86 Haseltonia, No.9, 2002

GRUSONIA PULCHELLA CLASSIFICATION AND ITS IMPACTS ON THE : MORPHOLOGICAL AND MOLECULAR EVIDENCE M. PATRICK GRIFFITH Rancho SantaAna Botanic Garden, 1500 N. CollegeAvenue, Claremont, CA 91711 michael. [email protected]

Summary. This report is an investigation into the relationships among members of the genus Grusonia sensu Anderson (2001) with an emphasis on examining the relationship of Gru- sonia pulchella within (Cactaceae). G.pulchella is morphologically and geographi- cally distinct from other Grusonia , and nrITS DNA sequence data suggest that it may rep- resent an independent evolutionary lineage from other grusonioid cacti. With the morphological, geographical, and molecular evidence considered, I propose to resurrect the generic concepts Knuth (1935), Grusonia Reichenbach ex Schumann (sensu Brit ton and Rose, 1919), and Micropuntia Daston (1946).

Introduction nia also include the genera Corynopuntia Knuth, Marenopuntia Backeberg, and Microp- The current circumscription of the Gruso- untia Daston (Anderson, 2001; Stuppy, 2002; nia pulchella (sand cholla) complex includes Wallace and Dickie, 2002; Gibson, ined.) several described taxa. The first was The G.pulchella (Engelm.) H. Rob. complex pulchella (Engelmann, 1863), whose type lo- is a morphologically variable taxon consisting cality is on the Walker River in . Several of scattered populations of small, terete- specimens from Utah and bordering Nevada stemmed opuntioid cacti found in the Great were described as new species under the new Basin of western North America (Benson, 1982; genus Micropuntia (Daston, 1946), i.e., Micro- Kartesz, 1987) (Fig. 1). Although of the puntia barkleyana, M. brachyrhopalica, and G. pulchella complex have long been consid- M. spectatissima. Benson's (1957) circumscrip- ered to possess affinities with other grusonioid tion of 0. pulchella included these three mor- cacti (Engelmann, 1863; Brit ton and Rose, photypes (as "aberrant forms") and notes that 1919; Benson, 1957, 1982; Anderson, 2001; the species' affinities are difficult to ascertain. Stuppy, 2002), aspects of the morphology, dis- Robinson (1973) combined sand cholla along tribution, and habit of G.pulchella suggest that with seven other species into the genus Gruso- it may represent an evolutionary lineage dis- nia, previously described by Reichenbach tinct from other opuntioids, as first suggested (1896). The most recent treatments of Gruso- by Daston (1946).

Figure I. Habitat of Grusoniapulchella: view south across the Columbus salt marsh from the ghost town/at luvial fan of Columbus, Esmeralda County, Nevada, USA. Haseltonia, No.9, 2002 87

Figure 2. in an alluvial fan above the Columbus salt marsh

Figure 3. Two specimens of Grusonia pulchella. Note enlarged fleshy underground areole- and glochid bearing structure. 88 Haseltonia, No.9, 2002

Grusonia pulchella (Fig. 2) differs morpho- Pinkava. Both species have been collected in logically from other members of the genus in a Nye County, Nevada. Based on locality data number of significant ways. These plants are from specimens cited in Benson (1982), the marked by an enlarged fleshy underground ranges of these two taxa do not appear to over- structure featuring areoles with prominent yel- lap. Furthermore, G. parishii occurs at eleva- low glochids (Fig. 3). Some authors term this tions below 900-1200 m, and G. pulchella is morphological feature a "tuberous root" (Ben- found at 1200-1500 m and higher (Benson, son, 1982;Anderson, 2001), but the areoles and 1969, 1982; Pinkava, 1999; Morefield, 2001). glochids present upon the upper portion of the All chromosome counts of G.pulchella show it structure provide evidence that it may be stem- to be diploid, 2n = 22 (Pinkava, 2002). derived (Brit ton and Rose, 1919) or perhaps a The habit of G.pulchella differs from that of true tuber (Jackson, 1928). The presence of an congeneric plants. The majority of Grusonia underground tuber would make this species species are easily identifiable to genus at a dis- unique among the grusonioids. Other Gruso- tance, as they form dense, spreading mats of nia species possess somewhat thickened roots branching, clavate cladodes (Benson, 1982; Ral- (Engelmann, 1859; Benson, 1982; Ralston and ston and Hilsenbeck, 1989, 1992; Powell, 1998; Hilsenbeck, 1989), but these do not approach de la Cerda-Lemus, 1999; Anderson, 2001), the proportions of the storage tuber of G. pul- which sometimes root adventitiously (Engel- chella. They also are derived entirely from root mann, 1859). Grusonia pulchella is not mat- tissue. Terminal stems of G. pulchella are often forming but is better described as a cushion or cylindrical rather than fully clavate (Brit ton a dense, low-growing single-trunked and Rose, 1919) and are rather flexible, much (Benson, 1982; Pinkava, 1999). The distal clado- like those of Britton & Rose, al- des of G. puichella do not root adventitiously though much reduced in size. Unlike most Gru- while attached, nor do they detach easily sonia species, G. pulchella has tubercles that (pers. obs.). are often inconspicuous (Benson, 1982), but Recent molecular analyses have provided this varies (Britton and Rose, 1919; Daston, much insight into the relationships among the 1946; Anderson, 2001). The areoles of G. pul- genera of Opuntioideae (Wallace and Dickie, chella bear persistent wool similar to the areo- 2002; Wallace and Gibson, 2002). Chloroplast lar wool of Pereskiopsis, whereas the areoles DNA sequences demonstrate a sister group re- of other Grusonia species have early-decidu- lationship of four species of Grusonia sensu ous wool or lack wool entirely (An- derson, 2001). Although displaying these subtle morphological affini- } ties with Pereskiopsis, sand cholla obviously lacks the overall habit ..-'"I

and persistent leaves of that genus. w... . Spines on the of G. pulchella ~ differ from those of other Gruso- i . ** nia species by being antrorsely barbed rather than retrorsely ( barbed (Parfitt, 1988; Pinkava, "'~ *' 1999). of G. pulchella are .O.~ ,* sufficiently distinct morphological- ly from other Grusonia seeds as to ~, * place them in the monotypic sub- ~C" genus Micropuntia sensu Stuppy (2002). One unique opinion even \ 0 considers possible affinities of G. 0 pulchella with (Philip- pi ex Weber) Schumann and Ptero- L Schumann (Daston, 1946). Grusonia pulchella is disjunct * Gruson;a pulchel/a 0 100 200 300 from other members of the genus 15)Gruson;a parish;; Grusonia (Fig. 4), which occur further to the southeast, through southernmost Nevada, , . Figure 4. Map showing the distribution of Grusonia pulchella and its most geographically proximate congener, G. parishii. The , and , and south range of G. parishii extends further south into . Other into Mexico (Benson, 1982; Pinka- Grusonia species occur further south and east, through the south- va, 2002; Gibson, ined.). The geo- western United States and into Mexico. Adapted from Benson, graphically nearest congener of 1982; Albee et al., 1988; Morefield, 2001; Pinkava, 2002; and Gib- sand cholla is G. tJari.\"hii (Orcutt) son. ined. Haseltonia, No.9, 2002 89

Anderson (2001) with a limited sampling of cho SantaAna Botanic Garden, Claremont, CA; (Engelm.) Knuth. Sampled Desert Botanical Garden, Phoenix, AZ; and SuI Grusonia in these studies include the type Ross State University Cactus Garden, Alpine, species G. bradtiana, along with G. clavata, G. TX. This study sampled 34 specimens, includ- marenae, and G. stanleyi. These two genera ing 2 specimens of G. pulchella, 7 specimens form a well-supported monophyletic group of other Grusonia species, including represen- with Pereskiopsis and Brit ton & tatives from all other described genera current- Rose. The relationship of G. pulchella within ly circumscribed within Grusonia sensuAnder- the Opuntioideae has not yet been elucidated. son (2001), and 25 specimens representing all Given the conflicting evidence of morphologi- other genera currently recognized in Opun- cal data towards the relationship of G. pulchel- tioideae (Table 1). DNA was extracted using 2X la within the Opuntioideae, there is a potential CTAB, followed by precipitation in cold iso- for molecular data to help resolve this species' propanol (Griffith and Porter, in press). All ex- taxonomic position. Toward this goal, 1 have tractions used approximately 0.5 g of epider- gathered DNA sequence data from the nrITS re- mal shoot tissue. Polymerase Chain Reaction gion for specimens of G.pulchella and putative (PCR) amplification of templates of the nuclear related taxa. ribosomal internal transcribed spacer region (nrITS) follows the methods outlined by Methods and Materials Columbus et al. (1998) using the primers ITS4 Live material was obtained through field- and ITS5 (White et al., 1990). Polyethylene gly- work or from live vouchered plantings at Ran- col precipitation (Morgan and Soltis, 1993) pu- rified all templates. Purified template amplifica- tions were sequenced directly with four primers, ITS2, ITS3, ITS4i, and ITS5i (White et RO ~ I 64 "-- Cylindropuntja tunicata al., 1990; Porter, 1997), using "big dye" chem-

Cylindropuntja whjppfej istry from Applied Biosystems Incorporated, ac- -Cyfjndropuntia caljlornjca cording to the manufacturer's specifications. Cyfjndropuntia acanthocarpa An Applied Biosystems Incorporated 3100 auto- -Cyfjndropuntia muitigeniculata mated DNA sequencer gathered all sequences Cyfjndropuntia ieptocaulis from PCR products. Genbank accessions from a Grusonja ciavata previous study (Hershkovitz and Zimmer, Grusonja invjcta 1997) provided 3 additional sequences repre- Grusonja marenae

-Grusonja aggeria senting outgroup taxa. Grusonia grahamjj Chromatograms from sequencing reactions 5] -Grusonia parishjj were assembled into contigs and edited using Cyfindropuntia ramosjssima Sequencher v4.1 (Gene Codes Corporation, Grusonia bradtiana Inc.). Consensus sequences were aligned man- QQ Grusonia pufchelfa Grusonia pulcheila ually with Se-Alv2.0a72 (Rambaut, 1996). Gaps

55 -Pereskiopsis pcrteri were treated as missing data. Informative indels -Pereskiopsis aquose were coded. Phylogenetic relationships among Pereskiopsis diguetjj these taxa were estimated using Fitch parsimo- - bcfiviana T ephrocactus aeroacanthus ny, in PAUP*v4.0B8 (Swofford, 1998). Estima- tions of confidence in the clades obtained by subulata these analyses were gathered through boot- strap analysis (Felsenstein, 1985) with 10,000 gonjianii

100 Consolee coralJicole pseudoreplicates, and through jackknifing (Far-

80 t-- Opuntia chaffeyi ris et al., 1996), also with 10,000 pseudorepli- I ~7 Opuntia erlnacea cates (63% deletion) as performed by PAUP*. Nopalea cocheniJJifera 84 174 -BrasiJiopuntia brasiliensis Results and Discussion inamoena 69 - Jongispina As a whole, Grusonia sensu Anderson Miqueliopuntie miquelii (2001) is not supported as a monophyletic Maihueniapoeppigii group, rather, it is apparently paraphyletic. The two specimens of G. pulchella form a well-sup- Figure s. A phylogenetic analysis of Grusonia pul- ported monophyletic group (99% bootstrap, chella and related taxa. Strict consensus of 809,300 88% jackknife) that is sister to the remaining most-parsimonious trees found by the Heuristic samples of Grusonia (Corynopuntia, Gruso- search of the nrITS DNA data. With uninformative characters excluded, length = 208; CI = 0.6667; RI = nia, and Marenopuntia) and all sampled mem- 0.8427; rescaled consistency = 0.6847. Bootstrap per- bers of the genus Cylindropuntia (Fig. 5). Al- centages above 50% are indicated above the branch- though this sister group relationship lacks es, and jackknife support above 50% is indicated strong statistical support, this topology was re- below the branches. covered in all 809,300 most-parsimonious 90 Haseltonia, No.9, 2002

Table 1. Sourcesof nrITS DNA sequencesfor specimensused in molecular analysis

Species Source* Voucher

Austrocylindropuntia subulata (Muehlenpfordt) Backeberg D B G 19900692

Brasiliopuntia brasiliensis (Schumann) Berger D B G 19900559

Consolea corallicola Small D B G 19970397

Cumulopuntia boliviana (Salm-Dyck) F. Ritter D B G 19709884

Cylindropuntia acanthocarpa (Engelm. & Bigelow) San Bernardino County, CA, USA Gri.ffith 211 (RSA)

Cylindropuntia echinocarpa (Engelm. & Bigelow) Knuth Clark County, NV, USA Gri.ffith 200 (RSA)

Cylindropuntia imbricata (Haworth) Knuth Nuevo Leon, Mexico Griffith 250 (RSA)

Cylindropuntia leptocaulis (de Candolle) Knuth , Mexico Gri.ffith 244 (RSA)

Cylindropuntia californica 0. Torrey & Gray) Knuth LosAngeles County, CA, USA Columbus s.n. (RSA)

Cylindropuntia ramosissima (Engelm.) Knuth Clark County, ~ USA Griffith 202-(RSA)

Cylindropuntia rosea (de Candolle) Backeberg Puebla, Mexico Griffith 187-(RSA)

Cylindropuntia tunicata (Lehmann) Knuth Coahuila, Mexico Griffith 256-

Cylindropuntia whipplei (Engelm. & Bigelow) Knuth SanJuan County, NM, USA Porter s.n. (RSA)

Grusonia aggeria (Ralston and Hilsenbeck)Anderson S R S U Powell6006 (SRSC)

Grusonia bradtiana (Coulter) Britton & Rose D B G 19850345

Grusonia invicta (Brandegee)Anderson R SA Griffith 218 (RSA)

Grusonia grahamii (Engelm.) H. Rob. S R S U Hardy 634 (SRSC)

Grusonia marenae (Parsons)Anderson D B G 19544980

Grusonia parishii (Orcutt) Pinkava R SA Wisura s.n. (RSA)

Grusonia pulchella(Engelm.) H. Rob. Churchill County, ~ USA Griffith 210 (RSA)

Esmeralda County, ~ USA Griffith 353 (RSA)

Grusonia villis (Rose) H. Rob. Coahuila, Mexico Griffith 246 (RSA)

Maihuenia patagonica (Philippi) Britt. & Rose Hershkovitz and Zimmer, 1997

Maihuenia poePPigii (Otto ex Pfeiffer) Hershkovitz and Zimmer, 1997 Philippi ex Schumann

Maihueniopsis atacamensis (Philippi) Ritter DBG20010112

Miqueliopuntia miquelii (Monville) F. Ritter D B G 19970129

Nopalea cochenillifera (L.) Salm-Dyck D B G 19970395

Opuntia chaffeyi Britt. & Rose D B G 19900238

Opuntia erinacea Engelm. & Bigelow Mono County, CA, USA Honer 658 (RSA)

Pereskiopsis aquosa (Weber) Britt. & Rose D B G 19970001

Pereskiopsis porteri (Brande. ex Weber) Britt. & Rose Hershkovitz and Zimmer, 1997

Pereskiopsis diguettii (Weber) Britt. & Rose Michoacan, Mexico Griffith 169 (RSA)

Pterocactus gonjianii Kiesling D B G s.n.

Quiabentia chacoensis Backeberg D B G 19850046

Tacinga inamoena (Schumann) Stuppy &Taylor DBG19990017

Tephrocactus aeroacanthus (Lemaire) Lemaire DBG20010115

Tunilla corrugata (Salm-Dyck) Hunt & Iliff D B G Hunt 66371

*D B G = Desert Botanical Garden, 1201 North Galvan Parkway, Phoenix, Arizona, USA; R SA = Rancho SantaAna Botanic Garden, 1500 North College Avenue, Claremont, California, USA; S R S U = Sui Ross State University Cactus Garden, Alpine, Texas, USA. Haseltonia, No.9, 2002 91

trees. In addition, the type species of Grusonia group. However, it would unnecessarily ob- (G. bradtiana) is sister to all sampled species scure the natural morphological diversity of of Cylindropuntia and Grusonia, with the ex- the lineage as well as the morphological cohe- ception of G. pulchella. The remaining Gruso- siveness of many of its members. nia species sampled form a monophyletic I am aware of the lack of strong statistical group, although this clade also lacks strong sta- support for many of the relationships suggest- tistical support. Since the paraphyly of Gruso- ed by the DNA sequence data. However, as nia sensuAnderson (2001) lacks such support, there is even less support for the monophyly of hypotheses that propose an affinity between G. Grusonia sensu Anderson (2001), I propose pulchella and other grusonioid cacti (Engel- the recognition of these older generic con- mann, 1863; Brit ton and Rose, 1919; Benson, cepts. Ongoing investigations involving addi- 1957, 1982; Anderson, 2001; Stuppy, 2002) tional DNA sequencing of nuclear and chloro- cannot be rejected by these data. Although the plast genes and anatomical work may further relationships between Cylindropuntia and elucidate these relationships, allowing for the Grusonia also lack strong statistical support, most perceptive possible. Further in- these data provide evidence that Cylindropun- vestigations may also elucidate whether specif- tia is nested within Grusonia sensu Anderson ic rank is warranted for the various morpho- (2001). The current circumscription of Gruso- types (Daston, 1946; Benson, 1957) currently nia (Anderson, 2001; Stuppy, 2002) therefore circumscribed within Micropuntia pulchella. taxonomically recognizes a non-monophyletic group. Nomenclatural Changes Although the ITS data do resolve differences between Cylindropuntia and Grusonia, the The following new combinations are need- sister group relationship of G. pulchella to all ed in support of this manuscript: other non-tropical North American terete- stemmed opuntioids may indicate a distinct Corynopuntia aggeria (Ralston and Hilsen- evolutionary lineage for the sand cholla. In the beck) M. P. Griffith, comb. nov. Basionym: context of recent tendencies to split the large Opuntia aggeria Ralston and Hilsenbeck, genus Opuntia sensu Benson (1982) into small- Madrofio 4: 226. 1989. er genera reflecting natural groups (Robinson, Corynopuntia emoryi (Engelm.) M.P. Griffith, 1973; Anderson, 1999,2001; Stuppy, 2002; Wal- comb. nov. Basionym: Opuntia emoryi En- lace and Dickie, 2002), I propose a solution to gelm., Proc.Am.Acad. 3: 303.1857. improve the taxonomy of the grusonioid cacti. Corynopuntia kunzei (Rose) M.P. Griffith, In the context of the morphological evidence, comb. nov. Basionym: Opuntia kunzei geographical separation, and the monophyly of Rose, Smithsonian Misc. Collect. 50: 505. the G. pulchella lineage, the recognition of 1908. these plants under the genus Micropuntia is Corynopuntia marenae (Parsons) M.P. Grif- supported. The generic concept of Grusonia fith, comb. nov. Basionym: Opuntia mare- may revert to that of Brit ton and Rose (1919), nae Parsons, Desert Pl. Life 8: 10. 1936 circumscribing only the type species G. brad- Micropuntia pulcbella (Engelm.) M.P. Griffith, tiana. Support for this narrow concept can comb. nov. Basionym: Opuntia pulcbella also be found in that species' morphological Engelm., Trans. Acad. Sci. St. Louis 2: 201. distinctness from other Grusonia species (Brit- 1863. ton and Rose, 1919; Anderson, 2001; Stuppy, 2002) and in the monophyly of the remaining Acknowledgements species. These remaining species of Grusonia I profusely thank Travis Columbus, Mark sensuAnderson (2001) should be treated under Porter, and Lee Lenz for ongoing advisement the genus Corynopuntia sensu Knuth (1935), and support. I gratefully thank Karen Walker including G. marenae (Parsons) Anderson (but and Charlie Butterworth for excellent com- excluding G. pulchella). Morphological sup- ments on an early draft of this manuscript. I am port for this generic concept exists in the form very grateful to Ann Hirsch, Myron Kimnach, of characters used to circumscribe Gruso- and two anonymous reviewers, who provided nia subg. Corynopuntia sensu Stuppy (2002), excellent feedback. I enthusiastically thank in addition to the characters discussed above. A Laura Sparks for excellent help in the laborato- summary of the generic circumscriptions pro- ry. Wendy Hodgson and Patrick Quirk (Desert posed here is presented in Table 2. The other Botanical Garden), John Dolan (Rancho Santa solution would be to include Corynopuntia, Ana Botanic Garden), and Mike Powell (SuI Cylindropuntia, Marenopuntia, and Microp- Ross State University Cactus Garden) were ex- untia into a broadly circumscribed Grusonia ceptionally generous in providing live material; (which has priority). I am not in favor of this not enough praise can be given to botanic gar- option, although this broad circumscription dens that maintain such collections for re- would certainly recognize a monophyletic search! Immense gratitude is due Naomi Fraga 92 Haseltonia, No.9, 2002

Table 2. Summary of generic circumscriptions presented here

Genus Included species Synapomorphies, Geography Corynopuntia Knuth c. aggeria (Ralston & Hilsenbeck) M.P.Griffith Mat-forming habit with growth to 50 cm (60 cm in c. marenae) com- C. agglomerata (Berger) Knuth posed of clavate segments (terete C. bulbispina (Engelm.) Knuth in c. marenae). Spines of retrorsely barbed, stiff. Deserts c. clavata (Engelm.) Knuth and grasslandsof Northern Mexico

C d t (B ) Kn th and Southwestern United States to .ume orum erger u Nye County, N evad a. c. emoryi (Engelm.) M.P.Griffith C.grabamii (Engelm.) Knuth C. invicta (Brandegee) Knuth C. kunzei (Rose) M.P.Griffith C. marenae (Parsons) M.P.Griffith c. moellerina Knuth c. parisbii (Orcutt) Knuth c. rejlexispina (Wiggins & Rollins) Backeberg c. scbottii (Engelm.) Knuth C. vi/is (Rose) H. Robinson

Grusonia Reichenbach G. bradtiana (Coulter) Brit ton & Rose Mat-forming habit with erect, ribbed, jointed growth to 1 m. Flowering areoles without glochids. Chihuahuan Desert of Coahuila, Mexico.

Micropuntia Daston M. pulchella (Engelm.) M.P. Griffith Cushion habit to 20 cm high; stor- age tuber with areoles, glochids. Spines of fruit antrorsely barbed, flexible. Great Basin of Nevada, Utah, and California, United States.

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