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Systematics |
Department of Biology, P. O. Box 400328, University of Virginia, Charlottesville, Virginia 22904-4328 USA
Received for publication October 18, 2004. Accepted for publication April 29, 2005.
ABSTRACT
Chloroplast and nuclear DNA sequence variation among populations and species was used to examine the phylogenetic history and hybridization of the North American Houstonia lineage. The ancestral species in the lineage do not show evidence of hybridization; however, the more recently derived species in eastern North America contain a wide degree of morphological and genetic variation both within and among species. Furthermore, there is a clear association between hybridization and polyploidy in the Houstonia lineage, with all potential hybrids occurring among species that contain polyploid populations. This suggests that polyploidy may break down species barriers and allow hybridization among lineages. These results indirectly support speciation models that involve genetic incompatibilities among species arising from gene silencing or genomic reorganization.
Key Words: Houstonia • hybridization • molecular phylogenetics • polyploidy • Rubiaceae • speciation
It has become increasingly apparent that hybridization has played a vital role in the evolution of many plant lineages (Stebbins, 1950
, 1980
; Grant, 1981
; Arnold, 1997
; Rieseberg, 1991
, 1998
). Often, hybridization may be identified based on morphological or ecological intermediacy. However, some hybrid species may show novel or transgressive characters (e.g., Rieseberg et al., 1999
; Schwarzbach et al., 2001
; Lexer et al., 2003
). Often hybridizing species share sympatric habitats, chromosome numbers, pollinators, and/or flowering phenology. In other cases, what appear to be morphologically and ecologically distinct species may show signs of current or historic hybridization based on molecular data. However, not all species that overlap in ecology and habitat show the signature of hybridization. That is, some sympatric species may be morphologically and molecularly distinct (Goodwillie, 1999
). Furthermore, some apparent hybrid zones may be composed of single species expressing a great deal of phenotypic variation due either to plasticity or few genes of large effect (Kornfield and Koehn, 1975
; Sage and Selander, 1975
; Kornfield et al., 1982
; Meyer, 1987
). Molecular population studies may clarify these patterns of reticulate evolution, distinguishing between past and present gene flow among species as well as differentiating between within-species variation and hybridization (Soltis et al., 1996
; Kornkven et al., 1998
; Mayer and Soltis, 1999
; Olsen and Schaal, 1999
; Comes and Abbott, 2001
; Floyd, 2002
). Specifically, hybridizing species would be paraphyletic or polyphyletic across multiple genes or genomes, whereas incomplete lineage sorting might lead to monophyly of one marker while a second marker would result in unresolved lineages (Wendel and Doyle, 1998
).
While evidence of hybridization can be obtained with molecular data, the question of why some lineages are prone to hybridization while others are not remains elusive. One proposed mechanism that may influence the propensity for hybridization among lineages is polyploidy (Grant, 1981
; see Levin, 2002
). It is well established that hybridization can result in polyploidy (e.g., Ainouche et al., 2003; Goldman et al., 2004
); however, crossing and cytogenetic studies have demonstrated that polyploids often hybridize more than their diploid counterparts (e.g., Paeonia, Stebbins, 1939
; Zauschneria, Clausen et al., 1945
; Eriophyllum, Mooring, 2001
, and Espinoza and Moor, 2002
).
One explanation for this apparent correlation is that polyploidy may break down species incompatibilities, allowing for gene exchange between previously isolated species. This is consistent with speciation models such as the Bateson-Dobzhansky-Muller model (Bateson, 1909
; Dobzhansky, 1937
; Muller, 1942
), particularly as explained by the duplication, degeneration and complementation model (DDC; Force et al., 1999
; Lynch and Force, 2000
; also see Werth and Windham, 1991
), as well as the genomic reorganization model (e.g., Wilson et al., 1974
, 1975
; Bush et al., 1977
). In all three models, mutations accumulate between species, which, when brought together in a hybrid offspring, cause inviability and/or infertility. However, doubling of the genome would negate the incompatibility by providing the hybrid offspring with functional genomes from both parents. Hence, it would be expected that hybridization would be more successful between recent polyploid species than among diploid species.
This study uses molecular analyses to investigate the relationship between polyploidy and apparent hybridization in the North American Houstonia L. (Rubiaceae) lineage, including Stenaria (Raf.) Terrell (Fig. 1). This lineage is composed of native herbs that vary in chromosome number and ploidy levels (Fig. 1; Lewis, 1962
; Terrell, 1996
; Church, 2003
). The basal members of the lineage are generally morphologically distinct species, while the more recently derived species overlap in both range and morphology (Terrell, 1996
). The most recent taxonomic treatments recognize 20 species of Houstonia (Terrell, 1996
) and five species of Stenaria (Terrell, 2001
) in North America. Species are distributed throughout much of North America, from Mexico, through the southern United States, and into southeastern Canada (Terrell, 1991
, 1996
, 2001
). Detailed morphological and cytological studies of this lineage have identified several morphologically intermediate polyploids (H. caerulea x H. serpyllifolia, H. longifolia x H. purpurea) (Lewis and Terrell, 1962
). However, crossing studies of these same species showed reduced seed set (H. serpyllifolia x H. caerulea) or small, inviable seeds (H. longifolia x H. purpurea) in intraspecific crosses of populations of unknown ploidy (Beliveau and Wyatt, 1999
). The goal of the present study is to determine which species are distinct, if the morphological variation among species is due to hybridization, and whether or not hybridization is limited to polyploid lineages. Specifically, we sampled multiple populations per species from throughout the range of the lineage to determine if there was any molecular evidence of hybridization among species.
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Plant samples
Leaf tissue was collected from 17 species and 74 populations of Houstonia and Stenaria (Appendix). The genus Houstonia includes four sections: Houstonia, Mullera, Amphiotis, and Ericotis. Sampling included all five species in Houstonia sect. Houstonia (H. caerulea, H. micrantha, H. procumbens, H. pusilla, and H. serpyllifolia), the single species in section Mullera (H. rosea), the four species and three varieties in section Amphiotis (H. canadensis, H. longifolia, H. ouachitana, H. purpurea var. calycosa, H. purpurea var. montana, H. purpurea var. purpurea), and five species in section Ericotis (H. acerosa, H. humifusa, H. palmeri, H. rubra, H. wrightii). Six species in Houstonia section Ericotis were not included due to their rarity in the USA. Population sampling occurred throughout the range of these species in the United States. Sampling was most intense for those species with high levels of morphological variation, all of which occur in eastern North America. Population sampling did not occur in Mexico or further south unless herbarium material was available. Field-collected leaf tissue was dried immediately in silica gel or frozen. Collections are listed in the Appendix by county and state for each species.
DNA isolation, amplification, and sequencing
Total DNA was isolated from leaf tissue using DNeasy plant mini kits as specified by the manufacturer (Qiagen, Valencia, California, USA). The internal transcribed spacer (ITS) regions 1 and 2 of the 18S–26S nuclear ribosomal DNA, and the trnL intron and trnS–trnG intergenic spacer of the chloroplast DNA genome were amplified via the polymerase chain reaction (PCR). Primers "its2" and "its5" were used to amplify the ITS1 region and "its3" and "its4" were used to amplify ITS2 (White et al., 1990
) using standard PCR techniques (see Church, 2003
). The trnL intron was amplified using Taberlet's primer pairs C and D (Taberlet et al., 1991
). The trnG–trnS intergenic spacer was amplified using primers trnS (GCU) and trnG (UCC) described by Hamilton (1999)
.
PCR products were cleaned and sequenced as outlined in Church (2003)
. All fragments were sequenced in both directions to verify all base calls. Ambiguous base calls were verified manually, and consensus sequences were assembled using the computer program Sequencher (Gene Codes Corp., Ann Arbor, Michigan, USA). Final verified consensus sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/; ITS1, DQ012667–DQ012734; ITS2, DQ012735–DQ012802; trnL, DQ012803–DQ012875; trnS–trnG, DQ012876–DQ012934). Alignments among sequences were made using the computer software package GCG (Wisconsin Package, version 10.0, Genetics Computer Group, Madison, Wisconsin, USA) and corrected manually.
Phylogenetic analysis
A South American member of the Hedyotidae, Oldenlandiopsis callitrichoides, was designated as the outgroup in each analysis. This species has been shown to be distinct from the North American Houstonia–Stenaria lineage based on previous molecular analyses (Church, 2003
) as well as morphological features (Terrell, 1991
).
Phylogenetic analyses were performed independently for the nuclear and chloroplast DNA data sets. To minimize analysis times, phylogenies were reconstructed separately for each of three major clades within the lineage (sections Houstonia and Mullera, section Amphiotis, section Ericotis plus Stenaria) based on previous molecular analyses (Church, 2003
). A combined analysis was also performed using a single sample from each species (except the highly variable H. purpurea and H. longifolia). The best models of evolution for the combined data set, each genome (nuclear and chloroplast DNA, all clades), and each clade per genome were determined using a hierarchical approach starting with the most complex model and comparing to ever-simpler models of evolution (see Church, 2003
). Maximum likelihood methods incorporating these models were then used to reconstruct phylogenies. To determine support for the tree topologies, bootstrapping was performed with 1000 replicates using the same maximum likelihood models. All analyses were performed in PAUP* version 4.0b8 (Swofford, 1999
).
Genetic distances within and among species were calculated using the program MEGA v. 2.1 (Kumar et al., 2001
). Distance calculations were corrected for the model of evolution for each sequence region. Given that not all models of evolution are available for use in MEGA, the best fit available model was used to infer genetic distances. Distances were calculated within and among all species as well as varieties.
RESULTS
Sequence variation
The chloroplast DNA trnL intron and the trnS–trnG intergenic spacer regions were sequenced in 73 individuals (H. wrightii 2 did not amplify), including four herbarium samples. The sequences resulted in an alignment of 973 base pairs, 238 of which were variable and 117 of these were parsimony informative. The trnS–trnG intergenic spacer was more variable than the trnL intron. The trnS–trnG spacer accounted for 476 base pairs, 57 distinct haplotypes (vs. 49 for trnL), 131 variable sites, and 65 parsimony informative characters. All subsequent analyses were performed using the combined trnS–trnG and trnL data.
Initially, the entire chloroplast data set was analyzed using maximum likelihood and the best model of evolution, which corresponded to a general time reversible model with gamma-distributed rate variation (shape parameter = 1.71) and invariable sites estimated at 0.322. This model was used to construct one tree, which was consistent with the genus level trees in previous phylogenetic analyses (Church, 2003
; results consistent with Fig. 1 on a per species level). Subsequently, the data were partitioned into the three major lineages (section Houstonia plus section Mullera, section Amphiotis, and section Ericotis plus Stenaria), and the most appropriate model of evolution was determined for each clade. This partitioning allowed for bootstrap analyses to be performed on each data set in a shorter amount of time. For each of the three clades, a submodel of the general time reversible model with invariant sites estimated (GTR + I), and rate distribution equal across sites (except in the section Houstonia, GTR + I + G) was determined to be the most appropriate model. Maximum likelihood trees were then reconstructed for each clade (Figs. 2a, 3a, 4a), and bootstrap analyses were performed.
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). The best model of evolution for this analysis corresponded to the Hasegawa-Kishino-Yano model (Hasegawa et al., 1985
) with gamma-distributed rate variation (shape parameter = 0.89), a transition/transversion ratio of 1.52, and 0 invariant sites. The resulting tree had high bootstrap support for two clades (sections Houstonia plus Mullera and section Amphiotis) with section Ericotis and the genus Stenaria being part of a basal grade. Subsequently, trees were reconstructed using maximum-likelihood for each of the three major lineages (Figs. 2b, 3b, 4b). The most appropriate models of evolution for each lineage corresponded to a submodel of the general time reversible model with invariant sites (GTR + I) estimated and rate distribution equal across sites.
The combined gene analysis included 19 samples (one per species except for H. longifolia and H. purpurea var. purpurea) for which both nuclear and chloroplast data were available. The topology of the tree is consistent with previous analyses (Church, 2003
) with the addition of Houstonia rosea (Fig. 1). The model of evolution corresponded to a submodel of the general time reversible with gamma-distributed rate variation. This analysis supports the separation of the data into three major clades (sections Houstonia and Mullera, section Amphiotis, section Ericotis plus Stenaria).
Sections Houstonia and Mullera
Four of the five species in Houstonia section Houstonia (H. caerulea, H. micrantha, H. pusilla, H. serpyllifolia) are very similar to one another morphologically (Terrell, 1996
) and genetically. The average genetic distance among these four species (0.016) is less than one-half that seen among species in the section Ericotis (0.05; H. rubra, H. humifusa, H. wrightii, H. acerosa, H. palmeri) (see Tables 1, 2). However, the morphological variation among species in the section Houstonia is also much less (Terrell, 1996
). Based on the nuclear data, two individuals of these species share a single haplotype (H. caerulea 7 and H. serpyllifolia 1; Fig. 2b). Phylogenies reconstructed from both the chloroplast DNA and nuclear data sets show moderate to high bootstrap support for a few individuals of both H. caerulea and H. serpyllifolia belonging to the opposite species clade (Fig. 2).
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The fifth species in the section Houstonia, H. procumbens, is quite distinct from the other four species based on molecular data as well as morphological (Terrell, 1996
) and cytological (Lewis, 1962
) data. Genetically, the average distance between this species and the remaining four in the section is about twice that between any one of the four previously mentioned species in this section (Tables 1, 2). These data, as well as the morphological and cytological data, suggest that this species is more similar to H. rosea (Fig. 2), which is the only species in the section Mullera.
The individual samples of H. rosea form a well-supported monophyletic clade in both the nuclear and chloroplast phylogenies (Fig. 2). This species is clearly distinct from the remaining Houstonia species and does not appear to be hybridizing with any other species.
Section Amphiotis
Most species within the section Amphiotis are polyphyletic based on both the nuclear and chloroplast data sets (Fig. 3). Within the chloroplast data, there is some sorting of lineages into geographic regions; however, these clades do not have substantial bootstrap support (Fig. 3a). The nuclear data show more diversity both within and among species (Table 2). Based on the nuclear data, the only distinct species appear to be H. ouachitana and H. canadensis (Fig. 3b). The three sampled populations of H. canadensis from Ohio and Virginia fall into one distinct clade (bootstrap 65%). The three individuals of H. ouachitana share one haplotype and form a separate distinct clade (bootstrap 81%).
The remaining two species in the section Amphiotis do not form distinct clades based on either chloroplast or nuclear data. Accessions of the three varieties of H. purpurea are found dispersed throughout both the nuclear and chloroplast trees (Fig. 3). Based on the chloroplast data, there is very little divergence among the three varieties (Fig. 3a). However, the nuclear data are more variable and result in some structure among the varieties. Based on the ITS tree, the three varieties of H. purpurea do not appear to be closely allied with one another (Fig. 3b). The most common variety, H. purpurea var. purpurea, was collected from North Carolina, Virginia, and Kentucky. These collections do not cluster together in the tree and hence no H. purpurea var. purpurea clade can be defined. Only one individual of H. purpurea var. calycosa was sampled. This collection, from Tennessee, appears most similar to the H. purpurea var. purpurea sample from Kentucky and the H. ouachitana samples, although there is no bootstrap support for this relationship. Houstonia purpurea var. montana is the only variety of H. purpurea that forms a distinct clade based on the nuclear data (Fig. 3b, bootstrap 100%).
The last species in the section Amphiotis, H. longifolia, is genetically quite variable. Although no varieties are named for this species, there are five population groups defined by Terrell (1996)
. These groups are separated based on morphological and geographic differences. The chloroplast sequence data do not differentiate these groups, and H. longifolia samples appear to be polyphyletic with H. purpurea (Fig. 3a). The nuclear sequence data provide greater resolution of some groups within H. longifolia (Fig. 3b). For instance, samples from the Northern group (collected in Vermont and Massachusetts), defined as the Great Lakes and northeastern USA, form a distinct clade (bootstrap 92%) based on the nuclear data set. The remaining samples, belonging geographically and morphologically to the tenuifolia and Appalachian groups, are not monophyletic based on the nuclear data. The polyphyly of H. longifolia and H. purpurea makes it difficult to determine if there is any genetic structuring to these species. However, based on an AMOVA, nearly 80% of the genetic variance occurred among regions (P < 0.001); therefore, there is some degree of regional variation within this species.
Section Ericotis and genus Stenaria
The species in the section Ericotis are closely allied with the genus Stenaria (Church, 2003
); however, the species in these lineages are morphologically and genetically distinct from one another and do not appear to hybridize (Terrell, 1996
; S. Church, personal observation). Within species genetic diversity in these lineages is low (Tables 1, 2), with multiple population samples forming monophyletic clades for most species. The distinctness of these species can be seen in the branch lengths as well as the high bootstrap values of the various species clades in the trees (Fig. 4). The exceptions to this include Stenaria nigricans and S. butterwickiae (Fig. 4a, S. nigricans forms two distinct clades, one of which includes S. butterwickiae). Also, H. wrightii and H. acerosa do not form distinct clades based on chloroplast and nuclear data, respectively (Fig. 4). However, the haplotypes of each of these species are distinct and do not fall within other species clades.
DISCUSSION
This study examines the phylogenetic history of the North American Houstonia lineage by sampling at the population level. The lineage can be divided into three distinct clades corresponding to (1) Houstonia sect. Ericotis and related Stenaria, (2) Houstonia sects. Houstonia and Mullera, and (3) Houstonia sect. Amphiotis. The species in and closely allied to the Houstonia section Ericotis (clade 1) are distinct, both morphologically and genetically. However, the remaining species, sections Houstonia and Mullera, and section Amphiotis, contain a wide degree of morphological and genetic variation both within and among species. Not surprisingly, the genetically similar species share morphological similarities. The result is that, at a minimum, four species in two sections are polyphyletic based on both the nuclear and chloroplast data. However, if this were due to incomplete lineage sorting, the nuclear and chloroplast regions, which evolve at different rates, might be incongruent, with species sorting into distinct lineages based on one or the other dataset. If hybridization were the cause of these shared haplotypes, it is more likely that both the nuclear and chloroplast data would show this pattern of shared haplotypes across species.
The species in the section Ericotis (H. acerosa, H. humifusa, H. palmeri, H. rubra, H. wrightii) and the genus Stenaria (S. butterwickiae, S. nigricans) are much more distinct from one another than most other species of Houstonia. The genetic distinctness of these species is not surprising based on the amount of morphological divergence among these species (see Terrell, 1996
). Floral and leaf morphology as well as habit vary from compact plants with reduced leaves and few, bright pink flowers (H. rubra) to large upright branching plants with over 50 flowers per individual (S. nigricans). Only S. nigricans and S. butterwickiae have any inconsistencies among species in the chloroplast phylogeny. The placement of S. butterwickiae within one of two S. nigricans clades cannot be confirmed based on the absence of S. butterwickiae from the nuclear data set. Among the remaining species, there is no apparent hybridization based on either morphological (Terrell 1996
) or genetic characters. This may be due to prezygotic isolating factors such as different flowering times (Terrell 1996
) or pollinators, as well as postzygotic factors that prevent the formation of viable hybrids. In some cases, differential chromosome numbers may separate species (Stenaria spp.: x = 9, 10; Houstonia spp.: x = 11). Polyploidy is not known to occur in any species in this group (Houstonia sect. Ericotis) other than H. acerosa (Lewis, 1962
); therefore varying ploidy levels also cannot account for the isolation among Houstonia species in the section. Alternatively, isolation among species with the same chromosome numbers may be due to high levels of divergence among species. The North American species of Houstonia and Stenaria are thought to have diverged from ancestral species in subtropical and temperate Mexico (see Lewis, 1962
, 1965
; Church, 2003
). The radiation of these species occurred as they spread north and east into North America. This suggests that the species that occur in the southwestern USA and Mexico (Houstonia sect. Ericotis) may be the ancestral species in the North American lineage, allowing more time for genetic isolation to have developed among species.
The more recently derived section Houstonia is composed of both distinct and potentially hybridizing species. Individuals of two species, H. caerulea and H. serpyllifolia, share both nuclear and chloroplast DNA haplotypes. Houstonia serpyllifolia is a polyploid species, while H. caerulea is known to have both diploid and polyploid populations. This variation in ploidy level might prevent hybridization between certain populations of these two species. However, these two species intergrade morphologically in the southern Appalachian Mountains, where tetraploid H. caerulea populations have been found (Lewis and Terrell, 1962
; Lewis and Oliver, 1970
; Terrell, 1996
). In this region, two H. caerulea individuals, from South Carolina and Virginia, have haplotypes identical to H. serpyllifolia individuals from the same regions. The observation of morphological intermediacy (Terrell, 1996
; S. Church, personal observation) as well as the nuclear and chloroplast DNA with similar patterns of intergradation, suggest that hybridization, not incomplete lineage sorting, may be occurring between these species.
The remaining four species of Houstonia sections Houstonia and Mullera are distinct from one another based on chloroplast data. However, H. pusilla and H. micrantha individuals share the same nuclear ITS haplotypes. Houstonia pusilla is distributed throughout eastern North America and is strictly diploid, while H. micrantha is restricted to the Mississippi River Basin and is strictly tetraploid. If these shared haplotypes were the result of hybridization, the same pattern of shared haplotypes would likely be seen in the chloroplast data. However, H. micrantha is distinct from H. pusilla based on the chloroplast data. Furthermore, the difference in ploidy level makes hybridization unlikely. Thus, these shared haplotypes are likely the result of incomplete lineage sorting.
The last two species in the clade, H. procumbens and H. rosea, are genetically quite distinct from the other species in the group. These two species do not appear to hybridize with any other species based on both molecular and morphological data. Currently, H. rosea is placed in a separate section within Houstonia (section Mullera); however, results from both the chloroplast and nuclear data suggest that H. procumbens and H. rosea may be more closely related than previous morphological evidence has suggested. These results are consistent with chromosome numbers in the genus, which have been shown to be an important correlate of phylogenetic relatedness (Lewis, 1962
; Church, 2003
). Both H. procumbens (tetraploid) and H. rosea (diploid) have a basic chromosome number of x = 7, while the other four species in the group have basic chromosome numbers of x = 8 (Lewis, 1962
). Therefore, the placement of H. procumbens in section Houstonia and H. rosea in section Mullera may not accurately reflect the relationships among these species. A more accurate taxonomic placement would include H. rosea in section Houstonia, sister to H. procumbens.
The remaining species in the Houstonia lineage, section Amphiotis, have even more complex patterns of apparent hybridization or incomplete lineage sorting based on the molecular data. There is very little resolution within this group based on the chloroplast data (Fig. 3a). This may be due to hybridization, in which these haplotypes are being transferred freely between species, or incomplete lineage sorting, in which these haplotypes are common to all species due to their presence in a recent common ancestor (Wendel and Doyle, 1998
). However, there are several well-resolved clades in the nuclear-DNA-based tree (Fig. 3b). Specifically, the nuclear data show three species-specific clades, H. ouachitana and H. purpurea var. montana are diploids with restricted ranges. The other clade contains H. canadensis, which is more widely distributed and not strictly diploid. Houstonia ouachitana is limited in distribution to five counties in Arkansas and two counties in neighboring Oklahoma (Terrell, 1996
), near the westernmost limits of H. purpurea and H. longifolia (Terrell, 1996
). The range of this species, along with its strict diploidy, may limit the opportunity and success of hybridization. Similarly, H. purpurea var. montana is also strictly diploid and restricted to a few mountain tops in North Carolina and Tennessee. The distinction of H. purpurea var. montana (the Roan Mountain bluet) from the other H. purpurea varieties might merit species status for this federally endangered plant. The uniqueness of the Roan Mountain bluet is further supported by previous crossing studies in which H. purpurea var. montana (diploid) crossed with H. purpurea var. purpurea (diploid or polyploid) failed to produce fertile seeds (Yelton, 1974
; but see Terrell, 1978
). Again, this lack of hybridization may be due to lack of other diploid populations in the same narrow range. Alternatively, H. canadensis forms a monophyletic clade based on ITS data, contains both diploid and tetraploid populations, and is widely distributed west of the Appalachians throughout the Tennessee Valley, Kentucky and into southern Ohio and Indiana. While the Ohio populations collected for this study appeared to have some morphological similarities with H. longifolia, the nuclear data do not support the idea of frequent hybridization between these species. Hybridizing species should show similar patterns of incongruence between species designations and clades in both nuclear and chloroplast data, whereas incomplete lineage sorting would lead to the observed pattern of resolution in one marker (these data, nuclear DNA) with a second marker (these data, cpDNA) resulting in unresolved lineages (Wendel and Doyle, 1998
).
While three species/varieties may not be hybridizing, the common H. purpurea varieties, var. purpurea and var. calycosa, along with H. longifolia, appear to be polyphyletic based on both nuclear and chloroplast data. Given the differential rate of evolution between the nuclear and chloroplast regions, the fact that neither marker can resolve these species into distinct lineages suggests that hybridization may be the underlying cause of the lack of resolution of these species. This is supported by field observations of morphological variation both within and among populations of these species (S. Church, personal observation). In particular, populations of these species overlap and intergrade throughout much of their range in the southern Appalachians and the Midwest and vary in ploidy level within and among populations (Lewis and Terrell, 1962
). These are the same population regions that are polyphyletic throughout both the nuclear and chloroplast trees. The only populations of these species that do not overlap in range with one another are those of H. longifolia in northeastern North America. In this region, H. longifolia is morphologically distinct. Collections from these northern populations form a monophyletic clade in the nuclear phylogeny (H. longifolia 1, 12, 13 from Massachusetts and Vermont). Previous samples from the same Massachusetts population are known to be diploid (Lewis and Terrell, 1962
). Thus the morphological, geographic and molecular data suggest that these northeastern populations of H. longifolia are not hybridizing with other species.
Polyploidy and hybridization
These data suggest that several species of Houstonia may be hybridizing freely with closely related lineages, while other species remain phylogenetically distinct. One clear correlate with this apparent dichotomy in hybridization is polyploidy. It has been proposed that polyploidy can break down species barriers and allow hybridization among taxa that are isolated in their diploid form (Stebbins, 1950
; Grant, 1981
). Polyploidy occurs in eight species (some polyploid species, others polyploid races) within the Houstonia lineage (H. caerulea, H. serpyllifolia, H. micrantha, H. procumbens, H. longifolia, H. purpurea, H. canadensis, H. acerosa; see Fig. 1). The four species for which there is both morphological and molecular evidence of hybridization are all polyploid (H. caerulea, H. serpyllifolia, H. longifolia, H. purpurea). In species with both polyploid and diploid populations (such as H. purpurea and H. longifolia), only the diploid lineages show no signs of hybridization at the molecular level (H. purpurea var. montana and the northeastern populations of H. longifolia). Another closely related species that does not show signs of hybridization is also strictly diploid (H. ouachitana; Lewis and Terrell, 1962
). These results are consistent with speciation models in which polyploidy breaks down species barriers by providing hybrids with two fully functional genomes.
The remaining four species in the genus that are known to be polyploid are H. canadensis, H. procumbens, H. micrantha, and H. acerosa. The latter species contains both diploid and polyploid populations and was not found to hybridize with any other recognized species, although B. L. Turner recognizes four morphogeographical varieties of this species, which show some degree of intergradation (Turner, 1995
; but see Terrell, 1996
). Houstonia micrantha (n = 16) and H. procumbens (n = 14) are both strictly polyploid species (Lewis and Moore, 1959
); however, their closest relatives, H. pusilla (n = 8) and H. rosea (n = 7), respectively, are strictly diploid. This inequality of chromosome numbers would likely prevent hybridization between these species. The last species with polyploid populations, H. canadensis, does not appear to hybridize with any other species, based on the nuclear data. However, morphological intermediates between this species and H. purpurea var. purpurea are known to occur, but the ploidy levels of the three populations sampled for the current study are not known.
Conclusions
The three major groups within the Houstonia lineage grade from well-resolved taxa to a mixture of poorly resolved and well-resolved lineages within a complex of related species. It appears that species boundaries are very distinct among ancestral lineages in Houstonia but less so among the more recently derived lineages. The recently derived lineages show some signs of hybridization as well as incomplete lineage sorting due to their recent radiation. There is clearly an association between hybridization and polyploidy in these species. While not all polyploids hybridize, all potential hybrids occur among species that contain polyploid populations. This suggests that polyploidy may break down species barriers to allow hybridization among lineages, indirectly supporting speciation models involving genetic incompatibilities among species due to gene silencing or genomic reorganization.
FOOTNOTES
The authors thank J. Murray, H. Wilbur, L. Galloway, and D. Carr, S. Kephart, and one anonymous reviewer for comments on the manuscript. We are very grateful to W. Lewis and E. E. Terrell for many thoughtful discussions throughout the completion of this work. We are also grateful to the following people for help in collecting leaf tissue: M. Brock, D. Church, M. Couvillion, E. Dale, J. Donaldson, S. Emery, C. & J. Freeman, R. Haynes, M. Heuhsen, J. Miller, J. Nelson, D. Nickrent, R. Paderewski, S. Vanderpool, L. Wolfe, and G. Yatskievych. We also thank the curators at the Missouri Botanical Gardens for allowing S. A. C. to examine and collect tissue from their specimens. This work was supported in part by a Miller-Jeffress Foundation grant to D. R. T. and two grants from the University of Virginia Society of Fellows to S. A. C. 
2 Author for correspondence (e-mail: schurch{at}gwu.edu
) current address: George Washington University, Department of Biology, 340 Lisner Hall, 2023 G St. NW, Washington, D.C. 20052 USA
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