DNA barcoding relationships and habitat description of Psorosa ferrugatella Turati, 1924 in Spain (Lepidoptera: Pyralidae, Phycitinae)

First DNA barcode sequences of Psorosa ferrugatella Turati, 1924 from the Iberian Peninsula is published and compared with other European Psorosa and closely related species. Three different habitats where P. ferrugatella inhabits are described.


Introduction
The genus Psorosa Zeller, 1846 belongs to the tribe Phycitini of the subfamily Phycitinae in the family Pyralidae.The world fauna of this genus includes 24 species (Slamka, 2019).Seven of them were reported for Europe (Leraut, 2014;Slamka, 2019).Psorosa species are distributed mostly in southern areas of the European region except P. nucleolella (Möschler, 1866) known from Central Europe.In the Iberian Peninsula, the genus was represented by two species: P. dahliella (Treitschke, 1832) and P. mediterranella Amsel, 1953until Corley (2019) added one specimen of Psorosa ferrugatella (Turati, 1924) from Portugal to the first record of a specimen captured in Granada on 14-VI-1914 and deposited in the Museum of Natural History in Vienna (Slamka, 2019), thus confirming its presence in the Iberian Peninsula.Recently, Girdley et al. (2020) recorded a preliminary data of P. dahliella and P. ferrugatella flying simpatrically in Murcia (Southeastern Iberian Peninsula) which were later updated in Garre et al. (2022).Even more recently, Ranki et al. (2022) have found P. ferrugatella in the Trebujena Marshes (Cádiz).
The biology of almost all species of the genus is poorly known with preimaginal stages and hostplants unknown and inhabiting in open arid habitats and various types of steppes.Different Psorosa species can be very similar in appearance, so that genitalia examination is often necessary for determination.

Morphological study
All specimens were examined externally to evaluate possible differences in their colouration and wing shape.Furthermore, they were dissected using standard procedures (Hausmann, 2001) with minor modifications.Male adult image (Figure 1A) was taken with a Nikon D70 digital camera and were zstacked using Zerene software.Morphology of genital structures (Figure 1B) were studied using a Zeiss Stemi 508 stereomicroscope with a Zeiss Axiocam ICc5 digital camera.All specimens are deposited in the Research Collection of Animal Biology (RCBA-UMU) in the Department of Zoology and Physical Anthropology of the Universidad de Murcia (Spain).

Molecular procedures
Psorosa specimens used for mitochondrial gene cytochrome oxidase subunit 1 (COI) sequencing are reported in Table I.For DNA extraction, two or three legs were removed from the specimens to sequence the 658 base-pair long barcode segment of the mitochondrial COI gene (cytochrome c oxidase 1, 5' terminus).The tissue samples were submitted to the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) to obtain DNA barcodes using the high-throughput protocol described in deWaard et al. ( 2008) which can be accessed at www.dnabarcoding.ca/pa/ge/research/protocols.The DNA extracts are currently stored at the CCDB, and the sequences are deposited in GenBank according to the iBOL data release policy (Table I).Sequences were compared with a reference library of Lepidoptera barcodes using the identification engine (BOLD-ID).The reference barcode database for Pyralidae used by BOLD-ID is continually validated by specialists to ensure accurate identifications and is particularly well parameterised due to a global campaign to barcode more than 2,563 species of the family (Ratnasingham & Hebert, 2007).Voucher data, images, sequences, and trace files are publicly available on the Barcode of Life Data System (BOLD) (Ratnasingham & Hebert, 2007).Sequence divergences for the barcode region were calculated using the Kimura 2-parameter (K2P) model and the degrees of interspecific genetic variation were calculated using the analytical tools of BOLD.All the new and related species sequences were downloaded and aligned with the CLUSTAL algorithm of the MEGA6 program (Tamura et al. 2013).In order to assess the COI divergences between P. ferrugatella and the other systematically related species from Europe, we included all sites with the pairwise deletion option (Table I).Our sequences of P. ferrugatella (BOLD:AEF6784; n=3 seqs) and the public ones of P. nucleolella (BOLD:AAU2037; n=1) and P. dahliella (BOLD:ACA9753; n=1) and, closely related species according to (Slamka, 2019): Alophia combustella (Herrich-Schäffer, 1852) (BOLD:ADK9057; n=2), Catastia kistrandella Opheim, 1963 (BOLD:AAI7381; n=3), Catastia marginea ([Denis & Schiffermüller], 1775) (BOLD:AAE9528; n=18), Rhodophaea formosa (Haworth, 1811) (BOLD:AAC8900; n=30), and Selagia spadicella (Hübner, 1796) (BOLD:AAE1543; n=24) were obtained from BOLD.Neighbour-Joining (NJ) and Maximum Likelihood (ML) trees were calculated to visualise similarity among selected species.All trees presented the same topology and were practically identical, therefore, only the ML tree is presented here (Figure 2).Due to the fact that one gene is too few for reasonable phylogenetic analysis (Gatesy et al. 2007), the trees presented here do not reliably illustrate evolutionary relationships among the sequenced taxa.For the parameter values considered (e.g., sensitivity to codon bias and unequal rates of evolution) the statistical inconsistency of Maximum parsimony (MP) method may occur and was not performed in this study.

Results and discussion
The P. ferrugatella specimens showed morphological traits typical of European individuals according to diagnosis in Slamka (2019).Previously P. dahliella specimens referred in Girdley et al. (2020) were re-identified as P. ferrugatella and sequenced to match correct identification.Integrating the evidence from COI mitochondrial DNA sequences and adult morphology, we conclude that the P. ferrugatella specimens collected in the wetlands of the protected landscape of Humedal del Ajauque and Rambla Salada (Murcia) are genetically different to those co-generic species previously sequenced from Europe based on mitochondrial data.Molecular data indicates significant divergence between P. ferrugatella with 2.1% mean distances to P. dahliella, 2.9% to P. nucleolella (Table II, Figure 2).
Divergence between Psorosa and the other closely related species varies between 4.2% and 8.1% (mean 6.6%; Table II) where the highest interspecific values were found between P. ferrugatella and P. dahliella with Alophia combustella (8.1% and 7.9%, respectively), whereas the lowest one was found between P. nucleolella and Selagia spadicella (4.2%).Differences among the other genera varies with the highest interspecific values between Rhodophaea formosa and Alophia combustella (8.2%) and the lowest one between Catastia marginea and Rhodophaea formosa with Selagia spadicella (5.9% and 6.1%, respectively) (Table II, Figure 2).The total number of nucleotide substitutions between species is 106 variable sites.In this sense, it seems that Psorosa is more closely related to Selagia (mean divergence: 5.3%) and with Catastia (mean: 6.3%) and Rhodophaea (mean: 6.5%).

Habitat description and biology
P. ferrugatella is a species previously known from across North Africa, with records from Morocco, Algeria, Tunisia, and Libya.The Portuguese specimen was recorded in the Algarve (Southwest of the Iberian Peninsula) at the salt marsh in the estuary of the Guadiana River, while the new specimens were recorded in a salt ravine in the protected landscape Humedal del Ajauque y Rambla Salada, in the La Llana beach in the Regional Park of Salinas y Arenales de San Pedro del Pinatar and in the Huerta de Alquerías in the plain of Murcia (Huerta de Murcia) (In the Southeast of the Iberian Peninsula).The landscapes of the riverside plain of the Guadalquivir River, in the furthest point south of the Iberian Peninsula where P. ferrugatella was recorded by Ranki et al. (2022), are characterised by the irregular regime of water inputs, which can go from flooding the marshland to turning it into a desert dryland.These circumstances, and the marine influence due to its proximity to the river mouth, produce soils with varied salinity content that characterise the vegetation.This habitat is characterised by a series of hyperhalophilic Mediterranean-Ibero-Atlantic thermomediterranean edaphohygrophilic vegetation (EH20) which forms estuaries, salt marshes and marshes with a mixture of salty and fresh waters which is represented by the communities Spartinetum maritimae, Puccinellio-Sarcocornietum perennis, Halimiono-Sarcocornietum alpini, Cistancho-Arthrocnemetum macrostachyi, Polygono-Limoniastretum monopetali and, on the edge of the estuaries, the halonitrophilic community, Cistancho-Suaedetum verae.Sometimes, a plantation of Polygono-Tamaricetum africanae may appear.The land use bordering the habitat are mainly agricultural fields (Figure 3A) (CMAOT, 2015).

B D A C
The beach of La Llana in the Salinas y Arenales de San Pedro del Pinatar Regional Park is integrated in a sand dune system composed of mobile sands (strandline, embryo, and mobile dunes) and consolidated sands (semi-fixed and fixes dunes) and alternating with these dune slacks are formed.This ridge of dunes delimits a salt marsh.The vegetation that colonises sand dunes is adapted to limiting factors such as sand burial, salt spray and xeric conditions, so few plant species can survive in these environments.The specimen was caught on the semi-fixed dunes in the domain of the plant association Loto cretici-Crucianelletum maritimae.Chamaephytes such as Teucrium dunense Sennen, Crucianella maritima L., Helichrysum stoechas subsp.caespitosum (C.Presl.)DC. and Ononis natrix subsp.ramosissima (Desf.)Batt. in Batt.& Trab.are the most characteristic species (Figure 3C).
The Huerta of Alquerías is an agricultural territory irrigated by the Segura River through an ancient network of canals.Citrus groves are the main crops and natural vegetation is restricted to the margins of paths, borders of crops, abandoned fields and banks of irrigation canals.Nitrophilous and ruderal species are predominant, besides some hygrophilous plants such as Tamarix canariensis Willd., Cynanchum acutum L., Phragmites australis (Cav.)Trin.ex Steud., Arundo donax L. and Apium nodiflorum (L.) Lag (Figure 3D).
The characteristics of these habitats suggest that Psorosa ferrugatella inhabits habitats that are not conditioned by the prevailing macroclimate, but to edaphic conditions, particularly soil moisture, salinity gradient and soil structure, that determine the selection of several plant communities of a nonclimax character.
In relation to the biology of the species, Slamka (2019) states that it flies from March to October in several generations, while in our territory sightings took place from early April to late June.Host plant and early stages are also unknown.
We emphasise again the combination of traditional morphological analysis and ecological traits with the additional dataset of DNA sequences for those taxonomic groups whose identification is particularly difficult due to the small size, handling and that it is mainly based on differences in the genitalia.

Figure 3 .
Figure 3. General images of the different habitats.A. Saltmarshes of Adventus near Trebujena, Cádiz, Spain (Photographed by Manuel Pozas).B. The beach of La Llana in the Salinas y Arenales de San Pedro del Pinatar Regional Park (Photographed by A. S. Ortiz).C. The Humedal del Ajauque y Rambla Salada (Photographed by M. Garre).D. Agricultural landscape of Huerta of Alquerías (Photographed by M. Garre).