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Research Article
Reinstatement of Leuctra biellensis Festa, 1942 (Plecoptera, Leuctridae)
expand article infoGilles Vinçon, Louis Boumans§, Jean-Luc Gattolliat|
‡ Unaffiliated, Grenoble, France
§ University of Oslo, Oslo, Norway
| Museum of Zoology, Palais de Rumine, Lausanne, Switzerland
¶ University of Lausanne, Lausanne, Switzerland
Open Access

Abstract

Both molecular and morphologic characters support the reinstatement of Leuctra biellensis Festa, 1942 as a valid species distinct from Leuctra nigra (Olivier, 1811). Genetic distances between L. biellensis and the different populations of L. nigra are around 9%, while intraspecific distances among L. nigra haploclades are less than 1%. Morphologically, the two species can be separated in male adult specimens by the shape of the two teeth on tergite VIII, by the lateral edges of tergites and by the distal expansion of the paraprocts. Leuctra biellensis occurs on the southern slope of the Alps in Italy and Switzerland (Ticino and Graubünden), while L. nigra has a wide distribution in Central and Northern Europe. As the type material of L. biellensis was lost, and to avoid future confusion between the two species, we designate as neotype a male imago collected at the type locality.

Key Words

Stoneflies, Switzerland, Italy, Alps, Leuctra nigra, neotype

Introduction

The SwissBOL (Swiss Barcode of Life) is an ongoing Project, started in 2011, which aims at inventorying the genetic biodiversity of all taxa occurring in Switzerland. Against the background of global warming and other anthropogenic pressures that are threatening freshwater aquatic biodiversity, Ephemeroptera (mayflies), Plecoptera (stoneflies) and Trichoptera (caddisflies) are considered as particularly vulnerable groups (Tierno de Figueroa et al. 2010, Conti et al. 2014, Errochdi et al. 2014). Between 2013 and 2015, DNA barcode sequences were obtained for 90 of the 112 stonefly species reported from Switzerland (Gattolliat et al. 2016). Complementary field searches were done in 2015 and 2016 to elucidate several cases of isolated specimens on the gene tree. First results showed high intraspecific genetic distances between some populations preliminary identified as Leuctra nigra (Olivier, 1811). It was hypothesized that two species were involved, one in the North of the Alps and one in the South (Gattolliat et al. 2016).

Leuctra biellensis Festa, 1942 was originally described from Val Chiobbia in the Piedmont, Northern Italy. This taxon was subsequently considered as a junior synonym of L. nigra by Consiglio (1967). The present reinstatement of L. biellensis is based both on genetic evidence and on distinctive morphological characters shared by all the specimens occurring along the southern slope of the Alps. New detailed comparative descriptions are given for L. biellensis and L. nigra.

The original description of Leuctra biellensis was based on a single male imago collected by F. Capra. Type material was reportedly deposited at Museo Civico di Storia Naturale “Giacomo Doria”, Genova, Italia (Festa 1942). However, we were unable to locate the type. According to the present curator of the entomological collection, it may be lost or may have never been deposited (Maria Tavano, comm. pers. 2015). In order to stabilize the nomenclatural concept of L. biellensis and to avoid future confusion between L. biellensis and L. nigra, we designate a neotype for L. biellensis, collected as close as possible to the type locality (article 75.3, ICZN 1999). The neotype and some of the topotypes were deposited at the Museo Civico di Storia Naturale “Giacomo Doria”, Genova, Italy.

Material and methods

Molecular study: DNA was extracted from specimens stored in the collection of the Museum of Zoology in Lausanne, using a non-destructive method allowing a posteriori morphological identification. 658 bp of the mitochondrial protein-coding gene cytochrome c oxydase subunit I (CO1) was amplified using the primers LCO1490 and HCO2198 (for details see Gattolliat et al. 2016).

Additional sequences were downloaded from the Barcode of Life Data System (BOLD) database, notably four sequences from Bavaria, projects Barcoding Fauna Bavarica (Hendrich et al. 2010) and Germany Malaise Trap 05 [GMGRC] (Geiger et al. 2016), two Belgian and four Norwegian sequences from the project ‘Norwegian Barcoding of Life [NorBOL] – Freshwater Insects’ (Boumans and Brittain 2012), and one more Swiss sequence from the project ‘West Palaearctic Plecoptera [WPPLE]’. All specimens are listed in Table 1. Sequences of Leuctra hippopus Kempny, 1899 and Leuctra pseudorosinae Aubert, 1954 were added to the data matrix as outgroups, as these have the most similar mitochondrial haplotypes according to Gattolliat et al. (2016).

Specimens used for the phylogenetic analysis of the mitochondrial gene CO1.

Species Sample ID Collectors Date Country Province Exact Site Lat / Lon Elev Accession number
Leuctra biellensis LN_TI_15 P. Baumann 5/30/2007 Switzerland Graubunden Cavagliasch, Cavaglia 46°21’45”, 10°02’43” 1690 GBIFCH00238522
Leuctra biellensis LN_TI_16 P. Baumann 5/30/2007 Switzerland Graubunden Cavagliasch, Cavaglia 46°21’45”, 10°02’43” 1690 GBIFCH00238523
Leuctra biellensis LN_TI_17 H. Vicentini 6/2/2006 Switzerland Ticino Quelle Vignino, Montagnola 45°58’06”, 8°55’03” 460 GBIFCH00238524
Leuctra biellensis LN_TI_18 H. Vicentini 6/2/2007 Switzerland Ticino Quelle Vignino, Montagnola 45°58’06”, 8°55’03” 460 GBIFCH00238525
Leuctra biellensis LN_TI_19 H. Vicentini 6/19/2010 Switzerland Ticino Quelle Pian Sagno Acquacalda 46°32’04”, 8°50’41” 1665 GBIFCH00238526
Leuctra biellensis EPT-344 R. Tester Ryf 6/1/2009 Switzerland Ticino Ticono, Mte. Ceneri 46°08’11”, 8°54’04” 680 GBIFCH00277633
Leuctra hippopus EPT-321 S. Knispel 4/7/2010 Switzerland Fribourg Ruisseau, Villargiroud 46°41’22”, 6°59’39” 795 GBIFCH00280313
Leuctra hippopus EPT-322 P. Stucki 4/16/2010 Switzerland Vaud Veyron, La Chaux 46°37’07”, 6°28’33” 540 GBIFCH00280288
Leuctra hippopus EPT-323 V. Lubini 4/1/2009 Switzerland Zürich Wehrenbach, Zürich 47°21’10”, 8°35’02” 500 GBIFCH00280295
Leuctra nigra NHMO-EPT_89 B. Koese & L. Boumans 3/18/2010 Belgium Limburg Rekem, Ziepbeek 50°55’28”, 5°39’15” 50 ca KY250738
Leuctra nigra NHMO-EPT_80 B. Koese & L. Boumans 3/18/2010 Belgium Limburg Rekem, Ziepbeek 50°55’28”, 5°39’15” 50 ca KY250735
Leuctra nigra NHMO-EPT_55 L. Boumans 5/12/2010 Norway Hedmark Galten, bekk til Galthaen 61°53’54”, 11°46’27” 655 KY250740
Leuctra nigra NHMO-EPT_1553 T. Ekrem 7/20/2010 Norway Finnmark Sør-Varanger, Sameti, Sametijohka 69°24’04”, 29°43’09” 45 KY250739
Leuctra nigra NHMO-EPT_982 L. Boumans, T. Ekrem 7/28/2010 Norway Finnmark Lebesby, Kunes, Austerelva 70°20’37”, 26°31’09” 10 KY250741
Leuctra nigra M200612 G. Vincon 6/20/2010 Switzerland Jura Jura Massif, Tramelan, Gruere lake tributaries 47°14’17”, 7°03’01” 1000 ca KY250737
Leuctra nigra NHMO-EPT_462 L. Boumans 5/26/2010 Norway Oppland O Heimdalsvatn, stream to outflow 61°53’54”, 8°53’51” 1095 KY250736
Leuctra nigra EPT-342 S. Knispel 5/18/2010 Switzerland Vaud Affl. Bressonne, Les Censières 46°34’24”, 6°42’50” 865 GBIFCH00277517
Leuctra nigra EPT-343 V. Lubini 5/21/2009 Switzerland St-Gallen Tüfenbach, Tüfi 47°19’21”, 9°12’44” 800 GBIFCH00277566
Leuctra nigra BC_ZSM_AQU_00196 M. Hess, U. Heckes & M. Franzen 7/3/2009 Germany Bavaria Vorderer Bayerischer Wald 48.909, 13.011 900 HM421988
Leuctra nigra BC_ZSM_AQU_00747 M. Hess & U. Heckes 7/16/2009 Germany Bavaria Mangfallgebirge 47.687, 12.056 990 HQ563171
Leuctra nigra BC_ZSM_AQU_001124 M. Hess & U. Heckes 4/10/2011 Germany Bavaria Hinterer Bayerischer Wald 48.94, 13.41 770 KY261279
Leuctra nigra BIOUG03760-E10 G. Sellmayer 6/8/2012 Germany Bavaria Bayerischer National Park 48.9509, 13.422 840 KY261521
Leuctra pseudorosinae EPT-532 J.-P. Reding 5/26/2014 Switzerland Vaud Ste Croix, Affluent Dénériaz, la Merla 46°50’55”, 6°31’33” 1200 GBIFCH00280264
Leuctra pseudorosinae EPT-533 J.-P. Reding 5/26/2014 Switzerland Vaud Ste Croix, Affluent Dénériaz, la Merla 46°50’55”, 6°31’33” 1200 GBIFCH00280174
Leuctra pseudorosinae EPT-534 J.-P. Reding 5/26/2014 Switzerland Vaud Ste Croix, Affluent Dénériaz, la Merla 46°50’55”, 6°31’33” 1200 GBIFCH00279996

The final data matrix included 25 COI sequences of 587–658 bp (no gaps or missing data). Analyses were conducted in MEGA7 (Kumar et al. 2016). We used uncorrected p distances to calculate genetic distances between haplotypes, and within and between major haploclades (Srivathsan and Meier 2012).

Tree topology was reconstructed using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrap, 1000 replicates).

Morphology: Ethanol-preserved specimens of L. nigra and potential L. biellensis from the collections of the Museum of Zoology in Lausanne (MZL), Delmastro (Del), Murányi (Mur), Ravizza (Rav) and Vinçon (Vin) were examined. Since 2016, all of Ravizza’s collection is housed in the MZL. Neotype, as well as five male and five female imagoes are deposited at the Museo Civico di Storia Naturale “Giacomo Doria”, Genova, Italy. The full list of examined specimens is given in the results section.

Results

Molecular study

The mitochondrial phylogeny clearly recovers L. nigra and L. biellensis as distinct monophyletic clades (bootstrap of 100%), with intraspecific distances below 1% (Table 2). Distances between haplotypes of L. biellensis and haplotypes of L. nigra range from 8.8 to 9.1%, while distances between these species and L. pseudorosinae and L. hippopus, are between 13 and 15% (Table 2). Distances between populations within L. nigra are always under 1% even in the case of geographically distant sites such as Finnmark in Norway and Bavaria in Germany (Table 2).

Estimates of evolutionary divergence between geographical clusters of Leuctra nigra and Leuctra biellensis using uncorrected p distances. Minimum and maximum distances are indicated in brackets.

L. nigra Germany & CH L. nigra Norway L. nigra Belgium L. biellensis L. hippopus L. pseudorosinae
L. nigra Germany & CH 0.000
(0.000–0.001)
L. nigra Norway 0.007 0.001
(0.002–0.010) (0.000–0.009)
L. nigra Belgium 0.004 0.004 0.000
(0.000–0.007) (0.002–0.009) (0.000)
L. biellensis 0.091 0.088 0.091 0.001
(0.090–0.094) (0.085–0.090) (0.090–0.092) (0.000–0.002)
L. hippopus 0.141 0.139 0.141 0.135 0.000
(0.140–0.143) (0.138–0.140) (0.141) (0.135) (0.000)
L. pseudorosinae 0.154 0.152 0.155 0.128 0.133 0.001
(0.153–0.157) (0.152–0.153) (0.155) (0.124–0.131) (0.131–0.136) (0.000–0.007)

Morphological re-examination and redescription

Leuctra nigra (Olivier, 1811)

Figs 1, 2a–g

Nemoura nigra Olivier 1811: 186

Leuctra acuminata Bengtsson, 1933 (syn. fide Brinck 1949).

Leuctra nigra Oliv., Mosely 1932: 27, figs 39–40, pl. V/26; Kühtreiber 1934: 76, fig. 55; Despax 1951: 140, fig. 61c-d; Illies 1955: 68, fig. 61; Aubert 1959: 65, figs 159–160; Mendl 1968: 70; Kis 1974: 87, 88, figs 37a–e; Ravizza and Vinçon 1998: 303, fig. 6a–f; Teslenko and Zhiltzova 2009, 223, figs 1235–1239; Lubini et al. 2012: 180, fig. 433, 196, fig. 477.

Locus typicus: France, Versailles.

Material examined

FRANCE: BRITANY: Finistère: Rosnoen, Toulencoat, 22.03.2001, 6♂ (Manach leg). VOSGES MASSIF: Vosges: SW Colmar, > Wasserbourg, Krebsbach, Fecht trib., Ill trib., 700 m, 17.05.1995, 10♂, 13♀; Ballon d’Alsace, Saut-de-la-Truite, 23.08.1996, 1♂, 8♀ (Vin). MASSIF-CENTRAL: Saône-et-Loire: Morvan Massif, Haut-Folon, 800 m, 14.07.1994, 5♂, 7♀ (Vin); Puy-de-Dôme: Forez Mountains, > Vertolaye, > Chansert Pass, Jasserie de la Croix Fossat, 1500 m, 17.07.1994, 3♂, 7♀; St Pierre-la-Bourlhonne, cross road to the Chansert Pass, Dore trib., 17.07.1994, 1♀; W. Clermont-Ferrand, between Pontaumur and La-Forêt, b., 21.04.2009, 1♂, 5♀ (Vin). Loire: Mont Pilat, Graix, 900 m, 17.07.1994, 1♀; Mont Pilat, Crêt de la Perdrix, Gier b., 1400 m, 17.07.1997, 3♀ (Vin). Ardèche: Mont Gerbier-de-Jonc, Loire spring, 16.06.2001, 2♀; Lachamp-Rafael, Bois de Cuze, peat bog, 1350 m, 15.06.2001, 4♂, 6♀ (Vin). Cantal: Plomb-du-Cantal Massif, > Super Lioran, 1300 m, 30.07.2004, 3♀ (Vin). Tarn: NW slope of Pic de Nord, Arnette trib., Thoré trib., 850 m, 1.05.1991, 4♂, 2♀ (Vin). Aude: > Citou, spring, trib. of Argent-double, 900 m, 1.07.1989, 1♀, 1.05.1991, 20♂, 3♀ (Vin). JURA MASSIF: Ain: La Vattay Ski Station, Faucille Pass, 1300 m, 30.05.1991, 1♂, 27.06.1995, 1♀, 13.07.1996, 1♀ (Vin); Valserine, Combe de Mijoux, Le Planet, 1200 m, 19.07.1991, 3♀; Valserine, Joux Verte, 19.07.1991, 2♀ (MZL); Doubs: Jougne, Swiss Border, Les Tavins, La Tavine, Orbe trib., 900 m, 28.06.1995, 1♀ (Vin). ALPS: Haute-Savoie: Montet Pass, > Vallorcine, Eau-Noire trib., 1450 m, 21.06.1991, 2♂, 15♀ (Vin); Savoie: Allevard, Val Pelouse, 1400 m, 7.06.1987, 10♂, 1♀; Saisie Pass, Nant Rouge trib., 1600 m, 22.05.1991, 1♂, 1♀ (Vin). Isère: Chartreuse Massif, < Charmette Pass, Tenaison b., Vence trib., 1200 m, 28.05.1988, 1♂ (Vin); Belledonne Massif, below Oursière Cascade, Doménon trib., brook, 1500 m, 4.06.1995, 1♂, 1♀; < Chamrousse, Fénérieux b., Prémol trib, between Prémol Forest House and Luitel Lake, 1200 m, 10.06.1985, 1♂, 3♀; < Chamrousse, Premol Forest House, Sonnant trib., 1100 m, 10.06.1985, 1♀, 24.05.1998, 1♀, 19.05.2013, 7♂, 5♀, 2.06.2013, 2♂, 1♀ (Vin). SWITZERLAND, JURA MASSIF: NW Tramelan, Gruère Lake, b. right side of the lake, 20.06.2012, 5♂, 11♀ (Vin). GERMANY: Fulda, 1981, 2♂, 1♀ (Zwick leg), SLOVAKIA: Vysoké Mountains, Tatry, Podspady, Javorinka r., 11.07.1967, 5♂, 5♀ (Steinmann & Mur).

Figure 1. 

Maximum Likelihood (ML) consensus tree reconstructed for 25 specimens of Leuctra spp. Tree drawn to scale, branch lengths measured in number of substitutions per site, deeper nodes labelled above branches with Maximum Likelihood bootstrap support.

Complementary description

Based on French specimens: Savoie, Allevard, Val Pelouse, 1400 m, 7.06.1987. Male (Fig. 2a–f): Tergite VI with two small triangular appendices pointing upwards and backwards in side view (Fig. 2b). Tergite VII with non-interrupted anterior margin and wide median bell-shaped membranous area. Tergite VIII with two strong teeth pointing upwards and backwards in side view (Fig. 2a, b, f); teeth sub-rectangular in dorsal view (Fig. 2f). Tergite IX with anterior margin interrupted on nearly half segment width, lateral edges triangular ending in sharp angles near anterior margin. Lateral lobes of paraprocts with a sclerotized hook-shaped expansion turning backwards and extending along distal part of specilla (Fig. 2c–d). Specilla straight in ventral and side views (Fig. 2c–d), slightly curved at their tip (Fig. 2c). Sternite IX: ventral vesicle racket-shaped (Fig. 2e). Female (Fig. 2g): subgenital plate with two rounded lobes separated by a small rounded lamella; a triangular sclerite placed between the 2 lobes is visible beneath transparent cuticle.

Figure 2. 

Leuctra nigra, from France, Allevard, Val Pelouse. Male: a = abdomen tip, dorsal view, b = lateral view; c = paraprocts lateral view, d = ventral view; e = ventral vesicle; f = tergite VIII top view. Female: g = subgenital plate, ventral view.

Leuctra biellensis Festa, 1942

Figs 1, 3a–g, 4

Leuctra biellensis, Consiglio 1962

Leuctra nigra Oliv., Fochetti and Tierno de Figueroa 2008: 259, fig. 160a–f.

Locus typicus: Alpe Finestre, Val Chiobbia, Piemonte (altitude 1700 m).

Material examined

Neotype (GBIFCH00235761): Italy, Pennine Alps : Biellese mountains, Oropa, torrents and brooks, 1200–1900 m, 06.1978–07.1978, Coll. C. Ravizza, 1♂ (Museo Civico di Storia Naturale "Giacomo Doria", Genova, Italy).

Topotypes (GBIFCH00235762): same data as neotype: 175♂, 153♀.

ITALY: Pennine Alps: Civiasco, (NO), 700 m, 25.04.1992, 16♂, 2♀; Valsesia, Morce brook, 500 m, 25.04.1975, 5♂, 1♀ (Rav); Biellese mountains, brooks, Elvo trib., 1100 m, 18.05.1981, 7♂, 1♀(Rav); Biellese mountains, Donato, Vione tor., 950 m, 21.05.1979, 16 larves (1 male nymph) (Rav); Andrate, Graglia, slow brook, Mombarone mount, 5.06.1978, ♂, ♀ (Rav). Aosta Valley, Dora Baltea trib., > Trovinasse, Colla della Lace, 1900 m, 11.07.2003, 1♂; > Trovinasse, Colla della Lace, 1700 m, 24.05.2003, 4♂, 6♀; Dora Baltea trib., Pont-St-Martin, > Carema, 500 m, 1.06.1991, 2♂, 4♀; Dora Baltea trib., > Nomaglio, road from Andrate to Biella, 600 m, 1.06.1991, 13♂, 9♀; 31.03.2000, 1♂, 1♀; E. Varallo, Civiasco, Sesia trib., b., 850 m, 2.06.1991, 2♂, 1♀; N. Varallo, > Cervarolo, 1500 m, 2.06.1991, 4♂, 1♀; < Cervarolo, > Piane del Alpe, b., 1200 m, 2.06.1991, 9♂, 6♀; W. Grevellona, Val Strona, > Campello Monti, 1500 m, 13.08.2005, 1♀; Val Strona, > Massiola, < Rosarolo, b., 750 m, 26.04.1999, 12♂, 3♀; Val Strona, > Omegna, b., 500 m, 24.05.2003, 2♂, 1♀; SW. Domodossola, Valle Anzasca, Macugnaga, Staffa, b., 1300 m, 2.06.1991, 16♂, 16♀; Valle Anzasca, Macugnaga, Prequartera, 800 m, 1.06.1991, 3♂, 3♀ (Vin). Cottian Alps: Ceres, Almesio, 750 m, 11.06.2001, 4♂, 3♀; Lemie, Val d’Ovarda, A. Milone, 1650 m, 20.06.2001, 1♂, 4♀; Locana, val Piantonetto, S. Giacomo, 1130 m, 26.06.2001, 1♀; Villafranca Piemonte, TO, Cappella della Missione, 260 m, 24.03.2002, 2♂; (Del); Graian Alps: Frassinato brook, Soana trib., 1000 m, 18.06.1992, 1♀ (Rav); Val Soana, 700 - 850 m, 11♂, 6♀ (Rav); Lis Pass, > Girardi, Stura di Lanza trib., b., 1100 m, 7.05.2000, 5♂, 2♀; 27.08.2005, 2♀; Val di Viu, Stura di Lanza trib., > Viu, Piazette, b., 1200 m, 1.04.2000, 3♂; Val di Viu, Stura di Lanza trib., < Viu, brook, 600 m, 22.04.2008, 3♂, 6♀; NE. Lanzo Torinese, > Corio, Pian d’Audi, Rio Malone trib., 865 m, 23.10.2000, 1♀; Aosta Valley, > Quincinetto, > Scalaro, 1500 m, 1.05.2009, 1♀; > Quincinetto, Rio della Folla, 1250 m, 11.07.2012, 1♀; > Quincinetto, road to Lecchia sup. and road to Scalaro (iron bridge), 1100 m, 1.05.2009, 1♂; Aosta Valley, > Champorcher, Valle della Legne, Ayasse trib., 1200 m, 6.05.2000, 1♂ (Vin). Rhaetian Alps: Po Valley, Rocchellaif, Po, 550 m, 1.05.1982, 4♂, 1♀ (Rav). Liguria: Calizzano brook, 900 m, 28.04.1976, 3♂ (Rav); (SV) Calizzano, Melogno Pass, Frassino trib. 800-1000 m, 27♂, 4♀ (Rav); Montenotte, Erro spring, 700-950 m, 4.05.1974, 48♂, 40♀ + 65♂, 58♀ (Rav); Mont San Giorgio, brook, 750 m, 16.04.1974, 10♂, 3♀ (Rav). Lombardia, Casargo, (CO), Varonne tor., 2.06.1973, 21♂, 16♀, Bucuarelli leg (Rav); PreAlps, San Michele (VA), Olocrene spring, 800 m, 8.07.1986, 2♂, 3♀(Rav).

SWITZERLAND: Ticino, Lepontine Alps: Ceneri Mount, > Isone, before the military base, 1.04.2015, 10♂, 5♀ (Vin); Valle Lucoresagguo, Piace Seguo, 1650 m, 23.05.1988, 28♂, 16♀ (Rav). Rhaetian Alps: Graubünden, Cavagliasch, Cavaglia, 1690 m, 30.05.2007, 2♂ (P. Baumann).

Complementary description

Male (Fig. 3a–f): Tergite VI with two small triangular appendices pointing upwards and backwards in side view (Fig. 3b). Tergite VII with non-interrupted anterior margin and wide median bell-shaped membranous area. Tergite VIII with two strong teeth pointing upwards and backwards (Fig. 3a, b) and nearly triangular in dorsal view (Fig. 3f). Tergite IX with anterior margin interrupted on nearly one third of segment width, lateral edges sub-triangular forming a wide strip along anterior margin (Fig. 3a). Lateral lobes of paraprocts with a sclerotized sickle-shaped expansion slightly bent towards distal part of specilla (Fig. 3c–d). Specilla straight in ventral and side views (Fig. 3c–d). Sternite IX: ventral vesicle racket-shaped (Fig. 3e). Female (Fig. 3g): subgenital plate with two rounded lobes separated by a small triangular lamella; a triangular sclerite placed between the 2 lobes is visible by transparency under the cuticle.

Figure 3. 

Leuctra biellensis from Swiss, Ceneri Mount, Isone. Male: a = abdomen tip, dorsal view, b = lateral view; c = paraprocts lateral view, d = ventral view; e = ventral vesicle; f = tergite VIII top view. Female: g = subgenital plate, ventral view.

Ecological preferences and distribution area

Leuctra biellensis is a crenophilic species occurring in springs and brooklets at various altitudes (260–1900 m). The flight period is mainly in spring and early summer (III–VII) but few adults also occur in autumn (VIII–X). Its distribution area widely covers the western part of the Italian Alps from the Rhaetian Alps to the Maritime Alps and also extends in Liguria (Fig. 4). Its occurrence in Switzerland is restricted to the southern slope of the Alps.

Figure 4. 

Distribution area of Leuctra biellensis. Red star = Type locality. White star = other localities with L. biellensis.

Discussion

Aubert (1954) was the first who challenged the validity of Leuctra biellensis, suggesting that it could be a junior synonym of Leuctra nigra, but without formally establishing the synonymy. In the same way, Illies (1966) also considered the species as doubtful but did not change its status. Consiglio (1962, 1967) likewise adopted an ambivalent attitude. He first considered L. biellensis to be a valid species (Consiglio 1962), then changed his mind and established the synonymy with L. nigra (Consiglio 1967, p. 18). This synonymy was then confirmed by Zwick (1973). However, neither Aubert nor Consiglio adduced the required morphological details that would have justified this synonymy.

Male and female imagos of these two species can be morphologically distinguished by the following characters. In the male, the two teeth of tergite VIII are sub-triangular in dorsal view in L. biellensis (Fig. 3f), instead of sub-rectangular in L. nigra (Fig. 2f); the lateral edges of tergite IX form a wide sclerotized strip on nearly half part of the anterior margin in L. biellensis (Fig. 3a), while they end in acute angles in L. nigra (Fig. 2a); the distal expansions of the paraprocts are sickle-shaped and not lying along the specilla in L. biellensis (Fig. 3d), while they are hook-shaped with their tip lying along the specilla in L. nigra (Fig. 2d). In the female, the lamella between the two lobes of the subgenital plate is triangular in L. biellensis (Fig. 3g) and rounded in L. nigra (Fig. 2g).

Leuctra biellensis is an alpine micro-endemic species only occurring in the western and internal part of the Alps like other cryptic species of Plecoptera (Ravizza and Vinçon 1998) or Trichoptera (Graf et al. 2015). The species is strongly crenophilic, with long flight period (III–X). Conversely, L. nigra is a central north-European species, mainly occurring in lentic biotopes and with shorter flight period (V–VIII) (Ravizza and Vinçon 1998). Both species were never collected together in the same locations and therefore L. biellensis can be considered as a sister species of L. nigra, inhabiting the internal slope of the Alps where it was probably isolated from L. nigra. The same applies to many other alpine species such as L. muranyii Vinçon and Graf, 2011 and L. juliettae Vinçon and Graf, 2011, two sister species of L. braueri Kempnyi, 1898, isolated in a restricted part of the eastern Alps (Vinçon and Graf 2011). Molecular tools associated with morphological characters are very promising to separate species in groups with recent diversification (Vitecek et al. 2017).

We found around 9% of distance between COI haplotypes of L. nigra and L. biellensis. While a 3.5% COI sequence distance has been proposed in the DNA barcoding literature as a likely maximal value for intraspecific divergence (Hebert et al. 2003; Zhou et al. 2010), higher intraspecific K2P and p values are not uncommon (e.g., Meier et al. 2006), and have also been reported for Plecoptera (Mynott et al. 2011; Boumans and Baumann 2012; Gill et al. 2015). Nonetheless, 9% distance is an unlikely intraspecific value. We are aware that species cannot be identified or described based on mitochondrial sequences alone. A major reason for this is that strongly divergent haploclades in some populations may result from hybridisation and subsequent mitochondrial introgression (Boumans and Tierno de Figueroa 2016). For this reason, we emphasise that the mitochondrial and morphological data converge to identify L. biellensis as a valid species distinct from L. nigra. Since our molecular study also contains Scandinavian samples, our results confirm the presence of L. nigra in this area and tend to confirm the synonymy made by Brinck (1949) of Leuctra acuminata Bengtsson, 1933 with L. nigra, especially as L. acuminata was established for specimens from Sweden originally identified as L. nigra.

Acknowledgements

We express our gratitude to Giovanni Delmastro (Carmagnola, Italy), Jean-Paul Reding (Neuchâtel, Switzerland) and Dávid Murányi (Budapest, Hungary) for their help and donation of comparative material, to J. Manuel Tierno de Figueroa (Granada, Spain), Wolfram Graf (Wien, Austria), Jean-Paul Reding for reviewing the manuscript. We also want to thank Sofia Wyler (SwissBOL), Albertine Roulet, Marion Podolak and Nicolas Hazi (MZL) for preparing the specimens stored in the MZL collection and sequencing them. We want to thank Lars Hendrich for giving us access to molecular data from the Project “Fauna Bavaria Barcoding” managed by the Zoologische Staatssammlung, München, Germany and Maria Tavano (Museo Civico di Storia Naturale “Giacomo Doria”, Genova, Italy) for hunting for the type material of Leuctra biellensis.

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