Evolution in fossil time series reconciles observations in micro- and macroevolution.

Extrapolating microevolutionary models does not always provide satisfactory explanations for phenotypic diversification on million-year time scales. For example, short-term evolutionary change is often modeled assuming a fixed adaptive landscape, but macroevolutionary changes are likely to involve changes in the adaptive landscape itself. A better understanding of how the adaptive landscape changes across different time intervals and how these changes cause populations to evolve has the potential to narrow the gap between micro- and macroevolution. Here, we analyze two fossil diatom time series of exceptional quality and resolution covering time intervals of a few hundred thousand years using models that account for different behaviors of the adaptive landscape. We find that one of the lineages evolves on a randomly and continuously changing landscape, whereas the other lineage evolves on a landscape that shows a rapid shift in the position of the adaptive peak of a magnitude that is typically associated with species-level differentiation. This suggests phenotypic evolution beyond generational timescales may be a consequence of both gradual and sudden repositioning of adaptive peaks. Both lineages are showing rapid and erratic evolutionary change and are constantly readapting towards the optimal trait state, observations that align with evolutionary dynamics commonly observed in contemporary populations. The inferred trait evolution over a span of a few hundred thousand years in these two lineages is therefore chimeric in the sense that it combines components of trait evolution typically observed on both short and long timescales.

Phylogenomic analyses reveal incongruences between divergence times and fossil records of freshwater snails in East Asia.

Fossils provide important insight into our understanding of phylogenetic history by serving as calibration points for divergence time estimation. However, uncertainties in the fossil record due to parallel evolution and convergent evolution can critically affect estimates of node ages. Here, we compare and contrast estimates of phylogenetic divergence with geologic and fossil history for two freshwater snail genera of the family Viviparidae in East Asia (Cipangopaludina and Margarya). Cipangopaludina species are commonly widely distributed species in East Asia, but extant Margarya species are endemic to the ancient lakes in Yunnan, China. According to some previous studies, parallel evolution or convergent evolution of shell morphology has occurred in the family several times which may affect divergence time estimation using fossil records. In this study, we used SNP data derived from ddRAD-seq loci to investigate population demographic history of both genera. Our results show a common pattern of lake endemic lineages diversifying from widely distributed lineages in the Miocene, and multiple colonization to a single ancient lake occurred in the Pleistocene. Our results indicate substantial incongruence among estimated phylogenomic divergence times, some fossil records, and formation ages of ancient lakes. These findings suggest some fossil records may be misidentified in these groups and highlight the need to carefully evaluate geological evidence and fossil records when using these for divergence time estimation.

Experimental Crossing Confirms Reproductive Isolation between Cryptic Species within Eulimnogammarus verrucosus (Crustacea: Amphipoda) from Lake Baikal.

Ancient lakes are known speciation hotspots. One of the most speciose groups in the ancient Lake Baikal are gammaroid amphipods (Crustacea: Amphipoda: Gammaroidea). There are over 350 morphological species and subspecies of amphipods in Baikal, but the extent of cryptic variation is still unclear. One of the most common species in the littoral zone of the lake, Eulimnogammarus verrucosus (Gerstfeldt, 1858), was recently found to comprise at least three (pseudo)cryptic species based on molecular data. Here, we further explored these species by analyzing their mitogenome-based phylogeny, genome sizes with flow cytometry, and their reproductive compatibility. We found divergent times of millions of years and different genome sizes in the three species (6.1, 6.9 and 8 pg), further confirming their genetic separation. Experimental crossing of the western and southern species, which are morphologically indistinguishable and have adjacent ranges, showed their separation with a post-zygotic reproductive barrier, as hybrid embryos stopped developing roughly at the onset of gastrulation. Thus, the previously applied barcoding approach effectively indicated the separate biological species within E. verrucosus. These results provide new data for investigating genome evolution and highlight the need for precise tracking of the sample origin in any studies in this morphospecies.

Species Radiations in the Sea: What the Flock?

Species flocks are proliferations of closely-related species, usually after colonization of depauperate habitat. These radiations are abundant on oceanic islands and in ancient freshwater lakes, but rare in marine habitats. This contrast is well documented in the Hawaiian Archipelago, where terrestrial examples include the speciose silverswords (sunflower family Asteraceae), Drosophila fruit flies, and honeycreepers (passerine birds), all derived from one or a few ancestral lineages. The marine fauna of Hawai'i is also the product of rare colonization events, but these colonizations usually yield only one species. Dispersal ability is key to understanding this evolutionary inequity. While terrestrial fauna rarely colonize between oceanic islands, marine fauna with pelagic larvae can make this leap in every generation. An informative exception is the marine fauna that lack a pelagic larval stage. These low-dispersal species emulate a "terrestrial" mode of reproduction (brooding, viviparity, crawl-away larvae), yielding marine species flocks in scattered locations around the world. Elsewhere, aquatic species flocks are concentrated in specific geographic settings, including the ancient lakes of Baikal (Siberia) and Tanganyika (eastern Africa), and Antarctica. These locations host multiple species flocks across a broad taxonomic spectrum, indicating a unifying evolutionary phenomenon. Hence marine species flocks can be singular cases that arise due to restricted dispersal or other intrinsic features, or they can be geographically clustered, promoted by extrinsic ecological circumstances. Here, we review and contrast intrinsic cases of species flocks in individual taxa, and extrinsic cases of geological/ecological opportunity, to elucidate the processes of species radiations.

The Hyalella (Crustacea: Amphipoda) species cloud of the ancient Lake Titicaca originated from multiple colonizations.

Ancient lakes are renowned for their exceptional diversity of endemic species. As model systems for the study of sympatric speciation, it is necessary to understand whether a given hypothesized species flock is of monophyletic or polyphyletic origin. Here, we present the first molecular characterization of the Hyalella (Crustacea: Amphipoda) species complex of Lake Titicaca, using COI and 28S DNA sequences, including samples from the connected Small and Large Lakes that comprise Lake Titicaca as well as from a broader survey of southern South American sites. At least five evolutionarily distant lineages are present within Lake Titicaca, which were estimated to have diverged from one another 12-20 MYA. These major lineages are dispersed throughout the broader South American Hyalella phylogeny, with each lineage representing at least one independent colonization of the lake. Moreover, complex genetic relationships are revealed between Lake Titicaca individuals and those from surrounding water bodies, which may be explained by repeated dispersal into and out of the lake, combined with parallel intralacustrine diversification within two separate clades. Although further work in deeper waters will be required to determine the number of species present and modes of diversification, our results strongly indicate that this amphipod species cloud is polyphyletic with a complex geographic history.

Diatoms do radiate: evidence for a freshwater species flock.

Due to the ubiquity and high dispersal capacity of unicellular eukaryotes, their often extraordinary diversity found in isolated and long-lived ecosystems such as ancient lakes is typically attributed to multiple colonization events rather than to in situ speciation. However, respective evolutionary studies are very scarce and the often high number of species flocks in ancient lakes across multicellular taxa raises the question whether unicellular species, such as diatoms, may radiate as well. Here, we use an integrative approach that includes molecular data from benthic diatom species of the genus Aneumastus endemic to ancient Lake Ohrid, fossil data obtained from the sediment record of a recent deep-drilling project and biogeographical information to test if this group, indeed, constitutes a species flock. Molecular-clock and phylogenetic analyses indicate a young monophyletic group of several endemic species. Molecular, fossil and biogeographical data strongly suggest a rapid intralacustrine diversification, which was possibly triggered by the emergence of novel habitats. This finding is the first evidence for a species flock in diatoms and suggests that in situ speciation is also a relevant evolutionary process for unicellular eukaryotes in isolated ecosystems.

Transcriptome-based phylogeny of endemic Lake Baikal amphipod species flock: fast speciation accompanied by frequent episodes of positive selection.

Endemic species flocks inhabiting ancient lakes, oceanic islands and other long-lived isolated habitats are often interpreted as adaptive radiations. Yet molecular evidence for directional selection during species flocks radiation is scarce. Using partial transcriptomes of 64 species of Lake Baikal (Siberia, Russia) endemic amphipods and two nonendemic outgroups, we report a revised phylogeny of this species flock and analyse evidence for positive selection within the endemic lineages. We confirm two independent invasions of amphipods into Baikal and demonstrate that several morphological features of Baikal amphipods, such as body armour and reduction in appendages and sensory organs, evolved in several lineages in parallel. Radiation of Baikal amphipods has been characterized by short phylogenetic branches and frequent episodes of positive selection which tended to be more frequent in the early phase of the second invasion of amphipods into Baikal when the most intensive diversification occurred. Notably, signatures of positive selection are frequent in genes encoding mitochondrial membrane proteins with electron transfer chain and ATP synthesis functionality. In particular, subunits of both the membrane and substrate-level ATP synthases show evidence of positive selection in the plankton species Macrohectopus branickii, possibly indicating adaptation to active plankton lifestyle and to survival under conditions of low temperature and high hydrostatic pressures known to affect membranes functioning. Other functional categories represented among genes likely to be under positive selection include Ca-binding muscle-related proteins, possibly indicating adaptation to Ca-deficient low mineralization Baikal waters.

Nuclear DNA content correlates with depth, body size, and diversification rate in amphipod crustaceans from ancient Lake Baikal, Russia.

Lake Baikal in Russia is a large, ancient lake that has been the site of a major radiation of amphipod crustaceans. Nearly 400 named species are known in this single lake, and it is thought that many more await description. The size and depth of Lake Baikal, in particular, may have contributed to the radiation of endemic amphipods by providing a large number of microhabitats for species to invade and subsequently experience reproductive isolation. Here we investigate the possibility that large-scale genomic changes have also accompanied diversification in these crustaceans. Specifically, we report genome size estimates for 36 species of Baikal amphipods, and examine the relationship between genome size, body size, and the maximum depths at which the amphipods are found in the lake. Genome sizes ranged nearly 8-fold in this sample of amphipod species, from 2.15 to 16.63 pg, and there were significant, positive, phylogenetically corrected relationships between genome size, body size, maximum depth, and diversification rate among these species. Our results suggest that major genomic changes, including transposable element proliferation, have accompanied speciation that was driven by selection for differences in body size and habitat preference in Lake Baikal amphipods.

Origin and diversification of Lake Ohrid's endemic acroloxid limpets: the role of geography and ecology.

BACKGROUND: Ancient Lake Ohrid, located on the Albania-Macedonia border, is the most biodiverse freshwater lake in Europe. However, the processes that gave rise to its extraordinary endemic biodiversity, particularly in the species-rich gastropods, are still poorly understood. A suitable model taxon to study speciation processes in Lake Ohrid is the pulmonate snail genus Acroloxus, which comprises two morphologically distinct and ecologically (vertically) separated endemic species. Using a multilocus phylogenetic framework of Acroloxus limpets from the Euro-Mediterranean subregion, together with molecular-clock and phylogeographic analyses of Ohrid taxa, we aimed to infer their geographic origin and the timing of colonization as well as the role of geography and ecology in intra-lacustrine diversification. RESULTS: In contrast to most other endemic invertebrate groups in Lake Ohrid, the phylogenetic relationships of the endemic Ohrid Acroloxus species indicate that the Balkan region probably did not serve as their ancestral area. The inferred monophyly and estimated divergence times further suggest that these freshwater limpets colonized the lake only once and that the onset of intra-lacustrine diversification coincides with the time when the lake reached deep-water conditions ca 1.3 Mya. However, the difference in vertical distribution of these two ecologically distinct species is not reflected in the phylogeographic pattern observed. Instead, western and eastern populations are genetically more distinct, suggesting a horizontal structure. CONCLUSIONS: We conclude that both geography and ecology have played a role in the intra-lacustrine speciation process. Given the distinct morphology (sculptured vs. smooth shell) and ecology (littoral vs. sublittoral), and the timing of intra-lacustrine diversification inferred, we propose that the onset of deep-water conditions initially triggered ecological speciation. Subsequent geographic processes then gave rise to the phylogeographic patterns observed today. However, the generally weak genetic differentiation observed suggests incipient speciation, which might be explained by the comparatively young age of the lake system and thus the relatively recent onset of intra-lacustrine diversification.

Origin and intra-island diversification of Sulawesi endemic Adrianichthyidae.

Although the family Adrianichthyidae is broadly distributed throughout East and Southeast Asia, 19 endemic species, over half of the family, are distributed in Sulawesi, which is an island in Wallacea. However, it remains unclear how this Adrianichthyidae biodiversity hotspot was shaped. In this study, we reconstructed molecular phylogenies for the Sulawesi adrianichthyids and estimated the divergence times of major lineages to infer the detailed history of their origin and subsequent intra-island diversification. The mitochondrial and nuclear phylogenies revealed that Sulawesi adrianichthyids are monophyletic, which indicates that they diverged from a single common ancestor. Species in the earliest branching lineages are currently distributed in the central and southeastern parts of the island, indicating that the common ancestor colonized Sula Spur, which is a large promontory that projects from the Australian continental margin, from Asia by oversea dispersal c.a. 20Mya. The first diversification event on Sulawesi, the split of the genus Adrianichthys, occurred c.a. 16Mya, and resulted in the nesting of Adrianichthys within Oryzias. Strong geographic structure was evident in the phylogeny; many species in the lineages branching off early are riverine and widely distributed in the southeastern and southwestern arms of Sulawesi, which suggests that oversea dispersal between tectonic subdivisions of this island during the late Miocene (7-5Mya) contributed to the distributions and diversification of the early branching lineages. In contrast, most species in the lineages branched off later are endemic to a single tectonic lake or lake system in the central Sulawesi, suggesting that habitat fragmentation due to the Pliocene collisions (c.a. 4Mya) among the tectonic subdivisions was the primary factor for diversification of the late branching, lacustrine lineages. Adrianichthys and some Oryzias in a certain late branching lineage are sympatric in Lake Poso, which indicates multiple colonizations of these distinct lineages into this tectonic lake. Thus, the diversification of Sulawesi adrianichthyids largely reflects the complex geological history of this island.

Cryptic diversity and speciation in an endemic copepod crustacean Harpacticella inopinata within Lake Baikal.

Ancient lakes are hotspots of species diversity, posing challenges and opportunities for exploration of the dynamics of endemic diversification. Lake Baikal in Siberia, the oldest lake in the world, hosts a particularly rich crustacean fauna, including the largest known species flock of harpacticoid copepods with some 70 species. Here, we focused on exploring the diversity and evolution within a single nominal species, Harpacticella inopinata Sars, 1908, using molecular markers (mitochondrial COI, nuclear ITS1 and 28S rRNA) and a set of qualitative and quantitative morphological traits. Five major mitochondrial lineages were recognized, with model-corrected COI distances of 0.20-0.37. A concordant pattern was seen in the nuclear data set, and qualitative morphological traits also distinguish a part of the lineages. All this suggests the presence of several hitherto unrecognized cryptic taxa within the baikalian H. inopinata, with long independent histories. The abundances, distributions and inferred demographic histories were different among taxa. Two taxa, H. inopinata CE and H. inopinata CW, were widespread on the eastern and western coasts, respectively, and were largely allopatric. Patterns in mitochondrial variation, that is, shallow star-like haplotype networks, suggest these taxa have spread through the lake relatively recently. Three other taxa, H. inopinata RE, RW and RW2, instead were rare and had more localized distributions on either coast, but showed deeper intraspecies genealogies, suggesting older regional presence. The rare taxa were often found in sympatry with the others and occasionally introgressed by mtDNA from the common ones. The mitochondrial divergence between and within the H. inopinata lineages is still unexpectedly deep, suggesting an unusually high molecular rate. The recognition of true systematic diversity in the evaluation and management of ecosystems is important in hotspots, as it is everywhere else, while the translation of the diversity into a formal taxonomy remains a challenge.

Mitogenomes do not substantially improve phylogenetic resolution in a young non-model adaptive radiation of freshwater gastropods.

BACKGROUND: Species flocks in ancient lakes, and particularly those arising from adaptive radiation, make up the bulk of overall taxonomic and morphological diversity in these insular ecosystems. For these mostly young species assemblages, classical mitochondrial barcoding markers have so far been key to disentangle interspecific relationships. However, with the rise and further development of next-generation sequencing (NGS) methods and mapping tools, genome-wide data have become an increasingly important source of information even for non-model groups. RESULTS: Here, we provide, for the first time, a comprehensive mitogenome dataset of freshwater gastropods endemic to Sulawesi and thus of an ancient lake invertebrate species flock in general. We applied low-coverage whole-genome sequencing for a total of 78 individuals including 27 out of the 28 Tylomelania morphospecies from the Malili lake system as well as selected representatives from Lake Poso and adjacent catchments. Our aim was to assess whether mitogenomes considerably contribute to the phylogenetic resolution within this young species flock. Interestingly, we identified a high number of variable and parsimony-informative sites across the other 'non-traditional' mitochondrial loci. However, although the overall support was very high, the topology obtained was largely congruent with previously published single-locus phylogenies. Several clades remained unresolved and a large number of species was recovered polyphyletic, indicative of both rapid diversification and mitochondrial introgression. CONCLUSIONS: This once again illustrates that, despite the higher number of characters available, mitogenomes behave like a single locus and thus can only make a limited contribution to resolving species boundaries, particularly when introgression events are involved.

The diversity of the antimicrobial resistome of lake Tanganyika increases with the water depth.

The presence of antimicrobial resistance genes (ARGs) in the microbiome of freshwater communities is a consequence of thousands of years of evolution but also of the pressure exerted by anthropogenic activities, with potential negative impact on environmental and human health. In this study, we investigated the distribution of ARGs in Lake Tanganyika (LT)'s water column to define the resistome of this ancient lake. Additionally, we compared the resistome of LT with that of Lake Baikal (LB), the oldest known lake with different environmental characteristics and a lower anthropogenic pollution than LT. We found that richness and abundance of several antimicrobial resistance classes were higher in the deep water layers in both lakes. LT Kigoma region, known for its higher anthropogenic pollution, showed a greater richness and number of ARG positive MAGs compared to Mahale. Our results provide a comprehensive understanding of the antimicrobial resistome of LT and underscore its importance as reservoir of antimicrobial resistance. In particular, the deepest water layers of LT are the main repository of diverse ARGs, mirroring what was observed in LB and in other aquatic ecosystems. These findings suggest that the deep waters might play a crucial role in the preservation of ARGs in aquatic ecosystems.

Diagnosis of the mechanisms of different types of discordances between phylogenies inferred from nuclear and mitochondrial markers.

In ancient freshwater lakes, an abnormally large species diversity is observed. The mechanisms that generated extremely high biodiversity in the ancient lakes have not been sufficiently studied and remain only partially known. Sequences of environmental changes in highly complex ecosystems such as Lake Baikal, may induce sophisticated combinations of microevolutionary processes. These processes are likely to result in unusual "patterns" of genetic variability of species. The most unusual patterns include the ones when speciation is followed by incomplete lineage sorting as well as mitochondrial or nuclear introgression. All these phenomena are diagnosed by comparing the topologies of phylogenetic trees inferred from molecular markers of evolution located in mitochondria and nuclei. Mitochondrial and nuclear introgression is a particularly interesting and complex case, which is the process of incorporating the gene alleles of one species into the gene pool of a sister species due to interspecific hybridization (introgressive hybridization). In many cases, existing methods for molecular phylogenetic analysis do not automatically allow the observed patterns of polymorphism to be explained and, therefore, cannot provide hypotheses that would explain the mechanisms which resulted to these patterns. Here we use adaptive dynamics models to study neutral molecular evolution under various scenarios of interaction between sister species and the environment. We propose and justify a set of criteria for detecting how two evolutionary trees may differ, with a special focus on comparing a tree inferred from nuclear DNA to one from mitochondrial DNA. The criteria react to branching pattern and branch lengths, including relative distances from ancestral lineages. Simulations show that the criteria allow fast and automated detection of various types of introgression, secondary breaches of reproductive barriers, and incomplete lineage sorting.

Old lake versus young taxa: a comparative phylogeographic perspective on the evolution of Caspian Sea gastropods (Neritidae: Theodoxus).

The Caspian Sea has been a highly dynamic environment throughout the Quaternary and witnessed major oscillations in lake level, which were associated with changes in salinity and habitat availability. Such environmental pressures are considered to drive strong phylogeographic structures in species by forcing populations into suitable refugia. However, little is actually known on the effect of lake-level fluctuations in the Caspian Sea on its aquatic biota. We compared the phylogeographic patterns of the aquatic Neritidae snail genus Theodoxus across the Pontocaspian region with refugial populations in southern Iran. Three gene fragments were used to determine relationships and divergence times between the sampled populations in both groups. A dated phylogeny and statistical haplotype networks were generated in conjunction with the analyses of molecular variance and calculations of isolation by distance using distance-based redundancy analyses. Extended Bayesian skyline plots were constructed to assess demographic history. Compared with the southern Iranian populations, we found little phylogeographic structure for the Pontocaspian Theodoxus group, with more recent diversification, homogeneity of haplotypes across the Pontocaspian region and a relatively stable demographic history since the Middle Pleistocene. Our results argue against a strong influence of Caspian Sea low stands on the population structure post the early Pleistocene, whereas high stands may have increased the dispersal possibilities and homogenization of haplotypes across the Pontocaspian region during this time. However, during the early Pleistocene, a more dramatic low stand in the Caspian Sea, around a million years ago, may have caused the reduction in Theodoxus diversity to a single lineage in the region. In addition, our results provide new insights into Theodoxus taxonomy and outlooks for regional conservation.

Recent lake expansion triggered the adaptive radiation of freshwater snails in the ancient Lake Biwa.

Lake expansion that leads to the formation of new habitats has potential to drive intralacustrine diversification. The ancient Lake Biwa in central Japan has historically experienced substantial changes in the lake size, and it provides a useful system for evaluating the role of lake-size fluctuations in the diversification of endemic fauna. Here, we used genome-wide DNA analyses and reconstructed the diversification history of the endemic freshwater snails belonging to the subgenus Biwamelania with respect to the geological history of Lake Biwa. We found that two genetically distinct snail lineages independently colonized Lake Biwa and they concurrently and rapidly radiated into 15 extant Biwamelania species. A combination of paleontological evidence and molecular dating technique demonstrated that the radiation of Biwamelania was tightly linked to the latest enlargement of the lake about 0.4 million years ago and suggested that increased ecological opportunity associated with the lake expansion drove the rapid adaptive radiation. We propose that the Biwamelania snails in Lake Biwa offer a promising new system for understanding the association between the geological history of the lake and rapid intralacustrine diversification.

Phylogenetic position and age of Lake Baikal candonids (Crustacea, Ostracoda) inferred from multigene sequence analyzes and molecular dating.

With 104 endemic species family Candonidae is one of the most diverse crustacean groups in Lake Baikal, yet their phylogenetic relationships and position in the family have not been addressed so far. Here, we study the phylogenetic position of Baikal candonids within the family and their evolutionary history using molecular markers for the first time since their original description. We choose 10 Baikal and 28 species from around the world, and three ribosomal RNA-s (18S, 28S, and 16S), and analyze individual and concatenated datasets using Bayesian Inference in MrBayes and BEAST. For molecular divergence time estimates, four fossil records are used to calibrate the root and three internal nodes. The 28S dataset is tested under the strict molecular clock, while for other data we use relaxed clocks. Resulting trees show incongruence between molecular and fossil divergence time estimates, with the former suggesting older ages. Strict molecular clock analysis results in narrower node age confidence intervals and younger time estimates than other analysis. All trees support at least two candonid lineages in Baikal, with two independent colonization events, and 28S suggests a major radiation between 12 and 5 Mya. This divergence time estimate mostly agrees with another, unrelated, ostracod group in the lake and other lake animals as well. Baikal candonid clades show a close phylogenetic relationship with Palearctic lineages, but their deep divergence is indicative of separate genera. Results also suggest a monophyly of tribes that today live exclusively in subterranean waters, and we offer several hypotheses of their evolutionary history.

Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod.

BACKGROUND: The Balkans are a major worldwide biodiversity and endemism hotspot. Among the freshwater biota, amphipods are known for their high cryptic diversity. However, little is known about the temporal and paleogeographic aspects of their evolutionary history. We used paleogeography as a framework for understanding the onset of diversification in Gammarus roeselii: (1) we hypothesised that, given the high number of isolated waterbodies in the Balkans, the species is characterised by high level of cryptic diversity, even on a local scale; (2) the long geological history of the region might promote pre-Pleistocene divergence between lineages; (3) given that G. roeselii thrives both in lakes and rivers, its evolutionary history could be linked to the Balkan Neogene paleolake system; (4) we inspected whether the Pleistocene decline of hydrological networks could have any impact on the diversification of G. roeselii. MATERIAL AND METHODS: DNA was extracted from 177 individuals collected from 26 sites all over Balkans. All individuals were amplified for ca. 650 bp long fragment of the mtDNA cytochrome oxidase subunit I (COI). After defining molecular operational taxonomic units (MOTU) based on COI, 50 individuals were amplified for ca. 900 bp long fragment of the nuclear 28S rDNA. Molecular diversity, divergence, differentiation and historical demography based on COI sequences were estimated for each MOTU. The relative frequency, geographic distribution and molecular divergence between COI haplotypes were presented as a median-joining network. COI was used also to reconstruct time-calibrated phylogeny with Bayesian inference. Probabilities of ancestors' occurrence in riverine or lacustrine habitats, as well their possible geographic locations, were estimated with the Bayesian method. A Neighbour Joining tree was constructed to illustrate the phylogenetic relationships between 28S rDNA haplotypes. RESULTS: We revealed that G. roeselii includes at least 13 cryptic species or molecular operational taxonomic units (MOTUs), mostly of Miocene origin. A substantial Pleistocene diversification within-MOTUs was observed in several cases. We evidenced secondary contacts between very divergent MOTUs and introgression of nDNA. The Miocene ancestors could live in either lacustrine or riverine habitats yet their presumed geographic localisations overlapped with those of the Neogene lakes. Several extant riverine populations had Pleistocene lacustrine ancestors. DISCUSSION: Neogene divergence of lineages resulting in substantial cryptic diversity may be a common phenomenon in extant freshwater benthic crustaceans occupying areas that were not glaciated during the Pleistocene. Evolution of G. roeselii could be associated with gradual deterioration of the paleolakes. The within-MOTU diversification might be driven by fragmentation of river systems during the Pleistocene. Extant ancient lakes could serve as local microrefugia during that time.

Predictors of shell size in long-lived lake gastropods.

AIM: To investigate shell size variation among gastropod faunas of fossil and recent long-lived European lakes and discuss potential underlying processes. LOCATION: Twenty-three long-lived lakes of the Miocene to Recent of Europe. METHODS: Based on a dataset of 1412 species of both fossil and extant lacustrine gastropods, we assessed differences in shell size in terms of characteristics of the faunas (species richness, degree of endemism, differences in family composition) and the lakes (surface area, latitude and longitude of lake centroid, distance to closest neighbouring lake) using multiple and linear regression models. Because of a strong species-area relationship, we used resampling to determine whether any observed correlation is driven by that relationship. RESULTS: The regression models indicated size range expansion rather than unidirectional increase or decrease as the dominant pattern of size evolution. The multiple regression models for size range and maximum and minimum size were statistically significant, while the model with mean size was not. Individual contributions and linear regressions indicated species richness and lake surface area as best predictors for size changes. Resampling analysis revealed no significant effects of species richness on the observed patterns. The correlations are comparable across families of different size classes, suggesting a general pattern. MAIN CONCLUSIONS: Among the chosen variables, species richness and lake surface area are the most robust predictors of shell size in long-lived lake gastropods. Although the most outstanding and attractive examples for size evolution in lacustrine gastropods come from lakes with extensive durations, shell size appears to be independent of the duration of the lake as well as longevity of a species. The analogue of long-lived lakes as 'evolutionary islands' does not hold for developments of shell size because different sets of parameters predict size changes.

Speciation patterns and processes in the zooplankton of the ancient lakes of Sulawesi Island, Indonesia.

Although studies of ancient lake fauna have provided important insights about speciation patterns and processes of organisms in heterogeneous benthic environments, evolutionary forces responsible for speciation in the relatively homogenous planktonic environment remain largely unexplored. In this study, we investigate possible mechanisms of speciation in zooplankton using the freshwater diaptomids of the ancient lakes of Sulawesi, Indonesia, as a model system. We integrate phylogenetic and population genetic analyses of mitochondrial and nuclear genes with morphological and genome size data. Overall, our results support the conclusion that colonization order and local adaptation are dominant at the large, island scale, whereas at local and intralacustrine scales, speciation processes are regulated by gene flow among genetically differentiated and locally adapted populations. In the Malili lakes, the diaptomid populations are homogenous at nuclear loci, but show two highly divergent mitochondrial clades that are geographically restricted to single lakes despite the interconnectivity of the lake systems. Our study, based on coalescent simulations and population genetic analyses, indicates that unidirectional hybridization allows gene flow across the nuclear genome, but prevents the introgression of mitochondria into downstream populations. We suggest that hybridization and introgression between young lineages is a significant evolutionary force in freshwater plankton.