Reinstatement of Leuctra biellensis Festa, 1942 (Plecoptera, Leuctridae)

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.


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).
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.

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).

Material examined. Neotype (GBIFCH00235761):
Italy, Pennine Alps : Biellese mountains, Oropa, torrents and brooks, 1200Oropa, torrents and brooks, -1900Oropa, torrents and brooks, m, 06.1978Oropa, torrents and brooks, -07.1978,Coll.C.  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 bellshaped 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.
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.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 (1962Consiglio ( , 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 andGraf, 2011 andL. juliettae Vinçon andGraf, 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.

Figure 1 .
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.

Table 1 .
Specimens used for the phylogenetic analysis of the mitochondrial gene CO1.

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.