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Article

Back from the Past: DNA Barcodes and Morphology Support Ablabesmyia americana Fittkau as a Valid Species (Diptera: Chironomidae)

by
Elisabeth Stur
1,*,
Fabio Laurindo da Silva
1,2 and
Torbjørn Ekrem
1
1
Department of Natural History, NTNU University Museum, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway
2
Department of Zoology, Institute of Biosciences, University of São Paulo, São Paulo 05508-090, Brazil
*
Author to whom correspondence should be addressed.
Diversity 2019, 11(9), 173; https://doi.org/10.3390/d11090173
Submission received: 9 August 2019 / Revised: 16 September 2019 / Accepted: 17 September 2019 / Published: 19 September 2019
(This article belongs to the Section Animal Diversity)
Browse Figures
Versions Notes

Abstract

:
Short, standardized gene fragments for species identification (DNA barcodes) have proven effective in delineating closely-related insect species, and can be critical characters to include in taxonomic studies. This is also the case for the species-rich and widely distributed fly family Chironomidae (non-biting midges). Inspired by observed genetic differences in partial COI gene sequences between North American and European populations of the chironomid Ablabesmyia monilis sensu lato, we investigated whether or not the morphology of male and female adults supported the distinction of more than one species. Our results support that the junior synonym Ablabesmyia americana is a valid species separate from A. monilis, and that A. monilis sensu stricto is distributed both in the Palearctic region and in North America. We provide re-descriptions of all of the major life stages of A. americana and of the adult female of A. monilis.

Graphical Abstract

1. Introduction

Over the last ten years, molecular approaches to analyze species boundaries have seen rapid development. Genetic divergences in the partial cytochrome c oxidase subunit 1 (COI) sequences (so-called DNA barcodes [1]) have proven effective for species recognition in several animal groups, including non-biting midges belonging to the family Chironomidae (Insecta, Diptera) (e.g., [2,3,4,5,6]). In several cases, DNA barcodes have also aided the detection of presumably cryptic species, whose distinction a posteriori could be confirmed by both morphology and nuclear genetic markers (e.g., [7,8]). Furthermore, DNA barcodes have been shown suitable to associate life stages in the family Chironomidae [9,10,11].
Ablabesmyia is a large and ecologically diverse genus in the subfamily Tanypodinae. About 90 species are known worldwide ([12] and citations within), many of them described from the Neotropical Region [13]. These small to medium size dipterans (1.00–6.00 mm) are found in a broad range of aquatic systems, from small streams and ponds to lakes and rivers. Larvae can live in freshwater sponge colonies [14]. The genus was erected by Johannsen [15] with Tipula monilis Linnaeus, 1758, as the type species. Historically Ablabesmyia had a wider concept, with most of its members placed in the Pentaneura group A of Edwards [16] and Johannsen [17]. Later, Freeman [18] and Roback [19] relegated Ablabesmyia to subgeneric status in Pentaneura (Philippi, 1865), while Fittkau [20] gave it full generic status. Ablabesmyia larvae are predators, and are recorded in many ecological studies (e.g., [21,22,23,24]). The genus is one of the most distinctive and well-defined genera within the tribe Pentaneurini, with adult males possessing a homologous complex of dorsomedial blades and lobes in their genitalia unique to the taxon [25,26].
The genus Ablabesmyia comprises four valid subgenera: Ablabesmyia Johannsen, Asayia Roback, Karelia Roback and Sartaia Roback. However, Oliveira, Silva and Gessner [13] reported inconsistencies in the establishment of these subgroups, and suggested at least one additional subgenus for Ablabesmyia, without promoting this taxonomic action. Identification of the adult Ablabesmyia is primarily based on features of the genitalia, as it is for many other members of Chironomidae. The morphological identification can be challenging, particularly for non-experts, and normally requires time-consuming dissections of the genitalia. In addition, some Ablabesmyia species lack diagnostic morphological traits in their adult life stage, and need associated larvae and pupae to be morphologically identifiable to species-level. Despite these obstacles, the taxonomy of Ablabesmyia has been investigated in multiple geographic regions, especially in the Neotropics (e.g., [13,14,27,28,29,30,31]), providing identification keys to species-level, as well as extensive reference collections. This makes Ablabesmyia a good model taxon to investigate the congruence between molecular and morphological traits.
Ablabesmyia americana was erected by Fittkau [20] as a species separate from A. monilis (Linnaeus, 1758) based on the inner features of the male genitalia, but without specifying the exact characters. Subsequently, Roback [25] compared North American with European specimens, and was unable to find any valid differences to justify the distinction of a separate species for the New World specimens, synonymizing A. americana with A. monilis. Through the sampling and DNA barcoding of A. monilis populations in North America and Europe, we discovered considerable genetic divergence across the northern Atlantic region. We were therefore inspired to investigate if this divergence could be supported by the morphology of larvae, pupae and adult life stages, and if the synonymy of Ablabesmyia americana and A. monilis can be supported.

2. Materials and Methods

The specimens included in this study were selected to represent taxa with a possible taxonomic conflict in the Ablabesmyia gr. monilis sensu Fittkau [20]. Fieldwork was conducted near and in the streams, rivers, fens, ponds and lakes of eastern, central and northern Norway, and Churchill, northern Canada (Manitoba). The material was initially collected for inventory studies and part of the International Barcode of Life initiative. Fourth-instar larvae and pupae were collected with hand nets, and adults sampled with sweep nets, emergence and Malaise traps. Specimens from Japan identified to A. monilis were kindly provided by Hiromi Niitsuma through Bohdan Bilyj. A small piece of tissue for DNA extraction was sampled under a stereomicroscope and sent to the Canadian Centre for DNA Barcoding at the University of Guelph (CCDB, www.dnabarcoding.ca) for DNA isolation, polymerase chain reaction (PCR) and sequencing. The remnants of nearly all of the sampled specimens were then macerated in potassium hydroxide (KOH) and mounted on permanent microscope slides in Euparal for species identification with a compound microscope. Measurements were taken according to Epler [32]. Morphological terminology and abbreviations follow Roback [25] and Sæther [33], supplemented by Kowalyk [34] for larval cephalic setation (head hairiness) and Silva et al. [35,36] for larval terminology. One slide-mounted male and one slide mounted female specimen from the Johannsen collection out of Ithaca dated prior to his 1905 work were analyzed. Seven additional slides with larvae and two with females were also examined, but these either did not have their date or their locality written on the labels. Material from the Johannsen collection is deposited in the Cornell University Insect Collection, Ithaca, New York, USA (CUIC). Other examined material is deposited in the NTNU University Museum, Norwegian University of Science and Technology, Trondheim, Norway (NTNU-VM); or the Centre for Biodiversity Genomics, University of Guelph, Canada.
Sequence information along with an image and collateral information for each voucher specimen were entered into the Barcode of Life Data System (BOLD) [37]. The detailed specimen records and sequence information, including trace files, are available in BOLD through the dataset ‘DS-ABLA, Ablabesmyia americana and A. monilis: A review’ with doi: 10.5883/DS-ABLA. GenBank accessions are given in Table 1.
DNA extraction and amplification followed standard protocols at the CCDB and used the primers LepF1 + LepR1 [38], or a cocktail of the Lep and Folmer, et al. [39] primers (C_LepFolF + C_LepFolR) [40]. Polymerase chain reaction (PCR) products were purified for bidirectional sequencing with BigDye 3.1 termination (Applied Biosystems, Foster City, CA, USA) at the CCDB. Alignments were implemented on amino acid sequences, and refined using the Muscle algorithm in MEGA X [41], and were trivial, since no indels were present. Only sequences > 300 bp were included in the final alignment.
Maximum likelihood (ML) tree searches were performed using the Tamura 3-parameter with gamma correction for rate heterogeneity (T92+G). This model was determined to be the most suitable model of nucleotide substitution for our dataset using the hierarchical likelihood ratio test in MEGA X. Bootstrap analyses were conducted with 1000 pseudoreplicates [42]. Neighbor-joining tree searches and analyses of intra- and interspecific genetic divergences were conducted using the Kimura-2-parameter (K2P) model [43] for easier comparison with most published DNA barcode investigations. We used Ablabesmyia aspera (Roback, 1959) and A. longistyla Fittkau, 1962 as outgroups in the analysis, and rooted the resulting tree with A. longistyla, as this species is morphologically most divergent from A. monilis sensu lato. The Barcode Index Numbers [44] equivalent to genetic clusters in our resultant tree were analyzed in BOLD to investigate the documented distribution of each genetic lineage.

3. Results

Mitochondrial DNA COI (mtDNA COI) sequences were recovered from 56 specimens of four Ablabesmyia species (Table 1). Most of the aligned sequences were 658 base pairs long (94.6%) with 164 variable sites (24.9%), and included 137 sites (83.5%) being possibly parsimony informative. The majority of the variable sites were in the third codon-position (Table 2). The sequences were highly AT-biased, particularly in the third position, with a combined average AT-composition of 89.2% (Table 2). A hierarchical likelihood ratio test of aligned sequences in MEGA X returned the Tamura 3-parameter with the gamma correction for rate heterogeneity (T92+G) as the best model (−lnL = 2435.952, BIC = 6045.744, AIC = 5096.616).
Average intra- and interspecific K2P-distances for the examined Ablabesmyia species were 3.7% and 12.5%, respectively (Table 3). Maximum intraspecific divergence was found in A. aspera (8.0%). The lowest interspecific divergences were observed between Ablabesmyia americana and A. monilis (average 10.0%). Intraspecific and interspecific distances generated by the ML model yielded similar results (data not shown). There were no identical mtDNA COI sequences between species, and all species were distinguishable by genetic deviation and character state variances (Figure 1).
The ML tree shows large barcode divergence between all sampled Ablabesmyia species. Ablabesmyia americana and A. monilis comprised well-supported monophyletic clusters, completely concurring with the morphological identifications (Figure 1). For comparison, a Neighbor-joining tree was also generated using the K2P model, and produced identical trees (data not shown). Bootstrap support showed minor deviation between analyses, but were always 90% or higher for all species groups, except for the principal Ablabesmyia aspera group, which was endorsed by 84% of the bootstrap pseudoreplicates in the NJ-analysis. Nucleotide sequences from specimens recognized morphologically as Ablabesmyia americana and A. monilis differed by a minimum of 10.0%. The adult male of Ablabesmyia americana presents a shorter, less curved aedeagal blade, and slightly weaker, inferior volsella in the male hypopygium (Figure 2A,B), whereas A. monilis exhibits a longer, more strongly curved dorsal lobe, and more conspicuous inferior volsella. Regarding adult females, Ablabesmyia americana has less than one-fourth dark sclerotized seminal capsules as opposed to about one-half to two-thirds sclerotization in A. monilis (Figure 2C,D).
The specimens determined as Ablabesmyia monilis by morphology build at least two distinct barcode clusters (Figure 1). However, we found no evident morphological characters that aid separation the species in these groups. Specimens of Ablabesmyia monilis were collected in Japan and Norway (Table 1, Figure 1), but one of the clusters (BIN BOLD:ABY9333) includes 327 records from eastern and western Canada in BOLD. Finally, specimens of Ablabesmyia aspera, used as an outgroup in our analysis, were grouped into three separate barcode clusters (BINs BOLD:AAF3626, BOLD:AAF3627, BOLD:AAF3638). Specimens of Ablabesmyia aspera of these three clusters were collected in Canada and Norway (Table 1), and exhibited a clear geographical structuring of genetic diversity across the distribution range, with one of the group composed only by species from Norway (BIN BOLD:AAF3626).

Taxonomy

Ablabesmyia americana Fittkau, 1962
Ablabesmyia americana Fittkau, 1962: 430 [20] (proposed new name for A. monilis Johannsen nec Linnaeus).
Ablabesmyia americana Sublette and Sublette, 1965: 148 [45] (taxonomic placement).
Ablabesmyia americana Roback, 1971: 375 [25] (proposed junior synonym of Ablabesmyia monilis Linnaeus).
Material examined. Type material: Lectotype (designated here, CUIC): Adult male with pupal and larval exuviae, USA, New York, Ithaca, Cascadilla Pond, 28.vii.1902, O.A.J. Lot 1507. 1 Paralectotype: Female with pupal and larval exuviae (CUIC), as previous except for Cascadilla Creek, 30.vii.1902, O.A.J. Lot 1508.
Additional material: Adult female (Specimen ID: HLC-27246, NTNU-VM 201786), CANADA, Manitoba, Churchill, Farnworth Lake, 10 Km SE Churchill, N 58.7°, W 94.05°, 21.vii.2007, leg. unknown. Adult female (Specimen ID: CHIR_CH268, NTNU-VM 201781), as previous except for Ramsey Creek, Malaise trap, N 58.73054°, W 93.78007°, 13 m a.s.l., 15-17.vii.2006, leg. T. Ekrem and E. Stur. Larva (Specimen ID: BIOG03304-G12, CBG), as previous except for Ontario, Algonquin Provincial Park, stream by Leaf Lake Sign, Drainage stream in swamp, N 45.5517°, W 78.2696°, 350 m a.s.l., 26.vi.2011. leg. G. Martin and O. Zaheer. Larva (Specimen ID: BIOG01725-B09) as previous.
Diagnostic characters. Ablabesmyia americana differs from Ablabesmyia monilis by the following characters. Adult male: Hypopygium with short, moderately curved aedeagal blade and inconspicuous inferior volsella. Adult female: Genitalia with seminal capsules less than one-fourth dark sclerotized. Immature stages: Indistinguishable from most of the described species of Ablabesmyia.
Adult female (n = 2, except where otherwise stated)
Size. Total length 2.83–3.04 mm. Wing length 2.3–3.0 mm. Total length/wing length 1.09–1.24. Wing length/profemur length 2.72–2.93.
General coloration. Head pale brown with darker occipital margin; pedicel and antenna brown; maxillary palp pale brown. Thorax pale brown. Wing membrane transparent with dark spots. Femur with one brown band, tibia with three brown bands. Abdominal tergites and genitalia brown.
Head. Temporal setae 20–26, irregularly multiserial. Eye ratio 1.60–1.99. Tentorium 188–221 μm long. Clypeus 157–163 μm long, 96–111 μm wide at largest part, bearing 30–32 setae. Cibarial pump 353–372 μm long. Lengths of palpomeres 1–5 (in μm): 65–68; 125–146; 175–198; 120–150; 212 (1). Antenna 690 (1) μm long, diameter of pedicel 98 (1) μm. AR 0.43 (1).
Thorax. Antepronotals 7–8. Acrostichals 70–86, double staggered row which diverges posteriorly to join the dorsocentral row of setae; dorsocentrals 58–64, biserial anteriorly and uniserial posteriorly; prealars 32–44; supraalar 2. Anapleural suture ratio 0.65 (1). Scutellum with transverse row of 20–24 setae and group of numerous fine anterior setae. Scutal tubercle indistinct.
Wing. Width 0.81–0.96 mm. Costa 2.1–2.4 mm long. VR 0.86–0.94. WW 0.36–0.37. Brachiolum with 3–4 setae. Squama with 38–42 setae.
Legs. Foreleg: Width at apex of tibia 66–70 μm, tibia with single, apical and pectinate spur 50 (1) μm long, with 9 (1) lateral teeth; ta1–3 with preapical pseudospurs. Mid leg: Width at apex of tibia 67–70 μm, tibia with two apical spurs 36–37; 61–67 μm long, with 4–6 lateral teeth; ta1–3 with preapical pseudospurs. Hind leg: Width at apex of tibia 65–71 μm, tibia with two apical spurs 33–36; 70–71 μm long, with 3–6 lateral teeth; comb not indistinct; ta1–3 with preapical pseudospurs. Claws slender, distally recurved and pointed and with large basal protuberance. Lengths and proportion of leg segments as in Table 4.
Genitalia (Figure 2C). Gonapophysis VIII elongated, 119–122 μm long. Coxosternapodeme 91–102 μm long. Postgenital plate broadly rounded. Cerci oval-quadrate, 73 (1) μm long, 58 (1) μm wide, with 18–24 elongated setae. Labia with inconspicuous microtrichia. Notum 177–179 μm long. Seminal capsules oblong, 56–58 μm long, 51 (1) μm wide, less than one-fourth dark sclerotized, with conical-shaped necks. Length ratio SCa/No 0.31–0.32.
Pupa (n = 2, except where otherwise stated)
Size. Abdomen 3.6–3.8 mm long in male.
General coloration. Exuviae (pupal skins) mostly pale brown without any distinctive pattern; thoracic horn brown.
Cephalothorax (Figure 3A). Wing sheath smooth, 1.2–1.3 mm long. Thoracic horn 441–450 μm long and 223–238 μm wide (Figure 3A). THR 1.98–1.89. Respiratory atrium filling the lumen cavity, reticulation of respiratory atrium indistinct, lumen homogeneous, preapical papilla and plastron plate absent, aeropyle tube sinuate with single membranous lobe. External membrane with few spinules basally interconnected, forming scales. Basal lobe covered with small spinules. Thoracic comb with 11–13 conical tubercles (Figure 3A).
Abdomen (Figure 3B–D). Tergite I with scar 201–210 μm long. Shagreen on tergites serially arranged in groups of 2–4 (Figure 3B), evenly distributed. Sternites I and II with sparse shagreen. Abdominal chaetotaxy as in Figure 3C. Abdominal segment VII with 4 LS-setae. A VIII with 5 LS-setae. Anal lobe 496–523 μm long, 383–387 μm wide (Figure 3D). ALR 1.28–1.36. Male genital sac not surpassing apex of anal lobe.
4th instar larva (n = 4, except where otherwise stated)
General coloration. Head yellow, postoccipital margin brown. Ligula pale yellow, with apex brown. Abdomen pale yellow. Procercus pale brown along anterior margin.
Head (Figure 4A). Length 787–899 μm, 422–519 μm wide; IC 0.51–0.59. Dorsally DP present, S5 and S8 postero-mesal to S7. Ventrally S9, S10 and SSm forming a right angle (Figure 4A).
Antenna (Figure 4B). Length 405–483 (3) μm, A1 346–410 μm long, with ring organ located 0.51–0.62 from base, A2 54–63 (3) μm long. Peg sensilla very small. Blade and accessory blade extending somewhat beyond flagellum. AR 5.22–5.82.
Maxilla (Figure 4C). Maxillary palp with three segments, palpomere three reduced; palpomeres 1–2: 61–78; 44–46 μm long; palpal formula 1+2; P1/P2 1.41–1.69 (3); A1/P1 4.98–6.14 (3); A2/P1 0.79–0.81. Ring organ located between palpomere 1 and 2.
Mandible (Figure 4D). Length 131–139 μm. Large, bluntly rounded inner tooth, partly overlying seta subdentalis. Sensillum campaniformium located 0.68–079 from apex. AMD 2.48–3.08.
Mentum and M appendage. Dorsomentum sclerotized, without teeth. Labial vesicles oblong. Pseudoradula with fine granulation, not arranged in distinct longitudinal rows, 131 (3) μm long.
Hypopharyngeal complex (Figure 4E,F). Ligula with 5 teeth, 86–122 μm long, 44–56 μm wide at base; row of teeth concave, middle and inner teeth smaller in size, outer larger; inner teeth slightly curved outward (Figure 4E). IO 15–22, MO 17–21. Paraligula bifid, 45–52 (3) μm long, inner tooth 26–33 (3) μm long. Pecten hypopharyngis with 15–20 subequal teeth, corner tooth and middle teeth slightly broader than remainder (Figure 4F).
Body (Figure 4G). Without fringe of swim-setae. Procercus 96–132 (3) μm long, 31–44 (3) μm wide, with 7 anal setae 594–740 (3) μm long. L/W 2.86–3.19 (3). Anal tubules slender, 230 (1) μm long. Posterior parapod 391–456 μm long. Claws simple (Figure 4G), some with small spines on inner and/or outer margin, 2 of smaller claws dark brown.
Ablabesmyia monilis (Linnaeus, 1758)
Tipula monilis Linnaeus 1758: 587 [46] (description of adults).
Ablabesmyia monilis Johannsen 1905: 142 [15] (description of adults and immature stages).
Pentaneura monilis Tokunaga 1937: 44 [15] (description of adults).
Ablabesmyia monilis: Fittkau 1962: 437 [20] (description of adults and immature stages).
Ablabesmyia monilis Roback 1971: 375 [25] (adult male description).
Material examined. Adult female: (Specimen ID: ATNA330, NTNU-VM 201789), NORWAY, Oppland, Rondane National Park, Skranglehaugen (P2), N 61.98141°, E 9.80480°, 1119 m a.s.l., 14–21.vii.2008, leg. T. Ekrem. Adult female: (Specimen ID: NO92, NTNU-VM 200850) as previous except for Trøndelag, Trondheim, Gjeddvatnet, N 63.38272°, E 10.60776°, 152 m a.s.l. 20.vi.2010, leg. J. K. Skei.
Adult female (n = 2, except where otherwise stated)
Size. Total length 3.63–3.77 mm. Wing length 2.7–3.3 mm. Total length/wing length 1.09–1.40. Wing length/profemur length 2.93 (1).
General coloration. Head pale brown with darker occipital margin; pedicel and antenna brown; maxillary palp pale brown. Thorax pale brown. Wing membrane transparent with dark spots. Femur with one brown band, tibia with three brown bands. Abdominal tergites and genitalia brown.
Head. Temporal setae 36–40, irregularly multiserial. Eye ratio 1.46–1.56. Tentorium 244–258 μm long. Clypeus 179–183 μm long, 130–139 μm wide at largest part, bearing 50–56 setae. Cibarial pump 381–409 μm long. Lengths of palpomeres 1–5 (in μm): 69–74; 141–183; 178–235; 136–150; 258–268. Antenna 722–813 μm long, diameter of pedicel 103–106 μm. AR 0.40–0.41.
Thorax. Antepronotals 11–15. Acrostichals 88–96, double staggered row which diverges posteriorly to join the dorsocentral row of setae; dorsocentrals 82–92, biserial anteriorly and uniserial posteriorly; prealars 36–50; supraalar 2. Anapleural suture ratio 0.65–0.67. Scutellum with transverse row of 18–22 setae and group of numerous fine anterior setae. Scutal tubercle indistinct.
Wing. Width 0.98–1.14 mm. Costa 2.6–3.3 mm long. VR 0.87–0.91. WW 0.34–0.37. Brachiolum with 2–3 setae. Squama with 46–78 setae.
Legs. Foreleg: Width at apex of tibia 91 (1) μm, tibia with single, apical and pectinate spur 72 (1) μm long, with 7 (1) lateral teeth; ta1–3 with preapical pseudospurs. Mid leg: Width at apex of tibia 71–86 μm, tibia with two apical spurs 33–44; 68–93 μm long, with 4–6 lateral teeth; ta1–3 with preapical pseudospurs. Hind leg: Width at apex of tibia 65–93 μm, tibia with two apical spurs 44–47; 75–90 μm long, with 4–6 lateral teeth; comb not indistinct; ta1–3 with preapical pseudospurs. Claws slender, distally recurved and pointed and with large basal protuberance. Lengths and proportion of leg segments as in Table 5.
Genitalia (Figure 2D). Gonapophysis VIII elongated, 118–130 μm long. Coxosternapodeme 110–111 μm long. Postgenital plate broadly rounded. Cerci oval-quadrate, 68–73 μm long, 62–63 μm wide, with 20–24 elongated setae. Labia with inconspicuous microtrichia. Notum 202–206 μm long. Seminal capsules oblong, 82–83 μm long, 60–61 μm wide, one-half to two-thirds sclerotized, with conical-shaped necks. Length ratio SCa/No 0.40–0.41.

4. Discussion

The analysis of partial COI sequences supports the separate species status of Ablabesmyia americana and A. monilis. Moreover, we could associate the species A. aspera and A. longistyla to molecular markers. The ML tree revealed that the majority of the analyzed species are distinctly separated by mtDNA COI sequences, and there are indications of morphologically cryptic lineages within A. monilis and A. aspera.
The presence of a barcode gap is a key concept in DNA barcoding [47]. When the amount of intraspecific genetic divergence is substantially smaller than the amount of interspecific genetic divergence, a barcode gap exists [48]. In our study, the pairwise interspecific genetic distances in the sampled Ablabesmyia species were clearly higher than the intraspecific divergences, demonstrating a barcode gap.
The average K2P distance values found for conspecific comparisons (3.7%) was discordant from those found in the literature, which range from 0.1% to 0.8% [49]. However, this disparity can be a result of cryptic diversity among our sampled species (see below). The mean cutoff value for species delimitation is likely higher in Chironomidae than in many other insect groups, and previous studies have found that morphological species fit poorly with Barcode Index Numbers [50]. For example, Lin, Stur and Ekrem [6] suggest a 4–5% threshold for genetically separate species of Tanytarsus (Chironominae), while Song, et al. [51] considered a 5–8% cutoff appropriate to discriminate species of Polypedilum (Chironominae). Herein, we refrain from suggesting an average threshold to delineate the species of Ablabesmyia. Yet any cutoff should be used with caution, since unusual deep intraspecific divergences can indicate cryptic species. Broader sampling throughout the distribution range of A. monilis would provide valuable data for the further analysis of the species boundary for this species. Nevertheless, data currently in BOLD shows that one genetic lineage of A. monilis sensu stricto (BOLD:ABY9333) is widely distributed in western Canada. Other BINs within A. monilis have records from Europe (BOLD:AAU0774, BOLD:AAF3633), Japan (BOLD:ADB8891), and central Norway (BOLD:ACE5418).
Ablabesmyia moniliformis was established by Fittkau [20] on the basis of Japanese species treated as Pentaneura monilis (Linnaeus, 1758) by Tokunaga [52], which he considered different from the European species [53]. However, Fittkau proposed this new name without providing a diagnostic description, neither made he reference to one, nor designated a holotype. Yet the proposed name satisfies the provisions of Article 13.1.2 of the International Code of Zoological Nomenclature, and the name is available and considered a nomen dubium [29]. Moreover, it seems that the Pentaneura monilis Tokunaga [52] type series may, in fact, comprises two or perhaps three species, including Ablabesmyia monilis, A. prorasha Kobayashi and Kubota, 2002 and A. jogancornua Sasa and Okazawa, 1991 [29]. Furthermore, Tokunaga [52] did not describe nor illustrate the form of the aedeagal complex, a crucial feature for the diagnosis of the Ablabesmyia species. This has prevented the identification of these species unequivocally, and is likely the reasons that the name moniliformis has been overlooked (or disregarded) by Japanese peers [53]. Based on our results, there is evidence supporting a separate cluster of A. monilis including records from Japan and Norway. This could represent one of the species in Tokunaga’s Ablabesmyia monilis specimens, and thus A. moniliformis. However, no morphological differences could be determined between these two barcode clusters. In addition, the material used by Tokunaga [52], deposited in the Kyushu University Museum, is deteriorated by the long-term storage in alcohol [29]. This prevents the comparison of our material with the Ablabesmyia moniliformis type series. The observed pattern could also be explained by the restricted geographical sampling of A. monilis throughout its distributional range. Therefore, additional material is needed to understand the variation between the specimens of A. monilis and closely-related species throughout their current distribution. Lastly, the female of Ablabesmyia alba, described by Chaudhuri, et al. [54] for India, resembles A. monilis by having seminal capsules with about one half to two thirds dark sclerotized, and may not be readily separable from this species. Further investigations using molecular data and morphological characters from all life stages will likely reveal the relationship of A. alba with A. monilis.
In addition to the divergences observed within A. monilis, there are two well-separated genetic lineages of A. aspera in Canada (BOLD:AAF3627 and BOLD:AAF3628), and a slightly divergent group with Norwegian specimens (BOLD:AAF3626), indicating cryptic species or divergence caused by geographical isolation in this group. Ablabesmyia aspera belongs to the monilis group sensu Roback, [19] and can be distinguished from all the species of that group by the longer, heavier aedeagal blades and the dorso-mesally approximated dorsal lobes, in natural position [19]. The species is widely distributed in Canada and the United States [25]. In Europe, Ablabesmyia aspera is only recorded from Norway [55].
DNA-aided species determination has become regular practice in various fields of study, including agriculture [56], bioengineering [57] and conservation biology [58]. Moreover, DNA barcodes are recognized as a powerful tool to separate species in a range of chironomid groups [5,6,10,11,51,59,60,61,62]. We found mtDNA COI valuable in exploring sequence diversity and distinguishing morphologically similar species within the genus Ablabesmyia. Furthermore, DNA barcodes indicate further cryptic species in Ablabesmyia, or deep intraspecific divergence caused by geographical isolation. We believe that broader geographical sampling and the inclusion of nuclear genes will strengthen our findings and provide additional evidence for the geographical structuring of genetic lineages in Ablabesmyia.

Author Contributions

Conceptualization, E.S., F.L.d.S. and T.E.; methodology, E.S, F.L.d.S. and T.E.; analysis, E.S., F.L.d.S. and T.E.; data curation, E.S. and T.E.; writing—original draft preparation, F.L.d.S.; writing—review and editing, E.S., F.L.d.S. and T.E.; visualization, E.S., F.L.d.S. and T.E.; project administration, E.S.

Funding

DNA barcode data in this publication were in part generated in collaboration with the Norwegian Barcode of Life Network (NorBOL), funded by the Research Council of Norway and the Norwegian Biodiversity Information Centre. F.L. Silva was supported by fellowships from the São Paulo Research Foundation (FAPESP - 2016/07039-8 and 2018/01507-5).

Acknowledgments

Thanks to the team at the Canadian Centre for DNA Barcoding for help with DNA barcode analysis, and to Paul Hebert for initiating the chironomid barcoding of Arctic midges in Churchill through a Natural Sciences and Engineering Research Council of Canada (NSERC) Polar Year Grant. Thanks also to James Liebherr at Cornell for providing a loan of Johannsen’s original material. The Norwegian Biodiversity Information Centre funded biosurveillance projects through the Norwegian Taxonomy Initiative, from which some of the specimens we analyzed originate. We would also like to thank Bohdan Bilyj and Hiromi Niitsuma for supplying us with specimens from Japan, and Martin Spies for providing us with valuable literature.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Maximum likelihood (ML) tree for species of Ablabesmyia, based on partial COI sequences (DNA barcodes) and using the Tamura 3-parameter model with gamma correction for rate heterogeneity. Numbers on branches are bootstrap values > 80%.
Figure 1. Maximum likelihood (ML) tree for species of Ablabesmyia, based on partial COI sequences (DNA barcodes) and using the Tamura 3-parameter model with gamma correction for rate heterogeneity. Numbers on branches are bootstrap values > 80%.
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Figure 2. Male. (A) Ablabesmyia americana, (B) Ablabesmyia monilis: Aedeagal blade. Female. (C) Ablabesmyia americana, (D) Ablabesmyia monilis: Seminal capsules.
Figure 2. Male. (A) Ablabesmyia americana, (B) Ablabesmyia monilis: Aedeagal blade. Female. (C) Ablabesmyia americana, (D) Ablabesmyia monilis: Seminal capsules.
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Figure 3. Ablabesmyia americana Fittkau. Pupa: (A) Thoracic horn with basal lobe and thoracic comb. (B) Shagreen on tergites. (C) Female abdominal segments with chaetotaxy, dorsal view. (D) Anal lobe and male genital sac, ventral view.
Figure 3. Ablabesmyia americana Fittkau. Pupa: (A) Thoracic horn with basal lobe and thoracic comb. (B) Shagreen on tergites. (C) Female abdominal segments with chaetotaxy, dorsal view. (D) Anal lobe and male genital sac, ventral view.
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Figure 4. Ablabesmyia americana Fittkau. Larva: (A) Head with chaetotaxy. Left: Ventral view, right: Dorsal view. (B) Antenna. (C) Maxillary palp. (D) Mandible. (E) Ligula and paraligula. (F) Pecten hypopharyngis. (G) Dark claws of posterior parapod.
Figure 4. Ablabesmyia americana Fittkau. Larva: (A) Head with chaetotaxy. Left: Ventral view, right: Dorsal view. (B) Antenna. (C) Maxillary palp. (D) Mandible. (E) Ligula and paraligula. (F) Pecten hypopharyngis. (G) Dark claws of posterior parapod.
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Table
Table 1. List of analyzed specimens with associated sample localities, Specimen IDs and GenBank accessions.
Table 1. List of analyzed specimens with associated sample localities, Specimen IDs and GenBank accessions.
TaxonCountryLife StageBOLD IDNTNU-VMGB Accession
Ablabesmyia americanaCanadaAdult10PROBE-11666 JF875917
Ablabesmyia americanaCanadaAdult10PROBE-12218 JF876334
Ablabesmyia americanaCanadaAdult10PROBE-12910 JF876913
Ablabesmyia americanaCanadaAdult10PROBE-16464 KR655494
Ablabesmyia americanaCanadaMale adultCHIR_CH10201777MK403766
Ablabesmyia americanaCanadaMale adultCHIR_CH174201778MK403744
Ablabesmyia americanaCanadaMale adultCHIR_CH228201779MK403740
Ablabesmyia americanaCanadaMale adultCHIR_CH267201780MK403767
Ablabesmyia americanaCanadaFemale adultCHIR_CH268201781MK403747
Ablabesmyia americanaCanadaMale adultCHIR_CH396201782MK403752
Ablabesmyia americanaCanadaMale adultHLC-27231201785KR433631
Ablabesmyia americanaCanadaFemale adultHLC-27246201786KR433665
Ablabesmyia americanaCanadaMale adultHLC-27252201790KR443910
Ablabesmyia asperaCanadaAdult10PROBE-10650 JF875206
Ablabesmyia asperaCanadaAdult10PROBE-11271 JF875757
Ablabesmyia asperaCanadaAdult10PROBE-12725 JF876793
Ablabesmyia asperaCanadaAdult10PROBE-12912 JF876915
Ablabesmyia asperaCanadaAdult10PROBE-13831 KR668901
Ablabesmyia asperaNorwayMale adultATNA539201788MK403756
Ablabesmyia asperaNorwayMale adultCH-OSF172143691JN285991
Ablabesmyia asperaNorwayMale adultCH-OSF174143693MK403755
Ablabesmyia asperaCanadaMale adultCHIR_CH638201783MK403753
Ablabesmyia asperaCanadaMale adultHLC-27096201784KR435725
Ablabesmyia asperaNorwayFemale adultSOE94124714HQ105015
Ablabesmyia longistylaNorwayMale adultTRD-CH185200177MK403739
Ablabesmyia monilisNorwayFemale adultATNA330201789HM421433
Ablabesmyia monilisNorwayFemale adultATNA538201787MK403749
Ablabesmyia monilisJapanFemale adultES358201791MK403757
Ablabesmyia monilisJapanMale adultES360201793MK403737
Ablabesmyia monilisJapanMale adultES361201794MK403738
Ablabesmyia monilisNorwayMale adultFinnmark213136311JF870860
Ablabesmyia monilisNorwayMale adultFinnmark214136312JF870861
Ablabesmyia monilisNorwayMale adultFinnmark258136355JN285993
Ablabesmyia monilisNorwayMale adultFinnmark458136554JN286007
Ablabesmyia monilisNorwayMale adultFinnmark508136603JN286012
Ablabesmyia monilisNorwayMale adultFinnmark568136663JF870930
Ablabesmyia monilisNorwayMale adultFinnmark721136814JN286021
Ablabesmyia monilisNorwayMale adultNO 104200861MK403758
Ablabesmyia monilisNorwayFemale adultNO 92200850MK403743
Ablabesmyia monilisNorwayMale adultRIN_CH13148138MK403765
Ablabesmyia monilisNorwayMale adultRIN_CH15148140MK403760
Ablabesmyia monilisNorwayMale adultRIN_CH29148153MK403748
Ablabesmyia monilisNorwayLarvaRIN_CH30148154MK403741
Ablabesmyia monilisNorwayLarvaRIN_CH32148156MK403754
Ablabesmyia monilisNorwayLarvaRIN_CH34148158MK403764
Ablabesmyia monilisNorwayLarvaRIN_CH50148174MK403759
Ablabesmyia monilisNorwayMale adultSOE10124308HQ105016
Ablabesmyia monilisNorwayMale adultSOE9124307HQ105017
Ablabesmyia monilisNorwayMale adultTRD-CH158200150MK403761
Ablabesmyia monilisNorwayFemale adultTRD-CH221200211MK403750
Ablabesmyia monilisNorwayMale adultTRD-CH292200278MK403763
Ablabesmyia monilisNorwayMale adultTRD-CH308200294MK403762
Ablabesmyia monilisNorwayFemale adultTRD-CH309200295MK403751
Ablabesmyia monilisNorwayMale adultTRD-CH325200310MK403742
Ablabesmyia monilisNorwayMale pupaTRD-CH427200410MK403745
Ablabesmyia monilisNorwayFemale adultTRD-CH8148261MK403746
Table
Table 2. Variable and informative sites, and average nucleotide composition in the aligned COI gene sequences.
Table 2. Variable and informative sites, and average nucleotide composition in the aligned COI gene sequences.
Nucleotide PositionVariable
Sites (%)		Informative Sites (%)T (%)C (%)A (%)G (%)AT (%)GC (%)
1st15.213.927.21626.430.453.646.4
2nd1.90.04327.413.61656.643.4
3rd82.986.1478.442.22.489.210.8
All24.9%83.5%39.117.327.416.266.533.5
Table
Table 3. Intra- and interspecific Kimura 2-parameter distances between Ablabesmyia species defined by morphology. N/A denotes species with only one specimen analyzed.
Table 3. Intra- and interspecific Kimura 2-parameter distances between Ablabesmyia species defined by morphology. N/A denotes species with only one specimen analyzed.
SpeciesAverage Intraspecific
K2-P Divergence		Maximum Intraspecific
K2-P Divergence		K2-P Divergence
to Nearest
Neighbor		Average Interspecific
K2-P Divergence
Ablabesmyia aspera3.7%8.0%10.8%12.6%
Ablabesmyia americana0.6%1.3%10.0%11.9%
Ablabesmyia longistylaN/AN/A14.4%16.2%
Ablabesmyia monilis4.2%8.6%10.0%12.5%
Table
Table 4. Lengths (in μm) and proportions of leg segments in Ablabesmyia americana Fittkau, female (n = 1–2).
Table 4. Lengths (in μm) and proportions of leg segments in Ablabesmyia americana Fittkau, female (n = 1–2).
Leg Positionfetita1ta2ta3
p1837924743450329
p2985–1126982–1125778508329
p3898–10751226–13811137657473
Leg Positionta4ta5LRBVSV
p12291530.802.152.37
p22271680.692.462.89
p32911810.822.242.16
Table
Table 5. Lengths (in μm) and proportions of leg segments for Ablabesmyia monilis (Linnaeus, 1758) (n = 1–2).
Table 5. Lengths (in μm) and proportions of leg segments for Ablabesmyia monilis (Linnaeus, 1758) (n = 1–2).
Leg PositionFetita1ta2ta3
p1113413831079661480
p21114–1397887–1413556–1028557–626386–456
p31163–11741177–1246876–1492500–908341–637
Leg Positionta4ta5LRBVSV
p13072100.782.172.33
p2261–297177–1960.63–0.731.85–2.442.73–3.60
p3230-380153–2240.74–1.201.82–2.631.62–2.67

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MDPI and ACS Style

Stur, E.; da Silva, F.L.; Ekrem, T. Back from the Past: DNA Barcodes and Morphology Support Ablabesmyia americana Fittkau as a Valid Species (Diptera: Chironomidae). Diversity 2019, 11, 173. https://doi.org/10.3390/d11090173

AMA Style

Stur E, da Silva FL, Ekrem T. Back from the Past: DNA Barcodes and Morphology Support Ablabesmyia americana Fittkau as a Valid Species (Diptera: Chironomidae). Diversity. 2019; 11(9):173. https://doi.org/10.3390/d11090173

Chicago/Turabian Style

Stur, Elisabeth, Fabio Laurindo da Silva, and Torbjørn Ekrem. 2019. "Back from the Past: DNA Barcodes and Morphology Support Ablabesmyia americana Fittkau as a Valid Species (Diptera: Chironomidae)" Diversity 11, no. 9: 173. https://doi.org/10.3390/d11090173

APA Style

Stur, E., da Silva, F. L., & Ekrem, T. (2019). Back from the Past: DNA Barcodes and Morphology Support Ablabesmyia americana Fittkau as a Valid Species (Diptera: Chironomidae). Diversity, 11(9), 173. https://doi.org/10.3390/d11090173

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