The Pyraloidea is one of the species-rich superfamilies of Lepidoptera and contains numerous economically important pest species that cause great loss in crop production. Here, we sequenced and annotated nine complete mitogenomes for Pyraloidea, and further performed various phylogenetic analyses, to improve our understanding of mitogenomic evolution and phylogeny of this superfamily. The nine mitogenomes were circular, double-stranded molecules, with the lengths ranging from 15,214 bp to 15,422 bp, which are comparable to other reported pyraloid mitogenomes in size. Gene content and arrangement were highly conserved and are typical of Lepidoptera. Based on the hitherto most extensive mitogenomic sampling, our various resulting trees showed generally congruent topologies among pyraloid subfamilies, which are almost in accordance with previous multilocus studies, indicating the suitability of mitogenomes in inferring high-level relationships of Pyraloidea. However, nodes linking subfamilies in the “non-PS clade” were not completely resolved in terms of unstable topologies or low supports, and future investigations are needed with increased taxon sampling and molecular data. Unexpectedly, Orybina Snellen, represented in a molecular phylogenetic investigation for the first time, was robustly placed as basal to the remaining Pyralidae taxa across our analyses, rather than nested in Pyralinae of Pyralidae as morphologically defined. This novel finding highlights the need to reevaluate Orybina monophyly and its phylogenetic position by incorporating additional molecular and morphological evidence.

Background Limited data are currently available on protozoan parasites of the genus Sarcocystis that infect their avian hosts within the order Anseriformes (waterfowl). To date, no Sarcocystis species has been recorded in ducks in China. Methods Leg muscles were sampled from 26 domestic ducks (Anas platyrhynchos) in China in 2021. Morphological characteristics of sarcocysts detected in the muscle tissue were described using light microscopy (LM) and transmission electron microscopy (TEM). Genomic DNA was extracted from single sarcocysts obtained from different ducks, and three genetic markers, 18S ribosomal DNA (18S rDNA), 28S ribosomal DNA (28S rDNA) and mitochondrial (mt) cytochrome oxidase subunit 1 (cox1), were amplified and cloned for sequence analyses. Results Sarcocysts were observed by LM in only three of the 28 samples (10.7%). These sarcocysts had a thick cyst wall with numerous brush-like villar protrusions (vps) of 3.8–4.3 μm in length (n = 30) on the cyst surface. TEM observation showed that the sarcocysts had lanceolated vps. Each vps narrowed in the stalk and contained a bundle of microtubules that extended into the ground substance. Comparisons of the new sequences with those deposited in GenBank showed that the most similar sequences were those of Sarcocystishalieti in the great cormorant Phalacrocorax carbo and European starling Sturnus vulgaris, and Sarcocystis calchasi in the domestic pigeon (Columba livia) at the 18S rDNA (99.1% identity); Sarcocystiswenzeli from the domestic chicken Gallus gallus at the 28S rDNA (95.9–96.0% identity); and Sarcocystis speeri from the opossum at the mtcox1 (98.2% identity). The new 18S rDNA, 28S rDNA and mitochondrial cox1 sequences shared up to 99.0%, 95.6% and 97.7% identity, respectively, with those of Sarcocystis spp. obtained from Anseriformes avian hosts. Phylogenetic analysis inferred from the sequences of the three genetic markers placed the organism within a group of Sarcocystis spp. obtained from avian or carnivorous intermediate hosts and avian, marsupial or carnivorous definitive hosts. Based on the morphological observation and molecular analyses, the organism found in the Chinese domestic ducks was regarded as a new species and named Sarcocystis platyrhynchosi n. sp. Conclusions Based on morphology and sequence analyses, the microcysts diagnosed in the domestic ducks examined in this study were named as a new species. This is the first record of Sarcocystis spp. from waterfowl in China. Sarcocysts of similar morphology occur frequently in different Anseriformes birds, and the relationships among these species need to be further clarified in future studies using more molecular markers. Graphical Abstract

Only 18S rDNA sequences of Sarcocystis spp. in South American camelids (SACs) are deposited in GenBank as references, and the definitive host of S. masoni in SACs is still unclear. Here, S. masoni sarcocysts detected in an alpaca (Vicugna pacos) in China were investigated with the aid of light (LM) and transmission electron (TEM) microscopy, and characterized using four genetic markers, i.e., 18S rDNA, 28S rDNA and ITS, and the mitochondrial cox1. Additionally, the life cycle of the parasite was completed via experimental animal infection. Under LM, S. masoni sarcocysts exhibited numerous 1.3–2.1 μm conical protrusions. Under TEM, the sarcocyst wall contained conical, cylindrical, or irregular-shaped villar protrusions, similar to type 9j. Two dogs (Canis familiaris) fed S. masoni sarcocysts shed sporocysts with a prepatent period of 8–9 days. The newly obtained 18S rDNA sequences showed 98.4–100% identity with those of S. masoni in SACs previously deposited in GenBank. Interestingly, the newly obtained sequences of 18S rDNA and mitochondrial cox1 shared 99.6–100% and 98.2–98.5% identity, respectively, with those of S. cameli in dromedary camels (Camelus dromedaries). Phylogenetic analysis based on sequences of 18S rDNA, 28S rDNA, or mitochondrial cox1 revealed that S. masoni has a close relationship with Sarcocystis spp. in ruminants. The relationship between S. masoni and S. cameli deserves to be further clarified in the future.

The Geometroidea is a large superfamily of Lepidoptera in species composition and contains numerous economically important pest species that cause great loss in crop and forest production. However, understanding of mitogenomes remains limited due to relatively fewer mitogenomes previously reported for this megadiverse group. Here, we sequenced and annotated nine mitogenomes for Geometridae and further analyzed the mitogenomic evolution and phylogeny of the whole superfamily. All nine mitogenomes contained 37 mitochondrial genes typical in insects, and gene organization was conserved except for Somatina indicataria . In S. indicataria , the positions of two tRNAs were rearranged. The trnR was located before trnA instead of after trnA typical in Lepidoptera, whereas the trnE was detected rarely on the minority strand (N‐strand). This trnR ‐ trnA ‐ trnN ‐ trnS1 ‐ trnE ‐ trnF newly recognized in S. indicataria represents the first gene rearrangement reported for Geometroidea and is also unique in Lepidoptera. Besides, nucleotide composition analyses showed little heterogeneity among the four geometrid subfamilies involved herein, and overall, nad6 and atp8 have higher nucleotide diversity and Ka/Ks rate in Geometridae. In addition, the taxonomic assignments of the nine species, historically defined by morphological studies, were confirmed by various phylogenetic analyses based on the hitherto most extensive mitogenomic sampling in Geometroidea.

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