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Home , Biffarius, Callianideidae, Callichirus major, Laomediidae, Thalassina, Thalassina anomala

invertebrate Systematics, 2002, 16, 839- 847

Molecular phylogeny of the mud and mud (Crustacea: : ) using nuclear 18S rDNA and mitochondrial16S rDNA

C. C. TudgeA,C and C. W CunninghamB

ABiology Department, American University, 4400 Massachusetts Ave. NW, Washington, DC 20016-8007, USA and Department of Systematic Biology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560-0163, USA. 8 Department of Zoology, Duke University, Durham, NC 27708-0325, USA. .. cTo whom correspondance should be addressed. Email: [email protected]

Abstract. Partial sequences of the 18S nuclear and 16S mitochondrial ribosomal genes were obtained for 14 species of thalassinidean (families , , Strahlaxiidae, Thalassinidae and ) and a further six species in related decapod infraorders (families Aeglidae, Astacidae, Lithodidae, Palinuridae, Raninidae and Scyllaridae). Maximum-likelihood and Bayesian analyses show equivocal support for the monophyly of the Thalassinidea, but show strong support for division of the infraorder into two major clades. This dichotomy separates representatives in the Upogebiidae, Laomediidae and Thalassinidae from those in the Strahlaxiidae and Callianassidae. The Laomediidae is shown to be paraphyletic, with the thalassinid species, squamifera, being placed on a branch between Axianassa and a clade comprising Jaxea and Laomedia, the three current laomediid genera. For a monophyletic Laomediidae, the family Axianassidae should be resurrected for the Axianassa.

Introduction relationships among four thalassinidean families and the (Fig. 1B). A period of 56 years elapsed before any The decapod infraorder Thalassinidea is a group of cryptic, further publications on the phylogenetic relationships within marine, burrowing shrimp-like or -like this obscure, but taxonomically large, infraorder emerged. that occur worldwide (with the exception of the coldest polar Poore (1994: 120) published a comprehensive revision ofthe waters) in mostly shallow (<200m) benthic habitats. Most members ofthe Thalassinidea based on morphology, including species form complex systems in soft sand or mud a new classification scheme, keys to families and genera and environments, but several taxa live in stony or coral-rubble a phylogenetic tree (reproduced in Fig. 1C to the family level areas and some even excavate in living coral and only). This tree was the first cladistic analysis ofthe infraorder sponge colonies (Dworschak 2000). There are currently as a whole and separated the 22 selected genera into the 528 species in 84 genera spread across 11 recognised currently recognised 11 families and three superfamilies families of the three superfamilies: Axioidea, Thalassinoidea (despite showing a dichotomy with only two major and Callianassoidea (Poore 1994; Dworschak 2000; clades). He also proposed a monophyletic origin for the P. Dworschak, personal communication). infraorder, with the Anomura being the closest sister-group. Borradaile (1903: 551), in his seminal work on the Several papers have been published employing cladistic classification of the Thalassinidea, presented a 'genealogical analyses of relationships among or within genera of certain tree' ofproposed relationships among 12 known genera in the thalassinidean families. These investigations cover the four families recognised at that time. His tree (reproduced in families and Calocarididae (Kensley 1989), Fig. lA with current family designations) separates these Callianideidae (Kensley and Heard 1991 ), Callianassidae genera into five major clades corresponding with the families (Staton and Felder 1995; Staton et al. 2000; Tudge et al. Axiidae (Axiopsis, Axius, Calocaris, Scytoleptus), 2000) and Ctenochelidae (Tudge et al. 2000). Laomediidae (Jaxea, Laomedia), Thalassinidae (Thalassina) In some cases, thalassinidean representatives have been and the two subfamilies, Callianassinae (, used as either ingroup or outgroup taxa for larger cladistic Callianidea, Glypturus) and Upogebiinae (Gebicula, analyses of various Decapoda; these include studies using ), of the Callianassidae. Later, Gurney (1938: 343) morphological characters by Martin and Abele (1986), used larval morphology to present an intuitive tree of Scholtz and Richter (1995) and Tudge ( 1997). Several

© CSIRO 2002 10.1 071/!S020 12 1445-5226/02/060839 840 C. C. Tudge and C. W. Cunningham

Cal li anassidae Upogebiidae Callianideidae

Anomura Axiidae Callianassidae Upogebiidae

(A) Pre-Homarine stern

Anomura (B)

Calocarididae

- Axiidae

'-- Strahlaxiidae AXJOIDEA

- Micheleidae

Thalassinidae I THALASS!NOIDEA - Laomediidae

'-- Upogebiidae Callianideidae - I CALLIANASSOIDEA '-- I Thomassiniidae

Ctenochelidae I I Callianassidae

(C) Fig. 1. (A) Genealogical relations between families in the Thalassinidea according to Borradaile (modified from Borradaile 1903). (B) Intuitive tree of relationships between some thalassinideans based on larval characters (modified from Gurney 1938). (C) Cladogram of phylogenetic relationships between II families of the Thalassinidea based on morphological characters (modified from Poore 1994). Note: the trees of both Borradaile and Poore have been modified to show taxonomic relationships at the family-level or higher. molecular phylogenetic analyses of various decapod Spears eta!. 1992; Crandall et al. 2000; Morrison et al. 2002; taxonomic groups have also included thalassinidean taxa for Perez-Losada et al. 2002). Similarly, the mitochondrial l6S comparison with anomurans (Spears and Abele 1988; Perez­ rDNA gene has been regularly used to investigate decapod Losada et al. 2002), astacideans (Crandall et al. 2000) and relationships (Cunningham et al. 1992; Crandall and -like decapods (Morrison et al. 2002). Also, see the Fitzpatrick 1996; Tam et al. 1996; Ovenden et al. 1997; synopsis of recent research into decapod phylogenetics by Kitaura et al. 1998; Tam and Kornfield 1998; Crandall et al. Schram (200 1) for a review of the different approaches and 1999, 2000; Schubart et al. 2000, 2001; Duffy et al. 2000; data sets being applied to the field. Morrison et al. 2002). Different regions of ribosomal genes The contribution of the small-subunit 18S ribosomal evolve at varying rates, making these useful molecular (r)DNA nuclear gene to phylogeny is well known markers across a broad taxonomic spectrum. and has been useful in investigating relationships across a This paper presents the results of a phylogenetic analysis wide variety of groups (Spears and Abele 1988, 1997, 1998; of partial sequences of mitochondrial 16S rDNA and nuclear Abele et al. 1989, 1992; Kim and Abele 1990; Abele 1991; 18S rDNA from 14 thalassinideans in five families Molecular phylogeny of the Thalassinidea S41

(Callianassidae, Laomediidae, Strahlaxiidae, Thalassinidae Materials and methods and Upogebiidae). Six other decapod taxa from the Specimen collection Astacidea, Palinura, Brachyura and Anomura are included in With the exception of the Raninoides specimen from 1991, for the analysis as outgroups. Similarities and differences to the genetic analysis were mostly collected over a 5-year period (I 995- 1999) from a variety of subtidal and intertidal locations previous evolutionary trees of the Thalassinidea, most worldwide (Table 1), but with an emphasis on coastal and the notably those of Borradaile (1903), Gurney (1938), Poore . The intertidal specimens were collected by ' yabby' pump, (1994) (Figs lA-C) and Tudge eta/. (2000), are discussed. digging or by hand and the subtidal specimens were trawled or dredged.

Table I. Specimens, collection, voucher and GenBank-sequence information Higher-level classification from Martin and Davis (200 I). Abbreviations: AMNH, American Museum of Natural History (New York); MY, Museum Victoria; Qld, ; USA, United States of America; USNM, National Museum ofNatural History (Washington DC); Vic, Victoria.

Astacidea Cambaridae (Girard, IS52). , USA, Jan. 199S. Voucher: AMNH 17874. GenBank sequences: AF436040 ( 16S), AF436001 (ISS) Palinura Palinuridae (Latreille, IS04). GenBank sequences: AF337966 (16S), U 191S2 (ISS) Scyllaridae Then us orienta/is (Lund, 1793). Gulf of Carpentaria, Qld, Australia, 1997. Voucher: not available Brachyura Raninidae Raninoides louisianensis Rathbun, 1933. Gulf of , USA, 1991. Voucher: T. Spears personal collection. GenBank sequences: AF436044 (16S), AF436005 (ISS) Anomura Aeglidae Aegla uruguayana Schmitt, 1942. San Antonio de Areco, Buenos Aires, Argentina, 10 Mar. 1996. Voucher: C. Cunningham personal collection. GenBank sequences: AF436051 (16S), AF436012 (ISS). Lithodidae Ciyptolithodes typicus Brandt, IS49. British Columbia, Canada. Voucher: S. Zaklan personal collection. GenBank sequences: AF425325 ( 16S), AF4360 19 (ISS) Thalassinidea Superfamily Thalassinoidea Thalassinidae Thalassina squamifera de Man, 1915. Town of 1770, Qld, Australia, 29 Dec. 1997. Voucher: MY J41662 Superfamily Callianassoidea Callianassidae Biffarius arenosus (Poore, 1975). Dunwich, Qld, Australia, 21 May 1997. Voucher: MY 140669 Biffarius delicatulus Rodrigues & Manning, 1992. Fort Pierce, , USA, 9 Jun. 1999. Voucher: USNM 309754 Callianassafllholi A. Milne Edwards, IS7S. Otago Harbour, New Zealand, 14 Nov. 1997. Voucher: MY J44SlS major (Say, ISIS). Isles Dernieres, Louisiana, USA, 27 Mar. 1996. Voucher: MY J39044. GenBank sequences: AF436041 ( 16S), AF436002 (ISS) Neocallichirus rathbunae (Schmitt, 1935). Fort Pierce, Florida, USA, S Jun. 1999. Voucher: USNM 309751 Neotrypaea californiensis (Dana, JS54). Marina del Rey, California, USA, 20 Jun. 1997. Voucher: A. Harvey personal collection. GenBank sequences: AF436042 ( 16S), AF436003 (ISS) Sergio mericeae Manning & Felder, 1995. Fort Pierce, Florida, USA, S Jun. 1999. Voucher: USNM 309755 Laomediidae Axianassa australis Rodrigues & Shimizu, 1992. Sao Sebastiao, Sao Paulo, , 20 Jul. 1997. Voucher: MY J44613 Jaxea nocturna Nardo, IS47. Loch Sween, Argyll, Scotland, 10 Nov. 1996. Voucher: MY 139045. GenBank sequences: AF436046 (16S), AF436006 (ISS) Laomedia healyi Yaldwyn & Wear, 1970. Western Port Bay, Vic., Australia, 16 Apr. 1997. Voucher: MY J40697 Upogebiidae Gebiacantha plantae (Sakai, 19S2). Yorke Island, Qld, Australia, 26 Feb. 199S. Voucher: MY J44914 Upogebia affinis (Say, ISIS). Fort Pierce, Florida, USA, 15 Nov. 1995. Voucher: MY J4066S. GenBank sequences: AF436047 (16S), AF436007 (ISS) Superfamily Axioidea Strahlaxiidae Neaxius glyptocercus von Martens, JS6S. Dunwich, Qld., Australia, 21 May 1997. Voucher: MY J39643 S42 C. C. Tudge and C. W. Cunningham

Molecular protocols 2058 bp. Our final alignments are available from the authors Tissue samples were preserved in 95% ethanol. DNA was isolated from (CWC). An ILD test (Farris et al. 1995) showed no fresh, frozen or ethanol-preserved specimens by grinding small significant incongruence (P > 0.05) and the two genes were fragments of muscle tissue in a buffer (0.1 M EDTA, 0.0 I M Tris pH 7.5, analysed separately and together. 1% SDS, Palumbi et al. 1991), followed by phenol-chloroform-isoamyl For both genes, the best-fit model found using ModelTest alcohol extraction and precipitation with 7.5 M ammonium acetate and cold isopropanol as described by Palumbi et al. ( 1991 ). Partial under maximum-likelihood was GTR + gamma + inv. sequences of mitochondrial 16S rDNA were amplified using rDNA Bootstrap consensus trees for both genes, analysed separately primers (LR-N-1339S, alias 16Sar, and LR-J-12SS7, alias 16 Sbr, for the maximum-likelihood bootstrap and Bayesian analyses, Simonet a/. 1994). Most of the nuclear ISS rDNA gene was amplified are presented in Fig. 2. There is weak support in the 18S using ISE-F (5'-CTGGTTGATCCTGCCAGT-3') and 1SSR3 (5'­ analysis for a monophyletic Thalassinidea (70% Bayes, 42% TAATGATCCTTCCGCAGGTT-3'). The amplification and sequencing of the two genes ( 16S and ISS) was identical (except where indicated) bootstrap). Although Bayesian analysis of the 16S gene and entailed the following regime. Polymerase chain reaction (PCR) showed rather strong support for a paraphyletic Thalassinidea was carried out on a Perkin Elmer GeneAmp PCR System 9600 (85%), there is little ML bootstrap support for a paraphyletic (www.pecorporation.com) using 50 11L reactions consisting of l 11L Thalassinidea (33%). The conflict between the 18S and 16S template DNA, 3 11L primers, 4.5 11L lO x reaction buffer, 5 11L Taq genes is not strong (also reflected by a non-significant ILD polymerase, 4 11L deoxynucleoside triphosphates, 5 11L MgCl2 and 24.5 11L water. Amplification involved 20 sec denaturation at 94°C, 1.5 min test ofP > 0.05) and they were combined for further analysis. annealing at 50°C (40°C for l6S) and 2.5 min of extension at 72°C for The ML analysis found a tree (Fig. 3) with Ln likelihood 35 cycles. The PCR product (3 11L) was visualised, via electrophoresis, - 8184.94 that weakly supports a paraphyletic on a 1% agarose gel using ethidium bromide staining. The remaining Thalassinidea (76% Bayes, 56% bootstrap). A monophyletic product (45 11L) was purified using the Wizard® PCR Preps Thalassinidea could not be rejected by an SH test. (www.promega.com) DNA Purification System. Applied Biosystems, Inc. (ABl) (www.appliedbiosystems.com) Prism BigDye Terminator Discussion Cycle Sequencing Ready Kits and an ABT Prism 3700 DNA Analyzer were used for sequencing. Sequences are available from the authors In these analyses, monophy1y of the Thalassinidea was (CWC). equivocal, being weakly supported by the 18S dataset only Sequence alignment and phylogenetic analysis (Fig. 2, left side) and not by the 16S dataset (Fig. 2, right side) Sequences were aligned using Clustal X (Thompson et al. 1994, 1997) or the combined analysis (Fig. 3). Poore (1994; Fig. I C) found with gap insertion and extension costs at I 0 and 5, respectively, and a monophyletic Thalassinidea based on two morphological regions of uncertain homology removed before phylogenetic analysis. synapomorphies (reduction of pleurobranchs on gills to seven Maximum-likelihood (ML) analyses using heuristic searches and TBR or less and presence of a setose lower margin on the propodus branch swapping were applied to the aligned sequences using PAUP* 4.0 (Swofford 200 I). For both maximum-likelihood and Bayesian and carpus ofpereopod 2), but a molecular study by Morrison analyses, we used the general-time-reversible (GTR) model to estimate et al. (2002) found strong support for non-monophyly of the the proportion of invariant sites and the alpha parameter of the gamma Thalassinidea (89% ML bootstrap support). The latter study distribution from the data (the best-fit model using ModelTest, Posada included more sequences ( 16S and 18S, as well as partial and Crandall 199S). Incongruence testing was carried out under the sequences of mitochondrial COil and nuclear 28S ribosomal parsimony criterion using the incongruence length difference (lLD) test (Farris et al. 1995), as implemented in PAUP* 4.0. DNA), but fewer thalassinidean taxa than the present study. The Bayesian analyses were performed using the program It is possible that the more rapidly evolving sequences used MRBAYES 1.11 (Huelsenbeck and Ronquist 200 I). MRBAYES uses a in the Morrison et al. (2002) study give a more reliable metropolis-coupled Markov chain Monte Carlo (MCMCMC) algorithm indication of relationships than just the two genes used in the to sample from the posterior distribution. Each Markov chain was present study. started from a random tree and run for 5 million cycles. The chain was sampled every 500 cycles in order to minimise the size of the output Between the Bayesian and the ML analyses there is no clear files and to ensure that these samples were independent. Four chains candidate for a sister-group to the Thalassinidea as a whole were run simultaneously with a 'temperature' of0.2. The initial40% of (Figs 2, 3). Interestingly though, the Bayesian analysis found cycles were discarded as burn-in and convergence was checked by strong (90%) support for a monophyletic sister-group to some ensuring that the likelihood values of sampled trees had approached a taxa in the Thalassinidea, including the spiny and slipper level distribution. Each analysis was performed twice to ensure that convergence was repeatable. lobsters (Panulirus and ), the anomurans (Aegla and Tests of alternative topologies were performed using the Cryptolithodes) and the brachyuran (Raninoides). Although Shimodaira- Hasegawa (SH) test (Shimodaira and Hasegawa 1999), this is intriguing, the weak support for this clade in which does not require that the hypotheses be designated a priori. bootstrapping (59%) suggests that further data may be Probability values were calculated in PAUP* 4.0 using 1000 RELL necessary to test this hypothesis. replicates to obtain a distribution. Within the Thalassinidea, we found two strongly supported Results major clades (Figs 2, 3). The first clade includes a After removing regions of uncertain alignment, our aligned monophyletic Callianassidae as the sister-group to a sequences consisted of 1731 base pairs (bp) of nuclear 18S representative in the axioid family, Strahlaxiidae (Neaxius rDNA and 327 bp of mitochondrial 16S rDNA for a total of glyptocercus ). Within the subfamily Callianassinae, the genus Molecular phylogeny of the Thalassinidea 843

18S 16S

Procambarus darldi

Panttlti·us argus 1001 I 85 100 I I 56 Thenus orienta/is 86 Cryptolitl10des typicus 22 73 100 I 941 47 1 Aeglauruguayana J?aninotdes /ouisitmensiJ

Upogebia a./finis 85 100 I 1100 33 100 I Gebiaamthapl antae I 95 100 100 hicmassa australis 97 Ja.xea nocturna 88 99 1001 1100 ~7 93 I Laomedia hea(vi j 97 70 83 42 Thnla.r.rtita squam(fi!ra NeartitJ' glyptocercus

100 100 I Sergio mericeae 100 99 I Neocallichirus rathbu11a 100 88 Callichirus major 100 93 79 100 1 Biflanits de!icatulus 64 88 1 Neo!IJ;paea californiel!si ! too 99 177 1 86 B(fjr1nit.r areno.m.r 60 100 I 1100 148 99 L CalliaJzaJ:rajilllO!i I 86

Fig. 2. A combined figure showing the opposed consensus trees for both Bayesian and maximum-likelihood analyses of the 18S data set (left side) and the 16S data set (right side). Percentage support for Bayesian posterior probabilities (upper) and maximum-likelihood bootstrap values (lower) are given for each node. Best-fit model = GTR +gamma + inv.

Biffarius is paraphyletic, with representatives of the genus monophyletic Callianassidae in this molecular analysis (Figs apparently grouping according to biogeography, uniting the 2, 3) is supported by at least 10 morphological characters (see American taxa Biffarius delicatulus and Neotrypaea family diagnosis in Poore 1994). In addition to Tudge et al. californiensis, and uniting the antipodean taxa, Biffarius (2000), a recent synopsis ofthe family Callianassidae has been arenosus and Callianassafilholi. These associations, although provided by Sakai (1999). His paper indicates a polyphyletic at first appearing enigmatic, should be viewed in light of family, extensively synonymises members of the comments by Tudge et al. (2000) that generic relationships Callianassoidea and has been considered of limited value in within the family Callianassidae should be treated with some helping to elucidate the relationships in this group of caution pending a re-diagnosis of the inclusive taxa, the thalassinideans (Tudge et al. 2000; Poore 2000). subfamily Callianassinae and the nebulous genus Callianassa The large clade constituted by the Callianassidae and in particular. The apparent biogeographic associations Strahlaxiidae in this molecular analysis (Figs 2, 3) is also between the four species mentioned above are contradictory supported by six morphological synapomorphies. Three of to those proposed for the same taxa in the morphological these synapomorphies can be found in the analysis of Poore analysis (Tudge et al. 2000), where the two Biffarius species (1994), but are also shared by representatives in several other are included in a monophyletic clade (only 59% supported) thalassinoid families (Callianideidae, Ctenochelidae, and C. filholi and N. californiensis are in another smaller Micheleidae and Thomassiniidae) for which tissue was not monophyletic clade ( 100% supported). The same available for this molecular analysis. These characters are ( 1) morphological analysis by Tudge et al. (2000) grouped these the presence of dense tufts of setae on abdominal somites 3- 5 four callianassine taxa into a monophyletic clade and indicated (2) the absence ofthe appendix intern a on male pleopod 1 and a comparable lack of resolution between this clade and the (3) a similar absence on male pleopod 2. Three additional callichirine taxa, Callichirus, Sergio and Neocallichirus. The morphological synapomorphies were found to support the 844 C. C. Tudge and C. W. Cunningham

r------Procambant.r darkti

,------Ranitzoides /ouisianenst:r Aegla Ltruguayana 90 100 59 97 Cryptolithodes (YpicLtY 100 Panulirus argus 100 T!temts orienrafi.s l/pogebiidae 100 Upogebia qffinis 100 Gebiacantha plantae Axianassidae .------Axianas.ra attsfrafl:r

Jaxea IIOC!llrlla Laomedia heaf.yi Strahlaxtidae .------Neaxi11sg/yp!ocercus

r------Ca!licltims mqjor 100 100 100 Sergio mericeae ~~ tOO 99 Neocallidnius rathbtmae Calliallassidae 90 Biffanits delicatu!tts Neotrypaea californiensis ·~~ Callimtassinae 85 Biffanils arenosuJ· Ca/lianassajil!toli ~~

Fig. 3. A composite of the consensus trees obtained from the Bayesian analysis and the single maximum-likelihood tree for the combined 16S and ISS data sets. Percentage support for Bayesian posterior probabilities (upper) and maximum-likelihood bootstrap values (lower) are given for each node. Best-fit model = GTR + gamma+ inv. Some higher taxonomic categories are provided on specific clades and the six outgroup taxa are indicated. Decapod images are modified from (top to bottom): Hobbs 1981 ; Rathbun 1937; Jara and Palacios 1999; Bliss 1982; Holthuis 1991 ; Rodrigues and Shimizu 1992; Sakai 1992; Yaldwyn and Wear 1970; Kensley eta!. 2000; Felder and Felgenhauer 1993; Hart 1982; Holthuis 1991. Note: images are not necessarily the same species as used in the analysis, but are representative of the genus, or a closely related genus. association of the Callianassidae with the Strahlaxiidae in an inclusion of Thalassina. This latter, alternative hypothesis, unpublished analysis by the senior author, which did not use although lacking any convincing morphological support (see the exact corresponding taxa. These characters are ( 4) a below), could not be rejected by an SH test of the molecular chelate pereopod 2 with the dactylus as long as the fixed finger data. The of the laomediids, suggested in the ( 5) the absence ofspiniform setae on the dactylus ofpereopods present analysis, resurrects an interesting issue on the 3 and 4 and ( 6) a row of lateral plumose setae on abdominal validity of the monotypic thalassinidean family, somite 2. Axianassidae Schmitt, 1924. Kensley and Heard (1990) The second major clade in Fig. 2 (left side only) and Fig. 3 succinctly summarised the history of the debate on whether includes three genera currently in the Laomediidae (Jaxea, the Axianassidae is a valid family in the Thalassinidea, and Laomedia, Axianassa), as well as representatives of the listed the supporters of the Axianassidae (Gurney 193 8; Thalassinidae and Upogebiidae. Within this clade, the Wear and Yaldwyn 1966; Goy and Provenzano 1979; Poore placement of the thalassinid, Thalassina squamifera, makes and Griffin 1979) and those who believe the genus the Laomediidae paraphyletic, or monophyletic with the Axianassa is one of five in the Laomediidae (de Man 1928; Molecular phylogeny of the Thalassinidea 845

Balss 1957; Le Loeuffand Intes 1974; Ngoc-Ho 1981). Our the 18S gene sequence data (Fig. 2, left side) and data support retention of the family Axianassidae for the unsupported by the 16S data (Fig. 2, right side) and the genus Axianassa, with Jaxea and Laomedia remaining in the combined analysis of both genes (Fig. 3). Borradaile (1903), Laomediidae. As well as the molecular evidence for a valid in his paper on thalassinidean classification, did not Axianassidae presented here (Figs 2, 3), we have identified implicitly state that the group is monophyletic, but inferred 10 morphological characters (six of them apomorphies) in this in his intuitive genealogical tree (Fig. lA) and Gurney which Axianassa differs from Jaxea and Laomedia. These (1938; Fig. lB) advocates paraphyly for the Thalassinidea he apomorphic characters are: (I) the linea thalassinica studied. Although a paraphyletic Thalassinidea has some displaced dorsally (possible autapomorphy); (2) absence of support in the present molecular analysis, the question of lateral lobes on abdominal somite 1; (3) linear epipods on the monophyly for this enigmatic group will require more gills; (4) reduction (or absence) of an exopod on maxilliped analysis of both morphological and molecular data sets to 3; (5) unequal chelae on pereopod I; and (6) dense tufts of resolve. lateral setae on abdominal somites 3- 5. A dichotomy within the Thalassinidea was previously The association of the Thalassinidae and the Laomediidae suggested by Poore (1994) based on morphological data, but (the 76% ML bootstrap supported node with Thalassina, neither of his major clades corresponds to ours. In contrast, Jaxea, and Laomedia in Fig. 3) is interesting, but lacks the dichotomy described by Gurney (1938; Fig. 1B) based on strong morphological support. In fact, only two larval characteristics is reminiscent of the relationships seen synapomorphies(?), the posterior margin of the carapace in our 16S tree and combined gene tree (Figs 2 (right side), with strong lateral lobes and both anterior and posterior teeth 3). This closer relationship of the thalassinidean families on the mandibular incisor, can be gleaned from the cladistic Laomediidae and Upogebiidae with the Anomura in analysis by Poore (1994 ). The former is shared with Gurney's proposed classification (Fig. 1B) is only weakly representatives of the Axioidea, whereas the latter character supported (under both Bayesian and ML analyses) here and is shared with members of the Thomassiniidae (once again the anomuran representatives are always part of a larger taxa missing from the current molecular analysis). Some decapod sister-clade (Figs 2, 3). Since our analysis only support, however, for this laomediid- thalassinid association, includes half of the families in the Thalassinidea, further is provided by studies of larval characters (Sankolli and sampling will be necessary to determine the composition of Shenoy 1979) and gill-cleaning structures and mechanisms these major clades, which appear with regularity in cladistic (Batang and Suzuki 1999; Batang et al. 2001 ). Although the analyses of this group. latter authors found gill-cleaning characters to be The three monophyletic superfamilies (Axioidea, conservative at the family level, there is still debate over their Thalassinoidea and Callianassoidea) indicated in the utility in phylogenetic studies (Poore 1994; Suzuki and classification of Poore (1994) are not maintained as McLay 1998). monophyletic entities in our analysis. In fact, in Poore's No unique morphological synapomorphies support the phylogeny (Fig. I C), these superfamilies are not adequately major monophyletic clade containing the Upogebiidae, represented by monophyletic clades at the same level either. Axianassidae, Thalassinidae and Laomediidae, seen in This discrepancy was noted by Martin and Davis (200 1), Figs 2, 3, even though there is strong molecular support in who followed Poore's revision in their recent work. Until both Bayesian and ML analyses. In the morphological better taxonomic congruence is achieved, and more of the analysis of Poore (1994; Fig. 1C), Upogebia, Thalassina and 11 families are represented in molecular analyses such as the Laomedia do not form a distinct clade, but three present study, direct comparisons will remain challenging. morphological characters variously ally these three taxa, which are part of a monophyletic clade (with other taxa) in Acknowledgments the current molecular analysis. These are (I) the rostrum This project was initiated when the senior author (CCT) was augmented with ridges (with the exception of the an Australian Research Council Postdoctoral Research Laomediidae), (2) a cylindrical carpus and propodus on Fellow (1996- 1999) at Museum Victoria, Melbourne, pereopod 1 (a symplesiomorphy shared with some axioids Australia, and the authors acknowledge the assistance of and some outgroup representatives) and (3) the loss or Gary Poore, Les Christidis, Janette Norman and Megan reduction of the male first pleopod (except in Thalassinidae) . Osborne from this institution for project support, access to This last morphological character is a possible laboratory facilities (CCT) and training in molecular synapomorphy shared with some callianassids (Poore 1994) techniques (CCT). The authors thank Cheryl Morrison, Bo or a symplesiomorphy also shared with some members of the Kong and Todd Oakley for technical support within the outgroup. Department of Zoology at Duke University. Also, Kristian In summary, the monophyly of the Thalassinidea Fauchald and Rafael Lemaitre (Department of Systematic suggested from a cladistic analysis of morphological Biology, National Museum of Natural History, Smithsonian characters (Poore 1994; Fig. 1 C) is only weakly supported by Institution) are thanked for supporting the Research 846 C. C. Tudge and C. W. Cunningham

Associate status of CCT during the completion of the Dworschak, P. C. (2000). Global diversity in the Thalassinidea project. The following colleagues are thanked for collecting, (Decapoda). Journal ofCrusta cean Biology 20 (Special number 2), assistance in collecting, or supplying specimens for this 238- 245. Farris, J. S., Kallersjo, M., Kluge, A. G., and Bult, C. (1995). Testing study: Katrin Berkenbush, David Brewer, Vania Coelho, significance of incongruence. Cladistics 10, 315- 319. Darryl Felder, Alan Harvey, Woody Lee, Rafael Lemaitre, Felder, D. L., and Felgenhauer, B. E. (1993). Morphology of the midgut­ Cheryl Morrison, Louise Nickel, Philip O'Neil, Gary Poore, hindgut juncture in the ghost shrimp Lepidophthalmus louisianensis John Salini, Jo Taylor and Stef Zaklan. The improvement of (Schmitt) (Crustacea: Decapoda: Thalassinidea). Acta Zoologica the manuscript by three anonymous reviewers is gratefully 74, 263- 276. Goy, J. W., and Provenzano, A. J. Jr. ( 1979). 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