Microtrichs of amphipod crustacea. Morphology and distribution

Microscopical cuticular structures of four amphipods, presumed to be sensory in function are described, using a scanning electron microscope. Gammarus sp, Liljeborgia sp and Orchomene sp showed very small peg‐like microtrichs emerging from cuticular pits of less than one micron in diameter. Single units, facing distally, were ranged in rows, several rows occupying a single integumental polygon. Density of rows and number of units per row were always higher in central areas. The distribution, organization and relationships with larger sensilla, suggest that microtrichs are specialized in detection of slow or weak water currents. Orchestia showed short sensilla coeloconica. The difference between this species and the other three may be related to its different habitat, which is terrestrial.     

Orchardgrass (<Emphasis Type="Italic">Dactylis glomerata</Emphasis> L.) EST and SSR marker development,annotation, and transferability

Orchardgrass, or cocksfoot [Dactylis glomerata (L.)], has been naturalized on nearly every continent and is a commonly used species for forage and hay production. All major cultivated varieties of orchardgrass are autotetraploid, and few tools or information are available for functional and comparative genetic analyses and improvement of the species. To improve the genetic resources for orchardgrass, we have developed an EST library and SSR markers from salt, drought, and cold stressed tissues. The ESTs were bi-directionally sequenced from clones and combined into 17,373 unigenes. Unigenes were annotated based on putative orthology to genes from rice, Triticeae grasses, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Of 1,162 SSR markers developed, approximately 80% showed amplification products across a set of orchardgrass germplasm, and 40% across related Festuca and Lolium species. When orchardgrass subspecies were genotyped using 33 SSR markers their within-accession similarity values ranged from 0.44 to 0.71, with Mediterranean accessions having a higher similarity. The total number of genotyped bands was greater for tetraploid accessions compared to diploid accessions. Clustering analysis indicated grouping of Mediterranean subspecies and central Asian subspecies, while the D. glomerata ssp. aschersoniana was closest related to three cultivated varieties.     

Impact of body melanisation on contrasting levels of desiccation resistance in a circumtropical and a generalist <Emphasis Type="Italic">Drosophila</Emphasis> species

We investigated the role of cuticular lipids, body melanisation and body size in conferring contrasting levels of desiccation resistance in latitudinal populations of Drosophila melanogaster and Drosophila ananassae on the Indian subcontinent. Contrary to the well known role of cuticular lipids in water proofing in diverse insect taxa, there is lack of geographical variations in the amount of cuticular lipids per fly in both the species. In D. ananassae, quite low levels of body melanisation are correlated with lower desiccation resistance. By contrast, increased levels of desiccation resistance are correlated with quite high melanisation in D. melanogaster. Thus, species specific cuticular melanisation patterns are significantly correlated with varying levels of desiccation resistance within as well as between populations and across species. Role of body melanisation in desiccation resistance is further supported by the fact that assorted dark and light flies differ significantly in cuticular water loss, hemolymph and dehydration tolerance. However, similar patterns of body size variation do not account for contrasting levels of desiccation resistance in these two Drosophila species. Climatic selection is evidenced by multiple regression analysis with seasonal amplitude of thermal and humidity changes (Tcv and RHcv) along latitude on the Indian subcontinent. Finally, the contrasting levels of species specific distribution patterns are negatively correlated with RHcv of sites of origin of populations i.e. a steeper negative slope for D. ananassae corresponds with its desiccation sensitivity as compared with D. melanogaster. Thus, evolutionary changes in body melanisation impact desiccation resistance potential as well as distribution patterns of these two Drosophila species on the Indian subcontinent.     

A revision of the <Emphasis Type="Italic">Heterospathe elegans</Emphasis> (<Emphasis Type="Italic">Arecaceae</Emphasis>) complex in New Guinea

Summary  Three closely affiliated species of Heterospathe Scheff. (H. elegans (Becc.) Becc., H. humilis Becc. and H. versteegiana Becc.) from New Guinea are revised. They are reduced to a single species which is divided into two subspecies, and the new combination H. elegans subsp. humilis (Becc.) M. S. Trudgen & W. J. Baker is made. The subspecies can be readily distinguished by their growth habit. Epitypes are designated for the three previously published names, as informative material on the habit is not included in the existing type specimens. A new, potentially related species is described as H. pullenii M. S. Trudgen & W. J. Baker.     

Variabilität, Verbreitung und Systematik der Arten und Unterarten in der Gattung Pungitius Coste, 1848 (Pisces, Gasterosteidae)

On the systematics and the evolution of variation and distributional patterns in the species and subspecies of the genus Pungitius Coste, 1848 (Pisces, Gasterosteidae) 1. In the genus Pungitius (similarly as in Gasterosteus) there seems to be of some interest the bony armature, especially from the zoogeographical point of view. These lateral bony shields are developed in the skin (instead of scales which are completely lacking in the family of Gasterosteidae) and are more or less present just as to the provenance of the individuals. 2. Preponderantly with respect to this bony armature several species and subspecies have been distinguished in the genus Pungitius, the variability, distribution and systematics of which are critically reviewed and compared in the present paper on the basis of museum material and data from the literature. 3. For the sake of adequate interpretation of modern distributional patterns there may be assumed an originally fully armoured type at the end of the Tertiary from which (after having escaped the ice by southward migration) during the Pleistocene period in several refugial areas new species or subspecies have derived, partially by reduction of the bony armature under freshwater conditions. During postglacial times these subspecies penetrated different areas but in some localities also produced mixed populations ofter having contacted within the same region. At least in some cases this may have led to subsequent hybridization. 4. The species P. platygaster in its modern distribution in the ponto-caspi-aralic region is now geographically isolated from the holarctic-circumpolarly distributed P. pungitius in its recent subspecies pungitius with reduced bony armature. Of this latter (originally fully armoured) species populations are assumed to have penetrated, at latest during the penultimate (= Riß) glaciation the Caspian Sea and subsequently the Black Sea and Lake Aral. These populations are the assumed ancestors of the nowaday still fully armoured P. platygaster. A decision as to the real degree of differentiation between the recent P. pungitius and P. platygaster has to be left to future crossbreeding experiments the results of which will possibly lead to a subspecies concept only. 5. In contrast to the foregoing the fully plated original type of P. pungitius underwent a serious reduction in its bony armature especially in the Southwest European refugium which led to a total loss of bony shields in the subspecies P. p. laevis. 6. As may be concluded from the now occurring types, this reduction seems to have been generally less pronounced in the East Asiatic refugium. We are confronted here, probably as a result of postglacial spread with the occurrence of three subspecies within the same area, i. e. the fullyarmoured P. p. sinensis on the one hand, and P. p. tymensis with reduced lateral plates (and also reduced lateral processes of the pubic bones in the pelvis girdle) and the holarctic-circumpolarly distributed P. p. pungitius with likewise reduced bony armature on the other hand. Of these subspecies the fully plated sinensis seems in its appearance to approach best what we perhaps may imagine as the original type of P. pungitius at the end of the Tertiary.     

The genus <Emphasis Type="Italic">Crataegus</Emphasis> L.: An ecological and molecular study

This study aimed at the indentification of the species and genotypes of the genus Crataegus in Syria and determination of the genetic relationships among them based on the analysis of genomic and chloroplast DNA (cpDNA) using ISSRs and CAPS techniques. Morphological characterization carried out on 49 Crataegus samples collected from different geographical regions of Syria revealed four Crataegus species: momogyna, C. sinaica, C. aronia and C. azarolus. In the dendrogram constructed for those samples based on ISSRs (20 primers), all samples that belong to C. monogyna were clustered in one cluster. Samples of the other three species were overlaped in another cluster. Two samples of these were the most distant from all other samples in the dendrogram and were suggested to represent hybrid species or subspecies. When CAPS technique was applied on four Crataegus samples that represent the four suggested species using 22 cpDNA regions and 90 endonucleases, no polymorphism was detected neither in amplification products sizes nor in restriction profiles. The inability of detection of variation in cpDNA among species suggested can be attributed to the low level of evolution of the cpDNA in the genus, and to the possibility that some of these species are either subspecies or hybrids since the cpDNA is inherited through one parent only.     

A radioactive assay allowing the quantitative measurement of cuticular permeability of intact <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

Cuticular penetration of five different ¹⁴C-labeled chemicals (benzoic acid, bitertanole, carbaryl, epoxiconazole and 4-nitrophenol) into Arabidopsis thaliana leaves was measured and permeances P (ms⁻¹) were calculated. Thus, cuticular barrier properties of A. thaliana leaves have been characterized quantitatively. Epoxiconazole permeance of A. thaliana was 2.79 × 10⁻⁸ ms⁻¹. When compared with cuticular permeances measured with intact stomatous and astomatous leaf sides of Prunus laurocerasus, frequently used in the past as a model species studying cuticular permeability, A. thaliana has a 48- to 66-fold higher permeance. When compared with epoxiconazole permeability of isolated cuticles of different species (Citrus aurantium, Hedera helix and P. laurocerasus) A. thaliana permeability is between 17- to 199-fold higher. Co-permeability experiments, simultaneously measuring ¹⁴C-epoxiconazole and ³H₂O permeability of isolated cuticles of three species (C. aurantium, H. helix and P. laurocerasus) showed that ³H₂O permeability was highly correlated with epoxiconazole permeability. The regression equation of this correlation can be used predicting cuticular transpiration of intact stomatous leaves of A. thaliana, where a direct measurement of cuticular permeation using ³H₂O is impossible. Water permeance estimated for A. thaliana was 4.55 × 10⁻⁸ ms⁻¹, which is between 12- and 91-fold higher than water permeances measured with isolated cuticles of C. aurantium, H. helix and P. laurocerasus. This indicates that cuticular water permeability of the intact stomatous leaves of the annual species A. thaliana is fairly high and in the upper range compared with most P values of perennial species published in the past.     

A new acuariid nematode, <Emphasis Type="Italic">Syncuaria mackoi</Emphasis> n. sp. (Spirurida), from <Emphasis Type="Italic">Ciconia nigra</Emphasis> (L.) (Ciconiiformes: Ciconiidae) in Europe

Syncuaria mackoi n. sp. is described, based on light and scanning electron microscopy, from the stomach of the black stork Ciconia nigra in Bulgaria. The new species is characterised by: body length 7.18–8.30 mm (males) and 10.13–11.40 mm (females); cordons anastomosing about the level of the middle of the muscular oesophagus and consisting of delicate cuticular plates; bifurcate deirids 7–9 μm long; lateral longitudinal cuticular band-like swellings; nine pairs of pedunculate caudal papillae, i.e. four precloacal and five postcloacal, the latter forming two groups (2 + 3); sixth pair of postcloacal papillae sessile; left spicule 854–907 μm long; right spicule 138–154 μm in length, with a curved distal end resembling a horn and a short lateral projection at some distance from its tip; and vagina vera provided with an anteriorly-directed blind appendix. The specimens identified as S. ciconiae (Gil’bert, 1927) by Macko (1964) from C. nigra in Slovakia are considered as belonging to S. mackoi. A pair of small cupolae situated dorsally and ventrally between bases of cordons of the new species is described for the first time in this genus; they are recognised as homologous to the pair of spines in the same position previously described in S. diacantha Petter, 1961 and S. plegadisi Digiani, 1999.     

Comparison of Defense Body Movements of <Emphasis Type="Italic">Apis laboriosa</Emphasis>, <Emphasis Type="Italic">Apis dorsata dorsata</Emphasis> and <Emphasis Type="Italic">Apis dorsata breviligula</Emphasis> Honey Bees

Defense behavior of three, free living giant (Megapis) honey bee subspecies, Apis laboriosa, A. dorsata dorsata and A. dorsata breviligula, was compared. Disturbed worker bees responded with characteristic dorso-ventral defense body twisting (DBT). Workers of A. laboriosa twisted the thorax by 55°, and the two other A. dorsata subspecies by about 10° more. A. laboriosa workers raised the tip of the abdomen by 90° and workers of the two other bee subspecies by about 20° higher. Differences in those traits were highly significant between A. laboriosa and both A. dorsata subspecies, but were not significant between those two subspecies. The whole cycle of DBT was the most vigorous in A. d. breviligula (0.11 s), and it was twice as vigorous as in A. d. dorsata (0.26 s) and trice as in A. laboriosa (0.32 s). A. laboriosa twisted the body together with wings folded over the abdomen, while the two A. dorsata subspecies raised the abdomen between spread wings. This supports the opinion to treat A. laboriosa as a separate species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CHEMICAL COMMUNICATION IN HAWAIIAN DROSOPHILA

We are interested in elucidating the extent to which lekking Hawaiian Drosophila species have diverged from their continental counterparts, which engage in sexual behavior at communal food sources, with regard to the chemical communication systems that the flies employ. Accordingly, we have analyzed flies from three closely related Hawaiian Drosophila species in the adiastola subgroup. These species are of interest because the males engage in a unique behavior: while courting, they raise their abdomens over their heads and emit anal droplets. Analysis of the flies' behavior, the hydrocarbons in males' anal droplets, and males' cuticular hydrocarbons suggest that females' responses to males may be mediated by cuticular pheromones and/or pheromones in males' extruded droplets that enable the females to distinguish conspecific from heterospeciflc males. Conversely, perception of cuticular hydrocarbons from conspecific females enables D. adiastola males to distinguish females from a closely related species from conspecific females. On the basis of these observations, we suggest that the adiastola subgroup species are unique among drosophilids in that they utilize an anal droplet-mediated pheromone communication system, some or all components of which are species specific. However, the lekking Hawaiian Drosophila species are similar to D. melanogaster and related continental species in that the Hawaiian flies employ a cuticular pheromone communication system, some components of which are sex and species-specific.     

Colonial and geographic variations in agonistic behaviour,cuticular hydrocarbons and mtDNA of Italian populations of <Emphasis Type="Italic">Reticulitermes lucifugus</Emphasis> (Isoptera,Rhinotermitidae)

Summary The subterranean termite, Reticulitermes lucifugus (Rossi), is found throughout Italy. The purpose of this study was to assess variations among colonies collected from the north to south of Italy. A multidisciplinary approach was used including behavioral tests, cuticular hydrocarbon analysis and partial sequencing of mitochondrial DNA. Results showed that Italian R. lucifugus populations were moderately aggressive and that aggressive behavior was unrelated to intercolonial geographic distance. Analysis of cuticular hydrocarbons demonstrated no qualitative variations between colonies, but quantitative differences were found according to caste and colony. Alkene content tended to decrease from north to south. Sequencing of mtDNA indicated kinship between two Tuscan populations and R. l. corsicus. This finding is suggestive of transtyrrenian distribution of the Corsican subspecies. Moreover, distance between haplotypes appears to be associated with intercolonial geographic distance.Received 25 March 2003; revised 3 October 2003; accepted 10 November 2003.     

Isozyme evidence on the specific distinctness and phylogenetic position of <Emphasis Type="Italic">Vicia incisa</Emphasis> (Fabaceae)

Vicia incisa is a taxonomically controversial species that has been also treated as a subspecies of V. sativa because of a great morphological similarity. The phylogenetic position of V. incisa is uncertain because various DNA markers have provided contradictory results. Isozymes of V. incisa encoded by 15 loci and resolved with the use of polyacrylamide gel electrophoresis (PAGE) are described and compared with those of seven related species belonging to sections Vicia, Sepium, Lathyroides and Pseudolathyrus in order to get new evidence about its taxonomic rank and phylogenetic position. Phylogenetic relationships are analyzed with maximum parsimony and neighbour joining methods. Vicia incisa is shown to differ from all three subspecies of V. sativa including, sativa, cordata and nigra, by alternate variants of ten isozymes out of 15 analysed. Instead, V. incisa has much more similarity to V. grandiflora and V. sepium by sharing eight isozyme variants which differ from the subspecies of V. sativa. The most parsimony and neighbour joining analyses of the isozyme variation placed V. incisa as basally linked to the V. grandiflora and V. sepium species couple in the clade of section Sepium (= sect. Atossa), while the subspecies of V. sativa together with V. lathyroides formed a separate clade of section Vicia. The isozyme data provide further support to the species status of V. incisa.     

Genetic diversity of flat-headed vole (<Emphasis Type="Italic">Alticola strelzowi</Emphasis> (Kastschenko, 1899)) inferred from cytochrome <Emphasis Type="Italic">b</Emphasis> variation

Variation of the cytochrome b gene fragment was examined in 27 flat-headed voles Alticola strelzowi from different parts of the species range. A total of 15 haplotypes were described, while the species is characterized by low levels of genetic differentiation and polymorphism. The haplotypes form three haplogroups, one of which corresponded to the subspecies A. s. strelzowi, and the other two, to A. s. desertorum. Based on different indices, the level of genetic polymorphism in the later subspecies was considered to be higher than in the first one. Phylogeographic analysis suggested post-glacial dispersal of flat-headed voles from a single refugium located in Western Altai. Using different techniques, relatively recent colonization of the Central Altai territory was demonstrated (subspecies A. s. strelzowi), which determined low level of genetic variation in this territory.     

Mapping species differences for adventitious rooting in a <Emphasis Type="Italic">Corymbia torelliana</Emphasis> × <Emphasis Type="Italic">Corymbia citriodora</Emphasis> subspecies <Emphasis Type="Italic">variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.     

On the morphology and taxonomic position of <Emphasis Type="Italic">Ungulaspis arctoa</Emphasis> (Osteostraci,Agnatha) from the lower devonian of the Severnaya Zemlya archipelago

An additional study of the cephalic shield and trunk scales of the holotype of Ungulaspis arctoa Afanassieva et Karatajūtė-Talimaa has shown that the exoskeleton surface is composed of dentin of at least two generations. Based on new data, the exoskeleton of Ungulaspis is reconstructed. A model for ontogenetic development of the exoskeleton of Ungulaspis arctoa is proposed. The superposition growth of osteostracan exoskeleton is described in detail for the first time using macro-rather than microremains. Using Osteostraci as an example, the existence of two (bipolar and unipolar) types of vertical growth of the exoskeleton in some groups of lower vertebrates is shown. The taxonomic position of Ungulaspis arctoa in the system of Osteostraci is discussed.     

Genetic population structure and morphological characters of Japanese psychrolutids of genus <Emphasis Type="Italic">Malacocottus</Emphasis> (Scorpaeniformes: Psychrolutidae)

The genetic population structure and the diagnostic characters of Malacocottus gibber from the Japan Sea and Malacocottus zonurus from the Okhotsk Sea and the northwestern Pacific were compared. Analysis of the nucleotide sequences of the mitochondrial control region revealed no genetic differences between the populations of M. gibber and M. zonurus, even though most individuals of both the species were found to be morphologically distinct. Most of the Malacocottus gibber specimens had the typical morphological characters of this species, namely the absence of an accessory spine on the preopercle of both sides and the absence of modified body scales above the lateral line. All the specimens of M. zonurus had accessory spines on both sides, and most of them had modified body scales. The results of this study suggest that M. gibber should be treated as a subspecies or a synonym of M. zonurus. The nested clade analysis and the analysis of molecular variance (AMOVA) showed that the Japanese Malacocottus fishes are genetically homogenous over their geographical range. The mismatch distribution of the Japanese Malacocottus fishes indicated that a sudden population expansion had occurred recently. The contrast in phylogeographic structures between the Malacocottus fish and the zoarcid Bothrocara hollandi—the most dominant deep-sea demersal fish in the Japan Sea—might be attributed to the differences in the depths of the habitats and larval ecology between these two fishes.     

New data on the morphology of <Emphasis Type="Italic">Spinitectus oviflagellis</Emphasis> Fourment, 1884 (Nematoda: Cystidicolidae) from the pyloric caeca of <Emphasis Type="Italic">Macrourus berglax</Emphasis> (Macrouridae) in the eastern Greenland Sea

Specimens of a little-known nematode, Spinitectus oviflagellis Fourment, 1884, the type-species of Spinitectus Fourment, 1884, were collected mainly from the pyloric caeca of a marine deep-water fish, the onion-eye grenadier Macrourus berglax Lacépède (a new host record), in the eastern Greenland Sea, North Atlantic Ocean. Studies using light and scanning electron microscopy revealed some taxonomically important, previously unreported features of S. oviflagellis, such as the detailed structure of the cephalic end, the position of the excretory pore and the presence of ventral pre-anal cuticular ridges (area rugosa) in the male, which indicated a certain degree of intraspecific biometrical variability in this species. S. oviflagellis is compared with similar congeneric species parasitising marine fishes.     

Hydrocarbon distribution and colony odour homogenisation in <Emphasis Type="Italic">Pachycondyla apicalis</Emphasis>

Summary Within and between individuals hydrocarbon (HC)-circulation was studied in Pachycondyla apicalis workers, using radioactive labeling. Newly synthesized HCs occurred both in the PPG and on the epicuticle in appreciable amounts, lesser quantities were found in the crop. The front basitarsal brush contained a greater amount of radiolabeled HCs than could be predicted from its surface area, suggesting preferential secretion to these organs. We propose that the newly synthesized HCs are secreted primarily to the front basitarsal brushes and are thereafter either distributed throughout the body surface, or cleared via the PPG and the alimentary canal.Using labeled HCs as a model, we tracked the time-dependent dispersion of cuticular lipids among 11 workers, one of which was prelabeled for 24 hours. Distribution among the recipients became progressively uniform, reaching near homogenization between 5–10 days. The mean HCs transfer of P. apicalis to the PPG was substantially lower compared to that of Camponotus fellah or Aphaenogaster senilis. In contrast, transfer to the cuticle in this species was superior. We attribute the low transfer to the PPG to the inefficacy of passive body contact characteristic of P. apicalis, as opposed to trophallaxis and/or allogrooming that typify the other two species. The higher occurrence of radiolabeled HCs in P. apicalis cuticle can be attributed to their accumulation in the basitarsal brushes. The impact of cuticular lipid transfer and formation of uniform colony odour, as opposed to the maintenance of an idiosyncratic caste-specific composition, are discussed.Received 5 September 2002; revised 17 January 2003; accepted 10 February 2003.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Polymorphism analysis of <Emphasis Type="Italic">csd</Emphasis> gene in six <Emphasis Type="Italic">Apis mellifera</Emphasis> subspecies

The complementary sex determination (csd) gene is the primary gene determining the gender of honey bees (Apis spp). In this study we analyzed the polymorphism of csd gene in six Apis mellifera subspecies. The genomic region 3 of csd gene in these six A. mellifera was cloned, and identified. A total of 79 haplotypes were obtained from these six subspecies. Analysis showed that region 3 of csd gene has a high level of polymorphism in all the six A. mellifera subspecies. The A. m. anatolica subspecies has a slightly higher nucleotide diversity (π) than other subspecies, while the π values showed no significant difference among the other five subspecies. The phylogenetic tree showed that all the csd haplotypes from different A. mellifera subspecies are scattered throughout the tree, without forming six different clades. Population differentiation analysis showed that there are significant genetic differentiations among some of the subspecies. The NJ phylogenetic tree showed that the A. m. caucasica and A. m. carnica have the closest relationship, followed by A. m. ssp, A. m. ligustica, A. m. carpatica and A. m. anatolica that were gathered in the tree in turn.