The Pollinating Fig Wasps Of Ficus Pumila

Mutualisms are one of the most important ecological interactions,with strong influences onalmost all levels in biological systems.Their long term persistence raises many challengingevolutionary questions.Figs and their pollinating wasps,with their great species richness anddiverse host life forms,are among the most tightly integrated pollination mutualisms known,andprovide a model system for developing and testing theories of mutualism evolution.The mutualisms of fig and fig wasps are both ancient and diverse.Results frommorphological studies suggested high species specificity between the two interacting partners.Initial molecular phylogenetic studies also suggested co-cladogenesis and coevolution at highertaxonomic levels.These studies led to the adoption of a predominant assumption of one host figtree to one pollinating wasp.However,more and more exceptions have been revealed by recent,especially molecular,studies and breakdowns in the one to one relationship may even representthe majority of relationships.In addition,the discovery of host switches of pollinating wasps andnatural hybrids between fig trees has further undermined the empirical basis of the one-to-one rule.Machado et al.(2005) have proposed a new evolutionary model,saying that the relationshipbetween fig and fig wasps is not strict-sense cospeciation between individual pairs of interactingfigs and wasps,but broad-sense coevolution between related groups,with host-shifting commonwithin groups of figs that are imperfectly defined genetically because of hybridization.However,their new model was based only on results from Ficus sections Pharmacosycea and Americana inthe Americas and may not be applicable elsewhere because some fig groups in other regions havedifferent relationships with their pollinators,with strict host specificity and cospeciation asdominant patterns.Consequently,the frequency in breakdowns of the one to one relationshipwithin different fig groups,in different regions,is essential for the understanding of the relativeimportance of the competing finer-scale cospeciation or broad-sense coevolution models.Wiebesia pumilae (Hill),originally described from Hong Kong,is the specific pollinator ofFicus pumila L.var.pumila and its variant var.awkeotsang,and shows no known morphological variation from place to place.However,the taxonomic status of F.pumila and W.pumilae areunresolved.The host was placed in Synoecia section Rhizocladus or Ficus section Ficus based ondifferent studies,while the pollinator was assigned to the genera Blastophaga or Wibesia bydifferent researchers.In addition studies in Taiwan and Fujian have found that some pollinatorsderived from var.pumila did not enter receptive figs of var.awkeotsang which suggested thatmore than one species of pollinator might be present.We employed three genes,COI (mitochondrial),Cytb (both mitochondrial and its NUMTssequences) and ITS2 (nuclear ribosomal) to investigate the extent of divergence,distributions andphylogenetic relationships within the Wiebesia pollinators of F.pumila in Southeastern China.Wesampled 331 individuals from 31 mainland and island populations.Using the softwareSTRUCTURE,ANeCA,PAUP and ARLEQUIN,we analysed the degree of genetic divergence,genetic diversity and demographic expansion dynamics.Integrating corresponding sequences inthe GenBank data base,we reconstructed phylogenetic trees.We also assessed the speciesspecificity of F.pumila and its pollinators,and estimated the influence of habitat differencesbetween mainland and islands on W.pumilae populations.The main results were as follows:1) The pollinators of F.pumila in Southeastern China showed deep genetic divergence,andcould be divided into three distinct clades.The genetic distances between any two clades exceededthose among conspecific individuals based on both mitochondrial and nuclear genes.Nomorphological differences were evident between the three clades.They should be recognized asthree cryptic species,named as Sp.A,Sp.B and Sp.C.The 331 wasp individuals were consistently placed into the three identical clades,irrespectiveof the genes and methods employed.All three clades were well defined genetically.Theprobabilities of assignment of individuals to each clade,assigned by STRUCTURE with1,000,000 repetitions,were all more than 95%.The three clades could not be linked togetherwithin the 95% statistical parsimony criterion by TCS integrated into ANeCA.The bootstrappercentages of the three monophyletic clades in different phylogenetic trees are all 99%.The divergences of COI between any two clades were more than ten times the averagedifferences within clades.The Kimura-2-parameter differences between clades were 6.63%, 10.94% and 10.69% respectively,while the average of those within each clades was only 0.57%.The distances between any two clades were not only beyond the standard COI barcoding thresholdfor animal species,but also higher than the known maximum found in similar studies on crypticfig wasps.The nuclear genes of W.'pumilae'also showed deep divergence.Different individuals sharedthe same ITS2 haplotypes within two clades,while the haplotypes in the three different cladesdiffered from each other by 7 to 13 mutations.Although two ITS2 haplotypes were detected in oneof the three clades,they were highly homogenized with just one different nucleotide out of 454bpsequences.Moreover one of these two haplotypes was limited to just one population.Thedistribution of ITS2 haplotypes coincided with the concerted evolution of the ribosome gene,further demonstrating interspecies differences among the three clades.NUMTs of Cytb verified complete isolation among the three clades.The recent NUMTshaplotypes derived from the individuals from different clades clustered with corresponding Cytbhaplotypes only within each clade in the combined phylogenetic trees,rejecting the possibility ofrecent gene flow among clades.2) The three cryptic pollinators of F.pumila are sister species.When clustered with the corresponding sequences of other pollinating fig wasps in GenBank,all the W.pumilae haplotypes were fixed in one monophyly with bootstraps of 99%,based on bothCOI and Cytb.The co-occurrence of NUMTs sequences converted from the same fragment ofmitochondrial genome,Cytb,in two of the three species,also suggested close relationship amongthem.In addition,the NUMTs haplotypes derived from different clades clustered together inoutgroups,illustrating their recent common ancestry.3) The three cryptic species display divergence in distribution and phenology.Habitatpreferences,geographical and perhaps temporal isolation could explain the co-existence of threepollinators in Southeastern China.Wiebesia'pumilae'Sp.A was widespread in almost all the northern populations,Sp.B wasdistributed mostly in southern populations,while Sp.C was mostly found in the northern islandpopulations.Their distributions suggested different habitat preferences,possibly related tolongitude,landforms,and mainland versus island environments.Habitat specialization and geographic barriers may have promoted the division and isolation of pollinators withoutequivalent host plant speciation.Sp.A and Sp.C displayed small differences in phenology,the former emerging about 10 daysearlier than the latter,independent of longitude.The difference in emergence time could havefavoured divergence among ancient populations of W.'pumilae'.4) Sp.A is distributed unequally in mainland and island populations,suggesting colonizationfrom the mainland to the islands.Sp.A was found in 27 of the 31 populations.Chi-square tests revealed a nonrandomdistribution of these populations,with two repartitioned groups mainly on the mainland andislands respectively,as defined by STUCTURE and ANeCA.AMOVA analysis revealedsignificant differences between mainland and island groups.Compared with the mainland group,the island populations displayed more obvious signs ofdemographic expansion.The island-specific haplotypes showed a typical star-like network withmuch more tip haplotypes.Recent or undergoing demographic expansion and bottleneck eventsin the island group were verified by both mismatch distribution analysis and neutrality tests,while Tajiam's D and Fs statistics did not deviate significantly from zero in the mainland group.The island group showed lower genetic diversity than the mainland,despite larger samplesizes of populations and individuals.Both the total and average population genetic diversities inthe islands were lower than in the mainland group,especially the mean number of pairwisedifferences.The maximum difference in mainland-specific COI haplotypes was 12 mutations,while that of the islands was just 3,indicating an ancient existence of mainland haplotypes.Furthermore,all the mainland specific haplotypes were settled in deep inner nodes inphylogenetic trees,further affirming their ancient origin.All island specific COI haplotypes were linked to two shared haplotypes with one mutation.The two shared haplotypes were distributed widely in both mainland and island populations,withthe highest frequencies among all haplotypes.The distribution pattern of these two sharedhaplotypes was in concordance with the characteristics of sea-land breezes in Hangzhou Bay.Many factors may have contributed to the mainland-island pattern of Sp.A,such as morefrequent extinction events on the islands due to greater population fluctuations and smallerpopulation sizes,individual radiations from mainland populations,colonization,expansion and independent evolution in the relatively isolated island habitats.5) There were potential glacial refugia for Sp.B and Sp.C,in Ningde and the southernregions.The southern populations of Sp.B and Sp.C had higher genetic diversity and more ancientspecific haplotypes than elsewhere.Four population-specific Sp.C COI haplotypes were detected,three in Ningde,one in Taohua.The three specific haplotypes in Ningde were distinguished fromeach other by 8 to 11 mutations,while that in Taohua was linked to the most frequently sharedhaplotype by 1 mutation.Mismatch distribution analysis and neutrality tests detected nosignificant recent expansion events in the Ningde population and all statistics showed that it was astable population that had existed for a long period.So Ningde and other southern regions werepotential glacial refugia of W.'pumilae'.The Wuyi Mountains there could provide protection forthe populations during glacial periods.After the last glacial maximum,wasps may haverecolonized north-eastern regions along the Wuyi Mountains.In conclusion,F.pumila is pollinated by three genetically distinguishable cryptic sisterspecies in Southeastern China.The three species coexist over much of the range of their host plant,with no evidence of hybridization,but vary in their relative abundance within the range of theirhost.Some differences in their biology were detected.