- Genetic and morphological support for possible sympatric origin of fish from subterranean habitats
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- Observation of the Catfish Chaetostoma microps Climbing in a Cave in Tena, Ecuador
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Please enable cookies in your browser to get the full Trove experience. Skip to content Skip to search. Physical Description xiv, p. Published Enfield, N. Language English View all editions Prev Next edition 4 of 4. Other Authors Trajano, Eleonora. Bichuette, Maria Elina. Kapoor, B. Edition 1st ed. Subjects Amblyopsidae. Hypogean fishes. Hypogean fishes -- Conservation. Fishes -- Ecophysiology. Fishes -- Adaptation. Fishes -- Habitat. Summary Subterranean fishes are among the most interesting and scientifically relevant organisms, possessing unique combinations of features as a result of evolution under a peculiar selective regime, with permanent darkness as a major directive force throughout the hypogean realm.
High divergence rates in relation to close epigean relatives, homoplastic evolution of several characters and the simplified nature of subterranean ecosystems, render subterranean organism excellent subjects for evolutionary and ecological studies.
Genetic and morphological support for possible sympatric origin of fish from subterranean habitats
Because of the special characteristics of this family environmental changes could pose a threat to their survival. A thorough knowledge of the ecology and biology of these animals is necessary for taking effective steps for their protection. In the subsequent chapters updated approaches, integrating new and previously known data, on general topics such as evolutionary genetics and development, behavior, biodiversity and conservation are presented.
A worldwide checklist with troglomorphic species represents a significant increase in relation to previous studies. Regional faunas, in totum or focusing on selected taxa, of countries such as China, India, Brazil, Mexico and the African continent are discussed in terms of biodiversity, natural history and distribution, and ecology.
A comprehensive chapter on the Amblyopsidae summarizes information on almost every aspect of the systematics and biology of this family. Thomas L. Circles and squares depict the results of eDNA analyses; stars and triangles represent known localities of the black or the white Proteus , respectively. We next analysed the same six samples for white Proteus eDNA.
Sources of hydrogeological and geological layers: refs 17 , 18 and Below we evaluate the basic parameters of our eDNA assay and its effectiveness in detecting Proteus in the laboratory as well as in different subterranean habitats. A suitable eDNA assay for Proteus must be able to detect trace amounts of highly diluted DNA released by potentially very small populations.
The qPCR technique is commonly used in achieving this goal 22 , When compared to classical PCR in combination with cloning and sequencing 27 , the real-time qPCR approach significantly improves the efficiency of eDNA detection and reduces the possibility of contamination as post-PCR analysis is omitted. Our tests showed that this method is suitable for detection of Proteus eDNA, including under the conditions encountered at karst springs.
A compromise between at least three factors is necessary for an optimal use of the eDNA assay for Proteus : 1 the time of sampling, 2 the amount of water filtered and 3 the lower detection limit of the method. The discharge from karst springs typically varies significantly through seasons in response to precipitation in the catchment area, with many springs inactive during the dry season 32 , As a consequence, sampling when water levels are optimal may be a challenge.
When water levels are very high, eDNA may become too diluted or dispersed for detection.
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The latter may have been the case in a few springs in Herzegovina, which therefore required a repeated sampling to detect the presence of Proteus eDNA. Another concern when sampling for Proteus eDNA is collecting water samples without disturbing the sediment at very low water levels, while higher water levels may increase sediment transport through the karst aquifer. Even though sediment potentially holds more eDNA 34 , 35 , it also prevents efficient filtration.
Furthermore, sediment can be a source of PCR inhibitors 36 , Monitoring PCR inhibition with the addition of synthetic DNA and corresponding primers and probes to each reaction, and diluting template DNA when inhibition is detected, is essential when analysing environmental samples for eDNA. Exposed DNA in water gradually decays predominantly due to the effects of UV-light, heat and decomposition by microorganisms 31 , 38 , Because of the absence of light and due to the relatively low and constant temperatures of groundwater inhabited by Proteus Supplementary Table S1 ; see also ref.
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As factors that affect DNA degradation may be less detrimental in subterranean streams, eDNA transport distance is likely increased in this habitat. Since eDNA transport in streams is greatly affected by discharge rates 41 , 42 , we timed the sampling, whenever and wherever it was possible, to the lowest water level and discharge rates that still allowed for efficient sampling. We believe this approach minimises the risk of not detecting a Proteus population due to an unknown variation in sequence.
This especially applies to the primer set designed to bind to the conserved 16S rRNA gene, which appears to be general enough to be used in detection of any population within as well as outside the known range of Proteus. The primer pair designed to amplify a fragment of the control region, on the other hand, targets a more variable region of the mitochondrial genome.
Therefore, the possibility that it could not bind efficiently to the hypothetical Montenegro population, which may be genetically distinct from all other known populations, cannot be excluded. Compared to classical approaches of visual surveying or trapping, the eDNA analysis substantially improves our ability to detect Proteus in groundwater. This is most clearly observable in the results of the Bela Krajina survey, where the number of known sites with the black Proteus more than doubled after a single sampling.
Although the number of sites used to validate the detection probability and accuracy of the method was low, laboratory tests see Supplementary Information and hydrogeological data strongly support the results obtained for springs with hitherto unknown status. Similar detection probabilities were reported for epigean aquatic vertebrate species e. Testing the usefulness of the eDNA assay in field research in Herzegovina resulted in the detection of Proteus at locations where it has not been previously recorded. Following our analyses, the presence of Proteus was visually confirmed by cave diving at the spring Kajtazovo Vrelo no.
Vlaho, pers. Lewarne, pers. The proximity of the two sites suggests that they may be hydrogeologically connected and therefore may share a common Proteus population. Nonetheless, preliminary results suggest that the two cave systems could be hydrogeologically isolated from each other B. Our results therefore favour the possibility that Proteus individuals are actually present at the sampled location and that the southern range of Proteus extends into the Dinaric Karst of Montenegro.
Next, using the eDNA assay, we report the discovery of new sites that may harbour the rare black Proteus , while its presence was visually confirmed in yet another one, which showed a faint trace of Proteus eDNA. The distance of the new easternmost site, the spring Planinec no. As in most aquatic cave animals, ranges of Proteus populations are probably historically determined and highly restricted by the boundaries of present-day subterranean hydrogeological networks It should be noted, however, that while the maximum span of the black Proteus range to the east, north and south was established in the present study, the extent of its distribution to the west remains unknown.
This conclusion is consistent with earlier predictions, when only one 9 or two sites 10 were known.
The finding is strongly supported by an existing intermittent hydrogeological connection 17 with Otovski Breg, a nearby site occupied by the white Proteus no. Rare cases of subterranean syntopic occurrence of closely related lineages are valuable for the study of the poorly understood mechanisms of speciation and differentiation within the subterranean realm e. The distribution of the black and white Proteus eDNA in Bela Krajina is in agreement with the existence of a potential reproductive barrier between these two lineages, at least regarding female mating preferences.
Similarly, if inter-lineage mating regularly occurred in the other direction, we would expect black Proteus eDNA to appear in the sample at Otovski Breg, a site which was found to harbour only white Proteus eDNA.
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Significantly, despite a low degree of sequence divergence between the two populations observed in the mitochondrial control region 14 , 15 , 45 , none of the comparative studies to date have detected any signs of their interbreeding, e. In combination with these observations, eDNA data suggest that the two populations may represent independent species, but additional analyses are needed to resolve the taxonomic status of the present as well as of other apparently monophyletic groups of Proteus.
In conclusion, we have demonstrated that the qPCR-based eDNA method can be utilised for a rapid detection of a rare subterranean species inhabiting karst groundwater. Furthermore, as suggested by the results of the Bela Krajina survey in particular, the qPCR-based approach enables us to assess the relative abundance of Proteus eDNA in groundwater over a small spatial scale.
Finally, we have shown that the eDNA approach can also be helpful in identifying potentially sympatric populations in the cryptic subterranean environment and therefore can be useful in the study of evolutionary history and taxonomy of subterranean taxa. Development of our methodology to detect traces of Proteus eDNA in water involved the following steps see Supplementary Information : 1 development of specific oligonucleotides for eDNA detection with qPCR, 2 testing the specificity of the oligonucleotides on tissue samples, 3 testing the lower detection limit of the method in laboratory conditions and 4 testing the performance of the method in nature, at three verified sites in Slovenia SYBR qPCR only.
Observation of the Catfish Chaetostoma microps Climbing in a Cave in Tena, Ecuador
Samples were collected in brand-new 5- or L plastic canisters and stored in a dark cool room until filtration. Water samples were filtered through sterile 0.
Up to four filter membranes were used per sample, depending on the degree of clogging by sediment and other particles in the water. For each template dilution, both target fragments were amplified in triplicate using separate plates for each primer pair.
A single well qPCR plate contained between four to 12 samples, so that individual samples were separated by at least one empty row and column. In addition, each plate included six negative controls double distilled and tap water from outside of Proteus range and two positive controls tissue DNA and eDNA extracted from the laboratory water tanks , for a comparison of melting curves.
Samples were scored positive for Proteus eDNA if at least two out of three replicate wells of at least one combination dilution-primer pair were positive by qPCR see Fig.