Copyright by Taylor Sultan Quedensley 2012

The Dissertation Committee for Taylor Sultan Quedensley Certifies that this is the approved version of the following dissertation:

MOLECULAR SYSTEMATICS OF THE MEXICAN TUSSILAGINIOID GROUP (: )

Committee:

Beryl Simpson Co-Supervisor

Robert Jansen Co-Supervisor

C. Randall Linder

David Hillis

James Mauseth MOLECULAR SYSTEMATICS OF THE MEXICAN TUSSILAGINIOID GENERA (ASTERACEAE: SENECIONEAE)

by

Taylor Sultan Quedensley B.S. Agr. Sci., M.S. Biology

Dissertation Presented to the Faculty of the Graduate School of The University of at Austin in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philospohy

The University of Texas at Austin August 2012

Dedication

This dissertation is dedicated to Don Mahoney and Dennis Breedlove. Their love for has inspired me for many years.

Acknowledgements

I am so grateful to Bob Jansen and Beryl Simpson for enabling me to be a botanist at The University of Texas at Austin and to pursue my academic and career goals.

I thank Mario Véliz (Universidad de San Carlos de ) for his support in the field and for the use of the BIGU Herbarium. In , I thank Jose Luis Villaseñor

(Universidad Nacional Autónoma de México), Jose Angel Villareal (Universidad

Autónoma Agraria Antonio Narro), M. Socorro González-Elizondo (Instituto Politécnico

Nacional), and Mario Ishiki (Colegio de la Frontera Sur) for assistance with fieldwork and specimen transport and export. I am grateful to Timmy Buxton (Cabrillo College) for his assistance in the field during multiple collecting trips. I also thank Taylor Nyberg and

Nicholas Wilhelm (The University of Texas at Austin) for assistance with laboratory components of this project, and Thomas Payne (CIMMYT) for providing lodging during research visits to Mexico City.

Leaf fragments for molecular studies were taken with permission from the following herbaria: BIGU, F, MEXU, TEX, US. I thank Beryl Simpson, Lindsay

Woodruff, and Thomas Wendt of the Resource Center for their support at the herbarium of The University of Texas at Austin. I am grateful to Donald Mahoney, David

Kruse-Pickler, and Mona Bourell of the San Francisco Botanical Garden for sending live material to The University of Texas at Austin and for allowing us to make voucher specimens from their garden. I am thankful to Robert Kowal (University of Wisconsin-

v Madison) and David Sutherland (University of Nebraska at Omaha) for sending living material to The University of Texas at Austin. Lastly, I thank Larry Gilbert and John

Crutchfield at the Brackenridge Field Laboratory for providing greenhouse space for growing living specimens included in this project.

I am very grateful to the following graduate students for all of their support over the past six years with my research and personal matters: Chris Blazier, Michael

Gruenstaeudl, Amanda Kenney, Scott Meadows, Teofil Nakov, Sandra Pelc, Erica

Schwarz, Roxi Steele, and Mao-Lun Weng. I thank David Des Marais with guidance on some aspects of the phylogenetic analyses and Eli Meyer for help with genome assembly and analysis.

Funding was provided by The University of Texas at Austin Graduate School,

College of Natural Sciences, Plant Biology Graduate Program, Sidney and Doris Blake

Professorship, and the Mexican Center. Additional funding was provided by the

American Society of Plant Taxonomists, the American Philosophical Society Lewis and

Clark Field Scholarship, the University of Hawaii at Manoa Department of , and the National Museum of Natural History at the Smithsonian.

I thank Dr. Billie Turner for his friendship and encouragement and for always believing in me. Without you my graduate school experiences would have had an empty feeling. I will always remember our coffee chats that meant so much to me for six years.

Finally, I wish to thank my mom Cindy, my wife Marnie, and my daughters Nomi

Xela and Lucille Xilone, for their unwavering support and love while I have been in graduate school. vi SYSTEMATICS OF THE MEXICAN TUSSILAGINIOID GENERA (ASTERACEAE: SENECIONEAE)

Taylor Sultan Quedensley, Ph.D. The University of Texas at Austin, 2012

Supervisors: Beryl Simpson & Robert Jansen

The Mexican tussilaginioid group consists of 13 diverse genera of sunflowers

(Asteraceae: Senecioneae) distributed from the USA to Panama, with most occurring in montane regions from Central Mexico to Guatemala. Presently, 140 species in 13 genera are recognized with many of these species considered to be endemic to threatened - forest or cloud forest ecosystems. Sixty-two species within the study group were included in a combined phylogenetic analysis of two regions of the nuclear ribosomal repeat, the internal and external transcribed spacers. Fifty-two of these taxa were analyzed in a phylogenetic framework for the first time. The results from the combined nrDNA analysis (62 species in 12 genera in the combined analysis) strongly support the monophyly of the Mexican tussilaginioid group, however, the topology and hypothesis testing using constraint models indicate that the genera Pittocaulon,

Psacaliopsis, and are not monophyletic. Telanthophora s.s. is monophyletic, although this is nested within Roldana s.s. Endemism is abundant among the clade with over half of the species restricted to relatively small geographic areas. Moreover,

vii most members of the group (ca. 120 species, or 87%) are present in montane regions under immense pressure from human land use practices at or above 1500 meters in

Mexico and Guatemala.

Two of the genera from my study group, Pippenalia and Psacaliopsis were taxonomically assessed based on their morphological characters and the nrDNA results.

A single species, Psacaliopsis purpusii, remains in the genus, while Pippenalia delphinifolia, Psacaliopsis macdonaldii, and P. pudica are transferred to Psacalium.

Funstonia gen. nov. is here erected a new genus encompassing a single species with two varieties.

Chloroplast genomes of atriplicifolium, Roldana aschenborniana,

R. barba-johannis, and Telanthophora grandifolia were sequenced with next generation sequencing in order to identify regions of variation and to compare the assemblies produced via de novo and reference-based methods. The reference-based assemblies were more complete than the de novo assemblies, and therefore the former sequences were utilized for phylogenetic analyses.

viii Table of Contents

List of Tables .… ...... xi List of Figures ...... xii List of Illustrations ...... xiii List of Appendices ...... xiv Chapter 1: Introduction to the Mexican tussilaginioid group (Asteraceae: Senecioneae) ...... 1 Chapter 2: Molecular phylogenetics of the endemic montane Mexican tussilaginioid group (Asteraceae: Senecioneae) ...... 10 Introduction ...... 11 Materials and Methods…...... 12 Taxon sampling and DNA isolation ...... ….12 DNA amplification, sequencing, and alignment ...... ….13 Phylogenetic analyses ...... ….15 Congruence between ETS and ITS ...... ….16 Alternative hypothesis testing ...... ….16 Results ...... 17 Phylogenetic analysis of ITS and ETS data ...... ….17 Congruence test……………………………………………………...18 Combined data analysis ...... ….18 Hypothesis testing ...... ….19 Discussion ...... 20 Psacaliopsis purpusii, Robinsonecio (Clade 1) ...... ….21 Psacaliopsis pinetorum/paneroi (Clade 2) ...... ….22 Digitacalia (Clade 3) ...... ….23 Arnoglossum, Barkleyanthus, Yermo (Clade 4) ...... ….24 Pippenalia, Psacaliopsis macdonaldii/pudica, Psacalium (Clade 5) .24 Roldana ‘Pericalia’ (Clade 6) ...... ….26 Pittocaluon bombycophole (Clade 7) ...... ….26 Pittocaulon praecox/vellatum, Villasenoria (Clade 8) ...... ….27 complex (Clade 9) ...... ….28 Roldana sundbergii complex (Clade 10) ...... ….29 Roldana platanifolia complex (Clade 11) ...... ….30 Roldana mixtecana/reticulata (Clade 12) ...... ….30 Telanthophora (Clade 13) ...... ….31 Other taxa not included in thirteen clades ...... ….32 Biogeographic implications ...... ….33 Conservation implications ...... 35

ix Conclusion ...... 35

Chapter 3: A reassessment of the Neotropical genera Pippenalia and Psacaliopsis (Asteraceae: Senecioneae) ...... 50 Taxonomical Background ...... 50 Materials and Methods ...... 55 Results and Discussion ...... 56 Key to the Mexican Tusslaginioid Group ...... 60 Taxonomic Treatment ...... 61

Chapter 4: Sequencing, assembly, and alignment of four chloroplast genomes .. 82 Introduction ...... 82 Materials and Methods ...... 85 Taxon sampling ...... ….85 Chloroplast DNA extraction, isolation, and genome amplification …85 SOLiD sample preparation and sequencing ………………………....86 Reference-based assembly ...... ….86 De novo assembly ...... ….87 Phylogenetic analyses ...... ….87 Comparisons of intergenic spacer regions ...... ….88 Comparisons of protein-coding regions ...... ….88 Results ...... 88 Reference-based assemblies ...... ….88 De novo assemblies ...... ….89 Sequence divergence of intergenic spacer regions ...... ….90 Sequence divergence of coding regions ...... ….90 Phylogenetic analyses ...... ….91 Discussion ...... 92 Reference-based vs. de novo assemblies ...... ….92 Phylogenetic implications ...... ….93 Future studies ...... ….93

References ...... 112

x

List of Tables

Table 2.1: Species and genera of the Mexican tussilaginioid group used for ITS/ETS combined data set analysis ...... 37 Table 2.2: Data set partitions with information on the number of informative/total characters contributed to the analyses ...... 38 Table 2.3: Results of the harmonic mean estimate in MrBayes to test the alternative topologies of the combined Bayesian Analysis results ...... 39 Table 3.1: Comparative morphology and geographic distribution of Funstonia pinetorum, Psacaliopsis purpusii, Psacalium delphinifolium, Psacalium macdonaldii, and Psacalium pudicum ...... 77 Table 4.1: Species, locality information, percentage of the chloroplast genome sequenced, and number of reads sequenced by the ABI SOLiD sequencer ...... 96 Table 4.2: Referenced-based assembly results ...... 97 Table 4.3: De novo assembly results ...... 98 Table 4.4: Contigs assembled de novo and compared against Jacobaea vulgaris chloroplast genome for sequence divergence ...... 99 Table 4.5: Analysis of intergenic spacer regions for the four Mexican Tussilaginioid taxa ...... 101 Table 4.6: Indels per species per intergenic region ...... 103 Table 4.7: Ten coding regions identified by Timme et al. (2007) as potentially informative ...... 104 Table 4.8: Data set partitions for the phylogenetic analyses ...... 105

xi List of Figures

Figure 2.1: Bayesian inference (BI) phylogram from ITS data set (144 accessions) ...... 40 Figure 2.2: Bayesian inference (BI) phylogram from ETS data set (109 accessions) ...... 43 Figure 2.3: Bayesian inference (BI) phylogram from reduced ITS data set (100 accessions) ...... 45 Figure 2.4: Bayesian inference (BI) phylogram from reduced ETS data set (100 accessions) ...... 47 Figure 2.5: Bayesian inference (BI) phylogram from combined ITS/ETS data set (100 accessions) ...... 48 Figure 3.1: Bayesian inference phylagram of the Mexican Tussilaginioid group based on a combined ITS/ETS data set of 53 taxa ...... 78 Figure 3.2: Distribution of the study taxa in Mexico ...... 79 Figure 3.3: Distribution of the study taxa in ...... 80 Figure 4.1: Contigs from de novo assembly blasted to the Jacobaea vulgaris chloroplast genome ...... 106 Figure 4.2: Histogram with sequence divergence of the intergenic spacers ...... 107 Figure 4.3: Histogram with sequence divergence of the intergenic spacers ...... 108 Figure 4.4: Bayesian inference (BI) phylogram from data set 1 (98,965 bp) ..... 109 Figure 4.5: Bayesian inference (BI) phylogram from data set 2 (40,826 bp) ..... 110 Figure 4.6: Bayesian inference (BI) phylogram from data set 3 (85,877 bp) ..... 111

xii List of Illustrations

Illustration 3.1: Ilustration of Funstonia pinetorum by Melissa Toberer ...... 81

xiii List of Appendices

Appendix 1.1 Descriptions of the genera based on the present study of the Mexican tussilaginioid group ...... 134 Appendix 2.1 Taxa included in seperate and combined nrDNA analyses with voucher information ...... 153 Appendix 3.1 Taxa included in the combined nrDNA analyses with voucher information ...... 159

xiv Chapter 1: Introduction to the Mexican

Tussilaginioid Group (Asteraceae: Senecioneae)

The Asteracaeae is the most species-rich family of flowering plants with ca.

24,000 ̶ 30,000 species in 1600 ̶ 2000 genera (Funk & Robinson, 2005; Funk et al., 2005;

Hind, 2007; Kadereit & Jeffrey, 2007). The Senecioneae, the most species-rich tribe with ca. 150 genera and 3,000 species (Nordenstam, 2003, 2007), consists of annual and perennial herbs, , , and a small number of epiphytes. It exhibits impressive variation in morphology, particularly with respect to growth form, leaf shape, type, and flower color (Barkley, 1985a). The basal chromosome number for the Senecioneae is purported to be n = 10 (e.g., Orduff et al., 1963; Nordenstam, 1977;

Robinson et al., 1997). The genus for which the tribe is named, L., is among the largest and most diverse genera worldwide, with 1000 ̶ 1250 species

(Nordenstam, 1978, 2007; Jeffrey 1986, 1992; Bremer, 1994; Coleman et al., 2003;

Pelser et al., 2010). Senecio s.l. has one of its principal centers of diversity in the montane regions of Mexico and Central America (Barkley, 1985a, 1985b, 1988).

The of the tribe Senecioneae dates from when Cassini first proposed a tribal classification of “Synathérées” in four volumes (Cassini, 1812, 1813a-c, 1814,

1816, 1817). The Senecioneae initially included Cacalia, Cineraria, Othonna, and

Senecio, but by the time of his final paper, Cassini (1829) recognized 35 genera in the tribe. Bentham (1873) was the first to recognize four subtribes in the Senecioneae:

Eusenecioneae, Othonneae, Tussilagineae, and Liabeae. Hoffman (1892) recognized three subtribes: Senecioninae, Othonninae, and Liabininae; he considered the 1 Tussilagininae to be a part of the Senecioninae. Liabeae was later recognized as a separate tribe (Robinson & Brettell, (1973a) and later Nordenstam (1977) recognized only two subtribes within the Senecioneae: Senecioninae, which included genera previously placed in Othonninae and Tussilagininae, and Blennospermatinae. At present, the following four subtribes are recognized: Senecioninae, Tussilaginiae (including former Blennospermatinae), Othonninae, and the monotypic Abrotanellinae (Nordenstam et al., 2009).

Four monophyletic ‘subclades’ are recognized in the Tussilaginieae, however the relationships among these subclades are unresolved (Pelser et al., 2007). The genera

Endocellion, Homogyne, Petasites, and form the first of the well-supported subclades. This subclade is restricted to temperate/boreal Eurasia with the exception of the morphologically variable Petasites frigidus that occurs in . A second subclade has a predominantly New World distribution and includes several genera including , , , Rainiera, and Tetradymia. These five genera are sister to the three genera formerly placed in the Blennospermatinae with both

Old and New World distributions. A third subclade consists of the Ligularia-

Cremanthodium-Parasenecio complex (Liu et al., 2006), that includes 11 genera, most of which occur in Asia. A fourth subclade includes two sister groups, well-supported by ITS and plastid data that are restricted to the New World (Pelser et al., 2007, 2010). One of the sister clades, the gynoxoid group (sensu Pelser et al., 2007, 2010), is restricted to

South America and includes small trees in the genera Aequatorium, Gynoxys,

Nordenstamia, and Paragynoxys. The other sister group includes a clade with 13 genera 2 that occurs in North and Central America. These genera have been referred to as the

Mexican tussilaginioid group, as ten of the 13 genera occur in Mexico (Barkley et al.,

1996).

The Mexican tussilaginioid group encompasses 140 species of herbs, suffruticose herbs, shrubs, small trees, and epiphytes that are distributed in the USA, Mexico, and

Central America. The species share the following characters: stigmatic surfaces united across at least the upper third of the inner face of the style branches; cylindrical anther collars; chromosome numbers n = ca. 30, principal phyllaries often with midrib thickened at base. Most of the species in the study group are concentrated in montane pine-oak or cloud forest ecosystems (Barkley 1985a, 1985b; Barkley et al., 1996). Endemism is relatively high in this group with ca. 60% percent of the species restricted to two or fewer states in Mexico, a single Central American country, or Guatemala plus the adjacent

Mexican state of . Appendix 1.1 provides a current overview of the genera and the species of the Mexican tussilaginioid group, recognizing a new genus and three species transferred to another genus, Psacalium.

Nordenstam (1977) informally recognized two complexes with their distributions concentrated in Mexico called the senecioid and cacalioid complexes based on previous studies of Pippen (1968) and Robinson & Brettell (1973e, 1974). The Mexican tussilaginioid group includes several segregate genera that were once included in Senecio s.l., based on their yellow, radiate heads (i.e., Barkleyanthus, Nelsonianthus, Pippenalia,

Pittocaulon, Psacaliopsis, Robinsonecio, Roldana, Telanthophora, and Villasenoria).

The other genera in the clade, referred to as the ‘cacalioid’ group, have discoid heads that 3 are not yellow were historically placed in the genus Cacalia s.l. (i.e., Arnoglossum,

Digitacalia, and Psacalium). Delimitations between Senecio and Cacalia have long been obscure (Wetter, 1983). Traditionally, Cacalia had been segregated from Senecio based on two floral characters, discoid heads and white to creamy corollas (Barkley &

Cronquist, 1974). When the generic name Cacalia was rejected as illegitimate (Waganitz,

1995; Brummitt, 1998), the term ‘cacalioid’ was no longer used and the ‘tussilaginioid’ complex was proposed to refer to the 13 genera focused on in the present study (Barkley,

1999; Nordenstam, 2007). Barkley et al. (1996) was the first to treat the Mexican tussilaginioid group from Mexico and Central America, although Arnoglossum and

Yermo, due to their geographic distribution in the USA, were not included in his treatment.

Several researchers have used morphological characters to treat the group taxonomically as a whole (Barkley 1985a, 1985b; Jeffrey, 1992; Barkley et al., 1996;

Nordenstam, 2007), or in part (i.e., Pippen 1964, 1968; Robinson & Brettell, 1973b,

1973c, 1973d, 1974; Robinson, 1974; Clark, 1996; Turner, 2005; Funston, 2008). Species delimitations within the Mexican tussilaginioid group have been largely based on morphological and anatomical characters. Kadereit & Jeffrey (1996) conducted an RFLP analysis using chloroplast DNA of New World Senecioneae, including Pittocaulon,

Roldana, and Telanthophora. Their results were the first from a phylogenetic analysis to demonstrate that these three species of the Mexican tussilaginioid genera belonged together with other New World Tussilagininae. Bain & Golden (2000) included

Barkleyanthus, Robinsonecio, Pittocaulon, and Psacalium in an analysis of the internal 4 transcribed spacer region (ITS) of nrDNA and demonstrated moderate support for these genera forming a monophyletic group. Pelser et al. (2007, 2010) inferred the phylogenetic relationships using nrDNA data from 13 accessions that included eight genera and 13 species of Mexican tussilaginioid group. Their data indicated strong support for the monophyly of this group and also demonstrated strong support for a South

American ‘gynoxoid’ clade (small trees from South America) as a sister group. Major clades in the Senecioneae were also supported by plastid sequence data, although it was noted that many taxa and clades were incongruent when comparing nrDNA and plastid data sets, including infrageneric relationships among the Mexican tussilaginioid group

(Pelser et al., 2010). Pelser et al. (2010) suggested a recent origin of this group (ca. 9.5–

12 mya), which corresponds to a period when the Asteraceae showed an explosion in diversity in Mexico and Central America indicated by pollen evidence (Raven &

Axelrod, 1974).

While most species in the Mexican tussilaginioid group have the chromosome number of n = 30, (i.e., Pippen, 1968; Keil & Stuessy, 1977; Strother, 1983; Strother &

Panero, 2001). Arnoglossum exhibits evidence of aneuploidy, (n = 30, 29, 28, 27, 26, 25;

Koyama, 1968, Robinson et al., 1997, Anderson, 1998). The ‘gynoxoid’ sister group has a haploid chromosome count of n = 40 (Turner et al., 1967; Powell & Cuatrecasas, 1970;

Hunziker et al., 1989; Robinson et al., 1997).

Most of the species within the Mexican tussilaginioid group occur at elevations above 1500 meters in cloud forests or pine-oak forests of Mexico and Guatemala, but two species of Roldana occur as far south as Costa Rica, and one of those species occurs also 5 in northern Panama. One species of Telanthophora occurs south to northern Panama.

Two genera, Arnoglossum and Yermo, occur in central and southern USA. The most diverse genera, Roldana (55 species), and Psacalium (49 species), have all but four species occurring in Mexico.

New World Asteraceae have been a focus of several recent studies related to endemism and conservation. On the Caribbean Islands where habitat loss is an ongoing concern for the preservation of biodiversity, 46 species in 15 genera of Senecioneae were used in conjunction with other endemic Asteraceae to promote conservation efforts in the region (Francisco-Ortega et al., 2008). In Chile, a rich and widespread Asteraceae flora with many genera having narrow geographic ranges was used to promote conservation, especially in the montane regions of that country (Moreira-Muñoz & Muñoz-Schick,

2007). In Mexico, several studies have utilized the Asteraceae, including members of the

Mexican tussilaginioid group, due to its abundance of species and relatively high levels of endemism in montane Mexico, to pin point regions of high biodiversity (Villaseñor et al., 2006; Villaseñor et al., 2007) and to promote conservation (Villaseñor et al., 1998;

González-Zamora et al., 2007). The Mexican tussilaginioid group is a model group of endemic plant species that could be useful for conservation biology studies.

Understanding the evolutionary relationships of this group is an initial step towards using these taxa to promote conservation efforts of these endemic taxa that predominantly occur in threatened ecosystems.

Taxonomic treatments and extensive field collections of the Mexican tussilaginioid group have provided a wealth of baseline data indicative of a 6 morphologically variable montane clade with a broad geographic distribution. Extensive fieldwork throughout Mexico and Central America would be required to sample properly the morphological diversity and broad geographic ranges within and between species in the group, especially in Psacalium and Roldana. Several species have a small number of representative vouchered material deposited in herbaria and many of these are named from only a single population (i.e., Nelsoninathus tapianus, Pittocaulon hintonii,

Psacalium calvum, P. cervinum, P. guatemalense, P. hintoniorum, P. matudae, P. mollifolium, P. nanum, P. pinetroum, P. putlanum, P. quercifolium, P. sharpie, Roldana glinophylla, R. hintonii, R. langlassei, R. mazatecana, R. mezquitlana, R. mixtecana, R. neogibsonii, R. sundbergii, R. tepopana. R. tlacotepecana, R. tonii. R. uxordecora,

Telanthophora bartlettii, T. jaliscana, T. sublaciniata, Yermo xanthocephalus).

Furthermore, population studies and hybridization experiments in a greenhouse will undoubtedly contribute to the knowledge of the relationships of these species.

This study focuses on the Mexican tussilaginioid group and their systematic relationships using nrDNA. A phylogeny of the group with high taxon sampling has not been previously constructed, and nuclear markers are an effective means of addressing generic and species delimitations among Asteraceae taxa. Although Pelser et al. (2007,

2010) utilized five plastid loci in a phylogenetic analysis, resolution is poor and there is little or no statistical support for the relationships. Two of the genera, Pippenalia and

Psacaliopsis, traditionally consisting of five species with ambiguous relationships, require a taxonomic assessment. The objectives of this dissertation research presented in the subsequent chapters are to: 7 Ch. 2) reconstruct a phylogeny of the Mexican tussilaginioid group using the nuclear

ribosomal external transcribed sequence (ETS) and the internal transcribed

sequence (ITS), test the monophyly of the Mexican tussilaginioid group, and test

the monophyly of Pittocaulon, Psacaliopsis, Roldana, and Telanthophora,

Ch. 3) complete a taxonomic reassessment of the genera Pippenalia and Psacaliopsis,

Ch. 4) sequence and assemble the chloroplast genomes of four taxa within the Mexican

tussilaginioid group in order to identify divergent regions of the chloroplast that

can be used for future phylogenetic studies and to compare the results from de

novo and reference-based assemblies.

Chapter 2 presents the phylogeny of the Mexican tussilaginioid group based on

ITS and ETS nrDNA sequence data using the ‘gynoxoid’ clade of South America as the outgroup. These two regions have been shown to be useful for fine-scale phylogenetic studies among the Asteraceae (e.g., Baldwin, 1992; Baldwin & Markos, 1998; Lee et al.,

2003; Roberts & Urbatsch, 2003; Sanz et al., 2008). The objectives of this component of the study were specifically to test the monophyly of the Mexican tussilaginioid group and the genera Pittocaulon, Psacaliopsis, Roldana, and Telanthophora. Moreover, this study was designed to sample most of the genera in the clade in order to establish generic delimitations among the Mexican tussilaginioid group.

Chapter 3 consists of a taxonomic reassessment of Pippenalia and Psacaliopsis, two morphologically diverse genera that consist of acaulescent herbs with vegetative and floral characters intermediate between the historical ‘senecioid’ and ‘cacalioid’

8 distinction. Prior to this study, Psacaliopsis consisted of five species. The purpose of this component of the study was to elucidate the relationships of Pippenalia and Psacaliopsis species using morphological characters, and to compare this analysis with the molecular phylogenetic relationships discussed in Chapter 2. Ninety-eight specimens were analyzed for this study and several morphological characaters and nrDNA data were compared to resolve the relationships of these two genera.

Chapter 4 compares the chloroplast genome sequences of four species;

Arnoglossuum atriplicifolium (L.) H. Rob., Roldana aschenborniana (S. Schauer) H.

Rob. & Brettell, R. barba-johannis (DC.) H. Rob. & Brettell, and Telanthophora grandifolia (Less.) H. Rob. & Brettell, in order to identify divergent regions of the plastid genomes that can be utilized in future phylogenetic studies within the Mexican tussilaginioid group. This chapter also compares the results between reference-based and de novo genome assemblies sequenced from the SOLiD next-generation sequencing platform.

9 Chapter 2: Molecular phylogenetics of the endemic montane

Mexican tussilaginioid group (Asteraceae: Senecioneae)

based on nrDNA ITS and ETS sequence data

INTRODUCTION

The Mexican tussilaginioid group (Asteraceae: Senecioneae) consists of 13 genera and 140 species distributed from the to Panama, with centers of diversity in Central Mexico and southern Mexico-Guatemala (Barkley 1985a, 1985b;

Barkley et al., 1996). This diverse group consists of small trees, shrubs, herbs, and epiphytes, most of which occur at elevations above 1500 meters in cloud forests or pine- oak forests. Endemism is relatively high in this group with ca. 60% percent of the species occurring in two or fewer states in Mexico, only in Guatemala, or in Guatemala and the adjacent Mexican state of Chiapas. These species and other endemic Asteraceae that occupy threatened montane ecosystems have been used to promote conservation efforts in Mexico (Villaseñor et al., 1998).

The Senecioneae is the largest tribe in the Asteraceae with respect to species numbers, with ca. 3000 species in 150 genera (Nordenstam, 2003, 2007). Species that comprise the Mexican tussilaginioid group are a part of subtribe Tussilaginieae, and these species are considered part of the ‘tussilaginoid’ group (Nordenstam, 2007). The species in the Mexican tussilaginioid group either have radiate capitula (‘senecionioid’ genera) or discoid capitula (‘cacalioid’ genera). Barkley et al. (1996) referred to the group as the

10 tussilaginioid genera of Mexico and Central America. Arnoglossum and Yermo, due to their occurrence only in the USA, were excluded from Barkley’s treatment.

Several researchers have treated the group taxonomically as a whole (Barkley

1985a, 1985b; Jeffrey, 1992; Barkley et al., 1996), or in part (i.e., Rydberg, 1924a,

1924b; Pippen 1964, 1968; Robinson & Brettell, 1973b, 1973d, 1974; Robinson, 1974;

Barkley & Janovec; 1996; Clark, 1996; Funston 2008). Generic and species delimitations within the Mexican tussilaginioid group have not been investigated using DNA sequence data with a thorough level of taxon sampling. Bain & Golden (2000) included four

Mexican tussilaginioid species Barkleyanthus salicifolius, Robinsonecio gerberifolius,

Pittocaulon praecox, and Psacalium peltatum in an analysis using the ITS region, and demonstrated moderate support for these genera, forming a monophyletic group. Pelser et al. (2007) inferred the phylogenetic relationships using ITS for 13 species in eight genera and their study was the first to demonstrate strong support for the monophyly of the

Mexican tussilaginioid group and strong support for the South American Tussilagininae

‘gynoxoid’ clade as the sister group. Pelser et al. (2010) later utilized ETS, ITS, and five chloroplast loci of 13 accessions that included all 13 genera and 13 species in the

Mexican tussilaginioid group that also confirmed the monophyly of the group and its sister relationship to the gynoxoid clade. Although major clades in the Senecioneae were also supported by plastid sequence data, it was noted that many taxa and clades were incongruent when comparing the nrDNA and plastid data sets (Pelser et al., 2010).

Sequence data were used from the internal transcribed spacer ITS and external transcribed spacer (ETS) nrDNA regions for phylogenetic analyses of the Mexican 11 tussilaginioid group. These two regions have been shown to be useful for fine-scale phylogenetic studies among the Asteraceae (i.e., Baldwin, 1992; Baldwin & Markos,

1998; Lee et al., 2003; Roberts & Urbatsch, 2003; Sanz et al., 2008). ETS and ITS sequence data combined have been demonstrated to yield better resolved evolutionary relationships with higher support than either alone (Baldwin & Markos, 1998; Clevenger

& Panero, 2000; Markos & Baldwin, 2001; Roberts, 2002; Roberts & Urbatsch, 2003).

The goals of this study were (1) to produce a phylogeny utilizing nrDNA sequence data of this diverse clade of montane endemic plants; (2) to test the monophyly of the Mexican tussilaginioid group, and (3) to test the monophyly of Pittocaulon,

Psacaliopsis, Roldana, and Telanthophora.

MATERIALS AND METHODS

Taxon sampling and DNA isolation. ⎯ Taxa examined are listed in Appendix

2.1 with voucher information and accession numbers for the DNA sequences submitted to Genbank. Genomic DNA was isolated using the DNeasy plant mini kit (Qiagen) from herbarium vouchers, silica-dried material from the field, and living material grown in a greenhouse at The University of Texas at Austin Brackenridge Field Laboratory. The ITS data set included 144 accessions representing 72 species, including 135 ingroup accessions (64 species in 12 genera) and nine outgroup accessions representing eight species. Twenty-four sequences were from previously published studies (Pelser et al.,

2007, 2011). The ETS data set included 119 accessions representing 69 species, including

114 ingroup accessions (64 species in 12 genera) and five outgroup accessions. Eighteen 12 sequences were from previously published studies (Pelser et al., 2007, 2011). For the combined ITS/ETS data set, 100 accessions representing 67 species were used, including

95 ingroup accessions (62 species in 12 genera) and five outgroup accessions. Fourteen sequences were from previously published studies (Bayer et al., 2002; Pelser et al., 2002,

2007, 2011). Not all of the accessions from the initial independent ITS and ETS analyses were used in the combined analyses because either not all accessions were succsessfully amplified for ITS or ETS, or only ITS or ETS sequences were available from Genbank.

Table 1 summarizes the number of species in each genus used in the combined analyses.

The sampled ingroup was selected to include species from all the representative genera, and to cover the geographic distribution and morphological variation among the genera.

Nelsonianthus was the only genus excluded in the combined data set and the ITS data set because suitable leaf material was unavailable for DNA extraction and only the ETS sequence was available from Genbank. For the 100 accessions used in the combined analysis, separate ITS and ETS analyses were also conducted.

Aequatorium and Gynoxys from South America were used as outgroups among the Tussilagininae as this ‘gynoxoid’ group is sister to the Mexican tussilaginioid genera

(Pelser et al., 2007), as well as Rugelia nudicaulis, a more distantly related Tussilagininae genus from eastern USA, and Senecio vulgaris, a non-tussilaginioid member of in the

Senecioninae was also included as an outgroup.

DNA amplification, sequencing and alignment. ⎯ Polymerase chain reactions

(PCR) were carried out for the nuclear ribosomal ETS region using 111 samples and the nuclear ribosomal ITS region for 120 samples. For the ETS region, PCR amplifications 13 were performed using the primer pairs ETS2 (Bayer et al., 2002) and 18S-ETS (Markos

& Baldwin, 1998). The ITS region was amplified using the primers ITS1A (Sharpe et al.,

2000) and ITS4 (White et al., 1990). Amplifications of ETS and ITS were conducted in

25 µL volumes containing 14.6 µL of ddH2O, 7.5 µL of FailSafe buffer – PreMix D

(EPICENTRE® Biotechnologies, Madison, Wisconsin), 0.25 µL of a 20 µM solution of each forward and reverse primer, 0.4 µL of Taq polymerase, and 2 µL of unquantified

DNA template. Reaction conditions were: one round of amplification consisting of denaturation at 94°C for 1 min 30 sec, annealing at 53°C for 30 sec, and extension at

72°C for 1 min 30 sec, followed by 29 cycles of 94°C for 30 sec, 53°C for 30 sec, and

72°C for 1 min 30 sec, with a final extension step of 72°C for 30 min. Amplifications were visualized on 1% agarose gels with ethidium bromide and a size standard to estimate fragment sizes and DNA concentrations.

Both the ETS and ITS regions for all taxa were cloned and sequenced in the analysis, and the ETS and ITS PCR products were cloned using the TOPO TA Cloning

Kit (Invitrogen, Carlsbad, , USA). Colonies were amplified using PCR in 25

µL volumes consisting of 16.1 µL of ddH2O, 8.0 µL of FailSafe buffer PreMix E, 0.2 µL of a 20 µM solution of each pUC-18 plasmid primer, 0.5 µL of Taq polymerase, and one colony. Reaction conditions were: one hot-start cycle at 95°C for 3 min 30 sec, followed by 35 cycles of denaturation at 95°C for 45 sec, annealing at 58°C for 45 sec, and extension at 72°C for 50 sec, with a final extension step of 72°C for 3 min.

Amplifications were visualized as above, and all PCR products were cleaned using Exo-

SAP by adding 2.25 µL of ddH2O, 0.25 µL of Exonuclease I (New England Biolabs, Inc., 14 Ipswich, Massachusetts), and 0.50 µL of Shrimp Alkaline Phosphatase (Promega Corp.,

Madison, Wisconsin) to each product. Sequencing was carried out at the Institute for

Cellular and Molecular Biology Core Facilities at The University of Texas at Austin.

Between 2 ̶ 5 clones per accession were sequenced for all species sampled for this study. For ITS, any clones from the analysis that differed in more than one nucleotide in the 5.8S region were not included to reduce the likelihood of including paralogous copies. Sequence data from Genbank were also used for the combined analyses of both data sets and the independent ETS and ITS analyses using additional taxa (Bayer et al.,

2002; Pelser et al. 2002, 2007, 2010). For the combined data sets, in instances where the clones were mixed, we selected two of the clones for the accessions sequenced for this analysis. Sequences from Genbank were direct sequenced from the nrDNA amplicons and not cloned.

Phylogenetic analyses. ⎯ Sequences were edited in Geneious Pro 4.0.4

(Drummund et al., 2006) and aligned using ClustalX v.1.8 (Thompson et al., 1997), and then manually adjusted in Mesquite (Madison & Madison, 2011). Five different data sets were analyzed. Data set 1 comprised 144 sequences and 72 species for ITS, and data set 2 comprised 119 sequences and 69 species of ETS. Data set 3 consisted of a reduced sampling of 100 sequences and 67 species for ITS and data set 4 included 100 sequences and 67 species for ETS. Data set 5 included a combined ITS/ETS of data sets 3 and 4.

Two approaches were utilized to analyze the data sets: Bayesian inference (BI) and maximum likelihood (ML). Both phylogenetic methods were used to analyze the separate and combined data sets. 15 Bayesian inference analyses were performed using Mr.Bayes v.3.1.2 (Ronquist &

Huelsenbeck, 2003). Substitution models were selected using the Akaike information criterion (AIC) in Modeltest v.3.06 (Posada & Crandall, 1998; http://darwin.uvigo.es/software/modeltest.html). The GTR+G model was determined to be the best model fitting the separate and combined ITS and ETS data sets. For the BI analyses, model parameters were estimated directly during runs, using four simultaneous chains and four million cycles each, sampling one every 1000 generations. Analyses were diagnosed for convergence using Tracer v1.4 (Rambaut & Drummond, 2007). For the ML analyses we used the RAxML, and the ML searches utilized the GTR+G model

(Randomized Axelerated Maximum Likelihood), this model being the only one implemented in RAxML (Stamatakis, 2006; Stamatakis et al., 2008). Statistical support was calculated by maximum likelihood in RAxML with 100 bootstrap replicates.

Congruence between ETS and ITS. ⎯ The incongruence length difference test

(ILD; Farris et al., 1994) was preformed in PAUP ver. 4.0b10 (Swofford, 2002) to test for the congruence between the ETS and ITS sequence trees.

Alternative Hypothesis Testing. ⎯ Bayesian topological hypothesis testing in

Mr.Bayes v.3.1.2 (Ronquist & Huelsenbeck, 2003) was utilized to test the monophyly of the three genera that were not strongly supported as monophyletic (Pittocaulon,

Psacaliopsis, and Roldana).

16 RESULTS

Phylogenetic analysis of ITS and ETS data. ⎯ The aligned ITS matrix for data set 1 was 827 bp in length and of 827 total characters in the aligned matrix, 304 (37%) were parsimony-informative, 418 (50%) were constant, and 105 (13%) were parsimony- uninformative. The tree resulting from the ITS Bayesian analysis with posterior probabilities and ML bootstrap values is presented in Fig. 2.1. Thirteen strongly supported clades in the Mexican tussilaginioid group were recovered. The aligned ETS matrix for data set 2 was 469 bp in length and of 469 total characters, 179 (38%) were parsimony-informative, 205 (44%) were constant, and 85 (18%) were parsimony- uninformative. The tree resulting from the ETS Bayesian analysis with posterior probabilities and ML bootstrap values is presented in Fig. 2.2 and shows 11 of the strongly supported clades in the Mexican tussilaginioid group. Both data sets support the monophyly of the Mexican tussilaginioid group and its sister relationship to the South

American gynoxoid group, however the ITS tree is more resolved with stronger support for many of the branches.

Separate analyses for ITS and ETS were also conducted for the 100 accessions where sequence data for both nrDNA regions were available. The aligned ITS matrix for data set 3 was 827 bp, of which 285 (34%) were parsimony-informative, 442 (53%) were constant, and 100 (13%) were parsimony-uninformative. The tree resulting from the ITS

Bayesian analysis of the 100 accessions with posterior probabilities and ML bootstrap values is presented in Fig. 2.3. Thirteen strongly supported clades in the Mexican tussilaginioid group were again recovered. The final aligned 3’ ETS matrix for data set 4 17 was 469 bp in length, of which 178 (38%) were parsimony-informative, 209 (45%) were constant, and 82 (17%) were parsimony-uninformative. The tree resulting from the ETS

Bayesian analysis with posterior probabilities and ML bootstrap values presented in Fig.

2.4 yielded 10 strongly supported clades in the Mexican tussilaginioid group.

Congruence test. ⎯ The incongruence length difference test (ILD) for the ITS and ETS data sets resulted in p = 0.19, and therefore both nrDNA sequence regions are not significantly incongruent.

Combined data analysis. ⎯ Table 2.2 summarizes sequence characteristics for the combined ITS/ETS data set and the independent ITS and ETS data sets. The phylogenetic tree resulting from the Bayesian Inference analyses for the combined

ITS/ETS data set with posterior probabilities and ML bootstrap values is presented in Fig.

2.5. The aligned, combined data set contains 1296 bp, of which 501 (38%) were parsimony-informative, 261 (21%) were constant, and 534 (41%) were parsimony- uninformative. Thirteen clades (1 ̶ 13) were identified in the tree produced from the BI analysis. Support for clades improved in the combined data set analysis (compare Figs.

2.1–2.4 with Fig. 2.5).

All analyses support the monophyly of the Mexican tussilaginioid group and the combined analysis and the ITS analyses strongly support 13 monophlyletic groups. The following description of the 13 clades focuses on the tree resulting from the combined analysis (Fig. 2.5).

Clade 1 includes Psacaliopsis purpusii and Robinsonecio gerberifolius (Bootstrap

(BS) = 98%; Posterior Probability (PP) = 1.0). A purported single taxon, Psacaliopsis 18 pinetorum/paneroi, is included in clade 2 (BS = 100%; PP = 1.0). Clade 3 includes the genus Digitacalia (BS = 100%; PP = 1.0). Clade 4 comprises the morphologically diverse genera Arnoglossum, Barkleynanthus, and Yermo (BS = 100%; PP = 1.0). Clade 5 includes Psacalium, Pippenalia, and the two discoid species in Psacaliopsis (BS = 99%;

PP = 1.0). Clade 6 comprises the discoid ‘Pericalia’ group with the addition of the radiate Roldana heracleifolia strongly supported as sister to the rest of the group (BS =

99%; PP = 1.0). Clade 7 includes two species of Pittocaulon with pubescent leaves and

Roldana eriophylla, which shares pubescent leaves, a chambered-pith, and the deciduous growth habit (BS = 99%; PP = 1.0). Pittocaulon praecox, P. vellatum, and Villasenoria comprise clade 8 (BS = 98%; PP = 1.0). Clade 9 is the Roldana petasitis complex

(Bootstrap = 65%; Posterior Probability = 1.0), clade 10 comprises the Roldana sundbergii complex (BS = 99%; PP = 1.0), and clade 11 comprises the Roldana platanifolia complex (BS = 100%; PP = 1.0). Clade 12 includes two species, Roldana mixtecana and Roldana reticulata (BS = 100%; PP = 1.0). Clade 13 includes the genus

Telanthophora (BS = 89%; PP = 1.0). There are 10 unresolved species of Roldana that are not included in the above 13 clades.

Hypothesis Testing. ⎯ Results of the Bayes topological hypothesis testing are summarized in Table 3. A log difference in the range of 3–5 is considered strong evidence in favor of the best tree estimate model (Fig. 2.3), whereas a log difference above 5 is considered very strong evidence for the alternative, or constraint model (Kass

& Raftery, 1995; Xie et al., 2011). The hypothesis of a monophyletic Roldana s.s. is

19 rejected (log difference = 17.46). The same is true for a monophyletic Pittocaulon s.s.

(log difference = 13.41) and a monophyletic Psacaliopsis (log difference = 108.37).

DISCUSSION

The separate and combined nrDNA phylogenies of the Mexican tussilaginioid group produced a more resolved phylogenetic tree than recovered to date (e.g., Pelser et al., 2007, 2010), with the results from the combined analysis demonstrating the highest support (Fig. 2.5). Thirteen major clades are strongly supported in the combined analysis

(Fig. 2.5) and the two separate ITS analyses (Fig. 2.1 and Fig. 2.3), although the ML bootstrap for Clade 9 is ≤ 65% in the combined analysis. These clades are not formally named in this study as more sequence data, including plastid markers, would be required to confirm the monophyly and relationships of these groups. The combined phylogeny strongly supports the monophyly of the Mexican tussilaginioid group and its sister relationship to the ‘gynoxoid’ group from South America. Descriptions of the thirteen supported clades as recovered in the combined analysis (Fig. 2.5) with information regarding morphological characters, geographical distribution, taxonomic implications, and purported number of species are provided below. Ten species of Roldana were unresolved in the analyses. Implications for future work are also suggested when relevant. Exhaustive studies with the inclusion of more taxa in several clades and the incorporation of a multigene plastid phylogeny are required to improve the understanding of this diverse montane plant group.

20 The most profound result of this study is the identification of the highly paraphyletic genus Roldana, previously thought to be a distinct, morphologically- circumscribed genus (Robinson & Brettell, 1974; Turner, 2005; Funston, 2008).

Psacaliopsis purpusii, Robinsonecio (Clade 1). ⎯ This clade consists of subalpine and alpine herbs with radiate yellow capitula, rosulate leaves with denticulate margins, and woody rhizomes. Clade 1 is strongly supported as sister to the rest of the

Mexican tussilaginioid group. Psacaliopsis purpusii has petiolate, peltate leaves that are orbicular and secondarily lobed. As this clade contains the type species of the genus, P. purpusii, it constitutes the genus Psacaliopsis with the impending transfer of two species to Psacalium and the erection of Funstonia gen. nov. (see discussions on Clades 2 and 5 below and Chapter 3). Robinsonecio has subsessile leaves that are ovate to spathulate.

Barkley & Janovec (1996) originally described two species of Robinsonecio and proposed its relationship to other tussilaginioid genera. Pruski (2012) used microcharacters to confirm their generic circumscription and placement among the tussilaginioid Senecioneae that comprise the Mexican tussilaginioid genera. Pruski

(2012) only examined material from three herbaria (F, MO, NY), which limited his capacity to make statements regarding distributions of the species. Pruski also failed to mention a specimen of Robinsonecio gerberifolius collected from Distrito Federal,

Lyonnet 395, which is deposited at NY where he examined material and also available to view in an online database. Any study of the genus must at least also include a thorough survey of material at BIGU, CAS, MEXU, TEX, and US.

21 Psacalioipsis purpusii occurs in pine-oak forests at 2200–2600 meters and has only been reported from and Puebla. Robinsonecio porphyresthes was not included in the study, but this species is morphologically and anatomically similar to R. gerberifolius (Barkley & Janovec, 1996; Pruski, 2012). Both species of Robinsonecio occur in alpine habitats and can occur above 4000 meters; R. gerberifolius occurs in central Mexico (Distrito Federal, Mexico, Puebla, Tlaxcala, and ), and in

Huehuetenango, Guatemala. R. porphyresthes is endemic to alpine habitats in

Tamaulipas, Mexico.

Psacaliopsis pinetorum/paneroi (Clade 2). ⎯ These two species are conspecific and will be treated as such in a taxonomical reassessment of Psacaliopsis H. Rob. &

Brettell (Chapter 3). Phenotypic plasticity is evident among herbarium specimens representative of Psacaliopsis pinetorum and P. paneroi. The centrally-peltate leaves have lobes that range from rounded to acute and undulate to shallowly lobed (up to ¼ way to center of the blade). Similarly, depending on the age of the plants collected and the amount of the material collected for each specimen, the number of capitula ranges from 5 to over 80 and levels of pubescence on the abaxial leaf surfaces is highly variable.

Morphologically, P. pinetorum is similar to P. purpusii from Clade 1 but the leaves of P. pinetorum are not secondarily lobed as in P. purpusii, the leaves are shallowly lobed with sinus depths to ¼ towards the center of the blade vs. sinus depths ½ to ¾ towards the center of the blade in P. purpusii, the capitula number 5–80+ in P. pinetorum vs. 5–18 in

P. purpusii, the phyllaries are in two subequal series vs. one series in P. purpusii, and the corolla tube 5–6 mm long in P. pinetorum vs. ca. 3.5 mm long in P. purpusii. 22 Psacaliopsis paneroi var. juxtlahuacensis is a variety based on a single population from

Oaxaca, Mexico, and the characters that define this variety are found in many herbarium specimens that have been determined as the alternate variety, P. paneroi var. paneroi.

When describing Psacaliopsis (Senecio) paneroi, Turner (1989) did not look at specimens of Senecio pinetorum. It is obvious that these two species are the same entity and S. pinetorum is the older name. Morphological differences and phylogenetic placement support this taxon as a new genus, Funstonia gen. nov. with two varieties.

Psacaliopsis paneroi var. paneroi became a taxonomic synonym of Psacaliopsis pinetorum var. pinetorum. The other variety P. paneroi var. juxtlahuacensis will become

Funstonia pinetorum var. juxtlahuacensis stat. nov. (see Chapter 3). This species occurs in pine-oak forests of central to southern Mexico, and it is also reported from and . Although it has not been collected in Guatemala, it is expected to reside there as well.

Digitacalia (Clade 3). ⎯ Pippen (1968) first proposed Digitacalia as a small genus of perennial herbs with leafy stems, discoid capitula with the corollas deeply cleft, and exothecial thickenings on the transverse walls. Digitacalia heteroidea was transferred to Roldana based on exothecial thickenings on the vertical walls in R. heteroidea compared to exothecial thickenings on the the transverse walls in the other species of Digitacalia, and larger capitula in a more lax capitulescence in R. heteroidea opposed to smaller capitula and a dense capitulescence in the species of Digitacalia

(Robinson & Brettell, 1974). Presently, R. heteroidea remains in Roldana (Turner, 2005;

Funston, 2008). In a recent treatment (Turner, 1990a), Digitacalia included five species 23 that occur in montane central and southern Mexico, but only two of the five species were included in the present study. Species delimitations are vague and based on variable morphological characters and few herbarium specimens (i.e., Turner, 1990a).The combined analysis indicates that the genus is a well-supported clade.

Arnoglossum, Barkleyanthus, Yermo (Clade 4). ⎯ This clade contains an assembly of three morphologically diverse genera, two of which are monotypic. Pelser et al. (2007, 2010) first demonstrated through molecular evidence that these three genera form a well-supported clade, and that Barkleyanthus diverged from the other two genera

3.7–4.8 million years ago (Pelser et al., 2010). Arnoglossum is strongly supported as the sister taxa to Yermo, and these two genera are strongly supported as a sister group to

Barkleyanthus. The entire clade is well-supported as the sister group to Clade 5.

Arnoglossum consists of nine species of herbs with discoid capitula that occur in the central and southeastern USA. This genus was originally treated as Cacalia in a North

American treatment (Pippen, 1978). Barkleyanthus salicifolius is a or small tree with radiate capitula that is widespread from southern to Honduras at 1000 ̶ 3000 meters. Yermo xanthocephalus is a small, suffruticose herb with radiate capitula endemic to Fremont, County, at 2000 meters. Arnoglossum and Yermo are the only two genera in the Mexican tussilaginioid group that are restricted to the USA.

Pippenalia, Psacaliopsis macdonaldii/pudica, Psacalium (Clade 5). ⎯

Pippenalia delphiniifolia was originally treated as a species in Odontotrichum (Rydberg,

1924b) based on its non-peltate, pinnatisected leaf blades. This species is unique in Clade

5 as it has radiate capitula and it is the only species with radiate capitula in the Mexican 24 tussilaginioid group that lacks a pappus. Psacalium was originally revised by Rydberg

(1924a), and subsequently revised by Pippen (1968) and more recently by Robinson

(1973d). Rydberg (1924a, 1924b) recognized the peltate leaved Psacalium as distinct from the non-peltate or subpeltate leaved Odonotrichum. In this and previous studies,

Odontotrichum is included within Psacalium (Pippen, 1968; Robinson, 1973d).

Psacaliopsis macdonaldii and P. pudica both share vegetative characters (basal peltate leaves with rounded lobes and hirsute pubescence of the leaf bases) and floral characters

(e.g., discoid capitula) with Psacalium, and therefore will be included with Psacalium in a future treatment. Pippenalia delphinifolia will also be transferred to Psacalium based on morphological affinities with those species in Psacalium with non-pelate or subpeltate leaves, including Psacalium cirsiifolium and P. palmeri (see Chapter 3). The nrDNA data also support Pippenalia as being closely related to Psacalium cirsiifolium and P. palmeri, and both of these species were previously placed in Odontotrichum along with

Pippenalia delphinifolia (Rydberg, 1924b), and P. cirsiifolium was the type species for the former Odontotrichum. With the impending transfer of the three aforementioned species to Psacalium, the genus now can be recognized as a monophyletic group.

“Psacalium (Pippenalia) delphinifolia” occurs at elevations of 2400 ̶ 3000 meters in pine-oak forests. “Psacalium (Psacaliopsis) macdonaldii is endemic to Oaxaca,

Mexico, and “Psacalium (Psacaliopsis) pudica” is endemic to the Sierra Cuchumatanes of Guatemala and has only been reported from the department of Huehuetenango and

Quiche. Psacalium, in the broadest sense with the inclusion of Odontotrichum (Robinson

& Brettell, 1973d) occurs in montane forests and meadows from southern Arizona to 25 Guatemala. Only one species, P. decompositum, occurs in the USA. Two species, P. guatemalense and P. pinetorum, are endemic to Guatemala.

Roldana ‘Pericalia’ (Clade 6). ⎯ In 1827, Cassini first proposed the genus

Pericalia, although it was not validly published until 1924 (Rydberg, 1924a). Pippen

(1964, 1968) treated Pericalia as a distinct entity with its discoid capitula with underground tubercles attached to the base of the stem. Pericalia, because its shared leaf and floral morphology, has been placed within Roldana (Robinson & Brettell, 1974;

Turner, 2005; Funston, 2008), although all three revisions recognize Pericalia as a morphologically distinct group of several Roldana species.

In the present study Pericalia (sensu Pippen, 1968) is monophyletic with the inclusion of Roldana heracleifolia, a species with radiate capitula. Roldana heracleifolia shares characters with R. mexicana and R. suffulta such as a single-stemmed herbaceous growth habit to 1 ̶ 3 meters tall, funnelform corollas ca. 8 mm long, and pubescent cypselae. Moreover, Turner (2005) suggested that R. sessifolia and R. michocana may be confused due to certain phenotypical constraints in particular geographic regions (i.e., forms with petiolate leaves), and this supports the present phylogenetic placement of these two species. Roldana mexicana has been treated as a variety of R. suffulta (Gibson,

1968) and this also supports the current placement of these two species within the

‘Pericalia’ clade. Roldana mexicana and R. suffulta species have glabrous cypselae and occur sympatrically in four central Mexican states. Roldana heteroidea and R. subpeltata should be also included in a future phylogenetic analysis to confirm the monophyly of this group. There is relatively high morphological variation among Roldana (Pericalia) 26 species, which can confound field identification of members of this clade (Turner, 2005).

More sampling across a wider geographic distribution should be included in future studies. Expanded investigations may lead to the resurrection of Pericalia as a genus.

With the inclusion of the radiate R. heracleifolia, the Pericalia group consists of seven species distributed in cloud forests and pine-oak forests of central Mexico, and five species of this group were included in the present study.

Pittocaluon bombycophole (Clade 7). ⎯ Pittocaulon contains five species with succulent stems, chambered piths, and seasonally deciduous leaves (Robinson & Brettell,

1973b; Clark, 1996). This clade contains two species in Pittocaulon that have pubescent leaves and 13 ̶ 20 involucral bracts, although a third species, P. hintonii, is purported to be related to these species as it shares characters with the previous two species but was not included in the phylogenetic analysis. Roldana eriophylla is strongly supported as a member of this clade, and further inspection of voucher specimens at TEX, MEXU, and

US, made it apparent that the material resembled other species in Pittocaulon with its succulent stems, chambered piths, and deciduous leaves. However, R. eriophylla only has eight involucral bracts, a feature not shared with the other two species in Clade 7. At this point it would not be warranted to transfer this species to Pittocaulon as Pittocaulon s.s. is not supported as monophyletic in the nrDNA trees (Figs. 2.2 ̶ 2.5). The three species formerly placed in Pittocaulon; P. bombycophole, P. filare and P. hintonii, occur in rocky, deciduous scrublands. Roldana eriophylla occurs in pine-oak forests. Two of the species, P. filare and P. hintonii, are narrow endemics. With the inclusion of R.

27 eriophylla, this group is purported to contain four species that occur from central to southern Mexico at elevations between 500 ̶ 1700 meters.

Pittocaulon praecox/vellatum, Villasenoria (Clade 8). ⎯ This clade consists of two very morphologically and geographically different taxa. The Pittocaulon praecox/vellatum complex includes succulents with chambered-piths and simple leaves that are deciduous prior to anthesis, and are associated with volcanic soils. Villasenoria orcuttii is a small tree to four meters in height with a solid pith and pinnately compound leaves. The phylogenies based on ITS/ETS data confirm the previously demonstrated well-supported relationship between P. praecox and Villasenoria orcuttii (Pelser et al.,

2010). Although Pelser et al. (2007) demonstrated data that P. bombycophole and P. praecox are not closely related, support values for the branches in the combined analysis were weak.

Pittocaulon praecox and P. vellatum occur sympatrically in central Mexico, and they differ only in a lack of pubescence just beneath the stem apices in the former

(Robinson & Brettell, 1973b). Pittocaulon praecox occurs from central Mexico to

Oaxaca and P. vellatum and its two varieties occur from central Mexico to Guatemala.

These species are most common in deciduous forests or scrublands between 1000 ̶ 2500 meters. Villasenoria occurs in Oaxaca and Veracruz and is the only genus in the Mexican tussilaginioid group restricted to elevations below 1100 meters in Mexico. (Clark, 1996,

1999).

Roldana petasitis complex (Clade 9). ⎯ This group includes nine species and five varieties (Funston, 2008) of mostly small shrubs or suffruticose herbs. Funston 28 (2008) & Turner (2005) disagreed in their species delimitations within Roldana. In particular, Funston (2008) treated several members of this complex as varieties. Turner

(2005) presented no evidence for his splitting of this group, and his classification of the genus as a whole is dubious. For the above reasons, Funston (2008) is followed with respect to the taxonomy and species delimitations. Roldana petasitis var. cristobalensis is based solely on the presence of eradiate capitula. Populations of Roldana anisophylla and

R. heterogama have been collected with radiate or eradiate capitula.

Although non-peltate leaves are most common among species in Clade 9, species may have peltate leaves (Roldana chapalensis, R. heterogama), may consist of individuals with peltate leaves or non-peltate leaves (R. angulifolia), or have non-peltate and peltate leaves on the same plant (R. petasitis var. oaxacana). Roldana gilgii is the only known member of the Senecioneae in the New World with white latex. Based on observations made in a greenhouse, the species within this complex are obligate outcrossers but F1 hybrids between Roldana petasitis var. oaxacana and R.p. var. petasitis can be readily made.

This complex is found in montane regions from northern Mexico () to

Central America, excluding . This complex contains ca. 12 ̶ 15 species, although more work involving morphological and phylogenetic analyses will be necessary to delimit species and varieties within this clade.

Roldana sundbergii complex (Clade 10). ⎯ This clade consists of five species, four of which have ovate to cordate leaves with callus denticulate margins and densely pubescent adaxial leaf surfaces. Roldana hintonii has elliptic to obovate leaves with 29 arachnoid pubescence on the veins of the adaxial leaf surfaces. Roldana aschenborniana is a widespread species that exhibits morphological variation throughout it range from northern Mexico to Guatemala. Two of the four specimens that Turner used in describing

R. sundbergii were noted by Gibson (1969) as possible hybrids. Roldana albonervia, R. aschenborniana, and R. barba-johannis are widespread from northeastern Mexico to

Honduras in pine-oak forest and cloud forest habitats most common at 2000–3200 meters. Roldana hintonii is endemic to the state of Mexico and is only reported from the vicinity of Temascáltepec in pine-oak and fir forests at 2100–3000 meters. Roldana sundbergii is endemic to Nuevo Leon from 800–2100 meters in tropical deciduous and pine-oak forests.

Roldana platanifolia complex (Clade 11). ⎯ The three representative species in this complex have 9 ̶ 13 phyllaries and palmatifid leaves, common to many species of

Roldana. Roldana platanifolia is a small herb from woody rhizomes with mostly basal leaves. Roldana grimesii and R. marquezii are small shrubs with cauline leaves distributed on the upper half of the stems. Roldana platanifolia occurs in pine-oak and fir forests of central Mexico from 2700–4100 meters. Roldana grimesii and R. marquezii occur in central Mexico in pine-oak forests from 1500–2700 meters. It is not known how many species occur in this group, although the number likely represents only a few species. Also, it would depend on whether one accepts Funston’s classification of a broad

R. grimesii, or Turner’s three separate species based on dubious distinctions.

Roldana mixtecana/reticulata (Clade 12). ⎯ These two species are similar with respect to their leaf morphology and radiate capitula that are arranged in corymbiform 30 cymes, uncommon among species of Roldana. Roldana mixtecana has more stems than

R. reticulata (3–5 vs. 1), the leaf blade margins are entire vs. serrate, and R. mixtecana is the only species in the Mexican tussilaginioid group with phyllaries in five graduated, dimorphic series, the outer three series rimmed with deep purple. The phyllaries are stramineous, a characteristic not found in other species of Roldana (Panero & Villaseñor,

1996). Panero & Villaseñor (1996) suggested a relationship of R. mixtecana to R. michoacana and R. sessifolia based on the shared small, angular leaves and the short and several-stemmed herbaceous habitat. These species differ from R. mixtecana in the presence of discoid capitula with white disc corollas. Funston (2008) suggested a relationship between R. mixtecana and R. hederifolia, although the latter species has discoid capitula and uniseriate phyllaries.

Roldana mixtecana has been reported from two districts in Oaxaca in pine-oak forests from ca. 2000 meters. Roldana reticulata is widespread at 2800–3600 meters in pine-oak forests in central Mexico. It is unknown how many species may fit into this clade.

Telanthophora (Clade 13). ⎯ Telanthophora was originally proposed by

Robinson & Brettell in 1974, and at that time it included 14 species of suffruticose herbs, shrubs, or small trees with solid piths and leaves restricted to just below the capitulescence. Clark (1996) revised the genus and her treatment included nine species.

Moreover, Clark (1996) indicated a phyletic relationship of Telanthophora with

Pittocaulon and Villasenoria based on morphological characters, although these relationships are not supported in the present study. In recent nrDNA phylogenies of the 31 Senecioneae, the relationship of Telanthophora to the remainder of the Mexican tussilaginioid genera is weakly supported based on ITS (Pelser et al., 2007) and ITS/ETS

(Pelser et al., 2010) data sets. According to the present study, Telanthophora is monophyletic, however, it is nested within species of Roldana. The small shrub with serrated leaves that is endemic to Oaxaca, T. liebmannii, is sister to the rest of Clade 13.

The subclade containing T. andrieuxii, T. jaliscana and T. uspantanensis contains species that are very similar with respect to their glabrous leaves and many aspects of the floral morphology. Telanthophora andrieuxii differs from the other two species in having eight involucral bracts as opposed to five to six. The subclade containing T. cobanensis and T. grandifolia consists of the two most widespread species in the genus, and each of which is represented by two varieties.

More collections of this genus need to be included in order to understand its evolutionary relationships better, especially the under-collected endemic species

Telanthophora bartlettii (Belize) and T. sublaciniata (Guatemala). Telanthophora bartlettii is restricted to below 1100 meters in the Maya Mountains of western Belize.

Moreover, more accessions from southern Central America should also be included in any future phylogenetic analysis. The nine species of Telanthophora occur in montane habitats and are common between 1200 and 2700 meters from northern Mexico to

Panama. Six species of Telanthophora were included in the study.

Other taxa not included in thirteen clades

With respect to Roldana, it is evident based on morphological variation and nrDNA data that this genus needs a thorough phylogenetic survey that would involve 32 extensive fieldwork, herbaria visits, and molecular work using nuclear and sufficient plastid data at both the species and population level, in order to elucidate further the relationships among this paraphyletic genus. The type species, Roldana lobata, is ambiguously placed within the Mexican tussilaginioid group, and it is morphologically and phylogenetically related to R. hartwegii, a widespread species. Although the combined analysis supported a clade including Roldana hartwegii and R. lobata, these two species are not discussed as a clade because of the lack of support for the relationship of R. ehrebergiana to R. lobata and R. hartwegii in the combined analyses, and their ambiguous placement in the separate ITS and ETS analyses. After further molecular analyses are conducted that include plastid data, only those species that formed a monophyletic group that included Roldana lobata would be able to be considered

Roldana s.s. Another unresolved species of Roldana, R. schaffneri, morphologically resembles Telanthophora in that it has terminal branches that terminate in and the leaves are pinnately-veined. Specimens of R. shaffneri have been incorrectly identified as Telanthophora grandifolia based on material observed at BIGU, MEXU,

TEX, and US. Roldana schaffneri is highly variable with respect to its morphology and it is widely distributed from central Mexico to . This phenotypic plasticity observed in herbarium specimens, and living material in the field and in a greenhouse, would make this and excellent project for a study using population genetics and species- level phylogenetics to place this species correctly in the clade.

Biogeographic implications. ⎯ Pelser et al. (2010) suggested a recent origin of the Mexican tussilaginioid group (9.5–15 mya), which corresponds to a period when the 33 Asteraceae showed an explosion in diversity in Mexico and Central America indicated by pollen evidence (Raven & Axelrod, 1974). Psacaliopsis purpusii/Robinsonecio (Clade 1) were sister to the remainder of the Mexican tussilaginioid group in a previous study using

ITS (Pelser et al., 2010), and the ITS data alone supported Robinsonecio as being sister the rest of Mexican tussilaginioid group (Pelser et al., 2007). In the present study, both the separate analyses and combined analysis also support Clade 1 with Psacaliopsis purpusii and Robinsonecio gerberifolius as being sister to the remaining Mexican tussilaginioid genera. Clade 1 is isolated at high elevations in Mexico and Guatemala from 2600–4200 meters. Species-rich genera (i.e., Psacalium and Roldana) have undergone adaptive radiation in montane Mexico and Guatemala, and the age of the origin of Psacalium is estimated to ca. 2.84–3.49 mya (Pelser et al., 2010).

Roldana is widespread, but high diversity in the mountains of central and southern Mexico imply an origin in these montane regions. Two species (R. heterogama and R. scandens) occur as far south as Costa Rica, and R. heterogama occurs in northern

Panama. With respect to Telanthophora, both the ITS and combined ITS/ETS data sets support T. liebmannii as sister to the rest of the genus. Telanthophora liebmannii is endemic to Oaxaca, suggesting a possible origin of the genus in southern Mexico, and subsequent radiations north and south.

Among the three species that occur near the Arizona–Mexico border;

Barkleyanthus salicifolius, Psacalium decompositum, Roldana hartwegii, only Clade 4 consisting of Arnoglossum, Barkleyanthus and Yermo colonized and diversified in the

USA. The most recent common ancestor between Arnoglossum (Midwest and the 34 Southeast USA) and Yermo (Fremont County, Wyoming) is estimated to have occurred

1.17–1.43 mya, suggesting a recent origin of the clade into the USA (Pelser et al., 2010).

The highlands of Guatemala share most of their species composition with southern Mexico. However, a few taxa at the species-level are endemic to Guatemala

(i.e., Psacaliopsis pudica, Psacalium guatemalense, P. pinetorum, Roldana sp. nov.,

Telanthophora sublaciniata). Two of the previously mentioned four species are described based on a single population. One species, Telanthophora bartlettii, is endemic to western Belize.

Conservation implications.⎯Montane forests of Mexico and Central America are under intense negative pressures from human land use practices and the effects of climate change. In Mexico, endemic montane Asteraceae, including many taxa within the

Mexican tussilaginioid group, have been used to illustrate regions of high biodiversity

(Villaseñor et al., 2006; 2007) and regions of high endemism (Villaseñor et al., 1998;

González-Zamora et al., 2007). The Mexican tussilaginioid group comprises an ecologically and morphologically diverse group of plants that are common in threatened montane ecosytems and once a clear understanding of their evolutionary relationships are fully known, these species may be used as tools towards assessing plant community richness in these ecosystems of Mexico and Central America.

CONCLUSION

A significant finding of this study is the establishment of 13 well-supported clades within the North and Central American Mexican tussilaginioid group using a large taxon 35 sampling that included many taxa sampled for the first time. A combined ITS and ETS nrDNA data set supports the monophyly of the Mexican tussilaginioid group. These data also support the monophyly of Telanthophora, but reject the monophyly of Pittocaulon,

Psacaliopsis, and Roldana. A sister relationship to the ‘gynoxoid’ group of South

America is resolved and well-supported (Bootstrap = 99%; Posterior Probability = 1.0).

Although this study provides a comprehensive phylogenetic analysis using nrDNA to elucidate relationships of the Mexican tussilaginioid group, field biologists are still left to cope with difficult taxonomic keys and an incomplete knowledge of morphological variation, phenotypic plasticity, and actual range distributions for the species. Robinson

& Brettell (1974), Barkley (1985a, 1985b, 1999), Jeffrey (1992) and Barkley et al.,

(1996) recognized morphological variation warranting the division of the tussilaginioid group in the United States, Mexico, and Central America. However, researchers such as

Jeffrey (1992) and Turner (2005) have provided little justification behind their recent species delimitations, new combinations, and broad taxonomic statements towards the species relationships among the group.

Increasing the number of populations for species complexes, adding more species of Roldana, and a thorough sampling of the diverse montane genus Psacalium (46 species) will provide more resolution into the evolution and the origins of montane endemism within this group.

36 Table 2.1. Represented species and genera of the traditional Mexican tussilaginioid group used for ITS/ETS combined data set analysis.

Genus No. of species sampled (total number of species in genus)

Arnoglossum 1 (9)

Barkelyanthus 1 (1)

Digitacalia 2 (5)

Nelsonianthus 0 (2)

Pippenalia 1 (1)

Pittocaulon 4 (5)

Psacaliopsis 5 (5)

Psacalium 4 (46)

Robinsonecio 1 (2)

Roldana 35 (55)

Telanthophora 7 (9)

Villasenoria 1 (1)

Yermo 1 (1)

37 Table 2.2. Data set partitions with information on the number of informative/total characters contributed to the analyses.

Data set Combined ITS ETS ITS (100 accessions) ETS (100 accessions)

# of accessions (# of species) 100 (67) 144 (72) 119 (69) 100 (67) 100 (67)

Total characters 1296 827 469 827 469

# of informative characters 501 304 179 285 178

% of informative characters 38% 37% 38% 34% 38%

Nucleotide substitution model GTR + G GTR + G GTR + G GTR + G GTR + G

38 Table 2.3. Results of the harmonic mean estimate in MrBayes to test the alternative topologies of the combined Baeysian Analysis results. Marginal likelihood (in natural log units) estimated using stepping- stone sampling based on 50 steps with 19500 generations (39 samples) within each step.

Genus Marginal likelihood best tree (ln) Marginal likelihood constraint model (ln) Difference (log units)

Pittocaulon -13246.18 -13232.77 13.41*

Psacaliopsis -13321.85 -13213.48 108.37*

Roldana -13207.82 -13225.28 17.46*

*A log difference above 5 is considered very strong evidence for the alterative model

39 Clade 4 -2 -1 I3) Arnoglossum plantagineum Arnoglossum atriplicifolium (

I8) ( -1 -2

Arnoglossum atriplicifolium -2 Yermo xanthocephalus 9 1 9 6 4 9 9 . Clade 1 Barkleyanthus salicifolius Barkleyanthus salicifolius Barkleyanthus salicifolius 0 0 1 1 -1 -2 0 0 1 -3 1 Clade 5 Clade 13 Robinsonecio gerberifolius Robinsonecio gerberifolius Robinsonecio gerberifolius -1 Clade 3 -2 -2 Clade 6 -1 Psacaliopsis pudica Psacalium palmeri Psacaliopsis macdonaldii -2 Psacalium megaphyllum -1 Pippenalia delphinifolia * Clade 2 -2 -1 -2 Psacalium peltatum Psacalium cirsiifolium -1 Pippenalia delphinifolia Psacalium cirsiifolium 0.2 0 1 0 0 I1) 1 0 1 ( I3)

1 -2 (

I6) -1 -2 ( I5)

-2 ( I4)

-2 ( 0

1 0 -2 0 0 -1 1 0 1 1 -2 0 -2 -1 1 1 Telanthophora grandifolia Roldana lobata 0 2 5 1 0 9 9 1 . Telanthophora grandifolia Telanthophora copeyensis Roldana suffulta Telanthophora cobanensis Telanthophora andrieuxii Roldana suffulta -1 -2 Roldana mexicana Roldana suffulta Telanthophora uspantanensis Roldana hartwegii Roldana suffulta Digitacalia crypta juxtlahuacensis Roldana suffulta Telanthophora uspantanensis Roldana suffulta Telanthophora cobanensis Digitacalia jatrophoides Telanthophora andrieuxii var. Roldana michoacana Digitacalia jatrophoides var. pentaloba 0 8 Digitacalia jatrophoides var. 0 1 8 6 Roldana sessifolia Telanthophora jalisciana 1 1 9 Gynoxoid group Gynoxoid Telanthophora liebmannii Roldana heracleifolia Roldana ehrenbergiana 9 8 Telanthophora liebmannii 9 8 . 3 8 0 0 1 Psacaliopsis purpusii Psacaliopsis purpusii 6 6 1 1 1 9 9 8 1 -1 1 9 6 4 Psacaliopsis paneroi Psacaliopsis pinetorum 1 1 9 9 9 -2 1 8 0 0 0 1 1 0 1 1 0 0 1 Gynoxys buxifolia 1 Gynoxys tolimensis Gynoxys soukupii Gynoxys buxifolia Aequatorium lepidotum Aequatorium asterotrichum 3 8 Gynoxys sodiroi .94 7 9 0 0 1 1 Rugelia nudicaulis 8 1 9 0 0 1 6 9 . 9 1 9 * Senecio vulgaris Figure 2.1. Bayesian inference (BI) phylogram from ITS data set (144 accessions). are discusses in the results for the combined data analysis. 1 1 40 Clade 8 -1 -3 -2 Villasenoria orcuttii Villasenoria orcuttii Villasenoria orcuttii I1) Clade 7 (

-1 I5) (

-2 -1 tzimolensis vellatum Clade 11 I4) (

var. var. -2 (I4)

I2) vellatum ( (I12) I2) I3)

(I6) ( ( -2

-2 -3 -1 -1 var. clone -1 (I14)

-3 -1 (I5) (I11) -2 (I8) 0

-3 1 0 (I15)

1 -2 (I8) -3 (I5) Pittocaulon vellatum (I3) Pittocaulon vellatum Pittocaulon bombycophole (I9) (I10) (I3) (I5) Roldana schaffneri (I1) Roldana schaffneri Pittocaulon filare Pittocaulon praecox Pittocaulon praecox Pittocaulon praecox -2 (I4) Pittocaulon praecox -1 Pittocaulon praecox Pittocaulon vellatum Roldana schaffneri Roldana schaffneri Pittocaulon vellatum var. 9 8 0.2 . 0 3 1 6 0 Roldana schaffneri 1 1 Roldana platanifolia 9 9 1 Roldana schaffneri Roldana schaffneri Roldana marquezii Roldana grimesii Roldana schaffneri 6 8 Roldana grimesii Roldana marquezii 8 9 . . Roldana marquezii Roldana marquezii Roldana grimesii Roldana marquezii Roldana grimesii Roldana lineolata Roldana lineolata 9 8 . Roldana uxordecora 0 0 1 1 0 0 1 1 Roldana eriophylla 8 1 0 9 1 0 1 1 6 9 0 . 1 9 5 1 9 5 8 .

Figure 2.1. (continued)

41 Roldana robinsoniana Roldana sp. nov. Roldana lanicaulis 1 Roldana mixtecana 100 Roldana reticulata Clade 12 Roldana gilgii Roldana heterogama-1 1 Roldana heterogama-2 98 Roldana heterogama-3 Roldana petasitis var. petasitis (I3 ) .93 Roldana petasitis var. oaxacana-1 (I3) Roldana petasitis var. oaxacana-1 (I4) .86 .96 Roldana gentryi Roldana chapalensis (I6) Roldana petasitis var. petasitis (I5) Roldana sp. hybrid (I3) Roldana petasitis var. cristobalensis Roldana petasitis var. sartorii (I5) Roldana petasitis* Roldana anisophylla (I5) Roldana petasitis var. oaxacana-2 (I3) Clade 9 .84 Roldana petasitis var. sartorii (I3) Roldana anisophylla (I3) Roldana sp. hybrid (I2) Roldana petasitis var. oaxacana-2 (I4) Roldana jurgensenii (I4) Roldana jurgensenii (I5) Roldana petasitis var. petasitis (I4) Roldana jurgensenii (I2) Roldana jurgensenii (I1) Roldana petasitis var. oaxacana-2 (I1) 1 Roldana petasitis var. sartorii (I1) Roldana chapalensis (I5) 82 1 Roldana angulifolia (I3) 89 Roldana angulifolia (I7) .98 Roldana metepecus (I8) Roldana metepecus (I12) Roldana metepecus (I10) Roldana acutangula Roldana sundbergii Roldana albonervia-1 Roldana hintonii (I5) 1 Roldana albonervia-2 1 92 94 .99 Roldana hintonii (I2) Clade 10 Roldana hintonii (I1) Roldana barba-johannis Roldana aschenborniana

0.2 Figure 2.1. (continued)

42 Clade 4 -1 Yermo xanthocephalus Arnoglossum atriplicifolium -3 -2 -1 Barkleyanthus salicifolius Barkleyanthus salicifolius Barkleyanthus salicifolius .99 1 98 Clade numbers 1 100 Clade 1 Clade 9 Clade 7 Clade 5 -3 -1 Roldana eriophylla -2 Pittocaulon bombycophole Clade 2 (E5) 1 -1 100 (E1) 0.2 (E1) -1 Robinsonecio gerberifolius -2 (E4) (E6) Pittocaulon filare

(E1) (E5) (E2)

-2 (E1) (E3) E1) (E1) Robinsonecio gerberifolius -1 E3) ( -2 ( -2 petasitis * Robinsonecio gerberifolius E4) cristobalensis oaxacana ( (E2) petasitis 87 sartorii var. . Gynoxoid group Gynoxoid -3 clone cristobalensis (E5) sartorii (E1) oaxacana var. var. -2 -1 Psacaliopsis pudica Psacaliopsis macdonaldii (E2) var. var. oaxacana var. var. var. Psacalium cirsiifolium var. * juxtlahuacensis sp. hybrid (E1) sp. hybrid (E2) Roldana angulifolia sp. hybrid (E3) Psacalium cirsiifolium Roldana chapalensis Roldana anisophylla Psacalium palmeri var. juxtlahuacensis 1 Roldana metepecus 100 juxtlahuacensis Rugelia nudicaulis Roldana petasitis Psacalium megaphyllum var. Nelsonianthus tapianus Pippenalia delphiniifolia .99 Pippenalia delphiniifolia Roldana anisophylla Roldana Roldana petasitis Roldana Roldana petasitis Roldana Roldana gentryi Roldana greenmanii var. Roldana heterogama Roldana petasitis 1 Roldana petasitis Roldana angulifolia 96 Roldana gilgii Roldana chapalensis Roldana heterogama Roldana heterogama Roldana chapalensis Roldana petasitis Roldana petasitis Roldana petasitis .91 1 Roldana petasitis Roldana jurgensenii 100 Gynoxys buxifolia 1 .99 1 Psacalium peltatum 89 100 90 1 .97 Psacaliopsis purpusii 95 Psacaliopsis purpusii Aequatorium asterotrichum Roldana hartwegii Psacaliopsis paneroi Gynoxys soukupii 1 Psacaliopsis pinetorum 1 .92 97 Figure 2.2. Bayesian inference (BI) phylogram from ETS data set (109 accessions). ETS set (109 accessions). from data (BI) phylogram inference 2.2. Bayesian Figure ≥ 0.80. values probability posterior Bayesian are the branches Numbers above ≥ 80%. values ML boostrap are the branches Numbers below analysis. data the combined for discusses in the results are Psacaliopsis paneroi 1 Psacaliopsis paneroi .83 .89 1 100 .95 1 100 1 88 . 88 . 0 .9 .93 Senecio vulgaris

43 1 Villasenoria orcuttii-1 Villasenoria orcuttii-2 99 99 Villasenoria orcuttii-3 1 1 Pittocaulon praecox-2 (E3) 94 Pittocaulon vellatum var. vellatum-2 Pittocaulon vellatum var. tzimolensis (E5) 1 Pittocaulon vellatum var. tzimolensis (E2) 100 Pittocaulon praecox-2 (E2) Clade 8 Pittocaulon praecox-1 (E3) Pittocaulon vellatum var. vellatum-1 (E1) Pittocaulon vellatum var. vellatum-1 (E2) Pittocaulon praecox-3 Pittocaulon praecox-2 (E1) Roldana suffulta-1 Roldana suffulta-2 (E5) 81 1 Roldana mexicana (E3) 94 1 Roldana mexicana (E4) Roldana heracleifolia Clade 6 .99 Roldana michoacana (E3) 1 Roldana sessifolia 98 Roldana michoacana (E4) Roldana lineolata Roldana lobata Roldana ehrenbergiana 1 Digitacalia jatrophoides var. jatrophoides .99 Digitacalia jatrophoides var. pentaloba 100 Digitacalia jatrophoides Clade 3 Digitacalia crypta 1 Roldana marquezii 1 100 Roldana grimesii 91 Roldana platanifolia Clade 11 Roldana schaffneri Roldana uxordecora Roldana acutangula 1 Roldana mixtecana 91 Roldana reticulata Clade 12 Roldana robinsoniana .88 Telanthophora cobanensis-1 91 Telanthophora cobanensis-2 Clade 13 (in part) Roldana sp. nov. Roldana lanicaulis Roldana sundbergii .81 1 Roldana albonervia clone #5 .94 Roldana barba-johannis .97 Roldana hintonii Clade 10 .98 Roldana albonervia clone #3 Roldana aschenborniana 1 Telanthophora liebmannii-1 100 Telanthophora liebmannii-2 .99 Telanthophora grandifolia-1 86 Telanthophora grandifolia-2 Telanthophora andrieuxii-1 Telanthophora uspantanensis-1 (E1) Clade 13 (in part) Telanthophora andrieuxii-2 Telanthophora uspantanensis -2 (E4) Telanthophora uspantanensis-1 (E5) Telanthophora uspantanensis-1 (E2) Telanthophora uspantanensis-2 (E2) Telanthophora jalisciana

0.2

Figure 2.2. (continued).

44

1 Senecio vulgaris 100 Rugelia nudicaulis .99 Aequatorium asterotrichum 1 Gynoxys buxifolia-1 Gynoxoid group 99 Gynoxys soukupii 1 Psacaliopsis purpusii-1 .95 100 Psacaliopsis purpusii-2 1 Robinsonecio gerberifolius-2 1 Robinsonecio gerberifolius-1 Clade 1 100 100 Robinsonecio gerberifolius-3 1 Psacaliopsis paneroi var. juxtlahuacensis 100 Psacaliopsis pinetorum Clade 2 Digitacalia crypta 1 1 .99 Digitacalia jatrophoides 100 100 .99 83 Digitacalia jatrophoides var. pentaloba Clade 3 97 Digitacalia jatrophoides var. jatrophoides 1 Arnoglossum atriplicifolium-1 1 100 Yermo xanthocephalus 100 1 Barkleyanthus salicifolius-1 Clade 4 .92 100 Barkleyanthus salicifolius-2 .99 1 Pippenalia delphinifolia-2 100 Pippenalia delphinifolia-1 88 .99 82 1 Psacalium cirsiifolium-1 .92 100 Psacalium cirsifolium-2 .99 93 Psacalium palmeri 100 1 Psacaliopsis macdonaldii Clade 5 .82 100 Psacaliopsis pudica .96 1 Psacalium megaphyllum 100 Psacalium peltatum 1 Roldana suffulta-1 100 .99 Roldana mexicana 1 96 Roldana suffulta-2 Roldana heracleifolia Clade 6 1 Roldana michoacana 81 Roldana sessifolia Roldana ehrenbergiana 1 Roldana hartwegii Roldana lobata 1 Roldana mixtecana 99 Roldana reticulata Clade 12

0.3

Figure 2.3. Bayesian inference (BI) phylogram from ITS data set (100 accessions). Sequences are identical to the sampling for the combined analysis. Numbers above the branches are Bayesian posterior probability this tree are in bold in Appendix 2.1. Clade numbers are discusses in the results for the combined data analysis.

45 Clade 12

1 Pittocaulon bombycophole 1 100 Pittocaulon filare Clade 7 97 Roldana eriophylla Pittocaulon praecox-2 (I2) .99 Pittocaulon praecox-2 (I4) Pittocaulon praecox-1 (I2) 1 92 Pittocaulon vellatum var. vellatum-1 (I1) Pittocaulon vellatum var. tzimolensis 100 Pittocaulon praecox-2 (I4) 1 .99 Pittocaulon vellatum var. vellatum-1 (I5) Clade 8 97 81 Pittocaulon vellatum var. vellatum-2 Villasenoria orcuttii-2 1 Villasenoria orcuttii-3 100 Villasenoria orcuttii-1 .99 1 Roldana grimesii 100 Roldana marquezii 93 Roldana platanifolia Clade 11 Roldana sp. nov. Roldana sp. hybrid (I2) Roldana hybrid (I3) Roldana anisophylla (I3) Roldana anisophylla (I5) .94 Roldana chapalensis (I6) .92 Roldana gentryi Roldana petasitis var. oaxacana-1 Roldana petasitis var. cristobalensis Roldana petasitis var. oaxacana-2 Roldana petasitis var. petasitis Roldana petasitis var. sartorii Clade 9 1 Roldana jurgensenii 86 Roldana angulifolia (I3) .99 Roldana angulifolia (I7) 87 Roldana chapalensis (I5) Roldana metepecus .95 Roldana heterogama-2 1 Roldana heterogama-1 99 Roldana heterogama-3 Roldana gilgii Roldana acutangula 1 Roldana albonervia .99 Roldana hintonii 1 Roldana barba-johannis Roldana aschenborniana Clade 10 Roldana sundbergii Roldana lanicaulis Roldana lineolata-1 Roldana shaffneri-1 Roldana robinsoniana Roldana uxordecora Telanthophora andrieuxii-2 .99 Telanthophora uspantanensis-2 .96 91 Telanthophora uspantanensis-1 Telanthophora andrieuxii-1 .99 Telanthophora jalisciana 1 Telanthophora grandifolia-1 .99 Telanthophora grandifolia-2 .99 1 100 Clade 13 81 Telanthophora cobanensis-1 95 Telanthophora cobanensis-2 1 Telanthophora liebmannii-2 100 Telanthophora liebmannii-1

0.3

Figure 2.3. (continued).

46

Senecio vulgaris Aequatorium asterotrichum .92 1 Gynobuxys buxifolia-1 Gynoxoid group 99 Gynoxys soukupii Rugelia nudicaulis 1 Psacaliopsis purpusii-1 100 Psacaliopsis purpusii-2 .86 Robinsonecio gerberifolius-2 1 Clade 1 Robinsonecio gerberifolius-1 100 .87 Robinsonecio gerberifolius-3 1 Psacaliopsis paneroi var. juxtlahuacensis 100 Psacaliopsis pinetorum Clade 2 1 Arnoglossum atriplicifolium 1 100 Yermo xanthocephalus 100 Clade 4 1 Barkleyanthus salicifolius-1 100 Barkleyanthus salicifolius-2 .97 Pippenalia delphiniifolia-2 88 Pippenalia delphiniifolia-1

1 Psacalium cirsiifolium-1 .94 1 98 Psacalium cirsiifolium-2 98 Psacalium palmeri .90 Clade 5 1 Psacaliopsis macdonaldii 100 Psacaliopsis pudica

.91 Psacalium megaphyllum Psacalium peltatum Roldana hartwegii Digitacalia crypta 1 Digitacalia jatrophoides 1 Digitacalia jatrophoides var. pentaloba Clade 3 100 Digitacalia jatrophoides var. jatrophoides

1 Pittocaulon bombycophole 1 100 Pittocaulon filare 82 Clade 7 Roldana eriophylla Pittocaulon praecox-2 (E2) Pittocaulon praecox-1 (E1)

.90 Pittocaulon praecox-1 (E3) Pittocaulon vellatum var. vellatum-1 (E1) 1 Pittocaulon vellatum var. vellatum-1 (E2) 99 Pittocaulon vellatum var. tzimolensis Clade 8 .99 Pittocaulon praecox-2 (E3) 1 95 92 Pittocaulon vellatum var. vellatum-2 Villasenoria orcuttii-2 1 94 Villasenoria orcuttii-3 100 Villasenoria orcuttii-1

0.2 Figure 2.4. Bayesian inference (BI) phylogram from ETS data set (100 accessions). Sequences are identical to the sampling for the combined analysis. Numbers in this tree are in bold in Appendix 2.1. Clade numbers are discusses in the results for the combined data analysis.

47

Roldana sp. nov. Roldana sp. htybrid (E1) Roldana sp. hybrid (E2)

.99 Roldana angulifolia (E1) .94 Roldana chapalensis (E3) 1 Roldana metepecus Roldana anisophylla (E1) Roldana anisophylla (E2) Roldana petasitis var. oaxacana-1 Roldana petasitis var. petasitis Roldana angulifolia (E5) Roldana chapalensis (E1) Clade 9 1 Roldana gentryi 96 Roldana petasitis var. cristobalensis Roldana petasitis var. oaxacana-2 Roldana petasitis var. sartorii .96 Roldana jurgensenii

.83 Roldana heterogama-2 1 Roldana heterogama-1 100 Roldana heterogama-3 Roldana gilgii Roldana acutangula

.98 Roldana albonervia .98 Roldana aschenborniana Roldana barba-johannis Clade 10 .83 .93 Roldana hintonii Roldana sundbergii

1 Roldana grimesii 1 100 Roldana marquezii 97 Clade 11 Roldana platanifolia Roldana lanicaulis 1 Roldana mixtecana 92 Roldana reticulata Clade 12 Telanthophora andrieuxii-2 Telanthophora andrieuxii-1 Telanthophora uspantanensis-1 Telanthophora uspantanensis-2

1 Telanthophora grandifolia-1 83 Telanthophora grandifolia-2 Clade 13 1 Telanthophora cobanensis-2 90 Telanthophora cobanensis-1 Telanthophora jalisciana

1 Telanthophora liebmannii-2 99 Telanthophora liebmannii-1 Roldana schaffneri-1 Roldana robinsoniana Roldana uxordecora Roldana suffulta-1

1 Roldana mexicana 88 Roldana suffulta-2 .99 1 Roldana michoacana Clade 6 97 Roldana sessifolia Roldana heracleifolia Roldana ehrenbergiana Roldana lineolata-1 Roldana lobata 0.2 Figure 2.4. (continued).

48 Pittocaulon praecox-2 (E2,I2) Pittocaulon praecox-2 (E3,I4) 1 Pittocaulon praecox-1 (E1,I2) Pittocaulon vellatum var. vellatum-1 (E1,I1) 1 Pittocaulon vellatum var. tzimolensis 100 Pittocaulon praecox-1 (E3,I3) Clade 8 1 .99 Pittocaulon vellatum var. vellatum-1 (E2,I5) 99 Pittocaulon vellatum var. vellatum-2 .81 Villasenoria orcuttii-2 1 97 Villasenoria orcuttii-3 100 Villasenoria ocuttii-1 Roldana sp. hybrid (E1,I2) Roldana sp. hybrid (E2,I3) Roldana angulifolia (E1,I3) .86 Roldana chapalensis (E3,I6) .97 Roldana metepecus Roldana anisophlylla (E1,I3) Roldana anisophlylla (E2,I5) Roldana petasitis var. oaxacana-1 Roldana petasitis var. petasits Roldana gentryi Roldana petasitis var. cristobalensis Clade 9 Roldana petasitis var. oaxacana-2 1 Roldana petasitis var. sartorii 99 Roldana jurgensenii .84 .97 Roldana angulifolia (E5,I7) Roldana chapalensis (E1,I5) 1 Roldana heterogama-2 1 Roldana heterogama-1 100 Roldana heterogama-3 Roldana gilgii Roldana acutangula 1 Roldana albonervia 1 83 Roldana hintonii .98 83 Roldana barba-johannis .81 88 Clade 10 1 Roldana aschenbergiana 99 Roldana sundbergii 1 Roldana grimesii 1 100 Roldana marquezii 100 Roldana platanifolia Clade 11 Roldana lanicaulis Roldana lineolata 1 Roldana mixtecana 100 Roldana reticulata Clade 12 Telanthophora andrieuxii-2 1 Telanthophora uspantanensis-2 .99 92 Telanthophora uspantanensis-1 Telanthophora andrieuxii-1 1 Telanthophora jalisciana 97 1 Telanthophora grandifolia-1 1 100 Telanthophora grandifolia-2 Clade 13 1 98 .96 Telanthophora cobanensis-2 89 Telanthophora cobanensis-1 1 Telanthophora liebmannii-2 100 Telanthophora liebmannii-1 Roldana schaffneri-1 Roldana robinsoniana Roldana uxordecora 0.4

Figure 2.5. (continued).

49 Chapter 3: A Reassessment of the Neotropical

Genera Pippenalia and Psacaliopsis (Asteraceae: Senecioneae)

The taxonomic assessment of Pippenalia McVaugh and traditional Psacaliopsis

H. Rob. & Brettell presented here includes morphological and molecular characters to elucidate the status of these endemic montane taxa. Pippenalia delphinifolia is transferred to Psacalium based on its leaf morphology and phylogenetic data that support this placement. A single species, Psacaliopsis purpusii, remains in Psacalioipsis. The nrDNA data places this species in a clade with Robinsonecio gerberifolius, another high elevation taxon with radiate yellow capitula and basal leaves. Funstonia gen. nov. is here described and includes a single species, Funstonia pinetorum (previously Psacaliopsis pinetorum), with two varieties. The nrDNA data strongly support Funstonia as a monospecific, monotypic genus (Fig. 3.1; Ill. 3.1). Psacaliopsis macdonaldii and P. pudica are transferred to Psacalium based on vegetative and floral morphological characters, and this is also supported by nrDNA data.

Taxonomical Background

The Senecioneae is the largest tribe in the Asteraceae with over 3000 species distributed worldwide (Nordenstam, 2003, 2007). The tribe is divided into four subtribes, one of which, the Tussilaginieae, includes the morphologically and ecologically diverse

Mexican tussilaginioid group (Robinson & Brettell, 1974; Barkley et al., 1996). The group consists of 13 genera and 140 species of herbs, shrubs, trees, and epiphytes that are a common floral component among montane ecosystems in Mexico and Guatemala

50 (Barkley, 1985a, 1985b; Barkley et al., 1996). The majority of the species are restricted to elevations above 1500 meters with their distributions ranging from the central and southeastern USA to northern Panama, although the group is concentrated in threatened montane ecosystems such as pine-oak and cloud forests of central and southern Mexico and Guatemala (Robinson & Brettell, 1974; Barkley 1985a, 1985b; Barkley et al., 1996).

Morphological studies (Robinson & Brettell, 1974; Barkley et al., 1996) and recent phylogenetic analyses (Pelser et al., 2007, 2010; Chapter 2) confirmed the position of

Pippenalia delphinfolia and Psacaliopsis purpusii among the Mexican tussilaginioid group.

The species that make up the Mexican tussilaginioid group share all or most of the following characters: stigmatic surfaces united across at least upper third of the inner face of the style branches; upper part of stamen filaments (anther collar) cylindrical; principal phyllaries often with midrib thickened at base; and chromosome numbers n = 30 (except n = 24 ̶ 30 in Arnoglossum). Historically, species in the Mexican tussilaginioid group were placed either in Senecio s.l., based on the presence of radiate capitula with yellow corollas or in Cacalia s.l., based on the presence of discoid capitula with white corollas.

The genera with radiate capitula were called the Mexican and Central American senecioid genera and those with discoid capitula were referred as the Mexican and

Central American cacalioid genera (Robinson & Brettell, 1974; Barkley, 1985a, 1985b).

The generic name Cacalia has been rejected (Waganitz, 1995; Brummitt, 1998), exemplifying a long-standing discordance between understanding species concepts within the Senecioneae and between the genera Cacalia and Senecio (Nash & Williams, 1976). 51 Based on morphological and anatomical differences, the Mexican tussilaginioid group has traditionally included the following genera, with the number of species in parentheses: Arnoglossum (7), Barkleyanthus (1), Digitacalia (5), Nelsonianthus (2),

Pippenalia (1), Pittocaulon (5), Psacaliopsis (5), Psacalium (46), Robinsonecio (2),

Roldana (55), Telanthophora (9), Villasenoria (1), and Yermo (1); (Pippen, 1968;

Robinson & Brettell, 1973b, 1973c, 1973d, 1973e, 1974; Robinson, 1974; McVaugh,

1972; Dorn, 1991). The gynoxoid group, which includes small trees from montane South

America in the genera Aequatorium, Gynoxys, Nordenstamia, and Paragynoxys, is strongly supported as the sister group to the Mexican tussilaginioid group (Pelser et al.,

2007, 2010; Chapter 2).

Pippenalia and Psacaliopsis (Asteraceae: Senecioneae) are genera that include acaulescent herbs with basal leaves. These two genera are distributed from central

Mexico to Honduras and El Salvador in pine-oak forests, elfin forests, and alpine habitats. Pippenalia is represented by a single species, P. delphinifolia, which has pedately-divided, subpeltate leaves and Psacaliopsis has traditionally consisted of five species with orbicular and centrally peltate leaves. Barkley (1985b) classified both of the genera as intermediates between Cacalia and Senecio, noting that the species had “the vegetative aspect of ‘Cacalia’ but with the superficial floral features of ‘Senecio.’

Pippenalia delphinifolia was originally described as Odontotrichum delphinifolium, but the type specimen was in fruit and no flowers were observed

(Rydberg; 1924b). McVaugh (1972) erected the genus Pippenalia based on the presence

52 of radiate capitula and the lack of a pappus, and Robinson & Brettell (1973d) transferred the remaining species of Odontotrichum to Psacalium.

Psacaliopsis originally included two species with strikingly different morphologies as described by Robinson & Brettell (1974). At the generic level,

Psacaliopsis was described as acaulescent herbs with basal, centrally peltate leaves and long petioles, with erect, radiate capitula and yellow corollas, or with nodding, discoid capitula and deep purple corollas. According to Robinson & Brettell (1974), morphologically Psacaliopsis was related to a group of herbs that included Psacalium and Pippenalia, which shared basal leaves and hirsute hairs on the leaf bases. Others have suggested that Psacaliopsis is most closely related to Roldana (Gibson, 1969; Funston,

2008). The type species, Psacaliopsis purpusii (Greenm. ex Brandegee) H. Rob. &

Brettell, was originally described as Senecio purpusii Greenm. ex Brandegee based on the presence of radiate capitula and yellow corollas. In his description, Greenman stated that

S. purpusii is most closely related to Senecio pinetorum Hemsl. Gibson (1969) revised

Senecio subg. Senecio sec. Palmatinervii, which included Senecio pinetorum and S. purpusii, and suggested a close relationship of these two species with many taxa that are presently in Roldana. Gibson discussed S. pinetorum as having either centrally peltate or non-peltate leaves, however, one specimen cited as S. purpusii [J. Rzedowski & McVaugh

207 (MICH)], was later determined to be Roldana tlacaopecana, an acaulescent plant with non-peltate leaves.

The second species placed in Psacaliopsis, P. pudica (Standl. & Steyerm.) H.

Rob. & Brettell, was originally described as Cacalia pudica based on the presence of 53 discoid capitula and purple corollas (Robinson & Brettell, 1974). Williams (1975) erected

Senecio subg. Senecio sec. Psacaliopsides, to include the anomalous P. pudica. In 1989,

Turner described Senecio paneroi, a species with radiate, yellow capitula that resembled

Psacaliopsis purpusii, except that its leaves were not secondarily lobed as in the latter species. Later, Turner (1990) described S. macdonaldii, a species with 1-2 nodding discoid capitula and deep purple corollas, which closely resembled P. pudica in most characters but had smaller leaves. Jeffrey (1992) transferred Senecio paneroi and S. macdonaldii to Psacaliopsis, but he provided no justification for this placement.

Pippenalia delphinifolia has the haploid chromosome number n = 30 (McVaugh,

1972; Correa & Pippen, 1978) and Psacaliopsis paneroi var. paneroi has a haploid chromosome number of n = 30 (Strother & Panero, 2001), which are similar to the other members of Mexican tussilaginioid genera that have chromosome counts. Pippenalia delphinfolia has senecioid type pollen grains (Bain et al., 1997), but there are no previous palynological studies recorded for Psacaliopsis. Within the Mexican tussilaginioid group

Digitacalia jatrophoides, Nelsonianthus epiphyticus, Pittocaulon praecox, Psacalium palmeri, P. peltatum, and Roldana lobata have been shown to have senecioid pollen grains, and Robinsonecio gerberifolius and Telanthophora cobanensis and T. grandifolia have helianthoid pollen grains (Bain et al., 1997).

A recent phylogeny using ITS, ETS, and five plastid loci indicated strong support for the close relationship between Pippenalia delphinifolia and Psacalium cirsiifolium.

(Pelser et al., 2010). Pelser et al. (2010) suggested a recent age for the most recent common ancestor of Pippenalia delphinifolia–Psacalium cirsiifolium to be 2.7–5.6 54 million years old. Nuclear ITS and ETS data also strongly supported a relationship between Psacaliopsis purpusii and Barkleyanthus salicifolius, although the plastid data did not support this relationship (Pelser et al., 2010). No other species of Psacaliopsis were included in that phylogenetic work and it did not shed light as to how the five species in Psacaliopsis were related or how they fit among the Mexican tussilaginioid group.

Phylogenetic evidence from Chapter 2 has elucidated the positions of Pippenalia and Psacaliopsis within the Mexican tussilaginioid group. The objectives of this study were to review all available herbarium material of Pippenalia and previously recognized

Psacaliopsis, and to produce a taxonomical assessment of these genera based on morphology, geographic distribution, and a phylogenetic analysis using the ribosomal repeat regions, ITS and ETS.

MATERIALS AND METHODS

The study of vegetative and floral morphological variation among the six species was conducted using 98 vouchered specimens of Pippenalia delphinifolia and the five previously recognized species of Psacaliopsis, including the two varieties of Psacaliopsis

‘paneroi’. Live plants cultivated in a greenhouse at the Brackenridge Filed Laboratory at

The University of Texas at Austin and plants in the field were also used to make morphological observations. Specimens were observed from the following herbaria:

BIGU, F, GH, MEXU, MICH, MO, NY, TEX, UC, and US. Digital photographs were also examined from BM, F, K, MEXU, NY, and US. When possible, types were observed 55 either as herbarium vouchers or as digital scanned images. Fieldwork was conducted in the Mexican States of and , and the Guatemalan Departments of

Huehuetenango and San Marcos. Herbarium abbreviations follow Index herbariorum

(Holmgen et al., 1990). Several species within the Mexican tussilaginioid group, including Psacaliopsis pinetorum, were observed during anthesis in a greenhouse and cypselae were collected following anthesis.

A phylogenetic analysis was conducted using 53 taxa (12 genera and 52 species in the Mexican tussilaginioid group), including Pippenalia delphinifolia the five published species of Psacaliopsis (Appendix 3.1). Gynoxys buxifolia was selected as the outgroup.

Methods for the DNA isolation, alignment, and the phylogenetic analyses are described in Chapter 2. Sequences for two taxa were from previously published studies (Pelser

2007, 2011). The GTR+G model was determined to be the best model fitting the combined ITS/ETS data set and this substitution model was selected using the Akaike information criterion (AIC) in Modeltest v.3.06 (Posada & Crandall, 1998; http://darwin.uvigo.es/software/modeltest.html).

RESULTS AND DISCUSSION

MORPHOLOGICAL RESULTS—Within the Mexican Tussilaginioid group,

Pippenalia delphinifolia has a unique combination of characters: acaulscent growth habit, radiate capitula, and the absence of a pappus. Table 3.1 summarizes the major morphological differences among the taxa previously recognized as Psacaliopsis according to their current taxonomical treatment. 56 HABIT AND VEGETATIVE MORPHOLOGY—Pippenalia delphinifolia is recognized as an acaluscent, scapose herb with fibrous roots and pedately-divided, subpeltate leaves.

The leaves of Pippenalia strongly resemble several species of Psacalium (e.g., P. cirsiifolium, P. napellifolium, P. nelsonii). The species of Psacaliopsis are acaulscent, scapose herbs arising from woody rhizomes with orbicular, centrally peltate, basal leaves.

Psacaliopsis purpusii is the only species in the genus with secondarily lobed leaves. The leaves of P. paneroi and P. pinetorum are extremely similar in shape, pubescence, and the variation in the leaf lobes, which range from being undulate to shallowly lobed with sinus depths to ¼ to the center of the blade, and the lobes range from rounded to acute.

Psacaliopsis macdonaldii and P. pudica have similar leaf morphology with respect to leaf shape and leaf lobe characteristics. However, the leaves of P. macdonaldii are larger than those of P. pudica. The leaves of these two species resemble several species of

Psacalium (sensu Rydberg 1924a).

FLORAL AND CYPSELA MORPHOLOGY—Pippenalia delphinifolia has a single radiate capitulum with golden yellow ray flowers and yellow disc flowers. Psacaliopsis paneroi, P. pinetorum, and P. purpusii have several to many radiate capitula with yellow ray and disc flowers. Psacaliopsis paneroi and P. pinetorum appear to have the same floral characters and phenotypic variation within these characters. Psacaliopsis macdonaldii and P. pudica have 1–2 nodding discoid capitula, with deep purple corollas;

P. macdonaldii has 1–2 capitula and P. pudica always have a single capitulum. These species are unique among the Mexican tussilaginioid group with respect to this

57 combination of characters. The nodding discoid capitula resemble those of several species of Psacalium with peltate leaves (sensu Rydberg, 1924a).

POLLINATION AND PHENOLOGY—It is purported that these plants are outcrossers, as this was observed for Psacaliopsis pinetorum, eight species of Roldana, three species of Telanthophora, two species of Psacalium, and Barkleyanthus salicifolius. Viable cypselae was evidence for cross-pollination. Barkley (1988) concluded that the widespread and diverse “aureoid assemblage” (Senecioneae: Senecioninae) in North

America were mostly outcrossers with few barriers to hybridization. He also stated that these species were commonly visited by ‘generalist’ insect pollinators, an observation also made in species of Psacalium, Roldana, and Telanthophora in the field and in a greenhouse, such as blowflies (Diptera: Callophoridae) and paper-nest wasps

(Hymneoptera: Vespidae).

Pippenalia has been collected in flower August to October. The species formerly in Psacaliopsis flower between June and November.

PHYLOGENETIC ANALYSES—Phylogenetic studies of the Mexican tussilaginioid genera based on nrDNA included the five purported species of Psacaliopsis (Chapter 2;

Fig 3.1). The aligned ITS/ETS data set is 1296 bp in length and represents 53 taxa with

52 ingroup species in 11 genera and one outgroup. Of 1296 total characters in the aligned matrix, 352 (27%) were parsimony-informative, 740 (57%) were constant, and 204 (16%) were parsimony-uninformative. The tree resulting from the Bayesian analysis of the 53 accessions with posterior probabilities and ML bootstrap values is presented in Fig. 3.1.

Gynoxys buxifolia is strongly supported as the outgroup. Psacaliopsis purpusii forms a 58 well-supported clade with Robinsonecio gerberifolius. This clade is sister to the rest of the Mexican tussilaginioid genera. Psacaliopsis paneroi var . juxtahuacensis and P. pinetorum form a well-supported clade. Psacaliopsis macdonaldii and P. pudica are within a clade that includes four species of Psacalium and Pippenalia delphinifolia. In order to support the monophyly of Psacalium, the two species of Psacaliopsis with discoid capitula and the anomalous Pippenalia delphinifolia will be transferred to

Psacalium.

DISTRIBUTION AND ECOLOGY—The study taxa occur in montane forests and alpine habitats at elevations ranging from 1800 ̶ 3600 meters and are distributed from central

Mexico to Honduras and El Salvador.

TAXONOMIC IMPLICATIONS— Based on the molecular and morphological data, the following taxonomic changes are made.Pippenalia delphinifolia is transferred to

Psacalium. As the type species, Psacaliopsis purpusii will remain in the genus. Based on observations of herbarium vouchers, including type specimens, Psacaliopsis paneroi is treated as a taxonomic synonym of P. pinetorum, and therefore P. pinetorum is the correct name for this taxon. The variety P. paneroi var. juxtlahuacensis becomes P. pinetorum var. juxtlahuacensis. Psacaliopsis macdonaldii and P. pudica, discoid species with deep purple corollas, are transferred to Psacalium. The ITS/ETS data also support this transfer, although plastid markers would be required to confirm this phylogeny.

A key to the Mexican tussilaginioid group reflecting these changes is provided below.

59 KEY TO THE MEXICAN TUSSILAGINIOID GROUP

1. Capitula discoid; corollas white, cream, yellow, or deep purple

2. Leaves chiefly basal, forming rosettes………………….………………….Psacalium

2. Leaves evenly distributed on stem

3. Plants short (less than 20 cm tall), corollas bright yellow...... Yermo

3. Plants taller (more than 50 cm tall), corollas white, cream, yellow, or purple

4. Capitula few, ecalyculate……………………………...………….Arnoglossum

4. Capitula many, calyculate

5. Corolla deeply lobed more than ½ of the tube…………….…...... Digitacalia

5. Corolla shallowly lobed less than ⅓ of the tube..…Roldana (Pericalia group)

1. Capitula radiate; corollas yellow

6. Scapose herbs with cauline leaves reduced and bract-like or absent

7. Leaves centrally peltate

8. Leaves secondarily lobed, adaxial surfaces strigose, mostly along the veins;

phyllaries in two subequal series………………………….…...….Psacaliopsis

8. Leaves not secondarily lobed, adaxial surfaces pilose with multicellular

trichomes expanded at the base; phyllaries in one series…………....Funstonia

7. Leaves not centrally peltate

9. Leaves ovate to spathulate; pappus bristles numerous……...…….Robinsonecio

9. Leaves laciniate; pappus absent...… ……………...…Psacalium delphinifolium

6. Herbs, shrubs, or small trees with cauline leaves prominent

10. Stems with chambered piths 60 11. Stems succulent; leaves absent during anthesis……………….…Pittocaulon

11. Stems woody; leaves present throughout season……..…..…...Barkleyanthus

10. Stems with solid piths

12. Epiphytic shrubs...... …………..… ………………………Nelsonianthus

12. Terrestrial suffrutescent herbs, shrubs, or small trees

13. Leaves pinnately compound…………………………...... …Villasenoria

13. Leaves simple

14. Leaves chiefly terminal on contracted stems………...... Telanthophora

14. Leaves distributed throughout the stems………………...…….Roldana

TAXONOMIC TREATMENT

PSACALIOPSIS H. ROBINSON & BRETTELL Phytologia 27: 408. 1974. —TYPE:

Psacaliopsis purpusii H. Rob. & Brettell

Perennial herb, rhizomatous. Stems striate, slender. Leaves basal, centrally peltate, blades orbicular, secondarily lobed. Margins entire with occasional denticulations. Lobe tips mucronate. Petioles slender, long, pubescent. Roots: thick, fibrous, from woody rhizomes. Capitulescences borne on long slender scapes, 1 ̶ 10 linear caliculae. Capitulescence of 5 ̶ 18 capitula, capitula radiate, erect. Receptacle epaleate.

Phyllaries in two subequal series, 12 ̶ 20 phyllaries per capitulum, 8 ̶ 20 mm in height, lanceolate with acute apices, margin of phyllaries with dense, glandular pubescence. Ray corollas yellow. Disc corollas yellow. Cypselae fusiform, glabrous, 5 ̶ 6 mm in height, ca.

1 mm wide; 6 ̶ 10 ribs. Both disk cypselae and ray cypselae, similar. 61

Psacaliopsis purpusii (Greenman ex Brandegee) H. Robinson & Brettell Phytologia 27:

408, 974.—Basionym: Senecio purpusii Greenman ex Brandegee Univ. Calif. Pub. Bot.

3: 393, 1909.—TYPE: MEXICO. Oáxaca: Cerro Verde, Jul 1908, C.A. Purpus 3140 holotype: (F!), isotype: (UC!).

Perennial herb 52 ̶ 60 cm tall, arising from woody rhizomes with thick roots.

Stems tomentose at base, becoming sparsely pilose early on scape, puberulent at scape apex. Petioles 10.5 ̶ 20.5 cm long, purple, puberulent to pilose. Petiole bases expanded and tomentose. Blades 5.5 ̶ 14.5 cm wide, abaxial surface tomentose, adaxial leaf surface strigose mostly along the veins, 7 ̶ 9 primary lobes with secondary lobes present. Primary lobes lanceolate, rounded to acute apices. Sinus depth up ½ ̶ ¾ length to center of blade.

Primary lobe width at base 0.6 ̶ 2.8 cm. Capitulescence of 2 ̶ 7 erect capitula, Capitula radiate, 1.5 ̶ 2.0 cm wide. Phyllaries 10 ̶ 12, in 1 series, 7 ̶ 10 mm long , sparsely strigose with glandular trichomes. Ray florets 10 ̶ 12, ligules yellow, 9 ̶ 10 mm long. Disk florets

>50, 10 ̶ 12 mm long, corollas yellow, apices blunt. Cypselae fusiform, 4 ̶ 6.5 mm long, 6

̶ 8 ribbed, glabrous; the pappus of many barbellate bristles 6 ̶ 8 mm long.

PHENOLOGY—Flowering June through November.

DISTRIBUTION AND HABITAT—This species occurs in Puebla and Oáxaca, Mexico, and is considered endemic (Fig. 3.1), growing in pine-oak forests between 2200 ̶ 2900 meters.

ADDITIONAL SPECIMENS EXAMINED—MEXICO. Oáxaca: Mpio. Tamazulapan,

Cerro Pericón al NW de San Pedro Nopala, 1 Jun 1985, P. Tenorio L. 8953 (F, MEXU, 62 TEX, US); Cerro Pericón al NW de San Pedro Nopala, 6 Jul 1986, P. Tenorio L. 11621 con A. Salinas T., A. Garcia. M. y D. Frame (F, MEXU); Cerro Pericón, 8-10 km north of

San Miguel Nopala on the road to Yosonuco, 4 Nov 1992, J.L. Panero 2607 con Patricia

Davila y P Tenorio L. (MEXU, TEX). Puebla: Mpio. Caltepec, Cerro El Gavilan de

Ejido, SW de Caltepec, 7 Nov 1984, P. Penoria L. 7996 con C. Romero y L. Tenorio M.

(MEXU, TEX). Municipio unknown: Cerro de los Gentiles, Aug 1909, C.A. Purpus 3843

(F, NY, UC, US).

DISCUSSION—Psacaliopsis purpusii morphologically resembles P. pinetorum, but differs in having secondarily lobed leaves (vs. leaves not secondarily lobed), phyllaries in one series (vs. phyllaries in two subequal series), and 5–18 capitula (vs. 9–45 capitula).

This species has few collections and more fieldwork is needed to determine the extent of this species’ range distributions.

FUNSTONIA Quedensley & Villaseñor gen. nov. (in prep).

Acaulescent, perennial herb 40 ̶ 80 cm tall with thick roots arising from woody rhizomes. Stems tomentose at base, becoming sparsely tomentose early on scape, puberulent at scape apex. Petioles 5 ̶ 17 cm long, purple, sparsely strigose. Petiole bases expanded and tomentose. Blades 5 ̶ 9 cm wide, abaxial surface variously pubescent, adaxial leaf surface pilose with multicellular trichomes tapering from broad bases, 8 ̶ 10 lobes, deltoid to rounded, rounded to acute apices. Sinus depth up to 1/3 length to center of blade. Lobe width at base 1.2 ̶ 3.2 cm. Capitulescence of 7 ̶ 50+ erect capitula. Capitula radiate, 0.8 ̶ 1.0 cm wide. Phyllaries 13 ̶ 16, in 2 subequal series, 10 ̶ 12 mm long, densely 63 hirsute. Ray florets 8 ̶ 10, ligules yellow, 10 ̶ 12 mm long. Disk florets 15 ̶ 30, 7 ̶ 9 mm long, corollas yellow, apices blunt. Cypselae fusiform, 3 ̶ 4 mm long, 6 ̶ 8 ribbed, glabrous; the pappus of many barbellate bristles 8 ̶ 10 mm long. .—TYPE: —MEXICO:

Oáxaca, Cordillera of Oáxaca, Nov-Apr 1980, Galeotti 2019 lectotype: (K); isolectotype:

(G!); photo; (MICH!).

Funstonia pinetorum (Hemsl.) Quedensley & Villaseñor comb. nov. (in prep). —

Basionym: Senecio pinetorum Hemsl. Biol. Cent.-Amer., Bot 2: 245, 1881.

Psacaliopsis pinetorum (Hemsl.) Funston & Villaseñor Ann. Missouri Bot. Gard. 95:

334 ̶ 335, 2008.

Roldana pinetorum (Hemsl.) H. Robinson & Brettell Phytologia 27: 423. 1974.

Senecio paneroi B.L. Turner Phytologia 67: 454. 1989.—TYPE: MEXICO: Guerrero: km

70-71 de la carretera Chilapa-Tlapa, E of Chilpancingo, 2 Nov 1986, J.L. Panero,

E.E. Schilling & B.E. Wofford 582 holotype: (TENN!); isotype: (MEXU!, TEX!).

Psacaliopsis paneroi (B.L. Turner) C. Jeffrey Kew Bull. 47: 55. 1992.

CHROMOSOME NUMBER—n = 30 (Strother & Panero, 2001).

PHENOLOGY—Flowering July through January.

DISTRIBUTION AND HABITAT—This species has been collected in the states of

Guerrero and Oáxaca, Mexico, and El Salvador and Honduras (Figs 3.1 and 3.2). It occurs in mixed conifer-oak forests, generally on steep slopes at elevations of 1800 ̶ 2700

64 meters. Funston (2008) reports that this species has been collected near Santa Ana, El

Salvador, although she does not cite a vouchered specimen.

REPRESENTATIVE SPECIMENS EXAMINED—MEXICO. Guerrero: Mpio. Atlamajalcingo del Monte, 16 km SW of Tototepec, 5 km SE of Quiahuitlazala, road from Tlapa, 2 Aug

1981, F. Lorea 1298 (MEXU). Mpio. Chilapa de Alvarez, Chilpancingo to Tlapa, 19 km east of Chilapa, microondas Pozo Largo at km 74, 12 Jan 2010, T. Sultan Quedensley

10193 (MEXU, TEX), Estación de microondas Pozo Largo, carretera Chilapa a Tlapa, 27

Dec 1993, J.I. Calzada 19000 (MEXU); km 72 along the highway between Chilpacingo,

Chilapa, and Tlapa, 18 Sep 1993, J.L. Panero 3315 con I. Calzada (CAS, MEXU, TEX);

Chilapa de Alvarez, km 130 along the highway between Chilpacingo, Chilapa, and Tlapa,

3 Jul 1994, J.L. Panero 3991 con I. Calzada (NY, TEX). Mpio. General Heliodoro

Castillo, La Guitarra, 26 Jan 1993, F.H. Belmont F. 49 (MEXU). Mpio. Leonardo Brava,

16 km SW of Filo de Caballo, along road from Filo de Caballo to Atoyac, 13 Jan 2010, T.

Sultan Quedensley 10197 (MEXU, TEX). Mpio. Tlacatepec, km SW from La

Hierbabuena, along highway between Filo de Caballo and Puerto del Gallo, 17 Aug 1985,

J.C. Soto Núñez 10034 (MEXU); along road from Puerto del Gallo to Puentecilla, 6.7 mi.

SW of junction with road to Atlixtac, 30 Nov 1984, D.M. Spooner 2843 (MEXU); along stream 35.5 km NW of Filo de Caballo along road to Atoyac, 11 Oct 1986, D.E.

Breedlove 65205 (CAS, MEXU, TEX, US). Municipio unknown: 12 km SW from El

Jilguero, 71 km SW from Filo de Caballo, along the road to Cerro Teotepec and Puerto del Gallo, 4 Oct 1986, J.L. Villaseñor 984 (UC); El Jilguero, 30 Oct 1998, N. Diego, B. 65 Ludlow & A. Acosta 8128 (MEXU); 3 km from Escalarilla along the road to Agua Fria, 2

Nov 1998, N. Diego, B. Ludlow & J.M Davila 8307 (MEXU). Oáxaca: Mpio.

Comaltepec, S. Comaltepec, 12 Nov 1988, L. López López 0318 (MEXU, UC). Mpio.

Macuiltianguis, ca. de Puerta del Sol, rumbo a San Pablo-Macuiltiangus a km 8, 10 Jan.

1981, R. Ortega O. & M. Ortiz T. 1621 (TEX). Mpio. de Zimatlán, Paraje Tierra Blanca,

8 km southeast of La Cofradía, community of San Pedro El Alto, pine-oak forest. A.

Niranda y O. Hernández 504 (MEXU); Noreste de la Navidad, 29 Oct 2003, Figueroa

Brito Sandra y Guzmán Rivera Flora Yadira 880 (MEXU). HONDURAS. Lempira: along trail from El Mojon to El Sucte, 31 Jan 1992, H. Thomas 159 (MO).

DISCUSSION—Based on Turner (1989), who did not see type material of Psacaliopsis pinetorun prior to the description of P. paneroi, observations in the field with J.L.

Villaseñor, and comparison of type material, we have determined that Psacaliopsis paneroi and its two varieties fall within the variation of P. pinetorum. This is the most wide spread species in the genus and it is expected to occur in pine-oak forest of Chiapas,

Mexico, and Guatemala, where it has not yet been reported.

KEY TO THE VARIETIES OF FUNSTONIA PINETORUM

1. Abaxial leaf surface densely pubescent (not arachnose) without punctate dots

………………………………………………………………. pinetorum var. pinetroum

1. Abaxial leaf surface arachnose with punctate dots...... F. pinetroum var. juxtlahuacensis

66 Funstonia pinetroum var. juxltahuacensis comb. nov. (Panero & Villaseñor)

Quedensley & Villaseñor in prep. Basionym: Psacaliopsis paneroi var.

juxtalahuacensis Panero & Villaseñor Brittonia 48: 81-83. 1996. Oáxaca: Mpio.

Santiago Juxtlahuaca, 2.3 km al S de la desviación a San Martín Peras sobre la

carretera a Coicoyán de Las Flores, 31 Oct 1994, J.L. Panero 5313 con E. Manrique

e I. Calzada. Holotype: (MEXU!); isotypes: (MSC,TEX!, UC!).

CHROMOSOME NUMBER—Unreported.

PHENOLOGY—Flowering November through January.

DISTRIBUTION AND HABITAT—Endemic to the mountains of western Oáxaca.

REPRESENTATIVE SPECIMENS EXAMINED—MEXICO. Oáxaca: Mpio. San Sebastián

Tecomaxtlahuaca, km 15 de la carretera Tecomaxtlahuaca-San Martín Peras, 12 Dec

2005, J.L. Panero 8871 con E. Schilling (TEX). Mpio. San Martín Peras. Loc. Km 30 de la carretera San Sebastián Tecomaxtlahuaca-Coicoyan de las Flores, 30 Nov 1994, J.I.

Calzada 19602 (TEX). Mpio. unkown: Lo. 13 km carretera Santos Reyes Tepejillo,

Llano El Meson, 15 Jan 1995, J.I. Calzada 19663 (MEXU, TENN, TEX);

DISCUSSION—This variety shares most of the characters with the nominate variety and is recognized by a fine arachnose pubescence on the abaxial leaf surfaces that makes the leaves appear shiny and punctate dots are present on the abaxial leaf surfaces. This combination of characters is consistent in all the specimens of P. pinetorum var. juxtlahuacensis observed and is not present in any of the material of P. pinetorum var. pinetorum. The other characters identified by Panero & Villaseñor (1996; i.e., open 67 capitulescence, sparsely pubescent petioles, sparsely pubescent capitulescence) distinguishing this variety it from Psacaliopsis pinetroum var. pinetorum were not consistent throughout the specimens observed for this study. Several specimens labeled as Psacaliopsis paneroi var. juxtlahuacensis have been annotated as P. pinetorum var. pinetorum because they do not have a shiny pubescence or puntate dot on the abaxial leaf surfaces. A key to the varieties of Funstonia pinetorum is provided.

PSACALIUM Cass.

Perennial herbs with mostly basal leaves; cauline leaves reduced, bract-like, or absent.

Leaves non-peltate (sensu Rydberg, 1924b) or peltate (sensu Rydberg, 1924a). Capiutla discoid. Disk corollas white, yellow (in P. matusae), or deep purple (in P. macdonaldii and P. pudicum). Pappus present or absent, or in one species, falling before maturity.

Type species Psacalium peltatum (Kunth) Cass.

This diverse genus consists of 49 species of acaulescent herbs with discoid capitula and white, cream, yellow, or purple corollas. Rydberg (1924a, 1924b) revised

Psacalium and Odontotrichum, both of which included plants with leaves centrally peltate or non-peltate to subpeltate, respectively. McVaugh (1972) transferred

Odontotrichum delphinifolium to Pippenalia, a new genus that contained this single species. Robinson & Brettell (1973d) revised Psacalium, which included the transfer of the remainder of the species previously in Odontotrichum to Psacalium. Although this genus has previously been noted to include seven species with purple flowers (Bremer, 68 1994; Arneberg et al., 2007), after reviewing the descriptions of those seven species during the present study, six of these species had purplish phyllaries or phyllary tips. One species, Psacalium nelsonii, was reported as having “corollas purplish tinted when dry.”

This purplish color in the dried corollas however is most likely a result of the oxidation of the corolla tissue. Therefore, no true purple-flowered Psacalium has been included in the genus prior to this study.

1. Psacalium macdonaldii (B.L. Turner) Quedensley & Villaseñor comb. nov. (in prep.)—Basionym: Senecio macdonaldii B.L. Turner Phytologia 69: 361 ̶ 363, 1990.

TYPE: MEXICO. Oáxaca: Mpio. Miahuatlán, 35 km. ESE of Miahuatlán, 5 km NE of

Santo Domingo Ozoltepec, Cerro Quiexobra, 3 Oct 1990, Andrew MacDonald 2992 holotype: (TEX!); isotype: (MEXU!).

Psacaliopsis macdonaldii (B.L. Turner) C. Jeffrey Kew Bull. 47: 55.

Plants acaulescent herbs, 35 ̶ 90 cm tall, with thick roots arising from woody rhizomes. Stems sparsely strigose at base, becoming sparsely hirsute early on scape, densely strigose at scape apex. Petioles 7 ̶ 18 cm long, purple, sparsely pilose. Petiole bases expanded and lanate. Blades 7.5 ̶ 9.5 cm wide, abaxial surface tomentose, adaxial surface glabrous with punctuate dots, 8 ̶ 11 lobes, lobes deltoid, acute apices. Sinus depth less than ¼ length to center of blade. Lobe width at base 1.3 ̶ 2.4 cm. Capitulescence of 1 ̶

2 nodding capitula, Capitula eradiate, 2.2 ̶ 3.4 cm wide. Phyllaries 14 ̶ 16, in 2 subequal series, 13 ̶ 15 mm long, sparsely strigose. Disk florets >50, 11 ̶ 15 mm long, corollas deep

69 purple, apices acute. Cypselae fusiform, 6 ̶ 8 mm long, 8 ̶ 10 ribbed, glabrous; the pappus of many barbellate bristles 7 ̶ 8 mm long.

CHROMOSOME NUMBER—Unreported.

PHENOLOGY—Flowering August through October.

DISTRIBUTION AND HABITAT—Psacalium macdonaldii is endemic to Oáxaca and has only been collected from two regions of this Mexican state (Fig. 3.2) and occurs in pine forests from 3300—3600 meters.

ADDITIONAL SPECIMENS EXAMINED—MEXICO. Oáxaca: Mpio. Miahuatlán,

Quiexobra, 7 Aug 1996, G.B. Hinton et al. 26794 (TEX). Mpio. San Pedro Mixtepec,

East of San José Pacifico, along the road to San Francisco Ozoltepec, 26 Jul 2002, L.

Schibli 124, con V. Chao y E. Correa. Mpio. San Juan Mixtepec, Cima del cerro Nube

Flan, 8 August 2008, S.H. Salas 6455 con E. Fuentes y P. Krasilnikov (TEX).

DISCUSSION—This species is closely related to Psacalium pudicum based on morphological similarity. The two species share an acaulescent growth habit with basal, peltate leaves and nodding, discoid capitula with 14—16 phyllaries in 2 subequal series and deep purple corollas. These species differ in that P. macdonaldii has 1—2 capitula, whereas P. pudicum has a single capitulum. Psacalium macdonaldii has larger leaves than P. pudicum, petioles 7—18 cm long and blades 7.5—9.5 cm wide in P. macdonaldii vs. petioles 5–8.5 cm long and blades 0.9–2.4 cm wide in P. pudicum. A phylogenetic analysis has also demonstrated a sister relationship between these two species (Fig. 3.1).

70 2. Psacalium pudicum (Standley & Steyermark) Quedensley & Villaseñor comb. nov.

(in prep.)—Basionym: Cacalia pudica Standley & Steyermark Field Mus. Bot. Ser. 23:

255. 1947.— TYPE: GUATEMALA. Huehuetenago: near Chemal, 9 Aug 1942, J.A.

Steyermark 48344 holotype: (F!).

Psacaliopsis pudica (Standley & Steyermark) H. Robinson & Brettell Phytologia 27:

408.

Pericalia pudica (Standley & Steyermark) Cautrecasas Brittonia 8: 157. 1957.

Senecio nubivagus L.O. Williams Phytologia 31: 441. 1975.

Plants 14.5–18.5 cm tall, acaulescent herbs with thick roots arising from woody rhizomes. Stems tomentose at base, becoming sparsely strigose early on scape, densely strigose at scape apex. Petioles 5–8.5 cm long, upper half purple, densely strigose. Petiole bases expanded and lanate. Blades 0.9–2.4 cm wide, abaxial surface tomentose, at least on veins, adaxial surface glabrous, 7–8 lobes, lobes triangular, rounded to acute apices.

Sinus depth up to ¼ length to center of blade. Lobe width at base 0.7–1.9 cm.

Capitulescence of one nodding capitulum, Capitula eradiate, 2.2–2.8 cm wide. Phyllaries

14–16, in 2 subequal series, 11–17 mm long , sparsely pilose. Disk florets >50, 12–14 mm long, corollas deep purple, apices acute. Cypselae fusiform, 7 ̶ 8 mm long, 6 ̶ 8 ribbed, glabrous; the pappus of many barbellate bristles 6 ̶ 8 mm long.

CHROMOSOME NUMBER—Unreported.

PHENOLOGY—Flowering July through November.

71 DISTRIBUTION AND HABITAT— Psacalium pudica is endemic to Sierra

Cuchumantanes in the departments of San Marcos and Huehuetenango in western

Guatemala (Fig 3.3). This species is restricted to alpine meadows above 3300 meters.

ADDITIONAL SPECIMENS EXAMINED—GUATEMALA. Huehuetenango: Mpio.

Todos Santos Cuchumatán, between Llano de San Miguel and Todos Santos

Cuchumatán, 29 Aug 1976, D.N. Smith 392 (F); La Torre, 25 Sep 1996, R. Morales, J.

Gálvez y M. Véliz 5718 (BIGU, TEX); Camino a La Torre, 27 Aug 1997, N. Hernández y

M. Véliz 6066 (BIGU, MEXU, TEX); Aldea Chichím, 11 Aug 1999, M. Véliz y R.

Morales 7222 (BIGU, MEXU, TEX); Camino a La Torre, 5 Sep 2001, M. Véliz 11397 con J. Véliz (BIGU, MEXU); La Torre, 5 Aug 2002, M. Véliz 12447 con J.C. Gálvez

(BIGU, MEXU); Camino a La Torre, 6 Aug 2003, M. Véliz 14027 con J. Véliz y R.

Morales (BIGU); Llano del Diablo, 18 Jul 2008, M. Véliz 20316 con L. Velásquez

(BIGU); Llano del Diablo, 18 August 2007, C. del Cid 41 y J. Vega (BIGU); near

Chémal. 9 Aug 1942, J.A. Steyermark 50329 (F); between Tojiah and Chemal at Km.

317.5 on Ruta Nacional 9 North, 30 Jul 1960, J.H. Beaman 3817 (MSU); Chemal at Km.

316.8 on Ruta Nacional 9 North, 4 Aug 1959, J.H. Beaman 3084 (MICG, NY, TEX, UC,

TEX). San Marcos: Mpio. Ixchiguán, Cerro Cotzic, 20 Nov 1976, D.N. Smith 509 (F).

Municipio unknown: Volcán Tajumulco, 7 Aug 2007, M. Véliz 10455 con M. Vásquez y

R. Luarca (BIGU).

DISCUSSION—This species was originally described as a Cacalia as it has discoid capitula and non-yellow corollas (Standley & Steyermark, 1947). It has smaller leaves 72 and a single nodding capitulum relative to the larger leaves and 1–2 nodding capitula of its purported sister species, Psacalium macdonaldii. Psacalium pudicum is abundant in alpine meadows and it occurs in areas where sheep and goat grazing is heavy. See discussion of P. macdonaldii above for species comparisons. Williams (1975) erected

Senecio subgenus Senecio section Psacaliopsides, which only included Psacaliopsis pudica, which was given a new name Senecio nubivagus by Williams because of the previously named Senecio pudicus Greene. However, Robinson & Brettell had already transferred Cacalia pudica to Psacaliopsis the previous year. Psacaliopsis macdonaldii and P. pudica share vegetative and floral similarities to Psacalium sensu Rydberg

(1924a), such as centrally peltate leaves and nodding capitula. but is similar with respect to floral morphology to other species in Odontotrichum (i.e., acaulescent growth habit and discoid capitula).

3. Psacalium delphinifolium (Rydb.) Quedensley & Villaseñor comb. nov. (in prep.)—

Basionym: Odontotrichum delphinifolium Bull. Torrey Bot. Club 51: 419. 1924.—

TYPE: Mexico. Huehuetenago: near Chemal, 9 Aug 1942, Rose 2390 holotype: (US!).

Pippenalia delphinifolia (Rydb.) McVaugh Cont. Univ. Herb. 9: 470. 1972.

Plants 40–50 cm tall. acaulescent herbs with fleshy fibrous roots. Stems lanate with pale brown trichomes at base, becoming pilose early on scape. Petioles 20–30 cm long, pilose above bases. Petiole bases expanded and lanate with pale brown trichomes. Blades subpeltate, the petiole inserted 1–2.5 mm from the basal sinus; 6–12 cm long, 15–20 cm 73 wide, pedately-divided, abaxial surface sparsely pilose, adaxial surface pubescent, especially near the veins at the base of the blade, 6–7 lobes, sinus depth more than ¾ length to the petiolar attachment of blade. Capitulescence of 1 erect capitulum, Capitula radiate, 6–9 cm wide. Phyllaries 13–21, in 2 subequal series, 9–15 mm long, blunt tips ciliate-fringed. Ray florets 12–18, ligules golden yellow, 2.2–3.5 cm. long. Disk florets

>180, 12–14 mm long, corollas yellow, apices acute. Cypselae fusiform, 4.5 ̶ 6 mm long,

15 ̶ 20 ribbed, glabrous; pappus absent.

CHROMOSOME NUMBER—n = 30 (Keil & Stuessy, 1977; Corea & Pippen 1978).

See discussion below.

PHENOLOGY—Flowering August through October.

DISTRIBUTION AND HABITAT—Psacalium delphinifolium occurs in pine-oak forests from 1900–2700 meters.

REPRESENTATIVE SPECIMENS EXAMINED—MEXICO. Aguascalientes: Mpio.

Calvillo, Sierra del Laurel, near the Jalsico-Aguascalientes border, ca. 10 mi southeast of

Calvillo, 26 Aug 1960, R. McVaugh 18427 (TEX). Chihuahua: Mpio. Guadalupe y

Calvo: Circa 212 km SSW of del Parral, 78.9 km SW of El Vergel on road to

Guadalope y Calvo, ca. 1.3 km s of bridge in Turachi, 25 Aug 1983, G. Nesom 4965

(TEX); top of Turachi Canyon, 3 mi S of bridge in Turachi, 49 mi SW of El Vergel, 22

Aug 1988, G. Neson 6510 with A. McDonald (TEX). Llano Grande, 13 May 1960, C.W.

Pennington 88, (TEX), Sierra Chinatú, 9 Oct 1959, D.S. Correll & H.S. Gentry 22951,

(TEX). Durango: Mpio. Canelas, on the road to Topia and Canelas, 5 km E of junction of this road with road to Topia (at Cuevacillas), 29 Jun 1992, R. Spellenberg & J. Bacon 74 11048, (TEX); Santiago Papasquiaro, 30 Aug 1991, J.L. Panero 2255 con S. González y

S. Acevedo (TEX). Mpio. Mezquital, 48 km west northwest of Huejuquilla El Alto,

Jalisco, on raod to Canoas, 21 Oct 1983, D.E. Breedlove 59032 with F. Almeda (TEX).

South of Santa Maria de Ocotán, 16 Sep 1986, M. González 1918 (TEX); 1 km al W de entroque al Llano Grande, camino al La Guajolota, 17 Jun 1985, M. González et al. 1728

(TEX). Mpio. Pueblo Nuevo, Sierra Madre Occidentale, along free highway 40 from

Durango to Mazatlán, 22 mi west of El Salto at km marker 135, 5 Aug 2009, T. Sultan

Quedensley 10133 (MEXU, TEX). Mpio. Tayoltita, 8 km al NE de Fraylecitos, 8 Jul

1984, P. Tenorio L. 6340 (TEX). : Mpio. Jocotepec, Ladera exps. NE que va a la puerta al la Chica, 7 Jun 1986, J.A. Machuca N. 1556 (TEX). Mpio. Mezquitic, Sierra

Huichola, Campamento Forestal Penitas, 24 Jul 1996, M. Flores Hdez. y R.O. Barrera R.

8 (TEX). Mpio. Tlajomulco, Cerro Viejo, ladera exposicion Norte, enfrente de San

Miguel Cuyutlan, 30 Jul 1986, J.A. Machuca N. 3879 (TEX). Zacatecas: Mpio.

Tlatenango, 27.6 km al NW de la carretera Jalpan-Guadalajara sobre la carretera a

Tlaltenango, 4 Oct 1995, J.L. Panero 6174 con C.C. Cleavinger (TEX). Municipio unknown, Brecha Jalpa-Tlaltenango, 2 Aug 1971, C. L. Díaz L. 2370 (TEX).

DISCUSSION— Pippenalia delphinifolia is in a well-supported clade with other

Psacalium species, warranting transfer to Psacalium based on leaf morphology and the nrDNA data (Fig. 3.1). Although McVaugh described the species as having peltate leaves, based on my observations in the field and among herbarium specimens at MEXU,

TEX, and US, the leaves blades are not centrally peltate and would be better described as subpeltate. The type species for Odontotrichum is Psacalium cirsiifolium, which has 75 leaves similar to Pippenalia delphinifolia. Although the haploid chromosome has been shown to be n = 30 in all published chromosomes count with in the Meixcan tussilaginioid group, However, it was noted by others that this number of 30 in

Pippenalia was not precise due to meiotic irregularities in the chromosome pairing (Keil

& Stuessy, 1997; Correa & Pippen, 1978).

76 Table 3.1. Comparative morphology and geographic distribution of Funstonia pinetorum,

Psacaliopsis purpusii, Psacalium delphinifolium, P. macdonaldii, and Psacalium

pudicum.

Funstonia pinetorum Psacaliopsis purpusii Psacalium delphinifolium P. macdonaldi P. pudicum

Leaf lobing Shallowly lobed Deeply and Pedately lobed Shallowly lobed Shallowly lobed to lobed midway secondarily lobed to the center of the blade Capitula 9–45 erect 5–18 erect 1 erect 1–2 nodding 1 nodding capitula capitula capitula capitulum Phyllaries 12–16 in two 13 in one series 13–21 14–16 in two 10–18 in two subequal series subequal series subequal series Number of ray 5–8 5–7 12–18 None None florets

Number of disc 20–27 20–50 180–230 50-80+ 50-80+ florets

Floret color Yellow Yellow Yellow Deep purple Deep purple

Cypselae 3–4 mm long 4–6.5 mm long 4–6.5 mm long 6–8 mm long 6–7 mm long

Geographical Central Mexico Oáxaca and Aguascalientes, Oáxaca, Mexico Huehuetenango to El Salvador Puebla, Mexico Chihuahua, and San Marcos, range and Honduras Durango, Guatemala Jalisco,Zacatecas

77

s m Yermo xanthocephalu

Arnoglossum atriplicifoliu Barkleyanthus salicifolius 1 100 Fig 3.1. Bayesian inference phylagram of the Mexican Tussilaginioid group based on group Tussilaginioid of the Mexican phylagram inference 3.1. Bayesian Fig the branches. above are probabilities ITS/ETSset of 53 taxa. Posterior data a combined in the species placed species in bold are The the branches. below are values Bootstrap this study. prior to or Psacliopsis Pippenalia tzimolensis vellatum jatrophoides var. var. pentaloba Robinsonecio gerberifolius var. 4 var. 0 . 0 Villasenoria orcuttii Psacalium pudicum Pscalium macdonaldii 1 oaxacana Pittocaluon bombycophole 100 pinetorum Psacalium palmeri

juxtlahuacensis Pittocaulon filare var. Psacalium megaphyllum var. Pittocaulon vellatum Roldana eriophylla var.

Psacalium cirsiifolium Pittocaulon praecox

1 Psacalium delphinifolium Psacalium peltatum 100 Pittocaulon vellatum Roldana lobata Telanthophora grandifolia Digitacalia jatrophoides Telanthophora cobanensis Digitacalia jatrophoides 1 Digitacalia crypta 1 .92 1

99 Roldana platanifolia Roldana reticulata 100 Roldana mexicana Telanthophora andrieuxii Telanthophora uspantanensis 100 Roldana heracleifolia Roldana uxordecora Roldana marquezii Roldana lineolata Roldana hartwegii Telanthophora liebmannii Roldana sessifolia 1 Roldana gilgii 97 1 100 Roldana ehrenbergiana Roldana heterogama Roldana metepecus Roldana acutangula 93 Roldana lanicaulis Roldana mixtecana Roldana hintonii Roldana petasitis Roldana robinsoniana 1 Roldana barba-johannis

1 1 Roldana aschenborniana 98

1 95 100 Roldana sundbergii Psacaliopsis purpusii

1 98

1 1 95 Funstonia pinetorum

1 1

1 Funstonia pinetorum 100 96 99 100 1

1 1 1 98 90 91 1 100

1 99 98 98 1 .96 .99 .89 100 .92 1 100 1 96 88 .94 Gynoxys buxifolia 88 .98

78 Puebla

Veracruz

Michoacan # + Funstonia pinetorum var. juxtlahuacensis # # # ^^ ^^ ^ ^^ ^ + # ^ ^ Funstonia pinetorum var. pinetorum ^ ++ * Oaxaca Chiapas * # Psacaliopsis purpusii Guerrero ^ ^#* * Psacalium macdonaldii

Figure 3.2. Distribution of species formerly placed in Psacaliopsis in Mexico

79 Belize

oo o Guatemala ^ Honduras

^ El Salvador Nicaragua ^ Funstonia pinetorum var. pinetorum o Psacalium pudicum

Figure 3.3. Distribution of species formerly placed in Psacaliopsis in Central America

80 Illustration 3.1. Funstonia pinetorum var. pinetorum. By Melissa Toberer.

81 Chapter 4: Sequencing, Assembly, and Alignment of Four Chloroplast Genomes

INTRODUCTION

The Asteracaeae is the most species-rich family of flowering plants with ca.

24,000 ̶ 30,000 species (Funk & Robinson, 2005; Funk et al., 2005; Hind, 2007; Kadereit

& Jeffrey, 2007). This diverse family consists of economically important food crops, ornamental plants and cut flowers, culinary and medicinal herbs, and several weedy species that have a negative economic impact on crop production. The Senecioneae, the largest tribe in the family with ca. 150 genera and 3,000 species (Nordenstam, 2003,

2007), exemplifies a wide breadth of ecological and morphological variation, especially with respect to growth form, leaf shape, inflorescence type, and flower color (Barkley,

1985a). Recent efforts in phylogenetics have helped resolve the tribal relationships within the Asteraceae (i.e., Panero & Funk, 2002; Funk et al., 2009), however, the Senecioneae is still ambiguously placed in the family (Panero & Funk, 2008). The Senecioneae is comprised of four subtribes: Blennospermatinae, Othonninae, Senecioninae, and

Tussilagininae (Pelser et al., 2007; Nordenstam et al., 2009). One of the monophyletic groups in the subtribe Tussilagininae, the Mexican tussilaginioid group (Barkley et al.,

1996; Pelser et al., 2007, 2010; Chapter 1), consists of 140 species in 13 genera and the taxa predominantly occur in pine-oak and cloud forests concentrated in montane regions of southern and central Mexico. Over half of the species are endemic to a narrow geographic range above 1500 meters in montane ecosystems that are threatened by human land use practices and the effects climate change. Pelser et al. (2010) suggested a

82 recent age for the Mexican tussilaginioid genera of ca. 9 mya, after which this group rapidly diverged and dispersed throughout the montane regions of Mexico and into

Central America.

Several studies have focused on the development of chloroplast markers to elucidate the evolutionary relationships of groups of species (Small et al., 1998; Shaw et al., 2005, 2007; Mort et al., 2007). Others have focused their search for useful phylogenetic markers from the chloroplast genome of Asteraceae (e.g., Panero & Crozier,

2003; Mort et al., 2007; Timme et al., 2007). Pelser et al. (2007, 2010) conducted a phylogenetic analysis of five chloroplast loci to resolve generic and species delimitations in the Senecioneae, including the Mexican tussilaginioid genera, and although their phylogenies provided some useful information pertaining to the relationships, they lacked resolution and strong statistical support.

Recently several chloroplast genomes in the Asteraceae have been sequenced

(Timme et al., 2007; Kumar et al., 2009; Dempewolf, 2010; Nie et al., 2012), including

Jacobaea vulgaris (Senecioneae; Doorduin et al., 2011). Whole chloroplast genomes are now commonly used to infer phylogenetic relationships at high taxonomic levels among taxa (i.e., Jansen et al., 2006; Jansen et al., 2007; Cronn et al., 2008; Parks et al., 2009; Whittall et al., 2010) and to identify phylogenetic markers useful at other levels (i.e., Timme et al., 2007; Daniell et al., 2006; Saski et al., 2007).

Currently, Jacobaea vulgaris is the only Senecioneae chloroplast genome sequenced (Doordin et al., 2011). Since the Senecioneae is the most species-rich tribe, other chloroplast genomes would enable research groups to identify variable regions in 83 the chloroplast genome that can be used to elucidate phylogenetic relationships within the tribe. Chloroplast genomes were sequenced for Arnoglossum atriplicifolium, Roldana aschenborniana, R. barba-johannis, and Telanthophora grandifolia (Asteraceae:

Senecioneae) with the SOLiD next generation sequencing platform to identify potential phylogenetic markers and to compare results between reference-based and de novo assemblies. Much of the genome sequencing with the SOLiD platform has been associated with de novo assembly methods of bacteria (i.e., Silva et al., 2010; Lee et al.,

2011; Ghosh et al., 2011). The chloroplast genome sequences of these four species were compared with Jacaobaea vulgaris, which provides a reference to which the reads from the sequencing of the four study taxa can be mapped and aligned. Although J. vulgaris is a member of the Senecioneae, it is in a different subtribe; Senecioninae.

Next generation sequencing can produce large amounts of data much quicker and cheaper than standard Sanger sequencing (Shendure & Ji, 2008; von Bubnoff, 2008;

Glenn, 2011), making it easy to sequence nuclear and organellar genomes. Chloroplast genomes have conservative rates of evolution (Wolfe et al., 1987), and previous studies have shown that Asteraceae chloroplast genomes are generally similar in gene order and genome organization (Timme et al., 2007; Kumar et al., 2009; Dempewolf, 2010;

Doorduin et al., 2011; Nie et al., 2012). However, it has been shown that the basal

Barnadesioideae do not have a 22 kb inversion present in all of the of higher Asteraceae, including members of the Senecioneae (Jansen & Palmer, 1987; Kim et al., 2005).

In addition to providing four chloroplast genome sequences, this is the first study utilizing the Applied Biosystems (ABI) SOLiD sequencing platform to sequence vascular 84 plant chloroplast genomes. The four genomes add to the number of Asteraceae chloroplast genomes already published (six). Two different methods for genome assembly were used, reference-based assembly with Jacobaea vulgaris as the reference in

BFAST, and de novo assembly in Velvet. Using the reference-based assembly aligned to the Jacobaea vulgaris genome, 42 intergenic spacer regions and 10 coding regions were analyzed for sequence divergence to identify potential markers in future phylogenetic studies among Senecioneae taxa. The reference-based assemblies are compared to the de novo assemblies.

MATERIALS AND METHODS

Taxon Sampling.⎯Four species in three Mexican tussilaginioid genera were selected for whole genome sequencing based on the nuclear phylogeny of the group

(Table 4.1; Chapter 2, Fig. 2.5). These taxa were cultivated in a greenhouse at the

Brackenridge Field Laboratory at The University of Texas at Austin. Vouchers were deposited at The University of Texas herbarium (TEX).

Chloroplast DNA extraction, isolation, and genome amplification.⎯Young leaves were removed from living plants and 10–12 g of tissue was placed in the dark for

24 hours for each isolation. Chloroplasts were isolated according to Jansen et al. (2005), and cpDNA was amplified with rolling circular amplification (RCA; Qiagen GmbH,

Hilden, ) using bacteriophage Phi29 polymerase and random hexamer primers

(Dean et al., 2001). An EcoRI restriction digest was performed and visualized with ethidium bromide on 1% agarose gel to confirm the purity and quantity of chloroplast 85 DNA.

SOLiD sample preparation and sequencing.⎯DNA libraries for each sample were prepared for SOLiD sequencing with the NEBNext® DNA Sample Prep Master

Mix Set 3 for SOLiD™. The MiniElute PCR Purification kit (Qiagen GmbH, Hilden,

Germany) was then used for the library preparation. SOLiD adapters were added and the

DNA samples were pipetted into Covaris® microtubes and sheared into fragments ranging from 100–1000 bp using a Covaris® Sonicator. To check if sonication was successful, an Agilent 2100 Bioanalyzer was used to measure to size of the sheared DNA fragments. The fragmented DNA was run on an E-Gel® SizeSelect™ gel (Invitrogen) and fragments ranging from 175–225 bp were selected for sequencing. Sequencing was performed with an ABI SOLiD™ sequencer at the Genome Sequencing and Analysis

Facility (GSAF) at The University of Texas at Austin. Short paired-end reads of 35 bp and 50 bp in length were recovered and these reads were implemented in both assemblies.

Reference-based assembly.⎯ Referenced-based assemblies were performed in

BFAST (http://bfast.sourceforge.net; Homer et al., 2009) using the Jacobaea vulgaris chloroplast genome (accession NC_015543) as the reference (Doordruin et al., 2011).

Sequences were quality filtered prior to assembly, excluding any pairs of reads in which one or both contained > 20 low quality (Phred score < 20) bases. Assembly of the remaining high-quality (HQ) reads was performed in color space (“-A 1”) with the default settings described in the BFAST documentation. Following assembly, SAM Tools

86 (Li et al., 2009) was used to convert the alignment output from BFAST into a consensus sequence in FASTA format.

De novo assembly.⎯ De novo assemblies were performed using the publicly available ABI SOLiD Pipeline v.2.0 (Life Technologies) based on Velvet v. 0.7.55

(Zerbino & Birney, 2009). Since this pipeline includes a quality filtering and error correction tool (SOLiD Accuracy Enhancement Tool, SAET v.2.2), raw reads were input.

The average insert length was set at 220 bp, insert length standard deviation at 50 bp, and seed size at 27, with all other parameters at the default settings. Following assembly, any short contigs (<1 kb) were discarded.

Phylogenetic analyses.⎯Referenced-based genome sequences of the four study taxa plus Jacobaea vulgaris were aligned in Geneious Pro 4.0.4 (Drummund et al., 2006) using Mauve (Darling et al., 2004), and then manually adjusted in Mesquite (Madison &

Madison, 2011). Bayesian inference (BI) analyses were performed using Mr.Bayes v.3.1.2 (Ronquist & Huelsenbeck, 2003). Substitution models were selected using the

Akaike information criterion (AIC) in Modeltest v.3.06 (Posada & Crandall, 1998; http://darwin.uvigo.es/software/modeltest.html). The GTR+G+I model was determined to be the best model fitting the three chloroplast data sets. Data set 1 included 98,965 bp, excluding both inverted repeat (IR) regions. Data set 2 consisted of the intergenic spacer regions and was 40,826 bp and included one of the copies of the IR region. Data set 3 consisted of 85,877 bp and included all 81 protein-coding regions. For the BI analyses, model parameters were estimated directly during runs, using four simultaneous chains

87 and four million cycles each, sampling one tree every 1000 generations. Analyses were diagnosed for convergence using Tracer v1.4 (Rambaut & Drummond, 2007).

Comparisons of intergenic spacer regions.⎯Forty-one intergenic spacer regions were determined to have sequence divergence ≥ 1%. These 41 regions were compared for sequence divergence between all five taxa in Geneious Pro 4.0.4

(Drummund et al., 2006). Comparisons were analyzed between the four ingroup taxa,

Telanthophora grandifolia and the two Roldana spp., and between Roldana aschenborniana and R. babra-johannis

Comparisons of protein-coding regions.⎯Ten coding regions were analyzed that were previously identified by Timme et al. (2007) as potential markers in phylogenetic analyses among Asteraceae taxa. Comparisons were analyzed between the four ingroup taxa, Telanthophora grandifolia and the two Roldana spp., and between

Roldana aschenborniana and R. babra-johannis

RESULTS

Reference-based assemblies.⎯Table 4.2 summarizes the results of the reference-based assemblies. The final four assemblies ranged from 150,064–150,542 bp in length and ambiguous nucleotides (Ns) ranged from 122–604. The GC content for all four genomes ranged 37.2%–37.3% and 99.6%–99.9% of the genomes are complete.

Gene content and gene order were identical in all four genomes and to Jacobaea vulgaris and the other five Asteraceae chloroplast genomes previously sequenced. There are 81 protein-coding genes, including 29 tRNA genes and four rRNA genes. A single run on 88 SOLiD and subsequent assembly yielded sufficient reads to map on average 99.775% of the complete chloroplast genome of the four Mexican tussilaginioid taxa. The average sequence divergence between the each of the four study taxa and the J. vulgaris reference genome was 2.4%.

De novo assemblies.⎯Table 4.3 summarizes the results from the de novo assemblies. For Arnoglossum atriplicifoilum, 572,749 reads were used after filtering and the final assembly consisted of 18 contigs and 122,541 bp with 24 Ns. For Telanthophora grandifolia, the final assembly included 12 contigs and 126,706 bp with 20 Ns, and was produced using 664,870 filtered reads. Roldana aschenborniana included 12 contigs and

126,351 bp with 22 Ns, with 693,721 filtered reads used to construct final assembly. The final assembly of R. barba-johannis contained 29 contigs and 115,340 bp with 32 Ns, and was contructed using 496,941 filtered reads. All of the contigs for the four species were blasted against the Jacobaea vulgaris chloroplast genome (Fig. 4.1).

Table 4.4 summarizes the level of sequence divergence between the contigs produced in the de novo assemblies and the identical regions in the Jacobaea vulgaris chloroplast genome sequence. The 100–300 bp flanking the 3’ and 5’ ends of the contigs were not included as because they had numerous ambiguous nucleotides. Sequence divergence of the contigs among the four species ranged from 0.7%–11.9%, however, the average sequence divergence among all of the contigs against Jacobaea vulgaris was 4.4%.

Although Roldana barba-johannis had the highest number of contigs (29) of the four species, this species had the lowest average sequence divergence from the Jacobaea vulgaris genome (3.55%). Arnoglossum atriplicifolium had the highest average sequence 89 divergence of 5.04%. The average sequence divergence between the reference genome and the de novo assemblies study taxa is 2% higher than between the reference genome and the reference-based assemblies. The de novo contigs had more gaps, which are either end gaps or gaps in the sequence, which implies potential insertions. Potential insertions in the contigs can only be confirmed with Sanger sequencing.

Sequence divergence of intergenic spacer regions.⎯All of the intergenic spacer regions for Arnoglossum atriplicifolium, Roldana aschenborniana, R. barba-johannis, and Telanthophora grandifolia were analyzed for sequence divergence and were compared to Jacobaea vulgaris. Regions that were equal to or less than 50 bp were ignored and those with sequence divergence levels ≥ 1% are presented (Table 4.5 and

Fig. 4.2). The intergenic spacer region trnE (UUC)-rpoB was not included in this analysis because the referenced-based assemblies of the four study genomes had Ns in this region.

Forty-three intergenic spacer regions were found to have sequence divergences ≥

1% and were compared among the the five taxa. Twenty-three of the intergenic regions were previously analyzed for sequence divergence in the Asteraceae (Timme et al., 2007;

Doordruin et al., 2011).

Six spacer regions, trnG (UCC)-trnT (GGU), ndhI-ndhG, 3’ trnK (UUU)-rps16, trnL (UAG)-rpl32, psaA-ycf3, trnH (GUG)-psbA, had ≥ 2% sequence divergence among the four ingroup taxa. Twelve of the intergenic spacer regions had indels (Table 4.6).

Sequence divergence of coding regions

Ten chloroplast genes identified by Timme et al. (2007) were examined for sequence divergence and all ten of the coding regions have less than 1% sequence 90 divergence (Table 4.7 and Fig 4.3). Alignment of all of the coding regions (81 genes,

86,021 bp) show 0.5% sequence divergence between all five species, 0.3% sequence divergence between the four ingroup species, and 0.2% sequence divergence between

Telanthophora and the two species of Roldana, and 0.2% sequence divergence between the two Roldana species.

Phylogenetic analyses

The characteristics for data sets 1–3 are summarized in Table 4.8. The aligned chloroplast data matrix for data set 1 is 98,965 bp in length and 313 (0.32%) were parsimony-informative, 96,934 (97.95%) were constant, and 1718 (1.73%) were parsimony-uninformative. The tree resulting from the Bayesian analysis of data set 1 with posterior probabilities values is presented in Fig. 4.4. Data set 2 consisted of 40,826 bp of intergenic spacer regions and 199 (0.49%) were parsimonious-informative, 39,770

(97.41%) were constant, and 857 (2.1%) were parsimony-uninformative, and the resulting tree is presented in Fig. 4.5. Data set 3 included the 81 protein-coding genes and consisted of 85,877 bp, with 148 (0.17%) parsimonious-informative characters, 84,782

(98.72%) were constant, and 947 (1.1%) were parsimony-uninformative. The resulting BI tree is presented in Fig. 4.6. All of the Bayesian tree topologies demonstrated similar relationships to that of the ITS/ETS data presented in Chapter 2.

91 DISCUSSION

The DNA sequences obtained through next generation sequencing enable researchers to use large data sets (e.g., entire genomes) to elucidate the evolutionary relationships among vascular plant taxa. Although this is the first study to use the ABI

SOLiD™ platform to sequence chloroplast genomes of flowering plants, several other studies have utilized the Illumina platform in a similar fashion (Givnish et al., 2010;

Meyers & Liston, 2010; Nock et al., 2011; Steele & Pires, 2011; Straub et al., 2011,

2012; Steele et al., 2012).

Reference-based vs. de novo assemblies.⎯ The results indicate that the reference-based assemblies are more complete than the de novo assemblies and readily provide a means for analyzing genome sequence comparisons among the study taxa. In an analysis by Steele et al. (2012) using Ilumina sequencing, contigs recovered from a de novo assembly ranged from 1–59. In the present study, the de novo assemblies resulted in

12–29 contigs and the contigs in all four assemblies included 115–126 kb of the chloroplast genome. The relatively large number of gaps present between the contigs and these gaps can be filled in with nucleotide sequence via Sanger sequencing. Gaps were also present within the contigs, gaps represent one of two possible situations. First, the gaps may represent insertions in the assembly not recovered in the reference-based methods. Second, the gaps can represent errors in the assembly process, which, like the former case, can only be confirmed with Sanger sequencing. It has been previously demonstrated that a hash size (kmer) value of 25–29 is optimal for maximizing the size of the contigs and retrieving the smallest number of contigs from the assembly (Robertson 92 et al., 2010), and this was supported in this study as a hash size of 27 was found tobe optimal for the de novo assemblies.

The major flowering plant orders have at least one chloroplast genome sequenced

(Straub et al., 2012) and referenced-based assemblies can be easily performed for many plant taxa. However, in cases where the chloroplast genome is highly rearranged (e.g.,

Geraniaceae, Guisinger et al., 2008; Poales, Givnish et al., 2010; Guisinger et al., 2010), de novo assembly remains the desired and efficient method.

Phylogenetic implications.⎯Based on the results from the phylogenetic analyses using data sets 1–3, there is evidence supporting the potential for the entire chloroplast genomes sequences to reconstruct a chloroplast phylogeny of the Mexican tussilaginioid genera. The six intergenic spacer regions with sequence divergences ≥ 2% have also been demonstrated to be phylogenetically informative in other studies (i.e., Daniell et al.,

2006; Saski et al., 2007; Timme et al., 2007; Doordruin et al., 2011). However, it maybe more practical and informative to use all of the intergenic spacer regions for phylogentic analyses.

Parks et al., (2009) demonstrated that the phylogenetic reconstruction of a recently diverged and closely related group of species is possible using large amounts of both coding and noncoding DNA sequence data. At very fine-scale taxonomic levels, such as in the Mexican tussilaginioid group, single-nucleotide polymorphisms (SNPs) may be used as DNA barcodes, as demonstrated by Nock et al. (2011). A previous study by Doorduin et al. (2011) demonstrated that at the population level in Jacobaeae vulgaris

(Senecioneae), there were more (SNPs) present in chloroplast intergenic and intronic 93 spacers than in the coding regions. Similarly, in the coding regions, they found more

SNPs in the introns than in the exons.

Future studies.⎯Even with the availability of entire chloroplast genome sequences, species delimitations within rapidly evolving and recent groups such as the

Roldana petasitis complex (Chapter 2, Clade 9) may not be resolvable using these methods. Future studies involving this complex and other Mexican tussilaginioid taxa utilizing whole chloroplast genome sequencing must involve a thorough sampling of all thirteen clades identified in Fig 2.5. Future evolutionary analyses of the Mexican tussilaginioid group, and other rapidly evolving, recently-diverged Asteraceae, should also include a selection of low copy nuclear markers. Studies have already evaluated the utility of low copy nuclear markers for phylogenetic analysis within the Asteraceae (i.e.

Mort & Crawford, 2004; Álvarez, et al., 2008; Steele et al, 2008). Recent studies have taken advantage of the capabilities of next-generation to also target the nuclear and mitochondrial genomes for phylogenetic marker selection and utility (i.e., Straub et al.,

2011, 2012; Steele et al., 2012).

At the time of this study, the cost for sequencing the chloroplast genome using the

ABI SOLiD™ sequencer, including the chloroplast DNA isolation and library prep, was ca. $350–$400 per sample. With regard to next-generation sequencing, as costs continue to decline and the accuracy of sequencing platforms continues to increase, large genomic data sets will be become more and more prevalent in phylogenetic studies. This study involved relatively expensive laboratory procedures such chloroplast DNA isolation and subsequent RCA and restriction digests. It is possible to use total genomic DNA 94 extractions for next-generation sequencing, which would reduce costs for sequencing per sample although it would require more reads to be analyzed by the sequencing platform.

95 Table 4.1. Species, locality information, percentage of the chloroplast genome sequenced, and number of reads sequenced by the ABI SOLiD sequencer.

Species Location # of reads sequenced

Arnoglossum atriplicifolium (L.) H. Rob Nebraska, USA ca. 3,000,000

Roldana aschenborniana (S. Schauer) H. Rob. & Brettell unknown, Mexico ca. 4,000,000

Roldana barba-johannis (DC.) H. Rob. & Brettell Chiapas, Mexico ca. 4,000,000

Telanthophora grandifolia (Less.) H. Rob. & Brettell Chiapas, Mexico ca. 6,000,000

96 Table 4.2. Referenced-based assemblies with the species, the total number of base pairs assembled, and the number of Ns present in the final assembly.

Species Total bp assembled GC content (%) number of Ns regions of Ns over 50 bp

Arnoglossum atriplicifolium 150,481 37.2 145 1

Telanthophora grandifolia 150,542 37.3 122 1

Roldana aschenborniana 150,262 37.3 384 3

Roldana barba-johannis 150,064 37.3 604 3

97 Table 4.3. De novo assemblies in Velvet with the species name, total base pairs assembled, total number of contigs, the average length of the contigs, the number of reads used in the assembly after the subsampling step, the N50, and the number of Ns present in the final assembly. Aa = Arnoglossum atriplicifolium, Ra =

Roldana aschenborniana, Rb = Roldana barba-johannis, Tg = Telanthophora grandifolia.

Species Total bp assembled contigs Avg. contig length number of reads used N50 number of Ns

Aa 122,541 18 6,808 575749 10,384 24

Tg 126,706 12 10,559 664870 21,581 20

Ra 126,351 12 10,529 693721 53,318 22

Rb 115,340 29 3,977 496941 5,189 32

98 Table 4.4. Contigs assembled de novo and compared against Jacobaea vulgaris chloroplast genome for sequence

divergence.

Arnoglossum atriplicifolium Roldana aschenborniana

Contig no. Length (bp) Sequence divergence (%) Contig no. Length (bp) Sequence divergence (%)

1 1534 2.9 1 2020 10.0

2 2905 4.3 2 1472 2.2

3 2833 8.1 3 7649 7.2

4 1032 8.7 4 33366 3.3

5 1428 7.9 5 1270 4.2

6 5966 2.4 6 5932 3.3

7 8009 3.9 7 3666 3.1

8 8273 5.7 8 50872 2.6

9 8660 3.6 9 1083 7.2

10 1449 6.6 10 4589 3.5

11 10391 4.2 11 6090 3.8

12 7740 4.8 12 505 6.5

13 14063 4.8

14 20011 1.6

15 7210 1.0

16 2066 7.9

17 2181 7.2

18 10138 5.2

Roldana barba-johannis Telanthophora grandifolia

Contig no. Length (bp) Sequence divergence (%) Contig no. Length (bp) Sequence divergence (%)

1 1301 1.2 1 5290 4.4

2 2060 1.5 2 3892 7.0

3 1067 9.0 3 1145 7.5

99 4 1204 11.9 4 13979 2.5

5 994 5.2 5 20419 4.2

6 542 2.4 6 1408 4.9

7 11520 2.6 7 10799 4.0

8 1226 5.2 8 21629 3.9

9 4934 5.5 9 35470 2.0

10 2931 2.9 10 7188 3.8

11 5186 2.4 11 1080 8.4

12 1609 2.7 12 2448 2.5

13 3907 3.9

14 1276 4.0

15 3492 3.8

16 6448 5.2

17 3633 3.3

18 4189 2.0

19 2682 2.0

20 2472 1.9

21 7526 3.5

22 2097 3.5

23 15062 1.8

24 1222 2.0

25 7106 0.7

26 5039 5.0

27 1283 0.8

28 4385 2.3

29 1435 1.6

100 Table 4.5. Analysis of intergenic spacer regions for the four Mexican tussilaginioid taxa. Aa =

Arnoglossum atriplicifolium, Ra = Roldana aschenborniana, Rb = Roldana barba-johannis, Tg =

Telanthophora grandifolia . Regions marked with * were analyzed by Timme at al., 2007 for sequence divergence. Regions marked with ** were analyzed by Dooruin et al. 2011 for sequence divergence.

Region Length (bp) Sequence divergence (%) 5 taxa ingroup (Tg, Ra, Rb) (Ra, Rb)

Whole chloroplast genome 150,689 0.9 0.7 0.5 0.5

All intergenic spacers 40,826 2.3 1.0 0.7 0.7 trnG (UCC)-trnT (GGU)* 175 3.3 2.6 2.1 3.1 ndhI-ndhG* 371 2.9 2.7 2.1 2.7

3’ trnK (UUU)-rps16*, ** 815 2.6 2.2 0.9 2.2 trnL (UAG)-rpl32* 792 2.6 2.0 1.7 2.1 psaA-ycf3 698 2.5 2.1 0.8 0.9 trnH (GUG)-psbA 249 2.3 2.4 2.8 3.6 ndhD-ccsA* 265 2.2 1.8 1.0 1.5 rpl32-ndhF* 1078 2.0 1.4 0.8 1.2 psbK-psbI 428 2.0 1.1 0.5 0.7 trnG (GCC)-trnfM (CAU)* 192 1.9 1.0 0.3 0.5 trnC (GCA)-petN* 415 1.8 1.5 0.6 0.2 petA-psaJ 760 1.8 1.1 0.4 0.5 psaJ-rpl33 437 1.8 1.1 0.3 0.5 rps18-rpl20** 290 1.7 1.6 0.7 0.3 rpl16-rps3 1210 1.7 1.5 0.8 1.0 trnS (GCU)-trnC (GCA) 762 1.7 1.4 1.0 1.0 petN-psbM*, ** 500 1.7 1.1 0.4 0.4 trnR (UCU)-trnG (UCC) 199 1.6 1.8 0.7 1.0 ndhI-ndhE 1126 1.6 1.4 0.7 1.0 psbM-trnD (GUC) 624 1.6 1.3 0.9 0.9

101 trnC (GCA)-psbM 1005 1.6 1.2 0.5 0.3 ycf3-trnS (GCU)*, ** 884 1.6 1.0 0.5 0.5 ndhE-psaC 242 1.6 0.8 0.6 0.8 trnL (UAA)-trnF (GAA)*, ** 351 1.6 0.7 0.2 0.3 ycf3-rps4 1286 1.5 0.9 0.6 0.5 ndhC-trnV (UAC)*, ** 766 1.4 1.2 0.7 0.4 psbA-matK 562 1.4 1.2 0.8 1.1 rps16-psbK 1384 1.4 1.0 0.7 0.8 trnM (CAU)-atpE* 207 1.4 0.8 0.3 0.0 ycf1-rps15* 211 1.3 1.4 1.3 1.9 rps16-trnQ (UUG)* 961 1.3 1.0 0.8 0.8 atpI-atpH 1145 1.3 1.0 0.4 0.6 psbI-trnS (GCU)* 138 1.2 1.1 1.4 2.2

3’trnK (UUU)-matK* 321 1.2 1.0 0.8 0.9 psbZ-trnG (GCC)* 294 1.2 0.7 0.2 0.3 trnY (GUA)-trnE (UUC)* 192 1.2 0.5 0.7 1.0 ndhC-atpE*, ** 455 1.1 0.5 0.2 0.1 trnD (GUC)-trnY (GUA)* 82 1.0 1.2 0.8 1.2 rpl36-infA* 115 1.0 0.9 0.0 0.0 trnT (UGU)-trnL (UAA)* 115 1.0 0.7 0.5 0.5

102 Table 4.6. Indels per species per intergenic region. Aa = Arnoglossum atriplicifolium, Ra = Roldana aschenborniana, Rb = Roldana barba-johannis, Tg = Telanthophora grandifolia .

Region Length (bp) Aa (indels) Tg (indels) Ra (indels) Rb (indels) trnG (UCC)-trnT (GGU) 199 0 0 0 1 ndhI-ndhG 371 1 0 1 1 trnL (UAG)-rpl32 792 0 1 1 1 trnH (GUG)-psbA 249 1 0 0 1 ndhD-ccsA 265 0 0 0 1 psbK-psbI 428 1 0 0 0 trnC (GCA)-petN 415 1 0 0 0 rpl16-rps3 1210 2 0 0 0 trnS (GCU)-trnC (GCA) 762 0 1 0 0 ndhI-ndhE 1126 1 0 1 1 trnC (GCA)-psbM 1005 1 0 0 0 ycf3-rps4 1286 1 0 0 0

103 Table 4.7. Ten coding regions identified by Timme et al. (2007) as potentially informative. Aa =

Arnoglossum atriplicifolium, Ra = Roldana aschenborniana, Rb = Roldana barba-johannis, Tg =

Telanthophora grandifolia .

Region Length (bp) Sequence divergence (%) 5 taxa ingroup (Tg, Ra, Rb) (Ra, Rb)

Whole chloroplast genome 150,689 0.9 0.7 0.5 0.5

All 81protein-coding 85,877 0.6 0.3 0.2 0.2 rps15 279 2.3 0.4 0.2 0.0 ycf1 5076 1.2 0.9 0.5 0.6 ndhF 2226 1.1 0.7 0.5 0.5 psbT 102 1.0 0.7 0.7 1.0 rpl32 165 1.0 0.6 0.0 0.0 psbH 5076 0.9 0.6 0.3 0.4 ccsA 960 0.9 0.4 0.1 0.2 matK 1512 0.8 0.5 0.3 0.4 accD 1446 0.8 0.3 0.05 0.0 petL 96 0.4 0.5 0.7 0.0

104 Table 4.8. Data set partitions for the phylogenetic analyses with information on the number and percentage of parsimonious informative character/total characters contributed to the analyses.

Data Set Genome (excluding both IRs) Intergenic Spacer Regions Coding Regions

Total characters 98,965 40,826 85,877

# of informative characters 313 199 148

% of informative characters 0.32 0.49 0.17

Nucleotide substitution model GTR+G+I GTR+G+I GTR+G+I

105

Arnoglossum atriplicifolium

Jacobaea vulgaris 150 kb 10 kb

Roldana aschenborniana

Jacobaea vulgaris 150 kb 10 kb

Roldana barba-johannis

Jacobaea vulgaris 150 kb 1010 kb kb

Telanthophora grandifolia

Jacobaea vulgaris 150 kb 10 kb 10 kb Figure 4.1. Contigs from de novo assembly blasted to the Jacobaea vulgaris chloroplast genome.

106

rpl36 - infA - rpl36

trnT (UGU) - trnL (UAA) trnL - (UGU) trnT

ndhC - atpE - ndhC

trnD (GUC) - trnY (GUA) trnY - (GUC) trnD

trnY (GUA) - trnE (UUC) trnE - (GUA) trnY

psbZ - trnG (GCC) trnG - psbZ

3’ trnK (UUU) - matK - (UUU) trnK 3’

psbI - trnS (GCU) trnS - psbI

atpI - atpH - atpI

rps16 - trnQ (UUG) trnQ - rps16

ycf1 - rps15 - ycf1

trnM (CAU) - atpE - (CAU) trnM (Rb), and rps16 - psbK - rps16

psbA - matK - psbA

9%J5?4:6D'

ndhC - trnV (UAC) trnV - ndhC

ycf3 - rps4 - ycf3 G8H,?#9%-' 3A/=?6#3S';<5=>'

R. barba-johannis

ndhE - psaC - ndhE 3A/R?9%:='

trnL (UAA) - trnF (GAA) trnF - (UAA) trnL

6#3F';<55>?6#3L';255>' (Ra),

ycf3 - trnS (GCU) trnS - ycf3

trnC (GCA) - psbM - (GCA) trnC

psbM - trnD (GUC) trnD - psbM ndhI - ndhE - ndhI

petN - psbM - petN

trnR (UCU) - trnG (UCC) trnG - (UCU) trnR

rpl16 - rps3 - rpl16 regions (Tg). Comparisons of 41 most variable intergenic

trnS (GCU) - trnC (GCA) trnC - (GCU) trnS

rps18 - rpl20 - rps18

psaJ - rpl33 - psaJ

petA - psaJ - petA

trnC (GCA) - petN - (GCA) trnC

psbK- psbI psbK-

trnG (GCC) - trnfM (CAU) trnfM - (GCC) trnG rpl32 - ndhF - rpl32

Figure 4.2. Histogram with sequence divergence of the intergenic spacer regions of of the intergenic Figure 4.2. Histogram with sequence divergence Arnoglossum atriplicifoilum, Roldana aschenborniana grandifolia Telanthophora Y-axis. on a scale of 0 to 4 and included the to sequence divergence percent identity

ndhD - ccsA - ndhD

psaA - ycf3 - psaA trnH (GUG) - psbA - (GUG) trnH

trnL (UAG) - rpl32 - (UAG) trnL

ndhI - ndhG - ndhI

3A/B?3A/2' 3’ trnK (UUU) - rps16 - (UUU) trnK 3’ trnG (UCC) - trnT (GGU) trnT - (UCC) trnG ve taxa ! ve All 4 ingroup taxa 4 ingroup Ra Rb vs Tg vs Ra vs Rb vs Tg

!"#$"%&' !"#$"%-' All Intergenic Spacer Regions Spacer Intergenic All ('

&'

0.0 0.5 +'

()*' Genome Whole 1.0 ,' 1.5

&)*' 2.0 -'

2.5 +)*' 3.0 3.5 ./01"'2"304"' ,)*' 4.0 Sequence Divergence (%) Divergence Sequence

107

2.5

2.0

1.5

1.0

0.5 Sequence Divergence (%) Sequence Divergence

0.0

rps15 ycf1 ndhE Whole Genome psbT rpl32 psbH Coding Regions ccsA matK accD Figure 4.3. Histogram with sequence divergence of the coding regions of Arnoglossum petL All !ve taxa atriplicifoilum, Roldana aschenborniana (Ra), R. barba-johannis (Rb), and Telanthophora !"#$"%&'4 ingroup taxa grandifolia (Tg). Comparisons of 10 coding regions suggested by Timme et al. (2007) as being potentially useful in a phylogenetic analysis. The plotted values were converted from Tg vs Ra vs Rb percent identity to sequence divergence on a scale of 0 to 2.5 and included on the Y-axis. Ra vs Rb The entire plastid genome (150,689 bp) and all of the coding regions (86,021 bp) are presented here for comparison.

108

Jacobaea vulgaris

Arnoglossum atriplicifolium

1

Telanthophora grandifolia

1

Roldana barba-johannis

1

Figure 4.4. Bayesian inference (BI) phylogram from data set 1 (Five species and 98,965 aligned bp). Numbers above the branches are Bayesian posterior probabilities. Roldana aschenborniana

0.03

109

Arnoglosum atriplicifolium

1

Telanthophora grandifolia

1

Roldana barba-johannis

1

Roldana aschenborniana

Figure 4.5. Bayesian inference (BI) phylogram from plastid data set 2 (40,826 aligned bp from the intergenic spacer regions). Numbers above the branches are Bayesian posterior probabilities.

Jacobaea vulgaris

110

Arnoglossum atriplicifolium

1

Telanthophora grandifolia

1

Roldana barba-johannis

0.91

Roldana aschenborniana

Figure 4.6. Bayesian inference (BI) phylogram from plastid data set 3 (85,877 aligned bp from the protein-coding regions). Numbers above the branches are Bayesian posterior probabilities. Jacobaea vulgaris

0.03

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133

Appendix 1.1. Descriptions of the genera based on the present study of the Mexican tussilaginioid group with chromosome numbers, distribution, and species lists. Asterisks indicate the type species.

Arnoglossum Raf.

Nine species of perennial, erect herbs. Leaves basal and cauline, petiolate, entire to lobed, non-peltate. Capitula few, discoid, ecalyculate. Corolla white, creamy, yellow, or purple, deeply lobed. Anthers with radial endothecium. Style branches truncate, penicillate, with continuous stigmatic areas. Cypselae oblong, ribbed, glabrous. Pappus bristles numerous. n = 30, 29, 28, 27, 26, 25 (Anderson, 1998; Robinson et al., 1997;

Koyama, 1968).

Distribution: central, eastern and south-eastern USA.

Species list:

Arnoglossum album L.C. Anderson

A. atriplicifolium (L.) H. Rob.

A. diversifolium (Torr. & A. Gray) H. Rob.

A. floridanum (A. Gray) H. Rob.

A. muehlenbergii (Sch. Bip.) H. Rob.

A. ovatum (Walter) H. Rob.

A. plantagineum* Raf.

A. reniforme (Hook.) H. Rob. 134 A. sulcatum (Fernald) H. Rob.

Robinson (1974) and Pippen (1978) recently produced revisions of the species of

Arnoglossum, although the 7 species treated by Pippen were treated as Cacalia. The genus restricted to the USA and species diversity for this genus is highest is the southeast

(i.e., Georgia and ).

Barkleyanthus H. Rob. & Brettell

One species, an erect, glabrous shrub or small tree; stems with chambered pith.

Leaves cauline, subsessile, entire, lanceolate to linear-lanceolate, non-peltate. Capitula many, radiate or discoid, flowers yellow, ecalyculate. Anthers with radial endothecium.

Style branches with continuous stigmatic areas. Cypselae elliptic-oblong, ribbed, sparsely pubescent. n = 30 (Ornduff et al., 1963; Turner & Flyr, 1966; Powell et al., 1974; Jeffrey,

1992; Robinson et al., 1997; Soto-Trejo et al., 2011).

Distribution: southern Arizona, Mexico, Guatemala, Honduras.

Species list:

Barkleyanthus salicifolius (Kunth) H. Rob. & Brettell

Robinson & Brettell (1974) first proposed the genus and this species is the most widespread and abundant taxon in the Mexican tussilaginioid group, occurring at a broad elevational range (1000-3000 meters) from the Arizona-Mexico border to Honduras. It is one of two genera that has chambered piths, the other being Pittocaulon. 135

Digitacalia Pippen

Five species of perennial, erect herbs. Leaves evenly distributed throughout the stems, petiolate, non-peltate, palmately lobed and veined. Capitula many, discoid, calyculate. Corollas white or purple, deeply lobed. Anthers ecaudate; endothecium polarized. Style branches truncate to obtuse with continuous stigmatic areas. Cypselae cylindrical, oblong, ribbed, glabrous or pubescent. Pappus bristles numerous. n = 30

(Pippen, 1968; Strother, 1983; Strother & Panero, 2001).

Distribution: Central and southern Mexico.

Species list:

D. chiapensis (Hemsl.) Pippen

D. crypta B.L. Turner

D. hintoniorum B.L. Turner

D. jatrophoides (Kunth) Pippen var. jatrophoides*

D. jatrophoides var. pentaloba B.L. Turner

D. napeifolia (DC.) Pippen

Pippen (1968) first named this small genus of herbs, but it was proposed as a distinct entity before that time (Pippen, 1964). Turner (1990a) provided a recent taxonomic treatment in which he described two species. His description of Digitacalia hintoniorum was based on a single specimen with slight variation in the number of leaf lobes, and in his discussion he states, “leaf shape and lobing is very variable in the 136 Digitacalia complex” as a basis for combining D. tridactylis into D. napeifolia.

Furthermore, Turner (1990a) recognized D. jatrophoides var. pentaloba based on midstem leaves mostly 7–lobed in var. jatrophoides vs. 5-lobed in var. pentaloba, and florets 8–10 per head in var. jatrophoides vs. 5–8 per head in var. pentaloba. He then stated, “occasional plants of var. pentaloba have characters which approach those of var. jatrophoides.” Turner’s conclusions were based on variable morphological characters and relatively few specimens. Most species of Digitacalia are under-collected and this genus requires a revision.

Funstonia gen nov.

One species, a perennial scapigerous herb arising from woody rhizomes. Leaves rosulate, petiolate, lobed, lobe sinus depth up to ¼ length to center of blade, palmately veined, adaxial leaf surface pilose with multicellular trichomes tapering from broad bases; leaf base pubescent. Capitula 8–45, radiate with yellow corollas, calyculate. Disc floret corolla shallowly lobed. Anthers with radial endothecium. Style branches with continuous stigmatic areas. Cypselae oblong, glabrous. Pappus bristles numerous. n = 30

(Strother & Panero, 2001).

Distribution: Central to southern Mexico, Honduras, El Salvador.

Species list:

Funstonia pinetorum (Hemsl.) Quedensley & Villaseñor comb. nov. var. pinetorum

Funstonia pinetorum var. juxtlahuacensis (Panero & Villaseñor) Quedensley &

Villaseñor 137

Funstonia is a new genus with a single species. Previously (Psacaliopsis) pinetorum, this species was closely allied to Psacaliopsis purpusii based on morphology.

However, F. pinetorum differs from P. purpusii in having shallowly lobed leaves, 9–45 capitula, and phyllaries in two subequal series. Psacaliopsis paneroi var. paneroi was identified as a new distinct species (Turner, 1989), and upon intensive study of herbarium specimens it was determined that the material of P. paneroi var. paneroi are identical to

P. pinetorum, which was described by Hemsley in 1908. Therefore, Psacaliopsis paneroi is a taxonomic synonym of P. pinetorum, and included in Funstonia.

Nelsonianthus H. Rob. & Brettell

Two species of small, epiphytic shrubs. Leaves cauline, non-peltate, arranged terminally on branches, petiolate, pinnately veined, Capitula numerous, radiate or discoid, flowers yellow, calyculate. Anthers sagittate to caudate; endothecium polarized.

Style branches obtuse; stigmatic areas continuous excluding basally. Cypselae oblong, ribbed, glabrous. Pappus bristles numerous. Chromosome counts unreported.

Distribution: Chiapas, Mexico and Guatemala.

Species list:

Nelsonianthus epiphyticus* H. Rob. & Brettell

N. tapianus (B.L. Turner) C. Jeffrey

138 Nelsonianthus was first erected by Robinson & Brettell (1973c). The two species have been rarely collected and it is locally extirpated from most former sites due to deforestation. More collections are essential in order to understand the present distributions of this genus and its conservation status.

Pittocaulon H. Rob. & Brettell

Five species of small trees with succulent stems and chambered piths. Leaves cauline, arranged terminally on branches, petiolate, non-peltate, deciduous prior to anthesis, palmately lobed and veined. Capitula many, radiate, flowers yellow, ecalyculate. Anthers with radial endothecium. Style branches with continuous stigmatic areas. Cypselae elliptic-oblong, ribbed, glabrous. Pappus bristles numerous. n = 30

(Turner et al., 1961; Strother, 1983; Jeffrey, 1992; Soto-Trejo et al., 2011).

Distribution: central and southern Mexico, Guatemala.

Species list:

Pittocaulon bombycohphole (Bullock) H. Rob. & Brettell

P. filare (McVaugh) H. Rob. & Brettell

P. hintonii H. Rob. & Brettell var. hintonii

P. hintonii var. cerrograndensis B.L. Clark

P. praecox (Cav.) H. Rob. & Brettell*

P. vellatum (Greenm.) H. Rob. & Brettell var. vellatum

P. vellatum var. tzimolensis (T.M. Barkley) B.L. Clark

139 This genus occurs predominantly on volcanic rock and it is the only genus flowering after the leaves senesce and fall from the plant. Pittocaulon was first circumscribed by Robinson & Brettell (1973b) and subsequently revised by Clark (1996).

Olson (2005) reviewed the anatomy and some physiological aspects of the genus.

Psacaliopsis H. Rob. & Brettell

One species, a perennial scapigerous herb arising from woody rhizomes. Leaves rosulate, petiolate, centrally peltate, secondarily lobed, lobe sinus depth ½–¾ length to center of blade, palmately veined, adaxial leaf surface strigose mostly along the veins; leaf base pubescent. Capitula solitary to many, radiate with yellow flowers, calyculate.

Disc floret corolla shallowly lobed. Anthers with radial endothecium. Style branches with continuous stigmatic areas. Cypselae oblong, glabrous. Pappus bristles numerous.

Chromosome counts unreported.

Distribution: Puebla and Oáxaca, Mexico.

Species list:

Psacaliopsis purpusii (Greenm. ex Brandegee) H. Rob. & Brettell

Prior to the present study, the genus consisted of five species of acaulescent herbs.

The presence of species with either radiate or discoid heads presents a classic problem in the delimitation of taxa within the Senecioneae. Based on morphological and phylogenetic evidence only a single species remains in the genus. Psacaliopsis purpusii occurs in pine-oak forests at 2200—2900 meters and species has been rarely collected 140 and it is locally extirpated from most former sites due to deforestation. More collections are essential in order to understand the present day distributions of this genus and its conservation status.

Psacalium Cass.

Forty-nine species of perennial scapose herbs. Leaves mostly basal, cauline leaved reduced, petiolate, non-peltate, subpeltate, or peltate, entire or lobed or pinnatisect; leaf bases pubescent. Capitula few to many, discoid, calyculate; one species with a solitary, radiate capitulum. Corolla white, creamy, yellow, or purple, deeply lobed, or shallowly lobed in P. macdonaldii and P. pudicum.. Anthers ecaudate; endothecium radial. Style branches with continuous stigmatic areas. Cypselae elliptic-obovate, ribbed, glabrous or pubescent. Pappus present or absent. n = 30 (De Jong & Longpre, 1963;

Pippen 1968; Keil & Stuessy, 1975, 1977; Strother, 1976; Strother, 1983; Turner, 1990b;

Turner & Zhao, 1992)

Distribution: Arizona, USA, Mexico, Guatemala.

Species list:

Psacalium amplifolium (DC.) H. Rob. & Brettell

P. amplum (Rydb.) H. Rob. & Brettell

P. beamanii H. Rob.

P. brachycomum (S.F. Blake) H. Rob. & Brettell

P. calvum (Brandegee) Pippen

P. cervinum (Rydb.) H. Rob. & Brettell 141 P. cirsiifolium (Zucc.) H. Rob. & Brettell

P. cronquistiorum B.L. Turner

P. delphinifolium (Rydb.) Quedensley & Villaseñor comb. nov. (in prep)

P. decompositum (A. Gray) H. Rob. & Brettell

P. eriocarpum (Hand.-Mazz.) S.F. Blake

P. filicifolium (Rydb.) H. Rob. & Brettell

P. globosum (B.L. Rob. & Fernald) H. Rob. & Brettell

P. goldsmithii (B.L. Rob.) H. Rob. & Brettell

P. guatemalense (Standl. & Steyerm.) Cuatrec.

P. guerreroanum B.L. Turner

P. hintoniorum B.L. Turner

P. holwayanum (B.L. Rob.) Rydb.

P. laxiflorum Benth.

P. macdonaldii (B.L. Turner) Quedensley & Villaseñor stat. nov. (in prep)

P. matudae H. Rob. & Brettell

P. megaphyllum (B.L. Rob. & Greenm.) Rydb.

P. mollifolium S.F. Blake

P. multilobum (Pippen) H. Rob. & Brettell

P. nanum Pippen

P. napellifolium (S. Schauer) H. Rob. & Brettell

P. nelsonii Rydb.

P. nephrophyllum (Rydb.) H. Rob. & Brettell 142 P. pachyphyllum (Sch. Bip.) Rydb.

P. palmeri (Greene) H. Rob. & Brettell

P. peltatum var. adenophorum S.F. Blake

P. peltatum var. conzattii (B.L. Rob. & Greenm.) Pippen

P. peltatum (Kunth) Cass.var. peltatum*

P. peltigerum var. hintonii Pippen

P. peltigerum var. latilobum Pippen

P. peltigerum (B.L. Rob. & Seaton) Rydb. var. peltigerum

P. pentaflorum B.L. Turner

P. perezii B.L. Turner

P. pinetorum (Standl. & Steyerm.) Cuatrec.

P. platylepis (B.L. Rob. & Seaton) H. Rob. & Brettell

P. poculiferum (S. Watson) Rydb.

P. pringlei (S. Watson) H. Rob. & Brettell

P. pudicum (Standl. & Steyerm.) Quedensley & Villaseñor stat. nov. (in prep)

P. purpusii (Greenm.) H. Rob. & Brettell

P. putlanum B.L. Turner

P. quercifolium H. Rob. & Brettell

P. radulifolium (Kunth) H. Rob. & Brettell

P. schillingii Panero & Villaseñor

P. sharpii B.L. Turner

P. silphiifolium (B.L. Rob. & Greenm.) H. Rob. & Brettell 143 P. sinuatum (Cerv.) H. Rob. & Brettell

P. tabulare Rydb.

P. tussilaginoides (Kunth) H. Rob. & Brettell

Psacalium now includes species traditionally placed in two genera:

Odontotrichum Zucc. with non-peltate or subpelate leaves, and Psacalium Cass., with centrally-peltate leaves (Rydberg, 1924a, 1924b). Robinson & Brettell (1973d) provided the most current taxonomic revision of the genus. This genus is abundant at middle to high elevations in pine-oak forests of Mexico.

Psacalium macdonaldii and P. pudica are here transferred from Psacaliopsis based on vegetative and floral morphology and phylogenetic data (see Chapter 3).

Pippenalia delphinifolia is also transferred to Psacalium (Chapter 3). This species was formerly recognized in Odontotrichum. However, it was elevated to genus-level status based on the presence of radiate capitula and the loss of the pappus. Since that time other species in Psacalium has been observed to lack a pappus (Robinson & Brettell, 1973d).

This taxon may have an intergeneric hybrid origin due to its meiotic irregularities (Keil &

Stuessy, 1977; Correa & Pippen, 1978) and variable fertility data (Correa & Pippen,

1978). For a review of the biology of Psacalium (Pippenalia) delphinifolia, see Correa &

Pippen (1978).

Robinsonecio T.M. Barkley & Janovec

144 Two species of perennial scapose herbs from thick rhizomes. Leaves rosulate, petiolate, ovate-lanceloate to spathulate, wooly, entire with denticulate margins. Capitula solitary to few (8), radiate, flowers yellow. Style branches with stigmatic areas continuous. Cypselae elliptic-oblong, glabrous or hirsute. Pappus bristles numerous. n =

30 (Stoutamire & Beaman, 1960).

Distribution: Mexico, Guatemala, in alpine habitats above 2800 meters.

Species list:

Robinsonecio gerberifolius* (Sch. Bip. ex Hemsl.) T.M. Barkley & Janovec

R. porphyresthes (T.M. Barkley) T.M. Barkley & Janovec

Robinsonecio porphyresthes is endemic to alpine meadows in Tamaulipas and R. gerberifolius is restricted to subalpine and alpine grasslands and open forest sites at elevation above 2600 meters to alpine peaks in central Mexico (Barkley & Janovec,

1996). Pruski (2012) recently reviewed the genus, although no new significant findings are presented in his manuscript, nor does Pruski discuss the relationship of Robinsonecio among the other related tussilaginioid genera.

Roldana La Llave

Fifty-five species of perennial suffruticose herbs, shrubs, and small trees. Leaves petiolate, peltate or non-peltate, palmately or pinnately veined, entire, lobate, or deeply lobed. Capitula many, radiate, disciform, or discoid, flowers yellow, creamy, or white, calyculate. Anthers with transitional endothecium. Style branches with continuous 145 stigmatic areas. Cypselae elliptic-obovate, ribbed, glabrous or pubescent. Pappus bristles numerous. n = 30 (Afzelius, 1924; Turner & Johnston, 1961; Turner et al., 1962; Ornduff et al., 1963; Turner & King, 1964; Turner & Flyr, 1966; Ornduff et al., 1967; Turner et al., 1973; Powell et al., 1974; Keil & Stuessy, 1975; Jansen & Stuessy, 1980; Strother,

1983; Sundberg et al., 1986; Turner & Barkley, 1989; Zhao & Turner, 1993; Strother &

Panero, 2001)

Distribution: Southern Arizona, USA, Mexico, Guatemala, El Salvador, Honduras,

Nicaragua, Costa Rica, Panama.

Species list:

Roldana acutangula (Bertol.) Funston

R. albonervia (Greenm.) H. Rob. & Brettell

R. aliena (B.L. Rob. & Seaton) Funston

R. angulifolia (DC.) H. Rob. & Brettell

R. anisophylla (Klatt) Funston

R. aschenborniana (S. Schauer) H. Rob. & Brettell

R. barba-johannis (DC.) H. Rob. & Brettell

R. chapalensis (S. Watson) H. Rob. & Brettell

R. ehrenbergiana (Klatt) H. Rob. & Brettell

R. eriophylla Greenm.) H. Rob. & Brettell

R. gentryi H. Rob. & Brettell

R. gilgii (Greenm.) H. Rob. & Brettell

R. glinophylla H. Rob. & Brettell 146 R. gonzaleziae (B.L. Turner) B.L. Turner

R. greenmanii H. Rob. & Brettell

R. grimesii (B.L. Turner) C. Jeffrey

R. guadalajarensis (B.L. Rob.) H. Rob. & Brettell

R. hartwegii (Benth.) H. Rob. & Brettell var. hartwegii

R. hartwegii var. carlomasonii (B.L. Turner & T.M. Barkley) Funston

R. hartwegii var. subcymosa (H. Rob.) Funston

R. hederifolia (Hemsl.) H. Rob. & Brettell

R. heracleifolia Hemsl.) H. Rob. & Brettell

R. heterogama (Benth.) H. Rob. & Brettell

R. heteroidea (Klatt) H. Rob. & Brettell

R. hintonii H. Rob. & Brettell

R. jurgensenii (Hemsl.) H. Rob. & Brettell

R. juxtlahuacana B.L. Turner

R. kerberi (Greenm.) H. Rob. & Brettell var. kerberi

R. kerberi var. calzadana (B.L. Turner) Funston

R. kerberi var. manantlanensis (R. Kowal) Funston

R. langlassei (Greenm.) H. Rob. & Brettell

R. lanicaulis (Greenm.) H. Rob. & Brettell

R. lineolata (DC.) H. Rob. & Brettell

R. lobata La Llave*

R. marquezii (B.L. Turner) C. Jeffrey 147 R. mazatecana B.L. Turner

R. mezquitlana (B.L. Turner) Funston

R. metepeca (B.L. Turner) C. Jeffrey

R. mexicana (McVaugh) H. Rob. & Brettell

R. michoacana (B.L. Rob.) H. Rob. & Brettell

R. mixtecana Panero & Villaseñor

R. neogibsonii (B.L. Turner) B.L. Turner

R. nesomiorum (B.L. Turner) C. Jeffrey

R. pennellii H. Rob. & Brettell var. pennellii

R. pennellii var. durangensis H. Rob. & Brettell

R. petasitis (Sims) H. Rob. & Brettell var. petasitis

R. petasitis var. critobalensis (Greenm.) Funston

R. petasitis var. oaxacana (Hemsl.) Funston

R. petasitis var. sartorii Sch. Bip. ex Hemsl.) Funston

R. platanifolia (Benth.) H. Rob. & Brettell

R. reticulata (DC.) H. Rob. & Brettell

R. sp. nov. (in prep)

R. robinsoniana (Greenm.) H. Rob. & Brettell

R. scandens Poveda & Kappelle

R. schaffneri (Sch. Bip. ex Klatt) H. Rob. & Brettell

R. sessifolia (Hook. & Arn.) H. Rob. & Brettell

R. subpeltata (Sch. Bip.) H. Rob. & Brettell 148 R. suffulta (Greenm.) H. Rob. & Brettell

R. sundbergii (B.L. Turner) B.L. Turner

R. tepopana (B.L. Turner) B.L. Turner

R. tlacotepecana Funston

R. tonii (B.L. Turner) B.L. Turner

R. uxordecora Quedensley & Villaseñor

Roldana is a diverse genus with species that are conspicuous montane floral elements in both pine-oak and cloud forests. Gibson (1969) revised Senecio sect.

Palmatinervii that included many of the present-day species of Roldana, and the two radiate species of Psacaliopsis (Gibson, 1969). Robinson & Brettell (1974) were the first to revise this diverse genus based on morphological characters and distributional data.

Nordenstam (1977) referred to this genus as “a rather broad and possibly heterogeneous concept.” The ‘Pericalia’ group has long been recognized as a separate entity based the discoid heads and mostly white corollas (Pippen 1964, 1968; Turner, 2005;). However, based mostly of vegetative morphology, Robinson & Brettell (1974) included the discoid

‘Pericalia’ species with Roldana. Funston (2008) recognized 48 species and 8 varieties, while Turner (2005) in a review of the Mexican species recognized 58 species, which only excluded Roldana scandens, an endemic of Costa Rica. Turner’s treatment is brief and provides no validation of his species delimitations. He also recognizes all of the varieties in Funston (2008) at the species level. Since that time two additional species have been discovered: R. uxordecora from Oaxaca, Mexico, and Roldana sp. nov. (in 149 prep.), the only riparian species in the Mexican tussilaginioid group and is from

Huehuetenango, Guatemala.

Telanthophora H. Rob. & Brettell

Nine species of suffruticose herbs, shrubs, and small trees. Leaves cauline, blades non-peltate, arranged terminally on branches, petiolate, pinnately veined, entire or lobate or deeply lobed. Capitula numerous, radiate or discoid, flowers yellow, calyculate.

Anthers with radial endothecium. Style branches obtuse with continuous stigmatic areas.

Cypselae elliptic-oblong, ribbed, glabrous. Pappus bristles numerous. n = 30 (Jeffrey,

1992; Sundberg et al., 1986; Powell & Powell, 1978).

Distribution: Mexico, Central America.

Species list:

Telanthophora andrieuxii (DC.) H. Rob. & Brettell

T. bartlettii. H. Rob. & Brettell

T. cobanensis (J.M. Coult.) H. Rob. & Brettell var. cobanensis

T. cobanensis var. molinae (H. Rob. & Brettell) B.L. Clark

T. grandifolia (Less.) H. Rob. & Brettell var. grandifolia

T. grandifolia var. serraquitchensis (Greenm.) B.L. Clark

T. jaliscana H. Rob. & Brettell

T. liebmannii (Buchinger ex Klatt) H. Rob. & Brettell

T. standleyi (Greenm.) H. Rob. & Brettell

T. sublaciniata (Greenm.) B.L. Clark 150 T. uspantanensis (J.M. Coult.) H. Rob. & Brettell

Telanthophora was originally circumscribed for 14 species with no varieties (H.

Rob. & Brettell 1974). Some of their species descriptions were based on only one or two specimens and there was little or no geographic variation with respect to many of the species in their treatment of the genus. Clark (1996, 2000) recognized nine species and four varieties in a treatment that clearly examined much of the broad geographic distribution representative of the genus.

Telanthophora guerckii (Hieron) Jeffrey is a combination proposed by Jeffrey

(1992) based on the species being “somewhat anomalous in Telanthophora” and that it is

“clearly ‘cacalioid’ and would be decidedly more anomalous if left in Senecio.” Due to scant evidence for Jeffrey’s placement, this species is not recognized in the genus. Clark

(2000) recognized several former species at the level of variety.

Villasenoria B.L. Clark

One species of small tree from tuberous roots. Leaves cauline, petiolate, pinnately compound. Capitula numerous, radiate, flowers yellow-orange, calyculate. Anthers with radial endothecium. Style branches apically truncate or subconical, stigmatic areas continuous. Cypselae oblong-cylindrical, glabrous. Pappus bristles numerous.

Chromosome counts unreported.

Distribution: Vera Cruz and Oaxaca, Mexico, at elevations below 1000 meters.

Species list: 151 Villasenoria orcuttii (Greenm.) B.L. Clark

This species was recently segregated from Telanthophora based on its pinnately compound leaves (Clark, 1999). This is the only genus that is restricted to elevations of

1110 meters and lower in Mexico.

Yermo Dorn

One species of small, perennial suffruticose herb from thick taproot. Leaves basal and cauline, entire, petiolate, lanceolate to obovate. Capitula numerous, discoid, corollas yellows, ecalyculate. Anthers auriculate. Style branches obtuse to truncate, penicillate, with continuous stigmatic areas. Cypselae elliptic-oblong, 10–veined, puberlous. Pappus bristles numerous. Chromosome counts unreported.

Distribution: Endemic to Fremont County, Wyoming, USA.

Species list:

Yermo xanthocephalus Dorn

This species is found at elevations ca. 2600 meters in Fremont County, Wyoming

(Dorn, 1991). Within the Mexican tussilaginioid group, this species is one of the narrowest endemics and has been collected in the field a relatively small number of times.

152 Appendix 2.1. Taxa included in seperate and combined nrDNA analyses with voucher information for the sequences of the external transcribed spacer (ETS) and internal transcribed spacer (ITS) of nuclear ribosomal DNA. Asterisks indicated sequences obtained from GenBank that were published in Pelser et al.

(2002*, 2007**, 2010***), and Bayer et al. (2002****). Herbarium abbreviations follow Index herbariorum (Holmgen et al., 1990). Cloned sequences are listed with abbreviations (E = ETS; I = ITS) and numbers. Genbank and cloned sequences in bold were used in the combined analyses (Fig. 2.5) and the reduced separate analyses (Figs. 2.3 and 2.4). An x represents missing data for either ETS or ITS. All cloned nrDNA sequences will be submitted to Genbank upon submission of the manuscript for publication.

Species; country, state or department, herbarium, collector and collection number, ETS Genbank accession number; ITS Genbank accession number. In parantheses, the Genbank numbers or clones number from the present study are listed. ETS is listed first.

Aequatorium asterotrichum B. Nord., Ecuador, Napo, S, Asplund 18263, (GU818111*; GU818489**),

Aequatorium lepidotum B. Nord., Ecuador, Carchi, MO, Palacios & Tipaz 10538, (x; EF538148**).

Arnoglossum atriplicifolium-1 (L.) H. Rob., USA, Ohio, MU, M.A. Vincent 3925, (GU818115***;

EF538154**). Arnoglossum atriplicifolium-2 (L.) H. Rob., USA, Tennessee, TEX, V.E. McNeilus 97-897,

(x; I6). Arnoglossum plantagineum Raf., USA, Iowa, MU, M.A. Vincent 5576, (x; EF538155**).

Barkleyanthus salicifolius-1 (Kunth) H. Rob & Brettell, Cultivated from Guatemala, Quetzaltenango, TEX,

T. Sultan Quedensley s.n. (E3; I5). Barkleyanthus salicifolius-2 (Kunth) H. Rob & Brettell, Mexico,

Chiapas, TEX, C. Santíz R. 585, (E1; I3, I8). Barkleyanthus salicifolius-3 (Kunth) H. Rob & Brettell,

Mexico, B, P. Genelle & G. Fleming 861, (GU818120***; x). Digitacalia crypta B.L. Turner, Mexico,

Guerrero, TEX, J.L. Panero 6186 (E1; I2). Digitacalia jatrophoides (Kunth) Pippen, Mexico, Oaxaca,

TEX, Panero 2330 & Salinas, (GU818152***; GU818545***). Digitacalia jatrophoides var. jatrophoides

(Kunth) Pippen, Mexico, Michoacan, TEX, J.M. Estebado 2111 (E3; I2). Digitacalia jatrophoides var. pentaloba B.L. Turner, Mexico, Puebla, TEX, B.L. Turner 80A-4c (E5; I10). Gynoxys buxifolia-1 (Kunth)

153 Cass., Ecuador, Napo/Cotapaxi border, TEX, J.L. Luteyn 13431 (E4; I1). Gynoxys buxifolia-2 (Kunth)

Cass., Ecuador, S, (x; EF538218*). Gynoxys sodiroi Hieron., Ecuador, Carchi, F, F. Hekker & W.H.A.

Hekking 10.285, (x; EF538219**). Gynoxys soukupii Cuatrec., Peru, L, Hutchison & Wright 5352,

(GU818174***; AF459963*). Gynoxys tolimensis Cuatrec., Ecuador, Carchi, B, B. Øllgaard & H. Balslev

8418, (x; EF538220**). Nelsonianthus tapianus (B.L. Turner) C. Jeffrey, Mexico, Chiapas, MO, I. Pérez &

L. Kendizabal 457, (GU818211**; x). Pippenalia delphinifolia-1 (Rydb.) McVaugh, Mexico, MO, D.E.

Breedlove 59032 & F. Almeda, (GU818627***; GU818230***). Pippenalia delphinifolia-2 (Rydb.)

McVaugh, Mexico, Durango, TEX, T.S. Quedensley 10133 (E3; I6). Pittocaulon bombycophole (Bullock)

H. Rob. & Brettell, Mexico, Guerrero, MEXU, J.L. Villaseñor s.n. (E4; I1). Pittocaulon filare (McVaugh)

H. Rob. & Brettell, Mexico, Zacatecas, TEX, T.S. Quedensley 10142 (E4; I7). Pittocaulon praecox-1

(Cav.) H. Rob. & Brettell, Mexico, Jalisco, US, H.H. Iltis et al. 31157, (E1, E3; I2, I3). Pittocaulon praecox-2 (Cav.) H. Rob. & Brettell, Cultivated from Mexico, Distrito Federal, TEX, T. Sultan Quedensley s.n. (E2, E3; I2, I4). Pittocaulon praecox-3 (Cav.) H. Rob. & Brettell, Cultivated from Mexico, MJG, R.

Greissl s.n., (GU818231***; x). Pittocaulon velatum var. tzimolensis (T.M. Barkley) B.L. Clark, Mexico,

Oaxaca, MEXU, C. Gallardo H. et al. 1477, (E2, E5; I14). Pittocaulon vellatum var. vellatum-1 (Greenm.)

H. Rob. & Brettell, Mexico, Jalisco, TEX, G. Flores M. et al 1819, (E1, E2; I1, I5). Pittocaulon velatum var. vellatum-2 (Greenm.) H. Rob. & Brettell, Mexico, Jalisco, MEXU, G. Flores M. et al 1819, (E4; I8).

Psacaliopsis macdonaldii (B.L. Turner) C. Jeffrey, Mexico, Oaxaca, TEX, G.B. Hinton et al. 26794, (E4;

I1). Psacaliopsis paneroi var. juxtlahuacensis Panero & Villaseñor, Mexico, Oaxaca, TEX, J.I.Calzada

19663, (E1, E3, E4; I4). Psacaliopsis pinetorum (Hemsl.) Funston & Villaseñor, Mexico, Guerrero, TEX,

T.S. Quedensley 10197, (E1; I2). Psacaliopsis pudica (Standl. & Steyerm.) H. Rob. & Brettell, Guatemala,

Huehuetenango, TEX, M. Véliz TEX, 7222, (E5; I1). Psacaliopsis purpusii-1 (Greenm. ex Brandegee) H.

Rob. & Brettell, Mexico, Oaxaca, TEX, J.L. Panero 2607 with Davila & Tenorio, (GU818235***;

GU818629***). Psacaliopsis purpusii-2 (Greenm. ex Brandegee) H. Rob. & Brettell, Mexico, Oaxaca,

TEX, J.L. Panero 2607 with Davila & Tenorio, (E3; I5). Psacalium cirsiifolium-1 (Zucc.) H. Rob. &

Brettell, Mexico, WIS, R.R. Kowal 3053, (GU818236***; EF538270**). Psacalium cirsiifolium-2 (Zucc.)

154 H. Rob. & Brettell, Mexico, Guerrero, MEXU, J.L. Villaseñor s.n., (E4; I2). Psacalium megaphyllum (B.L.

Rob. & Greenm.) Rydb., Mexico, Guerrero, MEXU, J.L. Villaseñor s.n., (E3; I1). Psacalium palmeri

(Greene) H. Rob. & Brettell, Mexico, Jalisco, TEX, J. Villa C. & H. Chávez L. 835, (E1; I1). Psacalium peltatum (Kunth) Cass., Mexico, Oaxaca, TEX, T.S. Quedensley 7076, (E5; I1). Robinsonecio gerberifolius-1 (Sch. Bip. ex Hemsl.) T.M. Barkley & Janovec, Mexico, Tlaxcala, TEX, R. Acosta P. 2449,

(E2; I1). Robinsonecio gerberifolius-2 (Sch. Bip. ex Hemsl.) T.M. Barkley & Janovec, Mexico, Mexico,

MEXU, J. Garcia P. 171, (E2; I4). Robinsonecio gerberifolius-3 (Sch. Bip. ex Hemsl.) T.M. Barkley &

Janovec, Mexico, Mexico, MO, J. Garcia P. 171, (GU818239***; GU818630***). Roldana acutangula

(Bertol.) Funston, Guatemala, Quetzaltenango, TEX, T.S. Quedensley 1817. Roldana albonervia-1

(Greenm.) H. Rob. & Brettell, Mexico, Jalisco, US, M. A. Wetter et al. 2043, (E3, E5; I9). Roldana albonervia-2 (Greenm.) H. Rob. & Brettell, Mexico, L, J. Garcia P. 969, (x; EF538291**). Roldana angulifolia (DC.) H. Rob. & Brettell, Mexico, Michoacan, MEXU, B. Farfán Heredia 334, (E1, E5; I3,

I7). Roldana anisophylla (Klatt) Funston, Mexico, Oaxaca, TEX, T.S. Quedensley 7082, (E1, E2; I3, I5).

Roldana aschenborniana (S. Schauer) H. Rob. & Brettell, cultivated from Mexico, TEX, T. Sultan

Quedensley s.n., (E2; I5). Roldana barba-johannis (DC.) H. Rob. & Brettell, Mexico, Oaxaca, TEX, T.S.

Quedensley 7039, (E5; I1). Roldana chapalensis (S. Watson) H. Rob. & Brettell, cultivated from Mexico,

TEX, T. Sultan Quedensley s.n., (E1, E2, E3; I5, I6). Roldana ehrenbergiana (Klatt) H. Rob. & Brettell,

Mexico, Puebla, MEXU, P. Tenorio L.et al. 8927, (E6; I4). Roldana eriophylla (Greenm.) H. Rob. &

Brettell, Mexico, Oaxaca, MEXU, C. Gallardo H. 1487 et al., (E4; I1). Roldana gentryi H. Rob. &

Brettell, cultivated from Mexico, TEX, T. Sultan Quedensley s.n., (E6; I3). Roldana gilgii (Greenm.) H.

Rob. & Brettell, Guatemala, Quetzaltenango, TEX, T.S. Quedensley 1987, (E1; I4). Roldana greenmanii H.

Rob. & Brettell, Mexico, Chiapas, MEXU, A. Espejo et al 963, (E2; x). Roldana grimesii (B.L. Turner) C.

Jeffrey, Mexico, Hidalgo, MEXU, I. Luna et al. 1863, (E1; I1, I3, I4, I5). Roldana hartwegii (Benth.) H.

Rob. & Brettell, Mexico, Nayarit, MEXU, G. Flores-Franco et al. 3190, (E3; I6). Roldana heracleifolia

(Hemsl.) H. Rob. & Brettell, Mexico, Zacatecas, MEXU, J.J. Balleza C. 9822 con M. Adame G., (E2; I9).

Roldana heterogama-1 (Benth.) H. Rob. & Brettell, Guatemala, Quetzaltenango, TEX, T.S. Quedensley

155 5114, (E1; I2). Roldana heterogama-2 (Benth.) H. Rob. & Brettell, cultivated from Guatemala,

Quetzaltenango, TEX, T.S. Quedensley s.n., (E5; I6). Roldana heterogama-3 (Benth.) H. Rob. & Brettell, cultivated from Mexico, Chiapas, TEX, T.S. Quedensley s.n., (E2; I4). Roldana hintonii H. Rob. & Brettell,

Mexico, Mexico, MEXU, J.L. Villaseñor 1328 et al., (E4; I1, I2, I5). Roldana jurgensenii (Hemsl.) H.

Rob. & Brettell, Guatemala, Quetzaltenango, TEX, T.S. Quedenlsey 5188, (E2; I1, I2, I4, I5). Roldana lanicaulis (Greenm.) H. Rob. & Brettell, Mexico, Oaxaca, TEX, T.S. Quedensley 7070, (E3; I2). Roldana lineolata-1 (DC.) H. Rob. & Brettell, Mexico, Oaxaca, TEX, T.S. Quedensley 7073, (E3; I2). Roldana lineolata-2 (DC.) H. Rob. & Brettell, Mexico, MU, R. Bye 11751, (x; EF538292*). Roldana lobata La

Llave, Mexico, Mexico, MEXU, J.L. Villaseñor R. 1517, (E5; I12). Roldana marquezii (B.L. Turner) C.

Jeffrey, Mexico, Puebla, MEXU, J.L. Contreras J. 4896, (E7; I3, I5, I8, I9, I10). Roldana metepeca (B.L.

Turner) C. Jeffrey, Mexico, Puebla, MEXU, J.L. Contreras J. 6763, (E1; I8, I10, I12). Roldana mexicana

(McVaugh) H. Rob. & Brettell, Mexico, Michoacan, MEXU, S. Zamudio 10059, (E3, E4; I1). Roldana michoacana (B.L. Rob.) H. Rob. & Brettell, Mexico, Michoacan, MEXU, J.L. Linares 4473, (E3, E4; I6).

Roldana mixtecana J.L. Panero & Villaseñor, Mexico, Oaxaca, TEX, J.I. Calzada 19488, (E4; I7).

Roldana petasitis var. cristobalensis (Greenm.) Funston, Mexico, Oaxaca, TEX, T.S. Quedenlsey 7040,

(E1, E5; I5). Roldana petasitis var. oaxacana-1 (Hemsl.) Funston, cultivated from Guatemala,

Quetzaltenango, TEX, T. Sultan Quedensley s.n., (E2; I3, I4). Roldana petasitis var. oaxacana-2 (Hemsl.)

Funston, Mexico, Oaxaca, TEX, T.S. Quedensley TQ 7049, (E2, E4; I1, I3, I4). Roldana petasitis var. petasitis (Sims) H. Rob. & Brettell, cultivated from Mexico, TEX, T. Sultan Quedensley s.n., (E1, E6; I3,

I4, I5). Roldana petasitis var. sartorii (Sch. Bip. ex Hemsl.) Funston, Mexico, Oaxaca, MEXU, J.J.

Calzada 20853, (E1, E5; I1, I3, I5). Roldana platanifolia (Benth.) H. Rob. & Brettell, Mexico, Hidalgo,

MEXU, N.P. Rodriguez C. 7., (E2; I4). Roldana reticulata (DC.) H. Rob. & Brettell, Mexico, Mexico,

MEXU, G.C. Tenorio 1846., (E5; I1). Roldana robinsoniana (Greenm.) H. Rob. & Brettell, Mexico,

Oaxaca, MEXU, J.J. Calzada 19895, (E2; I8). Roldana schaffneri-1 (Sch. Bip. ex Klatt) H. Rob. &

Brettell, Mexico, Guerrero, MEXU, N. Diego et al. 8543, (E2; I3). Roldana schaffneri-2 (Sch. Bip. ex

Klatt) H. Rob. & Brettell, Guatemala, Quetzaltenango, BIGU, T.S. Quedensley 1980, (x; I4, I5, I8).

156 Roldana schaffneri-3 (Sch. Bip. ex Klatt) H. Rob. & Brettell, Guatemala, Quetzaltenango, TEX, T.S.

Quedensley 5120, (x; I11, I12, I14, I15). Roldana sessifolia (Hook. & Arn.) H. Rob. & Brettell, Mexico,

Michoacan, F, E. Peréz & E. Garcia 1800, (E1; I4). Roldana sp. hybrid, Mexico, Oaxaca, TEX, T.S.

Quedensley 7077, (E1, E2, E3; I2, I3). Roldana sp. nova, Guatemala, Huehuetenango, TEX, T.S.

Quedensley 10188, (E6; I7). Roldana suffulta-1 (Greenm.) H. Rob. & Brettell, Mexico, L, Rzedowski

36569, (GU818246**; GU818631**). Roldana suffulta-2 (Greenm.) H. Rob. & Brettell, Mexico, Nayarit,

MEXU, G. Flores Franco 4188 et al., (E5; I1, I3, I4, I5, I6). Roldana sundbergii (B.L. Turner) B.L.

Turner, Mexico, Nuevo Leon, MEXU, J.A. Villareal & M.A. Carranza V-4204, (E3; I5). Roldana uxordecora Quedensley & Villaseñor, Mexico, Oaxaca, TEX, T.S. Quedensley 7050, (E3; I1). Rugelia nudicaulis Shuttlew. ex Chapm., USA, Tennessee, TENN, M.A. Feist, L.R. Phillippe, B.,Molano-Flores, D.

Busemeyer & C. Carroll 714 (ETS), L.R. Phillippe, J. Payne & P.B. Marcum 37061 (ITS),

(GU818247***; GU818632***). Senecio vulgaris L., Canada, Alberta, CANB, Bayer AB-95006 from

(ETS), New Zealand, CHR, S.J. Wagstaff 10.05.2002 (ITS), (AF319755****; EF538396**).

Telanthanthophora andrieuxii-1 (DC.) H. Rob. & Brettell, Mexico, Oaxaca, TEX, T.S. Quedensley 7083,

(E3; I3). Telanthanthophora andrieuxii-2 (DC.) H. Rob. & Brettell, Mexico, Tamaulipas, TEX, M.C.

Johnston 7402, (E2; I1). Telanthophora cobanensis-1 (J.M. Coult.) H. Rob. & Brettell, cultivated from

Mexico, TEX, T. Sultan Quedensley s.n., (E2; I5). Telanthophora cobanensis (J.M. Coult.) H. Rob. &

Brettell, Guatemala, Quetzaltenango, TEX, T.S. Quedensley 2649, (E2; I6). Telanthophora copeyensis

(Greenm.) H. Rob. & Brettell, Costa Rica, San Jose, U, M. Kapelle MK16, (x; EF538404**).

Telanthophora grandifolia-1 (Less.) H. Rob. & Brettell, cultivated, MJG, (GU818318***; EF538405**).

Telanthophora grandifolia-2 (Less.) H. Rob. & Brettell, cultivated from Mexico, TEX, T. Sultan

Quedensley s.n., (E6; I4). Telanthophora jalisciana (Buchinger ex Klatt) H. Rob. & Brettell, Mexico,

Guerrero, MEXU, C. Catalan H. 2352, (E1; I1). Telanthophora liebmannii-1 (Buchinger ex Klatt) H. Rob.

& Brettell, Mexico, Oaxaca, TEX, T.S. Quedensley 7084, (E5; I5). Telanthophora liebmannii-2 (Buchinger ex Klatt) H. Rob. & Brettell, Mexico, Oaxaca, TEX, L. Woodruff 197 with C. Todzia & A. Campos V., (E1;

I2). Telanthophora uspantanensis-1 (J.M. Coult.) H. Rob. & Brettell, Mexico, Oaxaca, TEX, T.S.

157 Quedensley 7068, (E1, E2, E5; I2). Telanthophora uspantanensis-2 (J.M. Coult.) H. Rob. & Brettell,

Mexico, Oaxaca, TEX, A. Campos V. 4389 con J.L. Panero, (E2, E4; I5). Villasenoria orcuttii-1 (Greenm.)

B.L. Clark, Mexico, Oaxaca, TEX, T.S. Quedensley 7086, (E5; I3). Villasenoria orcuttii-2 (Greenm.) B.L.

Clark, Mexico, Veracruz, TEX, E. Estrada M. 1003, (E1; I6). Villasenoria orcuttii-3 (Greenm.) B.L. Clark,

Mexico, XAL, L. Robles 389, (GU818324***; GU818726***). Yermo xanthocephalus Dorn, USA,

Wyoming, MO, L.C. Anderson 13691, (GU818327***; GU818727***).

158 Appendix 3.1. Taxa included in the combined nrDNA analyses with voucher information for the sequences of the external transcribed spacer (ETS) and internal transcribed spacer (ITS) of nuclear ribosomal DNA.

Asterisks indicated sequences obtained from GenBank that were published in Pelser et al. (2007*, 2010**).

Herbarium abbreviations follow Index herbariorum (Holmgen et al., 1990). Cloned sequences are listed with abbreviations (E = ETS; I = ITS) and numbers.

Species; country, state or department, herbarium, collector and collection number, ETS Genbank accession number; ITS Genbank accession number. In parantheses, the Genbank numbers or clones number from the present study are listed. ETS is listed first.

Arnoglossum atriplicifolium (L.) H. Rob., USA, Ohio, MU, M.A. Vincent 3925, (GU818115**;

EF538154*). Barkleyanthus salicifolius (Kunth) H. Rob & Brettell, Cultivated from Guatemala,

Quetzaltenango, TEX, T. Sultan Quedensley s.n. (E3; I5). Digitacalia crypta B.L. Turner, Mexico,

Guerrero, TEX, J.L. Panero 6186 (E1; I2). Digitacalia jatrophoides var. jatrophoides (Kunth) Pippen,

Mexico, Michoacan, TEX, J.M. Estebado 2111 (E3; I2). Digitacalia jatrophoides var. pentaloba B.L.

Turner, Mexico, Puebla, TEX, B.L. Turner 80A-4c (E5; I10). Gynoxys buxifolia (Kunth) Cass., Ecuador,

Napo/Cotapaxi border, TEX, J.L. Luteyn 13431 (E4; I1). Pippenalia delphinifolia (Rydb.) McVaugh,

Mexico, Durango, TEX, T.S. Quedensley 10133 (E3; I6). Pittocaulon bombycophole (Bullock) H. Rob. &

Brettell, Mexico, Guerrero, MEXU, J.L. Villaseñor s.n. (E4; I1). Pittocaulon filare (McVaugh) H. Rob. &

Brettell, Mexico, Zacatecas, TEX, T.S. Quedensley 10142 (E4; I7). Pittocaulon praecox (Cav.) H. Rob. &

Brettell, Cultivated from Mexico, Distrito Federal, TEX, T. Sultan Quedensley s.n. (E2; I2). Pittocaulon velatum var. tzimolensis (T.M. Barkley) B.L. Clark, Mexico, Oáxaca, MEXU, C. Gallardo H. et al. 1477,

(E2; I14). Pittocaulon vellatum var. vellatum (Greenm.) H. Rob. & Brettell, Mexico, Jalisco, TEX, G.

Flores M. et al 1819, (E2; I5). Psacaliopsis macdonaldii (B.L. Turner) C. Jeffrey, Mexico, Oáxaca, TEX,

G.B. Hinton et al. 26794, (E4; I1). Psacaliopsis paneroi var. juxtlahuacensis Panero & Villaseñor, Mexico,

Oáxaca, TEX, J.I.Calzada 19663, (E1; I4). Psacaliopsis pinetorum (Hemsl.) Funston & Villaseñor,

159 Mexico, Guerrero, TEX, T.S. Quedensley 10197, (E1; I2). Psacaliopsis pudica (Standl. & Steyerm.) H.

Rob. & Brettell, Guatemala, Huehuetenango, TEX, M. Véliz TEX, 7222, (E5; I1). Psacaliopsis purpusii

(Greenm. ex Brandegee) H. Rob. & Brettell, Mexico, Oáxaca, TEX, J.L. Panero 2607 with Davila &

Tenorio, (E3; I5). Psacalium cirsiifolium (Zucc.) H. Rob. & Brettell, Mexico, Guerrero, MEXU, J.L.

Villaseñor s.n., (E4; I2). Psacalium megaphyllum (B.L. Rob. & Greenm.) Rydb., Mexico, Guerrero,

MEXU, J.L. Villaseñor s.n., (E3; I1). Psacalium palmeri (Greene) H. Rob. & Brettell, Mexico, Jalisco,

TEX, J. Villa C. & H. Chávez L. 835, (E1; I1). Psacalium peltatum (Kunth) Cass., Mexico, Oáxaca, TEX,

T.S. Quedensley 7076, (E5; I1). Robinsonecio gerberifolius (Sch. Bip. ex Hemsl.) T.M. Barkley & Janovec,

Mexico, Tlaxcala, TEX, R. Acosta P. 2449, (E2; I1). Roldana acutangula (Bertol.) Funston, Guatemala,

Quetzaltenango, TEX, T.S. Quedensley 1817. Roldana aschenborniana (S. Schauer) H. Rob. & Brettell, cultivated from Mexico, TEX, T. Sultan Quedensley s.n., (E2; I5). Roldana barba-johannis (DC.) H. Rob.

& Brettell, Mexico, Oáxaca, TEX, T.S. Quedensley 7039, (E5; I1). Roldana ehrenbergiana (Klatt) H. Rob.

& Brettell, Mexico, Puebla, MEXU, P. Tenorio L.et al. 8927, (E6; I4). Roldana eriophylla (Greenm.) H.

Rob. & Brettell, Mexico, Oáxaca, MEXU, C. Gallardo H. 1487 et al., (E4; I1). Roldana gilgii (Greenm.)

H. Rob. & Brettell, Guatemala, Quetzaltenango, TEX, T.S. Quedensley 1987, (E1; I4). Roldana hartwegii

(Benth.) H. Rob. & Brettell, Mexico, Nayarit, MEXU, G. Flores-Franco et al. 3190, (E3; I6). Roldana heracleifolia (Hemsl.) H. Rob. & Brettell, Mexico, Zacatecas, MEXU, J.J. Balleza C. 9822 con M. Adame

G., (E2; I9). Roldana heterogama (Benth.) H. Rob. & Brettell, Guatemala, Quetzaltenango, TEX, T.S.

Quedensley 5114, (E1; I2). Roldana hintonii H. Rob. & Brettell, Mexico, Mexico, MEXU, J.L. Villaseñor

1328 et al., (E4; I1). Roldana lanicaulis (Greenm.) H. Rob. & Brettell, Mexico, Oáxaca, TEX, T.S.

Quedensley 7070, (E3; I2). Roldana lineolata (DC.) H. Rob. & Brettell, Mexico, Oáxaca, TEX, T.S.

Quedensley 7073, (E3; I2). Roldana lobata La Llave, Mexico, Mexico, MEXU, J.L. Villaseñor R. 1517,

(E5; I12). Roldana marquezii (B.L. Turner) C. Jeffrey, Mexico, Puebla, MEXU, J.L. Contreras J. 4896,

(E7; I3). Roldana metepeca (B.L. Turner) C. Jeffrey, Mexico, Puebla, MEXU, J.L. Contreras J. 6763, (E1;

I8).Roldana mexicana (McVaugh) H. Rob. & Brettell, Mexico, Michoacan, MEXU, S. Zamudio 10059,

(E3; I1). Roldana mixtecana J.L. Panero & Villaseñor, Mexico, Oáxaca, TEX, J.I. Calzada 19488, (E4; I7).

160 Roldana petasitis var. oaxacana (Hemsl.) Funston, Mexico, Oáxaca, TEX, T.S. Quedensley TQ 7049, (E2;

I1). Roldana platanifolia (Benth.) H. Rob. & Brettell, Mexico, Hidalgo, MEXU, N.P. Rodriguez C. 7., (E2;

I4). Roldana reticulata (DC.) H. Rob. & Brettell, Mexico, Mexico, MEXU, G.C. Tenorio 1846., (E5; I1).

Roldana robinsoniana (Greenm.) H. Rob. & Brettell, Mexico, Oáxaca, MEXU, J.J. Calzada 19895, (E2;

I8). Roldana sessifolia (Hook. & Arn.) H. Rob. & Brettell, Mexico, Michoacan, F, E. Peréz & E. Garcia

1800, (E1; I4). Roldana sundbergii (B.L. Turner) B.L. Turner, Mexico, Nuevo Leon, MEXU, J.A. Villareal

& M.A. Carranza V-4204, (E3; I5). Roldana uxordecora Quedensley & Villaseñor, Mexico, Oáxaca, TEX,

T.S. Quedensley 7050, (E3; I1). Telanthanthophora andrieuxii (DC.) H. Rob. & Brettell, Mexico, Oáxaca,

TEX, T.S. Quedensley 7083, (E3; I3). Telanthophora cobanensis (J.M. Coult.) H. Rob. & Brettell,

Guatemala, Quetzaltenango, TEX, T.S. Quedensley 2649, (E2; I6). Telanthophora grandifolia (Less.) H.

Rob. & Brettell, cultivated from Mexico, TEX, T. Sultan Quedensley s.n., (E6; I4). Telanthophora liebmannii (Buchinger ex Klatt) H. Rob. & Brettell, Mexico, Oáxaca, TEX, T.S. Quedensley 7084, (E5; I5).

Telanthophora uspantanensis (J.M. Coult.) H. Rob. & Brettell, Mexico, Oáxaca, TEX, T.S. Quedensley

7068, (E2; I2). Villasenoria orcuttii (Greenm.) B.L. Clark, Mexico, Oáxaca, TEX, T.S. Quedensley 7086,

(E5; I3). Yermo xanthocephalus Dorn, USA, Wyoming, MO, L.C. Anderson 13691, (GU818327**;

GU818727**).

161