First record and DNA barcoding of Donacaulaniloticus (Zeller, 1867) from the Iberian Peninsula (Lepidoptera: Crambidae)

Abstract Background Donacaulaniloticus (Zeller, 1867) is known from south-eastern Europe, Middle East and Turkey to Central Asia, northern India and China and widely distributed in North Africa (Morocco, Algeria, Libya and Egypt). New information Donacaulaniloticus (Zeller 1867) is recorded for the first time from the Iberian Peninsula and the first DNA barcode sequence is published and compared with other European and North American Donacaula species.

Donacaula Meyrick 1890 is known to occur in the Palearctic, Ethiopian, Oriental, Neotropical and Nearctic Regions, except in the Australian Region. The genus includes 31 described species, of which four occur in the Palearctic, four in the Ethiopian, two in the Oriental, nine in the Neotropical and twelve in the Nearctic Regions (Speidel 2005). Adults of Donacaula have high variability between individuals and between geographical populations with forewings that are light brown to yellowish-brown with some species having dark longitudinal lines and/or spots and apices of forewings can be pointed in females, but are always rounded in males (Speidel 2005).
Donacaula niloticus is known from south-eastern Europe, Middle East and Turkey to Central Asia, northern India and China and widely distributed in North Africa from Morocco to Egypt (Speidel 2005, Slamka 2008, Leraut 2012. In the Iberian Peninsula, two species of Doncaula have been recorded, D. forficella and D. mucronella according to Speidel (2005) and Vives-Moreno (2014), although Slamka (2008) and Leraut (2012) considered that D. mucronella is not present in the Iberian Peninsula. In relation to the biology of D. niloticus, the early stages are unknown, whilst the adults fly in several generations from April to September in riparian and steppe vegetation with nearby watercourses and lakes (Speidel 2005, Slamka 2008, Leraut 2012. In this article, we provide the first record and DNA barcoding of Donacaula niloticus (Zeller, 1867) collected from the saltmarshes of Adventus in Trebujena (Cádiz, Spain). The specimens collected have some consistent morphological features which differ from the other Donacaula species. In addition, mtDNA sequence(COI) were used to assess genetic divergence between the Donacaula species from Europe and North America.

Morphological study
During the sampling, one female specimen with a white discocellular spot and unpointed apex traits was collected (Fig. 1a). Additionally, one male with a dark brownish line from the costal margin to about the centre of the wing was photographed ( Fig. 1b and c).
The specimen was pinned, wings spread and dried. Their external characters were examined in order to evaluate possible differences in colouration and wing shape. Furthermore, it was dissected using standard procedures (Hausmann 2001) with minor modifications. The female adult image was taken with a Nikon D70 digital camera. Images were z-stacked using Zerene software. The female genitalia were studied using a Zeiss Stemi 508 stereomicroscope with a Zeiss Axiocam ICc5 digital camera. The specimen is deposited in the Research Collection of Animal Biology (RCBA-UMU) in the Department of Zoology and Physical Anthropology of the University of Murcia (Spain). First record and DNA barcoding of Donacaula niloticus (Zeller, 1867) from ...

Molecular study
For DNA extraction, two legs were removed from the specimen in order 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 (MZ920225).
Voucher data, images, sequences and trace files are publicly available on the Barcode of Life Data System (BOLD) (Ratnasingham and 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 of the new and related species sequences were downloaded and aligned with the CLUSTAL algorithm of the MEGA6 software (Tamura et al. 2013), including all sites with the pairwise deletion option. A phylogenetic hypothesis with Maximum Likelihood as an optimality criterion was generated using IQ-TREE v.1.6.12 (Nguyen et al. 2015). An alignment of 658 bps for 99 samples was partitioned into codon positions with ModelFinder software (Kalyaanamoorthy et al. 2017) and the 1st codon position was modelled with TN+F+G4; 2nd with TIM3e+G4; 3rd with TPM3u+F+I. Support values were calculated by 1,000 replications of both ultrafast bootstrap (UFBoot; Hogan et al. 2017) and Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT; Guindon et al. 2010), as well as approximate Bayes branch test (aBayes; Anisimova et al. 2011). In order to assess the COI divergences between Donacaula niloticus and the other Donacaula species from Europe and from North America, we included all sites with the pairwise deletion option. unipunctellus ( Robinson 1870) (BOLD:AAA4113; n = 11) from North America were obtained from the public database in BOLD. We selected Scirpophaga praelata (BOLD:AAB6864; n = 1), which is systematically related into subfamily Schoenobiinae as outgroup to root the trees.  First record and DNA barcoding of Donacaula niloticus (Zeller, 1867) from ...

Analysis
The COI divergences between Donacaula niloticus and seven Donacaula species from Europe and North America, including Scirpophaga praelata as additional Schoenobiinae species, are presented in Table 1.   All trees presented the same topology and were practically identical; therefore, only the ML tree is presented here with the branch tips collapsed since each of the groups of sequences correspond to a single BIN (Fig. 4). The complete tree can be consulted at Suppl. material 1. As one gene is insufficient for reasonable phylogenetic analysis (Gatesy et al. 2007), the tree presented here does not reliably illustrate evolutionary relationships amongst the sequenced taxa.

Discussion
The presence of Donacaula niloticus in the riparian vegetation near to the mouth of the Guadalquivir River into the saltmarshes of Adventus (Trebujena, Cádiz) is confirmed by integrating the evidence from COI mitochondrial DNA sequences and adult morphology and is genetically different to the other two European and six North American Donacaula species. Molecular data indicate significant divergence with large mean distances amongst Donacaula species (13.0%) with maximum distance between D. sordidellus and D. longirostrallus and minimum distance between D. melinellus and D. mucronella (7.6%). In   Fig. 4). Although the genetic differences between all the species of Donacaula are similar, the topology of the tree relates them and separates the other species of Schoenobiinae (Scirpophaga praelata).
Donacaula is associated with various semi-aquatic and marsh plants where their larval stages live in stems or roots or on exposed and non-submerged leaves of Phragmites australis, Glyceria spp. and Carex spp. These lepidopterans have developed various strategies and adaptations that have allowed them to stay in close proximity to water (Pabis 2018). D. niloticus larvae in the lower part of stems and their presence is indicated by a small round hole in the stem five or eight centimetres above the root. Pupation take place in the base of the stem (Speidel 2005). The landscapes of the riverside plain of the Guadalquivir river, in the furthest point south of the Iberian Peninsula where D. niloticus was collected, 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 (Fig. 3) (CMAOT 2015).
We emphasise here the importance of combining traditional morphological analysis and ecological traits with the additional dataset of DNA sequences for those taxonomic groups whose identification is particularly difficult and mainly based on differences in the genitalia.
This study has been supported by the Regional Excellence 19908-GERM-15 project of the Fundación Séneca (Regional Government of Murcia, Spain). Collecting permits were issued by the Environmental Authority of the Junta of Andalusia.