Identification And Molecular Pliylogeny Of The Pollinating Wasps And Nematodes

Nematodes are present in the symbiotic system of fig trees and pollinating fig wasps.However,related species of nematodes and fig wasps,and relationships between the both are not clear yet.In the present study,wasps and nematodes were collected from Ficus microcarpa,Ficus hirta,Fightiana,Fistulosa,F.fistulosa,F.hispida,F.curtipes,F.altissima and F.religiosa in Guangzhou and Xishuangbanna from 2016 to 2019.The species of fig wasps and nematodes were identified based on their morphological and molecular characteristics.The mitochondrial cytochrome C oxidase subunit I(mtDNA COI),ribosomal large and small subunit RNA(LSU rRNA,SSU rRNA)genes were used for amplification by PCR with corresponding primers,and PCR products were purified and then sequenced.The related phylogenetic trees based on above three gene sequences were constructed by using Mega 7.0,respectively.The phylogenetic relationships among species inferred from the trees were analyzed.Main results are as follows:1.Combining morphological and molecular characteristics,three genera and eight related pollinating fig wasps were identified,namely,Ceratosolen solmsi,C.fusciceps,C.emarginatus,C.armipes,Eupristina altissima,E.verticillata,E.koningsberg and Playscapa quadraticeps.Two other species,Ceratosolen sp.and Eupristina sp.were not identified to species level.2.A new species,Parasitodiplogaster religiosae n.sp.,was found and described from the syconia of Ficus religiosa.particularly slender and needle-like male spicule is its important morphological differentiation feature.Other main features include that male head is not constricted,stama without teeth is composed of three parts: short annular cheilostom,short tubular gynostom and funnel-shaped stegostom,anterior esophagus(anterior part + middle esophageal bulb)is muscular and the posterior one(isthmus + posterior esophageal bulb)glandular,nerve ring surrounds the middle isthmus,excretory pore opens behind the base of the posterior esophageal ball,sperm is amoeboid,gubernacul with blunt tip is slender,slightly bent ventrally,dorsally bending at the middle point,forming a elongated squid-shaped,tits ventral view is diamond-shaped,tail tip has a short mucron but not a bursa,9 pairs(P1-P8,P9d)of genital papillae are present at tail,P1,P2 and P3 are located in front of the cloaca,P4 just near the cloaca,P5 located behind the cloaca,P6 on the lateral,P7 and P8 are adjacent,almost at the level of the phasmids,P9 d is dorsal,near tail end.The cuticle,esophagus and stama of female are similar to that of male.The vagina is located at the posterior part of the body,a single ovary is present,oocytes are in single file,four large vaginal gland cells are observed,tail without mucron is conical.3.Six other related nematodes were identified based on combined morphological and molecular characteristics,namely: Schistonchus superbus,S.hirtus,Martininema(Schistonchus)fistulosus,Ficophagus(Schistonchus)microcarpus,Ficophagus(Schistonchus)altissimus,Ficophagus(Schistonchus)centerae.4.Sequence alignment and phylogenetic analysis showed that the placements of related nematodes are correct in the phylogenetic trees constructed based on rDNA SSU 18 S,LSU D2/D3 and mtDNA COI gene sequences,and the positions of related wasps are reasonable in the phylogenetic trees constructed based on the mtDNA COI gene sequences.Schistonchus hirtus from Ficus hirta has a closer genetic relationship with S.superbus from Ficus wightiana,and so does Ficophagus altissimus from Ficus altissima with Ficophagus microcarpus from Ficus microcarpa.A similar case was presented in the corresponding pollinating fig wasps,for example,Euptina altissima from Ficus altissima has a closer genetic relationship with E.verticillata from Ficus microcarpa.