2018

The 2018 full report (in Romanian) can be found here.

Obtaining egg parasitoids of Nezara viridula (rearing from wild or sentinel eggs, field collecting, loans from museums)

We acquired specimens collected from various habitats in 10 states (Bulgaria, South Korea, France, Greece, Japan, Republic of Moldova, Romania, Taiwan, Turkey and Ukraine). This includes specimens collected by the project’s team, received by the Invertebrate Diversity and Phylogenetics Laboratory for sorting and identification, specimens from several collections in Japan, and specimens on loan from Sophia Antipolis Institute that were collected in France using sentinel eggs of Nezara viridula. We also updated the list of the egg parasitoids of Nezara viridula with the publications from 2017 and 2018.

Molecular characterization of the egg parasitoids of Nezara viridula

Sampling and specimens

For the molecular characterization of the egg parasitoids of Nezara viridula and congeners that attack the eggs of other hosts we used about 100 specimens of Anastatus and Trissolcus from 10 countries: Bulgaria, South Korea, France, Greece, Japan, Republic of Moldova, Romania, Taiwan, Turkey and Ukraine (see table below).

Inter- and intraspecific genetic distances

We calculated mean inter- and intraspecific genetic distances using the K2P substitution model as it is frequently used in DNA barcoding studies. The alignment consisted of about 600 bp from the 5’ end of the COI mitochondrial gene (standard barcode region).

Mean intraspecific distances (first table below) vary from 0% in Anastatus ruficaudus and Anastatus sp. LF#1 to 7.5% in Anastatus giraudi (see table below).

Mean interspecific distances (second table below) vary from 6.5 % for the pair Anastatus oscari with Anastatus bernardito 13.8% for Anastatus giraudi with Anastatus sp. TK#2 and Anastatus gastropachae with Anastatus sidereus (see table below). Apparently, there is an overlap between the highest intraspecific value and the lowest interspecific value and there is no barcoding gap. However, Anastatus giraudi might be a species complex based on an a posteriori analysis of the specimens. In this case the maximum mean intraspecific genetic distance is 5.8% in Anastatus fulloi, and there is a barcoding gap. From the available data it seems that DNA barcoding will be an efficient tool for species delimitation and identification in the genus Anastatus.

Preliminary phylogenetic analyses

The tree below shows a ML tree of the COI gene for the genus Anastatus using Brasema (GenBank AN KR108721) as an outgroup. The tree was inferred in RAxML and is the best scoring tree out of 20 independent runs. The dataset was partitioned by codon position and support for the nodes was estimated with 1000 bootstrap pseudoreplicates.

Bootstrap support is 100% for almost all morphospecies except Anastatus giraudi where it is 72%, probably because of the presence of cryptic species. In Anastatus fulloi the bootstrap is 78%, but here the two specimens included in the analysis are from two distinct Japanese islands and the rather large genetic differentiation might be due to geographic variability (morphologically the specimens AnaJp0202 and AnaJp0301 are very similar). For the deeper nodes (interspecific relationships) the bootstraps are very low, as expected for COI.

Molecular species delimitation

We tried the PTP algorithm of species delimitation (https://mptp.h-its.org/#/tree) and used both the PTP and mPTP methods. With the first we obtained an evident oversplitting into 27 species, with evidently erroneous cases. For example Anastatus sidereus is broken into four species, every specimen being a distinct species; as this is a species with brachypterous and presumably stenotopic females high molecular divergence in the mitochondrial haplotypes is expected as cause. Anastatus sp. LF#2018-0 is split into four species, only two specimens out of five being conspecific. The mPTP method estimates 11 species. Anastatus sidereusis is one species but includes Anastatus formosanus and Anastatus sp. TK#2018-2. Anastatus sp. LF#2018-0 is one species, which based on morphology is expected. Since both algorithms give in this particular case different conflicting results, without knowledge of the system it is impossible to decide on the number of species based on this single locus species delimitation approach.