Discovery of a new Pseudalomya Telenga, 1930 (Hymenoptera, Ichneumonidae, Ichneumoninae) species from Taiwan and its implications for the systematic position of this genus

Ichneumonidae

Because Pseudalomya exhibits an intermediate morphology between two ichneumonine tribes Alomyini and Phaeogenini, it has generally been placed in both tribes (Laurenne et al. 2006;Quicke et al. 2009;Tereshkin 2009;Quicke 2015;Riedel 2019).Consequently, the tribal status of Pseudalomya remains a topic of debate.On one hand, this genus exhibits similarity with Alomyini because of the following characters: horned frons, enlarged vertex and genae, foramen with genae meeting (or almost meeting) ventrally, oval propodeal spiracle, metasomal tergite II without thyridium, and forewing with the second abscissa of M shorter than the first.On the other hand, Pseudalomya also exhibits similarity with Phaeogenini because of the following characters: two-segmented trochanter, two-spurred mid tibia, and metasomal tergite I with spiracles located at apical 0.3 (Laurenne et al. 2006;Tereshkin 2009;Riedel 2019).
Phylogenetic hypotheses reconstructed based on 28S D2-D3 rDNA sequences or combined (morphology and 28S) datasets suggested the tribal placement of Pseudalomya within the tribe Phaeogenini (Laurenne et al. 2006;Quicke et al. 2009).Consequently, the morphological similarities between Pseudalomya and Alomyini were interpreted as either symplesiomorphic traits or the result of convergent evolution (Quicke 2015).However, because of the lack of sampling of this genus in subsequent comprehensive phylogenetic studies (Bennett et al. 2019;Santos et al. 2021), the phylogenetic position of Pseudalomya remains incertae sedis (Santos et al. 2021).
In this study, we analyzed three Pseudalomya specimens that were newly obtained from the high mountains of central Taiwan.The specimens were collected during fauna inspection for the project SP110113: A survey for the selection of insect indicator species and their microhabitat usage in the Daxueshan area of Shei-Pa National Park.After morphological examinations, these specimens were discovered to be distinct from the known species of Pseudalomya.On the basis of morphological evidence, the specimens were subsequently validated as a new species, Pseudalomya truncaticornis sp.nov.This article describes the new species and presents a key to the global species of female Pseudalomya.In this study, the phylogenetic position of Pseudalomya was reassessed through multigene phylogenetic analyses.

Morphological examination
The morphological terms used in this study were identified from Broad et al. (2018).Measurements were performed with reference to Kikuchi and Konishi (2021).The following abbreviations were used in this study: OOL, ocello-ocular line; POL, postero-ocellar line; OD, ocellar diameter; PSI, propodeal spiracle index: major axis of propodeal spiracle/minor axis of propodeal spiracle; 1/M, the first abscissa of forewing M; 2/M, the second abscissa of forewing M; NI, nervellar index of hindwing: length of hindwing CU between M and cu-a/length of cu-a; T, metasomal tergite; S, metasomal sternite; and pS, posterior section of metasomal sternite.
The measurements in parentheses represent the measurements of the holotype.The cuticular microsculpture is described as per a study conducted by Eady (1968).The whole metasomal sternum was observed and dissected using a method developed by Kikuchi and Konishi (2021).The specimens were examined and measured using a microscope (Leica S8 APO; Leica Microsystems, Wetzlar, Germany) with a micrometer.Photographs were taken using a Leica DMC 5400 camera integrated into a Leica Z16 APO microscope equipped with the auto-stacking system Leica LAS V4.13 (all from Leica Microsystems).Line drawings were generated using Procreate (Savage Interactive, Hobart, Australia).All figures were edited and arranged into figure plates by using Adobe Illustrator CC and Photoshop CC (Adobe Systems, San Jose, CA, USA).The specimens and their photos have been deposited at the following institutes: NMNS, National Museum of Natural Science, Taichung, Taiwan; NARO, Institute for Agro-Environmental Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan; and SDEI, Senckenberg Deutsches Entomologisches Institut, Müncheberg, Germany.The Latin term ibidem, meaning "same as previous except as follows," was abbreviated as "ibid" to abridge information on the location of the materials examined.

Taxon sampling
To reassess the phylogenetic position of Pseudalomya, 52 operational taxonomic units (OTUs) from 38 ichneumonine genera were analyzed (Suppl.material 1).In addition, other ichneumonids belonging to the Ichneumoniformes group were included as outgroups (Suppl.material 1).These outgroups were selected from the dataset of Santos (2017) and included 24 species from 23 genera of Cryptinae, 11 species from 11 genera of Phygadeuontinae, one species from one genus of Agriotypinae (Agriotypus armatus), and one species from one genus of Microleptinae (Microleptes splendidulus).For OTUs whose sequences were obtained from an online database, species identification was double-checked using the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990) and the position of preliminary phylogenetic reconstruction.Chimera OTUs were constructed for genera lacking available sequences of multiple markers within individual species.

Molecular data collection and analysis
Total genomic DNA was extracted from the right midleg of each specimen by using a DNeasy Blood and Tissue Kit (Qiagen, Düsseldorf, Germany).Partial sequences of the mitochondrial cytochrome c oxidase I gene (COI) and two nuclear genes -D2-D3 regions of 28S ribosomal RNA gene (28S) and 18S ribosomal RNA gene (18S) -served as molecular markers for phylogenetic reconstruction.The sequences were retrieved from GenBank (Nation Center for Biotechnology Information).Target sequences were amplified through polymerase chain reaction (PCR).The primer pairs and conditions for PCR are listed in Suppl.material 2. The reaction volume was 15 µL: 5.1 µL of sterile distilled water, 0.6 µL of each (forward/reverse) primer (10 µM), 7.5 µL of GoTaq Green Master Mix (Promega, Madison, WI, USA), and 1.2 µL of DNA template.PCR products were purified and sequenced at Tri-I Biotech (Taipei, Taiwan).Sequences were edited using CodonCode Aligner v.10.0.2 (CodonCode Corporation, Dedham, MA, USA).
MAFFT v.7 (Katoh et al. 2019) was used for automated multiple sequence alignments.The default setting was used for the alignment of COI.By contrast, the E-INS-I algorithm was used for the alignment of the two nuclear genes (Santos 2017); the alignments of nuclear markers were manually optimized by removing regions with variable lengths and gaps.The translated alignment of COI was checked for stop codons by using MEGA v.11 (Tamura et al. 2021).All newly obtained sequences were uploaded on GenBank.The numbering of positions started from the gene's first nucleotide (full-length COI sequences served as references for numbering: GenBank accession JX131613 [Diadromus collaris] and MG923483 [Amblyjoppa sp.]).

Molecular phylogeny
To infer the phylogenetic position of Pseudalomya, phylogeny was reconstructed using the following four datasets: COI, 28S, 18S, and concatenated 18S+28S+COI.The concatenated dataset was first partitioned by gene and then, for the protein-coding COI, also by codon position (first plus second versus third).ModelFinder (Kalyaanamoorthy et al. 2017) with "partition merging" was used for searching the optimal partitioning scheme and substitution models under the Bayesian Information Criteria.
Male.Unknown.Etymology.The specific name "truncaticornis" is derived from the Latin "truncati-" (meaning "maimed" or "having appendages cut off") plus "cornis" (meaning "horned").It refers to the truncated apex of the horn on the frons in this species.Name is an adjective.
Remarks.Comparisons of the photos of holotypes and descriptions of congener species revealed the highest level of similarity between P. truncaticornis sp.nov.and P. nepalensis Riedel, 2019.However, unlike P. nepalensis, the new species had the black coloration in mesosoma and legs (reddish brown in P. nepalensis), the middle of face with sparse punctures with distance between punctures 2.0-3.0×their diameter (dense in P. nepalensis, distance less than 1.0× their diameter), and a short and apically truncated frontal horn (long and apically rounded in P. nepalensis).While variation in the color of the head was observed within P. truncaticornis sp.nov., given the disjunct geographical distributions between P. truncaticornis sp.nov.and P. nepalensis (Taiwan and the Himalaya, respectively) and the presence of morphological differences beyond mere coloration, P. truncaticornis sp.nov. is considered a distinct species.To the best of our knowledge, this is the first record of Pseudalomya in Taiwan.
No pseudogene, identifiable by the occurrence of stop codons in translated (amino acid) sequences, was detected in the protein-coding gene dataset.However, one 18S sequence from Pseudoplatylabus apicalis (GenBank accession KU753140) was eliminated because of its abnormal genetic distance in the 18S gene tree pretest.Table 1 presents the basic details of each aligned dataset.The information presented in this table includes the average length of unaligned sequences, length of aligned sequences, number of variable and parsimony-informative sites, and percentage of GC content.The datasets for the concatenated and individual markers are presented in Suppl.material 3.

Molecular phylogeny
Figs 6, 7 depict the ML phylogenetic trees reconstructed using the concatenated 18S+28S+COI dataset and the COI and 28S datasets, respectively.The 18S gene tree  could not satisfactorily resolve the phylogenetic relationships in the Ichneumoniformes group (see Suppl.material 4: fig.S3).All trees were rerooted using the outgroup Agriotypus armatus (Agriotypinae).The complete phylogenetic trees resulting from all datasets are presented in Suppl.material 4.

Discussion
The genus Pseudalomya has been sampled in previous phylogenies reconstructed by 28S data (Laurenne et al. 2006) or combined (28S + morphology) data (Quicke et al. 2009).Both hypotheses supported the placement of Pseudalomya within Phaeogenini.To the best of our knowledge, our study is the first to present COI-based and multigene molecular phylogeny that includes Pseudalomya.The results support the placement of Pseudalomya within Phaeogenini.However, the precise relationship of Pseudalomya with other members of Phaeogenini as reported in the literature and the present study differs because of between-study differences in taxon sampling and methodology.Pseudalomya was shown sister to Tycherus in the study of Laurenne et al. (2006), to an unidentified phaeogenine genus in the study of Quicke et al. (2009), to Dicaelotus sp. in our COI-based phylogenetic analysis (Fig. 7), and to Phaeogenes eguchii in our 28S-based and concatenated multigene phylogenetic analyses (Figs 6, 7).The aforementioned relationships are not highly supported, with the exception of that reported in Laurenne et al. (2006).
In our study, the Alomyini (Alomya debellator) is not recovered as a member of Ichneumoninae; this finding is congruent with the 28S-based hypothesis proposed by Laurenne et al. (2006) but incongruent with phylogenetic hypotheses reconstructed using morphological data (Gokhman 1992(Gokhman , 1995)), combined morphological and multigene data (Quick et al. 2009;Bennett et al. 2019), or genomic data (Santos et al. 2021).
Our findings, which were derived from phylogenetic analyses performed using universal genetic markers via Sanger sequencing, unveiled incongruent phylogenetic relationships within Ichneumoninae (Figs 6, 7).The tree reconstructed using the 18S dataset even failed to reveal subfamily-level relationships, likely because of to the conservativeness of this marker (see Suppl.material 4: fig.S3).These results imply probable constraints in phylogenetic inference using a restricted set of Sanger-based genetic markers or involving incomprehensive taxon sampling.However, our results do distinctly demonstrate the position of Pseudalomya as not belonging to Alomyini and clustered within Phaeogenini.Additionally, the alignment of COI sequences revealed that Pseudalomya lacked a unique insertion sequence (nucleotides 403-408) specific to Alomya (see Suppl.material 3: pseudalomya_ichneumoniformes_coi_withreference.fas),providing an additional character for differentiating between Pseudalomya and Alomyi-ni (at least Alomya).Considering the discrepancies between the literature and our study regarding the limitations of Sanger-based phylogenetic analyses, we refrained from the tribal reclassification of Pseudalomya.Comprehensive phylogenetic analyses based on genomic data are required to accurately determine the tribal status of Pseudalomya.
Lastly, this study indicates that the distribution of Pseudalomya extends from the Eastern Palaearctic region (Russia, Korea) and the Himalayas (Nepal) to Taiwan.The oriental species of Pseudalomya -P.nepalensis and P. truncaticornis sp.nov.-exhibit disjunct distributions between the Himalayas and Taiwan.This unique distribution pattern is also observed in vascular plants, vertebrates, and insects (e.g., Hsu and Yen 1997;Päckert et al. 2012;Niu et al. 2018).This pattern may be explained by longdistance dispersal, postglacial contraction, limited faunistic studies in the high-altitude intermediate regions, or the lack of material examined (Yen et al. 2000;Wang et al. 2013;Niu et al. 2018).Thus, further studies are required to comprehensively clarify the distribution of Pseudalomya.

Figure 2 .
Figure 2. Pseudalomya truncaticornis sp.nov.holotype (NMNS ENT 8836-1) A dorsal view of the head B anterior view of the head C lateral view of the head D foreleg E dorsal view of the mesoscutum F dorsal view of the propodeum G dorsal view of the metasomal tergites.Photographed by Hsuan-Pu Chen.

Table 1 .
Summary of each aligned and trimmed molecular dataset.The table presents information on the average length of unaligned sequences, length of aligned sequences, number of variable and parsimonyinformative (Pars-Inf ) sites, and percentage of GC content.