Inferring Species Networks from Gene Trees in High-Polyploid North American and Hawaiian Violets (Viola, Violaceae)

Keywords: Allopolyploidy, BEAST, homoeolog loss, low-copy nuclear gene, PADRE, single-molecule PCR, species network, Viola

Plant Material, In vitro Cloning of Homoeologs, PCR, and Sequencing

A total of 58 accessions representing 51 species and subspecies was sequenced for the low-copy nuclear gene GPI (glucose-6-phosphate isomerase; Table 2). Allexis batangae served as the outgroup (Tokuoka 2008). Each of the five high-polyploid violet lineages was represented by one accession, except for Boreali-Americanae, which was represented by two species (V. sagittata and the reportedly chromosomally divergent V. clauseniana; Clausen 1964). All major morphological groups occurring in North America were sampled, with dense sampling especially within the lineages putatively sister to the high polyploids (see Results section). DNA was extracted using a CTAB extraction protocol (Doyle and Doyle 1987). In most cases, DNA working solutions were made by diluting extractions 1:20, of which 1 μL was used per PCR reaction. For “difficult” DNA preparations, the obtained stock DNA solution was further cleaned using the DNeasy Blood & Tissue Kit (Qiagen, Düsseldorf, Germany), following the manufacturer's guidelines except omitting the first two steps.

Viola-specific primers (Table 3) were designed for a locus corresponding to exon 12 to exon 18 of the GPI gene in Arabidopsis (AB007647, NM123638) and with a length of ca. 2000 base pairs. In order to increase the chance of discovering all homoeologs, the GPI locus was amplified in two separate PCR reactions covering exon 12 to exon 16 (PCR1) and exon 13 to exon 18 (PCR2), respectively. Different approaches were used to amplify homoeologs in diploids, tetraploids, and high polyploids (Table 3). For diploids, PCR1 and PCR2 were performed using a single set of general primers each, and for tetraploids (sections Plagiostigma and Viola) using specific primers for their two homoeologs, CHAM and MELVIO. For the high-polyploid species, homoeologs were isolated in vitro by single molecule (sm) PCR at limiting dilution, where DNA concentration was so low that most of the reactions (approximately 60%) by pure chance did not receive any template molecules at all and thus produced no PCR product (see Kraytsberg and Khrapko 2005). Under such conditions, the positive reactions were most likely to have been initiated by a single template molecule. The smPCR protocol was performed in three steps. First, heteroduplex DNA was removed from the DNA template working solutions by 1 min denaturation at 85 °C followed by renaturation by slow cooling to room temperature over approximately 30 min. Second, optimal dilution of the DNA template was estimated in a PCR trial on a dilution series consisting of a 192 μL master mix divided into 16 reactions, in which the DNA template in reaction_{n + 1} was diluted 2:3 compared with reaction_n; hence, reaction₁₆ was 657 times more diluted than reaction₁. Finally, the highest DNA template dilution yielding a product, often a 1:25 to 1:50 dilution compared with reaction₁, was considered optimal and used for smPCR. smPCR was typically conducted with 32 replicates, that is, 384 μL master mix divided into 32 tubes. Forty PCR cycles generally amplified strong smPCR products. PCR products were visualized by electrophoresis on 1% agarose gels. Successful PCR products were diluted 1:10 and cycle sequenced directly using PCR primers with the BigDye 3.1 sequencing Kit (Applied Biosystems, Foster City, CA), then processed on a 3730 ABI DNA analyser (Applied Biosystems).

Phylogenetic Reconstruction

All sequence chromatograms were edited manually and subsequently aligned with MUSCLE, as implemented in Geneious version 5.3.5 under standard settings. Alignments were further optimized manually in order to ensure that all putatively homologous indels (i.e., having identical length and position, and > 80% sequence similarity for putative insertions) were consistently aligned. Indel characters were coded by using Simple indel-coding (Simmons and Ochoterena 2000) as implemented in the SeqState software (Müller 2005), except for length variation in polynucleotide motifs (one poly-A and one poly-AT in trnL-F); these were not coded. The final GPI alignment was 3602 bp long and contained 290 coded indels, of which 684 and 116 were parsimony informative, respectively (postedited from a MUSCLE-generated alignment that was 3569 bp long and contained 307 indels, of which 707 and 116 were parsimony informative). The final trnL-F alignment was 1309 bp long and contained 68 coded indels, of which 74 and 15 were parsimony-informative, respectively (post-edited from a MUSCLE-generated alignment that was 1145 bp long and contained 75 indels, of which 85 and 28 were parsimony informative). The GPI and trnL-F phylogenies were constructed using maximum parsimony (MP) and maximum likelihood (ML). MP analysis was performed with Tree analysis using New Technology (TNT) version 1.1 (Goloboff et al. 2008) with “Traditional Search,” Tree Bisection-Reconnection branch swapping, 10 replicates (sequence addition), and 10 trees saved per replication in effect. The consistency index (CI) and retention index (RI) were calculated. ML analysis was performed with Treefinder version of March 2008 (Jobb et al. 2004). Nucleotide substitution models for the exon and intron partitions were proposed by Treefinder based on the AICc model selection criterion. For GPI, three data partitions were defined and analyzed with different nucleotide substitution models, each with four rate categories: exons with HKY+Γ (A .284, C .198, G .207, T .311; TC = AG .354, TA = TG = CA = CG .073; alpha .366), introns with GTR+Γ (A .255, C .167, G .188, T .390; TC .256, TA .085, TG .099, CA .105, CG .130, AG .325; alpha 2.250), and coded indels with JC. The trnL-F data were analysed in the same way as GPI except that the entire nucleotide sequence was analysed with GTR+Γ (A .370, C .159, G .166, T .305; TC .331, TA .032, TG .146, CA .129, CG .120, AG .241; alpha .429) and, as for GPI, coded indels with JC. ML and MP bootstrapping were performed with the same settings as above using 1000 replicates. The GPI and trnL-F alignments and tree files are deposited at Dryad (doi:10.5061/dryad.68722) and TreeBASE (study number S11380).

Estimation of Divergence Times

We calibrated the phylogeny with four Viola seed fossils using a Bayesian relaxed clock as implemented in BEAST 1.5.4 (Drummond et al. 2006, Drummond and Rambaut 2007), from a reduced data set containing only species of the CHAM and MELVIO clades; CHAM and MELVIO denote the homoeologs present in the tetraploid lineages (sections) Melanium, Plagiostigma, and Viola of the northern hemisphere (cf. Marcussen et al. 2010). The data matrix was partitioned with respect to exon, intron, and coded indels, using the substitution models HKY+Γ, GTR+Γ, and the simple binary substitution model, respectively. The analyses used a speciation model that followed a Yule tree prior, with rate variation across branches uncorrelated and lognormally distributed. One Markov chain Monte Carlo (MCMC) chain was run for 37.5 million generations, with parameters sampled every 1000 step. A visually determined burn-in of 4 million generations was discarded. Effective sample sizes for all estimated parameters and node ages were well above 200, as recommended. Additionally, two shorter MCMC chains were run for 5 million generations to control for similar convergence to the long chain; these were not used further.

Seeds of Viola are easy to identify to genus, owing to a characteristic transverse cellular pattern of the inner surface of the testa (Van der Burgh 1987). Comprehensive morphological study of seeds across the entire genus has not been published to date, although Gil-ad (1997) used seed micromorphology in part to recognize distinct species and infer hybridization in Boreali-Americanae. At least 15 Viola morphospecies are known from the Eurasian Neogene (Dorofeev 1963, Mai 2001, Arbuzova 2005, Nikitin 2007). At least some of these can easily be assigned to currently recognized infrageneric groups based on unique traits and, in certain instances, show affinities with particular extant species. Four seed fossils were used for calibration of the phylogeny. Calibration 1 (appearance of the genus in the northern hemisphere) is linked to the almost synchronous appearance of several Viola seed morphotypes in Lower Miocene sediments (Dorofeev 1963) and the basal polytomy in the CHAM and MELVIO subclades, indicating rapid radiation (see Results section). The oldest fossil flora containing violet seeds, from Austria, has been dated to 17–18 Ma (Kovar-Eder et al. 2001). We used a lognormal prior probability that the basal node in either of the CHAM and MELVIO subclades was at least 18 myr old, with a 95% confidence interval of 5 myr (offset = 18; log(mean) = log(SD) = 0.6). Calibration 2 corresponded to the age of subsection Rostratae, which from the Upper Miocene (13.7–5.3 Ma) appears with several seed morphotypes or species (Van der Burgh 1987; Geissert et al. 1990). The oldest and most accurately dated seed fossil, attributed to the extant “V. canina” on morphological grounds (the irregularly folded testa), has been described from western Germany (Van der Burgh 1987) and dated to 9–10 myr old (Schäfer et al. 2004). We applied a lognormal prior probability that subsection Rostratae (node basal to V. laricicola, V. mirabilis, V. stagnina, V. striata, V. uliginosa) is at least 10 myr old, with a 95% confidence interval of 2 myr (offset = 10; log(mean) = 0; log(SD) = 0.4). Calibration 3 (age of the Eurasian subsection Viola) is based on the occurrence of three fossil seed species (Viola “sp. 1,” “sp. 2,” “sp. 3”) in Russian Pliocene (5.2–2.6 Ma) sediments (Arbuzova 2005). We used a lognormal prior probability that subsection Viola (node basal to V. chelmea, V. collina, V. hirta) is at least 5.2 myr old, with a 95% confidence interval of 2 myr (offset = 10; log(mean) = 0; log(SD) = 0.4). Calibration 4 corresponded to the appearance of the allo-octoploid V. palustris in Europe. European V. palustris is an allopolyploid of V. epipsila subsp. epipsila and V. pallens (data herein), and fossil seeds of this bog species, identifiable by their luster and splitting seed coats, are common in European Tertiary and Quartenary sediments (Van der Burgh 1983). Its oldest fossil, from Lower Pliocene (5.3–3.6 Ma) (Van der Burgh 1983, Arbuzova 2005), was used to constrain the epipsila-palustris node and the pallens-palustris node in each of CHAM and MELVIO subclades to being at least 3.6 myr old with identical lognormal prior probabilities (offset = 3.6; log(mean) = 0; log(SD) = 0.4). Finally, we put a normally distributed age constraint on the root height of 26 Ma (SD = 1.2), inferred from a comprehensive study of the genus (Marcussen et al. in preparation). The BEAST data files are available as online Appendix 1 (input .xml file) and online Appendix 2 (output .tree file) at Dryad (doi:10.5061/dryad.68722).

Reconstruction of the Most Parsimonious Allopolyploid Network

We used the computer software PADRE (Lott et al. 2009) to construct allopolyploid species networks from the GPI multilabeled tree. As an input “species tree” topology for PADRE, we used the maximum tree (in the terminology of Liu et al. 2010) topology for GPI, reconciled using the youngest coalescent age estimates for each clade previously obtained from BEAST (Fig. 3). The maximum tree minimizes the number of deep coalescences and has been demonstrated to be a consistent estimator of the species tree (Liu et al. 2010). Hence, in the tetraploids, the maximum tree topology was reconciled based on coalescent ages in the CHAM and MELVIO subclades, and in the high polyploids this was done based on ages in the four homoeolog subclades CNud, CPla, CRos, and MRos.

A challenge in the reconstruction of allopolyploid networks is to correctly identify polyploidizations even if the associated homoeologs have become “lost”, either due to gene deletion or our failure to detect them (e.g., due to primer mismatch). Two cases of putative homoeolog loss were observed in the GPI data: CRos in V. langsdorffii and V. tracheliifolia, and a hypothesized “MPla” in all the high polyploids (see Results section). To assess whether this absence was primary, that is, a result of the allopolyploid origin itself, or secondary, we generated four input tree files reflecting each of the four combinations of presence and absence of the CRos and “MPla” homoeologs. The four tree files were then analyzed separately in PADRE and the results were compared for the most parsimonious solution, that is, the one requiring the fewest polyploidizations and gene losses to explain the observed data. The four input tree files and resulting networks used for PADRE are available in online Appendix 3 at Dryad (doi:10.5061/dryad.68722).

Phylogenetic Reconstruction of Homoeologs

There is a general correspondence among chromosome number, ploidy level, and number of GPI homoeologs (Table 2). Diploids are mostly homozygous for GPI. In cases where more GPI alleles or homoeologs are found within the genome, each copy is either numbered (e.g., canadensis-1, canadensis-2), or given an appended letter code referring to the clade to which it belongs (C = CHAM clade, M = MELVIO clade). For the high polyploids, we have named the homoeologs resulting from the decaploidization (see below) CNud, CRos, CPla, and MRos, with reference to their closest sister clades, that is, CHAM-Nudicaules, CHAM-Rostratae, CHAM-Plagiostigma, and MELVIO-Rostratae, respectively (Fig. 1).

Results from the MP and ML analyses give congruent tree topologies for both GPI (Fig. 1) and trnL-F (Fig. 2). The GPI phylogenies (MP: 60 most parsimonious trees, CI = 0.74, RI = 0.92) confirm that the tetraploid sections Plagiostigma and Viola possess GPI homoeologs from two main clades, CHAM and MELVIO (Fig. 1), and the chloroplast trnL-F phylogenies (Fig. 2) (MP: 22 most parsimonious trees, CI = 0.84, RI = 0.86) correspond well to the CHAM clade in the GPI phylogeny. The CHAM and MELVIO subclade topologies are congruent (Fig. 1), with three exceptions: (i) sequences from species of section Chamaemelanium (2x) are only present in the CHAM clade, where they form a polytomy for GPI (Fig.1) and a more or less resolved clade for trnL-F (Fig. 2); (ii) sequences of V. vaginata, V. jalapaënsis, and V. primulifolia take well-supported but somewhat different positions in the CHAM and MELVIO subclades (Fig. 1); (iii) there is incomplete additivity of the CHAM and MELVIO subtree topologies for the high polyploids (see below; Fig. 1), suggesting either duplication or loss of homoeologs (or, alternatively, failure of detection). GPI homoeologs of the high polyploids formed four subclades, nested within clades containing sequences of the three sections Chamaemelanium (CNud), Plagiostigma (CPla), and Viola (CRos, MRos) (Fig. 1). Here the chloroplast is inherited from section Plagiostigma. Additional CHAM and MELVIO homoeologs are found in three of the high-polyploid species. The Chamaemelanium-derived homoeolog (CNud) is sister to a clade consisting of North American members of subsection Nudicaules. Of the section Viola-derived homoeologs, CRos is sister to a clade corresponding to the northern hemisphere subsection Rostratae s.lat., whereas the position of MRos was not resolved; CRos was not recovered in V. langsdorffii or in V. tracheliifolia. The homoeolog CPla, which is derived from the tetraploid section Plagiostigma, is sister to a large clade of North American species, the Primulifoliae lineage, but only the CHAM homoeolog is found in the high polyploids (Fig. 1). Three of the high polyploids possess further Plagiostigma-derived CHAM and MELVIO homoeologs, suggesting secondary polyploidy: V. grahamii harbors an additional Primulifoliae genome (CPri, MPri); V. langsdorffii contains additional genomes from V. epipsila-like and V. verecunda-like ancestors (Cepi, Mepi, Cver, Mver), and V. tracheliifolia possesses an additional genome from the V. macloskeyi/occidentalis complex (Cmac, Mmac). In all these cases, the chloroplast is inherited from the higher-ploid parent. Viola clauseniana has a deviant version of the Nudicaules homoeolog. Pseudogenized GPI homoeologs, identified by frameshifts and premature stop codons, are detected in the two high polyploids V. clauseniana (CRos) and V. grahamii (C_Pri). In addition, V. clauseniana possesses two pseudogenized GPI copies that are placed in the basal polytomy of the CHAM clade (Cghost1 and Cghost2); the higher frequency at which they are amplified using smPCR suggests that these occur in multiple copies in the genome and are paralogs rather than homoeologs.

In addition, the allo-octoploid origins of V. blanda and V. palustris, section Plagiostigma, are resolved. The two accessions of V. blanda (8x) both possess two V. renifolia-like (4x) GPI homoeologs (Cren, Mren) and the chloroplast sequence and two GPI homoeologs phylogenetically nested within the Primulifoliae lineage (4x) (Figs. 1 and 2). The two accessions of V. palustris (8x) are not monophyletic: the Alaskan sample (721 palustris) has homoeologs in common with the Pacific V. epipsila subsp. repens (4x; Cepi, Mepi) and V. macloskeyi/occidentalis (4x; Cmac, Mmac), whereas the Norwegian sample (933 palustris) shares homoeologs with the sympatric V. epipsila subsp. epipsila (4x; Cepi, Mepi) and northeastern North American V. pallens (4x; Cpall, Mpall), with which it also shares the chloroplast (Figs. 1 and 2).

Estimation of Divergence Times

A chronogram with average divergence time estimates and 10 calibration points denoted is presented in Figure 3. The four clades consisting of sequences from high-polyploid species (CNud, CPla, CRos, MRos) coalesce with their lower-ploid sister clades at 11.5 Ma (CPla) to 13.0 Ma (CRos). This narrow time interval with overlapping credibility intervals (8.6–16.1 Ma) suggests a single allopolyploidization event. Dating polyploidization events in general is not trivial, but as alleles/orthologs can be older but not younger than their species (Doyle and Egan 2010), a maximum age for the polyploidization is given by the youngest stem age estimate, that is, 11.5 (9.4–13.6) Ma (CPla). This value may be close to the actual date of allopolyploidization since the individual stem lineages are relatively short (0.6–2.7 Ma). The deviant coalescent age of 18.1 Ma for two “extra” GPI copies in V. clauseniana (Cghost1 and Cghost2) lends further support (see above) to the hypothesis that these are paralogs, not homoeologs, and therefore irrelevant in the reconstruction of allopolyploid relationships. The youngest coalescent age of the “western” and “eastern” high-polyploid lineages (Fig. 4) is 8.4 (6.2–10.7) Ma (CPla). For the “western” high polyploids, the youngest coalescence time for V. langsdorffii and V. tracheliifolia is 3.9 (1.0–7.1) Ma (MRos). This value is close to the inferred minimum age of the secondary polyploidization of V. tracheliifolia with the V. macloskeyi-occidentalis lineage at 3.7 (1.9–5.5) Ma (Mmac), whereas those associated with V. langsdorffii and the clades of V. epipsila and V. verecunda are inconsistently younger, 1.31 Ma (poor node support) and 2.2 (0.5–4.3) Ma, respectively. Coalescence times suggest allopolyploid origins in the last 3–4 Ma also for the high-polyploid V. grahamii (3.2 [1.3–5.4] Ma) and for the Plagiostigma octoploids V. blanda (3.4 [1.2–6.1] Ma) and the polyphyletic V. palustris (fossil used as calibration).

Reconstruction of the Most Parsimonious Allopolyploid Network

Our comparison of four scenarios reflecting all possible combinations of gene loss yielded a most parsimonious network that required 11 changes (2 gene losses and 9 polyploidizations), as compared with 12–15 changes in the other three scenarios (Table 4). The most parsimonious PADRE network implies loss of (or failure to detect) both CRos (in V. langsdorffii and V. tracheliifolia) and the hypothesized “MPla” (in all the high polyploids) and supports nine genome mergers in the ingroup (Table 4 and Fig. 4): (1) a tetraploidization basal to sections Viola + Plagiostigma, (2) a decaploidization basal to the high polyploids, individual allopolyploidizations marking the origins of (3) V. glabella (4x), (4 and 5) V. palustris (8x; two origins) and (6) V. blanda (8x), and three secondary allopolyploidizations within the high-polyploid clade marking the origins of (7) V. grahamii (14x), (8) V. langsdorffii (18x), and (9) V. tracheliifolia (14x). Two of these genome mergers (2 and 8), representing the origin of the decaploids and the origin of V. langsdorffii (18x), involve three lineages, and each can be inferred as two subsequent polyploidizations.

Allopolyploid Origin and Ancestry of the High-Polyploid Violets

We have shown that the high-polyploid violets mostly native to continental North America, Beringia, and Hawaii all possess genomes derived from three lower-ploid, sympatric lineages, corresponding to the North American lineage of section Chamaemelanium subsection Nudicaules (diploid), the Primulifoliae lineage within section Plagiostigma (tetraploid), and section Viola (tet- raploid) (Figs. 1 and 4). The most parsimonious species network suggests that the high polyploids are monophyletic at the decaploid level (Fig. 4 and Table 4). The decaploid level could only have been attained in two successive events of hybridization and genome duplication, but owing to the extinction of species at intermediate ploidy levels it is not possible to assess the sequence of individual hybrid combinations formed over the entire allopolyploidization process. In the following, we therefore refer to the decaploidization as a single event. The decaploids all have the Plagiostigma chloroplast (Fig. 2), showing that the Plagiostigma parent must have been the maternal parent, at least in the second hybridization. The occurrence of a distinct and deep-coalescent Nudicaules allele in V. clauseniana (Fig. 1) suggests that this particular polyploidization happened more than once, which seems to be the rule in polyploids (e.g., Soltis and Soltis 1999). Although the Boreali-Americanae and Pedatae lineages have remained at the decaploid level, the Mexicanae, Nosphinium, and Langsdorffianae lineages are all products of additional independent polyploid events incorporating genomes from various lineages within section Plagiostigma—once in Mexicanae (14x), once in Nosphinium (14x), and twice in Langsdorffianae (18x) (Fig. 4). Our fossil-calibrated phylogeny (Fig. 3) shows that the decaploidization may have happened 9–14 Ma and the secondary polyploidizations, probably less than 3–4 Ma. This suggests that the initial decaploid differentiated substantially over a period of a few million years before the individual secondary polyploidizations that ultimately generated the ancestors of the five modern-day high-polyploid lineages.

Beyond compelling phylogenetic evidence from the GPI locus, the triple-hybrid decaploid hypothesis outlined above is corroborated by chromosome numbers. Given the lowest chromosome number for the sections Chamaemelanium (2n = 2x = 12), Plagiostigma (2n = 4x = 24), and Viola (2n = 4x = 20), their raw decaploid would be expected to have 2n = 56( = 12 + 24 + 20), the 14-ploids 2n = 80( = 56 + 24), and the 18-ploids 2n = 104( = 56 + 24 + 24). This is indeed very close to the actual counts for these high-polyploid lineages, 2n = 54, 80, and 102, respectively (Table 1). In Langsdorffianae, slightly deviating counts of 2n = ca. 96 (Miyaji 1929; Sokolovskaya 1960, Sokolovskaya 1963; Volkova et al. 2003; Probatova et al. 2007) and 2n = ca. 120 (Taylor and Mulligan 1968) presumably reflect partly the great difficulty in counting many small chromosomes, and partly the wish to align counts with multiples of x = 12, the base number attributed to Langsdorffianae by early authors (Miyaji 1929; Clausen 1964). Counts of 2n = ca. 60 and 2n = ca. 72 (Sokolovskaya and Probatova 1986) are difficult to interpret in light of our results and could be errors or counts made on hybrids of V. langsdorffii with, for example, tetraploids (2n = 11x = 63) and octoploids (2n = 13x = 75).

In spite of the long time since the decaploidization took place, at a time when the parental sections themselves had differentiated only for about 8 Ma, hybrids between members of the three parental sections can still be made artificially and, in certain cases, are vigorous, especially intersectional crosses involving Rostratae and Plagiostigma (Gershoy 1928, Gershoy 1934). There are also reports of natural hybrids between Boreali-Americanae species and species of the other two lineages, but such hybrids are apparently rare (Russell 1955).

Diversification of the “Eastern” Decaploid Clade

The four decaploid species of the “eastern” decaploid clade (V. clauseniana, V. grahamii, V. pedata, V. sagittata; Fig. 4) are currently distributed in most of North America but do not reach the Pacific coast. The individual GPI homoeolog subclades have incongruent topologies for these species (Fig. 1), but the reconciled maximum tree (Fig. 4), constructed from the minimum coalescent ages, places the Pedatae lineage as sister to the other two lineages and Mexicanae nested within Boreali-Americanae. The isolated position of Pedatae is not surprising: its single species, V. pedata, differs sharply in having deeply divided leaves, often differently colored petals, lack of cleistogamy, and is the only violet known to be (partially) self-incompatible (Becker and Ewart 1990). The second lineage, Boreali-Americanae, largely sympatric with the former, consists of numerous morphotypes and ecotypes that are variably distinct, variably sympatric, and variably interfertile (Clausen 1962, Gil-ad 1997). In our opinion, this reflects primary diversification rather than secondary breakdown of species barriers as a result of disturbance as previously suggested (Clausen 1962. This would fit with the relatively young age of the complex (5.9 Ma) and the apparently simple genetics of the few characters that separate taxa (Brainerd 1913, Brainerd 1924). The Mexicanae lineage appears to have its origin in secondary allopolyploidization of a Boreali-Americanae species and a Primulifoliae species (section Plagiostigma) some 3.2 Ma. As previously mentioned, corresponding hybrids are not uncommon even among modern species (Russell 1955). The Mexicanae species are allopatrically distributed and particularly diverse in the high mountains of Mexico and Central America (but secondarily dispersed to northern South America).

Origin of the Hawaiian Violets Revisited

Previous studies based on rDNA have hypothesized that the Hawaiian violets, the Nosphinium lineage, were derived from within the amphi-Beringian Langsdorffianae complex (Ballard and Sytsma 2000). Our low-copy nuclear gene data contradict such a scenario. For the lineages in question, herein represented by the “western” high polyploids V. langsdorffii and V. tracheliifolia, rDNA has apparently been homogenized toward the MELVIO-section Viola genome in the polyploid phylogeny (Fig. 5), which means that phylogenetic signals from the seven or eight other diploid genomes present in the allopolyploid were not captured. Indeed, the homoeolog lineages of V. langsdorffii and V. tracheliifolia continue to form well-supported clades also with GPI. The two lineages may have split only about 4 Ma at the time of their respective allopolyploidizations, with the V. macloskeyi-occidentalis clade (V. tracheliifolia) and with the clades of V. epipsila and V. verecunda (V. langsdorffii). Hence, the Hawaiian violets are not derived from within the Langsdorffianae; however, the two lineages are in one sense phylogenetic “sisters” in that they share a most recent common ancestor—the western decaploid—besides three additional tetraploid ancestors that they do not share.

Our data provide regional biogeographic evidence that the now exclusively Hawaiian Nosphinium lineage originated, by allopolyploidy, somewhere near the Pacific coast of North America, as this is where its extant “sister” species all occur (V. macloskeyi, V. occidentalis, V. langsdorffii). The fact that these species all have pronounced boreal affinities continues to lend support to Ballard and Sytsma's (2000) idea that the Hawaiian violets have an “Arctic origin.” In a recent study, Havran et al. (2009) presented evidence that the ancestral Nosphinium violet first colonized the Maui Nui Complex of the Hawaiian archipelago. The emergence of this island, which later broke into four smaller islands (Maui, Moloka`i, Lāna`i, and Kaho`olawe), has been dated to 1.2–2.0 Ma (Price and Elliott-Fisk 2004) and thereby provides also a maximum age for the colonization event. However, the estimated age for the Nosphinium lineage based on our fossil-calibrated phylogeny is older, 3.7 (1.9–5.5) Ma, and the two time ranges are barely overlapping. Assuming these dates are accurate, this age discrepancy may be taken as evidence that the Nosphinium lineage persisted for some time on the American mainland, maybe up to 3–4 Ma, before dispersing to Hawaii. In Hawaii, it underwent rapid ecological and morphological radiation (Havran et al. 2009) while eventually becoming extinct on the mainland.

There is some evidence that polyploids are better adapted than diploids for establishment on oceanic islands (Harbaugh 2008, Soltis et al. 2009, Baldwin and Wagner 2010; see also Mummenhoff and Franzke 2007). Indeed, the highest incidence of polyploidy known is in the Hawaiian flora (Carr 1998). Hybridization and/or polyploidization shortly before dispersal to the Hawaiian Islands has been proposed for a number of angiosperm colonists outside of Viola (reviewed by Baldwin and Wagner 2010): hybrid origins may have aided their establishment or evolutionary success, by elevating genetic or genomic variation and potentially allowing for extensive recombination and expression of diverse phenotypes on which natural selection could act.

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