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 et al. (1997) 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 et al., 1997).
Although separated early as a genus in its own right, the recognition of Hystrix has been controversial ever since its establishment. Church (1967aChurch ( , 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),   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 et al. (1997) 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.

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 et al. (1991). 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 522 Zhang et al.   Figures  2 and 3, respectively.

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).
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, 2d, 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 Pseudoroegneria spicata (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 Genetic diversity in Hystrix and related genera 523 (E b ) chromosomes. No interstitial bands were observed (Figures 2o, 3o).

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 et al., 2002;Ellenskog-Staam et al., 2007;Fan et al., 2007). 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 spe-cies. 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 et al. (1999) 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 et al., , 2008. In this study, the C-banding patterns of the two taxa were similar to those of L. multicaulis, although 524 Zhang et al.  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(Dewey, , 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 Ns 1 Ns 2 , or NsXm genomes (Zhang and Dvorak, 1991;Wang et al., 1994;Sun et al., 1995;Anamthawat-Jónsson, 2005). The J (E) genome is from Thinopyrum and the Ns from Psathyrostachys. From this study it was indicated that two Genetic diversity in Hystrix and related genera 525