Molecular Phylogenetics and Evolution 139 (2019) 106553 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Calyx (con)fusion in a hyper-diverse genus: Parallel evolution of unusual flower patterns in Eugenia (Myrtaceae) T ⁎ Augusto Giarettaa, , Thais N.C. Vasconcelosa, Fiorella Fernanda Mazineb, Jair Eustáquio Quintino Fariac, Rodolfo Floresd, Bruce Holste, Paulo Takeo Sanoa, Eve Lucasf a USP – Universidade de São Paulo, Instituto de Biociências, Departamento de Botânica, Laboratório de Sistemática Vegetal, Rua do Matão, 277, CEP 05508-090 São Paulo, SP, Brazil b UFSCar – Universidade Federal de São Carlos, Campus Sorocaba, Rod. João Leme dos Santos km 110, 18052–780 Sorocaba, SP, Brazil c Universidade de Brasília, IB, Departamento de Botânica, Brasília 70910-900, DF, Brazil d Programa de Maestría en Biología Vegetal, Universidad Autónoma de Chiriquí, Chiriquí, Panama e Marie Selby Botanical Gardens, 811 South Palm Ave., Sarasota, FL 34236-7726, USA f Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom ARTICLE INFO ABSTRACT Keywords: Eugenia has a pantropical distribution and comprises ca. 1000 species found mostly in the Neotropics. Recent Calycorectes DNA based phylogenies show that unusual flower morphology of ‘eugenioid’ collections, e.g. fused calices that Homoplasy open by tearing, consistently emerged within Eugenia. These results emphasize a demand to revaluate flower Parallelism morphology in a phylogenetic context within the genus. A reassessment of calyx fusion in Eugenia and tradi- Plant systematics tionally related genera is here focused on clarification of the systematic relevance of this apparently recurrent Taxonomy characteristic. Twenty-four Eugenia species with some level of calyx fusion in the bud were newly used (one nuclear and four plastid markers) in conjunction with a representative sample of previously sequenced species to recover a time-calibrated Eugenia phylogeny of 86 accessions. Development of the fused calyx was analysed using scanning electron microscopy, differing patterns were re-coded and subsequently phylogenetic character reconstruction was performed. Eugenia was recovered as monophyletic including the traditionally segregated genera Calycorectes and Catinga. Ancestral character reconstruction uncovered free calyx lobes as the ancestral condition. Five development patterns leading to calyx fusion are reported in Eugenia including species with apparently six petals, which contrast with the standard tetramerous flowers. This condition is interpreted as the petaloid pattern, where two external fused calyx lobes cover the bud while two internal calyx lobes are free and petaloid. The fused calyx condition is homoplastic and evolved independently, several times in Eugenia, as did the different development patterns. Data presented here show that systematic incongruence resulting from multiple, independent origins of the fused calyx in Eugenia is further aggravated by an inability to distinguish parallelism and convergence within the recovered patterns. 1. Introduction 2005; Wilson, 2011) based in many combined sets of morphological and molecular evidence (Wilson, 2011). Myrteae is the most diverse Classical authors recognized two or three tribes of Myrtaceae, cir- tribe comprising half of the species of the family, with 51 genera and ca. cumscribed according to the presence of fleshy-berry or dry-capsular 2500 species (Wilson, 2011; WCSP, 2019) and includes the hyper-di- fruits (Candolle, 1828; Schauer, 1841; Niedenzu, 1898). Cladistic in- verse genus Eugenia L. sight based exclusively on morphology (Johnson and Briggs, 1984), Eugenia has a pantropical distribution and comprises ca. 1000 spe- molecular data (Gadek et al., 1996; Lucas et al., 2005; Wilson et al., cies, found mostly in the Neotropics (Wilson, 2011; Mazine et al., 2014; 2005) or both (Wilson et al., 2001), demonstrated that many characters 2016). Eugenia has high ecological importance (Staggemeier et al., presumed diagnostic in earlier classifications were, in fact, more 2017) and is the richest tree genus in some regions of the Brazilian homoplastic than previously appreciated. Myrtaceae is a large family Atlantic coastal forest (Oliveira‐Filho and Fontes, 2000). However, high with ca. 5500 species, and today classified into 17 tribes (Wilson et al., levels of homogeneity of taxonomically important characters such as ⁎ Corresponding author. E-mail address: [email protected] (A. Giaretta). https://doi.org/10.1016/j.ympev.2019.106553 Received 7 March 2019; Received in revised form 28 June 2019; Accepted 2 July 2019 Available online 06 July 2019 1055-7903/ © 2019 Elsevier Inc. All rights reserved. A. Giaretta, et al. Molecular Phylogenetics and Evolution 139 (2019) 106553 flowers and fruits make species identification notoriously difficult clades. Six additional Myrteae genera were used as the outgroup. The (Lucas and Bünger, 2015). At one hand, the classic Eugenia flower has internal transcribed spacer (ITS) of the ribosomal nucleus and the four free calyx lobes, enabling easy genus recognition (e.g. Berg, 1857; plastid regions psbA-trnH, rpl16, trnL-rpl32 and trnQ5′-rps16 were used. Landrum and Kawasaki, 1997; Sobral, 2003). On the other hand, col- In total, 149 new sequences are provided with the remaining obtained lections ‘eugenioid’ in every way apart from flowers with unusual from existing published (Lucas et al., 2007; Mazine et al., 2014, 2018; morphology, e.g. fused calices that open by tearing, consistently Bünger et al., 2016; Vasconcelos et al., 2017b) and unpublished work emerged within Eugenia (Mazine et al., 2014, 2018; Giaretta et al., (J.E. Faria, UB, Brasília, Brazil, unpubl. res.). The molecular sample of 2019) in DNA based phylogenies. These results emphasize a demand to 86 accessions is available in Appendix A. reassess flower morphology in a phylogenetic context within the genus. Fused calyx development patterns were assessed from floral buds in Previously, genera have been segregated from Eugenia on the basis as many development stages as possible, collected and conserved in of differences in degree of fusion and dehiscence of the calyx (Amshoff, 70% ethanol from field collections in Brazil and French Guiana. A 1951; McVaugh, 1969; Legrand and Klein, 1972). As a consequence, complementary survey was based on buds from recent herbarium col- genera such as Calycorectes O.Berg and Catinga Aubl., including species lection available at K; the material was rehydrated in boiling water for that resemble Eugenia in all but possession of a fused calyx (also de- 10 min, left to cool overnight and then preserved in 70% ethanol. A scribed as “calyx closed or almost closed in bud”, McVaugh, 1968) are total of 25 samples representing 18 species and all known variation taxonomically controversial. Some authors segregate Calycorectes from between fused and free calices in Eugenia were surveyed in a com- Eugenia based on calyx morphology whereas a ‘unified Eugenia’ is sup- parative development analysis. At least three species were assessed for ported by most contemporary authors (e.g. Holst, 2002; Flora do Brasil, each development pattern. The only exception is the longohypanthium 2020). pattern found only in a single species. Descriptions of later stages of bud A seminal molecular phylogenetic framework focused on infra- development were emphasized to facilitate recognition of development generic classification in Eugenia confirmed that the genus can only be patterns on pre-anthetic flowers, as these are easier to manipulate in monophyletic if fused calyx species previously identified within herbarium material. A list of analysed material using scanning electron Calycorectes are included (Mazine et al., 2014). The character of the microscopy (SEM) is provided in Appendix B. fused calyx conveniently diagnosed Eugenia sect. Schizocalomyrtus (Kausel) Mattos (Mazine et al., 2014; Giaretta et al., 2018) but the 2.2. DNA sequencing variable degree and pattern of calyx fusion suggest that closer scrutiny of developmental aspects of this character might allow more accurate Total DNA was extracted using QIAGEN® DNeasy® Plant Maxi Kits use. Vasconcelos et al. (2018) describe the most common pattern of from until 0.2 g of silica-gel dried leaf material generating 1.5 ml of calyx development in Eugenia species. However, there was no mention total DNA. Amplification and purification of DNA regions were per- to or descriptions of any of the unusual forms of calyx fusion in that formed according to protocols outlined in Lucas et al. (2007, 2011) and study. To fully understand the origins and significance of the closed (Shaw et al., 2007) for rpl32-trnL and trnQ5′-rps16. PCR conditions were calyx in Eugenia, a more representative morphological and molecular executed according to Bünger et al. (2016). Nucleotide sequencing sample is required; only four closed calyx species were used in the most follows the protocols outlined by Lucas et al. (2007). Sequences were recent phylogenetic study (Mazine et al., 2018). It is here estimated that assembled and aligned using MUSCLE (Edgar, 2004) and edited when ca. 40 species of Eugenia undergo some degree of calyx fusion, most of necessary using Geneious v7.9 (Kearse et al., 2012). DNA samples are which are included in what used to be recognized as Calycorectes. stored in the DNA Bank and Tissue Collections of Royal Botanic Gar- Phylogenetic relationships based on molecular data have been in- dens, Kew. vestigated