Systematic revision and molecular phylogenetics refine the generic classification of the bark louse family Stenopsocidae (Insecta: Psocodea: Psocomorpha)

Psocomorpha, as one of the suborders of the order Psocodea


Introduction
The paraneopteran insect order Psocodea is an inconspicuous group, containing more than 10000 valid species, and is divided into three suborders: Trogiomorpha, Troctomorpha, and Psocomorpha (Lienhard & Smithers, 2002;de Moya et al., 2021;Zhang, 2011).Recent progress based on molecular data resolved the phylogenetic position of par-asitic lice as the sister group of the family Liposcelididae in Psocodea (de Moya et al., 2021).Besides, many molecular phylogenetic studies recovered the relationships among the families or higher taxa of Psocodea (Johnson et al., 2004;Yoshizawa et al., 2006;Yoshizawa et al., 2014;Yoshizawa et al., 2018;de Moya et al., 2021).However, among the 37 families of 'Psocoptera' (Mockford, 2018), the intergeneric or interspecific phylogenies within a family have been poorly studied.The only case refers to Saenz Manchola et al. (2022), in which the phylogenetic relationships within the family Lachesillidae were reconstructed based on a dataset of 2060 orthologous genes.Yoshizawa and Johnson (2008) evaluated the higher level (subfamilies and tribes) classification within the family Psocidae based on a multi-loci phylogenetic study.
The bark louse family Stenopsocidae is a large group of the suborder Psocomorpha, currently comprising over 190 valid species in the world (Hopkins et al., 2023).It is noticeable that the fauna of Stenopsocidae is extremely rich in Asia, especially in China, from which 168 species have been described (Lienhard & Smithers, 2002;Li, 2002Li, , 2005;;Liang et al., 2013Liang et al., , 2014Liang et al., , 2017)).The adults of Stenopsocidae generally inhabit living trees, bamboos, etc., and are characterized by the abdomen with 2-3 ventral vesicles, and the forewing with pterostigma connecting to Rs by a crossvein and M connecting to areola postica cell by a crossvein (Fig. 1).
In the present study, we investigate the phylogenetic relationships within the family Stenopsocidae using DNA sequences from the mitochondrial genes and nuclear 18s rRNA gene.Combining the molecular phylogeny and morphological characters, we propose a new genus Neostenopsocus gen.n., and present a revised generic classification of Stenopsocidae.

2.
Material and Methods

DNA extraction, mitogenome sequencing and assembly
The genomic DNA was extracted from the thoracic muscle tissues of a single specimen using TIANamp Micro DNA Kit (TIANGEN, Beijing, China).For the sequencing li-brary for each specimen and used the Illumina NovaSeq platform to obtain 4 GB sequence data.Raw reads were trimmed of adapters using BWA (Burrows-Wheeler Aligner) software (Li & Durbin, 2010).All sequences were assembled mapping to the mitogenome as the reference sequence using Geneious Prime 2023 (Kearse et al. 2012), with the parameters sets as follows: 95% minimum overlap identity, 4 maximum ambiguity and a minimum overlap of 25 bp.Each mitochondrial contig was inputted into MI-TOS web service (Bern et al., 2013) for initial mitogenome annotation.The resultant gene boundaries were checked by alignment with homologous genes of the published mitogenomes (Genbank accession number: KX187004, MZ274186, MZ274207, MZ274208) and 18S rRNA (Genbank accession number: AY63049, AB856950, AY630491, AY630492) of psocids.The mitogenomes and 18S rRNA herein sequenced are deposited in Genbank (Table 1).

Phylogenetic analysis
In this study, we used mitochondrial genes (exclude the control region, intergenic spacers and tRNA genes, including the protein-coding genes and rRNA genes) and partial nuclear18S rRNA.The alignment of 13 protein-coding genes was based on amino acid sequences translated with the invertebrate mitochondrial genetic codon table using MUSCLE (Edgar, 2004) complied in MEGA 7.0 (Kumar et al., 2016).The rRNA genes were aligned using MAFFT 7.0 (Katoh & Standley, 2013) under iterative refinement method incorporating the most accurate local (E-INS-i) pairwise alignment information.
The BI analysis contained four simultaneous Markov chain Monte Carlo (MCMC) runs of 5 million generations for the average standard deviation of split frequency was below 0.01, and tree samples were outputted every 1000 generations with a burn-in of 25%.We estimated the best partitioning scheme and model for our dataset with PartitionFinder 2 (Lanfear et al., 2017).The concatenated dataset was partitioned into six subsets and the GTR + I + G model was selected (File S1).

Illustration and morphological study
Genitalic preparations were made by clearing the apex of the abdomen in a cold, saturated NaOH solution for 6 h.After rinsing the NaOH with water, the apex of the abdomen was transferred to glycerin for further dissection and examination.Observations were made using an Olympus CX-33 (Olympus Imaging Corporation, Tokyo) light microscope.Photographs and drawings were taken using a Sony Alpha 7II (Sony Corporation, Tokyo) digital camera attached to the Olympus CX-33.The figures were prepared with Adobe Photoshop 24.0.0 (Adobe, San Jose, CA, USA).The terminology follows Yoshizawa (2005).

Mitochondrial genome gene com position and general features
A total of 20 nearly complete mitochondrial genomes of Stenopsocidae were assembled.The mt genome of Stenopsocidae was determined to be a double-strand circular DNA molecule, including 37 typical genes: 13 PCGs, 22 tRNAs, and two rRNAs.In the 13 PCGs, ND2, COX1, COX2, ATP8, ATP6, COX3, ND3, ND6 and CytB are encoded on the major strand (J-strand), and ND5, ND4, ND4L and ND1 are encoded on the minor strand (Fig. 1).
The PCGs show the typical ATN start codons.The majority of PCGs also show the typical TAR (TAA/TAG) stop   codons, while the partial stop codon T for COX1, ND5 in all species.

Mitochondrial genetic arrange ment
All species of Stenopsocidae share a similar mitochondrial genetic arrangement with the infraorder Caeciliusetae, and show the trnE-trnS1 rearrangement which was considered to be an autapomorphy for Caeciliusetae by Saenz Manchola et al. ( 2021).Additionally, the family Stenopsocidae has two mitochondrial genetic arrangement types that were reported by Saenz Manchola et al. ( 2021) and Liu et al. (2017).Graphopsocus shares the trnM -trnI-trnC-trnQ gene block with the family Caeciliusidae (Fig. 2).Neostenopsocus gen.n., Malosteno psocus and Stenopsocus show the same mitochondrial genetic arrangement that was reported by Liu et al. (2017).In these three genera, trnM was located next to trnI.The trnI-trnM-trnC-trnQ gene block has not been found in the other taxa of the suborder Psocomorpha.

Phylogenetic relationship of Stenopsocidae
In the present study, we use the mitogenomes and 18S rRNA of 21 species of Stenopsocidae with additional 11 mitogenomes of other bark lice as outgroup to infer the phylogeny of the family Stenopsocidae.The aligned and cleaned full data matrix was 14738 bp (mitogenomic data: 13006 bp, 18S rRNA data: 1732 bp) in length (File S1).Phylogenetic analyses under ML and BI analyses showed the same topology and overall high nodal supports (Fig. 3).

Taxonomy
Order Diagnosis.Body length 2-3 mm, from postclypeus to wing tip length 3-5 mm.Antenna slightly longer than body length, not longer than wing length.Labrum with distal styli.Head with brown vertex.Forewing with several dark markings, proximal half with one or two brown marking between CuP and A, and distal half with brown markings at middle and also long distal margin, forming a large and a small V-shaped patterns, pterostigma general with brown markings on basal region.Forewing anterior margin rather sparsely setose, costal vein without seta from base to pterostigma; CuP glabrous, all other veins with single-row setae; pterostigma broad, with a distinct posterior angle.Abdomen with two ventral vesicles.Male genitalia: tip of aedeagal arch not exceeding parameres (aedeagal arch exceeding parameres in G. vietnamensis); endophallus with one pair of weakly sclerotized lobes.Female genital: spermathecal sac with a small pouch.
Distribution.The genus is widely distributed in all main zoogeographical regions of the world.
Diagnosis.Male body shorter than 3 mm, female body longer than 3 mm, from postclypeus to wing tip length 5-7 mm.Antenna slightly longer than body length, not longer than wing length.Labrum without distal styli.
Head with several dark brown markings.Wings transparent, without distinct markings (M.plurifasciatus with markings on wings), veins darker than wing margin.
Forewing costal vein with setae from base of anterior margin to pterostigma, all veins with single-row setae at least, but R, Rs+M, CuA+M, and CuA with two rows of setae, and basal membranous part of forewing with setae.Pterostigma broad, with a distinct posterior angle.Abdomen with three ventral vesicles.Abdomen with segments 8-9 indistinctly sclerotized.Male phallosome strongly sclerotized, endophallus with three lobes.Female spermathecal sac with a lateral pouch connecting to spermathecal duct.

Comparative morphology among genera of Stenopsocidae
Lienhard (1998) redescribed the four species of Stenopsocidae from Europe, and indicated several important diagnostic characteristics, i.e., the presence or absence of labral styli, the number of ventral vesicles, the ciliation of the forewing.Moreover, Stenopsocidae shows rich biodiversity in the Oriental region.All genera and most species of Stenopsocidae are living in this region.After examining many materials of Stenopsocidae from Asia, we consider the above characteristics to be the diagnostic features among the genera of Stenopsocidae.Several characteristics are reviewed in the following.Shape of the vertex (Fig. 4).The vertex in male is in a similar shape because of the presence of the large compound eyes.In females, with small compound eyes, the shape of vertex is different among the genera.In the genera Graphopsocus and Neostenopsocus gen.n., the vertex is rounded.In the genera Malostenopsocus and Stenopsocus, the vertex is extended and bulbed.The phylogenetic result implies that the round vertex is plesiomorphic for Stenopsocidae.
Presence or absence of labral styli (Fig. 5).The labral styli are present in Graphopsocus and Neostenopsocus  gen.n., but are absent in Stenopsocus and Malostenopsocus.In addition, the labral stylus has a round apex in the genus Graphopsocus, while has a sharp apex in Neosteno psocus.The presence of labral styli is considered to be plesiomorphic for Stenopsocids.Mockford (2000) interpreted that the presence of labral styli is plesiomorphic in caeciliusids.Considering the labral styli are present in some amphipsocids (Amphipsocidae) and absent in all members of Dasydemellidae, this character should be plesiomorphic in the infraorder Caeciliusetae.
The markings on forewing (Fig. 6).In Graphopsocus, several brown markings are generally present on forewing.In Malostenopsocus, the marking is often absent on forewing.In Neostenopsocus, the markings are often present near Rs or pterostigma.In Stenopsocus, the marking is often absent on forewing except for the pterostigma region.Graphopsocus can be easily distinguished from the other genera based on the markings covering most of forewing.However, some taxa of other families also share the similar distribution pattern of markings on forewing, i.e., Caecilius caligonoides (Ceaciliusidae) (see Mockford, 1969, fig. 153), Mesocaecilius elegans (Pseudocaeciliidae) (see Li, 2002, fig. 924).This condition is regarded to be highly homoplasious.
Forewing venation (Fig. 6).(i) shape of the pterostigma.In Graphopsocus, Malostenopsocus and Neostenopsocus, the pterostigma is broad with a distinct posterior angle.In Stenopsocus, the pterostigma is generally narrow, and the posterior angle of pterostigma is not obvious.(ii) shape of cell r5.Differences in shape are conferred on this cell by the curved Rsm.An essentially parallel-side cell r5 occurs in the genera Graphopsocus, Neostenopsocus gen.n. and Stenopsocus.The genus Malostenopsocus shows curved Rsm and broad base of Ciliation of forewing (Fig. 6).Veins of forewing except CuP are setose, with one-row setae.In Graphopsocus and Neostenopsocus, the basal half of forewing anterior margin and CuP are glabrous.In Malostenopsocus and Stenopsocus, the basal half of forewing anterior margin and CuP is pilosity.The glabrous anterior margin and CuP of forewing are considered to be plesiomorphic for Stenopsocidae.In addition, sparse setae are present in the basal membrane of forewing in Malostenopsocus.This condition is also present in the family Dasydemellidae.
Number of ventral abdominal vesicles.In Graphopsocus, two ventral vesicles are present.In Neostenopsocus gen.n., Malostenopsocus and Stenopsocus, three ventral vesicles are present.There can be no question that the condition of two ventral abdominal vesicles is plesiomorphic for Stenopsocidae.Turner (1974) and New (1987) considered the presence of abdominal vesicles to be associated with living open-live leaf surfaces.
Phallosome (Fig. 7).The phallosome consists of the parameres, the aedeagus, the phallobase and the endophallus (Yoshizawa, 2005).Mockford (2002) indicated only the endophallus shows promise of phylogenetic interpretation.The endophallus of Stenopsocidae consists of several lobes with or without minute sclerotized papillae.In Graphopsocus, the endophallus is simple, with weakly sclerotized portion.This condition is probably plesiomorphic for Stenopsocidae.In the other three genera, the endophallus is more complex than the bilobed endophallus in Graphopsocus.The sclerotized portion is present in the endophallus, which is divided into two parts: the upper part and the base part.In Malostenopsocus, the upper part is divided into two lobes, and the base part is undivided.In Stenopsocus, the upper and base parts are both partly divided.The new genus Neostenopsocus gen.n. shows the intermediate condition, in which the upper and base parts are both partly divided.
As discussed above, several plesiomorphic features in Stenopsocidae are revealed, i.e., round vertex, presence of labral styli, broad pterostigma with a distinct posterior angle, straight Rsm on the forewing, glabrous anterior margin and CuP on the forewing, presence of two abdominal vesicles and the bilobed endophallus with weakly sclerotized portion.The new genus Neostenopsocus gen.n. is supported by several apomorphies: (i) forewing mostly transparent, (ii) abdomen with three ventral vesicles; (iii) endophallus with partly divided upper and base parts of minute sclerotized papillae.

Intergeneric phylogeny of Steno psocidae
The present phylogenetic analyses confirm the monophyly of the family Stenopsocidae, which has been strongly supported based on the morphological characters and multi-loci phylogenetic studies (Yoshizawa, 2002;Yoshizawa et al., 2014).The paraphyly of Stenopsocus herein inferred agrees well with the previous result of the multi-loci phylogenetic study with less taxon sampling and a smaller dataset (Yoshizawa et al., 2014).However, Yoshizawa et al. (2014) did not reveal the sister group relationship between Stenopsocus and Malostenopsocus because these two genera are represented by only three species in this work.Malostenopsocus and Stenopsocus, herein recovered as sister group, share some similar characters: (i) head with bulbed vertex, (ii) absence of styli, (iii) abdomen with three ventral vesicles, (iv) forewing broad, with strongly curved posterior margin, (v) forewing CuP with one row of setae, (vi) genitalia generally weakly sclerotized, (vii) male endophallus complex with several areas composed of minute sclerotized papillae.As noted in Li (1993), Malostenopsocus can be distinguished from Stenopsocus based on: (i) basal membranous area of forewing with setae, (ii) posterior angle of pterostigma distinct, (iii) cell r5 in forewing with broad basal region (Rsm and Mm strongly curved), (iv) base part of endophallus without division.Thus, the generic status of Malostenopsocus is corroborated.
The phylogeny recovered a sister relationship between Neostenopsocus gen.n. and Malostenopsocus + Steno psocus.This clade is supported by several apomorphies: (i) labrum with styli, (ii) abdomen with three ventral vesicles; (iii) endophallus with several lobes of minute sclerotized papillae.The genus Graphopsocus is estimated to be the sister group of all other stenopsocid taxa, as the stem branch of Stenopsocidae in the present study.
Herein, we propose a new classification scheme for the family Stenopsocidae based on our phylogenetic analysis of molecular data and re-evaluation of morphological characters.In this scheme, Stenopsocidae is classified into four genera: Graphopsocus, Malostenopsocus, Neostenopsocus gen.n., and Stenopsocus.

Conclusion
We sequenced 20 new nearly complete mitochondrial genomes of Stenopsocidae, and reconstructed the intergeneric phylogeny of this family.A new genus, namely Neostenopsocus gen.n., is proposed based on the molecular phylogeny herein inferred.Most species of Stenopsocidae were described from the Oriental realm, and it is likely that unknown genera and species are still to be discovered in this region.Future fieldwork and taxonomic studies based on morphological and molecular data could lead to further insight into the diversity and phylogeny of this family.

Conflict of interest state ment
The authors declare no conflicts of interest. 8.
considered Stenopsocus to have close affinities with Caecilius, and designated Graphopsocus to be the typical genus of the family Stenopsocidae.Roesler (1944) placed the genus Stenopsocus Hagen, 1866 into Stenopsocidae and treated Graphopsocus as a subgenus of Stenopsocus.Stenopsocus was considered the typical genus of Stenopsocidae by Roesler (1944) and the subsequent studies.Based on the revision made by

Figure 3 .
Figure 3. Phylogenetic tree inferred from maximum likelihood analyses of the mitochondrial genes (the protein-coding sequences and rRNA genes) and the 18S rRNA gene.Numbers associated with each branch indicate ML bootstrap/Bayesian posterior probabilities.Side bars on the right are classification according to Yoshizawa et al. (2014), whereas those on the left reflect the revised classification proposed in the current study.

Table 1 .
Taxa included in this study with their number of Genbank accession number.

Table 2 .
Nucleotide composition of the mitogenomes in Stenopsocidae The monophyly of the family Stenopsocidae was recovered.Stenopsocus is divided into two monophylet- ic clades which both received strong supports, i.e., the "immaculatus-lachlani" clade and the clade comprising the other Stenopsocus species.The former clade is assigned to be the sister group of the genus Malostenopsocus, suggesting the paraphyly of Stenopsocus.As the "immaculatus-lachlani" clade includes S. immaculatus, the type species of Stenopsocus, this clade is considered to represent the true Stenopsocus.Thus, the latter clade represents a different genus from Stenopsocus, and here we propose a new genus Neostenopsocus gen.n. based on the above inferred relationship.Neostenopsocus gen.n. was assigned to be the sister group of Stenopsocus + Malostenopsocus.
Neostenopsocus gen.n.The generic name is a combination of Greek "neo-" (meaning "new") and "Stenopsocus", meaning the new genus of Stenopsocidae.Gender masculine.
Type species.Stenopsocus externusBanks, 1937: 259.Diagnosis.Body length from postclypeus to wing tip 4-7 mm.Antenna distinctly longer than body and forewing in length.Labrum with distal styli.Forewing with setose anterior margin and single-row setae on all veins except for CuP, CuP glabrous; pterostigma elongate, with a distinct posterior angle; Rsm slightly curved.Forewing pterostigma with variable brown markings, other regions transparent or yellowish.Genitalia from yellowish to dark brown.Male genitalia: tip of aedeagal arch not exceeding parameres.Female subgenital plate posteriorly convex; dorsal valve and ventral valve sclerotized, narrow; external valve reduced; spermathecal sac generally pear-shaped without pouch.Distribution.Oriental, Palaearctic and Australian regions.Etymology.Diagnosis.Body length from postclypeus to wing tip 4-7 mm.Antenna longer than body and forewing.Head with variable brown markings.Labrum without distal styli.Pterostigma transparent, partly brown or wholly brown.Forewing with setose anterior margin, all veins with single-row setae; pterostigma narrowly elongate, with indistinct or distinct posterior angle; Rsm slightly curved.Genitalia generally yellowish white, weakly sclerotized.Male genitalia: tip of aedeagal arch generally exceeding parameres; endophallus with complex sclerotized papillae regions.Female subgenital plate posteriorly convex; dorsal valve and ventral valve sclerotized; proximal portion of dorsal valve broad; external valve reduced; spermathecal sac generally pear-shaped.Distribution.Oriental and Palaearctic regions.