Confirmation of Shared and Divergent Genomes in the Glycine Tabacina Polyploid Complex (Leguminosae) Using Histone H3-D Sequences Author(S): Jeff J

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Conﬁrmation of Shared and Divergent Genomes in the Glycine tabacina Polyploid Complex (Leguminosae) Using Histone H3-D Sequences

Article in Systematic Botany · July 2000 DOI: 10.2307/2666688

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The user has requested enhancement of the downloaded file. Confirmation of Shared and Divergent Genomes in the Glycine tabacina Polyploid Complex (Leguminosae) using Histone H3-D Sequences Author(s): Jeff J. Doyle, Jane L. Doyle, A. H D. Brown, and Bernard E. Pfeil Source: Systematic Botany, 25(3):437-448. Published By: The American Society of Plant Taxonomists DOI: http://dx.doi.org/10.2307/2666688 URL: http://www.bioone.org/doi/full/10.2307/2666688

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Conﬁrmation of Shared and Divergent Genomes in the Glycine tabacina Polyploid Complex (Leguminosae) using Histone H3-D Sequences

JEFF J. DOYLE and JANE L. DOYLE L. H. Bailey Hortorium, Cornell University, Ithaca, New York 14853

A. H. D. BROWN and BERNARD E. PFEIL Centre for Plant Biodiversity Research, CSIRO Plant Industry, Canberra ACT2601, Australia

Communicating Editor: Alan Whittemore

ABSTRACT. Glycine tabacina, a wild perennial relative of soybean, comprises a widespread polyploid complex in Australia and islands of the Pacific Ocean. Data from a single-copy nuclear locus, histone H3-D, confirm the existence of two polyploid races. Plants of one of these (AABЈBЈ) are nonstoloniferous and have linear leaflets. One of the genomes of this race is that of an A-genome diploid, identified by the histone data most closely with a race of G. tomentella. Its other genome (BЈBЈ) was donated by a nonstoloniferous diploid species that is sister to all of the remaining B-genome species, which are stoloniferous. Plants of the second race of polyploid G. tabacina (BBBЈBЈ) are stoloniferous, have ovate leaflets, and combine a BЈ genome with a genome of the core B-genome diploid group. The likely source of the shared BЈ genome is a species previously referred to as G. sp. aff tabacina, that is here formally named Glycine stenophita.

Glycine Willd. subgenus Glycine comprises 16 plastomes (Fig. 1; Doyle et al. 1990b, 1990f). These named diploid (2n ϭ 40) species native to Austra- data support the hypothesis that the two polyploid lia, as well as several polyploid taxa, some of which forms share only one diploid genome. Lack of have colonized islands of the Pacific Ocean. One of cpDNA variation among several of the diploid taxa the widespread polyploid groups, the Glycine taba- having A-haplotypes precluded pinpointing the cina complex, is composed of two morphological maternal genome donor of the linear leaflet (AABB) forms (Costanza and Hymowitz 1987). One of these polyploid. In contrast, of the several chloroplast is stoloniferous with adventitious roots and has haplotypes found among accessions of the ovate ovate to lance-ovate mature leaflets, whereas the leaflet polyploid, six were identical to haplotypes other is nonstoloniferous and has linear mature occurring in the highly polymorphic B-genome dip- lealets. Artificial hybrids between the two forms are loid species group (Doyle et al. 1990d). This led to sterile, with meiotic pairing being about half of that the hypothesis that the BBB2B2 polyploid had at found in the usually fertile individuals produced in least six independent origins, presumably involving crosses between different populations of the same different B-genome diploid taxa (Doyle et al. 1990e). form. Based on artificial hybridizations between Further evidence as to the contributing genomes various diploid species and each of these polyploid of both types was available from restriction site forms (e.g., Singh et al. 1987, 1992) and on other maps of the biparentally inherited 18S–26S nuclear data (e.g., isozymes: Menacio and Hymowitz 1989), ribosomal gene family (nrDNA). Complex restric- Hymowitz et al. (1998) listed the genome formula tion fragment profiles suggested that both poly- of the ovate leaflet form as BBB2B2, and that of the ploid forms were fixed heterozygotes for nrDNA, linear leaflet form as AABB. These genome formu- that they shared one genome, and that the donor of lae were intended to signify that the AABB form this genome was a then-unnamed diploid species was an allopolyploid, whereas the BBB2B2 form was of the B-genome group (referred to as G. sp. aff. a partial or segmental allopolyploid, and that the tabacina by Doyle et al. 1990b, 1990d; here formally two forms shared one genome (Fig. 1). named as G. stenophita, see Appendix 1). It was also The two forms also differ consistently throughout hypothesized from these data that the maternal ge- their geographic range in their chloroplast DNA nome donor of the linear leaflet polyploid was the (cpDNA) haplotypes. Haplotypes of the ovate leaf- D4 diploid isozyme race of G. tomentella, a taxon let (BBB2B2) form belong to the B-plastome group, that has an A-type chloroplast haplotype (Doyle et whereas the linear leaflet (AABB) form has A-type al. 1990b). Ribosomal gene restriction maps in-

437 438 SYSTEMATIC BOTANY [Volume 25

mowitz et al. (1998) who suggested that G. canescens may be the source of the A-genome and G. micro-

phylla or G. latifolia the source of the B- and B2-genomes. Here we address the problem of genome origins in the G. tabacina polyploid complex using DNA sequences of histone H3-D, a single copy nuclear gene that has been shown to be useful in phylogenetic studies of the genus (Doyle et al. 1996, 1999a, 1999b).

MATERIALS AND METHODS

Ten accessions of the linear leaﬂet (AABB; here termed AABЈBЈ for reasons discussed below) polyploid were sampled, spanning its geographic range (Table 1). Four accessions of the ovate leaﬂet

(BBB2B2; henceforth termed BBBЈBЈ) polyploid were FIG. 1. Hypothesis of genomic constitutions and ori- also included in this study. The BBBЈBЈ polyploid gins of two G. tabacina polyploid types. Relationships was the focus of a companion study (Doyle et al. among diploid (2n ϭ 38 or 40) Glycine species are sum- 1999b) that used sequences of histone H3-D alleles marized from histone (Doyle et al. 1996 and unpublished from 40 accessions to test the hypothesis of multi- data) and nrDNA ITS (Nickrent and Doyle 1995; Kollipara ple origins of its maternal (B) genome originally et al. 1997; Singh et al. 1998) results. Shown to the right proposed on the basis of cpDNA results (Doyle et of the tree are chloroplast genome groups (Doyle et. al. Ј Ј 1990a, 1990c, 1990d; the chloroplast genome of G. pindanica al. 1990e). Four of these BBB B accessions were se- has not been studied) and nuclear genome designations lected on the basis of their B-genome histone H3-D (from Kollipara et al. [1997] and Singh et al. [1998], with homoeologue allele and cpDNA haplotype to de- the exception that the G. tomentella D4 isozyme group is termine the sequences of histone alleles at their pu- here assigned the genome symbol A4 to reflect its cyto- tative paternal (BЈ) homoeologous locus. Accessions genetic affinity with A-genome species). The nuclear ge- representing diploid species for which histone H3- nome of G. stenophita (previously called ‘‘G. sp. aff. G. ta- D sequences had not been obtained previously were bacina’’) has not been determined formally but is desig- also included to provide a more complete phylo- nated BЈ here; elsewhere it is referred to as B . The mor- 2 genetic context for the study (Table 1). Other se- phologies and hypothesized genome constitutions of the quences used in these phylogenetic analyses were two classes of G. tabacina polyploid are shown at far right, with arrows connecting them to their respective putative reported previously by Doyle et al. (1996, 1999a). nuclear (dashed arrows) and chloroplast (solid arrows) ge- Polymerase chain reaction (PCR) amplification nome donors. and direct cycle sequencing of PCR products were as described in Doyle et al. (1999a). In most cases, amplifications were performed with a 5Ј primer ferred for the ovate leaflet polyploid were consistent specific for histone H3-D (H3D61f) in combination with its second genome being a member of the B- with a general histone H3 3Ј primer (KV13). In ear- diploid group, but nrDNA variation in that group ly experiments, however, H3D61f was used in con- was insufficient for more precise identification of junction with a 3Ј primer specific for histone H3-D this donor. (H3D553r). These conclusions agreed well with hypotheses In most diploid accessions clean, readable se- based on artificial hybridizations: the linear leaflet quences were obtainable without cloning. Excep- (AABB) type is an allopolyploid in the strict sense tions were G. albicans and G. lactovirens. Cloning of the word, combining A- and B genomes, whereas was necessary to obtain interpretable sequences the ovate leaflet (BBB2B2) type combines two B ge- from these diploids and for the A and BЈ homoeo- nomes and so might be considered a segmental al- logues of most polyploid accessions; the TA or lopolyploid (Fig. 1; Singh et al. 1992; Hymowitz et TOPO-TA cloning kits (Invitrogen) were used ac- al. 1998). However, details of these hypotheses dif- cording to manufacturer’s specifications. The B-ho- fer from those of Singh et al. (1987, 1992) and Hy- moeologue from BBBЈBЈ polyploids was sequenced 2000] DOYLE ET AL.: GLYCINE TABACINA POLYPLOIDY 439

TABLE 1. Taxa and accessions sampled. 1All G. tabacina accessions reported here are polyploid. Designations following G. tomentella are diploid isozyme groups of this species complex. 2‘‘G’’ numbers are CSIRO Perennial Glycine Germplasm Collection numbers (vouchered at CANB); PI ϭ USDA Plant Introduction number. ‘‘A’’ or ‘‘B’’ after G. tabacina polyploid accessions indicate their chloroplast haplotype group. 3Abbreviations: Qld ϭ Queensland; NT ϭ Northern Territory; WA ϭ Western Australia; NSW ϭ New South Wales; Vic ϭ Victoria. 4B-homoeologue alleles are identical to alleles from diploid accessions G. sp. G1077 (GenBank AF093439), G. tabacina G1138 (AF093448), and G. latifolia G1137 (AF093434).

Speciesl Accession2 Provenance3 GenBank #4 G. albicans Tindale and Craven G2049 WA AF220375 G. arenaria Tindale G1305 WA U47396 G. argyrea Tindale G1420 Qld U47397 G. canescens F. J. Herm. G1232 NSW AF220372 G. clandestina Wendl. G1731 Qld U47398 G. curvata Tindale G1849 Qld U47399 G. cyrtoloba Tindale G1267 Qld U47400 G. falcata Benth. PI246591 Qld U47401 G. hirticaulis Tindale and Craven G1956 NT AF220373 G. lactovirens Tindale and Craven G2598 WA AF220376 G. latifolia Newell and Hymowitz G1137 NSW U47404 G. latrobeana Benth. G1251 Vic U47405 G. microphylla (Benth.) Tindale G1309 NSW U47407 G. pindanica Tindale and Craven G2939 WA AF220374 G. stenophita B. Pfeil and Tindale G1510 NSW AF093431 G1974 NSW AF093432 G2223 Qld AF093430 G2600 unknown AF093433 G. tabacina (Labill.) Benth. G1080 B NSW B: AF0934394 BЈ: AF220381 G1255 B NSW B: AF0934394 BЈ: AF220382 G1258 B New Caledonia B: AF0934344 BЈ: AF220383 G1298 A NSW A: AF220384 BЈ: AF220385 G1312 B NSW B: AF0934484 BЈ: AF220386 G1324 B New Caledonia B: AF0934484 BЈ: AF220387 G1326 A Japan A: AF220389 BЈ: AF220388 G1327 A Japan BЈ: AF220390 G1335 A Taiwan A: AF220392 BЈ: AF220391 G1430 A Qld A: AF220393 BЈ: AF220394 G1433 A NSW A: AF220396 BЈ: AF220395 G1828 A Qld A: AF220397 BЈ: AF220398 G1925 A Qld A: AF220399 BЈ: AF220400 G2206 A Qld A: AF220402 BЈ: AF220401 G2234 A Qld A: AF220403 BЈ: AF220404 G. tomentella Hayata (D3) G1403 Qld U47412 G. tomentella Hayata (D4) G1300 Qld AF220377 G1410 Qld AF220378 G1777 Qld AF220379 G2073 Qld AF220380 G. max (L.) Merr. A81-356022 cultivated U47406 440 SYSTEMATIC BOTANY [Volume 25 directly following preferential PCR amplifications 1.5.1 (Goloboff 1993; upgraded 1998) via Clados in which genomic DNA was digested with the re- (Nixon 1993b), followed by TBR branch swapping striction enzyme EcoR-I prior to amplification to on all equally most parsimonious trees. The pri- eliminate the BЈ-homoeologue (Doyle et al. 1999b). mary ratchet analysis included 20 iterations with Sequences of clones were obtained using either 300 cycles per ratchet (NONA settings: mult*1, histone primers or, in some cases, M13r or T7 prim- hold/1, with 10% of characters selected); branch ers targeting sites in the plasmid vector (pCR2.1; swapping continued until tree storage memory was Invitrogen). For diploid taxa, sequences were ob- exhausted. Bootstrap values were obtained for the tained in both directions to provide full overlap- resulting strict consensus tree using NONA, ping coverage of the ca. 500 bp region which con- WinClada (Nixon, pers. comm.), and DADA (Nixon tains all three introns, and hence nearly all of the 1993a). variation. For most polyploids, obtaining sequence in one direction was considered sufficient, given the RESULTS quality of the sequences and the homogeneity observed among alleles at each locus. Similarly, it was All polyploid accessions were eventually found not considered worthwhile to sequence multiple to possess two classes of histone H3-D sequences. clones in most cases. However, for some accessions Initially, histone H3-D amplification products using up to a dozen clones of a single type were pooled, two histone H3-D specific primers were obtained following screening for diagnostic length or restric- from one accession of each of the two types of poly- tion site variation. These pools were sequenced ploid G. tabacina and were sequenced directly. The with histone primers to obtain a sequence repre- sequence from the ovate leaflet (BBBЈBЈ) type, sentative of the entire class; plasmid primers could G1080, gave clear evidence of heterogeneity, consis- not be used because insertion of amplification prod- tent with its being a fixed heterozygote. Homoeol- ucts could have occurred in either orientation. ogous sequences from this and other ovate leaflet Sequences were edited using Sequencher 3.1 polyploids were obtained either by cloning or (for (GeneCodes, Inc.) software. Sequence alignment the B-homoeologue) by preferential amplification used the Clustal V algorithm (Higgins et al. 1992) and direct sequencing (Doyle et al. 1999b). as implemented in the MegAlign component of the In contrast, linear leaflet (AABЈBЈ) accession DNASTAR package. In order to explore the effect G1327 yielded a single clear sequence that was of alignment on phylogenetic results, gap and gap nearly identical to sequences of alleles from one of length parameters were varied as described else- its putative diploid progenitors, G. stenophita, (re- where (Doyle et al. 1996). ferred to in previous work as G. sp. aff. tabacina), a Phylogenetic analyses and estimates of sequence species sister to the remaining B-genome species divergence mainly were conducted using options (Doyle et al. 1990d, 1999a). No product from the available in PAUP* 4.0b2 (Swofford 1998). Gaps anticipated second (A-genome) homoeologue was were coded as ‘‘missing.’’ Numerous exploratory apparent. We therefore modified our amplification parsimony analyses were performed either on the and sequencing strategies, using the H3-D specific complete data matrix or, because of the many near- forward primer (H3D61f) and a more general his- ly identical sequences, on subsets of taxa. Thorough tone H3 reverse primer (KV13; Kanazin et al. 1996) analyses used the branch-and-bound algorithm or located 3Ј of H3-D specific primer H3D553r. With heuristic parsimony searches with numerous (100– H3D553r as a sequencing primer we obtained a 10,000) random addition replicates, retention of and readable sequence of this product, which again was searching on all equally most parsimonious trees, like alleles of G. stenophita. However, only a super- and tree bisection-reconnection (TBR) branch swap- imposed and unreadable sequence was obtained ping. Exploratory analyses used parameter values using the H3D61f primer, indicating that more than designed to limit search times (e.g., MAXTREES set one sequence was in fact present in this plant, and to retain smaller numbers of trees, nearest-neighbor suggesting that difficulties in amplifying and se- interchange [NNI] branch swapping). Various thor- quencing the remaining homoeologue were due to ough or ‘‘fast’’ bootstrap analyses were also per- the H3D553r primer. Inspection of sequences from formed using PAUP*. A-genome diploids revealed that the sequence com- In a second set of analyses, the ratchet method plementary to the H3D553r primer had two nucle- of Nixon (1999) was implemented as a search strat- otide substitutions at the 3Ј end relative to egy with the parsimony program NONA version H3D553r. Thus it appears that this primer amplifies 2000] DOYLE ET AL.: GLYCINE TABACINA POLYPLOIDY 441 the B-homoeologues preferentially. Attempts to The number of taxa was reduced by using a single eliminate the better-amplifying BЈ-homoeologue by accession each for G. stenophita (G1510) and G. to- predigestion of genomic DNA with a restriction en- mentella D4 (G1300) and by selecting representative zyme having a unique recognition site in this ho- sequences from each class of polyploid: both homoeologue (BsmA-I) were unsuccessful, presum- moeologues each from two AABЈBЈ accessions ably because this enzyme is sensitive to methyla- (G1326, G2234) and one BBBЈBЈ accession (G1255). tion and therefore does not digest plant genomic This reduced data matrix (24 OTUs) was subjected DNA efficiently. AABЈBЈ polyploid individuals to a branch-and-bound parsimony search in PAUP*, were therefore amplified with H3D61f and KV13 which identified seven equally most parsimonious and cloned; clones were screened with BsmA-I to trees (Fig. 3: length ϭ 243, consistency index with/ identify homoeologous classes and were sequenced without uninformative characters ϭ 0.88/0.81; reten- with H3D61f, KV13, or plasmid primers. tion index ϭ 0.81). The strict consensus topology was Aligned sequences within each homoeologous compatible with strict consensus topologies obtained class showed little differentiation. All BЈ sequences for searches using the entire data matrix. Results were very similar (0–1.8% Jukes-Cantor corrected from branch-and-bound bootstrapping of this data distances), regardless of whether they were ob- set agreed with less thorough analyses of the full tained from the AABЈBЈ or BBBЈBЈ polyploid. Se- data set, and strongly supported the affinities of each quences of the A-homoeologue differed by 0–3%. of the homoeologues of the polyploids (Fig. 3). As observed previously both for a broad sample of To summarize these results, accessions from both Glycine species (Doyle et al. 1996) and for a more polyploid types possessed sequences that grouped detailed survey of the B-genome (Doyle et al. with alleles from diploid G. stenophita (BЈ-genome) 1999a), nearly all variation was in introns. Both nu- as part of a relatively well-supported BЈ clade, with- cleotide substitutions and short length changes in a more strongly supported B-genome clade. Se- were observed. quences within the BЈ group were not identical, but Phylogenetic analyses of the entire data set pro- strict consensus trees showed few areas of resolu- duced large numbers of equally most parsimonious tion in this clade. Notably, there was no evidence of trees, in part, presumably, due to alternative reso- separate groupings of alleles from the two poly- lutions among the several nearly identical sequenc- ploid types, or for alleles of all polyploids relative es from each homoeologous class obtained from the to those of the diploid. polyploids. For this reason, and to test the effect of The second homoeologues of ovate leaflet poly- alignment order and parameters, several different ploids were also part of this strongly supported B- analyses were performed. genome clade, joining sequences from G. latifolia Thorough parsimony analyses (i.e., TBR branch and G. microphylla, members of the ‘‘core’’ B-ge- swapping on all equally parsimonious trees, nu- nome diploid species group (Doyle et al. 1999a). merous random addition sequences) were initiated This clade was sister to the clade that included G. on the entire (Ͼ50 taxon) dataset, but were aborted stenophita alleles and the BЈ-homoeologue from after saving several thousand equally most parsi- ovate leaflet polyploids. Details of relationships of monious trees. Strict consensus trees from these a broad sample of B-genome alleles from ovate leaf- analyses were consistent with ‘‘fast’’ bootstrap let polyploids with alleles from various core B-ge- trees for the same data sets. As reported for a nome accessions are discussed elsewhere (Doyle et smaller sample of Glycine species (Doyle et al. 1996), al. 1999b). topologies were not sensitive to differences in align- The second class of histone H3-D sequences in ment, but bootstrap support for clades varied linear leaflet polyploids joined alleles from diploid somewhat with alignment. An alternative parsi- accessions of the 2n ϭ 40 D4 isozyme group of G. mony approach was taken, implementing the very tomentella. Sequences from seven of the nine poly- fast and thorough ‘‘ratchet’’ search strategy of Nix- ploid accessions for which sequences of this class on (1999) in NONA (Goloboff 1993). Here, too, the were obtained formed an unresolved polytomy that search was stopped after many (Ͼ20,000) equally included alleles from these G. tomentella accessions. most parsimonious trees were identified. The strict Alleles of the remaining two polyploid accessions consensus topology was identical to those found in (G1298, G1430) formed a strongly supported group PAUP runs (Fig. 2). The various parsimony analyses sister to this clade. The entire group of alleles from agreed in identifying groups of sequences likely to the polyploid and from G. tomentella D4 dipoids be well-supported in complete searches. formed one of three unresolved components of the 442 SYSTEMATIC BOTANY [Volume 25

FIG. 2. Strict consensus topology for histone H3-D sequences identiﬁed in all parsimony analyses of the full data set, regardless of alignment strategy. Only taxa for which multiple accessions were sampled are given by their CSIRO ‘‘G’’ numbers; for cloned sequences, clone numbers are given after the accession number; for polyploids, ‘‘a’’, ‘‘b’’, or ‘‘b’’’ after the clone number refers to genome source. Numbers along branches are bootstrap values from a PAUP* ‘‘fast’’ bootstrap analysis. Shown to the right of the tree are the nuclear and chloroplast genome designations as in Fig. 1. relatively well-supported A-genome allele group on H-genome species (G. pindanica, G. arenaria, and G. shortest trees, the other two being the allele from hirticaulis) formed a well-supported clade, joined by G. latrobeana and a clade comprising alleles from G. a sequence fromaDgenome G. tomentella accession. argyrea, G. clandestina, and G. canescens. The overall This clade was strongly supported as sister to a topology, as well as the placement of the D4 iso- clade comprising sequences from the two I-genome zyme group of G. tomentella as part of the A-genome species (G. albicans and G. lactovirens), with this en- clade, is consistent with recent nrDNA ITS results tire group (D-, I-, and H-genomes) sister to the A- (Kollipara et al. 1997). genome group (Figs. 2, 3). In the thorough parsi- Species for which histone H3-D sequences were mony analysis performed on a subset of alleles not previously available also showed relationships from polyploids, alleles of the C-genome species (G. similar to those found in studies of the nrDNA ITS curvata and G. cytroloba) were well-supported as sis- region (Kollipara et al. 1997). Sequences from the ter to the A/D/H/I genome clade, and the F-ge- 2000] DOYLE ET AL.: GLYCINE TABACINA POLYPLOIDY 443

FIG. 3. Topology of one of seven equally most parsimonious trees from a reduced dataset, using representative accessions of G. tabacina polyploids, G. tomentella D4, and G. stenophita. Branch lengths are proportional to the amount of character change, with number of changes (using accelerated transformation optimization in PAUP*) given above each branch. Bootstrap values from a branch-and-bound bootstrap analysis (100 replicates) are shown in italic print below branches. Nodes without bootstrap values collapse in the strict consensus tree. Genome designations as in Fig. 1. nome species, G. falcata, was strongly supported as 1990b). The second genome of the ovate leaﬂet sister to the remainder of subgenus Glycine. polyploid is designated BiBi because it appears likely that several different diploid taxa with B-type

DISCUSSION genomes (B1B1,B2B2, etc.) contributed genomes to it Results from histone H3-D variation support the based on the presence, in different polyploid acces- hypothesis that the two classes of G. tabacina poly- sions, of at least six core cpDNA haplotypes found ploids are both fixed heterozygotes, and that they among diploids of the B-genome group (Doyle et share only one of their two genomes. Furthermore, al. 1990e). Although identifying cpDNA haplotypes the results support hypotheses from cpDNA and shared between diploids and polyploids was a sim- nrDNA restriction maps concerning the identities ple matter, determining the diploid taxa involved is of these various genomes. Genome designations made more complex by discordance between cp- DNA and histone H3-D variation in the diploids suggested for the two polyploids are A4A4BЈBЈ for (Doyle et al. 1999a). The data reported here for the the linear leaflet type, and BiBiBЈBЈ for the ovate leaflet type, for reasons detailed below. B-homoeologue are a subset from a thorough sur- The shared BЈBЈ genome is derived from G. sten- vey of this locus in the polyploids; the alleles at this ophita, a diploid whose position as sister to the re- locus have been shown to be identical with three maining members of the B-genome is indicated by of the 19 alleles found in core B-genome diploids histone H3-D topologies (Figs. 2, 3; Doyle et al. (Doyle et al. 1999b). 1996, 1999a), cpDNA haplotype trees (Doyle et al. Of the known diploid A-genome species, the

1990d), and by its lacking the adventitious roots most closely related to the A4A4 genome is the D4 characteristic of other B-genome diploids (Doyle et euploid isozyme race of G. tomentella. Glycine tomen- al. 1990d). This newly described species appears to tella is a complex that includes euploid and aneu- have been the paternal progenitor of both polyploid ploid cytotypes at both the diploid and tetraploid types, based on the absence of stenophita (BЈ) levels (2n ϭ 38, 40, 78, 80; M. Doyle et al. 1986). cpDNA haplotypes in either polyploid (Doyle et al. The D4 isozyme group also has the appropriate 444 SYSTEMATIC BOTANY [Volume 25 cpDNA haplotype (from the A-plastome group: the ovate leaflet polyploid may be a true allopoly- Doyle et al. 1990a) to be the maternal progenitor of ploid rather than a classic segmental polyploid. this polyploid, although this haplotype is shared by Concerning the linear leaflet polyploid types several other diploid taxa (Figs. 1–3). However, its (lacking adventitious roots) Hymowitz et al. (1998) nrDNA restriction map is the only one of over 150 concluded that these ‘‘are true allotetraploids and Glycine subg. Glycine accessions surveyed that may constitute any combination of A- and B-ge- matched that of the linear leaflet polyploid (Doyle nome species.’’ It is true that the cross involved in et al. 1990b). Singh et al. (1998) have assigned the forming the linear leaflet polyploid is broader than genome symbol D3 to this group. However this those that formed the ovate leaflet type. However, choice does not reflect the fact that the D4 isozyme as with the ovate leaflet polyploid, evidence to date race is more closely related cytogenetically to A- suggests that only a specific combination of ge- genome species than to other races of G. tomentella. nomes was involved. Once again, only G. stenophita– For example, meioses in hybrids between G. tomen- not ‘‘any’’ B-genome species, and not even a core tella (D4) and the A-genome species G. clandestina B-genome species–is implicated as pollen parent. show 14 bivalents (out of a possible 20) whereas With the resolution provided by histone H3-D data those between G. tomentella (D4) and G. tomentella that was not available from cpDNA restriction (D3; E-genome) show only four bivalents (Grant et maps, it is clear that a particular A-genome taxon al. 1984, 1986; M. Doyle et al. 1986). was involved as egg parent, and not other known Our genome hypotheses for polyploid G. tabacina A-genome diploid species (G. argyrea, G. canescens, differ from those of other workers (e.g., Singh et al. G. clandestina, or G. latrobeana). 1992). Hymowitz et al. (1998) state that the ovate We observed sequence variation at both homoe- leaflet type (with adventitious roots) G. tabacina ologous histone H3-D loci in both polyploids. For polyploid possesses genomes that ‘‘may be in any the B-homoeologue of the ovate leaf (BiBiBЈBЈ) poly- possible combinations (BBB1B1, BBB2B2,B1B1B2B2), ploid, this is consistent with the multiple origin involving only B-genome diploid species.’’ Their model hypothesized from cpDNA and from the hypothesis overlooks the fact that although numer- more extensive survey of histone H3-D alleles of B- ous core B-genome diploid taxa do indeed appear genome diploids and of this polyploid (Doyle et al. to be involved in the origin of the ovate leaflet poly- 1996, 1999a, 1999b). Sampling of G. stenophita has ploid, this group of species has contributed only not been extensive, but the grouping of the allele one of the two homoeologous genomes of the poly- from one accession of this species (G2223) with the ploid. All members of the ovate leaflet polyploid BЈ-homoeologue of only one G. tabacina BiBiBЈBЈ ac- derive their other (presumably paternal) genome cession (G1080) suggests that this locus may also from the earliest diverging member of the B-ge- provide evidence of multiple origins. Understand- nome group, G. stenophita. Thus, the ovate leaflet ing the origins of the A homoeologue of the AABЈBЈ polyploid includes a specific combination of ge- polyploid will require additional sampling of dip- nomes not hypothesized by these workers. More- loid G. tomentella. Unlike the B-homoeologue group over, G. stenophita does not appear to have been (Doyle et al. 1999a, b) we did not observe multiple used in any studies by the Hymowitz group, and subclades within the BЈ group in which alleles from thus does not have a genome designation. Although different polyploid accessions each grouped with a in our previous work (e.g., Doyle et al. 1990b) we different allele from a diploid accession and pro- refer to its genome as B2B2, this is not the same as vided strong evidence of multiple origins of the the B2B2 of Hymowitz et al. (1998), which instead polyploid. However, the divergent nature of the A- is a diploid stoloniferous, ovate leaflet form of dip- homoeologue alleles from two polyploid accessions loid G. tabacina (PI 373990 ϭ G1317). To clarify re- (G1298 and G1430) suggests that these alleles were lationships we therefore designate the genome of G. contributed by a genome donor separate from that stenophita ‘‘BЈ’’, in recognition of its unique position (or those) involved in other polyploid accessions. within the diploid B-genome group. Most acces- The relatively low level of sequence differentia- sions of G. stenophita have linear leaflets and are tion observed between diploids and polyploids is nonstoloniferous, and although they possess a sim- consistent with a very recent origin and range ex- ilar leaf venation to other B-genome species, this pansion of both polyploids. In the case of the B- taxon may in fact deserve a separate genome des- genome, all three alleles were identical with alleles ignation. Although detailed cytological studies in- sampled from diploid accessions (Doyle et al. volving G. stenophita have not yet been conducted, 1999a, 1999b), and the age of the taxon was esti- 2000] DOYLE ET AL.: GLYCINE TABACINA POLYPLOIDY 445 mated to be less than 30,000 years (Doyle et al. DNA. Australian Journal of Systematic Botany 3: 125– 1999b). The generally small differences between 136. diploids and polyploids at the BЈ or A H3-D histone , ,and . 1990c. Chloroplast DNA phy- loci could be due to artifacts of sampling or of PCR logenetic affinities of newly described species in Gly- cine (Leguminosae: Phaseoleae). Systematic Botany and cloning procedures. 15: 466–471. The geographical distributions of both G. tabacina , ,and . 1990d. Chloroplast DNA polyploids suggest that they are active colonizers. polymorphism and phylogeny in the B-genome of In Australia, the linear leaflet type has spread well Glycine subgenus Glycine (Leguminosae). American outside the rather narrow, nearly allopatric current Journal of Botany. 77: 772–782. ranges of its hypothesized progenitors (Doyle et al. , ,and . 1999a. Incongruence in the 1990b, 1990f). It is the more tropical of the two diploid B-genome species complex of Glycine (Legu- polyploid types, occurring both well north of these minosae) revisited: Histone H3-D alleles vs. chloro- diploids into the Northern Territory, and also south plast haplotypes. Molecular Biology and Evolution 16: 354–362. into New South Wales (Doyle et al. 1990b). The B- , ,and . 1999b. Origins, colonization, genome diploid progenitors of the ovate leaflet and lineage recombination in a widespread perennial polyploid (including G. stenophita) are plants of soybean species complex. Proceedings of the National temperate eastern Australia, and this polyploid for Academy of Sciences USA 19: 10741–10745. the most part is confined to their cumulative range. , , ,andJ.P.GRACE. 1990e. Multiple However, both polyploids have colonized islands of origins of polyploids in the Glycine tabacina complex the West-central Pacific (Taiwan, Ryukyu Is.). Inter- inferred from chloroplast DNA polymorphism. Pro- estingly, it is the ovate leaflet type that has colo- ceedings of the National Academy of Sciences USA nized islands of the tropical South Pacific (Vanuatu, 87: 714–717. , ,J.P.GRACE, and A. H. D. BROWN. 1990f. Fiji), despite having a more temperate distribution Reproductively isolated polyploid races of Glycine ta- in Australia (see Doyle et al. 1990b and 1990f for bacina (Leguminosae) had different chloroplast ge- details of ranges). nome donors. Systematic Botany 15: 173–181. Given these distributions, there is no evidence ,V.KANAZIN, and R. C. SHOEMAKER. 1996. Phylo- that the genetic distance of the original hybridiza- genetic utility of histone H3 intron sequences in the tion event has made any difference in colonizing perennial relatives of soybean (Glycine: Leguminosae). ability. Perhaps this is because although the linear Molecular Phylogenetics and Evolution 6: 438–447. leaflet type combines genomes that are more di- DOYLE, M. J., J. E. GRANT, and A. H. D. BROWN. 1986. verged from one another, there is greater genetic Reproductive isolation between isozyme groups of diversity within the ovate leaflet type due to mul- Glycine tomentella (Leguminosae), and spontaneous doubling in their hybrids. Australian Journal of Bot- tiple contributions from diverse B-genome diploids. any 34: 523–535. GOLOBOFF, P. A. 1993. Estimating character weights during ACKNOWLEDGEMENTS. The authors thank Donovan tree search. Cladistics 9:83–91. Bailey for assistance with phylogenetic analyses, Kevin GRANT,J.E.,P.GRACE,A.H.D.BROWN, and E. PUTIEVSKY. Nixon for making phylogenetic analysis programs avail- 1984. Interspecific hybridization in Glycine Willd. sub- able, and Lyn Craven for taxonomic insights. We are grate- genus Glycine (Leguminosae) Australian Journal of ful to two anonymous reviewers for helpful comments. Botany 32: 655–663. Work was supported by NSF DEB 9614984 to JJD. ,R.PULLEN,A.H.D.BROWN,J.P.GRACE,andP. M. GRESSHOFF. 1986. Cytogenetic affinity between the LITERATURE CITED new species Glycine argyrea and its congeners. Journal of Heredity 77: 423–426. COSTANZA,S.H.andT.HYMOWITZ. 1987. Adventitious HIGGINS, D. G., A. J. BLEASBY, and R. FUCHS. 1992. CLUS- roots in Glycine subgenus Glycine (Leguminosae): TAL V: Improved software for multiple sequence morphological and taxonomic indicators of the B ge- alignment. CABIOS 8: 189–191. nome. Plant Systematics and Evolution 158: 37–46. HYMOWITZ, T., R. J. SINGH, and K. P. KOLLIPARA. 1998. The DOYLE, J. J., J. L. DOYLE, and A. H. D. BROWN. 1990a. A genomes of Glycine. Plant Breeding Reviews 16: 289– chloroplast DNA phylogeny of the wild perennial rel- 317. atives of soybean (Glycine subgenus Glycine): congru- KANAZIN,V,T.BLAKE, and R. C. SHOEMAKER. 1996. Or- ence with morphological and crossing groups. Evo- ganization of the histone H3 genes in soybean, barley, lution 44: 371–389. and wheat. Molecular and General Genetics 250: 137– , ,and . 1990b. Analysis of a polyploid 147. complex in Glycine with chloroplast and nuclear KOLLIPARA,K.P.,R.J.SINGH,andT.HYMOWITZ. 1997. 446 SYSTEMATIC BOTANY [Volume 25

Phylogenetic and genomic relationships in the genus polyploid’ overlap considerably; however the more Glycine Willd. based on sequences from the ITS region useful characters in differentiating these species from of nuclear rDNA. Genome 40:57–68. each other (and other Glycine species) have been in- MENACIO,D.I.andT.HYMOWITZ. 1989. Isozyme variation dicated in bold type in the description. between diploid and tetraploid cytotypes of Glycine tabacina (Labill.) Benth. Euphytica 42:79–87. Glycine stenophita B.Pfeil and Tindale, sp. nov. NEWELL, C.A. and T. HYMOWITZ. 1978. A reappraisal of (Fig. 4).—TYPE: Australia, Australian Capital the subgenus Glycine. American Journal of Botany 65: Territory: Cultivated in greenhouse at CSIRO 168–179. Plant Industry, Black Mountain, Canberra; 27 NICKRENT,D.L.andJ.J.DOYLE. 1995. A molecular phylogeny of diploid Glycine (Fabaceae) based upon nu- Jul 1999, L.A. Craven & J.P. Grace 10083 (ϭ clear ribosomal ITS sequences. American Journal of G1974) (holotype: CANB; isotype: BRI, NSW). Botany 82: s153 (abstract). [Provenance: 38 km from Castlereagh High- NIXON, K. C. 1993a. DADA matrix editing program. Pub- way to Warrumbungle National Park, N.S.W., lished by author, L.H. Bailey Hortorium, 462 Mann Australia (310 20 ϭ S 1480 45 ϭ E),leg. Grace Library, Cornell University, Ithaca, New York, 14853. & Speer.] . 1993b. Clados computer software version 1.4. Pub- lished by author. L.H. Bailey Hortorium, 462 Mann Glycine stenophita B.Pfeil et Tindale: a G. latifolia Library, Cornell University, Ithaca, New York, 14853. Newell et Hymowitz stolonibus carentibus, foliolis . 1999. The Parsimony Ratchet, a new method for angustioribus usque ad 8 mm latis plerumque 7– rapid parsimony analysis. Cladistics 15: 407–414. 16-plo longioribus quam latioribus, pilis pro parte SINGH, R. J., K. P. KOLLIPARA,andT.HYMOWITZ. 1987. maxima albis differt. Polyploid complexes of Glycine tabacina (Labill.) Scrambling or climbing perennial herb, non-sto- Benth. and G. tomentella Hayata revealed by cytogenetic analysis. Genome 29: 490–497. loniferous. Stem hairs white, appressed to ascend- , ,and . 1998. The genomes of Glycine ing (rarely ascending-spreading), absent to sparse; canescens F. J. Herm., and G. tomentella Hayata of west- stipules to 3 mm long and 1.5 mm wide, not fused, ern Australia and their phylogenetic relationships in or sometimes fused on one side, the hairs white, the genus Glycine Willd. Genome 41: 669–679. appressed, absent to sparse. Petioles to 40 mm long, ,K.P.KOLLIPARA,F.AHMAD,andT.HYMOWITZ. the hairs white, appressed to ascending, absent to 1992. Putative diploid ancestors of 80-chromosome sparse. Leaves pinnately trifoliolate. Lateral stipels Glycine tabacina. Genome 35: 140–146. to 1.25 mm long and 0.5 mm wide; terminal stipels SWOFFORD, D. L. 1998. PAUP*. Phylogenetic Analysis Us- present, up to 1 mm long and 0.5 mm wide; lateral ing Parsimony (*and other methods). Sunderland, and terminal stipel hairs white, appressed to as- MA: Sinauer Associates. Appendix 1: Description of a new species of Glycine Willd. cending, absent (or nearly so). Petiolules to 1 mm The following is a diagnosis and description of a new long, the hairs white, ascending-spreading to Australian species of Glycine Willd. subgenus Glycine, spreading, sparse. Terminal rachis to 7 mm long, G. stenophita (Fig. 4). This species has been previously the hairs white, appressed, absent to sparse. Leaf- referred to as ‘Glycine sp. aff. tabacina’ (Doyle et al. lets to 80 mm long and 8 mm wide, narrowly ovate, 1990b, d; AABЈBЈ polyploid of this paper). The epi- narrowly elliptic or linear, the margin flat, the apex thet ‘stenophita’ is arbitrarily derived from two Greek acute and mucronulate; terminal leaflets usually words, stenos (narrow) and phitys (begetter), which slightly larger than lateral leaflets. Lateral leaflet allude to this species being one of the diploid parents length:width ratio (5-)7–12(-16) (usually around 10: of the ‘G. tabacina A-type polyploid’ cf. Doyle et al. 1). Terminal leaflet length:width ratio (6-)9–16(-20) (1990b). Characters that have been used to separate these species include the different ploidy levels, as (usually around 12:1). Leaflet venation brochidod- well as diagnostic electrophoretic mobility differences romous; secondary veins clearly visible; typically 0 of Endopeptidase (Enp) and Isocitric Dehydrogenase 40–80 from mid-vein; leaflet reticulation often (Idh) isozymes (Brown, unpub.). Many morphological clearly visible; moderately dense. Leaflet hairs: leaf- characters of this species and the ‘G. tabacina A-type let abaxial mid-vein and lamina hairs white, ap-

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FIG. 4. Top: Photograph of the holotype specimen of Glycine stenophita B. Pfeil and Tindale with scale bar. Bottom: the two tetraploid races of Glycine tabacina, with the linear-leaﬂet A-type on the left and the ovate-leaﬂet B-type on the right, both to the same scale as the G. stenophita specimen. 2000] DOYLE ET AL.: GLYCINE TABACINA POLYPLOIDY 447 448 SYSTEMATIC BOTANY [Volume 25

pressed to ascending, sparse; abaxial lamina hairs sometimes or partly twisted); the legume hairs usually confined to the minor veins; lamina hair white (or rarely pale rusty), appressed, sparse. density about equal to mid-vein hair density. Leaf- Seeds (3-)4–6(-7) per legume. let adaxial mid-vein and lamina hairs white, ap- Seed color uniform or mottled; overall barrel- pressed, absent to sparse; adaxial lamina hairs con- shaped but irregularly so (varying from a square, fined to the minor veins; lamina hair density less rectangle or parallelogram, to a truncated ellipse in than mid-vein hair density. side view, and from circular to elliptical in front Inflorescences loosely racemose, flowers spread view), with a slight point near the hilum; surface along the peduncle and clustering towards the usually rough (the perisperm adheres), tubercles apex; each bract subtending a single flower. Pedun- obscure and stellate or irregularly circular in out- cles to 100 mm long, the hairs white, appressed, line; surface reticulation indistinct. absent to sparse; bracts to 1.75 mm long and 0.5 Cytological Data. USDA 378705 (ϭ G2600) 2n mm wide, the hairs white, appressed, sparse. Ped- ϭ 40 (Newell and Hymowitz 1978) Unknown Ori- icels to 1 mm long, the hairs white, appressed to gin Representative Specimens Examined. ascending, sparse; bracteoles to 1.25 mm long and Australia. Queensland: Comet River, 2 km West of Comet, 14 0.5 mm wide, the hairs white, appressed, absent to Aug 1985, Grace, et al. 236 (BRI, CANB, K, NSW); sparse; bracteoles inserted below the base of the 20 km NW of Jondaryan, 11 Apr 1994, Fensham 1353 calyx. Calyx to 2.5 mm long and 2 mm wide, the (BRI); Kogan, 2 Apr 1979, McHarg 785 (BRI). New sinus between the 2 adaxial teeth usually to 0.5 mm South Wales: 12 km SE of Inverell, 9 Feb 1993, Dodds long (sometimes to 0.75 mm long); the calyx hairs 41 (NE); Warrabah National Park, 21 Feb 1990, Hosk- white, appressed, sparse. Corolla pink, pale purple ing 3767 (NE). Cultivated in A.C.T: 9 Mar 1990, or purple; the standard 7–8 mm long and 6–8 mm Grace 574 (ϭ G2217) (BRI, CANB, NSW), prove- wide, with lobes on the lower edge; wings 7–8 mm nance: 2.4 km East of Morven, Qld.; 25 Jul 1989, long; keel 5–6 mm long. Carpel: stigmatic hairs of- Grace 436 (ϭ G1651) (BRI, CANB, NSW), prove- ten present; style tapers gradually; tip not elongat- nance: 15 km from Narrabri on Newell Highway, ed; ovary hairs not forming a collar. N.S.W. ϾG ϭ numbers refer to accessions in the Mature chasmogamous legumes not seen. Cleis- CSIRO Perennial Glycine Germplasm Collection. togamous legumes solitary in leaf axils, 15 to 30 Distribution. Eastern Australia: on the Great mm long (usually shorter than 28 mm at dehis- Dividing Range in southern Queensland and north- cence), 3 to 4 mm wide, brown when mature, ini- ern N.S.W.; south from Rockhampton to Gilgandra, tially straight and remaining so after dehiscence (or east from Augathella to Gympie.

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