Description of two new species of Stigmaeopsis, Banks 1917 (Acari, Tetranychidae) inhabiting Miscanthus grasses (Poaceae)

We provided a new diagnosis of the genus Stigmaeopsis. Then we described two new species that are very closely related to Stigmaeopsis miscanthi (Saito) from Miscanthus spp. in Japan and China. The Japanese species, named Stigmaeopsis sabelisi Saito and Sato n. sp., was previously referred to as Stigmaeopsis miscanthi low aggressiveness form , and is characterized by its low male-to-male aggression behavior. The other species, Stigmaeopsis continentalis Saito and Lin n. sp., was found in China (Fujian district). Thus four species could be discriminated from the species inhabiting Miscanthus and reed grasses. However, the two new species and S. miscanthi resemble each other very closely, thus they are considered to be sibling species. To identify them several naive characters, geographic distribution and genetic data are needed. An updated key to all known species of Stigmaeopsis is also proposed.


Introduction
Stigmaeopsis celarius was first described by Banks (1917) from an introduced bamboo plant (Pleioblastus simonii) and established as the type species of the new genus Stigmaeopsis. Thirty-three years after the first description, this species was redescribed and Stigmaeopsis was synonymized with Schizotetranychus (Trägårdh 1915) by McGregor (1950). Much later, Saito et al. (2004) reinstated the genus Stigmaeopsis Banks (1917), described two new species and moved five others to the genus, formerly of the celarius species group of Schizotetranychus. Flechtmann (2012) added two new combinations, i.e. Stigmaeopsis malkovskii (Wainstein 1956) and Stigmaeopsis meghalayensis (Gupta and Gupta 1994). Furthermore,  described two more Stigmaeopsis species from bamboo plants. Thus we now recognize 11 species as belonging to this genus.
Almost all members of the Stigmaeopsis genus have characteristic nest-weaving habits (Saito 2010;. Considerable nest size variation exists between species on bamboo plants, which is correlated with species-specific variation in dorsal setal lengths (Mori and Saito 2013;. On the other hand, two Stigmaeopsis species are known to inhabit the perennial grasses, Miscanthus spp. and a reedgrass (Poaceae). In Stigmaeopsis miscanthi (Saito 1990), three forms, each of which possess different levels of male-to-male aggression (Saito 1995), were recently recognized (Sato et al. 2013a). The HG (males having high aggressiveness) and LW (males having low aggressiveness) forms of S. miscanthi have both been observed in Japan. The former is distributed over high temperature areas, and the latter over most low temperature areas with the exception of Hokkaido prefecture (Saito and Sahara 1999). However, there are very few morphological characters that can be used to identify these two forms other than the proportion of male leg development (armoured leg I in Saito and Sahara 1999;Sato et al. 2013a). Recently Sakamoto et al. (2017) found large differences between the LW, HG and a chinese (Cn) forms, suggesting that they are actually different species.
In the present study, we investigated the characteristics three forms in detail then discriminate two new species from S. miscanthi. Furthermore a new diagnosis of Stigmaeopsis was presented and a key to the world known species of Stigmaeopsis is also provided.

Materials and methods
The two mite species described in the present study were collected from their respective fields (see results) and preserved in glass tubes containing MA solution (Saito and Osakabe 1993). Several of these preserved specimens were then prepared into permanent specimens using the Canada balsam method described in Saito et al. (1993). The specimens used for dorsal setae measurements were taken from colonies reared in the laboratory after collection and mounted on slides using Hoyer's solution. A 10g weight was gently placed on each cover glass to flatten the mite bodies. The specimens were kept on a hot plate at 55°C for more than 7 days. The measurements were made from digital photographs taken with a digital camera (Sony Nex-7, Sony Corp., Tokyo, Japan) set up on a phase contrast microscope (Olympus BX63, Olympus Corp., Tokyo, Japan with 20x and 40x objective lens). Image J64 (provided by NIH, USA) for Macintosh (Apple Inc., USA) was used to measure seta and body lengths.

Description
Female -Body flattened and wide, straw to greenish yellow with small blackish green spots. Body size 485.4 ± 15.9 μm from tip of rostrum to end of hysterosoma and 347.1 ± 15.7 μm from middle of horizontal line connecting setae v2 bases to end of hysterosoma. Peritreme bent and slightly dilated at distal end (but varies with specimen conditions). Propodosoma well demarcated from hysterosoma. Dorsal propodosomal setae sc1, 10% shorter than distance between their bases ( Fig. 1, Table 1), and shorter than distance between bases of sc1 and c3. Bases of all dorsocentral hysterosomal setae (c1, d1, e1, f 1) placed approximately in a straight line and the pair of lines forms a V shape. Length of dorsocentral hysterosomal setae d1 subequal with distance between their bases, just reaching bases of f 1. Distance between bases of dorsolateral hysterosomal setae c2 longer than distance between bases of dorsolateral hysterosomal setae d2. Hysterosomal setae h3 exist ventrally. Lengths of dorsal setae and distances between their bases listed in Tables 1 and 2. Genital flap and area anterior to flap transversely striate (Fig. 1E). Distal segment of palpus has two simple setae and one spinneret (terminal sensilum), two eupathidia and one solenidion. Spinneret is conical in shape (Fig.  1B). Numbers of setae on leg segments presented in Figure 2 and Table 3.
Male -Body size 379.6 ± 39.6 μm from tip of rostrum to end of hysterosoma and 264.0 ± 26.7 μm from middle of horizontal line connecting setae v2 bases to end of hysterosoma. Lengths of dorsal setae and distances between their bases as in Tables 1 and 2. Femur-I with 9 tactile setae, one of which is tiny (dwarfed) and additional to those present in the female. Distal segment of palpus similar to female, spinneret slightly smaller (Fig. 1C). Aedeagus curved dorsally, weakly sigmoid (Fig. 1D). Numbers of setae on leg segments presented in Figure 3 and Table 3. Remarks -This species resembles Stigmaeopsis miscanthi but is distinguished from it by two morphological characters, as follows: the length of dorsal propodosomal setae sc1 shorter than distance between sc1 and c3 bases in S. sabelisi, but longer than that in S. miscanthi; The length of dorsocentral hysterodosomal setae d1 almost the same as distance between their bases in S. sabelisi, but much longer than that in S. miscanthi.

Stigmaeopsis continentalis
Male -Body size 415.9 ± 15.6 μm from tip of rostrum to end of hysterosoma and 302.4 ± 9.5 μm from middle of horizontal line connecting setae v2 bases to end of hysterosoma. Lengths of dorsal setae and distances between their bases as in Table 1. Femur-I with 9 normal setae, one of which is tiny (dwarfed) and additional to female setation. Numbers of setae and solenidia on distal segment of palpus presented in Fig. 4C and those on leg segments presented in Figures 5C-D, and Table 3. Aedeagus broadly curved dorsally, weakly sigmoid (Fig. 4D). Remarks -This species resembles both S. miscanthi and S. sabelisi but is distinguished from them by several characters as follows: the distance between the bases of dorsolateral hysterosomal setae c2 almost the same as that between the bases of dorsolateral hysterosomal setae d2 in S. continentalis, but the distance between the bases of c2 is longer than that between the bases of d2 in both S. sabelisi and S. miscanthi; in both S. continentalis and S. miscanthi, length of dorsocentral hysterosomal setae d1 are much longer than distance between their bases and extend past the bases of f 1, but setae d1 are almost the same length as distance between their bases in S. sabelisi.
Life type -This species lives within dense web nests (WN-c life type in Saito 1983) built over depressions on leaf adaxial surface (along midvein).

Key to the species of Stigmaeopsis (for adult female)
Due to the addition of two new species, the key to the Stigmaeopsis species in  should be changed as follows (following mite identification we recommend to refer to  Although S. meghalayensis does not satisfy several characters that represent Stigmaeopsis, we hold the decision until we can confirm the type specimen. The species marked with asterisk inhabit Miscanthus grasses and reedgrass (Poaceae), whereas all others inhabit Bambusoideae (Poaceae) plants. As shown in the above key, the lengths of dorsal setae are important characters for species identification in Stigmaeopsis. However, these setae are often broken and shortened in field collected mites, such that we must get our great attention to this possibility (Sakamoto et al. 2017). Specimens of newly emerged females from teleiocrysalis may help to avoid such troubles.
There are very few morphological differences between S. continentalis and S. miscanthi, thus they are considered to be cryptic species, such that geographic distribution (China in the case of the former and Japan in the case of the latter) and genetic information must be considered to distinguish S. continentalis from S. miscanthi. Furthermore, variation in the armored male morphology: S. miscanthi male leg I / leg III = 1.41; S. continentalis male = 1.32; S. sabelisi male = 1.29 (Saito and Sahara 1999;Sato et al. 2013a; in this study) may help with species identification, though males are rare in natural populations, due to their mortal combat tendencies.
From the above, we recognize 13 species of Stigmaeopsis. Although it is difficult to say that all species have been described, we reviewed several characteristics of Stigmaeopsis species in the genus diagnosis. From a morphological standpoint, no visible variations could be determined from the peritreme, aedeagus, striae of genital flap or those anterior to the flap (genital flap and area anterior to flap transversely striate) between congeneric species, which are useful characteristics for classifying other tetranychid species (Ehara 1999).
Almost all known species build nests over leaf depressions using silk threads (Saito et al. 2017; in this study) and live within them gregariously. Host plants are restricted to Poaceae distributed across Asian countries (India, Thailand, China (including Taiwan), Kazakhstan, Korea and Japan) other than artificial introduction.