FNZ 64 - Pisauridae (Arachnida: Araneae) - Methods and conventions
Vink, CJ; Dupérré, N 2010. Pisauridae (Arachnida: Araneae). Fauna of New Zealand 64, 60 pages.
(
ISSN 0111-5383 (print),
ISSN 1179-7193 (online)
;
no.
64.
ISBN 978-0-478-34722-7 (print),
ISBN 978-0-478-34723-4 (online)
).
Published 13 Jul 2010
ZooBank: http://zoobank.org/References/05FA0262-846F-4216-96B4-D5ED10D3A7D9
Methods and conventions
Collecting. Pisaurids can be collected by a variety of methods. The best method for collecting pisaurids is with a strong head torch at night (about two hours after sunset, when New Zealand species appear to be most active, CJV, pers. obs.). Light is reflected off the grate-shaped tapeta in the eyes and the spider’s presence is indicated by a bluish sparkle. Another useful method is daytime hand searching, either by looking for specimens directly on the ground or turning over rocks and logs. Females can often be found on nurserywebs at night; however, in the daytime, they can sometimes be found on or below their nurserywebs. Females can also be found in the daytime by searching the ground at the base of foliage that nurserywebs are attached to. Pitfall trapping is effective but unless the specimens are collected within a couple of days of being caught they can start to decay, which can make identification difficult. Decay can be prevented by the use of a good preservative such as propylene glycol, which also preserves DNA but can shrivel soft tissue (Vink et al. 2005).
Preservation. Pisaurids are best preserved in 70-75% ethanol. To ensure adequate DNA preservation, specimens should also be stored at -20°C or less (Vink et al. 2005). Spiders can be stored in 95-100% ethanol to preserve DNA but it is best to combine this with storage temperatures less than or equal -20°C (Vink et al. 2005). 95-100% ethanol makes specimens brittle and can be unsuitable for morphological examination.
Preparation. Specimens should be labelled with the locality, including area code (Crosby et al. 1976, 1998), and, if known, latitude and longitude, collection date, collector’s name and habitat data.
Most morphological features used for identifications can be seen under an ordinary dissecting microscope. When examining specimens in alcohol they should be rested in washed quartz sand or glass beads to provide support for the spider. External sclerites of the epigynum can be viewed in situ. However, if the abdomen is shrivelled the epigynum can be obscured or distorted. To see the structures clearly it is often necessary to remove hairs from the epigynum by scraping them with an entomological pin or pulling them out with fine forceps. 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.
Internal genitalia 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 illustrated for all species.
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 length of each species represent the smallest and largest individual of each sex found in all specimens examined. A mean body length and the standard error of the mean (s.e.m.) were calculated and the number of specimens measured is given. Intraspecific and interspecific mean body lengths were compared between males, females and species using the Student’s t-Test.
Types. New Zealand collections were searched and enquiries were made at overseas collections known to be associated with species’ authors for type specimens of New Zealand pisaurids.
In the descriptive part of this work, the status, repositories, and full label data for all type specimens examined are given. Label data are listed as follows: different labels are denoted by a solidus (/) and different lines on a label by a semicolon. All other punctuation is as it appears on the label. Additional information not included on the label is placed between square brackets.
Descriptions. For the new species, illustrations, measurements, and colour pattern descriptions were made from type specimens. For existing species, illustrations, measurements, and colour pattern descriptions were prepared from a non-type representative male and female specimen (with collection information shown).
Epigynal and male pedipalpal illustrations omit the setae for clarity.
Descriptions of colours are for alcohol-preserved specimens. It should be noted that colours and colour patterns can fade in older specimens that have not been stored away from light.
When possible, measurements were made with a micrometer ruler fitted to the eyepiece of a stereo microscope. However, longer measurements, such as body length, were made using a digital vernier calliper.
Characters diagnostic in other spider families (e.g., eye size and position) are not diagnostic for Pisauridae (Carico 1973) 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. Female internal genitalia were excised using a sharp entomological needle and cleared in lactic acid.
Map images were created using the geographic information system software ArcGIS 9 (ESRI).
Text conventions. The area codes of Crosby et al. (1976, 1998) are used in collection records.
The following acronyms for repositories are used:
AMNZ Auckland Museum, Auckland, New Zealand
BMNH Natural History Museum, London, United Kingdom
CMNZ Canterbury Museum, Christchurch, New Zealand
LUNZ Entomology Research Museum, Lincoln University, New Zealand
MNHN Muséum National d’Histoire Naturelle, Paris, France
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
Molecular biology. To construct a molecular phylogeny of New Zealand Pisauridae and to facilitate the identification of immature Dolomedes specimens, we used the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and the nuclear gene actin 5C. COI is one of the fastest evolving mtDNA genes and has been used to examine genetic differences between species in Dolomedes (Tanikawa & Miyashita 2008) and between species and populations in the closely related Lycosidae (Colgan et al. 2002, Vink & Paterson 2003, Chang et al. 2007, Hebets & Vink 2007). Actin 5C evolves more slowly and includes an intron of approximately 100 bp, which can vary between species (Vink et al. 2008a).
DNA was extracted from the muscle of one to three femurs of 58 specimens using a ZR Genomic DNA II Kit™ (Zymo Research) and each specimen was arbitrarily assigned a specimen code (Table 1). The primers initially used to amplify and sequence a 1054 bp COI fragment were LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) (Folmer et al. 1994) plus C1-N-2568 (5’-GCTACAACATAATAAGTATCATG-3’) (Hedin & Maddison 2001). However, we were able to PCR amplify a 1054 bp fragment for only five specimens, but a shorter COI fragment (850 bp) was successfully amplified and sequenced from the other specimens using C1-J-1718-spider (5’-GGNGGATTTGGAAATTGRTTRGTTCC-3’) (Vink et al. 2005) plus C1-N-2568. To amplify a 935 bp fragment of actin 5C, which included a 107 bp intron, we used the primers actin5C-F-229 (5’-AAGTATCCNATTGAGCATGGTATTG-3’) (Vink et al. 2008a) plus actin5C-R-1057 (5’-TTNGADATCCACATTTGTTGGAA-3’) (Vink et al. 2008a). PCR amplification of actin 5C was not always successful, presumably because the DNA had degraded. Vink et al. (2005) found that low copy genes, such as actin, could not be amplified from arachnid tissue that had not been preserved optimally for DNA preservation. PCR amplification was performed using i-StarTaq™ DNA Polymerase (iNtRON Biotechnology) in a Mastercycler® (Eppendorf) thermocycler with a cycling profile of 35 cycles of 94 ºC denaturation (30 s), 48 °C annealing (30 s), 72 ºC 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). Purified PCR fragments were sequenced in both directions at the Allan Wilson Centre Genome Service (Massey University). Sequence data were deposited in GenBank (ww.ncbi.nlm.nih.gov/GenBank/ – see Table 1 for accession numbers). Sequences were edited and compared to each other using Sequencher 4.6 (Gene Codes Corporation).
We also explored the phylogenetic utility of a section of the nuclear genome spanning the two ribosomal internal transcribed spacer regions (ITS1 and ITS2), all of the nuclear ribosomal RNA subunit 5.8S and small fragments of the flanking 18S and 28S. ITS1 and ITS2 have been used at the species and population level in spiders (Hedin 1997, Arnedo & Gillespie 2006, Vink et al. 2008b) including the related Lycosidae (Chang et al. 2007). However, in New Zealand Dolomedes, we occasionally observed double bands when visualising the PCR products and multiple peaks in the electropherograms of some specimens, which indicates there are multiple copies of ITS1 and ITS2 that have not evolved concertedly. Even if the separate copies were sequenced for each species, it is unlikely that we could be certain we were comparing homologous copies.