Convergent setal morphology in sand-covering spiders suggests a design principle
for particle capture: Electronic Supplementary Material
Rebecca P. Duncan, Kellar Autumn, Greta J. Binford*
Department of Biology
Lewis & Clark College
0615 SW Palatine Hill Rd
Portland, Oregon 97219
* Author to whom correspondence should be addressed. binford@lclark.edu, phone
(503)768-7653, fax (503)768-7658
1
Methods
(a) Taxon sampling and rationale for haplogyne outgroups
We surveyed hair morphology on the carapace of haplogyne outgroup taxa that were
selected to represent a broad phylogenetic range (ESM figure 1), including the most basal
family and at least one family in most major clades. The goal was to enable estimation of
the timing of evolutionary origin of hairlettes in this lineage. Our taxon sampling is
denser among taxa closely related to Sicarius. In the genus Loxosceles we sampled
representatives of all but one currently described species group.
(b) Specimen collection and care for non-Sicarius Haplogynes
All spiders were collected in the field at the locations indicated in ESM table 1. More
detailed collection localities are available from RPD or GJB. Loxosceles, Drymusa and
Scytodes were reared in the lab as described for Sicarius and Homalonychus, but were not
kept in sand.
(c) Sample preparation for comparative analyses of setal morphology and sand
attachment
As Loxosceles, Drymusa and Scytodes were important genera for other research, we
used molted carapaces from these genera to conduct all analyses (as described for
Sicarius and Homalonychus). For all other outgroups, we isolated the cephalothorax
from spiders previously preserved in 75% ethanol, subjected them to three washes in
100% molecular grade ethanol (at least three hours per wash), and dehydrated them in a
CPD2 critical point dryer (Ted Pella). After critical point drying, we mounted samples
on SEM stubs using either colloidal silver liquid (Ted Pella) or superglue (Devcon) and
2
let the adhesive set overnight. We dusted samples in fine sand (grade 0; ESM figure 3)
and applied acceleration as described before sputter coating and imaging in the SEM.
(d) Determining the particle sizes that attach to spiders
We separated sand bought from a hardware store into three size categories using a soil
sieve with 3 screens (Hubbard Scientific; mesh sizes 35, 60, 120). Categories were
named grades 0-2 according to size, where grade 0 contained the finest particles and
grade 2 contained the coarsest. We took small samples of each and measured the particle
sizes from images in ImageJ (NIH) to generate a particle size distribution as follows: For
grades 1 and 2 we cast a small sample of particles into a Petri dish and imaged under a
dissecting microscope (Nikon SMZ1500) with a digital camera (Nikon Coolpix 995).
For grade 0 we obtained a small sample on a SEM stub with double-stick tape (Scotch),
sputter coated it and imaged the sample by SEM because particles were too small to
measure accurately under a dissecting microscope. We took images at three
magnifications (500x, 1,000x and 7,000x) to show the full range of grain sizes in the
sample. We captured images at 1,000x and 7,000x in regions within the 500x images.
For all samples, we took the first image at a section of the Petri dish or SEM stub where
the particles were far enough apart to distinguish the edges of each grain. We captured
subsequent images at an equal number of frames from each.
To calculate the size distribution of particles from the images, we measured the length
at the longest part, and the width at the widest part perpendicular to the length
measurement of each grain. For grade 0 sand, sand grains >m in length were
measured from images at 500x magnification and sand grains between 0.5 and 10m
were measured and counted from images at 1,000x magnification. The smallest sand
3
grains were too small to measure accurately, so sand grains under 0.5m were counted
and not measured from images at 7,000x magnification. We divided the number of sand
grains by the area of each image to estimate the number of sand grains per square mm
and used these estimates to generate a frequency distribution of sand grains in three
categories: >10m, 0.5-10m, and <0.5m.
To determine which size range of sand could attach to the spiders, we placed live,
freshly molted and undusted Sicarius and Homalonychus representatives of various life
stages and from various populations in boxes containing different grades of sand. We
monitored sand capture by self-dusting over the course of four days, using a different
individual for each assay. We visualized spiders under a dissecting microscope (Nikon
SMZ1500) and assessed whether sand capture had occurred and if any cuticle was still
visible. We took images with a digital camera (Nikon Coolpix 995) before and 24 and 96
hours after placing spiders in sand. We assessed the degree to which ceramic
microspheres attach to Sicarius using the same protocol.
4
ESM Table 1. Species with representatives whose carapace setal morphologies were
surveyed by SEM. N is the number of individuals surveyed for each species.
Species
Homalonychus
selenopoides
Homalonychus
selenopoides
Homalonychus theologys
Collection locality (GPS information available upon request)
USA: Pima Co, AZ, Organ Pipe Cactus National Monument
(three localities)
USA: Pima Co, AZ,.Tucson Mountain State Park
N
5
Reference
Roth 1984
1
Roth 1984
2
Roth 1984
1
3
2
Roth 1984
Roth 1984
Sicarius albospinosa
Sicarius albospinosa
Sicarius albospinosa
Sicarius dolichocephalus
USA: San Diego co, CA, Anza-Borrego Desert State Park (two
localities)
USA: Imperial co, CA, SW of Hwy S2
USA: Palm Springs, CA, Boyd Research Center, Deep Canyon
SOUTH AFRICA: Northern Cape Province, Oorlogskloop
Nature Reserve
NAMIBIA: Brandberg Mountains
NAMIBIA: Gobabeb Research Station
NAMIBIA: Twyfelfontein, Wundergat
NAMIBIA: Ruacana Falls
1
3
1
3
Sicarius damarensis
NAMIBIA: Munsterland cave, Near Outjo
Sicarius rupestris
ARGENTINA: Rio Negro, Paso Córdoba
3
Sicarius terrosus
Sicarius patagonicus
Sicarius peruensis
ARGENTINA: San Luis, Parque Sierra de las Quijadas
ARGENTINA: Neuquin, Arroyito Dam
PERU: Tumbes, 19km North of Mancora
3
1
1
Sicarius peruensis
PERU: Quebrada Mogollon
1
Loxosceles spinulosa
Loxosceles spinulosa
Loxosceles spinulosa
Loxosceles spinulosa
Loxosceles vonwredei
NAMIBIA: Ruacana Falls
NAMIBIA: Windhoek, Arebusch Campsite
NAMIBIA: Munsterland cave, Near Outjo
SOUTH AFRICA: Northwest Province, Borakalo National Park
NAMIBIA: Uisib farm, Otjozondjupa
1
2
1
1
5
Loxosceles laeta
Loxosceles intermedia
ARGENTINA: Buenos Aires
ARGENTINA: Parque Nacional el Palmar
3
1
Loxosceles blanda
USA: Carlesbad, NM
3
Purcell, 1908
Purcell, 1908
Purcell, 1908
Lawrence,
1928
Lawrence,
1928
Holmberg,
1881
Nicolet, 1849
Simon, 1919
Keyserling,
1880
Keyserling,
1880
Purcell, 1905
Purcell, 1905
Purcell, 1905
Purcell, 1905
Newlands,
1980
Nicolet, 1849
Mello-Leitão,
1934
Gertsch and
Ennik, 1983
Scytodes sp.
Scytodes sp.
Scytodes sp.
Scytodes sp.
Drymusa serrana
PERU: Junin, Vitoc
PERU: Cuzco, Quillabamba
PERU: Lambayeque, Olmos, Along Rio Marañón, near El Mayo
PERU: Pevas
ARGENTINA: San Luis, Merlo
1
2
1
1
4
Diguetia canites
Plectreuris tristis
Plectreuris tristis
Dysdera crocata
Kukulkania arizonica
USA: Pima co, AZ, Brown Canyon, Baboquivari Mountains
USA: Pima co, AZ, Tucson Mountain Park
USA: Black Canyon City, AZ
USA: Multnomah co, OR, Portland
USA: Pima co, AZ, Catalina mountains, Rillito wash
3
1
1
2
1
Kukulkania arizonica
USA: Pima co, AZ, Catalina mountains, Sabino cree
1
Homalonychus theologus
Homalonychus theologus
Sicarius sp.
5
Goloboff &
Ramirez, 1991
McCook, 1889
Simon, 1839
Simon, 1839
Koch, 1838
Chamberlin &
Ivie, 1935
Chamberlin &
Ivie, 1935
ESM Figure 1. Phylogenetic tree of Haplogynes. Phylogenetic relationships between
Sicarius and other haplogyne taxa based on morphology. Asterisks mark the outgroups
represented in sand attachment and setal morphology comparative analyses. Tree is
based on Platnick et al., 1991
ESM Figure 2. Sand associates less with setae lacking hairlettes but still clumps
densely in setose regions of the carapace. To establish the role of setal morphology in
sand adhesion we dusted the cephalothorax or carapace of 8 haplogyne outgroups with
fine sand, applied acceleration as described in methods and compared them to dusted
Sicarius and Homalonychus using SEM. Sand adhered to all outgroups, but largely
associated with regions containing dense setae. Sand clumping occurred on setose
regions of the cuticle even when particles did not associate strongly with individual setae
(e.g. Loxosceles spinulosa, Plectreuridae, Filistatidae). The degree to which particles
adhered to individual setae varied, but was generally much less than the degree to which
they adhered to Sicarius and Homalonychus setae. Particles were often trapped between
or under setae (arrows, Scytodes, Drymusa, Dysderidae, Plectreuridae) and associated
more strongly with shorter setae that lay close to the cuticle than with longer setae
(arrow, Drymusa). n = 2 Filistatidae, 2 Dysderidae, 2 Plectreuridae, 3 Diguetidae, 4
Drymusa serrana, 5 Scytodes sp., 5 Loxosceles spinulosa, 5 Loxosceles vonwredei.
ESM Figure 3. Particle size distributions of grades 0, 1 and 2 sand. We separated
sand into three size categories using a soil sieve with three screens (mesh sizes 35, 60 and
120; Hubbard Scientific) and characterized the particle size distribution of each category
by measuring sand grains in a sample of each one. Categories were named grades 0-2
6
from finest to most coarse particle sizes. Grade 0 sand was used in all comparative
analyses of sand attachment.
ESM Figure 4. Fine particles attach to Sicarius and Homalonychus. We placed live
Sicarius in each size category of sand and in ceramic microspheres (3m; mean diameter =
40m) and allowed them to self-dust. We placed live Homalonychus in grades 1 and 0
sand. We monitored them over the course of 4 days to determine the range of particle
sizes that could stick to them. (a) Grade 2 sand did not adhere (n=4) to Sicarius. Grade
1 sand adhered to Sicarius (n=7), but did not completely mask the cuticle after 4 days.
Only grade 0 sand and the microspheres totally covered Sicarius (n=4 for each) to the
point that none of the cuticle was visible. (b) In Homalonychus, grade 1 sand adhered
(n=4), but a substantial amount of cuticle was visible after 4 days. Grade 0 sand
completely covered the cuticle in most individuals, leaving only small areas where the
colour of the cuticle showed through in one (n=4). In all individuals dusted with grade 0,
sand failed to cover a small region on the posterior slope of the carapace (arrowhead).
7