Genetic variation and putative hybridization inSalix alba andS. fragilis (Salicaceae): Evidence from allozyme data

Genetic variability was estimated by enzyme electrophoresis in 239 Belgian clones from theSalix alba-S. fragilis complex. This morphological complex suggested a high frequency of hybrids. To test this hypothesis, the clones were pooled as a single co-adapted species complex and secondly as belonging to either species, i.e. beingS. alba-like orS. fragilis-like. The standard genetic variability measures showed higher values for the complex than for the separate species. However, the observed mean heterozygosity was lower in the putative hybrid complex than for each of the species separately. The fixation indices were more variable at the species level and indicated that mostS. fragilis locations appeared fixed forlap-1, whereas no fixation occured inS. alba locations. Averaged at the regional (i.e. catchment) level, this difference between the two species remained and values ranged from 0.457 to 0.617 inS. alba and were much higher, fixed homozygous or monomorph inS. fragilis. Hierarchical F-statistics revealed that most of the differentiation occured at the lower levels of localities and tributaries and that there was no further differentiation between catchments. Tributaries which are 10–25km in length were proposed as the most likely entities for further examination of putative hybridization and events of allelic fixations. By considering the two abovementioned approaches of data input, it could be suggested that most of the allozyme differentiation was between the species and less between the regions.     

Genetic variation and natural hybridization betweenOrchis laxiflora andOrchis palustris (Orchidaceae)

Genetic divergence between population samples ofOrchis laxiflora and ofO. palustris from various European locations was studied by electrophoretic analysis of 25 enzyme loci. An average genetic distance of D_Nei = 1.24 was found between the two taxa, with 12 out of 25 loci showing alternative alleles (diagnostic loci). Genetic heterogeneity was observed within bothO. laxiflora andO. palustris, when northern and southeastern populations were compared, being lower in the former taxon (D = 0.06), than in the latter (D = 0.16). Karyologically, 2n = 36 was found for bothO. laxiflora andO. palustris. O. laxiflora andO. palustris produce hybrids, described asO. ×intermedia. Genotype analysis of several sympatric samples showed the presence of hybrid zones, including F₁ hybrids and, in low proportions, recombinant classes, putatively assigned to F_n and backcrosses, as well as a few introgressed individuals of both taxa. These data indicate that hybrids are only partially fertile, with a very limited mixing up of the two parental gene pools; this is also shown by the lack of significant lowering of genetic distances when sympatric and allopatric heterospecific samples are compared. Accordingly,O. laxiflora andO. palustris form a syngameon; nevertheless they can be considered as good taxonomic species, with virtually distinct gene pools, which evolve independently. The genetic variability inO. laxiflora andO. palustris is remarkably low ( _e = 0.05 and _e = 0.02, respectively). In particular, nearly complete absence of polymorphic loci was found inO. palustris from northcentral Europe. Two hypotheses are considered to explain the low genetic variability of this endangered species.     

Karyological differentiation and speciation in C. MediterraneanAnacamptis (Orchidaceae)

Anacamptis pyramidalis is a variable and wide-spread European-Mediterranean taxon. Beside a dominant cytotype with 2n = 36 it includes cytotypes with 2n = 54 and 63 in northern Tuscany (and the Eastern Pyrenees) and one with 2n = 72 on Malta. In contrast,A. urvilleana, formerly often misidentified and included inA. pyramidalis, is a monomorphic and distinct species, endemic to the Maltese Islands. It has 2n = 36, can be clearly separated by morphological and anatomical features and is isolated from partly sympatric populations ofA. pyramidalis with 2n = 72 by differences in chromosome number, flowering time and habitat preference.     

Genetic relatedness in the antCamponotus herculeanus. A comparison of estimates from allozyme and DNA microsatellite markers

Summary Genetic structure ofCamponotus herculeanus populations was studied using both traditional allozyme markers and recently discovered DNA microsatellites. Relatedness among worker nest mates was high in one study population (0.70), but lower in another (0.48). The estimates lower than 0.75, together with the multilocus genotype patterns, indicate either multiple mating by queens, or multiple queens reproducing in the colonies. These two could not be distinguished with the current data, although multiple matings seem a more probable explanation for the relatedness estimates bearing social biology of the species in mind. Inbreeding coefficients suggested random mating in both populations. Both allozyme and DNA microsatellite markers gave the same result concerning genetic parameters estimated, but the higher variability of the microsatellites resulted in more precise estimates of both parameters.     

Vegetative diversification and radiation in subtribeDendrobiinae (Orchidaceae): Evidence from chloroplast DNA phylogeny and anatomical characters

The data derived from a chloroplast DNA restriction site analysis of subtribeDendrobiinae (Orchidaceae) indicate that extreme vegetative diversification is concentrated in two limited parts of this group. Overlaying the vegetative character states onto the chloroplast DNA cladogram suggests that several xeromorphic, vegetative characters evolved in the lines leading to the above-mentioned clades. Several anatomical characters are also associated with xeromorphy. These vegetative and anatomical characters facilitated the establishment of this group in various dry habitats. On the other hand, the modifications of size and number of parenchymatous cells substantially contributed to the vegetative diversification. This fact implies that a simple structural adjustment can result in a major modification of growth habits in theDendrobiinae.     

A new species ofLiparis (Orchidaceae) from India

Liparis indiraii spec. nova from India is close toL. alata A. Rich. andL. atropurpurea Lindl.     

Vanda longitepala (Orchidaceae): a new species from Burma

Summary   Vanda longitepala, a new species of orchid from northern Burma is described and discussed.     

Habitat differentiation, hybridization and gene flow patterns in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. (Orchidaceae)

Detailed ecological, morphological and molecular analyses were performed in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. in Scandinavia. Comparisons were made with pure populations of either diploid ssp. fuchsii or tetraploid ssp. maculata. It was shown that mixed populations are the result of secondary contact between ssp. fuchsii and ssp. maculata. No patterns of recent and local autopolyploidization were found. Morphology and nuclear DNA markers (internal transcribed spacers of nuclear ribosomal DNA) showed that diploids and tetraploids from mixed populations have similar levels of differentiation to diploids and tetraploids from pure populations. Vegetation analyses, as well as analyses of environmental variables, revealed that diploid and tetraploid individuals in mixed populations are ecologically well differentiated on a microhabitat level. Diploids and tetraploids in pure populations have wider ecological amplitudes than they do in mixed populations. Triploid hybrids grew in intermediate microhabitats between diploids and tetraploids in the mixed populations. Plastid DNA markers indicated that both diploids and tetraploids may act as the maternal parent. Based on morphology and nuclear markers triploids are more similar to tetraploids than to diploids. There were indications of introgressive gene flow between ploidy levels. Plastid markers indicated that gene flow from diploid to tetraploid level is most common, but nuclear markers suggested that gene flow in opposite direction also may occur. Similar patterns of differentiation and gene flow appeared in localities that represented contrasting biogeographic regions. Disturbance and topography may explain why hybridization was slightly more common and the differentiation patterns somewhat less clear in the Scandinavian mountains than in the coastal lowland. An erratum to this article can be found at     

Genetic variation and hybridization in SwedishSchoenus (Cyperaceae)

Schoenus ferrugineus andS. nigricans have restricted distributions in Sweden and are almost exclusively confined to calcareous fen habitats. AtS. nigricans sites,S. ferrugineus is usually also present, and hybrids are frequently found. In this report, I used allozymes to estimate the amount of gene flow between the two species, and to compare the partitioning of genetic diversity in each of them. Thirteen loci were analysed at eight different enzyme systems. Seven loci were variable between or within the species. The two species had completely different alleles at two of the seven variable loci, whereas there was overlap at five loci. In all, 22 different alleles were found. Six of these alleles were confined toS. nigricans, and five alleles were confined toS. ferrugineus. Nei's genetic identity was 0.55.—InS. ferrugineus, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.0 (each polymorphic locus had two alleles). InS. nigricans, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.3.—The proportion of genetic diversity due to variation among sites (G _ST) was fairly similar in the two species, mean over loci = 0.12 inS. ferrugineus and 0.15 inS. nigricans. However, the proportion of genetic diversity due to variation among individuals within sites (G _IS) differed markedly between the two species, mean over loci = 0.54 inS. ferrugineus and 0.17 inS. nigricans. Accordingly, there was a much higher individual heterozygosity inS. nigricans than inS. ferrugineus. — Most hybrids were interpreted as F₁ hybrids. However, a small proportion, 0.5–1.6 %, were F_n hybrids or back-crosses.—On the Swedish mainland, all former occurrences ofS. nigricans are extinct, but viable hybrids are still present at a few sites in southernmost Sweden.     

Observations of the floral biology ofPrasophyllum odoratum (Orchidaceae,Spiranthoideae)

Prasophyllum odoratum is a vernal, nectariferous, terrestrial orchid that flowers profusely six to eight months following cyclical fires that disrupt sclerophyll woodlands. The morphology of the column and pollinarium is indicative of taxa placed within the subfam.Spiranthoideae. The orientation of the pollinaria to the stigma appears to prevent mechanical self-pollination. Both cross- and self-pollination appear to be effected by insects that forage within the brightly-colored, scented, non-resupinate flowers. Ants and drosophilid flies remove nectar, but do not appear to transport pollinaria between flowers. The primary pollinators are polytrophic flies in the fam.Syrphidae and opportunistic male bees in the genusLeioproctus (Colletidae). Approximately 52% of the flowers on a raceme set seed. The comparatively short floral tube ofP. odoratum reflects the dependence of this species on short-tongued insects to effect successful dispersal of pollinaria.     

Polyploidy and aneuploidy inOphrys,Orchis, andAnacamptis (Orchidaceae)

Studies on chromosome numbers and karyotypes in Orchid taxa from Apulia (Italy) revealed triploid complements inOphrys tenthredinifera andOrchis italica. InO. tenthredinifera there is no significant difference between the diploid and the triploid karyotypes. The tetraploid cytotype ofAnacamptis pyramidalis forms 36 bivalents during metaphase I in embryo sac mother cells. Aneuploidy was noticed inOphrys bertolonii ×O. tarentina with chromosome numbers n = 19 and 2n = 38. There were diploid (2n = 2x = 36), tetraploid (2n = 4x = 72), hexaploid (2n = 6x = 108) and octoploid (2n = 8x = 144) cells in the ovary wall of the diploid hybridOphrys apulica ×O. bombyliflora. Evolutionary trends inOphrys andOrchis chromosomes are discussed.     

New and noteworthyHabenaria spp. (Orchidaceae) from New Guinea and adjacent islands

5 new taxa ofHabenaria, namelyH. bougainvillae, H. elongata R. Br. var.leptophylla, H. ensigera, H. rechingeri andH. trichoglossa, are described and illustrated, with reference to affinities to related Australian and Indo-malayan species. The occurrence in New Guinea of severalHabenaria spp. typical for a savanna-like vegetation, led to look more thoroughly at these taxa:H. elongata R. Br. andH. ochroleuca R. Br., considered so far to be endemic in Northern Australia, andH. khasiana Hook. f., hitherto only known from southeastern Asia.Studies in the subtribeHabenariinae Bentham (Orchidaceae), 2.—Part 1: Candollea34, 357 (1979).Dedicated to Hofrat Prof. DrKarl Heinz Rechinger on the occasion of his 80th birthday. — On April 30, in the year 1927, the author and his brotherOtto Renz metKarl Heinz Rechinger on a small steam-boat in a stormy Aegaen Sea, travelling from Piraeus to the Northern Sporades Islands:Karl Heinz with destination to Chelidromia, the author andO. Renz to Skopelos. By this lucky chance a lasting friendship began.     

Studies in the embryology of the diandrous orchidCypripedium cordigerum (Cypripedieae,Orchidaceae)

The anther wall layers ofCypripedium cordigerum are six to eight. The glandular tapetum is 2- or 3-layered and its cells are uninucleate. Simultaneous cytokinesis results in decussate, isobilateral and tetrahedral pollen tetrads. Ripe pollen grains are 2-celled. The mature ovules are anatropous, bitegmic and tenuinucellate. Both the integuments are dermal in origin and 2-layered. The inner integument alone forms the micropyle. The female gametophyte is 6-nucleate and bisporic. The reduction of nuclei is due to the strike phenomenon. Double fertilization occurs. The primary endosperm nucleus divides to form two free endosperm nuclei. The mature embryo is undifferentiated. The cells ca, m and n contribute to the embryo. The suspensor is single-celled. The seed coat is formed entirely by the outer layer of the outer integument. There are three sterile and three fertile valves in the ovary. In the prefertilization stages these valves consist of parenchymatous cells with starch and raphides. After fertilization, the sterile valves develop sclerotic cells whereas the fertile valves remain parenchymatous. The pericarp structure and embryological features support the retention of tribeCypripedieae within theOrchidaceae.     

Genetic differentiation and speciation in Stomion (Goleoptera: Tenebrionidae): flightless beetles of the Galápagos Islands, Ecuador

Stomion is a swarm of 13 tenebrionid beetle taxa endemic to the Galápagos Islands. The taxa show marked morphological variation, and all are flighdess. They can be divided into three divergent morphological groups based on external sculpturing of the elytra. Eleven taxa were examined electrophoretically at 20 enzyme loci to measure the extent of genetic differentiation which accompanied morphological differentiation and speciation. Stomion are shown to be ideal candidates for allopatric and founder event speciation, with their limited dispersal ability, narrow distributions, and incidence of single-island endemics. Accordingly, individual heterozygosities were low in most taxa, and gene pool divergence was apparent among populations of single taxa occupying different islands. Both interspecific and intraspecific genetic identities were high, with an average 1 = 0.88 and I = 0.96, respectively. The relatively young age of the genus and the allopatric mode of speciation may account for high genetic relatedness. No clear relationship was found between allozyme phenotypes and the three morphological groups, thus, the evolution of these traits may have been facilitated by similar selection among islands of similar age and geography. The geographical distributions of the morphological groups can be used to suggest a general pattern of speciation for the group.     

Karyotype analysis and heterochromatin differentiation with Giemsa C-banding and fluorescent counterstaining inCephalanthera (Orchidaceae)

Detailed studies of the chromosomes of the three Austrian species of the genusCephalanthera showed them all to have basically similar karyotypes. BothC. damasonium (2n = 36) andC. longifolia (2n = 32) have three large and several classes of smaller chromosome pairs. The karyotype ofC. rubra (2n = 44) is composed of four large and several groups of smaller pairs. The heterochromatin in these species amounts to about 10% of total karyotype length. All the chromosomes have Giemsa-positive centromeres, but only a few have intercalary or terminal bands. Using differential fluorescent staining with DAPI/actinomycin D, quinacrine/actinomycin D (both A-T specific), and chromomycin A₃/distamycin A (G-C specific) three different types of major heterochromatic bands can be characterized in respect of their satellite DNA composition: highly A-T rich, slightly A-T rich, and very G-C rich. The chromosomes ofC. longifolia contain more A-T rich C-bands than those ofC. damasonium, while the latter's have more G-C rich heterochromatin. In both species several C-bands appear as secondary constrictions or gaps in the Feulgen-stained chromosomes, but most likely, in each species there is only one pair of chromosomes where the secondary constrictions function as nucleolus organizing regions. No major intraspecific variation could be observed except on one small chromosome pair ofC. longifolia which had a heteromorphic C-band in most individuals. Possible pathways of karyotype evolution involving polyploidy and Robertsonian events are discussed.     

Interspecific hybridization in natural populations ofCyrtandra (Gesneriaceae) on the Hawaiian Islands: Evidence from RAPD markers

Interspecific hybridization among Hawaiian species ofCyrtandra (Gesneriaceae) was investigated using randomly amplified polymorphic DNA (RAPD) markers. Thirty-three different primers were used to investigate interspecific hybridization for 17 different putative hybrids based on morphological intermediacy and sympatry with putative parental species. RAPD data provided evidence for the hybrid origin of all putative hybrid taxa examined in this analysis. However, the patterns in the hybrid taxa were not found to be completely additive of the patterns found in the parental species. Markers missing in the hybrid taxa can be attributed to polymorphism in the populations of the parental species and the dominant nature of inheritance for RAPD markers. Unique markers found within hybrid taxa require further explanation but do not necessarily indicate that the taxa are not of hybrid origin. The implications suggest that these interspecific hybridization events had, and continue to have, an effect on the adaptive radiation and conservation biology ofCyrtandra.     

Embryology and systematic position ofLiparis (Orchidaceae)

The embryology ofLiparis paradoxa andL. rostrata has been studied. The young anther wall consists of an epidermis, endothecium, three middle layers and secretory tapetum with uninucleate cells. In the mature anther, two or three sub-epidermal layers develop fibrous thickenings. The anther wall development corresponds to the massive type. Simultaneous cytokinesis results in decussate, isobilateral, linear, T-shaped and tetrahedral pollen tetrads. The pollinia are compact and at anthesis the pollen grains are 2-celled. Ovules are anatropous, bitegmic and tenuinucellate. Both the integuments are dermal in origin and 2-layered. Development of female gametophyte is of the monosporic, 8-nucleate type. Double fertilization occurs. The primary endosperm nucleus degenerates. The mature embryo is organized from the derivatives of tiers 1, 1, m, and n. Its development conforms to a variation of the Onagrad type. The seed coat is formed entirely by the outer layer of the outer integument. There are three sterile and three fertile valves in the ovary. In the prefertilization stages valves consist of parenchymatous cells. After fertilization, the sterile valves become sclerenchymatous whereas the fertile valves remain parenchymatous. The embryological characters support the disputed systematic position ofLiparis within subtribeLiparidinae ofEpidendreae.     

Pollination ofOphrys (Orchidaceae) in Cyprus

In the southern part of Cyprus the pollinator —Ophrys (Orchidaceae) relationships and its specifity have been investigated from the end of February until the middle of March 1986. 12Ophrys spp. were found. To date, only a single pollinator reference has been reported from this island. We found the following pollinators:Melecta tuberculata (Ophrys kotschyi),Eucera dimidiata (Ophrys flavomarginata),Eucera gaullei (Ophrys umbilicata),Eucera paulusi (Ophrys bornmuelleri),Anthophora erschowi (Ophrys elegans),Andrena torda (Ophrys sicula =O. lutea subsp.minor),Andrena cinereophila (Ophrys fusca, small-flowered),Andrena flavipes (Ophrys israelitica),Andrena morio (Ophrys iricolor andOphrys transhyrcana),Andrena bimaculata (Ophrys sphegodes aggr., probably formerly confused withO. transhyrcana). Most interestingly, it could be verified thatO. flavomarginata/O. umbilicata, O. bornmuelleri/O. levantina andO. transhyrcana/O. sphegodes aggr. (possiblyO. sintenisii) are different biospecies. This is a result of genetic isolation due to varying pollinators, and of differences in flower morphology.     

Genetic variation inOrchis papilionacea (Orchidaceae) from the Central Mediterranean region: Taxonomic inferences at the intraspecific level

The genetic differentiation ofOrchis papilionacea from Italy, Corsica, Sardinia, Sicily, and Greece was analyzed by isozyme studies. No significant differences at any of the 28 loci tested were observed between samples of the putative subspeciespapilionacea from Italy,grandiflora from Sicily andheroica from Greece, or by comparing sympatricpapilionacea-like andgrandiflora-like subsamples from Sardinia and Corsica. The genetic relationships found among the populations studied reflect more their geographic origin than their taxonomic assignment;papilionacea, grandiflora, andheroica are, therefore, at least in the study area, not to be considered as subspecies, but as simple morphs, controlled by few genes (possibly two) with Mendelian inheritance. The genetic divergence observed among the samples studied is very low and consistent with that of geographic populations of a single cohesive gene pool, linked by a high gene flow (Nm = 5.9).     

The embryology ofEpipogium roseum (Orchidaceae)

Development of pollen and female gametophyte inEpipogium roseum (D. Don)Lindl. has been investigated. The embryo sac conforms to the Apinagia type. The taxonomic position ofEpipogium within the family is discussed.