Ethics Statement

This investigation was conducted entirely in accordance with ethical standards and Croatian legislation. The work was approved by the Ethical Committee of the Faculty of Science, University of Zagreb.

The sample analyzed in this investigation comprises DNA sequences of individuals from all known Cobitis populations in the Adriatic watershed of Croatia and Bosnia and Herzegovina. Besides sequences used in taxonomic investigation of the Adriatic spined loaches [8], the sample was completed with two new locations and more sequences from populations that were represented with fewer samples in the mentioned investigation (Table 1). Above mentioned taxonomic investigation was based on various methods of phylogenetic reconstruction, morphological analyses, as well as comparison of phylogenetic models using Bayes factors [8] whereas in this investigation we have analyzed evolutionary history and population genetic structure using population genetic methods. Three genetic markers were analyzed: mitochondrial gene for cytochrome b (cyt b) and two nuclear genes: RAG1 gene and the first intron of the S7 gene, even though all three markers were not included in all analyses. Namely, different position and function of investigated markers, as well as their evolution inside the genus Cobitis, result in different credibility of phylogenetic and population genetic analyses. Cyt b gene, as a part of mitochondrial DNA (mtDNA), is inherited only maternally, has a faster mutational rate, lacks recombination and other mechanisms that would effectively erase mutations, and, thereafter, is phylogenetically more informative. RAG1 and S7 are nuclear genes, but inside S7 non-coding intron was analyzed, whereas RAG1 is a protein-coding gene. Since it was concluded that cyt b phylogenetic tree mostly resembles species diversification tree [8] and the only known mutation rate inside spined loaches, available for the molecular clock calibration, is the one of the cyt b gene, phylogenetic reconstruction, as well as divergence times and past changes in population size estimations were conducted based on cyt b data set. On the other hand, tests of genetic diversity and differentiation, as well as estimation of the gene flow among populations were based on all three genes in order to obtain the most reliable results based on both, mitochondrial and nuclear DNA, and reveal eventual differences. Effective population size estimations were conducted on cyt b data set, because the knowledge on mutation rate is also necessary for those calculations.

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Table 1. Sampling localities and number of specimens included in the investigation.

https://doi.org/10.1371/journal.pone.0131580.t001

Laboratory protocols for the DNA extraction, polymerase-chain reaction (PCR) and sequencing for new samples were the same as for samples used in taxonomic investigation and are described in [8]. Haplotype variants of nuclear genes in heterozygous individuals were reconstructed by a Bayesian statistical method implemented in PHASE 2.1 software [10], [11]. Phylogenetic reconstruction was conducted using Bayesian inference as implemented in MrBayes (version 3.1.2 [12]). Besides our sequences of cyt b gene, in the phylogenetic reconstruction and divergence time estimation we have also included haplotypes of C. elongata Heckel & Kner, 1858 and Italian C. bilineata [9], retrieved from the GenBank. The sequences of C. elongatoides Baçescu & Maier, 1969 and Sabanejewia romanica (Baçescu, 1943) [13] were used as outgroups. Two simultaneous runs were conducted. For each, Markov Chain Monte Carlo was run four times for three million generations with trees sampled every 100 generations. The best-fitting model of molecular evolution (GTR model) was selected by hierarchical likelihood ratio tests using MODELTEST software version 3.06 [14]. Average standard deviation of split frequencies approached 0 (0.000074) and the potential scale reduction factor approached 1 (1.002). Convergence between runs was high for all trace parameters, as investigated using Tracer v1.5.0 (software by Rambaut A, Suchard MA, Xie D & Drummond AJ; available from http://beast.bio.ed.ac.uk/Tracer). Divergence times between investigated species, as well as between phylogenetic lineages, were estimated by a Bayesian MCMC coalescent method, using Beast 1.7.0 software [15]. The rate homogeneity across phylogenetic lineages was assessed by the log-likelihood ratio test (LRT), comparing the likelihood of phylogenetic trees (reconstructed using maximum likelihood approach) with and without molecular clock enforcement in PAUP* (version 4.0b10 [16]). Since the likelihood scores were the same in both cases, we applied a strict molecular clock. Branch rates were drawn from an uncorrelated lognormal distribution and a Yule speciation prior with random starting tree. Substitution model applied was HKY with Gamma site heterogeneity model. We used default prior distributions for kappa, frequencies and alpha, whereas substitution rate parameters were unlinked across codon positions. The number of MCMC steps (the length of chain) was three millions. The molecular clock calibration was conducted based on the divergence rate of cyt b gene of 0.34% per lineage per million years (0.68% per pairwise divergence [17]). Phylogenetic reconstruction and divergence time estimation was based on cyt b data set only due to differences in three gene trees and conclusions of [8] that cyt b phylogenetic tree mostly resembles species diversification tree. Upon divergence time estimations based on the cyt b gene, we have used two estimated splitting-events as calibration points to assess the divergence rates of RAG1 and S7 first intron.

The level of intraspecific and intrapopulational genetic diversity was estimated by calculating several measures of DNA polymorphism for each genetic marker, using DnaSP v.5 [18]: number of polymorphic sites (S), haplotype diversity (Hd), average number of nucleotide differences (K) and nucleotide diversity (π). Furthermore, a frequency of each haplotype was calculated as a percentage of a certain haplotype in a population. For species distributed on more than one locality and presumably comprising more than one population (C. narentana and C. illyrica) intrapopulational genetic diversity was calculated for each population. Genetic differentiation analysis was employed in order to explore differences among populations of the same species. It comprised estimation of haplotype‒based statistics (χ² test, H_ST), as well as nucleotide sequence‒based statistics (K_ST, K_ST*, K_S*, Z, Z*) [19] and was conducted on all three investigated genes. The statistical significance of each statistic was ascertained by a permutation test. Changes in past effective population sizes were analyzed using Bayesian skyline plots (BSP), as implemented in Beast 1.7.0. The BSP model was employed on cyt b data set and it estimates the history of change in effective population size from the variability among sampled haplotypes, assuming a mutation rate of 0.34%/MY. The BSP settings were the same as used for divergence time estimations with the tree prior set to Coalescent: Bayesian Skyline.

Interactions among populations and species were investigated using maximum likelihood approach [20], [21] implemented in MIGRATE 3.2.1 [22]. We have calculated immigration rates between populations of species containing more than one population as mutation-scaled effective immigration rates and as the number of immigrants per generation. Migration rates were calculated separately for mitochondrial cyt b gene and nuclear data set (combined RAG1 and S7 first intron), due to the differences in the evolution of mitochondrial vs. nuclear DNA. Regarding nuclear data set, MIGRATE calculates two loci estimates independently, and multi-locus estimate takes into account the likelihood of each parameter at each locus. Same software was employed for the effective population sizes (Ne) estimations. Extrapolation of census population size (N) was based on formula N_e/N = 0.1–0.2 [23]. Since exact N_e/N value has not been estimated for spined loaches, we used current default value only to enable comparison with IUCN criterion C thresholds, taking into account drawbacks of such extrapolation [23]. Data on genetic diversity (intrapopulational) and differentiation (among populations) as well as interactions among populations of the same species, even though are not usually considered in extinction risk assessments, might be of great assistance in species’ future trend predictions and conservation decisions.

Evolutionary History of the Adriatic Spined Loaches

Time frames of important divergence events among the investigated Adriatic spined loaches correspond to recorded geological events. Separation of the “bilineata” subgroup from the ancestor that it shared with C. elongata (distributed in the Danube R. basin) occurred in the middle Miocene (11.3–16.4 MYA), which is concordant with the Dinarid Mountains uplift. The next evolutionary event characterized separation of main lineages inside the “bilineata” subgroup, which is dated to the second half of Miocene and possibly beginning of Pliocene. It is likely that separation and evolution of main groups and lineages of the Adriatic spined loaches was shaped by the evolution of Dinaric Lake system (DLS) and local tectonic activity. DLS is a system of tectonic lakes filled with freshwater and located inside the western Dinaric belt [24], [25]. It originated in the early Miocene and during its evolution lakes changed sizes and were temporary interconnected [26], [27]. Fossil evidence indicates that lakes belonging to DLS were inhabited by freshwater, highly endemic fauna [25], [28]. Even though fossil records of fish species from DLS were never investigated, our results reveal the main older diversification events inside spined loaches to also be connected with the so called spectacular Miocene radiation [28]. In accordance with consideration that DLS was a root for the evolution of snail genera Orygoceras, Emmericia and Melanoptychia [28], it is likely that evolution of DLS during favorable Miocene climatic conditions enabled diversification of cyprinid fishes. Nevertheless, diversification of the Adriatic spined loaches did not come to an end with the termination of DLS in lower Miocene or early Pliocene. The next important evolutionary event, represented by the origination of the majority of recent Adriatic species can be dated back to the period of the lower Pliocene and the beginning of the Pleistocene (1.5–3.4 MYA). There is geological evidence that tectonic activity in the investigated area was especially intense on the Pliocene/Pleistocene boundary, so it is likely that local tectonic events induced origination of the majority of the recent Cobitis species. It is interesting that even after that period a contact between the Zrmanja and the Italian C. bilineata populations was possible, resulting in the presence of the same haplotypes inside two isolated populations. Namely, the Po R. and the Zrmanja R. were periodically connected until the raising of the sea level in Holocene. Connections were formed during glaciations when sea level was much lower and the area of northern Adriatic desiccated [29]. Geological evidence demonstrated that historical Neretva and Cetina R. beds were significantly longer than recent [30], [31]. Based on our results, those two rivers not only had significantly longer beds, but were interconnected (constantly or periodically) until lower Pliocene (about 2.8 MYA). That is the period of the Pliocene optimum, with very warm climate conditions and sea level higher for 20–35 m comparing to recent conditions [32]. High sea level could have led to the final isolation of two rivers and provoked differentiation of two Cobitis species, even though it is also possible that intensive tectonic activity played a role in this segregation. Separation of C. illyrica and C. herzegoviniensis occurred concurrently and it could also be provoked by local tectonic activity.

Glaciations, that started in the final stage of Pliocene (Gelasina, 2.58 MYA [32]) further affected evolutionary history of the Adriatic spined loaches, most likely not as a diversification event, but by influencing on demographic history of species (as will be discussed later).

Intraspecific Genetic Diversity–Causes and Consequences

Pronounced differences among geographically closely located and phylogenetically related species in present pattern of DNA polymorphism imply different demographic history of each species after their separation from the common ancestor, but also points out different probabilities of viability and future survival.

Species C. jadovaensis contains extremely low genetic diversity, which together with skewed allele frequency distribution (great domination of one haplotype) is an indication of a strong evolutionary bottleneck. Therefore, even though C. jadovaensis is an old species (probably of the middle Pliocene origin), its intraspecific genetic diversity, that serves as a reservoir for coping with potential future environmental changes, was significantly reduced during an evolutionary bottleneck, which also reduces its viability potential and points out necessity of effective protection. Maximum likelihood estimation of its effective population size is even smaller than previously proposed N_e of 500 (50/500 rule [33]) and more recently revised N_e ≥ 1000 [26] that is considered sufficient to maintain the evolutionary potential.

Besides in C. jadovaensis, the lowest genetic diversity was noticed in Croatian population of C. bilineata and in C. herzegoviniensis. Observed haplotype frequency also implies the possibility of a bottleneck in an evolutionary history of spined loaches from the Zrmanja R. The bottleneck hypothesis could not be confirmed using estimations of BSPs because the possible bottleneck happened earlier in demographic history than caught by BSPs. Nevertheless, geological investigations provided evidences that region of northern Adriatic basin was affected by glaciations [34]. Furthermore, the same investigations did not reveal traces of glaciations in southern Dalmatia, which probably allowed unconstrained development of southern populations resulting in their recent, much higher genetic diversity. It is very interesting that diversity of phenotypes of C. jadovaensis and C. bilineata is also much smaller than for the species located slightly more to the south [8].

For C. jadovaensis and Croatian population of C. bilineata BSPs imply population stability and slight growth, respectively, in the most recent period of their demographic histories which is encouraging in conservational sense. Nevertheless, earlier evolutionary events reduced their genetic diversity and effective population sizes, making them extremely vulnerable in case of changed environmental conditions.

All measures of intraspecific cyt b polymorphism are also very small for C. herzegoviniensis, even though it is located further to the south. However, its lower intraspecific polymorphism is most likely a result of its younger divergence, as well as very narrow distribution on a small, unique area.

The pattern of DNA polymorphism observed in C. dalmatina (high level of DNA polymorphism, great number of haplotypes with similar frequencies), as well as timing of the onset of its intraspecific diversity imply that C. dalmatina was represented with a large population through longer time, even in periods that for geographically closely located C. bilineata and C. jadovaensis represented unfavorable conditions and it is likely that the strong selective pressure did not affect this species. At contrary, based on MSP, we can conclude that C. dalmatina underwent a population growth.

Besides unconstrained evolutionary development, distribution on greater number of localities is probably a cause of high genetic diversity of C. narentana. In understanding the evolutionary course of C. narentana and defining conservation priorities, it was important to find out whether samples from different localities represent populations that are genetically distinct. The genetic differentiation analysis, however, revealed that Neretvanian populations are not genetically distinct. Due to smaller sample size, the power of genetic differentiation analysis is questionable, so results should be observed with caution. Nevertheless, they are corroborated by the fact that several haplotypes (of all three investigated genes) were found on 2–5 locations and that all C. narentana populations are interconnected with migration events. It is interesting, however, that immigration rates and migration directions are not the same based on mitochondrial and nuclear DNA. Such locus-specific differences in gene flow could be explained by divergent selection [35] or smaller average coalescence time and maternal inheritance of mtDNA [36], but they could also be due to possible low power of performed analyses Even if the migration rates estimated in this investigation are not completely precise, Cobitis narentana is an excellent example of the importance of detecting such small modifications in intraspecific structure for adequate protection of a species. Namely, without knowledge on rate and direction of migrations, it would be reasonable to propose populations from Norin and Modro oko as conservation units with an argument that they contain the highest portion of the genetic diversity of the whole species and, thereafter, will most likely ensure its survival during modified conditions. However, detailed inspection of species structure implies that the Norin population is in fact a sink population receiving immigrants from three different populations (which probably caused its higher intrapopulational diversity). Protection of sink population is only effective through protection of source populations. Better candidates for higher protection regime are populations from Modro oko and Mislina. Namely, those populations also contain high level of genetic polymorphism and great number of haplotypes, but are sources of immigrants. Even though our data corroborate stability of C. narentana, reduction of size and diversity of Modro oko and Mislina populations, and especially their disappearance, would probably have negative effect on all other populations and significantly reduce probability of survival of the whole species, even if other populations remain undisturbed. Nevertheless, the most efficient conservation strategy for ensuring C. narentana survival would include conservation of the whole metapopulation, especially since we have estimated gene flow rates based on one mitochondrial and two nuclear genes. Including more genes into further investigations would enable more accurate estimations of the gene flow and intraspecific structure, but also finding finer scale intragenomic differences.

A completely different situation was recorded for C. illyrica. Contrary to Cetina and Neretva species, the high level of intraspecific genetic diversity of C. illyrica is primarily consequence of its splitting into three geographically distinct populations: Prološko blato, Krenica and Matica with Baćinska lakes. Based on the genetic differentiation analysis, those three populations are indeed genetically different. Furthermore, with the exception of one haplotype that was found in two populations, each population has its own cyt b haplotypes. Two hypotheses could explain structure of C. illyrica: simultaneous colonization of localities from a single population and their subsequent isolation, or fragmentation of historical population. In any case, historical connections that allowed migrations among populations are likely to have existed. Estimation of migration rates based on nuclear genes noticed connections among all three C. illyrica populations, as well as immigrations from Mostarsko blato. On the other hand, estimation based on mitochondrial DNA found restricted immigrations only from Krenica to Prološko blato population and absence of connections among any other populations. Contrary to situation with C. narentana, in order to ensure undisturbed evolutionary development of C. illyrica it is necessary to preserve all three populations as different entities with possibly independent future evolution. Cobitis illyrica is just another example that present taxonomy does not completely reflect biological diversity and that species are units restricted in space, but also in time.

Based on the definition of Evolutionary Significant Unit (ESU) of Fraser and Bernatchez [37], that ESU is a lineage demonstrating highly restricted gene flow from other such lineages within the higher organizational level of the species, three populations of C. illyrica should be defined and protected as ESUs, but among C. narentana populations we could not select ESUs. The same is with Management Units (MUs; defined by Waples [38] and Moritz [39] as populations connected by little or no contemporary gene flow, but not separated historically for very long periods of time), as well as other defined conservation units. Nevertheless, due to limited resources for species comprised out of more than one population it is important to decide whether some populations are more important for future survival of the whole species. Thereafter, for conservation purposes we find more adequate the original definition of Ryder [40], that ESU is a subset of more inclusive entity species, which possess genetic attributes significant for the present and future generation of the species in question. Units below species level that are of exceptional importance for future evolution and survival of the species in question, that may or may not be genetically different from other units (lineages, populations, subpopulations) should be recognized as conservation priorities. On the example of C. narentana it is clear that two populations actually represent “the important building blocks within the species” whose maintenance is necessary to keep the process of evolution not excessively constrained [41], even though they are not genetically distinct nor substantially reproductively isolated from other conspecific populations.

Conservation Priorities

Threats that can lead to an extinction of a certain species can be divided into two categories: anthropologic impacts (habitat loss and fragmentation, pollution, over-exploitation, introductions and translocations) and stochastic events associated with small population size [42]. Two types of extinction threats often interact causing an extinction vortex [43]. Considering future survival of the investigated spined loaches, it is fortunate that two types of extinction threats are not (yet) interacting in none of the species, e.g. species with small population sizes are under lesser human impact (C. jadovaensis, C. herzegoviniensis), whereas anthropologic impact is intense on species with greater population sizes (C. bilineata, C. dalmatina and C. narentana). Frankham et al. [23] concluded that long-term viability of populations with N_e <1000 is reduced because of the erosion of their ability to cope with environmental changes. Besides to C. jadovaensis, we believe that special conservation attention should be devoted to C. herzegoviniensis (given its extremely small distribution range, small genetic diversity, estimated effective population size close to 1000, and planned anthropological activities on its locality), but also to Prološko blato population. Even though C. illyrica does not seem to be endangered based on genetic criteria (estimated census size of the whole species between 25020 and 50040, BSP implying demographic growth), our results reveal separate evolutionary course of three populations comprised under that species. Effective population size of Prološko blato population is also close to 1000 pointing out greater risk of its long term survival. Even though maximum estimations of the effective population sizes of C. herzegoviniensis and C. illyrica in Prološko blato (profile likelihood at 95^th percentile) are larger (1931 and 2069, respectively) we still find it justified to request for special conservation attention because even those highest estimations, combined with small genetic diversity (especially in C. herzegoviniensis) do not guaranty population survival in case of planned anthropologic impact. Zrmanja population of C. bilineata also fulfils criteria to be recognized as an ESU. Due to its very small intrapopulational genetic polymorphism it should receive immediate and effective protection, even though whole species is considered LC and extrapolated census population size of Zrmanja population is higher than 10000.

Conclusions and Recommendations

The IUCN Red List of Threatened species, produced by the Species Survival Commission (SSC) of the World Conservation Union (IUCN; http://www.iucn.org), is considered as the most comprehensive resource detailing the global conservation status of plants and animals [2] and main initiator for many national and regional conservation actions [44], [45]. On the other hand, several authors have questioned its true value in understanding patterns of, and threats to, biodiversity [2], [46–48]. Our results confirm that molecular studies describing intraspecific genetic structure, as well as patterns of gene flow and evolutionary history of a species are fundamental to effective conservation efforts [49].

Considering that population mean genetic diversity is usually positively correlated with population size, but also with mean fitness [50], it is obvious that species with lower genetic diversity have less chance of future survival not only due to smaller effective population size, but also because of smaller capacity to adopt to changed environmental conditions. In that respect, C. jadovaensis seems to be in the worst position among all Adriatic spined loaches.

Our results corroborate usefulness of the effective population size, intraspecific and intrapopulational polymorphism measures and migration rates estimations for extinction risk assessments and conservation prioritizations. Population genetic measurements are able to detect differences among closely located and related species and estimate extinction risk even more accurately than current IUCN criteria. Furthermore, those measures can be obtained much faster than demographic parameters and with minimal population disturbance (only small tissue samples are needed), which both can be very valuable for small, endemic, yet endangered species. We believe that implementation of values of effective population size should be included into the IUCN criterion C, in order to avoid questionable extrapolations from effective to census population sizes [23]. That does not mean complete withdrawal of the census population size values, since they can be very useful when they are obtained either through demographic investigations or information on N_e/N ratio is available for taxonomic group in question. However, since the mentioned ratio differs greatly among taxa due to differences in demography and life history, effective population size could be used just as effectively, but more correctly for extinction risk assessments using criterion C. For example, if N_e of C. herzegoviniensis would be extrapolated into N using 0.1 value, number of mature individuals would fit it into VU category. However, if we use maximal default value of 0.2 [23], the same effective population size would fall out of the endangerment criteria. The possibility of mistake is even greater if N_e/N of spined loaches falls out of the currently proposed values, since estimates are available for only about 100 species [23]. Furthermore, our results speak in favor of inclusion of even more population genetic data in extinction risk assessments–since intrapopulational polymorphism is positively correlated with mean fitness, extinction risk could be estimated based on polymorphism measures also and we find justified to include them into the IUCN Red List categorization system, probably as an additional criterion. The objection that lower genetic diversity does not necessarily equate to elevation risk because it is usually the symptom of endangerment and not its cause [50], [51] does not reduce its possible usefulness in the estimation of the extinction risk for which it is a “symptom”. Furthermore, our example of the Adriatic spined loaches demonstrates that small genetic diversity may indeed be the cause of greater extinction risk. Namely, same anthropologic action will probably have more significant impact on C. jadovaensis and C. herzegoviniensis than on C. dalmatina and C. narentana precisely because of their smaller intraspecific diversity which, on the other hand, was not induced by demographic collapse, but by evolutionary history. Supplementary population genetic information (in particular information on intraspecific genetic structure and possible migration rates and directions) are also extremely useful for defining effective conservation measures and ensuring best possibility of survival for endangered species using financial resources in the most reasonable manner.