﻿Systematics of Lepidocyrtinusboneti Denis, 1948 (Collembola, Seirinae) reveals a new position for the species within Seirinae

﻿Abstract Seiraboneti Denis, 1948, comb. nov. is examined and redescribed based on syntypes and by a newly discovered Chinese population. Lectotype and paralectotypes were designated, and the type locality of the species has been fixed to Câuda, near Nhatrang, Vietnam. The species was first described in the genus Lepidocyrtinus, but based on morphological and molecular evidence it is here transferred to Seira. For the phylogenetic placement of Seiraboneticomb. nov., its mitogenome was included in a dataset comprising 19 species of Seirinae. Maximum Likelihood and Bayesian inferences clustered the species next to Seirasanloemensis Godeiro & Cipola, 2020 from Cambodia, forming a distinct Seira clade from the Old World, confirming the hypothesis of the existence of a different basal lineage of Seirinae in Southern Asia.


Introduction
Understanding the patterns of species diversity is a major goal for most of the researchers studying evolutionary biology. With this aim, an extensive comparison of phenotypic attributes across taxa and well-corroborated phylogenies are necessary (Simon et al. 2019). Seirinae Yosii, 1961 (sensu Zhang andDeharveng 2015) is an Entomobryidae subfamily with currently ca. 230 species in three genera (Bellinger et al. 1996(Bellinger et al. -2023. The first published molecular phylogeny regarding the Seirinae was based on three genes: two mitochondrial and one nuclear, and only three species of Seira Lubbock, 1870 and one of Lepidocyrtinus Börner, 1903 were sampled (Zhang et al. 2014). This study supported the Seirinae monophyly and a closer relationship of the subfamily with Lepidocyrtinae. Recently, these results were corroborated by a phylogeny based on morphological and barcode data (Zhang et al. 2019), as well as two studies with complete mitogenomes including 26 terminal taxa of the Entomobryidae (Godeiro et al. 2020a. Robust phylogenetic studies including the Seirinae are still scarce. Despite including representatives of the three current genera of Seirinae, these studies were focused only on species from the Neotropical region (Godeiro et al. 2020a, not sufficient to define infrageneric relationships. Only with the inclusion of species from several biogeographic regions we can have a clearer overview of the internal organization of the subfamily. In a study including data from an Asian Seirinae species in a phylogeny based only on mitogenomes from New World species, the Asian species appeared as a basal taxon to the entire New World group of Seirinae (Godeiro et al. 2020b).
The Asian continent has a great diversity of Seirinae species, whose species have been described during the last 80 years, but the morphological data originally described are mostly not sufficient for their comparison with other species (Denis 1948;Yosii 1959Yosii , 1961bYoshii and Suhardjono 1992;Bellinger et al. 1996Bellinger et al. -2023. Among the Asian taxa, Lepidocyrtinus boneti Denis, 1948 was described from Vietnam and Cambodia based on a few morphological characteristics such as the body color pattern, measurements of the body and appendices, bothriotricha pattern of the second to fourth abdominal segment, and morphology of the eyes and scales (Denis 1948). This species was never taxonomically revised, and it is only possible to know from the original description that it belongs to Seirinae due to the presence of heavily ciliated scales on body, the fourth abdominal segment with three bothriotricha, and the mucro falcate without a basal spine (Yoshii and Suhardjono 1992;Soto-Adames 2008;Cipola et al. 2018aCipola et al. , 2020Zhang et al. 2019;Godeiro et al. 2020a). A systematic study is necessary to reveal its position among the current Seirinae genera, as well as to provide a revision of its diagnostic characters for future interspecific comparisons.
Here we present a systematic study of Lepidocyrtinus boneti Denis, 1948 based on the lectotype and paralectotypes designated from syntypes, the species redescription, and a new record from China. We also transfer the species to Seira after a phylogenetic study using 19 other mitogenomes of Seirinae.

Materials and methods
Taxa sampling, sequencing, and mitogenome assembly The sequenced specimen belongs to the Chinese population of S. boneti comb. nov. and it was collected by NNG in October 2021 using an entomological aspirator. One specimen preserved in absolute alcohol was sent to Shanghai Yaoen Biotechnology Co., Ltd, China, where the DNA was extracted using TIANamp Micro-DNA extraction kit (Tiangen Co., Ltd, China). Libraries were constructed using KAPA Hyper Prep Kit (Roche) following custom procedures. Illumina NovaSeq platform was used to produce paired-end reads with 150 bp length. Approximately 10 Gb of data was delivered. NOVOPLASTY v. 3.8.3 (Dierckxsens et al. 2016) was used to assemble the mitogenome with kmer value = 33. MITOZ v. 2.4-alpha (Meng et al. 2019) was used to annotate and visualize the mitogenome. The final mitogenome sequence was submitted to the NCBI database, and the accession number is listed in Table 1.

Morphological analysis
The type material of Lepidocyrtinus boneti deposited at the Muséum National d'Histoire Naturelle, France, and specimens recently collected from China (Hainan island) were analyzed. Under a stereomicroscope Teelen XTL-207, Chinese specimens were bleached and diaphanized, first in 5% KOH and after in 10% lactophenol for 3 min/ each. Hoyer's liquid was used to mount the specimens between a slide and a glass coverslip Bellinger 1980, 1998). Mounted specimens were examined using a Leica DM2500 microscope. Illustrations were made with the help of an attached drawing tube and based on photographs taken with DMC4500 camera and LEICA APPLICATION SUITE v. 4.9. Specimens in ethanol gel were photographed using a Leica stereomicroscope S8AP0 attached to a Leica DMC4500 digital sight camera. Maps of species localities were made after Shorthouse (2010). The examined material is deposited at the collections of the Shanghai Natural History Museum (SNHM), Shanghai, China; Invertebrate collection of the Instituto Nacional de Pesquisas da Amazônia (INPA); and Muséum National d'Histoire Naturelle (MNHN), Paris, France. The terminology used in descriptions follows: clypeal chaetotaxy after Yoshii and Suhardjono (1992); labral chaetotaxy after Cipola et al. (2014a); labial papillae, maxillary palp and basolateral and basomedian labial fields after Fjellberg (1999), but using the Gisin's system (1964) for naming the chaetae rows; postlabial chaetotaxy after Chen and Christiansen (1993) and Cipola et al. (2018a); subcoxae outer chaetotaxy after Yosii (1959); trochanteral organ after Christiansen (1958b) and South (1961); unguiculus lamellae after Hüther (1986); male genital plate after Christiansen (1958a); and manubrial ventral formula after Christiansen and Bellinger (2000). The head dorsal chaetotaxy was described based on Mari-Mutt (1979) and that of the body based on Szeptycki (1979), both with additions of Soto-Adames (2008), Cipola et al. (2018a), and Zhang et al. (2019); and specialized chaetae (S-chaetae) after Zhang and Deharveng (2015). Symbols used to depict the chaetotaxy are presented in Fig. 3. Chaetotaxy data are all given by one side of body only, except for the head plate.
Note. On the basis of color pattern and morphological information extracted from the lectotype and type material (despite the poor state of conservation), we consider that both populations are conspecific.
Redescription. Body. Total length (head + trunk) of specimens 2.28-2.51 mm (n = 2 paralectotypes), lectotype 1.68 mm. Specimens whitish with brownish pigment on Ant I-IV; dark blue pigment forming a spot on Th III-Abd I laterally (rarely absent) and a smaller spot postero-laterally on Abd IV, coxae I, and femur III distally pigmented in dark-blue; eyepatches black ( Fig. 6). Scales heavily ciliated, oval or elongated and apically rounded (rarely truncate, pointed or irregular) present on Ant I to proximal 1/4 of Ant III, dorsal and ventral head, thorax, and abdomen, legs (except empodia), collophore anteriorly, both sides of the manubrium and dens ventrally; mac heavily ciliated apically, slightly foot shaped, rounded or acuminate; smooth microchaetae apically ramified or simple (Fig. 3).
Legs. Subcoxa I with one row of 3 chaetae and 2 psp; subcoxa II with an 'a' row of 8 chaetae, 'p' row of 4 chaetae and 3 psp; subcoxa III with one row of 8-10 chaetae, 1 anterior chaeta and 2 posterior psp ( Fig. 9A-C). Trochanteral organ with 16-18 spine-like chaetae, at least 2 anterior, 4 posterior, 5 internal, 1 apical and 4 distal arms (Fig. 9D). Anterior side of femurs II and III with 1 small proximal spine-like chaeta. Tibiotarsus outer  side distally with 1 tenent hair ciliated, apically capitate, and subequal to unguis outer length; inner side of tibiotarsus III with 1 smooth chaeta slightly longer than the unguiculus. Pretarsus with 1 minute anterior and 1minute posterior smooth chaetae (Fig. 9E). Unguis outer side with a pair of lateral teeth and 1 unpaired median tooth; inner side with 4 teeth, b.t. on proximal half, m.t. on distal one fourth and slightly longer than b.t., a.t. on distal one eighth and subequal to b.t. Unguiculus with all lamellae acuminate and smooth (ai, ae, pi, pe), except pe serrate and with a minute tooth on distal half (Fig. 9E).
Remarks. The present study increased substantially the morphological detailing of Seira boneti comb. nov. compared to the original description. Considering that most species of Seira from Asia are also poorly described (Denis 1948;Yoshii and Suhardjono 1992;Nguyen 2001;Cipola et al. 2018a), we compared S. boneti comb. nov. with other Asian species with 7 mac on Abd I (S. cinerea Yosii, 1966, S. nidarensis Baquero & Jordana, 2014, S. simbalwaraii Baquero & Jordana, 2015from India, and S. urbana Nguyen, 2001 from Vietnam) or 6 mac (S. hazrai Jordana, 2014 andS. prabhooi Baquero &Jordana, 2015 from India) ( Table 2). These species also resemble each other in dense head macrochaetotaxy (except for S. cinerea and S. urbana, which their head chaetotaxy are unknown), although S. nidarensis and S. hazrai differ from the others by the presence of Ps2 mac. They also resemble each other in Th II by PmA-B groups respectively with 6 or 7 and 3 mac (but differ from each other in PmC group), Th III with 13-14 inner mac apparently with the same homology (a1-5, m1i, p1i-1p, p2a-p2ea, p3), and Abd II-III respectively with 4 (a2, m3-3e, 3ep) and 1 (m3) inner mac, although this last segment was not described in S. cinerea. Seira boneti comb. nov. is more similar to S. urbana in body color pattern with a lateral spot on Th II-Abd II and another on And IV posteriorly, Th II anteriorly with some extra mac, and prelabral and labral chaetotaxy (see Nguyen 2001). Due to the similarities between these two species which coexist in the same region (Indochina), there is a risk they are synonyms. For this reason, we tried to consult the type material of S. urbana deposited at Vietnam Academy of Science and Technology, Vietnam, but the loan was not provided by the responsible (Dr. Anh T. T. Nguyen). Although the material was not obtained for a more rigorous comparison, based on the literature S. boneti comb. nov. differs from S. urbana in Th II anteriorly with 11-12 mac (8 in S. urbana), Abd IV with 12-13 inner mac (11 in S. urbana), unguiculus pe lamella serrated (apparently smooth in S. urbana), and collophore anteriorly with 3 proximal spine-like chaetae (4 in S. urbana).

Discussion
Whereas the Asian continent currently has almost 25% (~ 50 spp.) of the known richness of Seirinae (Bellinger et al. 1996(Bellinger et al. -2023, the moderate support values clustering S. boneti comb. nov. and S. sanloemensis are possibly related to the absence of other Asian taxa in the present phylogenetic analysis, mainly species morphologically similar to them, such as the six compared above with S. boneti comb. nov. (Yosii 1966;Nguyen 2001;Baquero et al. 2014Baquero et al. , 2015. This makes sense considering that phylogenetically related the Neotropical Seira species (e.g. S. atrolutea Arlé, 1939 Zeppelini, 2011, andS. mendoncae Bellini &Zeppelini, 2008) are morphologically similar in the pattern of dorsal chaetotaxy, such as head with at least 6 central mac (M1-2, S0-3) and 11 posterior mac (Pa1-5, Pm1-3, Pp1-3, Pp5) and Abd I with 5 mac (m2i-4) Zeppelini 2008, 2011;Cipola et al. 2014b;Godeiro and Bellini 2015). Such observation supports that this pattern of chaetotaxy on head and Abd I (Figs 7E, 8B) likely arose at least twice within Seirinae, once within derived Neotropical Seira species, and once within Seira from the Oriental region (e.g. S. boneti comb. nov. and S. sanloemensis) and in Lepidocyrtinus (Fig. 2). Consequently, if such a hypothesis is confirmed by further phylogenies, it may also indicate that there was a gradual gain of body macrochaetae in the Neotropical species of Seira, but the evolution of these characters is still unknown for Old World species.
From the recovered topology it is also possible to infer the evolution of other characters among the Seirinae. In Lepidocyrtinus, the developed lateral tooth on the unguis is likely a synapomorphy of the genus among the Seirinae, but it is not exclusive, as it is also present in other genera of Entomobryinae, such Acanthocyrtus Handschin, 1925,  Amazhomidia , Epimetrura Schött, 1925 andLepidocyrtoides Schött, 1917, suggesting the structure emerged more than once within Entomobryidae (Schött 1925;Cipola et al. , 2017Cipola et al. , 2018bCipola and Greenslade 2022). Still in Lepidocyrtinus, modified macrochaetae on dens represent an autapomorphy which appeared in the most derived taxa, as this characteristic is absent in the basal groups, like L. paraibensis (Bellini and Zeppelini 2009) (Fig. 2).
Molecular evidence justifies the transfer of S. boneti comb. nov. to Seira as found in the present study, clustering the species with another congener, S. sanloemensis. Such topology was also recovered in Bellini et al. (2023), although in this latter study the authors considered L. boneti as a Seira species based on preliminary data of the present study. Contrarily, the use of "Seira boneti" in the online database of Bellinger et al. (1996Bellinger et al. ( -2023 was possibly following the outdated classification of Yosii (1959Yosii ( , 1961a, in which Lepidocyrtinus was considered as a subgenus of Seira. This classification was updated in Godeiro et al. (2020a) based on species from the Neotropical region, which raised Lepidocyrtinus to the genus level again. Although many species in Bellinger et al. (1996Bellinger et al. ( -2023 were transferred back to Lepidocyrtinus following this new classification, this was not the case of L. boneti. In addition to the molecular evidence, morphologically this transfer can also be explained by the presence of head posterior macrochaetae (usually absent in Lepidocyrtinus), mesothorax normal (usually projected anteriorly in Lepidocyrtinus), and the absence of developed lateral teeth in the unguis (Fig. 9E), which is exclusive to Lepidocyrtinus compared with other Seirinae (see Cipola et al. 2020).
Our results corroborated that S. boneti comb. nov. belongs to an Old World Seira lineage (Fig. 2), although to reveal the natural groups and possibly to update new classifications, it is necessary to include more species from other continents, mainly European taxa of the Seira domestica group (Nicolet, 1842) (e.g., Cipola et al. 2018a), which is the type species of the genus. At this point, looking only to our phylogenetic tree, Seira needs to be better evaluated in the future, because the genus is either a nonmonophyletic group, or part of its taxa are classified improperly, and consequently it would be necessary to split the genus.

Conclusion
The present study redescribes Seira boneti comb. nov. Also, based on analyses including its mitogenome and 26 other sequences of Entomobryidae species, we surveyed its phylogenetic placement. This study is part of an on-going biogeographical and evolutionary study of the route of Seirinae global dispersion. To comprehend the evolutionary history of the subfamily we need comprehensive worldwide sampling and sufficient molecular markers. Available evidence suggests that the subfamily could be reorganized based on molecular data, especially given that our preliminary results grouped the two sampled Asian species into a distinct, ancestral clade relative to all New Word species of Seirinae.