CLUB CHOLLAS OF THE BIG BEND 3.0: REFINING SPECIES CONCEPTS AND DISTRIBUTIONS FOR THE CORYNOPUNTIA [OPUNTIA, GRUSONIA] SCHOTTII COMPLEX OF WESTERN TEXAS VIA NEW CHROMOSOME COUNTS, POLLEN STAINABILITY, AND MORPHOLOGIC DATA

Species level understanding of the Corynopuntia schottii complex C. aggeria, C. densispina, C. emoryi, C. grahamii, and C. schottii) within the USA has long been confused and misunderstood. Most recent taxonomic treatments are incomplete and/or ina ccur te regarding four of the species (excluding C. emoryi) The present study helps clarify all species concepts in t he Big Bend region of Texas through 97 chromosome counts, 186 pollen stainability measur ements, 272 new voucher specimens, and review of previously existing herbarium specim ens. Significant findings include (1) documentation of C. schottii as a hexaploid species that occurs from deep South Texas to just west of the Brewster County line and outside of Big Bend National P ark, (2) confirmation of the previously reported chromosome numbers for the remaining taxa, despite u ncovering widespread meiotic irregularities in C. grahamii and extremely plastic morphology of C. aggeria, (3) a range of fertility between and even within populations of all species, and (4) chromosomal and morphological evidence of hybridization and introgression in most taxa. D istinguishing characters, keys to the species (for both living and dried material) distribution maps, photographs of each taxon, and corrections to erroneously labeled published photographs are pr ovided. The Big Bend endemic Corynopuntia densispina (Ralston Hilsenb. J. Fenstermacher, comb. nov. is formally transferred from Opuntia/Grusonia. Evolutionary origins of the taxa and their larger geographi c and evolutionary context (including Mexico and the broader southwes tern USA) are discussed and avenues for future research are suggested. For over 100 years, taxonomic delineations within the Opuntioidiae hav been fraught with difficulty in large part due to a high level of interspecif ic hybridization, extensive vegetative dispersal and reproduction, paucity of taxonomically meaningful morphological characters, and environmentally influenced morphologic variability (Pinkava et al. 1998; Rebman Pinkava 2001; Powell Weedin 2004) Several of the most recent authorita tive taxonomic treatments involving Opuntia [sensu lato] occurring in the USA (e.g. Pinkava 2003; Powel l Weedin 2004; Hunt 2006) include statements to the effect that parts, or even the entirety, of this group is in need of critical study. For example in Texas, Powell and Weedin (2004) recogni zed 25 "normal" Opuntia (s.l. taxa as occurring in the Trans Pecos, while suspecting that betwe en 10 and 20 potentially recognizable species were overlooked; they further emphasized that the clas sifi tion of Opuntia (s.l. into species and varieties "remains little more than a goal for the future." Several recent molecular studies have attempted to elucid ate ntrafamilial relationships in Cactaceae (Nyffeler 2002; Wallace Dickie 2002; Griffith Porter 2009; Barcenas et al. 2011; Hernandez Hernandez et al. 2011; Majure et al. 2012c) however, de finitiv clarity at the generic and specific levels still remains elusive. Several recent f ield studies based on chromosome counts and morphologic observations have revealed new taxa and broadened un d rsta ding of taxon concepts Fenstermacher: Club chollas of the Big Bend 2 and evolutionary history (Baker et al. 2009; Donati 2010, 2011; Majure Ribbens 2012; Majure et al. 2012a, 2012b; Baker Cloud Hughes 2014; Breslin Donati 2014; Felge r t al. 2014) The resurgence of non molecular based research addresses recogni z d problems implicated in obscuring clarity of opuntioid systematics including inadequate rep licative chromosomal sampling, limited indepth, field based observations capturing detailed morphologic t raits and breeding system data, and limited distributional context of studies (Pinkava et al. 1998; Rebman Pinkava 2001; Barcenas 2004; Rebman 2006; Majure Ribbens 2012; Majure et al. 2012a, 2012b, 2012c; Bak er CloudHughes 2014) The present study adds new, non molecular insight into intrageneric relationships of the Big Bend Texas area club chollas. Data for the presen t study were accumulated over 10 years beginning in 2004 via field observations, chromosome counts, poll en stains, review of existing herbarium specimens, and new voucher collections. Taxonomic History The specific taxonomic conundrum focused upon here has engaged s peciali ts for over 60 years. Known from the 1950's as the " Opuntia schottii complex," (Anthony 1956) the group now comprises five taxa: Corynopuntia aggeria, C. densispina, C. emoryi, C. grahamii, and C. schottii, all five referred to herein as the C. schottii complex (CSC) These five species are part of a larger clade of small cylindroid cacti that over time have been variousl y placed in different genera, most recently within Grusonia while also giving support for a modified Corynopuntia concept (Barcenas 2015) Several recent studies, including the examination of seed mo rphol gy (Stuppy 2002) and molecular analyses (Griffith 2002; Griffith Porter 2009; Barcenas e t al. 2011; Barcenas 2015) while lacking repeatable phylogenies for the entire Cylindropuntia tribe (sensu Hunt 2006) have shown that within the concept of Corynopuntia one subgroup consistently segregates as monophyletic, separate from others that regularly shift affiliations. The subclades inv olved are most clearly resolved in Griffith (2002) Strict monophyletic interpretation in Griffith (2002) and Stuppy (2002) leads to placing all cylindroid cacti (including Cylindropuntia) into Grusonia. However, the amalgamation, as Griffith stated, obscures the natural diversity evident in the lineage, leading Griffith to resurrect older generic concepts including Micropuntia and Corynopuntia, also utilized by Stuppy (2002) Though a perfectly repeatable, monophyletic Corynopuntia has yet to be shown, the consistently segregating subgroup of club chollas includes the Big Bend species and does r ep atedly fall within Corynopuntia. Applying this generic designation, as opposed to Grusonia, would seem to be the most appropriate because the subclade is bound by both morphologic similarity and c onsistent segregation in genetic analyses (Griffith 2002; Griffith Porter 2009; Barcenas et al. 2011; Barcenas 2015) The two foundation taxa for the Big Bend club cholla complex, Corynopuntia [Opuntia] schottii and C. grahamii, were described by Engelmann (1856, 1859) based on collections made by Wright, Bigelow, and Schott during the 1851 53 U.S. and Mexico Boundary Survey. The western taxon, C. grahamii, was initially collected near El Paso and is known from sout hern New Mexico south along the Rio Grande into Texas's Big Bend  A) Corynopuntia [Opuntia] schottii was the eastern taxon, the type having been collected near the mouth of the Pecos River in present day Val Verde County, Texas. Its documented range in the USA ha s been thought to extend from south Texas upriver into the Big Bend region of southern Brewster County  B) Both taxa have been reported as ranging much further south of the international border than they do north of it (Hernandez et al. 2004; SEINET 2014) These two taxa have been variously treated as separ ate species (Engelmann 1856; Britton Rose 1919; Schulz Runyon 1930; Anthony 1956; Weni ger 1988; Ralston Hilsenbeck 1989; Anderson 2001; Pinkava 2003; Hunt 2006) or v arieties of O. schottii (Benson 1969, 1982; Correll Johnston 1970; Pinkava et al. 1985; Powell Weedin 2004; Powell et al. 2008) and not all authors have believed both taxa t o occur in the Big Bend (Weniger 1988) To keen in situ observers (Anthony 1954; Ralston 1987) field char a teristics easily distinguish Corynopuntia schottii and C. grahamii: both taxa have readily disarticulating distal stem segments (joints) but differ in distribution, phenology, joint growth origin, mound habit, and Fenstermacher: Club chollas of the Big Bend 3 Figure 1. Previously known range extents for the Corynopuntia schottii complex in the USA (Texas, New Mexico, Arizona) based on Pinkava (2003) and Powell, Weedin, Powell (2008) A. C. grahamii. B. C. schottii. C. C. emoryi. D. C. aggeria, dotted circle; C. densispina, solid oval.