Identification,systematics And Phylogeny Of The Genera In The Family Aphelenchoididae(Nematoda:Tylenchomorpha)

The family Aphelenchoididae Skarbilovich,1947 includes numerous species with morphological and ecological diversity.Among them,the pine wood nematode Bursaphelenchus xylophilus and the rice white tip nematode Aphelenchoides besseyi can cause serious damage to forestry and agriculture respectively,therefore,they attract great attention from scientists worldwide.However,most of the genera in Aphelenchoididae are still in confusions and ambiguities.In this study,ten species from three genera in Aphelenchoididae isolated from different habitats were identified and described by morphological and molecular characterisations.The results enriched the taxonomic information for this family.The combination of morphological characters of head region of Aphelenchoididae species with Bayesian concatenated tree based on ribosomal DNA(rDNA)loci clarified the phylogenetic evolution relationship of members in the family Aphelenchoididae.The results are as following:1.Identification of Aphelenchoides stellatus n.sp.Aphelenchoides stellatus n.sp.was isolated from packaging wood from Japan imported to Ningbo Port,China.The new species is characterised by a body length of 485-533 ?m(males)and 547-699 ?m(females).The cuticle is weakly annulated and there are four lines in the lateral field.The stylet is 9-11 ?m long and has small basal swellings.The excretory pore is located posterior to the nerve ring.Spicules smoothly curved,rose-thorn shaped.Apex and rostrum round,only slightly offset,dorsal limb 19-21 ?m long.Male tail bearing six(2+2+2)caudal papillae.The female spermatheca is axial and oblong with round sperm present in multiple rows.Both male and female tail pegs have 3-4 processes,appearing star-like under the scanning electron microscopy(SEM).The new species belongs to the Group 3 category sensu Shahina(1996)of Aphelenchoides species.Phylogenetic analyses based on full length ITS and 28S D2-D3 region of rDNA confirmed its status as a new species.2.Identification of Aphelenchoides rotundicaudatus n.sp.Aphelenchoides rotundicaudatus n.sp.was isolated from packaging wood from South Korea.The new species is characterised by a body length of 364-509 ?m(males)and 371-493 ?m(females).The cuticle is weakly annulated and there are four lines in the lateral field.The stylet is 8-9 ?m long and has small basal swellings.The excretory pore is located ca one body diam.anterior to median bulb.Spicules are small(10-13 ?m),apex and rostrum rounded and only slight offset.The male tail has six(2+2+ 2)caudal papillae.Both male and female tails are cylindrical with a broadly rounded terminus,often with a small region of thickened cuticle or a blunt peg ca 1 ?m long,particularly in the female.The new species belongs to the Group 1 category of Aphelenchoides species.Phylogenetic analyses based on partial 18S,28S D2-D3 and full length ITS of rDNA confirmed its status as a new species.3.Identification of Sheraphelenchus parabrevigulonis n.sp.and redescription of S.sucusSheraphelenchus parabrevigulonis n.sp.was isolated from pine wood packaging from Italy.The new species is characterised by a stylet length of 11 ?m,three lines in the lateral field,excretory pore at level with,or slightly posterior to,nerve ring,V=93.1,and spicules with long condyles and membrane-like rostrum in a shallow triangular shape.Another Sheraphelenchus population was isolated from onion bulbs from South Korea.The species was identified as S.sucus,which is characterised by a stylet length of 13 ?m,excretory pore at level of,or posterior to,median bulb,and V=80.9.The phylogeny trees based on rDNA 18S,28S D2-D3 and ITS full length sequences confirmed S.parabrevigulonis n.sp.as a new species and the correct identification of S.sucus.4.Identification of Pseudaphelenchus zhoushanensis n.sp.Pseudaphelenchus zhoushanensis n.sp.was isolated from a dead Pinus thunbergii at Changgang Mountain,Zhoushan Islands,Zhejiang Province,China.The new species is characterised by the small to medium body length,cuticle slightly annulated,presence of three lateral lines,stylet 9.0-10.7 ?m with small but conspicuous basal knobs,excretory pore located from same level as the metacorpus to slightly anterior to metacorpus,true bursa surrounding entire tail but inconspicuous,male tail conical with a single mucron,spicule with distinct condylus and rostrum strongly arcuate to a pointed end,female tail conical with annulation,strongly ventrally bent in distal part of tail,with terminus bluntly pointed or finely mucronate.Phylogenetic analyses using sequences of the 18S and 28S D2-D3 regions of rDNA confirmed the status of P.zhoushanensis n.sp.as a new species.Combining the molecular phylogenetic analyses,morphology and biology of P.zhoushanensis n.sp.and Tylaphelenchus jiaae indicates that T.jiaae is a member of Pseudaphelenchus to which it is herein transferred as P.jiaae n.comb.(=T.jiaae).5.Description of five unknown Aphelenchoides speciesSix Aphelenchoides populations were isolated from the different habitats in several countries and identified as five unknown species by morpholgical and molecular characters,including Aphelenchoides sp.12Asp,Aphelenchoides sp.14Asp,Aphelenchoides sp.18Asp,Aphelenchoides sp.AspX and Aphelenchoides sp.Asp25.All the unknown species belong to the Group 2 according to the category of Aphelenchoides species by the female tail shapes having a single mucro.The male of Aphelenchoides sp.18Asp is characterised by the spicules with bifurcated distal tip,which is a rare character never shown in Aphelenchoides species.There is a large number of members in Group 2 which make the distinguish of five unknown speices from the close related species is rather difficult.The phylogenetic analysis of Aphelenchoides species based on rDNA 18S and 28S D2-D3 sequences confirmed the status of these five species as unkown and undescribed new species.6.Morphology of the head region and phylogenetic relationships of AphelenchoididaeThe characters of the head region from six Aphelenchoides populations were observed using SEM.In total,40 types of head regions were clarified from 78 species from 11 genera of the family Aphelenchoididae according to the comparison of the head region characters,including oral aperture,inner labial sensilla,outer labial sensilla,cephalic sensilla,amphids apertures and annulations of head region.The concatenated phylogenetic analyses by combining 404 sequences of rDNA 18S and 28S D2-D3 revealed five major clades of Aphelenchoididae were clearly presented.Using the characters of the head region type is able to distinguish several closed species including Aphelenchoides besseyi and A.fujianensis.However,the types of head region in family Aphelenchoididae failed to match their phylogenetic relationships well.The molecular and SEM characterisations of the memebers in Aphelenchoididae need to be further explored,and the phylogenetic realtionship in different types of the head region for the members still need to be clarified.