcaline Phosphatase Phosphoglucomutase Galactosidase Glucose-phosphate isomerase Malate dehydrogenase Mannose-phosphate isomerase Peptidases Pyruvate kinase (*) with modifications n E.C. 3.5.4.4 2.7.3.3 4.2.1.1 1.6.*.* 3.1.3.1 2.7.5.1 5.3.1.9 code buffer ADA (*) D - Richardson et al (1986) APK (*) J- Richardson et al (1986) CA G (Cx2) - Richardson et al (1986) DIA E - Richardson et al (1986) APH G (Cx2) - Richardson et al (1986) PGM C - Richardson et al (1986) GPI GPI - Harris and Hopkinson (1976) GOT (*) J - Richardson et al (1986) MPI MPI - Harris and Hopkinson (1976) PEP (*) A - Richardson et al (1986) PK A - Richardson et al (1986) staining Richardson et al (1986) Harris and Hopkinson (1976) Gooding and Roslet (1979) Brewer and Sing (1970) Richardson et al (1986) Ayala et al (1972) Richardson et al (1986) Richardson et al (1986) Richardson et al (1986) Richardson et al (1986) Richardson et al (1986) Wallis et al (1984) Richardson et al (1986)

loci 1 1 1 1 1 2 1 1 1 1 2 1 1 1

4.1.2.13 ALDO (*) TEC - van Someren et al(1974)

3.2.1.23 GAL D - Richardson et al (1986)

Glutamate-oxaloacetate transaminase 2.6.1.1 5.3.1.8 3.4.1.1 2.7.1.40

1.1.1.37 MDH (*) GPI - Harris and Hopkinson (1976) Richardson et al (1986)

program ARLEQUIN (Roessli and Excoffier 2000) was used in order to perform the neutrality tests. RESULTS Allele frequencies are reported in the Appendix. Of 18 putative loci investigated by allozyme electrophoresis, eleven were found to be polymorphic according to the 99% criterion (Ada, Aldo, Aph-1, Ca, Dia, Gal, Gpi, Mdh-1, Mpi, Pep-1, and Pep-2). Four loci, which were polymorphic over a large geographic scale (Central Italy; Gentile and Sbordoni 1998) were monomorphic (Aph-2, Got-2, Mdh-2, and Pgm) at the micro-geographic scale of this study. Chi-square tests by locus indicated that the genotype constitution of populations remained constant over seven months for all but one locus. The only significant shift in the genotype frequencies (Chi2=22.394, v=2, p<<0.0001) occurred at the Mdh-1 locus, in the population of the Ramo Sulfureo (RSO). All pairs of polymorphic loci were tested for linkage disequilibrium. Only in four tests we observed statistically significant (p<<0.05) values of the correlation coefficient. These tests were Ada-Mdh-1, Ada-Pep-1, Mdh-1Mpi, and Pep-1-Pep-2, with R values of 0.24, 0.19, 0.18, and 0.26, respectively. Since correlation was very poor, all loci were considered independent genetic markers. Values of the fixation index (f) are reported in Table 2. Some loci showed genotype frequencies deviating from Hardy-Weinberg equilibrium, generally in the form of heterozygote deficiencies. Mean heterozygosity values are reported in Table 3. Values are low, ranging between 3 and 9 per cent. An overall analysis of F-statistics carried out on the replica samples allowed the evaluation of the proportion

Table 2 - FIS (Weir and Cockeham, 1984) values for each of the 18 loci investigated in the replica samples. The asterisk means p<0.05. The double asterisk means p<0.01. MEZ Ada Aldo Aph.1 Aph.2 Apk Ca Dia Gal Got.1 Got.2 Gpi Mdh.1 Mdh.2 Mpi Pep.1 Pep.2 Pgm Pk -0.027 --0.511* --------0.349 -------0.150 ------------GUA 0.686** --0.559** -----0.155 --0.786** -------0.067 --0.087 -0.067 -0.011 ----RSO 0.823** --0.517* ----0.786** ----------0.064 --0.197 --------LAV 0.709** --0.596** -----0.043 --------------0.654** ---------

of genetic variation due to inter-population and intrapopulation differences. FIT, FST and FIS estimates are 0.564, 0.284 and 0.390 (p[F-statistics not>0] <0.001) respectively, suggesting that the genetic variance observed is due to both inter-population and intra-population components. A jackknifed approach to F-statistics showed that the major contribution to genetic variation inter-populations is made by the sample from Mezzo-

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G. Gentile, S.M. Sarbu Table 4 - Above the diagonal: Neis (1978) genetic distance; p[D not >0] < 0.01 for all comparisons. Nm values are below the diagonal. Gene flow values were estimated from FST, by using (Weir and Cockerham, 1984). MEZ MEZ GUA RSO LAV **** 0.508 0.606 0.227 GUA 0.053 **** 1.536 4.958 RSO 0.032 0.020 **** 0.462 LAV 0.080 0.006 0.049 ****

Table 3 - Mean heterozygosity in the replica samples. Standard errors are indicated in brackets. A locus is considered polymophic if the frequency of the most common allele does not exceed 0.95. Pops. Mean no. of Percentage of alleles per loci polymorlocus phic 1.3 22.2 Mean heterozygosity observed MEZ 0.044 (0.019) GUA 1.6 38.9 0.089 (0.032) RSO 1.4 27.8 0.044 (0.02) LAV 1.3 22.2 0.033 (0.015) expected 0.055 (0.024) 0.129 (0.044) 0.083 (0.033) 0.074 (0.036)

giorno Cave (MEZ). In fact, after removing this sample, the FST value became equal to 0.180. After the neutrality tests, the hypothesis of neutrality could not be rejected for any of the loci scored. Thus, all of them were used to estimate gene flow among samples from FST values (Wright 1951). Pair-wise Nm values are reported in the Table 4. There is very little gene exchange between the population at MEZ and the other populations. Samples from Mezzogiorno Cave and from the Grotta del Fiume seem to represent independent gene pools. The geographic distance between the two cave systems is not prohibitive for the dispersal ability of A. dentiger, and there is no interruptions in the limestone layer where the two cave systems take place. So we hypothesize that the Sentino River may act as a geographic barrier. Gene exchange among the populations within the Grotta del Fiume is not homogeneous. Populations RSO and LAV are nearly isolated, as indicated by a very low level of gene flow. However, both these populations exchange genes with the population GUA. The population from the river room (RSO) seems to be the most isolated. Neis distance estimates are also reported in Table 4. Values observed are very low when compared with genetic distance values observed between other Androniscus populations (Gentile and Allegrucci 1999). DISCUSSION With regard to cave-dwelling organisms, numerous studies have shown that a high degree of genetic fragmentation is often found at large geographic scales (Crouau-Roy 1989; Kane et al 1994; Sarbu et al 1993;

Culver et al 1995; etc.). Surprisingly, our study shows that a high degree of genetic structure can occur in a single cave as well. In subterranean organisms, such a high level of genetic differentiation is not common at a microgeographic scale, having been observed only in few cases (see for a review Sbordoni et al 2003). In fact, FST values observed within the Grotta del Fiume are comparable in magnitude to values often observed in studies carried out over a large geographic area. (Caccone 1985; Sbordoni et al 1985; Kane et al 1992). A. dentiger from Grotta del Fiume exhibited a remarkable heterozygote deficiency, which also occurs if the different samples are considered separately. Furthermore, half of polymorphic loci showed heterozygote deficiency in at least one sample. These results are rather unexpected based on the previous observation that, of the many cave and surface populations of Androniscus investigated so far in Central Italy, very few showed evidence of deviation from the Hardy-Weinberg equilibrium. In fact, deviations, if observed, were reported only at few loci (Gentile and Sbordoni 1998), in few populations. The heterozygote deficit could be caused by a number of factors such as the Wahlund effect, underdominance, a combination of small population size and inbreeding, as well as the presence of a cryptic related taxon. In the following part of this paragraph, we discuss whether those factors may explain the data observed. The Wahlund effect is due to the occurrence of subunits, which are present within the samples, but not detected. It cannot be excluded that Androniscus may respond to micro-differentiation of the habitat, resulting in the presence of more than one sub-unit in the sampling area. However, the sampling strategy was designed to minimize this effect by collecting individuals in small and homogeneous sampling areas. We dont have strong evidence to support the occurrence of selection against heterozygotes (under-dominance), although it cannot be completely excluded. In case of selection, since heterozygote deficiency was observed at a number of loci, linkage disequilibrium would be reasonably expected between loci. However, pairwise tests between loci that resulted statistically significant were few, plus even significant R values observed indicated very poor correlation. Neither tests for neutrality allowed to reject the neutral hypothesis at any of the loci.

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The possibility that genetic heterogeneity among samples within the Grotta del Fiume is due to the presence of a closely related taxon is not likely either. Neis (1978) genetic distances between pairs of populations are on the whole rather small and similar in magnitude, suggesting that each of the four populations belong to the same species (Sbordoni 1982; Thorpe 1983). Most likely, a sympatric occurrence of sibling taxa would have been evident by a higher degree of genetic differentiation (Cobolli-Sbordoni et al 1990; Cesaroni et al 1992). As a matter of fact, high genetic distances between populations of A. dentiger, comparable to those observed between morphologically differentiated species of the same genus, have been already observed by Gentile and Allegrucci (1999), suggesting the existence of a group of sibling species in Central Italy. However, those distance values were one to two orders of magnitude higher than those observed between populations in the Grotta del Fiume. The combined effect of restricted home ranges, small effective population size, and inbreeding might explain the general heterozygote deficit and the low mean heterozygosity values observed. However, very low values of heterozygosity could be also due to a founder-effect. This appears unlikely because mean heterozygosity values estimated in the populations from Grotta del Fiume and Mezzogiorno Cave are comparable in magnitude to those observed in other cave populations from Central Italy (Gentile and Allegrucci 1999), where the occurrence of private alleles in many cases suggests long evolutionary times (Gentile and Sbordoni 1998). Admittedly, difficult to explain is the temporal allele frequency shift observed at the Mdh.1 locus. Even though the combination of restricted home ranges, small effective population size, and inbreeding may produce such a change in allele frequency, it seems unlikely that it can be observed after only one-two generations. It is also possible that allele frequency shift at this locus may be related to some form of selection and population turnover still uncovered. In fact, Mdh.1 locus was the only locus not consistent with neutrality when several populations of A. dentiger were investigated over a large geographic area in Central Italy (Gentile and Sbordoni 1998). As already mentioned, it has been possible, by using SIRA, to reveal that invertebrate communities of Grotta de Fiume, including Androniscus (see Fig. 2), are based on different trophic sources (Sarbu et al 2000). Indirect evidence of the use of different source of food comes also from the different color pattern exhibited by individuals found on guano and sulfidic sites, with most of the former showing a red-purple pigmentation (the red-purple pigmentation in Androniscus could be depending on the diet (Vandel 1960; Gentile, unpublished data)), whereas all the latter were white (not pigmented) (Gentile, unpublished data). On the whole, these observations do not fit very well with the generally accepted paradigm that

Figure 2- 13C and 15N values relative to A. dentiger samples from sulfidic sections (white circles) and non-sulfidic sections (black circles) of the cave. White diamonds indicate microbial mats from sulfidic rooms (redrawn after Sarbu et al 2000). The populations feeding on food indirectly based on photosynthesis (i.e. bat guano) are isotopically heavier, both with respect to carbon and to nitrogen, than the populations sampled in the sulfidic rooms.

caves are very homogeneous environments as a result of their habitat stability. Although preliminary, data in this paper suggest that this expectation may be not true when abundant and spottily trophic inputs transform a cave into a very patchy habitat, constraining sub-populations to the trophic resources regardless of the potential dispersal ability of individuals. Within this cave A. dentiger shows an evident contagious distribution, matching the distribution of the sources of food, where A. dentiger can reach a density of 10-50 individuals/m2 (unpublished data). This pattern has not been observed in other caves where food from the surface is scarcely available, generally consisting in small bits where only few individuals may be found. Thus, as a result of the scarcity of food individuals tend to maximize the exploration of the environment. Consistent with this scenario, the gene exchange between populations from Grotta del Fiume is less than should be expected considering the small geographic distances among sampling sites, and the absence of any apparent geographic barrier (Gentile and Sbordoni 1998). This scenario is strongly supported by data from stable isotopes, which have shown that sub-populations are strictly constrained by their food resources. These results need and deserve further investigation, which we will undertake by using microsatellite loci, which proved less susceptible to selection, and possibly by increasing the number of sampling sites in the Grotta del Fiume. However, data from this study showed that cave communities might be genetically highly structured even though geographic isolation is absent. This is an important outcome because it suggests that geographic isolation per se is not the most important determinant, for communities such as the one in the Grotta del Fiume to form and persist over time. These data are consistent

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G. Gentile, S.M. Sarbu

with the view that the stability of the distribution of the abundant sources of food within that cave could also be a very important issue. Within a cave, the abundance of guano and organic debris from the surface may not be continuously available for cave dwellers, being susceptible to great fluctuations over time. Chemoautotrophic bacteria inhabiting sulfidic habitat provide in turn a great deal of organic matter, continuously available. In agreement with this scenario is also the discovery of several endemic species of invertebrates that occur only in the sulfidic section of the Grotta del Fiume. All of them show features typical of cave-life adaptation, and are greatly differentiated with respect of their surface relatives. It is usually believed that long evolutionary time is needed for such a high degree of morphological differentiation to develop. This does not necessarily mean that long term evolution is responsible for the degree of genetic structure observed in A. dentiger. Actually, we believe that the absence of private alleles and the small genetic distance values between sub-populations would rather support short times. This is consistent with the geological history of the Grotta del Fiume. In fact, sulfidic environments of this cave would be once more continuous to one another due to the higher level of the phreatic zone. Isolated sulfidic lakes, as we know today, would have started forming only 10.000 years ago (Galdenzi, pers. communication), when the phreatic zone lowered. ACKNOWLEDGMENTS We gratefully thank V. Sbordoni for contributing to this research with his criticism and financial support. We thank the Consorzio Grotta di Frasassi for the logistic support given us. We thank T. Kane and R. Argano for their helpful comments to a previous version of this paper. REFERENCES Ayala, F.J., J.R. Powell, M.L. Tracey, C.A. Mourao, S. Perez-Salas. 1972. Enzyme variability in the Drosophila willistoni group IV. Genic variation in natural populations of Drosophila willistoni. Genetics 70: 113-139. Black, W.C., E.S. Krasfur. 1985. A FORTRAN program for the calculation and analysis of two-locus linkage disequilibrium coefficients. Theoretical and Applied Genetics 70: 491-496. Brewer, G.J., C.F. Sing. 1970. An introduction to isozyme techniques. New York: Academic Press Caccone, A. 1985. Gene flow in cave arthropods: a qualitative and quantitative approach. Evolution 41: 1198-1214. Cesaroni, D. , G. Allegrucci, V. Sbordoni. 1992. A narrow hybrid zone between two crayfish species from

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APPENDIX: Allele frequencies for the original and replica samples. MEZ1 MEZ2 GUA1 GUA2 RSO1 RSO2 LVR Ada (N) 1 2 3 Aldo (N) 1 2 Aph.1 (N) 1 2 Aph.2 (N) 1 24 24 25 25 24 25 25 1.000 1.000 1.000 1.000 1.000 1.000 1.000 21 24 25 24 23 25 24 0.048 0.208 0.800 0.646 0.739 0.860 0.479 0.952 0.792 0.200 0.354 0.261 0.140 0.521 22 24 25 25 24 25 25 0.977 1.000 1.000 1.000 1.000 1.000 1.000 0.023 0.000 0.000 0.000 0.000 0.000 0.000 23 20 25 25 24 25 23 0.000 0.000 0.500 0.560 0.146 0.320 0.826 0.891 0.950 0.500 0.440 0.854 0.680 0.174 0.109 0.050 0.000 0.000 0.000 0.000 0.000

14 Apk (N) 1 Ca (N) 1 2 3 Dia (N) 1 2 Gal (N) 1 2 10 Got.1 (N) 1 Got.2 (N) 1 Gpi (N) 1 2 Mdh.1 (N) 1 2 Mdh.2 (N) 1 Mpi (N) 1 2 3 24 24 21 25 22 23 24 24 25 25 25 25 21 24 23 25 25 24 24 24 25 25 24 25 24 24 25 25 25 25 24 24 25 25 25 25 24 24 25 25 24 25 19 24 25 25 25 25 22 24 25 25 24 25 0 -----24 25 25 25 25

G. Gentile, S.M. Sarbu Pep.1 25 (N) 1 2 3 25 Pep.2 (N) 1 2 25 3 Pgm (N) 1 25 Pk (N) 1 24 24 25 25 24 25 25 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.188 0.104 0.140 0.100 0.000 0.000 0.000 0.771 0.896 0.860 0.900 1.000 1.000 1.000 0.042 0.000 0.000 0.000 0.000 0.000 0.000 24 24 25 25 24 25 25 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.026 0.000 0.000 0.000 0.000 0.000 0.000 0.974 1.000 1.000 1.000 1.000 1.000 1.000 21 15 21 25 25 23 14 0.048 0.000 0.000 0.020 0.000 0.000 0.000 0.952 1.000 0.976 0.960 0.960 1.000 1.000 0.000 0.000 0.024 0.020 0.040 0.000 0.000 0.000 0.021 0.000 0.040 0.000 0.000 0.000 0.977 0.979 0.840 0.820 0.938 0.900 0.940 0.023 0.000 0.160 0.140 0.063 0.100 0.060 0 ---------------15 21 25 24 23 14 1.000 1.000 1.000 1.000 1.000 1.000 0.033 0.071 0.080 0.000 0.000 0.000 0.967 0.929 0.920 0.979 1.000 1.000 0.000 0.000 0.000 0.021 0.000 0.000

25

1.000 1.000 1.000 1.000 1.000 1.000 1.000

25

1.000 1.000 1.000 1.000 1.000 1.000 1.000

25

0.000 0.000 0.000 0.000 0.042 0.020 0.000 1.000 1.000 1.000 1.000 0.958 0.980 1.000

25

0.738 0.854 0.870 0.920 0.260 0.813 0.980 0.262 0.146 0.130 0.080 0.740 0.188 0.020

25

1.000 1.000 1.000 1.000 1.000 1.000 1.000

23

0.000 0.000 0.452 0.620 0.273 0.130 0.761 1.000 1.000 0.548 0.380 0.727 0.848 0.239 0.000 0.000 0.000 0.000 0.000 0.022 0.000