king2012

J OURNAL OF C RUSTACEAN B IOLOGY, 32(3), 465-488, 2012
DIVERGENT MOLECULAR LINEAGES AND NOT-SO-CRYPTIC SPECIES:

THE FIRST DESCRIPTIONS OF STYGOBITIC CHILTONIID AMPHIPODS
(TALITROIDEA: CHILTONIIDAE) FROM WESTERN AUSTRALIA
Rachael A. King 1,∗ , Tessa Bradford 2 , Andrew D. Austin 3 ,

William F. Humphreys 4 , and Steven J. B. Cooper 1
1 SouthAustralian Museum, North Terrace, Adelaide, South Australia 5000; and Australian Centre for Evolutionary
Biology and Biodiversity, School of Earth and Environmental Sciences, The University of Adelaide,
South Australia 5005, Australia
2 Australian Centre for Evolutionary Biology and Biodiversity, School of Earth and Environmental Sciences,
The University of Adelaide, South Australia 5005, Australia; and Catchment Biogeochemistry and Aquatic Ecology,
CSIRO Land and Water, Waite Campus, Urrbrae, South Australia 5064, Australia
3 Australian Centre for Evolutionary Biology and Biodiversity, School of Earth and Environmental Sciences,
The University of Adelaide, South Australia 5005, Australia

4 Western Australian Museum, Collections and Research Centre, Locked Bag 49, Welshpool DC, Western Australia 6986,
Australia; and School of Animal Biology, University of Western Australia, Nedlands, Western Australia, Australia
ABSTRACT
The Australian stygofauna comprises a unique and diverse assemblage of invertebrates, of which the amphipod crustaceans are a dominant
but poorly described element. Recent exploration of the Western Australian stygofauna, in particular the Yilgarn region of central Western
Australia, has shown evidence of great species diversity, with numerous individual calcrete aquifers found to contain unique assemblages
of invertebrate species. A recent fine-scale biodiversity initiative, using COI barcoding, of a single calcrete aquifer (Sturt Meadows) in the
Yilgarn region reported the presence of three divergent and morphologically cryptic stygobitic lineages of amphipods from Chiltoniidae,
which represent undescribed taxa. This paper details the subsequent systematic analysis of these COI lineages and presents a broader
phylogeny and detailed morphological analyses of the lineages. The report of cryptic species was not supported upon morphological
examination and three new species from three new genera (Scutachiltonia n. gen., Stygochiltonia n. gen., and Yilgarniella n. gen.) are
described from the Sturt Meadows calcrete aquifer. The three genera do not form a monophyletic group and are instead believed to
have evolved from separate colonisation events from distinct ancestors rather than from speciation events within the aquifer. This work
contributes to a broader research initiative, documenting the presence of a rich subterranean invertebrate fauna in the Yilgarn region.
K EY W ORDS: Amphipoda, COI, cryptic species, groundwater, mtDNA, stygofauna
DOI: 10.1163/193724012X626566
I NTRODUCTION largely by Crangonyctoidea (Bradbury and Williams, 1999;

Bradbury, 2000; Lowry and Stoddart, 2003; Väinölä et al.,
Within the last two decades, intensive exploration of Aus- 2008). Whilst abundant, the Australian stygobitic amphipod
tralian subterranean aquatic habitats and their associated fauna is poorly described; with estimates indicating that in
fauna has revealed a unique and highly diverse assemblage the western half of Australia only about 20% of known
of stygobitic invertebrates (stygofauna) (Humphreys, 2008; and identified stygobitic amphipods have been formally
Humphreys et al., 2009; Guzik et al., 2011a). Once con- described (28 species from four families – Bogidiellidae,
sidered a depauperate fauna, in comparison to subterranean Hadziidae, Melitidae, and Paramelitidae) and perhaps 20
diversity hotspots in Europe and North America, many times more remain to be discovered (Eberhard et al.,
major faunal groups (Mollusca, Nematoda, Oligochaetea, 2005; Finston et al., 2008; Guzik et al., 2011a). The
Hexapoda, and Crustacea) have now been found in a examination of molecular diversity in undescribed stygobitic
wide variety of geological habitats across Australia that amphipods has largely contributed to this estimate; with
include karst, larval tubes, alluvial sediments, fractured recent studies uncovering highly diverse mtDNA lineages
rock aquifers and subterranean carbonate deposits (calcrete equating to species endemic to individual aquifers (Cooper
aquifers) (Humphreys, 2008; Väinölä et al., 2008; Guzik et et al., 2007; Finston et al., 2007, 2008; Bradford et al.,
al., 2011a). 2010; Guzik et al., 2011b). Descriptive taxonomic work
Crustaceans are a dominant member of the Australian to formally identify species has lagged behind molecular-
stygofauna, with Amphipoda particularly well represented, based species discovery, in part due to a lack of specialised
∗ Corresponding author; e-mail: Rachael.King@samuseum.sa.gov.au
© The Crustacean Society, 2012. Published by Koninklijke Brill NV, Leiden DOI:10.1163/193724012X626566
466 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 3, 2012
taxonomists, but also due to the detection of cryptic species springs in South Australia (King, 2009a). Recent work, in-
(Finston et al., 2008). Adaptations to extreme conditions, cluding both molecular and morphological analyses, has de-
such as in stygobitic habitats, have been shown to lead scribed a highly diverse group and provided new species and
over large periods of time to phenotypic convergence with new genera as well as evidence of morphologically cryp-
the potential to confound morphological study (Wiens et tic species (King, 2009a, b; Murphy et al., 2009; King and
al., 2003; Lefébure et al., 2006; Trontelj et al., 2009; Leys, 2011). Whilst undescribed, stygobitic chiltoniid am-
Zakšek et al., 2009); within Amphipoda adaptations to phipods are known to exist in the Yilgarn region; populations
the stygobitic environment are typically the reduction or from several calcretes were sequenced and mistakenly iden-
loss of pigment and eyes, reduction of coxae, and the tified as belonging to Hyalidae, along with crangonyctoid
elongation of antennae, body segments and posterior legs amphipods by Cooper et al. (2007). Within the Sturt Mead-
(Bradbury, 2000). The potential for phenotypic convergence ows calcrete, three diverse sympatric chiltoniid amphipod
in stygobitic habitats, combined with typically small sample lineages were detected using mitochondrial and allozyme
sizes for stygofauna of a few individuals per collection, molecular markers (Bradford et al., 2010). Each purported
increases the difficulty in analysing morphological variation. species occurred in differing abundances across the calcrete
Under these conditions it is likely that morphology-only and each was reported to be more closely related to taxa in
identification approaches will lead to an underestimation of other regional calcretes than to each other. The lineages were
species diversity and a combination of techniques and data also reported to be cryptic species with no morphological
(molecular, morphological, biochemistry, and geographical) differences discerned (Bradford et al., 2010).
where available, are more appropriate to fully explore Evidence of high levels of diversity among morpho-
species boundaries and diversity of styogobitic amphipods logically cryptic species, coupled with the availability of
(Lang et al., 2003; Tomikawa et al., 2007; Flot et al., 2010). mtDNA sequence data and extensive material collected from
In Western Australia, the ancient Yilgarn craton (Fig. 1) the Sturt Meadows calcrete presented an opportunity for
is part of the Western shield, a region of stable continental subsequent systematic study, with the primary aim to ex-
crust thought to have been continually emergent from the amine and describe the new species from the Sturt Mead-
sea since the Palaeozoic, more than 250 million years ows calcrete. The availability of mtDNA COI sequence data
ago (Humphreys, 2001). This region was presumed to be from six Yilgarn calcrete chiltoniid populations including
deficient for stygobitic habitats until the 1998 discovery of Sturt Meadows (Cooper et al., 2007; Bradford et al., 2010),
stygofauna within numerous distinct subterranean calcrete as well as South Australian mound spring chiltoniid popu-
aquifers (hereafter referred to as calcretes) (Fig. 1). These lations (Murphy et al., 2009) meant that a comparatively
relatively thin (∼10 m deep) calcretes formed as carbonate broad molecular framework was possible for phylogenetic
deposited from groundwater flow along palaeo drainage examination and to inform taxonomic evaluation of the Sturt
channels during Pliocene aridification of inland Australia Meadows taxa. Taxonomic effort included the description
5-10 million years ago (Humphreys, 2001; Cooper et al., of species using morphological and molecular evidence, de-
2007), and are thought to have become aquatic refugia for tailed examination of phenotypic variation and an examina-
water dependent inland invertebrates as aridification altered tion of the phenomenon of cryptic species within the stygo-
the landscape from warm wet forests to arid and semi-arid bitic chiltoniid amphipods.
desert (Leys et al., 2003; Byrne et al., 2008). Complex
patterns of habitat connectivity and environmental gradients
both within calcretes and broadly across the Yilgarn region, M ATERIALS AND M ETHODS
as well as large and small historical landscape changes Morphological Methods
(aridification, geological movements, flooding events) have
combined to create highly structured subterranean systems Material from the Bradford et al. (2010) study was examined
and a richly diverse invertebrate fauna (Humphreys et for morphological differences, with 82 individuals identified
al., 2009). The stygobitic fauna of the Yilgarn has only to each of three purported species. With these individual
recently begun to be explored, yet the diversity of many samples frequently damaged through the mtDNA tissue
associated aquatic invertebrates (water beetles, copepods, extraction process, an additional 29 uncatalogued specimen
bathynellaceans, isopods, and crangonyctoid amphipods) lots from Sturt Meadows calcrete in the collections of the
within Yilgarn calcretes are high, with species restricted to Western Australian Museum (WAM) (15 samples) and The
single calcretes (Cooper et al., 2007, 2008; Leys and Watts, University of Adelaide (12 samples) were also examined for
2008; Guzik et al., 2009, 2011b). the three species (Table 1). Additional undescribed material
Access to an extensive Yilgarn bore field on the Sturt (20 samples) from 11 calcretes throughout the Yilgarn
Meadows pastoral property near Leonora in Western Aus- region from WAM were also examined for comparative
tralia has afforded opportunities to undertake comprehensive purposes (Table 2).
faunal surveys of the calcrete, and lead to the discovery of a Types were dissected along the left side and appendages
diverse group of invertebrates, including amphipods belong- illustrated with a drawing tube attachment to a Nikon Eclipse
ing to Chiltoniidae (Bradford et al., 2010). The Australian 80i microscope. All type material has been lodged with
chiltoniids are a small group of freshwater amphipods, his- the Western Australian Museum. The family and higher
torically represented by only two species in lowland sur- systematic treatment follows that outlined by Serejo (2003).
face water systems (creeks, dams, marshes) across south- All taxonomic descriptions in this work are attributed to
eastern Australia and two species from groundwater-fed R. A. King.
KING ET AL.: STYGOBIONT AMPHIPODS FROM WESTERN AUSTRALIA 467
Fig. 1. The calcretes of the Yilgarn region in Western Australia. Each palaeodrainage is labelled. Calcretes are as follows: 1, Mt Padbury; 2, Lake Violet;
3, Barwidgee; 4, Lake Mason; 5, Nambi; 6, Kaluwiri; 7, Depot Springs; 8, Yuinmerry; 9, Pinnacles; 10, Perrinvale; 11, Sturt Meadows; 12, Sons of Gwalia.
Molecular Methods nia murphyi King, 2009; Austrochiltonia dalhousiensis Zei-

Existing sequences from Sturt Meadows (from Bradford et dler, 1997; Wangiannachiltonia guzikae King, 2009; and
al., 2010), five additional Yilgarn calcretes (Lake Mason, Phreatochiltonia anophthalma Zeidler, 1991) (from Murphy
Lake Violet, Barwidgee, Mt. Padbury and Depot Springs) et al., 2009) were included in the analysis. Examination and
(from Cooper et al., 2007) and the four known species identification of specimens from all three Sturt Meadows lin-
from the South Australian mound springs (Arabunnachilto- eages included further sequencing of 11 specimens to ensure
Table 1. Chiltoniid amphipod material examined from the Sturt Meadows calcrete from the collections of the Western Australian Museum (WAM) (BES
field numbers, catalogue C numbers) and the University of Adelaide (SM field numbers). m = male; f = female; juv = juvenile.
No. of specimens Collection no. Latitude Longitude WAM accession no.

Yilgarniella sturtensis n. sp.
>30 (m, f, juv) BES 10241 28°42 0.9 S 120°50 27.42 E C49178
1 juv BES 10271 28°42 59.11 S 120°53 23.82 E C49179
2 (1 f, 1 m) BES 10280 28°41 54.13 S 120°53 36.24 E C49180
9 (1 m, 3 f, 5 juv) BES 10286 28°41 48.05 S 120°53 47.62 E C49181
4f BES 12029 28°42 0.83 S 120°53 36.13 E C49182
5 (1 m, 4 f) BES 12045 28°43 2.35 S 120°53 27.6 E C49183
1f BES 12057 28°41 51.32 S 120°54 2.2 E C49184
9 (3 m, 2 f, 4 juv) BES 12068 28°41 51.47 S 120°54 5.76 E C49185
Stygochiltonia bradfordae n. sp.
4 (2 f, 1 m, 1 juv) BES 10240 28°42 0.9 S 120°50 27.42 E C49186
4 (1 m, 3 f) BES 10262 28°41 47.72 S 120°53 36.56 E C49187
Scutachiltonia axfordi n. sp.
1 f, 2 m SM E13 28°41 56.57 S 120°54 18.18E C49188
1f BES 11998 28°42 14.04 S 120°53 32.28 E C49189
2m BES 12996 28°42 30.06 S 120°53 42.94 E C49190
1f SM F13 28°41 59.81 S 120°54 18.03 E C49191
1m SM N4 28°42 31.69 S 120°53 36.68 E C49192
1f BES 12994 28°42 36.47 S 120°53 35.45 E C49193
1m BES 11974 28°42 31.9 S 120°53 47.90 E C49194
1m SM E5 28°41 56.04 S 120°53 48.67 E C49195
2m SM B3 28°41 46.41 S 120°53 40.89 E C49196
1f SM W3 28°42 57.67 S 120°53 36.43 E C49197
1f SM E12 28°41 56.53 S 120°54 14.51 E C49198
1f SM E11 28°41 56.49 S 120°54 10.63 E C49199
that the identified morphospecies matched the molecular lin- and sequencing were performed as described in Cooper et
eages identified by Bradford et al. (2010). al. (2007). A ∼650 bp region of the mitochondrial Cy-
DNA was extracted from pereiopod tissue from 11 spec- tochrome oxidase subunit I (COI) gene was amplified us-
imens using the Gentra (Puregene) method for fresh tis- ing universal primers: M414 (forward, 5 -GGT CAA CAA
sue and preserved in absolute ethanol. PCR amplification ATC ATA AAG ATA TTG G-3 , alias LCOI490, Folmer et
al., 1994), M423 (reverse, 5 -TAA ACT TCA GGG TGA
CCA AAA AAT CA-3 , alias LCO2198, Folmer et al.,
Table 2. Chiltoniid amphipod material from additonal calcretes in the
Yilgarn region examined from field collections of the Western Australian
1994).
Museum (WAM). MEGA (Tamura et al., 2007) was used to align sequences,
for analyses of amino acids and to determine nucleotide
WAM no. Calcrete Latitude Longitude pairwise distances. These were calculated using two models
BES 10377 Barwidgee 27°8 14.89 S 120°56 57.95 E for comparison, the Kimura-2 and the Maximum Composite
BES 6650 Depot Springs 28°3 36.25 S 120°4 2.78 E Likelihood with Gamma distribution, both widely used in
BES 8382 Depot Springs 27°55 50.88 S 120°4 45.19 E crustacean studies (Lefébure et al., 2006; Pernet et al.,
BES 8407 Depot Springs 28°2 59.57 S 120°2 21.08 E 2010; Filipová et al., 2011). Baysian phylogenetic analyses
BES 8408 Depot Springs 28°3 36.25 S 120°4 2.78 E of aligned sequences were carried out using MrBayes
BES 8367 Kaluwiri 27°40 58.22 S 120°2 7.01 E 3.1.2 (Huelsenbeck and Ronquist, 2001). As in Bradford
BES 8393 Kaluwiri 27°40 58.22 S 120°2 7.01 E et al. (2010), the data set was partitioned by codon using
BES 8361 Lake Mason 27°32 24.03 S 119°37 27.41 E
independent models of sequence evolution for the first codon
BES 8363 Lake Mason 27°32 24.03 S 119°37 27.41 E
BES 6425 Lake Violet 26°40 29.53 S 120°13 55.2 E position (TrN + I + G), second codon position (K81uf + I)
BES 6434 Lake Violet 26°40 29.53 S 120°13 55.2 E and third codon position (HKY + G), which were with the
BES 9309 Mt. Padbury 25°41 41.93 S 118°4 46.49 E optimal models specified using ModelTest 3.7 (Posada and
BES 10317 Nambi 28°14 25.44 S 121°50 13.92 E Crandall, 1998), under the Alkaike Identification Criterion.
BES 10252 Perinvale 28°46 30.14 S 120°25 1.2 E Bayesian analyses were run using four chains for 10 million
BES 10253 Perinvale 28°46 30.14 S 120°25 1.2 E generations in two independent runs, sampling every 500
BES 6642 Pinnacles 28°15 26.78 S 120°7 36.84 E generations. The program Tracer 1.5 (Rambaut, 2003) was
BES 6643 Pinnacles 28°15 26.78 S 120°7 36.84 E
BES 8399 Pinnacles 28°12 43.74 S 120°2 36.53 E
used to evaluate convergence of distribution: the likelihood
BES 11830 Sons of Gwalia 28°56 3.3 S 121°18 4.39 E values converged after ∼1 million generations. A burnin of
BES 6657 Yuinmerry 28°32 55.03 S 119°5 28.07 E 2000 was chosen and a 50% consensus tree was constructed
from the remaining 18 001 trees.
R ESULTS equating possibly to separate species, were found within the

Depot Springs calcrete (at 12-16% divergence) as well as the
Molecular Results three from Sturt Meadows.
A 624 bp fragment of COI was obtained from 83 individ- The South Australian chiltoniid taxa did not form a mono-
uals. All COI sequences had an open reading frame, with phyletic group, instead four distinct lineages (93-100% BPP)
no evidence of insertions/deletions or anomalies in the pro- were evident that included the southern mound spring taxa
tein coding sequence, suggesting that they are all likely to (Ar. murphyi and W. guzikae) and two northern mound spring
be from functional mitochondrial COI. Phylogenetic anal- groups (Au. dalhousiensis and P. anophthalma), which
yses indicated that nine well supported and deeply diver- showed close affinities to Western Australian lineages: Au.
gent amphipod lineages exist within the sampled Yilgarn dalhousiensis from Dalhousie springs, SA to the lineage
calcretes that include Sturt Meadows, Lake Mason, Lake Vi- from Lake Mason calcrete, WA and P. anophthalma from
olet, Barwidgee, Mt. Padbury and Depot Springs (Fig. 2). Dalhousie Springs, SA to a Sturt Meadows lineage (= Scu-
These divergent molecular lineages were restricted in their tachiltonia n. gen.). Pairwise distances within lineages were
distribution to single calcretes. Nucleotide divergence pa- higher in the South Australian mound spring taxa (8-13%)
rameters, measured as pair wise distances (Tables 3, 4), were than in the stygofauna and the distances between lineages
slight within the stygofaunal lineages (0.2-1%), while pair was comparable to that of the stygofauna (17-35%) (Ta-
wise distances between stygofaunal lineages were between bles 3, 4).
9-39% (Table 3).
Strong support (100% Baysian Posterior Probability Morphological Results
(BPP)) was found for three distinct lineages that do not form Examination of the Sturt Meadow lineages did not uncover
a reciprocally monophyletic clade within the Sturt Meadows morphologically cryptic species, but instead supported the
calcrete (Fig. 2). The three lineages showed nucleotide di- recognition of three distinct morphospecies equating to three
vergence levels of 15-23%. Single lineages were observed in new species recognised here as three new chiltoniid genera
the majority of the Yilgarn calcretes (Lake Mason, Lake Vio- (Scutachiltonia n. gen., Stygochiltonia n. gen., and Yilgar-
let, Mt. Padbury, Barwidgee), however two distinct lineages, niella n. gen). Compared to the known epigean chiltoniid
Fig. 2. Posterior probability (50%) Bayesian consensus tree of Yilgarn chiltoniid amphipod COI sequences, posterior probabilities are listed on
corresponding branches. Star symbol indicates Sturt Meadows, WA groups. WA = Western Australia; SA = South Australia.
Table 3. mtDNA COI nucleotide pair wise distances between Yilgarn (WA) and South Australian stygobitic chiltoniid taxa. Values calculated via the
Kimura-2 (bold, bottom left) and Maximum Composite Likelihood (Gamma) models (plain, top right). * includes Sturt meadows taxa only; + includes Sturt
Meadows, and two putative Depot Springs species.
South Australian taxa Sturt Meadows (WA) Additional Yilgarn calcretes (WA)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1. Phreatochiltonia 0.345 0.198 0.320 0.220 0.150 0.220 0.358 0.358 0.231 0.252 0.267 0.312 0.250
2. Wangiannachiltonia 0.209 0.314 0.355 0.370 0.317 0.342 0.395 0.395 0.350 0.285 0.293 0.390 0.364
3. Au. dalhousiensis 0.139 0.202 0.263 0.201 0.228 0.224 0.292 0.292 0.215 0.256 0.231 0.240 0.133
4. Arabunnachiltonia 0.191 0.208 0.175 0.366 0.318 0.309 0.325 0.325 0.293 0.297 0.316 0.372 0.294
5. Yilgarniella 0.153 0.216 0.142 0.210 0.203 0.223 0.302 0.302 0.237 0.270 0.262 0.330 0.219
6. Scutachiltonia 0.115 0.190 0.156 0.189 0.148 0.199 0.372 0.372 0.176 0.216 0.220 0.263 0.264
7. Stygochiltonia SM* 0.147 0.207 0.151 0.189 0.151 0.138 na 0.219 0.108 0.116 0.261 0.268 0.242
8. Stygochiltonia (all)+ 0.152 0.202 0.155 0.187 0.159 0.139 na 0.235 na na 0.261 0.271 0.251
9. Mt. Padbury 0.202 0.234 0.187 0.202 0.187 0.207 0.150 0.167 0.252 0.269 0.386 0.359 0.269
10. Depot Springs sp. 2 0.152 0.211 0.149 0.182 0.165 0.126 0.093 na 0.172 0.158 0.261 0.289 0.222
11. Depot Springs sp. 3 0.163 0.185 0.167 0.185 0.175 0.144 0.095 na 0.180 0.122 0.259 0.273 0.282
12. Barwidgee 0.169 0.187 0.154 0.196 0.165 0.147 0.162 0.163 0.219 0.165 0.165 0.259 0.266
13. Lake Violet 0.194 0.243 0.167 0.214 0.202 0.177 0.178 0.180 0.216 0.181 0.182 0.175 0.274
14. Lake Mason 0.171 0.230 0.115 0.193 0.155 0.182 0.168 0.173 0.174 0.159 0.189 0.174 0.189
taxa, all three species from Sturt Meadows presented dis- sp. and currently thought to be endemic to Sturt Meadows, is
tinct evidence of stygobitic lifestyles: loss of eyes and pig- found in a somewhat patchy distribution across the calcrete
ment, elongation of antennae, elongation of pereiopods 6-7, and exhibits marked morphological characters (narrow elon-
elongation of the uropods. gate coxae 1-4, crenulated bases on pereiopods 5-7) mak-
Whilst all three species from Sturt Meadows have been ing it a robust, presumably well protected species in larger
sampled in sympatry (by Bradford et al., 2010) and S. brad- spaces within the calcrete. Styogochiltonia, represented by
fordae n. sp. and Y. sturtensis n. sp. found here in samples S. bradfordae n. sp., is the rarest species of the three within
BES 10241, 10262 (see Table 1)), morphology with support- the calcrete and presents the most characters of a stygo-
ing distributional data suggests each may inhabit a somewhat bitic lifestyle (narrow elongate body segments, coxae 1-7
distinct niche within the calcrete. Yilgarniella, represented reduced, basis of pereiopods reduced, pereiopods 6-7 very
by Y. sturtensis, is the most widespread of the three species long compared to pereiopod 5) and presumably is able to
within the calcrete and is the least specialised stygobite of inhabit smaller interstitial spaces compared with the other
the three, being closest in morphology to described epigean two species. Examination of the two distinct Depot Springs
species from southeastern Australia (coxae 1-4 unmodified molecular lineages indicates that they are congeneric with
and similar length to pereion segments, pereiopods 6-7 not Styochiltonia bradfordae.
extremely elongate compared to pereiopod 5). Based on
morphological examination, further species within Yilgar- S YSTEMATICS
niella are suspected in other calcretes across the Yilgarn (in-
cluding Nambi); sequence data from individuals from these Infraorder Talitrida Rafinesque, 1815
calcretes was not available for comparison. Scutachiltonia, Talitroidea s.s. Rafinesque, 1815
represented by the comparably large (5-6 mm) S. axfordi n. Chiltoniidae Barnard, 1972
Scutachiltonia King, n. gen.
Type Species.—Scutachiltonia axfordi King, n. sp.
Table 4. Pairwise distances within Yilgarn and South Australian stygob-
itic chiltoniid amphipod groups (A. dalhousiensis, Arabunnachiltonia, Mt. Diagnosis.—Eyes absent. Antenna 1 at least twice the size
Padbury, Depot Springs spp. 2 and 3 not included as too few sequences
available for comparision). ∗ includes Sturt meadows taxa only; + includes
of antenna 2. Coxae 1-3 long (at least twice as long as
Sturt Meadows, and two putative Depot Springs species. broad). Coxa 4 longer than broad, with a defined proximal
corner. Coxae 5-6 posterior lobe extending at least twice
Kimura-2 Maximum composite length of anterior lobe. Gnathopod 2 propodus in males
likelihood elongate (at least two times as long as wide). Pereiopod 5-
7 bases with postero-distal lobe; with distinct crenulation
Phreatochiltonia 0.0884 0.102031
Wangiannachiltonia 0.1109 0.130839 along posterior margins; anterior margins highly setose with
Yilgarniella 0.011 0.011244 up to ten robust setae. Pereiopods 6 and 7 about 1.5 times
Scutachiltonia 0.0046 0.004624 as long as pereiopod 5; carpus elongate and distinctly longer
Stygochiltonia SM∗ 0.0039 0.003874 than merus; propodus elongate and around 1.3 times as long
Stygochiltonia (all)+ 0.051 0.062 as carpus. Epimera 1-3 with postero-distal corners defined
Barwidgee 0.0029 0.002949 with a blunt spine.
Lake Violet 0.002 0.001957
Lake Mason 0.0035 0.003538 Etymology.—Named for the elongate coxae and large bases
of pereiopods 5-7, which together form long shields (latin:
“scuta”) and give the species a robust shape, and “chiltonia” Distribution.—Western Australia: Sturt Meadows calcrete
for its placement within Chiltoniidae. aquifer, situated on the Sturt Meadows Pastoral Property,
near Leonora.
Remarks.—Scutachiltonia includes “Clade 2” of Bradford
et al. (2010) and comprises the largest of the three species Description.—Holotype male (WAM C49169), length:
present at Sturt Meadows. It is unique in its possession of 5.3 mm. Head about as long as deep (Fig. 3A).
elongate coxae 1-4, pereiopods 5-7 with anterior margin Antenna 1 (Fig. 4B) 0.77 times body length, peduncular
strongly setose, and epimera 1-3 with postero-distal corner article 1 2.2 times as long as broad, inner lateral margin with
spines. No other similar morphotype has been found in mate- two robust setae; peduncular article 2 slightly longer than
rial examined from numerous regional calcrete aquifers. Ex- article 1, almost four times as long as broad; peduncular
tensive examination of the Sturt Meadows calcrete, together article 3 shorter than article 2, four times as long as broad;
with molecular evidence confirming a divergent monotypic flagellum three times longer than peduncle, of 22 articles,
group indicate that Scutachiltonia will remain monotypic with ventral aesthetascs on the proximal margins of the eight
and endemic to the Sturt Meadows calcrete. distal articles. Antenna 2 (Fig. 4A) about 0.4 times length of
Our results indicate that Scutachiltonia is more closely antenna 1; peduncular article 3 as broad as long, inner-distal
related to the South Australian mound spring amphipod margin with 2-3 robust setae; peduncular article 4 two times
P. anophthalma, than it is to the other Sturt Meadows as long as article 3, 3.5 times longer than broad, inner lateral
amphipods (Fig. 2). Whilst P. anophthalma is the only other margin with two robust setae, distal margin with one robust
described blind chiltoniid, the two species do not share seta; peduncular article 5 longer than article 4, 4.75 times as
any great morphological affinities and it is likely that as long as broad; flagellum 1.5 times longer than peduncle, of
more calcrete populations are analysed, their relationship 12 articles.
will become clearer. Mouthparts as for family (Fig. 4C-I) (see King, 2009b).
Scutachiltonia axfordi King, n. sp. Gnathopod 1 (Fig. 5A) coxa two times as long as broad,
Figs. 3-6 distal margin with seven short simple setae; basis ventral
margin with scattered simple setae; ischium, and merus
Morphological Diagnosis.—Antenna 1 greater than 3/4 distoventral corners with clusters of one or two simple
body length, flagellum three times longer than peduncle; setae; carpus with ventral-lateral lobe and row of 10 setulate
antenna 2 1/3 body length, flagellum at least 1.5 times longer setae becoming longer distally, distodorsal margin with long
than peduncle. Gnathopod 2 propodus (in males) two or setae; propodus 2.2 times as long as broad, subchelate,
more times as long as broad. Coxa 1-3 two times as long palm acute, distoventral corner with one robust seta at
as broad; coxa 4 elongate and with defined proximal corner. corner of palm, medial palm margin with short robust and
Pereiopods 5-6 bases longer than broad, with postero-distal long simple setae, distodorsal margin with long simple
lobe, entire posterior margin crenulate with associated short setae, inner face with three robust plumose setae; dactylus
setae, posterior margin with 7-10 robust setae concentrated with unguis, curved, fitting against palm, with proximal
distally; pereiopod 7 basis almost as long as broad, with plumose seta. Gnathopod 2 (Fig. 5B) coxa two times as
poster-distal lobe, entire posterior margin crenulate with long as broad, distal margin with five short simple setae;
associated short setae, posterior margin with 7-20 robust basis dorsal and ventral margins without setae; ischium
setae concentrated distally. Uropod 1 outer ramus with 2- and merus with few setae on ventral margins; propodus
3 robust setae along length, with distal cluster of 2-3 small two times as long as broad, subchelate, distoventral corner
setae and single long robust seta (half-length of ramus); marked by dactylar socket, palm margin with numerous
inner ramus with 1-3 robust setae in two rows along length, robust setae with subterminal spines. Pereiopod 3 (Fig. 5C)
with distal cluster of 4 small setae and single long robust seta coxa 2.2 times as long as broad, distal margin with six
(half-length of ramus). Uropod 2 outer ramus with 1-3 robust short simple setae; basis dorsal and ventral margins with
setae along length, with distal cluster of 2-3 small setae and few simple setae, distoventral corner with cluster of setae;
single long robust seta (half-length of ramus); inner ramus ischium distoventral corner with clusters of setae; merus
with 5-9 robust setae in two rows along length, with distal with slight distodorsal lobe, ventral margin with scattered
cluster of 4 small setae and single long robust seta (half- simple setae, distoventral corner with cluster of setae; carpus
length of ramus). Uropod 3 bi-articulate with second article ventral margin with robust setae and scattered simple setae;
minute (less than 1/4 length of first article). propodus dorsal margin with few setae; ventral margin with
Material Examined.—Holotype, WAM C49169, male, seven clusters of robust and simple setae; dactylus dorsal
5.3 mm, Sturt Meadows calcrete, Western Australia, BES margin with plumose seta, ventral margin with simple seta,
12986, Bore N4, 28◦ 42 36.396 S, 120◦ 53 31.74 E, coll. S. unguis present. Pereiopod 4 (Fig. 5D) coxa 1.4 times as long
Cooper, A. Allford, 4 Apr 2005. Allotype, WAM C49170, as wide, with distinct proximal excavation, distal margin
female, 5.6 mm, Sturt Meadows calcrete, Western Australia, with 19 short simple setae; basis dorsal and ventral margins
BES 11836, Bore E13, 28◦ 42 1.3314 S, 120◦ 54 12.996 E, with scattered simple setae, distoventral corner with cluster
coll. W. F. Humphreys, S. Cooper, R. Leys, A. Allford, of simple setae; ischium distoventral corner with cluster of
31 Mar 2005. Paratypes (two males, three females), WAM setae; merus with slight distodorsal lobe, ventral margin with
C49171, Sturt Meadows calcrete, Western Australia, BES scattered simple setae, distoventral corner with cluster of
12025, Bore E12, 28◦ 42 1.224 S, 120◦ 54 9.36 E, collected setae; carpus ventral margin with scattered robust and simple
by W. F. Humphreys, S. Cooper, J. Bradbury, M. Guzik, 25 setae; propodus ventral margin with seven clusters of robust
Sep 2004 (see Table 1 for additional material). and simple setae; dactylus dorsal margin with plumose seta,
Fig. 3. Scutachiltonia axfordi King, n. sp. A, Holotype male lateral view, WAM C49169; B, Allotype female, lateral view, WAM C49170. Scale bar:
0.5 mm.
ventral margin with simple seta, unguis present. Pereiopod 5 robust setae along length, distal end of dorsal margin with
(Fig. 5F) coxa posterior lobe with one short seta along distal one robust seta, ventral margin distinctly crenulated and with
margin; basis 1.4 times as long as broad, dorsal margin with 11 short simple setae along length. Pereiopod 7 (Fig. 5E)
seven robust setae along length, distodorsal margin with two coxa ventral margin with one short simple seta; basis 1.2
robust setae, ventral margin distinctly crenulated and with times as long as broad, dorsal margin with 10 robust setae
nine short simple setae along length. Pereiopod 6 (Fig. 5G) along length, distal end of dorsal margin with two robust
coxa posterior lobe with one short seta along distal margin; setae, ventral margin distinctly crenulated and with 10 short
basis 1.4 times as long as broad, dorsal margin with 10 simple setae along length; ischium dorsal margin with distal
Fig. 4. Scutachiltonia axfordi King, n. sp. holotype male, 5.3 mm, WAM C49169. A, Antenna 2; B, Antenna 1; C, Maxilla 2; D, Maxilla 1; E, Upper lip;
F, Mandible RHS; G, Mandible LHS; H, Maxilliped; I, Lower lip. Scale bars: (A-B) 0.2 mm, (C-I) 0.1 mm.
cluster of short robust setae; merus with slight postero-distal similar length to inner ramus, dorsal margin with two robust
lobe, dorsal margin with robust setae in five clusters, ventral setae along the length of the outer margin and four along
margin with robust setae in four clusters; carpus longer than the inner margin; outer ramus smaller than inner ramus,
merus, nine times as long as broad, dorsal margin with robust with four apical robust setae (distal-most seta as long as
setae in six clusters, ventral margin with robust setae in six half-length of ramus), with a row of two robust setae along
distal clusters. length; inner ramus with four apical robust setae (distal-
Pleopod 1 (Fig. 5H) similar to pleopods 2-3, unmodified most seta as long as half-length of ramus), with two rows
(compared to Chiltonia Stebbing, 1899), peduncle inner of robust setae along length, outer margin with five robust
margins with three distal retinacula (coupling hooks), inner setae, inner margin with four robust setae. Uropod 3 (Fig. 5J)
ramus of 10 articles, outer ramus of 13 articles. Uropod 1 bi-articulate, article 2 less than 0.25 length of article 1, distal
(Fig. 5L) peduncle two times longer than rami, dorsal margin margin with one short robust seta and one long robust seta
with two robust setae along the length of the outer margin apically. Telson (Fig. 5I) as long as broad, apically blunt,
and two along the inner margin; outer ramus with three with two apically divided robust setae and four simple setae
apical robust setae and a row of two robust setae along distally.
length; inner ramus with five apical robust setae (distal-most Allotype Female (WAM C49170).—Length: 5.6 mm
seta as long as half-length of ramus) and two rows of robust (Fig. 3B).
setae along length, outer margin with one robust seta, inner Similar morphology to male except for the following:
margin with two robust setae. Uropod 2 (Fig. 5K) peduncle Gnathopod 1 (Fig. 6A) propodus around two times as long
Fig. 5. Scutachiltonia axfordi King, n. sp. holotype male, 5.3 mm, WAM C49169. A, Gnathopod 1; B, Gnathopod 2; C, Pereiopod 3; D, Pereiopod 4; E,
Pereiopod 7; F, Pereiopod 5 (basis); G, Pereiopod 6 (basis); H, Pleopod 1; I, Telson; J, Uropod 3; K, Uropod 2; L, Uropod 1. Scale bars: (A-B, H-L) 0.1 mm,
(C-G) 0.2 mm.
Fig. 6. Scutachiltonia axfordi King, n. sp. allotype female, 5.6 mm, WAM C49170. A, Gnathopod 1; B, Gnathopod 2; C, Pereiopod 4 (basis); D, Pereiopod
5 (basis); E, Pereiopod 6 (basis); F, Pereiopod 7 (basis); G, Uropod 2; H, Uropod 1; I, Uropod 3. Scale bars: (A-B, G-I) 0.1 mm, (C-F) 0.2 mm.
as broad, inner face with three robust setae. Gnathopod 2 one robust seta along length; inner ramus with five apical
(Fig. 6B) similar to gnathopod 1 except propodus 2.4 times robust setae (distal-most seta as long as half-length of
as long as broad. Pereiopod 4 coxa (Fig. 6C) not as broad as ramus), with two rows of robust setae along length, outer
in male. Pereiopods 5-7 bases (Fig. 6D-F) not as broad as in margin with two robust setae, inner margin with two robust
male. setae.
Uropod 1 (Fig. 6H) peduncle dorsal margin with three Oöstegites present on coxae 2 to 5 to form the marsupium,
robust setae along the length of the outer margin and margins with scattered curved hooks.
three robust setae along the inner margin; outer ramus
Etymology.—Named for the Axford family of the Sturt
with four apical robust setae (distal-most seta as long as
Meadows pastoral property.
half-length of ramus) and one robust setae along length;
inner ramus with five apical robust setae (distal-most seta Variation.—Mature males and females were a similar size
as long as half-length of ramus) and two rows of robust in the samples studied. The number of robust setae on the
setae along length, outer margin with one robust seta, anterior margin of the bases of pereiopods 5-7 differs by one
inner margin with one robust seta. Uropod 2 (Fig. 6G) or two setae dependent on the size of the animal (smaller
peduncle dorsal margin with two robust setae on outer animals had less setae). The number of robust setae on the
margin and three robust setae on inner margin; outer ramus peduncles and rami of uropods 1 and 2 also differs with size
smaller than inner ramus, with three apical robust setae (larger individuals as illustrated, smaller individuals 2-3 less
(distal-most seta as long as half-length of ramus), with setae).
Remarks.—This species is distinctive primarily because of than half-length of ramus). Uropod 2 outer ramus with one
its elongate coxae and broad spinose bases of pereiopods or two robust setae along length, with distal cluster of up to
5-7. Individuals were fragile and many animals showed three small setae and a long robust setae (more than half-
damage, specifically loss of pereiopods, presumably from length of ramus); inner ramus with three to four setae in two
collection and sample processing. rows along length, with distal cluster of four to five setae
and one long robust seta (more than half-length of ramus).
Stygochiltonia King, n. gen. Uropod 3 bi-articulate with second article around 1/3 length
Type Species.—Stygochiltonia bradfordae n. sp. of first article.
Diagnosis.—Eyes absent. Antenna 1 at least twice the size Material Examined.—Holotype, WAM C49172, male,
of antenna 2. Coxae 1-3 short (about as long as broad). 3.4 mm, Sturt Meadows calcrete, Western Australia, BES
Coxa 4 broader than long, with poorly defined proximal 10241, 28◦ 42 0.9 S, 120◦ 50 27.42 E, coll. W. F. Humph-
corner. Coxae 5-6 posterior lobe reduced, not extending reys, S. Cooper, 16 Mar 2004. Allotype, WAM C49173,
much further than anterior lobe. Gnathopod 2 in males short female, 4.5 mm, collected with holotype. Paratypes (two
(1.5 or less times as long as wide). Pereiopod 5-6 bases with males, 1 female), WAM C49174, collected with holotype
postero-distal lobe; pereiopod 7 without postero-distal lobe. (see Table 1 for additional material).
Pereiopods 5-7 bases with subtle crenulation along posterior
Distribution.—Western Australia: Sturt Meadows calcrete
margin, concentrated proximally; anterior margin with up
aquifer, situated on the Sturt Meadows Pastoral Property,
to five robust setae. Pereiopods 6 and 7 at least twice as
near Leonora.
long as pereiopod 5, with numerous elongate robust setae
along anterior and posterior margins of all articles; carpus as Description.—Holotype male (based on WAM C49172),
long as merus; propodus elongate (1.7 to two times length length: 3.4 mm. Head about as long as deep (Fig. 7A).
of carpus). Epimera 1-3 with blunt squared postero-distal Antenna 1 (Fig. 8B) 0.8 times body length, peduncular
corners. article 1 three times as long as broad, inner lateral margin
Etymology.—“Stygo” for the stygobitic environment of the with two robust setae, distoventral margin with single robust
species as well as the typical stygobitic morphology of seta; peduncular article 2 shorter than article 1 (0.7 times
elongate pereiopods and reduced coxae, and “chiltonia” for as long), 2.5 times as long as broad; peduncular article 3
its placement within Chiltoniidae. shorter than article 2, three times as long as broad; flagellum
longer than peduncle (3.4 times), of 20 articles, with ventral
Remarks.—Stygochiltonia incorporates “clade 3” of Brad- aesthetascs on the proximal margins of the eight distal
ford et al. (2010) and represents the first chiltoniid amphi- articles. Antenna 2 (Fig. 8A) about 0.4 times length of
pod with all the archetypal subterranean morphological fea- antenna 1; peduncular article 3 broader than long, inner-
tures: loss of eyes; elongate antennae; narrow and elongate distal margin with two robust setae; peduncular article 4
pereionites; pereiopods 6 and 7 elongate compared to pereio- longer than article 3, three times longer than broad, inner
pod 5. It is unique in its possession of reduced coxae 1- lateral margin with two robust setae, distal margin with one
6, short gnathopod 2 propodus in males, pereiopod 7 basis robust seta; peduncular article 5 longer than article 4, four
without postero-distal lobe and epimera 1-3 postero-distal times as long as broad; flagellum longer than peduncle (1.6
corners blunt and squared. It is likely that individuals ex- times), of 9 articles.
amined from other calcrete sites within Western Australia Mouthparts as for family (Fig. 8C-I) (see King, 2009b).
(Depot Springs, Pinnacles), some noted by Bradford et al. Gnathopod 1 (Fig. 9A) coxa as long as broad, distal mar-
(2010) as potential sister species to S. bradfordae sp nov., gin with three short simple setae; basis distoventral corner
belong within Stygochiltonia. with cluster of simple setae; ischium, and merus distoven-
tral corners with clusters of setae; carpus with ventral-lateral
Stygochiltonia bradfordae King, n. sp. lobe and row of nine setulate setae becoming longer distally,
Figs. 7-10 distodorsal margin with long setae; propodus two times as
Morphological Diagnosis.—Antenna 1 greater than 3/4 long as broad, subchelate, palm acute, distoventral corner
body length, flagellum four times longer than peduncle; with one robust seta at corner of palm, medial palm margin
antenna 2 1/3 body length, flagellum two times longer than with short robust and long simple setae, distodorsal margin
peduncle. Gnathopod 2 propodus (in males) 1.5 times as with long simple setae, inner face with two robust plumose
long as broad. Coxae 1-3 as long as broad; coxa 1 narrowing setae; dactylus curved, fitting against palm, with proximal
distally, coxae 2-3 squared; coxa 4 broad (1.2 times as broad plumose seta. Gnathopod 2 (Fig. 9B) coxa as long as broad,
as long) and with undefined proximal corner. Pereiopods 5-6 distal margin with six short simple setae; basis distoventral
bases longer than broad, with postero-distal lobe, posterior corner with scattered simple setae; ischium and merus with
margin with indistinct crenulations, with five to seven short scattered setae on distoventral corners; propodus 1.5 times
setae along margin, posterior margin with three robust setae; as long as broad, subchelate, distoventral corner marked by
pereiopod 7 basis longer than broad, without postero-distal robust seta adjacent to dactylar socket, palm margin with nu-
lobe. Uropod 1 outer ramus with one to two robust setae merous robust setae with subterminal spines. Pereiopod 3
along length, with distal cluster of three small setae and a (Fig. 9C) coxa distal margin with seven short simple setae;
single long robust seta (more than half-length of ramus); basis dorsal and ventral margins with scattered simple se-
inner ramus with one or two setae along length, with distal tae, distoventral corner with clusters of setae; ischium dis-
cluster of four small setae and a single long robust seta (more toventral corner with clusters of setae; merus with slight
Fig. 7. Stygochiltonia bradfordae King, n. sp. A, Holotype male lateral view, WAM C49172; B, Allotype female, lateral view, WAM C49173. Scale bar:
0.5 mm.
distodorsal lobe, ventral margin with scattered simple setae, as broad, with indistinct proximal excavation, distal margin
distoventral corner with cluster of setae; carpus ventral mar- with six short simple setae; basis dorsal and ventral margins
gin with robust setae and scattered simple setae; propodus with scattered simple setae, distoventral corner with cluster
dorsal margin with one cluster of simple setae; ventral mar- of simple setae; ischium distoventral corner with cluster of
gin with four clusters of robust and simple setae; dactylus setae; merus with slight distodorsal lobe, ventral margin with
dorsal margin with plumose seta, ventral margin with sim- scattered simple setae, distoventral corner with cluster of se-
ple seta, unguis present. Pereiopod 4 (Fig. 9D) coxa as long tae; carpus ventral margin with scattered robust and simple
Fig. 8. Stygochiltonia bradfordae King, n. sp. holotype male, 3.4 mm, WAM C49172. A, Antenna 2; B, Antenna 1; C, Maxilla 2; D, Maxilla 1; E,
Maxilliped; F, Mandible RHS; G, Mandible LHS; H, Lower lip; I, Upper lip. Scale bars: (A-B) 0.2 mm, (C-I) 0.1 mm.
setae; propodus ventral margin with four clusters of robust distal lobe, dorsal margin with robust setae in four clusters,
and simple setae; dactylus dorsal margin with plumose seta, ventral margin with robust setae in three clusters; carpus as
ventral margin with simple seta, unguis present. Pereiopod long as merus, dorsal margin with robust setae in four clus-
5 (Fig. 9E) coxa posterior lobe without setae along margin; ters, ventral margin with robust setae in one cluster; propo-
basis 1.5 times as long as broad, dorsal margin with three dus longer than merus, dorsal margin with five clusters of
robust setae along length, distodorsal margin with one ro- robust setae, ventral margin with two clusters of simple se-
bust seta, ventral margin indistinctly crenulated and with tae; dactylus with plumose seta on ventral margin, unguis
seven short simple setae along length; ischium distodorsal present. Pereiopod 6 (Fig. 9F) coxa posterior lobe with one
margin with distal robust setae; merus with strong postero- seta along margin; basis 1.6 times as long as broad, dorsal
Fig. 9. Stygochiltonia bradfordae King, n. sp. holotype male, 3.4 mm, WAM C49172. A, Gnathopod 1; B, Gnathopod 2; C, Pereiopod 3; D, Pereiopod 4;
E, Pereiopod 5; F, Pereiopod 6; G, Pereiopod 7; H, Pleopod 1; I, Uropod 1; J, Uropod 2; K, Uropod 3; L, Telson. Scale bars: (A-B, H, I-L) 0.1 mm, (C-G)
0.2 mm.
margin with three robust setae along length, distal end of robust setae in four clusters, ventral margin with robust setae
dorsal margin with cluster of two robust setae, ventral mar- in two clusters; propodus longer than merus, dorsal margin
gin indistinctly crenulated and with five short simple setae with five clusters of robust setae, ventral margin with three
along length; ischium dorsal margin with distal robust setae; clusters of simple setae; dactylus with plumose seta on ven-
merus with strong postero-distal lobe, dorsal margin with ro- tral margin, unguis present. Pereiopod 7 (Fig. 9G) coxa ven-
bust setae in four clusters, ventral margin with robust setae tral margin with one short simple seta; basis 1.5 times as long
in three clusters; carpus as long as merus, dorsal margin with as broad, dorsal margin with four robust setae along length,
distal end of dorsal margin with two robust setae, ventral two robust setae. Telson (Fig. 10I) as long as broad with
margin indistinctly crenulated and with two short simple se- three pairs of setae at each dorsal distal corner.
tae along length; ischium dorsal margin with distal cluster Etymology.—Named for Tessa Bradford who first detected
of robust setae; merus with strong postero-distal lobe, dorsal the three Sturt Meadows lineages in her published study
margin with robust setae in five clusters, ventral margin with (Bradford et al., 2010).
robust setae in five clusters; carpus as long as merus, dor-
sal margin with robust setae in four clusters, ventral margin Variation.—Mature females were distinctly larger than ma-
with robust setae in two distal clusters; propodus longer than ture males. The holotype male is likely to be an early stage
merus merus, dorsal margin with five clusters of robust setae, male, one of only two found in collections of this rare
ventral margin with three clusters of simple setae; dactylus species. Whilst the penes were fully developed in both indi-
with plumose seta on ventral margin, unguis present. viduals, setation of the carpus of gnathopod 2 indicates only
Pleopod 1 (Fig. 9H) similar to pleopods 2-3, unmodified partial development from a juvenile morphotype. Although
(compared to Chiltonia), peduncle inner margins with two rare, this morphotype (mature male with setulate setae on the
distal retinacula (coupling hooks), inner ramus of five carpus of gnathopod 2) has been observed in populations of
articles, outer ramus of seven articles. Uropod 1 (Fig. 9I) other chiltoniid species (personal observation, RAK). Exam-
peduncle 1.5 times longer than rami, dorsal margin with ination of male specimens from the Depot Springs calcrete,
six robust setae along the length of the outer margin and a potentially con-generic species, showed fully developed
one seta along the inner margin; outer ramus with three males without this carpus setation. It is proposed that if later
stage males of S. bradfordae are found in future collections,
apical small robust setae and one long robust seta (longer
the propodus will be short (1.5 times length) and carpus free
than half-length of ramus) and a robust seta along length;
of the setulate setae, as seen in the Depot Springs males and
inner ramus with distal cluster of four robust setae (three
adult males of other chiltoniid species.
small and one twice size of others) and one long robust seta
(longer than half-length of ramus) and a seta along length. Yilgarniella King, n. gen.
Uropod 2 (Fig. 9J) peduncle similar length to inner ramus, Type Species.—Yilgarniella sturtensis sp. nov.
dorsal margin with three long robust setae along the outer
margin and one robust seta along the inner margin; outer Diagnosis.—Eyes absent. Antenna 1 at least twice the size
ramus slightly smaller than inner ramus, with apical cluster of antenna 2. Coxae 1-3 about 1.5 times as long as broad.
of two small robust setae and one long robust seta (longer Coxa 4 about as broad as long, with defined proximal corner.
than half-length of ramus), with one robust seta along length; Coxae 5-6 posterior lobe extending at least twice length of
inner ramus with distal cluster of four robust setae (three anterior lobe. Gnathopod 2 propodus in males elongate (two
small and one twice size of others) and one long robust times as long as wide). Pereiopod 5-7 bases with postero-
seta (longer than half-length of ramus), with two rows of distal lobe. Pereiopods 5-6 bases with subtle crenulation
along posterior margin, concentrated proximally; anterior
robust setae along length, outer margin with one robust seta,
margin with up to six robust setae. Pereiopod 7 basis
inner margin with two robust setae. Uropod 3 (Fig. 9K) bi-
posterior margin entirely crenulate, anterior margin with up
articulate, article 2 0.3 times length of article 1, distal margin
to six robust setae. Epimera 1-3 with blunt postero-distal
with one short robust seta and one long robust seta apically.
corners defined with a blunt spine.
Telson (Fig. 9L) longer than broad, apically blunt, with two
setae at each dorsal distal corner. Etymology.—Named for the Yilgarn region of Western
Australia where the specimens were sampled.
Allotype Female (WAM C49173).—Length: 4.5 mm
(Fig. 7B). Remarks.—Yilgarniella includes “clade 1” of Bradford et
al. (2010) and is a mostly unspecialised chiltoniid species
Similar morphology to male except for the following:
in terms of stygobitic morphology, looking much more like
Gnathopod 1 (Fig. 10A) carpus with ventral-lateral lobe
epigean chiltoniids; pereiopods 6 and 7 are not particularly
and row of 12 setulate setae becoming longer distally.
long, nor are their articles elongate compared to pereiopod
Gnathopod 2 (Fig. 10B) similar to gnathopod 1 except
5, and the coxae are not particularly reduced or enlarged.
propodus 2.6 times as long as broad. Pereiopods 5-7 bases
It is easily recognised by these and a combination of
(Figs. 10C-E) broader than in males. other characters: lack of eyes, elongate antennae, elongate
Uropod 1 (Fig. 10F) peduncle dorsal margin with eight gnathopod 2 propodus in males, bases of pereiopods 5-7
robust setae along length of the outer margin and one seta dorsal margin with few robust setae and ventral margin with
along the inner margin; outer ramus with two robust setae subtle to distinct crenulation, and pereiopod 7 basis with
along length; inner ramus with two rows of robust setae postero-distal lobe.
along length, outer margin with two robust setae, inner Individuals from the Nambi calcrete in Western Australia
margin with two robust setae. Uropod 2 (Fig. 10G) peduncle have been examined and determined to nominally be an
dorsal margin with four long robust setae along length of additional species of Yilgarniella. Further descriptive work
the outer margin and one robust seta along the inner margin; is ongoing.
outer ramus with two robust setae along length; inner ramus
with distal cluster of four robust setae (two small and two Yilgarniella sturtensis King, n. sp.
twice size of others) and one long robust seta (longer than Figs. 11-14
half-length of ramus), with two rows of robust setae along Morphological Diagnosis.—Antenna 1 greater than 3/4
length, outer margin with two robust setae, inner margin with body length, flagellum more than three times longer than
Fig. 10. Stygochiltonia bradfordae King, n. sp. allotype female, 4.5 mm, WAM C49173. A, Gnathopod 1; B, Gnathopod 2; C, Pereiopod 5 (basis); D,
Pereiopod 6 (basis); E, Pereiopod 7 (basis); F, Uropod 1; G, Uropod 2; H, Uropod 3. Scale bars: (A-B, F-H) 0.1 mm, (C-E) 0.2 mm.
peduncle; antenna 2 1/3 body length, flagellum at least 3 small setae and single long robust seta (about half-length
1.3 times longer than peduncle. Gnathopod 2 propodus of ramus). Uropod 2 outer ramus with 4-5 robust setae along
(in males) two times as long as broad. Coxae 1-3 1.5 length, with distal cluster of 3-4 small setae and single long
times as long as broad; coxa 4 slightly broader than long, robust seta (about half-length of ramus); inner ramus with
with defined posterior corner. Pereiopods 5-6 bases longer 5-7 robust setae in two rows along length, with distal cluster
than broad, with postero-distal lobe, ventral margin subtly of 3-5 small setae and single long robust seta (half-length of
crenulate (concentrated proximally) with associated short ramus). Uropod 3 bi-articulate, with second article around
setae, posterior margin with 2-5 robust setae concentrated 1/3 length of first article.
distally. Pereiopod 7 basis longer than broad, with postero- Material Examined.—Holotype, WAM C49175, male, 3.7
distal lobe entire margin subtly crenulate with associated mm, Sturt Meadows calcrete, Western Australia, BES 10262,
setae, posterior margin with five robust setae concentrated 28◦ 41 47.724 S, 120◦ 53 36.5634 E, coll. W. Humphreys, S.
distally. Uropod 1 outer ramus with 4-7 robust setae along Cooper, 18 Mar 2004. Allotype, WAM C49176, female, 4.7
length, with distal cluster of 3 small setae and single long mm, collected with holotype. Paratypes (two males, six fe-
robust seta (about half-length of ramus); inner ramus with 5 males, five juveniles), WAM C49177, collected with holo-
robust setae in two rows along length, with distal cluster of type (see Table 1 for additional material).
Fig. 11. Yilgarniella sturtensis King, n. sp. A, Holotype male lateral view, WAM C49175; B, Allotype female, lateral view, WAM C49176. Scale bar:
0.5 mm.
Distribution.—Western Australia: Yilgarn region (currently setae; peduncular article 4 longer than article 3, 2.2 times
only known from Sturt Meadows calcrete aquifer, situated longer than broad, inner lateral margin with two robust setae,
on the Sturt Meadows pastoral property, near Leonora). distal margin with one robust seta; peduncular article 5
longer than article 4, 4.5 times as long as broad; flagellum
Description.—Holotype male (WAM C49175), length: 3.7 longer than peduncle (1.3 times), of 11 articles.
mm. Head about as long as deep (Fig. 11A). Mouthparts as for family (Fig. 12C-I) (see King, 2009b).
Antenna 1 (Fig. 12A) 0.8 times body length, peduncular Gnathopod 1 (Fig. 13A) coxa 1.5 times as long as
article 1 2.4 times as long as broad, inner lateral margin with broad, distal margin with nine short simple setae; basis
two robust setae, distoventral margin with single robust seta; distoventral corner with cluster of simple setae; ischium,
peduncular article 2 shorter than article 1 (0.7 times as long), and merus distoventral corners with clusters of setae; carpus
2.8 times as long as broad; peduncular article 3 shorter than with ventral-lateral lobe and row of nine setulate setae
article 2, three times as long as broad; flagellum three times becoming longer distally, distodorsal margin with long
longer than peduncle, of 24 articles, with ventral aesthetascs setae; propodus two times as long as broad, subchelate, palm
on the proximal margins of the nine distal articles. Antenna acute, distoventral corner with one robust seta at corner
2 (Fig. 12B) about 0.4 times length of antenna 1; peduncular of palm, medial palm margin with short robust and long
article 3 as broad as long, inner-distal margin with robust simple setae, distodorsal margin with long simple setae,
Fig. 12. Yilgarniella sturtensis King, n. sp. holotype male, 3.7 mm, WAM C49175. A, Antenna 1; B, Antenna 2; C, Maxilla 2; D, Maxilla 1; E, Maxilliped;
F, Lower lip; G, Mandible RHS; H, Mandible LHS; I, Upper lip. Scale bars: (A-B) 0.2 mm, (C-I) 0.1 mm.
inner face with five robust plumose setae; dactylus with corner with cluster of setae; carpus ventral margin with
unguis, curved, fitting against palm, with proximal plumose robust setae and scattered simple setae; propodus ventral
seta. Gnathopod 2 (Fig. 13B) coxa 1.6 times as long as margin with five clusters of robust and simple setae; dactylus
broad, distal margin with seven short simple setae; basis dorsal margin with plumose seta, ventral margin with simple
distoventral corner with scattered simple setae; ischium and seta, unguis present. Pereiopod 4 (Fig. 13D) coxa 4 slightly
merus distoventral corner with scattered setae; propodus broader than long, with distinct proximal excavation, distal
two times as long as broad, subchelate, distoventral corner margin with 12 short simple setae; basis ventral margin
marked by robust seta adjacent to dactylar socket, palm with scattered simple setae, distoventral corner with cluster
margin with numerous robust setae with subterminal spines. of simple setae; ischium distoventral corner with cluster of
Pereiopod 3 (Fig. 13C) coxa 1.5 times as long as broad, setae; merus with slight distodorsal lobe, ventral margin with
distal margin with eight short simple setae; basis dorsal scattered simple setae, distoventral corner with cluster of
and ventral margins with scattered simple setae, distoventral setae; carpus ventral margin with scattered robust and simple
corner with clusters of setae; ischium distoventral corner setae; propodus ventral margin with five clusters of robust
with clusters of setae; merus with slight distodorsal lobe, and simple setae; dactylus dorsal margin with plumose seta,
ventral margin with scattered simple setae, distoventral ventral margin with simple seta, unguis present. Pereiopod
Fig. 13. Yilgarniella sturtensis King, n. sp. holotype male, 3.7 mm, WAM C49175. A, Gnathopod 1; B, Gnathopod 2; C, Pereiopod 3; D, Pereiopod 4; E,
Pereiopod 5; F, Pereiopod 6; G, Pereiopod 7; H, Uropod 3; I, Telson; J, Pleopod 1; K, Uropod 2; L, Uropod 1. Scale bars: (A-B, C-G) 0.2 mm, (H-I, J, K-L)
0.1 mm.
5 (Fig. 13E) coxa posterior lobe with one short seta along margin with distal robust setae; merus with strong postero-
margin; basis 1.4 times as long as broad, dorsal margin distal lobe, dorsal margin with robust setae in four clusters,
with two robust setae along length, distodorsal margin with ventral margin with robust setae in four clusters; carpus
two robust setae, ventral margin subtly crenulated and with as long as merus, dorsal margin with robust setae in three
seven short simple setae along length; ischium distodorsal clusters, ventral margin with robust setae in two clusters;
propodus longer than merus, dorsal margin with four clusters Similar morphology to male except for the following:
of robust setae, ventral margin with two clusters of simple Gnathopod 1 (Fig. 14A) carpus ventral-lateral lobe with
setae; dactylus with plumose seta on ventral margin, unguis row of 12 setulate setae; inner face with three robust setae.
present. Pereiopod 6 (Fig. 13F) coxa posterior lobe with one Gnathopod 2 (Fig. 14B) similar to gnathopod 1 except
seta along margin; basis 1.4 times as long as broad, dorsal propodus 2.4 times as long as broad. Pereiopod 4 (Fig. 14C)
margin with five robust setae along length, distal end of coxa broader than in male. Pereiopods 5-7 (Fig. 14D-F)
dorsal margin with cluster of robust setae, ventral margin slightly broader than in male.
subtly crenulated and with five short simple setae along Uropod 1 (Fig. 14H) peduncle dorsal margin with 10
length; ischium dorsal margin with distal robust setae; merus robust setae along the length of the outer margin and one
with strong postero-distal lobe, dorsal margin with robust along the inner margin; outer ramus with a row of seven
setae in four clusters, ventral margin with robust setae in robust setae along length. Uropod 2 (Fig. 14G) peduncle as
five clusters; carpus as long as merus, dorsal margin with long as inner ramus, dorsal margin with seven robust setae
robust setae in three clusters, ventral margin with robust along the length of the outer margin and one along the inner
setae in two clusters; propodus longer than merus, dorsal margin; outer ramus with a row of four robust setae along
margin with six clusters of robust setae, ventral margin with length; inner ramus with apical cluster of three robust setae
four clusters of simple setae; dactylus with plumose seta on and one long robust seta (about half-length of ramus), with
ventral margin, unguis present. Pereiopod 7 (Fig. 13G) coxa two rows of robust setae along length, outer margin with two
ventral margin with two short simple setae; basis 1.3 times robust setae, inner margin with three robust setae. Uropod 3
as long as broad, dorsal margin with five robust setae along (Fig. 14I) article 2 with one short robust seta, and two long
length, distal end of dorsal margin with two robust setae, robust seta apically. Telson (Fig. 14J) slightly broader than
ventral margin subtly crenulated and with eight short simple in male.
setae along length; ischium dorsal margin with distal cluster Oöstegites present on coxae 2 to 5, forming the mar-
of robust setae; merus with strong postero-distal lobe, dorsal supium, margins with scattered curved hooks.
margin with robust setae in five clusters, ventral margin with Etymology.—Named for the Sturt Meadows calcrete, where
robust setae in five clusters; carpus as long as merus, dorsal the species is found.
margin with robust setae in five clusters, ventral margin with
robust setae in three distal clusters; propodus longer than Variation.—Mature females were generally larger in size
merus, dorsal margin with eight clusters of robust setae, than mature males. Slight differences were noted in the
ventral margin with three clusters of simple setae; dactylus number of flagellar articles in antennae 1 and 2: larger
with plumose seta on ventral margin, unguis present. animals as illustrated, smaller individuals with 1-3 less
Pleopod 1 (Fig. 13J) similar to pleopods 2-3, unmodified articles. Setation of the pereiopods also differed slightly with
(compared to Chiltonia), peduncle inner margins with two size of the individuals with smaller animals having slightly
fewer setae.
distal retinacula (coupling hooks), inner ramus of six arti-
cles, outer ramus of 10 articles. Uropod 1 (Fig. 13L) pe-
duncle distinctly longer than rami, dorsal margin with five D ISCUSSION
robust setae along the length of the outer margin and four Diversity of stygobitic amphipods from Western Australia
along the inner margin; outer ramus with apical cluster of has recently been estimated to be high (Guzik et al., 2011a).
three small robust setae and one long robust seta (about half- The results presented here support this assertion, formally
length of ramus), a row of four robust setae along length adding a new stygobitic amphipod family (Chiltoniidae) for
and one inner robust seta; inner ramus with apical cluster of Western Australia and three new genera from the exploration
three small robust setae and one long robust seta (about half- of a single calcrete. Each of the three Sturt Meadows species
length of ramus) and two rows of robust setae along length, is endemic to that calcrete, corroborating the results of other
outer margin with three robust setae, inner margin with two studies within the Yilgarn region of central Western Aus-
robust setae. Uropod 2 (Fig. 13K) peduncle shorter than in- tralia that strongly suggest that calcretes exist as ‘closed is-
ner ramus, dorsal margin with three robust setae along the land habitats’ with sometimes highly structured populations
length of the outer margin and two along the inner margin; of locally endemic aquatic invertebrate species (Cooper et
outer ramus slightly smaller than inner ramus, with apical al., 2002, 2007, 2008; Leys et al., 2003; Guzik et al., 2008;
cluster of two small robust setae, with a row of five robust Leys and Watts, 2008; Bradford et al., 2010).
setae along length; inner ramus with apical cluster of five True cryptic stygobitic amphipod species have been re-
robust setae and one long robust seta (about half-length of ported in European subterranean environments (Lefébure
ramus), with two rows of robust setae along length, outer et al., 2007; Villacorta et al., 2008) and are an interesting
margin with three robust setae, inner margin with five ro- phenomenon because they suggest a selective pressure on
bust setae. Uropod 3 (Fig. 13H) bi-articulate, article 2 about morphology that is not reflected in mtDNA markers. How-
0.25 times length of article 1, distal margin with one short ever, the supposed morphologically cryptic nature of the
robust seta and three long robust setae. Telson (Fig. 13I) as Sturt Meadows chiltoniid amphipod lineages (Bradford et
long as broad, apically slightly blunt, with three pairs of se- al., 2010) was not upheld once detailed morphological ex-
tae around each dorsal distal corner. amination had taken place; the lineages were only cryptic
in so far as they are part of a largely unknown and un-
Allotype Female (WAM C49176).—Length: 4.7 mm described fauna. Nevertheless, Chiltoniidae are particularly
(Fig. 11B). well known as a difficult group in terms of species-level mor-
Fig. 14. Yilgarniella sturtensis King, n. sp. allotype female, 4.7 mm, WAM C49176. A, Gnathopod 1; B, Gnathopod 2; C, Pereiopod 4 (coxa); D, Pereiopod
5 (basis); E, Pereiopod 6 (basis); F, Pereiopod 7 (basis); G, Uropod 2; H, Uropod 1; I, Uropod 3; J, Telson. Scale bars: (A-B, C-F) 0.2 mm, (G-J) 0.1 mm.
phological variability (Zeidler, 1997; King, 2009a, b; King descriptive methods. One method identified species level
and Leys, 2011) and it is possible that cryptic species will limits as 10 times the intra-population COI divergence level
be found as more stygobitic species are sequenced and de- (Witt et al., 2006) and another as a static 0.16 substitu-
scribed. In these cases, it is likely that species boundaries tions per site for the COI gene, as measured by patristic
will be defined primarily by genetic divergence levels and distances but also more roughly by pairwise distance meth-
geographic isolation within a calcrete. ods (Lefébure et al., 2006). The lineages observed within the
The provisional delineation of crustacean species using Yilgarn calcretes largely meet both criteria for delimitation
nucleotide distance measures has been variously set in at- of species. Nucleotide distance measures have been found
tempts to qualify molecular data with traditional taxonomic to be highly variable within Amphipoda, ranging from 4 to
25% for species levels (Hogg et al., 2006; Witt et al., 2008); ACKNOWLEDGEMENTS
when they have been used within descriptions of species Kathleen Saint for technical support of molecular analyses. Julianne
and genera, they are often presented in conjunction with ad- Waldock for collection management support at the Western Australian
ditional data (e.g. morphological, geographical) (King and Museum. Peter, Flora and Paul Axford for their kind hospitality at Sturt
Meadows pastoral station and for showing a genuine interest in our
Leys, 2011). As the availability of molecular data increases, research of the stygofauna. Rae Young, Miranda and Phoebe Humphreys
the intrinsic value of distance measures for species delin- for supporting WH in the fieldwork and for kindly hosting the invaders from
eation, and the corresponding evolution of morphological SA. The morphological work presented here was supported by Australian
form, will be better determined. Biological Resources Study (ABRS) grant No. 208-61, while the molecular
data was generated as part of ARC Linkage grants LP0348753, LP0669062
Interpreting the evolutionary histories of calcrete inver- and LP100200494.
tebrates remains a complex task, dependent on variables
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J OURNAL OF C RUSTACEAN B IOLOGY, 32(3), 465-488, 2012

DIVERGENT MOLECULAR LINEAGES AND NOT-SO-CRYPTIC SPECIES:

Rachael A. King 1,∗ , Tessa Bradford 2 , Andrew D. Austin 3 ,

The University of Adelaide, South Australia 5005, Australia

I NTRODUCTION largely by Crangonyctoidea (Bradbury and Williams, 1999;

∗ Corresponding author; e-mail: Rachael.King@samuseum.sa.gov.au

Molecular Methods nia murphyi King, 2009; Austrochiltonia dalhousiensis Zei-

No. of specimens Collection no. Latitude Longitude WAM accession no.

R ESULTS equating possibly to separate species, were found within the

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