FNZ 70 - Periegopidae (Arachnida: Araneae) - Methods and conventions
Vink, CJ; Dupérré, N; Malumbres-Olarte, J 2013. Periegopidae (Arachnida: Araneae). Fauna of New Zealand 70, 41 pages.
(
ISSN 0111-5383 (print),
ISSN 1179-7193 (online)
;
no.
70.
ISBN 978-0-478-34740-1 (print),
ISBN 978-0-478-34741-8 (online)
).
Published 7 Mar 2013
ZooBank: http://zoobank.org/References/E8D9D21A-46FD-4E6B-9E79-DBE67BAE57D1
Methods and conventions
Collecting. Periegopids can be collected by a variety of methods. The best method for collecting periegopids is by searching under rocks and logs that are sitting on or partially embedded in soil on open ground within forest in areas with a good litter layer over well drained soil (Vink 2006). Periegopids have also been found under wooden discs (M. H. Bowie, pers. comm.) that are facsimiles for natural fallen logs (Bowie & Frampton 2004). Periegopids have also been collected by sieving and searching leaf litter. Specimens of P. suterii and P. australia have been caught in pitfall traps, but unless specimens are collected and preserved within a couple of days of being caught they can start to decay, which can make identification difficult. Decay of specimens caught in pitfall traps can be mitigated by the use of a good preservative such as propylene glycol, which also preserves DNA but can shrivel soft tissue (Vink et al. 2005). Malumbres-Olarte et al. (in press) used propylene glycol in their pitfall traps and found that useful DNA was preserved after two weeks in the field.
Preservation. Periegopids are best preserved long-term in 70–75% ethanol. To ensure adequate DNA preservation, specimens should also be stored at ≤ -20°C (Vink et al. 2005). Spiders can be stored in 95–100% ethanol to preserve DNA but as with lower ethanol concentrations, it is still best to combine this with storage temperatures ≤ -20°C (Vink et al. 2005). Preservation in 95–100% ethanol makes specimens brittle, potentially rendering them unsuitable for morphological examination.
Preparation. Specimens should be labelled with the locality, including area code (Crosby et al. 1976, 1998), latitude and longitude, collection date, collector’s name, habitat data, and collection method.
Most morphological features used in identification can be seen under an ordinary dissecting microscope. When examining a spider in ethanol it should be rested in washed quartz sand or glass beads to provide support for the specimen. This also allows the specimen to be positioned at any desired viewing angle. The features of the male pedipalp are best viewed by removing the left pedipalp at the junction between the trochanter and the femur, and viewed ventrally. In periegopids, as with other haplogyne spiders, there is no sclerotised external genitalia, only a slightly sclerotised plate, which makes distinguishing females from immature specimens difficult. Female internal genitalia were excised using a sharp entomological needle and were prepared for examination by placing the dissected genitalia in either lactic acid or 10% KOH solution for 1–3 hours at 37°C to dissolve soft tissue. Internal genitalia were only illustrated for P. suterii and P. australia.
Measurements. All measurements are in millimetres (mm). Where the measurements are expressed as a fraction, the numerator refers to the length of the structure and the denominator refers to its width. Measurements outside parentheses are for males and inside parentheses for females. The size ranges given for the body and carapace length of each species represent the smallest and largest individuals of each sex that we examined. The mean body length and carapace length were calculated and the number of specimens measured given.
Descriptions. For the new species, illustrations, measurements and colour pattern descriptions were made from the type specimen. For the existing species, illustrations, measurements, and colour pattern descriptions were prepared from a non-type representative male and female specimen (with collection information shown).
Descriptions of colours are for ethanol-preserved specimens. It should be noted that colours and colour patterns can fade in older specimens, particularly those that have not been stored away from light.
Measurements were made with a micrometer ruler fitted to the eyepiece of a Leica M125 stereo microscope.
Characters diagnostic in other spider families (e.g., eye size and position, leg spination) are not diagnostic for Periegopidae species and have not been included in the descriptions.
Illustrations. Specimens to be illustrated were first photographed with a Nikon Coolpix 950 digital camera attached to a SMZ-U Nikon dissection microscope. The digital photos were then used to establish proportions and the illustrations were detailed and shaded by referring back to the structure under the microscope.
Map images were created using the geographic information system software ArcMap 10 (ESRI).
Text conventions. The area codes of Crosby et al. (1976, 1998) are used in collection records.
The following acronyms for repositories are used:
AMNH American Museum of Natural History, New York, U.S.A.
LUNZ Entomology Research Museum, Lincoln University, New Zealand
MONZ Museum of New Zealand Te Papa Tongarewa, Wellington, New Zealand
NZAC New Zealand Arthropod Collection, Auckland, New Zealand
OMNZ Otago Museum, Dunedin, New Zealand
QMB Queensland Museum, Brisbane, Australia
Molecular biology. To construct a molecular phylogeny of New Zealand Periegopidae and to facilitate the identification of immature specimens, we used the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and the nuclear ribosomal RNA gene 28S. COI is one of the fastest evolving mtDNA genes and has been used to examine genetic differences between and among haplogyne spiders (e.g., Arnedo et al. 2001, 2009; Astrinet al. 2006; Starrett & Waters 2007; Binford et al. 2008; Dimitrov et al. 2008; Huber & Astrin 2009; Duncan et al. 2010) and New Zealand spider species (Vink & Paterson 2003; Vink et al. 2008, 2011a, b; Framenau et al. 2010; Vink & Dupérré 2010; Lattimoreet al. 2011; Malumbres-Olarte & Vink 2012). 28S was selected because it is a slow evolving gene (Hedin & Maddison 2001) and has been used in phylogenetic analyses of haplogynes (Bruvo-Mađaric et al. 2005; Binfordet al. 2008) as well as other spiders (e.g., Hedin & Bond 2006; Rixet al. 2008; Wang et al. 2008; Spagna et al. 2010). Eight specimens of P. suterii and three specimens of P. keani were sequenced for COI. Each specimen was arbitrarily assigned a specimen code (Table 1). A subset of four specimens, two from each species, was sequenced for 28S.
DNA was extracted non-destructively (see Paquin & Vink 2009) from either two to three legs using a ZR Genomic DNA™ Tissue Minipreps (Zymo Research). The primers used to PCR amplify and sequence COI fragments were either LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) (Folmeret al. 1994) plus C1-N-2776-spider (5’-GGATAATCAGAATANCGNCGAGG-3’) (Vinket al. 2005) or C1-J-1718-spider (5’-GGNGGATTTGGAAATTGRTTRGTTCC-3’) (Vinket al. 2005) plus C1-N-2776-spider. The two COI fragments were 1260 base pairs (bp) and 1055 bp long, respectively. Two different primer pairs were used to amplify and sequence two overlapping 28S fragments; 28S-B1 (5'-GACCGATAGCAAACAAGTACCG-3') (Bruvo-Mađaric et al. 2005) plus 28S-B2 (5'-GATTAGTCTTTCGCCCCTATA-3') (Bruvo-Mađaric et al. 2005) and 28Sa (5'-GACCCGTCTTGAAACACGGA-3') (Nunn et al. 1996) plus LSUR (5'-GCTACTACCACCAAGATCTGCA-3') (Rix et al. 2008). The two 28S fragments were 819–821 bp and 830–833 bp long, respectively. PCR amplification was performed using i-StarTaq™ DNA Polymerase (iNtRON Biotechnology) in a Mastercycler® (Eppendorf) thermocycler with a cycling profile of 35 cycles of 94ºC denaturation (30 s), 48°C (COI) or 60°C (28S) annealing (30 s), 72ºC extension (1 min) with an initial denaturation of 3 min and a final extension of 5 min. Excess primers and salts were removed from the resulting double-stranded DNA using a DNA Clean & Concentrator™ Kit (Zymo Research). Double bands were observed when 28S PCR products of Pk1 were visualised via gel electrophoresis; both bands were excised from the gel and prepared for sequencing using a Zymoclean™ Gel DNA Recovery kit. For Pk1, the primer pair 28S-B1 plus 28S-B2 amplified a fragment of 28S DNA that BLAST database searching indicated may have come from a ciliate protozoan. For Pk2, the primer pair 28Sa plus LSUR amplified a fragment of 28S DNA from a soil nematode in the family Cephalobidae. Amplification of 28S was attempted for specimens Ps5 and Ps6, but was not possible owing to contamination by a fungus that the primers preferentially annealed to. Contaminants were identified by BLAST searching their sequences (Altschul et al. 1997). Purified PCR fragments were sequenced in both directions at the Core Instrumentation Facility (University of California, Riverside, USA), the Massey Genome Service (Massey University, New Zealand), or Macrogen (Korea). Sequence data were deposited in GenBank (www.ncbi.nlm.nih.gov/GenBank/ – see Table 1 for accession numbers). Sequences were edited using Sequencher 4.6 (Gene Codes Corporation).