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Makar Molchanov
Makar Molchanov

Bufo Viridis !NEW!


The European green toad (Bufotes viridis) is a species of toad found in steppes, mountainous areas, semi-deserts, urban areas and other habitats in mainland Europe, ranging from far eastern France and Denmark to the Balkans and Western Russia. As historically defined, the species ranged east through the Middle East and Central Asia to western China, Mongolia and northwestern India, and south through Italy and the Mediterranean islands to North Africa. Following genetic and morphological reviews, 14 population (all largely or entirely Asian, except for the African and Balearic green toads) are now regarded as separate species. These species and the European green toad are placed in their own genus Bufotes, but they were included in Bufo.[2][3]




bufo viridis


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Bufotes viridis eats a variety of insects and invertebrates, mainly crickets, meal worms, small butterflies, earthworms, moths, beetles, ants, spiders[5] and caterpillars. There has also been a reported attack on a bat.[6]


We discovered a new lineage, endemic to Naxos (Central Cyclades), while coastal islands and Crete feature weak genetic differentiation from the continent. In continental Greece, both lineages, viridis and variabilis, form a hybrid swarm, involving massive mitochondrial and nuclear admixture over hundreds of kilometers, without obvious selection against hybrids.


Second, the mainland, especially of northeastern Greece and neighboring Turkey, adjacent to the Aegean Sea, constitutes a suture zone between Anatolian and Balkan lineages [7, 15, 26, 27]. This is also the case in green toads, where the mitochondrial sister taxa viridis and variabilis came into secondary contact in continental Greece [16, 17]. Since these two lineages originated after the split with B. siculus and B. balearicus, an analysis of their hybrid zone provides a valuable third point to examine the relationship between hybridizability and divergence time and thus speciation in this anuran radiation.


To better understand the terrestrial Aegean phylogeography of closely related vertebrates as well as to elucidate the genetic interactions of the presumably young lineages viridis and variabilis on the mitochondrial and nuclear levels, we conducted a fine-scale multilocus phylogeography of European green toads across their Aegean range. Specifically, (1) we tested whether any endemic lineages are found on Aegean islands and if lineage distributions and divergences can be attributed to past sea level changes. (2) We documented patterns of introgression between the continental lineages of viridis and variabilis, and compared them to other green toad hybrid zones [6, 18], in order to infer the timeframe of reproductive isolation under natural conditions.


For all remaining 687 green toads, we mito-typed PCR amplicons of the control region, using a novel enzyme-restriction procedure, designed with the NEBcutter 2.0 tool (New England Biolabs), which allowed to differentiate mitotypes between these two species. This procedure included: (1) PCR amplification as above; (2) enzymatic restriction by NlaIII (New England Biolabs) for 1 h at 37 C in 9 μL reaction volumes, containing PCR amplicons (3 μL), NEB4 buffer (0.7), BSA (0.1) and NlaIII (1 unit); (3) migration of digested products on a 2% agarose gel for 1 h 30 min at 100 V; and (4) scoring of restriction patterns on the gel: the digested PCR products of viridis featured three bands of ca. 190, 320 and 390 bp, while those of variabilis featured three bands of 120, 330 and 450 bp respectively.


Third, we genotyped eight microsatellite loci, cross-amplifying and polymorphic in both lineages (viridis, variabilis), successfully obtained from 635 individuals from 110 localities. These included the markers Bcalμ10, C203, C218, D210, D106, C205, C223, D105 [29]. Markers were amplified in multiplex PCRs (details in Additional file 1: Table S1) and run on a ABI3130 genetic analyzer. Alleles were scored using Genemapper 4.0 (Applied Biosystems) and genotypes checked for null alleles using micro-checker [30].


Insular diversity of the control region was highly variable between islands (Table 1), exhibiting particularly high values for Crete, where both variabilis and viridis haplotypes coexist (resulting in high п), and the lowest values for Naxos and Lesvos (each with a single fixed haplotype).


We identified 14 haplotypes of the nuclear tropomyosin (alignment: 555 bp), with some geographic associations. The eastern parts of the study area (variabilis) featured highly differentiated sequences shared by individuals from across W-Anatolia, Chios, Lesvos and Ikaria (green, light blue, blue, dark blue, black, pink, Additional file 2: Figure S1). As for mtDNA, Naxos green toads possessed a single tropomyosin haplotype (orange, Additional file 2: Figure S1). Individuals from southern Greece harbored haplotypes closely-related to a previously published B. viridis sequence (Genbank EU497619). However these haplotypes were also found in the eastern Aegean (loc. 117, 119), probably resulting from incomplete lineage sorting with the polymorphic variabilis lineage (see Discussion).


The other major aim of our study was to compare the patterns of hybridization between the Eurasian mitochondrial sister lineages viridis and variabilis with those in other diploid/diploid green toad contact zones of older divergence. As discussed by [19], the taxonomic status of the mtDNA lineages viridis and variabilis, whose distribution also clusters geographically [16, 17, 19], has remained unclarified. As we show here, no major (if any) reproductive barriers seems to separate the two gene pools. The focal region exhibits massive mitochondrial and nuclear admixture over hundreds of kilometers. This wide admixture supports that viridis variabilis natural hybrids are not selected against, suggesting poor or complete absence of prezygotic and probably no post-zygotic incompatibilities between these two lineages.


In conclusion, reproductive isolation seems to have accumulated gradually in green toads and may mainly stem from the additive effects of weak hybrid incompatibilities spread over the genome, rather than few important speciation genes. The evolution of reproductive isolation involves multiple interacting barriers in a continuous framework [1]. In the Palearctic green toad radiation, different stages along this continuum have evolved, which thus provides unique insights into the timeframe when gene flow might either promote complete isolation (through the evolution of pre-mating barriers, i.e. reinforcement) or (re-)merge gene pools. In the latter case, while gene flow cancels the intrinsic identity of incipient species, it enables new genetic combinations and thus increases the adaptive potential of populations, which may in turn promote future speciation processes (e.g. ecological speciation). In green toads, this temporal window seems quite narrow: only a million years separates a scenario of near panmixia (lineages of viridis / variabilis, this study) from a situation of complete reproductive isolation, potentially driven by reinforcement (B. siculus / B. balearicus; [18]). Between these extremes, the B. balearicus / B. viridis contact provides a presumably intermediate stage [6], with preliminary indications for ecological and / or behavioral adaptations keeping lineages apart (our unpublished data). Beyond intrinsic genetic causes, environmental and demographic features could affect this timeframe. Reinforcement can only drive divergence if secondary contacts are prolonged enough to allow a selective response to post-zygotic isolation. In Europe, such contacts primarily include the Mediterranean regions, where populations persisted during the Quaternary glaciations, rather than post-glacially colonized more northern latitudes, where secondary contacts could only be initiated since the Holocene. Nevertheless, this should not have affected our comparisons as shown in Fig. 3, since all population pairs were analyzed in supposedly refugial areas. Moreover, all three analyses ([6, 18], this study) were also highly comparable given that the same mitochondrial and nuclear markers have been used, both for estimating (relative) divergence and natural introgression.


Our multilocus phylogeographic analyses of Aegean green toads allowed to test biogeographic and speciation hypotheses with several new findings. First, we detected a new cryptic endemic lineage in the central Aegean (Naxos), along with strong population structure between other islands, mediated by the depth-dependent Pleistocene sea level dynamics around the Archipelago, and presumably involving human-driven colonization(s) during antique times. Second, we show that the young lineages viridis and variabilis massively admix across their secondary contact zone in mainland Greece, presumably driven by an eastern invasion of variabilis, contrasting with the patterns of introgression between more deeply diverged green toad lineages in other range parts. In green toads, post-zygotic reproductive isolation appears mediated by the time spent in allopatry, upon which gene flow either promotes or cancels the speciation process. This radiation thus provides an increasingly valuable anuran model system for speciation research (including allopolyploids in Asia, [68]), as it illustrates different time spans of secondary contacts along the speciation continuum.


Large genome size, including immense repetitive and non-coding fractions, still present challenges for capacity, bioinformatics and thus affordability of whole genome sequencing in most amphibians. Here, we test the performance of a single transcriptome to understand whether it can provide a cost-efficient resource for species with large unknown genomes. Using RNA from six different tissues from a single Palearctic green toad (Bufo viridis) specimen and Hiseq2000, we obtained 22,5 Mio reads and publish >100,000 unigene sequences. To evaluate efficacy and quality, we first use this data to identify green toad specific candidate genes, known from other vertebrates for their role in sex determination and differentiation. Of a list of 37 genes, the transcriptome yielded 32 (87%), many of which providing the first such data for this non-model anuran species. However, for many of these genes, only fragments could be retrieved. In order to allow also applications to population genetics, we further used the transcriptome for the targeted development of 21 non-anonymous microsatellites and tested them in genetic families and backcrosses. Eleven markers were specifically developed to be located on the B. viridis sex chromosomes; for eight markers we can indeed demonstrate sex-specific transmission in genetic families. Depending on phylogenetic distance, several markers, which are sex-linked in green toads, show high cross-amplification success across the anuran phylogeny, involving nine systematic anuran families. Our data support the view that single transcriptome sequencing (based on multiple tissues) provides a reliable genomic resource and cost-efficient method for non-model amphibian species with large genome size and, despite limitations, should be considered as long as genome sequencing remains unaffordable for most species. 041b061a72


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