Phylogenetic Analyses of Aurantioideae (Rutaceae) Based on Non-Coding Plastid DNA Sequences and Phytochemical Features

Abstract: Sequences of the plastid DNA atp B/ rbc L intergenic spacer and rps16 intron from 23 genera and 47 species of Rutaceae were used to resolve phylogenetic relationships in subfamily Aurantioideae. According to these, the subfamily is monophyletic, but its classical subdivision into tribes Clauseneae and Citreae is only justified if the genus Murraya s.s. (exclusive of the species segregated as Bergera , e.g., Murraya koenigii and M. siamensis ) and Merrillia are transferred to Citreae s.l. This conclusion is also well supported by phytochemistry, demonstrating accumulation of carbazoles in Bergera and Clausena , and of 8-prenylated coumarins and polyoxygenated flavonoids in Murraya s.s. and Merrillia. Formation of both carbazoles, as well as 8-prenylated coumarins, and polyoxygenated flavonoids in Micromelum suggests relationships between Clauseneae s.s. and Citreae s.l. The monophyly of several larger genera in both tribes is supported by relatively high bootstrap percentages and specific chemical profiles for e.g., Clausena, Micromelum, Glycosmis and Atalantia. In contrast, molecular, chemical, and other data show that none of the subtribes recognized within Aurantioideae reflect phylogenetic relationships. Only the clades with Clausena + Bergera, Murraya s.s. + Merrillia, and Citrus + Clymenia + Eremocitrus + Fortunella + Poncirus ("true Citrus fruit trees") are well supported by such data. Among the outgroup genera, Zanthoxylum (Rutoideae) and Toddalia (Toddalioideae) are much closer to each other than to Ruta (Rutoideae).     

Phylogenetic relationship of Clauseneae (Rutaceae) inferred from plastid and nuclear DNA data and taxonomic implication for some major taxa

Using sequences of the nuclear ribosomal ITS region as well as the chloroplast DNA trnL‐trnF and atpB‐rbcL regions, this study aims to provide further insight into the phylogenetic relationships within Clauseneae and its relationship to Citreae (Rutaceae). Using maximum likelihood (ML) and Bayesian inference (BI), we reconstructed the phylogeny of Clauseneae based on trnL‐F, atpB‐rbcL and ITS sequences. Our data matrix contained 91 accessions, representing 72 species and varieties from 31 genera and two outgroups, including new and extensive sampling of species and varieties representing four genera in the tribe Clauseneae. In the subfamily Aurantioideae, six major clades were resolved with strong support: 1) Micromelum clade: Micromelum; 2) Glycosmis clade: Glycosmis; 3) Bergera clade: Murraya sect. Bergera; 4) Clausena clade: Clausena; 5) Murraya clade: Murraya sect. Murraya + Merrillia; 6) Citreae clade: Citreae. Micromelum, Glycosmis, Clausena and Merrillia were confirmed as monophyletic. In contrast, Murraya s.l. was reconstructed as polyphyletic. Murraya sect. Bergera clustered with Clausena while Murraya sect. Murraya and Merrillia together formed a clade that is sister to the tribe Citreae. All members from Citreae were clustered into a natural group. The genus Micromelum was found to be primitive in this subfamily and more close to Glycosmis. Based on the phylogeny and morphological characters, we discuss the taxonomy of some members of Clauseneae and conclude that the current tribal and generic classification need further revision.     

Phylogenetic Relationships of Citrus and Its Relatives Based on matK Gene Sequences

The genus Citrus includes mandarin, orange, lemon, grapefruit and lime, which have high economic and nutritional value. The family Rutaceae can be divided into 7 subfamilies, including Aurantioideae. The genus Citrus belongs to the subfamily Aurantioideae. In this study, we sequenced the chloroplast matK genes of 135 accessions from 22 genera of Aurantioideae and analyzed them phylogenetically. Our study includes many accessions that have not been examined in other studies. The subfamily Aurantioideae has been classified into 2 tribes, Clauseneae and Citreae, and our current molecular analysis clearly discriminate Citreae from Clauseneae by using only 1 chloroplast DNA sequence. Our study confirms previous observations on the molecular phylogeny of Aurantioideae in many aspects. However, we have provided novel information on these genetic relationships. For example, inconsistent with the previous observation, and consistent with our preliminary study using the chloroplast rbcL genes, our analysis showed that Feroniella oblata is not nested in Citrus species and is closely related with Feronia limonia. Furthermore, we have shown that Murraya paniculata is similar to Merrillia caloxylon and is dissimilar to Murraya koenigii. We found that “true citrus fruit trees” could be divided into 2 subclusters. One subcluster included Citrus, Fortunella, and Poncirus, while the other cluster included Microcitrus and Eremocitrus. Compared to previous studies, our current study is the most extensive phylogenetic study of Citrus species since it includes 93 accessions. The results indicate that Citrus species can be classified into 3 clusters: a citron cluster, a pummelo cluster, and a mandarin cluster. Although most mandarin accessions belonged to the mandarin cluster, we found some exceptions. We also obtained the information on the genetic background of various species of acid citrus grown in Japan. Because the genus Citrus contains many important accessions, we have comprehensively discussed the classification of this genus.     

Pollen morphology of the subfamily Aurantioideae (Rutaceae)

The Aurantioideae is one of seven subfamilies of the Rutaceae consisting of two tribes, the Clauseneae, containing five genera, and the Citreae, with 28 genera. Each tribe contains three subtribes. The pollen morphology of the subfamily Aurantioideae is described and illustrated for the first time based on light and scanning electron microscopy. Five pollen types have been recognised in the subfamily, based mainly on aperture number and exine ornamentation. The pollen grains show a high degree of intergeneric variation. Pollen grains of Clauseneae are 3-colporate, microstriate or microstriato-reticulate, whereas pollen grains of Citreae are almost always 4/5 colporate with exines varying from microperforate to coarsely reticulate. Congruence between pollen types and the currently accepted classification is discussed, as well as the systematic implications of pollen morphology for the subfamily.     

Chemotaxonomic Relationship Between Murraya and Merrillia (Rutaceae)

Swingle^[14-15] divided Aurantioideac into two tribes, one of which, Clausereae was further divided into three subtribes, namely, Micromelinae, Clauseninae and Merrilliinae. Mic romelinae and Merrilliinae each have one genus, whereas Clauseninae has three genera. Morphologically, the Clauseneae is a natural tribe, the five genera are related in a linear sequence, starting with Micromelum as the most primitive and progressing in sequence to Glycosmis, Clausena, Murraya and Merrillia. Chemical studies also support this linear relationship, as revealed by the degree of oxygenation and complexity of the 3-methyl carbazole alkaloids, from CH₃ and C1₃ in Glycosmis to CHO and C₁₈ in Clausena and COOH and C₂₃ in Murraya^[20-21]. Distribution of flavonoids also indicates the progression from Clauseninae to Merrilliinae^[18-19]. Extensive work has been conducted on the chemistry and taxonomy of the genus Murraya ^{[1,5,7-13,16]}, and the data from these studies clearly indicate the presence of two distinct groups. Based on a combination of morphological and chemical differences, we agreed with Tanaka^[16-17] in dividing Murraya into two sections, i.e. section Murraya and section Bergera^[1]. However, our previous study^[1] has not touched on the relationship between the two sections. Tanaka^[16-17] placed section Bergera before section Murraya, and indicated that the former is close to Micromelum and the latter to Merrillia. Swingle^[14-15], on the other hand, put taxa of section Murraya ahead of those of section Bergera, presumably suggesting that plants of section Murraya are more primitive than those of section Bergera, this arrangement was followed by Huang^[2-3]. The two conflicting viewpoints would have direct bearings on the interpretation of the trends of biogenesis of prenylated indole and carbazole alkaloids, as well as on the weighing of the relative advancement of the morphological characters within the genus, such as in the assignment of indices of divergence and in the construction of Wagner Divergence Diagrams. Without more objective criteria, we find it difficult to select one of the two systems. In order to determine the relationship between the two sections of Murraya, we decided to study plants of related genera, with the hope that the chemical data may shed light on the problem. A plant that attracted our attention is Merrillia caloxylon (Ridley) Swingle. So far, only eupatorin and a few other flavonoids have been reported from the fruit of this species^[4,6]. Although Me. caloxylon belongs to Merrilliinae, a subtribe next to Clauseninae, Tanaka^[16-17] believes that it is close to Murraya section Murraya. Swingle^[15], also suggested that this species might have developed from the same stock that gave rise to Mu. paniculata. If their interpretations were accurate, we would expect that Me. caloxylon would also contain yuehchukene and 8-prenylated coumarins. The presence of the antiimplantation agent would not only open up a new source of the compound but also help us judge the relationship between the two sections of Murraya. It is in this context that we studied the chemical composition of Me. caloxylon. Indeed, root and stem bark of Me. caloxylon were found to contain the antiimplantation indole alkaloid yuehchukene (1), and the 8-prenylated coumarins sibiricin (II) and phebalosin (III), as well as 3-(3-methy1-buta-1,3-diene) indole (IV) and eupatorin (V.) Details on the chemical profiles are reported in another paper. Through this exercise, we have confirmed the close relationship between Merrilliinae and Murraya section Murraya, plants of both taxa contain yuehchukene and 8-prenylated coumarins, but no carbazole alkaloid. Root and stem bark of Me. caloxylon, like those of plants of section Murraya, are strawcolored to pale whitish. Its leaves also bear wings along the rachis an in Mu. alata, and the seeds are also villous. However, Me. caloxylon has long trumpetshaped flowers 55-60 mm long, much larger than those found in other rutaceous plants. Its fruit is ob long, up to 11 cm long and 8 cm across, bearing a thick and warty pericarp, exuding a very stick mucilage when cut, and containing numerous seeds (>30). The plant was known to exist in the Malay Peninsula and north Sumatra^[15], but, according to David Jones (per. comm.) of the University of Malaya, is now only available in cultivation in Malaysia and Singapore, a limitation to any further exploitation as an additional source of yuehchukene. Besides confirming the close relationship between Merrillia and section Murraya, we may also conclude that section Bergera is close to Glycosmis and Clausena, since they are known to contain carbazole alkaloids but no yuehchukene. Accordingly, we find Tanaka’s arrangement more acceptable: plants of section Bergera are more primitive than those of section Murraya, the former is close to Clausena whereas the latter (notably Mu. alata) to Merrillia. The relationship among the gonera with in Clauseneae may be illustrated as follow: Micromelum→ Glycosmis→Clausena→Murraya sect. Bergera→Murraya sect. Murraya→Marrillia. Accordingly, we may decide that the following character states are more primitive among plants of Murraya and Merrillia: root and stem bark dark brown, leaf rachis wingless, flower small, fruit purple-black with few seeds, and seed coat glabrous. In contrast, strawcolored or pale whitish bark, winged leaf rachis, large flower, red or yellow fruit with many seeds and villous seed coat can be regarded as more advanced characters. Acknowledgments Partial support was received from the World Health Organization Special Programme on Human Reproduction and the Kevin Hsu Research Fund (to YCK) and Commonwealth Science Council (to PPHB). The staff of the Singapore Botanic Gardens and the Forest Research Institute of Malaysia are thanked for their assistance in collecting plantmaterial.     

Phylogenetic relationships of the Aurantioideae inferred from chloroplast DNA sequence data

The tribes and subtribes of Aurantioideae, an economically important subfamily of the Rutaceae, have a controversial taxonomic history because of the lack of a phylogenetic framework. The rps16 and trnL-trnF sequences of the chloroplast were analyzed phylogenetically to construct an evolutionary history and evaluate the most recent classification system of Swingle and Reece (The Citrus Industry, volume 1 [1967]). Taxa representing tribes Citreae and Clauseneae and five of the six subtribes were sampled. Conflicts in the positions of some taxa between the rps16 and trnL-trnF trees are poorly supported. In all analyses, the Aurantioideae are monophyletic. The strict consensus tree of the combined analysis indicates that the two tribes along with the subtribes sampled are not monophyletic. The combined topology is not congruent with the widely used classification of Aurantioideae by Swingle and Reece. The tribes and subtribes are in need of revision.     

Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq

The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian “true citrus fruit trees” (genera Citrus, Poncirus, and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.     

DISTRIBUTION OF COAGULATION OF YOUNG SHOOT HOMOGENATES IN THE AURANTIOIDEAE

Distribution of coagulation of young shoot homogenates was surveyed in 430 accessions from the orange subfamily Aurantioideae. Representatives of four genera available for study (Clausena, Glycosmis, Aegle, Feronia) were coagulating taxa. The genus Citrus included both coagulating and noncoagulating taxa. Nineteen genera (Murraya, Merillia, Pamburus, Paramignya, Triphasia, Aeglopsis, Afraegle, Balsamocitrus, Feroniella, Swinglea, Atalantia, Citropsis, Hesperethusa, Pleiospermium, Severinia, Clymenia, Eremocitrus, Poncirus, Fortunella) were noncoagulating. The status of eight genera (Micromelum, Wenzelia, Monanthocitrus, Oxanthera, Merope, Luvunga, Burkillanthus, Limnocitrus) is not known. Homogenized tissue from coagulating taxa turned into a paste while tissue from noncoagulating taxa was thin and juicy. Coagulation did not take place when the pH of the grinding medium was 11 or higher but it would occur if the pH was lowered. Homogenates of noncoagulating taxa become coagulated below pH 5.2–5.3, but remained noncoagulating above this. Noncoagulating taxa contained an unidentified substance which inhibited coagulation of tissue from coagulating taxa. The mechanism of coagulation and noncoagulaton is not presently known. The significance of coagulation and its absence as a taxonomic and genetic marker is discussed.     

Phylogenetic relationships of the Aurantioideae (Rutaceae) based on the nuclear ribosomal DNA ITS region and three noncoding chloroplast DNA regions, atpB-rbcL spacer, rps16, and trnL-trnF

The tribes and subtribes of Aurantioideae, an economically important subfamily of the Rutaceae, have a controversial taxonomic history because a phylogenetic framework has been lacking. In order to construct an evolutionary history and evaluate the most recent classification system [Swingle and Reece 1967. The botany of Citrus and its wild relatives, in: The Citrus Industry, vol. 1, History, World Distribution, Botany, and Varieties. University of California, Berkeley, pp. 190-430], one nuclear and three noncoding chloroplast genes were sequenced and analyzed phylogenetically along with selected non-molecular characters. Taxa representing tribes Citreae and Clauseneae and their six subtribes were sampled. In all analyses Aurantioideae is monophyletic. The majority-rule consensus tree from the combined analysis indicates that the two tribes are not monophyletic. The combined topology is not congruent with the widely used classification of Aurantioideae by Swingle and Reece (1967). The tribes and subtribes are in need of revision.     

Pollen morphology supports the reinstatement of Bergera (Rutaceae)

Pollen morphology of 11 species of Murraya and Micromelum (Clauseneae: Rutaceae) was investigated using scanning electron microscopy (SEM) and palynological characters including pollen size, aperture number and exine ornamentation were evaluated. The results indicate that species in M. sect. Bergera and sect. Murraya have very distinct pollen morphology. Species in section Bergera have rugulate to foveolate exine, whereas the tectum in section Murraya is cross-striate. This supports the redefinition of the genus Murraya , retaining only the species of section Murraya , while M. sect. Bergera is better treated as a separate genus. From the palynological point of view, it is suggested that Bergera and Murraya s.s. are not closely related, with the latter being more close to Micromelum and Merrillia , a result in accordance with phytochemical, chromosomal and molecular evidence, thus supporting the reinstatement of the generic name Bergera L.     

真正柑桔果树群植物的分支学研究

本文用相容性分析方法(Compatability snalysis)分析了真正柑桔果树群(芸香科Rutaceae-柑桔亚科Aurantioideae-柑桔族(Citreae)-柑桔亚族(Citrinae)植物内各属间的分支学关系。给出了建立在7个相容性性状组成的最大族所决定的分支图。性状极性的确定使用了外群法。结果表明,柑桔属(Citrus L.)和多蕊桔属(Clymenia Swing)构成一个单系类群,他们的姐妹群是金柑属(Fortunella Swing.)。被认为起源于中国的3个属,柑桔属(Citrus)、金柑属(Fortunella)和积属(Poncirus Raf.)并未构成一个单系类群。本文还利用分支关系分析和讨论了真正柑桔果树群的种系发生关系。     

ESSENTIAL LEAF OILS IN REPRESENTATIVES OF THE AURANTIOIDEAE (RUTACEAE)

The distribution of essential leaf oils in representatives of 12 genera in the Aurantioideae has been studied by gas chromatography. Of the individual oil components, 40 were identified by retention times on two columns, augmentation, and some by IR spectrophotometry. Twelve groupings of components were established and these compared between the tribes Clauseneae and Citreae , among the subtribes clauseninae , balsamocitbinae , and citrinae , and between the “primitive” and the “near citrus group.” The essential oils conform to the botanical groupings, except for Pleiospermium in the citrinae and Murraya in the clauseneae. Fortunello crassifolia (Citbeae ), a possible garden hybrid, seems to be more closely related to the Clauseneae , the remote citroids, than to the near Citrus group. Atalantia also might be an intertribal hybrid with remote or other citroids. A specimen of Aeglopsis chevalieri (balsamocitbinae ) from Florida appears to be of hybrid origin with the parental source close to Citrus.     

DISTRIBUTION OF ENZYMATIC BROWNING OF YOUNG SHOOT HOMOGENATES IN THE AURANTIOIDEAE

Distribution of polyphenol oxidase-catalyzed browning of young shoot homogenates was studied in 428 accessions of the orange subfamily Aurantioideae. Browning and nonbrowning phenotypes were found in both tribes, namely Clauseneae and Citreae. The browning taxa had sufficient polyphenol oxidase activity and a presently unknown phenolic substrate, while the nonbrowning taxa were devoid of both. A third group of taxa contained the substrate only and they too were designated as browning since their homogenates turned brown when the enzyme from a known source was added. The color of homogenates was taxon-specific in most browning and nonbrowning taxa. Within the genus Citrus, 4 out of the 16 species recognized by Swingle were browning and 2 were nonbrowning. Exceptions noted within either group of taxa were presumably hybrids or mutants. The significance of enzymatic browning as an additional taxonomic criterion in studying species relationships and as a genetic marker in identificaton of hybrid taxa was discussed.     

部分柑桔属及其近缘属Giemsa C-带带型研究

本文应用Giemsa显带技术研究了枳属(Poncirus)、金柑属(Fortunella)和柑桔属(Citrus)16个分类群的染色体。枳属以末端带和着丝点带为主,金柑属与柑桔属主要以末端带为主;统计分析了各分类群每对染色体及全组染色体的异染色质含量,并列出其带型公式;探讨了金柑属的分类学地位;赞同把柚(C.grandis)作为柑桔属的基本种之一;根据异染色质含量的变化对柑桔属的带型演化进行了讨论。     

Phylogenetic Relationships and Evolution of the Androecia in Ruteae (Rutaceae)

Ruta, which belongs to tribe Ruteae, is the type genus of the subfamily Rutoideae and the family Rutaceae. Molecular systematic studies have shown that the genera in Ruteae are closer related to Aurantioideae than to most other genera of Rutoideae, some of the genera traditionally placed in Ruteae have been shown to be nested within the Aurantioideae clade, but the diagnostic characters for determining new patterns in the relationship are poor. In this study, we investigated the floral development of Boenninghausenia in Ruteae (sensu stricto), Haplophyllum in the basal position of Aurantioideae and Murraya in traditional Aurantioideae using scanning electron microscopy. The androecium of Boenninghausenia is obdiplostemony. As androecia in other genera within Ruteae (s.s.) are also obdiplostemonous, reconstruction of the ancestral state indicates that obdiplostemony is an ancestral character in this clade. Because the androecia of Haplophyllum and Murraya are also obdiplostemonous, obdiplostemony is also an ancestral character in Aurantioideae clade. The ancestral state reconstruction indicates this character can serve as a synapomorphy of the Ruteae-Aurantioideae clade. The results of our work also shed light on the evolution of the androecium in Rutaceae, as the obdiplostemony of this group is clearly derived from haplostemony in the ancestral genera in Rutaceae and has develop into polyandry by increasing antepetalous stamens.     

In vitro plant regeneration of Aristolochia indica through axillary shoot multiplication and organogenesis

Protocols for in vitro plant regeneration via axillary and adventitious shoot regeneration were established in an important medicinal plant, Aristolochia indica L. (Aristolochiaceae). Basal Murashige and Skoog's (MS) medium supplemented with 0.54 μM α-naphthaleneacetic acid (NAA) and 13.31 μM benzyladenine (BA) induced the maximum number of shoots (45-50) from shoot tip and nodal segment cultures. Phenolic accumulation in leaf and internodal stem derived callus cultured in MS medium containing NAA or 2,4-dichlorophenoxyacetic acid and BA or kinetin was controlled by the addition of 1.0 mg l^-1 phloroglucinol (PG) to the callus induction medium. Basal medium supplemented with 2.69 μM NAA, 13.31 μM BA and 1.0 mg l^-1 PG induced the best results in terms of shoot bud regeneration from leaf derived callus. Direct de novo development of shoots from leaf segments was achieved using 13.31 μM BA along with 50 mg l^-1 activated charcoal. The microshoots were rooted in White's medium supplemented with 2.46 μM indolebutyric acid. More than 85% of rooted plants survived in the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Taxonomic and phylogenetic relationships among the genera of the True Citrus Fruit Trees Group (Aurantioideae,Rutaceae) based on AFLP markers

Fifty-nine genotypes representing six genera of the True Citrus Fruit Trees Group (Aurantioideae, Rutaceae) were analyzed using AFLP technique to study their taxonomic and phylogenetic relationships. Fifteen primer combinations (out of 64 screened) were selected based on the polymorphism and quality of the bands produced by the primer and used in the present study. A total of 312 bands were obtained, of which 305 (97.8%) were polymorphic. The UPGMA tree of all genotypes was constructed based on the AFLP data using PAUP* beta version 4.0b8 software. Our AFLP molecular tree clearly confirms that the True Citrus Fruit Trees Group is monophyletic and supports the division of the group into genera mainly based on morphological characters, except that the genus Fortunella was nested within the genus Citrus cluster (as a monophyletic sub-branch). The subdivision of the genus Citrus into subgen. Papeda and subgen. Eucitrus as suggested by W. T. Swingle based on morphological characters and the point of view that C. medica, C. grandis and C. reticulata are the three basic species of the subgen. Eucitrus were also supported by our molecular data. In addition, contrary to the expectation based on morphological data, however, our molecular data demonstrated that C. ichangensis is more closely related to C. junos of the subgen. Eucitrus than to the C. hystrix of the subgen. Papeda. Furthermore, our study provided the first evidence that C. mangshanensis is basal to all the loose-skin citrus types. Finally, the taxonomic and phylogenetic relationships among the six genera and the important genotypes of the genus Citrus were dis-cussed in detail.     

Characterization of heterochromatin by sequential counterstain-enhanced fluorescence in three domestic bird species: Meleagris gallopavo, Columba livia domestica, and Anser anser L

The karyotypes of three avian species--Meleagris gallopavo, Anser anser L., and Columba livia domestica--were investigated by means of counterstain-enhanced fluorescence techniques (chromomycin A3/distamycin A/DAPI followed by DAPI/actinomycin D staining). A heterochromatin characterization of macro- and microchromosomes was performed. CMA3-positive (GC-rich) regions in the turkey included the telomeres of chromosomes 1, 3, 4, and Z. In the goose, the chromosome 2 was also CMA3-bright at the telomeres. The W chromosome possessed large amounts of CMA3-bright material on the short arm in both the turkey and the goose. Two types of centromeric heterochromatin were distinguished on acro- to telocentric chromosomes 6 to 14 in the pigeon. The microchromosomal heterochromation of the turkey and goose was GC-rich but had a high degree of variation. In the pigeon, several microchromosomes possessed predominantly AT-rich heterochromatin.