RESEARCH ARTICLE INVITED SPECIAL ARTICLE For the Special Issue: The Tree of Death: The Role of Fossils in Resolving the Overall Pattern of Plant Phylogeny Origin of Equisetum: Evolution of horsetails (Equisetales) within the major euphyllophyte clade Sphenopsida Andrés Elgorriaga1,5 , Ignacio H. Escapa1, Gar W. Rothwell2,3, Alexandru M. F. Tomescu4, and N. Rubén Cúneo1 Manuscript received 3 December 2017; revision accepted 5 June PREMISE OF THE STUDY: Equisetum is the sole living representative of Sphenopsida, a 2018. clade with impressive species richness, a long fossil history dating back to the Devonian, 1 CONICET, Museo Paleontológico Egidio Feruglio, Trelew, and obscure relationships with other living pteridophytes. Based on molecular data, the Chubut 9100, Argentina crown group age of Equisetum is mid- Paleogene, although fossils with possible crown 2 Department of Botany and Plant Pathology, Oregon State synapomorphies appear in the Triassic. The most widely circulated hypothesis states that the University, Corvallis, OR 97331, USA lineage of Equisetum derives from calamitaceans, but no comprehensive phylogenetic studies 3 Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA support the claim. Using a combined approach, we provide a comprehensive phylogenetic 4 Department of Biological Sciences, Humboldt State University, analysis of Equisetales, with special emphasis on the origin of genus Equisetum. Arcata, CA 95521, USA METHODS: We performed parsimony phylogenetic analyses to address relationships of 43 5 Author for correspondence (e-mail: [email protected]) equisetalean species (15 extant, 28 extinct) using a combination of morphological and Citation: Elgorriaga, A., I. H. Escapa, G. W. Rothwell, A. M. F. molecular characters. Tomescu, and N. Rubén Cúneo. 2018. Origin of Equisetum: Evolution of horsetails (Equisetales) within the major euphyl- KEY RESULTS: We recovered Equisetaceae + Neocalamites as sister to Calamitaceae + a clade lophyte clade Sphenopsida. American Journal of Botany 105(8): of Angaran and Gondwanan horsetails, with the four groups forming a clade that is sister to 1–18. Archaeocalamitaceae. The estimated age for the Equisetum crown group is mid- Mesozoic. doi:10.1002/ajb2.1125 CONCLUSIONS: Modern horsetails are not nested within calamitaceans; instead, both groups have explored independent evolutionary trajectories since the Carboniferous. Diverse fossil taxon sampling helps to shed light on the position and relationships of equisetalean lineages, of which only a tiny remnant is present within the extant flora. Understanding these relationships and early character configurations of ancient plant clades as Equisetales provide useful tests of hypotheses about overall phylogenetic relationships of euphyllophytes and foundations for future tests of molecular dates with paleontological data. KEY WORDS cladistics; combined-analysis; Equisetales; Equisetaceae; Euphyllophytina; fossil; phylogeny; sphenophytes. Equisetum L. is a genus of vascular plants comprising approximately early 20th century in terms of its phylogenetic placement in the plant 15 extant species with nearly worldwide distribution (Hauke, 1963, tree of life (Scott, 1900; Browne, 1908; Eames, 1936; Boureau, 1964; 1978; Husby, 2013; PPG I, 2016). Even though the morphology Bierhorst, 1971; Rothwell, 1999; Pryer et al., 1995, 2001; Karol et al., and anatomy of Equisetum have been documented in detail (e.g., 2010; Rothfels et al., 2015). Based on overall morphological differ- Bierhorst, 1971; Gifford and Foster, 1989; Evert and Eichhorn, 2012), ences, this genus has been divided into two subgenera, Equisetum the distinctiveness of this genus has vexed researchers since the L. and Hippochaete Milde (Hauke, 1963, 1978). More recently, American Journal of Botany 105(8): 1–18, 2018; http://www.wileyonlinelibrary.com/journal/AJB © 2018 Botanical Society of America • 1 2 • American Journal of Botany DNA- based phylogenetic analyses have placed E. bogotense, a spe- proposes that Equisetum belongs to a lineage from the Angaran cies morphologically allied with subgenus Equisetum, in equivocal and Gondwanan paleofloristic provinces, including species of positions (Des Marais et al., 2003; Guillon, 2004, 2007). Koretrophyllites Radczenko, Tschernovia Zalessky, Paracalamitina Despite the modest modern diversity of horsetails, paleobotani- Zalessky, Equisetinostachys Rasskasova, Cruciaetheca Cúneo et cal studies have long demonstrated that the genus Equisetum is the Escapa, and Peltotheca Escapa et Cúneo (Naugolnykh, 2004; Cúneo sole surviving representative of an extremely ancient and diverse and Escapa, 2006), a hypothesis partly supported by the similarity group, the Sphenopsida (Neuberg, 1964), which includes at least of reproductive structures of those fossils to teratological forms of 60 genera with a plethora of species (Boureau, 1964), and which modern Equisetum (Tschudy, 1939; Boureau, 1964; Naugolnykh, has a long evolutionary history that can be traced in time as far 2004). A third hypothesis proposes that both Equisetaceae and back as the Late Devonian (Gu and Shi, 1974; Wang et al., 2005). Calamitaceae originated during the Early Carboniferous, having Sphenopsids comprise two orders, the Sphenophyllales (Boureau, evolved independently since that time, perhaps sharing an ances- 1964) and the Equisetales (Boureau, 1964) (Stein et al., 1984; tor of archaeocalamitacean affiliation (Bierhorst, 1971; Page, 1972a; Stewart and Rothwell, 1993; Taylor et al., 2009), which reached a Gifford and Foster, 1989). peak in species richness during the Carboniferous period, when These previously developed hypotheses were mired in problems sphenopsids were some of the most common constituents of paleo- arising from the traditional view of the equisetalean fertile shoots tropical swamp forest ecosystems (Behrensmeyer et al., 1992; Cleal (i.e., strobili) as node–internode alternations and the homology et al., 2012). assumptions derived from that (e.g., Page, 1972b; Good, 1975). The oldest representatives of Equisetales, the archaeocalamites, However, a recent study combining developmental, genetic, mor- probably originated in the Early Carboniferous (Kidston, 1883; phoanatomical, and paleontological information, provides a new Chaphekar, 1965; Bateman, 1991), a time they are known from hu- perspective on the structure and morphological evolution of equi- mid environments of Europe and North America. Since that time, setalean reproductive structures, focusing on phytomers as mod- the group has exhibited a variety of growth architectures. These ular units of the shoots. Accordingly, the different reproductive include arborescent Calamitaceae with compact bracteate strobili, morphologies seen in the clade can be explained by the hierarchical from the northern hemisphere (Williamson and Scott, 1894; Good, expression of at least three different regulatory modules (Tomescu 1975; Feng et al., 2012), herbaceous Cruciaetheca Cúneo et Escapa et al., 2017), with the effects of each module being readily identi- and Tschernovia Zalessky from the Permian of South America fiable by distinct “structural fingerprints” (Rothwell et al., 2014; and Angara that had lax reproductive structures (Meyen and Rothwell and Tomescu, 2018). Accordingly, in the development of Menshikova, 1983; Cúneo and Escapa, 2006), and large Equisetites the Equisetum- type strobilus, the three regulatory modules are all Sternberg with multiple compact strobili on lateral branches, from activated, resulting in a stack of phytomers that lack node–inter- the European Triassic (Kelber and van Konijnenburg-van Cittert, node differentiation. In Peltotheca- type plants, the activation of two 1998). The unique morphology of modern horsetails, usually in- modules results in a determinate reproductive structure composed cluding bractless compact strobili on main aerial stems, as well of a series of fertile phytomers, while a single functioning module as numerous vegetative synapomorphies, dates at least from the produces Cruciaetheca- type plants having series of fertile phy- Jurassic, according to fossils from Patagonia (Channing et al., 2011; tomers alternating with vegetative ones as part of an indeterminate Elgorriaga et al., 2015), and probably from the Triassic (Stanich reproductive shoot (Tomescu et al., 2017). et al., 2009). Molecular- based phylogenetics has provided a broad array of A study based on molecular data (Sanderson, 2002) estimates hypotheses concerning the position of Equisetum among extant the divergence of extant Equisetum species to have occurred in vascular plants. In some of the earliest and most controversial posi- mid- Paleogene (late Eocene, ca. 40 Ma), with the main radiation tions for Equisetum recovered in such analyses, this genus is nested occurring during the Neogene (Des Marais et al., 2003). While that among modern ferns (i.e., “monilophytes” sensu Pryer et al., 2004), estimate may seem reasonable from analysis of living species, the either as sister to the Marattiales (Pryer et al., 2001, 2004; Wikström absence of a phylogenetic framework including well- preserved fos- and Pryer, 2005), or as sister to leptosporangiate ferns (Nickrent sils renders that hypothesis difficult to test. Fortunately, our under- et al., 2000). Subsequent analyses recovered Equisetum as sister to standing of Equisetum evolution has been enriched in the past years either an Ophioglossales + Psilotales clade (Grewe et al., 2013; Lu by the discovery of several