Phylogenetic relationships of Hechtia (Hechtioideae; Bromeliaceae)

José Luis Tapia Muñoz

This is the first phylogenetic analysis of the Megamexican Bromeliaceae genus Hechtia and includes 82.6 % of the known taxa. We used plastid (ycf1, rpl32-trnL intergenic spacer), and nuclear (PRK) DNA regions, as well as morphological characters. We generated 244 new sequences for a total of 62 taxa (including 12 species of the outgroup). Results of combined data using parsimony and Bayesian inference reveal the monophyly of Hechtia, as well as identify five well supported clades: (1) a clade (H. tillandsioides complex) as the sister group to the rest of Hechtia; (2) a clade including the species of the H. guatemalensis complex, distributed in Southern Megamexico; the remaining taxa of the genus are retained in a clade which consists of three well-supported clades; (3) the H. glomerata complex distributed in the Gulf of Mexico drainage; (4) a clade of two species (H. deceptrix and H. epigyna) that share an inferior ovary and are distributed north of the Tehuantepec Isthmus in the Si...

Phytotaxa 376 (6): 227–253 http://www.mapress.com/j/pt/ Copyright © 2018 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.376.6.1 Phylogenetic relationships of Hechtia (Hechtioideae; Bromeliaceae) IVÓN M. RAMÍREZ-MORILLO1,*, GERMÁN CARNEVALI1,5, JUAN P. PINZÓN2, KATYA ROMERO-SOLER1, N. RAIGOZA1, C. HORNUNG-LEONI3, R. DUNO1, J. L. TAPIA-MUÑOZ1 & I. ECHEVARRÍA4 1 Centro de Investigación Científica de Yucatán, A. C. Unidad de Recursos Naturales. Calle 43 # 130 x 32 y 34. Colonia Chuburná de Hidalgo. Mérida, Yucatán, CP 97205, Mexico. 2 Departamento de Botánica, Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Carretera MéridaXmatkuil, Km. 15.5, Apdo. Postal 4-115 Itzimná, CP 97100, Mérida, Yucatán, Mexico. 3 Universidad Autónoma del Estado de Hidalgo, Centro de Investigaciones Biológicas, Instituto de Ciencias Básicas e Ingeniería, Km 4.5 Carretera Pachuca-Tulancingo, Mineral de La Reforma, Hidalgo, CP 42184, Mexico. 4 Centro de Investigación Científica de Yucatán, A. C. Unidad de Bioquímica y Biología Molecular de Plantas. Calle 43 # 130 x 32 y 34. Colonia Chuburná de Hidalgo. Mérida, Yucatán, CP 97205, Mexico. 5 Orchid Herbarium of Oakes Ames, Harvard University Herbaria, 22 Divinity Avenue, Cambridge, Massachusetts 02138, U.S.A. *Corresponding author: Email: ramirez@cicy.mx Abstract This is the first phylogenetic analysis of the Megamexican Bromeliaceae genus Hechtia and includes 82.6 % of the known taxa. We used plastid (ycf1, rpl32-trnL intergenic spacer), and nuclear (PRK) DNA regions, as well as morphological characters. We generated 244 new sequences for a total of 62 taxa (including 12 species of the outgroup). Results of combined data using parsimony and Bayesian inference reveal the monophyly of Hechtia, as well as identify five well supported clades: (1) a clade (H. tillandsioides complex) as the sister group to the rest of Hechtia; (2) a clade including the species of the H. guatemalensis complex, distributed in Southern Megamexico; the remaining taxa of the genus are retained in a clade which consists of three well-supported clades; (3) the H. glomerata complex distributed in the Gulf of Mexico drainage; (4) a clade of two species (H. deceptrix and H. epigyna) that share an inferior ovary and are distributed north of the Tehuantepec Isthmus in the Sierra Madre Oriental; and (5) an internally poorly resolved clade with the remaining species containing several well-supported, geographically restricted clades. At this time it is uncertain what part of Megamexico was first invaded by the ancestor of Hechtia. Regardless, it becomes clear that from the original point of invasion in what is now Megamexico, it radiated into restricted geographical realms with secondary radiations occurring within them, which resulted in some recurrent particular evolutionary trends most likely associated with the invasion of dry, highly seasonal climates, or cooler areas subject to occasional frosts. Lateral inflorescences and flower morphology suggesting pollination syndromes other than melittophily (psychophily/trochilophily) have evolved more than once in Hechtia. Key words: Character evolution-Endemism-Megamexico Resumen Este es el primer análisis filogenético del género Megamexicano Hechtia (Hechtioideae; Bromeliaceae), el cual incluye al 82.6 % de los taxones conocidos. Usamos las regiones de ADN de plástidos (ycf1, rpl32-trnL espaciador intergénico) y nucleares (PRK), así como caracteres morfológicos. Generamos 244 nuevas secuencias para un total de 62 taxones (incluidas 12 especies del grupo externo). Los resultados de datos combinados usando parsimonia e inferencia Bayesiana revelan la monofilia de Hechtia, así como la identificación de cinco clados bien apoyados: (1) un clado (complejo H. tillandsioides) como el grupo hermano del resto de Hechtia; (2) un clado que incluye especies del complejo H. guatemalensis; los taxones restantes del género se agrupan en un clado el cual consta de tres linajes bien respaldados; (3) el complejo H. glomerata distribuido en la vertiente del Golfo de México; (4) un clado de dos especies (H. deceptrix y H. epigyna) que comparten un ovario inferior y se distribuyen al norte del Istmo de Tehuantepec en la Sierra Madre Oriental; y (5) un clado interno pobremente resuelto con las especies restantes que contienen varios clados bien respaldados y restringidos geográficamente. Actualmente es incierto el punto y la via de entrada del ancestro de Hechtia a Megaméxico, pero es muy probable que una vez que lo invadió, irradió a reinos geográficos restringidos con radiaciones secundarias dentro de ellos, lo que dio lugar a algunas tendencias evolutivas particulares recurrentes muy probablemente asociadas con la invasión de climas secos y altamente estacionales, o áreas más frías sujetas a heladas ocasionales. Las inflorescencias laterales y morfología floral que Accepted by Eric Gouda: 20 Oct. 2018; published: 22 Nov. 2018 227 sugiere otros síndromes de polinización diferentes a la melitofilia (psicofilia / troquilofilia), han evolucionado más de una vez en Hechtia. Palabras clave: Evolución de caracteres-Endemismo-Megaméxico Introduction The most recent classifications of Bromeliaceae using combined analysis of eight regions of plastid DNA, recognize eight lineages, each treated as a subfamily by Givnish et al. (2007, 2011, 2014). Of the three classic subfamilies recognized by Smith & Downs (1974), only Tillandsioideae and Bromelioideae are retrieved as monophyletic and nested within a broadly non-monophyletic Pitcairnioideae, a group held together by symplesiomorphies. Pitcairnioideae was thus recircumscribed as most of its original members now reside in several newly recognized subfamilies (e.g., Brocchinioideae, Lindmanioideae, Puyoideae, Hechtioideae, and Navioideae, which are related as follows: (Brocchinioideae, (Lindmanioideae, (Tillandsioideae, (Hechtioideae, (Navioideae, (Pitcairnioideae, (Puyoideae, Bromelioideae))))))). Givnish et al. (2014) further suggested that the family originated ca. 97.5 Ma in the Guayana Shield from where they radiated to other Neotropical areas 16–15.2 Ma, including a single long-distance dispersion event to Western Africa ca. 9.3 Mya. Previous phylogenetic analysis containing species of Hechtia Klotzsch (1835: 401) (i.e. Horres et al. 2000; Reinert, Russo & Salles 2003; Crayn, Winter & Smith 2004; Givnish et al. 2004, 2007, 2011) include less that 6 % (3 to 4) of the currently known species. Nevertheless, Hechtia has always been retrieved as monophyletic in studies at different taxonomic levels (Horres et al. 2000; Crayn et al. 2004; Givnish et al. 2004, 2007, 2011). Furthermore, Givnish et al. (2011) proposed that Hechtia arose ca. 16.6 Ma and invaded the current territory of Megamexico, where the ancestors of most modern species probably differentiated ca. 10.3 Ma. Currently, there is neither a modern systematic revision of the genus (the last one is that of Smith & Downs, 1974) nor a phylogenetic hypothesis of internal relationships. Hechtioideae comprises a single genus, Hechtia within which 75 species are currently recognized (Gouda et al. (cont.updated) and there is still several more awaiting nomenclatural recognition. Within Bromeliaceae, the subfamily is characterized by several features (sensu Givnish et al. 2007). These include capsular fruits, winged or almost naked seeds, dioecy, succulent leaves (see Givinsh et al. 2007) spiny or rarely entire foliar margins, and lack of stellate sclerenchyma. In addition, plants are terrestrial or lithophytic, growing over limestone, gypsum, or volcanic rocks. The flowers are fragrant (with the exception of flowers with putatively psychophilous or ornithophilous syndromes such as Hechtia rosea E. Morren ex Baker, H. iltisii Burt-Utley & Utley, and H. meziana L.B. Sm.), the pistillate ones with a simple-erect stigma and staminodia whereas staminate flowers bear stamens as well as pistillodes. Hechtia is restricted to and spans most of Megamexico III (Rzedowski 1991), a region encompassing all of the Chihuahuan and Sonoran deserts at the northern limit (Texas in U.S.A.), and extending southwards to northern Nicaragua (Nueva Segovia and Jinotega Departments; Figure 1), although most of the species (94 %) are restricted to Mexico proper (Ramírez and Jiménez 2012; Espejo-Serna 2012). The genus reaches its highest richness in the biogeographic provinces of Sierra Madre del Sur, Veracruzan, Chiapas Highlands, Pacific Lowlands, Balsas Basin, Transmexican Volcanic Belt, and Mexican Plateau (circumscribed as in Morrone 2014). About half of the species are restricted to a single province, whereas 35 % occur in two provinces. An additional 6 % are recorded from three or more provinces. When more than one, the provinces involved in the distribution of the taxa are always contiguous (Pech-Cárdenas 2015); suggesting long range dispersal is unlikely. Hechtia taxa occur at a wide range of elevations, from sea level to 2700 m. They occur in xerophytic shrublands (where they can be conspicuous floristic elements) as well as in Quercus-Pinus associations, or even in tropical dry forests, where they are less diverse. Currently, Hechtia is an insufficiently known taxonomic group. Advancement in its understanding has been hindered by its dioecy coupled with floral dimorphism and phenotypic plasticity. Also, the cumbersome, heavily spined plants that are often very large (e.g. Hechtia myriantha Mez; Espejo, López-Ferrari & Ramírez-Morillo 2005) have usually resulted in the accumulation of fragmentary herbarium specimens, difficulting the understanding of species variation and their boundaries. Many Hechtia species have been described from fruiting specimens because fruits are long lasting and relatively showy as opposed to the inconspicuous flowers lasting but one day; thus, plants are commonly collected in fruiting condition. In other, somewhat better cases, the type specimens are staminate; frequently the flowering period of staminate plants are longer, although individual flowers equally last one day. Additionally, sex ratios are usually male-skewed in many species, as it has been documented in Hechtia schottii Baker (Ramírez et al. 2008). A taxonomic consequence of the disparity in sexual morph representation in herbaria is that many species are 228 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. only partially known and the taxonomist´s task here consists in documenting the species to fill up voids of information. In many cases, it has been necessary to epitypify species whose types include only one sex, rendering the species ambiguously diagnosable (Ramírez-Morillo et al. 2012) or publish accounts of species where both sexual morphs as well as fruits are documented and reconciled (Ramírez, Jiménez & Treviño 2013; Ramírez et al. 2014; RamírezMorillo et al. 2016). FIGURE 1. Distribution of Hechtia (blue dots) in Megamexico, based on herbarium specimens (sensu Pech-Cárdenas 2015). Most modern Hechtia taxonomists have taken up the task to fully document morphologically their new species, as well as providing field observations to understand natural variation (Burt-Utley & Utley 1993; Burt-Utley et al. 2011; Espejo-Serna et al. 2008; Espejo-Serna et al. 2010; García-Ruiz et al. 2014; González-Rocha et al. 2014; LópezFerrari & Espejo-Serna 2013, 2014; Martínez-Correa et al. 2010; Ramírez-Morillo et al. 2014; Ramírez-Morillo et al. 2015; Ramírez-Morillo et al. 2016). In the course of these studies, we have been able to observe discontinuities in several relevant characters as well as identify species groups with distinctive biogeographies. There is variation in growth patterns (Ramírez-Morillo et al. 2014) that range from plants with monopodial architecture to others where sympodial growth may come along with precociously blooming rosettes (resulting in apparently lateral inflorescences) or these flowering terminally when rosettes are fully mature; these growth patterns are also associated with altitudinal and biogeographical distribution patterns as it will be discussed later. There is also variation in vegetative features such as rosette size, foliar margin and spination or the presence of stolons. Between taxa and clades within the genus, inflorescences are variable in architecture and flowers are equally variable in indument, as well as texture of sepals and petals, ovary position, and the morphology of fruits and seeds. Pollen is also variable in size and ornamentation of exine (Herrera 2016) whereas seed size and its external features are taxonomically informative (Fatzer 1989). Some anatomical features have been analyzed (Martínez-Correa et al. 2010; Flores 1975) and found potentially useful in disentangling the relationships within the genus. Several of the morphological groups within Hechtia also share distinctive, coherent biogeographical ranges (Pech-Cárdenas 2015). All this evidence lends clues to the evolutionary history of the genus that should reveal itself in phylogenetic analysis. PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 229 In this study we explore the monophyly and internal relationships of Hechtia with a taxonomic sampling far exceeding any previous study. To accomplish this, we use both morphological and molecular data of 82.6 % of the known species of the genus. We contextualize our sampling with representatives of the seven remaining bromeliad subfamilies to assess sister-taxa relationships. This study will lie the foundation for forthcoming articles aimed at further investigation of character evolution and an analysis of the factors driving the radiations of this Megamexico III-endemic clade. We aim at addressing the following questions:—is Hechtia a monophyletic assemblage of taxa when tested with a larger sampling and new evidence?;—what are the internal relationships of the group and what synapomorphies support clades identified by analysis?;—have lateral inflorescence, inferior ovary, and flower morphology suggesting the psychophily/trochyphily syndromes evolved one or several times in the genus? Materials and methods Taxa selection This study includes representatives of all subfamilies recognized by Givnish et al. (2007), accounting for a total of 62 taxa, 12 of which constitute an outgroup, and a total of 248 sequences, 244 new, four obtained from the GenBank (see Appendix 1). The outgroup includes taxa of the seven additional subfamilies: Bromelioideae (2 spp.), Brocchinioideae (1 sp.), Navioideae (1 sp.), Lindmanioideae (2 spp.), Pitcairnioideae (2 spp.), Puyoideae (1 sp.), and Tillandsioideae (3 spp.). To address questions regarding the internal relations of Hechtia, we selected 50 species (accounting for ca. 82.6 % of currently recognized taxa in the genus), including four undescribed yet well-characterized species from the Mexican States of Chiapas (CHIS), Guanajuato (GTO), and Tamaulipas (TAMPS), these are: Hechtia sp. TAMPS: Salto, TAMPS: Jaumave, GTO: Xichú, and CHIS: Comitán. Plant material Samples were taken from live plants that were field collected, obtained in exchange or purchased from trustworthy sources. Seed of several species were sown and grown at the Jardín Botánico Regional “Roger Orellana” of CICY and in private greenhouses. Material was obtained in Mexico, the USA, Guatemala, and Honduras. Silica gel-dried samples were obtained as exchange from the Marie Selby Botanical Gardens, the Rio de Janeiro Botanical Garden (Brazil), and the DNA bank of the Missouri Botanical Garden. Furthermore, private collectors or growers donated materials used in this study (see Appendix 1). DNA extraction, amplification and sequencing To yield a more robust phylogenetic tree than previous attempts, we chose three plastid DNA regions (rpl32-trnL intergenic spacer (UAG) (from here onwards referred to as rpl32-trnL), and two fragments of the ycf1 gene. We also sequenced a fragment of the low-copy, nuclear PRK gene. The rpl32-trnL region of circa 1,018 bp (Shaw et al. 2007) is found in the short single copy (SSC) region of the plastid DNA and several studies (e.g., Givnish et al. 2011) have demonstrated that it is useful in phylogenetic analysis. The ycf1 (plastid yeast cadmium factor protein 1 or TIC214), of approximately 5,500 bp (one of the longest genes of the plastid genome) is also mostly located in the SSC region and is the most variable coding plastid region in Angiosperms (Dong et al. 2015) even larger than that of matK (Neubig et al. 2009). Recently, Castello et al. (2016) and Pinzón et al. (2016) employed this region to resolve the relationships within the Tillandsia capillaris Ruiz & Pav. complex (Tillandsioideae) and Tillandsia utriculata L. complex, respectively, whereas Barfuss et al. (2016) successfully used it in a phylogenetic analysis that was the base for a taxonomic revision of Tillandsioideae. In these cases, larger absolute numbers of informative sites were found as compared to the plastid regions previously employed. Finally, the low-copy PRK (Phosphoribulokinase) gene has proven to be highly informative in Bromeliaceae (Schulte, Barfuss & Zizka 2009; Barfuss 2012). DNA extraction was performed with fresh or silica gel dried tissue. The DNeasy Plant Mini Kit (QIAGEN) was used following the manufacturer specifications. In a few cases, the Mini-prep 2× CTAB method (Vázquez-Lobo 1996, modified) was employed. To amplify the rpl32-trnL intergenic spacer the following primers were used: trnL(UAG) and rpl32-F (Shaw et al. 2007). The ycf1-a fragment was amplified with primers ycf1(part 2) whereas for the ycf1b fragment, the ycf1-4492f-br and ycf1-5440r-br primers were used (Barfuss et al. 2016; Castello et al. 2016). The nuclear PRK region was amplified with the following primers: prk-735f (Schulte et al. 2009), prk-630f, prk-890r, and prk-1057r (Barfuss 2012). 230 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. The following PCR protocol was employed for a final volume of 25 μl in each reaction: 2.5 μl Buffer 10x (Invitrogen), 1.85 μl MgCl2 50μM (Invitrogen), 0.4 μl dNTP 10X, 0.5 μl 10x each primer, 0.2 μl Taq DNA polymerase 5 U/μl (Invitrogen), 2 μl DNA, and the volume was completed with dH2O. To amplify the PRK region, 2.5 μl BSA 0.04 % was added to the solution. PCR reactions were conducted in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s (35 cycles for both ycf1 fragments), annealing at 62.5 °C (rpl32-trnL), 58 °C (both ycf1 fragments) and 62 °C (PRK) for 5 s, a period of 70 °C for 1.3 min, and a final extension at 70 °C for 7 min. To assess whether PCR reactions were successful, an agarose (1.5 %) gel electrophoresis was performed with ethidium bromide as a dye. To accomplish this, 5 μl of the PCR product were placed and photographed in an UV transiluminator. Samples were then sent to MACROGEN (South Korea; http://dna.macrogen.com) for sequencing. Primers for sequencing were the same as for amplification with the exception of the internal primers prk-735f and prk890r, used for sequencing nuclear PRK. Morphological characters Seven morphological characters were assessed and coded from the analysis of field populations, cultivated plants, material in spirits (70 % EtOH, 5 % glycerin; flowers and fruits), and herbarium specimens. Specimens from the following herbaria (acronyms according to Thiers, B., 2018) were available for analysis: ASU, B, BIGU, CHAPA, CICY, EAP, ENCB, F, GH, HGOM, HUH, IBUG, IZTA, K, LAGU, LL, MEXU, MHES, MICH, MO, NY, QMEX, SEL, TEFH, TEX, UAMIZ, US, W, WU, XAL, and ZEA. All structural characters employed are qualitative and discrete and are described as follows: (1) Growth pattern. Three different patterns were identified by Ramírez-Morillo et al. (2014) and were here codified as follows (see Appendix 2 for codification for each species): [0] SSP-Strict Sympodial Pattern: here, the apical meristem of a fully grown rosette produces an inflorescence; after fruiting the rosette dies, producing one or more offsets in a basal position, resulting in cespitose plants and then usually forming colonies consisting of several rosettes. [1] PMP-Pseudomonopodial Pattern: a rosette produces truly lateral inflorescences and the apical meristem remains indefinitely active, never making a transition into a floral meristem; thus, rosettes have indefinite vegetative growth, eventually becoming very large and flowering continuously as long as the rosette is alive. [2] SPFP- Sympodial with Precocious-Flowering Pattern: the inflorescence emerges from the center of a newly-forming shoot or rosette (thus precocious) that never fully develops; on occasion, fully developed rosettes flower simultaneously with the precocious ones. (2) Foliar margin. This character ranges from entire to serrulate (small teeth oriented to the apex of the leaf), to denticulate (minute teeth perpendicular to mid nerve) to spiny (spines antrorse and/or retrorse with hard, strong teeth): [0] Entire, [1] Serrulate, [2] Spiny, [3] Denticulate. (3) Style. It varies from absent with the stigmatic lobes emerging from the top of the ovary, to slightly developed or to conspicuous and elongated: [0] Absent, [1] Present. (4) Ovary position. It varies in Hechtia from completely inferior to completely superior, with intermediate states where the ovary is half-inferior or ¾ inferior: [0] Inferior, [1] Superior, [2] Partially inferior. (5) Flower fragrance. Its presence is very rare in Bromeliaceae, but all species of Hechtia (with the exception of those with red petals (the presumably hummingbird or butterfly-pollinated H. meziana, H. rosea, and H. iltisii) produce sweetly fragrant flowers (stronger in male flowers): [0] Absent, [1] Present. (6) Fruits. They vary from capsules with plumose, winged or naked seeds to fleshy fruits (berries), usually with naked seeds: [0] Capsules, [1] Berries. (7) Flower sex. Flowers have both sexual organs or they are either staminate or pistillate: [0] Hermaphrodite, [1] Unisexual. Sequences assembly, alignment, and coding of insertions/deletions The sequences generated were assembled with Geneious 7.1.3 (Kearse et al. 2012). Automated alignments of both our and GenBank sequences for each DNA region were produced with MUSCLE V3.8.31 (Edgar 2004) with the Mesquite V3.04 (Maddison & Maddison 2015) visual interface; these alignments were manually refined afterwards with the PhyDe V0.9971 editor (Müller et al. 2010). Indels were coded as binary characters following the simple coding method of Simmons & Ochoterena (2000), through SeqState V1.4.1 (Müller 2005). Information about taxa and sequences included in this study are shown on Appendix 1. PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 231 Phylogenetic analysis We initially conducted parsimony analyses (MP) of data from available sources of evidence: (1) seven morphological characters and 62 terminals; (2) each individual plastid DNA region (rpl32-trnL, rpl32-trnL +indels, ycf1-a, and ycf1-b) with 60–61 terminals; (3) nuclear PRK region with 58 terminals; (4) concatenated plastid regions with 62 terminals; (5) all plastid regions plus morphology; (6) all evidence (plastid and nuclear regions and morphology) with 62 terminals. Congruence between matrices was evaluated with the use of the Incongruence Length Difference test (ILD; Farris et al. 1995). This test was conducted in PAUP* V4.0a151 (Swofford 2003) with 1000 iterations per analysis. Phylogenetic reconstructions with Maximum Parsimony (MP) were performed with 500 iterations of the Parsimony Ratchet algorithm (Nixon 1999) through TNT v 1.5 (Goloboff, Farris & Nixon 2000). In all cases, Fitch parsimony (Fitch 1971) was implemented. Support of nodes was estimated with 1000 iterations of the bootstrap (BT) (Felsenstein 1985). Phylogenetic reconstructions were also performed under a Bayesian Inference (BI) approach for each the following data set: plastid, nuclear, and all evidence. Bayesian inference analyzes were performed with the use of MrBayes V3.2.6 (Ronquist & Huelsenbeck 2003) as implemented in the CIPRES Science Gateway platform (https://www.phylo.org/portal2/login!input.action). Nucleotidic substitution models were assessed with jModelTest 2.1.7 (Darriba et al. 2012) for each DNA partition. Substitution models best fitting the evolution of each nucleotidic partition were selected following the Akaike Information Criterion (AIC). The GTR + Γ model was identified as best for the evolution of the rpl32-trnL, ycf1-a, and ycf1-b regions whereas HKY + Γ was selected as the model best fitting the evolution of the PRK region. For the rpl32-trnL region, indels were treated as ‘restriction’ data, and thus, the ‘variable’ option was implemented. Two independent runs, each of 20.000.000 generations were performed; each run consisted of a “cold” and three “hot” chains. Each 1000 generations, a single tree was sampled. 25 % of the initial samples were discarded as burnin before data reached stationarity. Other parameters were set as for the default settings in Mr. Bayes. Convergence diagnosis was done in Tracer V1.6 (Rambaut et al. 2014) using the Effective Sample Sizes (ESS) values, which were all > 200. Clade Posterior Probabilities (PP) was assessed by a 50 % majority-rule consensus of trees retained after burn-in. Resulting tree was visualized and edited in Fig Tree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and Mesquite V3.04. An ancestral state reconstruction analysis was performed through parsimony using MESQUITE 2.75 (Maddison and Maddison 2015) for the same morphological characters used for the phylogenetic reconstruction (Appendix 2). Results Morphological characters and sequence alignments characteristics Table 1 resumes characteristics of the different sources of evidence employed in the analysis, including DNA regions (plastid and nuclear) and morphology. For each matrix, we indicated alignment size (pb), constant, variable, and parsimony-informative sites, number of resulting equally most-parsimonious trees and their length, number of taxa in each analysis, as well as consistency (CI) and retention (RI) indexes. DNA regions showed differences in variable and parsimony-informative sites, with the PRK nuclear region containing the highest number of variable sites and parsimony-informative sites, whereas the region with the lowest percentage of variable characters was rpl32-trnL and the ycf1-a region displayed the lowest percentage of parsimonyinformative characters. However, if we compare all plastid DNA regions with the nuclear PRK region, the percentage of variable sites is very dissimilar (16.2 % for plastid DNA, considering rpl32-trnL without indels; 49.3 % for PRK); it was even more different when considering the number of parsimony-informative characters (plastid DNA 6.8 %, 25.4 % for PRK). Coding of indels of the rpl32-trnL region resulted for this specific region in an increase of the parsimony-informative sites of only 1.2 %, but produced more trees, these most parsimonious (MPT´s); it also resulted in a decrease of the consistency and retention indexes. The addition of the seven morphological parsimony informative characters to the PRK matrix resulted in fewer trees, these shorter with higher CI and RI. Upon addition of the seven morphological characters to the plastid DNA regions matrix, the values of CI and RI increased, and the number of MPT´s decreased and shorter trees were found. In the case of the combined analysis, adding morphological data to all molecular evidence caused an increase on the number of maximally parsimonious trees, these shorter, and with lower values of CI. 232 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. PHYLOGENETICS OF HECHTIA Table 1. General statistics of the maximum parsimony phylogenetic reconstruction in Hechtia using DNA regions (plastid, nuclear, and morphology). Characteristics include alignment length, number and percentage of variable and parsimony-informative sites, taxa included in the analysis, number of MPTs, and their statistics (L, CI, and RI). Phytotaxa 376 (6) © 2018 Magnolia Press • 233 Marker Aligned size (bp) Constant sites (number/%) Variable sites (number/%) Parsimony informative characters (number/%) # taxa # trees Tree length CI RI rpl32-trnL 1165 995/85.4 % 170/14.6 % 68/5.8 % 61 1210 804 0.66 0.81 rpl32-trnL + indels 1179 995/84.4 % 184/15.6 % 82/7 % 61 9428 182 0.52 0.78 ycf1-a 906 760/83.9 % 146/16.1 % 49/5.4 % 60 5430 109 0.54 0.75 ycf1-b 904 749/82.9 % 155/17.1% 72/8 % 60 9236 136 0.58 0.78 PRK 1228 622/50.7 % 606/49.3 % 312/25.4 % 58 4683 797 0.56 0.75 Morphology 7 7/100% 7/100% 7/100% 62 1269 16 0.75 0.96 PRK + morphology 1235 622/50.4% 613/49.6% 319/25.8% 58 3455 778 0.58 0.78 cpDNA 2989 2504/83.8% 485/16.2% 203/6.8% 62 9392 448 0.52 0.75 cpDNA+morphology 2996 2504/83.6% 492/16.4% 210/7% 62 4821 412 0.59 0.82 All molecular evidence 4217 3126/74.1% 1091/25.9% 515/12.2% 62 1380 1741 0.72 0.79 All evidence 4224 3126/74% 1098/26% 522/12.3% 62 1838 1185 0.58 0.79 Phylogenetic analysis The majority rule consensus tree resulting from the parsimony analysis of the morphological evidence (not shown), retrieves some well-supported phylogenetic relationships, as follows: Brocchinia reducta Baker is sister to the rest of Bromeliaceae (100 % bootstrap support); internally Bromelioideae (here represented by two species of Bromelia L.) with 100 % of bootstrap support; Catopsis Griseb. as the sister group of Hechtia (a poorly supported relationship, 64 %), and a strongly supported clade constituted by Hechtia (a support of bootstrap of 100 %). Within Hechtia, there are only two clades, one constituted by Hechtia tillandsioides (André) L.B. Sm. and relatives (91 % bootstrap support) that is also retrieved in the strict consensus (not shown), and the poorly supported H. guatemalensis Mez Clade (73 % bootstrap support). This paucity of clades identified in this parsimony analysis is not surprising considering how few characters were included. However, the purpose of this analysis was not to resolve all the relationships of sampled taxa, but to assess the contribution of such characters and their congruence with molecular data. The mapping of the morphological characters on the strict consensus tree shows Hechtia to be supported by the characters: unisexual flowers, fragrant flowers, and the simple-erect stigma with sessile stigmatic lobes, the H. tillandsioides Clade is supported by the minutely serrulated foliar margin as well as central inflorescence, pedicellate flowers (pedicels thin), petals reflexed with the apices retrorsely curved and pendulous fruits with papiraceous carpels, whereas the Clade H. guatemalensis is characterized by 3/4 inferior ovary, central inflorescence as well as white petals. The most parsimonious trees recovered by the MP analysis of the nuclear PRK region (not shown) shows no important differences relative to that obtained through BI. In these trees, Clade B (H. tillandsioides complex) and Clade C (H. guatemalensis complex) are sister groups (BT: 93) and they are sister to the rest of Hechtia, which is a strongly supported relationship (BT: 99). Within Hechtia three strongly supported clades are identified: the H. glomerata Zucc. complex (Clade D; BT= 99), the H. epigyna Harms. complex (Clade E; BT= 100), and Hechtia (Clade F; BT=94). The best trees from the parsimony analysis of the combined plastid regions (not shown) show some relevant differences relative to the majority rule consensus tree retrieved by the Bayesian analysis of the same regions. The most parsimonious trees retrieve Brocchinia reducta as sister to the rest of Bromeliaceae (BT=100), which is resolved as a polytomy. Thus, the sister group of Hechtia is not identified here. Clades B and C are resolved in the same topology as in the Bayesian analysis. However, Clade D is not monophyletic in this analysis and Clade E is retrieved within Clade F (rest of Hechtia included in this study). The majority rule consensus tree from the Bayesian analysis shows better resolution than the most parsimonious trees. Figure 2 features a comparison of a 50 % majority rule of a Bayesian inference analyses of the combined plastid regions (left) and the nuclear PRK region (right). Both analyses retrieve Hechtia as monophyletic and strongly supported (PP=1, both analyses). Both analyses also identify the same major clades but in different, yet not incongruent, topologies. The PRK analysis identifies a (Pitcairnioideae(Bromelioideae(Puyoideae))) higly supported (PP=1) clade. However, in the plastid analysis there is a basal polytomy containing Hechtioideae, Tillandsioideae, and a moderately (PP=0.91) supported (Navioideae (Pitcairnioideae (Bromelioideae, Puyoideae))) clade (henceforth, the NPPB-clade). Within Hechtia the nuclear PRK region recovers a highly supported (PP=0.99) sister relationship for Clade B and Clade C. Within Hechtia, topologies for the remaining taxa are in agreement between both analyses in a highly supported clade (Clade F) (PP=1 in the plastid analysis; PP=1 in the nuclear analysis). In all cases, a polytomy formed by three clades (D, E, and F) is identified, each clade with the same species composition except by H. mapimiana López-Ferr. & Espejo that does not group in Clade F (it does with plastid evidence) and a clade formed by H. myriantha and Hechtia sp. TAMPS: Jaumave, that do not group with Clade D (as they do with the nuclear region). On the other hand, a sister group relationship of Clade B (Clade H. tillandsioides) and the rest of Hechtia is strongly supported (BT= 98; PP=0.99) in the plastid analysis (Figure 2, left). In the nuclear analysis, Clade B is highly supported (BT=93; PP=0.99) as sister to Clade C. The strict consensus parsimony tree (not shown) of combined data sets (morphology and molecular) presents less resolution for the relationships of the large clades, but within them, there are no topological differences with the 50 % majority tree performed with BI analysis; thus we have chosen to present the Bayesian inference tree (Figure 3), including values of posterior probabilities (PP) as well as bootstrap (BT) supports for clades retrieved from the MP analysis. In this combined analysis, the sister group of Hechtia (Clade A) is the NPPB-clade, albeit with very low support (PP=0.57). Hechtia (Clade A) is retrieved as monophyletic and strongly supported (BT=100; PP=1) and composed of two clades: the H. tillandsioides complex (Clade B) as sister to the rest of Hechtia, a poorly supported relationship (PP=0.86). 234 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. FIGURE 2. Left: A 50 % majority rule tree resulting from of a Bayesian analysis of the plastid DNA regions rpl32-trnL, ycf1(a,b). Posterior probabilities are above the branches. Right: A 50 % majority rule tree resulting from a Bayesian analysis of the nuclear region PRK. Posterior probabilities values are indicated above the branches. Clades are indicated with letters (A-F) and discussed in the text. PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 235 FIGURE 3. A 50 % majority rule tree resulting from a Bayesian analysis of the chloroplast (rpl32-trnL, ycf1(a, b), nuclear PRK region, and seven morphological characters of Hechtia. Bootstrap values are given above the branches, posterior probabilities below the branches. Clades are indictaed with letters (A-F) and discussed in the text. The Cortes Sea (Clade CSC); Tehuantepec Isthmus Clade (TIC), Mixteca Region Clade (MRC), and Tehuantepec-Mixteca Clade (TMC). 236 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. Following with the results of the 50 % majority tree performed with BI of all evidence, the rest of Hechtia consists of two clades: Clade C (BT=100; PP=1) is composed by the species in the H. guatemalensis complex, and is sister (a poorly supported relationship (PP=0.86)) to a highly supported (BT=100; PP=1) clade which is composed of an internal trichotomy, each clade highly supported and morphologically distinctive: the first is Clade D (BT=99; PP=1) composed of the species in the H. glomerata complex, forming two subclades: one consisting of H. elliptica L.B. Sm. +H. hernandez-sandovalii I. Ramírez, C.F. Jiménez & J. Treviño, the other including the rest of the complex. The second clade in the trichotomy is Clade E (BT=100; PP=1) composed by two species: H. deceptrix I. Ramírez & C. T. Hornung and H. epigyna; and the third, Clade F (BT=97; PP=1), with the rest of the species included in this analysis. Internally, Clade F lacks resolution at several levels but a few clades are readily indentified. The high-elevation, colorful H. matudae L.B. Sm. is sister (BT= 97; PP=1) to all of the other groupings within Clade F. This species was originally thought to belong to the H. podantha Mez complex but this relationship is not supported in any of the analyses. A few additional clades are also well-supported but hard to diagnose morphologically at this time although geographically structured. One clade with three species (H. cordylinoides Baker (H. flexilifolia I. Ramírez & Carnevali +H. nuusaviorum Espejo & López-Ferr.)) is highly supported (BT= 98; PP=1). Other well-supported (BT= 97; PP= 1) grouping includes H. aquamarina I. Ramírez & C.F. Jiménez, H. glauca Burt-Utley & Utley, H. lyman-smithii Burt-Utley & Utley, H. mooreana L.B. Sm., H. oaxacana Burt-Utley, Utley & García-Mend., H. roseana L.B. Sm., H. sphaeroblasta B. L. Rob. and H. tehuacana B.L. Rob. Another interesting, highly supported (BT= 96; PP=0.75) clade retrieved by the analysis is that composed of the species pair H. rosea and H. meziana. However, these two species are not retrieved as sisters in the combined Bayesian analysis of plastid DNA but they are with nuclear region PRK (BT=97; PP=1). Hechtia iltisii, the other species with an ornithophilous or psychophily syndrome, is never sister in any analysis of any of the two red-flowered species from the Tehuantepec area. Results of the evolutionary reconstruction of relevant morphological characters are depicted in Figure 4. The majority rule consensus tree emerging from the Bayesian inference analysis of the combined evidence (plastid and nuclear regions plus structural characters) was employed to assess these evolutionary patterns. Character states reconstructed were (Appendix 2) seven, three of which resulted synapomorphic for Hechtia, namely: simple-erect stigma/style lacking; presence of floral fragrance (evolved in parallel in some South American Tillandsia species), and unisexual flowers. The latter character state also shows parallel evolution in Catopsis. Another taxonomically relevant character in the Bromeliaceae, the type of fruit, was reconstructed, and capsular fruits, such as those in Hechtia, resulting plesiomorphic (reconstructions not shown). The character evolution reconstruction shows the SSP architectural pattern (sensu Ramírez et al. 2014) to be symplesiomorphic in Bromeliaceae and in Hechtia. On the other hand, the PMP architectural pattern is hypothesized to have evolved twice in Hechtia, once in Clade D, and again in H. epigyna (Clade E) from ancestors with a SSP growth pattern. Finally, precocious-flowering rossetes (the SPFP architectural pattern) apparently evolved at least three times within Hechtia (Clade F), most likely from SSP ancestors; however, the lack of resolution within this clade does not allow for a more accurate reconstruction of the evolution of this growth pattern. Inferior ovaries evolved at least twice within Bromeliaceae, once within Bromelioideae and again in Clade E (H. deceptrix and H. epigyna), whereas a ¾ inferior ovary evolved once within Clade C (the H. guatemalensis complex), in both cases from ancestors with superior ovaries. The reconstruction of foliar margin evolution showed that the ancestral state of Hechtia is ambiguous. However, a serrulate foliar margin is synapomorphic for Clade B, whereas a spiny foliar margin is either synapomorphic or symplesiomorphic for all remaining Hechtia (depending on which ancestral state Hechtia had). Discussion General considerations This is the first analysis of the internal relationships of Hechtia with a truly comprehensive data set (50 species out of 75 species in the genus; Ramírez-Morillo et al. 2016) and evidence from sequences of both plastid and nuclear DNA along with morphological data. The number of species included in the present analysis represents ca. 83 % of the accepted species in Hechtia. In contrast, previous phylogenetic analyses of Bromeliaceae only included 1–4 species (Reinert et al. 2003; Crayn et al. 2004; Givnish et al. 2004, 2006, 2007; Rex et al. 2009; Barfuss et al. 2005; Schulte et al. 2009; Horres et al. 2007; Schulte & Zizka 2008; Givnish et al. 2014). It is also noteworthy that the sampling for this study represents all the morphological groups we have identified to date in Hechtia, including taxa representing the three growth patterns found in the genus (sensu Ramírez et al. 2014). Furthermore, it also spans all PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 237 of the biogeographic areas occupied by the genus in Megamexico III. The sampling includes species representing the variation in characters of taxonomically and putatively ecological and evolutionary relevance, such as different leaf margins and texture, flowers (pedicellate vs sessile), putative pollination syndromes, and altitudinal preferences. Thus, the results that emerge from this analysis, expressed as well-supported relationships, can be thought of as a reflection of phylogenetic relationships throughout the genus. However, relationships within and between some clades are still unresolved; thus, additional evidence must be incorporated to the analysis to better assess the evolutionary history of Hechtia. FIGURE 4. Ancestral character state reconstructions of selected morphological characters using Maximum Parsimony, on the 50 % majority rule tree resulting from a Bayesian analysis of morphological and molecular evidence for species of Hechtia and selected outgroup. A. Growth pattern. B. Foliar margin. C. Ovary position. See Appendix 2 for a list of morphological characters used in the analysis. For this, we used the consensus tree of 50 % majority of Bayesian inference using the total evidence, and the reconstruction of the ancestral states was done through parsimony. 238 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. Phylogenetic relationships Sister relationships of Hechtia: Recent, well-supported phylogenetic analysis of Bromeliaceae (Givnish et al. 2014) strongly support the notion that Hechtia is monophyletic and sister to Clade NPPB (BT= 77; PP=96). In our analysis, however, support for a Clade NPPB-Hechtia relationship is weak (no bootstrap support and PP=0.65), a finding most likely explained by our use of fewer DNA regions and a smaller, less representative outgroup. Monophyly of Hechtia: All of the topologies retrieved from our analysis strongly support the monophyly of Hechtia. These results are consistent with previous studies (i.e. Rex et al. 2009; Givnish et al. 2007, 2011, 2014), all performed with a much smaller sampling of the genus. From the onset of the study, monophyly of Hechtia was expected because the genus is characterized within the Bromeliaceae by a distinctive combination of features, particularly the terrestrial, succulent habit, the unisexual, fragrant flowers, the entomophily, the dioecy, and the simple-erect stigma with sessile stigmatic lobes (Figure 5). This aggregation of character states is unique in the Bromeliaceae, which is a predominantly hermaphroditic clade, typically with odorless flowers, ornithophilous pollination syndromes, and a stigma usually with a well-developed and conspicuous style. As here circumscribed, members of Hechtia are rosette forming plants growing as terrestrials or lithophytes in volcanic, calcareous, or gypsophilous soils; their growth is cespitose or more rarely, rhizomatous. The inflorescences are mainly central (originating from a mature rosette or from a young one that stops its growth after blooming), or also lateral. Inflorescences are glabrous, waxy, or covered by white indumentum. The flowers are usually sessile, or with a short, thick pedicel (which may possibly be interpreted as an elongated receptacle holding nectariferous tissue, E. Gouda pers. comm.) or more rarely, provided with thin, long pedicels; petals are white, green, yellow, bluish to pale lilac, or red; sepals differs in texture and color from petals. Flowers last only one day, and both sexes produce a soft, sweet fragrance (excepting the red-flowered species). They usually attract bees, wasps, and flies of different species, but other flower visitors such as small beetles and butterflies are also lured by the abundant nectar and pollen produced by the flowers. Fruits are capsules with carpels of variable texture, ranging from hard to papyraceous; the seeds are fusiform, with nothing to aid in their dispersal but a pair of very tiny wings at both ends; this explains their low vagility and probably the high species-level endemism. Although unisexual flowers appear to be arisen twice in Bromeliaceae according our reconstruction of ancestral states [in Catopsis nutans (Sw.) Griseb.], this feature and dioecy are known elsewhere in the Bromeliaceae. Other Megamexican species of Catopsis (Tillandsioideae), Aechmea mariae-reginae H. Wendl. and a single species of Androlepis Brongn. ex Houllet are dioecious, and represent at least two independent origins of this state, since Catopsis belong to Tillandsioideae, and Aechmea maria-reginae and Androlepis are part of the same clade within Bromelioideae (Sass & Specht 2010). Also, unisexual flowers have been recorded in Dyckia Schult. & Schult. f. (Pitcairnioideae; Varadarajan 1986) but in these cases, plants are monoecious, and representing an independent origin as well from those in Hechtia, since Dyckia is well nested in Pitcairnioideae s. s. (Schütz et al. 2016); in Cryptanthus Otto & A. Dietr. there are plants with hermaphroditic and staminate flowers, a breeding system called andromonoecy (Ramírez-Morillo & Brown 2001). Our current understanding of phylogenetic relationships in the Bromeliaceae (e.g., Givnish et al. 2011, 2014) indicate that flower unisexuality (and breeding systems associated with it) evolved independently several times in the family from hermaphroditic ancestors. The evolutionary and ecological correlates of this fact are not understood at this time and they are found in various ecosystems (terrestrial and epiphytes, xeric sites to humid forests, low to high altitudes) and geographical areas (Megamexico III, Central America, and South America). However, it is noteworthy that all truly dioecious taxa are native from Megamexico III and Central America. In this regard, some species are extraordinary in being site-specific dioecious, such as Catopsis nutans, which presents perfect-flowered populations in Florida, USA but populations of the species are dioecious in Mexico (Benzing, Luther & Bennett 2000). The presence of floral fragrance is even more unusual in the family. All species of Hechtia produce diurnal flowers that last but a few hours. Most species produce strong to subtly sweet aromas, as well as abundant pollen (staminate flowers) and most commonly yellow colored nectar (both sexes). Stingless bees, as well as the introduced Apis mellifera L. (and a few, additional opportunistic insects) are the visitors and pollinators by excellence (RamírezMorillo et al. 2008). Also, Benzing (2000) discussed the possibility of beetle pollination in Hechtia owing to the nature of floral fragrance (e.g. aminoid odor). Furthermore, two closely related species, H. rosea and H. meziana, along with the unrelated H. iltisii, showed a reversion: all three species feature semi-tubular corollas of reddish colors, which fail to emit fragrances. These are most likely hummingbird or butterfly pollinated, a hypothesis yet to be formally tested. Givnish et al. (2007) suggest that entomophily is an ancestral pollinating syndrome in the family (recorded in Brocchinioideae, Navioideae, Lindmanioideae, Hechtioideae and Fosterella L.B. Sm. in Pitcairnioideae) and that ornithophily evolved from ancestors with insect pollination. A few other species of Bromeliaceae feature also fragrant flowers. For example, several species of the Diaphoranthema Beer and Phytarrhiza (Vis.) Baker subgenera of Tillandsia L. and the recently described tillandsioid genus Lemeltonia Barfuss & W. Till, display fragrant flowers of PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 239 diurnal anthesis, whereas taxa such as Stigmatodon Leme, G.K. Br. & Barfuss (Barfuss et al. 2016), along with genera and species such as Werauhia J.R. Grant and Encholirium glaziovii Mez feature highly fragrant flowers of nocturnal anthesis that are probably moth or bat pollinated. Bromeliaceae with fragrant flowers of diurnal anthesis are even more uncommon. Bromelia palmeri Mez, is an example where the yellow tubular flowers emit a sweet, menthol fragrance. FIGURE 5. Features of Hechtia. A. Rhizomatous rosettes of Hechtia roseana. B. Spinose foliar margins of Hechtia iltisii. Growth patterns (Ramírez et al. 2014): C. Hechtia malvernii with central inflorescence on mature rosette [SSP]. E. Sympodial with precacious blooming [SPFP] in Hechtia huamelulaensis. F. Pseudomonopodial [PMP] in Hechtia schottii. D. Pistillate flower showing simple-erect with sessile stigmatic lobes in Hechtia stenopetala. G. Staminate flowers of Hechtia schottii. H. Staminate flowers with a buttlerfly in Hechtia rosea. I. Capsular fruits of Hechtia roseana. J. Claviform seeds of Hechtia montana. K. Almost wingless, ellipsoid seeds of Hechtia tehuacana. L. Fusiform seeds of Hechtia aquamarina. Photograph credits: (A) Claudia Ramírez-Díaz. (C, I) K. Romero-Soler. (B, E) I. RamírezMorillo. (D) G. Romero-González. (G) G. Carnevali. (H) D. Carnevali. (F) C. Jiménez-Nah. (J, K, L) L. Can-Itzá and E. Gorocica. 240 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. The presence of a simple erect stigma, an apomorphy of Hechtia, has also been documented in species of several genera (Brown & Gilmartin 1989): in Cryptanthus (Bromelioideae), Brocchinia Schult. f. ex Schult. & Schult. f. (Brocchinioideae), Brewcaria L.B. Sm., Steyerm. & H. Rob., and Cottendorfia Schult. & Schult. f. (Navioideae), Fosterella (Pitcairnioideae), Catopsis, Guzmania Ruiz & Pav., Tillandsia, and Vriesea Lindl. (Tillandsioideae). This type of architectural morphology translates into a reduction of the stigmatic area, which could lead, in addition to a smaller area for pollen deposition, to a reduction of water loss (Brown & Gilmartin 1989), a condition that may be advantageous for a group of xeric plants like Hechtia. Species that belong to these genera and which present simple erect stigma are not all inhabitants of xeric sites, but all share bee pollinating syndromes (except perhaps the Guzmania species, most of which have ornithophilous characters), and it would be worthwhile to study this correlation. Recently, Barfuss et al. (2016) reported several additional architectural types in Tillandsioideae, all variations of the simple erect morphology, with variations in the position and conformation of the stigmatic lobes. Our preliminary study of stigmatic morphology based upon a relatively large sample of Hechtia, allowed us to identify several of the morphologies described by Brown & Gilmartin (1989) such as simple-erect for H. nuusaviorum as well as conduplicate-erect (sensu Leme 2009) for H. pringlei B. L. Rob. & Greenm. The small sample size of our preliminary exploration of this character evidences its high variability within Hechtia and warrants further research. The simple-erect stigma, on the other hand, has been reported for only a few genera besides Hechtia: Catopsis, Dyckia, Racinaea M.A. Spencer & L.B. Sm., and Tillandsia (Varadarajan 1986; Brown & Gilmartin 1989), all of them, probably representing independent origins of this feature. These preliminary results of stigmatic and style morphology suggests that a detailed, more exhaustive study of stigmatic morphology in Hechtia is likely to yield a variation greater than suspected and support the circumscription of already identified or novel groups within the genus. Internal relationships: Internally, there are three well supported clades within Hechtia: B, C and a trichotomy (formed by clades D, E, and F). Clade B, sister of the rest of the genus Hechtia, comprises four species corresponding to the H. tillandsioides complex. This clade includes H. caerulea (Matuda) L.B. Sm., H. lundelliorum L.B. Sm., H. purpusii Brandegee, and H. tillandsioides, species characterized by being rosetophilous herbs, with strict sympodial growth, with or without a well-defined, prostrate to suberect stem (H. caerulea); older plants feature long, pendent, narrow leaves, superficially conferring the plants a grassy aspect when seen from afar growing on vertical rocky walls (somewhat resembling species of the genus Pitcairnia L’Hér). The leaves have minutely serrate margins; adaxially the leaves are green, glaucous, and shiny or at the most have reddish margins whereas abaxially they are always whitelepidote; the apex is dry and often coiled. The flowers show thin pedicels; the petals are transversely convex, coiled/ reflexed, exposing the entire ovary (or pistillode), and stamens (or staminodes). The petal color varies from white (as in H. purpusii and H. lundelliorum) to pink (H. tillandsioides), lilac and caerulescent (as in H. caerulea), thin-textured petals; the fruits are erect when immature, then the pedicel bends down and the fruits are pendulous upon maturity with carpels becoming papiraceous and releasing minute seeds of ca. 4 mm long with two apical wings. Most species from this clade are terrestrial but some grown on rocky volcanic soils (as H. caerulea). They occur in low caducifolious forests, in some places always on vertical walls (as H. lundelliorum). This clade is distributed in the Transmexican Volcanic Belt, the Sierra Madre Oriental, and the Veracruzan provinces, always on the Gulf of Mexico slope (PechCárdenas 2015; Romero-Soler 2017). This clade is well characterized by its serrulate leaves, pedicelled flowers, these with transversely convex petals, and fruits pendulous with papiraceous carpels; its monophyly is also well supported by molecular features, along with a fairly circumscribed biogeographical distribution. The second clade, Clade C, is formed by species belonging to the so-called Hechtia guatemalensis complex, distributed in the southern portion of Megamexico III, in the Pacific Lowlands, Veracruzan, Mosquito, and the Chiapas Highland Provinces (in Belize [Holst et al., 2017], Guatemala, Honduras, El Salvador, and Nicaragua). This species aggregation is characterized by rosettes with strict sympodial growth, serrate leaves, very short spines, these hard and rigid and homogenous in size and shape along the margin, or when long, soft and flexible; upon exposure to the sun, the foliar blades develop red pigments resulting in a green-red pattern. The foliar surfaces are lustrous adaxially, whitelepidote abaxially. The flowers have green or red sepals, the corolla is spreading and the petals form a semi-cup; the star-like flowers have white petals and a ¾ inferior ovary; some species (H. guatemalensis) have pendulous mature fruits. The inflorescences develop quickly but flowers open a few at a time, thus making the blooming period to last for almost a month, more than any other species group in the genus where the inflorescence usually last just a week. This clade is diagnosed by a combination of characters: central inflorescences, flowers with ovary ¾ inferior, white flowers, and a distribution in Central America only, south of Motagua River close to the Guatemalan-Honduras border extending to northern Nicaragua. This clade includes the following species: H. malvernii Gilmartin, H. dichroantha Donn. Sm., and H. guatemalensis. PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 241 A third clade, Clade D, the so-called H. glomerata complex, includes all of the species with lateral inflorescences (with the exception of H. epigyna, discussed below), all floral parts are white lepidote and the petals are white. Lateral inflorescences are unusual and phylogenetically scattered in Bromeliaceae. They have been recorded in species of Greigia Regel and Disteganthus Lem. (Bromelioideae), and Dyckia (Pitcairnioideae; Benzing 2000), as well as occasionally in Tillandsioideae (e.g. Tillandsia complanata Benth.). Beyond the lateral inflorescences, species in this clade share several morphological features (Jiménez-Nah 2014): inflorescences are variously white-lepidote and the short-pedicelled flowers have always petals white in both sexes (except H. pretiosa Espejo & López-Ferr. that bears pink to magenta petals). The taxa of this clade are distributed in the Gulf of Mexico drainage in the Veracruzan, Tamaulipan, Yucatecan, and Sierra Madre Oriental biogeographical provinces, with a few species ranging into the neighboring provinces of the Mexican Plateau (H. argentea Zucc., H. glomerata, and probably H. texensis S. Watson, this last one not included in this analysis). An additional 3–4 undescribed species are known from the Chiapas Highlands provinces (Chiapas, as well as Honduras and Guatemala). The taxonomy of this group is highly complex and species are difficult to delimit and identify. Among the factors causing this complexity are the polycarpic growth of their rosettes; thus plants start to flowering when vegetatively still immature and coexist with much older, mature specimens bearing larger rosettes that have produced many, increasingly longer, more massive inflorescences. This results in populations containing plants on an immense array of sizes and degree of inflorescence branching. Hence, vegetative variation of fertile plants, at least sizes, may overlap among the closely related species, with the larger plants of vegetatively reduced species overlapping the dimensions of smaller plants of vegetatively large taxa. To make things worse, several species of the complex are only known from fragmentary specimens, rendering herbarium taxonomy a nightmare. Most of the diagnostic features of species are rosette characters and flower part sizes, along with fruit characters and geographical distribution. Clade E is formed by Mexican species with inferior ovary: Hechtia epigyna from the Tamaulipas biogeographical province, and H. deceptrix, recently described from Hidalgo (known from the Sierra Madre Oriental Province and neighboring areas of the Transmexican Volcanic Belt Province). Hechtia epigyna differs from H. deceptrix (RamírezMorillo et al. 2015) in its pseudomonopodial growth pattern, lateral inflorescence, flowers with lilac petals whereas H. deceptrix features flowers with white-greenish petals, strict sympodial growth pattern with central inflorescence. The placement of these two species in a well-supported clade, strongly suggests that lateral inflorescence evolved independently twice in Hechtia. Inferior ovary, on the other hand, evolved only in this clade within the genus, as well as independently in subfamily Bromelioideae. Clade F is a strongly supported aggregation containing most of Hechtia taxa (40 % of formally described species) and shows low resolution of internal phylogenetic relationships. Of the several morphological complexes characterized at the onset of this study, some resulted non-monophyletic. One such group is the so-called H. podantha complex. This is composed of species occurring at relatively high elevations of the Mexican Plateau that have central inflorescences emerging from a fully grown rosette (SSP growth pattern) and often feature conspicuously inflated inflorescence bracts (primary bracts) and short branches, usually shorter or as long as the primary bract length. Examples of such taxa are H. matudae, H. podantha, H. roseana, and H. tehuacana, all of which inhabit xerophytic shrublands, mostly at the higher elevations of the Mexican Plateau and Mexican Transvolcanic Belt. The species previously referred to the H. podantha complex were retrieved scattered within several clades within Clade F, with no structural or biogeographical evidence supporting its monophyly. Another example is the H. rosea complex, which encompasses the three species with tubular, red, fragrantless flowers, whose species fell in different clades: Hechtia rosea is sister to H. meziana (nuclear PRK and total evidence analyses) whereas the third species with this kind of floral morphology, H. iltisii is never retrieved as related to the species pair. However, the phylogenetic structure within Clade F features the phenomenon of geographically restricted clades, a recurrent theme in the evolution of Hechtia and other plant groups. Each clade represents a morphologically and ecologically diverse assemblage of species that invaded a particular area and radiated within it. There, they apparently would have filled several morphological space, pollination, and life history niches, often duplicating the patterns found in of different other clades of Clade F in different areas of Megamexico III. A discussion of this follows, clade by clade. One of the clades retrieved by the analysis is that composed by several taxa for NW Mexico, here referred to as “The Cortes Sea Clade” (CSC). This clade, represented in the analysis by six species, occurs around the mentioned sea in the states of Sonora, Baja California, Baja California Sur, Sinaloa, Nayarit, Durango, and Chihuahua. Central inflorescences are the norm here. A possible instance of hummingbird pollination was “invented” (evolved) within this clade, in H. iltisii, whereas the rest of the taxa feature the standard, bee pollinated, white flowers. Similarly, features of the H. podantha syndrome (henceforth, HPS) are found in H. mapimiana López-Ferr. & Espejo and H. subalata L.B. Sm., both high-elevation species. It seems that the HPS has been selected for dry, cold places and results in an 242 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. inflorescence with dense glomerules of flowers with fleshy (often yellow or green) petals, partially enveloped by inflated peduncle bracts. Presumably, this combination of characters affords protection to both young buds and flowers against dry and cold weather, particularly from frosts. Thus, this inflorescence structure is found in several, unrelated clades of Hechtia that grow at such high elevations. A second clade is composed of taxa that occur in the vicinity of the Tehuantepec Isthmus (TIC). All are inhabitants of hot, seasonally dry forests and feature the SPFP (sympodial with precocious-flowering pattern, SPFP) architecture. This growth pattern is here hypothesized as a way to gain a head-start with the flowering period and, later on, take advantage of the rainy season to release the seeds. The SPFP pattern is only found in Hechtia species from dry areas. Two species of this clade (H. rosea and H. meziana) feature presumably ornithophilous flowers, an independent “invention” (evolution) of this kind of floral model. Hechtia stenopetala Klotzsch, the type of the genus, is a member of this clade and the only one that grows on the Gulf of Mexico drainage in the dry areas of central Veracruz. The Tehuantepec Isthmus has served in the past as a two-way bridge for the passage of taxa from the Atlantic drainage to the Pacific watershed and vice versa. There are many examples of this mixture of floras (e.g. Pérez-García & Hágsater 2012). We will provide some examples from the Bromeliaceae. Tillandsia ionantha Planch. is a member of a Pacificdrainage clade of Tillandsia but there are populations in central Veracruz. On the other hand, Tillandsia streptophylla Schweidw. ex E. Morren is widespread on the Atlantic drainage from Honduras to Central Veracruz. However, there are isolated populations of the species in the southern area of the Tehuantepec Isthmus, particularly in the Chimalapas area. Another group within Hechtia, the Mixteca Region Clade (MRC Clade), is composed of several species that occur on both sides of the Central Valley of Oaxaca, particularly in the Mixteca Region. These species occur at relatively high elevations and in more mesic places than those of the previous clades, some occurring at the margins and on rocky outcrops of mesophilous forests. Albeit most taxa of this clade feature the SPFP architecture, at least two taxa feature SSP architecture: H. pringlei growing at intermediate to high elevations around the Central Valley of Oaxaca and H. pumila Burt-Utley & Utley, that grows in thorn scrub vegetation at middle elevations in the Mexican State of Guerrero. Within this clade, a subclade composed of three taxa, H. cordylinoides, H. nuusaviorum, and H. flexilifolia is noteworthy by displaying alternative strategies of niche occupation associated with cladogenesis. H. cordylinoides is sister to the other two species and morphologically very similar to H. flexilifolia whereas the sympatric H. nuusaviorum and H. flexilifolia are morphologically very different and grow side by side, even flowering more or less simultaneously. A final clade within Hechtia is that composed of taxa restricted to the relatively high elevation, extremely dry environments in the Tehuacán area and neighboring high western Mixteca Region (TMC). Species of this clade mostly display the SPFP growth pattern. However, H. roseana, restricted to the vicinities of the Valley of Tehuacán, has apparently evolved independently the Hechtia podantha syndrome. This clade is extremely variable in terms of vegetative structure. Species such as H. lyman-smithii are vegetatively reduced, densely cespitose, succulent, whereas H. roseana is a large plant that grows by means of elongated rhizomes that ramble among the surrounding vegetation searching for appropriate microniches where a new central-flowering rosette will root. At the other extreme of the variation is the gracile H. mooreana, which has an elongated, erect stem with the basic SPFP growth pattern. This clade is an exceptional example of the evolutionary potential and plasticity of Hechtia. Conclusions This study, with the largest taxonomic sampling of Hechtia to date, and with the use of plastid and nuclear DNA markers and morphological characters, provides conclusive evidence for the monophyly of Hechtia, and for it being composed of several highly supported clades. At this time it is uncertain what part of Megamexico was first invaded by the ancestors of Hechtia. However, as suggested by Givnish et al. (2011) it is most likely it happened either through long-range dispersal from the West Indies or via the southern Isthmus of Panama ca. 4.4–1.3 Ma. Regardless, it becomes clear that from the original point of invasion they radiated to occupy multiple biogeographical and ecological spaces in what is now Megamexico. In the meanwhile, they diversified morphologically and increased in species numbers. Today they consist in approximately 75 species-level-taxa. Nowadays, they grow from Nicaragua at its southern limits northward to Texas. In the course of its evolution, the biogeographically correlated phylogenetic structure of the genus suggests that particular regions were invaded at particular times and geographically confined smaller, separate radiations occurred, resulting in repeated PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 243 “inventions” (evolution) of critical evolutionary themes associated with the invasion of dry, highly seasonal climates, or cooler areas subject to frosts. Simultaneously, exploration of novel pollinations systems may have also played a role in the diversification of the genus. Speciation patterns suggests a mixture of niche conservatism and the creation of evolutionary novelties as concurrent or alternative evolutionary forces directing the diversification of the genus. Further ongoing macro-evolutionary research, with the inclusion of more taxa, more molecular markers, and structural characters, will help clarify the chronology of these evolutionary events and analyze the sequence of appearance of apomorphic novelties. Acknowledgments For assistance during field work in Mexico: Jacinto Treviño, Evelyn Rios, Laura Rodríguez, Sergio Terán, Gonzalo Guerrero, Luis Martínez, Demetria Mondragón, Prisciliano Flores, José Luis Chávez, Gerardo Salazar, Carolina Granados, María Cruz Flores, Adolfo Espejo, Javier García, Francisco Lorea, Carlos Durán, Ana Rosa López-Ferrari, Aniceto Mendoza, Jacqueline Ceja, Carlos Jiménez-Nah, Gregorio Castillo, Brian Sidoti, Virginia Rebolledo, Julián Bueno, Sofía Bueno, Ian Carnevali, Víctor Carnevali, Débora Carnevali, Oswaldo Suárez, William Cetzal, Sergio Zamudio, Luis Hernández Sandoval, Manuel Pool, Guadalupe Chuc, Francisco Chi, Filogonio May, Pablo Carrillo, Francisco Santana, Alejandro Zabalgoitia, Claudia Ramírez, Manuel González, Ivan Tamayo; in Honduras: Iliam and Daniel Rivera, Hermes Vega, Lilian Ferrufino, Paul and Margarita House, and Víctor Bocanegra; in Guatemala: Edgar Mó. The following people provided dried material on silica gel or help to obtain it: Pamela Hyatt, Dan Kinnard, Eloise Lau, Robert Kopstein, Ángel Lara, Raquel Monteiro, Rafaela Forzza, Claudine Mynssen, Andy Siekkinen, the Marie Selby Botanical Gardens and the Missouri Botanical Gardens. For maintaining the living collections at CICY: Clarisa Jiménez and Lilia Carrillo. To Lilia Can-Itzá and Alejandro Gorocica for images of seeds on the SEM. The senior author is indebted to the Elizabeth Bascom Fellowship and the Missouri Botanical Garden, the DAAD-ANUIES, Klarff Foundation, and Andrew Mellon Foundation, for financial support to study Bromeliaceae collections at herbaria in USA and Europe. To CONACyT for financial support (projects 67050 and 183281 to the first author). Thanks to curators of herbaria ASU, B, BIGU, CHAPA, CICY, EAP, ENCB, F, GH, HGOM, HUH, IBUG, IZTA, K, LAGU, LL, MEXU, MHES, MICH, MO, NY, PH, QMEX, SEL, TEFH, TEX, UAMIZ, US, W, WU, XAL, ZEA for allowing us borrowing their material. For access to databases and herbarium voucher images our thanks to the curators of herbaria B, CICY, MO, UAMIZ, particularly to Mary Merello, Jim Solomon, Gerrit Davidse, Tom Wendt, Adolfo Espejo, and Silvia Hernández-Aguilar. Special thanks to Gustavo Romero (HUH), Walter Till (WU), Rusty Russell (US) and the Peter Raven Library Staff at Missouri Botanical Garden, for their support on getting loan of herbarium specimens as well as specialized literature on Hechtia. We also acknowledge Walter Till, an anonymous reviewer and Eric Gouda, whose comments and suggestions improved this paper. References Barfuss, M.H.J., Samuel, R., Till, W. & Stuessy, T.F. 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Unpublished B.Sc. Thesis, Universidad Nacional Autónoma de México. 248 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. Appendix 1. Information for samples of species of Hechtia and the outgroup. Voucher information is listed as follows: taxon name, country, state or department, and locality, collector’s name and number, herbarium abbreviation. Herbarium abbreviations according to Thiers, B. (2018). GenBank numbers appear in the following order: rpl32-trnL, ycf1a, ycf1b, PRK; a dash indicates that the locus was not sequenced for the specimen. Sequences generated in this study are indicated with an asterisk*. Plants in cultivation without voucher are indicated as (l). Brocchinia reducta Baker. Venezuela, A.M. Piliackas s/n (RB), MF74097*/MF74103*/ MF74109*/MF74092*. Bromelia hemispherica Lam. Mexico, Jalisco, I. Ramírez 1806 (CICY), MF740978*/ MF741038*/MF741097*/ MF740921*. Bromelia karatas L. Mexico, Yucatán, I. Ramírez 1805 (CICY), MF740980*/MF741039*/ MF741098*/ MF740922*. Catopsis nutants (Sw.) Griseb. Mexico, s/l, J. Pinzón s/n (CICY), MF740981*/MF741040*/MF741099*/ MF740923*. Cottendorfia florida Schult. f. Brazil, s/l, S. C. de Sant ìAna 3692 (RB), MF740982*/MF741041*/ MF741100*,_. Encholirium magalhaesii L.B. Sm. Brazil, s/l, M. Moura 36 (RB), MF740983*, _ , MF741101*/ MF740924*. Lindmania longipes (L.B. Sm.) L.B. Sm. Venezuela, T. Givnish s.n. (WIS), HQ913783, _ , _ , _ . Lindmania guianensis (Beer) Mez. Venezuela, Bolívar, W. Till et al. 16018a (WU), _ , X753760/ KX753760, _ . Pitcairnia breedlovei L.B. Sm. Mexico, Chiapas, I. Ramírez 957 (CICY), MF741034*/MF741092*/ MF741151*/ MF740974*. Puya mirabilis (Mez) L.B. Sm. Argentina, s/l, G. Carnevali s/n (CICY), MF741035*/ MF741093*/ MF741152*/ MF740975*. Tillandsia dasyliriifolia Baker. Mexico, Yucatán, I. Ramírez s/n (CICY), MF741036*/ MF741094*/ MF741153*/ MF740976*. Tillandsia limbata Schltdl. Mexico, Veracruz, I. Ramírez 1464 (CICY), KU848483/MF741095*/ MF741154*/ MF740977*. Hechtia aquamarina I. Ramírez & C.F. Jiménez. Mexico, Puebla, I. Ramírez 1283 (CICY), MF740984*/ MF741042*/ MF741102*/ MF740925*. Hechtia caerulea (Matuda) L.B. Sm. Mexico, Mexico, I. Ramírez 1516 (CICY), MF740985*/ MF741043*/ MF741103*/ MF740926*. Hechtia cordylinoides Baker. Mexico, Oaxaca, I. Ramírez 1441 (CICY), MF740986*/ MF741044*/ MF741104*/ MF740927*. Hechtia deceptrix I. Ramírez & C.T. Hornung. Mexico, Hidalgo, I. Ramírez 1723 (CICY), MF740987*/ MF741045*/ MF741105*/ MF740928*. Hechtia dichroantha Donn. Sm. Guatemala, K. Romero 1169 (CICY), MF740988*/ MF741046*/ MF741106*, _ . Hechtia edulis I. Ramírez, Espejo & López-Ferr. Mexico, Chihuahua, I. Ramírez 1326 (CICY), MF740989*/ MF741047*/ MF741107*/ MF740929*. Hechtia elliptica L.B. Sm. Mexico, Coahuila, I. Ramírez 1347 (CICY), MF740990*/ MF741048*/ MF741108*/ MF740930*. Hechtia epigyna Harms. Mexico, Tamaulipas, I. Ramírez 1956 (CICY), MF740991*/ MF741049*/ MF741109*/ MF740931*. Hechtia flexilifolia I. Ramírez & Carnevali. Mexico, Oaxaca, G. Carnevali 7136 (CICY), MF740992*/ MF741050*/ MF741110*/ MF740932*. Hechtia fosteriana L.B. Sm. Mexico, Oaxaca, I. Ramírez 1747 (CICY), MF740993*/ MF741051*/ MF741111*/ MF740933*. Hechtia gayorum L.W. Lenz. Mexico, Baja California, I. Ramírez 2214 (l), MF740994*/ MF741052*/ MF741112*/ MF740934*. Hechtia ghiesbreghtii Lem. Mexico, Chiapas, G. Carnevali 7488 (CICY), MF740995*/ MF741053*/MF741113*/ MF740935*. Hechtia glauca Burt-Utley & Utley. Mexico, Michoacán, I. Ramírez 2215 (l), MF740996*/MF741054*/ MF741114*/ MF740936*. Hechtia glomerata Zucc. Mexico, Hidalgo, I. Ramírez 1495 (CICY), MF740997*/ MF741055*, _ , MF740937 *. Hechtia guatemalensis Mez. Honduras, I. Ramírez 1838 (CICY), MF740998*/ MF741056*/ MF741115*/ MF740938*. Hechtia hernandez-sandovalii I. Ramírez, C.F. Jiménez & J. Treviño. Mexico, Querétaro, I. Ramírez 1610 (CICY), PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 249 MF740999*/ MF741057*/MF741116*/MF740939*. Hechtia iltisii Burt-Utley & Utley. Mexico, Jalisco, I. Ramírez 1937 (CICY), MF741000*/MF741058*/ MF741117*/ MF740940*. Hechtia isthmusiana Burt-Utley. Mexico, Oaxaca, I. Ramírez 1536 (CICY), MF741001*/ MF741059*/MF741118*/ MF740941*. Hechtia ixtlanensis Burt-Utley. Mexico, Oaxaca, I. Ramírez 1379 (CICY), MF741002*/ MF741060*/ MF741119*/ MF740942*. Hechtia lepidophylla I. Ramírez. Mexico, Querétaro, I. Ramírez 1503 (CICY), MF741003*/ MF741061*/ MF741120*/ MF740943*. Hechtia lundelliorum L.B. Sm. Mexico, San Luis Potosí, I. Ramírez 1491 (CICY), MF741004*/ MF741062*/ MF741121*/ MF740944*. Hechtia lyman-smithii Burt-Utley & Utley. Mexico, Oaxaca, I. Ramírez 1038 (CICY), MF741005*/ MF741063*/ MF741122*/ MF740945*. Hechtia malvernii Gilmartin. Honduras, I. Ramírez 1842 (CICY), MF741006*/MF741064*/ MF741123*/ MF740946*. Hechtia mapimiana López-Ferr. & Espejo. Mexico, Durango, I. Ramírez 1317 (CICY), MF741007*/ MF741065*/ MF741124*/MF740947*. Hechtia matudae L.B. Sm. Mexico, Morelos, I. Ramírez 1918 (l), MF741008*/MF741066*/ MF741125*/ MF740948*. Hechtia melanocarpa L.B. Sm. Mexico, Guerrero, I. Ramírez 2217 (l), MF741009*/ MF741067*/ MF741126*/ MF740949*. Hechtia meziana L.B. Sm. Mexico, Chiapas, I. Ramírez 962A (CICY), MF741010*/ MF741068*/ MF741127*/ MF740950*. Hechtia michoacana Burt-Utley, Utley & García-Mend. Mexico, Michoacán, I. Ramírez 1105 (CICY), MF741011*/ MF741069*/MF741128*/MF740951*. Hechtia montana Brandegee. Mexico, s/l, I. Ramírez 2216 (l), MF741012*/MF741070*/ MF741129*/MF740952*. Hechtia mooreana L.B. Sm. Mexico, Guerrero, I. Ramírez 1976 (CICY), MF741013*/ MF741071*/ MF741130*/ MF740953*. Hechtia myriantha Mez. Mexico, Veracruz. I. Ramírez 1462 (CICY), MF741014*/ MF741072*/ MF741131*/ MF740954*. Hechtia nuusaviorum Espejo & López-Ferr. Mexico, Oaxaca, G. Carnevali 7133 (CICY), MF741015*/ MF741073*/ MF741132*/ MF740955*. Hechtia oaxacana Burt-Utley, Utley & García-Mend. Mexico, Oaxaca, I. Ramírez 1546 (CICY), MF741016*/ MF741074*/ MF741133*/MF740956*. Hechtia podantha Mez. Mexico, Hidalgo, I. Ramírez 1479 (CICY), MF741017*/MF741075 */ MF741134*/ MF740957*. Hechtia pretiosa Espejo & López-Ferr. Mexico, Guanajuato, I. Ramírez 2216 (l), MF741018*/MF741076*/ MF741135*/MF740958*. Hechtia pringlei B.L. Rob. & Greenm. Mexico, Oaxaca, I. Ramírez 1442 (CICY), MF741019 * / M F 7 4 1 0 7 7 * / MF741136*/MF740959*. Hechtia pumila Burt-Utley & Utley. Mexico, Guerrero, I. Ramírez 1742 (CICY), MF741020*/ MF741078*/ MF741137*/MF740960*. Hechtia purpusii Brandegee. Mexico, Veracruz, I. Ramírez 787 (CICY), MF741021*/ MF741079*/ MF741138*/ MF740961*. Hechtia rosea E. Morren ex Baker. Mexico, Oaxaca, I. Ramírez 924 (CICY), MF741022*/MF741080*/ MF741139*/ MF740962*. Hechtia roseana L.B. Sm. Mexico, Puebla, I. Ramírez 1293 (CICY), MF741023*/ MF741081*/ MF741140*/ MF740963*. Hechtia schottii Baker. Mexico, Yucatán, I. Ramírez 529 (CICY), MF741024*/MF741082*/ MF741141*/ MF740964*. Hechtia sphaeroblasta B.L. Rob. Mexico, Oaxaca, G. Carnevali 7152 (CICY), MF741029*/ MF741087*/MF741146*/ MF740969*. Hechtia stenopetala Klotzsch. Mexico, Veracruz, I. Ramírez 1269 (CICY), MF741030*/ MF741088*/MF741147*/ MF740970*. 250 • Phytotaxa 376 (6) © 2018 Magnolia Press RAMÍREZ-MORILLO ET AL. Hechtia subalata L.B. Sm. Mexico, Durango, I. Ramírez 1314 (CICY), MF741031*/ MF741089*/ MF741148*/ MF740971*. Hechtia tehuacana B.L. Rob. Mexico, Puebla, I. Ramírez 1566 (CICY), MF741032*/ MF741090*/ MF741149*/ MF740972*. Hechtia tillandsioides (André) L.B. Sm. Mexico, Puebla, I. Ramírez 1475 (CICY), MF741033*/ MF741091*/ MF741150*/MF740973*. Hechtia sp. 1, Mexico, Chiapas, Comitán, J. Pinzón 234 (CICY), MF741025*/ MF741083*/MF741142*/ MF740965*. Hechtia sp. 2, Mexico, Guanajuato, Xichú, I. Ramírez 1525 (CICY), MF741026*/ MF741084*/MF741143*/ MF740966*. Hechtia sp. 3, Mexico, Tamaulipas, Jaumave, I. Ramírez 1607 (CICY), MF741027*/ MF741085*/MF741144*/ MF740967*. Hechtia sp. 4, Mexico, Tamaulipas, Salto del Tigre, I. Ramírez 1599 (CICY), MF741028*/ MF741086*/MF741145*/ MF740968*. PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 251 Appendix 2. Matrix of morphological characters, with characters states and codification for all taxa included in the analysis. Taxon/Character and character state codification Brocchinia reducta Bromelia hemispherica Bromelia karatas Catopsis nutans Cottendorfia florida Encholirium magalhaesii Lindmania guianensis Lindmania longipes Pitcairnia breedlovei Puya mirabilis Tillandsia dasyliriifolia Tillandsia limbata Hechtia aquamarina Hechtia caerulea Hechtia cordylinioides Hechtia deceptrix Hechtia dichroantha Hechtia edulis Hechtia elliptica Hechtia epigyna Hechtia felixliifolia Hechtia fosteriana Hechtia gayorum Hechtia ghiesbreghtii Hechtia glauca Hechtia glomerata Hechtia guatemalensis Hechtia hernandezsandovalii Hechtia iltisii Hechtia isthmusiana Hechtia ixtlanensis Hechtia lepidophylla Hechtia lundelliorum Hechtia lymansmithii Hechtia malvernii Hechtia mapimiana Hechtia matudae Hechtia melanocarpa Hechtia meziana Hechtia michoacana Hechtia montana Hechtia mooreana Hechtia myriantha Hechtia nuusaviorum Hechtia oaxacana Growth pattern: (0) SSP; (1) SPFP; (2) PMP 0 0 Foliar margin: (0) Style: (0) entire; (1) serrulate; present; (1) (2) spiny; (3) absent denticulate Ovary position: (0) superior; (1) inferior; (2) partially inferior Floral fragrance: (0) absent; (1) present Fruit type: (0) berry; (1) capsule 0 2 0 0 2 1 0 0 1 0 Floral sex: (0) bisexual; (1) unisexual 0 0 0 0 0 0 2 0 0 2 0 0 0 0 1 0 0 0 0 0 ¿ 0 0 1 1 1 0 0 0 0 0 2 0 0 ¿ 1 0 0 0 0 0 2 0 3 0 0 0 0 0 0 0 0 0 ¿ 0 0 0 1 1 1 1 0 0 0 0 0 1 0 1 0 2 1 2 0 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 0 1 1 1 0 0 0 2 2 1 1 0 2 1 2 0 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 0 0 1 0 0 0 0 0 0 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0,1 1 1 1 1 2 2 1 0 1 1 1 0 1 1 2 0 1 2 2 2 2 1 2 1 1 1 1 1 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 1 1 0 1 2 1 0 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 2 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 1 1 1 1 252 • Phytotaxa 376 (6) © 2018 Magnolia Press 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ...continued on the next page RAMÍREZ-MORILLO ET AL. Appendix 2. (Continued) Taxon/Character and character state codification Hechtia podantha Hechtia pretiosa Hechtia pringlei Hechtia pumila Hechtia purpusii Hechtia rosea Hechtia roseana Hechtia schottii Hechtia sphaeroblasta Hechtia stenopetala Hechtia subalata Hechtia tehuacana Hechtia tillandsioides Hechtia sp. CHIS: Comitán Hechtia sp. GTO: Xichú Hechtia sp. TAMPS: Jaumave Hechtia sp. TAMPS: Salto Growth pattern: (0) SSP; (1) SPFP; (2) PMP 0 2 0 0 0 1 0 2 0 Foliar margin: (0) Style: (0) entire; (1) serrulate; present; (1) (2) spiny; (3) absent denticulate Ovary position: (0) superior; (1) inferior; (2) partially inferior Floral fragrance: (0) absent; (1) present Fruit type: (0) berry; (1) capsule 2 2 2 2 1 2 2 2 2 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 Floral sex: (0) bisexual; (1) unisexual 1 1 1 1 1 1 1 1 1 1 0 0 0 2 2 2 2 1 2 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 0 1 1 1 2 2 1 0 1 1 1 2 2 1 0 1 1 1 PHYLOGENETICS OF HECHTIA Phytotaxa 376 (6) © 2018 Magnolia Press • 253

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