Cladogenesis and reticulation in the Hawaiian endemic mints (Lamiaceae)

Cladistics Cladistics 19 (2003) 480–495 www.elsevier.com/locate/yclad

Cladogenesis and reticulation in the Hawaiian endemic mints (Lamiaceae) Charlotte Lindqvist,a,* Timothy J. Motley,b John J. Jeffrey,c and Victor A. Alberta b

a The Natural History Museums and Botanical Garden, University of Oslo, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway The Lewis B. and Dorothy Cullman Program for Molecular Systematics Studies, The New York Botanical Garden, Bronx, New York, NY 10458-5126, USA c Hakalau Forest National Wildlife Refuge, US Fish and Wildlife Service, 32 Kinoole Street, Suite 101, Hilo, HI 96720, USA

Accepted 17 September 2003

Abstract The Hawaiian endemic mints, which comprise 58 species of dry-fruited Haplostachys and fleshy-fruited Phyllostegia and Stenogyne, represent a major island radiation that likely originated from polyploid hybrid ancestors in the temperate North American Stachys lineage. In contrast with considerable morphological and ecological diversity among taxa, sequence variation in the nrDNA 5S non-transcribed spacer was found to be remarkably low, which when analyzed using standard parsimony resulted in a lack of phylogenetic resolution among accessions of insect-pollinated Phyllostegia and bird-pollinated Stenogyne. However, many within-individual nucleotide polymorphisms were observed, and under the assumption that they could contain phylogenetic information, these ambiguities were recoded as new character states. Substantially more phylogenetic structure was obtained with these data, including the resolution of most Stenogyne species into a monophyletic group with an apparent recent origin on OÕahu ( 6 3.0 My) or the Maui Nui island complex ( 6 2.2 My). Subsequent diversification appears to have involved multiple inter-island dispersal events. Intergeneric placements for a few morphotypes, seemingly misplaced within either Phyllostegia or Stenogyne, may indicate reticulation as one polymorphism-generating force. For a finer scale exploration of hybridization, preliminary AFLP fragment data were examined among putative hybrids of Stenogyne microphylla and S. rugosa from Mauna Kea, HawaiÕi, that had been identified based on morphology. Cladistic analysis (corroborated by multivariate correspondence analysis) showed the morphologically intermediate individuals to group in a strongly supported monophyletic clade with S. microphylla. Therefore, reticulation could be both historic and active in Stenogyne, and perhaps a force of general importance in the evolution of the Hawaiian mints. The relatively greater extent of lineage-sorted polymorphisms in Stenogyne may indicate selective differentiation from other fleshy-fruited taxa, perhaps through the agency of highly specialized bird pollinators that restricted gene flow with other Hawaiian mint morphotypes. Ó 2003 The Willi Hennig Society. Published by Elsevier Inc. All rights reserved. Keywords: Lamiaceae; Haplostachys; Phyllostegia; Stenogyne; Honeycreeper; Drepanidinae; Hawaiian Islands; DNA; 5S-NTS; AFLP; Cladogenesis; Phylogeny; Reticulation

Extensive morphological variation and divergent growth forms have long been known among oceanic island floras as compared to their continental relatives. In the Hawaiian Islands some examples of remarkable features among endemic plant taxa are woodiness, major seed and fruit alterations (including change of dispersal mode), lack of poisonous compounds, and presence of prickles, spines and other textural devices * Corresponding author. E-mail address: [email protected] (C. Lindqvist).

(Carlquist, 1980). Many Hawaiian plant groups display considerable morphological diversification, which is assumed to have occurred relatively recently because of the young age of the island chain (Carlquist, 1995). However, these radiations are usually contrasted with poor genetic divergence (e.g., Baldwin and Robichaux, 1995; Barrier et al., 2001; Helenurm and Ganders, 1985; Lowrey, 1995; Okada et al., 1997; Witter and Carr, 1988). One example occurs among the native Hawaiian lobelioids, which comprise 124 species in six genera (Buss et al., 2001) that all belong to a strongly supported

0748-3007/$ - see front matter Ó 2003 The Willi Hennig Society. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cladistics.2003.09.003

C. Lindqvist et al. / Cladistics 19 (2003) 480–495

monophyletic group representing a single dispersal event to the Hawaiian archipelago (Buss et al., 2001; Givnish et al., 1995; Givnish et al., 1996). Despite extensive diversity in morphological and ecological features, including homeotic variation in floral parts, only low phylogenetic resolution based on molecular evidence is found within Clermontia, the sister genus to the more sequence-diverged Cyanea (Albert et al., 1998; V.A. Albert, unpublished; see GenBank PopSet 11386030). In this paper, we show that the Hawaiian endemic mints represent another example in which broad morphological and ecological variation is contrasted with a strikingly low level of DNA sequence divergence. The native Hawaiian mints comprise a total of 58 species in three genera, Haplostachys, Phyllostegia, and Stenogyne (Sherff, 1935; Wagner, 1999a,b; Wagner and Weller, 1999; Wagner et al., 1999; Weller and Sakai, 1999). Haplostachys, with five species, of which four are extinct, was historically found mostly at low-mid elevations, in relatively dry habitats. The only extant species in this genus, Haplostachys haplostachya, is federally listed in the United States as endangered and its distribution has today been reduced to relatively small, restricted subpopulations in the xerophytic shrubland between Mauna Loa and Mauna Kea on HawaiÕi (Morden and Loeffler, 1999). The flowers of Haplostachys are fragrant and white, with a prominent lower corolla lip, and the species of this genus are the only endemic Hawaiian mints that bear dry nutlets. The genus Phyllostegia was recently treated by Wagner (1999a,b) and comprises 32 Hawaiian species. Two additional species have been described, one each from Tahiti and Tonga. One of the Hawaiian species, Phyllostegia variabilis, which is presumed extinct, was found in strand and coastal sites in the western end of the Hawaiian Island chain on Kure and Midway atolls and Laysan Island. The remaining species of Hawaiian Phyllostegia are widely distributed on all the extant high islands, primarily occurring in mesic to wet forest habitats. The species are perennial and can be herbs, lianas or subshrubs. The flowers of Phyllostegia species resemble those of Haplostachys in having fragrance and white (rarely pink or even red) corollas with expanded lower lips. They are usually arranged in terminal, racemose inflorescences, which sometimes are congested and unbranched (e.g., P. vestita). The 21 species of the genus Stenogyne are found on all of the extant high islands of the Hawaiian Island chain but exhibit their greatest diversity on Maui and HawaiÕi (Weller and Sakai, 1999). They are mainly perennial vines with scandent or decumbent stems and are found in lower elevation, mesic-wet forests to higher elevation, subalpine woodland. The flowers are axillary and odorless and the corollas are usually tubular with a reduced lower lip. The corollas of species in this genus display a range of sizes (from ca. 10–

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60 mm long) and colors (green and white to pink, red and dark maroon). Although the pollination and breeding system biology among the Hawaiian mints are only poorly known, flower morphology strongly indicates pollination predominantly by insects in Haplostachys and Phyllostegia and principally by birds in Stenogyne. Indeed, Hawaiian endemic honeycreepers (Drepanidinae) (Carlquist, 1980; Tarr and Fleischer, 1995) are known to visit a number of Stenogyne species (J.J. Jeffrey, pers. obs.). The Hawaiian mints were originally thought to be closely related to other fleshy fruited lamioid mints and were placed in the tribe Prasieae, which also contained the genera Gomphostemma and Prasium (Bentham, 1832–1836). However, more recent molecular phylogenetic studies have shown that the Hawaiian mints are a monophyletic group deeply nested within the dry-fruited and widely distributed genus Stachys (Lindqvist and Albert, 2002). Notably, it was hypothesized that the Hawaiian mints are products of one or more polyploid hybridization events involving bird and insect pollinated Western North American parents. In the present study, we further explored this hypothesized hybridization event and its potential impact on the molecular phylogenetic relationships and evolutionary history among the Hawaiian genera. We analyzed the variation within the rapidly evolving nontranscribed spacer of the 5S nuclear ribosomal array (5S-NTS), which has been used for a number of specieslevel studies in plants (e.g., Baum and Johnson, 1994; Cox et al., 1992; Cronn et al., 1996; Kellogg and Appels, 1995; Persson, 2000; Sastri et al., 1992; Udovicic et al., 1995; Weigend et al., 2002). Although being one of the fastest evolving nuclear loci known, published results from cladistic analyses of the 5S spacer have been relatively infrequent. One major problem is the nature of the evidence for phylogenetic hierarchy exhibited by the 5S array. Often 5S rDNA sequence heterogeneity has been identified among individual repeats, possibly caused by weak concerted evolutionary forces (Cronn et al., 1996; Kellogg and Appels, 1995). Also, lineage sorting of genetic polymorphisms may create incongruent phylogenetic estimations (see Wendel and Doyle, 1998). However, in a study of molecular phylogenetic relationships among rattan palm genera, the observed intraindividual polymorphism did not excessively interfere with phylogeny reconstruction since multiple clones obtained from individual species for the most part were resolved as monophyletic groups (Baker et al., 2000). Other studies have proven the 5S-NTS region useful for molecular phylogenetic hypotheses, and in terms of resolution and robustness it has even been shown to be superior to another more often used nuclear ribosomal locus, the internal transcribed spacer (ITS) of the 18S– 26S nrDNA repeat (e.g., Costello and Motley, 2001; Lindqvist and Albert, 1999; Persson, 2000).

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For a finer scale approach to the same issues, viz., reticulation and its impact on recoverable hierarchy, we employed the DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP; Vos et al., 1995) in a more restricted example. We investigated gene flow within Stenogyne, with emphasis on a putative hybrid swarm involving S. microphylla and S. rugosa on Mauna Kea. Stenogyne microphylla is a vining, minuteleaved, greenish-yellow flowered plant native to the dry, subalpine zones of Mauna Kea, Mauna Loa, and Hualalai on HawaiÕi (S. Carlquist 2092, F.R. Fosberg 41659, BISH) and Haleakala on Maui (Weller and Sakai, 1999). Stenogyne rugosa is an erect herb with generally larger leaves and flowers with reddish corollas that occurs at various altitudes on East Maui and HawaiÕi (Weller and Sakai, 1999). In areas of sympatry at high elevations of Maui and HawaiÕi, the two species appear to be hybridizing (Weller and Sakai, 1999). DNA fingerprinting techniques have often been used to detect genetic differences among closely related plant species or among different populations or varieties of a species (e.g., Escaravage et al., 1998; Hill et al., 1996; Kardolus et al., 1998; Krauss, 1998; Paran et al., 1998; QamaruzZaman et al., 1998; Ribeiro et al., 2002; Russell et al., 1999; Zerega et al., 2002). By using both DNA sequencing and fingerprinting markers, we hoped to investigate to what extent polymorphic character states at the 5S-NTS locus and variation in AFLP banding patterns could show evidence for phylogenetic hierarchy versus reticulation in the Hawaiian mints. Specific questions that we address include: (1) interrelationships among the Hawaiian mint genera, (2) the status of nucleotide polymorphism within genera for the 5S-NTS locus, which should correlate with hybridization, gene conversion, or lineage sorting rates, (3) the degree to which polymorphisms influence phylogeny estimation, and (4) the extent to which reticulation can be documented to have impacted the evolutionary history of Hawaiian mints at different hierarchical levels.

Materials and methods Plant material Most DNAs of Hawaiian mints were isolated from herbarium specimens held at BISH and NY (Holmgren et al., 1990). In a few cases fresh material, further dried in silica gel, was obtained during fieldwork. Included in the 5S-NTS study are a total of two accessions of Haplostachys, H. haplostachya and the extinct H. linearifolia, 42 Phyllostegia accessions representing 29 species and one presumed natural hybrid between P. glabra and P. grandiflora, and 28 accessions of Stenogyne representing 17 species and a putative natural hybrid between S. microphylla and S. rugosa. Also included are 23

accessions of Stachys belonging to the North AmericaHawaii clade (cf. Lindqvist and Albert, 2002), to which the Hawaiian mints belong. Furthermore, Stachys baicalensis and S. sylvatica are included, the latter of which was used to root the trees. See also Table 1 for a listing of the accessions used and their voucher information. For the AFLP study a total of 11 accessions of field collected Stenogyne were analyzed, representing the species S. calaminthoides, S. microphylla, S. rugosa, S. purpurea, a cultivated plant resembling S. angustifolia, and putative natural hybrids between S. microphylla and S. rugosa (see Table 2). The last accessions represented a range of morphological variation drawn from a larger sample of over 40 putative hybrid individuals from Halep
ohaku, Mauna Kea. Molecular methods DNA extractions, PCR amplifications, and automated DNA sequencing were performed as described in Lindqvist and Albert (2002). Because the Hawaiian mint 5S-NTS sequences appeared to be highly polymorphic using the universal (degenerate) 5S primers (as determined by observation of base intensities on chromatograms), mint-specific internal primers were used to exclude the possibility of contamination (see Lindqvist and Albert, 2002). Forward and reverse 5S-NTS sequences were edited and aligned for each accession using the software program Sequencher, version 3.1 (GeneCodes, Ann Arbor, MI, USA), and the consensus sequences were deposited in GenBank (see Table 1). For the AFLP analysis, DNA was extracted as described in Lindqvist and Albert (2002), the only exception being resuspension of the extracted DNA pellets in 20 ll 10 mM Tris. AFLP analysis was conducted according to the Applied Biosystems (Applied Biosystems, Foster City, CA, USA) plant mapping protocol with some minor modifications. Genomic DNA (ca. 0.3 lg) was digested by two restriction enzymes (EcoRI/MseI) and simultaneously ligated with EcoRI and MseI adapter sequences in a 6.6 ll reaction volume at 37 °C for 2 h. The ligated DNA was diluted 17-fold with TE buffer (20 mM Tris–HCl, 0.1 mM EDTA, pH 8.0). Preselective amplifications were performed using the EcoRI and MseI primers with one selective nucleotide and the following program: hold 74 °C 2 min; 20 cycles of 94 °C for 1 s, 56 °C or 30 s, 72 °C for 2 min; hold 60 °C 30 min. PCR products were diluted 19-fold with TE buffer and used as templates for the selective amplification using two primers, MseI plus three selective nucleotides and fluorescently labeled EcoRI plus three selective nucleotides. Selective amplification was performed using the following program: 94 °C for 2 min in the first cycle and for 1 s in subsequent cycles, 65 °C for 30 s, and 72 °C for 2 min, followed by reduction of the annealing temperature at each cycle by 1 °C for nine cycles; the annealing

C. Lindqvist et al. / Cladistics 19 (2003) 480–495

483

Table 1 Voucher and locality information and GenBank Accession Nos for the Hawaiian mint 5S-NTS sequences used in this study
Taxon

Haplostachys haplostachya (A. Gray) St. John Haplostachys linearifolia (Drake) Sherff Phyllostegia ambigua (A. Gray) Hillebr. Phyllostegia bracteata Sherff Phyllostegia brevidens A. Gray Phyllostegia electra C. Forbes Phyllostegia cf. Electra Phyllostegia floribunda Benth. Phyllostegia glabra (Gaud.) Benth. var. glabra

Phyllostegia glabra (Gaud.) Benth. var. lanaiensis Sherff Phyllostegia grandiflora (Gaud.) Benth. Phyllostegia glabra  grandiflora Phyllostegia haliakalae Wawra Phyllostegia hirsuta Benth.

Voucher informationa

Geographic locality

Optimized corolla tube charactersb Curvaturec

Lengthd

GenBank Accession

S. Perlman 14328 (NY)

HawaiÕi

0

2

AF308173

J.F. Rock 14025 (NY) 1. G. Clarke 688 (BISH) 2. R. Hobdy 3023 (BISH) B.H. Gagne s.n., 1981 (BISH) 1. K. Wood 3200 (BISH) 2. J. Griffin s.n., 1985 (BISH) K. Wood 2967 (BISH) J.J. Fay 156 (NY) J.D. Jacobi 1326 (BISH) 1. J. Obata s.n., 1990 (BISH)

West MolokaÕi HawaiÕi East Maui Maui East Maui HawaiÕi KauaÕi KauaÕi HawaiÕi OÕahu

0 0

3 0

0 0

0 0

0 0 0 0

0 0 0 3

AF308174 AF308175 AF308176 AF308177 AF308179 AF308178 AF308180 AF308188 AF308181 AF308182

2. K. Wood 3962 (NY) 3. W.L. Wagner 5761 (BISH) O. Degener 24160 (BISH)

West Maui MolokaÕi LanaÕi

0

3

AF308185 AF308184 AF308183

OÕahu OÕahu LanaÕi OÕahu OÕahu MolokaÕi OÕahu

0 0 0 0

3 0 0 0

0 0

0 0

AF308187 AF308186 AF308192 AF308189 AF308190 AF308191 AF308193

P. Welton 801 (BISH) J. Lau 3538 (BISH) D. Herbst 4048 (BISH) 1. E. Hosaka s.n., 1933 (NY) 2. J. Obata s.n., 1993 (BISH) Phyllostegia hispida Hillebr. L. Stemmermann 3973 (BISH) Phyllostegia kaalaensis St. John 1. J. Obata & R. Robichaux 407 (BISH) 2. S. Perlman 6117 (BISH) 3. W. Takeuchi & Paquin 3440 (BISH) 4. W. Takeuchi 941 (BISH) Phyllostegia kahiliensis St. John W.L. Wagner 5217 (BISH) Phyllostegia knudsenii Hillebr. K. Wood 2583 (BISH) Phyllostegia lantanoides Sherff J. Obata 86-624 (BISH) Phyllostegia macrophylla (Gaud.) Benth. 1. F.R. Warshauer 2862 (BISH) 2. S. Perlman 14184 (NY) Phyllostegia mannii Sherff F.R. Warshauer 2418 (BISH) Phyllostegia mollis Benth. O. Degener 20866 (NY) Phyllostegia parviflora (Gaud.) Benth. var. J. Obata s.n., 1990 (BISH) lydgatei (Sherff) W.L. Wagner Phyllostegia racemosa Benth. F.R. Warshauer 1447 (BISH) Phyllostegia renovans W.L. Wagner S. Perlman 10830 (BISH) Phyllostegia cf. renovans S. Perlman 13256 (BISH) Phyllostegia rockii Sherff C.N. Forbes 199 (BISH) Phyllostegia stachyoides A. Gray 1. J.S. Meidell 111 (BISH) 2. K. Wood 6280 (BISH) 3. F.R. Warshauer 1856 (BISH) Phyllostegia variabilis Bitter C. Lamoureux 1926a (BISH) Phyllostegia velutina (Sherff) St. John J. Griffin s.n., 1992 (BISH) Phyllostegia vestita Benth. St. John 22360 (NY) Phyllostegia warshaueri St. John S. Perlman 14185 (BISH) Phyllostegia wawrana Sherff S. Perlman 13690 (NY) Stenogyne angustifolia A. Gray 1. R. Hobdy 2451 (BISH) 2. O. Degener 19813 (BISH) Stenogyne bifida Hillebr. 1. F.R. Warshauer 2377 (BISH) 2. F.R. Warshauer 3028 (BISH) 3. K. Wood 6284 (BISH Stenogyne calaminthoides A. Gray C. Lindqvist & V.A. Albert 42 (NY) Stenogyne calycosa Sherff R. Hobdy 2553 (BISH) Stenogyne campanulata Weller & Sakai K. Wood 1790 (BISH) Stenogyne cranwelliae Sherff J. Davis 945 (BISH) Stenogyne haliakalae Wawra G.E. Olson 5 (BISH)

OÕahu OÕahu OÕahu KauaÕi KauaÕi OÕahu East Maui HawaiÕi MolokaÕi OÕahu OÕahu

AF308194 AF308195

0 0 0 0

0 0 0

0 0 0

0 0 0

0 0 0 0 0

0 0 0 0

—

AF308196 AF308197 AF308198 AF308199 AF308200 AF308201 AF308202 AF308203 AF308204

HawaiÕi KauaÕi KauaÕi East Maui West Maui MolokaÕi HawaiÕi Kure HawaiÕi HawaiÕi HawaiÕi KauaÕi HawaiÕi HawaiÕi MolokaÕi MolokaÕi MolokaÕi HawaiÕi

0 0 0 0 0 0 0 0

0 0 0 1 0 1 1 0

1

4

AF308205 AF308214 AF308206 AF308207 AF308208 AF308209 AF308210 AF308211 AF308212 AF308213 AF308215 AF308216 AF308218 AF308217 AF308219 AF308220 AF308221 AF308222

East Maui KauaÕi HawaiÕi Maui

1 0 0 1

3 0 0 4

AF308223 AF308224 AF308225 AF308226

—

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Table 1 (continued) Voucher informationa

Taxon

Stenogyne kaalae Wawra Stenogyne kamehamehae Wawra

Stenogyne kanehoana Degener & Sherff Stenogyne macrantha Benth. Stenogyne microphylla Benth.

Stenogyne cf. microphylla  rugosa Stenogyne purpurea H. Mann Stenogyne rotundifolia A. Gray Stenogyne rugosa Benth.

Stenogyne scrophularioides Benth. Stenogyne sessilis Benth.

Geographic locality

K. Nagata 1617 (NY) 1. P.K. Higashino 9461 (BISH) 2. W.L. Wagner 4801 (BISH) 3. W.L. Wagner 5888 (BISH) 1. J. Obata 331 (BISH) 2. J. Obata 356 (BISH) W. Mull & M. Mull s.n., 1980 (BISH) 1. F.R. Warshauer 2682 (BISH) 2. C. Lindqvist & V.A. Albert 35 (NY) C. Lindqvist & V.A. Albert 38 (NY) K. Wood 1772 (BISH) F.R. Warshauer 2545 (BISH) 1. C. Lindqvist & V.A. Albert 40 (NY) 2. B.H. Gagne s.n., 1975 (BISH) W.L. Wagner 5954 (BISH) 1. O. Degener 33639 (NY) 2. S.G. Weller 821 (BISH) 3. S. Perlman 15398 (BISH)

Optimized corolla tube charactersb c

d

GenBank Accession

Curvature

Length

0 1

0 5

1

5

1

4

Maui HawaiÕi

0

0

AF308234 AF308235

HawaiÕi

0

—

AF308236

KauaÕi Maui HawaiÕi

0 0 0

2 2 0

AF308237 AF308238 AF308240

0 0

3 3

OÕahu MolokaÕi West Maui East Maui OÕahu OÕahu HawaiÕi

Maui HawaiÕi HawaiÕi HawaiÕi Maui

AF308227 AF308228 AF308229 AF308230 AF308231 AF308232 AF308233

AF308239 AF308241 AF308244 AF308242 AF308243

*

See Lindqvist and Albert (2002) for voucher information of the Stachys sequences included. Herbaria abbreviations follow Holmgren et al. (1990). b Corolla tube characters as optimized with WinClada (Nixon, 2002) are recorded from Wagner et al. (1999); see also text. c Corolla tube curvature states are coded as follows: 0, straigth or nearly so; and 1, curved. d Corolla tube length is here the maximum recorded length according to Wagner et al. (1999), and is optimized additively with the following states: 0, 17 mm or less; 1, 19–20 mm; 2, 21–22 mm; 3, 24–25 mm; 4, 28–29 mm; and 5, 42–56 mm. a

Table 2 Voucher and locality information for Stenogyne accessions used in the AFLP study Taxon

Voucher information

S. S. S. S. S. S. S. S. S. S. S.

C. C. C. C. C. C. C. C. C. C. C.

calaminthoides LVA82 calaminthoides LVA349 purpurea LVA68 rugosa LVA63 rugosa LVA148 microphylla LVA85 microphylla LVA178 microphylla  rugosa LVA306 microphylla  rugosa LVA288 microphylla  rugosa LVA107 cf. angustifolia LVA32

Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist

& & & & & & & & & & &

V.A. V.A. V.A. V.A. V.A. V.A. V.A. V.A. V.A. V.A. V.A.

Albert Albert Albert Albert Albert Albert Albert Albert Albert Albert Albert

Geographic locality 82 (NY) 349 (NY) 68 (NY) 63 (NY) 148 (NY) 85 (NY) 178 (NY) 306 (NY) 288 (NY) 107 (NY) 32 (NY)

temperature was then maintained at 56 °C for the remaining 23 cycles. After screening several different primer combinations for selective amplification reactions, the following six combinations were chosen for all accessions: EcoRI + AAG combined with MseI + CTT, +CTC, and +CAC, respectively, and EcoRI + ACA combined with MseI + CTG, +CAC, and +CTC, respectively. The AFLP fragments were separated and visualized using a 5% Long Ranger gel (FMC Bioproducts, 191 Thomaston Street, Rockland, ME 04841) on an ABI Prism 377 DNA Sequencer (Applied Biosystems). Gel analysis was carried out with the Genescan

Saddle Road, HawaiÕi Kohala Mts., HawaiÕi Waimea district, KauaÕi KaÕ
u district, HawaiÕi Mauna Kea, HawaiÕi PuÕu L
aÕau, HawaiÕi Mauna Kea, HawaiÕi Mauna Kea, HawaiÕi Mauna Kea, HawaiÕi Mauna Kea Road, HawaiÕi cult., University of Hawaii Volcano Research Station, HawaiÕi

3.1 and Genotyper 2.1 software packages (Applied Biosystems). Analytical methods Edited 5S-NTS sequences were aligned using a reference alignment generated with the program MALIGN version 2.7 (Wheeler and Gladstein, 1994) and the parameter set described in Lindqvist and Albert (2002). The matrix of 97 taxa was subjected to non-additive parsimony analysis using NONA (Goloboff, 1998), as implemented through WinClada (Nixon, 2002). Two

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experiments were performed: analysis of the data with (i) within-individual polymorphic states coded as ambiguities (with standard IUPAC coding) and (ii) the polymorphisms recoded as representing new character states (with, e.g., A, T, G, C ¼ 0, 1, 2, 3; C&T ¼ 4; etc.). The latter treatment makes the assumption that possessing more than one nucleotide state per site within a tandemly repeated locus or between sister or homeologous chromatids may be phylogenetically informative. Both analyses were accomplished using the tree search criteria as described in Lindqvist and Albert (2002). Strict (‘‘nelsen’’) consensus trees were calculated by WinClada after ‘‘hard collapsing’’ unsupported nodes in all most-parsimonious trees. To estimate support for internal branches, parsimony jackknifing (Farris et al., 1996) was performed using the programs XAC and PAX (J.S. Farris, unpublished), the latter in the case of the recoded matrix, which included up to nine character states (the 10th being N, or ?). One thousand replicates, each performing SPR branch swapping with five random entry orders per replicate, were conducted. Furthermore, Branch Support analysis, based on comparing suboptimal trees with minimum length ones (Bremer, 1994), provided an additional strength measure less stringent than character resampling approaches (cf. Farris et al., 1996; C. Lindqvist and V.A. Albert, unpublished). Support calculations were automated by allowing NONA to save up to 10,000 additional optimal and suboptimal trees. Geographical and morphological character state optimizations (see Table 1) were performed using WinClada (Nixon, 2002). Data were treated as non-additive or additive (tube length), and unambiguous character-state optimizations were accomplished using a clipped subtree from the strict consensus of polymorphism-recoded trees. AFLP data were scored as the presence (1) or absence (0) of bands, and a binary matrix was constructed (see Appendix). Only AFLP fragments that could be unambiguously scored were included in the analysis. The matrix was subjected to phylogenetic analysis using NONA and the same options as for the 5S-NTS data. Population Aggregation Analysis, a procedure that searches for fixed differences within species (Davis and Nixon, 1992), was subjected to the AFLP data by merging terminals identified by us in the field as belonging to the same species into ‘‘units’’ (using WinClada; Nixon, 2002). We did not perform Population Aggregation Analysis on the 5S-NTS matrix because of two factors that could render the results spurious: (i) uncertainties in herbarium specimen identification, and (ii) vast differences in time of plant collection (from 1918 to 1998, and the potential for gene conversion to occur differentially during this period). Finally, a multivariate Detrended Correspondence Analysis (DCA) was performed to canonically correlate AFLP variation (PCORD for Windows, ver. 4, MjM Software Design, Gleneden Beach, OR, USA).

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Results 5S-NTS sequencing and analysis The Stachys 5S-NTS sequences varied from 389 to 402 base pairs (bp) in length, whereas the length of the Hawaiian mint sequences, obtained from specific, internal primers, varied from 286 to 287 bp. Cladistic analyses of the 97 5S-NTS sequences yielded 2021 trees with the length of 207, a consistency index ðCÞ of 0.73, and a retention index ðRÞ of 0.87 (Farris, 1989). The strict consensus tree (Fig. 1) revealed extremely low resolution among the Hawaiian mints with only few clades identified. The two Haplostachys species formed a clade sister to all remaining taxa, with Phyllostegia and Stenogyne unresolved with respect to each other. This lack of resolution is reflected by very low sequence divergence among the Hawaiian mints. In the 5S-NTS sequence matrix excluding the Stachys sequences, 330 characters of 364 total (91%) are uninformative as calculated with WinClada (Nixon, 2002; the 101 and 61 missing characters in the beginning and end of the matrix, respectively, were excluded). In the complete matrix including the Stachys sequences, of 526 characters total, 461 (88%) are uninformative. In contrast to the low 5S-NTS sequence divergence, a surprisingly high level of intra-individual nucleotide polymorphism was observed among the Hawaiian taxa after closer examination of base intensities on chromatograms. A comparison of the mean number of polymorphic sites (two-tailed t test) between Stenogyne and Phyllostegia (average of 2.9 and 4.2 polymorphic sites per accession, respectively) revealed no significant difference ðP ¼ 0:096Þ. However, all the Hawaiian genera together have significantly ðP ¼ 0:010Þ more polymorphic sites than the included Stachys taxa (the remaining taxa of the North America-Hawaii clade, cf. Lindqvist and Albert, 2002). Under the assumption that shared within-individual polymorphisms could contain phylogenetic information, i.e., if they were co-inherited on the same, sister, or homeologous chromatids, the ambiguities were recoded as new character states. Parsimony analysis of these data resulted in considerably more phylogenetic structure, and all Stenogyne taxa except for the two S. kanehoana accessions formed a monophyletic clade, which also included three accessions of P. kaalaensis as well as one accession of P. renovans (Fig. 2). There was an increase in internal clade structure, especially within Stenogyne, with a net gain of 15 subclades (Figs. 1 and 2). Phyllostegia gained three subclades but remained unresolved. The absence of mixed-generic clades (with the only exception of Stenogyne kanehoana, P. kaalaensis, and P. renovans; see Discussion) implied that polymorphism was attributable to sequence diversity occurring at single or homeologous 5S rDNA loci within these closely related

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Fig. 1. Strict consensus tree of 2021 most-parsimonious trees of Hawaiian mints and outgroups based on 5S-NTS data. A phylogram is shown with ACCTRAN optimized nucleotide changes above branches and parsimony jackknife and Branch Support (in parentheses) values below branches. The generic annotations of the Hawaiian taxa, Haplostachys, Phyllostegia, and Stenogyne are abbreviated H., P., and S., respectively.

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487

Fig. 2. Strict consensus tree of 1497 most-parsimonious trees of Hawaiian mints and outgroups based on 5S-NTS data with recoded nucleotide polymorphic sites. Phylogram shown with ACCTRAN optimized nucleotide changes above branches and parsimony jackknife and Branch Support (in parentheses) values below branches. The generic annotations of the Hawaiian taxa, Haplostachys, Phyllostegia, and Stenogyne are abbreviated H., P., and S., respectively. Stenogyne taxa are shown in bold.

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polyploids (e.g., Cronn et al., 1996; Kellogg and Appels, 1995). Polymorphic states accounted for the majority (16/ 30) of character changes that consistently supported the internal nodes of the Stenogyne clade, and all but one of these changes (a state linking the two individuals of S. angustifolia; Fig. 2) represented single-step transformations (e.g., C ! [CT] or G ! [AG]). This bias against multi-step transformations is consistent with the recoding model, which permits free change to or from polymorphic states even though some changes (e.g., A ! [CT] or [AG] ! [CT]) would require hidden nucleotide substitutions. Although recoding of the polymorphic characters seemed to increase phylogenetic structure within Stenogyne, the character state changes that supported the monophyly of Stenogyne involved non-polymorphic states. It is worth noting that monophyly of Stenogyne is apparent in many single parsimonious trees when the intra-individual polymorphisms are treated as ambiguities (not shown), and therefore, the polymorphism recoding can be interpreted as strengthening structure already suggested by some of the non-polymorphic characters. The most-parsimonious hypothesis for geographic origin of the Stenogyne clade (from Fig. 2) was OÕahu or the Maui Nui island complex (MolokaÕi, Maui, LanaÕi,

Fig. 3. Biogeographic history of the Stenogyne clade. Clipped subtree from the strict consensus tree in Fig. 2. Non-additive area states are shown with unambiguous optimization. The base of the clade optimizes to OÕahu or the Maui Nui complex (which includes the present-day islands MolokaÕi and Maui). There are three unambiguous forward dispersals from East Maui to the youngest island, HawaiÕi, plus multiple back dispersals from HawaiÕi to Maui Nui, OÕahu, and Kauai.

Fig. 4. Corolla evolution in the Stenogyne clade: corolla tube shape. As optimized non-additively, strongly curved corolla tubes have evolved four times from the straight or nearly straight condition.

Fig. 5. Corolla evolution in the Stenogyne clade: corolla tube length. Optimized additively as a quantitative character, the corolla tube in Stenogyne is primitively short but has evolved into longer forms several times. Longer measurements, i.e., states 4 and 5, tend to correlate with strong corolla tube curvature (see Fig. 4).

plus KahoÕolawe; Fig. 3), followed by numerous dispersal events. Optimization of floral morphology indicates that the most likely ancestral condition was flowers

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Fig. 6. A phylogram of one of three most-parsimonious trees based on AFLP data, arbitrarily oriented with Stenogyne calaminthoides basalmost. Number of ACCTRAN optimized nucleotide changes are shown above branches and parsimony jackknife values are shown below branches. Branches that collapse in the strict consensus tree are shown with stippled lines. The putative hybrid accessions are shown in bold. The numbers following taxon names refer to individual accessions (see Table 2).

489

5S spacer revealed only slight sequence divergence among the Hawaiian endemic mints, and only few clades were discerned within the three genera. Haplostachys was resolved as sister to all remaining taxa, for which only few clades were identified that largely consisted of different accessions of the same taxon (Fig. 1). This scant genetic variation exists in sharp contrast to the Hawaiian mintsÕ apparent morphological and ecological radiation reflected by radical differences in floral and fruit characters, as well as divergent leaf morphologies, which vary from linear in H. linearifolia to ovate (e.g., P. parviflora and Stenogyne calaminthoides), divided (e.g., P. rockii and juvenile S. kamehamehae), and minute (S. microphylla). Also, plant habit ranges from erect herbs to subshrubs and scandent lianas, and mint species can be found from low-elevation to subalpine habitats as well as in dry bushland to wet forest. Polymorphism and hybrid origins

Genetic divergence in the Hawaiian mints

Although only very low resolution was found in the Hawaiian mints with phylogenetic analyses of the 5S spacer, a surprisingly high level of within-individual nucleotide polymorphism was evident, which was significantly greater than in their parental lineage, Stachys. The polyploid hybrid origin presumed for the Hawaiian mints, between hummingbird- and insect-pollinated species of Stachys (Lindqvist and Albert, 2002),1 could in part explain the abundant intra-individual polymorphism within the 5S spacer. The occurrence of putative natural hybrids within Phyllostegia and Stenogyne (Wagner et al., 1999) could be additional evidence of a propensity for hybridization among the Hawaiian mints, perhaps displayed by the lack of monophyly among several infraspecific accessions (e.g., P. kaalaensis, S. rugosa, S. sessilis, and S. kamehamehae; Fig. 2). Although this study suggests that the polymorphism may have arisen from both relatively recent and ancient gene flow (possibly in part maintained by incomplete gene conversion), only cloning of individual array copies or repeat-specific amplifications would identify the nature of the polymorphism (e.g., Baker et al., 2000; Rauscher et al., 2002), i.e., to ascertain whether the variation occurs within or among alleles of single or perhaps homeologous array loci. In addition, these approaches could verify the assumption that shared within-individual polymorphisms contain phylogenetic information, although this seems already apparent from the strong hierarchic correlation (R > 75%) among all informative sites in the recoded analysis (Fig. 2). Indeed, although cloning or repeatspecific priming could be ideal to study gene flow and the

The nuclear 5S non-transcribed spacer represents one of the fastest evolving loci known in higher plants and it has therefore usually been used in phylogenetic analyses of plants at relatively low taxonomic levels. However, the

1 Note that with this taxon sampling, Stachys quercetorum is resolved as the sister taxon to the Hawaiian mints, whereas in this earlier study including more Stachys species but fewer Hawaiian mints, either S. quercetorum or S. chamissonis were seen to occupy this position.

with corolla tubes straight or nearly so (Fig. 4) with a maximum reported length 6 17 mm (Fig. 5). AFLP analysis The six AFLP primer pair combinations produced a total of 264 DNA fragments for the 11 accessions studied. Of these, 135 fragments (51%) were shared among more than one accession. Cladistic analysis conducted on the binary AFLP matrix (see Appendix), produced three most-parsimonious trees of length 405, a consistency index ðCÞ of 0.56, and a retention index ðRÞ of 0.41 (Fig. 6). The presumed hybrids all grouped in a strongly supported monophyletic clade with S. microphylla. Rather than grouping with some of their ostensive hybrids, the two S. rugosa accessions clustered either with or proximal to S. cf. angustifolia. Population Aggregation Analysis (Davis and Nixon, 1992) resulted in the same overall topology with similar support levels (data not shown). The DCA analysis, which produced groups largely resembling the phylogenetic results when the first and second axes of variation were co-plotted, is shown in Fig. 7. However, one S. rugosa (number 63) and one putative hybrid accession (number 107) can be seen to be outliers, highlighting the utility of employing a phylogenetic approach to the analysis of structure in these data.

Discussion

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Fig. 7. Detrended Correspondence Analysis (DCA) of AFLP data. The first and second axes, explaining 22.9 and 15.6% of the variation, respectively, are shown. The circles around groups of individual plots refer to parsimony jackknife supported clades in the strict consensus tree (see Fig. 6).

nature of evidence from polymorphisms for phylogenetic reconstruction, the results presented here demonstrate that subjecting direct-sequenced, recoded polymorphic data to cladistic analyses can be a tenable option when large numbers of herbarium specimens, and even endangered and extinct taxa, are studied. Phylogenetic patterns and intergeneric reticulation In the analysis that treated polymorphisms as nonambiguous states, most of Stenogyne was resolved as a discrete lineage occurring within a large polytomy of Phyllostegia species (Fig. 2). Optimization of geographic location for the accessions sampled indicates an origin for the Stenogyne clade on OÕahu or the former Maui Nui complex of volcanoes (Fig. 3), now separated into MolokaÕi, Maui, LanaÕi, and KahoÕolawe (Price and Clague, 2002). Forward dispersals to the younger island HawaiÕi are also indicated (at least three times), as are back dispersals from HawaiÕi to Maui Nui (three times) and from Maui Nui to OÕahu or KauaÕi (ca. three times; Fig. 3). The transgeneric positions of S. kanehoana, P. kaalaensis, and P. renovans (Fig. 2) likely reflect reticulation between the Stenogyne clade and elements of the unresolved entity Phyllostegia rather than evidence for 5S-NTS lineages with substantially different histories (which, as in the 5S rDNA paralogs of grasses, would yield tree branches with repeating species phylogenies; cf. Kellogg and Appels, 1995). Other possibilities for the unresolved placements of most taxa assigned to Phyllostegia include lineage sorting following ancient hybrid lineage formation or even homoplasy alone. S. kanehoana and P. kaalaensis are endemic to the WaiÕanae Mountains of OÕahu, and P. renovans is a KauaÕi endemic. Parsimony analysis with recoded polymorphisms

(see Fig. 2) revealed that the two S. kanehoana accessions included are resolved as a monophyletic group unresolved with Phyllostegia, whereas the accessions of P. kaalaensis group either (1) equivalently to S. kanehoana, (2) basal within Stenogyne or (3) like P. renovans, deeply within Stenogyne, adjacent to the S. purpurea/kaalae/angustifolia clade. Given the sympatry of S. kanehoana and P. kaalaensis, it seems possible that the two morphotypes represent the parental forms (perhaps sorted out from many generations) of a single hybridization event. The 5S-NTS data for S. kanehoana, for example, reveal an ostensibly recombinant locus that may be incompletely gene-converted, since nucleotide states at some sites are otherwise specific to Phyllostegia species or the greater Stenogyne clade (data not shown). Unfortunately, the direction of putative gene flow events cannot be discerned using only nucleotypic markers. Concerning the accessions identified as P. renovans and P. cf. renovans, the former represents a recently described taxon with post-flowering vegetative renewal features unique within the genus and suggested to be intermediate between Stenogyne and Phyllostegia (Wagner, 1999b). Furthermore, the species often grows with Stenogyne purpurea, with which our 5S-NTS data suggest P. renovans shares close (and therefore potentially hybrid) relationship (Fig. 2). The particular specimen we identify as Phyllostegia cf. renovans was suggested by Wagner (1999b) to be potentially different from P. renovans and worthy of further study. Our phylogenetic data support both of WagnerÕs assertions. Polymorphisms and cladogenetic processes The phylogenetic results based on recoded polymorphic ambiguities argue that the distribution of polymorphisms

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among the Hawaiian mints could be explained by different evolutionary mechanisms. The prevalence of these polymorphisms, although it cannot be distinguished using these data from ancestral polymorphism inherited from hybrid lineage origins, may suggest that many Hawaiian mints consist of interbreeding populations expressing microevolutionary processes such as gene flow, extinction, recolonization, founder effect, and genetic drift (e.g., Hanski, 1998). Sorting out of a genetically isolated lineage from within this hypothetical metapopulation may be demonstrated by the monophyly and hierarchical structure of most Stenogyne accessions (Fig. 2). If so, this cladogenesis has apparently occurred at a rate faster than the elimination of polymorphism by gene conversion (Cronn et al., 1996). It is also correlated with a switch from fragrant flowers with prominently lowerlipped, narrowly funnelform, mostly white-pink-colored corollas and inserted stamens (Haplostachys and Phyllostegia) to odorless flowers with lower-lip-reduced, longtubed, primarily red-colored corollas, mostly exserted stamens, and abundant nectar production (Stenogyne) (Wagner et al., 1999). Although bird pollination per se is part of the presumed hybrid heritage of the Hawaiian mints (Lindqvist and Albert, 2002), fixation of novel morphotypes after colonization of the Hawaiian Islands that were preferred by endemic honeycreepers could have driven the genesis of the Stenogyne clade by ensuring that its members would not normally interbreed with sympatric, insectpollinated mints. The phylogenetic results further suggest that Stenogyne flowers with shorter and basically straight corolla tubes (as in the basal-most species, S. bifida) may represent the original bird-pollinated condition in the Hawaiian Islands, and that flowers with longer and curved corolla tubes are evolutionarily derived (e.g., S. kamehamehae and S. calaminthoides; Figs. 4 and 5). The flowers of S. microphylla, which are of the more ancestral type (nearly straight corolla tubes 10–14 mm long; Wagner et al., 1999), are today visited by generalist nectar feeders such as the HawaiÕi ÔAmakihi (Hemignathus virens virens) and the IÕiwi (Vestiaria coccinea) (J.J. Jeffrey, pers. obs.). The IÕiwi, with its long (ca. 28 mm) arced bill (Smith et al., 1995), also pollinates a guild of Hawaiian flowers with large, analogously bent corolla tubes, including Stenogyne kamehamehae, S. calaminthoides (tubes up to 56 and 29 mm long, respectively; Wagner et al., 1999), and various endemic lobelioids (Campanulaceae) (Lammers and Freeman, 1986). Given the range of bill curvatures among less common and extinct honeycreepers (Carlquist, 1980; Tarr and Fleischer, 1995), it seems possible that greater pollinator specialization existed before human disturbance (Steadman, 1995), and that this might correlate with the internal subclade structure found within Stenogyne.

491

Reticulation within Stenogyne To investigate further the extent of hybridization and gene flow within the Hawaiian mints, we studied AFLP variation within Stenogyne, with emphasis on a putative hybrid swarm on Mauna Kea involving the two species S. microphylla and S. rugosa. For some years, unusual specimens of Stenogyne have been reported from Halep
ohaku at around 3000 meters elevation on Mauna Kea, immediately behind the Mauna Kea VisitorÕs Center on HawaiÕi (Weller and Sakai, 1999). Young S. microphylla plants typically establish themselves on mature trees of Sophora chrysophylla (Fabaceae). The habitat has been heavily disturbed by feral ungulate grazing (J.J. Jeffrey, pers. obs.). S. rugosa, perhaps a closely related species (Fig. 2), has also been collected at Halep
ohaku, and the unusual plants have been characterized as possible F1 and back-crossed hybrids with S. microphylla (Weller and Sakai, 1999). Indeed, S. microphylla and S. rugosa are known to occur together elsewhere, including PuÕu LÕau (Mauna Kea; W. Gagne 666, BISH, and pers. obs.), Mauna Loa, and Haleakal
a on Maui (Weller and Sakai, 1999). Putative hybrids have also been reported from elsewhere on Mauna Kea as well as Mauna Loa (Warshauer 3128, 3154, BISH; Weller and Sakai, 1999). Stenogyne rugosa appears to be more subject to ungulate predation than the more arboreal S. microphylla (pers. obs.), and the species may be a recent reoccurrence or recruit to the region now that ungulate grazing has been more successfully suppressed. On Mauna Kea, S. rugosa is most often found terrestrially, under the shade of Sophora trees and especially in ungulate exclosures, where its growth can be quite vigorous (pers. obs.). The putative hybrids could represent introgression of S. rugosa into otherwise stable populations of S. microphylla, since that speciesÕ arboreal habit provides natural herbivore protection and pure stands of it can be found at other elevations and locations on the mountain. Phylogenetic analysis of the primary (Fig. 6) and PAA-aggregated AFLP fragment data showed that the presumed hybrids all grouped in a strongly supported monophyletic clade with S. microphylla. Rather than grouping with some ostensive hybrid individuals, S. rugosa (one accession from Mauna Kea and another from a disjunct site in southern HawaiÕi) grouped either with or proximal to S. cf. angustifolia (Figs. 6 and 7). Some AFLP fragments were even shared between the putative hybrids and accessions of the large, pink-flowered Stenogyne calaminthoides, possibly indicating that gene flow with this geographically proximal taxon has occurred as well. However, such shared fragments, rare compared to overwhelming hierarchical evidence for hierarchy among other accessions, could only be interpreted as homoplasy by the parsimony analysis. Because the overall retention of observed similarity as

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synapomorphy ðRÞ is strikingly low for this few taxa (41%), a large proportion of the long branch length is homoplasy, which can be inferred to derive from introgression. Most importantly, the pattern of relationships inferred from cladistic analysis preliminarily suggest that (1) S. rugosa and S. cf. angustifolia either represent the same evolutionary taxon, are extremely similar, or are actively exchanging genes and (2) the direction of introgression appears to be from S. cf. angustifolia/S. rugosa into S. microphylla. The latter can be inferred from the fact that the putative hybrids, thus far grouping only with S. microphylla, occupy (at best) paraphyletic, intermediate positions between their presumed parents. S. rugosa may not even represent a fixed species, but possibly introgressants of hybrid genomes back into S. angustifolia. The fact remains that the two herbarium specimens sampled of S. angustifolia are very different in 5S-NTS sequence from either S. rugosa or S. microphylla (Fig. 2), but survey of just one locus cannot be expected to reveal extensive evidence for gene flow. In sum, the data may be supporting a present-day hybrid radiation including S. angustifolia, S. rugosa, and S. microphylla, although this should be investigated further with greater sampling and additional markers. It cannot be excluded, for example, that the morphological variation observed is occurring entirely at the population (intraspecific) level. Although the use of PCR-amplified DNA fragment data in parsimony analyses has been criticized (Backeljau et al., 1995), application of phylogenetic analysis to AFLP characters should be defensible so long as the data retain underlying evidence for hierarchy, as they obviously do in our preliminary Hawaiian mint sample (Fig. 6). This result corroborates other studies demonstrating the utility of AFLP data in phylogenetic reconstruction using the parsimony criterion (Kardolus et al., 1998; Zerega et al., 2002). The arguments of Backeljau et al. (1995) against the applicability of RAPDs (and by extension, AFLPs) are not compelling, since unknown but potential non-independence of characters is just as likely with morphological data (e.g., corolla tube color, corolla lobe color), which have frequently been subjected to parsimony analyses by other authors. Indeed, cladistic analysis is a means by which to evaluate the extent of character correlation, and with support analysis, the strength of recoverable hierarchy, even in the face of uncertain homology.

Conclusions The Hawaiian mints may provide a unique snapshot of microevolutionary processes that form macroevolutionary (cladogenetic) patterns (see Carroll, 2001). 5S-NTS data demonstrate the early split between Haplostachys and the fleshy-fruited genera, but do not permit resolution between Phyllostegia and Stenogyne. Despite some ad-

mixtures of likely ‘‘intergeneric’’ hybrids with Phyllostegia, Stenogyne is nonetheless supported as a discrete lineage of fleshy-fruited mints when polymorphisms are recoded as discrete states. Furthermore, insignificant differences in numbers of polymorphism between Phyllostegia and Stenogyne may reflect similar degrees of heterozygosity inherited from polyploid hybrid parental stock – the principal difference between these morphological genera could then be related to the extent to which more recent gene flow has obscured evidence for hierarchy. We therefore hypothesize that Stenogyne is a recent evolutionary lineage that might have differentiated from other fleshy-fruited mints through the agency of highfidelity bird pollinators that restricted gene flow with other Hawaiian mint morphotypes. The Stenogyne clade does not appear to be older than OÕahu or the Maui Nui complex (Fig. 3), i.e., 6 3.0 My (Price and Clague, 2002). This date is in keeping with the molecular clock estimate of Lindqvist and Albert (2002), which suggested that the entire Hawaiian mint lineage colonized the island chain between 2.6 and 7.4 My. The Hawaiian honeycreeper radiation has similarly been dated to 4–5 My from its most recent common ancestor (Fleischer et al., 1998). It also appears that cladogenesis within the Stenogyne lineage has proceeded more rapidly than gene conversion could eliminate polymorphic sites, which have then become hierarchically correlated with other informative sites. However, the evolutionary potential afforded by reticulation could be active in Stenogyne, which may be experiencing introgression between high elevation entities on Mauna Kea. Viewed at different hierarchical levels, reticulation can confound phylogeny reconstruction while generating evolutionarily variation. With highly heterozygous, polyploid forebears, the presumed hybrid ancestors of the Hawaiian mints may have had high potential to fix new and adaptive allelic combinations in small, founder populations which themselves became more (Stenogyne) or less (Phyllostegia) reproductively isolated by selective forces. It is possible that similar population-level processes underlie other major morphological radiations on islands, and perhaps otherwise (e.g., Carroll, 2001).

Acknowledgments The authors thank the BISH and NY herbaria and Patti Moriyasu (University of Hawaii Volcano Research Station) for providing plant material, Lyman Perry for permission to collect mints on Hawaiian state lands, and Steve Farris for permission to use the XAC and PAX parsimony jackknifing applications. This research was supported by the Lewis B. and Dorothy Cullman Foundation, the University of Alabama, the Centre for Tropical Biodiversity (Denmark), and the Research Council of Norway (Project No. 27741).

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Appendix. AFLP data matrix

S. S. S. S. S. S. S. S. S. S. S.

calaminthoides LVA82 calaminthoides LVA349 microphylla LVA85 microphylla LVA178 rugosa LVA148 rugosa LVA63 microphylla  rugosa LVA107 microphylla  rugosa LVA306 microphylla  rugosa LVA288 angustifolia LVA32 purpurea LVA68

1 0 0 0 0 0 0 1 0 0 0 0

1 1 1 1 1 0 1 1 1 1 1

0 0 0 0 0 0 0 0 1 0 0

1 1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 1 0 0

1 1 1 1 1 0 1 1 1 0 0

1 1 1 1 1 0 1 1 1 1 1

1 1 1 1 1 0 1 1 1 1 0

1 0 0 0 0 0 0 0 0 0 0 1 0

0 0 0 0 2 0 1 S. calaminthoides LVA82 1 S. calaminthoides LVA349 1 S. microphylla LVA85 1 S. microphylla LVA178 1 S. rugosa LVA148 1 S. rugosa LVA63 1 S. microphylla  rugosa LVA107 1 S. microphylla  rugosa LVA306 1 S. microphylla  rugosa LVA288 1 S. angustifolia LVA32 1 S. purpurea LVA68 1

1 1 1 1

0 0 0 1

0 0 0 0

0 0 1 0

0 0 0 0

1 1 0 1

0 0 0 0

0 0 0 0

1 1 1 1 0 1 1 1 1 1 1

0 0 0 0 0 1 0 0 0 1 0

1 0 0 0 0 0 0 0 0 0 0

0 0 1 1 1 1 1 1 0 1 1

1 0 0 0 0 1 0 1 0 0 1

0 0 1 0 0 0 0 0 0 0 0

0 0 1 1 0 0 1 0 0 0 1

1 0 0 0 0 0 0 1 0 0 0

6 0 0 0 0 0 0 0 1 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 0 0 1 6 0 1 0 0 0 0 0 0 1 0 0 0 0 2 1 0 0 0 0 1 0 0 0 0 0 0 0

S. S. S. S. S. S. S. S. S. S. S.

0 1 0 0 0 0 0 0 ? 0 0

0 0 0 0 1 0 0 0 ? 0 0

0 0 0 0 1 0 0 0 ? 0 0

0 0 1 1 1 1 1 0 ? 1 1

0 0 1 1 0 0 0 0 ? 0 0

1 1 1 1 1 1 0 1 ? 0 1

0 0 0 0 0 1 0 0 ? 0 0

0 0 0 1 1 1 0 0 ? 0 0

0 0 0 1 0 0 0 0 ? 0 0

S. S. S. S. S. S. S. S. S. S. S.

calaminthoides LVA82 calaminthoides LVA349 microphylla LVA85 microphylla LVA178 rugosa LVA148 rugosa LVA63 microphylla  rugosa LVA107 microphylla  rugosa LVA306 microphylla  rugosa LVA288 angustifolia LVA32 purpurea LVA68

S. S. S. S. S. S. S. S. S. S. S.

calaminthoides LVA82 calaminthoides LVA349 microphylla LVA85 microphylla LVA178 rugosa LVA148 rugosa LVA63 microphylla  rugosa LVA107 microphylla  rugosa LVA306 microphylla  rugosa LVA288 angustifolia LVA32 purpurea LVA68

S. S. S. S. S. S. S.

calaminthoides LVA82 calaminthoides LVA349 microphylla LVA85 microphylla LVA178 rugosa LVA148 rugosa LVA63 microphylla  rugosa LVA107

S. S. S. S.

microphylla  rugosa LVA306 microphylla  rugosa LVA288 angustifolia LVA32 purpurea LVA68

calaminthoides LVA82 calaminthoides LVA349 microphylla LVA85 microphylla LVA178 rugosa LVA148 rugosa LVA63 microphylla  rugosa LVA107 microphylla  rugosa LVA306 microphylla  rugosa LVA288 angustifolia LVA32 purpurea LVA68

5 1 1 1 0 1 1 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 0 0 0 1 0 0 0

0 0 0 0 0 0 0 0 0 0 1

1 1 0 0 0 1 0 0 ? 0 1

0 0 0 0 0 0 0 0 0 1 0

1 0 0 0 0 0 0 0 0 0 0

1 0 0 1 0 0 0 0 0 0 0

1 1 1 1 1 0 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 1

0 1 0 0 0 0 0 0 0 0 0

0 0 0 1 0 0 1 1 1 1 0

0 0 0 0 0 0 0 0 0 1 0

0 0 1 1 1 0 0 0 0 0 0

0 0 0 0 1 0 0 1 1 0 0

1 1 1 1 0 0 0 0 1 1 1

0 0 0 0 0 1 0 0 0 0 0

0 0 0 0 0 0 1 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 0 1 1 1

1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1

0 0 0 0 0 0 0

0 1 0 0 0 1 0

1 1 0 0 0 1 0

0 1 0 0 0 0 0

0 1 0 1 0 0 0

0 0 0 1 0 0 0

0 0 1 0 0 0 0

0 0 1 1 1 1 1 1 1 1 0

0 0 1 0 0 0 0 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1

0 1 0 1 1 0 0 0 0 1 0

1 1 0 0 1 0 0 0 0 1 0

1 1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 1

1 0 0 1 1 0 1 1 0 1 1

1 1 1 1 1 1 1 1 1 1 1

1 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 1

0 0 0 0 0 0 0 0 0 0 1

0 0 0 0 0 0 0 0 0 1 0

0 0 0 1 0 0 0 1 0 0 0

1 1 0 1 1 0 1 1 0 1 0

1 1 0 0 1 0 0 0 0 1 1

0 0 0 0 0 0 0 0 0 1 0

0 0 1 1 0 0 1 1 1 1 0

0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 1 0 0 0 0 0 0

1 1 1 1 1 1 1 0 0 0 1

1 1 1 1 1 1 1 1 1 1 1

1 1 0 0 1 1 1 1 0 1 1

0 0 0 0 1 1 0 1 0 1 1

0 1 0 0 0 0 0 0 0 0 0

1 1 1 1 0 1 1 1 1 0 1

0 0 0 0 0 0 0

0 0 1 0 1 0 1

1 0 0 0 0 0 0

0 0 0 0 0 0 0

1 1 0 0 1 0 1

1 0 0 0 0 0 0

0 1 0 0 0 0 0

0 0 0 0 0 0 1

1 0 0 0 1 0 0

0 0 1 0

1 1 0 0

0 0 1 1

0 0 0 1

1 0 1 1

0 0 0 1

0 0 1 0

0 0 0 0

0 0 1 1

1 0 0 1 1 1 0 0 1 0 1

0 0 0 0 1 0 0 0 0 0 0

1 1 0 0 0 0 0 0 0 0 0

0 0 0 1 0 0 1 0 0 0 0

1 1 1 1 1 1 1 1 ? 1 1

1 1 1 1 1 1 1 1 ? 1 1

1 0 1 0 0 0 1 0 ? 0 0

0 0 0 0 0 0 1 1 ? 1 0

1 0 0 0 0 0 0 0 ? 0 0

1 0 0 0 1 1 0 0 ? 1 0

1 1 0 0 1 0 0 0 ? 0 1

1 1 1 1 1 1 1 1 1 1 1 2 6 4 0 0 1 0 0 0 0 0 ? 0 0

2 0 0 0 0 0 1 0 0 1 0 0 0 7 0 1 0 0 0 0 0 0 0 0 0 0 1 2 0 1 1 1 0 1 1 1 1 0 1 1 1 7 0 0 0 0 0 0 0 0 2 0 0 0 1 2 2 0 0 0 0 0 1 1 0 1 ? 0 1

1 0 0 1 0 0 0 0 1 0 1

1 1 1 1 1 0 1 1 1 1 1

1 1 0 0 0 0 0 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1

1 1 0 1 0 0 0 1 0 1 0

1 1 0 1 1 1 1 1 1 1 1

0 0 0 0 1 0 1 0 0 0 0

1 1 0 0 0 0 1 1 0 1 0

1 1 0 1 0 0 1 1 0 1 1

0 0 0 0 0 0 0 0 0 0 1

0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 0 0 0 1 0 1 0

0 0 0 1 0 0 1 1 1 0 0

1 1 0 0 0 0 0 0 0 0 1

1 1 0 1 1 0 1 1 1 1 1

1 1 0 1 1 1 1 0 1 1 1

0 0 0 0 0 0 0 0 0 0 1

1 1 0 0 1 1 1 1 1 1 1

1 1 0 0 0 0 0 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 1 0 0 1

0 0 0 1 0 0 0 0 0 0 0

0 1 0 0 0 0 0 1 0 0 0

1 1 1 1 1 1 1 1 1 1 1

1 1 1 0 1 1 0 0 0 1 0

1 1 1 1 1 1 1 1 1 1 1

0 1 0 1 0 0 1 1 0 0 0

1 1 1 1 1 0 0

1 1 1 1 1 1 1

1 1 0 1 1 0 1

1 0 0 0 0 0 0

0 0 0 0 0 0 0

1 0 0 0 0 0 0

1 1 0 0 0 0 0

0 0 0 0 0 0 0

1 0 0 0 0 0 0

1 0 0 0

1 1 1 1

1 1 1 1

0 0 0 0

0 0 0 1

0 0 0 0

0 0 0 1

0 0 0 1

0 0 0 0

1 1 1 1 1 1 1 1 ? 1 1

1 1 1 1 1 1 1 0 ? 1 1

1 1 0 0 0 1 0 0 ? 1 1

1 1 1 1 1 1 1 1 ? 1 1

1 1 0 0 0 0 1 1 ? 0 1

1 1 0 0 0 1 0 1 ? 0 0

0 0 0 0 1 0 0 0 ? 1 0

0 0 0 0 0 0 0 0 ? 1 0

1 1 1 1 1 1 1 1 ? 1 1

3 0 0 0 0 0 0 0 0 0 0 1 0 8 0 1 1 1 0 0 1 0 0 1 1 1 1 3 0 0 0 1 1 0 0 0 0 0 0 1 1 8 0 0 0 0 1 0 0 0 3 0 0 0 0 2 3 0 0 1 1 1 0 1 1 1 ? 1 1

0 1 1 0 0 0 0 1 1 0 0

1 1 0 0 1 0 1 1 0 1 1

1 1 0 1 1 0 1 1 1 1 1

0 0 0 0 0 0 1 0 0 0 0

1 1 0 1 1 0 0 0 0 1 1

0 0 0 0 0 0 0 0 0 1 0

0 0 0 0 0 0 0 0 0 0 1

1 0 0 0 1 0 0 0 0 0 0

1 1 0 1 1 0 1 1 1 1 1

1 1 1 0 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 0 1 0

1 1 0 0 1 0 1 1 1 1 1

0 0 0 0 1 1 0 0 1 1 0

1 1 1 1 1 0 1 1 1 1 1

1 1 1 0 1 1 1 1 0 1 1

1 1 0 0 0 0 0 0 0 0 0

0 0 0 0 1 0 1 0 0 0 0

0 1 1 1 0 0 1 1 1 0 0

0 1 0 0 0 0 0 0 0 0 0

1 1 1 1 0 1 1 1 0 0 1

0 0 0 0 1 0 0 0 0 1 0

1 1 1 1 1 1 1 1 1 1 1

1 0 0 0 1 0 0 0 0 0 1

0 0 1 1 0 0 1 0 1 0 0

0 0 1 1 1 1 1 1 1 1 0

1 0 1 1 1 0 1 1 0 1 0

0 0 0 0 0 0 1

1 1 1 1 1 1 1

0 0 1 0 0 1 0

1 1 1 1 1 1 1

1 1 1 1 1 1 1

1 0 1 0 1 1 1

1 1 1 1 1 1 1

1 1 0 0 1 1 0

1 0 1 1 1 1 1

0 0 0 0

1 1 1 1

0 1 0 1

1 1 1 0

1 1 1 1

1 1 1 1

1 1 1 1

1 0 1 1

1 1 1 1

1 1 0 0 1 1 0 1 ? 1 1

0 0 0 0 0 0 0 0 ? 0 1

0 0 0 0 0 0 0 0 ? 0 1

1 0 0 1 0 0 0 0 ? 0 0

1 1 1 1 1 1 1 1 ? 1 1

0 0 0 0 0 1 1 0 ? 0 0

0 0 0 0 0 0 1 0 ? 0 0

1 1 0 1 0 1 1 0 ? 1 1

1 1 1 1 1 1 1 1 ? 1 1

4 0 1 0 0 1 0 0 0 0 0 0 0 9 0 1 1 0 0 1 0 0 0 0 1 1 1 4 0 1 1 1 1 1 1 0 1 1 1 1 1 9 0 1 0 0 0 0 0 0 4 0 0 0 0 2 4 0 1 1 1 1 1 1 1 1 ? 1 1

1 1 0 1 1 0 0 0 0 1 1

0 0 0 0 0 0 0 0 0 0 1

0 0 1 1 0 0 1 1 1 0 0

1 0 0 1 0 0 0 0 0 0 0

0 0 0 1 0 0 1 1 1 0 0

0 0 1 1 0 0 0 0 0 1 1

0 1 1 1 0 0 1 1 1 0 1

0 0 0 1 0 0 1 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1

1 1 1 0 1 1 1 1 1 1 1

0 0 0 0 0 0 1 0 1 0 0

1 1 0 0 1 1 1 1 1 1 0

0 0 0 0 1 0 1 0 0 0 1

0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 1 1 0 0 1 1 0

1 1 1 0 1 0 1 0 1 1 1

1 0 1 1 1 1 1 1 1 1 1

1 1 0 0 1 0 1 0 0 1 0

1 1 0 0 0 0 0 0 0 0 0

1 1 1 0 0 0 0 0 0 0 0

1 1 1 1 0 1 1 1 1 0 1

1 1 1 1 1 1 1 1 0 1 1

0 0 0 0 0 1 0 0 0 0 0

0 1 1 1 0 1 1 0 0 0 1

0 1 1 1 0 1 1 1 0 1 1

0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 0 1 0 0 0 0 0

1 0 0 0 0 1 0

0 0 1 1 0 0 0

0 0 0 0 0 0 0

1 0 1 1 0 1 1

0 0 1 0 0 1 1

1 0 0 0 0 0 0

0 0 0 0 0 0 0

1 1 0 0 0 0 0

1 1 1 1 1 1 1

0 0 1 1

1 1 1 0

0 0 0 1

1 1 1 1

1 1 1 1

0 0 0 1

1 0 0 1

0 0 0 0

1 1 1 1

1 1 1 1 1 1 1 1 ? 1 0

1 1 1 0 1 1 1 0 ? 1 1

0 0 0 1 0 0 0 1 ? 1 1

1 1 1 1 1 1 1 1 ? 1 1

0 0 0 0 1 0 0 0 ? 0 0

1 0 1 1 1 1 0 1 ? 1 0

0 0 0 0 1 0 0 0 ? 1 0

1 1 1 1 1 1 1 1 ? 1 1

0 0 1 0 0 0 0 0 ? 0 0

5 0 0 0 1 1 0 0 1 1 1 0 0 1 0 0 0 0 1 1 0 0 1 0 1 0 0 1 5 0 0 1 0 0 0 0 0 0 0 0 0 2 0 0 0 0 1 1 0 0 0 5 1 1 0 0 2 5 0 1 1 1 1 1 1 1 1 ? 1 1

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