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Peracarid monophyly and interordinal phylogeny inferred from nuclear small-subunit ribosomal DNA sequences (Crustacea: : ) Author(s): Trisha Spears, Ronald W. DeBry, Lawrence G. Abele, and Katarzyna Chodyla Source: Proceedings of the Biological Society of Washington, 118(1):117-157. 2005. Published By: Biological Society of Washington DOI: 10.2988/0006-324X(2005)118[117:PMAIPI]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.2988/0006- 324X%282005%29118%5B117%3APMAIPI%5D2.0.CO%3B2

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON 118(1):117±157. 2005. Peracarid monophyly and interordinal phylogeny inferred from nuclear small-subunit ribosomal DNA sequences (Crustacea: Malacostraca: Peracarida)

Trisha Spears, Ronald W. DeBry, Lawrence G. Abele, and Katarzyna Chodyla (TS, LGA, KC) Department of Biological Science, Florida State University, Tallahassee, Florida 32306-1100, U.S.A., [email protected], [email protected], [email protected] (RWD) Department of Biological Sciences, University of Cincinnati, P.O. Box 210006, Cincinnati, Ohio 45221-0006, U.S.A., [email protected]

Abstract.ÐPeracarids are a large group of malacostracan whose and phylogeny are uncertain. The present phylogenetic study of peracarids is, to our knowledge, the ®rst that includes full-length nuclear (n) small-subunit (SSU) ribosomal DNA (rDNA) sequences for representatives of every peracarid . Sequence length varied substantially (1807±2746 base pairs), and two variable regions (V4 and V7) contained long expansion seg- ments. Variable regions also exhibited signi®cantly greater heterogeneity in nucleotide frequencies than did core regions. Maximum-parsimony, maximum- likelihood, Bayesian, and distance phylogenetic estimates indicated a mono- phyletic Peracarida that excluded the and included the Thermosbaen- acea. This peracarid received strong support under Bayesian, maximum- parsimony, and distance analyses, but not maximum likelihood. Further, the thermosbaenacean does not occupy a position relative to other peracarids, as suggested by many morphology-based phylogenies. The phylo- genetic position of the Mysida within the Malacostraca remains uncertain, but a sister-group relationship between Mysida and (i.e., a mono- phyletic ) was consistently rejected, a result that also differs from those of most morphology-based phylogenies. High Bayesian clade-credibility support was obtained for an ( ϩ ϩ ) clade and a (Lophogastrida ϩ [ ϩ ]) clade. Within the pera- carid clade, internal branches were considerably shorter than terminal ones, so relationships among some peracarid lineages were equivocal. Data partitions corresponding to stem and loop regions in a secondary-structure model for nSSU rRNA had congruent phylogenetic signal but differed in nucleotide com- position and evolutionary model. Maximum parsimony and Bayesian phylo- genetic estimates based on the loop partition generally shared more nodes in trees inferred from combined data than did those based on stems, even though the stem partition had roughly twice as many characters.

Peracarids comprise roughly one-third of carids can be found in some of the most all crustaceans. The well over 21,000 de- remote regions (e.g., marine caves, ground- scribed are free-living, symbiotic, water, springs, and the deep sea). Support- or parasitic, and occur in marine, freshwa- ing this view is the observation that the ter, and even terrestrial . The current number of described peracarid species has accounting of peracarid species diversity is actually tripled in only the past 20 years. probably a gross underestimation, as pera- Peracarids were ®rst proposed as a su- 118 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON perorder within the class Malacostraca, sub- cently reviewed the historical debate about class , by Calman (1904), peracarid and phylogeny, and who united ®ve orders (Amphipoda, Cu- Richter & Scholtz (2001) also reviewed macea, Isopoda, Mysidacea, and Tanaida- pertinent issues concerning relationships cea) on the basis of several morphological among peracarids and other malacostracan features. Three new orders (, Spe- lineages. Monophyly for this superorder has laeogriphacea, and ) have been questioned as new orders have been been discovered since the mid-1950's, and discovered, and evolutionary relationships two orders (Mysida and Lophogastrida) are among the diverse peracarid lineages re- arguably suborders of the Mysidacea. Nine main poorly understood. Various morphol- orders of peracarids, at most, are currently ogy-based phylogenetic schemes (Fig. 1) recognized (Martin & Davis 2001; Appen- have been proposed, some of which are dix 1). computer-assisted cladistic analyses of mor- A short list of so-called peracarid fea- phological characters (e.g., Pires 1987, tures proposed by Calman (1909) includes Wagner 1994, Schram & Hof 1998, Wills a mandible with an articulated accessory 1998, Richter & Scholtz 2001), but little process (lacinia mobilis), a carapace (in phylogenetic consensus has emerged from most cases) that remains unfused with at these efforts. Watling (1999) attributed this least four of the posterior thoracic somites, lack of agreement to the immense dif®culty and a ®rst thoracic somite that is fused with of correctly assessing the homology of mor- the head. Peracarids also possess, uniquely phological features in peracarids, and he among crustaceans, a that is questioned the ef®cacy of performing cla- formed, in most cases, by oostegites de- distic analyses on seemingly homoplasious rived from thoracic epipods. Peracarids lack characters. More recent morphology-based a larval phase (e.g., nauplius or zoea), and cladistic studies have focused within spe- the young emerge from the brood pouch as ci®c peracarid lineages (e.g., Amphipoda: prejuveniles (i.e., mancas, in all but mysi- Vonk & Schram 2003, LoÈrz & Held 2004; daceans, thermosbaenaceans, and most am- Isopoda: Brandt & Poore 2003). phipods) that resemble adults to varying de- A glance at some of the alternative phy- grees. Unfortunately, synapomorphies for logenetic schemes (Fig. 1) illustrates major the Peracarida are rare because even the rel- points of contention. Of primary interest is atively few features suggested by Calman whether the Peracarida is monophyletic and are not applicable across all orders, and what orders are included within it. Some some of these features are found in non- (e.g., Monod 1924; Fig. 1c±g) consider the peracarids as well. For example, thermos- Thermosbaenacea to be a peracarid lineage; baeanaceans brood their young in a dorsal others place this in its own suborder, region that is formed from the carapace Pancarida (sensu Siewing 1958), that is sis- rather than in a ventral pouch formed from ter to the Peracarida (Fig. 1a, b, h), because oostegites as in most other peracarids. Fur- thermosbaenaceans have a different devel- ther, not all peracarids possess a carapace, opmental origin and location of the brood and the homology of this feature and many chamber. Controversy also exists as to others remains undetermined (see, e.g., whether the Mysidacea is a natural taxon Watling 1999). For these reasons, consid- and a peracarid lineage that includes mysids erable debate has surrounded the correct di- and lophogastrids (Fig. 1a±f, h), or whether agnosis of the superorder and its rightful mysids and lophogastrids are nonperacarid constituents, particularly since the discov- lineages that each merit ordinal status and ery of new orders. are more closely allied to the (Fig. Alternative taxonomic and phylogenetic 1g). scenarios.ÐHessler & Watling (1999) re- Taxonomic and phylogenetic uncertainty VOLUME 118, NUMBER 1 119 001). ) Siewing Fig. 1. Alternative phylogenetic hypotheses for selected extant malacostracan orders, with an emphasis on the Peracarida. Drawings modi®ed from (a (1963); (b) Pires (1987); (c) Wagner (1994); (d) Schram & Hof (1998); (e) Wheeler (1998); (f) Wills (1998); (g) Watling (1999); (h) Richter & Scholtz (2 120 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON also surrounds the enigmatic Spelaeogri- gate the control of body-segment organi- phacea and Mictacea, two taxa that are of- zation by the expression of the engrailed ten grouped together (Fig. 1b, c, h). A fairly segment-polarity gene (Scholtz et al. 1993, recent cladistic analysis (Shen et al. 1998) 1994) and homeotic, or Hox, gene expres- that included Spelaeogriphacea-like , sion (Panganiban et al. 1995, Averof & Pa- however, found the order Spelaeogriphacea tel 1997, and more generally, Schram & to be paraphyletic with respect to tanaids Koenemann 2004); studies of early embry- and cumaceans and not with mictaceans. onic cell lineages and cell fate (Dohle & Bowman et al. (1985), in their original di- Scholtz 1997; Gerberding & Scholtz 1999, agnosis of the Mictacea, noted similarities 2001; Sholtz 2000); and comparative ultra- of this order to mysidaceans, spelaeogri- tructural studies on the alimentary canal of phaceans, and thermosbaenaceans, and they malacostracans, including peracarids (e.g., intentionally selected the name Mictacea, Storch 1987, 1989; Oshel & Steele 1988; from the Greek ``miktos'' meaning mixed, Coleman 1990; Suh 1990; Ulrich et al. to highlight their observation that several 1991; De Jong & Casanova 1997a, 1997b; mictacean characters are found in other or- Kobusch 1998; Wallis & Macmillan 1998; ders of peracarids. Gutu (1998) later re- De Jong-Moreau et al. 2000; De Jong-Mo- moved halope Bowman & Iliffe, reau & Casanova 2001). 1985, from the Mictacea and placed it with More recently, various molecular ap- spelaeogriphaceans in a newly proposed proaches (primarily DNA sequencing) have peracarid order, Cosinzeneacea. That same been used to investigate questions about year, Gutu & Iliffe (1998) erected the Bo- systematics and within pera- chusacea, another new order of peracarids, carid orders (e.g., Amphipoda: France & to accommodate the Hirsutia (which Kocher 1996a, 1996b; Sherbakov et al. they removed from the Mictacea) and a 1999; Englisch & Koenemann 2001; En- newly described species Thetispelecaris re- glisch et al. 2003; LoÈrz & Held 2004; Cu- mix Gutu & Iliffe, 1998. Shortly thereafter, macea: Haye et al. 2004; Isopoda: Held Gutu (2001) proposed a sister-group rela- 2000; Michel-Salzat & Bouchon 2000; tionship between the Bochusacea and Tan- Dreyer & WaÈgele 2001, 2002; Mattern & aidacea. Here, we follow the classi®cation Schlegel 2001; Wetzer 2002; Lophogastri- of Martin & Davis (2001), which does not da: Casanova et al. 1998; Mysida: Meland recognize either the Cosinzeneacea or the & Willassen 2004; Mysidacea: Meland Bochusacea. 2003; Tanaidacea: Larsen 2001) and among Views also differ concerning the sister selected peracarids and other eumalacostra- group of peracarids (e.g., Thermosbaena- cans (Jarman et al. 2000). Lacking to date cea, Fig. 1a, b, h), the basal peracarid lin- is a molecularly based study of higher-level eage (e.g., either Mysidacea, Fig. 1c±f, h; malacostracan taxa that expressly focuses Amphipoda, Fig. 1a; or Isopoda, Fig. 1g), on peracarids and includes representatives and the of isopods (e.g., either of all nine putative peracarid orders. Amphipoda, Fig. 1c, d, f, or Tanaidacaea, The purpose of our study was to obtain Fig. 1a, b, h). Some have even proposed nucleotide-sequence data for the nuclear (n) (Nyland et al. 1987) that the Isopoda be re- small-subunit (SSU; also called 18S) ribo- moved from the Peracarida entirely. somal RNA (rRNA) gene (i.e., nSSU New and extremely promising avenues of rDNA) for representatives of all major mal- research are contributing to our understand- acostracan lineages and orders of peracarids ing of evolution and underlying homologies (Appendix 1) and to use a variety of phy- both in peracarids and in other malacostra- logenetic-inference methods on these data cans. Among these are studies in compar- (1) to assess peracarid monophyly (i.e., to ative developmental genetics that investi- determine which orders comprise a mono- VOLUME 118, NUMBER 1 121 phyletic peracarid clade, whether thermos- suborders (i.e., Amphipoda and Isopoda). baenaceans are included, whether the Mys- We omited the many additional species idacea are a monophyletic clade and wheth- from these suborders that are available in er they are included in the Peracarida or GenBank to keep the number of species to whether the Mysida and Lophogastrida are one that would permit computer-intensive in separate orders, either or both of which maximum-likelihood analyses. may be in the Peracarida) and (2) to infer Specimens were preserved in 95±100% phylogenetic relationships among peracarid ethanol for subsequent DNA extraction and orders. The inference of relationships with- kept at Ϫ80ЊC when possible. Voucher in orders of peracarids and among other specimens are maintained in the laboratory malacostracan lineages was not a major fo- of L. G. Abele, Department of Biological cus of this study, although some prelimi- Science, Florida State University, Tallahas- nary ®ndings are presented. We also com- see, Florida, with the exception of Spelaeo- pared phylogenetic estimates based sepa- griphus lepidops (in the laboratory of Les rately on stem and loop regions of the Watling, University of Maine, Darling Ma- nSSU rRNA molecule with those obtained rine Center, Walpole, Maine), and Thetis- for combined (i.e., stem ϩ loop) data, and pelecaris remex (in the laboratory of Thom- we explored, to some degree, the effect that as M. Iliffe, A&M University at Gal- stem and loop partitions with different char- veston, Texas). The authors acknowledge acteristics have on phylogenetic estimation the generosity of colleagues (Appendix 1) using rDNA data. who donated specimens, some of which are extremely rare and/or which required ex- Materials and Methods traordinary effort to collect. Molecular protocols.ÐGenomic DNA Selection of taxa.ÐSpecies included in was extracted from up to 0.5 g of muscle the present study are presented in Appendix tissue from live or ethanol-preserved ani- 1 according to the classi®cation of Martin mals. For extremely small specimens (e.g., & Davis (2001). Both molecular (Spears & thermosbaenaceans and mictaceans), either Abele 1999) and morphological (Richter & an entire individual or a substantial portion Scholtz 2001) studies support malacostra- of one was used in an extraction subsequent can monophyly, with the order to rinsing and sonication of the tissue in as a basal lineage. We therefore designated sterile distilled water to remove debris. Ex- the leptostracan sp. as the tractions were performed according to a in this study. At least one representative of phenol/chloroform/isoamyl alcohol proce- every malacostracan and peracarid order dure outlined by Kocher et al. (1989) or was included (Appendix 1), except for the with the G-NOME௡ DNA Kit (Q-BIOgene, syncarid order and the mono- Inc., Carlsbad, California) with a scaled- typic eucarid order Amphionidacea. Admit- down protocol (i.e., 500-␮l total volume). tedly, this sampling scheme fails to repre- After precipitation in 100% ethanol, DNA sent the vast species diversity among mal- pellets were washed with 70% ethanol, acostracans, but it was not our primary in- dried, suspended in 10±200 ␮l of sterile tent to discern relationships within this distilled water (depending on the amount of class of crustaceans, nor was it to infer re- starting tissue and the size of the DNA pel- lationships within the orders of peracarids. let), and stored at Ϫ20ЊC prior to gene am- We therefore included relatively few non- pli®cation. peracarids and at least one representative Standard protocols (see, e.g., Spears & from each of the nine putative orders of Abele 2000) were followed for (1) poly- peracarids. A few additional species were merase-chain-reaction (PCR) ampli®cation included for the more speciose peracarid (Saiki et al. 1988) of full-length nSSU 122 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON rDNA with a Perkin-Elmer DNA Thermal gions, which were mapped onto the multi- Cycler 480 (Perkin-Elmer, Foster City, Cal- ple alignment on the basis of Van de Peer ifornia); (2) puri®cation and cloning (when et al.'s (1997) model of nSSU rRNA sec- necessary) of PCR products; and (3) cycle ondary structure, and any necessary adjust- sequencing of ¯uorescently labeled DNA ments in the alignment were made by . templates with half reactions with the Ap- Portions of hypervariable and other ambig- plied Biosystems, Inc. (ABI; Foster City, uous regions in the alignment were omitted California) PRISM௡Big Dye௢ Terminator from subsequent phylogenetic analyses. We Ready Reaction Kit and a room-tempera- identi®ed the numbered nucleotide posi- ture ethanol/EDTA cleanup. Most peracar- tions corresponding to stem and loop data ids harbored endosymbionts that coextract- partitions by executing the alignment ®le in ed and coampli®ed when peracarid tissue the computer program PAUP* 4.0b10 and DNA were used, respectively; peracar- (Swofford 2002) and obtaining a data ma- id PCR products therefore required gel pu- trix of numbered characters. ri®cation and/or cloning before sequencing A partition-homogeneity test (also (Spears & Abele 2000). known as the incongruence-length-differ- We performed automated DNA sequenc- ence test; Farris et al. 1994, 1995) in ing using an ABI Model 373A Automated PAUP* (with the heuristic search option for Sequencing System (during the early phase 1000 replications and 10 random-sequence- of this study) and an ABI PRISM௡ 3100 addition replicates) assessed whether stem Genetic Analyzer (during the later phase). and loop partitions of the data contained Several brands of Taq polymerase (2.5 con¯icting phylogenetic signal. PAUP* Units) were used with equal success in 100- also yielded summary sequence statistics, ␮l PCR reactions to amplify both strands of such as uncorrected measures of sequence the entire nSSU rRNA gene, either as a sin- divergence, transition : transversion (Ti : Tv) gle PCR product or in overlapping frag- ratios between pairs of taxa (for assessment ments. Several DNA primers (available of saturation of transition substitutions; see, upon request) that are homologous to re- e.g., Knight & Mindell 1993), nucleotide gions dispersed throughout the nSSU rRNA frequencies, and tests for bias in nucleotide gene were used for both symmetric PCR frequencies across taxa. All partitions were ampli®cation and bidirectional sequencing examined for phylogenetic signal with the of double-stranded PCR products. test for skewness in a tree-length distribu- A consensus sequence was assembled for tion (Hillis & Huelsenbeck 1992) and the each species with Sequencher௢ 4.1. Appen- permutation-tail-probability test (Archie dix 1 lists the GenBank accession numbers 1989, Faith & Cranston 1991), both imple- of the sequences. mented in PAUP*. Sequence alignment and analysis.ÐA Phylogenetic analyses.ÐMaximum-par- multiple alignment of the nSSU rDNA se- simony (MP; Camin & Sokal 1965) analy- quences was generated by ClustalX 1.81 ses were performed on unordered and (Thompson et al. 1997) with a gap-opening equally weighted parsimony-informative penalty of 10.00, a gap-extension penalty of characters with the heuristic-search option 0.20, a 30% identity for delaying divergent in PAUP* for all three data partitions. Start- sequences, a transition weight of 1.00, and ing trees were obtained by random stepwise the ClustalW (Thompson et al. 1994) DNA- addition of sequences for 10,000 replicates weight matrix. The complete alignment is and with tree-bisection-reconnection (TBR) available from the ®rst author upon request. branch swapping on best trees only. The The secondary structure of a ribosomal option MULTREES was implemented; RNA molecule comprises double-stranded ``steepest descent'' was not implemented. (``stem'') and single-stranded (``loop'') re- Both delayed (DELTRAN) and accelerated VOLUME 118, NUMBER 1 123

(ACCTRAN) methods of mapping charac- partitions were homogeneous and combin- ter-state transformation on trees were used, able, but this test is susceptible to incorrect and the phylogenetic trees in both cases rejection or acceptance of congruence were compared on the basis of topology, among partitions under different conditions tree length, and consistency index (C.I.; (see, e.g., Hipp et al. 2004, and references Kluge & Farris 1969). MP estimates were therein). The computer program MrBayes similarly compared when alignment gaps 3.0b4 (Huelsenbeck & Ronquist 2002), were coded either as a ®fth character state which permits a simultaneous ML search or as missing data. on multiple data partitions with their re- We performed maximum-likelihood spective models of evolution, was therefore (ML; Felsenstein 1981) analyses on all un- used to investigate the effect, if any, of data ambiguously aligned characters using the partitions and model selection on phyloge- heuristic-search option in PAUP* with TBR netic inference. branch swapping and random stepwise ad- We conducted Bayesian analyses (see, dition of sequences to obtain starting trees e.g., Rannala & Yang 1996; Larget & Si- for 10 replicates. Modeltest version 3.06 mon 1999; Huelsenbeck et al. 2001, 2002) (Posada & Crandall 1998) with the Akaike using the Modeltest-generated model of Information Criterion (AIC) was used to se- evolution for (1) combined (``linked'') data, lect a model of nucleotide evolution (see, using both optimized and default initial set- e.g., Yang et al. 1994) resulting from hier- tings for model parameters (to determine archical log-likelihood-ratio tests for in- whether the speci®cation of optimized pa- creasingly complex models as outlined by rameter values improved Bayesian support Huelsenbeck & Crandall (1997) and start- for ); (2) ``unlinked'' stem and loop ing with a neighbor-joining (Saitou & Nei partitions, again using both optimized and 1987) topology that was inferred with Jukes default initial settings for model parameters & Cantor (1969) corrected distances. We (for the same reason); (3) stem and loop optimized model parameters for stem-, data separately, each without specifying pa- loop-, and combined-data partitions using a rameter values (to determine whether the successive-approximations approach (see, Bayesian consensus topologies based on the e.g., Swofford et al. 1996) implemented two data partitions were congruent with with PAUP*. Heterogeneity in nucleotide- each other and also with the Bayesian con- substitution rates among lineages was as- sensus topology for combined data and to sessed by a likelihood-ratio test (Felsenstein compare the clade-credibility values of 1981) comparing the best ML estimate of these topologies). MrBayes 3.0b4 (the most phylogeny (i.e., the one with the highest log current version) does not permit the user to likelihood, ln L) both with and without the specify correctly the proportion of invariant assumption of a molecular clock. sites for a given model of nucleotide sub- Because the ML method of phylogenetic stitution, so an unreleased beta version of inference as implemented in PAUP* uni- MrBayes, compiled by Fredrik Ronquist formly applies only a single model of nu- (Florida State University, Tallahassee, Flor- cleotide evolution (and its corresponding ida), was used to implement the cases parameter values) to a set of data, the ML above where parameter values had to be phylogeny estimated from the combined speci®ed. data partition according to one model of Negligible differences in clade support evolution may not be the most accurate re- were observed when either speci®c model- ¯ection of phylogeny if stem and loop par- parameter values or default settings were titions are best described by different mod- used. Bayesian analyses of separate stem els. The partition-homogeneity test was and loop partitions were therefore conduct- used to determine whether stem and loop ed with default parameter settings only. In 124 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON all cases, four Markov-Monte-Carlo chains bootstrapped pseudoreplicates (each with were run simultaneously for four million ®ve random-sequence-addition replicates) generations, and a sample tree was chosen was selected from the combined data and every 100 generations. Trees that were cho- used in heuristic searches. Heuristic search- sen once likelihood scores converged on a es of bootstrapped pseudoreplicates were stable value were used to create a 50%-ma- repeated under two different conditions so jority-rule consensus tree in PAUP*. In this that nodal support could be compared: once regard, a very conservative approach was with the optimized model-parameter values taken; trees chosen from the ®rst one mil- speci®ed a priori and a second time with lion generations were discarded as ``burn- estimated values. No signi®cant variation in in.'' clade support was found for these two cas- The minimum-evolution method using es; therefore, after stem and loop partitions LogDet- (or ``paralinear'') transformed dis- were bootstrapped separately, ML analyses tances (Lake 1994, Lockhart et al. 1994) were conducted with only the optimized pa- was implemented in PAUP* because this rameter values for each partition. Bayesian method is said to be less sensitive than MP clade-credibility values (P) for nodes in and ML to heterogeneity among taxa in nu- 50%-majority-rule consensus trees provided cleotide composition (although, unlike ML, an additional measure of support for phy- it presently cannot account for among-site logenetic relationships. heterogeneity in substitution rates). We Comparisons were made among optimal chose the value for the proportion of in- MP, ML, and several alternative phyloge- variant sites (I) that was optimized by ML netic hypotheses suggested by the peracarid using the successive approximations ap- literature (Fig. 1); we obtained the latter by proach. LogDet analyses were run with this implementing the ``enforce topological con- value of I under a variety of conditions straints'' option for a heuristic search in (e.g., removing I in proportion to nucleotide PAUP*. More general topological con- frequencies estimated from either all sites straints were used than the alternative phy- or constant sites only and both with and logenetic hypotheses shown in Fig. 1 (e.g., without adjustment for the mean number of isopods and tanaids would be constrained substitutions over all sites), after which to- to be sister taxa, but the positions of other pologies and tree scores were compared. taxa were allowed to vary). For MP, we im- These analyses were done for combined plemented Templeton's nonparametric test data (both with and without the inclusion of (Templeton 1983) and the Kishino-Hase- two long-branched taxa, Thetispelecaris re- gawa (K-H) parametric test (Kishino & mex and Tethysbaena argentarii), stem Hasegawa 1989) using PAUP*; for ML, we data, and loop data, so that comparisons implemented the one-tailed K-H test and could be made with other methods of phy- the nonparametric Shimodaira-Hasegawa logenetic estimation. (S-H) test (Shimodaira & Hasegawa 1999) Nodal support in MP trees was deter- (using RELL optimization with 1000 boot- mined from decay indices (i.e., the number strap pseudoreplicates), also using PAUP*. of character-state changes required to dis- solve a particular node; Bremer 1988, 1994) Results and nonparametric-bootstrap proportions (BP; Felsenstein 1985) with heuristic search Sequence and multiple-alignment analy- options described above with 1000 pseu- ses.ÐLength heterogeneity among the SSU doreplications for both MP (with 100 ran- sequences was substantial, ranging from dom-sequence-addition replicates) and dis- 1807 base pairs (bp) for three crustaceans tance analyses. For ML, which is more (the outgroup taxon Nebalia sp. and two computationally intensive, a sample of 200 peracarids, ingens and G. VOLUME 118, NUMBER 1 125 zoea) to 2746 bp for the valviferan isopod ders Mysida, Lophogastrida, and Mictacea metallica (Table 1). For all peracarid had the shortest V4 regions, and nonpera- crustaceans except the Mysida and Lopho- carid crustaceans and the Mysida and Lo- gastrida, nSSU rDNA sequences exceeded phogastrida had the shortest V7 regions. 1900 bp. All sequences were Amphipods were unique in having DNA in- similar in length for so-called ``core'' re- sertions in the V5 and V8 regions (Appen- gions of the SSU molecule (940±950 bp; dix 2). Table 1), presumably because of strong The average uncorrected sequence diver- functional constraints on evolution in this gence (calculated from data presented in region (see, e.g., Gerbi 1985). In contrast, Appendix 3) between orders of nonperacar- sequence length in ``variable'' regions of id malacostracans (excluding the outgroup the nSSU rRNA molecule showed a much Nebalia sp.) was relatively small and wider range (864±1803 bp; Table 1), and ranged from 3 to 5%. In contrast, the range an unambiguous alignment of data was dif- in average sequence divergence between or- ®cult and sometimes impossible for por- ders of peracarids (including the Mysida) tions of these regions. was much greater (9% between the Mysida Variable regions exhibited signi®cant and Isopoda and 30% between the spelaeo- heterogeneity in nucleotide frequencies (P griphacean Spelaeogriphus lepidops and the Ͻ 0.01; Table 1) compared to more homo- thermosbaenacean Tethysbaena argentarii). geneous core regions and contributed to the The average sequence divergence between signi®cant heterogeneity in nucleotide fre- the Mysida and nonperacarid taxa (exclud- quencies observed for the entire nSSU ing the outgroup Nebalia sp.) was only 6% rRNA molecule (P Ͻ 0.01; Table 1). This (range ϭ 4±8%), whereas that between the bias in nucleotide composition was re¯ect- Mysida and other peracarids was almost ed in the guanidine and cytosine content (% three times greater (17%). Spelaeogriphus G ϩ C) in different regions of the gene, lepidops was the most divergent taxon with variable regions having a narrower within the Peracarida and ranged from 27 range in % G ϩ C than core regions (Table to 30% in sequence divergence in pair-wise 1). The % G ϩ C content in variable re- comparisons with other taxa (Appendix 3). gions was not consistently higher for all Summary sequence statistics are shown taxa, however. For example, % G ϩ Cin in Table 2 for stem, loop, and combined variable regions was the same or lower than data used in ML and Bayesian analyses. that of core regions for the outgroup Ne- Nucleotide frequencies among these data balia sp., the three lophogastrid species, the partitions were homogeneous, although just Mysida, the mictacean Thetispelecaris re- barely for the combined data (P ϭ 0.06). In mex, and two of the three tanaids included contrast, all data partitions showed highly in this study. signi®cant heterogeneity in nucleotide fre- Comparisons of the sequence lengths of quencies when only parsimony-informative the eight variable regions (V1±V5 and V7± characters were considered (P Ͻ 0.05; Ta- V9) of the eukaryotic nSSU rRNA mole- ble 3). Nucleotide composition, re¯ected in cule revealed some interesting patterns (Ap- %Gϩ C content, was not uniform across pendix 2). The largest differences in se- data partitions and varied similarly whether quence length were con®ned to two so- all unambiguously aligned characters (Table called ``hypervariable'' regions (see, e.g., 2) or only parsimony-informative charac- Tautz et al. 1988; Hancock and Dover 1988, ters (Table 3) were considered; loop regions 1990), ranging from 226 to 879 bp and 86 had only 40% and 44% G ϩ C, respective- to 333 bp in the V4 and V7 regions, re- ly. spectively (Appendix 2). In general, non- Transition : transversion ratios for all peracarid crustaceans and the peracarid or- pairwise comparisons based on stem, loop, 126 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

Table 1.ÐLength (in nucleotides, nt) of the nuclear small-subunit (SSU) ribosomal RNA (rRNA) gene for crustaceans used in this study. Variable regions were identi®ed with the SSU secondary-structure model of Van de Peer et al. (1997). Classi®cation is according to Martin and Davis (2001). Percent G ϩ C content is given in parentheses (calculated with PAUP* [Swofford 2002] and with missing nucleotides deleted). 1 ϭ length approximate because of an indeterminate number of missing nucleotides; ␹2 ϭ chi-square test for homogeneity of nt frequencies; ** ϭ signi®cant P value at ␣ϭ0.01 with 78 degrees of freedom.

Taxon Entire SSU rRNA Core regions Variable regions

Leptostraca Nebalia sp. 1807 (51%) 943 (50%) 864 (48%) Stomatopoda Gonodactylus sp. 1818 (51%) 943 (50%) 875 (54%) empusa 1818 (51%) 943 (50%) 875 (54%) 1831 (51%) 943 (50%) 888 (53%) Spelaeogriphacea Spelaeogriphus lepidops 2046 (55%) 941 (53%) 1105 (58%) Thermosbaenacea Tethysbaena argentarii 2262 (53%) 940 (51%) 1322 (53%) Lophogastrida 1807 (46%) 941 (48%) 866 (43%) Gnathophausia zoea 1807 (46%) 941 (48%) 866 (44%) Eucopia sp. 1808 (46%) 941 (48%) 867 (44%) Mysida formosa 1817 (50%) 943 (50%) 874 (50%) integer 1816 (50%) 943 (51%) 873 (49%) Mictacea Thetispelecaris remex 1933 (50%) 941 (51%) 992 (49%) Amphipoda oceanicus 2261 (55%) 946 (53%) 1315 (56%) geometrica 2177 (53%) 950 (52%) 1227 (55%) sp. 2284 (51%) 948 (50%) 1336 (51%) Isopoda Paramphisopus palustris 2363 (54%) 945 (49%) 1418 (57%) racovitzai 2145 (52%) 944 (49%) 1201 (54%) 2746 (54%) 943 (49%) 1803 (57%) Tanaidacea dulongii 2002 (47%) 943 (50%) 1059 (44%) Paratanais malignus 2355 (47%) 942 (47%) 1413 (47%) Kalliapseudes sp. 2537 (54%) 942 (50%) 1595 (56%) Cumacea sculpta 2233 (54%) 940 (53%) 1293 (56%) Spilocuma salomani 20921 (52%) 9401 (50%) 11521 (55%) Euphausiacea Nyctiphanes simplex 1854 (51%) 943 (50%) 911 (53%) Meganyctiphanes norvegica 1853 (51%) 943 (50%) 910 (54%) Panulirus argus 1872 (51%) 942 (50%) 930 (53%) 1870 (51%) 943 (50%) 924 (53%) ␹2 (P) 0.00** 1.00 0.00** VOLUME 118, NUMBER 1 127 ␣ value at P ions. Characters loops]) based on ϩ 0.01** 704 314 678 0.06 1.121712** 1696 50 Ϫ signi®cant 1.26 (0.5±3.12) 0.24 (0.21±0.27) 0.28 (0.27±0.29) 0.22 (0.19±0.25) 0.26 (0.24±0.28) ϭ test statistic for skewness of a tree-length frequency ϭ 0.950073** 0.001** 1.00 1 597251 (35%) 101 (36%) 245 (32%) (36%) 40 0.25 (0.21±0.28) 0.21 (0.19±0.23) 0.19 (0.14±0.23) 0.35 (0.33±0.38) Ϫ 0.99 (0.00±2.57) transversions; g ϭ chi-square test for homogeneity of nt frequencies; ** ϭ 2 ␹ Stems Loops Combined transitions; Tv 453213 (64%) 433 (68%) (64%) 1.202141** 0.001** 0.90 1099 (65%) 55 0.24 (0.21±0.26) 0.31 (0.30±0.32) 0.24 (0.30±0.32) 0.21 (0.19±0.23) 1.59 (0.75±6.0) ϭ Ϫ permutation-tail-probability test (Archie 1989, Faith & Cranston 1991) for hierarchical structure with the heuristic- ϭ PTP ) P ( ) P ( 0.01 with 78 degrees of freedom. Table 2.ÐSummary statistics (obtained from PAUP* 4.0b10; Swofford 2002) for three data partitions (stems, loops, and combined [i.e., stems Adenine Cytosine Guanine Thymine 2 1 No. characters (% ofNo. combined) constant characters No. parsimony-uninformative variable characters No. parsimony-informative characters Mean nt frequency: ␹ PTP distribution (Hillis & Huelsenbeck 1992); search option and 1000 replications, each with 10 random sequence additions; a multiple alignment ofwere 27 used crustacean in nuclear maximum-likelihood, small-subunit ribosomal Bayesian, DNA and sequences distance after analyses. the Ti removal of ambiguously aligned nucleotide (nt) posit ϭ %GC Mean Ti : Tv ratiog (range) 128 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

Table 3.ÐSummary statistics (obtained with PAUP* 4.0b10; Swofford 2002) for parsimony-informative (P.I.) characters for three data partitions (stems, loops, and combined [i.e., stems ϩ loops]) based on a multiple alignment of 27 crustacean nuclear small-subunit ribosomal DNA sequences after the removal of ambiguously aligned nucleotide (nt) positions. Characters were used in maximum-parsimony analyses. Ti ϭ transitions; Tv ϭ ϭ transversions; g1 test statistic for skewness of a tree-length frequency distribution (Hillis & Huelsenbeck 1992); PTP ϭ permutation-tail-probability test (Archie 1989, Faith & Cranston 1991) for hierarchical structure with the heuristic-search option and 1000 replications, each with 10 random sequence additions; ␹2 ϭ chi-square test for homogeneity of nt frequencies; for chi-square test, * and ** ϭ signi®cant P value at ␣ϭ0.05 and 0.01, respectively, with 78 degrees of freedom.

Stems Loops Combined

# P.I. characters 433 245 678 Mean nt frequency Adenine 0.27 (0.21±0.34) 0.29 (0.20±0.35) 0.28 (0.22±0.30) Cytosine 0.26 (0.24±0.29) 0.20 (0.17±0.24) 0.24 (0.22±0.27) Guanine 0.26 (0.20±0.32) 0.24 (0.14±0.32) 0.25 (0.18±0.32) Thymine 0.21 (0.17±0.25) 0.27 (0.22±0.32) 0.23 (0.19±0.28) ␹2 (P) 0.0008** 0.02* 0.0000** %GC 52 44 49 Mean Ti : Tv ratio (range) 1.60 (0.75±16.00) 0.98 (0.00±2.57) 1.25 (0.50±3.00) Ϫ Ϫ Ϫ g1 1.84065** 0.944012** 1.093913** PTP (P) 0.001** 0.001** 0.001** and combined data averaged about 1.00 or of gaps as missing data or a ®fth character above (Tables 2, 3), suggesting that transi- state resulted in identical topologies; the tion substitutions were not saturated and consistency index and nodal support (i.e., that transitions and transversions could be bootstrap, or BP, values) were slightly high- weighted equally. Skewness tests and per- er when gaps were treated as a ®fth char- mutation-tail probability tests indicated sig- acter state. For simplicity, therefore, results ni®cant hierarchical structure for all three are reported for phylogenetic analyses per- data partitions (P Ͻ 0.01; Tables 2, 3), and formed on 678 parsimony-informative char- a partition-homogeneity test detected no acters (and stem and loop partitions of signi®cant phylogenetic incongruence be- these) with the DELTRAN option for char- tween stems and loops (P ϭ 0.456), sug- acter mapping and alignment gaps coded as gesting that these two partitions could be a ®fth character state. combined for subsequent phylogenetic Combined-data partition: The best MP analysis. Unequal stem and loop nucleotide estimate (Fig. 2a) resulted in a single tree composition (e.g., %G ϩ C, Table 2) and having 2796 steps, a C.I. of 0.4785, and a unequal nucleotide frequencies (e.g., Thy- retention index of 0.5357. The topology mine, Table 2) suggested, however, that nu- shows two major clades: clade B is a mono- cleotide substitution might proceed differ- phyletic Peracarida that excludes Mysida; ently in these two partitions. For these rea- clade A places the Mysida at the base of a sons, and because the partition-homogene- (Stomatopoda ϩ [ ϩ (Eucarida: ity test is not always an accurate method of Euphausiacea ϩ Decapoda)]) clade. The Is- assessing congruence among partitions (see, opoda are at the base of the peracarid clade, e.g., Hipp et al. 2004), phylogenetic anal- followed by the Tanaidacea, Cumacea, and yses were performed separately on stem the thermosbaenacean Tethysbaena argen- and loop partitions as well as on the com- tarii. A bifurcation follows next, resulting bined data. in a (Spelaeogriphacea ϩ Amphipoda) Maximum-parsimony (MP).ÐThe meth- clade and a (Mictacea ϩ Lophogastrida) od of character mapping and the treatment clade (Fig. 2a). Two trees one step longer VOLUME 118, NUMBER 1 129 00 lly 2796 steps; consistency index ϭ 50 based on an MP heuristic search of 1000 Ն 0.5357 (Farris 1989). Sidebars indicate orders of Crustacea. Node B indicates a monophyletic Peracarida clade as de®ned by the ϭ 0.4785; retention index Fig. 2. Phylogram (a) and (b) of the most parsimonious estimate of phylogeny for 27 crustaceans based on 678 parsimony-informative and equa weighted characters ofrandom nuclear sequence-addition small-subunit replicates. ribosomal Stemϭ DNA and by loop means data were of combined; PAUP* gaps 4.0b10 were (Swofford coded 2002). as The a heuristic-search ®fth character option state; was tree used length with 10,0 present study. In (b), numbers above branches indicate nonparametric-bootstrap (Felsenstein 1985) proportions pseudoreplicates, each with 100 random sequence additions; numbers below branches indicate decay indices (Bremer 1988, 1994). 130 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON than the MP result either switch the syn- carids. A sister-group relationship between carid Anaspides tasmaniae with the sto- the Isopoda and Amphipoda (as in Fig. 1c, matopod clade or place the Mysida as sister d, and f) was found in two trees that were to the peracarid clade. These differences are 49 steps longer than the best MP result indicated by a decay index of one for these (Figs. 2a, b), but these hypotheses were sig- nodes in Fig. 2b. Additional decay indices ni®cantly longer (P Ͻ 0.01) and rejected. Յ15 steps are also shown in Fig. 2b. Sup- In contrast, an Isopoda ϩ Tanaidacea clade port is strong (BP Ն 90; decay index ϭ 8; (as in Fig. 1a, b, and h) was found in three Fig. 2b) for a monophyletic Peracarida that trees that were only four steps longer than excludes Mysida but includes the thermos- the best MP tree, and this hypothesis was baenacean T. argentarii, so we reject the not signi®cantly worse (P Ն 0.25). A Spe- notion of a pancarid lineage apart from the laeogriphacea ϩ Mictacea clade (Fig. 1b, h, Peracarida. Similarly strong support (BP Ն and possibly c) was found in two trees that 90; decay indices Ն 10; Fig. 2b) is seen for were 23 steps longer than the MP tree, one individual stomatopod, decapod, euphausid, of which was signi®cantly worse (P ϭ mysid, lophogastrid, and amphipod clades. 0.04). A ``Bochusacea'' clade (sensu Gutu An isopod clade receives minimal (BP ϭ & Iliffe 1998) comprising the mictacean 68) and a tanaid clade slightly higher sup- Thetispelecaris remex ϩ Tanaidacea oc- port (BP ϭ 75), and yet both of these clades curred in a single tree that was also 23 steps have relatively high decay indices, of eight longer than the best MP tree, but this alter- and seven, respectively (Fig. 2b). A high native was rejected (P ϭ 0.05). A clade bootstrap proportion (94; Fig. 2b) is also comprising so-called ``relict'' cave-inhabit- seen for the sister-group relationship be- ing taxa (i.e., Spelaeogriphus lepidops ϩ tween the spelaeogriphacean Spelaeogri- Thetispelecaris remex ϩ Tethysbaena ar- phus lepidops and amphipods, but support gentarii) appeared in four trees that were is minimal for relationships among other 36 steps longer and signi®cantly worse (P peracarid orders and among nonperacarid Յ 0.01) than the MP tree. Finally, an MP malacostracans (e.g., BP Յ 75; decay in- search constrained for the best ML topolo- dices Յ 4; Fig. 2b). gy shown in Fig. 3a produced a tree that The branches are relatively long for tan- was signi®cantly longer (30 steps; P ϭ aids, the mictacean Thetispelecaris remex, 0.02) than the MP result. the basal amphipod Phronima sp., the spe- Stem-data partition: The MP analysis of laeogriphacean Spelaeogriphus lepidops, 433 P.I. characters produced nine equally the thermosbaenacean Tethysbaena argen- most-parsimonious trees (tree length ϭ tarii, both cumaceans (Spilocuma salomani 1647 steps; C.I. ϭ 0.4888; R.I. ϭ 0.2668). and Diastylus sculpta), and the branch lead- The strict-consensus topology (not shown) ing to the lophogastrid clade (Fig. 2a). In was similar to the best MP tree based on fact, most of the internal branches are short combined data (Fig. 2b); the two had 16 of compared to the terminal ones. Phylogenet- 25 nodes in common (10 of these within ic estimates may therefore re¯ect some un- the Peracarida, or clade B; Fig. 2b), but certainty (e.g., in the form of low nodal bootstrap support in the stem-based tree support) because synapomorphies for was lower for all but the Stomatopoda, De- clades are relatively few. capoda, Euphausiacea, Lophogastrida, and Tests of alternative phylogenetic hypoth- Amphipoda because of the fewer parsimo- eses suggested by Fig. 1 rejected (P Ͻ 0.05) ny-informative characters in the stem par- a monophyletic Mysidacea (ϭ Mysida ϩ tition. Other clades that were recovered in- Lophogastrida, as in Fig. 1a, b, c, e, f, and cluded the Eucarida, Tanaidacea, Amphi- h) but did not reject a sister-group relation- poda, Isopoda, Lophogastrida, Spelaeogri- ship between Mysida and remaining pera- phacea ϩ Amphipoda, and Thetispelecaris VOLUME 118, NUMBER 1 131

Table 4.ÐModels selected with Modeltest 3.1.0 (Posada and Crandall 1998) and optimized parameter values for stem, loop, and combined (stem ϩ loop) nuclear small-subunit ribosomal RNA partitions that were used in ML and Bayesian phylogenetic analyses for 27 crustaceans (Appendix 1). n ϭ number of unambiguously aligned nucleotide characters; GTR ϭ general-time-reversible model; TIM ϭ transition model; ⌫ϭgamma-distributed rates; I ϭ proportion of invariant characters; ␣ϭshape parameter for the gamma distribution of site-to-site variation; ⌸ϭnucleotide (A, C, G, T) frequency; R ϭ relative nucleotide-substitution rate.

Stems (n ϭ 1099) Loops (n ϭ 597) Combined (n ϭ 1696)

Model GTR ϩ⌫ϩI TIM ϩ⌫ϩI GTR ϩ⌫ϩI ln L Ϫ9340.20815 Ϫ5584.26694 Ϫ15034.30047 ⌸ A 0.23940 0.23939 0.24171 ⌸ C 0.30776 0.21413 0.27633 ⌸ G 0.24086 0.20087 0.22049 ⌸ T 0.21198 0.34562 0.26147 I 0.17241 0.29308 0.18268 ␣ 0.64814 0.81195 0.58821

RAC 0.86089 1.00000 0.74052

RAG 3.93819 3.48621 3.45876

RAT 1.19328 0.71170 0.86035

RCG 0.58694 0.71170 0.53632

RCT 2.93350 1.54514 1.97347

RGT 1.00000 1.00000 1.00000

remex (Mictacea) ϩ Lophogastrida, (see, tition had 43% fewer parsimony-informa- e.g., Fig. 2b). tive characters (Table 3). Loop-data partition: The MP analysis of Maximum likelihood (ML).ÐAnalyses 245 parsimony-informative characters pro- using the ML method of phylogeny esti- duced six equally most-parsimonious trees mation were based on 1696 aligned char- (tree length ϭ 1137 steps; C.I. ϭ 0.4688; acters (and stem and loop partitions of R.I. ϭ 0.2486). The strict-consensus topol- these) and used models of nucleotide sub- ogy (not shown) was similar to the best MP stitution selected by Modeltest with opti- tree based on combined data (Fig. 2b); the mized parameters (Table 4). two had 18 of 25 nodes in common (14 of Combined-data partition: Fig. 3a shows these within the Peracarida; node B; Fig. the best ML estimate (ln L ϭ 2b), but bootstrap support was lower in the Ϫ15034.30047) based on 1696 characters loop-based tree for all but the Euphausi- from combined stem and loop regions of acea, Lophogastrida, and Amphipoda be- the nSSU rRNA molecule with a general- cause of the relatively few parsimony-in- time-reversal (GTR) model of nucleotide formative characters in the loop partition. substitution (Lanave et al. 1984, Tavare The loop-based consensus topology had 1986, Rodriguez et al. 1990) accommodat- two more nodes in common with the MP ing unequal base frequencies, six types of topology for combined data than did the substitutions, gamma (⌫) distributed site-to- stem-based topology, and an MP analysis site variation in substitution rates, and a of bootstrapped loop data provided higher proportion of invariable (I) sites (i.e., GTR support (BP ϭ 80) for a monophyletic Per- ϩ⌫ϩI). Optimized parameter values are acarida (excluding Mysida) than did the provided in Table 4. A likelihood-ratio test stem data (BP ϭ 63). Further, the loop data comparing identical ML topologies esti- recovered more nodes (14) within the Per- mated with and without the assumption of acarida (i.e., node B; Fig. 2a) than did stem a molecular clock did not ®nd signi®cant data (10 nodes), even though the loop par- heterogeneity in nucleotide-substitution 132 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON ruent 50%- monophyletic 50 based on an and implemented Ն model using PAUP* 4.0b10 (Swofford I ϩ⌫ϩ 15034.30047) estimate of phylogeny for 27 crustaceans based on 1696 equally ϭϪ L Fig. 3. Phylograms showing the best maximum-likelihood (ML; ln weighted characters (stem and loop data combined) of nuclear small-subunit ribosomal DNA assuming a GTR 2002). The heuristic-search option was used with 10 random sequence-addition replicates. Sidebars indicate orders of Crustacea. Node B indicates a Peracarida clade as de®nedmajority-rule by consensus of the 30,001 present treesin when study. default MrBayes In parameter (b), 3.0b4 settings were the (Huelsenbeck used pair with & stem of Ronquist and numbers loop 2002); above partitions numbers (®rst) each linked below branch and (second) branches indicates unlinked Bayesian indicate posterior nonparametric probabilities bootstrap for (Felsenstein the 1985) cong proportions ML heuristic search of 200 pseudoreplicates, each with ®ve random sequence additions. VOLUME 118, NUMBER 1 133 rates among lineages [␭ϭϪ2 1978), so the combined data from MP and (Ϫ15038.39952 ϩ 15034.30047); ␭ϭ ML were reanalyzed after the sequences for 8.1981; ␣Ͻ0.01, 25 degrees of freedom). these two species were deleted. The result- For simplicity, therefore, only the results ing MP (length ϭ 2359 steps; C.I ϭ 0.5210; obtained without assumption of a molecular R.I. ϭ 0.3053) and ML topologies (ln L ϭ clock are presented. Ϫ13464.64692) (not shown) were more The best ML phylogeny (Fig. 3a) had 20/ congruent with respect to peracarid rela- 25 nodes in common with the MP tree, the tionships than were those in which the two most notable of which were (1) node B (in- taxa were included. In general, the Peracar- dicating a monophyletic Peracarida that ex- ida contained a (Lophogastrida ϩ (Spelaeo- cludes the Mysida), (2) node A (placing the griphacea ϩ Amphipoda) clade and a less- Mysida at the base of a [Stomatopoda ϩ resolved (Isopoda ϩ Cumacea ϩ Tanaida- (Syncarida ϩ [Euphausiacea ϩ Decapoda])] cea) clade (i.e., clades C and D, respective- clade), and (3) the (Spelaeogriphacea ϩ ly; Fig. 3b), and bootstrap support for Amphipoda) clade. Node B bifurcates into peracarid monophyly increased from 90 to (1) a Lophogastrida ϩ (Spelaeogriphacea ϩ 96 in the MP bootstrap-consensus tree once Amphipoda) clade (node C) having the the two problematic taxa were deleted. thermosbaenacean Tethysbaena argentarii Stem-data partition: Stem data were best as its sister taxon, and (2) a Tanaidacea ϩ represented by a GTR ϩ⌫ϩI model of (Isopoda ϩ Cumacea) clade (node D) hav- nucleotide substitution (the same as for the ing the mictacean Thetispelecaris remex as combined partition) for 1099 unambiguous- its sister taxon. These relationships do not, ly aligned characters with optimized param- however, receive strong bootstrap support eter values shown in Table 4. The best ML (Fig. 3b), and none of the alternative hy- estimate (ln L ϭϪ9340.20895; topology potheses described above for MP was re- not shown) did not recover a monophyletic jected (P Ն 0.06) by either the K-H or the peracarid clade, and differed most notably S-H test. The MP topology (Figs. 2a, b), from the best ML estimate for combined however, was signi®cantly worse (P ϭ data in (1) removing the mictacean Thetis- 0.02) than the ML tree by the K-H test only. pelecaris remex from the Peracarida and Because the uniform application of speci®c having it branch off the tree ®rst, after the model-parameter values to bootstrapped outgroup Nebalia sp., and (2) disrupting the pseudoreplicates may have contributed to Eucarida clade by placing the euphausids the lower nodal support in ML than in MP Nyctiphanes simplex and Meganyctiphanes trees, ML analyses were repeated on 100 norvegica as sister taxa to the Stomatopoda bootstrapped pseudoreplicates with the and not the Decapoda. Therefore, the only GTR ϩ⌫ϩI model and unspeci®ed mod- ``major'' node recovered from the smaller el-parameter values, but this approach did set of stem data was node D (Fig. 3b), and not improve BP values for any nodes. the stem-based ML tree had only nine of 16 Upon closer inspection, much of the dis- nodes within the peracarid clade in com- crepancy between the best MP and ML es- mon with the best ML estimate based on timates of phylogeny (Figs. 2, 3) could be the combined partition. Among these, how- accounted for by different positions within ever, was the Spelaeogriphacea ϩ Amphi- the Peracarida of the mictacean Thetispe- poda clade. lecaris remex and the thermosbaenacean Loop-data partition: Loop data were best Tethysbaena argentarii, two species with represented by a transition (TIM) model of relatively long branches in both trees. nucleotide substitution (a more restricted Long-branched taxa may have a destabiliz- model than GTR having only four substi- ing effect on phylogenetic inference, partic- tution types) with gamma distributed rates ularly under MP (see, e.g., Felsenstein (⌫) and a proportion of invariant sites (I) 134 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

(i.e., TIM ϩ⌫ϩI) for 597 unambiguously Combined data, with stem and loop par- aligned characters with optimized parame- titions unlinked: The topology of the 50%- ter values shown in Table 4. In contrast to majority-rule consensus of 30,001 trees the ML tree based only on stem data, the when stem and loop partitions were un- best ML estimate (ln L ϭϪ5584.26694; to- linked was highly congruent with that ob- pology not shown) for loop data did recover tained when these partitions were linked eucarid and peracarid clades, but the loop- (e.g., nodes A, B, C, and D appeared in based ML tree recovered fewer than half of both consensus topologies; Fig. 3b), except the 16 peracarid nodes seen in the com- that, in the unlinked analysis, the P values bined-data tree. The ML tree based on loop for clade B (peracarid monophyly) and sev- data alone also differed in two notable ways eral nodes therein were slightly lower. In from those based on stem or combined data: contrast, support for clade A (Mysida ϩ (1) the sister-group relationship between the Stomatopoda ϩ Syncarida ϩ Eucarida) and spelaeogriphacean Spelaeogriphus lepidops relationships therein were slightly higher. and the Amphipoda was disrupted (S. lepi- A Bayesian analysis performed after the dops was placed within the Tanaidacea, deletion of Thetispelecaris remex and Teth- making the latter paraphyletic) and (2) the ysbaena argentarii, as for MP and ML, cumacean clade was placed within the Is- yielded a consensus topology (not shown) opoda, making isopods paraphyletic. similar to that in Fig. 3b; i.e., peracarids Bayesian analyses.ÐBayesian analyses were monophyletic (P ϭ 1.00) and con- were performed on stem, loop, and com- tained clades C and D, each with P ϭ 0.71. bined partitions. Differences in clade cred- Eucarid monophyly was supported (P ϭ ibility values (P) were negligible for nodes 0.92), but relationships among the major in 50%-majority-rule-consensus trees malacostracan lineages were unresolved. whether model-parameter values for a given Stem-data partition: A Bayesian analysis data partition were speci®ed or not. Results of the 1099 characters in the stem partition are therefore presented for Bayesian anal- was as uninformative as the ML analysis. yses that were conducted when the default None of the major peracarid nodes in the settings were used. Bayesian consensus tree for combined data Combined data, with stem and loop par- (Fig. 3b) was recovered with P Ͼ 0.50 titions linked: Figure 3b shows the clade- when only stem data were used, nor was credibility values for nodes in the 50%-ma- the eucarid clade recovered. Instead, it pro- jority-rule consensus of 30,001 trees result- duced a major polytomy of crustacean or- ing from a Bayesian analysis and the GTR ders, except for the improbable pairing of ϩ⌫ϩI model (i.e., the same model used the Stomatopoda ϩ Euphausiacea (seen in the ML analysis of combined data) for also in the ML analysis based only on stem 1696 nucleotide characters. The Bayesian data), and this clade received surprising consensus topology was similar to MP and support (P ϭ 0.94). ML trees in excluding the Mysida from a Loop-data partition: Similar to the ML monophyletic Peracarida, which received estimate obtained with only loop data (597 signi®cant support (P ϭ 0.97; node B; Fig. characters), a Bayesian analysis (consensus 3b). The Bayesian and ML trees (Fig. 3b) topology not shown) supported peracarid were even more similar and had nodes C monophyly, and the credibility value for and D within the Peracarida in common. this clade (P ϭ 0.92) was comparable to These nodes received very high P values that seen with the combined data (see, e.g., (Ͼ0.90; Fig. 3b), and relationships within Fig. 3b, node B). Clade-credibility values node D (Lophogastrida ϩ [Spelaeogripha- were lower, however, for relationships with- cea ϩ Amphipoda]) were identical under in the peracarid clade and for those among these two methods of phylogeny estimation. nonperacarid orders. VOLUME 118, NUMBER 1 135

LogDet/paralinear distance analyses; shown) inferred from 1099 stem characters combined data.ÐTopologies were identical had 20/25 nodes in common with the com- regardless of the set of conditions under bined-data tree (Fig. 4a) and recovered which searches were conducted (i.e., nodes A and B. Relationships within clade whether the optimized proportion of invari- A were identical in the two cases, whereas ant sites [I ϭ 0.18268] was removed in pro- those within the peracarid clade differed portion to nucleotide frequencies estimated slightly, primarily because a rearrangement from all sites or only from constant sites, of Thetispelecaris remex disrupted clade C. both with and without adjustment for the The only sister-group relationship recov- mean number of substitutions over all ered between peracarid orders was the spe- sites). The resulting phylogram (minimum- laeogriphacean ϩ amphipod clade. evolution score ϭ 1.91057; Fig. 4a) shared Loop-data partition: The phylogeny (not the following with topologies estimated un- shown) inferred from 597 loop characters der MP (Figs. 2a, b), ML (Figs. 3a), and had slightly fewer (17/25) nodes in com- Bayesian analyses (Fig. 3b): (1) a mono- mon with the combined-data tree (Fig. 4a) phyletic peracarid clade that excluded mys- than did the stem-based distance estimate, ids but not lophogastrids (node B; Fig 4a); but it still recovered node B (indicative of (2) a clade comprising stomatopods, the peracarid monophyly). Node A (i.e., a clade syncarid, and eucarids, with mysids at the comprising mysids and nonperacarid mala- base (node A; Fig. 4a); (3) a (Lophogastrida costracans) was absent, as were eucarid and ϩ [Spelaeogriphacea ϩ Amphipoda]) clade tanaid clades, but the loop data did recover (node C; Fig. 4a); (4) a branching order that clade C (Lophogastrida ϩ [Spelaeogripha- was identical within amphipod, isopod, and cea ϩ Amphipoda]), a peracarid clade that tanaid clades; and (5) long branches for the was absent in the stem-based data. same peracarid taxa. The distance-based phylogeny differed Discussion most from the MP, ML, and Bayesian esti- mates with respect to relationships within Ours is the ®rst phylogenetic study of clade A; namely, the Eucarida clade was peracarids, to our knowledge, that includes disrupted, and stomatopods and not deca- full-length nSSU rDNA sequences for at pods were the sister taxa to euphausids. least one representative of every peracarid This relationship did not receive strong order, and from it we can conclude: (1) MP, bootstrap support (BP ϭ 54; Fig. 4b), how- ML, Bayesian, and distance analyses of the ever. Peracarid monophyly (clade B) re- combined (stem ϩ loop) data indicate that ceived higher support than under any other the superorder Peracarida is a monophyletic analytical method (BP ϭ 100; Fig. 4b). malacostracan lineage that includes the or- Support for clade C (BP ϭ 60; Fig. 4b) was ders Amphipoda, Cumacea, Isopoda, Lo- higher than in MP (Fig. 2b) but lower than phogastrida, Mictacea, Spelaeogriphacea, in Bayesian analyses (Fig. 3b), and support Tanaidacea, and Thermosbaenacea. (2) All was lacking for a (Tanaidacea ϩ [Isopoda phylogenetic estimates, regardless of meth- ϩ Cumacea]) clade that received high sup- od, refute monophyly of the Mysidacea; the port under Bayesian analyses (node D, Fig. order Mysida is excluded from the Peracar- 3b). The topology obtained when the long- ida, but its af®nity with nonperacarid mal- branched taxa Thetispelecaris remex and acostracans is equivocal (except in Bayes- Tethysbaena argentarii were deleted was ian analyses). (3) A fully resolved Peracar- completely congruent with that from the ida is lacking, in large part because of the original analysis when all taxa were includ- presence of short internal branches between ed. orders and much longer branches within the Stem-data partition: The phylogeny (not orders themselves. (4) ML and Bayesian 136 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON lsenstein es (Lake f Crustacea. 1.91057) of phylogeny for 27 crustaceans based on 1696 ϭ 0.18268 was removed in proportion to nucleotide frequencies estimated from constant sites while adjustment was made for ϭ I 50 based on a heuristic search of 1000 pseudoreplicates. Ն Fig. 4. Phylogram (a) and cladogram (b) showing the best estimate (minimum-evolution score Node B indicates a1985) monophyletic proportions Peracarida clade as de®ned by the present study. In (b), numbers above branches indicate nonparametric bootstrap (Fe the mean number of substitutions over all sites according to the heuristic-search option in PAUP* 4.0b10 (Swofford 2002). Sidebars indicate orders o equally weighted characters (stem and loop data combined) of nuclear small-subunit ribosomal DNA based on LogDet/paralinear-transformed distanc 1994, Lockhart et al. 1994) when VOLUME 118, NUMBER 1 137 analyses suggest two major clades within ceans within the Peracarida and refute the the Peracarida, one comprising (Lophogas- notion that they merit a separate eumala- trida ϩ [Spelaeogriphacea ϩ Amphipoda]) costracan superorder, Pancarida, as depicted and another comprising a less resolved (Is- in Fig. 1a, b, and h. The presence of ex- opoda ϩ Tanaidacea ϩ Cumacea); the (Spe- pansion segments (Table 1; Appendix 2) in laeogriphacea ϩ Amphipoda) clade is also the nSSU rRNA gene of Tethysbaena ar- supported by MP and distance analyses. (5) gentarii can also be viewed as evidence that The phylogenetic af®nities of the orders this species belongs in the Peracarida (see Thermosbaenacea and Mictacea with re- further discussion below), but we cannot re- spect to other peracarids remain uncertain. solve the phylogenetic position of T. argen- (6) Eumalacostraca, traditionally compris- tarii within the Peracarida. Thermosbaena- ing the suborders Eucarida, Syncarida, and ceans can be distinguished from other per- Peracarida, is not monophyletic and does acarids by the lack of oostegites and by the not include the Stomatopoda. Instead, the presence of a dorsal region for brooding Eucarida and Syncarida are sister taxa, their developing young that is formed from more related to the Mysida and Stomato- the carapace. In other peracarids, the brood poda than to the Peracarida. pouch, if present, is ventral and derived Molecules and morphology.ÐRichter & from oostegal plates, and our understanding Scholtz (2001) provide a concise and thor- of the evolution of this complex character ough summary, which we draw upon here, and its phylogenetic signi®cance is proba- of the issues relevant to malacostracan (in- bly incomplete. For example, Fryer (1964) cluding peracarid) evolution and of key thought the absence of the ``typical'' ventral morphological features associated with the peracarid brood pouch among sphaeromatid constituent groups. A bene®t of having a and epicarid isopods to be signi®cant, and molecularly based phylogenetic hypothesis Watling (1999) argued against the homol- is the ability to reevaluate hypotheses of ogy of oostegites and oostegal-plate for- morphological evolution. We do so below mation in peracarids. for some of the more contentious hypothe- Richter & Scholtz (2001) performed a ses and ``problematic'' taxa, but our efforts cladistic study of 93 characters for 19 mal- are hampered somewhat in two respects: (1) acostracans using parsimony and concluded the molecular estimate is not completely re- that thermosbaenaceans are the sister-group solved (i.e., not all nodes receive strong support), and (2) malacostracan and pera- of peracarids, citing embryological differ- carid phylogeny enjoy very little consensus ences (e.g., the number and arrangement of despite the vast body of literature on this ectoteloblasts). Some of their , topic, because different authors use differ- however, position thermosbaenaceans with- ent approaches to phylogeny reconstruction in the Peracarida. Frankly, few morpholog- and/or authors interpret characters differ- ical features ally thermosbaenaceans with ently. This latter point re¯ects concerns ex- peracarids. The most obvious is a lacinia pressed by Watling (1999) that the assess- mobilis in adults (see also Richter et al. ment of character homology has not re- 2002), but Richter & Scholtz (2001) also ceived suf®cient attention and that cladistic identi®ed a maxillipedal epipod that func- analyses of phylogenetically uninformative tions in ventilation (although Watling (not to be confused with parsimony unin- [1999] challenges the synapomorphic status formative) characters serve only to con- of this character, and this epipod is absent found our understanding of peracarid rela- in amphipods and mictaceans), the absence tionships. of epipods on thoracopods 2±8 (although Thermosbaenacea: Our ®ndings based on these are present in amphipods and lopho- nSSU rDNA clearly place thermosbaena- gastrids), paired entodermal plates, direct 138 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON development, and the presence of yolk on one that is not inferred from any explicit the posterior portion of the embryo. criteria of phylogeny reconstruction) that Mysida, Lophogastrida, and Eucarida: abandoned all previous notions of peracarid Questions concerning the phylogenetic af- monophyly. Watling (1983) proposed the ®nities among these taxa and their position removal of mysids and lophogastrids from within the Malacostraca have persisted for peracarids and their alliance with syncarids years. The Mysida and Lophogastrida are and eucarids, on the basis, in part, of fea- considered most frequently to be members tures of the carapace, compound eye, man- of a single order, the Mysidacea (Fig. 1 a± dible, blood system, and maxilliped. Mys- c, e, f, h), placed by Boas (1883) in the ids and lophogastids were in turn differen- Schizopoda along with the Euphausiacea tiated by more subtle differences in these (-like ``''). Calman's (1904, same characters. Calman (1904) also rec- 1909) revision of malacostracan classi®ca- ognized similarities (e.g., the ``caridoid fa- tion transferred mysidaceans to the super- cies'') among euphausids, lower decapods, order Peracarida and allied euphausids with mysids, and lophogastrids, but he did not decapods in the superorder Eucarida. Mys- use these to justify a close relationship idaceans as such have long been recognized among these taxa. as a ``connecting link'' between peracarids Watling (1999) revised his earlier view and other malacostracan groups (Siewing of peracarid evolution upon a critical reas- 1963: p. 99) or ``not too far removed from sessment of carapace, foregut, and oostegite the syncarid/eucarid ancestor of the Pera- homology, and a synthesis of evidence from carida'' (Dahl 1992: p. 344). Hence, this or- both morphology and developmental genet- der often appears in a basal position in per- ics. The resulting phylogenetic scheme re- acarid phylogenetic schemes (Fig. 1c, e, f, tained the more inclusive Eucarida and h). None of our best estimates of phylogeny placed mysids close to euphausids and (Figs. 2±4) supports mysidacean monophy- decapods (a view supported by Jarman et ly, however. Instead, all of our analyses de- al. [2000] on the basis of nuclear large-sub- ®nitively separate mysids and lophogastrids unit [nLSU] rDNA but not by our analyses and include only the latter within the Per- of nSSU rDNA, discussed further below). acarida. The Mysida, in contrast, are allied As shown in Fig. 1g, Watling (1999) pre- with eucarid, syncarid, and hoplocarid taxa, sents two major divisions within the Eu- albeit equivocally. malacostraca: a ``caridoid'' clade (Syncari- Among the features supporting the tra- da ϩ Lophogastrida ϩ Mysida ϩ Euphau- ditional view of mysidacean monophyly are siacea ϩ Decapoda), characterized by a nonrespiratory epipod on thoracopod 1 changes in the blood system and abdominal (Hessler 1983), the presence of lateral ex- musculature, and a second clade comprising tensions associated with bipartite cones of all remaining peracarids, characterized by the ommatidia (although bipartite cones are features associated with the mandibles, ce- seen in other peracarids), and the presence phalothoracic shield, and the manner by of a posterior tooth on the labrum (Richter which Hox-gene expression controls tho- & Scholtz 2001), but these latter characters racic-limb development. Our ®ndings (Figs. have been studied in relatively few pera- 2±4) do not support either of these two ma- carids. jor divisions, particularly with respect to the Our ®nding against mysidacean mono- alliance between lophogastrids and eucar- phyly is not entirely novel. Watling (1983) ids, and our ®ndings differ also with respect challenged the mysidacean af®nity with to relationships among the remaining pera- peracarids and completely revised eumala- carids in Watling's second clade. costracan classi®cation by proposing a We ®nd this phylogenetic con¯ict puz- ``pen and paper'' phylogenetic scheme (i.e., zling, especially considering the host of fea- VOLUME 118, NUMBER 1 139 tures cited by Watling in support of a close more primitive lineage. A preliminary relationship between lophogastrids, mysids, study (De Jong-Moreau et al. 2000) of the and eucarids. Watling's summary of euma- midgut and hepatopancreas of mysidaceans lacostracan carapace and foregut features also suggests monophyly for the group. (1999: tables I and II, respectively) does not Many of these early studies include rela- unequivocally justify the removal of lopho- tively few taxa, so claims of mysidacean gastrids or mysids from the Peracarida, monophyly on the basis of foregut mor- however. For example, a dorsal fold in the phology may be premature. These studies carapace is seen in lophogastrids, mysids, may indeed prove to be useful in elucidat- euphausiaceans, and dendrobranchiate ing phylogenetic relationships among per- shrimp, but it is also present in thermos- acarids and other malacostracans, but ad- baenaceans (Watling 1999: table I). Another ditional species must be studied for a better carapace feature, thoracic pleural folds, in- assessment of the distribution of foregut volves the ®rst thoracic somite in lopho- and midgut character states within and gastrids and mysids but also in thermos- among lineages. The same can be said for baenaceans and spelaeogriphaceans. Admit- preliminary yet promising studies on Hox- tedly, Watling makes a strong case for mov- and engrailed-gene expression, but the en- ing away from assessing relationships grailed studies on crustaceans to date solely on the basis of presence or absence (Scholtz et al. 1993, 1994; Scholtz 1995) of character states; he attempts to look for indicate that patterns of expression for this shared patterns of development for a given gene in mysids are more similar to patterns feature such as the carapace. Even the evo- observed in decapods than to those in am- lutionary pattern for carapace development phipods. proposed by Watling (1999: pp. 76±77) The summary phylogeny proposed by does not, however, preclude origin of two Richter & Scholtz (2001; Fig. 1h) and lines from a mysidacean-like ancestor, one based on a cladistic analysis of morpholog- leading to peracarids via lophogastrids and ical characters differs from Watling's another leading to eucarids via mysids. (1999) and ours in (1) retaining mysidacean Watling (1999) also cites aspects of fore- monophyly, (2) placing the Mysidacea gut morphology that distinguish mysids, eu- within the Peracarida, and (3) abolishing phausids, and decapods from thermosbaen- the Eucarida sensu stricto. In their scheme, aceans and other peracarids and place lo- decapods are basal to a ``Xenommacarida'' phogastrids in an intermediate position, but clade comprising syncarids, euphausids, his summary of foregut features (Watling pancarids, and peracarids and characterized 1999: table II) does not entirely substantiate by apomorphies associated with the om- this distinction. For example, euphausids matidia and thoracopods 2±8, but their (see, e.g., Suh 1990, Ullrich et al. 1991) character matrix (Richter & Scholtz 2001: lack an enlarged anterior foregut and cal- appendix) indicates that not all of these ci®ed teeth along dorsolateral ridges of the apomorphies are unambiguous, as the au- gastric mill, but these two features are pre- thors state. sent in mysids and decapods. In contrast, Isopoda, Cumacea, and Tanaidacea: All De Jong & Casanova's (1997a, 1997b) of our phylogenetic analyses place isopods comparative studies of the foregut suggest within the Peracarida, refuting Nyland et that lophogastrids, and not mysids, share al.'s (1987) opinion, based on myocardial more foregut features with decapods. A ultrastructure, that this order represents an more recent foregut study (De Jong-Moreau independent eumalacostracan lineage. Nor & Casanova 2001) supports mysidacean did any of our phylogenetic estimates group monophyly, however, placing lophogastrids isopods and amphipods as sister taxa (e.g., (speci®cally Gnathophausia gracilis) as the Schram's [1986] ``Edriophthalma''; see also 140 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

Schram & Hof 1998), although this rela- Our ®ndings consistently place amphi- tionship appears in many alternative hy- pods sister to spelaeogriphaceans (dis- potheses (Fig. 1c, d, and f). Instead, our cussed below), and these taxa occur with ®ndings invariably place isopods in a clade lophogastrids in a clade. This view is not with both tanaids (a relationship advocated consistent with the more recent phyloge- by several authorities; e.g., Fig. 1a, b, h) netic hypotheses set forth by Watling (1999; and cumaceans (similar to what was pro- Fig. 1g) and Richter & Scholtz (2001; Fig. posed by Siewing [1953, 1963] and similar 1h). Nor is it consistent with that of Poore to Watling's [1981] ``mancoid'' lineage, (in prep.), who con®rmed Wagner's (1984) which also included spelaeogriphaceans). cladogram (Fig. 1c) on the basis of parsi- Although support for this clade is strong in mony analyses of twice as many (93) mor- MP (Fig. 2b) and Bayesian (Fig. 3b) anal- phological characters. Watling (1999) yses, relationships within it remain equiv- placed amphipods in a basal position with ocal. Various characters have been ob- respect to other peracarids (excluding iso- served to support the Isopoda ϩ Tanaidacea pods) on the basis of the presence of a func- ϩ Cumacea relationship suggested by our tional maxillipedal epipod (even though the molecularly based phylogenetic estimates: epipod is lost in amphipods) and characters (1) a reduction of the oostegites upon molt- associated with the heart (which are not, ing after each brood (Siewing 1953); (2) a however, unambiguously apomorphic). reduced carapace and absence of epipods Richter & Scholtz (2001; Fig. 1h), in con- on thoracopods 2±8 (although this feature trast, place amphipods in a more derived is also seen in spelaeogriphaceans) (Hessler position with respect to other peracarids on 1983); and (3) a variable number of ecto- the basis of two unambiguous apomorphies: teloblasts (also seen in Mysida), a dorsally (1) a continuous anteroposterior degree of folded embryo, and similarities in midgut differentiation in the development of the formation and in the manca (Richter & segments bearing the ®rst and second an- Scholtz 2001). A close relationship among tennae and the mandibles from the nauplius these three taxa is also seen in one of Rich- to the postnauplius larva, and (2) the ab- ter & Scholtz's (2001) most parsimonious sence of a palp on the second maxilla. cladograms. Spelaeogriphacea: We ®nd no evidence Amphipoda: Consensus is good among cited in the morphological literature to sup- systematists that amphipods are a mono- port a Spelaeogriphacea ϩ Amphipoda phyletic clade within the Peracarida, and clade, yet this clade appeared consistently our molecularly based phylogenetic analy- in all of our phylogenetic estimates based ses (Figs. 2±4), including those with addi- on the nSSU rRNA data (Figs. 2±4). In- tional amphipod species (not shown), sub- stead, the Mictacea are proposed most often stantiate this view. We will therefore not re- as the sister group of spelaeogriphaceans view the several synapomorphies that char- (Fig. 1b, h, and possibly c). In support of acterize this clade (see, e.g., Dahl 1977). this latter view, Richter & Scholtz (2001) Additional evidence that amphipods are identi®ed two characters: the ®rst maxilli- peracarids is their having expanded nSSU ped lacking a palp and the absence of com- rRNA genes (Table 1), and evidence of pound . These two characters are hom- their monophyly is in the presence of ex- oplasious, however, with respect to am- pansion segments in the V5 and V8 regions phionids and bathynellaceans, respectively. of the nSSU rRNA molecule (Appendix 2), Future studies should include sequence data i.e., a molecular synapomorphy for this from additional species of spelaeogripha- clade. Considerable debate, however, has ceans to reveal whether the relationship surrounded the position of amphipods with- with amphipods persists, but we hope that in the Peracarida (Fig. 1). these preliminary ®ndings encourage fur- VOLUME 118, NUMBER 1 141 ther investigation into a morphological ex- exists!). Nevertheless, our preliminary re- planation for this intriguing relationship. sults suggest that a second novel order, Cos- The most recent morphology-based cladis- inzeneacea, erected by Gutu (1998) to in- tic study of peracarids (Poore, in prep.) re- clude the Spelaeogriphacea and Mictacea, veals no support for a spelaeogriphacean ϩ may not be a natural taxon, because of the amphipod relationship. We note, however, strong sister-group relationship between the that Richter & Scholtz (2001) identi®ed spelaeogriphacean Spelaeogriphus lepidops some character states shared by spelaeogri- and amphipods that appears in all of our phaceans and amphipods, although these phylogenetic estimates (Figs. 2±4). are symplesiomorphies and not synapomor- Syncarida: Complete understanding of phies. They include a second maxilla with the relationship between peracarids and oth- a vestigial or absent palp and the presence er malacostracans is somewhat hampered of yolk on the posterior portion of the em- by the lack of specimens suitable for mo- bryo. lecular study for rare but key taxa (e.g., the We can make some interesting observa- syncarid order Bathynellacea and the eu- tions, however, that taken together might carid order Amphionidacea). Among our suggest a possible evolutionary scenario molecularly based phylogenetic estimates, linking amphipods and spelaeogriphaceans: only Bayesian analyses provide support for (1) amphipods, although ecologically di- nodes delineating a mysid → hoplocarid → verse, are known from the same habitats to syncarid → eucarid branching order. Syn- which spelaeogriphaceans are restricted carids are an ancient lineage with a (groundwater and freshwater pools in record (see, e.g., Schram 1982, caves); (2) spelaeogriphaceans have an an- Briggs et al. 1993) and they are considered cient fossil record (discussed below), most often to be a somewhat isolated (Cal- whereas amphipods do not; and (3) amphi- man 1904) or primitive eumalacostracan pods are considered a highly derived pera- group. Dahl (1992), for example, cites the carid lineage with several autapomorphic absence of a dorsal fold in the carapace and features. However unlikely, it is perhaps thoracic morphology (with respect to ven- possible that amphipods arose from a spe- tilation) in syncarids as plesiomorphic fea- laeogriphacean-like ancestor that was once tures, and Oshel & Steele (1988) view the more widespread. syncarid foregut as plesiomorphic com- Mictacea, Bochuscaea, Cosinzeneacea: pared to that of eucarids and peracarids. Unfortunately, we cannot assess the validity Bayesian analyses of the molecular data of these three taxonomic categories, the support a close syncarid ϩ eucarid relation- species they comprise, or their phylogenetic ship, and Dahl (1992) notes a few features af®nities without additional sequence data shared by the Syncarida and Eucarida that from species of Mictocaris and Hirsutia. support this result, including naupliar eyes, Thetispelecaris remex has been classi®ed, antennular statocysts, and a proximal artic- along with Hirsutia, as a member of the ulation among thoracopods. Bochusacea (sensu Gutu & Iliffe 1998), a : The relationship of the Ho- novel taxonomic arrangement that has not plocarida (represented in the present study received much published scrutiny by sys- by the order Stomatopoda) with other mal- tematists. The more traditional view places acostracans has fostered considerable de- Hirsutia and Mictocaris within the Micta- bate as well (reviewed in Watling et al. cea. Because we follow the classi®cation of 2000), but two alternative views have gen- Martin & Davis (2001) here, we place T. erally prevailed. One considers hoplocarids remex within the order Mictacea (Appendix an independent offshoot that diverged early 1), but we acknowledge that this species is in malacostracan evolution (i.e., one that not a ``typical'' mictacean (if such a thing warrants its own subclass; see, e.g., Siewing 142 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

1963, Schram 1986, Martin & Davis 2001; lineage, the Phreatoicidea, is not basal to Fig. 1a, d, h). Another view considers the the more recent isopod families represented Hoplocarida a superorder within the sub- in our study (see Figs. 2a, 3a, 4a). Lopho- class Eumalacostraca, equivalent in rank to gastrids, on the other hand, have a relatively the Syncarida, Peracarida, and Eucarida. ancient () fossil record, and yet both Richter & Scholtz (2001), for example, lophogastrid families we included exhibit place stomatopods as the basal eumalacos- very short branches within their clade (Figs. tracan lineage and not as an independent 2a, 3a, 4a). Differences in evolutionary malacostracan subclass. In our study, only rates may therefore indeed exist among the Bayesian analyses of the molecular data taxa we used in this study, despite the like- permit an unequivocal assignment of sto- lihood-ratio test. matopods within the Malacostraca, but Highly divergent lineages may also be none of our phylogenetic estimates, regard- explained, in part, by a limited dispersal ca- less of the method used to generate them, pability (e.g., as for tube-dwelling tanaids places stomatopods basally with respect to and other deep-sea species) and/or patchy other ingroup malacostracans. distribution (e.g., the occurrence of spelaeo- Long branches.ÐLong branches appear griphaceans in isolated habitats in South in all peracarid orders except the Mysida America, South Africa, and Australia). (which we exclude from the Peracarida), Three orders, represented by Thetispelecar- the Lophogastrida, and the Isopoda. Long is remex, Tethysbaena argentarii, and Spe- branches are generally viewed as an indi- laeogriphus lepidops, all exhibit long cation that a lineage either has had an an- branches regardless of phylogenetic meth- cient divergence followed by an extended odology (Figs. 2a, 3a, 4a), but these never period of independent evolution or has an ``attract'' and form a clade in any of our accelerated rate of evolution. We can rule phylogenetic analyses, so they probably out the latter possibility because a likeli- represent remnants of quite different and hood-ratio test did not ®nd evidence of sig- ancient peracarid lineages that were once ni®cant heterogeneity in nucleotide-substi- more widespread. It has been suggested that tution rates among lineages for a given to- thermosbaenaceans (Wagner 1994) and spe- pology. Ancient divergences, in contrast, laeogriphaceans (Poore & Humphreys remain a likely explanation. For example, 2003) are Tethyan relicts that became the fossil record (Schram 1982, Briggs et stranded in groundwater as a result of ma- al. 1993) reveals that spelaeogriphaceans rine regression. Sequence data from addi- and tanaids extend to the and tional species in these three orders are need- that Permian cumaceans are not far behind, ed to assess this claim further and to estab- and all three of these lineages have rela- lish their af®nities with other peracarids. tively long branches in MP, ML, and dis- Long branches may be a source of sys- tance phylograms (Figs. 2a, 3a, 4a, respec- tematic error in phylogeny estimation (Fel- tively). The correspondence between line- senstein 1978, Anderson & Swofford age longevity (as indicated by the fossil re- 2004), particularly when parsimony is used. cord) and branch length is not upheld in all For example, clades having a single long cases, however. For example, fossil ther- branch may be misplaced and/or placed mosbaenaceans are known only from the more basally in a tree because synapomor- Quarternary, and amphipods only from the phies with their true sister group are eroded. mid-Tertiary, yet species from both groups This effect probably explains the lack of have relatively long branches in our trees. nodal support observed in Figs. 2a, 3a, and In contrast, the Isopoda, which are known 4a among many of the long-branched per- from the Carboniferous, have relatively acarid lineages. Not all of the long- short branches. Further, the oldest isopod branched peracarids are randomly attracting VOLUME 118, NUMBER 1 143 each other to produce spurious clades, how- neity in substitution rates) that could con- ever; i.e., many branches that are long, and found phylogenetic estimation when a sin- potentially could attract one another, do not gle model of evolution is used to describe (e.g., Thetispelecaris remex, Tethysbaena the combined data, as was done by the au- argentarii, and Spelaeogriphus lepidops, as thors. Another possible explanation for the discussed above). On the contrary, many discrepancy between the nuclear SSU and cases in which long branches do attract in LSU rDNA results might be differences in our phylograms (Figs. 2a, 3a, 4a) form taxonomic sampling. For example, the fo- quite ``sensible'' clades (e.g., the Amphi- cus of Jarman et al.'s (2000) study was eu- poda, Cumacea, and Tanaidacea clades). malacostracan taxa, so stomatopods were The presence of relatively short internal not included. The authors also included branches within the peracarid clade (node decapods from two families of dendrobran- B; Figs. 2a, 3a, 4a) is also potentially prob- chiate shrimp, whereas our study used se- lematic and can lower clade support. Our quences from a more derived decapod sub- ®ndings indicate, however, that certain order, the . These differences clades remain stable despite the short aside, some signi®cant phylogenetic simi- branches connecting their component taxa larity exists between the two studies, name- (e.g., the Isopoda and the larger Isopoda ϩ ly, that mysidacean monophyly is rejected Tanaidacea ϩ Cumacea clade; Figs. 2a, 3a, and that lophogastrids, not mysids, appear 4a). within a peracarid clade. Jarman et al. Comparisons among molecular stud- (2000) also suggested, as do we, close re- ies.ÐJarman et al. (2000) conducted MP lationships between amphipods and lopho- and ML analyses on partial nuclear large- gastrids and among isopods, cumaceans, subunit (nLSU) rDNA data to investigate and tanaids. eumalacostracan relationships, and their Some of our results are also consistent study included six of the nine putative per- with those of Meland's (2003) phylogenetic acarid orders. They found mysids to be the study of malacostracan lineages. Meland sister group of euphausids, and a syncarid likewise used nSSU rDNA but only for the was basal to this clade. This result contra- more variable V4±V7 regions of the SSU dicts ours, based on nSSU rDNA, that in- rRNA molecule. His study's primary focus dicated strong support for a monophyletic was relationships within the Mysida, so he Eucarida in which euphausids and decapods included only four of nine peracarid orders. are sister taxa and mysids are more basal. Nonetheless, both the Meland (2003) study The discrepancy between the two molecular and ours support the removal of the Mysida studies is perplexing, because nuclear SSU- from the Peracarida sensu stricto. and LSU rRNA genes are not truly inde- As stated above, our intent was not to pendent but are linked by the rRNA operon. infer relationships within peracarid orders, One might therefore expect them to yield but our ®nding that an asellote was ances- congruent gene trees, although the trees tral to a phreatoicid isopod, and that a val- might differ in nodal support. Indeed, nu- viferan was the most derived of the three, clear SSU and LSU rRNA partitions have agrees with the results of Dreyer and WaÈ- been combined in numerous phylogenetic gele (2002) and Meland (2003) for nSSU studies for diverse taxa, including crusta- rRNA and also with those of Wetzer (2002) ceans (e.g., Crandall et al. 2000, Oakley & based on mitochondrial 12S rDNA (al- Cunningham 2002). though this result changed when the author The stem and loop partitions of the nLSU explored the effects of different character rDNA data used in the Jarman et al. (2000) weighting and combining different gene study may have different characteristics partitions). (e.g., nucleotide composition or heteroge- Length heterogeneity and expansion seg- 144 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON ments.ÐThe presence of rapidly evolving nSSU rRNA genes can be clade speci®c expansion segments that contribute to ex- and phylogenetically informative. Expan- tremely long nSSU rRNA sequences has sions segments, when present, occur pri- been noted previously in peracarids but marily in the V4 and V7 regions of the mol- only for two of the nine putative orders (Is- ecule (Appendix 2). The one exception we opoda: Choe et al. 1999; Held 2000; Dreyer found was among amphipods, which pos- &WaÈgele 2001, 2002; Mattern & Schlegel sessed in addition expanded V5 and V8 re- 2001; Meland 2003; Amphipoda: Meland gions and which we interpret as a synapo- 2003). The present study adds representa- morphy for this group. tives from the Cumacea, Mictacea, Spelaeo- Jarman et al. (2000) mention that some griphacea, Tanaidacea, and Thermosbaena- of their partial nLSU rDNA eumalacostra- cea to the list of peracarid orders with ex- can sequences had expansion segments, but panded nSSU rRNA genes (Table 1; Ap- they did not provide details of how these pendix 2). were distributed among the taxa they se- The reason why expansion segments are quenced. found in the nSSU rRNA genes of some Our observation that mysids and lopho- taxa and not others is unknown, but our re- gastrids lack expansion segments (Table 1) sults (not shown) from PCR ampli®cation might be taken as evidence of evolutionary and DNA sequencing in this study indicate relatedness and viewed as justi®cation for a a correspondence between peracarid speci- monophyletic Mysidacea, but all of our mens that have an expanded nSSU rRNA phylogenetic analyses (Figs. 2±4) of mala- gene and the presence of an endosymbiont. costracan nSSU rDNA suggest otherwise A similar correspondence between expand- by conclusively separating these two taxa ed nSSU rRNA genes and endosymbionts and positioning the Lophogastrida, and not has been observed for certain branchiopod the Mysida, within the Peracarida sensu lineages (e.g., cyclestherid conchostracans stricto. Clearly, the absence of expansion and cladocerans; Spears & Abele 2000), but segments in the nSSU rRNA gene is a ple- the signi®cance of this ®nding remains un- siomorphy among malacostracans, as this is known. the condition in the outgroup Nebalia sp. Expansion segments can supplement and all other nonperacarid species included phylogenetic information contained in the in this study, except for the lophogastrids. more conservative core regions of the gene. Two alternative evolutionary scenarios This effect has been demonstrated for ser- could explain the absence of expansion seg- olid isopods (Held 2000) and some oniscid ments in lophogastrids: (1) an expanded isopods (Mattern & Schlegel 2001). Dreyer nSSU rRNA gene is a synapomorphy for and WaÈgele (2002) were also able to detect the Peracarida if this taxon excludes Mys- phylogenetic patterns in DNA insertions ida (i.e., an expanded nSSU rRNA gene and deletions among several families of iso- was present in the common ancestor of per- pods. Different species of the valviferan acarids) and this character state was sub- isopod Idotea were sequenced in the pre- sequently lost in the lophogastrid lineage, sent study (I. metallica) and in Meland and (2) lophogastrids represent the basal (2003; I. baltica), yet both had longer nSSU peracarid lineage (a view not inconsistent rDNA sequences than any other taxa in the with the fossil record), and the expansion respective studies. Meland sequenced only of nSSU rRNA genes occurred soon after from the V4 to the V7 regions of the gene, in peracarid evolution. Our results strongly but the lengths of the expansion segments reject mysidacean monophyly, but scenario in this region were comparable for the two (2) above would be consistent with a par- species of Idotea, further supporting the aphyletic Mysidacea that has Mysida fol- view that features of expansion segments in lowed by Lophogastrida at the base of the VOLUME 118, NUMBER 1 145

Peracarida. A decay index of one for the data were more congruent with those based Mysida node (Fig. 2b) indicates that this on combined data, even though stem data latter view is plausible. included almost twice the number of char- MP analyses of the bootstrapped molec- acters. The presence of a monophyletic per- ular data kept relationships among peracar- acarid clade that excluded mysids remained id lineages equivocal, for the most part a consistent feature, however, in all topol- (Fig. 2b). Bayesian analyses (Fig. 3b) pro- ogies whether they were inferred from only vided more resolution but still did not iden- stem or loop data, despite the smaller num- tify any particular group as being basal. Lo- ber of nucleotide characters in each of these phogastrids are basal, however, in clade C partitions. The same was true no matter of Fig. 3. Adding taxa to phylogenetic anal- what method of phylogenetic estimation yses can often reorder branches, so the in- was used. clusion of nSSU rDNA data for additional We therefore recommend against the a peracarid species, particularly mictaceans priori use of a single model of nucleotide and thermosbaenaceans, might result in a evolution (or of a single set of optimized phylogeny that ®rmly establishes a basal parameter values, for that matter) for nSSU peracarid lineage, possibly the Lophogas- rDNA when phylogenies are inferred, as trida. This result would in turn reveal which this practice can lead to the uniform appli- of the two scenarios of expansion-segment cation of inaccurate model-parameter val- evolution applies, although we favor the ues to stem and loop sequences. A more second hypothesis for two reasons: (1) am- suitable approach would be to assess fea- ple morphological evidence supports the tures relevant to nucleotide substitution Lophogastrida as a somewhat primitive tax- (e.g., nucleotide frequencies, among-site on (discussed above), and (2) we are aware heterogeneity in substitution rates) sepa- of no examples of species that have lost ex- rately for stems and loops to determine pansion segments once their presence was whether they differ. Otherwise, ML analy- established within a clade. ses of combined data may lead to an inac- Data partitions.ÐThe stem and loop par- curate or poorly supported estimate of phy- titions of the SSU molecule for peracarids logeny. This same argument applies to the studied herein have very different nucleo- use of maximum parsimony to infer rela- tide frequencies, but combining these data tionships (e.g., Conant & Lewis 2001, obscures the difference and may adversely affect accurate phylogeny estimation. In Buckley & Cunningham 2002), although general, LogDet/paralinear-distance analy- we did not explore alternative character sis of the combined data did not improve weighting for MP in the present study. phylogenetic resolution over that of MP and In contrast, Bayesian analyses are more ML, possibly because this method does not likely to produce an accurate re¯ection of account for among-site substitution-rate phylogeny when partitions differ in param- heterogeneity. We suspect that a combina- eter values, because this method can simul- tion of nucleotide compositional bias and taneously accommodate multiple models of substitution-rate heterogeneity throughout nucleotide evolution. The present study the nSSU rDNA gene contributes both to con®rms this point: the Bayesian consensus the differences we observed in the MP, ML, topology obtained by analysis of unlinked and distance estimates using combined data stem and loop partitions, each with its re- and to the lack of nodal support in the boot- spective model, was congruent with the ML strapped ML result. We found that the fast- phylogeny, but many more clades were sup- er-evolving loop partition was generally ported by high credibility values. Accuracy more informative than its stem counterpart, with the Bayesian method, however, still re- and Bayesian consensus trees based on loop quires correct model speci®cation, which is 146 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON no small matter (see, e.g., Buckley & Cun- with Siewing (1963), when he says, ``The ningham 2002 and references therein). Mysidacea, including the Lophogastrida, Several conclusions, as noted above, are in many respects original, representing emerge from the present study, and we be- the connecting link to other malacostracan lieve that the nSSU rRNA gene is a prom- groups, particularly to the Eucarida and ising molecular marker for inferring phy- Pancarida'' (p. 99). Although our ®ndings logenetic relationships among peracarid lin- indicate that the Mysidacea are not mono- eages but less so for superorders of euma- phyletic, they do suggest that mysids, and lacostracans. Additional nSSU rDNA probably lophogastrids, are both ``original'' evidence, particularly for species of micta- (to use Siewing's terms); i.e., mysids rep- ceans, spelaeogriphaceans, and thermos- resent the ancestral ``connection'' to non- baenaceans, will undoubtedly further clarify peracarid crustaceans, whereas lophogas- peracarid relationships, particularly if new trids are closer to the ancestor of peracarids. sequence data can break up some of the The distribution of expansion segments in long branches observed in the present the nSSU rDNA gene in malacostracans study. Improvement in the phylogeny will supports this view as well. Our ®ndings in turn permit a more thorough assessment contrast with those of Siewing (1963), of morphological- in this Richter & Scholtz (2001), and even Pires group. Data would be useful, too, from an (1987) in showing the Thermosbaenacea, or additional DNA marker that does not Pancarida in these authors' views, ®rmly evolve too rapidly and that is not linked to embedded within the Peracarida and not an- the rRNA operon. Such data would permit cestral to this clade. a comparison of independent ``gene trees,'' and congruence among trees could be taken Acknowledgments to re¯ect the ``true'' phylogeny. Our molecularly based phylogenetic We owe a great debt to colleagues (Ap- analyses suggest a scenario of peracarid pendix 1) who generously donated many of (and eumalacostracan) evolution not unlike the specimens (some of which are especial- that proposed by Richter & Scholtz's (2001) ly rare) used in this study. Collection of parsimony analysis of malacostracan mor- Thetispelecaris remex by Thomas M. Iliffe phological characters or even Siewing's was supported by NSF Biotic Systems and (1963) earlier assessment of morphology: Inventories Program (NSF 9870219) and ``The reconstruction of the natural system contributed to a DIVERSITAS-IBOY pro- of the Peracarida by means of homologies ject, ``Exploration and Conservation of An- shows that isopods and amphipods are end chialine Faunas.'' We could not have found points of two divergent lines of evolution the cave known as Punta degli Stretti on the which arise near the mysids. One of these island of Monte Argentario, , without lines comprises the Cumacea, Tanaidacea, Geoffrey Fryer's directions, and we are and Isopoda; the other line comprises only grateful for his excellent memory! The au- the Amphipoda'' (p. 98). This conclusion thors thank Rani Dhanarajan and coworkers parallels our ®ndings almost exactly, but of the Florida State University (FSU) Clon- whereas Richter & Scholtz (2001) and ing Facility, Steven Miller of the FSU Se- Siewing (1963) considered amphipods to be quencing Facility, and Ellyn Whitehouse closer to a monophyletic Mysidacea, we and Christopher Bacot (both formerly of the ®nd instead that amphipods (with spelaeo- FSU Sequencing Facility). The latter facil- griphaceans) are closer only to lophogas- ity acknowledges an upgrade made possible trids. Mysids, according to our results, have by NSF award DBI-00070316. Additional an af®nity with nonperacarid eumalacostra- support was received from the Alfred P. cans, but this result, too, is not inconsistent Sloan Foundation (RWD) and NSF VOLUME 118, NUMBER 1 147

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of models for nucleotide substitution used in (Woods Hole Marine Biological Laboratory, Depart- maximum-likelihood phylogenetic estima- ment of Marine Resources, Woods Hole, Massachu- tion.ÐMolecular Biology and Evolution 11: setts; 1996; GB AY781419) 316±324. Leach, 1814 (location and date unknown; gift from W. Kobusch; GB Associate Editor: Christopher B. Boyko AY781420) Order Mictacea Bowman, Garner, Hessler, Il- Appendix 1 iffe, & Sanders, 1985 Family Hirsutiidae Sanders, Hessler & Classi®cation (Martin and Davis 2001), GenBank Garner, 1985 accession numbers (GB), and collection data (site, Thetispelecaris remex Gutu & Iliffe, date, and collector and/or provider) for crustacean spe- 1998 (Basil Minns Blue Hole, Great Exuma Island, cies used in the present study. Bahamas; 20 Mar 2000; T. Iliffe; GB AY781421) Order Amphipoda Latreille, 1816 Class Malacostraca Latreille, 1802 Suborder Latreille, 1802 Subclass Phyllocarida Packard, 1879 Family Gammaridae Latreille, 1802 Order Leptostraca Claus, 1880 Gammarus oceanicus SegerstraÊle, 1947 Family Nebaliidae Samouelle, 1819 (Woods Hole Marine Biological Laboratory, Depart- Nebalia sp. (GB L81945) ment of Marine Resources, Woods Hole, Massachu- Subclass Hoplocarida Calman, 1904 setts; 1993; GB AY781422) Order Stomatopoda Latreille, 1817 Suborder Caprellidea Leach, 1814 Family Gonodactylidae Giesbrecht, 1910 Family Leach, 1814 Gonodactylus sp. (GB L81947) Caprella geometrica Say, 1818 (Woods Family Latreille, 1802 Hole Marine Biological Laboratory, Department of Squilla empusa Say, 1818 (GB L81946) Marine Resources, Woods Hole, Massachusetts; 1993; Subclass Eumalacostraca Grobben, 1892 GB AY781423) Superorder Syncarida Packard, 1885 Suborder Milne Edwards, 1830 Order Anaspidacea Calman, 2904 Family Ra®nesque, 1815 Family Anaspididae Thomson, 1893 Phronima sp. (location unknown; 1995; Anaspides tasmaniae Thomson, 1893 gift from M. Grygier; GB AY781424) (GB L81948) Order Isopoda Latreille, 1817 Superorder Peracarida Calman, 1904 Suborder Phreatoicidea Stebbing, 1893 Order Spelaeogriphacea Gordon, 1957 Family Amphisopodidae Nicholls, 1943 Family Spelaeogriphidae Gordon, 1957 Paramphisopus palustris (Glauert, Spelaeogriphus lepidops Gordon, 1957 1924) (Lake Monger, Perth, W. Australia; 31Њ55ЈS ( Cave, Table Mountain, South Africa; 23 Oct 15Њ50ЈE; amongst reeds on shore; 22 Mar 1995; D. 1993; gift from L. Watling; GB AY781414) Jones and G. D. F. Wilson; gift from G. D. F. Wilson Order Thermosbaenacea Monod, 1927 and the Australian Museum (P. 44487), Sydney; GB Family Thermosbaenidae Monod, 1927 Tethysbaena argentarii Stella, 1951 AY781425) (Punta degli Stretti Cave, Monte Argentario, Italy; 28 Suborder Latreille, 1902 Feb 1995; T. and F. Spears; GB AY781415) Family Latreille, 1802 Order Lophogastrida Sars, 1870 Asellus racovitzai (Williams, 1970) Family Lophogastridae Sars, 1870 (Newport Sulfur Spring, Wakulla Co., Florida; T. and Gnathophausia ingens Dohrn, 1870 F. Spears; 25 Mar 1995; GB AY781426) (Gulf of , 3552 m depth, DGOMBII station 1 Suborder Sars, 1882 (JSSD1), #20861; 3 Aug 2002; E. Escobar-Briones, F. Family Idoteidae Samouelle, 1819 Alvarez, and M. Wicksten; GB AY781416) Idotea metallica Bosc, 1802 (Woods Gnathophausia zoea Willemoes-Suhm, Hole Marine Biological Laboratory, Department of 1873 (Gulf of Mexico, 3410 m depth, DGOM station Marine Resources, Woods Hole, Massachusetts; 1990; 4 (JSSD4), #20859; 9 Aug 2002; E. Escobar-Briones, GB AY781427) F. Alvarez, and M. Wicksten; GB AY781417) Order Tanaidacea Dana, 1849 Family Sars, 1885 Suborder Tanaidomorpha Sieg, 1980 Eucopia sp. (DeSoto Canyon, Gulf of Family Tanaidae Dana, 1849 Mexico, 1870 m, 28Њ54.49ЈN, 87Њ39.47ЈW, station S36 (Audouin, 1826) (Al- rep.3; 12 Aug 2002; M. Wicksten; GB AY781418) varado Lagoon, western Gulf of Mexico, 30 Oct 2000; Order Mysida Haworth, 1825 gift from E. Escobar-Briones; GB AY781428) Family Haworth, 1825 Family Lang, 1949 Heteromysis formosa S. I. Smith, 1871 Paratanais malignus Larsen, 2001 VOLUME 118, NUMBER 1 153

(Frenchman's Bay, Bay, New South Wales, Superorder Eucarida Calman, 1904 Australia; date unknown; gift from K. Larsen and the Order Euphausiacea Dana, 1852 Australian Museum, Sydney; GB AY781429) Family Euphausiidae Dana, 1852 Suborder Apseudomorpha Sieg, 1980 Nyctiphanes simplex Hansen, 1911 Family Lang, 1956 (32Њ53ЈN, 117Њ17ЈW; 0±90 m; La Jolla Canyon, Cali- Kalliapseudes sp. (from shallow sandy fornia (eastern Paci®c); date unknown; R. Mc- beach, northern Gulf of Mexico, Ocean Springs, Mis- Connaughey; GB AY781433) sissippi; 1993; S. LeCroy; GB AY781430) Meganyctiphanes norvegica M. Sars, 1857 (location and date unknown; gift from W. Ko- Order Cumacea KroÈyer, 1846 busch; GB AY781434) Family Bate, 1856 Order Decapoda Latreille, 1802 Diastylus sculpta G. O. Sars 1871 (lo- Family Palinuridae Latreille, 1802 cation and date unknown; gift from L. Watling; GB Panulirus argus Latreille, 1804 (Gulf of AY781431) Mexico, Florida Keys; date unknown; gift from W. F. Family Scott, 1901 Herrnkind; GB AY781435) Spilocuma salomani Watling, 1977 Family Portunidae Ra®nesque, 1815 (surf zone, northern Gulf of Mexico, St. George Island, Callinectes sapidus Rathbun, 1896 Franklin Co., Florida; 1992; T. Spears and N. Segan- (northern Gulf of Mexico, off FSU Marine Lab, Flor- ish; GB AY781432) ida; 21 Sep 1996; C. L. Morrison; GB AY781436) 154 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON sed in Classi®cation 110 (53%) 110 (54%) 109 (54%) 109 (54%) 109 (55%) 114 (54%) 106 (54%) 108 (52%) 148 (53%) 124 (50%) 150 (57%) 50 (61%) 59 (61%) 58 (52%) 58 (52%) 58 (52%) 59 (61%) 59 (51%) 59 (61%) 103 (60%) 121 (56%) 103 (52%) 92 (52%) 92 (52%) 86 (49%) 86 (53%) 86 (51%) 216 (56%) 236 (56%) 234 (52%) 250 (64%) 207 (62%) 300 (64%) 0.01 with 78 degrees of freedom. 72 (58%) 72 (58%) 72 (59%) 72 (59%) 72 (59%) 84 (64%) 82 (59%) 72 (60%) 72 (61%) 72 (56%) 123 (52%) ␣ϭ value at P 232 (54%) 232 (55%) 233 (40%) 233 (40%) 233 (41%) 413 (55%) 332 (57%) 382 (53%) 554 (57%) 409 (56%) 879 (58%) signi®cant ϭ 68 (47%) 68 (47%) 69 (36%) 69 (36%) 69 (36%) 70 (57%) 70 (53%) 70 (41%) 68 (56%) 68 (52%) 68 (57%) C content is given in parentheses (calculated with PAUP* [Swofford 2002] and with missing nucleotides ϩ 209 (51%) 209 (52%) 207 (36%) 207 (37%) 208 (34%) 279 (52%) 242 (49%) 277 (49%) 234 (48%) 229 (46%) 242 (47%) 33 (54%) 32 (53%) 33 (54%)36 (64%) 212 (51%)38 (50%) 224 (57%) 68 (47%) 327 (47%) 64 (59%) 238 (53%) 67 (51%) 360 (58%) 72 (60%) 298 (59%) 68 (63%) 94 (56%) 74 (59%) 180 (55%) 59 (60%) 332 (53%) 59 (51%) 112 (51%) 59 (58%) 114 (58%) 127 (45%) 32 (38%) 32 (38%) 32 (38%) 35 (49%)35 (43%) 211 (48%)35 (49%) 211 (46%) 68 (48%)36 (59%) 211 (47%) 68 (53%)38 (58%) 39 (62%) 228 (50%) 69 (52%) 226 (47%) 72 248 (51%) (47%) 72 (54%) 90 72 (54%) (57%) 90 (49%) 167 59 (50%) (54%) 59 (62%) 111 61 (47%) (41%) 112 (48%) 129 (51%) 33 (54%) 33 (54%) 33 (60%) Chi-square test for homogeneity of nt frequencies; ** sp. ϭ 2 sp. ␹ sp. sp. 33 (45%) 210 (46%) 68 (49%) 229 (50%) 72 (51%) 89 (48%) 59 (54%) 104 (45%) Taxon V1 V2 V3 V4 V5 V7 V8 V9 Appendix 2.ÐLength (in nucleotides, nt) of the variable regions of the nuclear small-subunit (SSU) ribosomal RNA gene for crustaceans (Appendix 1) u this study. Variable (V) regionsis V1±V9 according (V6 is to absent Martin in ) and were Davis identi®ed with (2001). the Percent SSU secondary-structure G model of Van de Peer et al. (1997). Leptostraca Nebalia Stomatopoda Gonodactylus S. empusa deleted); Anaspidacea A. tasmaniae Spelaeogriphacea S. lepidops Thermosbaenacea T. argentarii Lophogastrida G. ingens G. zoea Eucopia Mysida H. formosa N. integer Mictacea T. remex Amphipoda G. oceanicus C. geometrica Phronima Isopoda P. palustris A. racovitzai I. metallica VOLUME 118, NUMBER 1 155 1.00 109 (42%) 108 (43%) 141 (55%) 131 (56%) 123 (57%) 116 (54%) 108 (52%) 108 (52%) 123 (57%) 1.00 57 (47%) 59 (58%) 60 (53%) 59 (58%) 58 (55%) 60 (60%) 60 (60%) 110 (61%) 110 (61%) 0.08 98 (51%) 225 (48%) 273 (47%) 290 (54%) 333 (57%) 252 (59%) 106 (56%) 106 (55%) 333 (57%) 1.00 69 (55%) 73 (58%) 72 (63%) 73 (60%) 72 (58%) 72 (59%) 73 (61%) 73 (61%) 72 (58%) 0.00** 257 (38%) 504 (47%) 656 (59%) 246 (53%) 292 (54%) 334 (51%) 237 (55%) 236 (56%) 292 (54%) 1.00 69 (48%) 68 (48%) 68 (46%) 68 (47%) 70 (49%) 67 (54%) 68 (49%) 68 (49%) 70 (49%) 0.15 239 (44%) 293 (47%) 275 (53%) 216 (50%) 258 (55%) 220 (55%) 226 (46%) 226 (49%) 258 (55%) 1.00 34 (48%) 35 (52%) 33 (57%) 33 (48%) 35 (40%) 33 (41%) 33 (54%) 33 (54%) 35 (40%) 33 (54%) 216 (51%) 68 (49%) 240 (53%) 72 (60%) 103 (50%) 59 (59%) 138 (52%) sp. Taxon V1 V2 V3 V4 V5 V7 V8 V9 ) P ( Appendix 2.ÐContinued. 2 Tanaidacea T. dulongii P. malignus Kalliapseudes ␹ Cumacea D. sculpta S. salomani Euphausiacea N. simplex M. norvegica D. sculpta Decapoda P. argus C. sapidus 156 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

Appendix 3.ÐPairwise comparisons of sequence divergence (uncorrected and expressed as the proportional amount of change per nucleotide position) for 1696 aligned characters (after regions of ambiguous alignment were excluded) of nuclear small-subunit ribosomal DNA for 27 crustaceans used in this study. Calculations were obtained with PAUP* (Swofford 2002), and missing nucleotides were omitted.

Taxon 1234567

1 Anaspides tasmaniae Ð 2 Kalliapseudes sp. 0.11410 Ð 3 Thetispelecaris remex 0.19022 0.21425 Ð 4 Asellus racovitzae 0.06563 0.10433 0.18863 Ð 5 Nebalia sp. 0.07238 0.13099 0.19373 0.08922 Ð 6 Gonodactylus sp. 0.03855 0.10859 0.18714 0.06504 0.06260 Ð 7 Tethysbaena argentarii 0.14075 0.16691 0.22115 0.13919 0.14917 0.13707 Ð 8 Tanais dulongii 0.16576 0.16312 0.23371 0.14289 0.17542 0.15963 0.20482 9 Panulirus argus 0.04284 0.10745 0.18976 0.06569 0.07304 0.03368 0.13900 10 Paramphisopus palustris 0.08151 0.10436 0.19516 0.05211 0.10202 0.07725 0.14528 11 Gnathophausia ingus 0.15736 0.18342 0.22604 0.16503 0.17484 0.15737 0.20497 12 Eucopia sp. 0.15843 0.18514 0.22676 0.16731 0.17629 0.15853 0.20814 13 Gammarus oceanicus 0.19919 0.21879 0.25759 0.19634 0.20836 0.19561 0.23146 14 Spilocuma salomani 0.15804 0.16691 0.22658 0.14837 0.16945 0.15624 0.19534 15 Gnathophausia zoea 0.15736 0.18342 0.22604 0.16503 0.17484 0.15737 0.20497 16 Heteromysis formosa 0.05574 0.12155 0.18664 0.07555 0.07792 0.04411 0.14824 17 Paratanais malignus 0.17797 0.17566 0.23891 0.16411 0.18764 0.17441 0.21878 18 Spelaeogriphus lepidops 0.27066 0.27203 0.30074 0.26643 0.27951 0.26624 0.28361 19 Neomysis integer 0.06183 0.12213 0.18850 0.07919 0.08041 0.05267 0.15064 20 Caprella geometrica 0.19283 0.21119 0.25678 0.18632 0.20013 0.18988 0.21466 21 Diastylis sculpta 0.19227 0.19497 0.26632 0.17890 0.20268 0.18870 0.21215 22 Callinectes sapidus 0.04917 0.10790 0.18871 0.07088 0.07751 0.04243 0.14393 23 Nyctiphanes simplex 0.04465 0.11347 0.18780 0.07421 0.07116 0.03672 0.14255 24 Meganyctiphanes norvegica 0.04223 0.11170 0.18791 0.06994 0.07121 0.03307 0.14140 25 Phronima sp. 0.23549 0.25198 0.27467 0.23454 0.24049 0.22941 0.24945 26 Idotea metallica 0.08477 0.10583 0.19555 0.05717 0.10526 0.07988 0.14319 27 Squilla empusa 0.03429 0.10557 0.18428 0.06323 0.05957 0.00490 0.13602

Taxon 8 9 10 11 12 13 14

8 Tanais dulongii Ð 9 Panulirus argus 0.15724 Ð 10 Paramphisopus palustris 0.15462 0.07970 Ð 11 Gnathophausia ingus 0.22127 0.15992 0.17766 Ð 12 Eucopia sp. 0.22249 0.16098 0.18061 0.00614 Ð 13 Gammarus oceanicus 0.23104 0.19995 0.19946 0.22262 0.22452 Ð 14 Spilocuma salomani 0.19861 0.15497 0.14262 0.20832 0.20754 0.23207 Ð 15 Gnathophausia zoea 0.22127 0.15992 0.17766 0.00000 0.00614 0.22262 0.20832 16 Heteromysis formosa 0.16709 0.05700 0.08896 0.16358 0.16463 0.19818 0.16529 17 Paratanais malignus 0.17302 0.17564 0.17086 0.22083 0.22509 0.23301 0.21503 18 Spelaeogriphus lepidops 0.27165 0.26832 0.27497 0.28893 0.29115 0.28447 0.29404 19 Neomysis integer 0.17637 0.06983 0.09259 0.16352 0.16337 0.20303 0.16136 20 Caprella geometrica 0.23490 0.19299 0.19317 0.21367 0.21551 0.08804 0.22604 21 Diastylis sculpta 0.21473 0.18872 0.17510 0.23781 0.23785 0.24438 0.19616 22 Callinectes sapidus 0.15794 0.02030 0.08008 0.15930 0.15985 0.19585 0.15642 23 Nyctiphanes simplex 0.16199 0.03979 0.08576 0.16350 0.16397 0.19643 0.15806 24 Meganyctiphanes norvegica 0.15958 0.03674 0.08275 0.16307 0.16353 0.19349 0.15688 25 Phronima sp. 0.26785 0.23509 0.23499 0.25251 0.25383 0.18084 0.26542 26 Idotea metallica 0.15243 0.07991 0.05713 0.18297 0.18530 0.19996 0.14420 27 Squilla empusa 0.15723 0.03185 0.07666 0.15574 0.15681 0.19449 0.15441 VOLUME 118, NUMBER 1 157

Appendix 3.ÐContinued.

Taxon 15 16 17 18 19 20 21

15 Gnathophausia zoea Ð 16 Heteromysis formosa 0.16358 Ð 17 Paratanais malignus 0.22083 0.18736 Ð 18 Spelaeogriphus lepidops 0.28893 0.27384 0.28990 Ð 19 Neomysis integer 0.16352 0.04352 0.18727 0.27080 Ð 20 Caprella geometrica 0.21367 0.19422 0.23669 0.27361 0.19726 Ð 21 Diastylis sculpta 0.23781 0.19069 0.24581 0.28939 0.19002 0.23312 Ð 22 Callinectes sapidus 0.15930 0.06209 0.17392 0.26900 0.07566 0.18765 0.18841 23 Nyctiphanes simplex 0.16350 0.05270 0.17867 0.27617 0.06552 0.19257 0.19226 24 Meganyctiphanes norvegica 0.16307 0.05029 0.17570 0.27205 0.06433 0.18962 0.19052 25 Phronima sp. 0.25251 0.23083 0.27584 0.29487 0.23628 0.18293 0.27570 26 Idotea metallica 0.18297 0.08978 0.16940 0.27071 0.09774 0.19800 0.17741 27 Squilla empusa 0.15574 0.04171 0.17261 0.26476 0.05394 0.18996 0.18512 Taxon 22 23 24 25 26 27

22 Callinectes sapidus Ð 23 Nyctiphanes simplex 0.04733 Ð 24 Meganyctiphanes norvegica 0.04428 0.00367 Ð 25 Phronima sp. 0.23304 0.23387 0.23279 Ð 26 Idotea metallica 0.08149 0.08781 0.08478 0.23809 Ð Ð 27 Squilla empusa 0.04123 0.03368 0.03003 0.22838 0.07745