Molecular phylogenetics of the Ronnbergia Alliance (Bromeliaceae, Bromelioideae) and insights into their morphological evolution
Graphical abstract
Introduction
Recent developments in the fields of phylogenetics, biogeography, and comparative biology have allowed the exploration of exciting evolutionary questions across many taxonomic groups. The Bromeliaceae have received special attention because of their clear shifts in adaptive regimes across lineages, high species diversity (ca. 3352 species), and almost exclusively Neotropical geographical distribution (Benzing, 2000, Bouchenak-Khelladi et al., 2015, Givnish et al., 2014). Consequently, recent studies have provided significant advances on the understanding of the phylogenetic relationships and major evolutionary events across the main lineages in the family (Crayn, 2004, Givnish et al., 2014, Givnish et al., 2011, Givnish et al., 2007, Givnish et al., 2004, Givnish and Sytsma, 2000, Quezada and Gianoli, 2011, Silvestro et al., 2014). However, at the generic level and below, the taxonomy and phylogenetic relationships of the Bromeliaceae are still poorly understood (Barfuss et al., 2005, Faria et al., 2004, Sass and Specht, 2010, Schulte et al., 2009). Different studies have approached this problem by focusing on the subfamily Bromelioideae, a monophyletic lineage plagued with generic delimitation problems, but at the same time highly attractive for evolutionary studies because of its high species diversity (ca. 936 species), large mosaic of phenotypic and adaptive variation, and high levels of narrow endemism across the Neotropics (Almeida et al., 2009, Evans et al., 2015, Faria et al., 2004, Heller et al., 2015, Horres et al., 2007, Sass and Specht, 2010, Schulte et al., 2009, Schulte et al., 2005, Schulte and Zizka, 2008, Silvestro et al., 2014, Smith and Downs, 1979, Sousa et al., 2007).
The problems of generic delimitation within Bromelioideae have been discussed since the early taxonomic treatments of Bromeliaceae. For instance, in the latest comprehensive revision of the family, Smith and Downs (1979) recognized that although characters such as inflorescence ramification, flower pedicels, and petal appendages are important to diagnose genera within Bromelioideae, they could occasionally occur in isolated species placed within the most artificial genera of the subfamily, such as Aechmea. Morphological studies later provided further evidence by showing that these “diagnostic” characters are prone to evolve rapidly and are only of limited taxonomic use (Brown and Terry, 1992, Faria et al., 2004, Schulte and Zizka, 2008). The limitations posed by the lack of unequivocal diagnostic characters have been progressively resolved with the advent of molecular phylogenetics. Recent studies, for example, have shown that the early-diverging lineages of Bromelioideae are generally tankless, terrestrial or lithophytic species, whereas the most recent and diverse lineage encompasses mostly tank-forming, epiphytic species (Evans et al., 2015, Givnish et al., 2011, Sass and Specht, 2010, Schulte et al., 2009, Silvestro et al., 2014). While in the past there have been only few changes in generic concepts for early-diverging Bromelioideae, generic delimitation in the tank-epiphytic clade, also known as the “Core Bromelioideae” (sensu Sass and Specht, 2010), has proven highly problematic and resulted in frequent taxonomic changes over the past decades (e.g. Brown and Leme, 2005, Leme and Kollmann, 2011, Smith and Kress, 1989, Smith and Spencer, 1992).
Phylogenetic resolution within the Core Bromelioideae is still insufficient to propose a thoroughly revised taxonomic classification; however, independent phylogenetic studies have revealed common patterns that can serve as a starting point for further investigation. While many genera of the Core Bromeliodeae have been shown to be non-monophyletic, a common pattern is that the several lineages within the Core Bromelioideae that have been identified by molecular phylogenetic studies, are often composed by species that share a similar and narrow geographical distribution (Faria et al., 2004, Sass and Specht, 2010, Schulte et al., 2009, Silvestro et al., 2014). Based on these studies, we have been able to identify and study in detail one of these lineages, which we call here the “Ronnbergia Alliance.” This study, therefore, aims to contribute to the understanding of the phylogeny and evolution of the Core Bromelioideae, which is an important step toward the revision of generic concepts within this bromeliad group.
Our prior definition of the Ronnbergia Alliance is based on a consensus of previous phylogenetic studies that show a small and well supported clade, frequently sister to the remaining Core Bromelioideae, that contains representative species of three taxonomic groups Ronnbergia, Hohenbergia subgenus Wittmackiopsis, and the Aechmea lingulata complex (sensu Siqueira Filho and Leme, 2006). Furthermore, it contains allied species of Aechmea subgenus Chevaliera and Aechmea subgenus Pothuava endemic to the rainforests of northwestern South America and southern Central America (Aguirre-Santoro et al., 2015, Givnish et al., 2011, Maia et al., 2012, Sass and Specht, 2010, Schulte et al., 2009, Schulte et al., 2005, Schulte and Zizka, 2008, Silvestro et al., 2014; Fig. 1). Except for the widespread species Aechmea lingulata, these taxonomic groups and allied species of Aechmea are characterized by their narrow endemism within three highly biodiverse regions of the Neotropics: (1) the Chocó-Tumbes-Magdalena region; (2) the central and northern Atlantic Forest and adjacent semi-deciduous forests and Caatinga; and (3) the Caribbean section that includes the Greater Antilles (except Hispaniola), the Cayman and Providencia islands, and the Yucatán Peninsula. In previous molecular phylogenetic studies, the Ronnbergia Alliance has received little attention and was only represented by few species. Therefore, we aim to assess the monophyly and phylogenetic relationships of the Ronnbergia Alliance by including a broad sampling of species with morphological and/or biogeographical affinities to the three main taxonomic groups and allied species that constitute this lineage.
The species that constitute these three taxonomic groups and allied species share many morphological affinities; yet they have rarely been placed together in taxonomic treatments. This lack of taxonomic recognition is due to the use of traditionally diagnostic characters for generic delimitation in Bromelioideae. For example, species of Ronnbergia and Aechmea subgenus Pothuava that co-occur in the forests of northwestern South America and southern Central America exhibit simple inflorescences, long-tubular subspreading corollas, and similar patterns of corolla coloration. However, these species have long remained in different genera because species of Aechmea subgenus Pothuava have petal appendages whereas those of Ronnbergia lack them. A well-resolved and highly supported phylogenetic reconstruction for the Ronnbergia Alliance will serve as a framework to assess the taxonomic value of morphological characters traditionally used as well as other so far neglected, characters. This is relevant because previous morphology-based phylogenetic analyses have suggested that overlooked characters such as corolla shape, ovary surface, stigma types, pollen ornamentation, and seed structure are good sources of potential diagnostic characters to define clades across Bromelioideae (Almeida et al., 2009, De Sousa et al., 2008, Faria et al., 2004). Moreover, traditional taxonomic characters such as sepal armature, petal appendages, ovule appendages, placentation, and pollen aperture should be revisited because they seem to be informative to diagnose major lineages within Bromelioideae but have been rarely explored in a phylogenetic context (Heller et al., 2015, Mez, 1896, Schulte and Zizka, 2008, Smith and Downs, 1979). In the present study, we explore some of these characters using ancestral character state reconstructions in order to reconstruct their evolutionary history and to assess their stability and diagnostic value. This approach will provide insights for future taxonomic restructuring of the Ronnbergia Alliance when a comprehensive phylogeny of the Core Bromelioideae is reconstructed and will also help detect traits that were important for the diversification of its lineages.
Our approach in this study can be summarized as two main objectives: (1) reconstruct the phylogenetic relationships of the Ronnbergia Alliance using multilocus DNA sequence data and a broad species sampling in order to test its monophyly, establish its placement within the Core Bromelioideae and identify its subclades; and (2) explore the taxonomic and evolutionary importance of 13 morphological characters that show potential as diagnostic characters for the Ronnbergia Alliance and its subclades for future taxonomic reorganization of the group. A better understanding of this lineage will also open new perspectives for future evolutionary and biogeographic studies.
Section snippets
Taxon sampling
A total of 168 terminals representing 129 species were included for the phylogenetic analyses (Supplementary Table A). Three species of Bromelia were chosen as outgroups because this genus has been identified as one of the early-diverging lineages within Bromelioideae (Evans et al., 2015, Givnish et al., 2011, Givnish et al., 2007, Schulte et al., 2009, Schulte and Zizka, 2008). In order to elucidate phylogenetic relationships within the Ronnbergia Alliance, we selected 94 terminals to
Phylogenetic analysis of the individual markers, cpDNA and nrDNA datasets, and congruence
Table 2 shows the general statistics of the alignments for the individual and combined datasets. The nuclear markers exhibited the greatest amount of variable and parsimony-informative sites. ETS was the most informative region among all the individual markers. In contrast, the plastid markers showed lower percentages of variable and parsimony-informative characters, matK-trnK being the most variable of the plastid markers.
The ML analyses of the individual markers resulted in topologies with
Monophyly, phylogenetic position, and diagnosis of the Ronnbergia Alliance
Although most of the independent molecular markers and the cpDNA dataset did not provide enough information to resolve the monophyly Ronnbergia Alliance, they did not offer a conflicting scenario against this hypothesis. This lack of resolution was the result of the low variation across the molecular markers, especially those of the plastid genome (Table 2), a phenomenon already discussed in previous molecular phylogenetic studies of Bromelioideae (Evans et al., 2015, Horres et al., 2007, Sass
Conclusions
Our comprehensive taxonomic sampling and use of six DNA sequence markers provided evidence to support the monophyly of the Ronnbergia Alliance, reveal the relationships within this clade, and explore the importance of selected morphological characters. These strongly supported results will serve as framework for future studies focused on proposing a stable and predictive classification of genera within the conflictive Core Bromelioideae. Moreover, the phylogenetic hypotheses showed that the
Acknowledgements
We are thankful to The New York Botanical Garden, Graduate Center, COLCIENCIAS, the American Society of Plant Taxonomists, The Explorers Club of America, and The Systematic Association and Linnaean Society of London for providing funds and resources to carry out this project. This research was partially funded by the NSF through grants to FAM (DEB-1343612, DEB-0818399) and Wayt W. Thomas (DEB-0946618). We also thank André Amorim, Eldis Becquer, Julio Betancur, Keron Campbell, Tracy Commock,
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