Hybridization in the Evolution of the Macaronesian Flora

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4085-4090, April 1996 Evolution Chloroplast DNA evidence of colonization, adaptive radiation, and hybridization in the evolution of the Macaronesian flora (oceanic island flora/Atlantic islands/biogeography/Angiosperms/Argyranthemum) JAVIER FRANCISCO-ORTEGA*, ROBERT K. JANSEN*t, AND ARNOLDO SANTOS-GUERRA: *Department of Botany, University of Texas, Austin, TX 78713-7640; and tJardin de Aclimataci6n de La Orotava, Puerto de La Cruz, Tenerife 38400, Canary Islands, Spain Communicated by Thomas N. Taylor, University ofKansas, Lawrence, KS, December 14, 1995 (received for review June 16, 1995)

ABSTRACT Most evolutionary studies of oceanic islands cene, when the first northern hemisphere glaciation and the have focused on the Pacific Ocean. There are very few exam- desertification of most of northern Africa led to massive ples from the Atlantic archipelagos, especially Macaronesia, extinction and migration of plants and animals (15). despite their unusual combination of features, including a Macaronesia, like other tropical and subtropical volcanic close proximity to the continent, a broad range of geological archipelagos, is under the influence of the trade winds. These ages, and a biota linked to a source area that existed in the winds, in combination with altitudinal gradients on the islands, Mediterranean basin before the late Tertiary. A chloroplast have produced several distinct ecological zones (15). Two main DNA (cpDNA) restriction site analysis of Argyranthemum climatic regions can be distinguished. The first is under the (Asteraceae: Anthemideae), the largest endemic genus of influence of humid and cool northeastern trade winds and plants of any volcanic archipelago in the Atlantic Ocean, was mainly covers a band in the north of the islands between 400 performed to examine patterns of plant evolution in Maca- and 1200 m. This region has three main ecological zones: ronesia. cpDNA data indicated that Argyranthemum is a humid lowland scrub, laurel forest, and heath belt (Table 1). monophyletic group that has speciated recently. The cpDNA In contrast, the second climatic region is not under the tree showed a weak correlation with the current sectional influence of the trade winds and is more arid. This area is classification and insular distribution. Two major cpDNA restricted to the southern slopes of the islands, in the north at lineages were identified. One was restricted to northern altitudes higher than 1200 m and in the coastal belt below 400 archipelagos-e.g., Madeira, Desertas, and Selvagens-and m. Four major ecological zones are recognized in the arid the second comprised taxa endemic to the southern archipel- climatic region: coastal desert, arid lowland scrub, Canarypine ago-e.g., the Canary Islands. The two major radiations forest, and high altitude desert (Table 1). In many instances, identified in the Canaries are correlated with distinct ecolog- the northeastern trade winds can also have an influence on the ical habitats; one is restricted to ecological zones under the southern slopes of some of the islands (18). This is especially influence of the northeastern trade winds and the other to true in the islands where the summit barely reaches 1200 m. In regions that are not affected by these winds. The patterns of these cases, the laurel forest can also spread toward the phylogenetic relationships in Argyranthemum indicate that southern slopes and a humid lowland scrub is found below that interisland colonization between similar ecological zones is forest in the south or west. the main mechanism for establishing founder populations. The high diversity of habitats appears to be one of the main This phenomenon, combined with rapid radiation into distinct factors responsible for the rich Macaronesian flora, which ecological zones and interspecific hybridization, is the pri- includes -700 endemic species (19). The largest endemic plant mary explanation for species diversification. genus (Argyranthemum) of all Atlantic Ocean volcanic islands occurs in these archipelagos (20, 21). Its 23 species provide the Oceanic islands provide model systems to assess organismic most spectacular example of speciation in Macaronesia (22), evolution following colonization and isolation. Classical stud- and at least one species is endemic to each of the major ies of the Galapagos finches (1) and Hawaiian Drosophila (2) ecological zones (Table 1). Thus, Argyranthemum is an ideal have had a major impact on the history of evolutionary thought group to examine patterns of speciation in Macaronesia. in the 19th and 20th centuries. Oceanic islands are relatively Further justification for studying this genus is that the closest simple systems where both patterns of dispersal and natural relatives (Chrysanthemum and Heteranthemis) have been selection can be more easily assessed than in continental identified from the continent (refs. 23-27; J.F.-O., R.K.J., systems. Most studies ofisland biota have been restricted to the A. Hines, and A.S.-G., unpublished data). Pacific Ocean archipelagos of Hawaii (3-6), Galapagos (7, 8), We used Argyranthemum as an example to assess the origin and Juan Fernandez (9-11). Few studies (12, 13) have focused and evolution of the Macaronesian flora. Phylogenies gener- on the Atlantic Ocean archipelagos that constitute the bio- ated from chloroplast DNA (cpDNA) data were used to geographical region known as Macaronesia (Fig. 1). These examine the role of colonization, adaptive radiation, and archipelagos have several unique features that make them hybridization in the evolution of endemic plants from the ideal systems for understanding the origin and evolution of Atlantic archipelagos. The approach is similar to previous island floras and faunas. Macaronesia comprises five major studies of endemic genera from three volcanic archipelagos archipelagos (Azores, Canaries, Cape Verde, Madeira, and from the Pacific Ocean: Dendroseris (9) and Robinsonia (10) in Selvagens) that exhibit a broad range of geological ages from the Juan Fernandez Islands; the silversword alliance (3) and 0.8 to 21 million years (14). The region is in close proximity to Cyanea (6) in the Hawaiian Islands; and Gossypium in the the continent, unlike most volcanic archipelagos. In addition, Galapagos (8) and Hawaiian (28) Islands. In all of these cases, the Macaronesian biota has been linked to one that spread cpDNA restriction site analyses were informative and provided from the Mediterranean basin in the late Miocene and Plio- evidence for rapid speciation, hybridization, and colonization in the flora of Pacific archipelagos. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: cpDNA, chloroplast DNA. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4085 Downloaded by guest on September 29, 2021 4086 Evolution: Francisco-Ortega et al. Proc. Natl. Acad. Sci. USA 93 (1996) weights from 1.01:1 to 2.5:1 of site gains/losses with ancestral states designated as Os (35). Bootstrap values (36) from 100 replicates were calculated to assess the amount of support for monophyletic groups. Maximum likelihood values were esti- mated for a subset of trees in the 15-taxon analysis using the RESTML program of PHYLIP (37). Estimates of nucleotide sequence divergence were also computed using equations 9 and 10 of Nei and Li (38). Values were calculated only for the 6-bp enzymes because of the availability of complete restriction maps. Sequence divergence was calculated among 11 taxa, including the four outgroups and seven Argyranthemum species from the three major lineages identified in the phylogenetic analyses of the complete data set.

RESULTS The 21 restriction enzymes identified '3190 restriction sites (978 from 6-bp enzymes and 2112 from 4-bp enzymes) in cpDNAs of each species. This represents 14,716 bp, or 11% of the chloroplast genome of each taxon. A total of 151 variable restriction sites was detected, 84 of which were phylogeneti- cally informative. Wagner parsimony yielded 54 equally parsimonious trees of 172 steps with a consistency index of 0.80 (excluding uninformative changes) and a retention index of 0.967. The trees provided strong support (14 characters, 100% bootstrap value) for the monophyly of Argyranthemum. Two FIG. 1. The Macaronesian islands (inset) and the distribution of major clades were resolved in the genus (Fig. 2A). One clade Argyranthemum in the Madeira, Selvagen, and Canary archipelagos. included taxa endemic to the Canaries (Canarian clade), Blackened islands are those where Argyranthemum occurs. whereas the second one comprised only species from Madeira, Selvagens, and Desertas (Madeiran clade). The Canarian clade Our investigation identified a strong and unexpected cor- consisted of two clades (A and B), which collapsed in the strict relation between the two major climatic regions of Macaro- consensus tree (Fig. 2A, dashed line). These three lineages nesia and the groups in the cpDNA phylogeny. Patterns of (one Madeiran and two Canarian) were detected in all 54 phylogenetic relationships in Argyranthemum indicate that equally parsimonious trees and their relationships were re- interisland colonization between similar ecological zones, solved in three different ways: (i) Madeira sister to the Canary adaptive radiation, and hybridization have all played an im- Islands, (ii) one of the two canary clades sister to the Madeira portant role in speciation in the genus. and the other Canarian clade, and (iii) an unresolved trichotomy. The bootstrap analysis provided weak support for the MATERIALS AND METHODS resolution of the trichotomy; the Madeiran lineage was sister to the Canarian taxa in only 12 of the 100 replicates. In One or more populations of each taxon of Argyranthemum, summary, Wagner parsimony did not resolve the basal rela- Heteranthemis, and Chrysanthemum were studied (see Fig. 2). tionships in Argyranthemum. Total genomic DNA was isolated from living material (29) and Weighted parsimony is preferable to Wagner parsimony purified in CsCl/ethidium bromide gradients. Restriction en- because of the known asymmetry in the probability of gains zyme digestions, agarose gel electrophoresis, transfer of DNA versus losses of restriction sites (33, 35). Thus we performed fragments to filters, labeling of probes by nick translation with weighted parsimony using a range of weights from 1.01:1.0 to 32p, and subsequent hybridization and autoradiography were 2.5:1.0 of gains/losses. All weighted parsimony analyses, even carried out as described elsewhere (30). Fourteen cloned with a low weight of 1.01 for gains, generated trees with the restriction fragments of lettuce cpDNA were used (31). Twen- Madeira lineage sister to the Canarian taxa (Fig. 2). The two ty-one restriction endonucleases were employed, including 14 equally parsimonious trees at a weight of 1.01 for gains had 6-bp enzymes (Ava I, Ava II, Ban II, Bgl II, BstNI, Cla I, Dra identical topologies to 2 of the 54 equally parsimonious I, EcoO109I,EcoRI, HincII, Nci I, Nsi I,Xba I, andXmn I) and Wagner trees; the only difference concerned the position ofA. 7 4-bp enzymes (BstUL, Hae III, Hha I, Hinfl, Msp I, Rsa I, and foeniculaceum, which collapsed into a large polytomy with other Taq I). Complete restriction site maps were constructed for Canarian taxa in one of the two trees (Fig. 2A, dashed arrow). each taxon for the 6-bp enzymes. The large number and small Weighted parsimony, and 23 of the Wagner trees, suggested size offragments generated by the 4-bp enzymes did not permit that the Madeira lineage may be sister to the Canarian taxa, but mapping. Mapped restriction sites for the 6-bp enzymes were support for this result was weak. We performed maximum used in phylogenetic analyses, and restriction site changes were likelihood analysis to determine which resolution of basal inferred from fragment patterns for the seven 4-bp enzymes. relationships is more likely. The maximum likelihood program Phylogenetic analyses were performed on two data sets; one is too intensive computationally to use all 55 taxa. Thus we included all 55 populations, and a second included only 15 taxa selected 11 Argyranthemum species representing all major from all major clades identified in the larger analysis. Both lineages and four outgroup taxa for these analyses (indicated Wagner (32) and weighted (33) parsimony were performed on in boldface type in Fig. 2A). Wagner and weighted parsimony a Macintosh Quadra 700 microcomputer using PAUP version analyses of these 15 taxa demonstrated that the reduction in 3.1.1 (34). The 55-taxon analysis used the TREE BISECTION the number of taxa had no effect on the major groups. Wagner RECONNECTION, MULPARS, ACCTRAN, and COLLAPSE options parsimony generated two equally parsimonious trees (data not with 100 random entries of the taxa. The 15-taxon data matrix shown) of 144 steps (consistency index of 0.867 and retention used the BRANCH AND BOUND, COLLAPSE, and ACCTRAN op- index of 0.940), one with Madeira sister to the Canarian taxa tions. Weighted parsimony was performed using a range of and the other with a Canary Island group sister to the Downloaded by guest on September 29, 2021 Evolution: Francisco-Ortega et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4087 Table 1. Distribution of Argyranthemum in Macaronesia Age, Altitude, Surface, No. of taxa per ecological zone* Island Myr m km2 CD ALS HLS LF HB PF HD Canary Islands El Hierro 0.8 1520 307 1 0 1 0 1 1 La Palma 2 2423 789 1 0 2 0 1 1 1 La Gomera 12.5 1484 425 2 0 1 1 0 Tenerife 12 3714 2355 3 2 3 1 1 2 1 Gran Canaria 14 1950 1625 3 1 2 1 0 1 Fuerteventura 21 807 1717 0 0 1 Lanzarote 15.5 670 717 0 0 1 Madeira Islands Madeira 5 1861 730 2 0 1 1 1 Deserta Grande 5 479 20 1 0 - Selvagen Islands Selvagem Pequena 12 49 0.5 1 Myr, million years. *Ecological zones (altitude ranges are given in parentheses) are as follows: CD, coastal desert (0-400 m); ALS, arid lowland scrub (400-600 m, only in zones which do not receive the influence of the northeastern trade winds); HLS, humid lowland scrub (400-500 m, only in zones which receive the influence of the northeastern trade winds); LF, laurel forest (600-1000 m, and only on northern face zones); HB, heath belt (1000-1200 m, only on northern face zones); PF, pine forest (1200-2000 m on northern face zones; 600-2000 on southern face zones); HD, high altitude desert (2000-3700 m). A - indicates that ecological zone is absent. Geographical and geological data were compiled from refs. 14, 16, 17, and 19. Each taxon is endemic to a particular zone and a particular island, the exception beingA. haemotomma, which is found both in Madeira and Deserta Grande. remaining taxa. Likelihood values were calculated for the two islands is reflected in the patterns of the cpDNA phylogeny most parsimonious topologies and 20 additional trees up to two (Fig. 2B): evolution of Argyranthemum in the northern archi- steps longer than the shortest trees. Maximum likelihood pelagos appears to have resulted from interisland colonization analysis requires the specification of the number of base pairs between the coastal areas, and radiation in the largest and in the recognition site of the enzymes. Our study used a most ecologically diverse island of Madeira. Each of the four combination of 4-bp and 6-bp enzymes. We performed the ecological zones other than the coastal desert on Madeira has likelihood estimates using both values. The two shortest trees been exploited by at least one taxon ofArgyranthemum (Table (data not shown, 144 steps) had the highest likelihood values, 1), and all of these taxa were derived from a single ancestor or regardless of whether 4 bp (tree 1, Ln = -7393.21524; tree 2, founder. Radiation apparently occurred rapidly without fur- Ln = -7395.15716) or 6 bp (tree 1, Ln = -10654,42282; tree ther enhancement of genetic variation from new colonizers, 2, Ln = - 10656.79832) was specified. Although the likelihood and it is likely that it followed a sequence from the coast of all 22 tree topologies did not differ significantly by the towards the rest of the island. Kishino and Hasegawa paired-sites test (39), the tree with the Argyranthemum in the Canary Islands had a more complex Madeira lineage sister to the Canarian taxa had the highest evolutionary history that involved more islands and at least likelihood and will be used hereafter. three ecological zones in each island (Table 1). This is re- The groups in the cpDNA tree did not correlate well with the flected in the cpDNAphylogeny. The two major clades in these current sectional classification in that no section was mono- islands were not correlated with insular distribution or sec- phyletic (Fig. 2A). There was also a poor correlation between tional classification (Fig. 2). However, there was a clear insular distribution and the cpDNA clades because none of the relationship between the cpDNA phylogeny and the ecology of major classes were restricted to a single island (Fig. 2B). Argyranthemum (Fig. 2B). Most taxa (22 of 25) from ecological cpDNA divergence in Argyranthemum (Table 2) ranged zones not influenced by the trade winds-i.e., coastal desert, from 0.056% (betweenA. pinnatifidum and A. haemotomma) to arid lowland scrub, pine forest, and high altitude desert-were 0.185% (between A. pinnatifidum and A. haouarytheum). Diver- restricted to clade A from the Canary Islands. The second gence between Argyranthemum species and continental genera clade (clade B) consisted primarily of taxa (12 of 18) that occur ranged from 0.155% (between A. maderense and H. viscidehirta) in ecological zones under the influence of the trade winds- to 0.298% (between A. haouarytheum and C. segetum). i.e., humid lowland scrub, laurel forest, and heath belt. The cpDNA phylogeny suggests that at least two independent introductions have occurred in Tenerife, La Palma, El Hierro, DISCUSSION and La Gomera, and that most interisland colonization oc- Colonization and Adaptive Radiation. The cpDNA phylog- curred between similar climatic regions. This finding is in eny (Fig. 2A) provides strong support for the monophyly of contrast with the pattern of ecological radiation in the Ha- Argyranthemum, in agreement with previous morphological waiian silversword alliance (40), where major ecological shifts (23) and DNA (27) evidence. This result, combined with the between wet and dry habitats accompanied speciation in each fact that the genus is absent from the continent, suggests that of the major island-endemic lineages. Argyranthemum has experienced a single colonization into Restriction of phylogenetic lineages to particular climatic Macaronesia. The cpDNA tree also resolves two major lin- regions has not been reported for other oceanic island groups. eages in genus (Fig. 2A). The first is restricted to the northern This correlation may occur in other groups in the Canary archipelagos in the islands of Madeira, Selvagens, and Deser- Islands because several genera [i.e., Crambe and Descurainia tas, and the second occurs in the Canary Islands. The northern (Brassicaceae), Cheirolophus (Asteraceae), and Limonium archipelagos are ecologically less diverse than the Canary (Plumbaginaceae)] have most of their taxa in the same habitat Islands. The island of Madeira has four ecological zones, on different islands (15). Our results support the hypothesis whereas Selvagem Pequena and Deserta Grande have one and that interisland colonization has taken place between areas two, respectively. The low number of ecological zones and with similar ecology. Downloaded by guest on September 29, 2021 4088 Evolution: Francisco-Ortega et al. Proc. Natl. Acad. Sci. USA 93 (1996) A B Ecological Taxon Section Island zone 2fru 1 fru frLIu c 2 fru Al 1 88 gra 68 -- fru LIuC 8 fru gra 3 lem fru foe Arg 4 tru par 2r hao hao 1 1 8Z2 fru 1 0) 3 fru purn C3 frUl 2 tru canl lid a) I lid 1. CO 1 d1ada jac CO 59 --- ada gra ada can Pre r,u c6 ada dug I- fil C- ] Mon ct esc CO 8 tbro gom - Sti Ut -- hao 2 u ten - foe Arg 1cal 2 web - Stig 76 ada pal - Pre bro - Sti ada Pre _-]1 ---ada erv ] 2 sve A 2 Arg

3 hie Sti 2 hie 2-~ mad L ]Arg I .. J.{....i . t...L L... t_ k I I.,.l 2 win - Sph ---b rCDI 2 dispiIl Stic -1 dis ]Arg (1;5)D MadeiraNl I-Il S -0 72 pin mnt Sph C t [)Deserta Grand e hae - Sti - CD tha -Arg Selvagem Pequ. - CD 5--- hae -Sti Madeira CD 15. Het vis Iberia and Morocco Morocco _, - Chr car 100°-° Chr cor Outgroup Mediterranean basin 1001_11 Chrseg Mediterranean basin FIG. 2. One of the two equally parsimonious trees from weighted parsimony analyses (weight of 1.01 in favor of site gains). Dashed lines in A indicate branches that collapse in the strict consensus tree of54 equallyparsimonious Wagner trees. Arrow inA indicates the branch that collapsed in the second weighted parsimony tree. Number of changes is given above each branch. Bootstrap value (100 replicates) >50 is shown below each branch. Taxa in boldface type were used in maximum likelihood comparisons. Taxa are coded as follows: ada (Argyranthemum adauctum subsp. adauctum), ada can (Argyranthemum adauctum subsp. canariae), ada dug (Argyranthemum adauctum subsp. dugourii), ada ery (Argyranthemum adauctum subsp. erythrocarpon), ada gra (Argyranthemum adauctum subsp. gracile), ada jac (Argyranthemum adauctum subsp. jacobifolium), ada pal (Argyranthemum adauctum subsp.palmensis), bro (Argyranthemum broussonetii subsp. broussonetii), brogom (Argyranthemum broussonetii subsp. gomerensis), cal (Argyranthemum callichrysum), Chr car (Chrysanthemum carinatum), Chr cor (Chrysanthemum coronarium), Chr seg (Chrysanthe- mum segetum), cor (Argyranthemum coronopifolium), dis (Argyranthemum dissectum), esc (Argyranthemum escarrei)fil (Argyranthemum filifolium), foe (Argyranthemum foeniculaceum), fru (Argyranthemum frutescens subsp.frutescens), fru can (Argyranthemum frutescens subsp. canariae), frufoe (Argyranthemumfrutescens subsp.foeniculaceum), fru gra (Argyranthemumfrutescens subsp. gracilescens),fru par (Argyranthemum frutescens subsp. parviflorum), fru pum (Argyranthemum frutescens subsp. pumilum),fru suc (Argyranthemum frutescens subsp. succulentum),fru 1 (Argyranthemum frutescens subsp. nov. 1), fru 2 (Argyranthemum frutescens subsp. nov. 2), gra (Argyranthemum gracile), hae (Argyranthemum haemotomma), hao (Argyranthemum haouarytheum subsp. haouarytheum), hao 1 (Argyranthemum haouarytheum subsp. nov. 1), hao 2 (Argyranthemum haouarytheum subsp. nov. 2), Het vis (Heteranthemis viscidehirta), hie (Argyranthemum hierrense), lem (Argyranthemum lemsii), lid (Argyranthemum lidii subsp. lidii), lid 1 (Argyranthemum lidii subsp. nov. 1), mad (Argyranthemum maderense), A 1 (Argyranthemum sp. nov. 1), A 2 (Argyranthemum sp. nov. 2),pin (Argyranthemumpinnatifidum subsp.pinnatifidum),pin mon (Argyranthemumpinnatifidum subsp. montanum),pin suc (Argyranthemumpinnatifidum subsp. succulentum), sun (Argyranthemum sundingii), sve (Argyranthemum sventenii), ten (Argyranthemum tenerifae), tha (Argyranthemum thalassophilum), web (Argyranthemum webbii), and win (Argyranthemum winteri). The distribution of the species among the five recognized sections (22) ofArgyranthemum (Arg),Monoptera (Mon), Preauxia (Pre), Sphenismelia (Sph), Stigmatotheca (Sti) is given (A), as is the geographic distribution among islands (B). Ecological zones in B are coded as in Table 1. Solid circles in B indicate ecological habitats not influenced by the trade winds. One conclusion that can be drawn from our results concerns remaining taxa, whereas taxa from Selvagens and Desertas the pattern of dispersal followed by Argyranthemum. In the occur in a more derived position (Fig. 2B). Thus, taxa from the Madeiran, clade,A. haemotomma from Madeira is sister to the northern archipelago may have followed a "stepping stone" Downloaded by guest on September 29, 2021 Evolution: Francisco-Ortega et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4089 Table 2. Nucleotide sequence divergence (equations 9 and 10 from ref. 38) expressed as percentage (above diagonal) and number of restriction site differences (below diagonal) between pairs of taxa ofArgyranthemum and continental relatives Taxa Chr car Chr cor Chr seg Het vis ada fru hae* hao 3 pin ten mad Chr car 0.012 0.080 0.128 0.209 0.233 0.217 0.282 0.225 0.225 0.177 Chr cor 3 0.048 0.128 0.209 0.233 0.217 0.282 0.225 0.225 0.177 Chr seg 9 6 0.144 0.209 0.249 0.234 0.298 0.235 0.241 0.193 Het vis 17 16 16 0.160 0.201 0.168 0.234 0.176 0.193 0.155 ada 24 23 26 20 0.104 0.104 0.152 0.121 0.112 0.160 fru 30 32 28 19 13 0.128 0.064 0.136 0.136 0.088 hae 24 23 25 19 12 16 0.177 0.056 0.120 0.072 hao 3 32 35 33 26 20 8 21 0.185 0.153 0.153 pin 25 26 27 22 13 16 7 23 0.128 0.118 ten 24 24 25 22 13 16 14 19 15 0.098 mad 24 23 25 21 7 17 14 21 13 13 Taxa are abbreviated in Fig. 1. *A. haemotomma from Deserta Grande. model (41). Alternatively, there could have been independent Argyranthemum hierrense and A. haouarytheum are also colonizations of Desertas and Salvagens from Madeira. In paraphyletic in the cpDNA tree (Fig. 2A) but morphologically either case, the north-to-south sequence Madeira-Desertas- (22) they are coherent taxonomic entities. They could also Selvagens is the prevailing direction of dispersal. In contrast, represent examples of ancient introgression and interspecific colonization between adjacent islands is not the predominant gene flow within the genus. In contrast, the paraphyly of the pattern in the Canary Islands. There is evidence of dispersal two subspecies ofA. broussonetii is likely due to the presence between Lanzarote and Fuerteventura, two adjacent islands of two independent evolutionary lineages. In each case, they situated furthest east. However, the two species involved (A. are also distinct in their morphology (J.F.-O. and A.S.-G., un- maderense and A. winteri) do not occur in the same lineage as published data), and each should be considered a distinct species. the taxa on the closest island, Gran Canaria (Fig. 2B). Indeed, Hybridization has generally been considered to play a minor the Gran Canarian taxa form a distinct group in clade B of the role in the evolution of insular plants (19, 45), even though Canarian lineage, that comprises taxa exclusively from the there is usually a lack of genic barriers to hybridization western islands. This suggests long distance dispersal ofArgy- between congeneric endemic species (46). In most cases, ranthemum between the western and the eastern Canary founder effect coupled with selection in different ecological Islands or, perhaps, extinction of taxa on the central islands. zones has been regarded as the main evolutionary force in Hybridization. The cpDNA phylogeny confirms earlier sug- volcanic archipelagos (46,47). However, hybridization has also gestions that hybridization has played an important role in the been implicated in the evolution of the silversword alliance, evolution ofArgyranthemum (42-44). The best example can be Bidens (Asteraceae), Scaevola (Goodeniaceae), and Pipturus found among the seven subspecies of the A. adauctum com- (Urticaceae) in the Hawaiian Islands (48-50). Thus it seems plex. Two subspecies occur in the pine forest (A. adauctum appropriate to suggest that interspecific hybridization has been subspp. canariae and dugourii); one is restricted to the arid a factor in the evolution of endemics in oceanic islands. lowland scrub of southern Gran Canaria (A. adauctum subsp. Sequence Divergence. The low levels of nucleotide sequence gracile), and the remaining four subspecies (A. adauctum divergence inArgyranthemum (Table 2) suggest that the genus and originated and radiated recently. cpDNA sequence divergence subspp. adauctum, erythrocarpon, jacobifolium, palmensis) values for oceanic island endemics and their mainland coun- are found in the heath belt area under the influence of the terparts is only known for the Hawaiian silversword alliance northeastern trade winds. The cpDNAphylogeny revealed that (3). Differentiation of this Pacific group from the closest the subspecies from the pine forest were part of clade A of the continental relatives is much higher than between Argyranthe- Canarian lineage (Fig. 2B), whereas those from the heath belt mum in Macaronesia and its mainland relatives (0.310-0.610 (except A. adauctum subsp. jacobifolium from Gran Canaria) in the silversword alliance/California tarweeds vs. 0.155-0.298 grouped in clade B. The three subspecies of A. adauctum in in Argyranthemum/mainland species), even though vigorous Gran Canaria represent a case of radiation into three of the intergeneric hybrids between the silversword alliance species major ecological zones of this island. This is not the case ofA. and their mainland relatives (Madia bolanderi and Raillardi- adauctum in the heath belt of Tenerife and La Palma, perhaps opsis muirii from California) can be generated (3). Fertile because early colonizers from this complex could have become hybrids between species ofArgyranthemum have been reported sympatric with the endemics A. webbii in La Palma or A. (42, 44, 51). We have also produced hybrids among various broussonetii in Tenerife. This could have led to the opportunity combinations of A. coronopifolium, A frutescens, and A. ma- for hybridization and the consequent enhancement of the derense. However, all attempted crosses between Argyranthe- variation and adaptation of A. adauctum. The two cpDNA mum and the two mainland relatives have failed to yield any groups of the A. adauctum complex differ by a minimum of 21 seeds (unpublished data). Thus, despite the fact that the restriction site changes. It is unlikely that convergent restric- chloroplast genomes of the Macaronesian genus and its main- tion site changes or ancestral polymorphisms could account for land relatives are less divergent than those of the silversword the separation of the subspecies of A. adauctum into the two alliance and its two closest North American genera, they are divergent clades. The coherence of the seven subspecies is apparently cross-incompatible. supported by five unique morphological characters (22). This In this study, we have demonstrated that despite its ecogeo- evidence, in combination with the known occurrence of hybrid graphical peculiarities, the Macaronesian flora has experi- swarms inArgyranthemum (42,43), indicates that introgression enced some of the same evolutionary phenomena as other from other species from the area ilnfluenced by the trade winds oceanic islands. A common pattern based on rapid morpho- is the most likely explanation for the paraphyly ofA. adauctum logical differentiation accompanied by little genetic diver- in the cpDNA tree. gence emerges for the evolution of the flora ofvolcanic islands. Downloaded by guest on September 29, 2021 4090 Evolution: Francisco-Ortega et al. Pro~c. Natl. Acad. Sci. USA 93 (1996) Our results, however, also show that interisland colonization 17. Carlquist, S. (1974) Island Biology (Columbia Univ. Press, New between similar ecological zones may be one of the primary York). factors involved in the evolution of the endemic flora of 18. Fernandez-Galvan, M. (1983) in Comunicacoes II Congreso In- In this is ternacional Flora Macaronesica, ed. Anonymous (Funchal, Ma- oceanic archipelagos. Argyranthemum particularly deira, Spain), pp. 269-293. evident in the Canary Islands where the two cpDNA clades are 19. Humphries, C. J. (1979) in Plants and Islands, ed. Bramwell, D. correlated strongly with the two major climatic areas. These (Academic, New York), pp. 171-199. two lineages evolved in parallel by exploiting different eco- 20. Hansen, A. & Sunding, P. (1993) Sommerfeltia 17, 1-295. logical zones. Occasionally these two climatic clades inter- 21. Howard, R. A. (1973) in Vegetation and Vegetational History of sected via hybridization-e.g., the seven subspecies of A. Northern Latin America, ed. Graham, A. (Elsevier, Amsterdam), adauctum-which may have provided a new source of varia- pp. 1-38. new 22. Humphries, C. J. (1976) Bull. Br. Mus. (Nat. Hist.) Bot. 5, tion. These hybrid genotypes may have provided oppor- 147-240. tunities for exploitation of the extremely diverse range of 23. Bremer, K. & Humphries, C. J. (1993) Bull. Nat. Hist. London habitats in Macaronesia. (Bot.) 23, 71-177. 24. Greger, H. (1977) in The Biology and Chemistry ofthe Compositae, John Clement and K. E. Holsinger assisted with the maximum eds. Heywood, V. H., Harborne, J. B. & Turner, B. L. (Academic, likelihood analysis. Technical assistance was provided by A. Strong London), Vol. 1, pp. 899-941. and A. Hines. We are grateful to G. J. Anderson, B. G. Baldwin, C. 25. Christensen, L. P. (1992) Phytochemistry 31, 7-49. Ferguson, J. Fuertes-Aguilar, A. L. Hempel, D. Levin, L. A. Prather, 26. Fancisco-Ortega, J., Crawford, D. J., Santos-Guerra, A. & J. F. 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