On the desmitracheate “micronetine” genus Nippononeta Eskov, 1992 (Araneae, Linyphiidae)

Abstract The desmitracheate system in a “micronetine” genus Nippononeta Eskov, 1992 is recognized for the first time in the present study. This makes the subfamilial placement of this genus problematic. A morphological study was conducted for Nippononeta kurilensis Eskov, 1992 (the type species of Nippononeta) and Nippononeta coreana (Paik, 1991). Characters of genitalia and tracheal system, as well as some somatic characters were studied in detail by using scanning electronic microscopy (SEM), and compared with those of Agyneta. Updated descriptions of the genus Nippononeta and its two species are presented. Putative synapomorphies for Nippononeta and Agyneta are provided, as well as some putative synapomorphies shared by the two genera. The results imply that both scaped epigynum and desmitracheate tracheal system are probably homoplastic. The placement of Nippononeta and Agyneta within Linyphiidae need to be resolved in future studies.


Introduction
Linyphiidae Blackwall, 1859 is the second largest spider family, including over 4,500 species (World Spider Catalog 2014). The genital characters are species-specific in linyphiids, and provide rich information for species identification, taxonomy, and phy-logenetic reconstruction (e.g. Hormiga 2000; Miller and Hormiga 2004;Hormiga and Scharff 2005;Tu and Hormiga 2011). Seven linyphiid subfamilies are recognized (Tanasevitch 2014); however, the results of phylogenetic analysis based on molecular data show that most of them are not monophyletic (Arnedo et al. 2009).
Classical taxonomy of Linyphiidae is often confusing because of the characters overlapping among groups. The currently accepted seven subfamilies are based on different characters. For example, the epigynum furnished with a scape carrying copulatory grooves and openings (referred to as "scaped epigynum" below) was used as the main diagnostic feature for Micronetinae Hull, 1920 (Millidge 1984;Saaristo and Tanasevitch 1996), and the desmitracheate tracheal system, in which the median trunks are extensively branched and extend into the prosoma, was used as the main diagnostic feature for Erigoninae (Blest 1976;Millidge 1984). However, both scaped epigynum and desmitracheate system can be also found in the "micronetine" genera: Tennesseellum Petrunkevitch, 1925, Agyneta Hull, 1911(including Meioneta Hull, 1920, now a junior synonym of Agyneta), and Anibontes Chamberlin 1924 (Millidge 1986;Hormiga 1994;Dupérré 2013). At the same time, neither of these two characters is found in some erigonines: Asthenargus Simon & Fage, 1922, Gongylidiellum Simon, 1884, and Ostearius Hull, 1911(Hormiga 2000Miller and Hormiga 2004). This makes the subfamilial placement of abovelisted genera problematic. A recent phylogenetic study of Linyphiidae based on molecular and morphological data (Arnedo et al. 2009) indicated that the "micronetines", as a paraphyletic group, together with the erigonines form the "micronetines+erigonines" clade. The morphology data suggested a single origin of the desmitracheate system, as a synapomorphy for Erigoninae, while the total evidence favored double origins.
Micronetinae is a large subfamily, which currently includes 1212 species placed in 91 genera (Tanasevitch 2014). After it was redefined by Saaristo and Tanasevitch (1996), Micronetinae has been extensively revised at genus level based on genital characters (e.g. Saaristo and Tanasevitch 2002a, b;Saaristo and Marusik 2004;Dupérré 2013;Sun et al. 2014). Comparative studies show that a series of transitions exist among the forms of scaped epigynum (Tu and Hormiga 2010). Five characters were proposed to accommodate interspecific variation in tracheal anatomy (Miller and Hormiga 2004;Arnedo et al. 2009). These imply a more complex pattern of the relationships among "micronetines" and erigonines than previously suggested. Accordingly, analyzing more groups that possess transitional characters between the typical "micronetine" and erigonine versions may help us infer the character evolution and resolve the phylogenetic relationships among linyphiid groups.
In the present study, we report another "micronetine" genus Nippononeta Eskov, 1992, having a scaped epigynum, but also a desmitracheate system. The genus Nippononeta was separated from Agyneta, the senior synonym of Meioneta (Eskov, 1992). We conducted a morphological study of Nippononeta. Characters of the genitalia and tracheal system, as well as some somatic characters, were documented with SEM images for N. kurilensis Eskov, 1992 (the type species of Nippononeta) and N. coreana (Paik, 1991), and compared with those of Agyneta, as well as those of Erigoninae. Putative synapomorphies were proposed for Nippononeta and Agyneta, which need to be tested in future studies.

Materials and methods
Specimens were examined and measured using a Leica M205A stereomicroscope. Male palps and epigyna were examined after they were dissected from the body. Left structures (e.g. palps, legs, etc.) were depicted. Embolic divisions were excised by breaking the membranous column which connects the suprategulum and the radix. Male palps and epigyna were cleared in methyl salicylate. Scanning electron microscopy (SEM) images were taken under a Hitachi S-3400N scanning electron microscope at the China Agricultural University. For SEM examination, the specimens were prepared following Álvarez-Padilla and Hormiga (2008). SEM images of the embolic division taken from the right palp were mirrored to match those taken from the left palp. All examined specimens are deposited in the College of Life Sciences, Capital Normal University, China (CNU). Terminology of genital and somatic characters follows Tu and Hormiga (2010) and Hormiga (2000)  Composition. The genus includes 24 species; the type species is Nippononeta kurilensis Eskov, 1992. Diagnosis (updated). Nippononeta species are similar to Agyneta in many genital characters and the desmitracheate system, but differ in the presence of a dorsal pattern on the abdomen, which is absent in most Agyneta. Male palps of Nippononeta can be distinguished from Agyneta by the conical elevation on the cymbium absent in the former (Fig. 1A), present in the latter; the presence of proximal cymbial fold (Fig. 1D) and the spine-like embolus thumb (Fig. 1G) in Nippononeta, absent in Agyneta. The scaped epigynum in Nippononeta can be distinguished by its narrowed epigynal basal part covered by a transversal fold, the well developed stretcher and remnant lateral , median branch of paracymbium (left) and outer margin fold continue with distal arm (lower) E-G embolic division E dorsal F ventral, arrow indicates basal hook of embolus G embolus, ventral, upper arrow indicates the last strongest spine of thumb; lower arrow indicates basal hook of embolus H anterior part of male abdomen, ventral, shows epiandrous gland spigots absent. ARP anterior radical process; AX apex of embolus; CRL cymbial retrolateral lobe; DTA distal tibial apophysis; E embolus; EC embolus column; EM embolic membrane; EP embolus proper; LC lamella characteristica; P paracymbium; PF proximal cymbial fold; PH pit hook; PHS pit hook sclerite; R radix; RTP retrolateral tibial process; T tegulum; TA terminal apophysis; TH thumb of embolus. lobes ( Fig. 2A-B), while in Agyneta the epigynal basal part is normal, the stretcher usually small or absent, but the lateral lobes are well developed bearing lateral pockets and copulatory openings (Tu and Hormiga 2010: fig. 6a).
Description (updated). Chelicerae of normal size, with narrowed fang base and stronger stridulatory ridges in the male than in the female (Fig. 3C-F). Female palpal claw absent (Fig. 2F). Tracheal system: Median trunks wider in diameter than lateral pair, highly branched and extending into prosoma (Fig. 2E); tracheoles with taenidia. Lateral pair and median trunks arising independently from spiracular atrium. Epiandrous gland spigots absent in the male (Fig. 1H). Spinnerets: PLS in females having the mesal cylindrical gland spigot base enlarged (Fig. 2G), the triplet formed by one flagelliform and two aggregate gland spigots present in males PLS (Fig. 2H). For other somatic characters and measurements see Eskov (1992).
Male palp (Fig. 1A-G). Tibia short, with serrated distal apophysis and pointed retrolateral process, sometimes with additional proximal process. Cymbium with small retrolateral lobe and proximal fold above paracymbial base. Paracymbium U-shaped, with median branch arising from inner margin. Distal suprategular apophysis modified into pit hook and hook sclerite. Embolic membrane large, with many papillae. Embolic division: radix boat shaped, connected to embolus by S-shaped membranous column. Embolus with pointed proper and serrated area, thumb modified into many spine-like projections, large apex and basal apophysis. Lamella characteristica usually splitting into two or three branches, at least one of them ribbon like with thread projections distally. Terminal apophysis composed by one large basal sclerite and one or two branches with papillae apex.
Epigynum (Figs 2A-D, 5). Epigynal basal part narrowed, covered by transversal fold formed by the tegument connecting to dorsal side of epigynum. Median plate absent and epigynal cavity dorsally opened. Scape sigmoid folded with well developed stretcher furnished with a pit; lateral lobes remnant; copulatory openings covered by folded scape.

Discussion
The putative synapomorphies based on genital characters suggest that the four desmitracheate "micronetine" genera: Nippononeta, Agyneta, Tennesseellum, and Anibontes have a common ancestor. Eskov (1992) erected the genus Nippononeta to allocate those species which were not consistent with several generic diagnoses of Meioneta (a junior synonym of Agyneta). Although Nippononeta and Agyneta have a desmitracheate system, the genital characters of both genera are of a typical "micronetine" type: male palpal embolic division has well developed lamella characteristica and terminal apophysis, and females have a scaped epigynum. At the same time, in most Erigoninae, lamella characteristica and terminal apophysis are secondarily lost (Hormiga 2000), and a scaped epigynum has never been documented. There are several putative synapomorphies based on genital anatomy in both genera. For Nippononeta, putative synapomorphies include: the embolus thumb modified into many spine-like projections (Fig. 1G), the presence of proximal cymbial fold (Fig.  1A), and the narrowed epigynal basal part covered by the transversal dorsal fold (Fig.  2B). For Agyneta, putative synapomorphies include: the presence of conical cymbial elevation, and the scaped epigynum with the stretcher remnant or absent, but a pair of well developed lateral lobes bearing lateral pockets and copulatory openings (Tu and Hormiga 2010: fig. 6a). Further, putative synapomorphies for (Nippononeta + Agyneta) clade are: the serrated distal tibial apophysis (Fig. 1D) and the absence of median plate (Fig. 2B). According to Dupérré (2013), the genitalia of Tennesseellum and Anibontes have the exact same configuration as that of Agyneta, only different in small details. Therefore, based on genital synapomorphies, Nippononeta should form the sister clade to the group including the other three "micronetine" genera (Agyneta, Tennesseellum, and Anibontes).
The results of phylogenetic analyses support the single origin of desmitracheate system (Arnedo et al. 2009;Hormiga 2000;Miller and Hormiga 2004). Four types of tracheal anatomy have been recognized in Linyphiidae: desmitracheate, haplotracheate, and two intermediate types (Miller and Hormiga 2004). Based on the examination for 121 linyphiids belonging to 98 genera, Blest (1976) found the desmitracheate system in 85 erigonines belonging to 65 genera. This tracheal system is addressed as an erigonine type, although there are eight erigonine species with the simple (haplotracheate) type. At the same time, 19 "micronetine" species belonging to 16 genera, and all other linyphiids examined by Blest (1976) had a haplotracheate system. Later studies found that the typical "micronetine" genera Agyneta, Tennesseellum, Anibontes (Millidge 1986;Hormiga 1994;Dupérré 2013) and Nippononeta also have a desmitracheate system. Although a phylogenetic analysis of linyphiids rendered "micronetines" paraphyletic, the morphological partition suggests that the desmitracheate system in linyphiids has a single origin (Arnedo et al. 2009), with secondary reduction to the haplotracheate system within erigonines (Miller and Hormiga 2004).
Furthermore, we find that, in addition to the desmitracheate system, some confirmed synapomorphies of erigonines (Hormiga 2000;Arnedo et al. 2009) such as the presence of triplet of spigots in male PLS (Fig. 2H), the absence of epiandrous gland spigots in males (Fig. 1H), and the absence of palpal claw in females (Fig. 2F) are shared not only with Nippononeta and Agyneta, but also with some haplotracheate "micronetines", e.g. Microneta viaria (Arnedo et al. 2009), Macrargus rufus, Maro sublestus, Oreonetides vaginatus and Ryojius sp. (Tu, unpublished data). This is consistent with the results of phylogenetic analysis demonsrating that "micronetines" are a paraphyletic group (Arnedo et al. 2009). The placement of Nippononeta and Agyneta within Linyphiidae and their relationships with other "micronetines" and erigonines need to be resolved in future studies.