An all-inclusive approach: A universal protocol for the successful amplification of four genetic loci of all Onscidea

Accounting more than 3,700 described species, Oniscidea is the largest and at the same time the only terrestrial isopod suborder inhabiting almost all terrestrial biomes. Despite the great effort dedicated on describing taxonomic diversity of Oniscidea, mainly employing morphology, there is still a considerable number of species/genera of uncertain generic/familiar assignment. Based on different morphological characters, alternative evolutionary relationships have been proposed to describe the diversity of Oniscidea at different phylogenetic levels. Accumulating morphological and genetic data are repeatedly challenging the monophyly of established taxa, undermining the validity of several morphological characters traditionally used in terrestrial isopod taxonomy, leading to often revisions of the current taxonomy of the Oniscidea . The use of genetic data facilitates the efforts to reconstruct the complex evolutionary history of the focal group by providing important data for the identification, delimitation, and description of species. The proposed protocol with universal PCR conditions and primers was used to successfully amplify COI, 16S, 28S and NAK loci in diverse Oniscidea taxa. The application of this protocol is anticipated to facilitate the generation of new genetic data and hence promote scientific research in Isopoda taxonomy, evolution, ecology, and other related fields.


SPECIFICATIONS
Herein we propose a universal easy and cost-effective way to produce genetic data for all Oniscidea. Multiple combinations and newly designed primers were tested under the same amplification conditions at a wide variety of taxa.
Using the same primers for all targeted taxa the same gene fragments are targeted and hence produced data could be used to generate new or enrich existing datasets. Trial registration; N/A Ethics; N/A Value of the Protocol; • Universal for all Oniscidea • Simple and easy to follow protocol • Sequencing of the most popular genetic markers for taxonomic and phylogenetic studies • Cost effective Description of protocol: Touchdown PCR was initially developed as a modification to traditional PCR aiming to increase the specificity and sensitivity of amplification [1][2][3] . The sensitivity of the proposed protocol was tested for four commonly targeted genetic loci for many taxonomically diverse taxa. The proposed protocol could be used to generate mitochondrial (16S, COI) and nuclear (28S, NaK) data.

Method details
The proposed final reaction volume is 20 μL and consists of 0.1 μL of Taq DNA Polymerase (5U/ μL), 2.4 μL of 25 mM MgCl 2 , 1X of Taq buffer A, 0.6 μL of 10 mM dNTPs, 0.6 μL of each primer (10 μM; Table 1 ) and > 2 ng of DNA template in case of COI and 16s or > 10ng in case of 28s and NAK. In any case the use of more than 100ng as DNA template should be avoided. The proposed protocol works effectively with samples of high DNA purity i.e.. A260/A280 rates over 1.5.
Thermocycling conditions for all four proposed genes should be as follows: The proposed protocol works efficiently even at very low DNA concentrations ( < 2ng), ill preserved or very old specimens. If aspecific products are amplified under these conditions an alternative, stricter thermocycling profile should be followed: a) 94 °C 10 min initial denaturation 1 cycle b) 94 °C 60s, 55 °C 60s, 72 °C 60s for 10 cycles c) 94 °C 60s, 50 °C 60s, 72 °C 60s for 10 cycles d) 94 °C 60s, 47 °C 60s, 72 °C 60s for 30 cycles e) 72 °C 10 min final extension 1 cycle f) Hold 10 °C PCR products may be visualized using a common agarose gel electrophoresis. Successfully amplified samples should be purified using any commercial purification protocol following the manufacturer's instructions before proceeding with sequencing. Cycle sequencing can be performed with the same primers used for the initial PCR reaction.

Validation
The effectiveness of the proposed protocol was tested for all available specimens, representing 142 species in 97 genera and 22 families, representing a diverse cross-section of the Oniscidea. Beyond terrestrial isopods, the same protocol was also successfully applied for representatives of the isopod orders Valvifera, Sphaeromatidea and Asellota.
Generated data were deposited in NCBI GenBank ( Table 2 ). A limited number of sequences produced from our group within the framework of other projects, using a very similar protocol, were already published within the framework of previews studies and their corresponding accession numbers are given in Table 1 . [ 8 , 9 ]. Although the new protocol using proposed thermocycling profile and primers were successfully tested in these cases too. Table 2 Species, locality of origin and GenBank accession numbers of individuals used for the protocol validation. Isolate codes with stars ( * ) indicate that they were previously published [ 8 , 9 ] Hemilepistus klugii (Brandt, 1833) Mongoloniscus Sphaerillodillo pubescens (Budde-Lund, 1885) Laureola hiatus Barnard, 1960 ON312037 - Armadillidium cavernarum Vandel, 1958 Schizidium fissum (Budde-Lund, 1885) Troglarmadillidium halophilum Sfenthourakis, 1993 Australiodillo bifrons (Budde-Lund, 1885) ( continued on next page ) Trichoniscus Tauroligidium stygium Borutzky, 1950 Typhloligidium cf. karabijajlae Borutzky, 1962 ( continued on next page )

Conclusion
Taking advantage of the universal and cost-effective nature of the proposed protocol a new potential for the production of genetic data could be foreseen. Based on this protocol retrieved data could be included in multiple datasets given that the same gene regions are targeted. Beyond the data published within the framework of this study, scientific research in related fields such as Isopod systematics, evolution, phylogeny e.t.c. could be facilitated by the application of the proposed protocol. It is worth noting that the majority of available specimens were collected more than two decades ago and a considerable number of them were ill-preserved for a long time (i.e., in 70% alcohol or formaldehyde). Hence, the absence of data in some cases might be attributed to the bad quality of extracted DNA or the lack of effort since we did not aim to sequence all loci for all specimens.

Data availability
All data were deposited in NCBI Genbank. Accession numbers are provided in the main text.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
All data were deposited in NCBI Genbank. Accession numbers are provided in the main text.