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Keywords:

  • adaptive radiation;
  • AFLPs;
  • Asteraceae;
  • geographical isolation;
  • hybridization;
  • Microseris;
  • selection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography

The disjunct allotetraploid lineage of the North American genus Microseris in New Zealand and Australia originated from one or a few diaspores after a single introduction via long-distance dispersal. The plants have evolved into four morphologically distinct ecotypes: ‘fine-pappus’, ‘coastal’, ‘murnong’, and ‘alpine’, from which the first two are grouped as Microseris scapigera, mainly from New Zealand and Tasmania, and the latter two as M. lanceolata, endemic to the Australian mainland. Three chloroplast (cp) DNA types were distinguished in each of the species, but their distribution, especially in M. lanceolata, showed discrepancies with ecotype differentiation. Here, we analyse the genetic structure of the nuclear (n) DNA among two plants of each of 55 New Zealand, Tasmanian, and Australian Microseris populations for amplified fragment length polymorphisms (AFLPs). The nuclear genetic structure is compared to geographical, ecotype, and cpDNA distribution, in order to resolve and illustrate the early process of adaptive radiation. The strongest signal in the AFLP pattern was related to geographical separation, especially between New Zealand and Australian accessions, and suggested an initial range expansion after establishment. The ecotypic differentiation was less-well reflected in the AFLP pattern, and evidence was found for the occurrence of hybridization among plants at the same geographical region, or after dispersal, irrespective of the cpDNA- and ecotypes. This indicated that the ecotype characteristics were maintained or re-established by selection. It also showed that genetic differentiation is not an irreversible and progressive process in the early stage of adaptive radiation. Our results illustrate the precarious balance between geographical isolation and selection as factors that favour differentiation, and hybridization as factor that reduces differentiation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography

Speciation of plant groups after long-distance dispersal of one or a few diaspores to isolated areas provide some of the most informative ‘natural experiments’ in evolutionary biology ( Baldwin et al., 1998 ). Classic examples are found in the oceanic island flora, including the Hawaiian silversword alliance ( Baldwin et al., 1990 ; Baldwin, 1997), the genus Dendroseris on the Juan Fernandez Islands ( Crawford et al., 1992 ; Sang et al., 1994 ), and various Macaronesian plant groups ( Böhle et al., 1996 ; Francisco-Ortega et al., 1996 ; Kim et al., 1996 ; Mes et al., 1996 ; Francisco-Ortega et al., 1997 ; Vargas et al., 1999 ). A few examples are also known from flora of island-like habitats on the continent, e.g. the Dendrosenecio of the tall mountains of eastern Africa ( Knox & Palmer, 1995). In these cases, rapid speciation under directional selection through adaptive radiation is observed ( Stuessy & Ono, 1998). This can be interpreted as the result of the elimination of competition from lineages specialized in particular resources, the increased competition with close relatives, and selection in response to alternative adaptive opportunities ( Givnish, 1997). Molecular phylogenetic analyses have shown that the conspicuous morphological variation in these plant groups is not necessarily paralleled by differences in overall genetic distances (e.g. Baldwin et al., 1998 ). This presumably reflects a relatively recent origin of the radiations, and a possible large effect of only a few genes affecting ecologically significant traits and reproductive isolation ( Givnish, 1997).

It is most likely that cases of long-distance dispersal, if they manage to get established at all, will pass through a phase in which there is very little genetic variation. The initial small and probable nonrepresentative samples of ancestral population genes present in the founder population, might further be decreased and sampled by random genetic drift, before differentiation through dispersal and disruptive selection takes hold (e.g. Ridley, 1993). The well-known examples cited above probably concern relatively late stages in this process, with multiple and strikingly different species occupying diverse niches. Two independent events of long-distance dispersal from the North American genus Microseris (Asteraceae, Lactuceae) illustrate earlier stages in the process. Microseris pygmaea D. Don. in Chile is a species in the very earliest stages of intraspecific differentiation ( Chambers, 1963; Bachmann et al., 1985 ; Van Heusden & Bachmann, 1992). Populations of M. scapigera (Forst.) Sch.-Bip. and M. lanceolata (Walp.) Sch.-Bip. in New Zealand and Australia are just at the crucial stage in which geographical and ecological differentiation are becoming sufficiently pronounced to overcome gene flow ( Sneddon, 1977). Here, we analyse the genetic structure among the populations of the Australasian Microseris that can be classified into a number of geographical groups and ecotypes, in order to get more insight into this early stage of the process of adaptive radiation.

The origin of the New Zealand and Australian Microseris involved a number of improbable events. The karyotype of the plants indicates that they are allotetraploid derivatives of a hybrid between an annual and a perennial species of the genus ( Chambers, 1955). A phylogenetic tree based on chloroplast (cp) DNA mutations suggests that the maternal parent belonged to a now extinct, early offshoot of the well-supported clade of annual, self-fertile Microseris ( Wallace & Jansen, 1990). The pollen parent was most likely a perennial, probably outcrossing, species, morphologically related to the present M. borealis (Bong.) Sch.-Bip. Judging from the present distribution of North American Microseris ( Chambers, 1955), the parents were ecologically distinct from each other. The allopolyploid hybrid has left no trace in North America. Its long-distance dispersal to the Southern hemisphere is best explained by a unique event involving one or a few achene(s). The age of the taxon is unknown, but reports from Captain Cook’s first voyage (1769; Ebes, 1988) and early European settlers (c. 1835; Gott, 1983) mention that Microseris was collected in New Zealand and was abundant in Australia at that time. According to the cpDNA tree ( Wallace & Jansen, 1990), the taxon originated before the onset of speciation of the present North American annual Microseris.

At present, Microseris occurs on both islands of New Zealand, in Tasmania and in south(east) Australia ( Sneddon, 1977; Vijverberg et al., 1999 ). Several ecotypes are recognized ( Table 1; Fig. 1; Sneddon, 1977; Gott, 1983; Vijverberg et al., 1999 ). A ‘coastal’ (C) ecotype characterized by waxy leaves grows in a number of isolated populations along the sea shores of New Zealand. A self-fertile ‘fine-pappus’ (F) type contains many hairy pappus parts (≈60) and is found in five small populations in Victoria (<200 individuals except for one) and a few larger ones in Tasmania. An ‘alpine’ (A) ecotype reproduces vegetatively by forming shoots from its roots and occurs in the mountains of south-east Australia at altitudes >1000 m above sea level. A ‘murnong’ (M) ecotype has tuberous roots and occurs in a relatively large number of populations in the lowlands of south(east) Australia. The latter type was widespread at the time of European settlement and used as a staple food of aborigines ( Gott, 1983). It now occurs as remnant populations in small fragmented areas only ( Prober et al., 1998 ). Plants of a few populations differ in the diagnostic character(s) from their most similar ecotype and are further indicated as ‘nontypical’ (nt). The ecotype characteristics are maintained in a common greenhouse environment ( Sneddon, 1977; own observations). On the basis of morphology, biogeography, and crossability data, two species are recognized (B. V. Sneddon, personal communication, Victoria University of Wellington, New Zealand: a revision of Australian Microseris for the Flora of Australia, volumes 37 and 38, Asteraceae 1 and 2, in preparation (A. E. Orchard [Ed.], Australian Biological Resource Study, Canberra, Australia): M. scapigera, that includes the ‘C’ and ‘F’ populations, and M. lanceolata, including the ‘A’ and ‘M’ ecotypes.

Table 1. Microseris scapigera and M. lanceolata populations investigated. Thumbnail image of
image

Figure . 1. Distribution of M. lanceolata populations examined. Population numbers follow ecotypes in geographical order: A=‘alpine’ ( Table 1), M=‘murnong’, 1=most northern population, and nt=a nontypical form of an ecotype. Symbols correspond to chloroplast types ( Vijverberg et al., 1999 ; Fig. 2): ▵=Mln-1, □=Mln-2, ○=Mln-3, and inline image=polymorphic for Mln-2 and Mln-3. Circles include geographical groups of particular cpDNA types: NSWne=north-east New South Wales, VIC=Victoria, and NSW/VIC=contact region between populations of cpDNA types Mln-2 and Mln-3.

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A comparative analysis of the cpDNA of New Zealand and Australian Microseris, based on restriction site mutations in the variable regions of the genome, confirmed the monophyly of the taxon as well as its close relationship to the North American annual Microseris ( Vijverberg & Bachmann, 1999; Vijverberg et al., 1999 ). The tree indicated rapid radiations in the history of the plant group, and confirmed the monophyly of M. lanceolata. Especially in M. lanceolata, the cpDNA types corresponded more with geographical distribution than ecotypic differentiation. This result raised the question whether processes such as introgression between ecotypes or parallel evolution of similar adaptations played a role in the evolution of Australian (and New Zealand) Microseris. Expanded nuclear (n) DNA investigations were needed to discriminate among these possible explanations.

Preliminary investigations of the nDNA indicated a very low level of genetic variation to be present in the New Zealand and Australian Microseris. A fine-scale restriction site analysis, using four-base recognizing enzymes, resolved eight mutations among six populations of the Australian mainland ( Van Houten et al., 1993 ). A cladistic treatment of these mutations showed similar results as found for the cpDNA, namely, a basal polytomy, with the ‘F’ populations least differentiated from the outgroup species, and the ‘A’ and ‘M’ populations as a more diverged clade. The restriction site analyses did not resolve enough mutations to determine the relationships among the sample of populations investigated here. The internal transcribed sequences (ITS) of the nuclear cistrons for ribosomal RNA lacked variation within the Australasian Microseris (unpublished results). Here, we use amplified fragment length polymorphisms (AFLPs) to analyse the genetic structure of the nDNA among plants of 55 New Zealand and Australian Microseris populations and a few North American and Chilean comparison species of Microseris. The genetic differentiation of the nuclear DNA of the plants was compared to geographical, ecotype, and cpDNA distribution, in order to obtain further insights into the ongoing evolution and speciation of this plant group.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography

Plant material

Achenes were collected from 55 natural populations of Microseris in New Zealand and Australia, covering the morphological variation and geographical distribution range of the two species, except for West Australia ( Table 1). Populations were classified on the basis of a few morphological characteristics (Introduction; Vijverberg et al., 1999 ) into ‘C’, ‘F’‘A’, and ‘M’ ecotypes, or as nt forms of one of these ecotypes. Population abbreviations in the text, figures, and tables, refer to their ecotypes in geographical order. A total of four C, five F, five Fnt, five A, five Ant, 30 M, and one Mnt populations were included in this study ( Table 1). Except for populations A1nt, A2nt and A3nt, A9 and A10, and F6nt and F10nt, populations were isolated from other known populations by at least a few kilometres. Achenes were germinated and seedlings planted and raised in a cool greenhouse in Amsterdam between 1992 and 1996. One to five plants per population were analysed, most of them derived from separate plants in the field. The North American annual species M. douglasii (DC.) Sch.-Bip., and M. elegans Green ex Gray, the perennials M. borealis and M. laciniata (Hook.) Sch.-Bip., and the Chilean annual M. pygmaea, each represented by two plants, were included for comparison ( Vijverberg et al., 1999 ).

AFLP analysis

Total DNA was isolated from fresh or frozen dried leaves using the cetyl-trimethyl-ammonium-bromide (CTAB) procedure described by Saghai-Maroof et al. (1984) and the extra purification steps by Vijverberg et al. (1999) . DNA content and purity were determined by measuring the optical densities at 260/280 nm, and DNAs were diluted to a final concentration of 12.5 ng μL–1 H2O. AFLPs were generated according to the protocol of KeyGene (Wageningen, The Netherlands) version 2.2, described by Vos et al. (1995) , with a few modifications. We digested half of the amount of DNAs (250 ng) used by Vos et al. (1995) with 5 U EcoRI and 3 U MseI. Ligation with 1.2 U T4 DNA ligase occurred overnight in a water bath that slowly cooled down from about 20–14 °C. Primer tests were performed with the primers supplied by the AFLP starter kit (GibcoBRL/Life Technologies, The Netherlands) and, due to densely distributed AFLP patterns obtained with the EcoRI +ACA and +AGC primers tested, with newly designed (pre)selective EcoRI primers containing one extra nucleotide. Pre-selective amplifications were performed with 5 μl 20x diluted ligation mixtures and the EcoRI (5′-GACTGCGTACCAATTC-3′) +AC or +AG primer in combination with the MseI (5′-GATGAGTCCTGAGTAA-3′) +C primer. Selective amplifications were performed with 5 μl 10x diluted pre-amplified DNAs, the EcoRI +ACAA or +AGCA primers, one of each of the MseI +CAA, +CAC, +CAG, +CAT, +CTA, +CTC, +CTG, or +CTT primers, and 0.15 U of Super Taq DNA polymerase (HT Biotechnology, UK). From these tests, the EcoRI +ACAA primer was chosen in combination with the MseI +CAG or +CTG primers to analyse all individuals. For the selective amplifications, EcoRI primers were γ32P-ATP labelled, using half of the activity (50 μCi) mentioned in the protocol of Vos et al. (1995) , and 25 U T4 polynucleotide kinase (GibcoBRL/Life Technologies, The Netherlands). Electrophoresis was performed with 4 μl AFLP+ loading dye mixtures loaded onto 6% polyacrylamide gels (Sequagel-6, Biozyme, The Netherlands) for 2.5 h at 55 W. After electrophoresis, gels were transferred to Whatman 3 mm filters and dried for 45 min at 80 °C and 15 min at 25 °C on a vacuum dryer. X-OMAT-AR5 films (Kodak, The Netherlands) were exposed to the dried gels for 4–48 h at room temperature.

Scoring and homology of AFLPs

The presence (1) or absence (0) of fragments was manually scored from the films and entered into a binary data matrix. The alignment between films was facilitated by the inclusion of two repeats of two reference plants on each gel, and by (largely) monomorphic fragments. Still, the alignment of fragments that were rarely present was difficult, and uncertain fragments were excluded.

Homology of a large part of the fragments of New Zealand and Australian Microseris was assumed on the basis of the close relationships of the plants, and the overall similarity of band patterns and intensities. Homology of the fragments of Australasian Microseris and North American and Chilean diploid species of the genus was more ambiguous, and band patterns and intensities differed, in part, certainly due to the ploidy difference. Only 12 fragments (3%) were shared between the Australasian Microseris and annual comparison species, and only two were monomorphic within the annuals. The AFLP patterns of the North American perennial Microseris were more similar to those of the Australasian ones, but also the number of shared fragments was low (16; 4%). Due to the low number of shared fragments and uncertainty about their homology, the North American and Chilean Microseris were excluded from the data analyses. The data set and a visualization of gels are available on request from the first author.

Data analyses

The AFLP data were analysed for similarities among individual plants, using NTSYS-PC 1.80 ( Rohlf, 1993), PAUP 4.0b2a ( Swofford, 1999), and SPSS 8.0 for Windows. The analyses were performed on a data set with usually two plants per population (113 individuals, 55 populations, 407 fragments). For populations A3nt, M2 and M30 only one plant was analysed, and for populations A6, M14, M16 and M17, that are polymorphic for cpDNA types ( Figs 1 & 2), two plants of each type were included when available. For about half of the populations, AFLP patterns of more than two (three to five) plants per population were available, and Mln-2 and Mln-3 ( Fig. 2; 76 individuals, 36 populations, 383 fragments). Finally, analyses were repeated with the fragments originating from the MseI + CAG or +CTG primers.

image

Figure . 2. Phylogenetic tree based on cpDNA variation of the New Zealand and Australian Microseris populations examined, summarized from Vijverberg et al. (1999) . Original tree is based on 55 restriction site mutations, most of them small indels located in the variable regions of the genome, tree length=60, and consistency index=0.86 (autapomorphies excluded). Tree shown here is simplified for structure within the outgroups (annual Microseris, not shown; 15 mutations), unique mutations for plants or populations (20 mutations), and some additional groupings within the Mln-2 and Mln-3 clades. The different cpDNA types as well as M. lanceolata are well-supported by the mutations indicated (see also Vijverberg & Bachmann, 1999). Population numbers follow ecotypes in geographical order: A=‘alpine’ ( Table 1), M=‘murnong’, F=‘fine-pappus’, C=‘coastal’, 1=most northern population, and nt=a nontypical form of an ecotype. CpDNA types follow species names: Msc=M. scapigera and Mln=M. lanceolata. Places of origin are: SEA=south-east Australia ( Fig. 1), VIC=Victoria, TAS=Tasmania, and NZ=New Zealand.

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From the binary data matrices, pairwise genetic similarities among plants were calculated, using Jaccard (1908) coefficients in NTSYS-PC or Nei & Li (1979) coefficients in PAUP. These coefficients were chosen because they avoid clustering on the basis of shared absences which were over-represented in the AFLP data, and are more likely to be nonhomologous than shared presences ( Wolfe & Liston, 1998). Clusters were formed via the unweighed pair-group method (UPGMA; Sneath & Sokal, 1973), and visualized as a phenogram. Branch support was assessed by a bootstrap analysis with 1000 replicates in PAUP.

Similarities among plants were also investigated via principal coordinate analyses (PCoA) ( Gower, 1966), using NTSYS-PC. For this, the eigenvectors with an eigenvalue >1 were extracted from the Jaccard similarity matrix, and the variation represented by the first four vectors was calculated. Results were depicted as scatter plots, with the individuals plotted on the first and second and third and fourth vectors, respectively. A minimum-length spanning tree was calculated from the same Jaccard similarity matrix, and superimposed over the individuals in the scatter plots to help detect local distortions (not shown).

Nuclear DNA types were defined on the basis of the results of the cluster and PCoA, and searched for type-specific AFLP fragments, using the FREQUENCIES option in SPSS. Similarly, groupings based on the nDNA types, combined with information about cpDNA types ( Fig. 2), geography ( Table 1), and ecotypes, were searched for specific AFLP fragments.

For some clusters, populations, or individuals of interest, their most similar individuals were resolved by sorting the part of the Jaccard similarity matrix in which these plants were involved. These sortings were performed to directly detect the most similar individuals, without effect of the choice, number and (inter) relationships, of the individuals included, that might be of influence in the cluster and PCoA.

The effect of geographical distances on the genetic similarities among plants of cpDNA type Mln-2 and Mln-3 was tested with a Mantel (1967) test, using Le Progiciel R v4.0d0 ( Casgrain & Legendre, 1998). These plants grow in a transect in south-east Australia ( Fig. 1), and their geographical distance matrix was calculated from the latitudes and longitudes of their origins. The matrix was compared to the Jaccard similarity matrix of the AFLP data of these plants, by calculating Spearman’s rank-correlation coefficient between the two matrices. The test was repeated, including the ‘M’ and ‘A’ populations separately, and evaluated for significance by 999 permutations.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography

New Zealand and Australian Microseris

For comparison, a summary of the phylogenetic tree based on cpDNA is given in Fig. 2. The tree shows all accessions of M. scapigera separated from those of M. lanceolata, and revealed three cpDNA types in each of the two species. The polytomy at the base of the tree, as well as the trichotomy in M. lanceolata, were interpreted as indicative for rapid radiations in the history of the taxon ( Vijverberg et al., 1999 ). While the same cpDNA type was present in all ‘C’ populations of M. scapigera, all three cpDNA types were found in the ‘F(nt)’ populations of that species and corresponded to geographical groupings (Msc-1 to Msc-3). In M. lanceolata, the three cpDNA types reflected more geographical distribution than morphological entities (Mln-1 to Mln-3; see also Fig. 1), and four populations from north-east Victoria (A6, M14, M16, M17) were polymorphic for cpDNA types Mln-2 and Mln-3.

The results of the AFLP data analyses, including 113 plants from 55 New Zealand and Australian Microseris populations and 407 fragments, are shown in Fig. 3. The MseI +CAG and +CTG primers resolved 178 and 229 of these fragments, respectively. Three quarters of the fragments were scored as present in only a minority of plants (<10%), whereas 7% were present in most of the individuals (>90%) and five were monomorphic for all plants included. The Jaccard similarities among the AFLPs of individual plants ranged between 0.26 and 0.98. The lowest value was found between New Zealand ‘F’ and north-west Victorian ‘M’ plants, the highest one between the two plants of ‘selfing’ populations. Plants of the ‘A’, ‘C’, ‘F’, and one third of the ‘M’ populations clustered per population. This includes three of the four populations that are polymorphic for cpDNA types Mln-2 and Mln-3 (A6, M14, and M16).

image

Figure . 3. Results of the AFLP data analyses, including 113 plants from 55 New Zealand and Australian Microseris populations and 407 fragments. Population numbers, cpDNA types, ecotypes, and places of origin, are explained in Table 1 and Figs 1 and 2. (a) UPGMA phenogram based on Nei and Li similarity coefficients among individuals, with percentages bootstrap support indicated along branches. (b) Scatter plots of the PCoA based on Jaccard similarity coefficients, with the individuals plotted on the first four vectors, indicated by their cpDNA types (see Figs 1 & 3a); NZ=all New Zealand populations, AUS=all Australian, including SEA, TAS and VIC populations, F7nt=deviating Tasmanian population, and “A”=cluster of the ‘alpine’ populations A5, A7, A9, and A10.

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The phenogram of the bootstrapped cluster analysis ( Fig. 3a) showed three groups that were clearly separated from the remaining individuals. These included: (1) ‘Ant’ and ‘Mnt’ populations of cpDNA type Mln-1 (2) the Victorian plus, less clearly grouped, two of the three Tasmanian ‘F(nt)’ populations and (3) all New Zealand accessions. The results generally corresponded to cpDNA types (see also Fig. 2), although they showed not such a clear separation of M. lanceolata and of accessions of cpDNA types Mln-2 vs. Mln-3. The nDNA showed additional pattern within cpDNA type Msc-1, separating population F7nt from Tasmania from the other members of this cpDNA type. The correspondence of the nDNA groups to ecotypes was less clear, showing three of the four types to be present in more than one group.

In the PCoA, 10 vectors were extracted with an eigenvalue >1, and the first four vectors represented a total of 20.5% of the variation. Scatter plots ( Fig. 3b) indicated the same groups as found in the cluster analyses ( Fig. 3a). The results confirmed a general congruency to the cpDNA pattern as well as a weaker correspondence to ecotypes (not indicated). The first vector separation showed that the clearest signal in the AFLP variation corresponded to the New Zealand vs. Australia, including Tasmania, separation. Within the New Zealand Microseris, no clear resemblance was found in the AFLP pattern to the clustering by cpDNA type, ecotype, breeding system ( Table 1), or island. Within the Australian Microseris, some more correspondence to cpDNA type, ecotype (see also Figs 3a & 4), breeding system or geographical area was indicated. The third (and first) vector clearly separated the populations of cpDNA type Mln-1, confirming their molecular similarities, despite the wide geographical and visible morphological separation of these ‘Ant’ populations from north-east New South Wales (A1nt to A4nt; Fig. 1) and ‘Mnt’ from Victoria (M20nt). Populations M20nt and F7nt from Tasmania, were exceptions to the general congruence of nuclear genetic similarities and other groupings. The fourth vector separated a cluster of four of the six ‘A’ populations (‘A’; see also Fig. 4). Additional structure among the populations of cpDNA types Mln-2 and Mln-3 was resolved in a separate analysis, and is described below. Against the geographical separation between New Zealand and Australia, the distinction between the two species M. scapigera and M. lanceolata (sensu Sneddon), mainly on the second vector, was weaker, while the geographical factor divided M. scapigera into two clearly separated groups. One of these groups included the Victorian and Tasmanian ‘F(nt)’ populations and the other one comprised all New Zealand accessions. The minimum-spanning tree (not shown) indicated distortion with respect to the two individuals of cpDNA type Mln-2 that were located near the Mln-1 group ( Fig. 3b, left plot), the isolated Victorian plant of cpDNA type Msc-1, and the deviating Tasmanian population F7nt. These individuals also showed similarities to their geographical relatives of the same cpDNA type.

image

Figure . 4. Results of the AFLP data analyses, including 76 plants from 36 south-east Australian Microseris populations of cpDNA types Mln-2 and Mln-3 and 383 fragments. Scatter plots of the PCoA based on Jaccard similarity coefficients are shown, with the individuals plotted on the first and second vectors, indicated by their cpDNA types: □=Mln-2 and ○=Mln -3, and ecotypes (upper plot): open=‘murnong’ and grey =‘alpine’, or places of origin (lower plot): open=NSW, light grey=NSW/VIC, dark grey=VIC, and black=VICnw/SA. Population numbers, cpDNA types, ecotypes, and places of origin, are explained in Table 1 and Figs 123. “A” denotes the cluster of populations A5, A7, A9, and A10, and A6 and A8nt indicate the remaining ‘alpine’ individuals.

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According to the Jaccard similarities, the AFLP patterns of populations of cpDNA type Mln-1 were most similar to those of population F7nt from Tasmania and a few ‘A’ populations from north-east Victoria. These Mln-1 populations showed not such a good similarity to their geographical nearest neighbours, i.e. to ‘M’ populations of cpDNA type Mln-2 from north-east New South Wales. The group of Victorian and Tasmanian ‘F(nt)’ populations, F1 to F5, F6nt, and F10nt, corresponded best to Victorian ‘M’ populations of cpDNA type Mln-3. The deviating Tasmanian population F7nt was, apart from population F6nt, most similar to north-east Victorian ‘A’ and ‘M’ populations of cpDNA type Mln-3. For the New Zealand populations, the most similar individuals could hardly be resolved, although, a slightly better correspondence to populations of cpDNA type Mln-1 and of Tasmania was found. In summary, the genetically and geographically distinct groups showed relatively high similarities to plants from north-east Victoria of cpDNA type Mln-3.

Apart from two bands that were uniquely present in all New Zealand accessions, the four nDNA clusters ( Fig. 3), excluding population F7nt, showed no diagnostic AFLPs. The groups based on nDNA results combined with other groupings also lacked type-specific AFLP fragments.

The results of the AFLP data analyses, including other subsets of two plants per population, when available, were most similar to the ones shown in Figs 3 & 4, confirming the results presented here. The results of the analyses that included the fragments of the MseI +CAG or +CTG primers separately were also similar to the ones described; however, bootstrap supports were reduced in some cases. This suggested that a better resolution among individuals can be obtained by using more than one primer combination and adding AFLP data.

Australian Microseris of cpDNA type Mln-2 and Mln-3

The results of the AFLP data analyses, including 76 plants from 36 south-east Australian Microseris populations of cpDNA types Mln-2 and Mln-3 ( Figs 1 & 2) and 383 AFLP fragments, are shown in Fig. 4. The Jaccard similarities ranged between 0.32 and 0.76, the former concerning the geographically most widely separated ‘M’ plants and the latter the two plants of some ‘A’ populations. The PCoA resolved six vectors with an eigenvalue >1, from which the first four represented a total of 13.9% of the variation. The scatter plots lacked a good divergence among individuals, but are especially considered here with respect to their congruency to other groupings. The clearest signal in the AFLP pattern corresponded to the separation of ‘M’ and ‘A’ (“A”; upper plot) ecotypes, except for populations A6 and A8nt. Population A6 is polymorphic for cpDNA types Mln-2 and Mln-3, and its nuclear genetic similarity to the ‘M’ populations supported the suggestion that hybridization has occurred between this ‘A’ and some ‘M’ populations. The ‘Ant’ population A8nt bears tubers at high altitudes, and this morphological similarity to the ‘M’ ecotype is supported by the AFLP pattern. The AFLP pattern also corresponded slightly to the cpDNA types along the first vector. As compared to the geography ( Fig. 1; Fig. 4, lower plot), a weak similarity to the AFLP pattern was found. This is best illustrated by the lack of overlap between the two extremes, i.e. the populations from New South Wales (open squares) vs. those from north-west Victoria/South Australia (black squares). The correspondence between the geographical distances and genetic similarities of the plants was also supported by significant (P < 0.001) correlations between the corresponding matrices, including all (r=–0.32) or only the ‘A’ (r=–0.83) or ‘M’ (r=−0.32) populations, respectively.

Based on the Jaccard similarities of AFLP data, the populations that are polymorphic for cpDNA types Mln-2 and Mln-3, i.e. A6, M14, M16, and M17, were most similar to geographically nearby located individuals, irrespective of their cpDNA- and ecotypes. This again indicated that hybridizations among populations of these two cpDNA types, including both the ‘A’ and ‘M’ ecotypes, occurred within the contact region between these populations in north-east Victoria. The hybrid nature of these plants was not visible in their morphology, which was that of the ‘A’ or ‘M’ ecotypes of the populations in which they occurred.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography

Detecting the early stage of adaptive radiation

The disjunct allotetraploid lineage of the North American genus Microseris in New Zealand and Australia is a good example of a taxon in an early stage of adaptive radiation (see Introduction). We have tried to resolve and illustrate this early process of speciation by studying the cp- ( Vijverberg & Bachmann, 1999; Vijverberg et al., 1999 ; Fig. 2) and nDNA ( Van Houten et al., 1993 ; this study) variation. The nuclear RFLPs and ITS-sequences resolved insufficient variation to reconstruct a nDNA phylogeny (unpublished results). The AFLP method used here, detected enough variation to estimate the nuclear genetic structure within the Australian and New Zealand Microseris (see Results, Figs 3 & 4). It was, however, too sensitive to allow for comparison with the North American relatives. Apart from this, the AFLP data indicated reticulate lineage relationships within the ingroup, which cannot accurately be depicted by a strictly bifurcating tree ( Wendel & Doyle, 1998). We analysed the AFLP data with descriptive methods and phenetic interpretations. The analyses include a distance-based clustering method, which allows to determine the bootstrap support for the different groups ( Fig. 3a), and multivariate analyses, that show additional resolution within and among clusters ( Figs 3b & 4). The results gave valuable information on the importance of geographical isolation, the measure of agreement with ecotype differentiation and cpDNA distribution, and the occurrence of dispersal within the range of the species.

The results of the AFLP data analyses show, apart from a few separated groups, little resolution among individuals and low vector percentages in the PCoAs ( Figs 3 & 4). This can be interpreted as the occurrence of relatively recent or still ongoing genetic exchange among most or all of the individuals included. Together with the sensitive method needed to detect cp- ( Vijverberg et al., 1999 ) and nDNA RFLPs ( Van Houten et al., 1993 ), and the lack of ITS-sequence variation (unpublished results), a low level of overall genetic distances within the New Zealand and Australian Microseris is indicated. This, combined with the ‘hard’ polytomies found at the base of the phylogeny reconstructions from the DNA variation ( Van Houten et al., 1993 ; Vijverberg et al., 1999 ), and visible ecotype differentiation (Table 1), suggests a relatively recent divergence and rapid radiation of the taxon. Similar conclusions have frequently been encountered in studies of adaptive radiation in plant taxa after long-distance dispersal to oceanic islands (e.g. Baldwin et al., 1990 ; Crawford et al., 1992 ; Sang et al., 1994 ; Kim et al., 1996 ; Baldwin, 1997). The low amount of overall genetic distances might reflect a relatively recent origin of the radiation ( Givnish, 1997) and the ‘hard’ polytomy, i.e. the fixed attachment of the polytomous node to its descendant node ( Maddison, 1989), is indicative for the process of rapid, multiple-speciation (e.g. Baldwin et al., 1990 ).

The importance of geographical isolation

The ‘hardest’ signal in the AFLP data is correlated with geographical distance ( Fig. 3). The cpDNA variation also corresponds most with geographical distribution ( Vijverberg et al., 1999 ; Figs 1 and 2). This result is likely to reflect dispersals that involve reduction or interruption of gene flow among populations. Dispersals, together with genetic drift in the early stages of local establishment, can sample the initial variation and result in a decrease of genetic variation within populations and an increase among populations (e.g. Ridley, 1993; Okada et al., 1997 ). Our molecular data illustrate this process by showing genetically relatively homogeneous groups at the different locations, and indicating dispersals between south-east Australia, Tasmania and New Zealand as well as on the Australian mainland. Geographic isolation, especially among islands, is found to be a fundamental stimulus in the speciation of oceanic island floras ( Stuessy & Ono, 1998), and seems to be the primary factor in the diversification of Macaronesian flora ( Baldwin et al., 1998 ). It is interesting to see the clear impact of geographical isolation on the genetic diversification also in the early stage of adaptive radiation studied here.

Selection for ecotype differentiation

The AFLP data show no significant separation of plants per ecotype ( Figs 3 & 4), and only weak support for the clustering per species (sensu Sneddon). This result is striking, given the morphological differentiation within the New Zealand and Australian Microseris ( Table 1). The incongruencies concern particularly the ‘F(nt)’ populations, from which some are more similar to populations of another ecotype ( Fig. 3), and the ‘A(nt)’ and ‘M(nt)’ populations that largely overlap ( Figs 3 & 4). The lack of a good reflection of the morphological differentiation in the nuclear genetic structure suggests, that the ecotype characteristics are maintained or re-established by selection without visible effect on the nDNA. This can possibly be explained by the involvement of only a few genes in the ecotype characteristics and/or an inefficient detection of these genes by the method used, i.e. the AFLP method approaches a random sampling of mostly neutral nDNA markers ( Van Eck et al., 1995 ; Vos et al., 1995 ). Morphological differentiation as a result of evolutionary changes at only a few genes is also reported for other plant taxa (e.g. Gottlieb, 1984; Baldwin, 1997) and is suggested to be one of the explanations for the low level of overall genetic distances usually found in adaptively radiated plant groups ( Givnish, 1997). A closer examination of the characters underlying the ecotypes of Australasian Microseris will have to confirm these conclusions, and a more detailed morphological analysis of the plant group is in progress at our lab.

The occurrence of parallel evolution of similar adaptations or hybridization among ecotypes was suggested by the incongruencies found between the cpDNA and ecotype distribution ( Vijverberg et al., 1999 ; Fig. 2) and detection of populations that are polymorphic for cpDNA types Mln-2 and Mln-3 (A6, M14, M16, M17). The results of the present AFLP study show evidence for the maintenance or re-establishment of ecotype characteristics in the presence of hybridization and/or after dispersal to another ecological region. The different processes are illustrated by a number of populations from which the best example is population A6. This population grows in north-east Victoria, the region in which populations of cpDNA types Mln-2 and Mln-3 as well as the ‘A’ and ‘M’ type occur ( Fig. 1), and is polymorphic for the two cpDNA types concerned. According to its AFLP pattern ( Fig. 4; Jaccard similarities), A6 is most similar to geographically nearby located ‘M’ individuals, indicating the influx of DNA from the other ecotype. At the same time, A6 expresses the ‘A’ morphology, suggesting selection for these ecotype characteristics. A similar example, possibly also involving cytoplasmic introgression, concerns the populations of New Zealand ( Figs 2 & 3). These include cpDNA types Msc-2 and Msc-3 as well as the ‘F’ and ‘C’ ecotypes, although their AFLP pattern is uniform. A third example is population F7nt from Tasmania ( Fig. 3), which illustrates hybridization in combination with dispersal. F7nt is, like the other Tasmanian populations F6nt and F10nt, of cpDNA type Msc-1 and the ‘F’ ecotype. On the basis of the nDNA, F7nt is similar to its Tasmanian relatives as well as to Victorian ‘M’ and ‘A’ populations of cpDNA type Mln-3 ( Fig. 3; Jaccard similarities). This result can be interpreted as secondary contact of F7nt with plants of different cpDNA- and ecotypes, involving an independent dispersal to Tasmania (back)crossing with the local populations, and/or selection for the local ecotype. Hybridization, and cytoplasmic introgression, have been reported for plants at higher taxonomic levels than the one investigated here ( Rieseberg et al., 1996 ; Wendel & Doyle, 1998). It is therefore not unexpected, that genetic exchange among New Zealand or Australian Microseris has been detected.

Two other Australian populations are likely to illustrate local adaptation after dispersal, without indication of hybridization upon arrival. A8nt from Mount Skene ( Fig. 1), is on the basis of its nDNA most similar to geographically nearby located ‘M’ populations ( Fig. 4; Jaccard similarities), whereas it shows morphological similarities to the ‘A’ and ‘M’ ecotype ( Table 1). This could result from a dispersal of a ‘M’ type to a mountain, followed by not yet completed selection for the ‘A’ characteristics. The second example concerns the Australian Mln-1 populations ( Figs 2 & 3), comprising ‘Ant’ from north New South Wales (A1nt to A4nt; Fig. 1) and a ‘Mnt’ from Victoria (M20nt). Here, long-distance dispersal is presumably followed by local selection through which ecotype characteristics have begun to change without changing the AFLP pattern. Ecological shifts are, apart from geographical isolation, a second major force in the adaptive radiation of oceanic island plants ( Stuessy & Ono, 1998), and appear to have been most prevalent in the speciation of the Hawaiian and Juan Fernandez Island floras ( Baldwin et al., 1998 ). The observed maintenance or re-establishment of ecotype characteristics within the New Zealand and Australian Microseris shows, that response to different ecological circumstances also play a role in the early stage of adaptive radiation of this plant group. This response is, however, not clearly reflected in the AFLP pattern at this stage, and not much effected by (incidental) influx of DNA of another ecotype.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography

The results of our study show that geographical isolation is the overriding factor that determines overall genetic differentiation in this lineage of Microseris at an early stage of adaptive radiation after long-distance dispersal. This indicates the importance of chance events, as a result of range expansion, in the initial stage of adaptive radiation. The ecotypes are less-well reflected in the AFLP pattern, and a number of cases illustrate the independence between morphological and genetic differentiation. The results point towards two aspects that seem to be important in the early stage of adaptive radiation: (1) that ecotype characteristics are maintained or re-established by an efficient selection in the presence of hybridization and (2) that dispersals over rather large distances can play a relatively important role in genetic exchange within the range of a species. The process of ongoing speciation that emerges from our study clearly illustrates the precarious balance between geographical isolation and selection as factors that favour differentiation, and at the same time hybridization that reduces differentiation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography

The authors would like to thank Ted H. M. Mes for fruitful discussion, and G. David E. Povel and Joost F. Duivenvoorden for advice with statistical analyses. This research was supported by the Life Science Foundation (SLW, grant number 805–38163), which is subsidized by the Netherlands Organization for Scientific Research (NWO).

Bibliography

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Bibliography
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