The parasitoid wasp Protaphidius nawaii parasitizes the aphid Stomaphis japonica, which is obligatorily attended by several species of ants of genus Lasius. Subgenus Lasius or Dendrolasius ants use different defense strategies to protect the aphids that they attend (Lasius, shelter building; Dendrolasius, aggressive attack). We performed molecular phylogenetic analysis based on partial mitochondrial DNA sequences of P. nawaii and found that the parasitoid wasp consists of two highly differentiated genetic lineages. Although these two lineages distributed sympatrically, one tends to parasitize aphids attended by ants of subgenus Lasius, and the other parasitizes aphids attended by ants of subgenus Dendrolasius. The two lineages of P. nawaii appear to exhibit different oviposition behaviors adapted to the different aphid-protection strategies of the two ant subgenera.
INTRODUCTION
In the face of the global biodiversity crisis (Brooks et al., 2006), cataloging of the earth's species is among the highest priorities. Several studies have highlighted the existence and importance of cryptic biodiversity, which may represent a substantial proportion of global biodiversity. Owing to the increasing amount of studies incorporating DNA-based techniques, the number of reported cryptic species is increasing (Beheregaray and Caccone, 2007).
Host specificity is one factor that generates parasitoid diversity (Hardy and Otto, 2014). In general, to increase the success rate of parasitism, parasitoids tend to specialize to specific hosts (Cheng, 1986; Godfray, 1994; Shaw, 1994; Althoff, 2003). Usually, characteristics of the host itself determines the host specificity of parasitoids, but sometimes, instead of the host, the surrounding environment determines host specificity of parasitoids. In particular, the host preference or specificity of parasitoids on aphids is determined by the attending ants of the host aphids. Sadeghi-Namaghi and Amiri-Jami (2018) found that the parasitoid wasp Lysiphlebus fabarum successfully attacks the aphids Acyrthosiphon gossypii and Brachycaudus cardui attended by particular ant species. In another case, the parasitoid wasps Paralipsis eikoae and Aclitus sappaphis, respectively, attack the host aphid Sappaphis piri attended by the ants Pheidole fervida and Lasius niger (Takada and Shiga, 1974; Takada and Hashimoto, 1985; Akino and Yamaoka, 1998).
The genus Protaphidius (Braconidae: Aphidiinae) is a group of parasitoid wasps parasitizing aphid genus Stomaphis. To date, only three species have been described: P. wissmannii from Europe (Starý, 1959), P. belokobylskiji from Russia (Davidian, 2007), and P. nawaii from Japan (Takada, 1983). Genus Protaphidius is characterized by a long pseudo-ovipositor that enables these wasps to parasitize host aphids hidden in crevices in tree trunks (Starsky, 1976). Previous studies confirmed that the genus Protaphidius is closely related to the genus Diaeretus (Shi and Chen, 2005), but the phylogenetic relationship within Protaphidius is unclear.
The aphid Stomaphis japonica (Aphididae: Lachninae) is attacked by the parasitoid wasp Protaphidius nawaii (Takada, 1983). Stomaphis japonica has an obligate mutualistic relationship with ants, and in Matsumoto (Nagano, Japan), where this study was conducted, it is attended mainly by ants of genus Lasius: Lasius (Lasius) japonicus, Lasius (Dendrolasius) fuji, and Lasius (Dendrolasius) nipponensis (Formicidae: Formicinae) (Yamamoto et al., 2015). Ants of the subgenera Lasius and Dendrolasius protect aphids in different ways. Those of subgenus Lasius build earthen shelters over both their trunk trails and the aphids they attend (Takada, 1983). These earthen shelters may protect the aphids from parasitoid wasps (Gibernau and Dejean, 2001). In contrast, ants of subgenus Dendrolasius rarely build earthen shelters, but they aggressively defend “bare” aphids. Because the subgenera Lasius and Dendrolasius employ different strategies to defend aphids, we hypothesized that P. nawaii may use different oviposition strategies to overcome the defense by Lasius or Dendrolasius. Therefore, there may be cryptic diversity in this parasitoid wasp determined by the ant species attending the aphids that it parasitizes.
Table 1.
Details of the DNA sequenced samples of the aphid-parasitizing wasp Protaphidius nawaii. ‘Lineage’ refers to the mtDNA lineage in Fig. 2. All samples were collected in Matsumoto (Nagano, Japan) in 2016.
To explore cryptic attending-ant-specific diversity in the parasitoid wasp P. nawaii, we undertook an extensive sampling of the wasps, performed molecular phylogenetic analysis, and identified two distinct wasp lineages, each specific to the aphids attended by ants of subgenus Lasius and to aphids attended by ants of subgenus Dendrolasius.
MATERIALS AND METHODS
Sample collection
Parasitoid wasps were sampled at seven sites in Matsumoto (Nagano, Japan) (Fig. 1). We collected all parasitized Stomaphis japonica mummies that we found on the trunks of 21 sawtooth oak trees (Quercus acutissima) from 5 June to 9 October 2016 (Table 1). All of the aphid colonies from which we collected parasitized mummies were attended by ants. The collected mummies were kept in the laboratory until the adult parasitoid wasps emerged. Forty-one wasps eventually emerged and were preserved in 70% ethanol. Three legs were cut from each wasp individual and preserved in 99.5% ethanol for molecular analysis. Attending ants from each colony were also collected and preserved in 99.5% ethanol for later identification by DNA barcoding. Voucher specimens, except for w85, w86, w88, w89, and w91, will be deposited as Hajimu Takada's Collection at the Hokkaido University Museum in Sapporo (Hokkaido, Japan).
DNA extraction, PCR amplification, and sequencing
We used a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) to extract DNA from the legs of the wasps and from the entire bodies of the ants, following the manufacturer's protocols. Then we amplified the mitochondrial COI gene by polymerase chain reaction (PCR) using Takara Ex Taq polymerase (Takara Bio, Shiga, Japan) and the primers Ron (5′-GGATCACCTGATATAGCATTCCC-3′) and Nancy (5′-CCTGGTAAAATTAAAATATAAACTTC-3′) for the parasitoid wasps, and Lasius-L (5′-TAYCCGCCATTAGCTTCAAA-3′) and Lasius-R (5′-TGAAATTAAGGATCCAATWGA-3′) for the ants (Maruyama et al., 2008). PCR was carried out for 30 cycles of 98°C for 10s, 50°C for 30s, and 72°C for 60s. Both strands of the amplified DNA were sequenced with a BigDye Terminator v1.1 Cycle Sequencing Kit (ABI, Weiterstadt, Germany) on an ABI 3130 Genetic Analyzer.
Fig. 1.
The seven sampling sites of this study (see Table 1 for details). Black and white circles indicate the sites where parasitoid Protaphidius nawaii wasps were collected (black: lineage A; white: lineage B). White circles (inside black) indicate the sites where both lineages A and B were collected. For lineages A and B, see Fig. 2.
Phylogenetic analysis
The mitochondrial COI sequences were edited and aligned with SeqScape v. 2.6 software (ABI, Weiterstadt, Germany). In the phylogenetic analysis of the wasps, we used the following sequences as outgroups (Aphidius matricariae EF077526, Lysiphlebus orientalis KC237748, Orussus abietinus EF032236). We constructed a maximum likelihood phylogenetic tree based on the Tamura 3-parameter model with MEGA7 software (Kumar et al., 2016), and determined the lineage of the wasps from each colony. The robustness of the tree was assessed by nonparametric bootstrapping with 500 replicates. The genetic distance (Kimura two-parameter (K2P) model) between the lineages was obtained by using MEGA7. The GenBank accession numbers of the COI gene sequences are listed in Table 1. To determine the species of the ants, we conducted a BLAST search on the NBCI website ( http://www.ncbi.nlm.nih.gov) for highly similar sequences to the ant DNA sequences.
RESULTS
Phylogenetic analysis
The partial COI gene of Protaphidius nawaii consisted of 422 nucleotide sites, of which 80 were parsimony informative. The topology of the maximum likelihood phylogenetic tree revealed two well-resolved lineages, designated A and B, within P. nawaii (Fig. 2). The genetic distance between the two lineages was 0.133–0.136 in K2P model. Both of the two lineages were collected within a limited area (Fig. 1), and even at the same sampling site (but from different tree) of 100 m between 614 m and 709 m. Both lineages distributed in the same altitudinal range (Table 1).
Species specificity of the parasitoid wasps
Twenty-one of the 23 specimens of lineage A, and two of the 18 specimens of lineage B emerged from aphids attended by Lasius (Lasius) japonicus ants, which build earthen shelters over the aphids they attend; two specimens of lineage A and 12 specimens of lineage B emerged from aphids attended by L. (Dendrolasius) fuji; and three specimens of lineage B emerged from aphids attended by L. (D.) nipponensis. L. (D.) fuji and L. (D.) nipponensis do not build earthen shelters over the aphids. In addition, one specimen of lineage B emerged from aphids attended by Camponotus obscuripes, which also does not build earthen shelters over the aphids (Table 1, Fig. 2). The P. nawaii wasps of lineage A were significantly more likely to emerge from aphids attended by Lasius (Lasius), and those of lineage B were significantly more likely to emerge from aphids attended by Lasius (Dendrolasius) or C. obscuripes (P < 0.05; Fisher's exact test).
Fig. 2.
Maximum-likelihood phylogenetic tree of Protaphidius nawaii based on a partial mitochondrial DNA sequence of the COI gene. Numbers beside the branches represent maximum-likelihood bootstrap values (as a percentage; only those >50% are shown). See Table 1 for specimen codes. The attending ant species are indicated by graphical symbols. The scale shows the nucleotide substitution rate of 0.05.
DISCUSSION
The results presented here show that the specificity of the parasitoid wasps studied is determined by the ant species attending the host aphids, rather than by the host itself. The results of the molecular analysis showed that two lineages of Protaphidius nawaii parasitoid wasps parasitized Stomaphis japonica, and each lineage had tendency to parasitize aphids attended by different ant subgenus (Lasius or Dendrolasius). The genetic distance between the two lineages was 0.133–0.136 (COI), which is sufficiently large compared with 0.03 (the standard interspecific COI genetic distances in insects; Hebert et al., 2003), or with 0.04 (COI genetic distance between sister species in Lysiphlebus, a genus of Aphidiinae; Petrovć et al., 2015). Thus, the two lineages of Protaphidius appear to be well supported mono-phyletic groups presumably equivalent to different species. The horizontal and elevational distribution of the two lineages largely overlapped (Fig. 1), indicating that the two lineages do not distribute allopatrically, but cohabit and partition a resource (aphids).
The defense strategies used by the attending Lasius and Dendrolasius ants against oviposition by the parasitoid P. nawaii wasp are both powerful, but different. Lasius (Lasius) ants defend the aphids by building shelters over them, whereas Lasius (Dendrolasius) ants defend the aphids not by building shelters but by aggressively attacking the parasitizing wasp. Note that L. (Dendrolasius) nipponensis sometimes builds rough shelters that cover only part of the aphid colony.
The correspondence of the two parasitoid wasp lineages to different attending ant species that use different defense strategies to protect aphids suggests that the two parasitoid wasp lineages may have adapted their oviposition behavior to circumvent these different defenses. Takada (1983) reported that P. nawaii parasitizes aphids protected by shelters by first inserting its antennae into the shelter to make a hole; then it inserts its pseudo-ovipositor through the hole to oviposit in a host aphid body (Fig. 3A). In contrast, when P. nawaii parasitizes bare aphids surrounded by numerous aggressive Dendrolasius ants, it stays at a distance from the aphids and uses its long pseudo-ovipositor to oviposit in a host aphid body (Maruyama et al., 2013) (Fig. 3B). These different oviposition behaviors so far reported may correspond to the two parasitoid wasp lineages revealed in this study. In the future, the morphological characteristics of the two wasp lineages should be examined along with their comparative oviposition success in aphids defended by Lasius (Lasius) or Lasius (Dendrolasius) ants.
As noted above, the different ways in which ants defend aphids may have led to the specificity of the two parasitoid lineages. However, the aphid defenses by each ant subgenus vary from situation to situation. Shelters constructed by ant subgenus Lasius collapse after rain and aphids are temporarily exposed. Additionally, the ant subgenus Dendrolasius sometimes covers some aphid individuals (but rarely covers all individuals). Such an occasional change in the way the ants defend aphids may have caused a partial discrepancy of the specificity of the two lineages of P. nawaii to the attending ant subgenera (w41 and w24 of lineage A parasitizing the Dendrolasius-attended aphids, and w42 and w55 of lineage B parasitizing the Lasius-attended aphids). In addition, P. nawaii of lineage B parasitized aphids attended by Camponotus obscuripes. This indicates that the lineage B of P. nawaii, which attacks aphids attended by the ant subgenus Dendrolasius, can also parasitize aphids attended by C. obscuripes, which does not build shelters.
Previous studies showed that the S. japonica-P. nawaii host-parasitoid system involves additional ectoparasitic hyperparasitoid wasp species Euneura stomaphidis and Dendrocerus stomaphis (Kamijo and Takada, 1983; Takada, 1983, 2009). Sanders and Van Veen (2010) showed that the relationship between aphids and their parasitoid and hyperparasitoid wasp communities varies with the presence of attending ants. As such, changes in the aphid parasitoid species (lineages) due to attending ant difference revealed in this study may affect the communities of the hyperparasitoids involved. Further investigation of this aphid–parasitoid wasp communities will shed light on the importance of ant association in the maintenance of host–parasitoid species-specific system.
ACKNOWLEDGMENTS
We thank to H. Takada for identifying the parasitoid wasps. This work was supported by Grants-in-Aid for a Research Fellow from the Japan Society for the Promotion of Science: KAKENHI Grant No. 26291090 to T. Itino and No. 16J09182 to T. Yamamoto.
AUTHOR CONTRIBUTIONS
TY and TI designed this study. HH and TY performed field study and molecular phylogenetic analysis. TK observed oviposition behavior of parasitizing wasps and took pictures. TY, YN and TI wrote the article and all authors contributed the final manuscript.
