DNA barcoding and a new taxonomic status of the Triaenodes ochreellus lefkas Malicky, 1974 (Insecta, Trichoptera) with new distribution data

In this paper new data on distribution and new taxonomic status of the caddisfly Triaenodes ochreellus lefkas are given. DNA barcoding data are also included into defining new status of the species Triaenodes lefkas stat. nov. Data from DNA barcoding analyses of 60 specimens from the genus Triaenodes from the BOLD database show certain taxonomic peculiarities in specimens of T. unanimis from Japan.


Introduction
The Leptoceridae family (long-horned) is one of the largest families of Trichoptera, with more than 1800 known species (Holzenthal et al. 2007;Morse 2020;Oláh 2016). It is cosmopolitan although its greatest biodiversity is in Asia (Morse 2020;Holzenthal et al. 2007). One of the most numerous genera within the family Leptoceridae is the genus Triaenodes McLachlan, 1865, with more than 230 described species (Malicky 2004(Malicky , 2005aMorse 2020). This genus is widespread and diverse on all continents, including Europe. Only three species of the genus Triaenodes have been found in Europe (Malicky 2004): T. bicolor (Curtis, 1834), T. ochreellus McLachlan, 1877and T. unanimis McLachlan 1877(Malicky 2004Morse 2020). Only the species T. ochreellus has two subspecies: T. ochreellus ochreellus and T. ochrellus lefkas Malicky, 1974(Ibrahimi et al. 2017Malicky 2004Malicky , 2005bMorse 2020). The adult has a thin body of small to medium size, with long antennae (Holzenthal et al. 2007). Larvae of Triaenodes species live in different types of habitats, especially in lentic aquatic biotopes, usually with aquatic vegetation (Ibrahimi et al. 2017;Wiggins 1978). They have elongated posterior legs with long setae, known as swimming paddles (Wiggins 1978). This morphological structure helps them to make rapid movements and swim among the aquatic plants that grow in lentic waters (Wiggins 1978).
In this study we present results of the DNA barcoding of the genus Triaenodes in Croatia, a review of some taxonomic points of this genus, new data on the distribution and taxonomic status of the taxon Triaenodes ochreellus lefkas.

Laboratory methods
Whole genomic DNA was extracted from legs using GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich, Germany) according to the manufacturer's specifications and eluted in 60 µl of elution buffer. Full-length mtCOI DNA barcode regions were amplified using LCO1490/HCO2198 (Folmer et al. 1994) primer sets. The 50 μl polymerase chain reactions (PCR) mixture contained 1 x Go Taq®Reaction Buffer (containing 1.5 mM MgCl2, Promega), 0.2 mM of each dNTP, 0.4 μM of each primer, 1.25 units of Go Taq®DNA Polymerase (Promega) and 5 µl of DNA eluate. PCR cycling conditions comprised an initial denaturation step (94°C for 2 min) followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s and elongation at 72°C for 90 s and a final extension step of 72°C for 7 min. Product purification and bidirectional sequencing was performed by Macrogen Inc. sequencing service (Seoul, South Korea) using amplification primers. Sequences were edited manually and aligned using the program BioEdit (Hall 1999). DNA sequences were submitted to Barcode of Life Data Systems (BOLD, Ratnasingham and Hebert 2007). BOLD ID and accession number of all specimens used in analyses are given in Table 1. Specimens from which DNA was extracted within this study are marked in bold in column Specimen ID in Table 1. To compare our mtCOI DNA barcode region with those species from the genus Triaenodes that occur in Europe, selected DNA barcode sequences from BOLD database (accessed on July 2017) were employed. A neighbor-joining and maximum likelihood gene tree was produced in MEGA 6 (Tamura et al. 2013), using the Kimura 2-parameter. Intraspecific and interspecific genetic distances, as uncorrected pdistances, were calculated using MEGA 6 (Tamura et al. 2013), using pairwise deletion. The number of hypothetical species within the data set was estimated according to the barcode gap (difference between inter-and intraspecific genetic distances) with the use of Automatic Barcode Gap Discovery, ABGD (Puillandre et al. 2012).
Macrophotography was performed with a Leica Wild MZ8 stereomicroscope and an Olympus SP-500 UZ digital camera, and was processed with the computer programme Olympus Quick Photo Camera 2.2. For determination of the collected specimens we used Malicky (2004). Systematic presentation follows Morse (2020)

Results
In figure 3 we are showing the relationships among the species of the genus Triaenodes, based on the 658 bp long fragment of the DNA barcode region. In this analysis we included 60 specimens from the genus Triaenodes from Europe and Asia (Tab. 1). Identical sequences were collapsed into unique haplotypes (Tab. 1, Fig. 3). In analysis of the genetic distance of barcode mtCOI within species, we eliminate p-distance value 0.0 of sequences that are not of the same length. Minimum intraspecific genetic distance between all Triaenodes species used in analysis is 0.002 (0.2%) recorded within T. unanimis (Tab. 2).
The most interesting results of our analyses relate to the species T. unanimis and T. ochreellus (Fig.  1). A specimen of T. unanimis (sample ID: RUSST219-12) and one of T. rufescens (sample ID: KJTRI665-13), both from Russia, have only 0.2% nucleotide sites different. Both of these specimens, presented as two different species in the BOLD database, group together ( Fig. 1) with 100% certainty. Triaenodes unanimis with sample ID OFTRI242-10 and OFTRI243-10 from Japan shows 8% difference as compared to the other specimens of T. unanimis, forming thus a separate branch in the phylogenetic tree (Fig. 3).
In analysis of species T. ochreellus two barcode sequences were available in databases and they represent same haplotype, which explains the p-distance value of zero. DNA barcode of T. ochreellus lefkas from Croatia (Fig. 2 A-D) (Sample ID: TTOCL_1) was analysed using the BOLD identification engine by a comparison with the full reference database of the DNA barcodes.
The identifying DNA barcode of T. ochreellus lefkas from Croatia (sample ID: TTOCL_1) turns out to have 90.48% similarity with the sequence obtained from the closest available reference sequences of T. ochreellus, which is from Spain (sample ID 09MNKK0409). The maximum intraspecific distance among Triaenodes species used in the analysis is 0.017 (1,7%) (Tab. 2). The discrepancy in the value of the maximum intraspecific genetic distance appears within T. ochreellus and it is 0.102 (10%) (Tab. 2). According to ABGD analyses, the resulting phylogenetic tree of the species T. ochreellus shows two branches, traditionally placed under subspecies: one as T. o. lefkas and one as T. o. ochreellus (Fig. 1). These two taxa have a disjunct distribution, T. ochreellus ochreellus is distributed in the west and T. ochreellus lefkas in south-east part of Europe (Fig. 4). Table 2. Values of the p-distance between groups of Triaenodes species and outgroup species for the barcode mtCOI region. In addition to this, during our study we found five new localities of T. ochreellus lefkas, two in Albania, Shkodër (Skadar) Lake and Shkumbin River, one in Montenegro, Skadar (Shkodër) Lake, and two in Croatia, the rivers Mislina and Cetina (Fig. 4).

Discussion
A new tool in the old practice of molecular taxonomy is DNA barcoding, proposed in 2003 (Hebert et al. 2003a(Hebert et al. , 2003b. This method, which in the case of animals uses a standard 648 bp long fragment of cytochrome c oxidase subunit 1 mitochondrial gene (mtCOI), along with the establishment of the Barcode of Life Data Systems (BOLD) (Ratnasingham & Hebert 2007) resulted in a new approach to species diversity. DNA barcoding is a very useful method in the study of biodiversity, taxonomy, phylogeny and phylogeography of different groups of organisms (e.g. Brehm et al. 2019;Cárdenas et al. 2013;Elías-Gutiérrez et al. 2008;Guo et al. 2016;Huemer et al. 2020;Léger et al. 2020;Tyagi et al. 2017;Yang et al. 2016), including Trichoptera (Kučinić et al. 2016(Kučinić et al. , 2019aSzivák et al. 2017;Santos et al. 2016;Valladolid et al. 2018Valladolid et al. , 2019Vitecek et al. 2020). In many studies, DNA barcoding has aided morphological identification of different taxa Kučinić et al. 2019bKučinić et al. , 2020, but also enabled the discovery of new species (Brehm et al. 2019;Dela Cruz et al. 2016;Graf et al. 2012;Kučinić et al. 2013;Léger et al. 2020;Tyagi et al. 2017;Vaglia et al. 2008).  Although there are no generally accepted values for genetic differences within DNA barcode region among various species, for animals it is considered that an intraspecific genetic distance of more than 2% between populations is high intraspecific variability within the same species (Hebert et al. 2003b). So, the genetic distance of more than 2% within Trichoptera species indicates a possibility of a different species, however, several studies on DNA barcode region show that this difference can be even over 8% (Graf et al. 2015;Zhou et al. 2007).
In this study specimen of T. unanimis (sample ID: RUSST219-12) and T. rufescens (sample ID: KJTRI665-13) both from Russia have only 0.2% nucleotide sites different, which is a value common within the same species. Also, in Fig. 3 we can see that they are grouping together with 100% certainty, indicating that they belong to the same species, T. rufescens.
We can assume a set of explanations: e.g. that the T. unanimis sequence (sample ID:RUSST219-12) is the product of contamination with the T. rufescens species, or that morphological identification of the specimen of T. unanimis is incorrect, or mislabeled.
Results of the ABGD analysis of T. unanimis support the separation of this species into 5 groups, possibly new species. In Fig. 3 we can notice that species T. unanimis shows a branching pattern with high values of nodes (minimum value is 90) indicating that this widespread species is formed of different taxa. For example, samples of T. unanimis shows with samples ID: OFTRI242-10 and ID: OFTRI243-10 from Japan show an 8% difference from other specimens of T. unanimis, forming a separate branch in the phylogenetic tree (Fig. 3). In future, taxonomic research into T. unanimis detailed morphological analyses of adult forms of all species potentially new to science should be included -from Russia, Sweden, Finland, Japan and other areas where T. unanimis is distributed.
DNA barcoded results of T. ochreellus lefkas from Croatia show high difference of about 10% from the T. ochreellus from Spain. This genetic difference between analyzed specimens and the populations to which they belong shows their interspecific relations, i.e. the state of a taxonomically separate species. This is supported by the fact that such a genetic distance is reported from various Trichoptera species (8. 06-15.65, Johanson 2007;8.05-21.7%, Pauls et al. 2010;8.2% Graf et al. 2015), which implies that these two taxa are also separate species.
Based on the phylogenetic species concept (PSC), developed independently by Eldredge & Cracraft (1980) and Nelson & Platnick (1981) and integrative taxonomy (e.g. Brehm et al. 2019;Cárdenas et al. 2013;Graf et al. 2012, Dela Cruz et al. 2016Léger et al. 2020;Valladolid et al. 2018;Vitecek et al. 2015aVitecek et al. , 2015bVitecek et al. , 2017 which includes molecular data, morphology data and distribution data, we elevate subspecies T. o. lefkas to the species level T. lefkas stat. nov. These two taxa have distinctly disjunct ranges with a distance more than 700 kilometers, without any contact zones between them (Fig. 4). The taxon T. lefkas is distributed only in the southeastern part of Europe, in the areas of southern Italy (only the province of Puglia), Greece, Albania, Montenegro, Croatia and Bosnia and Herzegovina Malicky 2005b;Oláh & Kovács 2014;Stanić-Koštroman et al. 2015) (Fig. 4), the finding in the River Cetina in Croatia being the north-westernmost instance of its distribution range (Fig. 4). Sipahiler for west Anatolia region in the Asian part of Turkey lists T. ochrellus (Sipahiler 2005) (Fig. 4), which we can assume applies to the then subspecies, and now species T. lefkas. In this study the distribution range of T. lefkas is expanded with new records from Croatia, Montenegro and Albania (Fig. 4). Before this study, in this area T. lefkas was found at one locality in Croatia (at the River Neretva, Kučinić et al. 2015;Malicky 2005b), one in Albania (Oláh & Kovács 2014) and two localities in Montenegro (Marinković-Gospodnetić 1981;Karaouzas et al. 2019). The nominate taxon Triaenodes ochreellus McLachlan, 1877 is distributed in Spain, France and Portugal (Gonzales & Menédez 2011;Malicky 2005b;Terra 1994) (Fig. 4).
It is also very interesting to find this species at one locality (Old Weston, Huntingdonshire) in Great Britain (Wallace 2016). The species was identified twice there, in 2010 and 2013, and it is considered that it was possibly introduced into Great Britain (Wallace 2016) (Fig. 4). Now T. ochreellus is a member of the fauna of Great Britain.
According to Malicky (2005b) T. lefkas has two generations, one in spring and summer and the other one in autumn which complies with our findings. The emergence of adults begins in April for first generation and the specimens of second generation were found in September and October (Malicky 2005b). This species also inhabits brackish aquatic habitats, as found in Italy (Corallini Sorcetti & Moretti 1984) and in Croatia Malicky 2005b). These biological features have not been established for the species T. ochreellus (Malicky 2005b).

Conclusion
This study contributes to the knowledge on the distribution of rare taxa of the genus Triaenodes in Southeastern Europe and at the same time to the integration of molecular analyses with ecological data and morphology for the validation of the taxonomic status of caddisfly species.