First instar nymphs of two peltoperlid stoneflies (Insecta, Plecoptera, Peltoperlidae)

The first instar nymphs of two peltoperlid stoneflies, i.e., Microperla brevicauda Kawai, 1958 of Microperlinae and Yoraperla uenoi (Kohno, 1946) of Peltoperlinae, were examined and described. Additionally, the phylogeny and groundplan of the first instar nymphs of Peltoperlidae and Plecoptera were considered. The first instar nymphs of M. brevicauda have a slender body with a prognathous head of typical shape; they represent a groundplan in Plecoptera. On the other hand, the first instar nymphs of Y. uenoi have a broad, cockroach-like body with an orthognathous and shortened head, the latter being regarded as a potential autapomorphy of Peltoperlinae. Such differences in body shape between the subfamilies are speculated to arise from heterochrony. The three-segmented cerci of Y. uenoi are characteristic to Systellognatha, whereas the four-segmented cerci of M. brevicauda were independently acquired within Microperlinae. The structure and distribution pattern of chloride cells in the first instar nymphs of Plecoptera were also discussed. The presence of coniform chloride cells is a potential groundplan of Arctoperlaria. One to two pairs of chloride cells are distributed on the first nine abdominal segments of M. brevicauda; this represents a groundplan character of Systellognatha. On the other hand, one to four pairs of chloride cells are found on the second to ninth abdominal segments of Y. uenoi; this distribution pattern may be an apomorphic groundplan of Peltoperlinae.

It has previously been suggested that studies of Plecoptera first instar nymphs could be a potential source of phylogenetic information that could contribute to clarifying phylogenetic relationships (Harper 1979;Sephton and Hynes 1982). To date, the data collected in this area has been fragmentary and a detailed study has yet to be conducted. In addition, while the taxonomy and morphology of adults, older nymphs, and egg structures from Peltoperlidae have been studied extensively (e.g., Stark and Stewart 1981;Isobe 1988, 1989;Stark and Nelson 1994;Sivec 2000, 2007;Stark et al. 2015;Chen 2020), information on peltoperlid hatchlings is entirely lacking.
Given this background, in the present study we examined and described, for the first time, the first instar nymphs of two Japanese peltoperlids, i.e., Microperla brevicauda Kawai, 1958(Kawai 1958 (Fig. 1A) of Microperlinae and Yoraperla uenoi (Kohno, 1946) (Kohno 1946) (Fig. 1B, C) of Peltoperlinae, as two representative species. We compared the data obtained to that from previous studies on other plecopterans with the aim of discussing the groundplan and phylogeny of Peltoperlidae within Plecoptera as well as reconstructing the groundplan of their first instar nymphs.
To observe chloride cells, some fixed specimens were stained with Mayer's acid haemalum for 1 h, mounted in distilled water, examined using an Olympus BX43 biological microscope, and finally photographed with a Pentax K-70 camera. Other fixed specimens were dehydrated in a graded ethanol series, immersed in acetone, and embedded in a Kulzer Technovit 7100 methacrylate resin in accordance with the protocol described by Machida et al. (1994). Serial, semi-thin sections at a thickness of 2 µm were cut using a Leica RM2235 semi-thin microtome equipped with a Leica TC-65 tungsten carbide knife. Sections were then stained with Mayer's acid haemalum for 1 h, 1% eosin Y for 1 h, and 1% fast green FCF 100% ethanol solution for 1 min, before being observed under the Olympus BX43 biological microscope and photographed with the Pentax K-70 camera.
For scanning electron microscopy, the fixed specimens were dehydrated in a graded ethanol series, naturally dried with HMDS (1,1,1,3,3,3-Hexamethyldisilazane) as described by Faull and Williams (2016), mounted on a stab, and then observed under a Hitachi TM-1000 scanning electron microscope at 15 kV without coating. Some mounted specimens were examined under the Olympus BX43 biological microscope and photographed with the Pentax K-70 camera.
The specimens examined in the present study have been deposited in the collection of the Faculty of Symbiotic Systems Science, Fukushima University.

Results
The present study follows the view of Matsuda (1976) on the abdominal segmentation on Plecoptera, i.e., that the sternum of the first abdominal segment is greatly reduced or absent. The definition of chloride cells, which are divided morphologically into four types, i.e., caviform, coniform, bulbiform, and floriform, follows that of Wichard et al. (1999).
Measurements of the first instar nymphs of Microperla brevicauda and Yoraperla uenoi are shown in Table 1. Description. Body slender, uniformly white, sparsely covered by long and short fine setae, without gill and ocelli ( Fig. 2A, B). Head prognathous, subtriangular ( Fig. 2A, B). Antenna nine-segmented, longer than twothirds of body length ( Fig. 2A, B; Table 1). Compound eye reddish-black with four ommatidia. Labrum near-ly semicircular, covering part of mandible (Fig. 2C). Maxillary coxopodites divided into distal cardo and proximal stipes (Fig. 2C); maxillary palp and endites of maxilla and lateral galea well developed, but mesal lacinia hardly visible externally (Fig. 2C). Labial coxopodites divided into proximal postmentum and distal prementum, but hardly recognizable in fixed specimen (Fig. 2C); labial palp and endites of labium, lateral paraglossa, and mesal glossa well developed (Fig. 2C). Pronotum rectangular, almost same width as the head, and wider than the abdomen ( Fig. 2A; Table 1). Lateral margin of mesonotum and metanotum less developed ( Fig.  2A). Thoracic appendage consisting of coxa, trochanter, femur, tibia, tarsus with three tarsomeres, and pretarsus with ungues (Fig. 2D); tibia longer than femur (Fig.  2D). Abdominal segments with a row of long fine setae along posterior margin of each tergum (Figs 2A, 4A). Coniform chloride cells approximately 10 µm in diameter and 1 µm in height distributed on lateral side of first nine abdominal segments; one pair on first, eighth, and ninth targa; two pairs on second to seventh segments, i.e., one pair on each of second to seventh terga and sterna (Fig. 4A, B, D, E). Less sclerotized supraanal lobe on hind margin of tenth tergum ( Fig. 2A). Cerci four-segmented, with a crown of long and short fine setae on  posterior margin of first three segments; short fine setae at apex of fourth segment ( Fig. 2A).
Thoracic appendage consisting of coxa, trochanter, femur, tibia, tarsus with three tarsomeres, and pretarsus with ungues (Fig. 3D); tibia almost identical in length to femur (Fig. 3D); two claws can be recognized from ventral view (data not shown). Abdominal segments with a row of long and short stout setae along posterior margin of each tergum, except first to third terga, covered by metanotum (Figs 3A, 4F); first terga without setae, second and third tergum with setae barely visible in section (data not shown). Coniform chloride cells approximately 10 µm in diameter and 1.5 µm in height distributed on posterior margin of second to ninth abdominal segments; one pair on second sternum; three pairs on third sternum; four pairs on fourth to seventh sterna; three or four pairs on eighth sternum; two pairs on ninth sternum (Fig. 4C, F, G). Cerci three-segmented, with a crown of long and short stout setae on posterior margin of first two segments; short stout setae on apex of third segment (Fig. 3A).
Notably, the cockroach-like body shape, which is regarded as an autapomorphy of Peltoperlidae (Zwick 1973(Zwick , 2000Uchida and Isobe 1989), was found in the first instar nymphs of Peltoperlinae but did not appear in those of Microperlinae. Given that the older nymphs of M. brevicauda have much broader, cockroach-like bodies (e.g., Shimizu et al. 2005) and that the full-grown embryos of Y. uenoi acquire the configuration of the first instar nymphs (Mtow and Machida 2018), such differences in the body shape of peltoperlid first instar nymphs could be interpreted as a result of heterochrony. In other words, morphogenesis of the cockroach-like body shape may commence by the later embryonic period in Peltoperlinae but occur only during the postembryonic stages in Microperlinae. Further detailed examination of the embryonic and postembryonic development of Peltoperlidae will be required to broaden our knowledge of nymphal shape morphogenesis in this family.
The chloride cells, which are known to have osmoregulatory functions (e.g., Wichard et al. 1999), of Antarctoperlaria nymphs are floriform type only (e.g., Zwick 1973), even in the first nymphal stage (Sephton and Hynes 1982), which is regarded as an apomorphic groundplan of Antarctoperlaria (e.g., Zwick 1973Zwick , 2000. On the other hand, the presence of three other types of chloride cells, i.e., caviform, coniform, and bulbiform, has been observed in arctoperlarian nymphs (e.g., Wichard et al. 1999;Tamura and Kishimoto 2010), but only the coniform type has been observed in hatchlings of Arctoperlaria (Berthélemy 1979;Kishimoto and Ando 1985) with the exception of euholognathan Notonemouridae, which has bulbiform type (Sephton and Hynes 1982). In the present study, the first instar nymphs of M. brevicauda and Y. uenoi had chloride cells of coniform type. Thus, the type of chloride cells found in M. brevicauda and Y. uenoi is apparently comparable to those found in euholognathan Brachyptera braueri Klapálek of Taeniopterygidae (Berthélemy 1979), as well as systellognathan Kamimuria tibialis (Pictet) of Perlidae (Kishimoto and Ando 1985) and Perlodes microcephalus (Pictet) of Perlodidae (Berthélemy 1979). Therefore, the coniform type of chloride cells may be regarded as a potential groundplan of Arctoperlaria. The bulbiform type of chloride cells found in Notonemouridae might be due to a secondary modification from the coniform type, because as Wichard et al. (1999) pointed out, the coniform type is regarded as the basic type of chloride cells.
In the present study, we also distinguished two distribution types of chloride cells in Peltoperlidae: (1) the first type, in which one to two pairs of chloride cells are distributed on the first nine abdominal segments, is found in M. brevicauda of Microperlinae; (2) the second type, in which one to four pairs of chloride cells are distributed on the second to eighth abdominal segments, is found in Y. uenoi of Peltoperlinae. Additional examination of chloride cells will be required to cover more lineages of Plecoptera in detail. However, given the distribution of the chloride cells on the abdomen of first instar nymphs, it may be meaningful that the first type has also been observed in Perlidae (Kishimoto 1983;Kishimoto and Ando 1985) and Perlodidae (Berthélemy 1979). Therefore, we could postulate the following scenario: (1) the first type is a groundplan character in Systellognatha, and (2) this groundplan feature was inherited by Microperlinae in Peltoperlidae; whereas, (3) the second type was acquired by Peltoperlinae as an apomorphic groundplan.

Conclusion
In the present study, we (1) examined and described the first instar nymphs of Peltoperlidae for the first time, (2) reconstructed the groundplan of first instar nymphs from Peltoperlidae and Plecoptera, and (3) demonstrated that data collected from first instar nymphs could provide a new basis for discussion and reconstruction of the groundplan and phylogeny of Plecoptera. To improve understanding of the plecopteran groundplan and phylogeny further, more detailed studies of first instar nymphs must be conducted; these should consider all major lineages of Plecoptera, especially the antarctoperlarian Diamphipnoidae and arctoperlarian Styloperlidae, on which information is entirely lacking.