The Kachin amber studied herein was obtained from amber mines near Noije Bum (26°20’N, 96°36’E), Hukawng Valley, Kachin State of northern Myanmar. The geologic age was dated as earliest Cenomanian (98.79±0.62 Ma), early Late Cretaceous (Shi et al. 2012; Yu et al. 2019). The studied amber piece was grounded with different-sized emery papers and polished with polishing grinding paste. Both the amber piece (ICJUST, No. CZT-PLE-MA13) and examined extant specimens are deposited in the ICJUST – Insect Collection of Jiangsu University of Science and Technology, Jiangsu Province, China.

Photographs were taken with a Canon EOS 6D digital camera equipped with a Canon MP-E 65 mm 5× macro lens. Line drawings were produced with the aid of an SDPTOP SZM45 stereomicroscope and ADOBE ILLUSTRATOR CS6. Photographs and drawings were adjusted and optimized with ADOBE PHOTOSHOP CS6.

Two methods of morphological phylogenetic analysis were used to evaluate the systematic placement of the new stonefly, including parsimony and Bayesian inference. The character list and data matrix were partially derived from a previously published dataset in Nelson (1984) (see Supplementary Files 1 and 2). To better accommodate the fossils, the invisible anatomical characters from Nelson (1984) were removed from the dataset. During the selection of taxonomic groups, the fossil impressions were excluded from the dataset because their outlines of bodies or fragmentary wings could not provide enough information for the phylogenetic analysis. Extinct taxa were restricted to the better-preserved Cretaceous stoneflies in Kachin amber, excluding the poorly described Pinguisoperla Chen, 2018 and the very likely synonym Burmaperla Jouault & Nel, 2022 of Largusoperla Chen, Wang & Du, 2018 (Jouault and Nel 2022). Another 24 extant genera of Arctoperlaria were also included in the analysis. The monophyly of Antarctoperlaria is well supported by the inner anatomical characters (Zwick 2000) and thus the inclusion of antarctoperlarian taxa in the phylogenetic analysis based on external morphological characters would generate bizarre tree topology. Meanwhile, the comparative discussion has excluded the new fossil in this study from Antarctoperlaria. Therefore, Antarctoperlaria is removed from the dataset. A putative ancestor with all plesiomorphic character states was regarded as the outgroup. A final matrix of 41 taxa with 85 external morphological characters was constructed (see Supplementary File 2).

The data matrix was subjected to a parsimony analysis with the program TNT 1.5 (Goloboff et al. 2008; Goloboff and Catalano 2016) using new technology search. Other parameters were set as default. On the calculated strict consensus tree, character state transformations were visualized using WINCLADA (Version 1.00.08). The Bayesian inference was performed with the software MRBAYES 3.2.7 (Ronquist et al. 2012). The parameters used in MRBAYES were generations = 10,000,000, samplefreq = 50, rates = gamma, burn-in = first 25% of sampled trees. A majority consensus tree was built with the remaining generations, and the posterior probability of each node was calculated. Convergence was checked with the average standard deviation of split frequency, which remained below 0.01. All tree files were adjusted and visualized in FIGTREE v. 1.4.2 (available from http://tree.bio.ed.ac.uk/software/figtree).

UFP – is used for the undetermined family in Perloidea. The following institutional abbreviation is used: ICJUST – Insect Collection of Jiangsu University of Science and Technology, Jiangsu Province, China. The following abbreviations of morphological characters are used: BL – body length (excluding antennae and cerci); HL – head length; HW – head width; ATL – antenna length (partially preserved); MPL – maxillary palp length; LPL – labial palp length; PL – pronotal length; PW – pronotal width; WL – wing pad length; FLL – foreleg length; MLL – midleg length; HLL – hind leg length; ABL – abdomen length; ABW – abdomen width; CL – cercus length (partially preserved); LFL – left foreleg; LML – left midleg; LHL – left hind leg; RFL – right foreleg; RML – right midleg; RHL – right hind leg; CE – compound eye; PR – pronotum; SM – submentum; GL – glossa; PG – paraglossa; CA – cardo; SS – stipes; LA – lacinia; LP – labial palp; MP – maxillary palp; AGR – abdominal gill remnant; CO – coxa; TR – trochanter; FE – femur; TI – tibia; TA – tarsi; CL – claw; T – tergum; ST – sternum; PP – paraproct.

The absence of well-developed abdominal gills as either lateral or anal appendages can easily exclude Kachinoperla gen. nov. from the suborder Antarctoperlaria (Benedetto 1974; Heckman 2008; Pessacq et al. 2020). The toothed lacinia and sharp-cusped mandibles of Kachinoperla indicate its grasping and holding predaceous food habit, which excludes its placement in the phytophagous Euholognatha (Fig. 6A–I) of Arctoperlaria (Stewart 2009). The placement of Kachinoperla in the other arctoperlariid infraorder Systellognatha is well supported by its predaceous type of mouthparts and slender palps, which are also known as diagnostic larval characters for the superfamily Perloidea (Fig. 7A–F) within Systellognatha (Zwick 1980). Conversely, in the other systellognathan superfamily Pteronarcyoidea (Fig. 7G–L), mouthparts of the three families, Peltoperlidae, Pteronarcyidae, and Styloperlidae, are all phytophagous (Uchida and Isobe 1989; Stewart 2009). Apart from the mouthparts, Kachinoperla can further be distinguished from Peltoperlidae by lacking the cockroach-like larval habitus which has a much wider thorax than the head and abdomen (Uchida and Isobe 1989). Kachinoperla differs from Pteronarcyidae in the absence of pronotal projections and the invisibility of notal contours between wing pads (Nelson 1988; Myers and Kondratieff 2017). Kachinoperla is also easily separated from Styloperlidae by the presence of giant tibial spurs, which are replaced by numerous trifurcate setae in Styloperlidae (Uchida and Isobe 1989). Thus, Kachinoperla seems to be closely related to Perloidea. However, the subequal glossae and paraglossae of Kachinoperla firmly prevent attribution to Perloidea, since the glossae much shorter than paraglossae is an apomorphic character defining the monophyly of Perloidea (Fig. 7A–F) (Zwick 2000). In addition to the proportion between glossae and paraglossae, the relative length between the maxillary and labial palps and the modification of their apical segments in both adult and immature stages are taxonomically informative, especially for higher taxa (families) and have been widely used in the classification of stoneflies (Stewart and Harper 1996; Stewart and Stark 2002; Jaihao and Phalaraksh 2011; Chen and Du 2018). Typical examples of apomorphically modified apical palpal segments are the circular and enlarged apical labial palpomere in Nemouridae (Fig. 6E) and the asymmetrically inserted, minute apical maxillary palpomere in Chloroperlidae (Fig. 7E–F). Characters of these palps are more useful in the fossil taxa because other characters of the fossils are not always well preserved and available for comparison. Apart from the subequal glossae and paraglossae, Kachinoperla is further excluded from the four extant families within Perloidea by the following diagnostic characters: from Perlidae by the absence of highly branched gills on sides and venter of all thoracic segments (Zwick 2000; Stewart and Stark 2002); from Chloroperlidae by the absence of asymmetrically inserted, minute apical maxillary palpomere, pronotum not oval, body not slender and metathoracic wing pads not parallel to the body axis (Zwick 2000; Stewart and Stark 2002); from Perlodidae by the absence of complicated pigmented pattern or chaetotaxy on the head and thoracic terga (Stewart and Harper 1996; Stewart and Stark 2002; Chen 2022b); and from Kathroperlidae by the absence of strongly elongated head and autapomorphic semiquadrate lacinia with no subterminal tooth (Zwick 2006; South et al. 2021). In conclusion, Kachinoperla undoubtedly belongs to the infraorder Systellognatha but cannot be attributed to any of the 17 extant stonefly families, including Perlidae and Peltoperlidae, which also comprise fossil records in Kachin amber (Chen et al. 2018; Chen and Xu 2020).

Larval descriptions are available for many extinct stoneflies of Systellognatha: Permian Palaeoperlidae, Tshekardoperlidae, and Tungussonympha (undetermined family in Perloidea, abbreviated as UFP below); Triassic Berekia Sinitshenkova, 1987 (UFP) and Triassoperla Lin, 1977 (UFP); Jurassic Derancheperla Sinitshenkova, 1990 (Perlodidae), Isoperlodes Sinitshenkova, 1992 (Perlodidae), Platyperlidae, Bestioperlisca Sinitshenkova, 1990 (UFP), Chloroperloides Sinitshenkova, 1985 (UFP), Perlisca Sinitshenkova, 1985 (UFP), Perlomimus Sinitshenkova, 1985 (UFP), Trianguliperla Sinitshenkova, 1985 (UFP), and Perlitodes Sinitshenkova, 1987 (UFP); and Cretaceous Dipsoperla Sinitshenkova, 1987 (Chloroperlidae), Ecdyoperla Sinitshenkova, 1998 (UFP), Savina Sinitshenkova, 1987 (UFP), and the Electroneuria (Perlidae) known from Kachin amber.

Almost all known larval fossils of Plecoptera are poorly preserved as fossil impressions and their descriptions and comparisons with other larvae or adults are restricted to the shapes and ratios of general body parts, whereas the more informative structures such as the mouthparts remain unclear (Sinitshenkova 1987). Apart from the apparent difference in age, Kachinoperla notably differs from the Permian Palaeoperlidae, Tshekardoperlidae, and Tungussonympha by the thoracic segments without clearly visible notal contours between wing pads, and furthermore from Tshekardoperlidae by each antennal segment not elongated (Sinitshenkova 1987; Sroka et al. 2018). Kachinoperla apparently differs from the Triassic Berekia and Triassoperla by the head much larger than the pronotum, pronotum trapezoidal, and wing pads broad and semicircular; in the two latter genera, the head is rounded and much smaller than the pronotum, the pronotum is respectively horseshoe-shaped and rectangular, and the wing pads are slender and near cylindrical (Lin 1977; Sinitshenkova 1987).

When compared with the Jurassic taxa, Kachinoperla differs from Derancheperla by the invisibility of notal contour between wing pads (Sinitshenkova 1990); from Isoperlodes by the pronotum near twice as long as the width, subequal in width to meso- and metanota (vs. pronotum length subequal to the width, much narrower than meso- and metanota), wing pads on mesonotum with circular lateral margins (vs. wing pads with angled anterolateral margins and near straight lateral margins), and by the invisibility of notal contour between wing pads (Sinitshenkova 1992); from Platyperlidae by the head broad and near trapezoidal (vs. head small and circular), and the invisibility of notal contour between wing pads (Sinitshenkova 1985, 1987); from Bestioperlisca by the wing pads not parallel to the body axis (vs. parallel), lateral pronotal margins not straight (vs. straight), and the femora at least 2.5 times wider than tibiae (vs. femora slightly wider than tibiae) (Sinitshenkova 1990); from Chloroperloides by the head broad and wider than pronotum (vs. head small and circular, much smaller than pronotum), and the wing pads large (vs. wing pads small) (Sinitshenkova 1985); from Perlisca by the head broad and wider than pronotum (vs. head small and circular, much smaller than pronotum), and the invisibility of notal contour between wing pads (Sinitshenkova 1985); from Perlomimus by the head broad and wider than pronotum (vs. head small and circular, much smaller than pronotum), thorax normal, meso- and metanota near as wide as pronotum (vs. thorax strikingly widened, meso- and metanota at least 1.5 times wider than pronotum) (Sinitshenkova 1985); from Trianguliperla by the head broad and much larger than the trapezoidal pronotum (vs. head triangular, much smaller than the elliptical pronotum), and the presence of large wing pads (vs. complete absence of wing pads) (Sinitshenkova 1985); and from Perlitodes by all wing pads with the same size (vs. posterior wing pads distinctly wider than anterior ones) and the invisibility of notal contour between wing pads (Sinitshenkova 1987).

When compared with the three Cretaceous genera known by fossil impressions, Kachinoperla can be distinguished from Dipsoperla by the head two times longer than pronotum (vs. head almost as long as pronotum), abdominal sterna complete (vs. abdominal sterna divided by a longitudinal median membrane) (Sinitshenkova 1987); from Ecdyoperla by the pronotum without posterior projections (vs. pronotum with projections on hind angles), and the wing pads long and semicircular (vs. wing pads short and rounded) (Sinitshenkova 1998); from Savina by the large head longer and wider than pronotum (vs. head small rounded, subequal in size and shape to pronotum), the wing pads broad (vs. wing pads slender), and the invisibility of notal contour between wing pads (Sinitshenkova 1987). The only well-preserved fossil stonefly exuvia is that of Electroneuria in Kachin amber, which provided the first chance to study the larval mouthparts and chaetotaxy of extinct stoneflies in a modern way. Electroneuria was attributed to the family Perlidae based on the predaceous mouthparts, long and slender palps, the presence of an occipital row of short spinules (although this character is also found in Perlodidae, see recent examples in Chen 2022b), the presence of thoracic gills, the invisibility of notal contour between wing pads, robust body, and long cerci (Sroka et al. 2018). The absence of thoracic gills (diagnostic of Perlidae) and the presence of projected abdominal tergum 10, which is an indication of a giant supra-anal process or epiproct in the male adult (reduced in Perlidae), are the most crucial characters separating Kachinoperla from Electroneuria (Zwick 2000; Sroka et al. 2018).

Apart from Electroneuria, all other stoneflies in Kachin amber were described based on adult morphology. Among these, all the male specimens of Perlidae were exclusively and certainly attributed to the subfamily Acroneuriinae Klapálek, 1914 by the presence of hammer on abdominal sternum 9, absence of projected hemiterga, and absence of hair brush on mesal areas of abdominal sterna (Sivec et al. 1988; Zwick 2000; Chen et al. 2018; Sroka et al. 2018; Chen 2019a, 2019b, 2020a, 2022c; Chen and Xu 2021; Jouault et al. 2022). Starkoperla Chen & Wang, 2020 is a perlid genus erected based on female morphology, which exhibits much longer and anteriorly located paraglossae than glossae, and the apomorphic elongation of the neck and cercal segments that can easily distinguish it from Kachinoperla (Chen and Wang 2020). Another genus of Perlidae with a female holotype is Burperla Chen, 2020, which is mainly characterized by the strikingly elongated maxillary and labial palps (longer than the head), RP vein reaching wing apex, and the broad and rounded subgenital plate with projected posterior margin (Chen 2020b). The non-elongated palps much shorter than the head exclude the close relationship between Kachinoperla and Burperla.

The two genera of Peltoperlidae, Zwickoperla Chen & Wang, 2020 and Borisoperla Chen & Xu, 2020, are characterized by the shortened head strongly inserted into the angulate prothorax (autapomorphy of Peltoperlidae) and share other similar characters with the extant peltoperlids (Chen and Xu 2020). The undescribed larvae of the two peltoperlids are expected to be cockroach-like as typical for the family (Zwick 2000) and which is apparently divergent from the regular larval habitus of Kachinoperla.

The subequal glossae and paraglossae of Kachinoperla are reminiscent of another extinct family Petroperlidae known from Kachin amber (Sroka et al. 2018; Sroka and Staniczek 2020). Petroperlidae is currently represented by four species in four genera, Petroperla mickjaggeri Sroka, Staniczek & Kondratieff, 2018, Lapisperla keithrichardsi Sroka, Staniczek & Kondratieff, 2018, Branchioperla ianstewarti Sroka & Staniczek, 2019, and Ovaloperla staniczeki Chen & Xu, 2022. The four species share the common characters distinguishable from Kachinoperla: the apical maxillary palpomere is subequal in length to previous segments but distinctly shortened in Kachinoperla; the labial palps are rather short, near half in the length of maxillary palps, whereas they are very long and about as long as maxillary palps in Kachinoperla; the apical labial palpomere is longer than previous segments (evident in the three former species but hardly visible in O. staniczeki) whereas it is shorter than previous segments in Kachinoperla; the pronotum has parallel lateral margins but it is trapezoidal in Kachinoperla (Sroka et al. 2018; Sroka and Staniczek 2020). Furthermore, the striking retention of highly-branched cervical and abdominal gills in the adult of B. ianstewarti suggests the existence of such developed gills in the larval stages of Petroperlidae, which is distinctly divergent from the state (absence of such highly-branched gills) in Kachinoperla.

Perspicuusoperlidae is another recently established extinct family from Kachin amber, which is regarded as the stem group of Euholognatha + Systellognatha (Chen 2022a). Although the maxillae and mandibles are vestigial in the female adult of Perspicuusoperlidae and are not available for comparison, the family was excluded from Systellognatha based on the unique egg structure and wing venation (Chen 2022a). On the contrary, Kachinoperla is well supported in Systellognatha based on its predaceous larval mouthparts (Zwick 1980). Moreover, the apical maxillary palpomere is distinctly shortened in Kachinoperla but remains unmodified in Perspicuusoperlidae; the labial palp is very long (about as long as maxillary palp) in Kachinoperla but it is short (length near ⅔ of maxillary palp) in Perspicuusoperlidae (Chen 2022a).

Maximum parsimony searches and Bayesian inference were conducted to investigate the systematic position of Kachinoperla (Fig. 8). The monophyly of the following extant families is supported in both analyses: Leuctridae, Capniidae, Notonemouridae, Taeniopterygidae, Nemouridae, Styloperlidae, Chloroperlidae, and Pteronarcyidae. A basal position of Scopuridae in Arctoperlaria was recovered by the external morphology, but the family has been widely supported as the sister-group of the superfamily Nemouroidea comprising Leuctridae, Capniidae, Notonemouridae, Taeniopterygidae, and Nemouridae (Zwick 2000). The monophyly of Nemouroidea is supported herein. The two extinct genera of Peltoperlidae were grouped with their extant relatives by several autapomorphies. As a highly diverse family both at present and during the Cretaceous, Perlidae was not supported as monophyletic. The Cretaceous perlids are scattered in the big clade of Systellognatha. The extinct Perspicuusoperlidae was recovered as a sister group of Systellognatha, which is in agreement with the speculation of Chen (2022a) that Perspicuusoperlidae is a stem group of Euholognatha + Systellognatha. The monophyly of another extinct family, Petroperlidae, was not supported in the analyses, but its close relationship with several extinct genera of Perlidae and the newly described Kachinoperla is suggested. Kachinoperla is recovered in a relatively basal position within Systellognatha but does not form any further terminal clades with other stoneflies known from Cretaceous Kachin amber. The main cause for several unclear relationships might be the lack of sufficient morphological characters available in these fossils.

Although larval characters of stoneflies can vary between different instars, the studied exuvia corresponds to the mature (last instar) larva, which has more stable and distinct characters than younger instars and is the research object of almost all larva-related taxonomic and phylogenetic studies in Plecoptera. During the comparison of stoneflies in Kachin amber, the main diagnostic characters include the size of mouthparts, the length of cercal segments, the shape of pronotum, and the body color. Abundant extant evidence (Figs 6–7; Stewart and Harper 1996; Stewart and Stark 2002; Jaihao and Phalaraksh 2011; Chen and Du 2018; Chen 2020c, 2022b; Chen et al. 2021) supports the tendency to retain the above-mentioned diagnostic characters from mature larva to adult stages. The body color in the character list (see Supplementary File 1) has two extreme character states (colorless or patterned) to distinguish the single entirely colorless Burperla from other taxa, which does not affect the comparison between Kachinoperla and other stoneflies. Therefore, the selected morphological characters in this study are reliable and comparable between larval and adult stages.

Based on the comparative discussions and phylogenetic analysis, Kachinoperla differs from any related extant and extinct taxa and is considered a basal group in Systellognatha according to its larval structures and other general external characters.

The hitherto five stonefly families known from Kachin amber of northern Myanmar, i.e., Perlidae, Petroperlidae, Peltoperlidae, Perspicuusoperlidae, and Kachinoperlidae, have exhibited both predaceous and phytophagous types of mouthparts, which suggests that the divergent evolution of feeding habits in stoneflies must have started earlier than the mid-Cretaceous. The various types of mouthparts and diverse feeding habits persist in extant Plecoptera, which allow different species to fill every conceivable major food niche in steams (Stewart 2009).

The coloration of Kachinoperla is composed of a bicolor head, a dark brown pronotum with pale spots, banded wing pads, banded femora of pale brown legs, dark brown abdominal segments, dark brown antennae, and cerci, which contrasts with the unpigmented larva (more likely to be an exuvia) of Electroneuria known also from Kachin amber. Such contrasting larval coloration might be non-functional, but it has apparently diverged from the common situation of aquatic insect larvae usually exhibiting subdued, cryptic coloration due to constraints imposed by the light environment of freshwater habitats (Hutchinson 1981). Coloration might be a product of sexual selection or Batesian mimicry, or even visual signals of aposematism to warm the potential predators (Foster et al. 2021). Even if Kachinoperla had aposematic larvae, it is not possible to examine the chemical compositions of defensive secretions or testing its unpalatability to predators. Such methodological difficulty in analyzing chemical defenses also exists in extant semi-aquatic insect orders including stoneflies (Newman 1991).

The dorsal ecdysial suture of Kachinoperla consists of a Y-shaped epicranial suture, and a longitudinal thoracic suture extended from the anterior of pronotum to the posterior of metanotum, which is identical to all extant stoneflies (Stewart and Stark 2002; Heckman 2008). This suggests that the emergence pathway of Plecoptera is highly conserved, i.e., the adult gradually emerges from the mature larva through the split dorsal ecdysial suture (Snodgrass 1947).

The giant larval wing pads of Kachinoperla are signals of macropterous adults in the family. All other adults reported from Kachin amber are also macropterous. The reduction or complete loss of wings is quite common in extant stoneflies but it is rarely reported from fossils. Except for the genetic factor (Nebeker and Gaufin 1967), numerous environmental factors are related to the brachypterous condition of stonefly adults, including higher altitudes (Hynes 1941, 1974; Brinck 1949), poor nutrition and low temperatures (Lillehammer 1976), long photoperiod that reduces larval growth (Khoo 1968), isolated habitats (Brinck 1949), more lentic water habitats such as lakes (Donald and Patriquin 1983), glaciation in mountains (Donald and Patriquin 1983), and other stimuli delaying the small larvae to emerge late in the season (Khoo 1964, 1968). The absence of wing reduction or loss in stoneflies from Kachin amber indicates that the situation in the ancient Burmese forest was, in principle, contrary to the above-mentioned environmental factors.

The well-developed swimming hairs on all larval legs of Kachinoperla as well as in Electroneuria are correlated in extant taxa with strong swimming ability to move upstream in order to avoid passive transportation by fast-flowing streams; such ability is variable among different taxonomic groups in Plecoptera (Hynes 1976; Otto and Sjöström 1986).

The strong and sharp larval claws of Kachinoperla are similar to those in Electroneuria (Sroka et al. 2018). Such developed larval claws could increase the area and reach for anchoring on the substratum and holding the prey during feeding (Jindal and Singh 2020), facilitating movement and clinging on rough surfaces, especially before emergence (Nelson 1991). This ability would have helped these larvae to explore different niches in the stream such as riffles, pools and swift velocity regions (Jindal and Singh 2020). The discovery of larval characters of Kachinoperla provides important evolutionary and paleoecological data for Plecoptera. Future discoveries of plecopteran immature stages will keep shedding light on the evolutionary history of stoneflies.