Research Article |
Corresponding author: Olavi Kurina ( olavi.kurina@emu.ee ) Academic editor: Vladimir Blagoderov
© 2015 Olavi Kurina, Erki Õunap, Kadri Põldmaa.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kurina O, Õunap E, Põldmaa K (2015) Two new Neuratelia Rondani (Diptera, Mycetophilidae) species from Western Palaearctic: a case of limited congruence between morphology and DNA sequence data. ZooKeys 496: 105-129. https://doi.org/10.3897/zookeys.496.9315
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Two new Mycetophilidae species, Neuratelia jabalmoussae sp. n. and Neuratelia salmelai sp. n. are described on the basis of material collected from Lebanon, Estonia and Finland. Detailed figures of male terminalia and photographs of general facies are provided along with discussions of their morphological distinction from sibling species. For the first time molecular characters are used to distinguish new fungus gnat species. Molecular analysis relies on cytochrome oxidase subunit one (COI) but has additionally been corroborated by information from the 28S and ITS2 regions of nuclear ribosomal DNA. Situations where morphological and molecular data provide conflicting evidence for species delimitation are discussed. A new country record from Georgia is provided for Neuratelia caucasica.
Mycetophilidae , Neuratelia , new species, Western Palaearctic, systematics, molecular analysis, COI, ITS2, 28S
The genus Neuratelia Rondani, 1856 forms a well delimited clade in the subfamily Sciophilinae (Mycetophilidae), as sister group to the remaining Sciophilinae (Borkent & Wheeler, 2013). According to
So far, alpha-taxonomy of fungus gnats has been carried out using traditional taxonomic methods, primarily morphological examination. Though in recent years nucleotide data have been implemented to address the phylogeny of this group (e.g.
The aim of this article is to publish taxonomic and faunistic information about Western Palaearctic Neuratelia specimens that the senior author has accumulated over recent years. Both morphological and molecular data were used for species delimitation. This resulted in describing two new species – one from Estonia and Finland and another from Lebanon.
The examined material of two new species was collected from Estonia and Finland using Malaise traps, and from Lebanon by light trapping, respectively. The Estonian locality lies at the herb rich edge of a mixed forest (Fig.
All specimens were stored initially in ethyl alcohol within which parts of them – after studying under a stereomicroscope Leica S8APO – are still preserved. For more detailed study of male terminalia, they were detached and macerated in a 10% solution of KOH, followed by neutralization and washing in distilled water. The remaining chitinous parts were thereafter inserted into glycerine for study, including black and white illustrations, and preserved as glycerine preparations in polyethylene microvials (see also
The habitus photos have been made in an alcohol medium using a Canon 7D camera with a Canon MP-E65 (F2.8 1–5×) lens (see
The material is deposited in the Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences [former Institute of Zoology and Botany], Tartu, Estonia (IZBE), in the Zoological Museum, University of Turku, Finland (ZMUT) and in the personal collection of J. Salmela, Rovaniemi, Finland (JSPC).
The genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany). Anterior segments of the abdomen that had been stored after dissection of genitalia were crushed and used for the extraction. This process was carried out following the manufacturer’s instructions for extraction of genetic material from mammalian tissue.
In total, one mitochondrial and two nuclear markers were sequenced. A 658-bp ’barcoding’ fragment from close to the 5’ terminus of the mitochondrial gene cytochrome C oxidase subunit 1 (COI), was amplified and sequenced using primers LCO1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and HCO2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) (
Consensus sequences were created with Geneious R7 (Biomatters Ltd., Auckland, New Zealand) or Sequencher 5.1 (Gene Codes, Ann Arbor, MI, USA). Sequences were double-checked by eye and aligned using ClustalW (
For COI, a neighbour-joining tree implementing Kimura 2-parameter model (a standard model analysing DNA barcode data, see e.g.
For the concatenated dataset, data were first divided into three subsets according to the markers used (COI, 28S and ITS). Thereafter, PartitionFinder 1.1.1 (
The morphology of studied material distinguished three previously known species of Neuratelia and a group of specimens, clearly delimited by characters of male terminalia. This group, represented by specimens from Estonia and Finland, resembles the widespread N. nemoralis (Meigen, 1818) and hereafter described and referred to as a new species – N. salmelai sp. n. In addition, another group of three specimens from different localities in Jabal Moussa Biosphere Reserve (Lebanon) had slight differences from N. caucasica Zaitzev, 1994 – a species only known from Caucasus. In the latter case, the species is described as N. jabalmoussae sp. n. but the morphological differences are diminutive underpinning the necessity of including DNA sequence data for species discrimination.
Sequencing the ‘barcode region’ of COI was successful for all specimens included in the current study. The success rate was lower for ITS2 and 28S rDNA, as all attempts to sequence 28S failed for one individual of N. nemoralis, and for a few specimens, only half of 28S or ITS was obtained (Table
Neighbour-joining tree of the COI ’barcode’ region of Neuratelia spp. Scale bar: Kimura 2-parameter genetic distance. Bootstrap supports are presented above the branches. Maximum likelihood analysis of the concatenated (COI, 28S, ITS2) dataset yielded a tree with similar topology, bootstrap supports for the divergencies obtained in this analysis are given below the branches. Support values inferior to 60 are not shown.
Voucher numbers, depositories and GenBank accession codes of studied Neuratelia specimens. COI: cytochrome oxidase subunit I; 28S: 28S rRNA; ITS2: internal transcribed spacer 2.
Species | Voucher No | Depository | COI | 28S | ITS2 |
---|---|---|---|---|---|
Neuratelia minor | IZBE0200274 | IZBE | KP715935 | KP715924 | KP715947 |
Neuratelia nemoralis | IZBE0200283 | IZBE | KP715936 | KP715925 | KP715948 |
Neuratelia nemoralis | IZBE0200284 | IZBE | KP715937 | KP715926 | KP715949 |
Neuratelia nemoralis | MYCE-JS-2013-0095 | ZMUT | KP715938 | KP715927 | KP715950 |
Neuratelia nemoralis | IZBE0200286 | IZBE | KP715939 | × | KP715951 |
Neuratelia nemoralis | IZBE0200285 | IZBE | KP715940 | KP715928 | KP715952 |
Neuratelia nemoralis | MYCE-JS-2013-0235 | ZMUT | KP715941 | KP715929 | KP715953 |
Neuratelia salmelai | IZBE0200253 | IZBE | KP715942 | KP715930 | KP715954 |
Neuratelia salmelai | MYCE-NV-2013-0131 | ZMUT | KP715943 | KP715931 | KP715955 |
Neuratelia jabalmoussae | IZBE0200250 | IZBE | KP715944 | KP715932 | KP715956 |
Neuratelia caucasica | IZBE0200255 | IZBE | KP715945 | KP715933 | KP715957 |
Neuratelia caucasica | IZBE0200262 | IZBE | KP715946 | KP715934 | KP715958 |
Holotype. 1♂, LEBANON, Kesrouane Mar Elias, 34°03'06,9"N, 35°46'00,5"E, 1138 m a.s.l., at light, 27.v.–4.vi.2012, J. Kullberg leg. (IZBE0200250, slide mounted in Euparal with terminalia in glycerine). Paratypes. 1♂, LEBANON, Kesrouane Mar Geryes, 34°03'20,9"N, 35°44'28,9"E, 749 m a.s.l., at light, 26.v.–2.vi.2012, J. Kullberg leg. (IZBE0200251, in alcohol with terminalia in glycerine);1♂, LEBANON, Kesrouane Ghbele, 34°03'25,5"N, 35°43'02,5"E, 884 m a.s.l., at light, 26.v.–30.v.2012, J. Kullberg leg. (IZBE0200252, in alcohol).
Male (Fig.
Head dark brown, with numerous pale to yellowish setae. Three ocelli in a shallow and wide triangular arrangement, with laterals separated from eye margins by a distance slightly more than their own diameter. Face conical, about 0.8 times as wide as maximum height; clypeus rectangular, about 0.6 times as wide as high; both brown, setose, with setae on clypeus stronger than those on face. Mouthparts yellow. Palpus five segmented, yellowish with apex of fifth segment brownish. Ratios of three apical palpal segments 1.0: 1.68–1.85, 1.77 [1.85]: 1.97–2.00, 1.98 [2.00]. Scape and pedicel light brown to brown, flagellomeres light brown, with short yellowish setae. First flagellomere yellowish at basal third. Flagellum evenly tapering; first flagellomere clavate, 2.9–3.1 times as long as broad apically, 2–13 flagellomeres cylindrical, fourth flagellomere about 1.7–2.5 times as long as broad, apical flagellomere slightly conical, 4.8–5.4 times as long as broad at base.
Thorax. All parts brown to dark brown, all setae yellow to light brownish. Mesonotum with evenly arranged numerous setae. Scutellum wholly setose with about 10 stronger setae along the margin, not arranged to distinct pairs. Antepronotum with 8–9 and proepisternum with 4–7 setae of unequal size, laterotergite with 22–26 setae and mediotergite with ca 14–16 setae medially on lower part. Other pleural parts bare. Halteres pale yellow, setose.
Legs. All coxae yellow, basally infuscated. All trochanters brown. All femora and tibiae yellow, tarsi seem considerably darker because of dense setae. Foretibia with 1–2 ad, 0–2 d and 2–3 pd. Midtibia with 4–7 a, 2–3 d, 1–2 av and 2–4 pd. Hind tibia with 7–8 a, 1–2 ad (1 at apex), 4–6 d, 0–1 pd, 5 p and with a posterior apical comb of setae. Ratio of femur to tibia for fore-, mid- and hind legs: 0.86–0.94, 0.9 [0.9]; 0.77–0.86, 0.82 [0.77]; 0.71–0.75, 0.73 [0.75]. Ratio of tibia to basitarsus for fore-, mid- and hind legs: 0.91–0.94, 0.93 [0.94]; 1.26–1.3, 1.27 [1.3]; 1.51–1.54, 1,53 [1.51].
Wing hyaline, length 5.0–5.03, 5.02 [5.03] mm (n=3). All veins brown, costal and radial veins somewhat darker. Both surfaces of veins setose, except bare bM-Cu and r-m. Wing membrane with micro- and macrotrichia on both surfaces. Costa reaches very little from R5 to M1. Sc reaches costa at about one sixth between Rs and tip of R1. R5 sinuate. Rs about as long as crossvein r-m. M1 basally obsolete: observable vein begins distally from middle of R1. Cubital fork begins proximally from apex of Sc.
Abdomen with tergites brown and sternites yellowish. Tergites 6–7 somewhat darker. Terminalia (Figs
Male terminalia of N. jabalmoussae sp. n., dorsal view (7) and ventral view (8). Scale bar = 0.1 mm. Abbreviations: cerc = cerci; gc = gonocoxite; gc vam = ventroapical margin of gonocoxite; gc vl = ventroapical lobe of gonocoxite; gst db = dorsal branch of gonostylus; gst ib = internal branch of gonostylus; gst mb = medial branch of gonostylus; gst vb = ventral branch of gonostylus; tg 9 = IX tergite.
Male terminalia of N. jabalmoussae sp. n. (9, 10), N. caucasica Zaitzev, 1994 (11, 12) and N. minor (Lundström, 1912) (13, 14). 9, 11, 13 internal view of gonostylus 10, 12, 14 ventral view of ventral branch of gonostylus. Scale bars = 0.1 mm. Abbreviations: gst db = dorsal branch of gonostylus; gst ib = internal branch of gonostylus; gst mb = medial branch of gonostylus;gst vb = ventral branch of gonostylus; vb il = internal lobe of ventral branch of gonostylus; vb ll = lateral lobe of ventral branch of gonostylus; vb ml = medial lobe of ventral branch of gonostylus.
Female. Unknown.
Unknown.
The species is named after the type locality in Jabal Moussa Biosphere Reserve, Lebanon; the specific epithet is a noun in genitive case.
Neuratelia jabalmoussae sp. n. is very similar to N. caucasica, into which it also runs to in the key by
GEORGIA. 2♂♂ 2♀♀, Surami, 42°01'34,2"N, 043°29'52,5"E, 941 m a.s.l., sweeping, O. Kurina leg. 18.v.2012 (IZBE0200255– IZBE0200258, in alcohol); 2♂♂ 1♀, Borjomi, 41°50'9,2"N, 043°19'56,7"E, 936 m a.s.l., sweeping, O. Kurina leg. 21.v.2012 (IZBE0200259, ♂, on pin with terminalia in glycerine; IZBE0200260, IZBE0200261, in alcohol); 3♂♂ 4♀♀, Mtirala NP, near visitor centre, 41°40'20,7"N, 041°52'31,8"E, 465 m a.s.l., sweeping, O. Kurina leg. 20.v.2013 (IZBE0200262, ♂, slidemounted in Euparal with terminalia in glycerine; IZBE0200263–IZBE0200268, in alcohol); 1♂ 2♀♀, Kintrishi NP, 41°45'11,7"N, 041°58'38,4"E, 453 m a.s.l., sweeping, O. Kurina leg. 22.v.2013 (IZBE0200269–IZBE0200271, in alcohol).
Having been described from North Caucasus (Krasnodarsk region), the species has not been recorded since and the studied material represents the first records from Georgia. According to male terminalia the species is close to N. minor and N. jabalmoussae sp. n.
SLOVAKIA. 1♂, NP Muránska planina, Murán 3.5 km NE, sweeping, 48°45'46,5"N, 020°04'55,9"E, 483 m a.s.l. 30.v.2009, O. Kurina leg. (IZBE0200272, on pin with terminalia in glycerine; earlier published in
Neuratelia minor was described and figured by
Holotype. 1♂, ESTONIA. Palupõhja, Kaha (ME 57), Malaise trap, 58°25'54,68"N, 026°14'28,90"E, 29.vi.–8.vii.2009, V. Soon leg. (IZBE0200253, slide mounted in Euparal with terminalia in glycerine). Paratypes. 1♂, FINLAND. Lkor: Sodankylä, Kaita-aapa, Malaise trap, 67°50'45,5"N, 026°33'17,6"E, 5.vi.–3.vii.2012, J. Salmela leg. (IZBE0200254, in alcohol with terminalia in glycerine); 1♂, FINLAND. Lkoc: Kittilä, Kielisenpalo, Malaise trap, 68°01'16,6"N, 025°03'46,9"E, 26.vi–24.vii.2007, J. Salmela leg. (MYCE-NV-2013-0093 in ZMUT, in alcohol with terminalia in glycerine); 1♂, FINLAND. Lkoc: Kittilä, Vuotsonperän-jänkä, Malaise trap, 67°37'15,9"N, 025°26'43,6"E, 25.vi.–24.vii.2009, J. Salmela leg. (MYCE-NV-2013-0131 in ZMUT, in alcohol with terminalia in glycerine); 1♂, FINLAND, Lkor: Sodankylä, Pomokaira 67°52'19,2"N, 026°12'46,8"E, 11.6.-10.7.2013, J. Salmela leg. Malaise trap Salix swamp with seepages (DIPT-JS-2014-0199 in JSPC, in alcohol).
Male (Figs
Head and thorax of N. salmelai sp. n. Scale bar = 0.5 mm. Abbreviations: anepst = anepisternum; aprnt = antepronotum; clyp = clypeus; cx = coxa; fc = face; flgm = flagellar segment; hlt = halter; ltg = laterotergite; mtepst = metepisternum; mtg = mediotergite; oc = ocellus; ped = pedicell; plp = palpal segment; preepst = preepisternum; proepst = proepisternum; sc = scutum; scp = scape; vrt = vertex; wg = wing.
Head (Fig.
Thorax (Fig.
Legs. All coxae yellow with basal fourths brown. In case of two paratypes, cx3 entirely light brownish with darker basal half. All trochanters brown. All femora and tibiae yellow, tarsi seem darker because of dense setae. Foretibia with 2–3 ad, 1–3 d and 2–3 pd. Midtibia with 6–10 a, 0–4 d, 4–5 av and 2–3 pd. Hind tibia with 8–10 a, 1–2 ad (1 at apex), 7–8 d, 5–7 p and with a posterior apical comb of setae. Ratio of femur to tibia for fore-, mid- and hind legs: 0.86–0.91, 0.88 [0.91]; 0.72–0.87, 0.80 [0.87]; 0.72–0.77, 0.75 [0.72]. Ratio of tibia to basitarsus for fore-, mid- and hind legs: 0.81–1.00, 0.9 [0.9]; 1.22–1.33, 1.27 [1.22]; [1.66].
Wing hyaline, length 4.1–5.0, 4.52 [4.49] mm (n=4). All veins brown, costal and radial veins somewhat darker. Both surfaces of all veins setose. Wing membrane with micro- and macrotrichia on both surfaces. Costa reaches very little from R5 to M1. Sc reaches costa at about quarter between Rs and tip of R1. R5 sinuate. Rs about as long as crossvein r-m. M1 basally obsolete: observable vein begins distally from middle of R1. Cubital fork begins proximally from apex of Sc.
Abdomen with tergites brown to dark brown and with sternites yellow to brownish yellow. Terminalia (Figs
Female. Unknown.
Unknown.
The species is named in honour of Dr. Jukka Salmela, who kindly provided us the material collected from Finland.
Following the key by
FINLAND. 1♂, Lkor: Savukoski, Törmäoja, Malaise trap, 67°50'48,5"N, 029°28'20,8"E, 14.vi–10.vii.2012, J. Salmela leg. (MYCE-JS-2013-0095 in ZMUT, in alcohol with terminalia in glycerine); 1♂, Lkor: Sodankylä, Tarmpomapää, Malaise trap, 67°59'14,0"N, 025°55'09,4"E, 1.–29.ii.2009, J. Salmela leg. (MYCE-JS-2013-0235 in ZMUT, in alcohol with terminalia in glycerine); 1♂, Ab: Turku, Pomponrahka, Malaise trap, 2011, J. Salmela leg., (IZBE0200286, in alcohol with terminalia in glycerine). ESTONIA. 2♂♂, Matsalu NP, Matsalu village, window trap, 58°44'04"N, 023°42'42"E, 29.v–17.vi.2009, I. Süda leg. (IZBE0200275, IZBE0200276, in alcohol); 7♂♂, Palupõhja, Kaha (ME 57), Malaise trap, 58°25'54,68"N, 026°14'28,90"E, 31.v–15.vi.2009, V. Soon leg. (IZBE0200277– IZBE0200282, in alcohol; IZBE0200283, slide mounted in Euparal). SLOVAKIA. 1♂, NP Slovenský raj, Javorina Mt., sweeping, 48°53'23,1"N, 020°15'20,8"E, 1112 m a.s.l., 4.vi.2009, O. Kurina leg (IZBE0200284, in alcohol with terminalia in glycerine; earlier published in
This is a widely distributed species in the Palaearctic region (
This study combines for the first time the results of morphological and molecular analyses for delineating species of fungus gnats. As a common practice in insect taxonomy, we relied on characters of male genitalia and the mitochondrial COI barcoding, respectively. In one case, however, these two types of data provided conflicting evidence for species delimitation in the fungus gnat genus Neuratelia. Therefore, additional characters were sought by sequencing also the 28S and ITS2 regions of the nuclear ribosomal DNA. The latter is becoming increasingly applicable in delimitation of insect taxa (e.g.
Taxonomic work on insects has mostly been carried out on the basis of morphological examination. In many cases where studying external characters fail to yield unequivocal results, genital morphology has been proven to be valuable source of additional information (
The situation with N. salmelai and N. nemoralis is, however, much more intriguing. Regarding these species, there are five COI barcode haplotypes in our data matrix that have a maximum 1% genetic distance. Specimens of N. salmelai and N. nemoralis are impossible to distinguish from each other on the basis of barcode data, as the holotype of N. salmelai from Estonia carries COI sequence that is identical to that of two specimens of N. nemoralis from two different regions of Finland. A Finnish paratype of N. salmelai, on the other hand, has COI sequence identical to that of a specimen of N. nemoralis from Slovakia. Such a situation has been called ‘barcode sharing’ in literature (e.g.
In contrast to the failure of genetic markers to distinguish N. salmelai and N. nemoralis, their male terminalia were remarkably different. The differences are more pronounced than among the other three species included in this study. Most likely these taxa represent recently diverged species that still share the genetic diversity of their common ancestor. The evolution of insect genitalia can be more rapid than diversification of commonly studied markers (
The study was supported by the grants 9174 and 8583 of the Estonian Science Foundation by institutional research funding (IUT21-1, IUT20-30 and IUT20-33) of the Estonian Ministry of Education and Research and the European Union through the European Regional Development Fund (Centre of Excellence FIBIR). We are grateful to Dr. P. Vilkamaa (Helsinki, Finland) and Dr. J. Salmela (Turku, Finland) for providing material collected from Lebanon and Finland, respectively. Prof. T. Saigusa (Fukuoka, Japan) and Dr. J. Kjærandsen (Tromsø, Norway) are thanked for invaluable comments on the species. We are grateful to J. Kullberg (Helsinki, Finland) for collecting the material of N. jabalmoussae and for sending the photographs of collecting locality in Lebanon and to Prof. T. Tammaru (Tartu, Estonia) for intermediating this. We thank R. Puusepp (Tartu, Estonia) for technical assistance with sequencing. Dr. R. Davis (Tartu, Estonia) kindly checked the English language of the manuscript.