In most species of Kosteletzkya, a flower persists for only a single day; by early afternoon it has already begun to wither. Cross-pollinations were therefore performed by hand in the morning, shortly after the flowers had fully opened. As has been reported for Kosteletzkya pentacarpos (Ruan et al. 2011), most species of Kosteletzkya recurve their styles later in the day and push their stigmas into the pollen mass, thereby effecting self-pollination in the absence of any earlier exogenous pollination. This creates a problem when controlled crosses are attempted because it is impossible to be certain that successful seed-set was due to pollen from the other experimental parent rather than from the same flower. In the case of the larger, sturdier flowers found in some other Malvoideae, the pre-anthesis removal of the anthers solves this problem. This has been done, for example, in Hibiscus sect. Furcaria (Menzel and Wilson 1961), Hibiscus sect. Muenchhusia (Fabricius, 1763) O. J. Blanchard, 1988 (Wise and Menzel 1971), and Gossypium (Wilson and Stapp 1985). However the same technique was found to be too traumatic and inefficient for smaller flowers such as those of Kosteletzkya.

A solution to this problem was found in the use of halves of gelatin capsules to separate the male and female parts of the flowers (Fig. 1a). In this technique a hole is punctured through the apex of a capsule-half using a dissecting needle that has been heated in a flame. A razor blade is then used to cut up one side of the capsule-half wall and over the top, passing through the previously cut hole. It is then possible to 1) insert the closed tines of a straight forceps into the open end of the capsule-half, 2) allow the tines to spread the razor-cut slit, 3) slip the whole unit over the base of the style branches distal to the anther mass, and then 4) allow the capsule-half to close, effectively isolating the stigmas and pollen mass from one another (Fig. 1b). In the present study each such unit was attached to a flower immediately after a cross-pollination, and the procedure proved to be highly successful in preventing self-pollination. The only drawback was that extra care had to be taken when watering the plants in order to avoid deforming or dissolving the capsule-halves.

The gelatin-capsule device could not be used with one of the species. Kosteletzkya borkouana Quézel, 1957 is effectively an obligate selfer because the stigmas have usually already recurved into the pollen mass by the time the corolla opens in the morning. To make matters more difficult, this species has the smallest flowers of any Kosteletzkya. Nevertheless it was necessary to visit the plants at 0300-0400h to carefully cut away the unopened corolla and remove the mercifully few pre-dehiscence anthers. And because the time was still hours away from the anthesis of any of the other species, actual manual cross-pollination of the emasculated flower had to await a later visit.

While most of the species that were studied were short-day, fall-and-winter-flowering plants, three of them flowered in the late summer (Kosteletzkya pentacarpos) or early fall (Kosteletzkya ramosa Fryxell, 1977 and Kosteletzkya reclinata Fryxell, 1977). To make these three available to a greater variety of other potential crossing partners, beginning in early summer plants of several of the other species were put on carts, moved daily at 1700h into an adjoining darkened room, and retrieved the next morning. Within three or four weeks, flower-bud initiation was evident. Early flowering was thereby induced so as to coincide with the flowering of the three late-summer-early-fall species.

In general, crosses worked in both directions. It appeared to make no difference in the success of an attempted cross whether a participant in the cross was the ovule-parent or pollen-parent, so no tabulated distinction is made here concerning the direction of the crosses reported. As a matter of insurance, however, the actual practice was that whenever two plants were crossed in which the size of the flowers, or more especially the style lengths, were considerably different, the smaller of the pair was used as the pollen recipient, on the theory that pollen adapted to traversing a short style might be challenged by a longer style (Williams and Rouse 1990, Sorensson and Brewbaker 1994, Tiffin et al. 2001).

Voucher specimens of most of the plants used as parents of crosses in this study, as well as specimens of the hybrids themselves, are deposited at the University of Florida Herbarium (FLAS), Florida Museum of Natural History, Gainesville, Florida, USA. In a few cases the vouchers for the parents are either the original wild-collected specimens that were the seed sources for the greenhouse plants, or they are specimens from the same seed source but grown in other years.

Pollen was stained with Cotton Blue in lactophenol. Each pollen slide was made from a single flower. Normal-sized, fully and deeply blue-stained grains were treated as “stained” and are expressed here as a percent of the total number of grains on a slide. At least three and usually five or more slides were counted for each hybrid combination. The majority of the species that participated as parents in the stainability evaluations were themselves counted and their stainabilities were found to range from 97 to 100 percent. Later in the hybridization program space was at a premium and the few replicates of hybrid plants that could be grown were used almost solely as a source of young flower buds for meiotic samples, so for some of the later-produced hybrids no fruit or pollen data were obtained. By that time, however, the general patterns of fruit-set and pollen stainability were already evident.

Recurvature of the styles is a problem for controlled pollinations, but it is a boon for fruit-set purposes because, with one exception, it was theoretically possible to let the greenhouse hybrids pollinate themselves and use those results rather than resorting to manual self-pollination. In actual practice, however, the plants were usually hand-pollinated anyway, as a part of the routine of nearly daily visits to the greenhouse.

The exception is hybrid progeny in which Kosteletzkya tubiflora (de Candolle, 1824) O. J. Blanchard & McVaugh, 1978 is one parent. This species bears distinctive yellow-and-red tubular flowers with an exserted staminal column and style (Figure 6G), and they are almost certainly bird-pollinated in the plant’s native setting. The related Kosteletzkya thurberi A. Gray, 1887 with structurally similar flowers, was reported to be visited by the Bumblebee Hummingbird (Atthis heloisa [Lesson and DeLattre, 1839]; reported as “Selasphorus heloisa”) in northern Mexico (Van Devender et al. 2004). Unlike the rest of the species, Kosteletzkya tubiflora has protogynous flowers that remain open and nectar-producing for two days or more. On the first day the stigmas are receptive and the anthers remain undehisced. By the next day the staminal column has elongated, exserting the now-dehiscent anthers to the earlier position of the stigmas. At this point, the stigmas may or may not remain receptive, but they do not recurve in the absence of pollination. As a consequence, the hybrids involving this species required hand-pollination.

Fruits will set in Kosteletzkya when as few as one of the five ovules has been fertilized, and no distinction is made here as to the number of seeds in a set fruit. When an unfertilized spent flower falls, part of the pedicel remains attached to the plant, readily marking the former presence of a flower. Percent fruit-set is simply the proportion of fruit-bearing pedicels out of a total number of post-flowering pedicels.

All chromosome counts and observations of chromosome pairing were made from pollen mother cells (PMCs) at meiotic metaphase I. Details of methods of collection, fixing, staining, and preservation of meiotic material can be found in Bates and Blanchard (1970) and Blanchard (2012). It was seldom possible to obtain preparations in which all of the chromosomes were in the same plane of focus. This was not a problem for microscopic examination and interpretation, but it usually yielded less-than-satisfactory photographs (Figure 2a–c), especially of hybrids with numerous univalents that were not constrained at the metaphase plate (Fig. 2b); hence the extensive use here of camera lucida drawings. For simplicity of presentation, and because of the scale of this study, cytological outcomes are expressed in bivalent-equivalents in which the occasional quadrivalent is converted to two bivalent-equivalents, and the occasional trivalent is expressed as a single bivalent-equivalent. Unpaired chromosomes (univalents) were encountered in all crosses between different ploidy levels (e.g. Fig. 4a–d), as well as when the parents of a cross are genetically substantially divergent (e.g. Fig. 2b, 3b, 5a), and these too were counted, at least earlier in the investigation. Again, however, for the sake of clarity, they are not tabulated, but should be understood to have been present. For most hybrids at least five PMCs were examined; in more that half of the cases, more than 10 were examined.

All seven available New World species were involved in the crossing program (an eighth species, Kosteletzkya thurberi, was unavailable), and all interspecific crosses that were attempted were successful and comprised 17 of the 21 possible pairwise combinations among the seven species. Hybrid plants generally grew as vigorously as their parents under greenhouse conditions. Pollen stainability among them ranged from 15 to 98 percent, while fruit-set ranged from 2 to 66 percent. Low or high values in one measure did not necessarily correspond with those of the other measure. For instance, the combination Kosteletzkya blanchardii Fryxell, 1977 × Kosteletzkya ramosa had 90 percent pollen stainability but only 7 percent fruit-set.

Despite wide morphological differences among the New-World species, nearly complete chromosome pairing, as indicated by the average number of bivalents, was found in each of the 15 hybrid combinations that were examined meiotically (Table 3). Average values ranged from 18.5 to 19 bivalents out of a possible 19. As an example, Fig. 3c shows a meiotic metaphase figure from the hybrid Kosteletzkya depressa (Linnaeus, 1753) O. J. Blanchard, Fryxell et D. M. Bates, 1978 × Kosteletzkya tubiflora, whose parental species are dramatically different in habitat, morphology and floral adaptations. Kosteletzkya depressa is a lowland, bee-pollinated plant with a small, white-to-pink, rotate corolla (petals 0.8–1 cm long), included staminal column, and a green calyx (Fig. 6B); Kosteletzkya tubiflora is an upland, apparently bird-pollinated plant with a large, yellow, tubular corolla (petals 2.5–3 cm long), exserted staminal column and a pink-to-red calyx (Fig. 6G).The two species also differ markedly in fruit and seed characteristics.

All three possible hybrids among the three known African diploid species Kosteletzkya adoensis (A. Richard, 1847) Masters, 1868, Kosteletzkya buettneri and Kosteletzkya grantii (Masters, 1868) Garcke, 1880 were obtained, although these offspring were not as robust as the New-World hybrids. Like the New-World diploid species, the three African parent species differ considerably in habit, leaf shape, inflorescence form and details of flowers (see Figs 6H, L, O and P), fruits and seeds. However in dramatic contrast to the diploid New-World hybrids, pollen stainability in the African diploid hybrids ranged from 0 to 1 percent, fruit-set was 0 percent, and average chromosome pairing ranged from 2.0 to 9.1 out of a potential 19 bivalent-equivalents. A meiotic metaphase I of the hybrid Kosteletzkya adoensis × Kosteletzkya grantii is shown in Fig. 5a.

Hybrids between African diploids and African tetraploids were generally obtained with more difficulty, particularly in the case of the diploid Kosteletzkya adoensis, in which, to cite the most extreme example, several hundred cross-pollinations with Kosteletzkya begoniifolia (Ulbrich, 1917) Ulbrich, 1924 resulted in only a single viable seed. Nevertheless, altogether six of the possible 12 diploid-tetraploid hybrids were eventually produced. As might be expected in hybrids between ploidy levels, pollen stainability was low (0-2 percent in the four interspecific combinations that were sampled for this characteristic) and their fruit-set was likewise low (0 percent in the same four combinations). Average chromosome pairing in these six hybrids varied widely. Two approached the potential maximum of 19, forming 17.9 to 18.8 pairs (Figs 4a and 4c), while the other four ranged in average from 3.9 to 13.1 bivalent-equivalents (Fig. 4b).

No diploid-hexaploid hybrids could be obtained despite numerous cross-pollinations.

The African polyploids could be crossed fairly easily among themselves, and eight of the ten possible hybrids were produced. In the five cases where pollen stainability and fruit-set were determined, stainability ranged from 5 to 97 percent, whereas fruit-set for four of the hybrids was 0 percent, while for a fifth (Kosteletzkya begoniifolia × Kosteletzkya rotundalata) it was 63 percent. Of the four tetraploid-tetraploid hybrids that were examined cytologically, one had bivalent-equivalents approaching the maximum possible 38, whereas the other three ranged from 3.5 to 24.6 bivalent-equivalents (Fig. 2b). Of the three tetraploid-hexaploid hybrids examined cytologically, two averaged over 37 pairs out of a potential maximum of 38 (Fig. 4d), while the third averaged only 6.5 bivalent-equivalents.

Eleven of the 21 possible diploid-diploid trans-Atlantic combinations were produced. At least four failed attempts involved Kosteletzkya adoensis as one potential parent. One combination that was obtained, Kosteletzkyagrantii × Kosteletzkya depressa, produced flower buds that aborted between meiosis and flowering, making pollen stainability and fruit-set data impossible to obtain. In another combination, Kosteletzkya adoensis × Kosteletzkya depressa, though the plants flowered, they were weak-stemmed and slow-growing, and produced only a feeble root system. The 10 surviving hybrids had pollen stainabilities and fruit-sets that were intermediate, on average, between those of New World diploid-diploid crosses and those of the African diploid-diploid crosses, and ranged from 0 to 74 percent pollen stainability and 0 to 14 percent fruit-set. However, depending on which of the African diploids participated, the pollen-stainability outcomes were different: in the seven African-New World crosses involving Kosteletzkya buettneri, stainability ranged from 26 to 74 percent; in the two crosses involving Kosteletzkya grantii, stainability was zero percent in both cases; and in the single cross involving Kosteletzkya adoensis, the result was seven percent pollen stainability. Outcomes of chromosome pairing observations were even more distinctly different depending on which African parent was involved. In all six combinations in which Kosteletzkya buettneri was the African parent, pairing closely approached the maximum possible 19 (see for example Fig. 3d). However when Kosteletzkya grantii or Kosteletzkya adoensis were involved, the pairing in the three hybrids that were examined cytologically ranged from 1.2 to 11.4 bivalent-equivalents (see for example Fig. 3b).

In the case of trans-Atlantic crosses between African tetraploids and New-World diploids there was again a bimodal pattern. In three of the seven hybrid combinations examined meiotically, chromosome pairing approached the maximum 19; in the other four the range was 4.3 to 10.9.

As was the case for African-African crosses, no diploid-hexaploid trans-Atlantic combinations could be obtained despite numerous crossing attempts.

In clear contrast to the nearly perfect chromosome pairing (18.5–19 bivalent-equivalents) in all of the 17 diploid New-World hybrids, the three African diploids contain chromosome sets with only low-to-modest affinity among themselves (2.0, 3.1 and 9.1 bivalent-equivalents). A consequence of this is mirrored in the negligible pollen stainability (0 to 1 percent) and zero fruit-set in hybrids among the three African species. This has prompted the designation here of three distinct genomes among the African diploids: A for the Kosteletzkya adoensis genome, B for the Kosteletzkya buettneri genome and G for the Kosteletzkya grantii genome.

Considering chromosome-pairing relationships in these genomic terms, it is apparent that only one genome is shared by all of the New World species. More interestingly, the trans-Atlantic crosses between the African diploid Kosteletzkya buettneri and six different species from the New World show a nearly perfect pairing in each, consisting on average of 18.9 to 19 bivalent-equivalents. Clearly then, the one New-World genome must be B. Indirect support for this comes from the fact that New-World species recognize only 7.7 to 11.4chromosomes in the G genome, and only 1.2 chromosomes in the A genome—a pattern similar to crosses of these same two genomes directly with Kosteletzkya buettneri itself. These results, of course, indicate a direct connection between the African and New World parts of the genus. Simply stated, the African Kosteletzkya buettneri appears to be more closely related to the New World species than it is to its two African diploid congeners.

Allopolyploidy is an important mechanism for speciation in plants (Otto and Whitton 2000, Soltis et al. 2004, Wood et al. 2009), and polyploid series among related species are often found to have resulted from this process. In the Malvaceae: Malvoideae, allopolyploidy has been extensively documented in Gossypium (Endrizzi et al. 1985) and in Hibiscus sect. Furcaria (Wilson 1994). In Hibiscus sect. Furcaria, all of the 41 genomically studied polyploid species are allopolyploids; in Gossypium, all of the five known polyploid species are allopolyploids. On this basis I have hypothesized that the polyploid species in Kosteletzkya will prove to be allopolyploids as well.

In classic allopolyploidy, the production of an interspecific hybrid is the first step leading to a new species, yet in an examination of over 2800 herbarium specimens comprising all 17 species in the genus, no plants were found that might have been considered natural hybrids. In a way, this is not a surprise. The New-World species, though relatively easily inter-crossable, are at present largely geographically allopatric, and even where they are in geographic proximity they are kept separate elevationally (e.g. Kosteletzkya depressa and Kosteletzkya tubiflora in western Mexico) or by flowering season (e.g. Kosteletzkya depressa and Kosteletzkya pentacarpos in western Cuba and southern Florida). In contrast, while the African diploids are broadly sympatric, though perhaps often ecologically separated, experiments reported here have shown that hybrids among them are more difficult to obtain and weaker—conditions likely to pertain in the wild as well. In either hemisphere the hybrids themselves would obviously be transitory, living for a few years and then likely vanishing unrecognized and leaving few if any progeny.

Nevertheless, there appears to be clear evidence of allopolyploidy in Africa. The experimental diploid-tetraploid hybrid between Kosteletzkya grantii (2x) and Kosteletzkya begoniifolia (4x) averaged 17.9 bivalent-equivalents out of a possible 19, and likewise the diploid-tetraploid hybrid between Kosteletzkya buettneri (2x) and Kosteletzkya borkouana (4x) averaged 18.8 out of 19. The reverse crosses (Kosteletzkya buettneri × Kosteletzkya begoniifolia and Kosteletzkya grantii × Kosteletzkya borkouana) yielded averages of 3.9 and 7.0 respectively. This suggests that the G genome but not the B genome is present in the tetraploid Kosteletzkya begoniifolia, and the B genome but not the G genome is present in the tetraploid Kosteletzkya borkouana. Neither of these two diploids was combined in this study into a hybrid with the tetraploid Kosteletzkya rotundalata but this species shows nearly complete chromosome homology with Kosteletzkya begoniifolia—an average of 37.0 pairs out of a potential 38—suggesting a close relationship between the two, and indirectly indicating that the G genome is also present in Kosteletzkya rotundalata. Finally, the tetraploid Kosteletzkya semota was combined with both the B-bearing Kosteletzkya borkouana and the G-bearing Kosteletzkya rotundalata, producing on average only 3.5 and 11.3 pairs respectively, from which it can be reasonably concluded that Kosteletzkya semota contains neither B nor G genomes. This is further suggested by crosses of Kosteletzkya semota with the African diploids Kosteletzkya buettneri and Kosteletzkya grantii as well as with three New-World B-genome diploids, in which all five results ranged between 4.3 and 13.1 bivalent-equivalents.

When the tetraploids Kosteletzkya begoniifolia and Kosteletzkya borkouana were crossed with one another, the resulting hybrids averaged 24.6 bivalent-equivalents out of a possible 36, which indicates that they share a genome. That genome cannot be either G or B since it is shown above that neither is shared by the two tetraploids. This shared, unknown Kosteletzkya borkouana-Kosteletzkya begoniifolia-Kosteletzkya rotundalata genome cannot be present in Kosteletzkya semota because many fewer that a full set of 19 chromosomes were detected in Kosteletzkya semota by these other tetraploids.

Although one might invoke some undiscovered or now-extinct genome as the postulated shared genome, the most obvious suggestion is that it is the extant genome A. It was therefore particularly frustrating that hundreds of cross-pollinations between the A-bearing Kosteletzkya adoensis and the tetraploids Kosteletzkya begoniifolia and Kosteletzkya borkouana produced only a single viable seed—from the first of these two combinations—and that seed yielded a severely stunted, deformed, non-reproductive plant. This made it impossible to introduce an A genome into either of the two tetraploids to the extent that it could reach meiosis and seek out a possible genomic match. On the other hand, the putative hybrids that would have had to form in nature as a first step in the allopolyploid production of the postulated AAGG and AABB tetraploids are, respectively, the combinations AG (Kosteletzkya adoensis × Kosteletzkya grantii), and AB (Kosteletzkya adoensis × Kosteletzkya buettneri). Both of these hybrids were indeed produced experimentally. The first had 1 percent pollen stainability, 0 percent fruit-set and produced an average of 2.0 bivalent-equivalents out of a potential 19; the second had 0 percent pollen stainability, 0 percent fruit-set, and an average of 3.1 bivalent equivalents. Fig. 5a illustrates a meiotic metaphase I of the AG hybrid.

Remarkably, one day I found in the greenhouse a normal-appearing fruit on a branch of the otherwise profoundly sterile diploid hybrid AG plant. It contained three well-formed seeds, all of which subsequently germinated and produced vigorous plants which themselves yielded abundant fruit when selfed. This restoration of fertility strongly suggested that a spontaneous doubling of chromosomes had occurred in at least a small part of the hybrid plant, thereby creating identical pairs of chromosome sets and permitting full synapsis in meiotic prophase I, with the result that meiosis and gamete formation were able to proceed to normal completion. Examination of meiotic metaphase in one of these plants indeed showed 38 pairs of chromosomes (Fig. 5b). The conclusion: a combination of experimental and accidental events had resulted in a new, fully fertile tetraploid. A test of this would be a cross between this artificial tetraploid and one of its wild putative counterparts, Kosteletzkya begoniifolia or Kosteletzkya rotundalata. Both of these test hybrids were obtained, and they showed averages of 36.9 and 36.6 bivalent-equivalents respectively out of a potential 38. Fig. 5c shows a meiotic figure illustrating the combination Kosteletzkya begoniifolia × Kosteletzkya artificial tetraploid.

This settled two important matters: 1) that the artificial tetraploid corresponded genomically to these wild tetraploids, and 2) that the identity of the elusive shared genome was indeed A. The latter was also separately verified by subsequent backcrossing of the artificial tetraploid to each of its two diploid parents. These crosses with Kosteletzkya adoensis and with Kosteletzkya grantii each yielded an average pairing of 19 out of 19 potential pairs. Interestingly, Kosteletzkya adoensis, which had been so intractable in attempts at crossing it with the two wild tetraploids, crossed fairly readily with their home-made counterpart, and the offspring grew and flowered well.

In summary, these results suggest that the genomic makeups of the tetraploids Kosteletzkya begoniifolia, Kosteletzkya rotundalata and Kosteletzkya borkouana are respectively AAGG, AAGG and AABB.

Since it is generally observed that most allopolyploids arise via unreduced gametes (de Wet 1971, Ramsey and Schemske 1998), and since the actual initiation of an allopolyploid event is rarely witnessed, it is noteworthy that the spontaneous polyploidization reported here was apparently due not to unreduced gametes but to somatic doubling. Part of the plant—perhaps only a single flower—must have arisen from a chromosome doubling in a somatic apical initial or an early derivative. A contrary interpretation would require the unlikely independent production, within only a single flower and no other flowers on the plant, of a minimum of six unreduced gametes—three eggs and three sperm—which then would have to meet by chance and go on to result in a single capsule bearing three fertile seeds out of a potential five.

Despite numerous attempts, no hybrids could be produced between the single African hexaploid Kosteletzkya racemosa and any of the diploids, either African or New-World, however the hexaploid did cross with all four tetraploids. With Kosteletzkya begoniifolia and Kosteletzkya borkouana it averaged 37.1 and 37.8 chromosome pairs respectively out of a possible 38 (Figure 4d), whereas with Kosteletzkya semota it showed 6.6 pairs out of a possible 38. (Meiotic material from the hybrid Kosteletzkya rotundalata × Kosteletzkya racemosa was not obtained.) Now that genomic makeups are known for the tetraploids Kosteletzkya borkouana and Kosteletzkya begoniifolia it can be stated with reasonable certainty that the genomic constitution of the hexaploid is AABBGG. This is because both AAGG (Kosteletzkya begoniifolia) and AABB (Kosteletzkya borkouana) tetraploids were shown separately to find nearly perfect correspondence with two sets of chromosomes in the hexaploid.

The hexaploid could theoretically have arisen in nature in one of two ways. A triploid ABG hybrid could have formed between an AABB tetraploid and a GG diploid, or between an AAGG tetraploid and a BB diploid, in both cases followed by chromosome doubling. No persuasive evidence at present strongly favors either scenario as being more likely, but the Kosteletzkya buettneri-like narrower leaves and depressed fruit, plus the Kosteletzkya begoniifolia-like dark petal bases and larger seeds that together characterize the hexaploid, hint that the combination AAGG × BB might be the better candidate. The matter is complicated by the fact that while both of the relevant diploids occur in the geographical vicinity of the two known occurrences of Kosteletzkya racemosa in southern Congo-Kinshasa and northwestern Zambia, none of the tetraploids are currently known to do so.

The preceding discussion is not meant to imply that Kosteletzkya adoensis, Kosteletzkya buettneri and Kosteletzkya grantii are the direct, immediate sources of the genomes A, B and G that are found in the polyploids, but rather that the lineages that gave rise to these three modern diploids also contributed their genomes relatively recently to the polyploids. The two interspecific hybrids that are the diploid counterparts, AB and AG, of the natural tetraploids whose genomic makeups are known, i.e. Kosteletzkya borkouana (AABB) and Kosteletzkya begoniifolia/rotundalata (AAGG), are only somewhat similar morphologically, not identical, to these tetraploids. Likewise the artificial tetraploid that is the full genomic counterpart of the wild species Kosteletzkya begoniifolia is similar to the latter, but distinguishable from it. Finally, both the triploid combination Kosteletzkya begoniifolia-Kosteletzkya buettneri (ABG), and the triploid combination Kosteletzkya borkouana-Kosteletzkya grantii (also ABG), look similar, but not identical, to the hexaploid Kosteletzkya racemosa (AABBGG).

Kosteletzkya semota, the third genomically distinctive tetraploid (recall that Kosteletzkya rotundalata and Kosteletzkya begoniifolia are genomically alike), appears to show little affinity with any of the three known diploid genomes. In effect the hexaploid Kosteletzkya racemosa offered all three known genomes to Kosteletzkya semota in a cross, but the 37-38 chromosomes of the latter could only recognize, on average, 6.6 Kosteletzkya racemosa chromosomes—the equivalent of about one-third of a genome. There were similar outcomes when semota participated in crosses with other tetraploids discussed above, yielding bivalent-equivalents ranging from 3.5 to 11.3, indicating at best a low-to-modest level of chromosomal homology with any of the other known genomes. Interestingly however, Kosteletzkya semota was shown to share over two-thirds of a genome (13.1 pairs) in a cross directly with the diploid Kosteletzkya grantii. These varied results leave uncertainties about the evolutionary position Kosteletzkya semota, so it seems best to give it a provisional designation of XXYY, which recognizes the species as distinctive and assumes an allopolyploid origin. At least one of its diploid progenitors, and its constituent genome, remains undiscovered or, more likely, extinct.

The decision to convert trivalents and quadrivalents to bivalent-equivalents (see Materials and methods) was intended to make Table 3 easier to read and interpret, but it is worth considering whether this conversion might have led to bias. In polyploid hybrids, multivalent associations sometimes indicate pairing within genomes (autosyndesis), which in turn suggests autopolyploidy—an interpretation that conflicts with the hypothesis of allopolyploidy that I have posited here. In the present case however, such an explanation is highly unlikely. Of the 618 meiotic cells examined, only nine had a single trivalent and only 18 others had a single quadrivalent. Moreover, 24 of these 27 multivalents occurred in diploid-diploid hybrids, and therefore could not be attributed, by definition, to autosyndesis. The three exceptions, involving one trivalent and two quadrivalents, were all found in hybrids in which the artificial tetraploid was one parent, and since I have shown here that this plant was derived from an interspecific diploid-diploid hybrid, its multivalents cannot be interpreted as indicating autopolyploidy.

Figure 6 depicts a reconstruction of the postulated genomic-phytogeographic history of the genus Kosteletzkya based on the cytogenetic evidence presented here. It assumes that the degree of chromosome pairing in the experimental hybrids can be used as a rough relative measure of the degree of evolutionary divergence of the parents of a cross. In support of this assumption, I note that in the well-studied malvaceous genus Gossypium, the African genomes A, B and E show pairing relationships among themselves (data from Konan et al. 2009) that are similar to those among the A, B, and G genomes of Kosteletzkya, andin the case of Gossypium the extent of the evolutionary divergence suggested by the degree of chromosome pairing is supported by both morphological evidence (Fryxell 1971) and molecular evidence (Cronn and Wendel 2004).

The reconstruction shown here indicates the reticulate nature of the evolution of Kosteletzkya in Africa, and also emphasizes that all of the early events in the history of the genus took place on the African continent. The lineage giving rise eventually to the B and G genomes is shown as separating from the A-genome lineage early in the evolution of the genus. This B-G branch itself branched in a more recent step, leading eventually to the extant African diploids Kosteletzkya buettneri and Kosteletzkya grantii respectively, while the A genome eventually gave rise to the extant diploid Kosteletzkya adoensis. The rest of the African diversification occurred at the polyploid level, and initially involved two separate interspecific hybridizations, each of which involved an A-genome plant—one in combination with a B-genome plant and the other with a G-genome plant. Following a doubling of the chromosome complements in these two hybrids, and subsequent evolution at the tetraploid level, the two resulted in three extant species: Kosteletzkya borkouana with genomic makeup AABB, and Kosteletzkya begoniifolia and Kosteletzkya rotundalata, each with AAGG. A cross between one of these tetraploids and a diploid bearing the third genome, followed by doubling, produced the hexaploid Kosteletzkya racemosa having a genomic makeup of AABBGG. Of the two possible hybrid combinations that might have led to this hexaploid, I have illustrated the one in which the tetraploid partner is from the Kosteletzkya begoniifolia-Kosteletzkya rotundalata (AAGG) lineage, since this alternative seems better supported by morphological evidence. The two postulated early-diverging genomes that led to the formation of the tetraploid Kosteletzkya semota have been designated here as XXYY, but in the depiction in Fig. 6, one of its genomes is tentatively shown as having its origin in the Kosteletzkya grantii lineage, since the cross Kosteletzkya semota × Kosteletzkya grantii yielded 13.1 chromosome pairs, the equivalent of more than 2/3 of a genome.

The atypically high level of fruit-set—63 percent—in the African tetraploid-tetraploid cross Kosteletzkya begoniifolia × Kosteletzkya rotundalata contrasts dramatically with the zero percent seen in all twelve of the other African hybrids for which there are fruit-set data (Table 3). This and the pairing evidence imply that the two parents diverged relatively recently from a common tetraploid ancestor, and this, too, is suggested in Fig. 6.

Finally, the history of Kosteletzkya in the New World was set into motion by a relatively recent dispersal to the New World of a B-genome-bearing Kosteletzkya buettneri ancestor, followed by a rapid radiation to yield the seven known diploids in that hemisphere. A similar pattern, in which one among several African genomes is also found in the New-World, can be seen in Gossypium (Endrizzi et al. 1985, Wendel and Cronn 2003) and in Hibiscus sect. Furcaria (see Menzel et al. 1983), and in both cases trans-Atlantic dispersals to the New World have been invoked.

In reporting on chromosome numbers in Kosteletzkya, I used the numbers evidence to suggest that Africa was the birthplace of the genus (Blanchard 2012). The data newly presented here add strength to this contention. The genomic and ploidy profiles of the African half of the genus are so strikingly more deep and complex than in the New World half as to lead almost inevitably to the view that, despite similar levels of morphological diversity, the New World taxa are of a much more recent origin.

Within the diversity of the New-World Kosteletzkya species there is little likelihood that any as-yet-undiscovered polyploids exist. Any interspecific New-World hybrid that formed would contain two B genomes, and in the event of a chromosome doubling, there would be four closely similar sets of chromosomes entering prophase I of meiosis. The result, barring strong preferential pairing, would be multivalent associations, confused and uneven segregation at anaphase, and consequently much reduced fertility.

An extension of this idea may explain why genome A, rather than one of the other two African genomes, is the one that is shared among the tetraploids Kosteletzkya begoniifolia, Kosteletzkya borkouana and Kosteletzkya rotundalata. While there is some disagreement about whether or not allopolyploidy occurs more commonly in hybrids between parents with a greater genetic distance between them (Chapman and Burke 2007, Buggs et al. 2008, Paun et al. 2009, Buggs et al. 2011), the situation in Kosteletzkya suggests that distance may count. Of the three pairwise combinations among the African diploids, only the two combinations AB and AG, have truly low levels of chromosome pairing—averaging only about 2 or 3 bivalents out of a potential 19. It is these two combinations that have been shown here to have given rise to the tetraploids Kosteletzkya borkouana and Kosteletzkya begoniifolia/Kosteletzkya rotundalata respectively. The third combination, BG, averages 9.1 bivalents—the equivalent of nearly half a genome. If a hybrid of the latter were to have formed and experienced a doubling of its chromosomes it would be much more likely than the other two to suffer serious meiotic problems. There is no present-day evidence of such a polyploid, and its existence would not be expected.

The suggestion that a Kosteletzkya carrying a B genome made its pre-Columbian way across the Atlantic calls for an evaluation of the dispersal capabilities of the group. Stephens (1966) discussed the matter for Gossypium, which presents a similar problem of an amphi-Atlantic distribution of its A genome. Hochreutiner, in his revision of the genus Hibiscus (1900), considered the winged fruits of species of Hibiscus sect. Pterocarpus Garcke, 1849 and found no dispersal function for the wings since the capsules of Hibiscus dehisce in place to release their seeds. Instead he suggested that the wings mimicked those of the fruits of an East-African Pavonia in which, however, the wing-bearing mericarps actually individually enclose the seeds and could therefore presumably act as windborne disseminules. He later elaborated further on winged fruits in the Malvoideae (1913). Mattei (1917) rejected Hochreutiner’s interpretation and also extended the discussion to Kosteletzkya, saying that in both Hibiscus sect. Pterocarpus and in Kosteletzkya the capsule-valves separate from the fruiting axis, so that it was possible that incompletely laterally disarticulated capsule-valve pairs could become windborne and carry seeds with them. Hochreutiner later (1924) conceded that his earlier comments about mimicry had been weak, but he maintained that in his experience all of the capsule-valves in these plants separated at the same time from the axis, not in groups. He proposed instead that the wings were organs for dehiscence of the capsule, not for seed dissemination, and cited other not really comparable examples from elsewhere in the Malvoideae.

My experience with Kosteletzkya indicates that both Mattei and Hochreutiner were correct. Adjacent capsule-valves in Kosteletzkya often do cohere in pairs or threes and separate together from the fruiting axis, carrying one or two seeds with them (Blanchard in Verdcourt and Mwachala 2009). This can easily be observed when one harvests seeds by manually stripping a fruiting branch of its mature capsules. Many capsules fall completely apart with this rough handling, but some do not. Equally importantly, I found that it was not uncommon, when working in the greenhouse or collecting the plants in the wild, to discover that single or coherent capsule-valves had attached to clothing. The fruits of nearly all Kosteletzkya have bristly, sometimes hooked hairs either covering the whole surface or confined to the valve margins. These certainly are responsible for the adhesion, and I have no doubt that they may cling to fur and feathers as well, and aid in the dispersal of the seeds. To the extent that the wings help the bristles to project from the general fruit surface and therefore to be better exposed to passers-by, the wings aid in dispersal, but hardly in the form of windborne “flight.”

Kosteletzkya pentacarpos has been shown (as Kosteletzkya virginica [Linnaeus, 1753] A. Gray, 1849) to have an air space within the seed that permits it to float (Poljakoff-Mayber et al. 1992), and the same species has been credited with considerable salt tolerance and a seed coat that remains impermeable to water for some time (Poljakoff-Mayber et al. 1994). These features may aid in salt-marsh-to-salt-marsh dispersal as they apparently do in Hibiscus moscheutos Linnaeus, 1753, with which Kosteletzkya pentacarpos often shares habitat (Kudoh et al. 2006), and it is possible that the same characteristics could enhance the prospects of a trans-Atlantic crossing.

Dispersal over considerable distances appears to have been accomplished by several Kosteletzkya species. Known contemporary distributions suggest that Kosteletzkya adoensis has jumped from the African mainland to Madagascar, which is a minimum of 800 miles from the nearest known mainland population. It also appears to have dispersed westward from its main center in East Africa to the mountains of Cameroon (1100 miles) and the mountains of Sierra Leone (a further 1350 miles), with no known occurrences of the species—and no montane habitats—in the intervening areas. Kosteletzkya begoniifolia has made a similar jump from montane East Africa to Cameroon (1050 miles), and Kosteletzkya borkouana has dispersed from eastern Democratic Republic of the Congo and East Africa 1300 miles across a considerable expanse of the Sahara to the Borkou region of northern Chad (Blanchard 2013). In the New World, Kosteletzkya depressa has spread to the Cayman Islands and throughout the Greater Antilles, presumably from a mainland source (see Howard 1973), and Kosteletzkya pentacarpos has spread from the United States to Cuba and Bermuda. Moreover, if the latter turns out not to have been transported to Eurasia by human agency, it must have made the trans-Atlantic trip by long-distance dispersal, as did the carrier of the ancestral B-genome, but in this case dispersing from west to east.

It is worth noting, however, that some of these dispersal feats may have been aided in Africa by paleoclimatic cycles that provided geographically more benign intervening conditions (Quézel 1978, de Menocal 2011). In the case of Kosteletzkya borkouana, for example, the Sahara Desert was apparently largely vegetated at times during the interval from 15000 to 5000 years ago (de Menocal et al. 2000), and may have afforded the plant an opportunity to disperse by much shorter hops to northern Chad from East Africa. In the case of the Caribbean-island immigrants, on the other hand, the time frame in which these plants could have dispersed without having undergone appreciable subsequent divergence would have been too recent to attribute their dispersal to the narrowed or bridged ocean gaps known to have occurred earlier in the Cenozoic, so some sort of island-hopping seems to be the only plausible explanation for the island distributions of Kosteletzkya depressa and Kosteletzkya pentacarpos (see Pindell 1994).

Two Kosteletzkya species were unavailable for inclusion in the hybridization experiments reported here: Kosteletzkya thurberi from northwestern Mexico and the rare Kosteletzkya batacensis, endemic to the Philippine island of Luzon.It can be reasonably predicted that Kosteletzkya thurberi is a diploid of genomic makeup BB like all other New-World Kosteletzkya species. Kosteletzkya batacensis, on the other hand, remains a complete mystery. On the basis of general morphology—particularly of the fruits—the plant seems to belong in Kosteletzkya, although it is unique in the genus in being an annual. However its restricted range and its remote geographical location in relation to the rest of the genus would make any further speculation on its relationships almost reckless. There was an active maritime trade between Manila and Mexico for centuries, but suggestions of a Mexican origin for the plant (Merrill 1912, 1918, Borssum-Waalkes 1966), although appealing, are not supported by what is known of the several extant Mexican taxa (Blanchard 2008).