A new vertebrate species native to the British isles: Bufo spinosus Daudin, 1803 in Jersey

Recent molecular and morphological studies have shown that Bufo bufo and B. spinosus are genetically distinct and morphologically diagnosable across a relatively narrow contact zone in northern France and should be regarded as different species. However, the species identity of the neighbouring populations of Bufo on the British Channel Island of Jersey has not been investigated. We here present new molecular (a mtDNA RFLP assay plus sequences of the nuclear RAG1 gene) and morphological evidence that these populations are to be assigned to B. spinosus, and can thus be considered an addition to the native British herpetofauna. Jersey toad populations are declining and have a distinct breeding ecology compared to other populations in mainland Britain. We discuss the results in the light of amphibian conservation efforts in Jersey.Fieldwork and sampling in Jersey was conducted under licence from the States of Jersey Environment Department and with support from Amphibian and Reptile Conservation. Other funds were provided by grants CGL2008-04271-C02-01/BOS and CGL2011-28300 (Ministerio de Ciencia e Innovación, Ministerio de Economía y Competitividad, Spain, and FEDER) and PPII10-0097- 4200 (Junta de Comunidades de Castilla la Mancha and FEDER) to IMS, who is currently funded by Project “Biodiversity, Ecology and Global Change”, co-financed by North Portugal Regional Operational Programme 2007/2013 (ON.2–O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).Peer Reviewe

Morphological and Molecular Characterization of Orchid Fruit Development

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species Erycina pusilla and compare it to two terrestrial orchid species: Cynorkis fastigiata and Epipactis helleborine. Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While C. fastigiata and E. helleborine develop a lignified layer at the valve boundaries, E. pusilla fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes INDEHISCENT (IND)/HECATE 3 (HEC3), REPLUMLESS (RPL) and SPATULA (SPT)/ALCATRAZ (ALC) in E. pusilla, and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (AG) /SHATTERPROOF (SHP) orthologs can also be formed in E. pusilla, and that the expression of HEC3, RPL, and SPT can be associated with dehiscence zone development similar to Arabidopsis. Our expression analysis also indicates differences, however, which may underlie fruit divergence

A common toad hybrid zone that runs from the Atlantic to the Mediterranean

We document the distribution of the common toad Bufo bufo and the spined toad B. spinosus at their contact zone across France with data from a mitochondrial DNA RFLP assay, complementing similar work including nuclear markers in the northwest and southeast of France and in Italy. We also reconstruct geographical clines across the species’ contact zone in central France. Bufo bufo is found in the north-eastern half of France. Bufo spinosus is found in the south-western complement. The contact zone they form runs from the Atlantic coast near Caen, France, to the Mediterranean coast near Savona, Italy, and has a length of over 900 km. In central France B. bufo and B. spinosus engage in a hybrid zone with a unimodal genetic signature. Hybrid zone width is ca. 10 km at mitochondrial DNA and averages at 61 km for four nuclear loci. The hybrid zone is distinctly asymmetric with a signature of B. spinosus in B. bufo and not the other way round. We attribute this observation to B. bufo moving southwards at the expense of B. spinosus, with introgression in the direction of the advancing species. We noted substantial geographic variation in characters for species identification. Morphological species identification performs well in France, but breaks down in Italy. Mitochondrial DNA is inconclusive in south-eastern France and Italy. The nuclear genetic markers perform consistently well but have not yet been applied to the zone in full. Possible, but surely heterogeneous ecological correlates for the position of the hybrid zone are mountains and rivers.Peer Reviewe

Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (Bufo bufo and B. spinosus) in the northwest of France

Background: Hybrid zones are regions where individuals of two species meet and produce hybrid progeny, and are often regarded as natural laboratories to understand the process of species formation. Two microevolutionary processes can take place in hybrid zones, with opposing effects on population differentiation. Hybridization tends to produce genetic homogenization, reducing species differences, whereas the presence of mechanisms of reproductive isolation result in barriers to gene flow, maintaining or increasing differences between taxa. Results: Here we study a contact zone between two hybridizing toad species, Bufo bufo and B. spinosus, through a combination of molecular (12 polymorphic microsatellites, four nuclear and two mitochondrial SNP markers) and morphological data in a transect in the northwest of France. The results show largely concordant clines across markers, defining a narrow hybrid zone of ca. 30 km wide. Most hybrids in the centre of the contact zone are classified as F2 or backcrossed individuals, with no individuals assigned to the F1 hybrid class. Conclusions: We discuss the implications of these results for our understanding of the evolutionary history of these species. We anticipate that the toad contact zone here described will become an important asset in the study of hybrid zone dynamics and evolutionary biology because of its easy access and the abundance of the species involvedPeer reviewe

A common toad hybrid zone that runs from the Atlantic to the Mediterranean. Supplementary Material

We document the distribution of the common toad <i>Bufo bufo </i>and the spined toad <i>B. spinosus </i>at their contact zone across France with data from a mitochondrial DNA RFLP assay, complementing similar work including nuclear markers in the northwest and southeast of France and in Italy. We also reconstruct geographical clines across the species’ contact zone in central France. <i>Bufo bufo </i>is found in the north-eastern half of France. <i>Bufo spinosus </i>is found in the south-western complement. The contact zone they form runs from the Atlantic coast near Caen, France, to the Mediterranean coast near Savona, Italy, and has a length of over 900 km. In central France <i>B. bufo </i>and <i>B. spinosus </i>engage in a hybrid zone with a unimodal genetic signature. Hybrid zone width is ca. 10 km at mitochondrial DNA and averages at 61 km for four nuclear loci. The hybrid zone is distinctly asymmetric with a signature of <i>B. spinosus </i>in <i>B. bufo </i>and not the other way round. We attribute this observation to <i>B. bufo </i>moving southwards at the expense of <i>B. spinosus</i>, with introgression in the direction of the advancing species. We noted substantial geographic variation in characters for species identification. Morphological species identification performs well in France, but breaks down in Italy. Mitochondrial DNA is inconclusive in south-eastern France and Italy. The nuclear genetic markers perform consistently well but have not yet been applied to the zone in full. Possible, but surely heterogeneous ecological correlates for the position of the hybrid zone are mountains and rivers

Additional file 4 of Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (Bufo bufo and B. spinosus) in the northwest of France

Appendix IV. SNP data in GenePop format [20]. Populations are in the order 1–17, as in Table 1. (TXT 9 kb

Additional file 2 of Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (Bufo bufo and B. spinosus) in the northwest of France

Appendix II. Result of Structure analysis, showing the deltaK graph towards the selection of K = 2. (TIF 49 kb

Additional file 1 of Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (Bufo bufo and B. spinosus) in the northwest of France

Appendix I. Genetic polymorphisms in a transect across 17 Bufo spinosus and B. bufo populations in northwestern France, estimated for 12 microsatellite loci: Na–number of alleles, Ho–observed heterozygosity, He–expected heterozygosity and the size range of alleles in base pairs. Data for populations 1–3, 16 and 17 are from [18]. (XLSX 17 kb

Exploring the evolutionary origin of floral organs of Erycina pusilla, an emerging orchid model system

Background: Thousands of flowering plant species attract pollinators without offering rewards, but the evolution of this deceit is poorly understood. Rewardless flowers of the orchid Erycina pusilla have an enlarged median sepal and incised median petal (‘lip’) to attract oil-collecting bees. These bees also forage on similar looking but rewarding Malpighiaceae flowers that have five unequally sized petals and gland-carrying sepals. The lip of E. pusilla has a ‘callus’ that, together with winged ‘stelidia’, mimics these glands. Different hypotheses exist about the evolutionary origin of the median sepal, callus and stelidia of orchid flowers. Results: The evolutionary origin of these organs was investigated using a combination of morphological, molecular and phylogenetic techniques to a developmental series of floral buds of E. pusilla. The vascular bundle of the median sepal indicates it is a first whorl organ but its convex epidermal cells reflect convergence of petaloid features. Expression of AGL6 EpMADS4 and APETALA3 EpMADS14 is low in the median sepal, possibly correlating with its petaloid appearance. A vascular bundle indicating second whorl derivation leads to the lip. AGL6 EpMADS5 and APETALA3 EpMADS13 are most highly expressed in lip and callus, consistent with current models for lip identity. Six vascular bundles, indicating a stamen-derived origin, lead to the callus, stelidia and stamen. AGAMOUS is not expressed in the callus, consistent with its sterilization. Out of three copies of AGAMOUS and four copies of SEPALLATA, EpMADS22 and EpMADS6 are most highly expressed in the stamen. Another copy of AGAMOUS, EpMADS20, and the single copy of SEEDSTICK, EpMADS23, are most highly expressed in the stelidia, suggesting EpMADS22 may be required for fertile stamens. Conclusions: The median sepal, callus and stelidia of E. pusilla appear to be derived from a sepal, a stamen that gained petal identity, and stamens, respectively. Duplications, diversifying selection and changes in spatial expression of different MADS-box genes shaped these organs, enabling the rewardless flowers of E. pusilla to mimic an unrelated rewarding flower for pollinator attraction. These genetic changes are not incorporated in current models and urge for a rethinking of the evolution of deceptive flowers. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0938-7) contains supplementary material, which is available to authorized users

Morphological and molecular characterization of orchid fruit development

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species Erycina pusilla and compare it to two terrestrial orchid species: Cynorkis fastigiata and Epipactis helleborine. Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While C. fastigiata and E. helleborine develop a lignified layer at the valve boundaries, E. pusilla fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes INDEHISCENT (IND)/HECATE 3 (HEC3), REPLUMLESS (RPL) and SPATULA (SPT)/ALCATRAZ (ALC) in E. pusilla, and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (AG) /SHATTERPROOF (SHP) orthologs can also be formed in E. pusilla, and that the expression of HEC3, RPL, and SPT can be associated with dehiscence zone development similar to Arabidopsis. Our expression analysis also indicates differences, however, which may underlie fruit divergence