Isogenus nubecula
Creators: name: Davy-Bowker, John
name: Hammett, Michael J.
name: Mauvisseau, Quentin
name: Sweet, Michael J.
Publisher: name: Zenodo
Year: 2018
Subjects: Biodiversity
Taxonomy
Animalia
Arthropoda
Insecta
Plecoptera
Perlodidae
Isogenus
Isogenus nubecula

description: Isogenus nubecula (Newman, 1833)   
 is a rare stonefly species that has reportedly become extinct in many European countries from which it had historically been known (Davy-Bowker, 2003). Whilst Derka et al., (2002) described I. nubecula as a species originally recorded from many localities in Western and Central Europe. Zwick (1992) noted its widespread disappearance in the second half of the 20th century. In central and eastern Europe, Claessens (1981) found no records since 1936 from Austria, Belgium, Switzerland and the Netherlands, and Landa et al., (1997) considered that I. nubecula had probably also become extinct in the Czech Republic and Slovenia. Surviving European populations are few in number and occur mainly in eastern Europe in the Lafnitz and Rába rivers in South-Eastern Austria and western Hungary (Graf & Kovács, 2002; Kovács & Ambrus, 2001); the River Tisza in Hungary (Juhász et al., 1998; Kovács & Ambrus, 2001); the River Jelesna, Slovakia where a single nymph was found in 1998 (Derka et al., 2002); Northrhine-Westfalia in Germany (Graf et al. 2009); and in central Sweden (Hoffsten, 2003). Isogenus nubecula may potentially also occur in Finland, Estonia, Latvia and Lithuania.   
 Within the United Kingdom (UK), I. nubecula was first recorded in 1959 from the River Dee at Bangor-on- Dee in North Wales (Hynes, 1963). This site has a stone road bridge and a large within-channel island (Fig. 1a). The substratum consists of large gravel and cobbles, much of which is unstable due to high water velocity. A detailed account of the UK records of I. nubecula between its discovery in 1959 and 2003 is provided by Davy- Bowker (2003). In summary, its continued presence on the River Dee was confirmed in the surveys carried out in 1981 and 1982 by Mills and Andrew (1984) where its maximum range was recorded at nine of thirteen sites surveyed over a 45km stretch of the river. In 1992, the range of I. nubecula had reduced significantly, to just two of its former sites (Bowker, 1993), however in 1993 it was found at five sites (Bowker, 1995), though this reduced again two years later and by 1995 only one nymph was found at one site (Tanner, 1997). Despite repeated surveys conducted in 1997 (Tanner, 1997); 1998 (Millband, 1998); 1999 (Millband, 1999); 2003 and 2004 (Davy-Bowker, 2003; Hammett & Wallace, 2005); 2007 (Davy-Bowker et al., 2007) and unpublished surveys carried out by John Davy-Bowker in 2012 and 2015, no further specimens of I. nubecula were found on the River Dee. The September 1995 record was therefore the last record of I. nubecula in the UK. Its occurrence from a single river and none of its tributaries, its decline in range, and subsequent disappearance provided the basis for Macadam (2015) in his review of the stoneflies of Great Britain to assign I. nubecula the international threat category of ‘Critically Endangered’.   Materials and methods  
 On the 12th March 2017, the River Dee at Bangor-on-Dee (Fig. 1a) was visited and sampled by John Davy-Bowker in an attempt to find I. nubecula nymphs. Sampling and bankside sorting was carried out for about two hours and all large Perlodidae nymphs found were transferred to sample containers with river water and oxygenation. Nymphs of different Perlodidae species, including I. nubecula (Fig. 1b) were kept in separate containers and aerated until adult rearing.   
A follow up survey was also performed by Mike Hammett on the 15th March 2017, at Bangor-on-Dee and also at Erbistock a short distance upstream. The same procedure was followed, but over a 20 minute time period at both sites, with all large Perlodidae nymphs being kept in separate containers and aerated until adult rearing.  
 Following the completion of adult rearing, one of the prospective I. nubecula adults was preserved in>95% ethanol and sent to the University of Derby for DNA extraction and sequencing. All lab equipment was disinfected prior to doing any lab work with 10% bleach and 100% ethanol. Pipettes, tubes and tips were sterilised for 20 min under UV light before use. Extraction was completed using the Qiagen DNeasy Blood and Tissue kit following the manufacturer’s instructions and a polymerase chain reaction (PCR) was performed using primers (Forward) LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' and (Reverse) HC02198: 5'- TAAACTTCAGGGTGACCAAAAAATCA-3' (Folmer et al., 1994) which amplified the mitochondrial gene cytochrome oxidase 1.   
 The DNA extraction and sequencing procedure was as follows. PCR was performed in a 25 µL total volume with 12.5 µL of 2x PCRBIO Ultra Mix Red (PCRBIOSYSTEMS), 1 µL of each primer (10µM), 9.5 µL of ddH2O and 1 µL of DNA template. The PCR programme included an initial 1 min denaturation at 95ºC, 35 cycles of denaturation at 95 ºC for 1 min, annealing at 40 ºC and elongation at 72 ºC for 1 min and 30 s. A final elongation step of 7 min at 72 ºC was added at the end of the PCR (Folmer et al. 1994). A negative control was added during the PCR to ensure the absence of contamination.   
PCR products were confirmed by electrophoresis on 2% agarose gel stained with 3 µL of GelRed= Nucleic Acid Gel Stain, Biotium. Product sizes were checked by comparing amplified DNA to 5 µL of PCRBio Ladder IV (PCRBIOSYSTEMS). 15 µL of PCR product added with 2 µL of Forward primer were then sent for sequencing by Eurofins Genomics UK.  
 Multiple sequences were created from the same individual to ensure no technical errors occurred during sequence amplification, these were aligned using Geneious Pro R6 and a representative sequence was submitted to GenBank under the accession number MF801622. A phylogenetic tree was developed following a global alignment with free end gap and using the Neighbor-Joining method within the Geneious Pro R6 software and using Tamura- Nei’s Genetic Distance Model on the cytochrome oxidase subunit I (COI) fragments from stonefly species retrieved from the NCBI database. This phylogenetic tree (Fig. 2) illustrates where I. nubecula sits in relation to a range of other available COI sequences for stonefly species found or previously recorded from the UK (Appendix. 1).   Results and discussion  
 Following subsequent adult rearing, definitive species identifications were then possible using the key of Hynes (1977). It was confirmed that I. nubecula has been rediscovered at Bangor-on-Dee. However, numbers of I. nubecula nymphs were low (Table 1). The second species was identified as Perlodes mortoni (Klapálek, 1906). (Table 1).   
 The rediscovery of I. nubecula makes this species one of the rarest stoneflies in the UK and Europe and its rediscovery is therefore of international significance. The River Dee population is important at a European level, as this record highlights the westernmost point in Europe where the species is found, with the next nearest populations occurring in Austria and western Hungary, Slovakia and central Sweden.   
 We draw readers attention to the importance of further monitoring of species such as I. nubecula, as stoneflies are an important food source for many species in river systems from fish to many birds (during different life stages). Moreover, they have an essential functional role inside river trophic webs as gatherer-collectors, scrappers, shredders or predators. The conservation of species such as I. nubecula is therefore important on many levels. The sequencing of the COI gene of I. nubecula (submitted to the NCBI database) can now allow for the development of novel tools for surveying this species.   
 In this context, further monitoring could be assisted with the development of eDNA techniques targeted for I. nubecula. eDNA is a relatively new tool available to conservationists, ecologists and environmental managers alike (Thomsen & Willerslev, 2014) and can significantly aid in understanding a species home range with reduced physical surveying needs and reduced costs (Mauvisseau et al., 2017). Access to this DNA sequence and a greater comprehension of the phylogenetic relationships (Fig. 2) between close relative species are indeed crucial for developing species specific primers. Furthermore, public available repositories already contain COI sequences from various organisms. Supplementing the public database with DNA sequences from this endangered species will enhance our knowledge about phylogenetic relationships in the Perlodidae family. Our sequencing results place I. nubecula closest to the genera Isoperla and Diura. The close genetic relatedness we observe between Isogenus, Isoperla, and Diura is what we would expect within the same family.   
 Finally, we suggest that I. nubecula is also a potential candidate for a captive breeding program, whereby, if successful, individuals could be re-introduced into previously known and still suitable habitats through the River Dee catchment and surrounding area. This could make a significant contribution to safeguarding the future survival of this species in Europe.
description: Published as part of Davy-Bowker, John, Hammett, Michael J., Mauvisseau, Quentin & Sweet, Michael J., 2018, Rediscovery of the critically endangered ' scarce yellow sally stonefly' Isogenus nubecula in United Kingdom after a 22 year period of absence, pp. 295-300 in Zootaxa 4394 (2) on pages 295-298, DOI: 10.11646/zootaxa.4394.2.12, http://zenodo.org/record/1197763