Annals of Botany Page 1 of 15 doi:10.1093/aob/mcv005, available online at www.aob.oxfordjournals.org Phylogenetic analysis in Myrcia section Aulomyrcia and inferences on plant diversity in the Atlantic rainforest Vanessa Graziele Staggemeier1,2,*, Jose´Alexandre Felizola Diniz-Filho1,Fe´lix Forest2 and Eve Lucas2,3 1Departamento de Ecologia, Laborato´rio de Ecologia Teo´rica e Sı´ntese, ICB, Universidade Federal de Goia´s, 74001-970, Goiaˆnia, GO, Brazil, 2Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK and 3Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK * For correspondence. E-mail [email protected] Received: 21 June 2014 Returned for revision: 31 July 2014 Accepted: 23 December 2014 Downloaded from Background and Aims Myrcia section Aulomyrcia includes 120 species that are endemic to the Neotropics and disjunctly distributed in the moist Amazon and Atlantic coastal forests of Brazil. This paper presents the first comprehensive phylogenetic study of this group and this phylogeny is used as a basis to evaluate recent classifica- tion systems and to test alternative hypotheses associated with the history of this clade. http://aob.oxfordjournals.org/ Methods Fifty-three taxa were sampled out of the 120 species currently recognized, plus 40 outgroup taxa, for one nuclear marker (ribosomal internal transcribed spacer) and four plastid markers (psbA-trnH, trnL-trnF, trnQ-rpS16 and ndhF). The relationships were reconstructed based on Bayesian and maximum likelihood analyses. Additionally, a likelihood approach, ‘geographic state speciation and extinction’, was used to estimate region- dependent rates of speciation, extinction and dispersal, comparing historically climatic stable areas (refugia) and unstable areas. Key Results Maximum likelihood and Bayesian inferences indicate that Myrcia and Marlierea are polyphyletic, and the internal groupings recovered are characterized by combinations of morphological characters. Phylogenetic relationships support a link between Amazonian and north-eastern species and between north-eastern and south- eastern species. Lower extinction rates within glacial refugia suggest that these areas were important in maintaining at University of Maryland on March 20, 2015 diversity in the Atlantic forest biodiversity hotspot. Conclusions This study provides a robust phylogenetic framework to address important ecological questions for Myrcia s.l. within an evolutionary context, and supports the need to unite taxonomically the two traditional genera Myrcia and Marlierea in an expanded Myrcia s.l. Furthermore, this study offers valuable insights into the diversifi- cation of plant species in the highly impacted Atlantic forest of South America; evidence is presented that the lowest extinction rates are found inside refugia and that range expansion from unstable areas contributes to the highest levels of plant diversity in the Bahian refugium. Key words: Aulomyrcia, Atlantic forest, diversification rates, diversity patterns, extinction, GeoSSE, Marlierea, Myrcia, Myrtaceae, speciation. INTRODUCTION section occurs primarily in rainforests from two of the most ‘The distinction between Myrcia and Marlierea is a somewhat threatened biomes in the world, with 80 species in Amazonia nebulous one, and it is probable that Marlierea comprises a and related forests of Central and northern South America and phylogenetically diverse assemblage of species which have 40 species in the Atlantic coastal rainforests of Brazil (Lucas been arbitrarily assigned to the genus because of the character et al., unpubl. res.). According to Lucas et al. (2011),represen- of the irregularly splitting calyx.’ (McVaugh, 1956,page166). tatives of this clade are characterized by a closed calyx and a Myrcia s.l. (sensu Lucas et al., 2007) comprises 753 species combination of flower, inflorescence and venation characters (Govaerts et al.,2014); representing the second-largest (Supplementary Data Fig. S1), such as: (1) inflorescence axes Neotropical genus of Myrtaceae after Eugenia (1038 species; emerging from a single terminal whorl and primary axes irregu- Govaerts et al.,2014). Taxonomic classification in Myrcia has larly or asymmetrically branched; (2) calyx lobes free to par- traditionally emphasized characters of the calyx and mode of tially fused, irregularly tearing vertically through the opening of the flower (McVaugh, 1956; Berg, 1855–1856); as hypanthium; (3) hypanthium extended into a tube flared beyond described, molecular studies show that these traits do not group the ovary, but inconspicuous after deep tearing; and (4) a (usu- species naturally (Lucas et al.,2007, 2011). ally) bilocular ovary, with two ovules per locule. The most recent phylogenetic analysis of Myrcia s.l. (Lucas As currently circumscribed, Myrcia s.l. encompasses four et al., 2011) recovered nine major clades that have been treated genera that were recognized during most of the 20th century: as nine informal groups. Myrcia section Aulomyrcia (Lucas Calyptranthes, Gomidesia, Myrcia and Marlierea. However, et al., unpubl. res.) represents one of these groupings. This earlier studies (Lucas et al.,2011) have shown that the first VC The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Page 2 of 15 Staggemeier et al. — Phylogeny and diversity of Myrcia section Aulomyrcia two genera are monophyletic and nested in the paraphyletic obtained from other studies (Lucas et al.,2007, 2011;MF Myrcia and Marlierea, indicating that the current circumscrip- Santos, USP, Sa˜o Paulo, Brazil, unpubl. res.; C. Wilson, RBG tion of these genera needs to be re-evaluated. McVaugh (1956) Kew, UK, unpubl. res.). suspected the artificiality of Marlierea but maintained the ge- nus in his studies on Tropical American Myrtaceae based on the presence of persistent bracts and the abortion of the primary DNA sequencing axis of the inflorescence. Total DNA was extracted mainly from 0Á3 g of silica- The importance of Myrcia s.l. as a structural and ecological gel-dried leaf material (in some cases from 0Á2 g when using component of the Atlantic rainforest of Brazil makes this group herbarium specimens) using a modified version 2Â cetyltri- a good proxy for the diversity of other important angiosperm methyl ammonium bromide (CTAB) protocol (Doyle and groups in the Atlantic rainforest (Murray-Smith et al., 2009), Doyle, 1987). Total DNA was further purified for long-term stor- one of the biodiversity hotspots (Myers et al.,2000). Within age in the RBG Kew DNA and Tissue Collections by equilib- this hotspot there are areas of higher angiosperm diversity and rium centrifugation in caesium chloride–ethidium bromide areas of elevated Myrcia diversity (Murray-Smith et al.,2009) gradients (1Á55 g ml–1)followedbybutanolextraction of ethid- that may correspond to refugia (Carnaval and Moritz, 2008). ium bromide and dialysis to remove caesium chloride. Downloaded from Refugia (Haffer, 1969) are thought to act as ‘museums’ for the Amplification and purification of target DNA regions were exe- maintenance of biodiversity due to their expected lower extinc- cuted according to the protocols outlined in Lucas et al. (2007, tion rates and/or higher speciation rates, when compared with 2011) using internal primers for some regions when necessary unstable areas, resulting in a positive net diversification bal- (Appendix). PCR conditions are listed in Supplementary Data ance. Refugia can act as ‘safe havens’ (Keppel et al.,2012) Table S2. Sequencing was performed according to Lucas et al. where biota would be protected for a long time, contributing (2007). DNA sequences were assembled using MUSCLE http://aob.oxfordjournals.org/ important pieces of information for a better understanding (Edgar, 2004) and edited in GENEIOUS 7.0.2 with subsequent of the evolutionary processes that may have driven modern manual adjustments when necessary. Any doubtful base calls species diversity. and all phylogenetically informative base changes were com- This study aimed to test the monophyly of Myrcia section pared with the general consensus and individually checked. A Aulomyrcia and further test its phylogenetic placement in the paralogous copy of ITS was identified in Myrcia riodocensis and genus and to examine phylogenetic relationships within this this sequence was excluded. All sequences have been deposited section. This phylogenetic framework was then used as a basis in GenBank and the DNA samples are deposited in the RBG to make inferences on the evolutionary history of Myrcia Kew DNA and Tissue Collections (Appendix). Due to the inheri- at University of Maryland on March 20, 2015 section Aulomyrcia, especially concerning speciation and tance of the plastid genome as a single linked unit, the resulting extinction dynamics along the Atlantic forest. four plastid DNA matrices were combined into a single dataset of 3477 bp. The molecular datasets, nuclear and combined plastid regions, were then analysed independently and simultaneously. MATERIAL AND METHODS Taxonomic and molecular sampling Phylogenetic analyses We sampled 93 taxa of tribe Myrteae, including 83 Myrcia s.l., of which 55 were from Myrcia section Aulomyrcia, encompass- Phylogenetic reconstruction using maximum likelihood ing as much as possible of the morphological and geographical (ML) and Bayesian inference was performed on all datasets. variation observed in the group (Appendix; Supplementary