New faunistic and taxonomic insights on little known Crambidae from forested habitats of Italian Peninsula (Lepidoptera

We provided new distribution data and taxonomic notes concerning eleven Crambidae species new or little known for the Italian fauna. Most records were collected in Calabria, the southernmost region of peninsular Italy, where forested habitats were surveyed during last years. Some accessory data were mined from Barcode of Life Data System (BOLD) and were used to refine the distribution of treated species. In detail, we found three species new for Italian fauna: Evergestis subfuscalis (Staudinger, 1870), Udea languidalis (Eversmann, 1842), and Hyperlais siccalis (Guenée, 1854). Three species not recorded in peninsular Italy so far: Cynaeda gigantea (Wocke, 1871), Evergestis dumerlei Leraut, 2003, and Catoptria staudingeri (Zeller, 1863). Four species not recorded in southern Italy so far, Evergestis infirmalis (Staudinger, 1870), C ynaeda dentalis ([Denis & Schiffermüller], 1775), Paratalanta pandalis (Hübner, 1796), Hodebertia testalis (Fabricius, 1794). Evergestis sophialis (Fabricius, 1787) was for the first time recorded in Calabria. Barcoding analysis allowed us to confirm the status of Evergestis subfuscalis as bona species, eastern vicariant of E. mundalis . Furthermore, we underlined the needs of a revision of the Udea fimbriatralis species group as barcodes of western and eastern European specimens appear to be intermingled, suggesting the presence of one unique variable species. In conclusion, the importance of the Italian peninsula as a bridge between western and eastern European faunas was remarked.

This paper listed the most interesting findings for Italian fauna concerning Crambidae species as the result of surveys carried out in the main forest type of the Calabria Region, the southernmost territory of Italian Peninsula.In detail, we surveyed (1) beech and Acer-dominated forests of the Pollino National Park, (2) Calabrian black pine forests and Alnus-dominated woodlots of the Sila National Park, (3) silver-fir forests of the Regional Park of the Serre Mountains, (4) chestnut woodlots of the Catena Costiera Mountains, and (5) mixed forest of Calabrian black pine and beech of the Aspromonte National Park.Notes concerning species range, biology and taxonomy were also provided.

Methods
Samplings were carried out using UV LED light traps (INFUSINO et al., 2017) powered by portable batteries, under weather conditions favourable to moth activity and trap efficiency, i.e. low wind speed, temperatures near the average of a given period, low or absent moonlight, low or absent rain.Exact locations of sampling sites were reported in the list of species.Further records available in the public repository Barcode of Life Data System (BOLD) were utilized to refine the range of species and their presence in Italy.
Identification was carried out using specialised literature concerning European Crambidae (GOATER et al., 2005;LERAUT, 2012;SLAMKA, 2006SLAMKA, , 2008SLAMKA, , 2013)).Most difficult species were dissected, and genitalia were mounted on microscope slides.Nomenclature followed the Fauna Europaea available online at www.faunaeur.org.Specimens and slides of genitalia were preserved in the Lepidoptera collection of the Research Centre for Forestry and Wood, Rende, Italy.
Some specimens were submitted to DNA barcoding, an analysis of the 658bp long sequence of the mitochondrial 5' cytochrome oxidase gene, subunit 1 (COI), using the standard procedures of the Canadian Centre for DNA Barcoding (CCDB).Obtained sequences were blasted in the BOLD data systems (RATNASINGHAM & HEBERT, 2007), to compare sequences with those available.For barcoded specimens we reported: the sample ID, the length of the obtained COI sequences (bp), the Barcode Index Number (BIN) to which they belong and their statistics, i.e. average (Av.dist.)and maximum percentage distances (Max.dist.)among sequences belonging to the same BIN and percentage distance from the Nearest Neighbor (NN), and, finally, the name and the BIN of the NN.

CRAMBIDAE ODONTIINAE
Cynaeda gigantea (Wocke, 1871) Original records -Italy, Calabria, Saracena, Serra Ambruna, 1035 m, 39.8234ºN-16., 2006).In Italy it was only recently reported from the North (LERAUT, 2012), but the exact locality was unspecified.These are the first records from peninsular Italy.Calabrian specimens were collected by light traps in two localities of the Pollino National Park with dry and sunny habitats, typical for this species, and where larvae can find their foodplants Echium and Onosma (SLAMKA, 2006).
Identification was based on wing pattern (Fig. 1) and morphology of genitalia (Fig. 12).It can be easily distinguished as the phallus has two cornuti, whereas in C. dentalis ([Denis & Schiffermüller], 1775), the only congeneric species known to fly in the same range, cornuti are absent (SLAMKA, 2006).
It was found from Europe to Central Asia, Middle East, North Africa, Madeira and Mediterranean Islands, absent from the northernmost European regions (LERAUT, 2012).In Italy was reported from the North, Latium, Sicily and Sardinia (PINZARI et al., 2010).This is the first record from South Italy, where it was collected by light traps in a prairie of the Pollino National Park, and in a chestnut woodlot and a sunny grassland of the Catena Costiera Mountains.Larvae feed on Echium, Anchusa and Onosma (SLAMKA, 2006).Identification was based on the wing pattern (Fig. 2).

EVERGESTINAE
Evergestis dumerlei Leraut, 2003Original record -Italy, Calabria, Spezzano Piccolo, Righio, 1341 m, 39.3153ºN-16.5273ºERecorded from Morocco, Spain, France (LERAUT, 2012) and Italy where it is known from the surroundings of Bolzano, Alto Adige (HUEMER, 2012).These are the first records from peninsular Italy.Calabrian specimen was found during daytime in a humid grassland of the Sila National Park.Larval foodplants are probably Brassicaceae (SLAMKA, 2006).For this species it is reported one generation per year from July to October (SLAMKA, 2006;LERAUT, 2012), however the finding of late-May suggests the presence of two generations per year, the first in Spring and the second in Summer-early Autumn.
Identification was based on wing pattern (Fig. 3) and DNA barcoding which showed a low intraspecific variability among sequenced specimens (Table I), and a high similarity between Calabrian and Central Italy samples (99.85%).LERAUT (2012).This is the first record from Italy, where it was collected by light traps in three localities of the Pollino National Park, within beech forest stands.Immature stages are unknown (GOATER et al., 2005).
Identification was based on the morphology of genitalia and DNA barcoding, as wing pattern (Fig. 4) is not enough to separate it confidently from E. mundalis (Guenée, 1854).Male genitalia are characterized by rather short parallel-sided valvae with rounded apex, and by the distal portion of phallus slender than in E. mundalis, with an oblique row of strong cornuti near base (GOATER et al., 2005) (Fig. 13).However, DNA barcoding was decisive for species identification as also morphology of genitalia is questioned leading some authors to consider E. subfuscalis a synonym of E. mundalis (LERAUT, 2012).In fact, the COI sequence of Calabrian specimen showed a marked genetic distance from those of E. mundalis from France (Table I) but share the same BIN with two Iranian and two Greek specimens, demonstrating definitively its eastern affinity, and confirming also the existence of an eastern species distinct from E. mundalis (Fig. 18).
In Italy E. mundalis is known from Central and North regions (BASSI et al., 1995), whilst none record is available for southern regions, so far.Recently, several specimens regarded as E. mundalis were reported from Central Italy (PINZARI et al., 2010).It should be interesting to verify the exact identity of these specimens and to assess the distribution of the mundalis / subfuscalis species pair along the Italian Peninsula.It is known from South Europe, eastwards to southern Siberia (GOATER et al., 2005).In Italy it was only reported from the North and the South (BASSI et al., 1995).This is the first record from Calabria, where it was collected by light traps in a locality of the Pollino National Park, within an ecotonal habitat between a dry and sunny calcareous slope and a young Acer forest.Immature stages on Descurainia sophia (L.) Webb ex Prantl.and Sisymbrium spp.(GOATER et al., 2005).
Identification was based on the morphology of male genitalia, with valvae, uncus and gnathos clearly different from those of the similar E. infirmalis (Staudinger, 1870), and with the phallus bearing two dense clusters of long cornuti in E. sophialis and two clusters of small cornuti in E. infirmalis (GOATER et al., 2005) (Fig. 14).Wing pattern is sometimes useless, especially in worn specimens as the male we recorded (Fig. 5).According to PINZARI et al. (2010), it is known from Greece, European Russia, Turkey, Syria and Italy, where it was found in Central Apennine.In Calabria it was collected by light traps in the same locality and during the same night of E. sophialis, i.e. within an ecotonal habitat between a dry and sunny calcareous slope and a young Acer forest of the Pollino National Park.Early stages are unknown (GOATER et al., 2005).

Evergestis infirmalis (Staudinger
Identification was based on the morphology of male genitalia, as wing pattern (Fig. 6) is not always enough to separate it confidently from E. caesialis.(Herrich-Schäffer, 1849).In male genitalia of E. infirmalis the main distinctive features from E. caesialis are the slightly bulbous uncus, the slender gnathos without evident teethes, the valva dilated at base and tip, and the distal portion of the spinulose phallus bearing two clusters of small cornuti (GOATER et al., 2005).The dissected male genitalia of the Calabrian specimen perfectly matched this description (Fig. 15).DNA barcode of the dissected specimen has a great homogeneity with other Italian specimens deposited in BOLD that belong to the same BIN (Table I) (Fig. 19), demonstrating their conspecificity.Recorded from South-East Europe, Turkey, North Iran and Turkmenistan (SLAMKA, 2013).This is the first record for Italy.Calabrian specimen was collected in a small clearing within an Acer forest on calcareous substratum, in the Pollino National Park.Preimaginal stages are unknown (SLAMKA, 2013).

PYRAUSTINAE
Identification was not easy as the congeneric U. fimbriatralis, also not mentioned for the Italian fauna so far, has similar wing pattern and genitalia of both species are rather similar and quite variables (SLAMKA, 2013).According to MALLY & NUSS (2011), based on molecular and morphological evidences, fimbriatralislanguidalis compose a strictly related species-pair the former having a western and the latter an eastern range (SLAMKA, 2013).LERAUT (2012) considers languidalis the eastern subspecies of fimbriatralis.Here we used the name languidalis for our specimen as markings on wings are well developed (Fig. 7), and a small thorn is present on the distal part of the phallus (Fig. 16), both characters attributable to U. languidalis (SLAMKA, 2013).The great intra-BIN genetic variability justifies the taxonomic uncertainty present in this species group (Table I).We can hypothesize the presence of only one variable species, but the question deserves further studies.
Tropical and subtropical species, probably resident in the Mediterranean Basin (SLAMKA, 2013), also known in northern Italy and Sicily (BASSI et al., 1995).This is the first record for southern Italy, where it was collected by light traps in a chestnut orchard.Larva feeds mainly on Asclepiadaceae (SLAMKA, 2013).Identification was based on the wing pattern (Fig. 9).
Known from France, Spain and Morocco (GASTÓN et al., 2015), this is the first record from Italy where it was collected by light traps in a locality of the Pollino National Park within a dry and sunny prairie surrounded by broadleaved forests, on calcareous substratum to which it appears to be associated (COURTOIS, 1986).Early stages feed on Iberis pinnata L. (LHOMME, 1935).
Identification was based on the morphology of male genitalia and DNA barcoding, as wing pattern (Fig. 10) is not always enough to separate it from congeneric species.Male genitalia perfectly match those figured in LERAUT (2012) and in GASTÓN et al. (2015) (Fig. 17).Barcoded specimens belong to a BIN from which the nearest neighbor has a marked genetic distance and belong to a not specified Hyperlais species (Table I , 2008), this is the first record from continental Italy where it was collected by light traps in a small clearing within a beech forest of the Aspromonte National Park, and two shrublands of the Sila National Park within a Calabrian black pine forest.Early stages feed on mosses (SLAMKA, 2008).Identification was based on the wing pattern (Fig. 11).

Discussions and conclusions
In this paper we provided new distribution data concerning eleven Crambidae species new or little known for the Italian fauna.Most interesting findings concerned three species new for Italian fauna, namely Evergestis subfuscalis, Udea languidalis, and Hyperlais siccalis.In addition, Cynaeda gigantea, Evergestis dumerlei, and Catoptria staudingeri were not previously recorded from peninsular Italy, and four species were not recorded in southern Italy so far, namely Evergestis infirmalis, Cynaeda dentalis, Paratalanta pandalis, and Hodebertia testalis.Evergestis sophialis was for the first time recorded in Calabria.
Most of listed species have in Italy the western boundary of their range (Cynaeda gigantea, Evergestis subfuscalis, E. caesialis, E. infirmalis, Udea languidalis), but Evergestis dumerlei, Hyperlais siccalis and Catoptria staudingeri have there the eastern boundary of their range.The presence of eastern and western elements in the Italian fauna, especially along the peninsular territory, is the result of the complex biogeographic history of this geographic area and is one of the reasons that make lepidopteran fauna of Italy the richest of Europe.
Barcoding analysis allowed us to confirm the status of Evergestis subfuscalis as bona species and eastern vicariant of E. mundalis and enabled us to underline the needs of a revision of the Udea fimbriatralis species group as haplotypes of western and eastern Europe of species belonging to it appear to be not well differentiated and intermingled, suggesting the presence of a unique variable species.