Protelenomus Kieffer is a derived lineage of Trissolcus Ashmead (Hymenoptera, Scelionidae), with comments on the evolution of phoresy in Scelionidae

a derived lineage of


Introduction
Scelionid parasitoids of hemipteran eggs are the subject of active study, driven by the economic damage caused by a variety of bug pests, and by recent works that accelerate further advancement. These include progress in the taxonomy and systematics of these parasitoids, which underlie accurate identification and classification (Talamas et al. 2017;Talamas et al. 2019;Tortorici et al. 2019;Talamas et al. 2021). Protelenomus

Molecular analysis
Genomic DNA was extracted using a TIANamp Micro DNA Kit (Tiangen Biotech (Beijing), Co.., Ltd), following the nondestructive DNA extraction protocol described in Taekul et al. (2014). Four molecular markers were amplified: two nuclear ribosomal (18S and 28S D2-3), one mitochondrial protein (COI), and one single-copy nuclear protein (wingless). Polymerase chain reactions were performed using Tks Gflex DNA Polymerase (Takara) with primer pairs shown in Table 1 and conducted in a T100 Thermal Cycler (Bio-Rad). Thermocycling conditions consisted of an initial denaturing step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s and an additional extension at 72 °C for 5 min. Amplicons were directly sequenced in both directions with forward and reverse primers on an Applied Biosystems (ABI) 3730XL by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.
(Guangzhou, China). Chromatograms were assembled with Geneious 11.0.3. The assembled sequence was translated to amino acids using the invertebrate mitochondrial code to check for stop codons and frame shifts and was compared via BLAST against the GenBank database to check for contamination and pseudogenes (e.g., nuclear mitochondrial DNA, NUMT) as implemented in Geneious 11.0.3. The sequences generated from this study are deposited in GenBank (accession numbers are shown in Suppl. material 1).

Phylogenetic analysis
Multiple sequence alignments for each gene were performed with MAFFT v7.490 (Katoh and Standley 2013) by the E-INS-i strategy for 18S and 28S, and the L-INS-i strategy for COI and wingless. Maximum likelihood phylogenetic analyses were conducted in IQ-TREE (v. 2.1.3) (Minh et al. 2020) following the methodology of Chen et al. (2021). Eight partitions were specified in the original concatenated alignment: one for each ribosomal gene and three for each codon position in COI and wingless (Chernomor et al. 2016). ModelFinder was employed to determine the best nucleotide substitution model for each partition and to merge partitions to increase overall model fit (Kalyaanamoorthy et al. 2017). Branch support was estimated with 1000 ultrafast bootstrap replicates (Hoang et al. 2018). Ten independent tree searches were conducted, and we present the tree with the greatest log-likelihood score. Maruzza japonica Mineo was selected as the outgroup.

Imaging
Photographs of live specimens were taken with a Canon 5D Mark III (Tokyo, Japan) camera with a 100 mm macro lens. Multifocal images of mounted specimens were made using a Nikon SMZ25 microscope with a Nikon DS-Ri 2 digital camera system and a Macropod Microkit photography system. All image stacks were rendered using Helicon Focus. Scanning electron micrographs were produced using a Phenom Pro Desktop SEM. Images were post-processed with Adobe Photoshop CS6 Extended.

Results
The phylogenetic analysis retrieved Trissolcus siliangae Yan, Chen & Talamas sp. nov. embedded within Trissolcus, as the sister taxon to a clade comprising (T. vindicius + T. cultratus) + (T. corai + (T. japonicus + T. plautiae)) ( Fig. 6). Treatment of Protelenomus as a derived lineage of Trissolcus is also supported by a morphological character, the subacropleural sulcus. Talamas et al. (2017) proposed that this sulcus had value for circumscribing Trissolcus and noted that it was present in all Palearctic Trissolcus except for T. exerrandus Kozlov & Lê. Notably, a preliminary phylogenetic analysis indicates that T. exerrandus does not belong in Trissolcus, although its destination is presently unclear. In their synonymy of Latonius Kononova, Vasiliţa et al. (2021) reported that T. planus (Kononova) does not have a subacropleural sulcus. In the figures provided in the revision of Protelenomus by Veenakumari et al. (2019), the subacropleural sulcus is present in T. anoplocnemidis (Ghesquière), T. gajadanta (Veenakumari), T. maasai (Veenakumari), T. tibialis (Veenakumari), and T. zulu (Veenakumari), whereas it is clearly absent in T. flavicornis (Kieffer). This character can be difficult to assess in species with coarse sculpture and from images with glare on the specimens. In T. siliangae sp. nov., the subacropleural sulcus is clearly visible in Figs 3F and 4B. Given that this sulcus appears to be absent in some highly derived species, we consider that it may still be a synapomorphy for  (2021)).
Epinomus anoplocnemidis Ghesquière, 1948  Legs. Color: coxae and distal tarsomeres dark brown to black, otherwise yellow to light brown. Anteroventral area of hind femora: not covered by setae. Femur and tibia not enlarged. Basitarsi of fore leg with a row of densely stout bristles at basal half. Claws well developed, curved.
Male. Unknown. Diagnosis. Moniliform antennae in females are rare in Scelionidae, shared in Trissolcus by T. siliangae, T. flavicornis, T. gajadanta, and T. planus; these species also have a single papillary sensillum on each clavomere. Care should be taken to count the antennomeres (11 in females, 12 in males) so that female specimens are not mistaken for males. Clavomeres that are only slightly wider than the preceding flagellomeres are more common, found in many species of the former Protelenomus and in more typical Trissolcus such as T. sipioides.
Trissolcus siliangae has a laterally invaginated metapostnotum, as in T. hullensis (Johnson 1985), which is found in a minority of Trissolcus species. In Veenakumari et   al. (2019), the metapostnotum in T. flavicornis appears to extend medially, separating the propodeum from the metanotum until it reaches the vicinity of the lateral margin of the metascutellum (see figures 27, 29, and 31 of that publication). We find this to be the case for T. gajadanta as well, based on examination of a specimen from Ivory Coast (Fig. 5). Trissolcus siliangae can thus be separated by the combination of the moniliform antennae, claval formula (1-1-1-1), and lateral invagination of the metapostnotum. Additionally, T. siliangae can be separated from the very similar T. gajadanta by the striation on T2: robust in the anterior ⅔ of the tergite in T. gajadanta and only weakly present in T. siliangae; and by the robust parapsidal lines in T. gajadanta, which are not indicated in T. siliangae. Notably, the posterior head in T. gajadanta has two concavities lateral to a dorsoventral median ridge, with the dorsal part of the occipital carina located low on the posterior head (Fig. 5B).
Etymology. This species is named after one of its collectors, Dr. Siliang Wang, for her discovery of this species.

Discussion
The phenomenon of phoresy has been documented in a variety of scelionids: Paratelenomus anu Rajmohana, Sachin & Talamas (Rajmohana et al. 2019), Thoronella Masner (Carlow 1992), Synoditella Muesebeck (Lanham and Evans 1958), Sceliocerdo Muesebeck (Brues 1917), Scelio Latreille (Ramachandra Rao  (Orr et al. 1986;Arakaki et al. 1995), and Trissolcus. These taxa are distantly related within Scelionidae and present examples of evolutionary convergence. Phoresy occurs in Trissolcus, Paratelenomus, and some Telenomus that parasitize heteropteran eggs and are part of a scelionid radiation on Hemiptera (Chen et al. 2021). The scelionids not associated with Hemiptera are more distantly related and they also attack more distantly related hosts: Orthoptera, Mantodea, Lepidoptera, and Odonata, although it should be noted that phoretic parasitoids of Lepidoptera can be found within Telenomus (Arakaki et al. 1995). Detailed examination of this assortment of relationships may yield information on the selection pressures for phoresy, which may include finding the eggs or reaching the eggs at a stage sufficiently early for parasitoid development to occur, or to have a competitive advantage. In Trissolcus, interspecific competition is common, and being the first parasitoid may yield an advantage for intrinsic (larval) competition. Phoresy in Trissolcus is worth further examination, both in terms of behavioral studies that will illuminate its benefits, and further phylogenetic analysis to determine if it has evolved more than once in the genus, and to identify sister taxa to phoretic lineages. Ultrafast bootstrap support values indicated by colored circles at nodes. Some nodes were not annotated due to short branch lengths.