Introduction

Ploidy has a long tradition of utility for illuminating species boundaries, hybrid zones, and interspecific relationships among plants (e.g., Stace 2000). Knowing the ploidal levels of taxa used in phylogenetic analyses can also aid in detecting potential hybridization events through incongruence in reconstructions using biparentally inherited nuclear loci (Ionta et al. 2007, Soltis et al. 2008). Researchers have frequently used cytological data to help understand species evolution and delimitations in the nopales or prickly pear cacti, i.e., the genus Opuntia (Pinkava and McLeod 1971, Pinkava et al. 1973, 1977, 1985, Weedin and Powell 1978, Pinkava and Parfitt 1982, Weedin et al. 1989, Pinkava et al. 1992, Powell and Weedin 2001, 2004). Subfamily Opuntioideae (Opuntia s.l., as previously recognized; Benson 1982) is known to have the highest number of polyploids in Cactaceae (Cota and Philbrick 1994, Pinkava 2002), and Opuntia s.s. is well known for interspecific hybridization (e.g., Grant and Grant 1982, Griffith 2003) and subsequent genome duplication (Pinkava 2002, L.C. Majure (LCM), R. Puente (RP), P. Griffith (PG), W.S. Judd (WSJ), P.S. Soltis (PSS), D.E. Soltis (DES) unpubl. data).

The significance of polyploidy in plant evolution and speciation has long been recognized (Stebbins 1940, 1950, 1971; Swanson 1957, DeWet 1971, Harlan and DeWet 1975, Grant 1981, Leitch and Bennett 1997, Ramsey and Schemske 1998, Adams and Wendel 2005, Tate et al. 2005, Doyle et al. 2008, Soltis and Soltis 2009, Jiao et al. 2011). As stated by Stebbins (1950), p. 369), “polyploidy … is one of the most rapid methods known of producing radically different, but nevertheless vigorous and well-adapted genotypes.” Polyploidy also is considered one of the unequivocal means of true sympatric speciation (Futuyma 1998, Otto and Whitton 2000) and is considered to be common in plants (Stebbins 1940, DeWet 1971, Ramsey and Schemske 1998, Tate et al. 2005). For example, virtually all major clades of angiosperms have undergone one or more episodes of genome duplication (Soltis and Soltis 2009). Likewise, polyploidy is very important throughout Cactaceae (Pinkava 2002), and within Opuntia s.s., polyploids previously have been recorded in Opuntia humifusa (Raf.) Raf., 1820, and relatives (Bowden 1945a, b, Pinkava et al. 1985, Powell and Weedin 2004, Baker et al. 2009a, b, Majure and Ribbens in press) of the Humifusa clade (sensu LCM, RP, PG, WSJ, PSS, DES unpubl. data).

There are currently six species recognized in the Humifusa clade, Opuntia abjecta Small, 1923, Opuntia humifusa, Opuntia macrorhiza Engelm., 1850, Opuntia pottsii Salm-Dyck, 1849, Opuntia pusilla (Haw.) Haw., 1812, and Opuntia tortispina Engelm. & J.M. Bigelow, 1856 (Pinkava, 2003; LCM unpubl. data). The Humifusa clade is distributed widely from the western U.S. and northern Mexico (represented by Opuntia macrorhiza s.l., Opuntia pottsii, and Opuntia tortispina) and throughout the eastern U.S. including the upper Midwest (e.g., Michigan, Minnesota, Wisconsin) and southern Ontario (Benson, 1982; represented by Opuntia abjecta, Opuntia humifusa s.l., Opuntia macrorhiza s.l., and Opuntia pusilla).

Opuntia humifusa s.l. is composed of numerous morphological entities that have been recognized in certain taxonomic treatments as different species (see Small 1933). Throughout its range, Opuntia humifusa s.l. has been divided into as many as 14 taxa (Britton and Rose 1920, Small 1933, Benson 1982, Majure and Ervin 2008). Thus, Opuntia humifusa s.l. is occasionally referred to as a species complex (Doyle 1990). Currently, two taxa are recognized in Opuntia humifusa s.l. (Opuntia humifusa var. ammophila (Small) L.D. Benson and Opuntia humifusa var. humifusa; Pinkava 2003). Likewise, Opuntia macrorhiza has been divided into as many as 11 taxa (see Benson 1982). Opuntia macrorhiza was previously considered a variety of Opuntia humifusa (see Benson 1962; see Table 1 for synonyms of Opuntia humifusa s.l. and Opuntia macrorhiza s.l. sampled in this study), Opuntia pottsii was considered a variety of Opuntia macrorhiza, and Opuntia tortispina was placed in synonymy with Opuntia macrorhiza (Benson 1982).

Opuntia pusilla has been divided into several species: Opuntia drummondii Graham, 1841, Opuntia frustulenta Gibbes, 1858, Opuntia impedita Small, 1923, Opuntia pes-corvi LeConte, 1857, and Opuntia tracyi Britton, 1911 (Britton and Rose 1920, Small 1933); however, Benson (1982) placed them in synonymy under the name Opuntia pusilla. Opuntia triacantha (Willd.) Sweet, 1826, also has been divided into several species, i.e., Opuntia abjecta of the Florida Keys, Opuntia militaris Britton & Rose, 1919, of Cuba, and Opuntia triacantha from different parts of the Greater and Lesser Antilles (Britton and Rose 1920), but all of these have since been placed in synonymy within Opuntia triacantha (Benson 1982). Phylogenetic and morphological studies have indicated that Opuntia abjecta is not even in the same clade as Opuntia triacantha (LCM, WSJ unpubl. data) and so here is treated as Opuntia abjecta.

Contributing to the confusing taxonomic history of this clade is the high degree of morphological variation exhibited by most taxa, the lack of complete sampling throughout the range of the clade, the absence of cytological and phylogenetic evidence, reliance on poorly prepared and sparse herbarium collections (Majure and Ervin 2008, LCM unpubl. data), and hybridization and polyploidy (Benson 1982, Rebman and Pinkava 2001). Careful examination of morphological characters across the geographic range of the widely distributed Opuntia humifusa s.l. and Opuntia macrorhiza s.l. reinforces the hypothesis that hybridization may have preceded the origin of geographical morphotypes, because morphological characters displayed by certain taxa appear to be introgessive between Opuntia humifusa s.l. and Opuntia macrorhiza s.l. (Table 2). For instance, Opuntia cespitosa Raf., 1830, from the eastern U.S. and recently recognized by Majure and Ervin (2008), has yellow tepals that are basally tinged crimson- to orange-red, a characteristic typical of Opuntia macrorhiza and occasionally Opuntia tortispina from western North America (Benson 1982, Pinkava 2003, Powell and Weedin 2004), but the spine characters of Opuntia cespitosa are typical of Opuntia humifusa s.l. (see Majure and Ervin 2008).

Although chromosome counts have been reported for many of the Opuntia taxa from the southwestern U.S. and other areas (Stockwell 1935, Spencer 1955, Pinkava and McLeod 1971, Pinkava et al. 1973, 1977; Weedin and Powell 1978, Pinkava and Parfitt 1982, Pinkava et al. 1985, Weedin et al. 1989, Pinkava et al. 1992, Powell and Weedin 2001, Pinkava 2002, Negrón-Ortiz 2007, Segura et al. 2007, Baker et al. 2009a, b), few chromosome counts have been reported for taxa of Opuntia in the eastern and midwestern U.S. (Majure and Ribbens in press), and most of those taxa belong to the Humifusa clade. Bowden (1945a, b), Hanks and Fairbrothers (1969), Doyle (1990), and Baker et al. (2009 a, b) have all made counts of members of the Humifusa clade from the eastern U.S. Bowden (1945a, b), Doyle (1990), and Baker et al. (2009a) recorded diploid (2n = 22) and tetraploid (2n = 44) material of Opuntia humifusa from the eastern U.S., and Bowden (1945a) recorded tetraploid (2n = 44) material of Opuntia impedita (currently syn. of Opuntia pusilla). Hanks and Fairbrothers (1969) recorded an aneuploid number for Opuntia humifusa (2n = 17, 19) likely in error, since aneuploids are very rare in Cactaceae (Pinkava 2002). Majure and Ribbens (in press) recorded tetraploids of Opuntia humifusa s.l. and Opuntia macrorhiza s.l. from the Midwest, suggesting that the northernmost populations of those taxa are polyploid. Opuntia macrorhiza, Opuntia pottsii, and Opuntia tortispina have all been counted extensively in the southwestern U.S. (Pinkava and McLeod 1971, Pinkava et al. 1973, Pinkava et al. 1977, Pinkava et al. 1992, Pinkava et al. 1998, Powell and Weedin 2001, Powell and Weedin 2004), where Opuntia macrorhiza and Opuntia pottsii have been recorded exclusively as tetraploids, and Opuntia tortispina has been recorded as either tetra- or hexaploid.

Chromosome counts reported for species in the Humifusa clade do not encompass all of the taxa within the range of the clade nor the wide distributions exhibited by several of the more common species. To further our understanding of species complexes and the evolution of polyploids within those complexes, cytological data are needed from the entire distribution of a given species (Babcock and Stebbins 1938, Stebbins 1942, Stebbins 1950). Thus, an in-depth study of the distribution of cytotypes and correlations between cytotypes and morphology is desperately needed in order to aid in the delimitation of potentially unrecognized and cryptic species and to elucidate relationships in the Humifusa clade.

Here we present chromosome counts for all taxa considered to be part of the Opuntia humifusa complex and all taxa of the Humifusa clade (LCM, WSJ, PSS, DES, unpubl. data) and provide counts throughout most of the known ranges of all taxa to determine the geographic structure of ploidy and differences in ploidy among morphologically distinct taxa. We also reconstruct a phylogeny of diploid and polyploid members of the Humifusa clade based on nrITS data to investigate the relationship between geographic distribution and evolutionary relationships. We provide counts for another common species in the southeastern U.S., Opuntia stricta (Haw.) Haw., 1812, because it has been hypothesized to hybridize with members of the Humifusa clade (Benson 1982). In addition, ploidy of the putative hybrid between Opuntia abjecta and Opuntia stricta, i.e., Opuntia ochrocentra Small, 1923, was analyzed. Ploidy determinations of the Humifusa clade, coupled with morphological character analysis and further molecular phylogenetics, will aid in the delimitation of species in the group and in determining the origin and evolutionary significance of polyploidy in this clade.

Material and methods

Chromosome counts – Methods follow those of Majure and Ribbens (in press). Briefly, root tips were collected from early morning throughout early afternoon and placed in 2mM 8-hydroxyquinoline (Soltis 1980) for up to 8 hours at 4°C or in N₂O(Kato 1999) for 1 hour and then fixed in a 3:1 solution of absolute ethanol: glacial acetic acid for 2 to 24 hours. Root tips then were placed in 70% ethanol for at least 2 hours and digested in 40% HCl for 5-10 minutes (depending on the size of the root) at room temperature. Squashes were performed in 60% acetic acid and stained with 1% aceto-orcein dye and viewed on a Zeiss Photomicroscope III (Carl Zeiss, Oberkochen, Germany). To confirm each count, at least three to five metaphase cells were counted per specimen. These multiple counts per sample alleviated concerns regarding endomitosis, which has been reported in the allopolyploid (4x), Opuntia spinosibacca M.S. Anthony, 1956, (Weedin and Powell 1978), tetraploid Opuntia pusilla (Bowden 1945b), as well as in many other angiosperms (e.g., Barrow and Meister 2003, Tate et al. 2009, I. Jordan-Thaden, pers. comm.). We counted chromosomes of 277 individuals of the Humifusa clade, 14 individuals of Opuntia stricta s.l., three samples of the putative hybrid Opuntia ochrocentra, and two individuals of the putative hybrid Opuntia alta Griffiths, 1910. Generally, only one accession per population was counted.

Taxonomy – Taxa used for ploidy analysis are listed in Appendix 1. Species delimitations within Opuntia humifusa s.l. and Opuntia macrorhiza s.l. are problematic, so we recognize both Opuntia humifusa and Opuntia macrorhiza as broadly circumscribed (Table 1). Thus, we have arranged our counts of plants within these two species (see Appendix 1) according to their various segregates to determine whether the morphological variation of these segregate entities (Table 2) is correlated with cytotype and/or geographical and phylogenetic patterns.

Cytogeographic analysis – We mapped the localities for all of the individuals for which we determined ploidy (277 in number) and incorporated previous counts (n = 41) (Bowden 1945a, Pinkava and McLeod 1971, Pinkava et al. 1973, Weedin and Powell 1978, Pinkava and Parfitt 1982, Pinkava et al. 1985, Weedin et al. 1989, Doyle 1990, Pinkava et al. 1992, Pinkava et al. 1998, Powell and Weedin 2001, Baker et al. 2009a, b; Majure and Ribbens in press) to cover the majority of the geographic distribution of each taxon. This allowed us to explore the geographic boundaries of the different ploidal levels encountered in this clade and construct hypotheses regarding polyploid formation and speciation.

Phylogenetic analysis – We generated sequences from the nuclear ribosomal internal transcribed spacer (nrITS: White et al. 1990) for a sample of diploid (n = 6) and polyploid taxa (n = 8) of the Humifusa clade from the eastern and western U.S. (Table 3). Opuntia basilaris Engelm. & J.M. Bigelow, 1856, was used as an outgroup based on previous analyses of Opuntia (LCM unpubl. data). A phylogenetic analysis of these data was carried out to determine whether the geographic distribution of ploidy (as determined here) was correlated with the evolutionary history of the clade. We carried out a Maximum Likelihood analysis using RAxML (Stamatakis 2006) running 10000 bootstrap pseudoreplicates under 25 rate categories and the GTR+Γmodel of molecular evolution.

Results

The base chromosome number for Cactaceae has been well established as x = 11 (Remski 1954, Pinkava and McLeod 1971, Lewis 1980, Pinkava et al. 1985, Pinkava 2002), and we saw no deviation from this in our counts (Appendix 1). Out of 318 counts of the Humifusa clade, including 41 from the literature, 210 (66%) were polyploid and 108 (34%) were diploid. Diploid (2n = 2x = 22) and tetraploid (2n = 4x = 44) Opuntia humifusa s.l. and Opuntia macrorhiza s.l. were discovered (Fig. 1A-D, I-J, L). Diploid Opuntia humifusa s.l. is restricted entirely to the southeastern U.S., whereas diploid Opuntia macrorhiza s.l. is restricted entirely to the southwestern U.S. (eastern Texas (see Appendix 1) and southeastern New Mexico (M. Baker and D.J. Pinkava pers. comm.)). Tetraploid members of Opuntia humifusa s.l. and Opuntia macrorhiza s.l. are much more widely distributed throughout the U.S. than are their diploid relatives (Fig. 2). Tetraploids of Opuntia humifusa s.l. are found from Massachusetts south to the southeastern U.S. where they abut the distribution of diploid taxa and throughout the eastern and midwestern U.S. Tetraploid Opuntia macrorhiza s.l. is distributed throughout parts of the Great Plains through the midwestern U.S., most of the southwestern U.S., parts of the Rocky Mountains, and the upper Sierra Madre Occidental in Sonora, Mexico (Fig. 2).

Diploid, triploid, and tetraploid populations of Opuntia pusilla were discovered (Fig. 1E-G) throughout its restricted range in the southeastern U.S. (Fig. 3). Interestingly, with the exception of two populations, polyploid individuals (3x and 4x) were mostly confined to the coastline, although diploid populations were much more widespread throughout the interior part of the distribution of the species (Fig. 3). Of the three examples of Opuntia abjecta sampled from the Florida Keys, one was diploid (Fig. 1H), and two were tetraploid. Opuntia tortispina (southwestern U.S.) was hexaploid in six and tetraploid in one of the populations examined (see Fig. 2 for hexaploid distribution).

Individuals of Opuntia stricta sampled from the southeastern U.S. were all hexaploid. Samples included members of the taxa considered by some (Anderson 2001) to be Opuntia dillenii (Ker-Gawl.) Haw., 1819, and Opuntia stricta. Three individuals of the putative hybrid Opuntia ochrocentra from two localities in the Florida Keys were pentaploid (Fig. 1K), and the putative hybrid Opuntia alta was hexaploid.

Maximum likelihood analysis of ITS data reveals that the Humifusa clade is made up of two well-supported subclades. One is restricted to the southeastern U.S. and includes polyploid members of Opuntia pusilla and Opuntia abjecta, and the other includes southwestern diploid Opuntia macrorhiza and all other polyploids pertaining to Opuntia humifusa s.l., Opuntia macrorhiza s.l., and Opuntia tortispina. There is no further resolution within the tree at the species level using ITS (Fig. 4). Species relationships within these two clades are further resolved with the addition of other loci (LCM unpubl. data), however, that is beyond the scope of this study.

Discussion

Opuntia macrorhiza has only been recorded previously as tetraploid (Pinkava et al. 1971, 1973, 1977, 1992, 1998; Powell and Weedin 2001, 2004; Pinkava 2003). These are the first reports of diploid Opuntia macrorhiza and likely represent descendants of those progenitors from which tetraploid Opuntia macrorhiza s.l. and other polyploids arose. Likewise, this is the first report of diploid and triploid Opuntia pusilla, which was formerly known only from tetraploid counts (Bowden 1945a).

Diploid members of Opuntia humifusa s.l. (e.g., represented by the segregate taxa Opuntia ammophila Small, 1919, Opuntia austrina Small, 1903, Opuntia lata Small, 1919, in this study; see also Appendix 1) exhibit high levels of morphological variability but each is diagnosable morphologically, which suggests that these segregate taxa may need to be recognized at the species level. Likewise, diploid material of Opuntia macrorhiza s.l. from eastern Texas (e.g., Opuntia xanthoglochia Griffiths, 1910, in this study; see also Appendix 1) and southeastern New Mexico is morphologically distinct from tetraploid material of Opuntia macrorhiza s.l., which may also justify the recognition of Opuntia xanthoglochia and Opuntia macrorhiza as separate species.

Our hexaploid counts of Opuntia stricta are consistent with those of Pinkava et al. (1992) and Negrón-Ortiz (2007).In contrast, Spencer (1955) reported Opuntia stricta from Puerto Rico to be diploid. Other authors have also found Spencer’s counts from Puerto Rico to be inconsistent with more recent counts (e.g., Negrón-Ortiz 2007 for Consolea Lem., 1862).

Our three pentaploid counts of Opuntia ochrocentra support the proposed hybrid origin of this species between hexaploid Opuntia stricta (2n = 66) and diploid Opuntia abjecta (2n = 22) through unreduced gametes of Opuntia abjecta. Opuntia ochrocentra also exhibits intermediate morphological characters (e.g., growth form, spine characters) that further support its hybrid origin (LCM unpubl. data).

Diploid refugia and polyploid formation – Polyploidy is very common within the Humifusa clade, occurring in 66% of the samples reported here. Most researchers that have studied Opuntia cytologically have found polyploid taxa (e.g., Bowden 1945a, Weedin and Powell 1978, Pinkava et al. 1985, Doyle 1990, Segura et al. 2007, Baker et al. 2009a, b, Majure and Ribbens in press, but see Spencer 1955). All diploids in our analysis were restricted to either the southeastern or southwestern (eastern Texas and southeastern New Mexico) U.S., and the polyploid individuals were found nearly everywhere in between as well as north of these two diploid “refugia.” The disjunct pattern observed here in the Humifusa clade and in other studies between the southeastern U.S. and the southwestern U.S. is thought to have occurred as a result of the disruption of a semi-arid zone along the Gulf Coast region during the mid-Pleistocene (Webb 1990, Althoff and Pellmyr 2002). These two areas likely served as glacial refugia for a variety of animals and plants (e.g., Remington 1968, Davis and Shaw 2001, Al-Rabab’ah and Williams 2002, Althoff and Pellmyr 2002, Soltis et al. 2006, Waltari et al. 2007, Whittemore and Olsen 2011) and may have promoted current species richness and genetic diversity in southern populations (Hewitt 2000). Specifically, Swenson and Howard (2005) identified southeastern Texas and northern Florida as Pleistocene refugia for animal and plant species. Species from these regions subsequently came into contact following the last glacial maximum and formed hybrid zones at contact areas expanding out from these refugia. Swenson and Howard (2005) also hypothesized “post-glacial routes of expansion” from these proposed diploid refugia (e.g., Fig. 1, G & H in Swenson and Howard 2005). Those post-glacial routes and diploid contact zones are consistent with the current distributions of polyploid taxa within Opuntia humifusa s.l. and Opuntia macrorhiza s.l. The restricted diploid and widespread polyploid distribution pattern has been recorded in many other plants and is a common pattern seen in polyploid complexes (Babcock and Stebbins 1938, Stebbins 1950, 1971, DeWet 1971, Lewis 1980, Grant 1981, Parfitt 1991).

The seemingly disjunct southeastern New Mexico diploid population of Opuntia macrorhiza s.l. may represent a mere extension of the eastern Texas diploid refugium, which has since been mostly replaced by polyploid taxa. Alternatively, a diploid extension may still exist but was not detected due to the lack of cytological data for populations from east Texas to southeastern New Mexico (Fig. 2). Diploid taxa of other clades (e.g., Opuntia polyacantha Haw. var. arenaria (Engelm.) Parfitt, 1819) are coincidentally found near the same region (Pinkava 2002, 2003), however, suggesting that a third diploid refugium, i.e., in southeastern New Mexico-western Texas, may need to be recognized.

Pinkava (2003) suggested that an Opuntia humifusa - Opuntia macrorhiza - Opuntia pottsii complex originated along the east coast of the U.S. and spread westward to Arizona, where it came into contact and hybridized with Opuntia polyacantha and formed the mostly hexaploid Opuntia tortispina. From our data, this scenario is plausible in that Opuntia tortispina has morphological characters representative of both Opuntia polyacantha and Opuntia macrorhiza and is found where populations of diploid and tetraploid Opuntia macrorhiza s.l. and diploid Opuntia polyacantha come into contact. However, considering the two diploid refugia suggested by our analyses and what is known about the historical biogeography of the southeastern U.S. (e.g., Webb 1990), it is likely that the Humifusa clade originated in the southwestern U.S. and adjacent northern Mexico, then dispersed eastward into the southeastern U.S. The arid habitat along the coast of the Gulf of Mexico during the mid-Pliocene to early Pleistocene would have been interrupted during the mid-Pleistocene, creating the disjunction and promoting the genetic divergence among diploid populations we see today (Fig. 4). Taxa from these two diploid refugia would have come back into contact and formed the widely successful polyploids of the Midwest and eastern U.S. (Fig. 5). This scenario is further corroborated by phylogenetic analyses, where eastern U.S. polyploids of Opuntia humifusa s.l. are resolved in a clade with the southwestern diploid Opuntia macrorhiza (Fig. 4). The lower frequency of diploids encountered in western populations of the Humifusa clade also suggest that those diploid populations may be older (see Stebbins 1971, p. 157) than those of the southeastern U.S.; however, this could merely be a bias resulting from more limited sampling of western populations.

The various morphotypes of tetraploid Opuntia macrorhiza in the western U.S. likely arose from southwestern diploid populations but subsequently spread in all directions after formation. Tetraploid Opuntia macrorhiza appears to have arisen numerous times, given that several morphotypes exist throughout its range. However, only two diploid morphotypes are known to exist (eastern Texas and southeastern New Mexico), suggesting that other ancestral diploids may have since gone extinct or have not yet been found, or that polyploid taxa exhibiting unique, derived characters were partly responsible for the origin of certain morphotypes, which have no diploid counterparts.

Stebbins (1971) suggested that there are several degrees of maturation of polyploid complex formation (i.e., initial, young, mature, declining, relictual), which may be deduced by comparing the relative geographic distribution of polyploids versus diploids. By these criteria, Opuntia humifusa s.l. and Opuntia macrorhiza s.l. may represent a mature polyploid complex. The diploid taxa are less common than polyploids and are largely restricted in distribution, whereas the polyploid taxa are much more widespread. Stebbins (1971) also proposed that mature polyploid complexes are relatively young, derived during the Plio- or Pleistocene epochs. This scenario would place polyploid formation in the Humifusa clade at the same time as Pleistocene megafauna. Thus, frequent environmental disturbances associated with glacial and interglacial cycles could have mediated the repeated contact of divergent diploid taxa leading to polyploid formation. Migrating herbivores would have then dispersed those polyploid products over large geographic areas (Jansen 1986). Divergence time estimation of the Humifusa clade places the origin of the clade in the late Pliocene to early Pleistocene (LCM, RP, PG, WSJ, PSS, DES unpubl. data), in agreement with this scenario. The occurrence of only polyploid individuals in previously glaciated areas of the U.S. provides further evidence for their subsequent spread into those available niches following the last glacial maximum.

Many polyploid populations of Opuntia humifusa s.l. and Opuntia macrorhiza s.l., especially in the eastern U.S., are largely isolated from one another and from diploid populations, suggesting that polyploid formation is not ongoing, at least on such a large scale as during the Pleistocene or immediately after the last glacial maximum. In contrast, polyploids in Opuntia pusilla are mostly sympatric with diploids in the Gulf of Mexico region and are represented by triploids and tetraploids. Polyploids of Opuntia pusilla also do not share the wide geographic distribution of those polyploids derived from Opuntia humifusa s.l. and Opuntia macrorhiza s.l. These observations suggest that the polyploids of Opuntia pusilla may have formed only recently, do not share comparable dispersal agents, or lack the obvious adaptive advantages of those polyploids derived from Opuntia humifusa s.l. and Opuntia macrorhiza s.l.

Many polyploid populations of Opuntia humifusa s.l. and Opuntia macrorhiza s.l.occupy northerly distributions and thus have a very high tolerance to cold temperatures. The hexaploid Opuntia fragilis (Nutt.) Haw., 1819 (not in the Humifusa clade) similarly inhabits areas of northern North America (Parfitt 1991, Loik and Nobel 1993, Ribbens 2008, Majure and Ribbens in press), with diploid relatives (e.g., Opuntia polyacantha var. arenaria) restricted to the southwestern U.S. (Parfitt 1991, Pinkava 2002). Thus, certain polyploid taxa appear to be more cold-resistant than their southerly diploid relatives (and presumed progenitors). Opuntia humifusa s.l. from northern areas of its distribution can withstand temperatures of -20°C (Nobel and Bobich 2002). However, the cold tolerance of diploid taxa has not been tested. Certain polyploid taxa of the Humifusa clade may therefore be better adapted to adverse environmental conditions than their diploid progenitors, which may partly explain their wide distribution relative to their diploid counterparts.

Agamospermy – The tetraploid Opuntia cespitosa (an entity within Opuntia humifusa s.l.; see Table 1) produces viable seed in the absence of outcrossing (Majure pers. obsv.), so this taxon is either self-compatible, which is common in Cactaceae (Rebman and Pinkava 2001), or agamospermous. Agamospermy is commonly associated with polyploidy (Stebbins 1950, DeWet and Stalker 1974, Harlan and DeWet 1975, Lewis 1980, Grant 1981, Whitton et al. 2008) and has been reported in numerous polyploid Opuntia species as well (Reyes-Agüero et al. 2006, Felker et al. 2010), including Opuntia humifusa s.l. and Opuntia stricta (Naumova 1993). Agamospermy would account for the high level of morphological variation observed among polyploid populations, as a result of the maintenance of a specific genotype within a given population through the lack of recombination (DeWet and Stalker 1974). Some agamic complexes also have wider distributions than their diploid progenitors (Babcock and Stebbins 1938, Stebbins 1950), as do certain polyploid taxa in this study.

Autopolyploidy vs. Allopolyploidy – The mechanism by which Opuntia polyploids are formed (auto- vs. allopolyploidy) is unclear. Unreduced gametes have frequently been found in meiotic analyses of Cactaceae (e.g., Pinkava et al. 1977, Pinkava and Parfitt 1982, Pinkava et al. 1985). Unreduced gamete formation coupled with interspecific hybridization (allopolyploidy) likely is a major factor in polyploid formation within the genus, given that Opuntia is renowned for hybridization (Benson 1982, Grant and Grant 1982, Pinkava 2002, Griffith 2004, LCM, RP, PG, WSJ, PSS, DES unpubl. data). It is probable that unreduced gamete formation within a single species (autopolyploidy) also plays a role in the formation of polyploids. Autopolyploids have been discovered in Cactaceae (Pinkava et al. 1985, Sahley 1996, Hamrick et al. 2002) and may be more common than is suspected.

Opuntia humifusa as currently circumscribed consists of numerous morphological entities, which are either diploid or tetraploid; those populations differing in ploidy are generally geographically well separated from one another. It is evident from our phylogenetic analysis (Fig. 4) that Opuntia humifusa is polyphyletic. Considering morphological and genetic data, it is likely that tetraploid Opuntia humifusa is of allopolyploid origin. However, the pattern in Opuntia pusilla is different, with populations of diploids found in close proximity to populations of triploids and tetraploids (Fig. 3). This evidence, plus morphological similarity among ploidal levels, suggests possible formation of autopolyploids. This same pattern is seen in other autopolyploid taxa (Lewis 1967, Nesom 1983), although there are exceptions to this pattern (Stebbins 1950, Soltis 1984, Husband and Schemske 1998). Molecular phylogenetic analysis (Fig. 4) and morphological characters (LCM, RP, PG, WSJ, PSS, DES unpubl. data; see Fig. 1E-G) of Opuntia pusilla also do not support an interspecific hybrid origin for the different ploidal levels herein observed for this species, although more variable molecular markers, cytogenetic work, and more detailed morphological analyses are needed to appropriately address this question.

Morphological correlations with polyploids – Some polyploid taxa in the Humifusa clade share morphological characters with diploids and other polyploids, suggesting that they may be derived from hybridization (Table 2). Opuntia nemoralis Griffiths, 1913, (Fig. 1J; an entity within Opuntia humifusa s.l.; see Table 1) shares spine color and orientation, cladode color, and glochid color of tetraploid Opuntia macrorhiza (from Arkansas), although, it possesses small and easily disarticulating cladodes, retrorsely-barbed spines, and the pile forming growth form and yellow flowers of Opuntia pusilla (Fig. 1E-G). Opuntia cespitosa (Table 1), as mentioned above, exhibits the red-centered flowers, glaucous-gray cladodes, and dark glochids (Fig. 1I) of tetraploid Opuntia macrorhiza (Fig. 1D), as well as the spine characters of diploid Opuntia humifusa s.l. (= Opuntia ammophila, Opuntia austrina, Opuntia lata; Table 2).

Throughout the distribution of the most common polyploid taxa, there also are polyploid populations that appear to be introgessive products of hybridization with other polyploids. For instance, in Michigan, Wisconsin, and western Illinois, certain populations display characters of both Opuntia cespitosa and tetraploid Opuntia macrorhiza (see Majure 2010, Fig. 1). In Bibb County, Alabama, populations appear to be intermediate between Opuntia cespitosa and Opuntia pollardii Britton & Rose, 1908, (tetraploids of Opuntia humifusa s.l.; see Table 1), with the red-centered flowers and rotund cladodes of Opuntia cespitosa, but the yellowish glochids and light green cladode color of Opuntia pollardii. In Fayette County, Tennessee, plants appear intermediate between Opuntia humifusa s.s. (i.e., tetraploid Opuntia humifusa represented by the type collection) and Opuntia cespitosa, having the yellowish glochids of tetraploid Opuntia humifusa s.s. and the spine characters of Opuntia cespitosa. Each one of the areas in which these intermediate plants occur appears to be a region of secondary contact, where polyploid taxa have introgressed to form new polyploid morphotypes that exhibit characters of both of the putative parents.

In the eastern U.S., most populations are represented by only one morphotype and thus appear to be morphologically stable (except for typically variable characters such as spine number; see Rebman and Pinkava 2001), indicating that hybridization is not ongoing among genomically distinct polyploid taxa. In contrast, in central Arkansas and populations farther west, more than one species and/or morphotype may be encountered within a given population. Also, in many coastal populations throughout the southeastern U.S., more than one species may be encountered, and putative hybrid taxa are sometimes observed.

Conclusions

Members of the Humifusa clade are found throughout most of the continental U.S., with no obvious breaks or disjunctions in distribution patterns until detailed analyses of chromosome number were carried out. Our analyses indicate that diploid taxa in the Humifusa clade are presently confined to the southwestern and the southeastern U.S., which likely represent Pleistocene refugia for these taxa. Polyploid taxa of Opuntia humifusa s.l. and Opuntia macrorhiza s.l. were likely formed when diploids from these two refugia came into contact during interglacial cycles of the Pleistocene. This scenario is supported further by phylogenetic analyses, in which two clades correspond to these two diploid refugia, and polyploid taxa are found in either clade. Polyploid taxa likely also contributed to the diversity of polyploid morphotypes through secondary contact and introgression with other polyploids. After the end of the last glacial maximum, open niches would have been readily available for colonization by polyploid taxa produced towards the leading edge of the expansion and distribution of the Humifusa clade. These polyploids subsequently dispersed throughout most of the continent and occupied all suitable habitats available after glacial retreat, accounting for the distribution that we see today. Distributional success was enabled by the extreme cold tolerance displayed by many of the polyploid taxa, which allowed them to colonize more northern areas presumably unsuitable for diploid taxa.