Genetic diversity and phylogeny in Hystrix (Poaceae, Triticeae) and related genera inferred from Giemsa C-banded karyotypes.

The phylogenetic relationships of 15 taxa from Hystrix and the related genera Leymus (NsXm), Elymus (StH), Pseudoroegneria (St), Hordeum (H), Psathyrostachys (Ns), and Thinopyrum (E) were examined by using the Giemsa C-banded karyotype. The Hy. patula C-banding pattern was similar to those of Elymus species, whereas C-banding patterns of the other Hystrix species were similar to those of Leymus species. The results suggest high genetic diversity within Hystrix, and support treating Hy. patula as E. hystrix L., and transferring Hy. coreana, Hy. duthiei ssp. duthiei and Hy. duthiei ssp. longearistata to the genus Leymus. On comparing C-banding patterns of Elymus species with their diploid ancestors (Pseudoroegneria and Hordeum), there are indications that certain chromosomal re-arrangements had previously occurred in the St and H genomes. Furthermore, a comparison of the C-banding patterns of the Hystrix and Leymus species with the potential diploid progenitors (Psathyrostachys and Thinopyrum) suggests that Hy. coreana and some Leymus species are closely related to the Ns genome of Psathyrostachys, whereas Hy. duthiei ssp. duthiei, Hy. duthiei ssp. longearistata and some of the Leymus species have a close relationship with the E genome. The results suggest a multiple origin of the polyploid genera Hystrix and Leymus.

Introduction

Hystrix Moench is a small perennial genus of the tribe Triticeae (Poaceae). Moench (1794) established the genus Hystrix with Hy. patula Moench as the type-species through its distinctive morphological character of either lacking glumes entirely or, if present, of possessing long setaceous awn-shaped ones. Since then, 11 species have been included in Hystrix (Hitchcock, 1951; Bor, 1960; Tzvelev, 1976; Kuo, 1987; Osada, 1993). Baden revised the genus and recognized six species, one of which is divided into three subspecies within Hystrix. All are tetraploids (2n = 4x = 28) except for Hy. californica, which is an octaploid (2n = 8x = 56). The natural distribution of Hystrix is disjunct with two species in North America (Hy. patula and Hy. californica), and the remainder in Central and Eastern Asia (Löve, 1984; Baden ).

Although separated early as a genus in its own right, the recognition of Hystrix has been controversial ever since its establishment. Church (1967a, 1967b) reported that Hy. patula had a close affinity to species of the Elymus canadensis complex and treated Hy. patula as E. hystrix L. Consequently, Dewey (1982) and Löve (1984) recognized the genus Hystrix as a section of Elymus. However, Jensen and Wang (1997) reported that two species of Hystrix, Hy. coreana and Hy. californica, shared the genome of Leymus (NsXm), and so transferred Hy. coreana from Hystrix to Leymus. Based on the results of studies on meiotic pairing and genomic in situ hybridization (GISH), Zhang reported that Hy. patula possesses the ElymusStH genome, whereas Hy. duthiei ssp. duthiei and Hy. duthiei ssp. longearistata contain the LeymusNsXm genome. However, based on results of GISH and southern genomic hybridization, Ellenskog-Staam reported that Hy. coreana, Hy. longearistata and Hy. duthiei contained the Ns1Ns2 genomes, while Hy. patula contained the StH genomes, and Hy. komarovii most likely had a variant of the StH genomes. Although the varied genomic constitution of Hystrix species has been reported, morphological similarities have likewise occurred, such as the loosely tufted caespitose, relatively high culms, broadly lanceolate leaves and the obsolete, reduced, or setaceous glumes. Thus, there is every indication that genome relationships and genetic diversity among Hystrix species need to be further investigated.

Karyotype analysis is considered to be an important method in genome analysis. Giemsa C-banded karyotyping stains constitutive heterochromatin, this resulting in unique banding patterns of individual chromosomes. This process thus provides more accurate evidence for identifying homologous chromosomes in karyologically similar species, thereby complementing studies of genome evolution among related species (Morris and Gill, 1987). Baden undertook a pilot study on Giemsa C-banding patterns in Hy. patula, Hy. komarovii and Hy. coreana, and the results showed Hy. coreana as having large and conspicuous telomeric bands, different from the C-banding patterns of Hy. patula and Hy. komarovii. In this study, we investigated the genetic diversity among Hystrix species, as well as the phylogenetic relationship between Hystrix and its relatives (including related genera and their diploid ancestors) by using the Giemsa C-banded karyotype. The specific objectives were: (a) to report the Giemsa-C banded karyotypes of 15 perennial taxa in Triticeae representing nine genera; (b) to estimate genetic diversity among these perennial species; (c) to compare C-banding patterns among the diploid and tetraploid species; and (d) to explore the possible diploid ancestor of Hystrix species.

Materials and Methods

Plant material

A total of 15 taxa in Triticeae were used in this study, including four taxa of Hystrix (2n = 4x = 28), three species of Leymus (2n = 4x = 28, NsXm genome), two species of Elymus (2n = 4x = 28, StH genome), two species of Pseudoroegneria (2n = 2x = 14, St genome), two species of Psathyrostachys (2n = 2x = 14, Ns genome), Hordeum bogdanii (2n = 2x = 14, H genome), and Thinopyrum bessarabicum (2n = 2x = 14, Eb genome) (Table 1). All seed material was collected in the field by the authors of this paper or kindly provided by the American National Plant Germplasm System (Pullman, Washington, USA) and Dr. S. Sakamoto (Kyoto University, Japan). Voucher specimens were deposited in the Herbarium of the Triticeae Research Institute, Sichuan Agricultural University, China (SAUTI).

Table 1

- Species of Hystrix and other closely related genera used in Giemsa-C banding analysis.

Species	2n	Genome	Accession n.	Origin
Pseudoroegneria libanotica (Hack.) D. R. Dewey	14	St	PI 228391	Ardabil, Iran
Pseudoroegneria spicata (Pursh) Á. Löve	14	St	PI 232138	Montana, United States
Hordeum bogdanii Wilensky	14	H	Y 1508	Xinjiang, China
Psathyrostachys huashanica Keng ex Kuo	14	Ns	ZY 3157	Shanxi, China
Psathyrostachys juncea (Fisch.) Nevski	14	Ns	PI 430871	Former Soviet Union
Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve	14	Eb	PI 531711	Crimea, Ukraine
Elymus sibiricus L.	28	StH	ZY 3041	Sichuan, China
Elymus canadensis L.	28	StH	PI 236805	Canada
Leymus arenarius (L.) Hochst.	28	NsXm	PI 272126	Alma-Ata, Kazakhstan
Leymus racemosus	28	NsXm	PI 478832	Montana, United States
Leymus multicaulis (Kar. & Kir.) Tzvelev	28	NsXm	PI 440325	Dzhambul, Kazakhstan
Hystrix patula Moench	28	StH	PI 372546	Ottawa, Canada
Hystrix coreana (Honda) Ohwi	28	NsXm	W6 14259	Vladivostack, Russian Federation
Hystrix duthiei (Stapf) Bor ssp. duthiei	28	NsXm	ZY 2004	Sichuan, China
Hystrix duthiei ssp. longearistata (Hack.) Baden, Fred. & Seberg	28	NsXm	ZY 2005	Tokyo, Japan

Giemsa C-banding analysis

All seeds were germinated in Petri dishes on moistened filter paper at 22 °C. Root tips from the germinating seeds were pre-treated in ice-cold water for 24-28 h, fixed in ethanol: acetic acid (3:1, v/v) for 24 h at room temperature, and then stored in the refrigerator. Each root tip was squashed in a drop of 45% acetic acid.

The Giemsa C-banding technique followed the procedure of Gill . Metaphase cells with a complete chromosome complement were photographed, five cells being subsequently analyzed for each material. Idiograms so constructed were based on chromosome lengths, similarities in their morphology, banding patterns and relative arm-ratios. Chromosomes were arranged from the longest to the shortest and were designated with the Arabic numerals 1-7 in diploids and 1-14 in tetraploid species.

Results

The Giemsa-C banded metaphase chromosomes in representative species of Hystrix and relatives are shown in Figure 1. The C-banded karyotypes and ideograms of all the taxa in Hystrix and related genera are shown in Figures 2 and 3, respectively.

Figure 1

Giemsa-C banded metaphase chromosomes in representative species of Hystrix and relatives. a.Hystrix patula. b.Hy. duthiei ssp. longearistata. c.Hy. coreana. d.Leymus arenarius.e.L. racemosus.f.Pseudoroegneria libanotica. g.Hordeum bogdanii. h.Psathyrostachys juncea. Bar = 10 μm.

Figure 2

C-banded karyotypes in 15 taxa of Hystrix, Elymus, Leymus,Pseudoroegneria, Hordeum, Psathyrostachys and Thinopyrum. a.Hystrix patula. b. Hy. duthiei ssp. duthiei. c.Hy.duthiei ssp. longearistata. d.Hy. coreana. e.Elymus sibiricus. f.E. canadensis. C-banded karyotypes in 15 taxa of Hystrix, Elymus, Leymus,Pseudoroegneria, Hordeum, Psathyrostachys and Thinopyrum. g.Leymus arenarius. h.L. racemosus. i.L. multicaulis. j.Pseudoroegneria spicata. k.Pse. libanotica. l.Hordeum bogdanii. m.Psathyrostachys juncea. n.Psa. huashanica. o.Thinopyrum bessarabicum.

Figure 3

Ideograms in 15 taxa of Hystrix, Elymus, Leymus,Pseudoroegneria, Hordeum, Psathyrostachys and Thinopyrum. a.Hystrix patula. b.Hy. duthiei ssp. duthiei. c.Hy.duthiei ssp. longearistata. d.Hy. coreana. e.Elymus sibiricus. f.E. canadensis. g.Leymus arenarius. h.L. racemosus. i.L. multicaulis. j.Pseudoroegneria spicata. k.Pse. libanotica. l.Hordeum bogdanii. m.Psathyrostachys juncea. n.Psa. huashanica. o.Thinopyrum bessarabicum.

C-banding in Hystrix species

There were small telomeric bands in all of the 14 chromosomes in Hy. patula most of which with minor intercalary bands (Figure 1a). Large centromeric bands were also present in both arms of chromosomes 7, 8 and 9 (Figures 2a, 3a).

Hy. duthiei ssp. duthiei and Hy. duthiei ssp. longearistata revealed similar basic C-banding patterns. All the 14 chromosomes presented minor to small terminal and centromeric C-bands, except for chromosome 2 of Hy. duthiei ssp. duthiei, where centromeric bands were absent (Figures 2b, 2c). In Hy. duthiei ssp. longearistata, nine of the fourteen chromosomes contained minor interstitial bands, whereas these were present in only four of the 14 chromosomes in Hy. duthiei ssp. duthiei (Figures 3b, 3c).

Large terminal bands in all the 14 chromosomes were characteristic of the C-banding pattern in Hy. coreana. Except for a minor interstitial band in chromosome 2, no centromeric or interstitial bands were encountered in this species (Figures 1c, 3d).

C-banding in Elymus (StH), Pseudoroegneria (St) and Hordeum (H) species

Giemsa-C banded karyotypes in two Elymus species containing the StH genome were comprised of terminal, interstitial and a few centromeric bands. In E. sibiricus, small to medium terminal C-bands were observed in one or both arms of all the chromosomes, besides rather large interstitial bands in chromosomes 9-13 (Figures2e, 3e). In E. canadensis, all the chromosomes presented terminal C-bands in one or both arms. Furthermore, distinct bands were located near the centromere in both arms of chromosome 10, besides two pairs of chromosomes containing centromeric bands (Figures 2f, 3f). The banding pattern of E. canadensis is similar to that of Hy. patula.

In Pseudoroegneriaspicata (St) and Pse. libanotica (St), the C-banding patterns were rather similar, being characterized by large terminal bands in both arms or only in the short arm of all the seven chromosomes. Centromeric bands were found in chromosomes 1, 2 and 7 in Pse. spicata (Figures 2j, 3j), whereas in Pse. libanotica these were observed in chromosomes 1, 4, 6 and 7 (Figures 2k, 3k).

In H. bogdanii (H), small to medium interstitial bands were present in one or both arms of all the chromosomes, all of which showed terminal bands (Figures 2l, 3l).

C-banding in Leymus (NsXm), Psathyrostachys (Ns) and Thinopyrum (E) species

Although distinct terminal bands were present in the chromosomes of L. arenarius and L. racemosus, they were rather faint in those of L. multicaulis. All the 14 chromosomes of L. arenarius and L. racemosus, with the exception of chromosome 10 of L. arenarius, presented large terminal or interstitial C-bands and the absence of centromeric bands (Figures 2g, 3g; 2h, 3h). Small to medium terminal, interstitial and centromeric bands were found in the chromosomes of L. multicaulis (Figures 2i, 3i).

The C-banding patterns of the two Psathyrostachys (Ns) species were characterized by diagnostic terminal or interstitial bands in all the seven chromosomes (Figures 2m, n; 3m, n). One satellited chromosome (chromosome 6) and one chromosome with centromeric C-bands (chromosome 1) were observed in Psa. juncea (Figures 2m, 3m). Large terminal C-bands were observed in one or both arms of all the chromosomes in Psa. huashanica, although there were no centromeric bands (Figures 2n, 3n).

Distinct terminal and centromeric C-bands were noted in one or both arms of the seven Th. bessarabicum (Eb) chromosomes. No interstitial bands were observed (Figures 2o, 3o).

Discussion

Relationships among Hystrix, Elymus and Leymus

Cytological and molecular studies showed that species of Hystrix differed as to genomic constitution (Jensen and Wang, 1997; Mason-Gamer ; Zhang and Zhou, 2006; Zhang ; Ellenskog-Staam ; Fan ). Hy. patula, the type species of Hystrix, shared the StH genome of Elymus, whereas Hy. coreana, Hy. duthiei ssp. duthiei, Hy. duthiei ssp. longearistata and Hy. californica contained the NsXm genome of Leymus. In this study, the Giemsa C-banding patterns of four taxa of Hystrix were different. Furthermore, the C-banding patterns of Hy. patula were similar to those of E. canadensis and E. sibiricus. Darkly stained centromeric bands were observed in all the three species, although these were absent in the remaining Hystrix species. The results were consistent with those of chromosome pairing and GISH, hence suggesting a close relationship between Hy. patula and the Elymus species and a distant one between Hy. patula and the other species of Hystrix.

C-banding patterns of Hy. duthiei ssp. duthiei and Hy. duthiei ssp. longearistata were characterized by minor terminal and centromeric bands in almost all of the 14 chromosomes, thus displaying a certain degree of similarity with those of L. multicaulis. Nevertheless, there were differences in the number of minor interstitial bands. Hy. duthiei ssp. longearistata revealed 23 terminal bands, whereas Hy. duthiei ssp. duthiei only 16. Zhou reported a certain morphological divergence and sterility barrier between the two taxa due to a difference in distribution and habitat. From previous cytological and molecular studies on our part, it was shown that the NsXm genomes of Hy. duthiei ssp. duthiei and Hy. duthiei ssp. longearistata were the same as those of the genus Leymus (Zhang , 2008). In this study, the C-banding patterns of the two taxa were similar to those of L. multicaulis, although less centromeric and more interstitial bands were found in the latter. This indicated that Hy. duthiei ssp. duthiei and Hy. duthiei ssp. longearistata were closely related to L. multicaulis, which is congruent with results from cytological and molecular studies.

Jensen and Wang (1997) reported that Hy. coreana contained the NsXm of Leymus and so transferred the species to this genus. In this study, Hy. coreana revealed distinct terminal bands in all the 14 chromosomes, which similar to the banding patterns of L. arenarius and L. racemosus, and consistent with cytological and molecular studies.

Relationships between tetraploids and their diploid ancestors

From studies on chromosome pairing, there are indications that the St and H genome in Elymus originated from Pseudoroegneria and Hordeum, respectively (Dewey, 1967, 1971). In this study, C-banding diversity was observed among Elymus (including Hy. patula), Pseudoroegneria and Hordeum. Distinct terminal C-bands were observed, for example, in Pseudoroegneria (St), these being absent in tetraploid Elymus (StH) species. Similar results were found in E. trachycaulus (2n = 4x = 28, StH) and Pse. spicata (St) (Morris and Gill, 1987). These results suggested the occurrence of chromosomal re-arrangement between the St and H genomes in polyploidization events during the speciation process.

Previous cytological and molecular studies showed that species of Leymus have either JN, or Ns1Ns2, or NsXm genomes (Zhang and Dvorak, 1991; Wang ; Sun ; Anamthawat-Jónsson, 2005). The J (E) genome is from Thinopyrum and the Ns from Psathyrostachys. From this study it was indicated that two Psathyrostachys (Ns) species, besides L. arenarius (NsXm), L. racemosus (NsXm) and Hy. coreana (NsXm), had large terminal bands. However, the C-banding patterns of Th. bessarabicum (Eb), L. multicaulis (NsXm), Hy. duthiei ssp. duthiei (NsXm) and Hy. duthiei ssp. longearistata (NsXm) were characterized by centromeric and terminal bands. From the results it could be inferred that Hy. coreana and some species of Leymus were closely related to the Ns genome of Psathyrostachys, whereas for Hy. duthiei ssp. duthiei, Hy. duthiei ssp. longearistata and a part of Leymus species this was so with the E genome. The present data are consistent with previous cytological and molecular data, thereby suggesting large genetic diversity within the genera Hystrix and Leymus, and the multiple-origin of the polyploid genera Hystrix and Leymus (Sun ; Anamthawat-Jónsson and Bödvarsdóttir, 2001; Yang ; Zhang and Zhou, 2006).

The use of C-banding in the phylogeny of Triticeae

Giemsa C-binding has been widely used in chromosome identification, genetic mapping and studies on genome evolution of Triticeae species, ever since it was first reported (e.g., Morris and Gill, 1987; Gill ; Linde-Laursen and Baden, 1994). The basic C-banding patterns of Pse. spicata, H. bogdanii, Psa. juncea, Psa. huashanica, L. racemosus, L. multicaulis, Hy. coreana, and Hy. patula, as exposed in the present study, are consistent with the findings from previous studies on C-banding (Linde-Laursen and von Bothmer, 1986; Morris and Gill, 1987; Wei ; Baden ; Wang ; Ge ). These findings imply that the C-banding technique is relatively stable and repeatable. The C-banding analysis undertaken in this study revealed genetic diversity and phylogenetic relationships among species from nine genera in Triticeae, consistent with available data on chromosome pairing and molecular evidence. Thus, the Giemsa C-banding technique can be used as a supplementary method for analyzing the genomic constitution of wild species, especially the Triticeae.