Australian
The literature on
For example, the taxonomy of
The advancement in molecular techniques, especially the introduction of allozyme technique to study interspecific hybridisation in
The objective of the present study was to consider all available data, morphological and molecular (mitochondrial and nuclear genes) of the valid eight
Sixty female specimens of
Localities with coordinates and the GenBank accession numbers for
Sites | |
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Water body |
Latitude | Longitude | GenBank accession numbers | ||
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COI | 16s | 28s | ||||||||
1 | Yanga National Park | New South Wales (NSW) | 26/10/2010 | NSYAN | Lake | 34°42′19.04′′S | 143°35′30.73′′E | KC020663–66 | KC154331–34 | — |
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2 | Yanga National Park, Mercedes | New South Wales (NSW) | 26/10/2010 | NSMER | Lake | 34°22′44.94′′S | 143°47′12.80′′E | KC020667–71 | KC154336–40 | KC154371-72 |
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3 | Yanga National Park, Steam Engine | New South Wales (NSW) | 26/10/2010 | NSSTE | Lake | 34°28′58.26′′S | 143°40′33.20′′E | KC020662 | KC154330 | — |
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4 | Parramatta | New South Wales (NSW) | Sample collected in 1986 by Moreno Julli. Since then, it was cultured and maintained in a laboratory | SYDCB | Lake | 33°47′26.00′′S | 151°0′28.00′′E | KC154279–83 | KC154341–45 | KC154358–62 |
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5 | Mannum, Mandina | South Australia (SA) | 26/10/2009 | SAMAN | Flood plain | 36°16′29.99′′S | 139°54′40.41′′E | KC154284-85 | KC154326–29 | KC154363–65 |
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6 | Snuggery | South Australia (SA) | 20/10/2009 | SASNU | Flood plain | 36°33′45.00′′S | 140°0′7.88′′E | KC020660 | KC154322–25 | KC154366–70 |
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7 | Myponga Reservoir | South Australia (SA) | 2008–2010 | SAMYP | Reservoir | 35°24′10.06′′S | 138°25′43.14′′E | KC154270–75 | — | — |
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8 | South Para Reservoir | South Australia (SA) | 2008–2010 | SASPA | Reservoir | 34°41′47.28′′S | 138°51′41.63′′E | KC154276–78 | — | — |
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9 | Chowilla | South Australia (SA) | 10/02/2011 | SACHOW | Flood plain | 34°3′33.76′′S | 140°45′44.48′′E | KC020655–59 | — | — |
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10 | Woorabinda | South Australia (SA) | 15/10/2009 | SAWOO | Flood plain | 30°0′59.90′′S | 134°32′1.88′′E | KC020661 | — | — |
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11 | Runaway Perth | Western Australia (WA) | 10/03/2010 | WARPER | Pond | 31°56′11.10′′S | 115°58′31.78′′E | KC020651–54 | — | — |
Eggs and young adults from a total of 60 ovigerous females were teased out from the brood chamber and stored in new 0.5 mL microtubes containing 90% denatured ethanol for genetic analysis. The rest of the body was transferred onto a slide containing Polyvinyl Alcohol (PVA) and left in a Greiner petri dish for 24 h. After 24 h equilibration, the specimen was dissected using tungsten needles and examined under a compound microscope for further detailed morphological analysis. Digital photographs were taken using Olympus BX51 microscope under high resolution using polarizing light and composite line drawings were made from these photographs for different parts of the specimen. Inbuilt imaging software Image J was used to calculate sizes of body parts.
In addition to light microscopy, specimens were prepared for SEM as described in Berner and Rakhmatullaeva [
DNA extraction was performed on eggs or young adults with DNeasy Tissue kit (Qiagen Inc.) according to the manufacturer’s instructions.
Polymerase chain reaction was subsequently utilized to amplify the COI gene with Folmer primer pair (LCO 1490 and HCO 2198) [
16s rDNA mitochondrial genes were amplified using 16s1 5′-CCGGAATTCCGCCTGTTTATCAAAAACA-3′ and 16s2 5′-CCCAAGCTTCTCCGGTTTGAACTCAGAT-3′ (modified Simon et al. 1994) [
The PCR products were run in 2% agarose gels containing 10
Both forward and reverse DNA sequences were analyzed and aligned using Clustal W application implemented in software Bioedit ver. 7.0.0. [
The sequences are deposited in GenBank under accession numbers KC154268–KC154287; KC020651–KC020671 for COI gene fragments; KC154322–KC154345 for 16s gene fragments; and KC154358–KC154372 for 28s gene fragments.
COI gene sequences for
Abbreviations used for museum collections are as follows: AMS = Australian Museum Sydney, Australia; NHMUO = Natural History Museum, University of Oslo, Norway; NMCL = Naturkunde-Museum, Coburg, Germany; SAM = South Australian Museum, Australia.
Ten key morphological characteristics were used in the present study and are summarized in Table
Comparisons of selected morphological characters of eight
Character |
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Size of adult female in mm | 1–1.4 | 0.75–0.95 | 0.9–1.2 | 0.5–0.9 | 0.7–1 | 0.8–1 | 0.5–0.86 | 0.7–1 |
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Posterior |
Row of fine spinules punctuated partially or entirely with 10 to 12 spines | Approx. 1/3rd of the posterior end shows spines projecting from the edge of the carapace | Long flexible setules followed by a row of fine thin spines interrupted in between with fine spiniform setules rising parallel from the edge | acute and spine shaped | Long flexible setules followed by a row of fine thin spines interrupted in between with fine spiniform setules rising parallel from the edge | Is convex shaped with clear and coarse reticulation and serrated spines at the edge of the carapace. It ends on posterior side at a posterodorsal angle with blunt spine | Row of thin hairs continues till the rear edge, where it reaches its highest point near the junction of the posterodorsal angle. Posterodorsal angle shows presence of spine which is laid almost parallel to its edge | |
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Reticulation and sculpture of carapace | Evenly sized polygonal in shape with heavy edges and dotted surfaces | Distinctly reticulated with relatively large raised nearly hexagonal meshes | Distinctly reticulated with relatively large flattened polygonal meshes | Coarser reticulation on the surface with nearly hexagonal structure | Is irregular in shape | Thick regular hexagonal reticulation | Strong polygonal reticulation | Cross-linking regular polygonal structure |
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Postabdomen | Moderately long and wide that tapers gradually in width towards the terminal claw | Narrow and half the length compared to the maximum body width | Broad and almost half in length compared to the maximum body width | Narrow and of medium size with rounded end | Broad with the securiform (ax) shape | Broad with two small obtuse and horn shaped angles or abdominal appendages at the posterior end | Postabdominal shape towards the end has been mentioned as wide (Lilljeborg 1900b) [ |
Moderately long and wide that tapers gradually in width towards the terminal claw |
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Anal denticles | 7–10 | 8–10 | 9-10 | 8–10 | 9–11 | 7–9 | 7–14 | 8–10 |
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Total number of setulated setae |
6 |
4-5 |
4 |
— | — | — | — | — |
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Number of setae on exopodite V | 4 | 3 | 4 | — | 4 | — | 4 | — |
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Middle pecten | 21 long and stouter teeth | Presence of fine setules | With heavier and slightly longer spinules, compared to proximal pecten | — | Presence of larger spinules | — | Presence of larger spinules | Form 1 toothed pecten variety with a central pecten on the claw showing 18 to 24 finely setulated pectens |
Australia: slide of a dissected parthenogenetic female from Parramatta Lake near Sydney, NSW (33°47′26′′S/151°00′28′′E), deposited in the South Australia Museum (SAM). Dr. John Chapman (Office of Environment and Heritage, NSW, Sydney) collected this species in 1986 from Parramatta Lake in Sydney and since then it has been cultured and maintained in a laboratory (Moreno Jully, OEH, NSW, personal communication, 23 June 2014). Sample specimens of the species were provided by Dr. Tsuyoshi Kobayashi (OEH, NSW) on 26/10/2010. Genetic Reference number: COI + 16s = SYDCB001 − 05. Five ephippial females (undissected) stored in a vial containing 70% denatured alcohol deposited in the SAM, accession number SAM C7024.
((a)–(c)) SEMs of fornix. (a) Absence of denticles; (b) smooth extended hooks; (c) smooth small denticles. ((d)–(f)) SEMs of the rostral pore. (d) Flat; (e) bulging; (f) flat.
((a)–(c)) SEMs of reticulation on the carapace. (a) Polygonal reticulation; ((b) and (c)) hexagonal reticulation. ((d)–(f)) Line drawing of Trunk Appendage V for the three species.
Trunk limb I with setulated setae on endites two to five (E2 to E5). Two ejector hooks situated on the anterior side of the limb. Trunk limb II with five endites E1 to E5 consisting of seven setulated setae and two soft setae; the gnathobase bears five setae and E2 bears one long and one short accessory spines. Trunk limb III consists of one exopodite and five endites. Trunk limb IV with eight setulated setae. Trunk limb V with a small sized exopodite bearing four setae of which two are long and two are small in size (Figure
Postabdomen slightly tapered and obliquely truncated distally. There are about eight anal denticles (Figure
SEMs of postabdomen and denticles of the three species.
Trunk limb I with setulated setae on endites 2 to 5 (E2 to E5), and two setae on E5. Trunk limb II comprises five endites E1 to E5, with four brush setae on gnathobase and two short accessory spines on E2. Trunk limb III exopodite consists of four setulated setae on the distal end and 2 small setulated setae on the lateral end. There are five endites on trunk limb III of which endites 1 to 4 are highly reduced and E5 bears a number of gnathobasic filtering setae. Trunk limb IV has five setulated setae at the distal end. Posterior end has two setae. Trunk limb V has a small sized exopodite bearing four setae of which two are long and two is smaller in size (Figure
Postabdomen is almost half in length compared to the maximum body width. There are eight recurved anal denticles (Figure
Trunk limb I with setulated seta on endites 2 to 5 (E2 to E5); E5 shows the presence of three setae. Trunk limb II comprises of five endites E1 to E5: the gnathobase bears five brush setae and E2 bears two long accessory spines. Trunk limb III exopodite consists of three setulated setae on distal end and one small setulated seta on the lateral end. Trunk limb IV has four setulated setae in the distal end and the posterior end has a single seta. Trunk limb V has a small sized exopodite bearing three setae of which the distal and proximal one are the longest compared to middle setae (Figure
Postabdomen is almost half the length compared to maximum body width. There are nine recurved anal denticles (Figure
Basic statistics and selected substitution models for mtDNA sequences are shown in Table Clade 1 and Clade 2 comprising Clade 3 comprising
Sequence information for the different gene fragments without the outgroup. Fragment length in base pair, number of variable sites (
Gene fragments | Length |
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Model test |
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mtDNA | ||||
16s | 540 | 75 | — | K81uf + I |
COI | 645 | 148 | — | K81uf + G |
cmtDNA | 1185 | 220 | — | TVM + G |
Nuclear | ||||
28s | 575 | 479 | — | GTR + I + G |
Minimum and maximum cmt mtDNA (upper line in each cell) and 28s (bold bottom line in each cell) raw pairwise divergence within and between
Species |
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0-1 (0-1) |
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10 (13–15) |
0-1 (0-1) |
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14 (24-25) |
15 (26-27) |
0 (0) |
Phylogenetic trees inferred from concatenated mtDNA gene sequences for
Tree indicating the phylogenetic relationship inferred from 28s gene sequences for
Analysis of COI sequences between Australian
Minimum and maximum COI raw pairwise divergence within and between species with ML corrected divergence value inside the closed bracket () of the
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0 (0) | |||||||||||||||
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20 (80) | 0 (0) | ||||||||||||||
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15-16 |
18-19 |
0–3 (0–4) | |||||||||||||
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15 (58) | 18 (73) | 16-17 |
0 (0) | ||||||||||||
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17 (68) | 17 (83) | 16-17 |
15 (51) | 0 (0) | |||||||||||
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17 |
18-19 |
15–17 |
15 |
6 |
0-1 |
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18 (64) | 20 (79) | 17–19 |
14 (46) | 15 (43) | 14 |
0-1 |
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18 (64) | 20 (79) | 17-18 |
14 (46) | 15 (43) | 14 |
0-1 |
0 (0) | ||||||||
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17-18 (64) | 20 (89) | 18-19 |
16 (67-68) | 15-16 (78) | 16 |
17 (73) | 17 (73-74) | 0 (0) | |||||||
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17 |
19 (86) | 16-17 |
17 (64-65) | 18 |
17–19 |
19 |
19 (70-71) | 13 (31-32) | 0–2 (0–2) | ||||||
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19 (79) | 21 (103) | 18–21 |
17 (82) | 16 (92) | 16 (84) | 15-16 (87) | 16 (88) | 16-17 (72) | 19 (69) | 0 (0) | |||||
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20 |
21 (101) | 18–21 |
17 (79-80) | 16 (90) | 16 |
16 |
16 (85-86) | 16 (70) | 19 (67-68) | 3 (3) | 1 (1) | ||||
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19 |
20 (90–92) | 20-21 |
19 (68–70) | 17 |
17-18 |
18-19 (74–76) | 18-19 (74–76) | 18 (59–61) | 18 (55–58) | 14-15 (37–39) | 14-15 (35–37) | 0–2 (0–2) | |||
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18 (71) | 20 (96) | 20-21 |
19 (74) | 16 (84) | 17-18 |
19 (80) | 19 (80) | 18-19 (64-65) | 18-19 (61-62) | 15 (43) | 14 (41) | 4-5 (5-6) | 0 (0) | ||
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19 (68) | 20 (92) | 20-21 |
19 (71) | 17 (81) | 17 (73-74) | 19-20 |
19-20 (77) | 18 (61-62) | 18 (58-59) | 14 (40) | 13-14 (37-38) | 2-3 (2-3) | 4-5 (5-6) | 0 (0) | |
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17-18 |
19-20 (96–99) | 17–20 |
17-18 (74–78) | 15-16 |
16-17 |
18-19 |
18-19 (80–83) | 17-18 (65–68) | 18-19 (62–65) | 14-15 (43–47) | 14-15 (41–45) | 11-12 (18–23) | 11-12 |
10-11 |
0–3 |
Maximum likelihood analysis of COI gene for
Molecular analysis of the published COI sequences available from GenBank including the sequences generated from this study (Figures
The specimens of
The lack of sufficient taxonomic detail for Australian
Despite close morphological similarities of
There are a total of 11 unmounted specimens, approximately 30 years old, of
Phylogenetic relationships among species of
Phylogenetic analysis of mt COI sequences for
In this work an integrated approach was used for achieving the goal of species identification and discrimination of
The author declares that there is no conflict of interests regarding the publication of this paper.
The author would like to thank the Sir Mark Mitchell Research Foundation, ANZ Holsworth Grant, and ABRS Travel Bursary for financial support. The author appreciates very much the helpful suggestions on the paper from Dr. John Jennings and Dr. Tsuyoshi Kobayashi. The author also extends sincere thanks to SA Water, Microbiology Department, for granting the permission and providing the necessary facilities to carry out the genetic work. The author greatly appreciates the helpful suggestions on the English from Ms. Samantha-Ann Schneider. The author would like to thank the reviewers for their comments that helped improve the paper. Thanks also go to all the staff at Adelaide Microscopy, for there assistance with the SEM.