Pamela Puppo

Development of phylogenetic markers from single-copy nuclear genes for multi locus, species level analyses in the mint family (Lamiaceae).

The use of single copy nuclear markers is of increasing importance in plant phylogenetics. The generally higher level of variability compared to chloroplast DNA and the ability to use incongruence in a multilocus analysis to determine reticulation patterns makes these kinds of sequence based markers especially useful for species level investigations. However, the prevalence of gene duplication that results from the high frequency of polyploidization events during the evolution of higher plants can impede marker development especially for groups lacking model organisms. Here, we present the strategy and results of marker development for phylogenetic analysis in Micromeria, using publicly available DNA sequences and ESTs from related genera from Lamiaceae, subfamily Nepetoideae. By eliminating markers with signatures of duplication during four steps of marker development, we were able to select 19 primer pairs that resulted in orthologous products for all the species studied. This corresponds to 23% of the initial 84 primer pairs designed. Using an initial sampling of eight individuals, we tested the markers for support of phylogenetic hypotheses related to the evolution of Micromeria on the Canary Islands. While some hypotheses were supported by all markers, an east west split, with a closer relationship between the species of Tenerife and Madeira on one hand and the ones from Gran Canaria and the eastern islands on the other is supported by 12 markers but contradicted by the remaining seven. This indicates that reticulation and inter-island gene flow played a role in the evolution of Micromeria.

Molecular phylogenetics of Micromeria (Lamiaceae) in the Canary Islands, diversification and inter-island colonization patterns inferred from nuclear genes☆

Here we reconstruct the evolutionary history of Micromeria in the Canary Islands using eight nuclear markers. Our results show two centers of diversification for Micromeria, one in the eastern islands Gran Canaria and Lanzarote, the other in the western islands, Tenerife, La Palma and El Hierro. Suggested directions of inter-island colonization are the following: Gran Canaria to Lanzarote and La Gomera; Tenerife to La Palma (from the paleoisland of Teno), to El Hierro (from the younger, central part), and to La Gomera and Madeira (from the paleoislands). Colonization of La Gomera probably occurred several times from Gran Canaria and Tenerife. The taxonomic implications of these results are discussed. Incongruence among the different markers was evaluated and, using next generation sequencing, we investigated if this incongruence is due to gene duplication.

Genetic structure of Micromeria (Lamiaceae) in Tenerife, the imprint of geological history and hybridization on within-island diversification

Abstract Geological history of oceanic islands can have a profound effect on the evolutionary history of insular flora, especially in complex islands such as Tenerife in the Canary Islands. Tenerife results from the secondary connection of three paleo‐islands by a central volcano, and other geological events that further shaped it. This geological history has been shown to influence the phylogenetic history of several taxa, including genus Micromeria (Lamiaceae). Screening 15 microsatellite markers in 289 individuals representing the eight species of Micromeria present in Tenerife, this study aims to assess the genetic diversity and structure of these species and its relation with the geological events on the island. In addition, we evaluate the extent of hybridization among species and discuss its influence on the speciation process. We found that the species restricted to the paleo‐islands present lower levels of genetic diversity but the highest levels of genetic differentiation suggesting that their ranges might have contracted over time. The two most widespread species in the island, M. hyssopifolia and M. varia, present the highest genetic diversity levels and a genetic structure that seems correlated with the geological composition of the island. Samples from M. hyssopifolia from the oldest paleo‐island, Adeje, appear as distinct while samples from M. varia segregate into two main clusters corresponding to the paleo‐islands of Anaga and Teno. Evidence of hybridization and intraspecific migration between species was found. We argue that species boundaries would be retained despite hybridization in response to the habitats specific conditions causing postzygotic isolation and preserving morphological differentiation.

Genetic diversity and differentiation patterns in Micromeria from the Canary Islands are congruent with multiple colonization dynamics and the establishment of species syngameons

BackgroundEspecially on islands closer to the mainland, such as the Canary Islands, different lineages that originated by multiple colonization events could have merged by hybridization, which then could have promoted radiation events (Herben et al., J Ecol 93: 572–575, 2005; Saunders and Gibson, J Ecol 93: 649–652, 2005; Caujapé-Castells, Jesters, red queens, boomerangs and surfers: a molecular outlook on the diversity of the Canarian endemic flora, 2011). This is an alternative to the scenario where evolution is mostly driven by drift (Silvertown, J Ecol 92: 168–173, 2004; Silvertown et al., J Ecol 93: 653–657, 2005). In the former case hybridization should be reflected in the genetic structure and diversity patterns of island species. In the present work we investigate Micromeria from the Canary Islands by extensively studying their phylogeographic pattern based on 15 microsatellite loci and 945 samples. These results are interpreted according to the hypotheses outlined above.ResultsGenetic structure assessment allowed us to genetically differentiate most Micromeria species and supported their current classification. We found that populations on younger islands were significantly more genetically diverse and less differentiated than those on older islands. Moreover, we found that genetic distance on younger islands was in accordance with an isolation-by-distance pattern, while on the older islands this was not the case. We also found evidence of introgression among species and islands.ConclusionsThese results are congruent with a scenario of multiple colonizations during the expansion onto new islands. Hybridization contributes to the grouping of multiple lineages into highly diverse populations. Thus, in our case, islands receive several colonization events from different sources, which are combined into sink populations. This mechanism is in accordance with the surfing syngameon hypothesis. Contrary to the surfing syngameon current form, our results may reflect a slightly different effect: hybridization might always be related to colonization within the archipelago as well, making initial genetic diversity to be high to begin with. Thus the emergence of new islands promotes multiple colonization events, contributing to the establishment of hybrid swarms that may enhance adaptive ability and radiation events. With time, population sizes grow and niches start to fill. Consequently, gene-flow is not as effective at maintaining the species syngameon, which allows genetic differentiation and reproductive isolation to be established between species. This process contributes to an even further decrease in gene-flow between species.

Erratum to: Development and characterization of 16 microsatellite markers for Micromeria (Lamiaceae) from Tenerife (Canary Islands, Spain) using 454 sequencing

Micromeria presents eight species in Tenerife, and from these, three are endangered or critically endangered. In this paper we use a 454 run from M. hyssopifolia to develop and characterize 16 new microsatellite markers and test them using 19 individuals from two populations of this species. The number of alleles per locus varied from 2 to 10 and observed heterozygosity ranged from 0.125 to 0.900. Most loci successfully amplified in the threatened species M. densiflora, M. glomerata and M. rivas-martinezii and were also polymorphic. These loci will be valuable tools for assessing the genetic diversity of these endangered species.

Development of microsatellites in the Hawaiian endemic palm Pritchardia martii (Arecaceae) and their utility in congeners.

PREMISE OF THE STUDY Pritchardia (loulu palm) is the seventh largest flowering plant genus in the Hawaiian archipelago, and many species are of high conservation concern. The island radiation has produced many cryptic species complexes across fine ecological gradients. Microsatellite primers were optimized to investigate genetic diversity of Pritchardia martii (Gaudich.) H. Wendl. and species boundaries among the Hawaiian Pritchardia. METHODS AND RESULTS Six new loci and three previously described loci for a closely related genus, Phoenix, were optimized and tested. Five loci were polymorphic, and 72 alleles were detected across loci within P. martii. The five loci were also polymorphic across the other 27 currently recognized Pritchardia species. • CONCLUSIONS These results indicate the utility of these microsatellite markers for understanding the conservation genetics of P. martii and species delimitation and hybridization across the genus.

Revision of section Calceolaria (Calceolariaceae) in Chile

Calceolaria is characterized by its bilabiate generally yellow flowers with the lower lip saccate and two stamens. Section Calceolaria has near 10 species all herbaceous and hygrophilous with a particular staminal morphology. The thecae are dimorphic and the connective is elongated in various ways. In Chile, only two species of this section, C. pinnata and C. tripartita, were known from the different flora publications since the 18 th century. Moreover, the use of invalid species names such as C. scabiosifolia Roem. & Schult. contributed to the misidentification of the material of this section, further complicating its taxonomy. The aim of the present work is to make the first revision of the species of this section present in Chile as well as presenting a thorough revision of the literature that makes reference to them. A key for the species identification is included as well as descriptions, illustrations, comments on their distribution, and their conservation status in the country according to the IUCN methodology.

Using a new RAD-sequencing approach to study the evolution of Micromeria in the Canary islands

As found in other oceanic islands, the Canary Islands include a large number of single island endemic species, some of which form clades that are broadly distributed within the archipelago. The genus Micromeria (Lamiaceae), for instance, includes groups of morphologically similar but ecologically diverse species on each island, representing a great model to investigate niche shifts and adaptation within the Canary Archipelago. Previous attempts to reconstruct phylogenetic relationships within the genus did not lead to robust phylogenies, presumably due to introgression and/or incomplete lineage sorting. In this study, we use a newly developed RAD-sequencing method to improve phylogenetic resolution and to better understand relationships among the Canary Island endemic Micromeria. Overall, we obtained 3571 loci that were genotyped for a total of 46 individuals of Micromeria. Our data reconstructed a highly resolved phylogeny, and corroborated the latest species reclassification of the M. varia s.l. species complex, the taxonomically most complicated group within the genus. Furthermore, taxa occupying similar ecological conditions in different islands, were shown to be closely related. This is the case of taxa from the laurel forest from La Gomera and Gran Canaria, suggesting that the laurel forest likely worked as a filter, only allowing the establishment of colonizers already pre-adapted to these conditions. We also found introgression between these species so it is also possible that the genes that facilitated the adaptation to laurel forest were swapped between Gran Canaria and La Gomera. The observations obtained in this study also allowed us to explain the role of introgression in the origin of M. varia s.l. species complex.

Challenges for assessing vertebrate diversity in turbid Saharan water-bodies using environmental DNA

The Sahara desert is the largest warm desert in the world and a poorly explored area. Small water-bodies occur across the desert and are crucial habitats for vertebrate biodiversity. Environmental DNA (eDNA) is a powerful tool for species detection and is being increasingly used to conduct biodiversity assessments. However, there are a number of difficulties with sampling eDNA from such turbid water-bodies and it is often not feasible to rely on electrical tools in remote desert environments. We trialled a manually powered filtering method in Mauritania, using pre-filtration to circumvent problems posed by turbid water in remote arid areas. From nine vertebrate species expected in the water-bodies, four were detected visually, two via metabarcoding, and one via both methods. Difficulties filtering turbid water led to severe constraints, limiting the sampling protocol to only one sampling point per study site, which alone may largely explain why many of the expected vertebrate species were not detected. The amplification of human DNA using general vertebrate primers is also likely to have contributed to the low number of taxa identified. Here we highlight a number of challenges that need to be overcome to successfully conduct metabarcoding eDNA studies for vertebrates in desert environments in Africa.

New Species of Calceolaria (Calceolariaceae) from Northern Peru

Abstract. Two new species of Calceolaria L. from northern Peru belonging to section Teucriifoliae Kraenzl. are described, illustrated, and compared with their most closely related relatives. These new taxa grow in the province of Bolívar, department of La Libertad, in Peru. Calceolaria chibulensis C. Romero, Bussmann & Puppo grows at elevations between 2400 and 2500 m; C. comulcana C. Romero, Bussmann & Puppo grows at elevations between 3900 and 4200 m.