A taxonomic revision of the genus Stempellinella (Diptera: Chironomidae)

The species of the chironomid genus Stempellinella are revised, described, and figured, and keys to known larvae, pupae, and male and female adults are presented. Five species new to science are diagnosed and described. An emended generic diagnosis of Stempellinella is given and morphological differences from its putative sister genus Zavrelia are discussed. The partial cytochrome oxidase subunit I (COI) gene sequences (DNA barcodes) of 10 Stempellinella species are presented and the variation in genetic distances within and between species is compared. The results indicate that DNA barcodes are suitable molecular markers for identification of Stempellinella species. The following life stages and species are diagnosed and described: the larva, pupa, male, and female of S. brevis, S. chambiensis, S. ciliaris, S. edwardsi, S. fimbriata sp. n., S. flavidula, S. leptocelloides, S. saltuum, and S. truncata; the pupa, male, and female of S. reissi and S. tamaseptima; the larva, pupa, and male of Stempellinella boltoni sp. n.; the pupa and male of S. lamellata sp. n.; the male and female of S. coronata; and the male of S. apicula, S. brevilamellae, S. distincta sp. n., and S. sublettorum sp. n. Two pupal types are described but not formally named. The following changes of combination are made: Stempellina chambiensis and Stempellina truncata are transferred to the genus Stempellinella. Zavrelia inopinata is listed as a new junior synonym of Stempellinella ciliaris.


Introduction
Non-biting midges of the genus Stempellinella are small to minute (1-3 mm) dipterans with immatures living in unpolluted lotic and lentic freshwaters. The genus was erected by Brundin (1947) based on species in Edwards' (1929) ''saltuum group'' of the genus Stempellina Thienemann and Bause in Bause (1913). Since then, new species have regularly been added to the genus, either directly as species in Stempellinella (Casas and Vilches-Quero 1991;Inoue et al. 2004;Guo and Wang 2005) or as species erroneously placed in Stempellina or Tanytarsus (Freeman 1958;Webb 1969;Sasa 1980). Stempellinella cuneipennis (Edwards, 1929), originally described on an adult female, belongs to the genus Neozavrelia and was transferred to this genus by Ekrem (2006). Including the taxonomic changes and the species new to science below, Stempellinella now contains 18 formally described species world wide.
Systematically, Stempellinella is placed in the subtribe Zavreliina of the tribe Tanytarsini (Diptera, Chironomidae, Chironominae) (Ekrem and Saether 2000;Saether and Roque 2004). Brundin (1948) presented the first thorough morphological analysis of the immature stages and grouped Stempellinella with Zavrelia Kieffer and Bause in Bause, 1913 in the Zavrelia-group, morphologically distinct from the Stempellina-group in all life stages. This is also the preliminary conclusion of more recent phylogenetic analyses based on morphological and molecular data (T. Ekrem, in preparation).
The genus has been recorded from all continents except the Antarctic, with most species found in the Holarctic region. The larvae and pupae live in springs, brooks, rivers, and lakes where the larvae construct small, straight, transportable cases of sand, silt, and detritus. While some species appear to be generalists with respect to habitat choice, others have been found only in certain environments, and seem to have specific living requirements. Thus, several Stempellinella species might work well as biological indicators in freshwater biomonitoring.
The main aim of this study was to revise and describe species in the genus Stempellinella with morphometrics and figures, and to present identification keys to the four major life stages of as many species as possible. Although a stereo microscope usually is necessary to spot Stempellinella larvae in benthos samples, they are relatively easy to rear in the laboratory if caught with cases at 4th instar. Thus, the immature stages of some species could be identified and described through individual rearings. Life stages of other species were associated by their partial COI gene sequences (Table I). The use of short DNA sequences to discriminate between and to identify species (DNA barcoding) has been suggested as an effective tool in biodiversity studies (e.g. Hebert et al. 2003) and also found to be functional for identification of all stages of Chironomidae (Ekrem et al. 2007). As additional tools for identification, 24 DNA barcodes of 10 Stempellinella species are here presented (Table I). These sequences and the accompanying specimen data are available through the Barcode of Life Data Systems (BOLD, www.barcodinglife. org).

Material and methods
Field work was conducted in Canada, Germany, Norway, Luxembourg, and South Africa to supplement material on loan from collections and colleagues in Europe, North America, South America, Africa, and Asia. Live benthos and drift samples were usually brought back to the laboratory where the small larvae (in their cases) were sorted out and isolated under a stereo microscope. Single larvae were kept in small 262 cm boxes together with water and some sediment for rearing purposes. The emerged adults were fixed with 96% ethyl alcohol together with their cast larval and pupal exuviae. The larval exuviae usually remained in the larval cases after pupal emergence. Alcohol-preserved specimens were dissected, the bodies cleared in 8% KOH or in the case of DNA extraction, in a buffered solution with the enzyme protease-K (Qiagen DNeasy tissue extraction kit), and mounted in Euparal. DNA extraction, PCR amplification and sequencing were carried out as described by Ekrem and Willassen (2004), but the primers LCO1490 (59-  and HCO2198 (59-  were used for amplification of partial cytochrome oxidase subunit I (COI) gene sequences. PCR used annealing  at least reaching antennal segment 2, usually apex of distal Lauterborn organ or beyond. Lauterborn organs large, bulbous, placed alternately on antennal segment 2. Proximal Lauterborn organ on short pedicel near base of segment 3, distal organ on short to moderately long pedicel apically on segment 2. Labral seta SI pectinate, bases fused; SII plumose on tall pedestal; chaetae pectinate to plumose; SIII simple, SIV present. Labral lamella well-developed comb. Pecten epipharyngis consisting of three long, slender, triangular scales. Premandible with two to four teeth, well-developed brush. Premandibular teeth only slightly inclined relative to main axis of premandible. Mandible with pale dorsal tooth; apical and three inner teeth brown. Seta subdentalis long, curved, usually reaching well beyond apical mandibular tooth. Seta interna consisting of four strongly plumose branches. Pecten mandibularis with well-developed lamellae, outer lamella stronger than rest. Mentum with rounded median tooth; six pairs of lateral teeth equal or subequal in size laterad; first pair often more or less fused with median tooth. Ventromental plates fan-shaped with obvious striation, medially at most reaching lateral margin of second lateral tooth. Mentum slightly wider than ventromental plate. Seta submenti placed medially to posteromedian corner of ventromental plate. Postoccipital plate little to moderately developed, continuous or split.
Body: anterior parapods with numerous simple spines; posterior parapods with [12][13][14][15][16][17][18] simple claws. Two pairs of conical anal tubules present. Supraanal seta strong and long. Procercus about as long as wide with two short and four long anal setae, short setae set individually and separate from the common base of the long setae.

Comments
The genus morphologically most similar to Stempellinella is probably Zavrelia. However, there are a few characters which can be used to separate the genera in most life stages (Table II). The larvae and pupae of Stempellinella are particularly similar to those of Zavrelia, and combinations of characters are needed to separate Stempellinella and Zavrelia species in these life stages.

Diagnostic characters
Stempellinella apicula can be separated from other species in Stempellinella by the following combination of characters. Adult male with broadly triangular frontal tubercles; AR ca 0.6; superior volsella somewhat rectangular with apex drawn into an anteromedially directed point; anal point long with spinulae and microtrichia between well-developed crests; gonostylus with obvious posteromedially pointed apex.
Legs ( Figure 1D): fore tibia with 12 mm long spur; mid and hind tibiae with wellseparated, [12][13] mm long tibial combs, one mid tibial comb with 35 mm long spur, one hind tibial comb with 32 mm long spur; mid tarsus 1 without sensilla chaetica; pulvilli absent. Lengths and ratios of leg segments in Table III. Hypopygium (Figure 1E, F): anal tergite 95 mm long with transverse anal tergite band; three strong median setae at some distance from anal point base, one lateral seta, 22 apical setae; anal point 43 mm long, basally 7 mm broad with long, well-developed crests which end close to anal point apex; 10 small spinulae and microtrichia between anal crests; microtrichia-free area on each side of anal point base. Gonocoxite 80 mm long; gonostylus 60 mm long; HR 1.33. Superior volsella (Figure 1F) somewhat rectangular with apex drawn into an anteromedially directed point, four dorsal and two median setae on setiger, superior volsella otherwise bare; digitus absent; median volsella 30 mm long, straight, posteromedially directed, stem simple with numerous simple ca 20 mm long lamellae which reach two-thirds length of inferior volsella; inferior volsella 60 mm long, slightly club-shaped, with several distal setae, dorsal surface without microtrichia.

Etymology
The species is named after my good colleague Mike Bolton who made the type material available for this study.

Diagnostic characters
Stempellinella boltoni can be separated from other Stempellinella species by the following combination of characters: adult male with AR 0. 8-0.95; anal point long, triangular, with [11][12][13][14][15][16] strong double spinulae between well-developed anal crests; strong median setae on anal tergite; lateral anal tergite setae absent; setiger of superior volsella strongly angled, narrow basally, width shorter than horizontal distance between superior and median volsellae; median volsella 45 mm long with microtrichose, palmate stem. Pupa with conical cephalic tubercles and moderately wrinkled frontal apotome; thorax completely brown pigmented, slightly granulated and wrinkled anterodorsally; thoracic horn comparatively short and broad (ca 280 mm long) with strong chaetae along dorsal margin; median antepronotal not on tubercle; one lateral seta on abdominal segment III and IV taeniate, anterolateral patches of shagreen on tergites VIII-IX only; pleura apparently without shagreen. Larva with 35 mm long, slightly curved spur apically placed on antennal pedestal; AR ca 0.9; premandible with three teeth; S3 split in two long and several small branches.
Colour: head and thorax brown with somewhat darker scutal stripes, eyes dark brown, abdomen and legs pale yellow-brown.
Wing: not measurable since all males were pharate.
Larva (n55, unless otherwise stated). Total length ca 2-2.5 mm, house ca 3 mm. Head capsule yellow, brown postoccipital rim and teeth on mandible and mentum. Live individuals not examined.
Body: anterior parapods with long, simple spines; hind parapods with 15 simple hooks; L2 apparently simple; anal segment with four 60 mm long anal tubules; supraanal seta strong, 245 mm long; procercus with two short (ca 140 mm long) and four long (ca 550 mm long) anal setae, the short setae situated individually, and not on the common base of the long setae.

Remarks
Stempellinella boltoni is very similar to S. fimbriata and S. tamaseptima in the adult male, but can be separated from the former by the more broadly triangular anal point and the broader, blunt apex of the superior volsella, and from the latter by the longer distance between superior and median volsellae (longer than width of setiger base in S. boltoni). The three species can also be separated by their COI gene sequences (Table IV).
The species has been recorded from two localities in Ohio, USA. The larvae build transportable cases of comparatively coarse sand and detritus.

Diagnostic characters
Stempellinella brevilamellae can be separated from other species in Stempellinella by the following combination of characters. Adult male with well-developed, conical frontal tubercles; AR ca 0.8; superior volsella somewhat rectangular with medially pointed apex; anal point long with spinulae and microtrichia between well-developed crests; median volsella 50-55 mm long, branched, covered with microtrichia.

Remarks
Stempellinella brevilamellae has been recorded only from its type locality in China (Guo and Wang 2005). The immature stages and their habitat remain unknown, but the larvae probably construct small, straight transportable cases of sand and detritus like the morphologically similar species in South Africa and Japan.

Diagnostic characters
Stempellinella brevis can be separated from other Stempellinella species by the following combination of characters. Adult male with wing length ca 1.3 mm, 3.2 times longer than broad; AR about 0.8; frontal tubercles large, conical; anal point with several (4)(5)(6)(7)(8)(9)(10) small spinulae between u-shaped crests; few (4-7) median tergite setae of which a few small sometimes are placed close to anal point; median volsella without microtrichiae, with fan of simple lamellae. Adult female with AR 0. 26-0.32; vaginal floor large, covering more than half of vagina ventrally; spermathecal ducts longer than notum and rami combined; rami as long as notum; diameter of seminal capsules lower than length of notum; coxosternapodeme without anterolateral lobe. Pupa with moderately developed, conical cephalic tubercles; thoracic horn long and thin with numerous small chaetae scattered on middle one-third; posterior precorneal somewhat shorter than anterior and posterior precorneals; thorax with scattered sculpturing anteriorly; anterior dorsocentrals of same length as posterior dorsocentrals; point patches on tergite II small, starting at level of seta D5; anal lobe with [15][16] taeniae. Larva with moderate (ca 24 mm long), digitiform spur on antennal pedestal; AR 0. 77-0.87; S3 split in three or four branches; distal Lauterborn organ pedicel twice as long as basal Lauterborn organ pedicel.

Remarks
A lectotype is designated to stabilize the species' nomenclature for the future.
Stempellina ciliaris Goetghebuer, 1944 was regarded as a junior synonym of S. brevis by Brundin (1949), but examination of the type material of both species revealed that these are morphologically separable.
Stempellinella brevis is widespread in the West Palaearctic region above), and the specimens labelled S. brevis from North America that I have seen were all misidentified specimens of S. fimbriata sp. n. described below. Thus, the record in Fauna Europaea for the Nearctic region is doubtful ). The species is very similar to S. saltuum in the adult male, but can be separated by the smaller wing length to wing width ratio, lower AR, and fewer median tergite setae. Stempellinella brevis is most similar to S. flavidula in the pupal and larval stages, but can be separated from this species by smaller point patches on tergite III, continuous point patches on TVI, and the absence of shagreen on pleurae VI and VII in the pupa; longer antennal pedestal spur and longer apical Lauterborn organ pedicel in the larva.
The larvae of S. brevis build small, straight transportable cases of detritus, and have been found in brooks, streams, rivers, and lakes. Brundin (1949) found larvae of S. brevis from shore to a depth of 18 m in the oligohumic Lake Innaren and characterized the species as eurybath in subarctic lakes. Dittmar (1955) recorded the species in a stream in Sauerland, and I have found the species in both low-and high-altitude streams, brooks, and rivers in southern Norway.

Diagnostic characters
Stempellinella chambiensis can be separated from other Stempellinella species by the following combination of characters: male imago with AR ca 0.5; anal point long, thin, with comparatively low crests; base of anal point with two to four median tergite setae and numerous groups of three or four long microtrichia; lateral anal tergite seta present; median volsella medially directed, without microtrichia, with short, anally curved lamellae. Female imago with AR 0. 25-0.30; VR 1.35-1.64; well-developed vaginal floor, covering ca onethird of vagina ventrally, with setae; cerci shorter than tergite IX; spermathecal ducts as long as notum (excluding rami). Pupa with strongly granulose frontal apotome and thorax; thoracic horn with numerous small but strong chaetae on distal two-thirds; hook row with ca 25 teeth; few but long points in patches on abdominal tergites III-VI; segment VIII with two (or sometimes three) lateral taeniate setae. Larva with large spur on antennal pedestal; AR lower than 0.8; antennal segment 5 as long as antennal segment 4; antennal blade extending far beyond apex of antennal segment 5; Lauterborn organs large with minute pedicels; premandible with three teeth; S3 split in three branches.
Colour: cleared specimens pale brown with somewhat darker scutal stripes and postnotum, eyes red with darker ventral margin.

Remarks
As Freeman (1958, p 353) noted, several of the syntypes belonging to Thienemanniella chambiensis and T. trivittata must have been wrongly labelled at some point. I largely agree with Freeman's (1956Freeman's ( , 1958 decision in designing and sorting the true types, with the exception that I will not regard the female, but the male from Kabasha as paralectotype. The female specimen is not included in Goetghebuer's (1935, p 52) original description, but the male is and should be regarded as belonging to the original type series. I have also examined the two additional specimens listed by Freeman, one male from Amadi, Sudan and one male from Nelspruit in Transvaal. Neither of these is conspecific with Stempellinella chambiensis, and both fit the diagnosis of Stempellina given in Cranston et al. (1989).
Males of Stempellinella chambiensis are quite similar to the males of the Palaearctic S. edwardsi, the Japanese S. coronata, and the Neotropic S. lamellata, but can be separated from all these species by the above combination of diagnostic characters. The pupal exuviae are perhaps most similar to those of S. leptocelloides, but can be separated from these by the more granulose frontal apotome and thorax, and the shorter frontal tubercles. The species has so far been recorded from Africa, Southeast Asia, Australia, and South America typically in and along slow-flowing rivers and streams with a pH of around 7. The Tri Ping Fung area (China) was at the time of sampling a small primary forest on limestone formations. The river water was clear and the river bank was lined with large boulders and coarse gravel (E. J. Fittkau, personal communication). Stempellinella chambiensis larvae were quite numerous in sandy and stony parts of the Rio Bento Gomes (Stempellinella in Stur 2000), and occurred at the spring as well as in the fourthorder stream. The Rio Bento Gomes shows large seasonal fluctuations in the water level, and S. chambiensis appeared to be most numerous in periods of reduced water flow at the end of August (Stur 2000). The two Australian localities where S. chambiensis has been sampled are quite different. The stream in Yabba Creek north of Brisbane is small and shaded, while Walsh River east of Dimbulah lays in a more tropical, dry area and has less vegetation lining its banks (M. Baehr, personal communication). The Fazzari stream in São Paulo State, Brazil is a first-order stream surrounded by a well-preserved riparian forest. The water has a high level of dissolved oxygen, low conductivity, and temperatures ranging from 15 to 21uC, while the stream bed is characterized by a predominance of organic material in the bottom substrate (F. Roque, personal communication). The larvae of S. chambiensis build small transportable cases of sand grains and organic particles as in the remainder of the species in the genus.
Compared with the other species in Stempellinella, S. chambiensis has an extraordinarily wide distribution. Thus, it is tempting to question the conspecificity of the sampled populations. However, I have not been able to observe any morphological differences between the sampled specimens large enough to be diagnostic, and therefore choose to treat the examined specimens as one species until observed genetic variation between the geographically separate populations proves otherwise. ( E7, 4 May 1999, 1 June 1999, 15 June 1999, 29 June 1999, 13 July 1999, 27 July 1999 August 1999, I. Schrankel; 1 LP(") (VM, To169) Schlennerbach, emergence, 12 June 2005, P. Martin.

Diagnostic characters
Stempellinella ciliaris can be separated from other species in Stempellinella by the following combination of characters. Adult male with AR ca 0.80; superior volsella with medially pointed apex and two median setae; anal point long and slightly triangular with long, strong crests which end far distal on the anal point, several long spinulae between crests; lamellae of median volsella almost reaching apex of inferior volsellae. Adult female with large seminal capsules, diameter as large or larger than length of notum excluding rami and considerably larger than length of cercus; small vaginal floor, covering ca one-quarter of vaginal opening ventrally; gonocoxapodeme strongly curved. Pupa with low, broad conical cephalic tubercles; chaetae distributed evenly on distal two-thirds of thoracic horn; shagreen on pleura IV; anterior patches or transverse band of points on tergite VII. Larva with moderate (ca 20 mm long), digitiform spur on antennal pedestal; antennal segment 3 inserted subapically on segment 2; antennal blade reaching beyond apex of distal Lauterborn organ; AR 0. 87-0.97, 0.91; AAR about 1.20; S3 split in three branches; distal Lauterborn organ on longer pedicel.
Body: anterior parapods with long, simple spines; hind parapods with [14][15][16][17][18]16 simple hooks; L2 apparently simple; anal segment with four narrow anal tubules, 58 mm long; supraanal seta strong, ca 235 mm long; procercus ca 24 mm long with two short (ca 150 mm long) and four long (ca 575 mm long) anal setae, the short setae situated individually, and not on the common base of the long setae.

Remarks
The type material was collected in Meiergraben near Lunz by F. Gouin and A. Thienemann 1941A. Thienemann -1943, and Thienemann's field notes (in ZSM) indicate that they found numerous ''Zavrelia-type'' larvae which were reared. At least one male and one female from this rearing were sent to Goetghebuer for identification. A male and a female on one pin with the labels ''Stempellina ciliaris n. sp.'' and ''Meiergraben (Autr 1943 D r Thienemann)'' in Goetghebuer's handwriting, but without a type label, were found in the Goetghebuer collection in RBINS. These two specimens fit the original description of S. ciliaris (Goetghebuer 1944), except that the male antenna is lost, the female antenna has five flagellomeres and the label bears the name of Thienemann instead of Gouin. However, the male antenna could have been lost after description, and the female antenna has a long first flagellomere with two circles of setae and the paralectotype first antennal flagellomere has both median and distal chaetae. Thus, the flagellum probably has erroneously been interpreted as six-segmented (see Goetghebuer 1944, Figure 2b). Thienemann and Gouin worked closely together in Lunz (A. Thienemann, field notes; E. J. Fittkau, personal communication) and it is reasonable to believe that Goetghebuer got material collected by both Thienemann and Gouin for identification. The two individuals were most certainly examined by Goetghebuer before the description of S. ciliaris and are considered to be part of the type material despite the small discrepancies with the original description. The male is best preserved and is here designated lectotype to stabilize nomenclature. There are three slides with four males, one female, four pupae, nine pupal exuviae, and one larva, and an alcohol vial with two males, one female, three pupal exuviae, and one larva in ZSM which bear the sample number and dates from Thienemann's and Gouin's notes from Meiergraben. Six of the pupal exuviae are conspecific with S. ciliaris, while six are conspecific with S. flavidula. The pupal exuviae conspecific with S. ciliaris are thus not regarded as part of the type series, since they cannot be directly linked to the male and female types in the Goetghebuer collection.
Stempellinella ciliaris was listed as a synonym of S. brevis by Brundin (1949), and also treated as such by Thienemann (1950), but examination of the type material of both species revealed that these are morphologically distinct.
Zavrelia inopinata was described as larva from two localities in Romania (Botnariuc and Cindea-Cure 1954). The type material has not been located and is probably lost (V. Tatole, personal communication). However, the original description is very detailed and well figured, and it is clear to me that the species must be a junior synonym of S. ciliaris. The description of the type localities also fits the localities in which S. ciliaris is found (springs and spring brooks).
Stempellinella ciliaris has been recorded from Austria, France, Germany, Luxembourg, and Romania, but previous records of S. flavidula or S. brevis could actually be S. ciliaris, as these species are quite similar in the adult and immature stages. The abovedescribed species can be separated from S. flavidula by longer spines on the anal point and a longer median volsella in the adult male; a smaller vaginal floor and shorter seminal ducts in the adult female; smaller cephalic tubercles, more chaetae on the thoracic horn and presence of anterior point patches on tergite VII in the pupa; and a higher AAR and longer antennal blade in the larvae. The observed differences in the adult female and larva are small and might not be diagnostic as more material is examined.
The larvae of S. ciliaris build small, straight, transportable cases of sand and detritus, and are typically found in calcareous springs and spring brooks. The pupae of this species have been observed to use the posterolateral spur of the abdominal segment VIII to aid splitting of the thorax at adult emergence (Ekrem 2005). The species is a recorded host for larvae of the water mite Atracides fonticolus (K. Viets) in a calcareous spring in Luxembourg (Stur et al. 2005

Diagnostic characters
Stempellinella coronata can be separated from other Stempellinella species by the following combination of characters: male imago with AR ca 0. 5-0.6; anal point long, thin, with comparatively very weak crests; small median anal tergite setae sometimes present close to anal point base; anal point base with numerous groups of three or four strong microtrichia; lateral anal tergite seta absent; median volsella posteriorly curved, without microtrichia, with fan of simple, anally directed lamellae (Inoue et al. 2004, Figure  5B). Female imago with AR 0. 25-0.37; VR 1.58-1.70; well-developed vaginal floor, covering ca one-half of vaginal opening ventrally, with setae; seminal capsules small (diameter ca half of notum length); spermathecal ducts longer than notum excluding rami.

Redescription
Adult male is thoroughly described by Inoue et al. (2004), and only a few additional observations are added here: hypopygium ( Figure 11A) without lateral seta on anal tergite, with two median setae and ca 16 apical setae including lateral setae on anal point.

Remarks
Females were associated with males by 98.7-100% similarity in 666 base pairs of the mitochondrial COI gene.
Stempellinella coronata is most similar to the Afrotropical S. chambiensis and the Palaearctic S. edwardsi (see Remarks under S. chambiensis). The species has been recorded so far only from Japan where it inhabits clear, unpolluted pools in the upper to mid reaches of the Ohta River system (Inoue et al. 2004). Although immature stages are unknown, the larvae are likely to build small transportable cases of sand grains and organic particles like the remainder of the species in the genus.

Diagnostic characters
Stempellinella distincta can be separated from other Stempellinella species by the following combination of characters: adult male with AR 0. 7-0.9; anal point with numerous strong spinulae between well-developed anal crests; strong median setae on anal tergite; lateral anal tergite setae present; setiger of superior volsella long, narrow, strongly curved medially; median volsella 40-50 mm long with microtrichose, palmate stem.
Colour: as male.
Legs: as male. Genitalia ( Figure 13I): tergite IX slightly triangular, about 45 mm long; sternite VIII with 18 setae, of which two to four are placed on vaginal floor; vaginal floor large, covering one- half of vaginal opening ventrally; gonapophysis VIII single lobe with long posteromedially directed microtrichia; gonocoxite IX with one seta; gonocoxapodeme slightly curved; coxosternapodeme well developed with obvious anterior and posterior lobes. Notum including rami 129 mm long, notum alone ca 60 mm long. Seminal capsules ovoid, diameter 48 mm with 165 mm long spermathecal ducts. Postgenital plate triangular. Cercus 39 mm long.
Body: anterior parapods with long, simple spines; hind parapods with 14 simple hooks; L2 apparently simple; anal segment with four narrow anal tubules; supraanal seta strong, ca 225 mm long; procercus ca 30 mm long with two short (ca 150 mm long) and four long (ca 550 mm long) anal setae, the short setae situated individually, and not on the common base of the long setae.

Remarks
Lectotype designation here to promote future nomenclatural stability.
The larvae of Stempellinella edwardsi, early instars in particular, can be difficult or impossible to separate from those of S. leptocelloides, and the characters used in the key below overlapped in material of S. edwardsi and S. leptocelloides from Canada and Norway examined by G. A. Halvorsen (personal communication).
Stempellinella edwardsi is a common species in clean lakes, dams, and slow-flowing parts of rivers and streams in Europe. The species has been recorded also in the East Palaearctic and Nearctic regions (Makarchenko et al. 2005; Saether and Spies 2004; above). The larvae and pupae of S. edwardsi live in sand and silt where they build their small houses of fine sand and silt particles. Typically the building material is much finer that that used by the other European members of this genus.
Colour: head and thorax brown with somewhat darker scutal stripes, eyes dark brown, abdomen and legs pale yellow-brown.
Cephalothorax ( Figure 16A, B): cephalic tubercle well developed, conical, 25-40, 35 mm long; frontal setae taeniate, 160-225, 188 mm long; pedicel sheath tubercle absent. Thoracic horn , 322 mm long with some 5 mm long chaetae distributed dorsally on distal half, most in middle; precorneals taeniate, arranged in slight triangular pattern, the two anteriormost setae situated closer to each other than to the third, anterior and posterior precorneals ca 230 mm long, longer than median precorneal (165-175, 171 mm long); one taeniate median antepronotal ca 240 mm long on low tubercle, two lateral antepronotals (one sensillum basiconicum); two pairs of fine dorsocentrals, setae of each pair equally strong, 25-40, 35 mm long. Extensive fields of granulation and fine sculpturing present on thorax, a few stronger granules present along median suture line. Prealar tubercle well developed, wide; nose of wing sheath strong.
Larva (n55, unless otherwise stated). Total length ca 1.9 mm, house ca 2.3 mm. Head capsule yellow, brown postoccipital rim and teeth on mandible and mentum. Live individuals not examined.
Body: anterior parapods with long, simple spines; hind parapods with 14-18 simple hooks; L2 apparently simple; anal segment without or with very short anal tubules; supraanal seta strong, 245 (n53) mm long; procercus with two short (ca 130 mm long) and four long (ca 525 mm long) anal setae, the short setae situated individually, and not on the common base of the long setae.

Remarks
Stempellinella fimbriata is very similar to S. tamaseptima in the adult male, but can be separated by the differently shaped superior volsella, and the longer distance between superior and median volsellae (longer than width of setiger base in S. fimbriata). The pupae of S. fimbriata show some variation in the extension of the point patches on the abdominal tergites II-VI which can be more extensive than seen in Figure 16C.
The species has been recorded from many streams and small rivers throughout North America (see above) and also from the sublittoral and profundal zones in Canadian lakes (G. A. Halvorsen, personal communication (Edwards). Goetghebuer (1937-54) key.

Diagnostic characters
Stempellinella flavidula can be separated from other species in Stempellinella by the following combination of characters. Adult male with medially pointed superior volsella apex; anal point long and slightly triangular with long crests which end far distal on the anal point, several short spinulae between crests; lamellae of median volsella reaching two-thirds length of inferior volsella; obvious microtrichose wart basally on inferior volsella. Adult female with large seminal capsules, diameter as large as or larger than length of notum excluding rami and considerably larger than length of cercus; spermathecal duct considerably longer than notum including rami; vaginal floor large, covering one-third to one-half of vaginal opening ventrally; gonocoxapodeme almost straight. Pupa with large, broad conical cephalic tubercles; strong chaetae dorsally on thoracic horn mid section; shagreen on pleurae III-VII; point patches on tergite VI transversely divided; anterolateral patches of shagreen on tergite VII. Larva with moderate (ca 18 mm long), digitiform spur on antennal pedestal; antennal blade reaching apex of distal Lauterborn organ; AR ca 0. 8-1.0; S3 split in three or four branches; distal Lauterborn organ pedicel about twice as long as proximal Lauterborn organ pedicel; premandible with two teeth.
Larva (n55, unless otherwise stated). Total length ca 2.5 mm, case ca 3 mm. Head capsule brown, somewhat darker postoccipital rim and teeth on mandible and mentum. Live individuals not examined.
Body: anterior parapods with long, simple spines; hind parapods with [14][15][16]15 simple hooks; L2 simple; anal segment with four narrow anal tubules, 58 mm long; supraanal seta strong, ca 230 mm long; procercus ca 30 mm long with two short (ca 130 mm long) and four long (ca 500 mm long) anal setae, the short setae situated individually, and not on the common base of the long setae.

Remarks
A lectotype is here designated to promote future nomenclatural stability.
Stempellinella flavidula has been recorded from Austria, the British Isles, Corsica, Germany, Ireland, Luxembourg, Poland, Slovakia, and Switzerland above), but previous records of the species might have been of S. ciliaris as this species is quite similar in the adult and immature stages. The larvae of S. flavidula build small, straight transportable cases of sand and detritus, and are typically found in calcareous springs, spring brooks, and small streams. The species has been found to be host to several water mite species in springs in Luxembourg (Stur et al. 2005

Etymology
The species name is an adjectival form of the Latin noun ''lamella'', meaning a small plate, and refers to the exceptionally long lamellae of the median volsella.

Diagnostic characters
Stempellinella lamellata can be separated from other Stempellinella species by the following combination of characters: male imago with AR ca 0.5; anal point long, thin, without crests; base of anal point with two median tergite setae and numerous groups of strong microtrichia; lateral anal tergite seta present; median volsella medially directed, without microtrichia, with long, anally curved lamellae which reach past apex of inferior volsella. Pupa with strongly granulose frontal apotome and thorax; thoracic horn with numerous small chaetae on distal two-thirds; hook row with ca 40 teeth; long points in patches on abdominal tergites III-VI, point patches on tergites IV-VI p-shaped; segment VIII with three lateral taeniae.

Remarks
Stempellinella lamellata is quite similar to the Palaearctic S. edwardsi, the Japanese S. coronata, and S. chambiensis in the southern hemisphere, but can be separated from all these species by the above combination of diagnostic characters. The description of the pupa is based on tentatively associated pupal exuviae from Nova Friburgo, and should be treated with caution. However, their close resemblance to the pupae of S. chambiensis, only differing in the number of lateral taeniae on segment VIII, the width and the number of teeth in the hook row in segment II, and the slightly larger cephalic tubercles supports the association since the males of S. chambiensis and S. lamellata are very similar morphologically. The species has so far only been recorded from streams in Brazil and Bolivia. Although the larvae still remain unknown, they probably build small transportable cases of sand grains and organic particles as in the remainder of the species in the genus, and most probably have morphological characters similar to those of S. chambiensis (e.g. premandible with three teeth). (  Stempellinella leptocelloides (Webb) (Poole and Gentili 1997). Giłka (2005) taxonomic placement. Stempellinella cf. leptocelloides. Epler (2001) identification key to larva.

Diagnostic characters
Stempellinella leptocelloides can be separated from other Stempellinella species by the following combination of characters: adult male with AR 0. 8-1.0; anal point widely triangular with numerous strong orally directed spinulae between well-developed anal crests; small median tergite setae at anal point base; lateral anal tergite setae present; anal tergite band weakly Tshaped; setiger of superior volsella oval with medially pointed apex; median volsella thin, 30 mm long, stem simple, bare, lamellae simple. Adult female with AR 0.29; none to two setae on well-developed vaginal floor; spermathecal ducts longer than notum and rami combined; rami slightly shorter than notum; diameter of seminal capsules shorter than notum. Pupa with long, almost tubular cephalic tubercles and moderately wrinkled frontal apotome; thorax completely brown pigmented and extensively granulated, median antepronotal on well-developed tubercle; one lateral seta on abdominal segment III and IV taeniate; point patches on tergites IV-V usually separated both longitudinally and horizontally, giving the impression of four point patches; anterolateral patches of shagreen on tergites VII-IX. Larva with 18-25 mm long, triangular spur on antennal pedestal, AR 0. 7-0.9, antennal segment 2 long with proximal Lauterborn organ pedicel at one-sixth length; premandible with three teeth; S3 long, simple.
Larva (n55, unless otherwise stated). Total length ca 2.6 mm, larval case ca 2.7 mm. Head capsule yellow, brown postoccipital rim and teeth on mandible and mentum. Live individuals not examined.
Body: anterior parapods with long, simple spines; hind parapods with 14-16 simple hooks; L2 apparently simple; anal segment with anal tubules 50 mm long; supraanal seta strong, 225 mm long; procercus 25 mm long with two short (ca 130 mm long) and four long (ca 550 mm long) anal setae, the short setae situated individually, and not on the common base of the long setae.

Remarks
The holotype, allotype, and seven paratypes (3"", 4RR) have been mounted on microscope slides. Unfortunately, the type material was partly destroyed in the mail upon return to INHS. The holotype, one female paratype, and one male paratype were missing from the damaged package when it arrived in Illinois (C. Favret, personal communication). However, since there is no doubt of the species identity and all paratypes are conspecific, a neotype is not designated at this point.
Stempellinella leptocelloides has a characteristic anal point, and adult males can easily be distinguished from all other Stempellinella by this feature. Female adults, pupae and larvae are most similar to species in the edwardsi species group, but can be separated from most of these by the diagnostic characters given above. The larvae of Stempellinella edwardsi, in particular early instars, can be difficult or impossible to separate from those of S. leptocelloides and the characters used in the key below overlapped in material of S. edwardsi and S. leptocelloides from Canada and Norway examined by G. A. Halvorsen (personal communication).
Stempellinella leptocelloides has been recorded from many streams, small rivers and lakes throughout North America (see above), and is probably widely spread in the Nearctic Region. The larvae build straight, transportable cases of fine, pale silt specked with a few dark silt particles.

Diagnostic characters
Stempellinella reissi can be separated from other Stempellinella species by the following combination of characters: adult male with AR about 0.7; frontal tubercles large, conical; anal point with several (11)(12) small spinulae between v-shaped anal point crests; four or five strong median tergite setae placed far from anal point; superior volsella strongly bent medially, square distally; median volsella short, stout, without microtrichiae, with fan of simple and pectinate lamellae. Adult female with AR 0.31; vaginal floor large, concave, covering about half of vaginal opening in ventral view; spermathecal ducts longer than notum and rami combined; seminal capsules as wide as length of notum; rami and notum equally long, combined shorter than 100 mm; more than 30 setae on sternite VIII, no setae on vaginal floor. Pupa with well-developed, conical cephalic tubercles; frontal apotome with some large granulation; thoracic horn long and thin with numerous small chaetae scattered on most of horn; anterior precorneal somewhat shorter than median and posterior precorneals; thorax quite smooth; dorsocentrals short, about 30 mm; anal lobe with 10-14 taeniae.
Colour: head pale brown with dark pedicels and eyes; thorax with pale brown ground colour, dark brown scutal stripes, brown postnotum, preepisternum, and median anepisternum, scutellum pale, halteres pale; abdomen and legs pale brown.

Remarks
Stempellinella reissi has been recorded from the Sierra Nevada in Spain and at the river Tech in the eastern Pyrenees, France, and probably has a restricted distribution to high elevation areas in central and southern Europe (see Casas and Vilches-Quero 1991 for additional information on the type locality). The males of this species can easily be separated from all other Stempellinella species by its characteristic median volsella which is quite similar to the median volsella seen in the genus Zavrelia. Bare eyes and a transverse median tergite band are, however, characteristics for species in Stempellinella. The pupa of S. reissi keys to the genus Zavrelia in the key to the Chironominae of the Holarctic region (Pinder and Reiss 1986), but can be separated from all known Zavrelia species by the coarsely granulose frontal apotome and only scattered shagreen on the pleurae (pleurae in Zavrelia have dense shagreenation with points in rows). ( (Goetghebuer, 1921). Goetghebuer (1937-54) description, key.

Diagnostic characters
Stempellinella saltuum can be separated from other Stempellinella species by the following combination of characters: adult male with wing length ca 1.5 mm, 3.6 times longer than broad; AR about 1.3; frontal tubercles large, conical; anal point with several (7)(8)(9)(10)(11)(12)(13)(14) small spinulae between v-shaped crests; numerous (11)(12)(13)(14)(15)(16) median tergite setae of which a few small setae are usually placed close to anal point; superior volsella oval with posteromedially directed apex; median volsella without microtrichiae, with fan of simple lamellae. Adult female with AR 0.27; vaginal floor small, concave following lateral margins of vagina; spermathecal ducts almost as long as notum and rami combined; rami as long or longer than notum; diameter of seminal capsules lower than length of notum. Pupa with welldeveloped, broadly conical cephalic tubercles; thoracic horn long and thin with numerous small chaetae scattered on distal two-thirds; median precorneal shorter than anterior and posterior precorneals; thorax with scattered sculpturing anteriorly; anterior dorsocentrals usually longer than 50 mm, longer than anterior dorsocentrals; anal lobe with 18-25 taeniae in anal fringe. Larva with moderate (ca 24 mm long), digitiform spur on antennal pedestal; AR 1.19-1.48; S3 split in three or four branches; both Lauterborn organs on short pedicels.
Larva (n54, unless otherwise stated). Total length not measurable, larval case ca 3 mm long. Head capsule brown, somewhat darker postoccipital rim, teeth on mandible and mentum. Live specimens not examined.
Body: anterior parapods with long, simple spines; hind parapods with 16 simple hooks; L2 apparently simple; anal tubules not visible in larval exuviae; supraanal seta strong, 315 mm long; procercus ca 30 mm long with two short (ca 200 mm long) and four long (ca 600 mm long) anal setae, the short setae situated individually, and not on the common base of the long setae.

Remarks
A lectotype is here designated for future stabilization of taxonomy. Although the measurements and ratios of the male in the original description are written in singular, I regard it as very likely that Goetghebuer examined all the specimens with the identical locality labels ''Meirelbeke, 6.5.20'' when describing the species. Which exact specimen was used for the original description is not certain, thus all the four male adults in RBINS are considered part of the type material.
Stempellinella saltuum has been recorded from Austria, Belarus, Belgium, Germany, Poland, Romania, and Sweden , but I have only seen material from Belarus and Belgium, and cannot confirm the other records in Fauna Europaea. The species is very similar to S. brevis in the adult male, but can be separated by the v-shaped anal point crests and the numerous median tergite setae. The larva and pupa are apparently most similar to those of S. ciliaris, but S. saltuum can be separated from this species by the longer thoracic horn, longer anterior dorsocentrals and more taeniate setae in the anal fringe in the pupa, and by a higher AR and longer distal Lauterborn organ pedicel in the larva.
The larvae of S. saltuum build small, straight transportable cases of detritus, and are found in floodplain streams and brooklets.

Etymology
The species is named in honour of Mary and Jim Sublette for their great contributions to Chironomidae research.

Diagnostic characters
Stempellinella sublettorum can be separated from other Stempellinella species by the following combination of characters: adult male with AR ca 0.9; anal point with several welldeveloped, often trifid, spinulae in an area which extend from between well-developed anal crests to the anal tergite itself; strong median tergite setae present; lateral anal tergite setae absent; setiger of superior volsella comparatively large, elongate oval, straight or slightly concave median margin, with broad base; median volsella 50 mm long, conspicuously bent posteriad at half length, stem microtrichose, distally palmate.

Remarks
Stempellinella sublettorum can easily be separated from all other Stempellinella species by its microtrichose, conspicuously bent median volsella. The species has so far only been recorded from its type locality in New Brunswick.

Diagnostic characters
Stempellinella tamaseptima can be separated from other Stempellinella species by the following combination of characters: adult male with AR 0. 6-1.0; anal point with numerous strong spinulae between well-developed anal crests; strong median setae on anal tergite; lateral anal tergite setae absent; setiger of superior volsella roughly rectangular with concave median margin, base wider than horizontal distance between superior and median volsellae; median volsella 35-40 mm long with microtrichose, palmate stem. Adult female with AR 0. 31-0.35; few or no setae on vaginal floor; seminal capsules 54-60 mm in diameter; spermathecal ducts longer than notum and rami combined, and usually longer than 200 mm; notum longer than rami; coxosternapodeme with obvious anterolateral lobe. Pupa with separate point patches on abdominal tergites III-VI; one lateral seta on abdominal segment III and IV taeniate, large anteromedian patch of shagreen on tergite VII; anterolateral patches of shagreen on tergites VIII-IX.

Remarks
The slide box containing the type material (A43) appears to be missing from the Sasa collection at NSMT (T. Kobayashi, personal communication) and the type material was therefore unavailable for examination. The females described here are tentatively associated with the males. Both sexes were found at the locality in Kawasaki and the females are morphologically different from the other Stempellinella species recorded in Japan. Unfortunately no pupae were available for this study, and the original pupal description differs considerably from the typical Stempellinella pattern. For instance, no Stempellinella species has so far been seen with the abdominal tergal armament and setation described for S. tamaseptima (Sasa 1980, Plate 22), a pattern found to be relatively constant in both Stempellinella and Zavrelia. Thus, there is a chance that Sasa's description of the pupa is inaccurate, and that most of the deviations from closely related Stempellinella species (e.g. S. fimbriata and S. truncata) are due to misinterpretations by Sasa. The material used by Sasa long), digitiform spur on antennal pedestal; AR ca 0.9; proximal Lauterborn organ placed on 5 mm long pedicel, distal organ on [15][16][17][18][19][20] mm long pedicel; antennal blade just reaching apex of distal Lauterborn organ; S3 split in three branches; premandible with three teeth.
Larva (n54, unless otherwise stated). Total length ca 1.8 mm, larval case ca 2 mm. Head capsule brown, dark brown postoccipital rim and teeth on mandible and mentum. Live individuals with light yellow body.

Remarks
The species fits the diagnostic characters for the genus Stempellinella, and is hereby transferred to this genus. The male specimens from Natal and eastern Transvaal differ from specimens from the Western Cape Province by having a broader wing, lower AR, narrower antennal plume and somewhat higher crests of the male anal point (on equal-sized specimens). These differences are here regarded as intraspecific, but future molecular data or immature stages from the northern populations (which were unavailable for this study) might recognize the two populations as separate species.
Stempllinella truncata is a common species in clear, fast-flowing streams and smaller rivers in mountainous areas of South Africa. The water is neutral or slightly alkaline (pH [7][8], and the stream beds typically consist of boulders, cobbles, and some bedrock. The larvae and pupae of S. truncata live in sand and silt in quiet backwater sections where they build their small cases of sand grains and organic particles.

Diagnostic characters
Stempellinella sp. A can be separated from other Stempellinella species by the following combination of characters: pupa with moderately developed, broadly conical cephalic tubercles; thoracic horn long and thin with numerous small chaetae scattered on middle one-third; thorax with scattered sculpturing dorsally; anterior dorsocentrals slightly longer than posterior dorsocentrals; point patches on tergite II small, starting just anterior to seta D5; tergite six with anterolateral shagreen patches in addition to large point patches; anal lobe with 19-20 taeniae.

Remarks
Stempellinella sp. B is recorded only from Jilin Province in China and Minnesota in the USA. The only observable difference between the material from these two widely separate localities is that the Chinese specimen is slightly larger and somewhat less sculptured on the thorax and frontal apotome. These small differences are here regarded as intraspecific. A formal name is not given since only pupal skins are known, and it is uncertain if these correspond to a Stempellinella species known only from the adult.

Discussion
There seems to be sufficient morphological and COI variation to separate known species in Stempellinella. Pairwise COI distances are considerably shorter within species than between species, and DNA barcodes appear to be good markers for species identification. It should be noted, however, that few populations were sampled for the molecular data, and that higher intraspecific haplotype variation can be expected as specimens from other geographical areas are analysed. Species of the genus Stempellinella can be partly assembled in two putative species groups based on a few objective synapomorphies in the adult males: the species of the edwardsi group are recognized by having costa ending proximal to apex of vein M 3+4 , a T-shaped anal tergite band, and spurs in both tibial combs on the mid and hind tibiae. The species of the truncata group all have a microtrichose and branched stem of the median volsella. The remaining species have no obvious group-forming character in common, but Stempellinella reissi clearly separates from the rest by having short, stout median volsella with broad leaf-shaped lamellae in the adult male hypopygium. This structure is similar to that seen in species of the genus Zavrelia, but the lamellae of the median volsella in the Zavrelia species I have examined are not as broadly leaf-shaped as the lamellae in S. reissi.
The phylogenetic relationships of the species and putative species groups in Stempellinella are interesting also in a zoogeographical perspective since members of this genus are found on all continents except Antarctica. Preliminary results based on morphological and molecular data strongly indicate a monophyletic Stempellinella and monophyletic edwardsi and truncata species groups, but the analyses need broader taxonomic sampling and further refinement before publication.