Molecular systematics of the Kakaducarididae (Crustacea: Decapoda: Caridea)

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Molecular Phylogenetics and Evolution 46 (2008) 1003–1014 www.elsevier.com/locate/ympev

Molecular systematics of the Kakaducarididae (Crustacea: Decapoda: Caridea) Timothy J. Page a,*, John W. Short b, Christopher L. Humphrey c, Mia J. Hillyer a, Jane M. Hughes a a

Australian Rivers Institute, Griffith University, Nathan Campus, Qld. 4111, Australia b BioAccess Australia, PO Box 261, Narangba, Qld. 4504, Australia c Environmental Research Institute of the Supervising Scientist, GPO Box 461, Darwin, NT 0866, Australia Received 23 June 2007; revised 20 December 2007; accepted 22 December 2007 Available online 1 January 2008

Abstract The systematic relationships of the freshwater shrimp family, Kakaducarididae, were examined using mitochondrial and nuclear DNA sequences. Combined nuclear (18S rDNA, 28S rDNA, Histone) and mitochondrial (16S rDNA) analyses placed the kakaducaridid genera, Kakaducaris and Leptopalaemon, as a strongly supported clade within the Palaemonidae, in a close relationship with the genus Macrobrachium. Monophyly of the Australian Kakaducarididae was strongly supported by the molecular data. Estimated net divergence times between Kakaducaris and Leptopalaemon using mitochondrial 16S rDNA equate to a late Miocene/Pliocene split. Within Leptopalaemon, each locality was distinct for mitochondrial COI haplotypes, suggesting long-term isolation or recent genetic bottlenecks, a lack of contemporary gene flow amongst sites and a small Ne. Mitochondrial groupings within Leptopalaemon were largely congruent with several previously recognised morphotypes. Estimated net divergence times between L. gagadjui and the new Leptopalaemon morphotypes equate to a split in the late Pliocene/early Pleistocene. The hypothesis that the Kakaducarididae is comprised of relict species in specialised ecological niches is not supported by the molecular data, which instead suggest a relatively recent origin for the group in northern Australia, sometime in the late Miocene or Pliocene. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Leptopalaemon; Kakaducaris; Palaemonidae; Kakaducarididae; Nuclear DNA; Mitochondrial DNA; COI; 16S; 18S; 28S; Histone; Phylogenetics

1. Introduction Kakadu National Park and surrounds, which include the ancient Arnhem Land ‘‘stone country” of eroded sandstone plateaux and escarpments (Fig. 1), host unique biotic assemblages, including a significant component of endemic freshwater macro-crustaceans (Finlayson et al., 2006). These endemics include a genus of phreatoicid isopods with exceptional species diversity (Finlayson et al., 2006) and two genera of palaemonoid shrimp currently assigned to the family Kakaducarididae (as defined by Bruce,1993). *

Corresponding author. Fax: +61 7 3735 7615. E-mail address: t.page@griffith.edu.au (T.J. Page).

1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.12.020

Apart from the Texan cave species, Calathaemon holthuisi (Strenth, 1976), which was provisionally assigned to the Kakaducarididae by Bruce (1993), the Kakaducarididae is comprised of only two described species, viz. Leptopalaemon gagadjui Bruce and Short, 1993, and Kakaducaris glabra Bruce, 1993. Both species are recorded from upland, permanent streams in the north-western portion of the Arnhem Land plateau (Fig. 1). The former species is widely distributed, whereas the latter is restricted to a single locality. Unlike Macrobrachium Bate, 1868, which is the dominant shrimp genus in northern Australia and other tropical regions of the world, both kakaducaridid shrimps are gregarious, active during daylight, and seek shelter only when disturbed. By contrast, epigeal species of

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T.J. Page et al. / Molecular Phylogenetics and Evolution 46 (2008) 1003–1014

Fig. 1. Map of kakaducarid sampling sites on the Arnhem Land plateau. The black outline is the boundary of Kakadu National Park. See Table 1 for site details.

Macrobrachium are typically solitary, aggressive and nocturnal, seeking shelter during daylight. Of the thirteen species of Macrobrachium known from Australia (Short, 2004), one species, M. bullatum Fincham, 1987, is common in upland habitats in the Northern Territory and is often sympatric with L. gagadjui. Bruce (1993) initially erected the Kakaducarididae as a subfamily of the Palaemonidae, but in an addendum in the same paper, elevated the taxon to familial rank to ‘‘maintain parity” with a new classification of the Caridea by Chace (1992). Bruce (1993) distinguished the Kakaducarididae on the basis of the highly modified mouthparts, which appeared to form a filter-feeding apparatus. Apart from Calathaemon holthuisi, filtratory mouthparts had not previously been recorded in the Palaemonidae, the largest and most diverse shrimp family (Davie, 2002). Interestingly, filter-feeding behaviour is well represented in the Atyidae, the other major family of shrimps found in tropical freshwaters. Bruce (1993) provisionally included Calathaemon holthuisi in the Kakaducarididae on the basis of its similar filtratory mouthparts. However, he did so with some reservation and his definition of the family did not encompass the morphology of the species. In particular, he noted that ‘‘the appendix masculina of Calathaemon does not show the characteristic features of Kakaducaris or Leptopalamon”. Apart from the filtratory mouthparts and unique form of the appendix masculina in the latter two genera, the Kakaducarididae and Palaemonidae are otherwise highly similar in general morphology. Palaemonids in general, and Macrobrachium in particular, have been much studied, due largely to their ubiquity

and importance in aquaculture. Despite this, they remain a challenge to characterise due to their highly conservative morphology (Short, 2004) and a high level of morphological homoplasy (Short, 2000; Liu et al., 2007). A recent large-scale taxonomic revision of Australian Macrobrachium found that some of the characters traditionally used to define palaemonid taxa, including genera, were sometimes variable within species (Short, 2004). This highlights the difficulty of palaemonid identification at many different systematic levels (Bruce and Short, 1993; Walker and Poore, 2003). Genetic methods can provide another view to help interpret patterns of biodiversity (Liu et al., 2007). Molecular phylogenetics has been used recently to understand the evolutionary relationships of Macrobrachium in Australia and throughout the world (e.g. Murphy and Austin, 2003, 2004, 2005; de Bruyn et al., 2004; Liu et al., 2007), and for larger-scale caridean shrimp relationships (e.g. Porter et al., 2005). However, the Kakaducarididae have not been included in any of these studies. The results of many molecular studies have highlighted inconsistencies at the generic-level (Murphy and Austin, 2003), and revealed examples of cryptic species (de Bruyn et al., 2004). This study was conducted in conjunction with an ongoing morphological study of the group by one of the present authors (JWS), representing a taxonomic feedback loop similar to that described by Page et al. (2005) for a molecular–morphological study of the Caridina indistincta complex. The present molecular study investigates systematic relationships of the Kakaducarididae using both mitochondrial and nuclear DNA, and in particular will test: (1) the

T.J. Page et al. / Molecular Phylogenetics and Evolution 46 (2008) 1003–1014

monophyly of the Kakaducarididae, including new study material; (2) evidence of a sister-group relationship between the Kakaducarididae and Palaemonidae; (3) the hypothesis that the Kakaducarididae is an ancient group comprised of relict taxa; (4) the hypothesis that Leptopalaemon and Kakaducaris represent two homogeneous natural groups sufficiently distinct to be recognised as genera; (5) whether there is very low gene flow and high net divergence between populations; (6) the hypothesis that the new kakaducaridid morphotypes represent cryptic species. 2. Materials and methods 2.1. DNA extraction and sequencing Kakaducaridid study material was collected from 12 sampling sites in Kakadu National Park and neighbouring Arnhem Land, the Northern Territory, Australia (Table 1 and Fig. 1). Material of Calathaemon holthuisi, originally described from Ezell’s Cave, San Marcos, Texas, and provisionally assigned to the Kakaducarididae by Bruce (1993), was not available for study. Two of the Arnhem Land sites studied were from the central Arnhem Land plateau, indicating that the family is not confined to the north-western edge of the plateau as previous records indicated. Most of the study material originated from a field collecting program initiated by one of the present authors (CLH) in the late 1990s. This program involved staff from the Environmental Research Institute of the Supervising Scientist (ERISS) as well as Kakadu National Park rangers. Collecting was largely done on an opportunistic basis in conjunction with regular fieldwork in target areas. Material representing six of the sampling sites was obtained from the Queensland Museum (QM) collection and the remainder from the collections of ERISS. All material was preserved in 70–100% ethanol at the time of capture or soon thereafter. For the QM material, fifth pleopods were removed for DNA extraction, except for one small specimen of Kakaducaris glabra where a whole pereiopod was used. For the ERISS material, DNA was extracted from whole specimens. DNA was also extracted from tail tissue of two species of palaemonid shrimp (Macrobrachium) (Table 1). Total genomic DNA was extracted from ethanol-preserved tissue using a CTAB/phenol–chloroform protocol modified from Doyle and Doyle (1987). Following homogenisation in 700 lL extraction buffer (0.5 M Tris–HCl, pH 8.0, 2 M NaCl, 0.25 M EDTA, 0.05 M CTAB), samples were incubated overnight with 5 lL of Proteinase K (20 mg/ml) at 65 °C. Proteins and lipids were removed with a series of phenol/chloroform extractions and the resultant pellets precipitated in isopropanol (80 °C) and resuspended in ddH2O. Five genes were targeted for amplification and sequencing, two of which are mitochondrial DNA (mtDNA): cytochrome c oxidase I (COI, also known as COX1) and 16S ribosomal DNA (16S); while three are nuclear DNA (nDNA): 18S ribosomal DNA (18S), 28S ribosomal

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DNA (28S) and Histone (H3). These genes were chosen because they were known to be informative at a variety of phylogenetic levels (Crandall et al., 2000; Porter et al., 2005). COI is effective in discriminating taxa at the population and species-level, but less so at higher phylogenetic levels (DeSalle et al., 2005; Lefe´bure et al., 2006). We used the mitochondrial 16S rDNA gene because it is the most densely sampled gene region on GenBank for Palaemonoidea (76 species, June 2007), and because it can recover both deeper relationships, such as genera within a family, as well as shallower species-level differences (Lefe´bure et al., 2006). 16S has often been used for very largescale, deep phylogenies, in particular for prokaryotes and microscopic eukaryotes (Beiko et al., 2005), and also shows promise as a potential barcode (Chu et al., 2006). Both of the highly conserved, nuclear large and small subunit RNAs (28S/18S) and Histone (H3) are popular for deeplevel crustacean phylogenetics (Crandall et al., 2000; Porter et al., 2005), and the portions of each gene that we have chosen are well represented on GenBank for Palaemonoidea (18S: 22 species; 28S: 25; H3: 6 species; June 2007). The following primer sets were used to isolate and amplify a fragment of each target gene using the polymerase chain reaction: COIf-L and COIa-H (Palumbi et al., 1991), 16S-F-Car and 16S-R-Car (von Rintelen et al., 2007), 18Sai and 18Sb3.0 (Whiting et al., 1997), 28S-Rd1a and 28S-Rd4b (M.F. Whiting pers. comm. in Crandall et al., 2000) and H3-F and H3-R (Colgan et al., 1998). Reactions contained 0.25 lL of 10 mM dNTP’s (Bioline; London, UK), 1 lL 50 mM MgCl2, 1.25 lL of 10 polymerase reaction buffer, 0.275 U of Taq polymerase (all Fisher Biotec), 0.5 lL each of two primers (10 lM), 0.5 lL (5–80 ng) of DNA template, adjusted to a final volume of 12.5 lL with ddH2O. The PCR cycling conditions for all reactions consisted of an initial denaturing period at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min, followed by a final chain extension at 72 °C for 5 min. Prior to sequencing, product yield, specificity and potential contamination were checked by agarose gel electrophoresis. Target product was purified using Exo-SAP, with 0.25 lL of Exonuclease I (Fermenta), 1 lL of Shrimp Alkaline Phosphatase (SAP) (Promega) and 5 lL PCR product incubated at 37 °C for 35 min and then 80 °C for 20 min. Purified samples were stored at 4 °C until sequenced. Sequences were generated on an ABI 3130xl capillary auto sequencer at Griffith University using the BigDye Terminator v1.1 Cycle sequencing kit. Cycle sequencing conditions followed the standard protocol but used half the suggested reaction volume. Sequences were edited using Sequencher 4.1.2 (Gene Codes Corporation). 2.2. Dataset construction and sequence alignment A total of 62 kakaducaridid and two palaemonid specimens were sequenced, with differing numbers for each gene

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Table 1 Kakaducaridid shrimp specimens sequenced for this study, with Genbank accession numbers Site No.

Sample site (catchment)

Latitude (S)

Longitude (E)

N

Mitochondrial DNA COI (N)

16S (N)

18S (N)

28S (N)

H3 (N)

Kakaducaris glabra

6

12° 54.76’

132° 56.00’

4

EF588295(2)

EF588318(2)

EU249463(2)

EU249464(2)

EU249461(2)

Leptopalaemon gagadjui

1

Lightening Dreaming Ck (Namarrgon Gorge) (SAC), NT, Australiaa Arnhem escarpment (Oenpelli outlier) (EAC), NT, Australiaa Unnamed creek (Namarrgon Gorge) (SAC), NT, Australiab Nourlangie plateau south of Namarrgon Gorge (SAC), NT, Australiab Upper Barramundie Ck. (SAC), NT, Australiaa Freezing Gorge (trib.of Koolpin Creek)(SAC), NT, Australiab Upper Katherine River (KC), NT, Australiab Upper Mann River (MC), NT, Australiab

12° 21.62’

133° 05.49’

4

EF588289(4)

EF588307(2)

12° 55.15’

132° 58.75’

4

EF588302(1)

132° 54.06’

8

EF588305(2), EF588306(1) EF588304(1)

EF588298(1)

12° 59.90’

EF588279(2), EF588280(2) EF588281(8)

EF588298(2)

EF588302(1)

13° 19.27’

132° 26.25’

2

EF588296(1)

EF588315(2)

13° 29.41’

132° 05.35’

3

EF588314(2)

EF588298(2)

EF588303(1)

13° 24.84’

133° 11.04’

7

EF588285(2), EF588286(1) EF588282(7)

EF588308(1)

EF588298(1)

EF588299(1)

13° 17.94’

133° 32.34’

8

EF588309(1)

EF588298(2)

EF588300(1)

Arnhem plateau (Oenpelli outlier) (EAC), NT, Australiaa Magela Ck (South arm) (EAC), NT, Australiaa Nourlangie plateau N. of Namarrgon Gorge (SAC), NT, Australiab Nourlangie plateau N. of Namarrgon Gorge (SAC), NT, Australiaa

12° 22.96’

133° 23.05’

5

12° 48.60’

132° 59.80’

3

12° 51.76’

132° 59.47’

10

EF588291(2), EF588292(8)

EF588313(2)

EF588297(1)

EF588301(1)

12° 54.20’

132° 57.30’

4

EF588293(3)

EF588312(3)

EF588297(1)

EU249465(2)

7 8

9 10 11 12

Leptopalaemon sp. 1 Leptopalaemon sp. 2 Leptopalaemon sp. 3

2 3 4

5

EF588287(6), EF588288(1), EF588290(1) EF588283(4), EF588284(1) EF588294(3)

Nuclear DNA

EU249459(2)

EU249459(2)

EF588310(2) EF588311(1)

EU249459(2)

Catchment abbreviations: EAC, East Alligator River; KC, Katherine River; MC, Mann River; SAC, South Alligator River. Queensland Museum registration numbers (in order of museum samples): W24751, W24755, W16550, W24754, W26845, W24748. a Specimen sources: P. Davie (Queensland Museum). b Authors.

T.J. Page et al. / Molecular Phylogenetics and Evolution 46 (2008) 1003–1014

Taxa

T.J. Page et al. / Molecular Phylogenetics and Evolution 46 (2008) 1003–1014

(Table 1). Numerous sequences of the five genes were added from GenBank to our datasets to provide phylogenetic context (Table 2). Two main datasets were constructed to investigate phylogenetic relationships at different levels (Table 3). The first dataset, ‘‘Leptopalaemon COI”, employed only the fastevolving COI gene to differentiate relatively shallow divergences between populations of Leptopalaemon. The second dataset, ‘‘Palaemonidae Combined”, includes one mitochondrial gene (16S) and three nuclear genes (18S/28S/ H3) to place the kakaducaridids into a deeper systematic context in relation to their putative close relative, the Palaemonidae. Each of the four genes in this second dataset were also analysed independently, including extra palaemonid taxa which do not have exemplar sequences of all four genes (Table 3). COI and H3 sequences were aligned without gaps. The remaining three regions were ribosomal DNA and constituted both highly conserved and highly variable regions, which can be challenging to align (Gillespie et al., 2005), especially as there is no universally accepted method for alignment (Chu et al., 2006). To avoid incorrectly aligned

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sites, all ribosomal sequences were first aligned with ClustalX (Thompson et al., 1997) at default values of 15/6 for gap opening/extension, and then run through Gblocks version 0.91b (Castresana, 2000) (parameters = minimum number for conserved position: 2/3 of sequences; minimum number for flanking position: 2/3 of sequences; maximum number contiguous non-conserved positions: 8; minimum length of block: 8; allowed gap positions: with half). Blocks of poorly aligned sites identified by Gblocks were excluded from analyses. A hypervariable region of the 28S D2 expansion segment, encompassing helices 2 g to 3–1 (sensu Gillespie et al., 2005), was excluded from the analyses (see Section 3 below). 2.3. Phylogenetic and distance analyses The best-fit models of molecular evolution (Akaike Information Criterion) were selected separately for every separate gene region within each dataset and for the combined dataset as a whole, using Modeltest version 3.06 (Posada and Crandall, 1998) in PAUP* version 4.0 b10 (Swofford, 2002). A partition homogeneity test was carried

Table 2 Palaemonid and other shrimp sequences included in this study Taxa

Palaemonidae Creaseria morleyi Cryphiops caementarius Macrobrachium australiense Macrobrachium brasiliense Macrobrachium equidens Macrobrachium lar Macrobrachium latidactylus Macrobrachium mammillodactylus Macrobrachium olfersi Macrobrachium platycheles Macrobrachium potiuna Macrobrachium rosenbergii/dacqueti # Macrobrachium sp. MLP123 Macrobrachium trompii Palaemon intermedius* Palaemon elegans Palaemonetes atrinubes Outgroups Atyidae Atyoida bisulcata Typhlatya pearsi Apheidae Lysmata debelius Lysmata wurdemanni Penacidae Penaeus semisulcatus

GenBank accession numbers Mitochondrial

Nuclear DNA

16S

18S

28S

H3

DQ079710A DQ079711A EF588317B AY377839D AY282773E EF588316B AY282770E AY282776E AY377848D AY377850D AY377851D NC006880F DQ079720A AY377852D AF439516G DQ079729A AF439520G

DQ079746A DQ079747A AY374177C AY374169C AY374174C AY374168C AY374171C AY374173C AY374176C AY374175C DQ079756A AY374166C DQ079754A AY374172C AY374170C DQ079764A AY374178C

DQ079784A DQ079785A AY374145C AY374146C AY374149C AY374155C AY374156C AY374158C AY374159C AY374161C DQ079797A AY374144C DQ079795A AY374165C AY374154C DQ079807A AY374150C

DQ079671A DQ079672A EU249460B

DQ681278H DQ079735A

DQ079738A DQ079770A

DQ079774A DQ079813A

DQ079661A DQ079702A

DQ079718A DQ079719A

DQ079752A DQ079753A

DQ079793A DQ079794A

DQ079681A DQ079682A

DQ079731A

DQ079766A

DQ079809A

DQ079698A

EU249462B

DQ079685A DQ079683A

DQ079696A

Sources of sequences: APorter et al. (2005); Bthis study; CMurphy and Austin (unpublished); DMurphy and Austin (2005); EMurphy and Austin (2004); F Miller et al. (2005); GMurphy and Austin (2003); HPage et al. (2007). # Macrobrachium rosenbergii has been split into 2 species (Wowor and Ng 2007). Mitochondrial sequence is from M. dacqueti. Nuclear sequences may be from either M. rosenbergii or M. dacqueti. * Macrobrachium intermedium has been redescribed as Palaemon intermedius (Walker and Poore 2003).

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Table 3 Different datasets, molecular models and tree scores for analyses conducted in this study Dataset

Genes

Leptopalaemon COI Palaemonidae combined Palaemonidae Palaemonidae Palaemonidae Palaemonidae

16S 18S 28S H3

Fig.

COI 16S/18S/28S/H3

2 3

16S 18S 28S H3

4a 4b 4c 4d

Molecular models from Modeltest

TVM+G HKY+I+G/SYM+I+G/GTR+G/GTR+I+G/ TVM+I+G (combined) TrN+I+G SYM+I+G GTR+I+G GTR+I+G

Tree scores ML

Bayesian

Parsimony steps (no. of trees)

1056.10 8570.97

1110.74 8372.76

105 (65) 1397 (1)

4105.06 2347.76 2187.62 1418.74

4150.12 2408.47 2254.68 1449.79

887 (2) 357 (22) 345 (6) 210 (6)

Models: HKY, Hasegawa-Kishino-Yano; GTR, General Time Reversible; SYM, Symmetrical; TrN, Tamura-Nei; TVM, Transversion; +I, proportion of invariable sites; +G, gamma distribution of site-to site variation; ML, maximum likelihood.

out in PAUP* for the Palaemonidae Combined dataset to test for significant differences in phylogenetic signals between genes. We carried out three different forms of phylogenetic inference on both of the datasets; maximum likelihood analysis (PHYML version 2.4.4; Guindon and Gascuel, 2003), Bayesian (MrBayes version 3.1.2; Huelsenbeck and Ronquist, 2001) and parsimony (PAUP*). The individual gene analyses within the Palaemonidae Combined dataset were analysed with maximum likelihood. MrBayes parameters were: 2 million generations, trees sampled every 100 cycles, dataset partitioned by gene within each dataset, 50% burn in, and two runs of four chains heated to 0.2. For maximum likelihood analyses, a single model of evolution was selected for each dataset. Both parsimony (full heuristic, 100 random repetitions) and maximum likelihood analyses were bootstrapped 1000 times. 3. Results 3.1. Kakaducarididae sequences The following numbers of kakaducaridid shrimp sequences were produced; COI: 57 Leptopalaemon, 2 Kakaducaris; 16S: 20 Leptopalaemon, 2 Kakaducaris; 18S: 10 Leptopalaemon, 2 Kakaducaris; 28S: 7 Leptopalaemon, 2 Kakaducaris; H3: 3 Leptopalaemon, 2 Kakaducaris (Table 1 for all GenBank accession numbers). COI sequences produced were 400 base pairs (bp) long and correspond to positions 999–1398 of the palaemonid Macrobrachium dacqueti (as Macrobrachium rosenbergii, cf. Wowor and Ng, 2007) mitochondrial genome (Accession No. NC006880; Miller et al., 2005). 16S sequences were 455 bp and equate to positions 11,390–11,843 of the M. dacqueti genome. 18S sequences were 481 bp, which is equal to positions 1–480 of the ‘‘M. rosenbergii” sequence AY374166 (Murphy and Austin, unpublished); 28S sequences were 706 bp (positions 1–682 of the ‘‘M. rosenbergii” sequence AY374144, Murphy and Austin, unpublished); and H3 were 328 bp. A hypervariable region of the 28S D2 expansion segment, corresponding to positions 425–682 of ‘‘M. rosenbergii” sequence AY374144 (Murphy and Austin, unpublished),

was excluded because it could not be reliably aligned (see Section 2). All Modeltest-derived models are displayed in Table 3. Chi-square tests of homogeneity of base frequencies across ingroup taxa from all of the phylogenetic datasets found no significant differences (P > 0.99 for all). Pairwise partition homogeneity tests were carried out between each of the four genes in the Palaemonidae Combined dataset. All of the comparisons amongst the nuclear genes were non-significant, but all of the comparisons between each of the nuclear genes and 16S were significant (P = 0.01–0.03). When the outgroup taxa were removed (i.e. non-kakaducaridids or palaemonids) and the tests rerun between 16S and the nuclear genes, they were all non-significant (P = 0.20–0.50). As the only conflict between genes was caused by outgroup 16S sequences, all genes were combined into a single dataset. The three forms of phylogenetic inference were largely congruent for each dataset, with Bayesian posterior probabilities frequently higher than the other analyses’ bootstrap values (see Table 3, for all tree scores). 3.2. Relationships within Leptopalaemon and Kakaducarididae No COI haplotypes are shared amongst Leptopalaemon sites (Fig. 2), with each population forming a separate clade or having a unique haplotype, thus suggesting longterm isolation, a lack of contemporary gene flow amongst sites and a small Ne, as well as possible founder effects or recent genetic bottlenecks. The maximum percentage difference between any two Leptopalaemon COI sequences is 5.5% uncorrected P-distance (7.38% with Modeltest model, and 1.54%/1.64%, respectively, for 16S). This difference reflects the most divergent clade within Leptopalaemon, which includes sites 4 and 5 (L. sp. 3), as well as site 3 (L. sp. 2) (Fig. 2). There is also a clade formed by sites 9 and 10, the two most western populations of L. gagadjui, including the type locality. Net divergence using a correction for within-clade polymorphism between sites 3/4/5 and the remaining Leptopalaemon is 2.13% P-distance (2.96% Modeltest model; 2.23% K2P model [as per Costa

T.J. Page et al. / Molecular Phylogenetics and Evolution 46 (2008) 1003–1014

and Modeltest distances using both rates, ±SE), and therefore the late Miocene or Pliocene. Again, as stated above, these calculations provide a rough estimate only.

Site 1 93/96 91

Site 7 Site 8 Site 9

69/87 48

Leptopalaemon gagadjui

Site 10

89/89/81

1009

3.3. Relationships between the Kakaducarididae and Palaemonidae

Site 11 96/100 88 85/63 37

Site 12

Leptopalaemon sp. 1

Site 2

88/96/77

Leptopalaemon sp. 2

Site 3

90/99/84 99/100 99

Site 4

Leptopalaemon sp. 3

Site 5

Kakaducaris glabra 0.01 substitutions/site

Fig. 2. Maximum likelihood phylogram of Leptopalaemon COI sequences (maximum likelihood bootstrap/Bayesian posterior probabilities values above node, and parsimony bootstrap values below) (nodes with <60% for two of three values have been collapsed).

et al., 2007]). Given a divergence rate of 1.4% per million years (derived from a homologous 30 COI region of alpheid shrimp; Knowlton and Weigt, 1998), these equate to 1.45– 2.22 (±SE) million years since separation (or 1.71–2.52 million years using 16S rates below), which would place the divergence sometime in the late Pliocene or early Pleistocene. As alpheids (and crabs, used below for a 16S estimate of net divergence times between Leptopalaemon and Kakaducaris) are not a closely related group, these calculations provide a rough estimate to place divergences into a geological epoch. Preliminary morphological investigations by two of the present authors (JWS, CLH) recognised two, new, easilydiscernible morphotypes, one at sites 2, 4 and 5 and bearing a resemblance to Kakaducaris glabra, and the other at site 3 and resembling Leptopalaemon gagagdjui. The COI data demonstrated conclusively that both of the new morphotypes should be assigned to Leptopalaemon and that the new Kakaducaris-like morphotype actually comprised two highly cryptic species of Leptopalaemon, one at site 2 in the East Alligator River catchment and the other forming a clade comprising sites 4 and 5 in the South Alligator River catchment. The other previously identified morphotype at site 3 in the East Alligator River catchment was well supported by the COI data. Some geographical structure was also revealed within sites currently assigned to Leptopalaemon gagagdjui, particularly sites 9 (the type locality) and 10 which may form a distinct western group. Leptopalaemon and Kakaducaris sequences are well differentiated from each other in both COI (maximum = 14.50% P-distance; 32.67% Modeltest; 16.53% K2P) and 16S analyses (16S maximum = 4.86% P-distance; 6.13% Modeltest). Calculated net divergence times between Leptopalaemon and Kakaducaris, using the more appropriate, conserved 16S fragment (0.65% and 0.9% per million years; Schubart et al., 1998; Sturmbauer et al., 1996), equates to 4.55–8.08 million years (full range of P-distance

Combined analyses of three nuclear and one mitochondrial gene (Fig. 3 and Table 3) placed the Kakaducarididae (Leptopalaemon and Kakaducaris) in a close relationship with the Palaemonidae, and with Macrobrachium in particular (Fig. 4). The Kakaducarididae falls within a strong clade containing Macrobrachium and Cryphiops caementarius Dana, 1852. The close relationship between the latter two taxa was noted by Short (2004). These three taxa then form a higher-level clade with Creaseria morleyi (Creaser, 1936) relative to another strong palaemonid clade, which contains representatives of the genus Palaemon Weber, 1795 (and Palaemonetes Heller, 1869 in other analyses). This again highlights the degree of genetic diversity within Macrobrachium and its close and probable paraphyletic relationship to the Kakaducarididae and Cryphiops. Individual gene analyses (Fig. 4 and Table 3) were considerably less resolved than the combined analyses. The Kakaducarididae (‘‘K” in Fig. 4) were recovered as a clade in all analyses. Palaemon and Palaemonetes formed a clade in both the 16S and 18S analyses. Similar to the combined analysis, the Kakaducarididae (Leptopalaemon and Kakaducaris), Macrobrachium, Cryphiops caementarius and Creaseria morleyi formed a clade in both the 28S and H3 analyses, and was strongly supported in the latter. The long branch leading to the Kakaducarididae in Fig. 3 is due to an apparent higher rate of divergence for nuclear rDNAs (18S/28S) relative to other palaemonids. Kakaducaris glabra

100/100 100

Leptopalaemon sp.3

89/100/79 96/62/99

88/47/77

Kakaducarididae

Leptopalaemon gagadjui

Macrobrachium australiense Macrobrachium lar Cryphiops caementarius

98/100/99

Macrobrachium sp.MLP123

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Palaemonidae

Macrobrachium potiuna

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Creaseria morleyi Palaemon elegans

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Lysmata debelius Lysmata wurdemanni

100/100 100

Typhlatya pearsei

Outgroups

Atyoida bisulcata Penaeus semisulcatus

0.05 substitutions/site

Fig. 3. Maximum likelihood phylogram of kakaducaridid and palaemonid combined 16S/18S/28S/H3 sequences (maximum likelihood bootstrap/Bayesian posterior probabilities values above node, and parsimony bootstrap values below) (nodes with <60% for two of three values have been collapsed).

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(a) 16S rDNA

(c) 28S rDNA

(b) 18S rDNA K.glabra L.gagadjui L.sp.3 M.australiense M.lar M.equidens M.mammillodactylus C.caementarius M.spMLP123 M.dacqueti M.platycheles M.trompii M.latidactylus Molfersii M.potuina M.brasiliense Cr.morleyi P.intermedius Pa.atrinubes P.elegans

K

P

(d) Histone K.glabra L.gagadjui L.sp.3 M.australiense M.lar M.equidens M.mammillodactylus C.caementarius M.spMLP123 M.dacqueti/rosenbergii M.platycheles M.trompii M.latidactylus Molfersii M.potuina M.brasiliense Cr.morleyi P.intermedius Pa.atrinubes P.elegans

K

K.glabra L.gagadjui L.sp.3 M.australiense M.lar M.equidens M.mammillodactylus C.caementarius M.spMLP123 M.dacqueti/rosenbergii M.platycheles M.trompii M.latidactylus Molfersii M.potuina M.brasiliense Cr.morleyi P.intermedius Pa.atrinubes P.elegans

K.glabra

K

P

K

L.sp.3 M.australiense M.lar C.caementarius

P

M.potuina

P

M.spMLP123 Cr.morleyi P.elegans Atyoida bisulcata Typhlatya pearsei

OG

Fig. 4. Maximum likelihood bootstrap cladograms of individual gene analyses of kakaducaridid and palaemonid combined 16S/18S/28S/H3 sequences: (a) 16S rDNA, (b) 18S rDNA, (c) 28S rDNA, (d) Histone. Nodes with <60% for two of three support values (maximum likelihood bootstrap/Bayesian posterior probability/parsimony bootstrap) have been collapsed. Nodes with >80% for all three values have thicker lines. ‘‘K” = Kakaducarididae, ‘‘P” = Palaemonidae, ‘‘OG” = outgroup.

Whereas kakaducaridid 16S and H3 sequences appear to be within the range of fairly typical Macrobrachium sequences, the kakaducaridid nuclear ribosomal sequences are distinct, as has also been reported in myriapods (Giribet and Wheeler, 2001). The mean divergences between Macrobrachium and kakaducaridid nuclear rDNA sequences (P-distances–18S: maximum 11.81%, mean 9.84%; 28S: 9.39%, 8.07%) are about double that of within Macrobrachium alone (18S: maximum 8.02%, mean 4.75%; 28S: 8.13%, 4.39%). This is in contrast to the mean divergence between Macrobrachium and kakaducaridid 16S sequences (P-distances: 11.34%), which falls well within the range of divergence amongst Macrobrachium alone (maximum: 17.19%, mean: 9.85%). 4. Discussion 4.1. Monophyly of Australian Kakaducarididae The monophyly of Australian Kakaducarididae, including the new study material, was strongly supported by the molecular data. The two endemic Australian genera, Lept-

opalaemon and Kakaducaris, were recovered as a clade in all analyses. This clade was strongly supported in both the combined and individual gene analyses with the exception of 16S. 4.2. Relationships between Australian Kakaducarididae and the Palaemonidae The endemic Australian genera, Leptopalaemon and Kakaducaris, represent a well-defined lineage of palaemonoid shrimps, whether one is considering DNA, morphology, behaviour, ecology or geography. However, the molecular data did not support a sister-group relationship between the Palaemonidae and Kakaducarididae (Leptopalaemon and Kakaducaris). The combined analyses using three nuclear and one mitochondrial gene placed Leptopalaemon and Kakaducaris as a well supported clade within the Palaemonidae in a close relationship to Macrobrachium. However, the distinctiveness of the kakaducaridid lineage from Macrobrachium was highlighted by the relatively high rate of divergence in 18S/ 28S nuclear rDNAs.

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Although Kakaducaris and Leptopalaemon have diverged considerably from Macrobrachium in nuclear genes and morphology, the molecular data strongly suggest that the common ancestor of Leptopalaemon/Kakaducaris would have been more similar to Macrobrachium than one of the other palaemonine genera currently occurring in northern Australia, e.g. Palaemon or Palaemonetes. Macrobrachium are likely to have occurred in northern Australia since at least the Miocene (Short, 2000; Murphy and Austin, 2004). Representatives of the genus would have been well established in upland freshwaters well before the estimated time of ancestral splitting of Kakaducaris and Leptopalaemon in the late Miocene/Pliocene. Macrobrachium is a likely source of isolated divergent taxa, including Leptopalaemon/Kakaducaris and Cryphiops, in much the same way that the widespread and morphologically conservative atyid shrimp genus, Caridina, is the likely source for isolated cave genera in the Northern Territory (Page et al., 2007). 4.3. The Kakaducarididae as an ancient group comprised of relict taxa Bruce (1993) suggested a close phylogenetic relationship between the two Australian genera, Leptopalaemon and Kakaducaris and the North American cave species, Calathaemon holthuisi. He also hypothesised that the Kakaducarididae was comprised of ‘‘relict species in specialised ecological niches”. An ancient origin for the Kakaducarididae is not supported by our molecular data, which strongly suggest that the two Australian genera originated in the Miocene or Pliocene from a Macrobrachium-like ancestor. The close similarity of the filtratory mouthparts between Leptopalaemon/Kakaducaris and Calathaemon must therefore be considered an example of homoplasy rather than synapomorphy. 4.4. Generic status of Leptopalaemon and Kakaducaris Sequences obtained for the putative Leptopalaemon species were well differentiated from Kakaducaris glabra in both COI and 16S analyses. By contrast the maximum difference between any two Leptopalaemon sites was much lower in both COI and 16S. Some recent meta-analyses have looked at molecular variation in COI across many taxonomic levels within Crustacea. Costa et al. (2007) found an average of 19.75% (K2P model) sequence difference between genera within a decapod family. However, the range of genetic difference between genera reported by Costa et al. (2007) was very wide (11.27–49.93%). The K2P difference of 16.53% between Leptopalaemon and Kakaducaris is therefore toward the lower end of the range reported by Costa et al. (2007). At the very least, the molecular data indicate that the putative Leptopalaemon species are a highly homogeneous

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group genetically and are well separated from Kakaducaris glabra. An ongoing morphological study by one of the present authors (JWS) has so far revealed that the two nominal genera are much closer than previously thought and possibly only one significant morphological character separates the two genera, viz. the number of arthrobranch gills above the third maxilliped. 4.5. Gene flow and net divergence between populations The COI haplotypes at each locality were unique, indicating a lack of contemporary gene flow, long-term isolation, recent genetic bottlenecks or founder effects, and a small Ne. Net COI divergence between the most divergent Leptopalaemon clade (sites 3, 4, 5) and the remaining Leptopalaemon sites was 2.13% P-distance (2.96% Modeltest; 2.23% K2P). Using a rate of 1.4% per million years this equates to divergence sometime in the late Pliocene or early Pleistocene. By far the most divergent kakaducaridid population was Kakaducaris glabra at site 6. As previously mentioned, net COI divergence of K. glabra from the Leptopalaemon sites was 14.5% P-distance (32.67% Modeltest; 16.53% K2P). This equates to a much earlier divergence in the late Miocene or Pliocene. 4.6. Cryptic speciation Preliminary morphological investigations by two of the present authors (JWS, CLH) recognised two, new, easilydiscernible morphotypes, one bearing resemblance to Kakaducaris glabra, with reduced body spination and rostral length, while the other resembled Leptopalaemon gagagdjui, but had a reduced number of rostral teeth and shorter rostrum. The COI data demonstrated conclusively that both of the new morphotypes should be assigned to Leptopalaemon rather than Kakaducaris and that there was significant population structure that may equate to cryptic speciation. Furthermore, the COI data revealed that the new Kakaducaris-like morphotype may actually comprise two highly cryptic species of Leptopalaemon, one at site 2 in the East Alligator River catchment and the other forming a clade comprising sites 4 and 5 in the South Alligator River catchment. The other previously identified morphotype at site 3 in the East Alligator River catchment was well supported by the COI data. Some geographical structure was also revealed within sites currently assigned to Leptopalaemon gagagdjui, particularly sites 9 (the type locality) and 10 which may form a distinct western group. COI divergence between putative Leptopalaemon species varied between 2.13% and 5.5%, which is considerably lower than the universal COI crustacean species threshold of 16% proposed by Lefe´bure et al. (2006). Costa et al. (2007) suggested a similar threshold for decapod species within a genus (mean K2P distance 17.16%), but also reported a mean intraspecific distance of 0.46% (maximum

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2.57%), which is much smaller than the levels displayed by Leptopalaemon (K2P mean 2.93%, maximum 5.46%). Simple thresholds to delimit taxa are unlikely to be effective because of an overlap between inter and intra-taxon diversity (Moritz and Cicero, 2004; Meyer and Paulay, 2005) and group-specific rates of divergence (DeSalle et al., 2005; Lefe´bure et al., 2006), which will depend on each group’s effective population size (Nielsen and Matz, 2006). Leptopalaemon may comprise a relatively young complex of species, which can be a challenge to identify using the ‘‘DNA barcode” approach which has recently gained momentum (Hebert and Gregory, 2005). DNA barcodes are likely to be useful for well-known, well-sampled groups for which many reference sequences are available, but less effective for poorly known groups (Meyer and Paulay, 2005), such as the Kakaducarididae, and for completely new taxa, which will require other corroborating information (Nielsen and Matz, 2006). As the putative Leptopalaemon species are also allopatric, one cannot easily tease apart species differences from local adaptation and genetic drift within a species (Bickford et al., 2007). A nuclear DNA barcode could provide an independent and wider view compared to the rather restricted perspective offered by mitochondrial DNA alone (Dasmahapatra and Mallet, 2006). The case for recognising the new morphotypes as full biological species, rather than as divergent populations of L. gagadjui, is strengthened by the highly conservative morphology of L. gagadjui over its relatively broad geographic range, including sites in close proximity to the new morphotypes. The highly dissected topography of the Arnhem Land plateau is also likely to have promoted allopatric speciation.

porary gene flow between collection sites and also identified sufficient geographic structure within Leptopalaemon to equate to cryptic speciation. Additional cryptic speciation may also have been uncovered compared to the new morphotypes so far recognised. Although qualified by a lack of a comparable COI divergence rate estimates for freshwater Palaemonoidea, the COI data were also useful in providing net divergence estimates (based on a homologous 30 region of alpheid shrimp) between the most divergent Leptopalaemon clade and other putative Leptopalaemon species. The late Pliocene/early Pleistocene estimate for divergence within Leptopalaemon, in combination with an estimate for net divergence between Leptopalamon and Kakaducaris in the late Miocene/Pliocene (using the more appropriate, conserved 16S gene fragment), provides compelling evidence, at the very least, that the kakaducaridid lineage is relatively modern and does not comprise relict species. The COI data were least effective in providing a definitive answer to the question of whether or not Leptopalaemon and Kakaducaris should be regarded as separate genera. The degree of COI divergence between the two nominal genera was towards the lower end of the range found between genera in a recent meta-analysis (Costa et al., 2007). However, COI divergence between the two nominal genera was over double that within Leptopalaemon alone. Ultimately, the status of the two nominal genera will be reviewed using a combination of both molecular and morphological evidence in a taxonomic revision of the Kakaducarididae currently in progress. Similarly, the results of this study will be used in the revision to review the familial status of the Kakaducarididae and in delimiting cryptic species within Leptopalaemon.

4.7. Conclusions

Acknowledgments

The benefits of using a broad approach in molecular systematics are readily apparent from the results of this study. In this study, five different genes, including three nuclear (18S rDNA, 28S rDNA, Histone) and two mitochondrial (16S rDNA and COI) were used to examine relationships at different taxonomic levels. A combined dataset comprising 18S, 28S, Histone and 16S sequences conclusively demonstrated that the Kakaducarididae (Leptopalaemon and Kakaducaris) is a monophyletic group and was highly effective in revealing the systematic position of the lineage within the Palaemonoidea. In particular, the combined analyses placed the group within the Palaemonidae in a position close to, but distinct from Macrobrachium and Cryphiops. By comparison, the individual gene analyses were less definitive about the relationships with Macrobrachium and Cryphiops. Based on the commonly used 16S gene fragment alone, the Kakaducarididae would have been difficult to separate from the latter two genera. At the lowest level, the COI mitochondrial data provided valuable information regarding the lack of contem-

We thank the rangers of Kakadu National Park (Parks, Australia), under the keen supervision of Greg Spiers, for collections of shrimp samples from the Nourlangie plateau district. The traditional owners of the other districts of Kakadu National Park as well as Arnhem Land also granted access to their land for sampling. We thank Peter Davie of the Queensland Museum for lending material and allowing tissue samples to be taken. Mike Scanlon (Department of Conservation and Land Management, Western Australia) and Rafi Mazor (University of California) provided comparative palaemonid specimens. Jon Mallatt (Washington State University) helped in aligning sequences with RNA secondary structures and Greg Edgecombe (Natural History Museum, London) helped in finding literature. We thank Renee Bartolo from ERISS for providing the base map in Fig. 1, and two anonymous reviewers for greatly improving the manuscript. Funding was provided by the Environmental Research Institute of the Supervising Scientist (Northern Territory) and the Australian Rivers Institute (Griffith University).

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