Historic Museum Samples Provide Evidence for a Recent Replacement of Wolbachia Types in European Drosophila melanogaster.

Wolbachia is one of the most common bacterial endosymbionts, which is frequently found in numerous arthropods and nematode taxa. Wolbachia infections can have a strong influence on the evolutionary dynamics of their hosts since these bacteria are reproductive manipulators that affect the fitness and life history of their host species for their own benefit. Host-symbiont interactions with Wolbachia are perhaps best studied in the model organism Drosophila melanogaster, which is naturally infected with at least 5 different variants among which wMel and wMelCS are the most frequent ones. Comparisons of infection types between natural flies and long-term lab stocks have previously indicated that wMelCS represents the ancestral type, which was only very recently replaced by the nowadays dominant wMel in most natural populations. In this study, we took advantage of recently sequenced museum specimens of D. melanogaster that have been collected 90 to 200 yr ago in Northern Europe to test this hypothesis. Our comparison to contemporary Wolbachia samples provides compelling support for the replacement hypothesis. Our analyses show that sequencing data from historic museum specimens and their bycatch are an emerging and unprecedented resource to address fundamental questions about evolutionary dynamics in host-symbiont interactions. However, we also identified contamination with DNA from crickets that resulted in co-contamination with cricket-specific Wolbachia in several samples. These results underpin the need for rigorous quality assessments of museomic data sets to account for contamination as a source of error that may strongly influence biological interpretations if it remains undetected.

An integrative approach highlights the discrepancy in the genetic, phenotypic, and presumptive taxonomic structure of Phoxinus (Actinopterygii, Leuciscidae, Phoxininae) in Bulgaria.

The present drainage network of Bulgaria is the result of a complex Neogene and Quaternary evolution. Karst, which has developed on 23% of the territory, further complicates the hydrological pattern. Fresh waters of Bulgaria drain into the Black Sea and the Aegean Sea basins and can be roughly divided into the Danube (Middle and Lower Danube), non-Danube Black Sea, East Aegean, and West Aegean hydrological regions. Phoxinus, a small leuciscid fish, has a mosaic distribution in all four of these regions, inhabiting small mountainous and semi-mountainous streams. Based on morphology, it was identified as three species, Phoxinus phoxinus in the Danube, Phoxinus strandjae in the non-Danube, and Phoxinus strymonicus in West Aegean region. Later, molecular data revealed Phoxinus csikii and Phoxinus lumaireul in the Middle Danube and P. csikii in the Lower Danube. Phoxinus has been the focus of many studies, showing a high molecular and morphological diversity, which is not entirely consistent with previous morphology-only-based taxonomic concepts. In this study, molecular (a mitochondrial marker and a nuclear marker) and morphological data from both historical and recently sampled collections were analysed to assess the applicability of the integrative approach in Phoxinus. The results showed a significant influence of the complex paleo- and recent hydrology on the currently observed genetic structure of the considered populations and species. Furthermore, the study also demonstrated a strong influence of phenotypic plasticity on the morphological analysis of Phoxinus and the lack of a clear differentiation between P. csikii and P. strandjae. A barcoded specimen was designated as neotype to fix the species named P. strandjae in the current taxonomic concept. Finally, a significant discordance between genetically delimited clades and phenotypic groups did not allow a proper delineation of the species distributed in Bulgaria, demonstrating that more molecular markers are needed for further taxonomic study of the Phoxinus complex.

Phylogeography of the land snail genus Orcula (Orculidae, Stylommatophora) with emphasis on the Eastern Alpine taxa: speciation, hybridization and morphological variation.

BACKGROUND: The Central and Southern European mountain ranges represent important biodiversity hotspots and show high levels of endemism. In the land snail genus Orcula Held, 1837 nine species are distributed in the Alps and a few taxa inhabit the Carpathians, the Dinarids and the Western Black Sea region. In order to elucidate the general patterns of temporal and geographic diversification, mitochondrial and nuclear markers were analyzed in all 13 Orcula species. We particularly aimed to clarify whether the Alpine taxa represent a monophyletic group and if the local species diversity is rather the result of isolation in geographically separated Pleistocene glacial refuges or earlier Tertiary and Quaternary palaeogeographic events. In order to test if patterns of molecular genetic and morphological differentiation were congruent and/or if hybridization had occurred, shell morphometric investigations were performed on the Orcula species endemic to the Alps. RESULTS: The phylogenetic trees resulting from the analyses of both the mitochondrial (COI, 12S and 16S) and the nuclear (H4/H3) data sets revealed three main groups, which correspond to the three subgenera Orcula, Illyriobanatica and Hausdorfia. The reconstruction of the historic geographic ranges suggested that the genus originated in the Dinarides during the Middle Miocene and first colonized the Alps during the Late Miocene, giving rise to the most diverse subgenus Orcula. Within the latter subgenus (including all Alpine endemics) almost all species were differentiated by both molecular genetic markers and by shell morphometrics, except O. gularis and O. pseudodolium. CONCLUSIONS: The present study confirms the importance of the Alps as biodiversity hotspot and origin center of land snail diversity. The species diversity in the subgenus Orcula was likely promoted by Miocene to Pliocene palaeogeographic events and the insular distribution of preferred limestone areas. In some cases, speciation events could be linked to the divergence of populations in glacial refuges during the Pleistocene. Sporadic contact between geographically separated and reproductively not yet isolated populations led to intermixture of haplogroups within species and even hybridization and mitochondrial capture between species.

Revision of the Garra species of the Hajar Mountains in Oman and the United Arab Emirates with the description of two new species (Teleostei: Cyprinidae).

The Garra species inhabiting the Hajar Mountains are revised and five species are recognised, two of which are newly species described herein. Garra barreimiae, from the United Arab Emirates (UAE) and Northern Oman, is distinguished from other Garra species in the Hajar Mountains by a strongly mottled flank pattern with individual or series of midlateral orange scales, a white dorsal-fin tip, an orange spot at the upper operculum, and more gill rakers on the lower limb of the first gill arch. Garra barreimiae shawkahensis is a synonym of G. barreimiae. Garra gallagheri, from the Wadi Bani Khalid drainage in Oman, is distinguished by flank-scale margins the same colour or slightly darker than the centre of the scales. Garra longipinnis, from the interior wadis in the Central Hajar Mountains in Oman, is distinguished by flank-scale margins being clearly darker than the centre of scales. The original description of G. longipinnis was based on a few individuals with very large fins, which are here considered aberrant. Garra shamal, new species, from the coastal drainages around Muscat in Oman, is distinguished by a strongly mottled flank pattern usually without orange midlateral scales, a white dorsal-fin tip, no orange spot at the upper operculum, and middle caudal-fin rays and membranes the same colour or slightly darker than the rest of the fins in colouration. Garra sharq, new species, from the Wadi Kabbah drainage and a few interior springs in Oman, is distinguished by a strongly mottled flank pattern with individual or series of orange midlateral scales, no orange spot at the upper opercle, and dorsal-fin tip and membranes between central caudal-fin rays the same colour as the rest of the fins. All five species are well differentiated genetically and form distinct mitochondrial clades with between 2.1 and 9.2% differences (p-distances) in the mitochondrial COI.

Investigating gene flow between the blind cavefish Garra barreimiae and its conspecific surface populations.

Cave-dwelling taxa often share the same phenotypic modifications like absence of eyes and pigmentation. These "troglomorphic characters" are expressed in the populations of Garra barreimiae from the Al Hoota Cave and nearby Hoti Pit in Northern Oman. Surface morphotypes of this cyprinid species are common throughout the distribution area. Very rarely individuals with intermediate phenotypes can be found. In the present study, potential gene flow between cave and surface populations was tested and population structure within five sampling sites was assessed. Overall, 213 individuals were genotyped at 18 microsatellite loci. We found that the cave populations have lower genetic diversity and are clearly isolated from the surface populations, which seem to be sporadically in contact with each other. The results indicate a recent genetic bottleneck in the cave populations. Thus, it can be assumed that during climatic changes the connection between cave and surface water bodies was disjoined, leaving a subpopulation trapped inside. Nevertheless, occasional gene flow between the morphotypes is detectable, but hybridisation seems only possible in cave habitat with permanent connection to surface water. Individuals from surface sites bearing intermediate phenotypes but cave genotypes imply that phenotypic plasticity might play a role in the development of the phenotype.

Characterization of 19 new microsatellite loci for the Omani barb Garra barreimiae from 454 sequences.

BACKGROUND: Garra barreimiae is a cyprinid fish from the southeastern Arabian Peninsula, which inhabits regularly desiccating wadis and survives in isolated ponds or underground. In 1984 a cave-dwelling population was found in the Al Hoota cave system and previous genetic analyses revealed some differentiation with limited gene flow between the surface populations and the cave population. Since no suitable markers are available for evaluation of gene flow between the cave population and the adjacent surface populations, we focused on designing and establishing novel microsatellite markers from next generation sequencing data. FINDINGS: 19 microsatellite markers containing di- and tetranucleotide simple sequence repeats were developed from 454 sequences. Forty-four individuals from two surface populations (Wadi Al Falahi and Misfat Al Abriyeen) of G. barreimiae (sampling permission number 13/2012, export permission number 29/2012) were used for analyses and characterization of the loci. On average, the number of alleles per locus is 7.6 (range: 2-20). Two markers displayed indication of linkage disequilibrium in both populations (DL6X, 9XNC). Significant deviation from Hardy-Weinberg equilibrium was observed at four loci in the Misfat Al Abriyeen population (2PUM, 88CM, 1EHE, 3Z7M) and at two loci in the Wadi Al Falahi population (QLIM, 3 N43). Three of the microsatellite loci were significant for null alleles in one of the two populations (Misfat Al Abriyeen: CJHG; Wadi Al Falahi: PH8A, 3ROZ). Expected and observed heterozygosities ranged from 0 to 95.0% respectively from 0 to 95.8% (Wadi Al Falahi) and from 0 to 89.1% respectively from 0 to 95.0% (Misfat Al Abriyeen). Fourteen of these markers were successfully cross-amplified in G. rufa. CONCLUSION: This 19 microsatellite loci provide a useful tool to understand the structure and genetic differences of populations. Moreover, these markers will help to evaluate species delimitation in G. barreimiae and potentially even in related species.

Paraphyly and budding speciation in the hairy snail (Pulmonata, Hygromiidae).

Delimitation of species is often complicated by discordance of morphological and genetic data. This may be caused by the existence of cryptic or polymorphic species. The latter case is particularly true for certain snail species showing an exceptionally high intraspecific genetic diversity. The present investigation deals with the Trochulus hispidus complex, which has a complicated taxonomy. Our analyses of the COI sequence revealed that individuals showing a T. hispidus phenotype are distributed in nine highly differentiated mitochondrial clades (showing p-distances up to 19%). The results of a parallel morphometric investigation did not reveal any differentiation between these clades, although the overall variability is quite high. The phylogenetic analyses based on 12S, 16S and COI sequences show that the T. hispidus complex is paraphyletic with respect to several other morphologically well-defined Trochulus species (T. clandestinus, T. villosus, T. villosulus and T. striolatus) which form well-supported monophyletic groups. The nc marker sequence (5.8S-ITS2-28S) shows only a clear separation of T. o. oreinos and T. o. scheerpeltzi, and a weakly supported separation of T. clandestinus, whereas all other species and the clades of the T. hispidus complex appear within one homogeneous group. The paraphyly of the T. hispidus complex reflects its complicated history, which was probably driven by geographic isolation in different glacial refugia and budding speciation. At our present state of knowledge, it cannot be excluded that several cryptic species are embedded within the T. hispidus complex. However, the lack of morphological differentiation of the T. hispidus mitochondrial clades does not provide any hints in this direction. Thus, we currently do not recommend any taxonomic changes. The results of the current investigation exemplify the limitations of barcoding attempts in highly diverse species such as T. hispidus.