Evolution of the outer ovule integument and its systematic significance in Melastomataceae

Evolution of the outer ovule integument and its systematic significance in Melastomataceae Abstract Ovule characters have been shown to be informative at higher taxonomic levels and are therefore of interest in angiosperm systematics. In this study, we aimed to describe the evolution of the outer ovule integument thickness in Melastomataceae and to evaluate its systematic and biological significance. This character was investigated in 137 species, and data from the literature were compiled for 22 additional species, totalling 159 taxa. Integument origin was studied in selected species to clarify whether different conditions were associated with different ontogenetic origins. The most recent common ancestor of Melastomataceae probably had an ovule with a two-cell-layered outer integument. A multilayered outer integument evolved independently at least six times in the family. Moreover, reversals to the two-cell-layered plesiomorphic condition have also occurred more than once. The two-cell-layered and multilayered outer integuments have different ontogenetic origins, enhancing the taxonomic usefulness of this character. The thickness of the outer ovule integument is of systematic significance in Olisbeoideae, Rupestrea, Melastomateae and Miconieae. The multilayered outer integument may have some adaptive significance for Miconieae and Mouriri, groups with endozoochoric fleshy fruits. Evidence of a multiplicative testa was observed in a few species with both types of integuments and it may be associated with a massive seed coat found in large seeds. INTRODUCTION The ovule is the developmental precursor of the seed and, as such, plays an essential role in the complex and successful reproduction process in spermatophytes (Bouman, 1984; Gasser, Broadhvest & Hauser, 1998; Linkies et al., 2010; Endress, 2011). In angiosperms, important reproductive events such as the female gametophyte and egg cell development, pollen tube attraction and guidance, double fertilization, and embryo and endosperm development all occur in the ovule (Fuentes & Vivian-Smith, 2009; Endress, 2011). The ovule can be morphologically understood as the megasporangium (or nucellus) covered by one or two integuments (Linkies et al., 2010). Gymnosperms typically have ovules with a single integument, whereas the ovules of angiosperms are commonly bitegmic (Gasser et al., 1998; Endress, 2011). After fertilization, these integuments give rise to the seed coat, which in angiosperms comprises a testa and a tegmen, derived from the outer and inner integuments, respectively. The seed coat can be responsible for embryo protection and seed dispersal, and it is a key factor in the regulation of seed germination (Windsor et al., 2000; Kelley & Gasser, 2009). Despite the remarkable adaptive significance of the seed coat, the thickness (i.e. number of cell layers) of the integuments seems to have been a relatively stable character in angiosperm evolution and therefore to be informative at higher taxonomic levels (Davis, 1966; Tobe, 1989; Endress, 2011). Ovule integuments may consist of two or more cell layers (Bouman, 1984; Endress, 2011). Two-cell-layered integuments are characterized as dermal integuments because they develop exclusively from the dermal layer of the ovule primordium. In contrast, multilayered integuments may be of dermal origin or may derive from dermal and subdermal layers (subdermal integuments) (Bouman, 1984; Shamrov, 2000; Endress, 2011). Dermal or subdermal integuments can become multiplicative and increase in thickness by periclinal divisions during both ovule and seed development (Corner, 1976; Bouman, 1984). The occurrence of ovules with a two-cell-layered inner integument is one of the embryological characters that define the order Myrtales (Tobe & Raven, 1983a; Tobe, 1989). On the other hand, the thickness of the outer integument is a variable character in the order (Tobe & Raven, 1983a). In Melastomataceae, one of the two largest families in Myrtales (Dahlgren & Thorne, 1984; Conti et al., 1997), the outer ovule integument may consist of two or more cell layers (Ziegler, 1925; Subramanyam, 1942, 1944, 1948, 1951; Etheridge & Herr, 1968; Corner, 1976; Tobe & Raven, 1983a; Medeiros & Morretes, 1996; Cortez & Carmello-Guerreiro, 2008; Caetano et al., 2013; Ribeiro, Oliveira & Silveira, 2015). However, the evolutionary history of this attribute in the family has not previously been studied. Moreover, despite the importance of integument characters for systematics, the scarcity of the data available for the family hampers their use in the delimitation of clades and understanding their relationships and their evolutionary and ecological significance. Melastomataceae (including Memecylaceae currently treated as subfamily Olisbeoideae) comprises c. 5400 species in 170 genera and is one of the larger families of angiosperms, with considerable species richness in the New World (Renner, 1993; Conti et al., 1997; Stone, 2006; MELNet, 2007; Goldenberg et al., 2015). Despite the uncontroversial monophyly of the family, relationships in Melastomataceae are not fully understood and improving their resolution is still a work in progress (Clausing & Renner, 2001; Fritsch et al., 2004; Michelangeli et al., 2004, 2013; Penneys et al., 2010; Goldenberg et al., 2012; Rocha et al., 2016a, b; Veranso-Libalah et al., 2017). Although the latest review for the family based on morphology and anatomy (Renner, 1993) recognized nine tribes, recent studies based on DNA sequence data have proposed the establishment of new ones and considerable changes to the delimitation of others (Clausing & Renner, 2001; Fritsch et al., 2004; Michelangeli et al., 2004, 2011, 2013; Goldenberg et al., 2008, 2012, 2015; Amorim, Goldenberg & Michelangeli, 2009; Penneys et al., 2010; Penneys & Judd, 2011, 2013; Rocha et al., 2016a, b). At least 15 major lineages are currently known in the family, although not all of them are recognized at the tribal level (Goldenberg et al., 2015). In this sense, knowledge of potentially diagnostic attributes for specific clades of Melastomataceae, such as the thickness of the outer ovule integument, may be of great significance. Therefore, the objective of the present study was to describe the evolution of this character in Melastomataceae, assessing its systematic and biological significance and clarifying whether different conditions (two-cell-layered and multilayered outer integument) are associated with different ontogenetic origins. Additionally, we also investigated the evolutionary association between multilayered outer integument and fleshy fruits. MATERIAL AND METHODS Taxon sampling The thickness of the ovule outer integument was examined in 137 species belonging to 14 of 15 major lineages currently recognized in Melastomataceae (sensuGoldenberg et al., 2015). The only tribe not sampled in the present study was Astronieae. We also included published data for 22 additional species for a total of 159 taxa (Table 1) (Ziegler, 1925; Subramanyam, 1942, 1944, 1948, 1951; Etheridge & Herr, 1968; Corner, 1976; Medeiros & Morretes, 1996; Caetano, 2010; Caetano et al., 2013; Ribeiro et al., 2015). As outgroups, we added nine species of the CAP clade, a strongly supported lineage sister to Melastomataceae and formed by Crypteroniaceae, Alzateaceae and Penaeaceae s.l. (including Oliniaceae and Rhynchocalycaceae; Conti et al., 2002; APG IV, 2016; Berger et al., 2016). Embryological data for these species were obtained from literature reports (Table 1; Tobe & Raven, 1983b, 1984a, b, c, d, 1987a, b). Table 1. List of the Melastomataceae species analysed, status of the character observed and literature data; fruit type (dry or fleshy) is also given Groups    Species*  Character state  Reference†  Fruit type  Reference†  Outgroups  Crypteroniaceae  Axinandra zeylanica  Two-layered (2–3)  1  Dry  19  Crypteronia paniculata  Two-layered (2)  2  Dry  19  Dactylocladus stenostachys  Two-layered (2–3)  3  Dry  19  Alzateaceae  Alzatea verticillata  Two-layered (2–4)  4  Dry  19  Penaeaceae  Olinia emarginata  Multilayered (3–4)  5  Fleshy  19  Olinia ventosa  Multilayered (3–5)  5  Fleshy  19  Penaea mucronata  Two-layered (2)  6  Dry  19  Rhynchocalyx lawsonioides  Two-layered (2)  7  Dry  19  Saltera sarcocolla  Two-layered (2)  6  Dry  19  Olisbeoideae    Memecylon heyneanum  Two-layered (2)  8  Fleshy  20      Memecylon umbellatum  Two-layered (2)  9  Fleshy  9      Memecylon sp.  Two-layered (2)  9  Fleshy  20      Mouriri acutiflora  Multilayered (5–6)  PS  Fleshy  20      Mouriri cearensis  Multilayered (5–6)  PS  Fleshy  20      Mouriri guianensis  Multilayered (5–6)  PS  Fleshy  20      Votomita guianensis  Two-layered (2–4)  PS  Fleshy  20  Kibessieae    Pternandra azurea  Two-layered (2)  9  Fleshy  21    Pternandra coerulescens  Two-layered (2)  PS  Fleshy  21  Henrietteeae  Bellucia grossularioides  Two-layered (2)  PS  Fleshy  22    Bellucia mespiloides  Two-layered (2)  PS  Fleshy  22    Henriettea ramiflora  Two-layered (2)  PS  Fleshy  22    Henriettea saldanhae  Two-layered (2)  PS  Fleshy  22    Henriettea succosa  Two-layered (2)  PS  Fleshy  22  Bertolonieae  Bertolonia mosenii  Two-layered (2)  PS  Dry  23  Blakeeae  Blakea trinervia  Two-layered (2)  9  Fleshy  23    Blakea multiflora  Two-layered (2)  PS  Fleshy  24    Chalybea calyptrata  Two-layered (2)  PS  Fleshy  24    Chalybea ecuadorensis  Two-layered (2)  PS  Fleshy  24  Dissochaeteae  Dissochaeta divaricata  Two-layered (2)  PS  Fleshy  25    Oxyspora paniculata  Two-layered (2)  10  Dry  25    Sonerila wallichii  Two-layered (2)  11  Dry  26  Cyphostyleae  Allomaieta grandiflora  Two-layered (2)  PS  Dry  27  Triolena clade  Triolena amazonica  Two-layered (2)  PS  Dry  28    Triolena obliqua  Two-layered (2)  PS  Dry  28    Triolena paleacea  Two-layered (2)  PS  Dry  26  Cambessedesia clade  Behuria glutinosa  Two-layered (2)  PS  Dry  29  Cambessedesia espora  Two-layered (2)  PS  Dry  23    Dolichoura spiritusanctensis  Two-layered (2)  PS  Dry  29    Huberia consimilis  Two-layered (2)  PS  Dry  29    Merianthera bullata  Two-layered (2–3)  PS  Dry  29    Merianthera parvifolia  Two-layered (2–3)  PS  Dry  29  Rhexieae  Arthrostemma ciliatum  Two-layered (2)  PS  Dry  30    Rhexia mariana  Two-layered (2)  12  Dry  30  Microlicieae  Chaetostoma armatum  Two-layered (2)  13  Dry  23    Microlicia cordata  Two-layered (2)  PS  Dry  31    Microlicia euphorbioides  Two-layered (2)  PS  Dry  23    Microlicia fasciculata  Two-layered (2)  PS  Dry  23    Poteranthera pusilla  Two-layered (2)  PS  Dry  32    Rhynchanthera grandiflora  Two-layered (2)  PS  Dry  23  Melastomateae  Marcetia alliance  Acanthella sprucei  Multilayered (3)  PS  Dry  30  Aciotis purpurascens  Multilayered (3)  PS  Dry  30  Acisanthera hedyotoidea  Multilayered (3)  PS  Dry  30  Acisanthera quadrata  Multilayered (3)  PS  Dry  30  Appendicularia thymifolia  Multilayered (3)  PS  Dry  30  Comolia microphylla  Multilayered (3)  PS  Dry  30  Ernestia glandulosa  Multilayered (3)  PS  Dry  30      Macairea radula  Multilayered (3)  PS  Dry  30  Marcetia taxifolia  Multilayered (3)  PS  Dry  30  Nepsera aquatica  Multilayered (3)  PS  Dry  30  Sandemania hoehnei  Multilayered (3)  PS  Dry  30  Siphanthera hostmannii  Multilayered (3)  PS  Dry  33  Core Melastomateae  Brachyotum ledifolium  Two-layered (2)  PS  Dry  30  Desmoscelis villosa  Two-layered (2)  PS  Dry  30  Heterocentron elegans  Two-layered (2)  PS  Dry  30  Melastoma malabathricum  Two-layered (2)  14  Fleshy  30  Monochaetum calcaratum  Two-layered (2)  9  Dry  30  Monochaetum ensiferum  Two-layered (2)  15  Dry  30  Monochaetum meridense  Two-layered (2)  PS  Dry  30  Osbeckia aspera  Two-layered (2)  8  Dry  26  Osbeckia brachystemon  Two-layered (2)  8  Dry  26  Osbeckia stellata  Two-layered (2)  8  Dry  25  Pterolepis glomerata  Two-layered (2)  PS  Dry  30  Tibouchina clavata  Two-layered (2)  PS  Dry  30  Tibouchina clinopodifolia  Two-layered (2)  PS  Dry  30  Tibouchina laevicaulis  Two-layered (2)  PS  Dry  30  Tibouchina nodosa  Two-layered (2)  9  Dry  30  Tibouchina semidecandra  Two-layered (2)  15  Dry  30  Merianieae    Axinaea dentata  Two-layered (2–3)  PS  Dry  34  Axinaea floribunda  Multilayered (3)  PS  Dry  34  Axinaea grandifolia  Two-layered (2–3)  PS  Dry  34  Axinaea minutiflora  Two-layered (2–3)  PS  Dry  34  Graffenrieda gracilis  Two-layered (2)  PS  Dry  33  Graffenrieda harlingii  Two-layered (2)  PS  Dry  28  Graffenrieda latifolia  Two-layered (2)  PS  Dry  33  Macrocentrum repens  Two-layered (2)  PS  Dry  35  Meriania sclerophylla  Multilayered (3)  PS  Dry  33  Meriania subumbellata  Multilayered (3)  PS  Dry  33  Meriania urceolata  Two-layered (2–3)  PS  Dry  33  Miconieae  Eriocnema clade  Eriocnema acaulis  Multilayered (3)  PS  Dry  31  Eriocnema fulva  Multilayered (3)  PS  Dry  31  Physeterostemon thomasii  Two-layered (2)  PS  Dry  36  Miconia I  Miconia dodecandra  Multilayered (3)  PS  Fleshy  23  Miconia superba  Multilayered (3)  PS  Fleshy  35  Miconia II  Miconia centrodesma  Multilayered (3)  PS  Fleshy  23  Leandra + Ossaea  Leandra reversa  Multilayered (3)  PS  Fleshy  23  Ossaea capillaris  Multilayered (3)  PS  Fleshy  28  Tococa  Tococa guianensis  Multilayered (3)  PS  Fleshy  35  Caribbean  Charianthus alpinus  Two-layered (2)  PS  Fleshy  37  Charianthus nodosus  Two-layered (2)  PS  Fleshy  37  Tetrazygia crotonifolia  Multilayered (3)  PS  Fleshy  38  Tetrazygia discolor  Two-layered (2)  PS  Fleshy  37  Tetrazygia elaeagnoides  Multilayered (3)  PS  Fleshy  37    Monopodial Clidemia  Clidemia mortoniana  Multilayered (3)  PS  Fleshy  39  Clidemia spectabilis  Multilayered (3)  PS  Fleshy  39  Conostegia  Conostegia bigibbosa  Two-layered (2)  PS  Fleshy  40  Conostegia brenesii  Two-layered (2)  PS  Fleshy  40  Conostegia consimilis  Two-layered (2)  PS  Fleshy  40  Conostegia friedmaniorum  Two-layered (2)  PS  Fleshy  40  Conostegia icosandra  Two-layered (2)  PS  Fleshy  40  Conostegia lasiopoda  Two-layered (2)  PS  Fleshy  40  Conostegia monteleagreana  Two-layered (2)  PS  Fleshy  40  Conostegia schlimii  Two-layered (2)  PS  Fleshy  40      Conostegia speciosa  Two-layered (2)  PS  Fleshy  40  Conostegia subcrustulata  Two-layered (2)  PS  Fleshy  40  Conostegia xalapensis  Two-layered (2)  PS  Fleshy  40  Mecranium, Anaectocalyx and allies  Mecranium acuminatum  Multilayered (3)  PS  Fleshy  41  Mecranium latifolium  Multilayered (3)  PS  Fleshy  41  Miconia ciliata  Multilayered (3)  PS  Fleshy  39  Tococa platyphylla  Multilayered (3)  PS  Fleshy  28  Miconia III  Leandra subseriata  Multilayered (3)  PS  Fleshy  28  Miconia crocea  Multilayered (3)  PS  Fleshy  28  Miconia denticulata  Multilayered (3)  PS  Fleshy  28  Miconia goniostigma  Multilayered (3)  PS  Fleshy  28  Miconia papillosa  Multilayered (3)  PS  Fleshy  28  Miconia stenobotrys  Multilayered (3)  PS  Fleshy  42  Miconia theizans  Multilayered (3)  PS  Fleshy  28  Miconia IV (Miconia discolor subclade)  Miconia budlejoides  Multilayered (3–4)  PS  Fleshy  23  Miconia cabucu  Multilayered (6–7)  16  Fleshy  23  Miconia capixaba  Multilayered (3–4)  PS  Fleshy  43  Miconia castaneiflora  Multilayered (4)  PS  Fleshy  43  Miconia chartacea  Multilayered (3–4)  PS  Fleshy  23  Miconia cubatanensis  Multilayered (5)  PS  Fleshy  23  Miconia discolor  Multilayered (3–4)  PS  Fleshy  23  Miconia fasciculata  Multilayered (3)  PS  Fleshy  43  Miconia hyemalis  Multilayered (3–4)  PS  Fleshy  43  Miconia lymanii  Multilayered (4)  PS  Fleshy  43  Miconia pepericarpa  Multilayered (3)  17  Fleshy  43  Miconia sclerophylla  Multilayered (4)  PS  Fleshy  43  Miconia IV  Miconia albicans  Multilayered (3)  18  Fleshy  23  Miconia donaeana  Multilayered (3)  PS  Fleshy  43  Miconia fallax  Multilayered (3)  17  Fleshy  23  Miconia laevigata  Multilayered (3)  PS  Fleshy  28  Miconia latecrenata  Multilayered (3)  PS  Fleshy  23  Miconia leucocarpa  Multilayered (3)  PS  Fleshy  23  Miconia minutiflora  Multilayered (3)  PS  Fleshy  23  Miconia rubiginosa  Multilayered (3)  PS  Fleshy  23  Miconia rufescens  Multilayered (3)  PS  Fleshy  33  Miconia serrulata  Multilayered (3)  PS  Fleshy  28  Miconia stenostachya  Multilayered (3)  PS  Fleshy  23    Miconia V  Leandra rufescens  Multilayered (3)  PS  Fleshy  35  Miconia ceramicarpa  Two-layered (2)  PS  Fleshy  35  Miconia chamissois  Multilayered (3)  PS  Fleshy  23  Miconia ibaguensis  Multilayered (3)  PS  Fleshy  23  Miconia inaequidens  Multilayered (3–4)  PS  Fleshy  23  Miconia nervosa  Multilayered (3)  PS  Fleshy  28  Miconia paucidens  Multilayered (4)  PS  Fleshy  23  Miconia prasina  Multilayered (3)  PS  Fleshy  35  Miconia pusilliflora  Multilayered (3–4)  PS  Fleshy  23  Miconia rugosa  Multilayered (3–4)  PS  Fleshy  35    Clidemia  Clidemia hirta  Multilayered (3)  PS  Fleshy  23    Leandra s.s. + Pleiochiton  Leandra aurea  Multilayered (3)  PS  Fleshy  23  Leandra cardiophylla  Multilayered (3)  8  Fleshy  42  Leandra melastomoides  Multilayered (3)  PS  Fleshy  23  Ossaea amygdaloides  Multilayered (3)  PS  Fleshy  23  Ossaea confertiflora  Multilayered (3)  PS  Fleshy  23  Pleiochiton ebracteatum  Multilayered (3)  PS  Fleshy  23  Unplaced species  Phainantha laxiflora  Two-layered (2)  PS  Dry  35  Rupestrea johnwurdackiana  Multilayered (4–5)  PS  Dry  44  Groups    Species*  Character state  Reference†  Fruit type  Reference†  Outgroups  Crypteroniaceae  Axinandra zeylanica  Two-layered (2–3)  1  Dry  19  Crypteronia paniculata  Two-layered (2)  2  Dry  19  Dactylocladus stenostachys  Two-layered (2–3)  3  Dry  19  Alzateaceae  Alzatea verticillata  Two-layered (2–4)  4  Dry  19  Penaeaceae  Olinia emarginata  Multilayered (3–4)  5  Fleshy  19  Olinia ventosa  Multilayered (3–5)  5  Fleshy  19  Penaea mucronata  Two-layered (2)  6  Dry  19  Rhynchocalyx lawsonioides  Two-layered (2)  7  Dry  19  Saltera sarcocolla  Two-layered (2)  6  Dry  19  Olisbeoideae    Memecylon heyneanum  Two-layered (2)  8  Fleshy  20      Memecylon umbellatum  Two-layered (2)  9  Fleshy  9      Memecylon sp.  Two-layered (2)  9  Fleshy  20      Mouriri acutiflora  Multilayered (5–6)  PS  Fleshy  20      Mouriri cearensis  Multilayered (5–6)  PS  Fleshy  20      Mouriri guianensis  Multilayered (5–6)  PS  Fleshy  20      Votomita guianensis  Two-layered (2–4)  PS  Fleshy  20  Kibessieae    Pternandra azurea  Two-layered (2)  9  Fleshy  21    Pternandra coerulescens  Two-layered (2)  PS  Fleshy  21  Henrietteeae  Bellucia grossularioides  Two-layered (2)  PS  Fleshy  22    Bellucia mespiloides  Two-layered (2)  PS  Fleshy  22    Henriettea ramiflora  Two-layered (2)  PS  Fleshy  22    Henriettea saldanhae  Two-layered (2)  PS  Fleshy  22    Henriettea succosa  Two-layered (2)  PS  Fleshy  22  Bertolonieae  Bertolonia mosenii  Two-layered (2)  PS  Dry  23  Blakeeae  Blakea trinervia  Two-layered (2)  9  Fleshy  23    Blakea multiflora  Two-layered (2)  PS  Fleshy  24    Chalybea calyptrata  Two-layered (2)  PS  Fleshy  24    Chalybea ecuadorensis  Two-layered (2)  PS  Fleshy  24  Dissochaeteae  Dissochaeta divaricata  Two-layered (2)  PS  Fleshy  25    Oxyspora paniculata  Two-layered (2)  10  Dry  25    Sonerila wallichii  Two-layered (2)  11  Dry  26  Cyphostyleae  Allomaieta grandiflora  Two-layered (2)  PS  Dry  27  Triolena clade  Triolena amazonica  Two-layered (2)  PS  Dry  28    Triolena obliqua  Two-layered (2)  PS  Dry  28    Triolena paleacea  Two-layered (2)  PS  Dry  26  Cambessedesia clade  Behuria glutinosa  Two-layered (2)  PS  Dry  29  Cambessedesia espora  Two-layered (2)  PS  Dry  23    Dolichoura spiritusanctensis  Two-layered (2)  PS  Dry  29    Huberia consimilis  Two-layered (2)  PS  Dry  29    Merianthera bullata  Two-layered (2–3)  PS  Dry  29    Merianthera parvifolia  Two-layered (2–3)  PS  Dry  29  Rhexieae  Arthrostemma ciliatum  Two-layered (2)  PS  Dry  30    Rhexia mariana  Two-layered (2)  12  Dry  30  Microlicieae  Chaetostoma armatum  Two-layered (2)  13  Dry  23    Microlicia cordata  Two-layered (2)  PS  Dry  31    Microlicia euphorbioides  Two-layered (2)  PS  Dry  23    Microlicia fasciculata  Two-layered (2)  PS  Dry  23    Poteranthera pusilla  Two-layered (2)  PS  Dry  32    Rhynchanthera grandiflora  Two-layered (2)  PS  Dry  23  Melastomateae  Marcetia alliance  Acanthella sprucei  Multilayered (3)  PS  Dry  30  Aciotis purpurascens  Multilayered (3)  PS  Dry  30  Acisanthera hedyotoidea  Multilayered (3)  PS  Dry  30  Acisanthera quadrata  Multilayered (3)  PS  Dry  30  Appendicularia thymifolia  Multilayered (3)  PS  Dry  30  Comolia microphylla  Multilayered (3)  PS  Dry  30  Ernestia glandulosa  Multilayered (3)  PS  Dry  30      Macairea radula  Multilayered (3)  PS  Dry  30  Marcetia taxifolia  Multilayered (3)  PS  Dry  30  Nepsera aquatica  Multilayered (3)  PS  Dry  30  Sandemania hoehnei  Multilayered (3)  PS  Dry  30  Siphanthera hostmannii  Multilayered (3)  PS  Dry  33  Core Melastomateae  Brachyotum ledifolium  Two-layered (2)  PS  Dry  30  Desmoscelis villosa  Two-layered (2)  PS  Dry  30  Heterocentron elegans  Two-layered (2)  PS  Dry  30  Melastoma malabathricum  Two-layered (2)  14  Fleshy  30  Monochaetum calcaratum  Two-layered (2)  9  Dry  30  Monochaetum ensiferum  Two-layered (2)  15  Dry  30  Monochaetum meridense  Two-layered (2)  PS  Dry  30  Osbeckia aspera  Two-layered (2)  8  Dry  26  Osbeckia brachystemon  Two-layered (2)  8  Dry  26  Osbeckia stellata  Two-layered (2)  8  Dry  25  Pterolepis glomerata  Two-layered (2)  PS  Dry  30  Tibouchina clavata  Two-layered (2)  PS  Dry  30  Tibouchina clinopodifolia  Two-layered (2)  PS  Dry  30  Tibouchina laevicaulis  Two-layered (2)  PS  Dry  30  Tibouchina nodosa  Two-layered (2)  9  Dry  30  Tibouchina semidecandra  Two-layered (2)  15  Dry  30  Merianieae    Axinaea dentata  Two-layered (2–3)  PS  Dry  34  Axinaea floribunda  Multilayered (3)  PS  Dry  34  Axinaea grandifolia  Two-layered (2–3)  PS  Dry  34  Axinaea minutiflora  Two-layered (2–3)  PS  Dry  34  Graffenrieda gracilis  Two-layered (2)  PS  Dry  33  Graffenrieda harlingii  Two-layered (2)  PS  Dry  28  Graffenrieda latifolia  Two-layered (2)  PS  Dry  33  Macrocentrum repens  Two-layered (2)  PS  Dry  35  Meriania sclerophylla  Multilayered (3)  PS  Dry  33  Meriania subumbellata  Multilayered (3)  PS  Dry  33  Meriania urceolata  Two-layered (2–3)  PS  Dry  33  Miconieae  Eriocnema clade  Eriocnema acaulis  Multilayered (3)  PS  Dry  31  Eriocnema fulva  Multilayered (3)  PS  Dry  31  Physeterostemon thomasii  Two-layered (2)  PS  Dry  36  Miconia I  Miconia dodecandra  Multilayered (3)  PS  Fleshy  23  Miconia superba  Multilayered (3)  PS  Fleshy  35  Miconia II  Miconia centrodesma  Multilayered (3)  PS  Fleshy  23  Leandra + Ossaea  Leandra reversa  Multilayered (3)  PS  Fleshy  23  Ossaea capillaris  Multilayered (3)  PS  Fleshy  28  Tococa  Tococa guianensis  Multilayered (3)  PS  Fleshy  35  Caribbean  Charianthus alpinus  Two-layered (2)  PS  Fleshy  37  Charianthus nodosus  Two-layered (2)  PS  Fleshy  37  Tetrazygia crotonifolia  Multilayered (3)  PS  Fleshy  38  Tetrazygia discolor  Two-layered (2)  PS  Fleshy  37  Tetrazygia elaeagnoides  Multilayered (3)  PS  Fleshy  37    Monopodial Clidemia  Clidemia mortoniana  Multilayered (3)  PS  Fleshy  39  Clidemia spectabilis  Multilayered (3)  PS  Fleshy  39  Conostegia  Conostegia bigibbosa  Two-layered (2)  PS  Fleshy  40  Conostegia brenesii  Two-layered (2)  PS  Fleshy  40  Conostegia consimilis  Two-layered (2)  PS  Fleshy  40  Conostegia friedmaniorum  Two-layered (2)  PS  Fleshy  40  Conostegia icosandra  Two-layered (2)  PS  Fleshy  40  Conostegia lasiopoda  Two-layered (2)  PS  Fleshy  40  Conostegia monteleagreana  Two-layered (2)  PS  Fleshy  40  Conostegia schlimii  Two-layered (2)  PS  Fleshy  40      Conostegia speciosa  Two-layered (2)  PS  Fleshy  40  Conostegia subcrustulata  Two-layered (2)  PS  Fleshy  40  Conostegia xalapensis  Two-layered (2)  PS  Fleshy  40  Mecranium, Anaectocalyx and allies  Mecranium acuminatum  Multilayered (3)  PS  Fleshy  41  Mecranium latifolium  Multilayered (3)  PS  Fleshy  41  Miconia ciliata  Multilayered (3)  PS  Fleshy  39  Tococa platyphylla  Multilayered (3)  PS  Fleshy  28  Miconia III  Leandra subseriata  Multilayered (3)  PS  Fleshy  28  Miconia crocea  Multilayered (3)  PS  Fleshy  28  Miconia denticulata  Multilayered (3)  PS  Fleshy  28  Miconia goniostigma  Multilayered (3)  PS  Fleshy  28  Miconia papillosa  Multilayered (3)  PS  Fleshy  28  Miconia stenobotrys  Multilayered (3)  PS  Fleshy  42  Miconia theizans  Multilayered (3)  PS  Fleshy  28  Miconia IV (Miconia discolor subclade)  Miconia budlejoides  Multilayered (3–4)  PS  Fleshy  23  Miconia cabucu  Multilayered (6–7)  16  Fleshy  23  Miconia capixaba  Multilayered (3–4)  PS  Fleshy  43  Miconia castaneiflora  Multilayered (4)  PS  Fleshy  43  Miconia chartacea  Multilayered (3–4)  PS  Fleshy  23  Miconia cubatanensis  Multilayered (5)  PS  Fleshy  23  Miconia discolor  Multilayered (3–4)  PS  Fleshy  23  Miconia fasciculata  Multilayered (3)  PS  Fleshy  43  Miconia hyemalis  Multilayered (3–4)  PS  Fleshy  43  Miconia lymanii  Multilayered (4)  PS  Fleshy  43  Miconia pepericarpa  Multilayered (3)  17  Fleshy  43  Miconia sclerophylla  Multilayered (4)  PS  Fleshy  43  Miconia IV  Miconia albicans  Multilayered (3)  18  Fleshy  23  Miconia donaeana  Multilayered (3)  PS  Fleshy  43  Miconia fallax  Multilayered (3)  17  Fleshy  23  Miconia laevigata  Multilayered (3)  PS  Fleshy  28  Miconia latecrenata  Multilayered (3)  PS  Fleshy  23  Miconia leucocarpa  Multilayered (3)  PS  Fleshy  23  Miconia minutiflora  Multilayered (3)  PS  Fleshy  23  Miconia rubiginosa  Multilayered (3)  PS  Fleshy  23  Miconia rufescens  Multilayered (3)  PS  Fleshy  33  Miconia serrulata  Multilayered (3)  PS  Fleshy  28  Miconia stenostachya  Multilayered (3)  PS  Fleshy  23    Miconia V  Leandra rufescens  Multilayered (3)  PS  Fleshy  35  Miconia ceramicarpa  Two-layered (2)  PS  Fleshy  35  Miconia chamissois  Multilayered (3)  PS  Fleshy  23  Miconia ibaguensis  Multilayered (3)  PS  Fleshy  23  Miconia inaequidens  Multilayered (3–4)  PS  Fleshy  23  Miconia nervosa  Multilayered (3)  PS  Fleshy  28  Miconia paucidens  Multilayered (4)  PS  Fleshy  23  Miconia prasina  Multilayered (3)  PS  Fleshy  35  Miconia pusilliflora  Multilayered (3–4)  PS  Fleshy  23  Miconia rugosa  Multilayered (3–4)  PS  Fleshy  35    Clidemia  Clidemia hirta  Multilayered (3)  PS  Fleshy  23    Leandra s.s. + Pleiochiton  Leandra aurea  Multilayered (3)  PS  Fleshy  23  Leandra cardiophylla  Multilayered (3)  8  Fleshy  42  Leandra melastomoides  Multilayered (3)  PS  Fleshy  23  Ossaea amygdaloides  Multilayered (3)  PS  Fleshy  23  Ossaea confertiflora  Multilayered (3)  PS  Fleshy  23  Pleiochiton ebracteatum  Multilayered (3)  PS  Fleshy  23  Unplaced species  Phainantha laxiflora  Two-layered (2)  PS  Dry  35  Rupestrea johnwurdackiana  Multilayered (4–5)  PS  Dry  44  *Authors for species names can be found in Supporting Information Appendix S1. †References: 1, Tobe & Raven (1983b); 2, Tobe & Raven (1987a); 3, Tobe & Raven (1987b); 4, Tobe & Raven (1984a); 5, Tobe & Raven (1984b); 6, Tobe & Raven (1984c); 7, Tobe & Raven (1984d); 8, Subramanyam (1942); 9, Corner (1976); 10, Subramanyam (1951); 11, Subramanyam (1944); 12, Etheridge & Herr (1968); 13, Ribeiro et al. (2015); 14, Subramanyam (1948); 15, Ziegler (1925); 16, Medeiros & Morretes (1996); 17, Caetano (2010); 18, Caetano et al. (2013); 19, Dahlgren & Thorne (1984); 20, Morley (1976); 21, Maxwell (1981); 22, Penneys et al. (2010); 23, Martins et al. (2009); 24, Penneys & Judd (2013); 25, Jie & Renner (2007); 26, Triana (1871); 27, Michelangeli et al. (2011); 28, Wurdack (1980); 29, Goldenberg et al. (2012); 30, Michelangeli et al. (2013); 31, Cogniaux (1891); 32, Rocha et al. (2016b); 33, Wurdack (1973); 34, Cotton, Borchsenius & Balslev (2014); 35, Berry et al. (2001); 36, Amorim et al. (2009); 37, Michelangeli et al. (2004); 38, Judd & Ionta (2013); 39, Almeda (2009); 40, Kriebel (2016); 41, Skean (1993); 42, Michelangeli et al. (2008); 43, Caddah (2013); 44, Goldenberg et al. (2015); PS, present study. View Large Anatomical analysis We sampled ovules from pre-anthesis buds or anthetic flowers. The samples were obtained from fixed material later preserved in ethanol or from rehydrated herbarium specimens (Appendix 1; Smith & Smith, 1942). The thickness of the outer ovule integument was characterized by counting the number of integument cell layers in transverse and/or longitudinal sections of the median region of the ovule. The micropylar region was not considered for this definition because in this area the integumental cells may divide periclinally, forming additional layers (Subramanyam, 1942, 1948, 1951; Corner, 1976; Medeiros & Morretes, 1996; Caetano et al., 2013; Ribeiro et al., 2015). To assess the origin of multilayered outer ovule integument, some species were selected for observation of the initiation and development of the integuments (whether dermal or subdermal according to Bouman, 1984): Acisanthera quadrata Pers. (Melastomateae), Clidemia hirta (L.) D.Don, Leandra aurea (Cham.) Cogn., Miconia fallax DC., M. leucocarpa DC., M. pepericarpa Mart. ex DC. and Ossaea confertiflora (DC.) Triana (Miconieae). In these plants, we analysed ovules from flower buds in different developmental phases and from anthetic flowers. The material was fixed in 2% glutaraldehyde and 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 6.8, for 24 h (modified from McDowell & Trump, 1976). All samples were embedded in resin (Leica Microsystems, Heidelberg, Germany, and Heraeus Kulzer GmbH, Germany) and transversally and longitudinally sectioned with an AO Spencer 820 (GMI Inc., Ramsey, MN, USA) or RM 2245 (Leica) rotary microtome with a steel blade. Sections of c. 5.0 μm were adhered to glass slides and stained with 0.05% Toluidine Blue in 0.1 M sodium phosphate buffer, pH 6.8 (O’Brien, Feder & McCully, 1964). Observations and photomicrographs were made with a DM 5000 light microscope coupled to a DFC 390 (both Leica Microsystems) digital camera or with a Zeiss Axioplan compound microscope with a Nikon DXM1200C digital camera. The outer ovule integument was classified as two-cell-layered when formed by two cell layers or variations on this condition (two or three or two to four cell layers in the same ovule) or multilayered when consisting of three or more cell layers (Tobe, 1989; Endress, 2011). Phylogenetic reconstruction Tree inference and divergence time estimation were performed under a Bayesian framework implemented in BEAST 1.8.0 (Drummond et al., 2012). Molecular data for the species of interest were gathered from the NCBI database (GenBank), where the most common markers available across our sampled taxa were selected. The molecular data set includes six plastid and two nuclear markers, the plastome being represented by three intergenic spacers (accD-psaI, psbK-psbI and trnS-trnG), two protein-coding genes (ndhF and rbcL) and one intron (rpl16), and nuclear DNA being represented by two ribosomal spacers (nrETS and nrITS). GenBank accessions for all sequences included in this analysis are available in Supporting Information Appendix S2. Phylogenetic inference was performed on 160 terminals, including nine species from the Crypteroniaceae–Alzateaceae–Penaeaceae (CAP) clade treated as the outgroup (Conti et al., 2002; Berger et al., 2016). Individual loci were aligned with MAFFT 7.3 (Katoh & Standley, 2013) using the G-INS-i strategy. The best DNA partitioning scheme and models were estimated with PartitionFinder2 (Lanfear et al., 2012) under the Bayesian information criterion (BIC). The best scheme resulted in five partitions (accD-psaI, ndhF plus rpl16; nrETS; nrITS; psbK-psbI plus trnS-trnG; rbcL). The GTR+G model was recovered as the best fit for all partitions. The molecular clock prior was set to the lognormal uncorrelated model and the tree prior was set to the birth-death model. Fossil and secondary calibration constraints were placed in three nodes: (1) Melastomataceae crown (prior = lognormal, mean 1.5, SD 1, offset 56), based on the Palaeocene leaf fossil Melastomites montanensis R.W.Brown (Brown, 1962); (2) Rhexia Gronov. + Arthrostemma Pav. ex D.Don crown (prior = lognormal, mean 1.5, SD 1, offset 23), based on Miocene seed fossils (Collinson & Pingen, 1992); and (3) CAP clade crown (prior normal, mean 52.7, SD 6), based on the estimated age recovered in a wider analysis of Myrtales (Berger et al., 2016). We performed three independent runs of 30 million generations each, sampling every 1000 generations, and the stable posterior distributions of the independent runs were combined with LogCombiner v.1.8.0. Convergence was assessed using Tracer v.1.6 (Rambaut et al., 2014), and runs were considered satisfactory with effective sample size (ESS) values greater than 200. A maximum clade credibility tree was generated with TreeAnnotator v.1.8.0. Overall, both topology and divergence times were congruent with recently published hypotheses (Penneys & Judd, 2005, 2013; Stone, 2006; Goldenberg et al., 2008, 2015; Penneys et al., 2010; Caddah, 2013; Michelangeli et al., 2013; Kriebel, Michelangeli & Kelly, 2015; Meirelles, 2015; Berger et al., 2016; Reginato, 2016; Rocha et al., 2016a). Ancestral state reconstruction and correlated evolution Ancestral state reconstruction of the outer integument thickness in Melastomataceae was inferred using stochastic character mapping. This character was treated as discrete and unordered, i.e. all transitions among the states of the character can occur at equal probabilities. The test for the correlated evolution of the outer integument thickness and fruit type in this family was performed using Pagel’s model for binary characters (Pagel, 1994; Pagel & Meade, 2006). The fruit type considered (dry or fleshy) was based on literature (see Table 1). All analyses were carried out using the packages ape (Paradis, Claude & Strimmer, 2004) and phytools (Revell, 2012) in the R 3.2.5 environment (http://www.r-project.org/). To visualize phenotypic evolution on the trees, we mapped character histories across phylogenetic trees using a colour scheme (Revell, 2013). RESULTS Integument initiation and development The multilayered outer integument was derived from both dermal and subdermal layers in Acisanthera quadrata, Clidemia hirta, Leandra aurea, Miconia fallax, M. leucocarpa, M. pepericarpa and Ossaea confertiflora. In all seven studied species, the initiation and development of the integuments occur simultaneously by means of periclinal divisions of epidermal cells located at the base of the ovule primordium, characterizing it as a subdermal integument (Fig. 1A–E). Figure 1. View largeDownload slide Longitudinal sections showing the initiation (indicated by arrows) and development of the integuments in Melastomataceae species with a multilayered outer ovule integument. A–B, Leandra aurea. C, Clidemia hirta. D, Miconia pepericarpa. E, Ossaea confertiflora. F, L. aurea. G–H, M. fallax. I, L. aurea. J, C. hirta. K, L. aurea. L, Miconia leucocarpa. M, M. pepericarpa. N, M. fallax. Figure 1. View largeDownload slide Longitudinal sections showing the initiation (indicated by arrows) and development of the integuments in Melastomataceae species with a multilayered outer ovule integument. A–B, Leandra aurea. C, Clidemia hirta. D, Miconia pepericarpa. E, Ossaea confertiflora. F, L. aurea. G–H, M. fallax. I, L. aurea. J, C. hirta. K, L. aurea. L, Miconia leucocarpa. M, M. pepericarpa. N, M. fallax. The outer integument grows more rapidly than the inner one and extends beyond the apex of the nucellus first (Fig. 1F–H). However, when the megagametophyte starts its development, both integuments already cover the entire nucellus, forming a ‘zig-zag’ micropyle (Fig. 1I, J). Starting from this stage, the integuments extend, accompanying the growth of the rest of the ovule until its maturity (Fig. 1K–N). Outer ovule integument thickness In ten of the 14 major lineages sampled, the species had only ovules with a two-cell-layered outer integument (Table 1; Fig. 2): Kibessieae (Fig. 3A), Henrietteeae (Fig. 3B, C), Bertolonieae (Fig. 3D), Blakeeae (Fig. 3E), Dissochaeteae, Cyphostyleae (Fig. 3F), Triolena Naudin clade (Fig. 4A), Cambessedesia DC. clade (Fig. 4B–D), Rhexieae (Fig. 4E) and Microlicieae (Fig. 4F). Figure 2. View largeDownload slide Stochastic mapping of outer ovule integument thickness onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using six plastid and two nuclear makers. Red corresponds to the multilayered status, and blue to the two-cell-layered status. Changes in colour along the branches indicate transitions between characters states. Mya, million years ago; Pli, Pliocene; Qa, Quartenary. Figure 2. View largeDownload slide Stochastic mapping of outer ovule integument thickness onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using six plastid and two nuclear makers. Red corresponds to the multilayered status, and blue to the two-cell-layered status. Changes in colour along the branches indicate transitions between characters states. Mya, million years ago; Pli, Pliocene; Qa, Quartenary. Figure 3. View largeDownload slide Transverse (A–D and F) and longitudinal (E) sections showing ovules of Melastomataceae species belonging to tribes that exhibit exclusively a two-cell-layered outer integument. A, Pternandra coerulescens (Kibessieae). B, Bellucia grossularioides (Henrietteeae). C, Henriettea ramiflora (Henrietteeae). D, Bertolonia mosenii (Bertolonieae). E, Chalybea ecuadorensis (Blakeeae). F, Allomaieta grandiflora (Cyphostyleae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 3. View largeDownload slide Transverse (A–D and F) and longitudinal (E) sections showing ovules of Melastomataceae species belonging to tribes that exhibit exclusively a two-cell-layered outer integument. A, Pternandra coerulescens (Kibessieae). B, Bellucia grossularioides (Henrietteeae). C, Henriettea ramiflora (Henrietteeae). D, Bertolonia mosenii (Bertolonieae). E, Chalybea ecuadorensis (Blakeeae). F, Allomaieta grandiflora (Cyphostyleae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 4. View largeDownload slide Transverse (A, C and E–F) and longitudinal (B, D) sections showing ovules of Melastomataceae species belonging to tribes or clades that exhibit exclusively a two-cell-layered outer integument. A, Triolena obliqua (Triolena clade). B, Cambessedesia espora (Cambessedesia clade). C, Merianthera bullata (Cambessedesia clade). D, Merianthera parvifolia (Cambessedesia clade). In C and D, punctual periclinal divisions may give rise to a third layer in certain regions of the outer integument (arrows). E, Arthrostemma ciliatum (Rhexieae). F, Microlicia cordata (Microlicieae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 4. View largeDownload slide Transverse (A, C and E–F) and longitudinal (B, D) sections showing ovules of Melastomataceae species belonging to tribes or clades that exhibit exclusively a two-cell-layered outer integument. A, Triolena obliqua (Triolena clade). B, Cambessedesia espora (Cambessedesia clade). C, Merianthera bullata (Cambessedesia clade). D, Merianthera parvifolia (Cambessedesia clade). In C and D, punctual periclinal divisions may give rise to a third layer in certain regions of the outer integument (arrows). E, Arthrostemma ciliatum (Rhexieae). F, Microlicia cordata (Microlicieae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. In the Cambessedesia clade, although the outer integuments of Merianthera bullata R.Goldenb., Fraga & A.P.Fontana and M. parvifolia R.Goldenb., Fraga & A.P.Fontana are mostly two-cell-layered, punctual periclinal divisions led to the formation of a third cell layer in some regions of the ovule and indicate a multiplicative integument in Merianthera Kuhlm. (Fig. 4C, D). Ovules with a multilayered outer integument occur in some species of the remaining groups: Olisbeoideae, Melastomateae, Merianieae and Miconieae (Table 1; Fig. 2). Rupestrea johnwurdackiana (Baumgratz & D’El Rei Souza) Michelang., Almeda, & R.Goldenb., not yet formally placed in any tribe in the family, also has a multilayered outer integument with four to five cell layers, possibly because of a multiplicative origin (Table 1; Figs 2, 5A, B). Figure 5. View largeDownload slide Longitudinal (A–E) and transverse (F–G) sections showing ovules of Rupestrea and Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. A–B, multilayered integument consisting of four to five cell layers in Rupestrea johnwurdackiana. C, two-cell-layered integument of Votomita guianensis (Olisbeoideae) with a thicker calazal region (arrow). D, multilayered integument of Mouriri cearensis (Olisbeoideae). Two-cell-layered integument in species of core Melastomateae (Melastomateae). E, Heterocentron elegans; F, Tibouchina clavata; G, multilayered integument with three cell layers in Aciotis purpurascens (Marcetia alliance, Melastomateae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 5. View largeDownload slide Longitudinal (A–E) and transverse (F–G) sections showing ovules of Rupestrea and Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. A–B, multilayered integument consisting of four to five cell layers in Rupestrea johnwurdackiana. C, two-cell-layered integument of Votomita guianensis (Olisbeoideae) with a thicker calazal region (arrow). D, multilayered integument of Mouriri cearensis (Olisbeoideae). Two-cell-layered integument in species of core Melastomateae (Melastomateae). E, Heterocentron elegans; F, Tibouchina clavata; G, multilayered integument with three cell layers in Aciotis purpurascens (Marcetia alliance, Melastomateae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. In Olisbeoideae, the outer ovule integument is two-cell-layered in Memecylon L. and in Votomita guianensis Aubl. (Fig. 5C). In the latter taxon, this integument is multiplicative and its cells undergo periclinal divisions, forming up to four cell layers in the chalazal region (Fig. 5C). On the other hand, in all species of Mouriri Aubl. sampled the outer integument is multilayered, with four to six cell layers (Fig. 5D). In Melastomateae, the outer integument is two-cell-layered in species of core Melastomateae (Fig. 5E, F). It is multilayered with three cell layers in species of the Marcetia DC. alliance (Figs 5G, 6A, B). Figure 6. View largeDownload slide Transverse (A and C–F) and longitudinal (B) sections showing ovules of Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. Multilayered integument with three cell layers in species of the Marcetia alliance, Melastomateae: A, Ernestia glandulosa; B, Nepsera aquatica. Outer integument thickness in Merianieae: C, Graffenrieda harlingii; D, Axinaea dentata; E, Meriania urceolata; F, Meriania sclerophylla. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 6. View largeDownload slide Transverse (A and C–F) and longitudinal (B) sections showing ovules of Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. Multilayered integument with three cell layers in species of the Marcetia alliance, Melastomateae: A, Ernestia glandulosa; B, Nepsera aquatica. Outer integument thickness in Merianieae: C, Graffenrieda harlingii; D, Axinaea dentata; E, Meriania urceolata; F, Meriania sclerophylla. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. In Merianieae, a two-cell-layered outer integument was observed in Graffenrieda gracilis (Triana) L.O.Williams, G. harlingii Wurdack (Fig. 6C), G. latifolia (Naudin) Triana and Macrocentrum repens (Gleason) Wurdack. An originally two-cell-layered integument, although with a third cell layer formed in some regions, occurs in Axinaea dentata E.Cotton (Fig. 6D), A. grandifolia (Naudin) Triana, A. minutiflora E.Cotton and Meriania urceolata Triana (Fig. 6E). In Axinaea floribunda (Naudin) Triana, Meriania sclerophylla (Naudin) Triana (Fig. 6F) and M. subumbellata Cogn., the outer integument is multilayered, with three cell layers. Most species of Miconieae have ovules with multilayered outer integuments (Table 1) (Fig. 7A–C). The thickness of this integument ranges from three to seven cell layers, but three layers were most frequent (Table 1) (Fig. 7A). More than three cell layers were observed only in species of the Miconia Ruiz & Pav. IV clade (Fig. 7B) and in the Miconia V grade (Fig. 7C) (Table 1), with evidence of a multiplicative integument. The two-cell-layered condition was observed only in the Eriocnema Naudin and Conostegia D.Don clades and in the Caribbean and Miconia V grades. In the Eriocnema clade, the outer integument is two-cell-layered in Physeterostemon thomasii Amorim, Michelangeli & Goldenb. (Fig. 7D), but multilayered with three cell layers in Eriocnema acaulis Triana (Fig. 7E) and E. fulva Naudin. All species belonging to the Conostegia clade have ovules with a two-cell-layered outer integument (Fig. 7F, G). In the Caribbean grade, Charianthus alpinus (Sw.) R.A.Howard, C. nodosus (Desr.) Triana (Fig. 7H) and Tetrazygia discolor (L.) DC. (Fig. 7I) have two-cell-layered outer integuments, whereas in Tetrazygia crotonifolia (Desr.) DC. (Fig. 7J) and T. elaegnoides (Sw.) DC. it is multilayered, with three cell layers. In the Miconia V grade, a two-cell-layered outer integument was observed only in M. ceramicarpa (DC.) Cogn. Figure 7. View largeDownload slide Longitudinal (A–C and G) and transverse (D–F and H–J) sections showing ovules of Melastomataceae species belonging to the Miconieae tribe. A, multilayered integument with three cell layers in Clidemia hirta. B, multilayered integument with five cell layers in Miconia cubatanensis. C, multilayered integument with three to four cell layers in Miconia inaequidens. D, two-cell-layered integument in Physeterostemon thomasii. E, multilayered integument with three cell layers in Eriocnema acaulis. Two-cell-layered integument in species of the Conostegia clade: F, Conostegia lasiopoda; G, Conostegia subcrustulata. H, two-cell-layered integument in Charianthus nodosus. I, two-cell-layered integument in Tetrazygia discolor. J, multilayered integument with three cell layers in Tetrazygia crotonifolia. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 7. View largeDownload slide Longitudinal (A–C and G) and transverse (D–F and H–J) sections showing ovules of Melastomataceae species belonging to the Miconieae tribe. A, multilayered integument with three cell layers in Clidemia hirta. B, multilayered integument with five cell layers in Miconia cubatanensis. C, multilayered integument with three to four cell layers in Miconia inaequidens. D, two-cell-layered integument in Physeterostemon thomasii. E, multilayered integument with three cell layers in Eriocnema acaulis. Two-cell-layered integument in species of the Conostegia clade: F, Conostegia lasiopoda; G, Conostegia subcrustulata. H, two-cell-layered integument in Charianthus nodosus. I, two-cell-layered integument in Tetrazygia discolor. J, multilayered integument with three cell layers in Tetrazygia crotonifolia. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Ancestral state reconstruction Stochastic mapping analyses indicate the most recent common ancestor of Melastomataceae as probably having an ovule with a two-cell-layered outer integument. Ovules with a multilayered outer integument evolved independently at least six times: (1) in the ancestor of Mouriri; (2) in the ancestor of Rupestrea R.Goldenb., Almeda & Michelang.; (3) in the ancestor of the Marcetia alliance; (4) in the ancestor of a clade in Merianieae; (5) in Axinaea floribunda; and (6) in the ancestor of Miconieae. However, multiple reversals from multilayered to two-cell-layered integuments apparently also occurred independently in Merianieae in Meriania urceolata and in Miconieae in Physeterostemon R.Goldenb. & Amorim, in a small clade in the Caribbean grade, in the Conostegia clade and in Miconia ceramicarpa (Fig. 2). A test for correlated evolution showed that outer ovule integument thickness is not significantly correlated with fruit type in the family (Fig. 8; likelihood ratio: 2.1037; P = 0.35). DISCUSSION Different ontogenetic pathways gave origin to the outer integument in Melastomataceae Multilayered ovule integuments may originate from the dermal layer and become thicker by periclinal division of epidermal cells (dermal integuments), or from both the dermal and the subdermal layers (subdermal integuments) (Bouman, 1984; Endress, 2011). At least in Miconieae and in the Marcetia alliance (Melastomateae), the multilayered outer integument is classified as ‘subdermal’ because it derives from the dermal and subdermal layers of the ovule primordium (Bouman, 1984; Shamrov, 2000). This pattern contrasts with the dermal origin typically observed in species with a two-cell-layered outer integument (Bouman, 1984; Endress, 2011), a condition that is found in most Melastomataceae. Although dermal and subdermal integuments can become multiplicative and produce multilayered integuments, the different ontogenetic patterns observed support the distinct nature of these two character states (two-cell-layered and multilayered outer integument) and their use in the delimitation of clades in Melastomataceae. Outer ovule integument thickness as a taxonomic marker for Melastomataceae clades During the evolutionary history of Melastomataceae, the thickness of the outer ovule integument has apparently remained relatively stable, enabling us to recognize many clades in the family. Ovules with a two-cell-layered outer integument are probably a plesiomorphic condition in Melastomataceae and occur in most of the analysed lineages. However, ovules with a multilayered outer integument evolved independently in Mouriri, Rupestrea, the Marcetia alliance, Merianieae and Miconieae. Moreover, reversals to the plesiomorphic two-cell-layered integument condition have also occurred more than once. In Olisbeoideae, Mouriri differs from Memecylon and Votomita Aubl. by having a multilayered outer integument. Molecular data support each of these genera as monophyletic (as well as three other genera in the subfamily) and show that the Neotropical genera Mouriri and Votomita are sister groups (Stone, 2006, 2014). Additional data on the outer integument thickness of Lijndenia Zoll. & Moritzi, Spathandra Guill. & Perr. and Warneckea Gilg are necessary to establish whether the thick outer integument found in Olisbeoideae is a single condition observed in Mouriri or if it is homoplastic. Regardless, this character state could be used to separate Mouriri from Votomita from an embryological standpoint. The presence of a multilayered and considerably thick outer integument characterizes and delimits Rupestrea. This genus of two species was positioned as sister to Microlicieae in our analysis, although recently placed with low support as sister to the Microlicieae + Melastomateae + Rhexieae clade (Goldenberg et al., 2015). In Melastomateae, the three-layered outer integument is a synapomorphy for the Marcetia alliance as species in core Melastomateae and the closely associated tribes Rhexieae and Microlicieae have two cell layers, although Rupestrea has a multilayered integument. Microlicieae, Rhexieae, the Marcetia alliance and core Melastomateae form a large monophyletic group and share stamens with elongated pedoconnectives and capsular fruits, but their relationships are still being debated (Michelangeli et al., 2013; Rocha et al., 2016a). So far, the Marcetia alliance has been characterized by tetramerous flowers (with some exceptions), a tetralocular ovary (or reduced to two or three locules), absence of crown hairs on the ovary apex, and by cochleate, ovate or lacrimiform seeds (Michelangeli et al., 2013; Rocha et al., 2016a). The considerable variation in the outer ovule integument thickness and the absence of a broad sampling in Merianieae limit the utility of our data for systematic conclusions at this time. In this tribe, although the shape of the calyx and stamens has been traditionally used in taxonomy, these floral characters have proved to be poorly informative for the circumscription of the more diverse genera (Mendoza-Cifuentes & Fernández-Alonso, 2010, 2012). In this respect, embryological characters such as the one tested here can provide important information for clade delimitation. The variation detected in Merianieae indicates that this character should be considered and more extensively investigated in future systematic studies on the tribe. Finally, in Miconieae, despite the considerably frequent multilayered condition, ovules with a two-cell-layered outer integument delimit the genus Physeterostemon and two clades. Molecular data indicate that Physeterostemon is a well-supported monophyletic genus sister to Eriocnema, the two forming a clade sister to the remaining Miconieae (Amorim et al., 2009; Goldenberg et al., 2015). Physeterostemon also differs from Eriocnema by its inferior ovary (Cogniaux, 1891; Fritsch et al., 2004; Goldenberg & Amorim, 2006; Amorim et al., 2009; Amorim, Jardim & Goldenberg, 2014). Both Eriocnema and Physeterostemon have capsular fruits, different from the fleshy fruits found in other Miconieae (Amorim et al., 2009). In the Caribbean grade, the clade with Charianthus D.Don and Tetrazygia discolor differs from the remaining species of Tetrazygia Rich. ex DC. by having a two-cell-layered outer integument. This clade stands out from the grade because of its biogeography, consisting of endemic species of the Lesser Antilles (Michelangeli et al., 2008), but the relationships in the group remain undefined (Penneys & Judd, 2005; Goldenberg et al., 2008). The two-cell-layered condition of the outer ovule integument can be seen as a synapomorphy for the Conostegia clade because its sister group, the monopodial Clidemia D.Don clade, has a multilayered outer integument. The Conostegia clade, which included species of Clidemia, Conostegia and Miconia (currently recognized as Conostegia) (Kriebel, 2016), is well supported by molecular analyses, but thus far no morphological or anatomical character has been found to diagnose the group (Kriebel et al., 2015). Relationship between the multilayered outer integument and fleshy fruits in Melastomataceae Although we hypothesized that the outer ovule integument thickness would be correlated with fruit type, the results failed to achieve significance. Nonetheless, ancestral state reconstructions show a pattern in which a multilayered outer integument tends to occur more frequently among clades with fleshy fruits, such as the Miconieae and Mouriri (Fig. 8). Although a lack of significance in this kind of analysis might be related to the small number of changes in character states over the phylogeny (Felsenstein, 1985), the results may also indicate a more complex scenario for the evolution of this anatomical character. Figure 8. View largeDownload slide Mirror composite trees showing the stochastic mapping of outer ovule integument thickness (A) and fruit type (B) onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using molecular makers. Tree A (outer ovule integument thickness): blue corresponds to the two-cell-layered status, and red to the multilayered status. Tree B (fruit type): blue corresponds to the dry status, and red to the fleshy status. Figure 8. View largeDownload slide Mirror composite trees showing the stochastic mapping of outer ovule integument thickness (A) and fruit type (B) onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using molecular makers. Tree A (outer ovule integument thickness): blue corresponds to the two-cell-layered status, and red to the multilayered status. Tree B (fruit type): blue corresponds to the dry status, and red to the fleshy status. Fleshy fruits in Melastomataceae are mainly dispersed by endozoochory by birds, but also by other animals such as rats, bats, monkeys and fishes (Magnusson & Sanaiotti, 1987; Renner, 1989; Stiles & Rosselli, 1993; Ellison et al., 1993; Galetti & Stotz, 1996; de Figueiredo & Longatti, 1997; Garcia, Rezende & Aguiar, 2000; Lapenta & Procópio-de-Oliveira, 2008; Maruyama, et al., 2013). Since the seed testa is the mechanical layer responsible for the protection of the embryo in the family (Corner, 1976; Cortez & Carmello-Guerreiro, 2008), the presence of more layers in the outer ovule integument may correspond to more sclerified layers in the seed coat, which in turn could make this seed coat thicker and more rigid. In Miconia albicans (Sw.) Triana, for example, the three-layered outer ovule integument gives rise to the testa, which consists of an exotesta of sclerified palisade cells covering the meso- and endotesta with cuboidal sclerified cells (Cortez & Carmello-Guerreiro, 2008). Endozoochory has been frequently related to a resistant seed coat (Baskin & Baskin, 2014), responsible for the protection of the embryo and of other seed components during passage through the digestive tract of animals (Boesewinkel & Bouman, 1984; Mohamed-Yasseen et al., 1994). Thus, the presence of an additional layer in the outer ovule integument may have been an important evolutionary step, particularly in Miconieae and Mouriri. Apart from these clades, endozoochoric fleshy fruits have also evolved independently in Blakeeae, Dissochaeteae and Henrietteeae (Renner, 1989; Stiles & Rosselli, 1993; Clausing, Meyer & Renner, 2000; Penneys et al., 2010), all tribes with a two-cell-layered outer ovule integument, proving that the multilayered condition is not strictly associated with fleshy fruits. In addition to the multilayered outer integument, the presence of a multiplicative integument can also lead to thicker seed coats, as reported for Memecylon (Olisbeoideae) (Corner, 1976). In Blakea trinervia L. (Blakeeae), the seed coat is apparently non-multiplicative (Corner, 1976), but we do not know whether this pattern is repeated through the entire tribe. There is no anatomical information on the seed coat structure for Dissochaeteae and Henrietteeae. The relationship between ovules with a multilayered outer integument and fleshy fruits seems to occur in the sister group of Melastomataceae. Crypteroniaceae, Alzateaceae and Penaeaceae mostly have ovules with a two-cell-layered outer integument and capsular fruits (Meijer, 1972; Tobe & Raven, 1983b, 1984a, c, d, 1987a, b; Conti et al., 2002). The only exception is Olinia Thunb. (Penaeaceae), with multilayered outer integument and fleshy fruits (Rao & Dahlgren, 1969; Tobe & Raven, 1984b; Conti et al., 2002). Multiplicative outer integument and large seeds in Melastomataceae Indications of a multiplicative integument were observed in Votomita (Olisbeoideae), in species of the Miconia discolor subclade and of the Miconia V grade (Miconieae), in Merianthera (Cambessedesia clade) and in Rupestrea. In general, ovules that become large seeds have multiplicative integuments, which give rise to thicker and massive seed coats (Corner, 1976; Boesewinkel & Bouman, 1995). We presume that this relationship also exists in different lineages of Melastomataceae, independent of the initial condition, as it was found in species with multilayered and two-cell-layered outer ovule integuments. Olisbeoideae are known to have few ovules that will develop into one or several large seeds (Morley, 1976, 1999; Bremer, 1981; Clausing & Renner, 2001). In this subfamily, in addition to the multiplicative integument detected in Votomita, the same structure has been reported for Mouriri and Memecylon (Corner, 1976). In Miconieae, a multiplicative integument consisting of more than three cell layers is a recurrent condition exclusively detected in species belonging to the Miconia discolor subclade (Miconia IV clade) and the Miconia V grade. The Miconia discolor subclade and some species of the Miconia V grade are notable for their fruits with few large seeds (Goldenberg, 1999; Martins et al., 2009; Caddah, 2013; Ocampo & Almeda, 2013; Ocampo, Michelangeli & Almeda, 2014), which differ from fruits with numerous minuscule seeds observed in most other species of Miconieae (Baumgratz, 1985; Groenendijk, Bouman & Cleef, 1996; Bécquer, Michelangeli & Borsch, 2014; Ocampo et al., 2014). A similar relationship between multiplicative integument and seed size occurs in Rupestrea, with a few large seeds in the ovary (Goldenberg et al., 2015). CONCLUSIONS We studied the evolution of the outer ovule integument thickness in Melastomataceae, reporting this condition in a large number of species. In addition, we showed that two-cell-layered and multilayered integuments have different ontogenetic origins. The results indicate that the outer ovule integument thickness is relatively stable in the family, but the variations observed in Olisbeoideae, Rupestrea, Melastomateae and Miconieae are of systematic value for the groups. Finally, there is a tendency of association between the multilayered integument and fleshy fruits in Miconieae and Mouriri, and between multiplicative integument and large seeds in a few members of the family. SUPPORTING INFORMATION Appendix S1. List of the species of Melastomataceae analysed in this study, information source for its systematic position, data source and herbarium and voucher data. Appendix S2. GenBank accession numbers for sequences included in the phylogenetic analyses based on plastid and nuclear markers. ACKNOWLEDGEMENTS We thank the Structural Botany Laboratory of the New York Botanical Garden for technical support, Ricardo Kriebel and Julien Bachelier for useful suggestions on the manuscript, and Fabiana dos Santos Oliveira and Lucas Simon Torati for help in obtaining some samples. We also thank an anonymous reviewer for valuable comments that improved the manuscript. This study was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp – process numbers 2008/10793-0, 2010/15077-0 and 2013/08945-4). S.P.T. is indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico (process number 303493/2015-1) for the fellowship received. Portions of the research were also funded by the National Science Foundation (DEB-0818399). REFERENCES Almeda F. 2009. Melastomataceae. In: Davidse G, Sousa M, Knapp S, Chang F, eds. Flora Mesoamerica . Mexico City: Universidad Nacional Autonoma de Mexico, 180– 337. Amorim AM, Goldenberg R, Michelangeli FA. 2009. 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Abstract

Abstract Ovule characters have been shown to be informative at higher taxonomic levels and are therefore of interest in angiosperm systematics. In this study, we aimed to describe the evolution of the outer ovule integument thickness in Melastomataceae and to evaluate its systematic and biological significance. This character was investigated in 137 species, and data from the literature were compiled for 22 additional species, totalling 159 taxa. Integument origin was studied in selected species to clarify whether different conditions were associated with different ontogenetic origins. The most recent common ancestor of Melastomataceae probably had an ovule with a two-cell-layered outer integument. A multilayered outer integument evolved independently at least six times in the family. Moreover, reversals to the two-cell-layered plesiomorphic condition have also occurred more than once. The two-cell-layered and multilayered outer integuments have different ontogenetic origins, enhancing the taxonomic usefulness of this character. The thickness of the outer ovule integument is of systematic significance in Olisbeoideae, Rupestrea, Melastomateae and Miconieae. The multilayered outer integument may have some adaptive significance for Miconieae and Mouriri, groups with endozoochoric fleshy fruits. Evidence of a multiplicative testa was observed in a few species with both types of integuments and it may be associated with a massive seed coat found in large seeds. INTRODUCTION The ovule is the developmental precursor of the seed and, as such, plays an essential role in the complex and successful reproduction process in spermatophytes (Bouman, 1984; Gasser, Broadhvest & Hauser, 1998; Linkies et al., 2010; Endress, 2011). In angiosperms, important reproductive events such as the female gametophyte and egg cell development, pollen tube attraction and guidance, double fertilization, and embryo and endosperm development all occur in the ovule (Fuentes & Vivian-Smith, 2009; Endress, 2011). The ovule can be morphologically understood as the megasporangium (or nucellus) covered by one or two integuments (Linkies et al., 2010). Gymnosperms typically have ovules with a single integument, whereas the ovules of angiosperms are commonly bitegmic (Gasser et al., 1998; Endress, 2011). After fertilization, these integuments give rise to the seed coat, which in angiosperms comprises a testa and a tegmen, derived from the outer and inner integuments, respectively. The seed coat can be responsible for embryo protection and seed dispersal, and it is a key factor in the regulation of seed germination (Windsor et al., 2000; Kelley & Gasser, 2009). Despite the remarkable adaptive significance of the seed coat, the thickness (i.e. number of cell layers) of the integuments seems to have been a relatively stable character in angiosperm evolution and therefore to be informative at higher taxonomic levels (Davis, 1966; Tobe, 1989; Endress, 2011). Ovule integuments may consist of two or more cell layers (Bouman, 1984; Endress, 2011). Two-cell-layered integuments are characterized as dermal integuments because they develop exclusively from the dermal layer of the ovule primordium. In contrast, multilayered integuments may be of dermal origin or may derive from dermal and subdermal layers (subdermal integuments) (Bouman, 1984; Shamrov, 2000; Endress, 2011). Dermal or subdermal integuments can become multiplicative and increase in thickness by periclinal divisions during both ovule and seed development (Corner, 1976; Bouman, 1984). The occurrence of ovules with a two-cell-layered inner integument is one of the embryological characters that define the order Myrtales (Tobe & Raven, 1983a; Tobe, 1989). On the other hand, the thickness of the outer integument is a variable character in the order (Tobe & Raven, 1983a). In Melastomataceae, one of the two largest families in Myrtales (Dahlgren & Thorne, 1984; Conti et al., 1997), the outer ovule integument may consist of two or more cell layers (Ziegler, 1925; Subramanyam, 1942, 1944, 1948, 1951; Etheridge & Herr, 1968; Corner, 1976; Tobe & Raven, 1983a; Medeiros & Morretes, 1996; Cortez & Carmello-Guerreiro, 2008; Caetano et al., 2013; Ribeiro, Oliveira & Silveira, 2015). However, the evolutionary history of this attribute in the family has not previously been studied. Moreover, despite the importance of integument characters for systematics, the scarcity of the data available for the family hampers their use in the delimitation of clades and understanding their relationships and their evolutionary and ecological significance. Melastomataceae (including Memecylaceae currently treated as subfamily Olisbeoideae) comprises c. 5400 species in 170 genera and is one of the larger families of angiosperms, with considerable species richness in the New World (Renner, 1993; Conti et al., 1997; Stone, 2006; MELNet, 2007; Goldenberg et al., 2015). Despite the uncontroversial monophyly of the family, relationships in Melastomataceae are not fully understood and improving their resolution is still a work in progress (Clausing & Renner, 2001; Fritsch et al., 2004; Michelangeli et al., 2004, 2013; Penneys et al., 2010; Goldenberg et al., 2012; Rocha et al., 2016a, b; Veranso-Libalah et al., 2017). Although the latest review for the family based on morphology and anatomy (Renner, 1993) recognized nine tribes, recent studies based on DNA sequence data have proposed the establishment of new ones and considerable changes to the delimitation of others (Clausing & Renner, 2001; Fritsch et al., 2004; Michelangeli et al., 2004, 2011, 2013; Goldenberg et al., 2008, 2012, 2015; Amorim, Goldenberg & Michelangeli, 2009; Penneys et al., 2010; Penneys & Judd, 2011, 2013; Rocha et al., 2016a, b). At least 15 major lineages are currently known in the family, although not all of them are recognized at the tribal level (Goldenberg et al., 2015). In this sense, knowledge of potentially diagnostic attributes for specific clades of Melastomataceae, such as the thickness of the outer ovule integument, may be of great significance. Therefore, the objective of the present study was to describe the evolution of this character in Melastomataceae, assessing its systematic and biological significance and clarifying whether different conditions (two-cell-layered and multilayered outer integument) are associated with different ontogenetic origins. Additionally, we also investigated the evolutionary association between multilayered outer integument and fleshy fruits. MATERIAL AND METHODS Taxon sampling The thickness of the ovule outer integument was examined in 137 species belonging to 14 of 15 major lineages currently recognized in Melastomataceae (sensuGoldenberg et al., 2015). The only tribe not sampled in the present study was Astronieae. We also included published data for 22 additional species for a total of 159 taxa (Table 1) (Ziegler, 1925; Subramanyam, 1942, 1944, 1948, 1951; Etheridge & Herr, 1968; Corner, 1976; Medeiros & Morretes, 1996; Caetano, 2010; Caetano et al., 2013; Ribeiro et al., 2015). As outgroups, we added nine species of the CAP clade, a strongly supported lineage sister to Melastomataceae and formed by Crypteroniaceae, Alzateaceae and Penaeaceae s.l. (including Oliniaceae and Rhynchocalycaceae; Conti et al., 2002; APG IV, 2016; Berger et al., 2016). Embryological data for these species were obtained from literature reports (Table 1; Tobe & Raven, 1983b, 1984a, b, c, d, 1987a, b). Table 1. List of the Melastomataceae species analysed, status of the character observed and literature data; fruit type (dry or fleshy) is also given Groups    Species*  Character state  Reference†  Fruit type  Reference†  Outgroups  Crypteroniaceae  Axinandra zeylanica  Two-layered (2–3)  1  Dry  19  Crypteronia paniculata  Two-layered (2)  2  Dry  19  Dactylocladus stenostachys  Two-layered (2–3)  3  Dry  19  Alzateaceae  Alzatea verticillata  Two-layered (2–4)  4  Dry  19  Penaeaceae  Olinia emarginata  Multilayered (3–4)  5  Fleshy  19  Olinia ventosa  Multilayered (3–5)  5  Fleshy  19  Penaea mucronata  Two-layered (2)  6  Dry  19  Rhynchocalyx lawsonioides  Two-layered (2)  7  Dry  19  Saltera sarcocolla  Two-layered (2)  6  Dry  19  Olisbeoideae    Memecylon heyneanum  Two-layered (2)  8  Fleshy  20      Memecylon umbellatum  Two-layered (2)  9  Fleshy  9      Memecylon sp.  Two-layered (2)  9  Fleshy  20      Mouriri acutiflora  Multilayered (5–6)  PS  Fleshy  20      Mouriri cearensis  Multilayered (5–6)  PS  Fleshy  20      Mouriri guianensis  Multilayered (5–6)  PS  Fleshy  20      Votomita guianensis  Two-layered (2–4)  PS  Fleshy  20  Kibessieae    Pternandra azurea  Two-layered (2)  9  Fleshy  21    Pternandra coerulescens  Two-layered (2)  PS  Fleshy  21  Henrietteeae  Bellucia grossularioides  Two-layered (2)  PS  Fleshy  22    Bellucia mespiloides  Two-layered (2)  PS  Fleshy  22    Henriettea ramiflora  Two-layered (2)  PS  Fleshy  22    Henriettea saldanhae  Two-layered (2)  PS  Fleshy  22    Henriettea succosa  Two-layered (2)  PS  Fleshy  22  Bertolonieae  Bertolonia mosenii  Two-layered (2)  PS  Dry  23  Blakeeae  Blakea trinervia  Two-layered (2)  9  Fleshy  23    Blakea multiflora  Two-layered (2)  PS  Fleshy  24    Chalybea calyptrata  Two-layered (2)  PS  Fleshy  24    Chalybea ecuadorensis  Two-layered (2)  PS  Fleshy  24  Dissochaeteae  Dissochaeta divaricata  Two-layered (2)  PS  Fleshy  25    Oxyspora paniculata  Two-layered (2)  10  Dry  25    Sonerila wallichii  Two-layered (2)  11  Dry  26  Cyphostyleae  Allomaieta grandiflora  Two-layered (2)  PS  Dry  27  Triolena clade  Triolena amazonica  Two-layered (2)  PS  Dry  28    Triolena obliqua  Two-layered (2)  PS  Dry  28    Triolena paleacea  Two-layered (2)  PS  Dry  26  Cambessedesia clade  Behuria glutinosa  Two-layered (2)  PS  Dry  29  Cambessedesia espora  Two-layered (2)  PS  Dry  23    Dolichoura spiritusanctensis  Two-layered (2)  PS  Dry  29    Huberia consimilis  Two-layered (2)  PS  Dry  29    Merianthera bullata  Two-layered (2–3)  PS  Dry  29    Merianthera parvifolia  Two-layered (2–3)  PS  Dry  29  Rhexieae  Arthrostemma ciliatum  Two-layered (2)  PS  Dry  30    Rhexia mariana  Two-layered (2)  12  Dry  30  Microlicieae  Chaetostoma armatum  Two-layered (2)  13  Dry  23    Microlicia cordata  Two-layered (2)  PS  Dry  31    Microlicia euphorbioides  Two-layered (2)  PS  Dry  23    Microlicia fasciculata  Two-layered (2)  PS  Dry  23    Poteranthera pusilla  Two-layered (2)  PS  Dry  32    Rhynchanthera grandiflora  Two-layered (2)  PS  Dry  23  Melastomateae  Marcetia alliance  Acanthella sprucei  Multilayered (3)  PS  Dry  30  Aciotis purpurascens  Multilayered (3)  PS  Dry  30  Acisanthera hedyotoidea  Multilayered (3)  PS  Dry  30  Acisanthera quadrata  Multilayered (3)  PS  Dry  30  Appendicularia thymifolia  Multilayered (3)  PS  Dry  30  Comolia microphylla  Multilayered (3)  PS  Dry  30  Ernestia glandulosa  Multilayered (3)  PS  Dry  30      Macairea radula  Multilayered (3)  PS  Dry  30  Marcetia taxifolia  Multilayered (3)  PS  Dry  30  Nepsera aquatica  Multilayered (3)  PS  Dry  30  Sandemania hoehnei  Multilayered (3)  PS  Dry  30  Siphanthera hostmannii  Multilayered (3)  PS  Dry  33  Core Melastomateae  Brachyotum ledifolium  Two-layered (2)  PS  Dry  30  Desmoscelis villosa  Two-layered (2)  PS  Dry  30  Heterocentron elegans  Two-layered (2)  PS  Dry  30  Melastoma malabathricum  Two-layered (2)  14  Fleshy  30  Monochaetum calcaratum  Two-layered (2)  9  Dry  30  Monochaetum ensiferum  Two-layered (2)  15  Dry  30  Monochaetum meridense  Two-layered (2)  PS  Dry  30  Osbeckia aspera  Two-layered (2)  8  Dry  26  Osbeckia brachystemon  Two-layered (2)  8  Dry  26  Osbeckia stellata  Two-layered (2)  8  Dry  25  Pterolepis glomerata  Two-layered (2)  PS  Dry  30  Tibouchina clavata  Two-layered (2)  PS  Dry  30  Tibouchina clinopodifolia  Two-layered (2)  PS  Dry  30  Tibouchina laevicaulis  Two-layered (2)  PS  Dry  30  Tibouchina nodosa  Two-layered (2)  9  Dry  30  Tibouchina semidecandra  Two-layered (2)  15  Dry  30  Merianieae    Axinaea dentata  Two-layered (2–3)  PS  Dry  34  Axinaea floribunda  Multilayered (3)  PS  Dry  34  Axinaea grandifolia  Two-layered (2–3)  PS  Dry  34  Axinaea minutiflora  Two-layered (2–3)  PS  Dry  34  Graffenrieda gracilis  Two-layered (2)  PS  Dry  33  Graffenrieda harlingii  Two-layered (2)  PS  Dry  28  Graffenrieda latifolia  Two-layered (2)  PS  Dry  33  Macrocentrum repens  Two-layered (2)  PS  Dry  35  Meriania sclerophylla  Multilayered (3)  PS  Dry  33  Meriania subumbellata  Multilayered (3)  PS  Dry  33  Meriania urceolata  Two-layered (2–3)  PS  Dry  33  Miconieae  Eriocnema clade  Eriocnema acaulis  Multilayered (3)  PS  Dry  31  Eriocnema fulva  Multilayered (3)  PS  Dry  31  Physeterostemon thomasii  Two-layered (2)  PS  Dry  36  Miconia I  Miconia dodecandra  Multilayered (3)  PS  Fleshy  23  Miconia superba  Multilayered (3)  PS  Fleshy  35  Miconia II  Miconia centrodesma  Multilayered (3)  PS  Fleshy  23  Leandra + Ossaea  Leandra reversa  Multilayered (3)  PS  Fleshy  23  Ossaea capillaris  Multilayered (3)  PS  Fleshy  28  Tococa  Tococa guianensis  Multilayered (3)  PS  Fleshy  35  Caribbean  Charianthus alpinus  Two-layered (2)  PS  Fleshy  37  Charianthus nodosus  Two-layered (2)  PS  Fleshy  37  Tetrazygia crotonifolia  Multilayered (3)  PS  Fleshy  38  Tetrazygia discolor  Two-layered (2)  PS  Fleshy  37  Tetrazygia elaeagnoides  Multilayered (3)  PS  Fleshy  37    Monopodial Clidemia  Clidemia mortoniana  Multilayered (3)  PS  Fleshy  39  Clidemia spectabilis  Multilayered (3)  PS  Fleshy  39  Conostegia  Conostegia bigibbosa  Two-layered (2)  PS  Fleshy  40  Conostegia brenesii  Two-layered (2)  PS  Fleshy  40  Conostegia consimilis  Two-layered (2)  PS  Fleshy  40  Conostegia friedmaniorum  Two-layered (2)  PS  Fleshy  40  Conostegia icosandra  Two-layered (2)  PS  Fleshy  40  Conostegia lasiopoda  Two-layered (2)  PS  Fleshy  40  Conostegia monteleagreana  Two-layered (2)  PS  Fleshy  40  Conostegia schlimii  Two-layered (2)  PS  Fleshy  40      Conostegia speciosa  Two-layered (2)  PS  Fleshy  40  Conostegia subcrustulata  Two-layered (2)  PS  Fleshy  40  Conostegia xalapensis  Two-layered (2)  PS  Fleshy  40  Mecranium, Anaectocalyx and allies  Mecranium acuminatum  Multilayered (3)  PS  Fleshy  41  Mecranium latifolium  Multilayered (3)  PS  Fleshy  41  Miconia ciliata  Multilayered (3)  PS  Fleshy  39  Tococa platyphylla  Multilayered (3)  PS  Fleshy  28  Miconia III  Leandra subseriata  Multilayered (3)  PS  Fleshy  28  Miconia crocea  Multilayered (3)  PS  Fleshy  28  Miconia denticulata  Multilayered (3)  PS  Fleshy  28  Miconia goniostigma  Multilayered (3)  PS  Fleshy  28  Miconia papillosa  Multilayered (3)  PS  Fleshy  28  Miconia stenobotrys  Multilayered (3)  PS  Fleshy  42  Miconia theizans  Multilayered (3)  PS  Fleshy  28  Miconia IV (Miconia discolor subclade)  Miconia budlejoides  Multilayered (3–4)  PS  Fleshy  23  Miconia cabucu  Multilayered (6–7)  16  Fleshy  23  Miconia capixaba  Multilayered (3–4)  PS  Fleshy  43  Miconia castaneiflora  Multilayered (4)  PS  Fleshy  43  Miconia chartacea  Multilayered (3–4)  PS  Fleshy  23  Miconia cubatanensis  Multilayered (5)  PS  Fleshy  23  Miconia discolor  Multilayered (3–4)  PS  Fleshy  23  Miconia fasciculata  Multilayered (3)  PS  Fleshy  43  Miconia hyemalis  Multilayered (3–4)  PS  Fleshy  43  Miconia lymanii  Multilayered (4)  PS  Fleshy  43  Miconia pepericarpa  Multilayered (3)  17  Fleshy  43  Miconia sclerophylla  Multilayered (4)  PS  Fleshy  43  Miconia IV  Miconia albicans  Multilayered (3)  18  Fleshy  23  Miconia donaeana  Multilayered (3)  PS  Fleshy  43  Miconia fallax  Multilayered (3)  17  Fleshy  23  Miconia laevigata  Multilayered (3)  PS  Fleshy  28  Miconia latecrenata  Multilayered (3)  PS  Fleshy  23  Miconia leucocarpa  Multilayered (3)  PS  Fleshy  23  Miconia minutiflora  Multilayered (3)  PS  Fleshy  23  Miconia rubiginosa  Multilayered (3)  PS  Fleshy  23  Miconia rufescens  Multilayered (3)  PS  Fleshy  33  Miconia serrulata  Multilayered (3)  PS  Fleshy  28  Miconia stenostachya  Multilayered (3)  PS  Fleshy  23    Miconia V  Leandra rufescens  Multilayered (3)  PS  Fleshy  35  Miconia ceramicarpa  Two-layered (2)  PS  Fleshy  35  Miconia chamissois  Multilayered (3)  PS  Fleshy  23  Miconia ibaguensis  Multilayered (3)  PS  Fleshy  23  Miconia inaequidens  Multilayered (3–4)  PS  Fleshy  23  Miconia nervosa  Multilayered (3)  PS  Fleshy  28  Miconia paucidens  Multilayered (4)  PS  Fleshy  23  Miconia prasina  Multilayered (3)  PS  Fleshy  35  Miconia pusilliflora  Multilayered (3–4)  PS  Fleshy  23  Miconia rugosa  Multilayered (3–4)  PS  Fleshy  35    Clidemia  Clidemia hirta  Multilayered (3)  PS  Fleshy  23    Leandra s.s. + Pleiochiton  Leandra aurea  Multilayered (3)  PS  Fleshy  23  Leandra cardiophylla  Multilayered (3)  8  Fleshy  42  Leandra melastomoides  Multilayered (3)  PS  Fleshy  23  Ossaea amygdaloides  Multilayered (3)  PS  Fleshy  23  Ossaea confertiflora  Multilayered (3)  PS  Fleshy  23  Pleiochiton ebracteatum  Multilayered (3)  PS  Fleshy  23  Unplaced species  Phainantha laxiflora  Two-layered (2)  PS  Dry  35  Rupestrea johnwurdackiana  Multilayered (4–5)  PS  Dry  44  Groups    Species*  Character state  Reference†  Fruit type  Reference†  Outgroups  Crypteroniaceae  Axinandra zeylanica  Two-layered (2–3)  1  Dry  19  Crypteronia paniculata  Two-layered (2)  2  Dry  19  Dactylocladus stenostachys  Two-layered (2–3)  3  Dry  19  Alzateaceae  Alzatea verticillata  Two-layered (2–4)  4  Dry  19  Penaeaceae  Olinia emarginata  Multilayered (3–4)  5  Fleshy  19  Olinia ventosa  Multilayered (3–5)  5  Fleshy  19  Penaea mucronata  Two-layered (2)  6  Dry  19  Rhynchocalyx lawsonioides  Two-layered (2)  7  Dry  19  Saltera sarcocolla  Two-layered (2)  6  Dry  19  Olisbeoideae    Memecylon heyneanum  Two-layered (2)  8  Fleshy  20      Memecylon umbellatum  Two-layered (2)  9  Fleshy  9      Memecylon sp.  Two-layered (2)  9  Fleshy  20      Mouriri acutiflora  Multilayered (5–6)  PS  Fleshy  20      Mouriri cearensis  Multilayered (5–6)  PS  Fleshy  20      Mouriri guianensis  Multilayered (5–6)  PS  Fleshy  20      Votomita guianensis  Two-layered (2–4)  PS  Fleshy  20  Kibessieae    Pternandra azurea  Two-layered (2)  9  Fleshy  21    Pternandra coerulescens  Two-layered (2)  PS  Fleshy  21  Henrietteeae  Bellucia grossularioides  Two-layered (2)  PS  Fleshy  22    Bellucia mespiloides  Two-layered (2)  PS  Fleshy  22    Henriettea ramiflora  Two-layered (2)  PS  Fleshy  22    Henriettea saldanhae  Two-layered (2)  PS  Fleshy  22    Henriettea succosa  Two-layered (2)  PS  Fleshy  22  Bertolonieae  Bertolonia mosenii  Two-layered (2)  PS  Dry  23  Blakeeae  Blakea trinervia  Two-layered (2)  9  Fleshy  23    Blakea multiflora  Two-layered (2)  PS  Fleshy  24    Chalybea calyptrata  Two-layered (2)  PS  Fleshy  24    Chalybea ecuadorensis  Two-layered (2)  PS  Fleshy  24  Dissochaeteae  Dissochaeta divaricata  Two-layered (2)  PS  Fleshy  25    Oxyspora paniculata  Two-layered (2)  10  Dry  25    Sonerila wallichii  Two-layered (2)  11  Dry  26  Cyphostyleae  Allomaieta grandiflora  Two-layered (2)  PS  Dry  27  Triolena clade  Triolena amazonica  Two-layered (2)  PS  Dry  28    Triolena obliqua  Two-layered (2)  PS  Dry  28    Triolena paleacea  Two-layered (2)  PS  Dry  26  Cambessedesia clade  Behuria glutinosa  Two-layered (2)  PS  Dry  29  Cambessedesia espora  Two-layered (2)  PS  Dry  23    Dolichoura spiritusanctensis  Two-layered (2)  PS  Dry  29    Huberia consimilis  Two-layered (2)  PS  Dry  29    Merianthera bullata  Two-layered (2–3)  PS  Dry  29    Merianthera parvifolia  Two-layered (2–3)  PS  Dry  29  Rhexieae  Arthrostemma ciliatum  Two-layered (2)  PS  Dry  30    Rhexia mariana  Two-layered (2)  12  Dry  30  Microlicieae  Chaetostoma armatum  Two-layered (2)  13  Dry  23    Microlicia cordata  Two-layered (2)  PS  Dry  31    Microlicia euphorbioides  Two-layered (2)  PS  Dry  23    Microlicia fasciculata  Two-layered (2)  PS  Dry  23    Poteranthera pusilla  Two-layered (2)  PS  Dry  32    Rhynchanthera grandiflora  Two-layered (2)  PS  Dry  23  Melastomateae  Marcetia alliance  Acanthella sprucei  Multilayered (3)  PS  Dry  30  Aciotis purpurascens  Multilayered (3)  PS  Dry  30  Acisanthera hedyotoidea  Multilayered (3)  PS  Dry  30  Acisanthera quadrata  Multilayered (3)  PS  Dry  30  Appendicularia thymifolia  Multilayered (3)  PS  Dry  30  Comolia microphylla  Multilayered (3)  PS  Dry  30  Ernestia glandulosa  Multilayered (3)  PS  Dry  30      Macairea radula  Multilayered (3)  PS  Dry  30  Marcetia taxifolia  Multilayered (3)  PS  Dry  30  Nepsera aquatica  Multilayered (3)  PS  Dry  30  Sandemania hoehnei  Multilayered (3)  PS  Dry  30  Siphanthera hostmannii  Multilayered (3)  PS  Dry  33  Core Melastomateae  Brachyotum ledifolium  Two-layered (2)  PS  Dry  30  Desmoscelis villosa  Two-layered (2)  PS  Dry  30  Heterocentron elegans  Two-layered (2)  PS  Dry  30  Melastoma malabathricum  Two-layered (2)  14  Fleshy  30  Monochaetum calcaratum  Two-layered (2)  9  Dry  30  Monochaetum ensiferum  Two-layered (2)  15  Dry  30  Monochaetum meridense  Two-layered (2)  PS  Dry  30  Osbeckia aspera  Two-layered (2)  8  Dry  26  Osbeckia brachystemon  Two-layered (2)  8  Dry  26  Osbeckia stellata  Two-layered (2)  8  Dry  25  Pterolepis glomerata  Two-layered (2)  PS  Dry  30  Tibouchina clavata  Two-layered (2)  PS  Dry  30  Tibouchina clinopodifolia  Two-layered (2)  PS  Dry  30  Tibouchina laevicaulis  Two-layered (2)  PS  Dry  30  Tibouchina nodosa  Two-layered (2)  9  Dry  30  Tibouchina semidecandra  Two-layered (2)  15  Dry  30  Merianieae    Axinaea dentata  Two-layered (2–3)  PS  Dry  34  Axinaea floribunda  Multilayered (3)  PS  Dry  34  Axinaea grandifolia  Two-layered (2–3)  PS  Dry  34  Axinaea minutiflora  Two-layered (2–3)  PS  Dry  34  Graffenrieda gracilis  Two-layered (2)  PS  Dry  33  Graffenrieda harlingii  Two-layered (2)  PS  Dry  28  Graffenrieda latifolia  Two-layered (2)  PS  Dry  33  Macrocentrum repens  Two-layered (2)  PS  Dry  35  Meriania sclerophylla  Multilayered (3)  PS  Dry  33  Meriania subumbellata  Multilayered (3)  PS  Dry  33  Meriania urceolata  Two-layered (2–3)  PS  Dry  33  Miconieae  Eriocnema clade  Eriocnema acaulis  Multilayered (3)  PS  Dry  31  Eriocnema fulva  Multilayered (3)  PS  Dry  31  Physeterostemon thomasii  Two-layered (2)  PS  Dry  36  Miconia I  Miconia dodecandra  Multilayered (3)  PS  Fleshy  23  Miconia superba  Multilayered (3)  PS  Fleshy  35  Miconia II  Miconia centrodesma  Multilayered (3)  PS  Fleshy  23  Leandra + Ossaea  Leandra reversa  Multilayered (3)  PS  Fleshy  23  Ossaea capillaris  Multilayered (3)  PS  Fleshy  28  Tococa  Tococa guianensis  Multilayered (3)  PS  Fleshy  35  Caribbean  Charianthus alpinus  Two-layered (2)  PS  Fleshy  37  Charianthus nodosus  Two-layered (2)  PS  Fleshy  37  Tetrazygia crotonifolia  Multilayered (3)  PS  Fleshy  38  Tetrazygia discolor  Two-layered (2)  PS  Fleshy  37  Tetrazygia elaeagnoides  Multilayered (3)  PS  Fleshy  37    Monopodial Clidemia  Clidemia mortoniana  Multilayered (3)  PS  Fleshy  39  Clidemia spectabilis  Multilayered (3)  PS  Fleshy  39  Conostegia  Conostegia bigibbosa  Two-layered (2)  PS  Fleshy  40  Conostegia brenesii  Two-layered (2)  PS  Fleshy  40  Conostegia consimilis  Two-layered (2)  PS  Fleshy  40  Conostegia friedmaniorum  Two-layered (2)  PS  Fleshy  40  Conostegia icosandra  Two-layered (2)  PS  Fleshy  40  Conostegia lasiopoda  Two-layered (2)  PS  Fleshy  40  Conostegia monteleagreana  Two-layered (2)  PS  Fleshy  40  Conostegia schlimii  Two-layered (2)  PS  Fleshy  40      Conostegia speciosa  Two-layered (2)  PS  Fleshy  40  Conostegia subcrustulata  Two-layered (2)  PS  Fleshy  40  Conostegia xalapensis  Two-layered (2)  PS  Fleshy  40  Mecranium, Anaectocalyx and allies  Mecranium acuminatum  Multilayered (3)  PS  Fleshy  41  Mecranium latifolium  Multilayered (3)  PS  Fleshy  41  Miconia ciliata  Multilayered (3)  PS  Fleshy  39  Tococa platyphylla  Multilayered (3)  PS  Fleshy  28  Miconia III  Leandra subseriata  Multilayered (3)  PS  Fleshy  28  Miconia crocea  Multilayered (3)  PS  Fleshy  28  Miconia denticulata  Multilayered (3)  PS  Fleshy  28  Miconia goniostigma  Multilayered (3)  PS  Fleshy  28  Miconia papillosa  Multilayered (3)  PS  Fleshy  28  Miconia stenobotrys  Multilayered (3)  PS  Fleshy  42  Miconia theizans  Multilayered (3)  PS  Fleshy  28  Miconia IV (Miconia discolor subclade)  Miconia budlejoides  Multilayered (3–4)  PS  Fleshy  23  Miconia cabucu  Multilayered (6–7)  16  Fleshy  23  Miconia capixaba  Multilayered (3–4)  PS  Fleshy  43  Miconia castaneiflora  Multilayered (4)  PS  Fleshy  43  Miconia chartacea  Multilayered (3–4)  PS  Fleshy  23  Miconia cubatanensis  Multilayered (5)  PS  Fleshy  23  Miconia discolor  Multilayered (3–4)  PS  Fleshy  23  Miconia fasciculata  Multilayered (3)  PS  Fleshy  43  Miconia hyemalis  Multilayered (3–4)  PS  Fleshy  43  Miconia lymanii  Multilayered (4)  PS  Fleshy  43  Miconia pepericarpa  Multilayered (3)  17  Fleshy  43  Miconia sclerophylla  Multilayered (4)  PS  Fleshy  43  Miconia IV  Miconia albicans  Multilayered (3)  18  Fleshy  23  Miconia donaeana  Multilayered (3)  PS  Fleshy  43  Miconia fallax  Multilayered (3)  17  Fleshy  23  Miconia laevigata  Multilayered (3)  PS  Fleshy  28  Miconia latecrenata  Multilayered (3)  PS  Fleshy  23  Miconia leucocarpa  Multilayered (3)  PS  Fleshy  23  Miconia minutiflora  Multilayered (3)  PS  Fleshy  23  Miconia rubiginosa  Multilayered (3)  PS  Fleshy  23  Miconia rufescens  Multilayered (3)  PS  Fleshy  33  Miconia serrulata  Multilayered (3)  PS  Fleshy  28  Miconia stenostachya  Multilayered (3)  PS  Fleshy  23    Miconia V  Leandra rufescens  Multilayered (3)  PS  Fleshy  35  Miconia ceramicarpa  Two-layered (2)  PS  Fleshy  35  Miconia chamissois  Multilayered (3)  PS  Fleshy  23  Miconia ibaguensis  Multilayered (3)  PS  Fleshy  23  Miconia inaequidens  Multilayered (3–4)  PS  Fleshy  23  Miconia nervosa  Multilayered (3)  PS  Fleshy  28  Miconia paucidens  Multilayered (4)  PS  Fleshy  23  Miconia prasina  Multilayered (3)  PS  Fleshy  35  Miconia pusilliflora  Multilayered (3–4)  PS  Fleshy  23  Miconia rugosa  Multilayered (3–4)  PS  Fleshy  35    Clidemia  Clidemia hirta  Multilayered (3)  PS  Fleshy  23    Leandra s.s. + Pleiochiton  Leandra aurea  Multilayered (3)  PS  Fleshy  23  Leandra cardiophylla  Multilayered (3)  8  Fleshy  42  Leandra melastomoides  Multilayered (3)  PS  Fleshy  23  Ossaea amygdaloides  Multilayered (3)  PS  Fleshy  23  Ossaea confertiflora  Multilayered (3)  PS  Fleshy  23  Pleiochiton ebracteatum  Multilayered (3)  PS  Fleshy  23  Unplaced species  Phainantha laxiflora  Two-layered (2)  PS  Dry  35  Rupestrea johnwurdackiana  Multilayered (4–5)  PS  Dry  44  *Authors for species names can be found in Supporting Information Appendix S1. †References: 1, Tobe & Raven (1983b); 2, Tobe & Raven (1987a); 3, Tobe & Raven (1987b); 4, Tobe & Raven (1984a); 5, Tobe & Raven (1984b); 6, Tobe & Raven (1984c); 7, Tobe & Raven (1984d); 8, Subramanyam (1942); 9, Corner (1976); 10, Subramanyam (1951); 11, Subramanyam (1944); 12, Etheridge & Herr (1968); 13, Ribeiro et al. (2015); 14, Subramanyam (1948); 15, Ziegler (1925); 16, Medeiros & Morretes (1996); 17, Caetano (2010); 18, Caetano et al. (2013); 19, Dahlgren & Thorne (1984); 20, Morley (1976); 21, Maxwell (1981); 22, Penneys et al. (2010); 23, Martins et al. (2009); 24, Penneys & Judd (2013); 25, Jie & Renner (2007); 26, Triana (1871); 27, Michelangeli et al. (2011); 28, Wurdack (1980); 29, Goldenberg et al. (2012); 30, Michelangeli et al. (2013); 31, Cogniaux (1891); 32, Rocha et al. (2016b); 33, Wurdack (1973); 34, Cotton, Borchsenius & Balslev (2014); 35, Berry et al. (2001); 36, Amorim et al. (2009); 37, Michelangeli et al. (2004); 38, Judd & Ionta (2013); 39, Almeda (2009); 40, Kriebel (2016); 41, Skean (1993); 42, Michelangeli et al. (2008); 43, Caddah (2013); 44, Goldenberg et al. (2015); PS, present study. View Large Anatomical analysis We sampled ovules from pre-anthesis buds or anthetic flowers. The samples were obtained from fixed material later preserved in ethanol or from rehydrated herbarium specimens (Appendix 1; Smith & Smith, 1942). The thickness of the outer ovule integument was characterized by counting the number of integument cell layers in transverse and/or longitudinal sections of the median region of the ovule. The micropylar region was not considered for this definition because in this area the integumental cells may divide periclinally, forming additional layers (Subramanyam, 1942, 1948, 1951; Corner, 1976; Medeiros & Morretes, 1996; Caetano et al., 2013; Ribeiro et al., 2015). To assess the origin of multilayered outer ovule integument, some species were selected for observation of the initiation and development of the integuments (whether dermal or subdermal according to Bouman, 1984): Acisanthera quadrata Pers. (Melastomateae), Clidemia hirta (L.) D.Don, Leandra aurea (Cham.) Cogn., Miconia fallax DC., M. leucocarpa DC., M. pepericarpa Mart. ex DC. and Ossaea confertiflora (DC.) Triana (Miconieae). In these plants, we analysed ovules from flower buds in different developmental phases and from anthetic flowers. The material was fixed in 2% glutaraldehyde and 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 6.8, for 24 h (modified from McDowell & Trump, 1976). All samples were embedded in resin (Leica Microsystems, Heidelberg, Germany, and Heraeus Kulzer GmbH, Germany) and transversally and longitudinally sectioned with an AO Spencer 820 (GMI Inc., Ramsey, MN, USA) or RM 2245 (Leica) rotary microtome with a steel blade. Sections of c. 5.0 μm were adhered to glass slides and stained with 0.05% Toluidine Blue in 0.1 M sodium phosphate buffer, pH 6.8 (O’Brien, Feder & McCully, 1964). Observations and photomicrographs were made with a DM 5000 light microscope coupled to a DFC 390 (both Leica Microsystems) digital camera or with a Zeiss Axioplan compound microscope with a Nikon DXM1200C digital camera. The outer ovule integument was classified as two-cell-layered when formed by two cell layers or variations on this condition (two or three or two to four cell layers in the same ovule) or multilayered when consisting of three or more cell layers (Tobe, 1989; Endress, 2011). Phylogenetic reconstruction Tree inference and divergence time estimation were performed under a Bayesian framework implemented in BEAST 1.8.0 (Drummond et al., 2012). Molecular data for the species of interest were gathered from the NCBI database (GenBank), where the most common markers available across our sampled taxa were selected. The molecular data set includes six plastid and two nuclear markers, the plastome being represented by three intergenic spacers (accD-psaI, psbK-psbI and trnS-trnG), two protein-coding genes (ndhF and rbcL) and one intron (rpl16), and nuclear DNA being represented by two ribosomal spacers (nrETS and nrITS). GenBank accessions for all sequences included in this analysis are available in Supporting Information Appendix S2. Phylogenetic inference was performed on 160 terminals, including nine species from the Crypteroniaceae–Alzateaceae–Penaeaceae (CAP) clade treated as the outgroup (Conti et al., 2002; Berger et al., 2016). Individual loci were aligned with MAFFT 7.3 (Katoh & Standley, 2013) using the G-INS-i strategy. The best DNA partitioning scheme and models were estimated with PartitionFinder2 (Lanfear et al., 2012) under the Bayesian information criterion (BIC). The best scheme resulted in five partitions (accD-psaI, ndhF plus rpl16; nrETS; nrITS; psbK-psbI plus trnS-trnG; rbcL). The GTR+G model was recovered as the best fit for all partitions. The molecular clock prior was set to the lognormal uncorrelated model and the tree prior was set to the birth-death model. Fossil and secondary calibration constraints were placed in three nodes: (1) Melastomataceae crown (prior = lognormal, mean 1.5, SD 1, offset 56), based on the Palaeocene leaf fossil Melastomites montanensis R.W.Brown (Brown, 1962); (2) Rhexia Gronov. + Arthrostemma Pav. ex D.Don crown (prior = lognormal, mean 1.5, SD 1, offset 23), based on Miocene seed fossils (Collinson & Pingen, 1992); and (3) CAP clade crown (prior normal, mean 52.7, SD 6), based on the estimated age recovered in a wider analysis of Myrtales (Berger et al., 2016). We performed three independent runs of 30 million generations each, sampling every 1000 generations, and the stable posterior distributions of the independent runs were combined with LogCombiner v.1.8.0. Convergence was assessed using Tracer v.1.6 (Rambaut et al., 2014), and runs were considered satisfactory with effective sample size (ESS) values greater than 200. A maximum clade credibility tree was generated with TreeAnnotator v.1.8.0. Overall, both topology and divergence times were congruent with recently published hypotheses (Penneys & Judd, 2005, 2013; Stone, 2006; Goldenberg et al., 2008, 2015; Penneys et al., 2010; Caddah, 2013; Michelangeli et al., 2013; Kriebel, Michelangeli & Kelly, 2015; Meirelles, 2015; Berger et al., 2016; Reginato, 2016; Rocha et al., 2016a). Ancestral state reconstruction and correlated evolution Ancestral state reconstruction of the outer integument thickness in Melastomataceae was inferred using stochastic character mapping. This character was treated as discrete and unordered, i.e. all transitions among the states of the character can occur at equal probabilities. The test for the correlated evolution of the outer integument thickness and fruit type in this family was performed using Pagel’s model for binary characters (Pagel, 1994; Pagel & Meade, 2006). The fruit type considered (dry or fleshy) was based on literature (see Table 1). All analyses were carried out using the packages ape (Paradis, Claude & Strimmer, 2004) and phytools (Revell, 2012) in the R 3.2.5 environment (http://www.r-project.org/). To visualize phenotypic evolution on the trees, we mapped character histories across phylogenetic trees using a colour scheme (Revell, 2013). RESULTS Integument initiation and development The multilayered outer integument was derived from both dermal and subdermal layers in Acisanthera quadrata, Clidemia hirta, Leandra aurea, Miconia fallax, M. leucocarpa, M. pepericarpa and Ossaea confertiflora. In all seven studied species, the initiation and development of the integuments occur simultaneously by means of periclinal divisions of epidermal cells located at the base of the ovule primordium, characterizing it as a subdermal integument (Fig. 1A–E). Figure 1. View largeDownload slide Longitudinal sections showing the initiation (indicated by arrows) and development of the integuments in Melastomataceae species with a multilayered outer ovule integument. A–B, Leandra aurea. C, Clidemia hirta. D, Miconia pepericarpa. E, Ossaea confertiflora. F, L. aurea. G–H, M. fallax. I, L. aurea. J, C. hirta. K, L. aurea. L, Miconia leucocarpa. M, M. pepericarpa. N, M. fallax. Figure 1. View largeDownload slide Longitudinal sections showing the initiation (indicated by arrows) and development of the integuments in Melastomataceae species with a multilayered outer ovule integument. A–B, Leandra aurea. C, Clidemia hirta. D, Miconia pepericarpa. E, Ossaea confertiflora. F, L. aurea. G–H, M. fallax. I, L. aurea. J, C. hirta. K, L. aurea. L, Miconia leucocarpa. M, M. pepericarpa. N, M. fallax. The outer integument grows more rapidly than the inner one and extends beyond the apex of the nucellus first (Fig. 1F–H). However, when the megagametophyte starts its development, both integuments already cover the entire nucellus, forming a ‘zig-zag’ micropyle (Fig. 1I, J). Starting from this stage, the integuments extend, accompanying the growth of the rest of the ovule until its maturity (Fig. 1K–N). Outer ovule integument thickness In ten of the 14 major lineages sampled, the species had only ovules with a two-cell-layered outer integument (Table 1; Fig. 2): Kibessieae (Fig. 3A), Henrietteeae (Fig. 3B, C), Bertolonieae (Fig. 3D), Blakeeae (Fig. 3E), Dissochaeteae, Cyphostyleae (Fig. 3F), Triolena Naudin clade (Fig. 4A), Cambessedesia DC. clade (Fig. 4B–D), Rhexieae (Fig. 4E) and Microlicieae (Fig. 4F). Figure 2. View largeDownload slide Stochastic mapping of outer ovule integument thickness onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using six plastid and two nuclear makers. Red corresponds to the multilayered status, and blue to the two-cell-layered status. Changes in colour along the branches indicate transitions between characters states. Mya, million years ago; Pli, Pliocene; Qa, Quartenary. Figure 2. View largeDownload slide Stochastic mapping of outer ovule integument thickness onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using six plastid and two nuclear makers. Red corresponds to the multilayered status, and blue to the two-cell-layered status. Changes in colour along the branches indicate transitions between characters states. Mya, million years ago; Pli, Pliocene; Qa, Quartenary. Figure 3. View largeDownload slide Transverse (A–D and F) and longitudinal (E) sections showing ovules of Melastomataceae species belonging to tribes that exhibit exclusively a two-cell-layered outer integument. A, Pternandra coerulescens (Kibessieae). B, Bellucia grossularioides (Henrietteeae). C, Henriettea ramiflora (Henrietteeae). D, Bertolonia mosenii (Bertolonieae). E, Chalybea ecuadorensis (Blakeeae). F, Allomaieta grandiflora (Cyphostyleae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 3. View largeDownload slide Transverse (A–D and F) and longitudinal (E) sections showing ovules of Melastomataceae species belonging to tribes that exhibit exclusively a two-cell-layered outer integument. A, Pternandra coerulescens (Kibessieae). B, Bellucia grossularioides (Henrietteeae). C, Henriettea ramiflora (Henrietteeae). D, Bertolonia mosenii (Bertolonieae). E, Chalybea ecuadorensis (Blakeeae). F, Allomaieta grandiflora (Cyphostyleae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 4. View largeDownload slide Transverse (A, C and E–F) and longitudinal (B, D) sections showing ovules of Melastomataceae species belonging to tribes or clades that exhibit exclusively a two-cell-layered outer integument. A, Triolena obliqua (Triolena clade). B, Cambessedesia espora (Cambessedesia clade). C, Merianthera bullata (Cambessedesia clade). D, Merianthera parvifolia (Cambessedesia clade). In C and D, punctual periclinal divisions may give rise to a third layer in certain regions of the outer integument (arrows). E, Arthrostemma ciliatum (Rhexieae). F, Microlicia cordata (Microlicieae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 4. View largeDownload slide Transverse (A, C and E–F) and longitudinal (B, D) sections showing ovules of Melastomataceae species belonging to tribes or clades that exhibit exclusively a two-cell-layered outer integument. A, Triolena obliqua (Triolena clade). B, Cambessedesia espora (Cambessedesia clade). C, Merianthera bullata (Cambessedesia clade). D, Merianthera parvifolia (Cambessedesia clade). In C and D, punctual periclinal divisions may give rise to a third layer in certain regions of the outer integument (arrows). E, Arthrostemma ciliatum (Rhexieae). F, Microlicia cordata (Microlicieae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. In the Cambessedesia clade, although the outer integuments of Merianthera bullata R.Goldenb., Fraga & A.P.Fontana and M. parvifolia R.Goldenb., Fraga & A.P.Fontana are mostly two-cell-layered, punctual periclinal divisions led to the formation of a third cell layer in some regions of the ovule and indicate a multiplicative integument in Merianthera Kuhlm. (Fig. 4C, D). Ovules with a multilayered outer integument occur in some species of the remaining groups: Olisbeoideae, Melastomateae, Merianieae and Miconieae (Table 1; Fig. 2). Rupestrea johnwurdackiana (Baumgratz & D’El Rei Souza) Michelang., Almeda, & R.Goldenb., not yet formally placed in any tribe in the family, also has a multilayered outer integument with four to five cell layers, possibly because of a multiplicative origin (Table 1; Figs 2, 5A, B). Figure 5. View largeDownload slide Longitudinal (A–E) and transverse (F–G) sections showing ovules of Rupestrea and Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. A–B, multilayered integument consisting of four to five cell layers in Rupestrea johnwurdackiana. C, two-cell-layered integument of Votomita guianensis (Olisbeoideae) with a thicker calazal region (arrow). D, multilayered integument of Mouriri cearensis (Olisbeoideae). Two-cell-layered integument in species of core Melastomateae (Melastomateae). E, Heterocentron elegans; F, Tibouchina clavata; G, multilayered integument with three cell layers in Aciotis purpurascens (Marcetia alliance, Melastomateae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 5. View largeDownload slide Longitudinal (A–E) and transverse (F–G) sections showing ovules of Rupestrea and Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. A–B, multilayered integument consisting of four to five cell layers in Rupestrea johnwurdackiana. C, two-cell-layered integument of Votomita guianensis (Olisbeoideae) with a thicker calazal region (arrow). D, multilayered integument of Mouriri cearensis (Olisbeoideae). Two-cell-layered integument in species of core Melastomateae (Melastomateae). E, Heterocentron elegans; F, Tibouchina clavata; G, multilayered integument with three cell layers in Aciotis purpurascens (Marcetia alliance, Melastomateae). Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. In Olisbeoideae, the outer ovule integument is two-cell-layered in Memecylon L. and in Votomita guianensis Aubl. (Fig. 5C). In the latter taxon, this integument is multiplicative and its cells undergo periclinal divisions, forming up to four cell layers in the chalazal region (Fig. 5C). On the other hand, in all species of Mouriri Aubl. sampled the outer integument is multilayered, with four to six cell layers (Fig. 5D). In Melastomateae, the outer integument is two-cell-layered in species of core Melastomateae (Fig. 5E, F). It is multilayered with three cell layers in species of the Marcetia DC. alliance (Figs 5G, 6A, B). Figure 6. View largeDownload slide Transverse (A and C–F) and longitudinal (B) sections showing ovules of Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. Multilayered integument with three cell layers in species of the Marcetia alliance, Melastomateae: A, Ernestia glandulosa; B, Nepsera aquatica. Outer integument thickness in Merianieae: C, Graffenrieda harlingii; D, Axinaea dentata; E, Meriania urceolata; F, Meriania sclerophylla. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 6. View largeDownload slide Transverse (A and C–F) and longitudinal (B) sections showing ovules of Melastomataceae species belonging to tribes that exhibit variation in outer integument thickness. Multilayered integument with three cell layers in species of the Marcetia alliance, Melastomateae: A, Ernestia glandulosa; B, Nepsera aquatica. Outer integument thickness in Merianieae: C, Graffenrieda harlingii; D, Axinaea dentata; E, Meriania urceolata; F, Meriania sclerophylla. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. In Merianieae, a two-cell-layered outer integument was observed in Graffenrieda gracilis (Triana) L.O.Williams, G. harlingii Wurdack (Fig. 6C), G. latifolia (Naudin) Triana and Macrocentrum repens (Gleason) Wurdack. An originally two-cell-layered integument, although with a third cell layer formed in some regions, occurs in Axinaea dentata E.Cotton (Fig. 6D), A. grandifolia (Naudin) Triana, A. minutiflora E.Cotton and Meriania urceolata Triana (Fig. 6E). In Axinaea floribunda (Naudin) Triana, Meriania sclerophylla (Naudin) Triana (Fig. 6F) and M. subumbellata Cogn., the outer integument is multilayered, with three cell layers. Most species of Miconieae have ovules with multilayered outer integuments (Table 1) (Fig. 7A–C). The thickness of this integument ranges from three to seven cell layers, but three layers were most frequent (Table 1) (Fig. 7A). More than three cell layers were observed only in species of the Miconia Ruiz & Pav. IV clade (Fig. 7B) and in the Miconia V grade (Fig. 7C) (Table 1), with evidence of a multiplicative integument. The two-cell-layered condition was observed only in the Eriocnema Naudin and Conostegia D.Don clades and in the Caribbean and Miconia V grades. In the Eriocnema clade, the outer integument is two-cell-layered in Physeterostemon thomasii Amorim, Michelangeli & Goldenb. (Fig. 7D), but multilayered with three cell layers in Eriocnema acaulis Triana (Fig. 7E) and E. fulva Naudin. All species belonging to the Conostegia clade have ovules with a two-cell-layered outer integument (Fig. 7F, G). In the Caribbean grade, Charianthus alpinus (Sw.) R.A.Howard, C. nodosus (Desr.) Triana (Fig. 7H) and Tetrazygia discolor (L.) DC. (Fig. 7I) have two-cell-layered outer integuments, whereas in Tetrazygia crotonifolia (Desr.) DC. (Fig. 7J) and T. elaegnoides (Sw.) DC. it is multilayered, with three cell layers. In the Miconia V grade, a two-cell-layered outer integument was observed only in M. ceramicarpa (DC.) Cogn. Figure 7. View largeDownload slide Longitudinal (A–C and G) and transverse (D–F and H–J) sections showing ovules of Melastomataceae species belonging to the Miconieae tribe. A, multilayered integument with three cell layers in Clidemia hirta. B, multilayered integument with five cell layers in Miconia cubatanensis. C, multilayered integument with three to four cell layers in Miconia inaequidens. D, two-cell-layered integument in Physeterostemon thomasii. E, multilayered integument with three cell layers in Eriocnema acaulis. Two-cell-layered integument in species of the Conostegia clade: F, Conostegia lasiopoda; G, Conostegia subcrustulata. H, two-cell-layered integument in Charianthus nodosus. I, two-cell-layered integument in Tetrazygia discolor. J, multilayered integument with three cell layers in Tetrazygia crotonifolia. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Figure 7. View largeDownload slide Longitudinal (A–C and G) and transverse (D–F and H–J) sections showing ovules of Melastomataceae species belonging to the Miconieae tribe. A, multilayered integument with three cell layers in Clidemia hirta. B, multilayered integument with five cell layers in Miconia cubatanensis. C, multilayered integument with three to four cell layers in Miconia inaequidens. D, two-cell-layered integument in Physeterostemon thomasii. E, multilayered integument with three cell layers in Eriocnema acaulis. Two-cell-layered integument in species of the Conostegia clade: F, Conostegia lasiopoda; G, Conostegia subcrustulata. H, two-cell-layered integument in Charianthus nodosus. I, two-cell-layered integument in Tetrazygia discolor. J, multilayered integument with three cell layers in Tetrazygia crotonifolia. Outer integument cell layers are indicated by four-point stars and inner integument cell layers by three-point stars. Nu, nucellus; OI, outer integument; II, inner integument; VB, vascular bundle. Scale bars = 10 μm. Ancestral state reconstruction Stochastic mapping analyses indicate the most recent common ancestor of Melastomataceae as probably having an ovule with a two-cell-layered outer integument. Ovules with a multilayered outer integument evolved independently at least six times: (1) in the ancestor of Mouriri; (2) in the ancestor of Rupestrea R.Goldenb., Almeda & Michelang.; (3) in the ancestor of the Marcetia alliance; (4) in the ancestor of a clade in Merianieae; (5) in Axinaea floribunda; and (6) in the ancestor of Miconieae. However, multiple reversals from multilayered to two-cell-layered integuments apparently also occurred independently in Merianieae in Meriania urceolata and in Miconieae in Physeterostemon R.Goldenb. & Amorim, in a small clade in the Caribbean grade, in the Conostegia clade and in Miconia ceramicarpa (Fig. 2). A test for correlated evolution showed that outer ovule integument thickness is not significantly correlated with fruit type in the family (Fig. 8; likelihood ratio: 2.1037; P = 0.35). DISCUSSION Different ontogenetic pathways gave origin to the outer integument in Melastomataceae Multilayered ovule integuments may originate from the dermal layer and become thicker by periclinal division of epidermal cells (dermal integuments), or from both the dermal and the subdermal layers (subdermal integuments) (Bouman, 1984; Endress, 2011). At least in Miconieae and in the Marcetia alliance (Melastomateae), the multilayered outer integument is classified as ‘subdermal’ because it derives from the dermal and subdermal layers of the ovule primordium (Bouman, 1984; Shamrov, 2000). This pattern contrasts with the dermal origin typically observed in species with a two-cell-layered outer integument (Bouman, 1984; Endress, 2011), a condition that is found in most Melastomataceae. Although dermal and subdermal integuments can become multiplicative and produce multilayered integuments, the different ontogenetic patterns observed support the distinct nature of these two character states (two-cell-layered and multilayered outer integument) and their use in the delimitation of clades in Melastomataceae. Outer ovule integument thickness as a taxonomic marker for Melastomataceae clades During the evolutionary history of Melastomataceae, the thickness of the outer ovule integument has apparently remained relatively stable, enabling us to recognize many clades in the family. Ovules with a two-cell-layered outer integument are probably a plesiomorphic condition in Melastomataceae and occur in most of the analysed lineages. However, ovules with a multilayered outer integument evolved independently in Mouriri, Rupestrea, the Marcetia alliance, Merianieae and Miconieae. Moreover, reversals to the plesiomorphic two-cell-layered integument condition have also occurred more than once. In Olisbeoideae, Mouriri differs from Memecylon and Votomita Aubl. by having a multilayered outer integument. Molecular data support each of these genera as monophyletic (as well as three other genera in the subfamily) and show that the Neotropical genera Mouriri and Votomita are sister groups (Stone, 2006, 2014). Additional data on the outer integument thickness of Lijndenia Zoll. & Moritzi, Spathandra Guill. & Perr. and Warneckea Gilg are necessary to establish whether the thick outer integument found in Olisbeoideae is a single condition observed in Mouriri or if it is homoplastic. Regardless, this character state could be used to separate Mouriri from Votomita from an embryological standpoint. The presence of a multilayered and considerably thick outer integument characterizes and delimits Rupestrea. This genus of two species was positioned as sister to Microlicieae in our analysis, although recently placed with low support as sister to the Microlicieae + Melastomateae + Rhexieae clade (Goldenberg et al., 2015). In Melastomateae, the three-layered outer integument is a synapomorphy for the Marcetia alliance as species in core Melastomateae and the closely associated tribes Rhexieae and Microlicieae have two cell layers, although Rupestrea has a multilayered integument. Microlicieae, Rhexieae, the Marcetia alliance and core Melastomateae form a large monophyletic group and share stamens with elongated pedoconnectives and capsular fruits, but their relationships are still being debated (Michelangeli et al., 2013; Rocha et al., 2016a). So far, the Marcetia alliance has been characterized by tetramerous flowers (with some exceptions), a tetralocular ovary (or reduced to two or three locules), absence of crown hairs on the ovary apex, and by cochleate, ovate or lacrimiform seeds (Michelangeli et al., 2013; Rocha et al., 2016a). The considerable variation in the outer ovule integument thickness and the absence of a broad sampling in Merianieae limit the utility of our data for systematic conclusions at this time. In this tribe, although the shape of the calyx and stamens has been traditionally used in taxonomy, these floral characters have proved to be poorly informative for the circumscription of the more diverse genera (Mendoza-Cifuentes & Fernández-Alonso, 2010, 2012). In this respect, embryological characters such as the one tested here can provide important information for clade delimitation. The variation detected in Merianieae indicates that this character should be considered and more extensively investigated in future systematic studies on the tribe. Finally, in Miconieae, despite the considerably frequent multilayered condition, ovules with a two-cell-layered outer integument delimit the genus Physeterostemon and two clades. Molecular data indicate that Physeterostemon is a well-supported monophyletic genus sister to Eriocnema, the two forming a clade sister to the remaining Miconieae (Amorim et al., 2009; Goldenberg et al., 2015). Physeterostemon also differs from Eriocnema by its inferior ovary (Cogniaux, 1891; Fritsch et al., 2004; Goldenberg & Amorim, 2006; Amorim et al., 2009; Amorim, Jardim & Goldenberg, 2014). Both Eriocnema and Physeterostemon have capsular fruits, different from the fleshy fruits found in other Miconieae (Amorim et al., 2009). In the Caribbean grade, the clade with Charianthus D.Don and Tetrazygia discolor differs from the remaining species of Tetrazygia Rich. ex DC. by having a two-cell-layered outer integument. This clade stands out from the grade because of its biogeography, consisting of endemic species of the Lesser Antilles (Michelangeli et al., 2008), but the relationships in the group remain undefined (Penneys & Judd, 2005; Goldenberg et al., 2008). The two-cell-layered condition of the outer ovule integument can be seen as a synapomorphy for the Conostegia clade because its sister group, the monopodial Clidemia D.Don clade, has a multilayered outer integument. The Conostegia clade, which included species of Clidemia, Conostegia and Miconia (currently recognized as Conostegia) (Kriebel, 2016), is well supported by molecular analyses, but thus far no morphological or anatomical character has been found to diagnose the group (Kriebel et al., 2015). Relationship between the multilayered outer integument and fleshy fruits in Melastomataceae Although we hypothesized that the outer ovule integument thickness would be correlated with fruit type, the results failed to achieve significance. Nonetheless, ancestral state reconstructions show a pattern in which a multilayered outer integument tends to occur more frequently among clades with fleshy fruits, such as the Miconieae and Mouriri (Fig. 8). Although a lack of significance in this kind of analysis might be related to the small number of changes in character states over the phylogeny (Felsenstein, 1985), the results may also indicate a more complex scenario for the evolution of this anatomical character. Figure 8. View largeDownload slide Mirror composite trees showing the stochastic mapping of outer ovule integument thickness (A) and fruit type (B) onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using molecular makers. Tree A (outer ovule integument thickness): blue corresponds to the two-cell-layered status, and red to the multilayered status. Tree B (fruit type): blue corresponds to the dry status, and red to the fleshy status. Figure 8. View largeDownload slide Mirror composite trees showing the stochastic mapping of outer ovule integument thickness (A) and fruit type (B) onto the resulting phylogenetic tree of Melastomataceae from the Bayesian analysis using molecular makers. Tree A (outer ovule integument thickness): blue corresponds to the two-cell-layered status, and red to the multilayered status. Tree B (fruit type): blue corresponds to the dry status, and red to the fleshy status. Fleshy fruits in Melastomataceae are mainly dispersed by endozoochory by birds, but also by other animals such as rats, bats, monkeys and fishes (Magnusson & Sanaiotti, 1987; Renner, 1989; Stiles & Rosselli, 1993; Ellison et al., 1993; Galetti & Stotz, 1996; de Figueiredo & Longatti, 1997; Garcia, Rezende & Aguiar, 2000; Lapenta & Procópio-de-Oliveira, 2008; Maruyama, et al., 2013). Since the seed testa is the mechanical layer responsible for the protection of the embryo in the family (Corner, 1976; Cortez & Carmello-Guerreiro, 2008), the presence of more layers in the outer ovule integument may correspond to more sclerified layers in the seed coat, which in turn could make this seed coat thicker and more rigid. In Miconia albicans (Sw.) Triana, for example, the three-layered outer ovule integument gives rise to the testa, which consists of an exotesta of sclerified palisade cells covering the meso- and endotesta with cuboidal sclerified cells (Cortez & Carmello-Guerreiro, 2008). Endozoochory has been frequently related to a resistant seed coat (Baskin & Baskin, 2014), responsible for the protection of the embryo and of other seed components during passage through the digestive tract of animals (Boesewinkel & Bouman, 1984; Mohamed-Yasseen et al., 1994). Thus, the presence of an additional layer in the outer ovule integument may have been an important evolutionary step, particularly in Miconieae and Mouriri. Apart from these clades, endozoochoric fleshy fruits have also evolved independently in Blakeeae, Dissochaeteae and Henrietteeae (Renner, 1989; Stiles & Rosselli, 1993; Clausing, Meyer & Renner, 2000; Penneys et al., 2010), all tribes with a two-cell-layered outer ovule integument, proving that the multilayered condition is not strictly associated with fleshy fruits. In addition to the multilayered outer integument, the presence of a multiplicative integument can also lead to thicker seed coats, as reported for Memecylon (Olisbeoideae) (Corner, 1976). In Blakea trinervia L. (Blakeeae), the seed coat is apparently non-multiplicative (Corner, 1976), but we do not know whether this pattern is repeated through the entire tribe. There is no anatomical information on the seed coat structure for Dissochaeteae and Henrietteeae. The relationship between ovules with a multilayered outer integument and fleshy fruits seems to occur in the sister group of Melastomataceae. Crypteroniaceae, Alzateaceae and Penaeaceae mostly have ovules with a two-cell-layered outer integument and capsular fruits (Meijer, 1972; Tobe & Raven, 1983b, 1984a, c, d, 1987a, b; Conti et al., 2002). The only exception is Olinia Thunb. (Penaeaceae), with multilayered outer integument and fleshy fruits (Rao & Dahlgren, 1969; Tobe & Raven, 1984b; Conti et al., 2002). Multiplicative outer integument and large seeds in Melastomataceae Indications of a multiplicative integument were observed in Votomita (Olisbeoideae), in species of the Miconia discolor subclade and of the Miconia V grade (Miconieae), in Merianthera (Cambessedesia clade) and in Rupestrea. In general, ovules that become large seeds have multiplicative integuments, which give rise to thicker and massive seed coats (Corner, 1976; Boesewinkel & Bouman, 1995). We presume that this relationship also exists in different lineages of Melastomataceae, independent of the initial condition, as it was found in species with multilayered and two-cell-layered outer ovule integuments. Olisbeoideae are known to have few ovules that will develop into one or several large seeds (Morley, 1976, 1999; Bremer, 1981; Clausing & Renner, 2001). In this subfamily, in addition to the multiplicative integument detected in Votomita, the same structure has been reported for Mouriri and Memecylon (Corner, 1976). In Miconieae, a multiplicative integument consisting of more than three cell layers is a recurrent condition exclusively detected in species belonging to the Miconia discolor subclade (Miconia IV clade) and the Miconia V grade. The Miconia discolor subclade and some species of the Miconia V grade are notable for their fruits with few large seeds (Goldenberg, 1999; Martins et al., 2009; Caddah, 2013; Ocampo & Almeda, 2013; Ocampo, Michelangeli & Almeda, 2014), which differ from fruits with numerous minuscule seeds observed in most other species of Miconieae (Baumgratz, 1985; Groenendijk, Bouman & Cleef, 1996; Bécquer, Michelangeli & Borsch, 2014; Ocampo et al., 2014). A similar relationship between multiplicative integument and seed size occurs in Rupestrea, with a few large seeds in the ovary (Goldenberg et al., 2015). CONCLUSIONS We studied the evolution of the outer ovule integument thickness in Melastomataceae, reporting this condition in a large number of species. In addition, we showed that two-cell-layered and multilayered integuments have different ontogenetic origins. The results indicate that the outer ovule integument thickness is relatively stable in the family, but the variations observed in Olisbeoideae, Rupestrea, Melastomateae and Miconieae are of systematic value for the groups. Finally, there is a tendency of association between the multilayered integument and fleshy fruits in Miconieae and Mouriri, and between multiplicative integument and large seeds in a few members of the family. SUPPORTING INFORMATION Appendix S1. List of the species of Melastomataceae analysed in this study, information source for its systematic position, data source and herbarium and voucher data. Appendix S2. GenBank accession numbers for sequences included in the phylogenetic analyses based on plastid and nuclear markers. ACKNOWLEDGEMENTS We thank the Structural Botany Laboratory of the New York Botanical Garden for technical support, Ricardo Kriebel and Julien Bachelier for useful suggestions on the manuscript, and Fabiana dos Santos Oliveira and Lucas Simon Torati for help in obtaining some samples. We also thank an anonymous reviewer for valuable comments that improved the manuscript. This study was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp – process numbers 2008/10793-0, 2010/15077-0 and 2013/08945-4). S.P.T. is indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico (process number 303493/2015-1) for the fellowship received. Portions of the research were also funded by the National Science Foundation (DEB-0818399). REFERENCES Almeda F. 2009. Melastomataceae. In: Davidse G, Sousa M, Knapp S, Chang F, eds. Flora Mesoamerica . Mexico City: Universidad Nacional Autonoma de Mexico, 180– 337. Amorim AM, Goldenberg R, Michelangeli FA. 2009. 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