Molecular phylogeny and taxonomy of the genus Disocactus (Cactaceae), based on the DNA sequences of six chloroplast markers

Abstract: 
 The genus Disocactus is native to Mexico and Central America and includes 11 species in four subgenera: D. subg. Ackermannia, D. subg. Aporocactus, D. subg. Disocactus and D. subg. Nopalxochia. Phylogenetic reconstruction was carried out with data from DNA sequences using the maximum parsimony and Bayesian inference criteria to explore the monophyly of the genus, its subgenera and its position within Hylocereeae. Six chloroplast markers (matK, psbA-trnH, rpl16, trnL-F, trnQ-rps16 and ycf1) were sequenced in ten species of Disocactus, 17 representatives from the remaining genera of Hylocereeae and five members of outgroups (Acanthocereus, Lemaireocereus and Pereskia). Our phylogenetic analysis supports neither the monophyly of Disocactus as it is currently defined nor that of the subgenera. The clade Disocactus s.str. was recovered for 13 species, including Epiphyllum anguliger, E. crenatum and E. lepidocarpum. Three subclades were observed within this clade, and three well-supported sister-species relationships were recovered: D. eichlamii and D. quezaltecus; D. biformis and D. nelsonii; and D. ackermannii and D. phyllanthoides. Disocactus speciosus subsp. aurantiacus was not recovered in the clade of D. speciosus. Epiphyllum and Pseudorhipsalis are identified as sister clades of Disocactus. Based on the obtained results, a new circumscription for Disocactus is proposed. Citation: Cruz M. Á, Arias S. & Terrazas T. 2016: Molecular phylogeny and taxonomy of the genus Disocactus (Cactaceae), based on the DNA sequences of six chloroplast markers. — Willdenowia 46: 145–164. doi: http://dx.doi.org/10.3372/wi.46.46112 Version of record first published online on 1 April 2016 ahead of inclusion in April 2016 issue.


Introduction
The genus Disocactus Lindl. (Cactaceae, Cactoideae, Hylocereeae) includes species with terrestrial, climbing and epiphytic growth forms. These species exhibit diurnal, brightly coloured flowers and may be found from central Mexico to Central America (Barthlott & al. 2015). However, as in other groups of cacti, the delimitation of the genus has been modified over the years, as a reflection of an oscillating taxonomic history. In 1845, Lindley proposed Disocactus as a monotypic genus for Cereus biformis Lindl., a species native to Guatemala, with morphological characteristics that were sufficiently distinct from other cacti, such as flat stems and small pink flowers with few perianth segments. Later, Britton & Rose (1923) added another species, which was also from Guatemala (Table 1). In subsequent years, four species were added to Disocactus: two corresponded to taxa that were transferred from other genera (Rhipsalis Gaertn. and Pseudorhipsalis Britton & Rose), and two represented monotypic genera (Bonifazia Standl. & Steyerm. and Chiapasia Britton & Rose). Therefore, Bravo-Hollis (1978) recognized six species in Disocactus (Table 1) based on the classification of Buxbaum (1958).
In the 1990s, the genus Disocactus underwent two major modifications in terms of its delimitation and the 146 Cruz & al.: Molecular phylogeny and taxonomy of Disocactus number of included species. Barthlott (1991a) modified the delimitation by proposing a more inclusive genus that consisted of species that share a distribution in Central America and are characterized by diurnal flowering. Barthlott (1991a) transferred the genera Aporocactus Lem., Heliocereus Britton & Rose, Nopalxochia Britton & Rose and Wittia K. Schum. to Disocactus, which consisted of a total of 15 species. The diagnostic characteristics of the genus recognized by Lindley (1845) were lost (e.g. flat or ribbed stems, stems with or without spines, large or small flowers with intense or white colouration, a thick pericarpel with ribs and spines or one that is thin and bare), which generated uncertainty about the relationship with Pseudorhipsalis. For example, Barthlott (1991a, b) recognized Pseudorhipsalis as a separate genus based on its small white flowers and the smaller size of its pollen grains. Kimnach (1993) restricted the limit of the genus and believed that the species included in Disocactus should exhibit a flower with a narrow receptacle without conspicuous podaria and without spines or hairs. Therefore, he included Pseudorhipsalis because he believed that its morphological characteristics were insufficient to maintain it in a separate genus ( Table 1).
The most recent classification by Hunt & al. (2006) revisits Barthlott's proposal (1991a, b) and the taxonomic changes made by Bauer (2003). These authors recognized 11 species of Disocactus that exhibit intensely coloured flowers (yellow to red) and diurnal flowering. They transferred D. amazonicus (K. Schum.) D. R. Hunt to the genus Pseudorhipsalis, and certain species or varieties were classified as subspecies (Bauer 2003).
Various subgenera or sections have been recognized within Disocactus. Barthlott (1991a) grouped the species in D. subg. Ackermannia (K. Schum.) Barthlott, D. subg. Aporocactus (Lem.) Barthlott, D. subg. Disocactus, D. subg. Nopalxochia (Britton & Rose) Barthlott and D. subg. Wittia Barthlott ( Table 2; Fig. 1). Among these subgenera, D. subg. Disocactus and D. subg. Wittia are characterized by flat stems and a bare or nearly bare pericarpel; the difference between these two subgenera is the size of the flower. In D. subg. Wittia the flowers are short in length (2.5 -3 cm) and the stamens are inserted in two series, whereas in D. subg. Disocactus the flowers are larger (3 -6 cm), and the stamens are inserted in one series. In the remaining subgenera, the pericarpel exhibits spines, scales or visible areoles. In particular, D. subg. Aporocactus exhibits stems with 8 -11 ribs, areoles Table 1. Summary of specific placement in Disocactus and other related genera based on previous taxonomic contributions. Lindley (1845) Britton & Rose (1923) Bravo-Hollis (1978) Barthlott (1991a, b) Kimnach (1993) Hunt & al. (2006)  with spines and slightly zygomorphic flowers measuring 7 -10 cm. Conversely, the stems in D. subg. Ackermannia have fewer ribs (3 -7), are rarely flat, and exhibit areoles with spines and actinomorphic or slightly zygomorphic flowers measuring 8 -20 cm. Disocactus subg. Nopalxochia displays flat stems, areoles with or without spines, and pale pink actinomorphic flowers measuring 8 -10 cm. Kimnach (1993)  Wittia exhibit erect or expanded tepals up to the middle section of the flower and the stamens are inserted in two series. However, the apex of the tepals in D. sect. Wittia is tinted blue and the stamens are nearly exerted, with thin filaments, whereas the tepals in D. sect. Disocactus range from pink to purple and the stamens are exerted, with thick filaments. Conversely, D. sect. Macranthi and D. sect. Pseudorhipsalis share tepals that are expanded from the base, with stamens inserted in a series. Nevertheless, D. sect. Macranthi may display more than one flower (5 -6 cm) per areole, and the nectary occupies two-thirds of the floral length, whereas in D. sect. Pseudorhipsalis the flowers are small (less than 3 cm), with one flower emerging per areole and a small nectary (one-third of the floral length). Finally, Hunt & al. (2006) recognized four of the subgenera (D. subg. Ackermannia, D. subg. Aporocactus, D. subg. Disocactus and D. subg. Nopalxochia) proposed by Barthlott (1991a) (Table 2; Fig. 1).
The relationship between Disocactus and the remainder of cacti from the tribe Hylocereeae has not yet been examined in depth. Britton & Rose (1923) proposed that Disocactus and Epiphyllum Haw., along with seven other genera (Chia pasia, Eccremocactus Britton & Rose, Epiphyllanthus A. Berger, Nopalxochia, Schlumbergera Lem., Wittia and Zygocactus K. Schum.), comprise the subtribe Epiphyllinae, which, according to these authors, is characterized by the presence of phylloclades (with a cylindrical base) that are primarily free of spines and develop regular flowers. However, Buxbaum (1958) proposed that Disocactus, Pseudorhipsalis and five other genera (Bonifazia, Chiapasia, Lobeira Alexander, Nopalxochia and Wittia) comprise the subtribe Disocactinae, which is characterized by phylloclades that branch from cylindrical stems, brightly coloured flowers with a diurnal anthesis, a scaly pericarpel that is nearly bare and a generally scaly receptacle tube. Under the most recent classification (Hunt & al. 2006 (Nyffeler 2002;Arias & al. 2005;Bárcenas & al. 2011;Hernández-Hernández & al. 2011). However, all agree that Disocactus is resolved within a clade that corresponds to the tribe Hylocereeae, but its exact position is uncertain.
Recently, phylogenetic analyses based on DNA sequences have tested the relationships between the taxa and, consequently, their actual delimitations. Increasing evidence based primarily on chloroplast molecular markers indicates that some tribes and genera that had been delimited using only morphological data are not monophyletic, such as Grusonia Rchb. f. ex Britton & Rose (Griffith 2002), Pachycereus (A. Berger) Britton & Rose (Arias & al. 2003), Mammillaria Haw. (Butterworth & Wallace 2004), Peniocereus (A. Berger) Britton & Rose (Arias & al. 2005), Pereskia Mill. (Edwards & al. 2005), Rebutia K. Schum. (Ritz & al. 2007), Pfeiffera Salm-Dyck (Korotkova & al. 2010 -Sánchez & al. 2013). Phylogenetic analyses including sequences from Disocactus have been unable to clearly establish the monophyly of the genus or its relationships with the other members of Hylocereeae because these analyses were not intended to discern the phylogenetic relationships of this genus (Nyffeler 2002: three spp.;Bárcenas & al. 2011: ten spp.;Hernández-Hernández & al. 2011: eight spp.), and sample sizes were insufficient. In the topologies produced by Nyffeler (2002) and Bárcenas & al. (2011), only one sister relationship was recovered between Hylocereus and Selenicereus, whereas Disocactus formed part of the polytomy of the tribe Hylocereeae. However, in the analyses performed by Hernández-Hernández & al. (2011), Epiphyllum diverged early within Hylocereeae, whereas Table 2. Summary of infrageneric groups in Disocactus according to recent taxonomic studies. Barthlott (1991a) Kimnach (  Disocactus was a sister to the clade that includes the genera Hylocereus, Selenicereus and Weberocereus. Due to the above issues and the discrepancy between taxonomists regarding the number of species within Disocactus (Table 1) caused by poor morphological delimitation of the genus, the question arises of whether Disocactus is monophyletic. The objectives of this study were threefold: (1) to carry out a phylogenetic reconstruction of the genus Disocactus using six chloroplast molecular markers (matK, psbA-trnH, rpl16, trnL-F, trnQ-rps16 and ycf1) to evaluate whether Disocactus and the subgenera recognized by Hunt & al. (2006;D. subg. Ackermannia, D. subg. Aporocactus, D. subg. Disocactus and D. subg. Nopalxochia) are monophyletic groups; (2) to understand the position of Disocactus within Hylocere eae; and (3) to generate a taxonomic proposal for Disocactus based on these results.

Taxon sampling
The present study included ten of the 11 species of Disocactus according to the classification proposed by Hunt & al. (2006) and incorporated a comprehensive sampling of ten terminals for five subspecies of D. speciosus (Cav.) Barthlott and two for D. ackermannii (Haw.) Ralf Bauer, D. biformis (Lindl.) Lindl., D. macranthus (Alexander) Kimnach & Hutchinson and D. phyllanthoides (DC.) Barthlott. Representatives from Epiphyllum, Hylocereus, Pseudorhipsalis, Selenicereus and Weberocereus were included to test the monophyly of the genus because they were recovered as part of a single clade in previous phylogenies (Nyffeler 2002). Outgroups were sampled based on previous phylogenetic analyses of the family (Nyffeler 2002;Arias & al. 2005;Hernández-Hernández & al. 2011) and included representative species of Acanthocereus Britton & Rose (Lodé 2013), Lemaireocereus Britton & Rose and Pereskia. This study involved 315 new sequences; their voucher information and GenBank accession numbers are listed in the Appendix.

Amplification and sequencing
Phylogenetic reconstructions for Cactaceae have mainly employed chloroplast molecular markers. Nuclear markers suitable for cacti have yet to be developed because the existing nuclear markers provide a lower number of informative sites compared with chloroplast markers, and they do not exhibit polymorphism as has been shown for ITS in other groups of cacti, e.g. Lophocereus (A. Berger) Britton & Rose (Hartmann & al. 2001), Mammillaria (Harpke & Peterson 2006), Hylocereus and Selenicereus (Plume & al. 2013). A combination of different chloroplast markers is necessary to increase the resolution of the phylogeny (Korotkova & al. 2010). Within the diverse chloroplast markers, it has been proposed that introns outperform spacers and coding regions in terms of phylogenetic utility (introns > spacer sequences > coding regions) (Korotkova & al. 2011). Six molecular markers were used: two introns (rpl16 and trnL-F), two spacers (psbA-trnH and trnQ-rps16) and two coding regions (matK and ycf1).
Collected samples were dried with silica gel and stored at −20 °C. Total DNA was extracted using the EZ-10 Spin Column Plant Genomic DNA Minipreps kit (Bio Basic Inc., Ontario, Canada) according to the manufacturer's protocol, but the incubation time in tissue lysis buffer was modified to 120 minutes. Total DNA was stored at −20 °C. Specific regions were amplified using a standard polymerase chain reaction (PCR) protocol in a total volume of 25 µL containing 2.5 µL of 10× buffer, 0.5 µL of dNTPs (10 mM), 1 µL of BSA, 0.3 µL of each primer (10 mM The trnL-trnF region was amplified using the primers "C" and "F" designed by Taberlet & al. (1991). The PCR programme employed for this region consisted of 2 min at 94 °C, 29 cycles of 30 s at 94 °C, 30 s at 52 °C and 1 min at 72 ºC, and a final extension of 7 min at 72 °C. The psbA-trnH intergenic spacer was amplified using the primers psbA (Sang & al. 1997) and trnH (Tate & Simpson 2003), with initial denaturation of the DNA for 2 min at 94 °C, followed by 29 cycles of 30 s at 94 °C, 30 s at 52 °C and 1 min at 72 °C, and a final extension of 7 min at 72 °C. The trnQ-rps16 intergenic spacer was amplified using the primers trnQ2 (Korotkova & al. 2010) and rps16x1 (Shaw & al. 2007), with initial denaturation of the DNA for 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 1 min at 55 °C and 1 min at 72 °C, and a final extension of 10 min at 72 °C. The ycf1 region was amplified using the primers ycf1-4182F and ycf1-5248R (Franck & al. 2012), with initial denaturation of the DNA for 3 min at 94 °C, followed for 35 cycles of 1 min at 94 °C, 1 min at 50 °C and 2 min at 72 °C, and a final extension of 5 min at 72 °C. The unpurified PCR products were sequenced at the University of Washington (UW-High Throughput Genomics Center, http://www.htseq.org/). All regions were sequenced using amplification primers. Sequence assembly and editing was carried out using Sequencher 4.8 (GeneCode).

Phylogenetic analysis
The sequences were aligned using MAFFT (Katoh & al. 2002), and the alignments were then manually inspected. In all of the alignments, the extremes were eliminated due to the presence of ambiguities. Insertions and deletions (indel) were coded following the simple indel coding method suggested by Simmons & Ochoterena (2000) using the Indel Coder option of SeqState v. 1.40 (Müller 2005). Maximum parsimony (MP) phylogenetic analyses were carried out for each of the markers to demonstrate their congruence (trees with similar topologies) and concatenate them in a single matrix and to identify markers that maximize the resolution of the tree. MP phylogenetic analysis with the concatenated marker matrix without hotspots (highly variable regions) was carried out using PAUP (Swofford, 2002). A heuristic search with 15 000 replicates was conducted; the tree bisection and reconnection (TBR) branch-swapping algorithm was employed, and Pereskia lychnidiflora DC. was designated as an outgroup. A second MP phylogenetic analysis was performed with the concatenated marker matrix and coded indels and without hotspots using the above parameters. For branch support, 10 000 jackknife (JK) replicates and 10 000 bootstrap (BS) replicates were employed. Bayesian inference (BI) analysis was carried out using MrBayes 3.2 (Huelsenbeck & Ronquist 2001). The data were partitioned, and each partition used GTR+I+G as a substitution model, pursuant to the results obtained with jModeltest2 (Darriba & al. 2012). Two independent runs were conducted with four chains each, and six million generations were run, with the chains sampled every 1000 generations. Chain convergence was observed using Tracer (Rambaut & Drummond 2009). The first 20 % of trees were discarded as the burn-in. The remaining trees were then used to build a 50 % majority rule consensus tree. The alignment is available at http://purl.org/phylo/treebase/phylows/ study/TB2:S18804.

Results
Six markers were amplified for all ingroup and outgroup species, with two exceptions: rpl16 was not obtained for Epiphyllum lepidocarpum (F. A. C. Weber) Britton & Rose, and ycf1 was not obtained for E. pumilum Brit-ton & Rose and E.thomasianum (K. Schum.) Britton & Rose. The aligned matrix of the six concatenated plastid sequences and the coded indels was 5853 bp in length, only 14 % of which consisted of variable sites, and half of those variable sites were parsimony-informative (PI) sites (411). The cytosine-guanine content was lower than the adenine-thymine content. Four markers presented regions that were difficult to align; these regions corresponded to polyA or polyT sequences. In the rpl16 intron, an inversion was identified in the sequences of Disocactus flagelliformis (L.) Barthlott, D. martianus (Zucc.) Barthlott and all of the sequences of Acanthocereus, Hylocereus, Lemaireocereus, Peniocereus, Selenicereus and Weberocereus. The ycf1 region contributed 41 % of the observed indels, whereas the psbA-trnH spacer contributed 29 % of the indels, despite being a short region. This characteristic is directly related to the disparity in the length of the marker sequence ( Table 3). The results obtained from the MP and BI analyses were congruent; the same number of clades was recovered with high branch support. MP and BI yielded trees with similar, although not identical, topologies, due to the degree of resolution that each method provides.
Using the MP criteria, 57 equally parsimonious trees were obtained, with a length (L) of 1296 steps, showing a consistency index (CI) of 0.72 and a rescaled consistency (RC) index of 0.57. The strict consensus tree recovered three main groups. The H1 group includes clades D (95 % JK, 95 % BS), P (100 % JK, 100 % BS) and E (100 % JK, 100 % BS), whereas the H2 group consists of clades A (100 % JK, 100 % BS) and HSW (100 % JK, 100 % BS), and the H3 group includes three species of Acanthocereus (100 % JK, 100 % BS) (Fig. 2). Clade D defines the genus Disocactus and contains eight species previously classified therein plus three species of Epiphyllum, which are recovered in two subclades with moderate or no support. Subclade 3D (97 % JK, 86 % BS) is sister to the remaining included species, whereas subclade 2D (100 % JK, 100 % BS) forms part of the second divergent event in the genus. Subclade 1D (-JK, -BS) is the last to diverge and includes four species. Epiphyllum anguliger (Lem.) H. P. Kelsey & Dayton was recovered as sister species of sub- clade 2D and E. lepidocarpum was recovered as a sister taxon to this relationship; however, both hypotheses lack support. Epiphyllum crenatum (Lindl.) G. Don is nested between the previous relationship and subclade 1D. Disocactus speciosus (ten terminals) was the only species recovered as non-monophyletic. Clade P, which includes two species of Pseudorhipsalis, and clade E, which consists of six species of Epiphyllum whose relationships exhibit moderate-to-high support, form a sister clade to Disocactus (Fig. 2).
The BI analysis showed that the H1, H2 and H3 groups were recovered with high support (Fig. 3). Two subclades were recovered in addition to the three subclades observed in the MP analysis. Subclade 4D consists of Disocactus macranthus and Epiphyllum crenatum, with 0.63 PP support. Subclade 5D includes E. anguliger and E. lepidocarpum, with a support of 0.68 PP, and forms part of the basal polytomy along with subclade 2D. Unlike MP, subclade 3D in the BI analysis is recovered between subclades 1D and 4D. In addition, within the H1 group, clade E is sister to clade D (0.78 PP) and clade P is sister to both clades E and D (0.99 PP).
The H1 group in both reconstructions is supported by four molecular synapomorphies, three of which correspond to substitutions and one to an indel. Clade D is defined by a single combination of seven synapomorphies, six of which are substitutions (matK, trnQ-rps16, rpl16 and ycf1) and one is an indel (rpl16). Moreover,  (Table 4).

Discussion
Our phylogenetic analyses did not recover Disocactus or the subgenera recognized by Hunt & al. (2006) as monophyletic. One group of 11 species of Disocactus belongs to the H1 group, which includes D. biformis (type species) and is defined here as Disocactus s.str., whereas the two analysed species of D. subg. Aporocactus are nested in the H2 group. Therefore, the genus Aporocactus should be re-established, and Disocactus must be re-circumscribed (see below). Based on its placement in group H1, Disocactus is more closely related to Epiphyllum and Pseudorhipsalis than to the remaining genera of Hylocereeae, as discussed below.

The contribution of the the markers and coded indels
The region matK provided the lowest number of informative sites. This region was also less variable in other groups of Cactaceae, such as in the genus Pfeiffera (Korotkova & al. 2010), the tribe Rhipsalideae (Korotkova & al. 2011) and the tribe Cacteae (Vázquez-Sánchez & al. 2013). Two sites supported the relationships at the genus level (214 for the genus Pseudorhipsalis, 706 for the genus Disocactus; Table 4), which shows that matK may, to a lesser degree, contribute information to resolve relationships at the genus level and not only at the family level. Conversely, markers with high evolu-tionary rates, such as the introns rpl16 and trnL-F and the spacer regions psbA-trnH and trnQ-rps16 (Borsch & Quandt 2009;Korotkova & al. 2011), provided 31 % of informative sites and allowed the establishment of relationships at primarily the genus and species levels in the genera Epiphyllum and Pseudorhipsalis. The region ycf1 contributed 49 % of informative sites. This region is also highly variable in other groups of cacti, such as Opuntia (L.) Mill. (Majure & al. 2012), Harrisia Britton (Franck & al. 2013) and Copiapoa Britton & Rose (Larridon & al. 2015). The region ycf1 produced the tree with the highest resolution, supporting the relationships at the genus and species levels; however, it did not individually resolve the relationships between close species. Moreover, the coded indels provided 13 % of the PI sites. The incorporation of coded indels in phylogenetic reconstructions is a frequent practice in Cactaceae (e.g. Nyffeler 2002; Butterworth & Wallace 2004;Korotkova & al. 2010Korotkova & al. , 2011Calvente & al. 2011a, b;Franck & al. 2013;Vázquez-Sánchez & al. 2013;Bonatelli & al. 2014). Furthermore, their inclusion improved the resolution and support values for the tree compared with the tree that did not include the coded indels (data not shown), as previously suggested (Simmons & al. 2001).

Genus Disocactus s.str.
Based on our results, Disocactus consists of 13 known species, including Epiphyllum anguliger, E. crenatum and E. lepidocarpum (Fig. 2, 3). None of the three subgenera recognized by Hunt & al. (2006) (Table 2) was recovered; however, three subclades were recognized with some support and three sister species relationships were observed. Subclade 1D includes the relationship between D. eichlamii (Weing.) Britton & Rose and D.  Fig. 2, 3), and a poor resolution is observed within this group, which we consider to be a reflection of the morphological issues presented by the species. Our results partially support the proposal of Bauer (2003), as only three of the five subspecies were recovered in the clade mentioned above. The variability of the stems, spines and flowers shown by these three subspecies is high, and they are only distinguished by subtle morphological differences (Bauer, 2007). Their shared morphological characteristics include stems with rigid spines, large red or orange flowers (12 -17 cm) and a pericarpel and receptacle tube with rigid spines. They are broadly distributed in W Mexico (Durango to Oaxaca) and C Guatemala (the Sierra de los Cuchumatanes), growing in Pinus forests and tropical montane cloud forest (Fig. 4). Disocactus speciosus subsp. aurantiacus (Kimnach) Ralf Bauer was not recovered as part of the group that includes the three subspecies mentioned above in either analysis (MP or BI) (Fig. 2, 3, subclade 1D). This position in the topologies is congruent with the morphological differences exhibited by D. speciosus subsp. aurantiacus, which is characterized by flexible and pendulous stems as well as longer hairs, instead of spines, on the stems and flowers. This taxon is distributed in the tropical montane cloud forests of El Salvador, Honduras and Nicaragua (Fig. 4). Bauer (2003) included it in D. speciosus because it also exhibits ribbed stems and large flowers (12.5 -15.5 cm). Although our results do not completely clarify the relationship of this taxon within subclade 1D (Fig. 2, 3), the molecular autapomorphies revealed in this study (substitution in ycf1, one indel in psbA-trnH, and rpl16; Table 4) in addition to the morphological characteristics listed above indicate that it should be recognized as a separate species, as was originally proposed (Kimnach 1974;Barthlott 1991a). Hunt & al. (2006) classified D. speciosus and D. ackermannii as part of D. subg. Ackermannia because they both develop large red or orange flowers (8 -20 cm). However, we indicate herein that the recognized groups of D. speciosus are independently related, but never to D. ackermannii (Fig. 2, 3, subclades 1D, 3D). In subclade 2D, Disocactus biformis was recovered as a sister taxon to D. nelsonii (Britton & Rose) Linding. with strong support (100 % JK, 100 % BS MP; 1 PP BI). These species share morphological characteristics including expanded external tepals, a scaly pericarpel and hairs measuring less than 0.5 cm, which allowed Kimnach (1993) to suggest the closeness of the species corroborated in this study. Disocactus biformis, D. eichlamii, D. nelsonii and D. quezaltecus are native to Guatemala, Honduras and Mexico (Chiapas) and are exclusive to tropical montane cloud forests (Fig. 4). Although these four species are included in the section or subgenus Disocactus (Table 2) because they share phylloclades, flowers, and fruits without spines, our results reveal that they do not form a clade. Five indels (two in trnL-F and three in psbA-trnH) plus five substitutions (one in matK, one in psbA-trnH, trnQ-rps16, rpl16 and ycf1) maintain the two groups in independent subclades (Table 4; Fig. 2, 3).
Subclade 3D supports the relationship among Disocactus ackermannii, D. ×kimnachii G. D. Rowley and D. phyllanthoides (97 % JK, 86 % BS MP; 1 PP BI; Fig. 2, 3), in contrast to the proposal made by Hunt & al. (2006). These authors believed D. ackermannii to be more closely related to D. macdougallii (Alexander) Barthlott (not included in this analysis) and D. speciosus, which together comprise D. subg. Ackermannia, whereas D. phyllanthoides was maintained in Nopalxochia (Table  2). However, we identified a relationship that was more consistent with the taxonomic treatment by Bravo-Hollis (1978): with the exception of D. ×kimnachii (a taxon not recognized as a species by Hunt & al. 2006), the two remaining species and D. macdougallii comprise the genus Nopalxochia. Disocactus ackermannii and D. phyllanthoides are native to tropical montane cloud forests in E and S Mexico (Veracruz to Chiapas; Fig. 4), whereas D. ×kimnachii is found only in Costa Rica. The initial hypothesis about the origin of D. ×kimnachii proposed that it is a hybrid between a member of Heliocereus or Nopalxochia and Epiphyllum crenatum (Kimnach 1984), whereas a subsequent hypothesis suggested that the putative parents include Epiphyllum crenatum subsp. kimnachii (Bravo ex Kimnach) U. Guzmán and a member of the group "Heliocereus" (Bauer 2003). Under this proposal and according to our results, D. phyllanthoides may be one of the parents. The three taxa are characterized by stems that branch close to the base, a pericarpel and receptacle tube with colourful scales that increase in size towards the perianth, and areoles with trichomes and flexible spines.
Three terminals of Disocactus speciosus aff. subsp. cinnabarinus from Chiapas (Mexico) were recovered with strong support (96 % JK, 99 % BS, MP; 1 PP BI; Fig. 2, 3). Neither the MP nor BI analysis recovered this taxon as part of D. speciosus, where the terminal from D. speciosus subsp. cinnabarinus from Chimaltenango (Guatemala) is nested, close to the type locality (Weingart 1910). The terminals from Chiapas (Arias 1666, Cruz 18, Cruz 22) were initially identified as D. speciosus subsp. cinnabarinus due to their shared floral morphology (Bravo-Hollis 1978; Bauer 2003), and it was even indicated that the observed differences were due to variations between populations (Bauer 2007). However, the adult stems of the Chiapas samples exhibit a consistent difference in the presence of phylloclades. This morphological difference was observed by Standley & Steyermark (1944) and was used as a basis for describing Heliocereus heterodoxus Standl. & Steyerm. for the region limited by the Tacana Volcano between Mexico (Chiapas) and Guatemala (San Marcos). We identified two indels in the markers psbA-trnH and ycf1 ( Table 4) that are exclusive to these three terminals. Based on these molecular characteristics and the morphological difference mentioned above, we believe that the three terminals do not correspond to D. speciosus subsp. cinnabarinus; therefore, we propose that it be recombined as D. speciosus subsp. heterodoxus (Standl. & Steyerm.) M. Á. Cruz & S. Arias.
Epiphyllum anguliger, E. crenatum and E. lepidocarpum were recovered as part of Disocactus s.str. with strong support (Fig. 2, 3). This relationship is supported by seven shared substitutions, which we recognize as the molecular synapomorphies of the clade Disocactus ( Table 4). The results do not allow discrimination of the closest relationships between these three species within Disocactus because the two topologies (MP and BI) are incongruent. However, we may recognize a possible relationship between E. crenatum and subclade 3D, which includes Nopalxochia sensu Bravo-Hollis (1978). This hypothesis is based on the shared presence of scales on the pericarpel and the receptacle tube, which are larger towards the perianth, as well as flexible spines. The generation of hybrids has also been reported for both species (D. ackermannii & D. phyllanthoides) (Anderson 2005). In addition, the distribution of E. crenatum complements the species of subclade 3D in México (Veracruz, Oaxaca and Chiapas), although E. crenatum extends towards Guatemala and Honduras. Although the relationship between E. anguliger and E. lepidocarpum is uncertain, the flexible spines observed on the stems and flowers with two sets of stamens are morphological characters shared with Disocactus (Bauer 2009). Epiphyllum anguliger is distributed in W Mexico (Guerrero, Jalisco, Nayarit and Oaxaca), whereas E. lepidocarpum is native to Costa Rica (Fig. 4).

Disocactus and sister genera
The topology of both phylogenies (MP and BI) is highly congruent: the tribe Hylocereeae consists of three groups (Fig. 2, 3). The subtribe Epiphyllinae, as redefined here, includes the genera Disocactus (Clade D), Epiphyllum (Clade E) and Pseudorhipsalis (Clade P), whereas the subtribe Hylocereinae consists of Aporocactus (Clade A), Hylocereus, Selenicereus and Weberocereus (HSW). Acanthocereus represents the third group. This result is partially consistent with the subtribes proposed by Buxbaum (1958) and the clades recovered in the phylogenetic analyses of Peniocereus by Arias & al. (2005). The main difference between the two subtribes is that phylloclades are more common in Epiphyllinae, whereas ribs are more common in Hylocereinae. In addition, both phylogenies indicate that Disocactus is more closely related to Epiphyllum and Pseudorhipsalis.
Within the subtribe Epiphyllinae, Disocactus is never recovered as a sister genus to Pseudorhipsalis. In the MP reconstruction, Disocactus is a sister to Epiphyllum and Pseudorhipsalis, whereas in the BI reconstruction, Pseudorhipsalis is sister to Disocactus and Epiphyllum. This classification is consistent with Bauer's argument (2002), which indicates that the distribution, habitat and certain morphological characteristics differ between Pseudorhipsalis and Disocactus. Pseudorhipsalis is distributed from Mexico to Bolivia, but its centre of diversity is found in Costa Rica and Panama, where it grows in low-elevation zones (below 650 m) and hot, humid regions. The primary stem of Pseudorhipsalis is easily distinguished from the secondary stem due to its cylindrical shape; its areoles are close-set, and its flowers are very small (0.7 -2.5 cm), funnel-shaped or rotate and are characterized by a short flowering period. The flowers are not oriented towards the light, and the pollen is small (45 -55 µm) and polycolpate. In contrast, Disocactus is distributed from NC Mexico to Nicaragua, where it grows in elevated (above 1200 m) tropical montane cloud forests. The primary stem of Disocactus may only be distinguished from the secondary stem in certain species; the distance between the areoles is greater; and the flowers may be small (5.5 -6 cm) to large (16 cm) and funnel-shaped. The flowers spend several days in anthesis and are mainly oriented towards the light, which results in a curve in the receptacle tube when they grow on pendulous stems; the pollen is large (70 -100 µm) and tricolpate. These morphological differences favour the hypothesis suggested in the BI analysis, in which Disocactus is the sister genus to Epiphyllum, which is distributed from Mexico to Argentina, although its greatest diversity is found between S Mexico and Costa Rica in low zones or at high elevation (0 -2300 m) in hot and temperate regions (Bauer 2003). These two genera share phylloclades with a cylindrical base, flowers larger than 2.5 cm, and a scaly pericarpel and receptacle tube. Moreover, our results allow us to exclude Aporocactus from Disocactus and recognize it as an independent genus. Nevertheless, its relationship with the subtribe Hylocereinae merits its own study because only the BI phylogenetic analysis showed high support.
Based on the data from cpDNA sequences, this study provided significant evidence indicating that certain floral morphological characteristics that are used to delimit Diso cactus have arisen more than once in Hylocereeae. The diurnal, funnel-shaped and intensely coloured (reds and others) flowers are not exclusive to Disocactus (Fig. 1). Based on these data, we hypothesize a convergent origin for the nocturnal, long, funnel-shaped flowers with light colouration (white) found in Disocactus and Epiphyllum. Therefore, supplementary studies are required to clarify the relationship between Disocactus and its sister genera, as well as its possible origin within the subtribe Epiphyllinae (as it is recognized here). A comparative morphological study of vegetative (e.g. shape and succulence of cotyledons in seedlings; stem shape during the juvenile stage) and reproductive structures (e.g. number of sets of stamens; seed size and micro-ornamentation) is needed to explore the observed synapomorphies and their transformation within the subtribe. The origin of the epiphytic growth within Hylocereeae also needs to be explored based on climbing shape, including the change in stem growth from indeterminate to determinate. A top priority is to combine our molecular data with a set of morphological attributes in a total evidence analysis to corroborate the phylogenetic relationships obtained here and recover the currently unresolved terminal nodes. We also aim to understand the evolution of the highlighted characteristics in subtribe Epiphyllinae.

Taxonomy
Based on phylogenetic analysis presented here, the 14 species accepted in Disocactus are listed below. Five new combinations are included. In addition, one taxon of presumably hybrid origin is indicated (Kimnach 1984