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16 July 2013 Molecular and morphological methods reveal cryptic diversity and three new species of Nearctic Micropsectra (Diptera:Chironomidae)
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Abstract

We used an integrative-taxonomy approach to help resolve taxonomic issues within the genus Micropsectra (Diptera:Chironomidae). We used partial cytochrome c oxidase subunit I (COI) and nuclear carbamylphosphate synthetase (CAD) sequences and morphological data to provide a framework for better understanding of North American species in this group of nonbiting midges. As part of our results, we describe 3 new species: Micropsectra neoappendica, n. sp., Micropsectra penicillata, n. sp., and Micropsectra subletteorum, n. sp., and 1 species new to the north-central USA, Micropsectra xantha (Roback). Two of the species, M. neoappendica n. sp. and M. subletteorum n. sp., initially appeared to be morphologically identical to species known from the Palearctic. However, molecular data indicated that they were genetically distinct, and reexamination of adult and pupal morphology revealed slight but consistent diagnostic differences. The implications of using species-level identifications for cryptic-species complexes in biological monitoring and conservation management are briefly discussed with reference to our findings. Our results emphasize the importance of using molecular tools in conjunction with traditional morphological techniques when studying Chironomidae diversity, especially when relying on diagnoses from other geographic regions.

Chironomidae taxonomy and systematics for several genera are arguably more advanced in the Palearctic region than in the Nearctic. Therefore, keys and species descriptions used for many Nearctic Chironomidae often are drawn from Palearctic literature. A common experience among users of taxonomic keys for many aquatic invertebrates is to end with a name that fits the specimen in question, but to find slight discrepancies when comparing species descriptions and the specimen. These discrepancies may or may not indicate that the specimens are heterospecific. Many transAtlantic species do bear a strong resemblance to one another, but it is often unknown whether Nearctic and Palearctic counterparts are truly conspecific (Epler 2001).

The fact that many species descriptions are based primarily on morphology of the adult male, often without incorporating other life-stages, adds to this problem, especially because closely related species can exhibit strong similarities in this life stage. For example, Townes (1945) originally synonymized the Nearctic Polypedilum (Polypedilum) flavum (Johannsen, 1905) with the Palearctic species Polypedilum (Polypedilum) convictum (Walker, 1856) because of morphological similarity of the adult males. However, the immature stages are quite different (Epler 2001). This example emphasizes the importance of including multiple life stages in a description whenever possible.

Molecular techniques, such as deoxyribonucleic acid (DNA) barcoding, may provide alternative means for differentiating species. These techniques are especially useful for separating morphologically cryptic species (Sinclair and Gresens 2008, Pauls et al. 2010, Carew et al. 2011, Takano et al. 2011) and associating multiple life stages (Carew et al. 2005, Willassen 2005, Ekrem et al. 2007, 2010a, Zhou et al. 2007, Stur and Ekrem 2011). DNA barcoding is an invaluable tool that aids in species identification and evaluation of whether morphologically similar transAtlantic species are truly conspecific (Sinclair and Gresens 2008).

One of the most species-rich genera within Chironomidae is Micropsectra Kieffer, 1909, which comprises ≥140 named species known from the Holarctic region and a few species known from areas outside the Holarctic (see Stur and Ekrem 2006). Some Micropsectra are found in oligotrophic or mesotrophic lakes, but most species in the genus occur in lentic areas of springs and streams (Säwedal 1982). For example, Stur and Wiedenbrug (2006) found high abundance and diversity of Micropsectra species in 2 groundwater spring communities in Berchtesgaden National Park, Germany. The new species described below come from similar spring-fed stream habitats. These specific habitat preferences make the genus quite important in biological monitoring studies (Stur and Ekrem 2006), but habitat preferences are most informative when used at the species level. For example, the rapid bioassessment protocol published by Barbour et al. (1999) lists tolerance values for many Nearctic Chironomidae genera and species, partitioned by region. The values assigned to the genus Micropsectra differ significantly by region, and range from 1.4 to 7 on a 10-point scale, where 0 indicates extreme sensitivity. Species-level sensitivities for the Micropsectra in our study were not presented by Barbour et al. (1999). Unfortunately, not enough information is currently available for Nearctic species in the genus to provide an accurate assignment of pollution tolerance of the genus or to assign tolerance values to species within Micropsectra. Further knowledge of habitat preference and identification tools for the genus will help resolve this problem.

Presently, species within Micropsectra are divided into 4 species groups, including atrofasciata, attenuata, notescens, and recurvata groups. However, these groups are based primarily on European fauna and have to be reconsidered as more species from other parts of the world are described. Moreover, all recent revisions of the genus Micropsectra have been confined primarily to major species groups recognized from the Palearctic (Reiss 1969a, Säwedal 1976, 1981, 1982, Stur and Ekrem 2006, 2008, Ekrem et al. 2010b). Some Micropsectra species are thought to be Holarctic in distribution, but close examination of some Palearctic and Nearctic populations can reveal genetic and morphological differences large enough to be regarded as diagnostic at the species level.

We used a combination of morphological and molecular approaches to help refine species-group classifications within Micropsectra and began an examination of morphological and molecular differences between some cryptic species within the genus. We attempted to provide a framework for a better understanding of North American Micropsectra and, subsequently, the genus as a whole. We used sequences from mitochondrial cytochrome c oxidase subunit I (COI) and nuclear carbamylphosphate synthetase (CAD) genes to assess whether morphologically similar specimens from Nearctic and Palearctic populations are conspecifics with a transAtlantic distribution or whether sufficient genetic variation exists to classify these specimens as distinct species. We also used the resulting CAD phylogenies to discuss whether morphological traits can be used independently to define species groups within Micropsectra.

Materials and Methods

Sampling and data collection

Larval Micropsectra specimens were obtained from the benthos of 5 streams in Minnesota, USA, during late winter/early spring 2010. All specimens were held in constant-temperature incubators at 5°C to approximate stream temperatures and allowed to develop to adulthood in mass rearings, resulting in a cumulative total of 99 adult Micropsectra with associated pupal exuviae. Rearing chambers were typically checked twice per day to ensure that emerging adults were associated with the appropriate exuviae. Upon emergence, adults and associated exuviae were stored in individual vials and preserved in 95% ethanol. Unfortunately in nearly all cases, larval exuviae could not be retrieved. The antennae, head, wings, legs, and hypopygium were dissected. The head and hypopygium were cleared in 10% KOH and slide mounted in Euparal® with the pupal exuviae, legs, wings, and antennae, for species identification. The thorax and abdomen were slide-mounted after DNA extraction (described below). On the few occasions where >1 fly of the same sex emerged between observations, the adults and exuviae were stored in the same vial until dissection. Adults were mounted on individual slides as described above, but both exuviae were mounted on a single, separate slide. All specimens were identified and separated into 4 morphotypes. A subset of 46 specimens collected from Minnesota streams was selected for molecular analyses (Table S1; available online from:  http://dx.doi.org/10.1899/12-020.1.s1 (10.1899_12-020.1.s1.doc)). Specimens were selected to include representatives from each Micropsectra morphotype. In addition, 1 Tanytarsus specimen was sequenced to include among the outgroup species.

The full COI data set consisted of 291 individuals, including 5 outgroup species and 44 Micropsectra species (31% of known Micropsectra species). Of these, 4 species (43 individuals) were collected in Minnesota: Micropsectra neoappendica n. sp., Micropsectra penicillata n. sp., Micropsectra subletteorum n. sp., and Micropsectra xantha (Roback, 1955) (Table S1). The remaining sequences were acquired from GenBank, and included 3 taxa of Tanytarsus and 2 taxa of Paratanytarsus, which served as outgroups (Table S1). A 2nd reduced COI data set that conformed to the CAD data set (described below) also was generated and analyzed. The CAD data set consisted of 82 individuals from 37 species (33 Micropsectra and 4 outgroup taxa; 27% of known Micropsectra species). Twelve individuals were from the above-named Micropsectra taxa. The remaining sequences were acquired from GenBank, and included 2 Paratanytarsus and 2 Tanytarsus species that served as outgroups. Voucher and sequence information for all specimens collected are also accessible in BOLD project “Nearctic Micropsectra (NAMIC)” and the BOLD data set “Holarctic Micropsectra (DS-HOLMIC)” ( http://boldsystems.org). Remaining DNA extracts are stored at the Museum of Natural History and Archaeology, Norwegian University of Science and Technology (VM). A detailed record of all taxa, including collection localities and GenBank accession numbers, is available in Table S1.

DNA extraction, amplification, and sequencing

A GeneMole® instrument (Mole Genetics AS, Lysaker, Norway) was used to extract DNA from most specimens, following standard protocol for the MoleStrips DNA Tissue kit. DNA was extracted from remaining specimens using protocol for the Qiagen DNeasy tissue extraction kit (Qiagen, Oslo, Norway), with the exception that less elution buffer (150 µL) than recommended was used because of small specimen sizes. Polymerase chain reaction (PCR) amplification and sequencing was carried out on partial COI and CAD genes using the primer pairs and PCR programs described in table 1 of Ekrem et al. (2010b). We used HotStar Taq (Qiagen) when amplifying COI and TaKaRa HS ExTaq (Takara Bio Inc., Otsu, Shiga, Japan) for amplification of CAD. PCR products were purified using ExoSAP-IT® (Affymetrix Inc., Cleveland, Ohio) and sent to an external sequencing service (Eurofins MWG Operon, Ebersberg, Germany). All gene products were sequenced bidirectionally, and forward and reverse sequences were aligned and manually edited using Sequencher 4.8 (Gene Codes Corp., Ann Arbor, Michigan). Conflicting or ambiguous base calls were initially given the appropriate International Union of Biochemistry ambiguity symbol. Initial ambiguities were checked after alignment and corrected, if possible, with reference to trace files. Consensus sequences were assembled and initially aligned in BioEdit (version 7.0.5.3; Hall 1999) for inspection. Introns in CAD sequences were recognized by the guanine thymine–adenine guanine (GT–AG) rule (Rogers and Wall 1980) and eliminated from the alignment because these areas exhibited extreme variation among species. All CAD sequences had intron placement at position 725 and no indication was seen of paralogous gene copies. CAD exons were aligned by their amino acids in MEGA5 (Tamura et al. 2011; available from:  www.megasoftware.net) using the Clustal-W algorithm with the BLOSUM weight matrix and gap opening costs of 10 and gap extension costs of 0.1 and 0.2 in pairwise and multiple alignments, respectively. Alignment of COI sequences was trivial because no indels were observed within the sequences.

Tree-based species delimitation and phylogenetic analyses

Phylogenetic analysis of the initial full COI data set was inferred with neighbor joining (NJ) and maximum likelihood (ML) methods in MEGA5. The Kimura 2-parameter (K2P) substitution model was used for the NJ analysis to infer evolutionary distances because it has been a standard substitution model in other barcode studies, thus allowing comparisons among studies. Although this method is commonplace in barcoding studies, recent authors have questioned the strict use of the K2P model (Srivathsan and Meier 2012). This concern prompted us to include ML analyses with 500 bootstrap replicates using the General Time Reversible model with a discrete Gamma distribution and invariable sites (GTR+G+I), which was the most appropriate nucleotide substitution model for our data set based on the Bayesian (BIC) and corrected Akaike Information Criterion (AICc) in the ML model selection feature of MEGA5.

The phylogenetic relationships among species in Micropsectra were further explored using CAD because of its greater ability to reconstruct lower-level phylogenies in Diptera (Winterton et al. 2007, Winkler et al. 2009, Ekrem et al. 2010b). The best-fit model of substitution was identified using a ML criterion in MEGA5. BIC indicated that the Tamura 3-parameter model with a discrete Gamma distribution and invariant sites (T92+G+I) was the most appropriate model, whereas AICc indicated that the GTR+G+I model best fit our CAD data set. Therefore, we used the GTR+G+I model for all ML and Bayesian analyses because the T92 model is not incorporated in RAxML (version 7.4.2; Stamatakis 2006) or MrBayes (version 3.1.2; Huelsenbeck and Ronquist 2001;  http://mrbayes.sourceforge.net/). The best-fit partition scheme was found using PartitionFinder (version 1.0.1; Lanfear et al. 2012) and contained 2 partitions in the nucleotide sequences: 3rd position and 1st+2nd position. Multiple tree-building methods were used to assess whether phylogenetic results were robust to different analytical assumptions. ML analysis with 500 bootstrap replicates was run on the partitioned data set in RAxML using the software raxmlgui (Silvestro 2012). Bayesian analysis (BA) was done on the partitioned data set with MrBayes. For BA, we specified flat prior probabilities and ran 2 parallel analyses, each with 4 chains, for 5 million generations, sampling every 1000th generation and using a 10% burn-in. We examined the trace files with Tracer 1.5 (Rambaut and Drummond 2008;  http://tree.bio.ed.ac.uk/software/tracer/) to verify that Markov Chain Monte Carlo (MCMC) convergence had occurred and that the estimated sample size (ESS) had reached an acceptable level before summarizing the data from the sampled trees in a consensus. Trees created by BA were imported and modified for presentation in MEGA5. A reduced COI data set, corresponding to the same individuals analyzed for CAD, also was analyzed phylogenetically with ML using the GTR+G+I substitution model and 500 bootstrap replicates in MEGA5. Last, a concatenated data set containing COI and CAD sequences was constructed and analyzed with both RAxML and MrBayes with the following 5 partitions identified by PartitionFinder: COI 1st position, COI 2nd position, COI 3rd position, CAD 1st+2nd position, CAD 3rd position.

Species descriptions and specimen deposition

Most of the morphological terminology and abbreviations used here follow Sæther (1980), with the following exceptions: taeniae, introduced by Langton (1994), is used to describe the flattened setae found on the pupal exuviae and the term setiger (Spies 1998) describes the setae-bearing area of the superior volsella on the male hypopygium. The following abbreviations also are used: P♂  =  associated pupal exuviae and adult male; P♀  =  associated pupal exuviae and adult female; LhcP♂  =  associated larval head capsule, pupal exuviae, and adult male. Measurement techniques follow those outlined by Soponis (1977), with the exception of the gonocoxite length, which was measured along the limb's longest access, as described by Stur and Spies (2011). Lengths of the genital volsellae were measured along their median margin, and the anal point was measured from the anterior ends of the anal crests to the tip of the anal point. Mensural data are presented as ranges, followed by the mean and number of observed specimens.

Holotypes and most paratypes are deposited in the University of Minnesota Insect Collection in St Paul, Minnesota, USA (UMSP). Additional paratypes are deposited in VM. We also examined type material and other reference material of closely related taxa from the following collections (abbreviations correspond to those used in text): The Natural History Museum, London, UK (BMNH); Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, Ontario, Canada (CNC); private collection of Peter H. Langton, Londonderry, Northern Ireland (PHL); UMSP; VM; Natural History Collections, Bergen Museum, University of Bergen, Norway (ZMBN); and Zoologische Staatssammlung München, Munich, Germany (ZSM).

Results

Tree-based species delimitation

All Nearctic specimens collected for our study formed distinct monophyletic groups at the species level in analyses of the COI, CAD, and the concatenated COI/CAD data, and distinctly separated the 4 Nearctic Micropsectra from morphologically similar Palearctic counterparts (Figs 1, 2, Figs S2, S3; available online from:  http://dx.doi.org/10.1899/12-020.1.s2 (10.1899_12-020.1.s2.pdf) and  http://dx.doi.org/10.1899/12-020.1.s3 (10.1899_12-020.1.s3.pdf)). This result is particularly notable for 2 of the Nearctic species, M. neoappendica n. sp. and M. subletteorum n. sp., which are very similar morphologically to species known from the Palearctic. Micropsectra subletteorum n. sp. is closely allied morphologically with many species within the notescens species group. Males of M. neoappendica n. sp. fit the species description of the Palearctic Micropsectra appendica Stur and Ekrem, 2006 almost perfectly. Only after divergence patterns were discovered with molecular analyses of COI and CAD markers, did subsequent examination reveal subtle but corroborating morphological characters that can be used to differentiate M. neoappendica and M. appendica (see Species Description below).

Fig.1. 

Full mitochondrial cytochrome c oxidase subunit I (COI) maximum likelihood (ML) phylogeny of 291 individuals from 49 species. Taxa denoted with ▪ were collected in Minnesota and are described/redescribed in our study. Numbers on branches indicate ML bootstrap support values. Values within highly supported (>90%) species clades were removed for ease of interpretation. Low bootstrap support values are included for the higher-level relationships to indicate the inability of COI sequences to resolve relationships between species and species groups clearly, particularly when compared to nuclear carbamylphosphate synthetase (CAD) sequences (Fig. 2). The scale bar indicates branch lengths as number of substitutions per site corrected by the General Time Reversible model with a discrete Gamma distribution and invariable sites model (GTR+G+I).

i2161-9565-32-3-892-f01.tif

Fig.2. 

Carbamylphosphate synthetase (CAD) phylogeny of 82 individuals from 37 species, based on Bayesian (BA) and maximum likelihood (ML) analysis. Taxa denoted with ▪ were collected in Minnesota and are described/redescribed in our study. Shaded boxes enclose current or proposed species groups. Values on branches indicate BA posterior probabilities and ML bootstrap support values, respectively. The scale bar indicates branch lengths as number of substitutions per site corrected by the General Time Reversible model with a discrete Gamma distribution and invariable sites model. ** indicates discrepancies between the BA and ML analysis (e.g., Micropsectra roseiventris was sister to remaining in-group taxa in BA, but sister to Paratanytarsus taxa in ML).

i2161-9565-32-3-892-f02.tif

NJ and ML analysis yielded very similar results for the analysis of our COI data. Other than minor differences in bootstrap support values, no notable differences were found, and all species clusters were well resolved in both analyses. However, because of recent contention regarding the use of K2P and strictly NJ approaches (Srivathsan and Meier 2012), the results based on the ML analysis are the focus of our discussion. COI ML analyses of the full and the reduced data sets formed strongly supported species clusters. All bootstrap support values for species clusters in the analysis of the full data set were >90% (Fig. 1). A similar trend was seen in the reduced COI ML and NJ/K2P analyses, with all species clusters with bootstrap support > 90%, except Micropsectra sofiae Stur and Ekrem, 2006 (bootstrap support  =  65%).

Phylogenetic analyses

CAD phylogenies from ML and BA yielded similar species-group patterns, with no notable differences other than support values for certain clades (Fig. 2). One such difference was found in the support for the Micropsectra kurobemaculata–Micropsectra lindrothi clade. BA indicated strong support for this clade and placed these 2 species together (posterior probability [pp]  =  95%) and as sister to the remaining species within the notescens group (pp  =  99%), whereas ML analysis indicated much weaker support for these placements (bootstrap support values <50%). We choose to leave these 2 species as members of the notescens group at this point, but recognize that as more species are included in analyses, a more appropriate placement may exist for M. kurobemaculata (Sasa and Okazawa, 1992) and M. lindrothi Goetghebuer, 1931. Another difference between BA and ML analysis was in the placement of Micropsectra roseiventris (Kieffer, 1909). BA placed this species as sister to the remaining in-group taxa (Fig. 2), with a pp  =  0.66. ML analysis, on the other hand, weakly indicated that this taxon is sister to Paratanytarsus (bootstrap support  =  0.44). Further analysis of the relationship between Paratanytarsus and Micropsectra is beyond the scope of our study, but warrants future research.

Analysis of the concatenated COI/CAD data yielded results that were similar to the CAD phylogeny with a few discrepancies (Fig. S2). Most notable is the placement of the lacustris group within the phylogeny. The CAD phylogeny depicts this group as sister to the atrofasciata group, whereas the combined COI/CAD phylogeny indicates this group is sister to the atrofasciata, notescens, and recurvata groups.

Analyses yielded strongly supported atrofasciata and notescens species groups and a paraphyletic recurvata group, results that correspond well with conclusions of Ekrem et al. (2010b) (Fig. 2). Micropsectra lacustris Säwedal, 1975, here designated as a member of the newly re-erected lacustris group was previously considered a member of the recurvata group. We could not confirm the finding of a paraphyletic attenuata group found by Ekrem et al. (2010b) because CAD sequences are not currently available for Micropsectra pharetrophora Fittkau and Reiss, 1999, the specimen accounting for paraphyly in this group. Results for the species described in our study show M. neoappendica n. sp. as sister to M. appendica in the atrofasciata group, M. penicillata n. sp. as sister to M. lacustris in the lacustris group, M. subletteorum n. sp. as sister to M. insignilobus Kieffer, 1924 in the notescens group, and M. xantha as sister to M. recurvata Goetghebuer, 1928 in the recurvata group (Fig. 2).

Description of Species

  • Micropsectra neoappendica Anderson, Stur & Ekrem, new species

  • (Figs 3, 4A)

  • Fig.3. 

    Micropsectra neoappendica n. sp., male hypopygium.

    i2161-9565-32-3-892-f03.tif

    Fig.4. 

    Pupal abdominal segments III–V. A.—Micropsectra neoappendica, n. sp. B.—Micropsectra appendica.

    i2161-9565-32-3-892-f04.tif

    Type material

    Holotype.—

    P♂. USA, Minnesota: Cook County, Fiddle Creek, Forest Rd. 152/Lima Grade, stream access ∼100 m NW of South Brule River bridge near edge of road, 47°56′35″N; 90°26′17″W, 505 m, larva collected 11.iv.2010, A. M. Anderson (UMSP, on slide; VM, DNA extract; voucher ID: ALY29).

    Paratypes.—

    8 P♂, 11 P♀. All data as for holotype (7 P♂, 10 P♀ UMSP; 1 P♂, 1 P♀ VM), voucher ID and GenBank accession codes in Table S1.

    Diagnostic characters.—

    The following combination of characters separates this species from others currently in the atrofasciata group: Adult male. AR ∼1.27, LR1 ∼1.52. Anal point long with high, slightly curved crests, with long, rounded point. Elevated hump anterior to base of anal point; hump typically with small rounded point at tip. Superior volsella roundish. Digitus short, thin, and triangular. Median volsella club-shaped with numerous spoon-shaped lamellae on distal ⅓; length of median volsellae ∼123 µm. Pupa. Total length ∼4.6 mm. Thoracic horn relatively short, ∼306 µm with chaetae on distal ⅔. Distance between anterior and posterior dorsocentral setae pairs approximately equal. Nose of wing sheath moderate to strong. Patches of TIV consisting of long longitudinal spines extending nearly to the posterior end of tergite. Patches of TV consisting of spinules and points in a similar pattern to TIV. Anal fringe with ∼30 setae.

    Etymology.—

    The species epithet neoappendica refers to the striking similarity that this Nearctic species has with its putative sister species in the Palearctic, Micropsectra appendica.

    Description

    Adult male.—

    Measurements and ratios in Table 1.

    Table 1. 

    Measurements and ratios for adult males of Micropsectra species. Lengths are in µm unless indicated. Data are presented as ranges, followed by the mean and the number of specimens observed in parentheses. VR  =  venarum ratio, AR  =  antennal ratio, LR  =  leg ratio, BV  =  “Beinverhältnisse”, BR  =  bristle ratio, SV  =  “Schenkel-Schiene-Verhältnis”, HR  =  hypopygium ratio. Morphological terminology and abbreviations follow Sæther (1980).

    i2161-9565-32-3-892-t01.tif

    Coloration.—

    Thorax with light green to light brown ground color, dark brown scutal stripes, preepisternum, postnotum, and median anepisternum; scutellum and halteres greenish to light brown; abdomen green to light brown; head with dark brown antennal pedicel, black eyes, and light brown palps; legs light brown.

    Head.—

    Antenna with 13 flagellomeres. Frontal tubercles present (8–11, 9 µm), temporal setae in 1 row; palpomere 3 with 2 sensilla clavata in subapical pit.

    Wing.—

    Subcosta and media bare, brachiolum with 2–4 setae, squama bare.

    Legs.—

    Pulvilli present, nearly ½ as long as claws. Fore tibia with spur (17–25, 22 µm); mid and hind tibial combs ∼15 µm long; middle tarsomere Ta1 with 4–5 sensilla chaetica.

    Hypopygium (Fig. 3).—

    Anal tergite with separate, posteriorly directed anal tergite bands; 4–5, 4.3 median tergite setae situated on an elevated hump anterior to anal point base; 9–11, 9.5 ventral apical setae. Anal point long with high, slightly curved anal crests, apex long and rounded; large knob present between crests; microtrichia-free area at base, except lateral of anal point. Superior volsella round with 6–8, 7 dorsal setae and 3–4 median setae on setiger, 1 strong setae on stem; microtrichia fields located dorsally on stem and ventrally on setiger. Digitus small, thin, triangular, not extending past margin of superior volsella. Median volsella club-shaped, comparatively long and thick, with setiform and spoon-shaped lamellae; spoon-shaped lamellae on distal ⅓; stem nearly reaching apex of inferior volsella. Inferior volsella without dorsoapical or dorsomedian swellings, numerous setae at tip. Inner margin of gonocoxite with 3–4 strong setae.

    Pupa.—

    Measurements given in Table 2.

    Table 2. 

    Measurements of pupal exuviae and pupal structures of Micropsectra species. Lengths in µm unless indicated. Data are presented as ranges, followed by the mean with the number of specimens observed in parenthesis. DC  =  dorsocentral setae. Morphological terminology and abbreviations used here follow Sæther (1980), except taeniae (Langton 1994), is used to describe the flattened setae found on the pupal exuviae.

    i2161-9565-32-3-892-t02.tif

    Coloration.—

    Pupal exuviae brownish with dark brown apodemes; cephalothorax, TVIII and anal lobe darker brown.

    Cephalothorax.—

    Cephalic tubercles present, small, conical; pedicel sheath tubercle weakly developed. Thoracic horn relatively short with numerous long chaetae on distal ⅔; precorneals arranged in triangular pattern, the 2 anterior-most setae situated closer to each other than to the 3rd; 1 median and 2 lateral antepronotals (1 seta, 1 sensillum basiconicum); 2 pairs of dorsocentrals, anterior and posterior pair approximately equidistant from each other. Coarse granulation dorsally on anterior ½ of thorax. Prealar tubercle absent; nose of wing sheath moderate to strong.

    Abdomen (Fig. 4A).—

    TII almost covered by shagreen except for 2 mediolateral and 1 posteromedian oval patches; pedes spurii B on segment II obvious; hook row width ∼½ as long as segment width. Spines of TIII in large, laterally curved patches in posterior ½ of tergite, shagreen extensively distributed lateral and anterior to spine patches and in between. Patches on TIV consisting of spinules in anterior, oval patches with spines heavily distributed in longitudinal, lateral extensions; shagreen present lateral to the anterior patches. Patches on TV similar in shape to those in TIV, however distinctly shorter and consisting only of spinules and points. Patches on TVI similar in shape to those of TIV and TV, but somewhat shorter than patches of TV and consisting only of shagreen. Tergite TVII bare or with weak anterolateral shagreen patches. TVIII with weak anterior shagreen patches. Segment I with 3 D, 1 V, and 2 L setae; segment II with 3 D, 4 V, and 3 L; segment III with 5 D, 4 V, and 3 L; segment IV with 5 D, 4 V, 1 L, 2 lateral taeniae; segment V with 5 D, 4 V, 3 lateral taeniae; segment VI with 5 D, 4 V, 4 lateral taeniae; segment VII with 5 D, 4 V, 4 lateral taeniae; segment VIII with 1 dorsal taenia, 1 ventral taenia, 5 lateral taeniae; anal tergite with 1 dorsal taenia. Two pairs of small sensorial setae medially on TII–VII; 1 pair of O-setae present anteriorly on segments II–VII. Anal lobe with 27–33, 30 long taeniae in 1 row. Posterolateral comb of segment VIII narrow with 3–5 apical teeth, median tooth often longer than the rest.

    Taxonomic remarks

    Adult males of M. neoappendica are quite similar morphologically to M. appendica, and thus, fit well in the atrofasciata group. Two characters that help to differentiate these 2 species include the comparatively shorter median volsella of M. neoappendica (120–125, 122.5 µm than the 130–144, 137 µm reported in Stur and Ekrem 2006) and the shape of the elevated hump anterior to the anal point base (Fig. 3). In the specimens we have examined, this hump appears less curved in M. neoappendica and typically exhibits a small rounded point or hump at the tip of the elevated region. Setae extending from the gonocoxite and the seta on the stem of the superior volsella appear to be somewhat stronger than those on M. appendica (Fig. 3). The 2 species are best differentiated in the pupal stage. Pupal exuviae of M. neoappendica can be separated by the placement of the dorsocentrals, where the distance between anterior and posterior pairs is approximately equal, as opposed to having a much greater separation of the anterior pair (∼4× greater) than the posterior pair as seen in M. appendica. In addition, the thickness of DC3 and DC4 is similar for M. neoappendica, whereas 1 thick and 1 thin setae are noted for M. appendica. The nose of the wing sheath of M. neoappendica is typically quite strong, whereas it is generally weak in M. appendica. Spine patches of TIV and TV further differentiate the species, with long longitudinal spines on TIV of M. neoappendica extending nearly to the posterior end of the tergite and spinule/point patches of TV distinctly shorter than spine patch of TIV, with patch tapering off ∼⅔ the length of the tergite (Fig. 4A). Conversely, longitudinal spines of TIV of M. appendica typically taper off approximately at the mid-point of the tergite, with much shorter spines and spinules extending to the posterior end of the tergite; the spine/spinule patch of TV is subequal in size and pattern to TIV patches (Fig. 4B). Last, pupae of M. neoappendica seem to have fewer anal lobe taeniae than M. appendica (27–33, 30 as compared to 35–42, 38 as described in Stur and Ekrem 2006). Males of M. neoappendica are also quite similar to Micropsectra tusimalemea Sasa and Suzuki, 1999, but can be separated by the differentiating characters for M. appendica described in Stur and Ekrem (2006).

    Ecology and distribution.—

    Micropsectra neoappendica is known from a shallow stretch of a small coldwater trout stream in northeastern Minnesota, USA.

  • Micropsectra penicillata Anderson, Stur & Ekrem, new species

  • (Fig. 5A–H)

  • Fig.5. 

    Micropsectra penicillata n. sp., male. A.—Hypopygium. B.—Pupal abdominal segments II–VI, dorsal view. C.—Anal point, lateral view. D.—Pupal frontal apotome. E.—Posterolateral combs of pupal segment VIII. F.—Pupal thorax. G.—Pupal thoracic horn. H.—Pupal abdominal segments VIII–IX.

    i2161-9565-32-3-892-f05.tif

    Type material

    Holotype.—

    P♂, USA, Minnesota: Cook County, South Brule River, Forest Rd. 152/Lima Grade, 15 m upstream of bridge, 47°56′35″N; 90°26′17″W, 499 m, larvae collected 11.iv.2010, A. M. Anderson, (UMSP, on slide; VM, DNA extract; voucher ID: ALY71).

    Paratypes.—

    11 P♂, 7 P♀. All data as for holotype, voucher IDs and GenBank accession in Table S1; 1♂, USA, Colorado: Lake Co., East Fork Arkansas River, 3041 m, 9–30.viii.1984, J. S. Herrmann, originally determined by J. Sublette as M. recurvata, (UMSP); 3♂, USA, Michigan: Alcona Co., Mikado, Huron National Forest, White Cedar Swamp, Pine River Campground, 8.v.1983, P. Hudson, (UMSP); 1 P♂, USA, South Carolina: Oconee Co., Horse Pasture River, 17.iii.1979, P. L. Hudson, (UMSP); 1♂, USA, South Carolina: Oconee Co., small stream near Devils Fork Landing, Jocassee Reservoir, 19.ii.1982, P. L. Hudson, (UMSP); 1 P♂, USA, Tennessee: Sevier Co., Gatlinburg, LeConte Creek, 4.v.1980, P. L. Hudson (UMSP); 1♂, Canada, Alberta: Hotchkiss, 16.viii.1961, F. L. Kessel-Light; 2♂, Canada, Manitoba: Churchill, Ramsey Creek, 58°43′50″N; 93°46′48″W, 13 m; adult collected with malaise trap over brook, 23–24.viii.2006, T. Ekrem and E. Stur, (VM, voucher IDs CHIR_CH9, CHIR_CH119); 4♂, Canada, Ontario: Caledon Hills, Woodrow Farm Stream, Trap 4, 29.viii–5.ix.1980, (UMSP).

    Diagnostic characters.—

    The following combination of characters separates this species from all other Micropsectra species: Adult male. AR ∼1.3. LR1 ∼1.5. Anal point slender with pointed tip. Superior volsella finger-shaped. Digitus absent. Stem of median volsella long and s-shaped, with numerous narrow, leaf-like lamellae, often held tightly together as in a fine-tipped painting brush. Inferior volsella tapered towards apex and laterally curved, with ∼6–8 distal setae (Fig. 5A). Pupa. Total length ∼4.33 mm. Thoracic horn relatively short, ∼336 µm with numerous chaetae on distal ⅔ (Fig. 5B). Anterior pair of dorsocentrals placed further from each other than posterior pair (Fig. 5F). Long, strong, longitudinal spines on TIV; spinules and points on TV, similar in shape to TIV (Fig. 5B). 23–31, 28 anal fringe setae (Fig. 5H).

    Etymology.—

    The species epithet penicillata (brush-like), refers to the median volsella in the male hypopygium, which has the appearance of a painter's brush or a small tail.

    Description

    Adult male.—

    Measurements and ratios in Table 1.

    Coloration.—

    Thorax with light green to light brown ground color, dark brown scutal stripes, preepisternum, postnotum, and median anepisternum; scutellum and halteres greenish to light brown; abdomen green to light brown; head with dark brown antennal pedicel, black eyes, and light brown palps; legs light brown.

    Head.—

    Antenna with 13 flagellomeres. Frontal tubercles present, 5–12, 8 µm; temporal setae in 1 row; palpomere 3 with 2 sensilla clavata in subapical pit.

    Wing.—

    Subcosta and media bare, brachiolum with 3 setae, squama bare.

    Legs.—

    Pulvilli present, reaching half length of claws. Fore tibia with spur 20–35, 28 µm long, middle and hind tibial combs 12–22, 18 µm and 12–22, 19 µm, respectively. Middle tarsomere Ta1 with 2–4, 3 sensilla chaetica.

    Hypopygium (Fig. 5A).—

    Anal tergite with tergal bands separate, posteriorly directed, not reaching crests of anal point; 3 median tergite setae anterior to anal point base; 7–8 ventral apical setae. Anal point with long, curved anal crests; apex of anal point long, slender with tip pointed; small knob between crests present; superior volsella finger-shaped; setiger with rounded apex and bent in median direction, 4–6, 5 dorsal and 2 median setae on setiger, 1 strong seta on stem; stem of superior volsella lacking field of microtrichia; digitus absent; median volsella long, s-shaped, with numerous narrow, leaf-like lamellae extending from distal end, often held tightly together; tip reaching far beyond apex of superior volsella. Inferior volsella narrowing toward distal end, with ∼6–8 distal setae, tip seemingly bifed and curved medially. Inner margin of gonocoxite with 3–4 strong setae.

    Pupa.—

    Measurements in Table 2.

    Coloration.—

    Pupal exuviae brownish with dark brown apodemes; cephalothorax, TVIII and anal lobe darker; large pigment-free area dorsally in posterior half of thorax.

    Cephalothorax.—

    Cephalic tubercles present, comparatively small, cone-shaped (Fig. 5D); pedicel sheath tubercle weakly developed; thoracic horn fairly short with numerous chaetae on distal ¾, chaetae ∼⅓ to ½ length of thoracic horn (Fig. 5G); precorneals arranged nearly in a line, with 1 setae offset, the 2 anterior-most setae situated closer to each other than the 3rd (Fig. 5G); 1 median antepronotal, 2 lateral antepronotals (1 sensillum basiconicum); 2 pairs of dorsocentrals (DCs), anterior pair placed further from each other than posterior pair and with thin setae as compared to the thicker setae of posterior pair, DC1 longer than DC2, DC3 and DC4 typically nearly equal in length (Fig. 5F). Some granulation present along median suture line of thorax. Prealar tubercle absent; nose of wing sheath weakly developed.

    Abdomen.—

    TI bare. TII almost covered by shagreen except 2 lateral round patches and 1 posteromedian oval patch; pedes spurii B on TII obvious; hook row ∼½ as long as segment width. Spines of TIII in large, laterally curved patches in posterior ½ of tergite; shagreen extensively distributed lateral and anterior to spine patches with numerous points between patches. Patches of TIV consisting of spinules in anterior, oval, slightly diagonal patches with long spines in longitudinal, lateral extensions, with a slight lateral, posterior curve; few points present laterally of longitudinal spines. Patches of TV similar in shape to TIV, somewhat shorter, and consisting only of spinules and points. Patches of TVI consisting only of shagreen, similar to patches TIV and TV in shape, but often much shorter (Fig. 5B). Tergite TVII and TVIII with small, anterior, oval shagreen patches, with patches of TVIII somewhat larger and more distinct than TVII in some specimens. Segment I with 3 D and 1 V setae; segment II with 3 D, 5 V, 3 L; segment III with 5 D, 4 V, 3 L; segment IV with 5 D, 4 V, 1 L, 2 lateral taeniae; segment V with 5 D, 4 V, 3 lateral taeniae; segment VI with 5 D, 4 V, 4 lateral taeniae; segment VII with 5 D, 4 V, 4 lateral taeniae; segment VIII with 1 dorsal taenia, 1 ventral taenia, 5 lateral taeniae; anal tergite with 1 dorsal taenia (Fig. 5B, H). Two pairs of small sensorial setae medially on TII–VII; 1 pair of O-setae present anteriorly on sternites II–VII. Anal lobe with evenly convex lateral margins, fringe with long taeniae in 1 row (Fig. 5H). Posterolateral comb of segment VIII with 3–6, 4 apical teeth longer than rest (Fig. 5E).

    Taxonomic remarks

    Micropsectra penicillata exhibits morphological characteristics that tentatively place this species as a member of the Palearctic recurvata group (sensu Säwedal 1981), with the exception of the lack of a digitus, an atypical character for the current recurvata group. Specimens from this species have been collected from various localities throughout North America but have never been formally described. The Sublette collection at UMSP contains adult male specimens of this species from various localities including 1 adult male from Colorado, USA, that was previously identified as M. recurvata because of morphological similarities. Several others from the collection were left undetermined. Adult males can be differentiated from M. recurvata by the absence of the digitus in M. penicillata, and appearance of the median volsella lamellae, which lack the spoon-shaped appearance of many Micropsectra species, including M. recurvata, and instead appear individually as thin leaves that are often held tightly together in a cluster, giving an appearance similar to a thin, pointed, round paintbrush or pointed tail (Fig. 5A), showing most morphological similarity to M. lacustris (see discussion below). Pupae of M. penicillata differ from M. recurvata by the presence of long longitudinal spines on tergite IV (Fig. 5B) as opposed to a short spine patch in M. recurvata. Males of M. penicillata also share morphological similarities with M. lacustris, and phylogenetic analyses based on CAD nuclear gene sequences indicate a sister relationship between these 2 species (Fig. 2). Micropsectra penicillata can be differentiated from M. lacustris based on a distally tapered and laterally curved inferior volsella, the absence of a digitus, and a comparatively longer median volsella (102–137, 122 µm compared to 74–79 µm reported by Säwedal (1975) for M. lacustris). The median volsella lamellar setae of M. penicillata are often held together more tightly than those of M. lacustris, which tend to appear more fanned. Pupae of the 2 species are differentiated based on the pattern of tergite armament; specifically, M. penicillata does not exhibit long longitudinal spines on TV (Fig. 5B) as seen in M. lacustris (see fig. 6 by Säwedal 1975). Further, Säwedal indicates that M. lacustris has 2 L and 2 LS setae on TIV, whereas M. penicillata has 1 L and 2 LS setae.

    Ecology and distribution.—

    Micropsectra penicillata is currently known from stream localities from Colorado eastward in the USA and from Manitoba and Ontario, Canada.

  • Micropsectra subletteorum Anderson, Stur & Ekrem, new species

  • (Fig. 6A–G)

  • Fig.6. 

    Micropsectra subletteorum n. sp., male. A.—Hypopygium. B.—Pupal abdominal segments II–VI, dorsal view. C.—Posterolateral combs of pupal segment VIII. D.—Pupal frontal apotome. E.—Pupal thorax. F.—Pupal thoracic horn. G.—Pupal abdominal segments VIII–IX.

    i2161-9565-32-3-892-f06.tif

    Type material

    Holotype.—

    P♂, USA, Minnesota: Washington Co., Valley Creek, Belwin Conservancy, 44°55′09″N; 92°47′54″W, 218 m, larva collected 3.iii.2010, A. M. Anderson, (UMSP, on slide; VM, DNA extract; voucher ID: ALY95).

    Paratypes.—

    10 P♂, 4 P♀. All data as for holotype, voucher ID and GenBank accession codes in Table S1 (9 P♂ UMSP, 1 P♂ VM); 6♂, USA, Minnesota: Goodhue Co., Trout Brook, 44°32′97″N; 92°48′07″W, 229 m, adults collected 30.iii.2008, R. W. Bouchard. (UMSP, on slide; VM, DNA extracts); 6 P♀, Cook Co., South Brule River, Forest Rd. 152/Lima Grade, 15 m upstream of bridge, 47°56′35″N; 90°26′17″W, 499 m, larvae collected 11.iv.2010, A. M. Anderson, (UMSP, on slide; VM, DNA extracts); 1 P♀, Cook Co., Fiddle Creek, Forest Rd. 152/Lima Grade, stream access ∼100 m NW of South Brule River bridge near edge of road, 47°56′35″N; 90°26′17″W, 505 m, larva collected 11.iv.2010, A. M. Anderson (VM); 1♂, USA, Ohio: Ottawa Co., Lake Erie, Put-in-Bay, Gibraltar Island, 17.vi.1980, P. L. Hudson (UMSP); 1♂, USA, Wisconsin: Burnett Co., Spring Brook, 11 mi E, 4 mi S of Siren, WI; 45°43′N; 92°09′W, 17.iv.1966, Dean Hansen, (UMSP LRII-64); 1♂ Burnett Co., Spring Brook, 11 mi E, 4 mi S of Siren, WI, 45°43′N; 92°09′W, 16.iv.1966, Dean Hansen (UMSP, S73-183); 1♂, Canada, Manitoba: Churchill, Ramsey Creek, 58°43′50″N; 93°46′48″W, 13 m, adult collected with malaise trap, 23–24.viii.2006, T. Ekrem and E. Stur (VM, voucher ID: CHIR-CH219); 1♂, Canada, Ontario: Renfrew Co., White Lake, 23.vi.1968, R. D. MacDonald (CNC).

    Diagnostic characters.—

    The following combination of characters separates this species from other Micropsectra species: Adult male. AR ∼1.6, LR1 ∼1.53. Anal point long and slender, with pointed tip. Superior volsella finger-shaped, field of microtrichia on stem absent. Digitus slender with slight curve, often extending past median margin of superior volsella. Median volsella long, parallel-sided with medially directed spoon-shaped lamellae. Inferior volsella with dorsoapical swelling. Gonostylus relatively narrow and tapering distally. Pupa. Total length ∼5.4 mm. Thoracic horn relatively short, ∼450 µm long with numerous long chaetae on distal i2161-9565-32-3-892-e01.gif; chaetae length ∼⅓ to ½ length of horn. Abdominal segment IV with 1 L setae and 2 lateral taeniae. Patches of TIV and TV consisting of spinules in anterior, oval patches, with shagreen lateral and posterior. Anal lobe with ∼44 setae.

    Etymology.—

    This species is named in honor of Jim and Mary Sublette in recognition of their contribution to Chironomidae taxonomy and systematics and the large number of Nearctic Micropsectra specimens they collected and prepared over the years. Many of these specimens provided a wealth of information for our research and are referenced throughout our paper.

    Description

    Adult male.—

    Measurements and ratios in Table 1.

    Coloration.—

    Thorax dark brown with yellowish to light brown halteres; abdomen dark brown; head with dark brown antennal pedicel, black eyes, and light brown palps; legs dark brown.

    Head.—

    Antennae with 13 flagellomeres. Frontal tubercles small 5–10 µm; temporal setae in 1 row; palpomere 3 with 2–3 sensilla clavata in subapical pit.

    Wing.—

    Macrotrichia on entire wing except for most basal part and fields between c and r4+5. Subcosta and media bare, distal ⅓ of cubitus and distal ½ of post cubitus bare; brachiolum with 3 setae, squama bare.

    Legs.—

    Pulvilli present, ∼⅓ as long as claws. Fore tibia with 22–38, 30 µm spur, mid and hind tibial combs, respectively 17–25, 21 and 17–25, 22 µm long; middle tarsomere Ta1 with 3–5, 4 sensilla chaetica.

    Hypopygium (Fig. 6A).—

    Anal tergite with tergal bands separate, posteriorly directed, reaching crests of anal point; 3–7, 5 median tergite setae anterior to anal point base; anal tergite without hump in front of anal point crests; 8–12, 10 ventral apical setae. Anal point with long, slightly curved anal crests; apex long, slender, and pointed; knob between crests absent; microtrichia between crests absent; microtrichia surrounding base of anal point. Setiger of superior volsella with 4–7, 5 dorsal and 2 median setae, 1 strong seta on stem; dorsal microtrichia on stem absent, field of microtrichia present ventrally on setiger, anterior to base of digitus. Digitus finger-like and slender with slight curve, often reaching past median margin of superior volsella. Median volsella long, parallel sided, with caudally directed setiform and spoon-shaped lamellae, on distal ⅓–¼; tip generally not extending beyond superior volsella. Inferior volsella with moderately developed median, dorsoapical swelling and bearing numerous distal setae. Inner margin of gonocoxite with 3 strong setae.

    Pupa.—

    Measurements in Table 2.

    Coloration.—

    Pupal exuviae light brownish with dark brown apodemes; cephalothorax, TVIII and anal lobe darker; large pigment-free area dorsally in posterior ½ of thorax.

    Cephalothorax.—

    Cephalic tubercles present, cone-shaped, 30–51, 38 µm; pedicel sheath tubercle absent (Fig. 6D). Thoracic horn 350–580, 450 µm long with numerous comparatively long chaetae on distal i2161-9565-32-3-892-e02.gif; precorneals arranged in triangular pattern, the 2 anterior-most setae situated closer to each other than the 3rd, posterior precorneals longer than the other 2 (Fig. 6E); 1 median and 2 lateral antepronotals (1 sensillum basiconicum); 2 pairs of dorsocentrals, anterior pair shorter and weaker than posterior pair, DC1 longer than DC2, DC3 and DC4 of similar length. Some granulation present along median suture line. Prealar tubercle present, roundish; nose of wing sheath weak to absent.

    Abdomen (Fig. 6B, G).—

    TI bare. TII covered by shagreen except for 1 posteromedian oval patch and 2 small mediolateral patches; pedes spurii B on TII obvious; hook row >½ as long as segment width. Spines of TIII in large, laterally curved patches in posterior ½–⅔ of tergite, shagreen extensively distributed lateral and anterior to spine patches, also covering much of the region between patches. Patches of TIV consisting of anterior, ovoid patches; shagreen present lateral and posterior to spine patches. Spine patches and shagreenation of TV similar to TIV. TVI consisting only of shagreen, similar to shagreenation in TIV and TV, except shagreen does not extend laterally in the posterior region as in TIV and TV. TVII with weak shagreenation in a pattern resembling that of TVI. TVIII with lateral oval shagreen patches. Segment I with 3 D and 1 V setae; segment II with 3 D, 4 V, and 3 L; segment III with 5 D, 4 V, and 3 L; segment IV with 5 D, 4 V, 1 L, and 2 lateral taeniae; segment V with 5 D, 4 V, and 3 lateral taeniae; segment VI with 5 D, 4 V, and 4 lateral taeniae; segment VII with 5 D, 4 V, and 4 lateral taeniae; segment VIII with 1 dorsal taenia, 1 ventral taenia, and 5 lateral taeniae; anal tergite with 1 dorsal taenia. One pair of O-setae present anteriolaterally on sternites II–VII. Anal lobe with long taeniae in 1 row on lateral margins (Fig. 6G). Posterolateral comb of segment VIII with 5–6 marginal teeth (Fig. 6C).

    Taxonomic remarks

    Micropsectra subletteorum is closely allied with many species in the notescens group, particularly M. apposita (Walker, 1856), Micropsectra brundini Säwedal, 1979, Micropsectra contracta Reiss, 1965, Micropsectra insignilobus, Micropsectra junci (Meigen, 1818), Micropsectra lindebergi Säwedal, 1976, Micropsectra nigripila (Johannsen, 1905), and Micropsectra notescens (Walker, 1856). The primary characteristic distinguishing adult males of M. subletteorum from the aforementioned species is that M. subletteorum lacks a field of microtrichia on the basal part of the superior volsella and lateral teeth on the anal tergite (Fig. 6A). The following characteristics also are useful for distinguishing males of M. subletteorum from M. brundini, M. insignilobus, and M. lindebergi: a comparatively higher LR1, and stronger anal point crests (see tables 1–3 in Säwedal 1979). The gonostylus of M. subletteorum also is gradually narrowing towards the distal end (Fig. 6A) and the AR is lower than seen in the 3 aforementioned species. Säwedal (1979) indicates that the low number of sensilla chaetica of M. brundini (0–2) also may help differentiate M. brundini from other notescens group species. However, we have observed specimens of M. brundini from the PHL collection that have up to 4 sensilla chaetica, indicating this character may not be reliable. Micropsectra subletteorum can be further differentiated from M. insignilobus by having a comparatively longer median volsella (68–85 µm compared to 40–59 µm). Micropsectra subletteorum differs from M. junci and M. nigripila by having a comparatively longer digitus that often extends to or past the margin of the superior volsella (Fig. 6A). The digitus of M. subletteorum also exhibits more curvature than that of M. notescens, M. contracta, and M. apposita. Micropsectra subletteorum is further differentiated from M. notescens by lacking the ball-shaped swelling at the tip of the inferior volsella (see fig. 10 by Säwedal 1976) and having a longer median volsella, 67–85, 74 compared to 38–64 µm (Säwedal 1976). The short, blunt-tipped anal point of M. nigripila also differentiates males of the 2 species, and the rounded tip of the superior volsella (Fig. 6A) sets M. subletteorum apart from the more triangular superior volsella of M. contracta and M. apposita.

    Pupae of M. subletteorum can be easily differentiated from M. contracta and M. apposita by the absence of semi-long longitudinal spines posterior to the oval spinule patch on TIV (Fig. 6B). The species differs from M. insignilobus and M. lindebergi by exhibiting 2 LS and 1 L setae on TIV rather than 3 LS setae. The thoracic horn of M. subletteorum also is shorter than in these 2 species, M. nigripila, and the parthenogenetic Micropsectra silvesterae Langton, 1998, (∼450 µm as compared to 680 µm for M. insignilobus and M. lindebergi; 624–947, 825 µm for M. nigripila (Oliver and Dillon 1994); and 440–835, 672 µm for M. silvesterae; Säwedal 1976, Oliver and Dillon 1994, Langton 1998). Further, thoracic horn setae start basally for both outer and inner lateral margins, and filament length is ⅓–½ the length of the thoracic horn as compared to ⅕–⅙ for M. insignilobus and M. lindebergi (see Säwedal 1976); the particularly long thoracic horn with short chaetae of both M. nigripila and M. silvesterae also serves to differentiate M. subletteorum from these species (cf. fig. 10 by Oliver and Dillon 1994, fig. 2C by Langton 1998). Micropsectra subletteorum also can be distinguished from M. silvesterae by the presence of only 1 median antepronotal seta as compared to 2 in M. silvesterae (Langton 1998). Micropsectra subletteorum differs from pupae of M. junci by lacking the finger-like prealar tubercle in front of the wing sheath. Micropsectra subletteorum can be further differentiated from M. insignilobus and M. junci in that it has 40–47, 44 anal lobe filaments, as compared to 53–60 in M. insignilobus and 62–73 in M. junci. Pupae of M. subletteorum are perhaps most similar to M. notescens, and few reliable characters have been found to separate the 2. The thoracic horn length and setae arrangement is perhaps the best distinguishing character at present, with the thoracic horn of M. subletteorum typically shorter than M. notescens. Säwedal (1976) reported the average length of the thoracic horn of M. notescens as 660 µm, but we have seen specimens with thoracic horn length as low as 525 µm, which falls within the range we report for M. subletteorum. Thoracic horn setae appear to begin slightly lower on the inner lateral margin for M. subletteorum, covering i2161-9565-32-3-892-e05.gif to i2161-9565-32-3-892-e04.gif as compared to ⅔ for M. notescens.

    Ecology and distribution.—

    This species is common in small groundwater-dominated trout streams throughout Minnesota and has been found in other lotic and lentic localities in the eastern half of the USA and Canada.

  • Micropsectra xantha (Roback, 1955)

  • (Figs 7A–G, 8A, B)

  • Fig.7. 

    Micropsectra xantha, male. A.—Hypopygium. B.—Pupal abdominal segments II–VI, dorsal view. C.—Superior, median, and inferior volsellae. D.—Pupal frontal apotome. E.—Pupal thorax. F.—Pupal thoracic horn. G.—Pupal abdominal segments VIII–IX.

    i2161-9565-32-3-892-f07.tif

    Fig.8. 

    A.—Superior volsella, Micropsectra xantha. B.—Anal point, M. xantha. C.—Superior volsella, Micropsectra connexa. D.—Anal point, M. connexa.

    i2161-9565-32-3-892-f08.tif

    Calopsectra (Micropsectra) xantha Roback, 1955: 4

    Type material

    Neotype (here designated).—

    P♂, USA, Minnesota: Goodhue Co., Cold Spring Brook, spring pool off of County Rd. 68, ∼1 km N of Hwy 60, 44°17′46″N; 92°25′35″W, 256 m, larvae collected 25.iv.2010, A. M. Anderson (UMSP, on slide; VM, DNA extract; voucher ID: ALY52).

    Additional material examined.—

    13 P♂, 17 P♀, 1 LhcP♀. All data as for neotype, voucher IDs and GenBank accession codes in Table S1 (12 P♂, 1 P♀ UMSP; 13 P♂, 1 P♀ VM); 3♂, 1 P♂, USA, New York: Cattaraugus Co., Allegany State Park, adult collected with light trap 28.v–3.vi.1963, W. W. Wirth, (UMSP, 573–676). 1♂, USA, Virginia: Fairfax Co., Falls Church, Holmes Run, adult collected with light trap 31.v.1961, W. W. Wirth, (UMSP); 7♂, USA, Wisconsin: Burnett Co., Spring Brook, 11 mi E, 4 mi S of Siren, WI, 45°43′N; 92°09′W, adults collected with sweep net 13.x.1967, Dean Hansen (UMSP); 3♂, Burnett Co., Spring Brook, 11 mi E, 4 mi S of Siren, WI, 45°43′N; 92°09′W, adults collected with light trap 7.vi.1968, Dean Hansen (UMSP); 1♂, Canada, Quebec: Lewis Co., 31.v.1929, (UMSP).

    Diagnostic characters.—

    The following combination of characters separates this species from other species of Micropsectra: Adult male. AR ∼1.3, LR1 ∼1.6. Anal point long and slender, with pointed apex. Superior volsella finger-shaped. Digitus long and narrow, extending beyond median margin of superior volsella. Median volsella moderately long and s-shaped, with numerous long, medially directed spoon-shaped lamellae. Inferior volsella swollen at tip, with ∼10 strong setae (Fig. 7A). Pupae. Total length ∼5.3 mm. Thoracic horn relatively long, ∼507 µm with numerous chaetae covering distal i2161-9565-32-3-892-e03.gif; thoracic horn chaetae ∼⅓ length of horn (Fig. 7F). Abdominal segment IV with 3 lateral taeniae. Patches of TIV and TV consisting of spinules in anterior, oval patches, with shagreen lateral and posterior (Fig. 7B). Anal fringe with ∼43 setae (Fig. 7G).

    Etymology.—

    Roback (1955) does not include information regarding the name choice for this species in his original description, however, the Greek term xanthos means yellow and was probably given in reference to the coloration of this species.

    Description

    Adult male.—

    Measurements and ratios in Table 1.

    Coloration.—

    Ground color of head, thorax, abdomen, and legs light yellow or green; antennal pedicel, scutal stripes, preepisternum, postnotum, and median anepisternum darker yellow; eyes black.

    Head.—

    Antenna with 13 flagellomeres. Frontal tubercles present as small dots (3–5, 4 µm); temporal setae in 1 row; palpomere 3 with 2–3 sensilla clavata in subapical pit.

    Wing.—

    Subcosta and media bare, brachiolum with 2 setae, squama bare.

    Legs.—

    Pulvilli present, nearly ½ the length of claws. Fore tibia with 28–38, 33 µm spur; middle and hind tibial combs 15–24 and 15–27 µm respectively; middle tarsomere Ta1 with 3–4 sensilla chaetica.

    Hypopygium (Fig. 7A).—

    Anal tergite with tergite bands separate, posteriorly directed, not reaching crests of anal point; 2–4, 3 median tergite setae on slightly elevated ridge; 8–10, 9 ventral apical setae. Anal point with long, pointed apex; anal crests slightly curved; knob between anal crests absent; large microtrichia-free area around base. Setiger of superior volsella with 5–7, 6 dorsal setae and 2 median setae; 1 seta on stem; a small field of dorsal microtrichia on stem of superior volsella, and a large field ventrally on setiger. Digitus long and narrow, reaching well beyond the median margin of the superior volsella. Median volsella long and s-shaped, long, medially directed setiform and spoon-shaped lamellae, ∼10–15 numerous spoon-shaped lamellae extending from tip. Inferior volsella greatly swollen at tip, appearing somewhat ball-shaped and bearing ∼10 strong setae. Inner margin of gonocoxite with 2–3 strong setae.

    Pupa.—

    Measurements given in Table 2.

    Coloration.—

    Pupal exuviae light brownish with darker brown apodemes; cephalothorax, TVIII, and anal lobe darker brown.

    Cephalothorax.—

    Cephalic tubercles present, small, conical; pedicel sheath tubercle well developed (Fig. 7D). Thoracic horn fairly long with numerous chaetae covering distal i2161-9565-32-3-892-e06.gif; thoracic horn chaetae ∼⅓ length of thoracic horn (Fig. 7F); precorneals arranged nearly in a line, with 1 seta somewhat offset, and the 2 anterior-most setae situated closer to each other than the 3rd, anterior precorneal typically shorter than the other 2; 1 median antepronotal, 2 lateral antepronotals (1 sensillum basiconicum); 2 pairs of dorsocentrals, anterior pair often shorter than posterior pair. Some granulation present along median suture line of thorax. Prealar tubercle present, roundish; nose of wing sheath weak to moderate.

    Abdomen (Fig. 7 B, G).—

    TI bare. TII almost covered by shagreen except for 1 posteromedian oval patch; pedes spurii B on TII obvious; hook row >½ as long as segment width. Spines of TIII in laterally curved patches in posterior ½ of tergite, shagreen extensively distributed lateral and anterior to spine patches, and covering most of the area between patches. Patches of TIV consisting of spinules in anterior, oval patches; shagreen lateral and posterior to patches. TV patches and shagreenation similar to TIV. Patches of TVI consisting only of shagreen; shagreen in anterior region of tergite somewhat larger than those in mid and posterior region, resembling shape of spinule patches in TIV and TV. TVII with shagreen in anterior region. TVIII with small anterolateral shagreen patches. Segment I with 3 D, 1 V setae; segment II with 3 D, 4 V, 3 L; segment III with 5 D, 4 V, 3 L; segment IV with 5 D, 4 V, 3 lateral taeniae; segment V with 5 D, 4 V, 3 lateral taeniae; segment VI with 5 D, 4 V, and 4 lateral taeniae; segment VII with 5 D, 4 V, and 4 lateral taeniae; segment VIII with 1 dorsal taenia, 1 ventral taenia, and 5 lateral taeniae; anal tergite with 1 dorsal taenia. One pair of O-setae anteriolaterally on sternites II–VII. Anal lobe with fringe of laterally curved taeniae in 1 row. Posterolateral comb of segment VIII with 4–6 teeth longer than the rest.

    Taxonomic remarks.—

    The holotype could not be located at the Academy of Natural Sciences Natural History Museum in Philadelphia, USA (ANSP) (J. Weintraub, ANSP, personal communication). However, as noted by Säwedal (1981), the hypopygium of the holotype was poorly preserved. In addition, as discussed below, males of Micropsectra xantha are very similar morphologically to Micropsectra connexa (Kieffer, 1906). There are no known paratypes of M. xantha and, therefore, to stabilize future taxonomy of the species, we find it necessary to designate a neotype for M. xantha.

    As noted by Säwedal (1982), the adult male of M. xantha is very similar to M. connexa (see additional nomenclature notes on this species by Stur and Ekrem 2006) and warrants placement in the Palearctic recurvata group. We have examined the male holotype of M. connexa, located in BMNH, and confirm that the most reliable difference between M. xantha and M. connexa is in the anal point. The anal point of M. xantha is slender and parallel-sided toward the tip, whereas in M. connexa, the anal point is broader and cone-shaped toward the tip (Fig. 8B, D). In addition, the anal point crest is stronger in M. xantha than in M. connexa. We also have noted differences in the superior volsella (Fig. 8A, C). Micropsectra xantha appears to have a broader superior volsella with a long, narrow digitus that is generally <⅓ the width of the superior volsella. The superior volsella of M. connexa is comparatively narrower with a wider digitus that is ½–⅓ the width of the superior volsella. Last, the seta on the stem of the superior volsella appears more basal on M. connexa than M. xantha.

    Stur and Ekrem (2006) place M. connexa with the attenuata group. Here, we refute this placement, and instead find a more appropriate placement in the recurvata group, together with M. xantha, based on the combination of a long and s-shaped median volsella, long anal point (Fig. 8D), and a digitus that extends past the margin of the finger-shaped superior volsella (Fig. 8C). We attempted to amplify both standard and short (130 base pair [bp]) COI-fragments from specimens of M. connexa found in the Sublette collection (UMSP; all originally labeled as M. xantha) to provide additional evidence that M. xantha and M. connexa are truly separate species. However, we were not successful in obtaining a usable sequence, probably because of the age (>40 y) of the specimens available to us. Micropsectra xantha is also quite similar to M. digitata Reiss, 1971, however the digitus in M. digitata is missing and the anal point is wider and more rounded at the tip.

    Ecology and distribution.—

    The original holotype was collected from a small, cold spring west of Concord, Massachusetts, USA (Roback 1955). In the original description, Roback mentions the water temperature of the spring varied from 7–10°C, with a mean temperature of 9°C. The neotype is known from a southeastern Minnesota spring with very similar thermal characteristics. Specimens are also known from various other localities in the eastern ½ of the USA and Canada.

    Discussion

    New Nearctic Micropsectra and cryptic diversity

    This small survey of only 5 Minnesota streams, each visited on only 1 sampling occasion, yielded 3 new species of a single chironomid genus, a clear indication that many Nearctic chironomid species are left to be discovered and described. These new species more than double the number of Micropsectra previously known from Minnesota with 24 species now known from the Nearctic. Without the aid of molecular techniques or examination of multiple life stages, 2 of the species described in this work, M. neoappendica and M. subletteorum, probably would have gone unnoticed if identified using existing Palearctic keys and species descriptions, thereby satisfying the definition of cryptic species by Bickford et al. (2007). This fact is particularly important for M. neoappendica, which, to date, has been found only in 2 neighboring, pristine streams in northeastern Minnesota. The 3 other species described/redescribed in our work have been collected by the authors at multiple Nearctic localities or were found in the large set of Micropsectra material collected and curated by Jim Sublette, now at UMSP. To contrast—and to point out potential habitat differences between the appendica/neoappendica cryptic species complex (the Palearctic counterpart of M. neoappendica)—M. appendica is described as having a wide geographical distribution, with collections made from various types of ponds, rivers, brooks, streams, and springs (Stur and Ekrem 2006). This broad distribution suggests different habitat preferences between the 2 species and potential differences in pollution sensitivity. Habitat partitioning by cryptic species, such as suggested here, is not uncommon and is known for various cryptic invertebrates (e.g., Tarjuelo et al. 2001, Folino-Rorem et al. 2009) and vertebrates (e.g., Davidson-Watts et al. 2006). Therefore, considering that cryptic species may exhibit different responses to environmental conditions, use of a single species, rather than a cryptic species-complex, will allow much more accurate biological monitoring and is important for appropriate and accurate conservation and management programs (Bickford et al. 2007).

    Molecular species delimitation and phylogeny

    As pointed out by Stur and Spies (2011), including species-specific DNA sequences as a standard component of new species descriptions has clear advantages that extend beyond the ability of recognizing cryptic diversity. These advantages include improved consistency in appropriate use of species names (this paper, Stur and Spies 2011), association of multiple or unknown life stages (Zhou et al. 2007, Ekrem et al. 2010a, Pauls et al. 2010, Stur and Ekrem 2011), and an additional set of characters that can be used to identify and establish relationships among species.

    Our results emphasize that COI is an effective tool for species delimitation and recognition of cryptic species, but we choose not to discuss relationships among species based on this gene. COI has a weak phylogenetic signal and is unsuitable for inference of species relationships in the Chironomidae (Ekrem et al. 2007, 2010b). The COI trees generated in our analyses did not correspond well with our knowledge of Micropsectra phylogeny and many of the relationships between species had low (<50%) bootstrap support (Figs 1, S3). Furthermore, in the COI phylogenies of both the full (Fig. 1) and reduced (Fig. S3) data sets, the 2 outgroup Paratanytarsus species were nested within the Micropsectra, rendering this genus paraphyletic. Thus, we recognize the power of COI for species delimitation and that it can be quite effective at identifying higher relationships in certain taxa, but we have chosen not to focus on interpreting results from our COI or combined COI/CAD data set to avoid misinterpretations of the data with regard to phylogenetic relationships. Based on results of our analyses and those of others (Winterton et al. 2007, Winkler et al. 2009, Ekrem et al. 2010b), we think that CAD is most reliable for reconstruction and interpretation of phylogenetic relationships for the Chironomidae and some other dipteran families. Results based on our combined COI/CAD data did largely conform to our CAD-only results (Figs 2, S2). The few discrepancies that exist between the 2 methods of analysis probably are the result of conflicting phylogenetic signals from COI that partially mask more reliable species-group hypotheses based on the CAD marker alone. Consequently, all discussion of phylogenetic relationships is based on the nuclear CAD gene, shown by others to have a strong phylogenetic signal for estimating genus-level relationships within Chironomidae (Ekrem et al. 2010b).

    Revision and expansion of Micropsectra species groups

    Palearctic species of Micropsectra have traditionally been subdivided into species groups to aid in identification, species revisions, and classification. Recent authors have questioned the appropriateness and accuracy of these groups (Giłka 2001, Giłka and Paasivirta 2008, Ekrem et al. 2010b). Present molecular and morphological evidence shows a well supported atrofasciata group (e.g., Ekrem et al. 2010b, Stur and Ekrem 2006), but lines of evidence supporting monophyletic attenuata, recurvata, and notescens groups are less distinct (e.g., Giłka 2001, Giłka and Abramczuk 2006, Giłka and Paasivirta 2008, Ekrem et al. 2010b), particularly considering the recent synonymy of Krenopsectra and Parapsectra with the Micropsectra (Ekrem et al. 2010b). Additional problems with the groups become apparent when attempting to place Nearctic species in the Palearctic-derived classification system.

    Most Nearctic Micropsectra have not yet been described (Reiss 1995, our study). Many species awaiting description and some that have been described do not conform to the current species-group classification, which is based primarily on the adult male (e.g., Micropsectra spinigera Reiss, 1995, Micropsectra borealis (Kieffer, 1922), Micropsectra radialis Goetghebuer, 1939, and the parthenogenetic species Micropsectra sedna Oliver, 1976, and M. silvesterae). This situation highlights the need for a major revision of the genus and expanded species-group system.

    In our study and in the study by Ekrem et al. (2010b), molecular evidence indicated a paraphyletic recurvata group, with former recurvata group members M. recurvata and M. lacustris falling into genetically distinct clades (Figs 2, S2; figs 3, 4 by Ekrem et al. 2010b). If chironomid taxonomists choose to retain current species groups, perhaps Säwedal's original classification of a separate lacustris group (Säwedal 1975) was more appropriate than his later placement of M. lacustris in the recurvata group (Säwedal 1981). Molecular evidence from our CAD data set suggests placement of M. penicillata as sister species to M. lacustris. Micropsectra penicillata shares similarities with the recurvata group, as pointed out by preliminary classification of the undescribed species by J. Sublette in his personal collection (now part of UMSP), but distinct differences, which we pointed out in the taxonomic remarks for the species (see above) also exist. These morphological differences, combined with molecular evidence (Figs 2, S2), indicate an alternative grouping for the species.

    A further drawback of the existing species-group classification is that it is based primarily on the morphological characteristics of the adult male, and it was founded before molecular data were commonly used in systematics. The current species-group classification does not extend to immature stages (Pinder and Reiss 1986). Most chironomid descriptions historically have been based on the adult male, but descriptions of immature life stages are becoming increasingly important, particularly for purposes of biological monitoring and studies of species diversity (e.g., Ekrem et al. 2010a, Zhou et al. 2010).

    Chironomid pupal exuviae are a simple and effective method for assessing species diversity (e.g., Ferrington et al. 1991, Calle-Martinez and Casas 2006, Raunio et al. 2007, Ruse 2011), particularly since descriptions of pupal exuviae have become more common and readily available. We think that this life stage may provide important features that, combined with characters of the adult male and molecular data, may help resolve future species-group concepts. For example, when examining Fig. 2 (our study) and fig. 3 by Ekrem et al. (2010b), the exuviae of all members of the atrofasciata group that have described exuviae have long longitudinal spine patches on abdominal tergite IV. Conversely, exuviae known for the notescens group, with the exception of M. contracta and M. apposita, have short anterior patches consisting of spinules and points. Micropsectra contracta and M. apposita have semilong spines (none long enough to be synonymous with spine-type from the atrofasciata group) extending partway down tergite IV. Members of a reduced recurvata group, including M. recurvata and M. xantha, also fit the exuviae generalization of the notescens group, forming a recurvata–notescens clade. Adult male morphology clearly separates recurvata group members from the notescens group members, but we have not yet identified pupal characters that can be used to separate them. Furthermore, if the lacustris group is re-established with M. penicillata as a member, this group fits better with a combined lacustris–atrofasciata clade (Fig. 2). As with the recurvata–notescens clade, members of the lacustris–atrofasciata clade have similar spine patch patterns on the exuviae, with known species in this clade exhibiting long longitudinal spine patches on tergite IV. Adult males separate the lacustris group from the atrofasciata group, but no definite pupal characters distinguish the groups at this point. The results of our analysis of the concatenated data placed the lacustris group as sister to the atrofasciata, notescens, and recurvata groups (Fig. S2) rather than to the atrofasciata group. Placement of this group should become clearer as more species and markers are added to the data set. However, most importantly, all species groups we propose are strongly supported in both CAD and combined COI/CAD analyses.

    To expand our ideas further and to express the need for a revision and expansion of current species groups, M. radialis and M. borealis form a separate group in our phylogeny (Fig. 2) and that of Ekrem et al. (2010b). Moreover, the morphologies of the adult male and pupa are markedly different from those of other Micropsectra species mentioned here. Thus, these species do not to conform to any of the current species groups or the proposed system described above. Säwedal (1981) mentioned constructing a borealis group, based on redescription of M. borealis (Säwedal and Willassen 1980), but apparently, no subsequent treatment of the group was completed. Säwedal and Willassen (1980) suggested that M. borealis and M. radialis form a monophyletic clade with the atrofasciata group based largely on similarities of the superior volsella. We dispute placement of these species in the atrofasciata group. Instead, we propose erecting the borealis group, which currently includes M. borealis and M. radialis as members based on genetic similarity, morphological similarities of the male genitalia, and reasons outlined by Stur and Ekrem (2006).

    The former Parapsectra and Krenopsectra taxa were recently synonymized with Micropsectra (Ekrem et al. 2010b). When considering the former Parapsectra, all taxa less one form a monophyletic group. Only M. mendli (Reiss, 1983) falls outside of this group and forms its own clade (Fig. 2). Therefore, we suggest formation of a nana group that currently includes Micropsectra nana (Meigen, 1818), Micropsectra bumasta (Giłka & Jażdżewska, 2010), Micropsectra chionophila (Edwards, 1933), and Micropsectra uliginosa (Reiss, 1969).

    We are hesitant to place Micropsectra acuta Goetghebuer, 1934 firmly in the attenuata group (Fig. 2). The adult male of M. acuta shows similarities to other species in the attenuata group, and molecular results clearly indicate a linkage, but pupal morphology is somewhat different in that M. acuta lacks a laterally curved patch of longitudinal spines on tergite III as seen in the other attenuata-group species. In addition, M. acuta has no lateral taeniae on any tergite and has only lateral setae (a trait unusual for Micropsectra), and tergite III has only 4 setae rather than the usual 5. These differences lead us to think that as more Micropsectra species are described and sequenced for appropriate markers, M. acuta could form a new species group in Micropsectra, probably with Micropsectra fallax Reiss, 1969 and Micropsectra nohedensis Moubayed & Langton, 1896, both previously placed in Krenopsectra.

    Last, our analyses suggest that the placement of M. roseiventris is unique with respect to the rest of the Micropsectra, perhaps rendering the genus paraphyletic as currently classified (Figs 2, S2). Further research at the molecular and morphological levels is needed to confirm the placement and stance of this species in relation to the rest of the Micropsectra.

    Our results clearly suggest a change in the current species-group organization in Micropsectra. They indicate that a combination of morphological characters from adult male and pupal exuviae and molecular data from protein-coding markers is an appropriate approach to divide large Chironomidae genera into taxonomically useful groups that reflect likely genealogical relationships.

    Conclusions

    Our research demonstrates strong concordance between morphological species concepts, DNA barcodes, and CAD sequence data. We have used partial CAD sequences to confirm the species-divergence patterns detected with COI DNA barcodes. COI barcodes were not robust enough to relay information about relationships between species of Micropsectra, but concordance between CAD results and adult and pupal morphology was very strong. This strong concordance of CAD and morphological data is especially important considering that morphological phylogenetic analyses are of limited value for assessing species-level phylogenies of Chironomidae because of a high level of homoplasy in morphological data sets (Ekrem 2003, Stur and Ekrem 2006). As Micropsectra species continue to be described, revealing new morphological character combinations, and as the DNA barcode database for the genus is expanded, particularly with regard to Nearctic species and those in other less-studied localities, the taxonomic framework for Micropsectra will have to be reconsidered. This need for reconsideration probably will be a trend throughout other major genera of Chironomidae and, perhaps, other large families of aquatic insects. We anticipate further shifts and alterations in the current species-group classification system and a continued need for chironomid taxonomists to revisit the appropriateness of these groups. With recent and forthcoming advances in molecular taxonomy, taxonomic work that combines forces of molecular and morphological methods will rapidly advance our understanding of phylogenetic relationships in species-rich genera, such as Micropsectra, emphasizing the need for taxonomists to use all data available and to be well versed in both molecular and morphological techniques.

    Key to Known Nearctic Micropsectra Adult Males

    This key adopts several couplets given by Säwedal (1976) or Stur and Ekrem (2006), but some species and diagnostic characters have been added. Males of the Micropsectra sedna and M. silvesterae are unknown and, therefore, are not included in the key. Both of these species are parthenogenetic. We have elected not to arrange the key by species group at this time considering that many of the species included in the key either are not known in multiple life stages or are not available for inclusion in molecular analyses. We found it was necessary to format the last couplet in this key differently than previous couplets because of the extreme similarity in these 3 notescens-group species. AR  =  antennal ratio, LR  =  leg ratio. Morphological terminology and abbreviations follow Sæther (1980).

    1. Superior volsella deeply divided; anal point long and toothed (fig. 1 by Reiss 1995)M. spinigera Reiss, 1995

    1′. Superior volsella undivided; anal point not toothed2

    2. Setiger of superior volsella more or less circular (Fig. 3), sometimes with straight median margin3

    2′. Setiger of superior volsella triangular or finger-like (Fig. 5A), never with straight median margin6

    3. Mid and hind tibiae each with one obvious spur; digitus digitiform, extending well beyond median margin of superior volsella (fig. 2 by Säwedal and Willassen 1980)M. borealis (Kieffer, 1922)

    3′. Mid and hind tibiae without spurs; digitus digitiform or triangular, occasionally pointed, sometimes extending slightly beyond median margin of superior volsella (Fig. 3; fig. 12A, B by Stur and Ekrem 2006)4

    4. Median volsella large, thick, club-shaped; knob between crests of anal point present; digitus short, never reaching margin of median volsella (Fig. 3)M. neoappendica n. sp.

    4′. Median volsella small, thin, never club-shaped; knob between crests of anal point absent; digitus extending to or beyond margin of median volsella5

    5. Apex of median volsella not extending to or beyond that of inferior volsella (fig. 12A by Stur and Ekrem 2006)M. logani (Johannsen, 1928)

    5′. Apex of median volsella reaches to or beyond that of inferior volsella (fig. 7 by Oliver and Dillon 1994)M. polita (Malloch, 1919)

    6. Median volsella with leaf-like lamellae, sometimes held tightly together (Fig. 5A; fig. 8 by Säwedal 1976) or large and spatulate (fig. 3 by Reiss 1969a)7

    6′. Median volsella with spoon-shaped lamellae (Figs 6A, 7A, C)9

    7. Lamellae of median volsella large and spatulate; superior volsella triangular; digitus reduced, never reaching margin of median volsella (fig. 3 by Reiss 1969a)M. attenuata Reiss, 1969

    7′. Lamellae of median volsella leaf-like; superior volsella finger-shaped; digitus either absent or extending to or past margin of superior volsella8

    8. Lamellae of median volsella often held tightly together, appearing brush- or tail-like; digitus absent; median volsella long (103–138 µm) (Fig. 5A)M. penicillata n. sp.

    8′. Lamellae of median volsella not as above; digitus long, often extending to or past margin of superior volsella; median volsella short (23–31 µm) (fig. 8 by Säwedal 1976)M. lindrothi Goetghebuer, 1931

    9. Median volsella long and sinuous or s-shaped (Figs 5A, 7A)10

    9′. Median volsella of varying length, more-or-less straight and parallel sided (Fig. 6A)15

    10. Median volsella nearly as wide as inferior volsella; inferior volsella strongly curved medially, gradually narrowing to tip (fig. 1 in Oliver and Dillon 1994)M. dives (Johannsen, 1905)

    10′. Median volsella never as wide as inferior volsella; inferior volsella may or may not curve medially or narrow to tip11

    11. Inferior volsella either very slender or swollen at tip, appearing somewhat ball-shaped12

    11′. Inferior volsella of normal width14

    12. Inferior volsella very slender, distal part bent medially (fig. 3 by Reiss 1974)M. recurvata Goetghebuer, 1928

    12′. Inferior volsella swollen at tip, appearing somewhat ball-shaped (Fig. 7A)13

    13. Anal point slender and parallel-sided towards tip (Fig. 8B); superior volsella broad with long, narrow digitus; digitus <⅓ width of superior volsella (Fig. 8A)M. xantha (Roback, 1955)

    13′. Anal point relatively broad and cone-shaped toward tip (Fig. 8D); superior volsella comparatively narrow; digitus >⅓ width of superior volsella (Fig. 8C)M. connexa (Kieffer, 1906)

    14. Superior volsella finger-like (fig. 1 by Säwedal 1981); LR1  =  1.38–1.40; AR  =  1.57–1.73 (Säwedal 1981)M. tori Säwedal, 1981

    14′. Superior volsella comparatively triangular (fig. 2 by Oliver and Dillon 1994); LR1  =  1.50–1.69; AR  =  0.73–1.46M. geminata Oliver and Dillon, 1994

    15. Digitus short, rarely reaching margin of superior volsella (fig. 11 by Reiss 1969b, figs 22, 24 by Säwedal 1976); if digitus reaches close to margin of superior volsella, gonostylus is thick and stout and anal tergite with large lateral tooth or inferior volsella is enlarged at tip16

    15′. Digitus long, extending to margin of superior volsella or slightly beyond17

    16. Anal point short, triangular with blunt tip; inferior volsella enlarged at tip; digitus not extending beyond margin of superior volsella (fig. 7 by Oliver and Dillon 1994)M. nigripila (Johannsen, 1905)

    16′. Anal point long or acute; inferior volsella not enlarged at tip17

    17. Median volsella of medium length (57–78 µm); AR  =  1.04–1.49; gonostylus thick and stout, narrowing towards tip (fig. 24 by Säwedal 1976); anal tergite with lateral toothM. junci (Meigen, 1818)

    17′. Median volsella short (28–36 µm); AR  =  0.50–0.73; gonostylus not thick and stout, but may narrow towards tip; anal tergite without lateral toothM. nana (Meigen, 1818)

    18. Superior volsella triangular (fig. 17 by Säwedal 1976); anal tergite with small lateral tooth; middle tarsomere Ta1 with 5–7 sensilla chaeticaM. apposita (Walker,1856)

    18′. Superior volsella finger-shaped (Fig. 5A; fig. 2 by Säwedal 1976), more-or-less parallel at base; anal tergite with or without lateral tooth; middle tarsomere Ta1 with ≤5 sensilla chaetica19

    19a. Gonostylus narrowing toward tip (Fig. 6A); LR  =  1.52–1.54; anal tergite without lateral tooth; superior volsella lacking field of microtrichia on basal part; AR  =  1.47–1.70; digitus often extending to or past margin of superior volsella; median volsella 68–85 µmM. subletteorum n. sp.

    19b. Gonostylus not narrowing toward tip (fig. 2 by Säwedal 1976); LR  =  1.34–1.51; anal tergite with small lateral tooth; few microtrichia on basal part of superior volsella; AR  =  1.47–1.83; digitus extending to or past margin of superior volsella; median volsella  =  40–59 µmM. insignilobus Kieffer, 1924

    19c. Gonostylus not narrowing toward tip (fig. 2 by Säwedal 1976); LR  =  1.37–1.41; anal tergite with small-to-medium lateral tooth; large group of microtrichia on basal part of superior volsella; AR  =  1.75–2.14; digitus extending to or past margin of superior volsella; median volsella  =  63–78 µmM. brundini Säwedal, 1979

    Key to Known Nearctic Micropsectra Pupae and Pupal Exuviae

    This key adopts several couplets given by Säwedal (1976), Langton (1991), or Stur and Ekrem (2006), but several species and diagnostic characters have been added. Pupae of Micropsectra connexa, M. spinigera, and M. tori are unknown and, therefore, are not included in the key. We have elected not to arrange the key by species group at this time because many of the species included in the key either are not known in multiple life stages or are not available for inclusion in molecular analyses. Morphological terminology and abbreviations follow Sæther (1980).

    1. Abdominal segments II–V each with 4–7 lateral setae + taeniae (fig. 3 by Säwedal and Willassen 1980)M. borealis (Kieffer, 1922)

    1′. Abdominal segments II–V each with 3 lateral setae + taeniae (Fig. 5B)2

    2. Tergite II with shagreen in 2 triangular patches that are not connected anteriorlyM. sedna Oliver, 1976

    2.′ Tergite II extensively covered with shagreen, leaving only a median and usually lateral rounded patches free of shagreen (Fig. 5B)3

    3. Tergites III–VI with similarly paired, anterior point patches (fig. 10.54E by Pinder and Reiss 1986)M. nana (Meigen, 1818)

    3′. Tergite III with posterior spine patches (Fig. 4A, B)4

    4. TIV with long longitudinal spine patches extending from anterior spinule patches (Fig. 3A, B)5

    4′. TIV with only short anterior patches consisting of spinules and points (Fig. 6B)9

    5. Longitudinal spine patches of TIV extend ∼½ the length of the tergite; a row of fine shagreen extends posteriorly from each spine patch (fig. 20 by Säwedal 1976)M. apposita (Walker, 1856)

    5′. Longitudinal spine patches of TIV extend well over halfway down length of tergite; shagreenation not extending posteriorly from spine patches6

    6. TIII with 1 lateral setae and 2 lateral taeniaeM. logani (Johannsen, 1928)

    6′. TIII with 3 lateral setae; no taeniae7

    7. Longitudinal patches of TV consisting of shagreen, points, spinules, and often spines; spines, if present, similar in size to those of TIV; total patch length subequal to that of TIV (fig. 11 by Oliver and Dillon 1994)M. polita (Malloch, 1919)

    7′. Longitudinal patches of TV consisting only of spinules and points; length of patches less than those of TIV (Figs 4A, 5B)8

    8. Distance between anterior and posterior pairs of dorsocentrals is approximately equal; nose of wing sheath moderate to strongM. neoappendica n. sp.

    8′. Distance between anterior pair of dorsocentrals 2–3× greater than that of posterior pair and anterior pair thinner than posterior pair (Fig. 5F); nose of wing sheath weak to moderateM. penicillata n. sp.

    9. Prealar tubercle with finger or nose-like projection (fig. 5 by Reiss 1969a, fig. 25 by Säwedal 1976)10

    9′. Prealar tubercle without finger-like projection11

    10. Anal lobe with 23–31 lateral taeniae; tergites IV and V almost entirely covered with shagreen and spinules; the spinules of the anterior transverse patches similar in size, but more dense and darker than elsewhere on tergites (fig. 7 by Reiss 1969a)M. attenuata Reiss, 1969

    10′. Anal lobe with 46–52 (Langton 1991) or 62–73 (Säwedal 1976) lateral taeniae; tergites IV and V with distinct patches of spines or spinules that are stronger than surrounding shagreen (fig. 16 by Säwedal 1976)M. junci (Meigen, 1818)

    11. Tergite VI with a single, crescent-shaped anterior point patch; pleura without shagreen (fig. 5 by Oliver and Dillon 1994)M. dives (Johannsen, 1905)

    11′. Tergite VI with paired anterior point patches or only with paired longitudinal shagreenation; pleura with or without shagreen12

    12. Tergite IV with 3 lateral taeniae13

    12′. Tergite IV with 1 or 2 lateral taeniae16

    13. Shagreen absent between spine patches of TIII (fig. 146J by Langton 1991)M. recurvata Goetghebuer, 1928

    13′. Shagreen present between spine patches of TIII (Fig. 6B)14

    14. Tergite III with spinules (<40 µm) in paired, comparatively longitudinal patches, approximately equal in size to those on TIV and TV, the greater part of the spinule patch anterior to seta D5 (fig. 148C by Langton 1991); pedicel sheath tubercle weakly developedM. insignilobus Kieffer, 1924 and M. brundini Säwedal, 1979

    14′. Tergite III with spines (>40 µm) or spinules in paired patches that curve in lateral direction posteriorly, if spinules (<40 µm) present, they are noticeably larger than those on TIV and TV; the greater part of spine patch posterior to seta D5 or approximately equally distributed above and below seta D5 (Fig. 6B; fig. 148I by Langton 1991); pedicel sheath tubercle usually well developed15

    15. Anal lobe with >43 lateral taeniae (48–70, Langton 1991; 43–56, Säwedal 1976); thoracic horn >450 µm (495–710 µm, Langton 1991; 450 µm, Säwedal 1976)M. lindrothi Goetghebuer, 1931

    15′. Anal lobe with <48 taeniae; thoracic horn 378–553 µm; moderate nose on wing sheathM. xantha (Roback, 1955)

    16. Thoracic horn setae covering apical ⅔ and diminishing in length towards tip; setae short, less than ⅕ of horn length (fig. 10 by Oliver and Dillon 1994; fig. 2C by Langton 1998)17

    16′. Thoracic horn setae covering apical i2161-9565-32-3-892-e07.gif and not diminishing significantly in length toward tip; setae long, ∼⅓ of horn length (Fig. 6F; fig. 4 by Oliver and Dillon 1994)18

    17. Cephalothorax with 2 median antepronotal setae; wing sheath without nose; anal lobe with 48–66 taeniaeM. silvesterae Langton, 1998

    17′. Cephalothorax with 1 median antepronotal seta; wing sheath with small nose; anal lobe with 22–52 taeniaeM. nigripila (Johannsen, 1905)

    18. Tergite VI with paired anterior point patches (fig. 3 by Oliver and Dillon 1994); anal lobe with 31–36 taeniaeM. geminata Oliver and Dillon, 1994

    18′. Tergite VI consisting only of shagreen (Fig. 6B); anal lobe with 40–47 taeniaeM. subletteorum n. sp.

    Taxonomic note: Micropsectra brundini was described from oligotrophic lakes in Greenland (Säwedal 1979) and is morphologically similar to M. lindebergi and M. insignilobus in both the adult male and pupal stages; females of this species are not yet known. The type material of this species could not be located at ZSBS (M. Spies, ZSBS, personal communication). We attempted unsuccessfully to locate type material of this specimen at various other museums. However, we have examined material identified as M. brundini in the collections of Peter Langton, Claus Lindegaard, and the Sublette material at UMSP and confirm that this species is morphologically very similar to M. lindebergi and M. insignilobus. Values for the AR seem to be the most consistent and obvious differences used to separate M. brundini from M. lindebergi (1.75–2.14 and 1.31–1.58, respectively), and a longer median volsella aids in differentiation from M. insignilobus (63–78 vs 40–59 µm). Säwedal (1979) lists other characters that may aid in differentiation, but overlap between these species, particularly M. brundini and M. insignilobus, is considerable. Resolution of these taxonomic issues is beyond the scope of our paper, but we think sampling of localities known to produce M. brundini and subsequent molecular analyses of fresh specimens could help assess relationships among these species.

    Acknowledgements

    We thank Jim Sublette, Peter Langton, Trond Andersen, Martin Spies, Claus Lindegaard, Godtfred Anker Halvorsen, and Will Bouchard for loan or donation of material. We also give many thanks for the following funding sources that allowed this work to be completed: the USA–Norway Fulbright Foundation, the University of Minnesota Dayton Fund Research Fellowship, the Norway–America Association Norwegian Marshall Fund Award, a Philanthropic Educational Organization (P.E.O.) Scholar Award from the International Chapter of the P.E.O. Sisterhood, and a Torskeklubben of Minneapolis Graduate Fellowship. We also thank the team at the Canadian Centre for DNA Barcoding in Guelph for help with entering data in BOLD. Last, we thank Len Ferrington, Ralph Holzenthal, Roger Moon, Bruce Vondracek, and 3 anonymous referees for their comments and critique of our manuscript.

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    The Society for Freshwater Science
    Alyssa M. Anderson, Elisabeth Stur, and Torbjørn Ekrem "Molecular and morphological methods reveal cryptic diversity and three new species of Nearctic Micropsectra (Diptera:Chironomidae)," Freshwater Science 32(3), (16 July 2013). https://doi.org/10.1899/12-026.1
    Received: 15 February 2012; Accepted: 1 May 2013; Published: 16 July 2013
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