1
Supplemental Material
PSEUDOGENES AND EXCLUDED SEQUENCES
Results–Pseudogenes of ndhF were found via direct sequencing of PCR products in Rhodolirium speciosum (Herb.) Ravenna (GB: KC217409) and Phycella australis
Ravenna (GB: KC217397). These sequences exhibit a stop codon at position 145-147 and share a 97-aa deletion spanning positions 1,483-1,773 of our ndhF nucleotide alignment.
Two pseudogenized copies of ndhF were identified through cloning in Famatina maulensis, Fmau-ndhF1 (GB: KC217380) and Fmau-ndhF2 (GB: KC217381). Fmau- ndhF1 exhibits a stop codon at position 649-651 and a deletion at 717-722, while Fmau- ndhF2 has a stop codon at position 1,057-1,059 of the ndhF alignment.
An unusual 3′ndhF sequence was obtained by direct sequencing of Rhodophiala ananuca-1 (GB: KC217413), which does not show a stop codon in the amino acid alignment, but has an unusually high substitution rate, as well as numerous non- synonymous substitutions and a deletion between positions 1,614-1,619 of the alignment.
Cloning of the ndhF PCR product from this sample and sequencing of 4 colonies revealed the non-pseudogenized copy only, which was used in subsequent analyses.
Through direct sequencing of the trnL(UAA)-F(GAA) PCR product from the same R. ananuca sample, we obtained a symplesiomorphic sequence (GB: KC217491), which contains unusual features such as insertions between positions 50-53 (shared with
Cyrtanthus), 7 bp after position 149 (autapomorphic), and 780-785 (shared with
Cyrtanthus, Lycoris radiata (L’Hér.) Herb., Pancratium canariense Ker Gawl., Griffinia 2
Ker Gawl., Worsleya procera (Lem.) Traub, Phycella, Placea, Rhodolirium, and Traubia modesta), and a 110-bp autapomorphic deletion between positions 192-301 of the trnL(UAA)-F(GAA) alignment. Finally, the 3′ycf1 sequence from Rhodophiala splendens-1
(GB: KC207537) has an unusually high substitution rate with numerous autapomorphic non-synonymous substitutions; however, no stop codon was detected in the amino acid alignment. These sequences possessed extremely long branches relative to the other sequences in ML trees of each region (not shown). Moreover, these seven excluded sequences appear in basal positions relative to sequences from related species or from different samples of the same species. The only exception to this observation is Fmau- ndhF1, which appears embedded in the clade formed by Phycella and Placea, in agreement with the position of Famatina maulensis based on other markers.
Discussion–Pseudogenes are usually defined as sequences of genomic DNA that are derived from functional genes and exhibit degenerative features such as premature stop codons and frameshift mutations that prevent their expression, although some highly divergent sequences are functional by being involved in the regulation of gene expression and by generating genetic diversity (Balakirev and Ayala 2003). In the case of the unusual trnL(UAA)-F(GAA) sequence from R. ananuca, we hesitate to call it a pseudogene, because most of this region is non-coding. Nonetheless, pseudogenized trnF genes, although extremely rare, have been reported in other groups, including Annonaceae,
Brassicaceae, Asteraceae, Solanaceae, and Juncaceae (reviewed in Poczai and Hyvönen
2011). We were unable to detect a stop codon in the R. splendens-1 3′ycf1 sequence, perhaps because this is just a partial sequence and does not span the whole length of this gene. 3
On the other hand, ndhF pseudogenes have frequently been reported in a diverse array of angiosperms, including Orchidaceae subfamily Epidendroideae (Neyland and
Urbatsch 1996), Anacampseros L. (Anacampserotaceae; Applequist and Wallace 2001),
Catalpa Scop. (Bignoniaceae; Li 2008), and Erodium L’Hér. ex Aiton (Geraniaceae;
Blazier et al. 2011). The complete absence of the ndhF gene from the chloroplast genome has been reported in Gnetales and Pinaceae (Wakasugi et al. 1994; Braukmann et al.
2009), in several parasitic lineages (e.g., de Pamphilis and Palmer 1990; Haberhausen and Zetsche 1994), and in Amaryllidaceae tribe Eucharideae (Meerow 2010). The latter example suggests that the American lineage of Amaryllidaceae is susceptible to the loss of this gene. Within Clade A, ndhF might have been pseudogenized in two independent events given the pattern of deletions, the first involving the most recent common ancestor of Phycella australis and Rhodolirium speciosum, and the second involving Famatina maulensis, where there are two ndhF pseudogenes. The unusual 3′ndhF sequence obtained from Rhodophiala ananuca-1 might constitute a third event of ndhF pseudogenization within Hippeastreae; however, because a “normal” copy was also detected in this sample, we might be dealing with a duplication of the gene and its transfer to the nucleus (Baldauf and Palmer 1990; Gantt et al. 1991; Millen et al. 2001).
We currently do not have an explanation for these odd cpDNA sequences and hesitate to provide one without further experimental evidence. Other studies have explained the presence of pseudogenes in the chloroplast genome by invoking functional transference to the nucleus, functional replacement by another gene, intermolecular recombination, and complex processes of structurally mediated illegitimate recombination (e.g., Ansell et al. 2007; Blazier et al. 2011). 4
POSITIVE SELECTION TESTS
Materials and Methods–A detection analysis of positive selection was performed for both coding cpDNA regions in DataMonkey (Delport et al. 2010a). Codon alignments for 3′ycf1 and ndhF were determined in Geneious Pro, considering only complete, non- identical sequences. After removal of indels, these alignments were used to infer a maximum likelihood (ML) tree for selection analysis for each locus, using the following approach. Each codon alignment, its respective ML tree, and the codon model of evolution selected by the CodonTest method (Delport et al. 2010b) were used to detect alignment-wide evidence for positive selection with the PARRIS method (Scheffler et al.
2006) at p = 0.05.
Results–The final codon alignments consisted of 56 sequences and 582 codons for ndhF, and 72 sequences and 691 codons for 3′ycf1. CodonTest selected the K81 model (Kimura 1981) with substitution code 012210 for the ndhF data set and a model with substitution code 012010 for 3′ycf1. Trees inferred by RAxML for these codon alignments agreed in topology and had similar branch lengths to those estimated for the complete matrices. Neither codon alignment showed evidence of positive selection at p <
0.05 (ndhF: LRT = 0.1, p = 0.951; 3′ycf1: LRT = 3.32, p = 0.190). Under the null (‘nearly neutral’) model (M1), DataMonkey estimated a mean dN/dS ratio of 0.366 for ndhF and
0.725 for 3′ycf1.
RECOMBINATION DETECTION ANALYSES 5
Materials and Methods–Tests to detect recombination in the expanded ITS alignment of Hippeastreae were conducted with the Recombination Detection Program 4
(RDP4; Heath et al. 2006; Martin et al. 2010). RDP4 implements several non-parametric recombination detection methods for identifying and analyzing recombination signals
(Martin et al. 2010). Besides identifying putative recombinant sequences, RDP4 also infers the most likely recombination breakpoints in the sequence, and putative major (i.e., a sequence closely related to that from which the greater part of the recombinant’s sequence may have been derived) and minor (i.e., a sequence closely related to that from which sequences in the proposed recombinant region may have been derived) parental sequences for each recombination event. Breakpoint polishing, checking for misalignments, and phylogenetic evidence for recombination signals were options that were invoked. The highest acceptable p value was set to 0.05 with Bonferroni correction applied for multiple comparisons.
Two data sets were analyzed: 1) the complete matrix with 91 non-identical sequences including outgroups (Table 2); and 2) a reduced set of 69 sequences restricted to Clade B, where a recombination signal was detected by Meerow (2010). When a signal was detected in the preliminary scan step, the guidelines to test and refine recombination hypotheses found in the RDP3 manual (Martin et al. 2010) were followed.
Results–No signal of recombination was detected for the complete data set.
However, after the refinement of the preliminary hypotheses, two putative recombination events were inferred in the data set restricted to Clade B (Fig. S9) by the Sister-Scanning
Method (SiScan; Gibbs et al. 2000). 6
The first event involves Rhodophiala bifida subsp. granatiflora as the major parent, and possibly Hippeastrum cipoanum as the minor parent, although the program noted the possibility of misidentification of the second parent. This event gave rise to the sequences of Eithea blumenavia and most core-Rhodophiala. The program suggested that
Zephyranthes cearensis, Famatina herbertiana, and Rhodophiala ananuca were also daughter sequences of this event, but these were rejected based on their p-values and guidelines to refine recombination hypotheses in the RDP3 manual. However, it is still possible that these sequences are also involved in this event, which could have been masked by more recent recombination events (Martin et al. 2010).
The second recombination event gave rise to Hippeastrum cipoanum; an unknown sequence, possibly R. bifida subsp. granatiflora, was identified as the major parent, and
Hippeastrum striatum as the minor parent.
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USA 91: 9794–9798. 99 Zephyranthes challensis - Zephyranthes andina Habranthus andalgalensis - Zephyranthes citrina Zephyranthes pulchella - 61 Zephyranthes macrosiphon Zephyranthes smallii Zephyranthes chlorosolen 76 Zephyranthes candida Zephyranthes seubertii Zephyranthes puertoricensis Zephyranthes albiella Zephyranthes longistyla Famatina cisandina Famatina andina Rhodophiala phycelloides Rhodophiala a . advena Rhodophiala advena Rhodophiala bagnoldii Rhodophiala tiltilensis - Rhodophiala sp. 69 Rhodophiala splendens2 Rhodophiala montana - Rhodophiala araucana Rhodophiala ananuca2 Rhodophiala splendens1 Famatina herbertiana - Zephyranthes treatiae Zephyranthes rosea Zephyranthes carinata Zephyranthes orellanae Zephyranthes morrisclintii - Zephyranthes drummondii Zephyranthes clintiae Zephyranthes simpsonii Zephyranthes atamasco - 53 Habranthus immaculatus 52 Haylockia americana Sprekelia howardii Phycella sp. Phycella a . cyrtanthoides - Phycella angustifolia Placea davidii Placea lutea - Rhodolirium speciosum Phycella australis Famatina maulensis 84 Placea a . ornata Placea ornata Placea germainii - - Placea arzae Phycella scarlatina 93 Rhodolirium montanum - Rhodolirium andicola Rhodolirium laetum Traubia modesta Zephyranthes bifolia Habranthus sp. - Habranthus robustus - Habranthus brachyandrus - Habranthus martinezii - Habranthus tubispathus Habranthus pedunculosus 58 - Sprekelia formosissima 51 Zephyranthes lifolia Zephyranthes mesochloa Rhodophiala bi da subsp. bi da Rhodophiala bi da subsp. granatiora Eithea blumenavia Hippeastrum reticulatum - Hippeastrum brasilianum Hippeastrum sp. Hippeastrum puniceum Hippeastrum parodii Hippeastrum evansiae 52 Hippeastrum psittacinum Hippeastrum morelianum 63 Hippeastrum striatum Hippeastrum papilio Hippeastrum traubii 99 Zephyranthes cearensis 61 Grinia parviora - Grinia espiritensis - Lycoris radiata - Grinia hyacinthina - Lycoris aurea - Worsleya procera Pancratium canariense Cyrtanthus herrei Cyrtanthus carneus Cyrtanthus obliquus
FIGURE S1. Single most-parsimonious tree derived from trnL(UAA) intron data set. Values above branches correspond to parsimony jackknife frequency di erence (GC MP-JK) values > 50%. Clades subtending terminals in bold denote groups of identical sequences. “-“ = JK < 50%. Habranthus sp. Habranthus robustus Zephyranthes bifolia Habranthus brachyandrus 56 Habranthus martinezii Habranthus tubispathus Habranthus pedunculosus Sprekelia formosissima Zephyranthes lifolia Famatina herbertiana Famatina cisandina Famatina andina Rhodophiala phycelloides Rhodophiala a . advena Rhodophiala advena Rhodophiala bagnoldii Rhodophiala tiltilensis 61 Rhodophiala sp. Rhodophiala montana Rhodophiala araucana Rhodophiala ananuca2 Rhodophiala splendens1 Zephyranthes puertoricensis Zephyranthes albiella 62 Zephyranthes citrina Zephyranthes pulchella Zephyranthes macrosiphon Zephyranthes smallii Zephyranthes chlorosolen Zephyranthes candida Zephyranthes seubertii 94 Habranthus immaculatus Haylockia americana Hippeastrum sp. Hippeastrum puniceum Hippeastrum parodii Hippeastrum evansiae Hippeastrum traubii Hippeastrum psittacinum 93 Hippeastrum morelianum Hippeastrum striatum Hippeastrum papilio Hippeastrum reticulatum Rhodophiala bi da subsp. bi da Rhodophiala bi da subsp. granatiora Sprekelia howardii Zephyranthes challensis Zephyranthes andina Zephyranthes longistyla Zephyranthes mesochloa Zephyranthes cearensis Zephyranthes treatiae Zephyranthes rosea Zephyranthes carinata Zephyranthes orellanae Zephyranthes morrisclintii Zephyranthes drummondii Zephyranthes clintiae Zephyranthes simpsonii Zephyranthes atamasco Habranthus andalgalensis Eithea blumenavia Hippeastrum brasilianum Phycella scarlatina Phycella sp. Phycella a . cyrtanthoides Phycella angustifolia Placea davidii Placea lutea Placea a . ornata - Placea ornata Placea germainii Placea arzae Rhodolirium speciosum Phycella australis Famatina maulensis 85 58 Gri nia parviora Gri nia espiritensis Gri nia hyacinthina Lycoris aurea Worsleya procera Rhodolirium montanum Rhodolirium andicola Rhodolirium laetum Traubia modesta Lycoris radiata Pancratium canariense Cyrtanthus herrei Cyrtanthus carneus Cyrtanthus obliquus
FIGURE S2. Strict consensus tree of 17 most-parsimonious trees derived from trnL(UAA)-F(GAA) intergenic spacer data set. Values above branches correspond to parsimony jackknife frequency di erence (GC MP-JK) values > 50%. Clades subtending terminals in bold denote groups of identical sequences. “-“ = JK < 50%. 61/64 Zephyranthes challensis Zephyranthes andina 85/88 Habranthus andalgalensis 62/63 Zephyranthes citrina Zephyranthes candida Zephyranthes seubertii 85/90 Zephyranthes smallii 62/68 Zephyranthes pulchella 99/99 Zephyranthes macrosiphon Zephyranthes chlorosolen 99/* Zephyranthes puertoricensis Zephyranthes albiella Zephyranthes treatiae Zephyranthes rosea Zephyranthes carinata 75/82 Zephyranthes orellanae Zephyranthes morrisclintii 84/87 Zephyranthes drummondii Zephyranthes clintiae Zephyranthes atamasco Zephyranthes simpsonii 61/67 Eithea blumenavia Hippeastrum reticulatum Sprekelia howardii Zephyranthes mesochloa Zephyranthes cearensis Famatina cisandina Rhodophiala tiltilensis 52/58 Rhodophiala splendens2 Rhodophiala araucana 62/67 Rhodophiala sp. 61/80 Rhodophiala montana Rhodophiala a . advena Rhodophiala advena 70/74 */* 60/74 Rhodophiala ananuca1 Rhodophiala ananuca2 Rhodophiala bagnoldii Famatina herbertiana 85/86 Zephyranthes longistyla Zephyranthes avissima Habranthus pedunculosus 85/93 85/87 Habranthus martinezii Habranthus tubispathus Habranthus sp. 80/86 94/96 Habranthus robustus Habranthus brachyandrus Sprekelia formosissima Zephyranthes lifolia Zephyranthes bifolia Hippeastrum sp. 50/80 94/95 Hippeastrum puniceum Hippeastrum parodii Hippeastrum evansiae Hippeastrum psittacinum 98/99 97/98 Hippeastrum morelianum Hippeastrum striatum */* Hippeastrum papilio Hippeastrum traubii 85/90 Rhodophiala bi da subsp. bi da Rhodophiala bi da subsp. granatiora Hippeastrum brasilianum 98/98 Habranthus immaculatus Haylockia americana 99/* Famatina maulensis 84/94 Rhodolirium speciosum Phycella australis 97/99 Phycella sp. 62/67 Phycella angustifolia Phycella a . cyrtanthoides 56/77 Placea davidii 99/* Placea a . ornata -/62 Placea ornata -/76 Placea germainii Placea lutea Phycella scarlatina Rhodolirium montanum */98 Rhodolirium andicola Rhodolirium laetum Traubia modesta Outgroups
FIGURE S3. Strict consensus of 4 most-parsimonious trees derived from 3’ycf1 data set. Values above branches correspond to parsimony jackknife frequency di erence (GC MP-JK) values > 50%, followed by maximum-likelihood bootstrap absolute frequency (ML-BS) values > 50% for all nucleotide characters. Clades subtending terminals in bold denote groups of identical sequences. Outgroups are not shown.”*” = 100 JK or BS; “-“ = JK or BS < 50%. Rhodophiala ananuca1 54/59 Rhodophiala ananuca2 Rhodophiala bagnoldii 55/76 Rhodophiala phycelloides Rhodophiala a . advena Rhodophiala advena Famatina herbertiana Famatina cisandina Rhodophiala tiltilensis Rhodophiala sp. 77/76 Rhodophiala montana Famatina andina Rhodophiala splendens1 Rhodophiala splendens2 Rhodophiala araucana Zephyranthes treatiae Zephyranthes rosea Zephyranthes carinata Zephyranthes orellanae 85/86 Zephyranthes morrisclintii Zephyranthes drummondii Zephyranthes clintiae Zephyranthes atamasco Zephyranthes simpsonii 61/63 Zephyranthes puertoricensis Zephyranthes albiella Zephyranthes minima Zephyranthes candida Zephyranthes pulchella 55/57 Zephyranthes citrina Zephyranthes seubertii Zephyranthes smallii Zephyranthes macrosiphon Zephyranthes chlorosolen 55/56 74/88 Zephyranthes longistyla Habranthus andalgalensis Sprekelia howardii Zephyranthes challensis Zephyranthes andina Zephyranthes mesochloa Zephyranthes cearensis 61/85 Habranthus martinezii Habranthus tubispathus 57/65 Habranthus sp. Habranthus robustus 60/62 Habranthus pedunculosus Habranthus brachyandrus Sprekelia formosissima 63/73 Zephyranthes lifolia Zephyranthes bifolia 98/99 Rhodophiala bi da subsp. bi da Rhodophiala bi da subsp. granatiora Hippeastrum sp. Hippeastrum parodii 61/63 Hippeastrum puniceum Hippeastrum evansiae Hippeastrum traubii 61/64 Hippeastrum psittacinum 61/64 Hippeastrum morelianum Hippeastrum striatum Hippeastrum papilio 52/67 Habranthus immaculatus Haylockia americana 94/96 Eithea blumenavia Hippeastrum reticulatum Hippeastrum brasilianum Phycella angustifolia Phycella a . cyrtanthoides Placea davidii Placea a . ornata Phycella sp. 62/71 Phycella scarlatina Placea ornata Placea germainii 94/97 Placea lutea Placea arzae 86/88 Rhodolirium montanum 97/99 Rhodolirium andicola Rhodolirium laetum Traubia modesta Outgroups
FIGURE S4. Most-parsimonious tree derived from ndhF data set. Values above branches correspond to parsimony jackknife frequency di erence (GC MP-JK) values > 50%, followed by maximum-likelihood bootstrap absolute frequency (ML-BS) values > 50% for all nucleotide characters. Clades subtending terminals in bold denote groups of identical sequences. Outgroups are not shown. “-“ = JK or BS < 50%. Zephyranthes citrina Zephyranthes pulchella 61/66 Zephyranthes macrosiphon Zephyranthes smallii Zephyranthes chlorosolen 62/64 Zephyranthes puertoricensis Zephyranthes albiella Zephyranthes candida Zephyranthes seubertii 94/70 99/74 Zephyranthes challensis Zephyranthes andina Habranthus andalgalensis Zephyranthes longistyla Habranthus sp. Habranthus robustus Zephyranthes bifolia Habranthus brachyandrus Habranthus martinezii 83/87 Habranthus tubispathus 57/61 Habranthus pedunculosus Sprekelia formosissima Zephyranthes lifolia Zephyranthes mesochloa Hippeastrum sp. Hippeastrum puniceum -/- Hippeastrum parodii Hippeastrum evansiae 63/64 Hippeastrum traubii Hippeastrum psittacinum Hippeastrum morelianum Hippeastrum striatum Hippeastrum papilio Famatina cisandina Famatina andina Rhodophiala phycelloides Rhodophiala a . advena Rhodophiala advena Rhodophiala bagnoldii Rhodophiala tiltilensis Rhodophiala sp. Rhodophiala montana Rhodophiala araucana 86/88 Rhodophiala ananuca2 Famatina herbertiana Rhodophiala splendens2 Rhodophiala splendens1 99/77 97/82 Habranthus immaculatus 60/61 Haylockia americana Sprekelia howardii Zephyranthes treatiae Zephyranthes rosea Zephyranthes carinata Zephyranthes orellanae Zephyranthes morrisclintii Zephyranthes drummondii Zephyranthes clintiae Zephyranthes simpsonii Zephyranthes atamasco Hippeastrum reticulatum Rhodophiala bi da subsp. granatiora Rhodophiala bida subsp. bida Zephyranthes cearensis Eithea blumenavia Hippeastrum brasilianum Phycella sp. Phycella a . cyrtanthoides Phycella angustifolia Placea davidii Placea a . ornata Placea ornata Placea germainii 94/95 Placea arzae Famatina maulensis Rhodolirium speciosum Phycella australis Phycella scarlatina Placea lutea 92/90 Rhodolirium montanum */* Rhodolirium andicola Rhodolirium laetum Traubia modesta 61/76 Gri nia parviora 62/65 Gri nia espiritensis Gri nia hyacinthina Worsleya procera Lycoris aurea Lycoris radiata Pancratium canariense Cyrtanthus herrei Cyrtanthus carneus Cyrtanthus obliquus
FIGURE S5. Strict consensus tree of 11 most-parsimonious trees derived from trnL(UAA)-F(GAA) data set. Values above branches correspond to parsimony jackknife frequency di erence (GC MP-JK) values > 50%, followed by maximum-likelihood bootstrap absolute frequency (ML-BS) values > 50% for all nucleotide characters. Clades subtending terminals in bold denote groups of identical sequences. ”*” = 100% JK or BS; “-“ = JK or BS < 50%. Zephyranthes puertoricensis
Zephyranthes seubertii
Zephyranthes candida Zephyranthes albiella Zephyranthes simpsonii
Zephyranthes atamasco Zephyranthes treatiae
Zephyranthes rosea
ZEPHYRANTHES Mexico/Texas-1 ZEPHYRANTHES Mexico/Texas-2
Rhodolirium speciosum
Famatina maulensis Zephyranthes lifolia Zephyranthes Zephyranthes longistyla Habranthus andalgalensis CORE-RHODOPHIALA RhodoliriumRhodolirium montanum andicola
Zephyranthes citrina PLACEA-PHYCELLA Habranthus immaculatus Habranthus sp. Habranthus robustus
CORE-RHODOPHIALA
Haylockia americana
Eithea blumenavia Eithea Zephyranthes bifolia Zephyranthes cearensis Zephyranthes
HabranthusHabranthus tubispathus martinezii Sprekelia formosissima Sprekelia howardii RHODOPHIALA BIFIDA ZEPHYRANTHES ANDINA S.L.
Habranthus brachyandrus
Rhodolirium laetum Hippeastrum reticulatum Hippeastrum brasilianum Traubia modesta
Zephyranthes mesochloa
Habranthus pedunculosus
root
FIGURE S6. Hybridization network derived from strict consensus trees resulting from analyses of cpDNA1 and equivalent ITS matrix (89 terminals) collapsed at nodes with GC MP-JK and ML-BS < 90% (HybNet1). Outgroups are not shown. Blue lines denote reticulation nodes and black lines are tree nodes. Branch lengths have no direct meaning; they have been modi ed to make the diagram more easily readable. Zephyranthes atamasco simpsonii Zephyranthes
Zephyranthes candida Zephyranthes seubertii
Zephyranthes rosea
Zephyranthes treatiae Zephyranthes albiella Zephyranthes puertoricensis
Habranthus andalgalensis
Zephyranthes longistyla Habranthus robustus Habranthus sp.
CORE-RHODOPHIALA Habranthus martinezii
CORE-HIPPEASTRUM
Zephyranthes lifolia
Habranthus brachyandrus
Habranthus tubispathus
ZEPHYRANTHES Mexico/Texas-2
RhodoliriumPhycella speciosum australis Zephyranthes bifolia
PLACEA-PHYCELLA ZEPHYRANTHES ANDINA S.L.
Zephyranthes cearensis ZEPHYRANTHES Mexico/Texas-1 RHODOPHIALA BIFIDA Eithea blumenavia Famatina maulensis Hippeastrum reticulatum Sprekelia formosissima Haylockia americana Sprekelia howardii Zephyranthes citrina
Rhodolirium andicola Habranthus pedunculosus Zephyranthes mesochloa Rhodolirium montanum Habranthus immaculatus
Hippeastrum brasilianum
Rhodolirium laetum
Traubia modesta
root
FIGURE S7. Hybridization network derived from strict consensus trees resulting from analyses of cpDNA1 and equivalent ITS matrix (89 terminals) collapsed at nodes with GC MP-JK and ML-BS < 70% (HybNet3). Outgroups are not shown. Blue lines denote reticulation nodes and black lines are tree nodes. Branch lengths have no direct meaning; they have been modi ed to make the diagram more easily readable. Zephyranthes puertoricensis
Zephyranthes albiella
Zephyranthes seubertii Zephyranthes candida Zephyranthes
Zephyranthes simpsonii Zephyranthes Zephyranthes atamasco Zephyranthes treatiae Zephyranthes
ZEPHYRANTHES Mexico/Texas-2 Zephyranthes rosea Zephyranthes
Habranthus robustus
Zephyranthes citrina Zephyranthes Habranthus sp. ZEPHYRANTHES ANDINA S.L. CORE-HIPPEASTRUM Habranthus martinezii
Habranthus brachyandrus Habranthus tubispathus ZEPHYRANTHES Mexico/Texas-1
Zephyranthes bifolia Zephyranthes lifolia
HippeastrumZephyranthes reticulatum cearensis Rhodolirium speciosum Eithea blumenavia Phycella australis PLACEA-PHYCELLA
Zephyranthes mesochloa
Famatina maulensis Sprekelia howardii Sprekelia formosissima Habranthus immaculatus HabranthusHaylockia pedunculosus americana CORE-RHODOPHIALA Hippeastrum brasilianum
Rhodolirium laetum
Traubia modesta Rhodolirium andicola RHODOPHIALA BIFIDA Rhodolirium montanum
root
FIGURE S8. Hybridization network derived from strict consensus trees resulting from analyses of cpDNA2 and equivalent ITS matrix (87 terminals) collapsed at nodes with GC MP-JK and ML-BS < 70% (HybNet4). Outgroups are not shown. Blue lines denote reticulation nodes and black lines are tree nodes. Branch lengths have no direct meaning; they have been modi ed to make the diagram more easily readable. Eithea blumenavia 473 646 Rhodophiala bi da p = 6.97 E-3 subsp. granatiora Unknown (Hippeastrum cipoanum?) Famatina andina Rhodophiala bi da p = 1.78 E-4 subsp. granatiora Unknown (Hippeastrum cipoanum?) Rhodophiala advena Rhodophiala bi da p = 1.78 E-4 subsp. granatiora Unknown (Hippeastrum cipoanum?) Rhodophiala araucana Rhodophiala bi da p = 1.78 E-4 subsp. granatiora Unknown (Hippeastrum cipoanum?) Rhodophiala bagnoldii Rhodophiala bi da p = 1.78 E-4 subsp. granatiora Unknown (Hippeastrum cipoanum?) Rhodophiala montana Rhodophiala bi da p = 1.78 E-4 subsp. granatiora Unknown (Hippeastrum cipoanum?) Rhodophiala phycelloides Rhodophiala bi da p = 1.78 E-4 subsp. granatiora Unknown (Hippeastrum cipoanum?) Rhodophiala splendens Rhodophiala bi da p = 1.78 E-4 subsp. granatiora Unknown (Hippeastrum cipoanum?) Hippeastrum cipoanum 445 640 Unknown p = 9.61 E-3 (Rhodophiala bi da subsp. granatiora?) Hippeastrum striatum
FIGURE S9. Partial schematic plot of alignment of ITS across Clade B indicating the presence of two recombination events detected by RDP4. The recombinant sequences are labelled above the bars, putative major parents to the left, and minor parents to the right. The p-values are Bonferroni corrected for multiple comparisons. Putative recombination breakpoint positions in the recombinants are indicated for the rst sequence of each event.