Phylogenetic relationships among loliginid squids (Cephalopoda: Myopsida) based on analyses of multiple data sets

Zoological Journal of the Linnean Society (2000), 130: 603–633. With 6 figures doi:10.1006/zjls.2000.0242, available online at http://www.idealibrary.com

Phylogenetic relationships among loliginid squids (Cephalopoda: Myopsida) based on analyses of multiple data sets FRANK E. ANDERSON∗ Laboratory of Molecular Systematics, Smithsonian Institution, 4210 Silver Hill Road, Suitland, MD 20746, U.S.A. Received October 1998; accepted for publication January 2000

The phylogenetic relationships among the loliginid squids, a species-rich group of shallowwater muscular squids, have been investigated recently using several approaches, including allozyme electrophoresis and analyses of morphological and DNA sequence data, yet no consensus has been reached. This study examines the effects of combining multiple data sets (morphology, allozymes and DNA sequence data from two mitochondrial genes) on estimates of loliginid phylogeny. Various data combinations were analysed under three maximum parsimony weighting schemes: equal weights for all characters, successive approximations and implicit weights parsimony. When feasible, support for branches within trees was assessed with nonparametric bootstrapping and decay analysis. Some ingroup relationships were consistent across all analyses, but relationships among outgroup taxa and basal ingroup taxa varied. Combining data increased bootstrap support for several nodes. Methods that downweight highly variable characters (i.e. successive approximations and implicit weights parsimony) produced very similar trees which included two major clades: a clade consisting of all species sampled from American waters (except Sepioteuthis), and a clade of several east Atlantic species (Loligo forbesi Steenstrup, Loligo vulgaris Lamarck and Loligo reynaudi d’Orbigny) plus several Indo-West Pacific species in the genera Uroteuthis and Loliolus. The Sepioteuthis species occupied a basal position within Loliginidae, but Sepioteuthis itself was not always monophyletic. The position of a clade of a few Lolliguncula species and Loligo (Alloteuthis) also varied across analyses. A new loliginid classification is proposed based on these findings.  2000 The Linnean Society of London

ADDITIONAL KEY WORDS:—Loliginidae – total evidence – morphology – 16S-cytochrome c oxidase I – allozymes – phylogeny – maximum parsimony. CONTENTS

Introduction . . . . . Material and methods . Data collection . . Data analyses . . . Results . . . . . . Analyses of individual

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∗ Present address: Department of Zoology and Center for Systematic Biology, Southern Illinois University, Carbondale, IL 62901, U.S.A. E-mail: [email protected] 0024–4074/00/120603+31 $35.00/0

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Combined data analyses . Discussion . . . . . . . Overview of findings . . Systematics and a proposed Summary . . . . . . Acknowledgements . . . . References . . . . . . . Appendix 1 . . . . . . . Appendix 2 . . . . . . . Appendix 3 . . . . . . . Appendix 4 . . . . . . . Appendix 5 . . . . . . .

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INTRODUCTION

The cephalopod taxon Loliginidae is an economically important group of neritic squids numbering forty to fifty described species (Brakoniecki, 1986; Nesis, 1987). Several loliginid species are important targets of fisheries and some species have served as important model systems for neurophysiological research (e.g. Young, 1938). In addition, many loliginids form critical links in coastal food webs as mesopredators, feeding on crustaceans, fish, and other cephalopods and, in turn, serving as prey for larger animals (Fields, 1965; Recksiek & Frey, 1978). Despite the abundance of loliginids in continental shelf habitats and their commercial and scientific importance, loliginid classification and phylogeny remain confused (Voss, 1977; Roper 1983, 1985). There have been several recent attempts to alleviate this confusion. Natsukari (1983, 1984) and Brakoniecki (1986) used morphological characteristics—primarily features of the sucker rings and hectocotylus (the modified arm males use to pass spermatophores to females)—to erect a number of generic and subgeneric groupings within Loliginidae. Unfortunately, their classifications disagreed on some important points. Vecchione et al. (1998) evaluated several morphological characters in an effort to reach consensus on loliginid classification, but the affinities of several species (including some commercially important species in American waters and Loligo forbesi in the east Atlantic) remained problematic. Anderson (1996) attempted a preliminary cladistic analysis of loliginids using 48 morphological characters. These data were insufficient for resolving relationships within the ingroup, despite use of successive approximations (Farris, 1969) and safe taxonomic reduction (Wilkinson, 1995) approaches. Some support for particular clades (Alloteuthis Wu¨lker, Loliolus Steenstrup, Sepioteuthis Blainville and Uroteuthis sensu Vecchione et al. [1998]) was found, but relationships within and among these groups could not be determined. Comparative biochemical techniques also have been used to investigate relationships among loliginids. Brierley and Thorpe (1994) and Brierley et al. (1996, 1997) used allozyme electrophoresis to evaluate taxonomic issues within Loliginidae, such as the status of the genus Loligo Lamarck, but only eight species were sampled, limiting the informativeness of these studies. Anderson (2000) gathered sequence data for regions of two mitochondrial genes (the 16S ribosomal RNA gene and the cytochrome c oxidase subunit I gene) from 19 loliginid species. The sequences were analysed separately and in combination using maximum parsimony and maximum

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T 1. Overview of analytical approach. EW=equal weights parsimony, G=Goloboff fit (implicit weights) parsimony (k=2), SW=successive weighting (based on the maximum RCI value of all characters on all equal weight parsimony trees). Full=all taxa, reduced=only taxa with DNA sequence data. 1=bootstraps were not performed due to the large number of MPTs found in the full analyses. 2=the MANOB approximation was used when analysing allozyme data with other data Data set Morphology only Allozymes 16S, COI, combined DNA data DNA-morphology DNA-morphology-allozymes

Taxon set

Analysis type

Bootstrap?

Full Only allozymes Reduced Reduced, full Reduced, full

EW, G, SW MANAD EW, G, SW EW, G, SW EW, SW2

No1 Yes Yes No1 No1

likelihood. While these analyses show some interesting patterns, support for several clades was not strong, and the analyses are not useful for elucidating the phylogenetic positions of the many species for which DNA data are lacking. In this study, all available data—morphology, allozyme data for 21 loci and sequence data from two mitochondrial genes—were used to investigate relationships within Loliginidae. The data sets were analysed separately and in combination using maximum parsimony under three weighting schemes: equal weights, implicit weights parsimony (Goloboff, 1993) and successive approximations (also known as ‘successive weighting’; Farris, 1969). Incongruence among data sets was examined using the incongruence length difference test (Farris et al., 1994, 1995). Nonparametric bootstrapping (Felsenstein, 1985) and decay analysis (Bremer, 1988) were used to determine levels of nodal support and to study the effects of combining data sets. Results from this study were compared to results of maximum likelihood analyses of the sequence data alone, and a phylogenetic classification of most described loliginid species is proposed. Several hypotheses will be addressed. First, do combined analyses of multiple data sets support monophyly of Loliginidae? Second, are traditionally recognized genera (e.g. Alloteuthis, Loligo, Lolliguncula, Loliolus, Sepioteuthis and Uroteuthis) monophyletic? Finally, are the results presented here based on maximum parsimony analyses congruent with results based on maximum likelihood analysis of a combined DNA sequence data set?

MATERIAL AND METHODS

Data collection Table 1 presents an overview of the analyses. Taxon names used henceforth in the text conform to those of Vecchione et al. (1998). The data collected for each taxon in the analysis are shown in Table 2. Morphological data Sixty-one morphological characters were coded based on examination of museum specimens and review of relevant literature. Data were collected for most described loliginid species (a total of 40) and 13 putative outgroup taxa, including two sepiolid

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T 2. All outgroup and ingroup taxa and the data set(s) coded for each in this study Taxon

Morphology

16S

COI

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X

X X

Allozymes

Outgroup taxa Ctenopteryx sicula Chiroteuthidae Euprymna sp. Euprymna scolopes Grimalditeuthis bonplandi Grimpoteuthis sp. Helicocranchia pfefferi Joubiniteuthis portieri Mastigoteuthis latipinna Moroteuthis robusta Pickfordiateuthis Taonius borealis Rossia Octopus

X X X X X X

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Ingroup taxa Loligo (Alloteuthis) africana Loligo (Alloteuthis) media Loligo (Alloteuthis) subulata Loligo bleekeri Loligo forbesi Loligo gahi Loligo ocula Loligo opalescens Loligo pealei Loligo plei Loligo reynaudi Loligo roperi Loligo sanpaulensis Loligo surinamensis Loligo vulgaris Loliolus affinis Loliolus beka Loliolus hardwickei Loliolus japonica Loliolus kobiensis Loliolus sumatrensis Loliolus uyii Lolliguncula argus Lolliguncula brevis Lolliguncula diomedeae Lolliguncula mercatoris Lolliguncula panamensis Sepioteuthis australis Sepioteuthis lessoniana Sepioteuthis sepioidea Uroteuthis bartschi Uroteuthis chinensis Uroteuthis duvauceli Uroteuthis edulis Uroteuthis etheridgei Uroteuthis noctiluca Uroteuthis pickfordae Uroteuthis reesi Uroteuthis sibogae Uroteuthis singhalensis Uroteuthis vossi

X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

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X

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squids, eight oegopsid squids and two octopods (Appendices 1–3). Morphological data for the oegopsid taxon Chiroteuthidae were primarily collected from representatives of Chiroteuthis d’Orbigny, but sequence data (see below) was collected from a representative of the chiroteuthid species Valbyteuthis danae Joubin. This data set is a modified version of a previously published matrix (Anderson, 1996); several characters were added, a few others were removed (mainly because they could be coded for very few taxa), and five new outgroups were added to increase concordance with the molecular sequence data set. In addition, several missing data points in Anderson (1996) were coded. Allozyme data Two published allozyme data sets were examined. One set (Brierley & Thorpe, 1994) included four loliginid species: Loligo gahi d’Orbigny, Loligo forbesi, Loligo vulgaris and Loligo (Alloteuthis) subulata (Lamarck). The other (Brierley et al., 1997) included eight species (Uroteuthis edulis Hoyle, Uroteuthis chinensis Gray, Uroteuthis bartschi Rehder, Sepioteuthis lessoniana Lesson and the four species mentioned above). As Brierley and Thorpe’s (1994) set is a subset of the Brierley et al. (1997) set, only Brierley et al.’s (1997) data were reanalysed. Molecular sequence data As part of another study (Anderson, 2000), approximately 650 base pairs (bp) of the cytochrome c oxidase subunit one (COI) gene and 500–600 bp of the 3′ end of the 16S ribosomal RNA gene were PCR amplified and directly sequenced for representatives of 19 loliginid species. The sequences were initially aligned with CLUSTAL in Sequence Navigator (ABI Prism) using default parameters. The COI alignment required no modification, and no gaps were introduced. The 16S alignment was modified by eye using a test version of MANIA, a manual sequence editor (D. L. Swofford and D. J. Eernisse, unpublished). Adjustments were generally minor, but some regions (corresponding to sites 11–17, 33, 45–49, 242–278, 308–327, 341–348 and 370–371 in the Genbank Octopus sequence) could not be aligned with confidence across all sequences, even with reference to a Sepia officinalis (cuttlefish) 16S rRNA secondary structure (Gutell et al., 1993; Bonnaud et al., 1994). These alignment-ambiguous areas were culled from the 16S data prior to analysis. The COI and 16S data were analysed seperately and combined into one data set, with most taxa represented by a culled 16S sequence and a COI sequence from the same individual. For Octopus and Moroteuthis, a 16S sequence from EMBL (Octopus cyanea accession number=X79570, Moroteuthis sp. accession number=X79581) and COI sequences from Octopus tetricus and Moroteuthis robusta were combined to form two composite sequences. Some other taxa are only represented by a 16S sequence (Euprymna sp.) or a COI sequence (Taonius borealis, Valbyteuthis danae/‘Chiroteuthidae’, Joubiniteuthis portieri, Mastigoteuthis latipinna). EMBL 16S sequences for some taxa were combined with other 16S sequences from the same species to produce consensus sequences (accession numbers: Sepioteuthis lessoniana=X79572, Loligo forbesi=X79584, Loligo vulgaris=X79586) (Bonnaud et al., 1994). The accession numbers for all other sequences are: COI (Genbank AF075386–AF075419; AF000056 for Octopus tetricus, AF075417 for Euprymna scolopes) and 16S (Genbank AF110072–AF110100; EMBL number for Euprymna sp.=X79593).

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Data analyses Partitioned molecular and morphological data analyses Each data set (except the allozyme data; see below) was analysed in three ways using test versions of PAUP∗ 4.0 d60–63, written by D. L. Swofford. First, each data set was analysed using maximum parsimony with all characters unordered and equally weighted. Each equal weights heuristic search consisted of either 100 or 500 random addition sequence replicates, with zero-length branches not collapsed and ten trees held at each step of random addition. The most-parsimonious trees (MPTs) resulting from the equal weights parsimony analysis were used to reweight the characters in the data matrix based on the maximum value of the rescaled consistency index (RCI) for each character (Farris, 1989). The reweighted data matrix was analysed using the same search strategy as in the equal weights analysis. Multiple rounds of successive approximation were employed until no change in either MPT number or character weights were observed. Each data set (except the allozyme data) was also analysed using implicit weights parsimony (Goloboff, 1993) with k set at two and using the same search parameters listed above. When the morphological data were analysed alone, the MAXTREES limit (50 000 trees) was hit in the equal weights analysis. In this case, ten replicate analyses using different random starting seeds (1–10) were performed to find as many MPTs as possible. Strict consensus cladograms from each replicate were combined to form a ‘grand strict’ consensus tree (see Anderson, 1996 for details). The allozyme data consist of frequencies of alleles at 22 loci for eight species. For these analyses, the allozyme frequency data were retained, and the MANAD frequency parsimony criterion (as implemented in FREQPARS; Swofford & Berlocher, 1987) was used when the allozyme data were analysed alone. All possible dichotomously-branching trees for the allozyme data matrix were generated with PAUP∗ 4.0, input into FREQPARS and evaluated under the MANAD criterion. When combining the allozyme data with other data sets, the MANOB approximation (Berlocher & Swofford, 1997) in PAUP∗ was used as a MANAD estimator. This approach allows allelic frequency information to be retained when analysing combined data sets. To assess the accuracy of the MANOB approximation, these data were analysed alone using MANOB in PAUP∗ 4.0, and the results were compared with those from the MANAD (FREQPARS) analysis. The data were bootstrapped using the MANOB approximation to assess support for particular groups. The allozyme data could not be analysed under implicit weights parsimony (test versions of PAUP∗ do not allow implicit weighting of stepmatrix characters). Combinability tests The incongruence length difference (ILD) test (Farris et al., 1995) was used to evaluate the combinability of the different data partitions. Eight ILD tests were performed to examine incongruence between the following pairs of data sets: 16SCOI, 16S-allozymes, 16S-morphology, COI-allozymes, COI-morphology, combined DNA data-allozymes, DNA data-morphology, and morphology-allozymes. For each comparison, all taxa missing data from one or both partitions were culled prior to analysis (resulting, for example, in only eight taxa being retained in any ILD comparison involving allozymes). One hundred ILD replicates (using branch-andbound searches for comparisons involving the allozyme data, heuristic searches as

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described above for all other comparisons) were used to estimate the null distribution. The maximum number of trees saved was set at 100 during comparisons between the DNA data sets and morphology, due to the large number of MPTs found when the morphological data were used. Finally, all tests were run both with uninformative characters excluded, as recommended by Cunningham (1997), and with these characters included. The overall significance level for the ILD tests was adjusted for multiple comparisons using the sequential Bonferroni correction (Holm, 1979; Rice, 1989; but see Stewart-Oaten, 1995). Combined analyses Three data partition combinations were analysed under equal weights and successive approximations parsimony. The 16S and COI data sets were combined into a single DNA data set. The morphological data were then added to the DNA data set. Finally, the allozyme data were added to the DNA-morphology data set. In the analyses of all four data sets (=DNA-morphology-allozymes), there were 61 morphological characters (27 binary, 34 multistate, with a total of 191 character states; all characters parsimony-informative), 657 COI characters (246 parsimonyinformative, 349 constant), 488 16S characters (78 parsimony-informative, 313 constant) and 21 allozyme characters (all multistate, total of 117 character states; 10 parsimony-informative characters under MANOB approximation). The DNA and DNA-morphology data sets were also analysed under implicit weights parsimony. Allozyme and DNA data were combined under one taxon name if they came from the same ingroup species. Within the DNA-morphology and DNA-morphologyallozymes data combinations, two taxon sets were analysed. In one, all taxa (a total of 55—all 53 taxa included in the morphological data matrix, plus an unidentified representative of the sepiolid genus Euprymna Steenstrup [16S data only; from Bonnaud et al., 1994] and Uroteuthis etheridgei Berry, a putative species that is morphologically indistinguishable from Uroteuthis chinensis; see Yeatman and Benzie [1994] and Anderson [2000]) were included in the analyses, even if only one type of data (morphology) was coded. These matrices will be referred to as ‘full’ matrices. In the other set, only taxa for which DNA data (either 16S, COI, or both) had been coded were retained. These are the ‘reduced’ matrices. During analyses of the full data matrices, large numbers of equally parsimonious trees were found, and it was impossible to determine whether the analysis had found all MPTs. In these situations, it was necessary to use the heuristic search options NCHUCK and CHUCKSCORE to estimate the length of the MPTs before using a regular search to find all of the MPTs. For the initial searches, 500 or 1000 random addition sequence replicates (other parameters as described earlier) were performed with NCHUCK set at 10 and CHUCKSCORE set at 1 (for equal weights or successive weights parsimony) or −20000 (for implicit weights parsimony). These settings cause no more than 10 trees with lengths greater than or equal to 1 (or −20000) to be retained. Any subsequent searches that yielded trees with scores worse than those found in the initial ‘chucking’ analyses were discarded. In some cases, NCHUCK and CHUCKSCORE were used to reduce the search time of regular searches by setting CHUCKSCORE at a value slightly higher than the value of the shortest tree(s) found in the initial chucking analysis. Despite these efforts, it is possible that not all most-parsimonious trees were found. When feasible, support for internal nodes was assessed with nonparametric

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bootstrapping (Felsenstein, 1985) (100 bootstrap replicates, each replicate consisting of a heuristic search of either 10 or 100 random sequence addition replicates with search parameters as listed above). For two bootstrap analyses (the Goloboff fit bootstrap of the combined molecular data set and the equal weights bootstrap of the DNA-morphology-allozymes data set), only ten random sequence addition replicates were performed per bootstrap replicate (due to limited computing time). AutoDecay (Eriksson, 1998) and PAUP∗ were used to calculate decay indices for the equal weights reduced DNA and DNA-morphology trees using the same search protocol described above. The NEXUS file used to run all analyses (and text versions of the COI and 16S alignments) are available from the author upon request.

RESULTS

Due to the large number of analyses performed, only a few representative trees can be shown. Descriptive statistics (total number of trees found, tree length, consistency index, retention index, and rescaled consistency index) for all analyses are listed in Appendix 4. Strict consensus trees from particular analyses are shown; descriptions for strict consensus trees resulting from all other analyses are listed in Appendix 5.

Analyses of individual data sets Morphological data Equal weights parsimony analysis of the morphological data alone yielded 50 000+ trees (the MAXTREES limit). All ten replicates of the equal weights analysis using different random seeds yielded strict consensus trees that were identical to one another, suggesting (but not guaranteeing) that the topologies of the individual strict consensus trees accurately reflect a strict consensus of all MPTs. Successive weighting of each replicate set of 50 000 trees from equal weights parsimony analysis yielded a total of 13 370 unique trees (strict consensus, Fig. 1). Goloboff fit analysis of this data set yielded 675 best trees.

Allozyme data Both the MANAD (FREQPARS) and the MANOB (PAUP∗) analyses yielded three most-parsimonious trees for the eight-taxon allozyme data set (strict consensus, Fig. 2). The branch-and-bound bootstrap analysis revealed low support (<50%) for all nodes except the Uroteuthis edulis–Uroteuthis chinensis sister pair.

Molecular sequence data Results of the separate 16S and COI analyses are described in Appendices 4 and 5.

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Figure 1. Strict consensus cladogram of 13 370 equally-parsimonious trees resulting from successive approximations analysis of the morphology data set.

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Figure 2. Strict consensus cladogram of three most-parsimonious trees from MANAD and MANOB frequency parsimony analyses of the allozyme data set, with MANOB bootstrap support values above 50% shown.

T 3. Results of incongruence length difference tests for comparisons among all data partitions. ILD tests with parsimony-uninformative characters 1 included and 2 excluded Comparison 16S-allozymes 16S-morphology COI-allozymes COI-morphology 16S-COI Combined DNA-allozymes Combined DNA-morphology Allozymes-morphology

P value1

P value2

0.10 0.02∗ 0.32 0.67 1.0 0.25 0.25 0.95

0.62 0.08 0.51 0.87 1.0 0.32 0.50 0.59

∗ Statistically significant at the P<0.05 level. No tests were statistically significant at the P<0.05 level after sequential Bonferroni correction.

Combined data analyses Incongruence length difference tests None of the 16 comparisons yielded statistically significant results at the P<0.05 level after sequential Bonferroni correction (Table 3). One comparison (16S-morphology, all characters included) was statistically significant without sequential Bonferroni correction (Table 3).

Molecular data Results of the combined DNA analyses are described in Appendices 4 and 5. Successive weighting and Goloboff fit analysis (k=2) of the combined DNA data

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Figure 3. Maximum likelihood phylogram resulting from analysis of the DNA data set under the GTR+
+I model (−ln L=9105.26591767; modified from Anderson, 2000). The Octopus and Moroteuthis terminals are composites: the COI and 16S data are from different individuals within these genera.

set produced the same tree. The phylogram resulting from ML analysis of the combined DNA data (GTR+
+I model; Anderson, 2000) is shown in Figure 3. Molecular data plus morphological data The equal weights parsimony analysis of the full DNA-morphology data resulted in 39 375 trees. Three rounds of successive weighting yielded 6615 trees (strict

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Figure 4. Strict consensus cladogram of 6615 trees from a successive approximations analysis of the full DNA-morphology data matrix.

consensus, Fig. 4). The Goloboff fit analysis of this data set resulted in 243 trees. Equal weights parsimony analysis of the reduced DNA-morphology data set resulted in three trees. Successive weighting and Goloboff fit analysis of this data set resulted

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in one tree each differing only within the Sepioteuthis clade (successive weighting tree with bootstrap support values from several analyses shown in Fig. 5). Molecular data plus morphological data plus allozyme data Equal weights parsimony analysis of the full DNA-morphology-allozymes data set yielded 39 375 trees. Successive weighting analysis of this data set yielded 50 000+ trees. Equal weights parsimony analysis of the reduced data set resulted in two trees, and one round of successive weighting of the reduced data set yielded one tree. No Goloboff fit analyses were performed.

DISCUSSION

Overview of findings A few major points can be made regarding these results. First, neither the morphological data set nor the allozyme data set alone appears to be particularly useful for resolving relationships among loliginid species. Second, incongruence among data sets does not appear to be an important consideration, but topological incongruence among trees from different analyses is present. Third, the lack of resolution found in most equal weights parsimony analyses suggests that phylogenetic signal is being overwhelmed by noise near the base of the ingroup, possibly hinting at a rapid radiation early in loliginid history. Fourth, for all of the combined data sets, attempts to objectively downweight highly variable characters (via successive weighting or implicit weighting) cause analyses to converge on an overall tree topology that is similar to that found in the maximum likelihood analysis of the molecular data alone. Finally, addition of data had a profound effect on levels of bootstrap support, appreciably increasing support for many nodes (Fig. 5). Analysis of the morphological data alone reveals more support for some groups than found in Anderson (1996), but general confusion regarding basal relationships within Loliginidae remains. Phylogenetic signal (as measured by the g1 statistic: Hillis & Huelsenbeck, 1992) is present in the morphological data set (Anderson, 1996), but the large number of MPTs results in low resolution of the strict consensus topology. Successive weighting analyses support loliginid monophyly and provide some ingroup resolution, particularly within Uroteuthis, but some groups (e.g. Loliolus) are not supported and basal relationships are not resolved (Fig. 1). Implicit weights analysis yields a strict consensus tree that is even more resolved (not shown; Appendix 5), but several relationships contradict findings based on the molecular data set, particularly regarding the positions of Sepioteuthis and Loligo (Alloteuthis). The frequency parsimony analysis of Brierley et al.’s (1997) allozyme data set shows that these data do not strongly support any particular set of relationships among the eight loliginid taxa sampled other than the close relationship between the Uroteuthis edulis and Uroteuthis chinensis samples (Fig. 2). The usefulness of this data set seems extremely limited, in part because only a fraction of the described species in the family has been sampled. The allozyme data also do not seem to contribute much insight to any of the combined analyses (Appendix 5), and will not be considered further. Incongruence among characters from any two data sets is not statistically significant

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Figure 5. The single tree from a successive weighting analysis of the reduced DNA-morphology data matrix. The implicit weights tree has the same topology, except within Sepioteuthis, where S. sepioidea and S. australis are sister taxa. Numbers on branches are as follows: above the branch, left to right: (a) bootstrap support for branch from equal weights analysis of the reduced morphological data alone, (b) bootstrap support from equal weights analysis of the DNA-morphology data set, (c) bootstrap support from equal weights analysis of DNA-morphology-allozyme data; below the branch, left to right: (a) bootstrap support from implicit weights analysis of morphological data alone, (b) bootstrap support based on implicit weights analysis of the DNA-morphology data set. Branches with no numbers have bootstrap support values of less than 50% for all analyses. Branches with single numbers above (or below) have the same level of bootstrap support (100%) across all data combinations.

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under the ILD test (Farris et al., 1995) when  is adjusted for multiple comparisons using the sequential Bonferroni correction (Holm, 1979; Rice, 1989), although some comparisons should be considered marginally significant (e.g. 16S and morphology; Table 3). There is a great deal of topological incongruence among certain partitions and analyses, however. Particularly important instances of topological incongruence within the ingroup include (1) a consistent lack of resolution at the base of the ingroup in several analyses, (2) the status of the Sepioteuthis species (Sepioteuthis monophyly in some trees, para- or polyphyly in others), (3) the position of the clade represented by Alloteuthis subulata and Lolliguncula mercatoris in the reduced analyses (near the base of the ingroup or with Sepioteuthis in some analyses, nested between the Loligo forbesi clade and the Uroteuthis clade in others), and (4) the position and status of Loliolus (within a paraphyletic Uroteuthis in some analyses, sister group to Uroteuthis in others, and even paraphyletic in some trees; Appendix 5). Some topological incongruence can be attributed to lack of resolution in certain trees (particularly in equal weights trees), but other instances of incongruence seem more distressing. Most instances of topological incongruence are correlated with groups with low bootstrap support values and decay indices. For example, only seven clades are supported by decay indices of four or greater in the equal weights analysis of the reduced DNA-morphology data set (Uroteuthis+Loliolus [4], Loligo forbesi+L. vulgaris+L. reynaudi [5], Lolliguncula mercatoris+Alloteuthis subulata [5], Uroteuthis etheridgei+U. chinensis [9], Euprymna sp.+Euprymna scolopes [10], L. vulgaris+L. reynaudi [14], and Octopus+Grimpoteuthis [23]), and these clades are found in nearly all trees. All other groups are supported by decay indices of three or less. The presence of topological incongruence among trees from different analyses despite the absence of significant character incongruence may be due to low data decisiveness, resulting in weak support for particular conflicting clades in different data sets (Goloboff, 1991; Davis et al., 1998). Despite these problems, some relationships are found in almost all trees. For example, a clade of ‘American’ loliginids, a Uroteuthis–Loliolus clade and a clade of three East Atlantic species (Loligo forbesi, L. vulgaris and L. reynaudi) are found in nearly all partitioned and combined data trees (Appendix 5). Apart from these groups, no clades of more than two species are found in all analyses. Both objective differential weighting methods used here (implicit weights parsimony and successive weighting) seem to converge on a reasonably stable general hypothesis of relationships in which only the positions of a few clades vary (Fig. 6), with some nodes strongly supported in bootstrap analysis (Fig. 5). These two methods produced tree topologies similar to that found in a maximum likelihood analysis of the combined DNA data alone (Fig. 3). All three methods—maximum likelihood, successive weighting and implicit weights parsimony—take character reliability into account in some way (character reliability being used here as some inverse function of the amount of homoplasy) (Farris, 1969; Felsenstein, 1973; Goloboff, 1993; Swofford et al., 1996). In these analyses, highly homoplastic characters do not play as great a role in the estimation of relationships as less homoplastic characters do. The implicit weights, successive weighting and maximum likelihood analyses generally support a number of relationships that are concordant with suggestions made by other authors, including (1) monophyly of the family Loliginidae (Vecchione et al., 1998; Anderson, 2000), (2) a basal position within the family for the Sepioteuthis species (Vecchione et al., 1998), (3) a clade of American species and a Japanese

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Figure 6. A tree diagram depicting a conservative estimate of loliginid phylogeny based on the analyses presented here and a proposed taxonomic scheme for the group. All decapod outgroups are shown as ‘other decapods’; monophyly of particular non-loliginid decapod taxa is not implied. The position of Alloteuthini is variable; in some trees, it is found between Sepioteuthis and the remainder of Loliginidae. Bioluminescent species within Loliolini are denoted with asterisks.

species (Brakoniecki, 1986), and (4) a clade comprised of two subclades—one consisting of three east Atlantic species (Loligo forbesi, L. vulgaris and L. reynaudi) and the other consisting of several bioluminescent Indo-West Pacific species (Uroteuthis) and a group of non-luminescent species with a peculiar ‘palisade-like’ hectocotylus (Loliolus) (Fig. 6).

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T 4. A suggested phylogenetic taxonomic scheme for the cephalopod family Loliginidae. Areas of uncertainty are highlighted with question marks (?’s). Species bearing bioluminescent organs are shown in square brackets; the designation Uroteuthis could be provisionally used for these species (see text) Loliginidae (Lesueur, 1821) Sepioteuthinae Naef, 1921 Sepioteuthis Blainville, 1824 australis, lessoniana, sepioidea Loligininae Naef, 1921 Lolligunculini (new taxon) Heterololigo Natsukari, 1984 bleekeri Doryteuthis Brakoniecki, 1986 non Naef, 1921 gahi, ocula, opalescens, pealei, plei, sanpaulensis, surinamensis, roperi Lolliguncula Steenstrup, 1881 brevis, diomedeae, panamensis Loligo Lamarck, 1798 forbesi, vulgaris, reynaudi Alloteuthini (new taxon) Alloteuthis Wu¨lker, 1920 africana, media, subulata Afrololigo Brakoniecki, 1986 mercatoris, argus? Loliolini (new taxon) Loliolus Steenstrup, 1856 affinis, beka, japonica, kobiensis, hardwickei, sumatrensis, uyii [bartschi, chinensis, duvauceli, edulis, etheridgei, noctiluca, pickfordae, reesi, sibogae, singhalensis, vossi]

Combining different sets of taxa and phylogenetically informative data into a simultaneous analysis can affect profoundly both estimates of relationships (Gauthier et al., 1988; Wiens & Reeder, 1995) and measures of nodal support (e.g. bootstrap support or Bremer/decay indices). Here, the sequential addition of data to equal weights and implicit weights analysis of the reduced data matrix increased bootstrap support for several nodes. In the equal weights bootstrap analyses of the reduced DNA, DNA-morphology and DNA-morphology-allozymes data sets, bootstrap support for some nodes increased as the morphological and allozyme data sets were added (Fig. 5). Support for most ingroup nodes increased only marginally; these increases may be attributable to sampling error (i.e. too few bootstrap replicates). For the Goloboff fit bootstrap analyses of the reduced DNA and reduced DNA-morphology data sets, support for several nodes increased markedly when morphological data were added (Fig. 5). Only a few nodes experienced a decrease in bootstrap support when data were added, and most decreases were minimal. Systematics and a proposed classification When characters are differentially weighted using either successive approximations or implicit weights parsimony, a fairly consistent estimate of phylogeny is produced, providing a backbone hypothesis of loliginid phylogeny. This phylogeny can provide the basis for a phylogenetic classification, where names are applied only to monophyletic groups (De Quieroz & Gauthier, 1990). Below, suggested modifications of Vecchione et al.’s (1998) classification will be introduced in an effort to recognize only monophyletic taxa while attempting to maximize stability and minimize confusion. A summary of the proposed classification is shown in Table 4 and is mapped onto a conservative phylogeny in Figure 6.

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The position of Loliginidae within Cephalopoda Some proposed relationships between Loliginidae and other cephalopods are not supported by these analyses. J. Z. Young (1991) and Brierley et al. (1996) suggested that Ctenopteryx Appello¨f, a monotypic genus of oceanic oegopsid squids with unique trabeculate (ribbed) fins, is closely related to Loliginidae based on neural anatomy and allozyme data, respectively. In contrast, Berthold and Engeser (1987) proposed that Loliginidae, Pickfordiateuthidae, Sepiolidae and Sepiidae form a monophyletic group to the exclusion of all oegopsid squids and expanded the taxon name Myopsida (usually reserved for Pickfordiateuthidae+Loliginidae; see below) to encompass this group. Analyses herein do not support either of these suggestions, although some analyses support a close sepiolid-loliginid relationship (Appendix 5). Unfortunately, the identity of the extant sister group of Loliginidae remains unknown. The sister taxon of Loliginidae may be an oegopsid group such as Ommastrephidae; more muscular oegopsids need to be sampled to evaluate this possibility. The status of the monogeneric family Pickfordiateuthidae has also been a subject of debate. Brakoniecki (1996) suggested including Pickfordiateuthis Voss within Loliginidae. Others recognize the possibility that Pickfordiateuthis may be more closely related to sepiolids than loliginids, or that it may be a group of neotenous Lolliguncula species (M. Vecchione, pers. comm.). Analyses of the full data matrices suggest that Pickfordiateuthis is more closely related to sepiolids than to loliginids, but data on Pickfordiateuthis are extremely limited. I suggest that the name Myopsida be reserved for a Pickfordiateuthis+Loliginidae clade while recognizing that further work may establish conclusively that these two groups are not sister taxa. Status of Loligininae and Sepioteuthis Within Loliginidae, the monophyly of the traditional subfamily Loligininae (all loliginid taxa except Sepioteuthis) seems secure. The monophyly of Sepioteuthis is not certain—in some equal weights trees, the positions of the Sepioteuthis species are variable (Appendix 5)—but in most trees the Sepioteuthis species are the basal taxa of a monophyletic Loliginidae. In the interest of stability, the monophyly of Sepioteuthis and validity of the name should be provisionally accepted, pending further study. American loliginines plus Loligo bleekeri Keferstein A clade of all non-Sepioteuthis American species plus a species from Japanese waters (Loligo bleekeri) is present in most trees (Appendix 5). A name for this clade seems justified and potentially useful. The suffix ‘-ini’ can be combined with the name Lolliguncula (a genus within this clade) to yield the taxon name Lolligunculini for this suprageneric clade within the subfamily Loligininae. Within Lolligunculini are two subclades that correspond roughly to groups previously recognized by Brakoniecki (1986) and others. The name Lolliguncula should be retained for the group of Lolliguncula species in American waters (Lolliguncula brevis, L. diomedeae and L. panamensis, with the possible exception of L. argus Brakoniecki and Roper; see below). The subgenera Loliolopsis (consisting only of L. diomedeae) and Lolliguncula (all other Lolliguncula species) also could be retained, pending further study. The status of Lolliguncula argus is unclear. L. argus is a poorly known, recently described species from the tropical east Pacific (Brakoniecki & Roper, 1985). This species is found to be closely related to the other Lolliguncula species in American

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waters in some trees, but is inferred to be part of a clade of East Atlantic species in other trees (Appendix 5). Distributional data and earlier work on morphology (Brakoniecki, 1986) suggest that L. argus is a member of the Lolliguncula clade described above. However, in light of the variable position of the species, the taxonomic status of L. argus must remain uncertain. Brakoniecki (1986) suggested applying the name Doryteuthis to all American species other than Lolliguncula and Sepioteuthis sepioidea (Blainville). The name Doryteuthis could be retained as a generic designation for the clade comprised of the species gahi, ocula, opalescens, pealei, plei, sanpaulensis, surinamensis, and roperi. Unfortunately, this use of the name Doryteuthis could cause confusion due to discrepancies between Naef’s and Brakoniecki’s definitions of this group and problems in the designation of types (M. Vecchione, pers. comm.). However, there is precedence for the use of the name as proposed here (Brakoniecki, 1986), and using Doryteuthis avoids the creation of a new name for this clade. The Japanese species Loligo bleekeri is the first offshoot within Lolligunculini and it does not fall within either of the two clades discussed above. The taxonomic status of this species, which Brakoniecki placed in his genus Doryteuthis, can be resolved by resurrecting the genus name Heterololigo proposed by Natsukari (1983) (with bleekeri as the type species), yielding the combination Heterololigo bleekeri. East Atlantic loliginines A clade of two east Atlantic species (Loligo forbesi and Loligo vulgaris) plus an east Atlantic and Indian Ocean species (Loligo reynaudi) was consistently found in most analyses, and has moderately high equal weights bootstrap support (Fig. 5). Brakoniecki (1986) foreshadowed this finding by proposing that Loligo (Loligo) be reserved for a clade comprised of Loligo forbesi, L. vulgaris and L. reynaudi. Vecchione et al. (1998) made a similar suggestion, although they considered the position of Loligo forbesi unresolved and did not include it in their Loligo (Loligo). Vecchione et al.’s Loligo (including several American species, Loligo bleekeri, species traditionally classified in Alloteuthis and Loligo vulgaris) is paraphyletic with respect to the Indo-West Pacific Uroteuthis and Loliolus species. Restricting the name Loligo to the clade comprised of Loligo forbesi, L. vulgaris and L. reynaudi would be prudent, and may provide a temporary solution to the problem of the variable phylogenetic position of another East Atlantic clade (see below). Support was found for the monophyly of Vecchione et al.’s Loligo (Alloteuthis) (although this clade is not part of Loligo as defined above). This three-species clade appears to be sister to a clade comprised of Lolliguncula mercatoris and possibly Lolliguncula argus (Fig. 6). Brakoniecki (1986) proposed the genus Afrololigo, consisting of Lolliguncula mercatoris and Lolliguncula abulati, a species not studied here. These analyses support the resurrection of Afrololigo, possibly as a genus within a new taxon, Alloteuthini, comprised of Afrololigo (mercatoris and possibly abulati and argus) and species traditionally grouped in Alloteuthis (africana, media and subulata) (Fig. 6). The position of Alloteuthini differs across trees from different weighted analyses (Appendix 5). Typically, it is nested between Loligo (as defined above) and the Uroteuthis clade as part of a paraphyletic east Atlantic assemblage (Fig. 5). In some trees, however, Alloteuthini is nested between Sepioteuthis and Lolligunculini (e.g. Fig. 4). Anderson (2000) noted similar variability in rooted and unrooted maximum likelihood analyses of the DNA sequence data alone, suggesting either that this clade

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is a ‘rogue’ taxon or that the inferred position of the root itself (and the species of Sepioteuthis) is problematic. At present, the position of this clade cannot be resolved with confidence. Indo-West Pacific loliginines Little support was found for the monophyly of Vecchione et al.’s Uroteuthis—a group comprised of all bioluminescent loliginids—although monophyly of this group cannot be statistically rejected (Anderson, 2000). The name Uroteuthis is provisionally retained here for all bioluminescent loliginids until the status of this taxon can be reviewed in greater detail. If Uroteuthis is found to be paraphyletic with respect to Loliolus, the name Loliolus could be applied to all taxa in this clade. The genus Loliolus appears to be monophyletic based on most analyses, so the generic designation Loliolus can retained for affinis, beka, hardwickei, japonica, kobiensis, sumatrensis and uyii—all taxa with a unique ‘crest’ on the hectocotylus (Vecchione et al., 1998). The subgeneric names Loliolus (for affinis and hardwickei) and Nipponololigo (for all other Loliolus species) can be used as suggested by Vecchione et al. (1998), pending verification of monophyly of each of these taxa. The clade including all Loliolus and Uroteuthis species is monophyletic, regardless of the status of Uroteuthis, and is here designated Loliolini (based on the genus name with priority, Loliolus).

Summary The results presented here are concordant with analyses of the combined DNA data set using maximum likelihood (Anderson, 2000; Fig. 3). Within the ingroup, the only major difference between the maximum likelihood tree based on the molecular data alone and the successive weighting or implicit weights analyses of all data is the position of Alloteuthini. If this clade is actually more closely related to the east Atlantic and Indo–West Pacific loliginids, Brakoniecki’s (1986) assertion that the widening of the Atlantic played a critical role in separating the east and west Atlantic faunas would be supported. If the true position of this clade is instead between Sepioteuthis and Lolligunculini, it is unclear how this distribution could be explained without invoking multiple dispersal events and disruptions of gene flow. Despite the recent development of morphological (Anderson, 1996, and herein), allozyme (Brierley et al., 1997) and DNA sequence data sets (Anderson, 2000) for loliginid squids—not to mention more than a century of taxonomic work— phylogenetic relationships within Loliginidae remain somewhat confused. Strong support for any particular pattern of relationships among generic-level clades within the family is generally lacking (Fig. 5). This may stem from the realities of the loliginid radiation. If the major loliginid lineages underwent a rapid radiation tens or hundreds of millions of years ago, their relationships will be difficult to unravel, because all phylogenetic methods have difficulty with phylogenies that exhibit short internal branches with long branches leading to extant taxa (Maley & Marshall, 1998). If the phylogeny of the group is tightly correlated with major marine tectonic events spanning tens of millions of years, as Brakoniecki (1986) suggested, another explanation of the relatively weakly supported relationships among some of the generic-level clades is required. It seems possible that, for particular branches deep

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within the tree, rapidly evolving sites may be nearly randomized, while more slowly evolving sites may not have produced enough informative changes. Summary statistics for different COI codon positions seem to support this assertion: third positions have a lower ensemble rescaled consistency index (0.093) than do either first positions (0.116) or second positions (0.447) on the equal weights COI tree, and there is far less variation at first and second position sites (65 and 77 variable positions, respectively) than at third position sites (208 variable positions). Unfortunately, morphological data are also relatively uninformative regarding relationships among generic-level clades, but they are reasonably useful for distinguishing these clades. Increased taxon sampling and additional data— molecular, morphological, behavioral and developmental—will be necessary to elucidate relationships within loliginids, and between loliginids and other taxa, with any confidence.

ACKNOWLEDGEMENTS

I thank John Pearse, Janet Voight, Mike Vecchione and two anonymous reviewers for their helpful criticism of the manuscript, and David Swofford for advice and for allowing the use of test versions of PAUP∗ 4.0. The Laboratory of Molecular Systematics (National Museum of Natural History, Smithsonian Institution) and John Huelsenbeck provided access to computational facilities. Financial support for this work was provided by the Earl and Ethel Myers Marine Biology and Oceanographic Trust, the American Museum of Natural History Lerner-Grey Fund for Marine Research, the UC Santa Cruz Department of Biology and the Friends of Long Marine Laboratory. This work is a portion of a doctoral dissertation submitted to the Department of Biology, University of California, Santa Cruz.

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APPENDIX 1

Morphological data matrix with characters and character states and a list of material examined. Polymorphic taxa were coded as separate states (as suggested by Wiens, 1995), since frequency information was rarely available or difficult to interpret. The matrix contains a total of 61 characters (27 binary, 34 multistate) and a total of 191 character states. N’s denote characters that are ‘not applicable’ for particular taxa, and ?’s denote missing data. N’s were treated as missing in all analyses. Ingroup taxa

Loligo (Alloteuthis) africana Loligo (Alloteuthis) media Loligo (Alloteuthis) subulata Loligo bleekeri Loligo forbesi Loligo gahi Loligo ocula Loligo opalescens Loligo pealei Loligo plei Loligo reynaudi Loligo roperi Loligo sanpaulensis Loligo surinamensis Loligo vulgaris Loliolus affinis Loliolus beka Loliolus hardwickei Loliolus japonica Loliolus kobiensis Loliolus sumatrensis Loliolus uyii Lolliguncula argus Lolliguncula brevis Lolliguncula diomedeae Lolliguncula mercatoris Lolliguncula panamensis Sepioteuthis australis Sepioteuthis lessoniana Sepioteuthis sepioidea Uroteuthis bartschi Uroteuthis chinensis Uroteuthis duvauceli Uroteuthis edulis Uroteuthis noctiluca Uroteuthis pickfordae Uroteuthis reesi Uroteuthis sibogae Uroteuthis singhalensis Uroteuthis vossi

1 2 3 4 5 6 0 0 0 0 0 0 . . . . . . 11110111120110?0120178100N0011000002121001?210000111110000000 11?1011112012210120178100N001100000212100????????111110000000 11?1011112012210120178100N00110000021210010210000111110000000 11?1011??2?????0?20100?00N0001010?0200110????????1?1110000000 11?1011132?110?0?20111?01N0001010?02001?0????????1?1110000000 11?1011112011010020017N01N00010100020011010210000101110000000 11?1011112?11110021210N01N0001010?02001?0????0???101110000000 11?1011112011020020200N00N00010100020010410210000101110000000 1111011112011110021200N01N00010100020011410210?00101110000000 1111011112011110020110N01N0001010002001101021?000101110000000 11?1011212?31010?20178?00N0001010?02001?0102100001?1110000000 11?1011112011010?20210N01N00010100020011010210000101110000000 11?1011122011010020017N01N000101000200110????????101110000000 11?1011112011110?21210N00N0001010?02001?01020?000101110000000 11?10111120100?0020111?00N0001010?02001?010210000101110000000 11?10111221100?0022354N400???1010102101001?210000101110000000 11?1011152011010?20154N0011101010?02001?0102100001?1110000000 1101011122110010022354N00022?1010102101001?210000101110000000 11?1011112011010020154N001110101000200100102100001?1110000000 11?101112200NN10020154N00102110100020010010210000101110000000 11?101115203??10020154N00102110100020010?102?00001?1110000000 11?101112200NN10020154N0011101010002001?0????????101110000000 11?10111120110?0?3008??10N11110000021110310201000101110000000 11?1011122111010020437N30N00010100021110110201100101110000000 11110111220110?0020135N10N0001010002111011?201100101110000000 11?1011022?110?0?20133301N2211000?0211102102010001?1110000000 11?101112211122002003?N10N00010100021110210201100101110000000 11?1011002111010020111200N0001010001??10010210000101110000000 11?1011002111010020111200N0001010001??10011210000101110000000 1101011112110010020111200N0021000001??10010210000101110000000 11210111121110?0020101000N00010130020211010210000111110000000 11?1011102111110020122000N110101300200100102100001?1110000000 11?1011112111010020111100N22010130020010010210000101110000000 11?1011112111110020122100N220101300200110102?0?001?1110000000 11?101111200??00022311?10N???10130021010011210000101110000000 11?1011012?110?0?201?????N???10130020011?????????1?1110000000 11?1011112111010?20111?10N00010130020011?10210000101110000000 11?10111121110?0020122100N0001013002001101?210?001?1110000000 11?1011142111110020111100N00010130020011011200?00101110000000 11?1011012011010?2011110?N???1013?020011?????????1?1110000000

COMBINED-DATA PHYLOGENETICS OF LOLIGINID SQUIDS Outgroup taxa

Chiroteuthidae Ctenopteryx Euprymna Grimalditeuthis Grimpoteuthis Helicocranchia Joubiniteuthis Mastigoteuthis Moroteuthis robusta Octopus Pickfordiateuthis pulchella Rossia Taonius

627

1 2 3 4 5 6 0 0 0 0 0 0 . . . . . . 12?1011100NNNNN0N0NNNNNNNN???100??1???0??0N1N??2113001010?010 ???1110NN0NNNNN0N0NNNNNNNN???1011003??10?1200??00100010001000 00?1210NN0NNNNN1N1NNNNNNNN???1102000???0?????????101101020000 ???201110NNNNNNNN0NNNNNNNN???1000?1???0??0N1N??111300102000N0 NN?000NNNNNNNNNNN0NNNNNNNN???0NN0005NN00NNNNN??NN0N11?03200N1 ???1011??1?4?????0NNNNNNNN???10???04??0??0N3N??001300002111N0 11?1311240NNNNN0N0NNNNNNNN???1000?0???00?0N1N??11130010100000 ???1010NN0NNNNN0N0NNNNNNNN???1000?0???00?0N1N??2113001010?010 11?1010NN3N2N??0?0NNNNNNNN???1000?02000??1200??01120010000000 ???000NNNNNNNNNNN4NNNNNNNN???0NN002NNN00?0N?N??100N01?03200N1 1101012?31?????0?21266N00N00112N0?00??10?1100??00101110000000 00?1211??0NNNNN1N1NNNNNNNN???12N0000???0?????????101101020000 ???10112????NN1000NNNNNNNN???1000004??00?0N3N??001100002111N0

APPENDIX 2

Character codings and descriptions for morphological data. Additional sources of information that pertain to certain systems or taxa include: spermatophores (Hess, 1987); Pickfordiateuthis pulchella (Voss, 1953; Brakoniecki, 1996); Loligo (Alloteuthis) africana (Adam, 1950) Loligo pealei, Loligo ocula, Loligo plei and Loligo roperi (Cohen, 1976); Loligo sanpaulensis and Loligo gahi (Brakoniecki, 1984); Loligo chinensis (Natsukari and Okutani, 1975; Nateewathana, 1992); Loligo edulis, Loligo beka, Loliolus affinis, Loligo sumatrensis (Nateewathana, 1992); Loligo surinamensis (Voss, 1974); Loligo kobiensis/Loliolus rhomboidalis (Burgess, 1967); Loligo sibogae (Adam, 1954; Natsukari, 1976); Loligo pickfordae, Loligo duvauceli, Loligo singhalensis (Adam, 1954); Loliolus (Lu et al., 1985); Sepioteuthis (Lu and Tait, 1983); Lolliguncula argus (Brakoniecki and Roper, 1985); Lolliguncula panamensis (Berry, 1911; Brakoniecki, 1980); Uroteuthis bartschi (Adam, 1954; Rehder, 1945; Voss, 1963); Loliolopsis diomedeae (Berry, 1929); Chiroteuthidae (Roper and Young, 1967); Grimalditeuthis (Young, 1972); Joubiniteuthis (Young and Roper, 1969); Mastigoteuthis (Nesis, 1987); Helicocranchia (Voss, 1980); Taonius (Voss, 1980); Octopoda (Nesis, 1987). Characters are grouped by system; numbers refer to the position of the character in the data matrix. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Radula rachidian tooth cusps (0=one/1=three) Radula first lateral teeth cusps (0=one/1=two/2=one large and two small) Brachial cartilages (0=absent/1=fibrous type/2=hyaline type) Tentacles (0=absent/1=present/2=present in larvae only) Arm sucker rows (0=two/1=four/2=both two and four/3=six on I-III, four on IV) Chitinous sucker rings (0=absent/1=present) Arm sucker rings (0=smooth/1=with teeth/2=both) Arm sucker teeth position (0=all around sucker/1=on distal edge/2=both) Arm sucker teeth shape (0=sharp/1=square or rounded and blunt/2=low, wide and flat/3= small, low and round/4=both sharp and square/rounded and blunt/5=both square or rounded and low, wide and flat) Club morphology (0=many tiny suckers/1=two rows in carpus/2=four rows in carpus/3=no marginals or distinct dactyl) Central club sucker size (0=much larger than marginals/1=similar to marginals) Central manus sucker teeth (0=absent/1=present/2=modified as hooks/3=both absent and present/4=large hooklike teeth) Central manus sucker teeth shape (0=blunt/1=pointed/2=both blunt and pointed) Central manus sucker teeth pattern (0=uniform/1=alternating small and large/2=both patterns seen) Marginal manus sucker teeth shape (0=blunt/1=pointed/2=both blunt and pointed) Retractile tentacles (0=absent/1=present) Club trabeculae (0=one per marginal sucker/1=two per marginal sucker) Hectocotylized arms (0=no hectocotylus/1=left dorsal arm/2=left ventral arm/3=right ventral arm/4=third right or left arm) Hectocotylus dorsal row portion (0=distal suckers modified to tip/1=central suckers modified/ 2=all modified)

628

F. E. ANDERSON

20. Hectocotylus ventral row portion (0=no modification/1=distal suckers modified to tip/2= central suckers modified/3=all modified/4=both no modification and distal suckers modified to tip found in different individuals) 21. Dorsal row modification type (0=small suckers with large columnar pedicel/1=tiny suckers with long triangular pedicels/2=fat, conical suckerless papillae/3=long, thin suckerless papillae/ 4=fused crest/5=tiny papillae/6=small suckers and pedicels/7=both tiny suckers with long triangular pedicels and fat, conical suckerless papillae/8=both tiny suckers with long triangular pedicels and long, thin suckerless papillae) 22. Ventral row modification type (0=small suckers with large columnar pedicels/1=tiny suckers with long triangular pedicels/2=fat, conical suckerless papillae/3=long, thin suckerless papillae/ 4=fused crest/5=no suckers or pedicels/6=suckers embedded in swelling/7=both small suckers with large columnar pedicels and tiny suckers with long triangular pedicels/8=both tiny suckers with long triangular pedicels and fat, conical suckerless papillae) 23. Size of suckers in modified region (0=about the same in both rows/1=ventral row sucker stalks longer/2=dorsal row sucker stalks longer/3=dorsal row stalks longer proximally, ventral row stalks longer distally) 24. Length of hectocotylus (0=same length as unmodified third arm/1=longer than unmodified third arm/2=shorter than unmodified third arm/3=both ‘0’ and ‘1’ seen in different individuals/ 4=hectocotyluses similar in length to the unmodified third arm and shorter hectocotyluses seen in different individuals) 25. Puffy ridge between sucker rows in modified portion of hectocotylus (0=absent/1=present) 26. Fused crest in ventral row of hectocotylus (0=without suckers/1=with suckers) 27. Male II arm suckers (0=no modification relative to female equivalents/1=proximal suckers enlarged/2=all suckers enlarged) 28. Male III arm suckers (0=no modification relative to female equivalents/1=proximal suckers enlarged/2=all suckers enlarged) 29. Male right IV arm suckers (0=no modification/1=suckers enlarged/2=suckers reduced) 30. Buccal membrane (0=absent/1=present) 31. Buccal membrane lobes (0=seven/1=eight/2=no lobes) 32. Buccal lappet suckers (0=absent/1=present) 33. Photophores on ink sac (0=absent/1=one round photophore/2=one U-shaped photophore/ 3=two bean-shaped photophores) 34. Pores on ink sac (0=absent/1=present) 35. Pairs of fins (0=one pair/1=two pairs/2=no fins) 36. General fin shape (0=subterminal round/1=longitudinally oval/2=terminal rhomboid or transverse/3=oval and trabeculate/4=apical/5=subterminal ‘paddles’) 37. Anterior fin edge (0=nearly straight/1=convex) 38. Posterior fin edge (0=straight/1=convex/2=concave and much longer than anterior edge) 39. Accessory nidamental glands (0=absent/1=present) 40. Raised ridge on ventral mantle surface of males (0=absent/1=present) 41. Spermatophore placement (0=on buccal membrane/1=near base of left gill on mantle wall/ 2=on buccal membrane and left gill/3=on buccal membrane and right gill/4=both on buccal membrane and near base of left gill) 42. Spermatophore cement body (0=single/1=divided) 43. Cement body ratio (0=oral component larger than aboral component/1=oral component approximately equal in size to aboral component/2=oral component smaller than aboral component) 44. Spermatophore ejaculatory apparatus (0=long and simple/1=simple, mostly in cap/2=spiral filament/3=divided filament) 45. Oral component of cement body (0=not divided/1=divided) 46. Size of cement body (0=short/1=long) 47. Shape of aboral cement body component (0=barrel or cup-shaped/1=tubular) 48. Sperm mass secondary coiling (0=absent/1=present/2=polymorphic) 49. Cement body secondary coils (0=absent/1=present) 50. Gladius (0=absent/1=present) 51. Conus (0=absent/1=unfused ventral folding/2=primary conus/3=secondary conus) 52. Oviducts (0=both oviducts developed/1=only left oviduct developed) 53. Cornea (0=fully open/1=open through pore)

COMBINED-DATA PHYLOGENETICS OF LOLIGINID SQUIDS

54. 55. 56. 57. 58. 59. 60. 61.

629

Nuchal cartilage (0=absent/1=present) Digestive gland (0=single/1=paired) Mantle/funnel locking apparatus (0=straight/1=oval/2=mantle and funnel fused/3=absent) Mantle cavity (0=undivided/1=three chambers/2=ventral septum) Eyes with photophores (0=no/1=yes) Spacious closed coelom containing ammonium chloride solution (0=absent/1=present) Antitragus (tubercle on posterior side of funnel cartilage) (0=absent/1=present) Suckers (0=stalked/1=sessile)

APPENDIX 3

Material examined for morphological data in total evidence analyses. Material examined are listed by species name in alphabetical order. The sex and approximate dorsal mantle length, when known, are listed for each specimen examined. Abbreviations: CAS=California Academy of Sciences, NMNH=United States National Museum of Natural History, UMML=University of Miami Invertebrate Museum, F=female, M=male, J=juvenile (sex not determined), U=sex undetermined, DML=dorsal mantle length. Ingroup taxa Loligo (Alloteuthis) africana Adam – NMNH 727426 (1 M, 56 mm DML), NMNH BCF Table 6IX 6E2-218 9-6-63 (2 M, 78 and 71 mm DML), UMML 1757 (1 F, 45 mm DML; 1 M, 58 mm DML) Loligo (Alloteuthis) media (Lamarck) – NMNH 817475 (3 F, 56, 64 and 67 mm DML; 2 M, 42 and 50 mm DML), UMML 1251 Loligo (Alloteuthis) subulata (Lamarck) – UMML 1252 (2 M, 100 and 101 mm DML), NMNH 817534 (1 F, 70 mm DML) Loligo bleekeri Keferstein – NMNH 332905 (1 J, 40 mm DML), UMML 1211 (2 M, 36 and 38 mm DML) Loligo budo Wakiya and Ishikawa – UMML 1212 (1 F, 170 mm DML; 1 M, 190 mm DML) Loligo forbesi Steenstrup – NMNH (1 F, 133 mm DML) Loligo gahi d’Orbigny – UMML 2087 (1 F, 72 mm DML), UMML 2090 (2 F, 90 and 91 mm DML; 1 M, 69 mm DML) Loligo ocula Cohen – UMML 1683 (2 M, 53 and 62 mm DML), NMNH 727095 (2 M, 87 and 127 mm DML) (paratypes), NMNH 727096 (1 F, 89 mm DML) Loligo opalescens Berry – NMNH 33176 (1 M, 143 mm DML) Loligo patagonica Smith – UMML 1231 (1 F, 83 mm DML) Loligo pealei Lesueur – NMNH 730069 (2 M, 85 and 95 mm DML), NMNH 730531, NMNH 730183 (1 M, 206 mm DML), NMNH 814169 (1 F, 136 mm DML), NMNH 814191 (1 M, 90 mm DML; 1 J, 83 mm DML) Loligo plei (Blainville) – NMNH 574548 (1 M, 105 mm DML), NMNH 576456 (4 M; 146, 154, 195 and 217 mm DML), NMNH 813979 (2 M, 181 and 260 mm DML), NMNH 814288 (1 F, 120 mm DML; 1 M, 105 mm DML), NMNH 814316 (1 M, 198 mm DML), NMNH 814317 (1 M, 213 mm DML), NMNH 814318 (1 M, 197 mm DML), NMNH 814315 (1 M, 163 mm DML), NMNH 574320 (1 M, 169 mm DML), NMNH 574180 (2 M, 215 and 277 mm DML) Loligo reynaudi d’Orbigny – UMML 1233 (1 M, 175 mm DML), UMML 1234 (1 M, 95 mm DML) Loligo roperi Cohen – NMNH 575874 (1 M, 53 mm DML), UMML 933 (1 F, 38 mm DML; 2 M, 41 and 43 mm DML) (paratypes), UMML 1798 (1 M, 55 mm DML), UMML 72777 (1 M, 77 mm DML) (holotype) Loligo sanpaulensis Brakoniecki – UMML 1813 (2 M, 144 and 150 mm DML) (paratypes) Loligo surinamensis Voss – UMML 2053 (1 F, 92 mm DML), UMML 31.2023 (2 F, 76 and 88 mm DML) Loligo vulgaris Lamarck – UMML 1240 (1 M, 210 mm DML), UMML 1241 (1 F, 43 mm DML), UMML 1597 (1, 137 mm DML) Loliolus affinis Steenstrup – CAS 030250 (2 M, 21 and 25 mm DML) Loliolus beka Sasaki – UMML 1209 (1 F, 55 mm DML), UMML 1210 (1 M, 59 mm DML) Loliolus hardwickei Steenstrup – CAS 030251 (1 M, 40 mm DML), NMNH 817822 Loliolus japonica Hoyle – NMNH 727551 (2 M, 75 and 77 mm DML), NMNH 332903 (3 M, 58, 70 and 77 mm DML), UMML 1224 (2 M, 61 and 68 mm DML), UMML 1226 (1 F, 60 mm DML)

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F. E. ANDERSON

Loliolus kobiensis Hoyle – UMML 31.2203 (1 F, 87 mm DML; 1 M, 76 mm DML) Loliolus sumatrensis d’Orbigny – NMNH 817821 (1 F, 52 mm DML), NMNH 817820 (1 F, 53 mm DML; 2 M, 48 and 50 mm DML) Loliolus uyii Wakiya and Ishikawa – CAS 035049, UMML 1239 (1 F, 94 mm DML; 1 M, 69 mm DML) Lolliguncula diomedeae (Hoyle) – CAS 030492 (2 M, 38 and 41 mm DML), NMNH 576907 (2 F, 90 and 93 mm DML), NMNH 730085, UMML 31.697 (1 F, 102 mm DML), UMML (2 F, 95 and 104 mm DML), UMML 1799 (1 M, 83 mm DML) Lolliguncula argus Brakoniecki and Roper – CAS 030252 (2 F, 43 and 43 mm DML; 1 M, 39 mm DML) Lolliguncula brevis (Blainville) – CAS 030491 (2F, 42 and 43 mm DML), NMNH 884122 (1 M, 66 mm DML) Lolliguncula mercatoris Adam – UMML 1244 (1 M), UMML 31.790 (1 M, 15 mm DML) UMML 31.2550 (1 M, 21 mm DML) Lolliguncula panamensis Berry – CAS 030157 (1 M, 44 mm DML), CAS 030495 (2 F, 86 and 105 mm DML) Sepioteuthis australis (Quoy and Gaimard) – NMNH 816311 (1 F, 102 mm DML) Sepioteuthis lessoniana Lesson – CAS 030624 (2 F, 93 and 105 mm DML), NMNH 297637 (2 M, 127 and 166 mm DML), NMNH CH6-7 (1 M, 155 mm DML) Sepioteuthis sepioidea (Blainville) – CAS 030428 (1 M, 72 mm DML), NMNH 574199 (1 M, 123 mm DML), NMNH 576881 (1 M, 101 mm DML), NMNH 9548 (2 M, 99 and 106 mm DML), NMNH 576877 (1 M, 110 mm DML), NMNH 814382 (1 F, 119 mm DML) Uroteuthis bartschi Rehder – CAS 030485 (1 M, 104 mm DML), NMNH 575388 (1 M, 122 mm DML), UMML 1255 (2 F, 119 and 121 mm DML) Uroteuthis chinensis Gray – UMML PJ-102 (2 F, 75 and 107 mm DML), UMML PJ- 110 (1 F, 92 mm DML) Uroteuthis duvauceli d’Orbigny - NMNH 817827 (2 F, 100 and 123 mm DML), NMNH 817829 (1 M, 126 mm DML), NMNH 727560 (1 F, 110 mm DML), NMNH 727561 (2 M, 70 and 93 mm DML), NMNH 817823 (1 M, 66 mm DML), CAS 084583 Uroteuthis edulis Hoyle – NMNH 814158 (4 M, 127, 133, 136 and 142 mm DML), CAS 030539 (2 M, 99 AND 107 mm DML) Uroteuthis etheridgei Berry – UMML 1220 (1 F, 90 mm DML; 1 M, 104 mm DML) Uroteuthis noctiluca Lu et al. – NMNH 00813974 (1 F, 68 mm DML; 3 M - 50, 51 and 56 mm DML) Uroteuthis reesi Voss – UMML 1803 (1 M, 62 mm DML) Uroteuthis sibogae Adam – NMNH 575813 (1 F, 123 mm DML; 1 M, 139 mm DML) Uroteuthis singhalensis Ortmann – UMML 31.2323 (1 M, 140 mm DML), UMML 2168 (1 M) Uroteuthis vossi Nesis – UMML 1259 (2 M, 65 and 78 mm DML) Outgroup taxa Chiroteuthis veranyi (Fe´russac) – NMNH 815873 (1 U, 150 mm DML) Chiroteuthis sp. – NMNH 816118 (1 U, 190 mm DML) Ctenopteryx sicula (Ve´rany) – NMNH 728929, NMNH 727721, NMNH 730695 (1 U, 68 mm DML), NMNH 728935 (2 U, 21 and 45 mm DML), NMNH 730696 (1 U, 75 mm DML), NMNH 730697 (1 M, 81 mm DML), NMNH 730698 (1 F, 52 mm DML) Euprymna moresi (Verrill) – CAS 021433 (1 F, 31 mm DML; 1M, 33 mm DML) Euprymna scolopes Berry – CAS 030512 (2 U, 24 and 28 mm DML), CAS 030751 (1 U, 30 mm DML) Grimalditeuthis bonplandi (Ve´rany) – NMNH 574985 (1 U, 93 mm DML), NMNH 815472 (1 U, 137 mm DML), NMNH 815473 (1 U, 100 mm DML) Joubiniteuthis portieri ( Joubin) – NMNH 815474 (1 U, 57 mm DML) Mastigoteuthis agassizi Verrill – NMNH 577022 Mastigoteuthis atlantica Joubin – NMNH 884061 (1 F, 72 mm DML) Mastigoteuthis hjorti Chun – NMNH 728953 (2 F, 32 and 155 mm DML) Mastigoteuthis magna Joubin – NMNH 728943 (1 M, 103 mm DML) Moroteuthis robusta (Dall in Verrill) – CAS 030111 (partial specimen, total length 9 ft., 7 inches), CAS 035031 (1 U, 300+ mm DML) Pickfordiateuthis pulchella Voss – UMML 1948 (20 mm DML) Rossia macrosoma (delle Chiaje) – NMNH 730594 (1 M, 41 mm DML) Rossia pacifica Berry –CAS 030356 (2 F, 30 and 30 mm DML), CAS 081003 (1 U, 50 mm DML), NMNH 332807 (2 M, 31 and 35 mm DML)

COMBINED-DATA PHYLOGENETICS OF LOLIGINID SQUIDS

631

Rossia pacifica diegensis – NMNH 214376 (1 M, 22 mm DML) (syntype) Taonius pavo (Lesueur) – NMNH 728975 (3 M, 157-231 mm DML) Taonius sp. – NMNH 815742 (1 U, 236 mm DML)

APPENDIX 4

Descriptive statistics for all analyses. Abbreviations are: MOR=morphology, DNA=16S-COI, DMF=full DNA-morphology, DMR=reduced DNA-morphology, DMAF=full DNA-morphologyallozymes, DMAR=reduced DNA-morphology-allozymes, e=equal weights, s=successive weights, g=Goloboff fit (implicit weights) Analysis MORe MORs MORg Allozymes 16Se 16Ss 16Sg COIe COIs COIg DNAe DNAs DNAg DMFe DMFs DMFg DMRe DMRs DMRg DMAFe DMAFs DMARe MDARs

# MPTs

TL

CI

RI

RCI

50K+ 13,370 675 3 280 3 5 1 1 1 2 1 1 39,375 6615 243 3 1 1 39,375 50K+ 2 1

211 — 47.36 174.91 336 — 57.994 1740 — 108.547 2088 — 165.786 2326 — 210.50589 2265 — 207.817 2504.646 — 2407.766 —

0.607 — — 0.925 0.640 — — 0.289 — — 0.343 — — 0.363 — — 0.368 — — 0.402 — 0.401 —

0.738 — — 0.700 0.535 — — 0.355 — — 0.371 — — 0.412 — — 0.393 — — 0.415 — 0.393 —

0.448 — — 0.647 0.342 — — 0.102 — — 0.127 — — 0.150 — — 0.144 — — 0.167 — 0.158 —

APPENDIX 5

Strict consensus tree descriptions (see Appendix 4 for descriptions of abbreviations). Description of strict consensus topology for ingroup taxa MORe

MORg

((((((duvauceli, chinensis, (noctiluca, (hardwickei, affinis)), edulis, (vossi, pickfordae), sibogae, singhalensis, reesi, bartschi, (( japonica, beka), (uyii, (sumatrensis, kobiensis))), forbesi, vulgaris, reynaudi, opalescens, (pealei, ocula, surinamensis), plei, gahi, roperi, sanpaulensis, bleekeri, (((brevis, (argus, mercatoris)), panamensis), diomedeae), ((subulata, media), africana), ((lessoniana, australis), sepioidea)), (Pickfordiateuthis pulchella, (Rossia, Euprymna))), Ctenopteryx, Moroteuthis), (Helicocranchia, Taonius)), (((Chiroteuthis, Mastigoteuthis), Joubiniteuthis), Grimalditeuthis)), (Octopus, Grimpoteuthis)) (((((((((((((opalescens, pealei), bleekeri, (reynaudii, ((media, subulata), africana))), plei, ((sanpaulensis, ((argus, ((brevis, diomedeae), panamensis)), mercatoris)), gahi)), ((vulgaris, (((duvauceli, (((edulis, chinensis), sibogae), (((vossi, pickfordae), noctiluca), singhalensis), reesi, bartschi)), (sepioidea, (lessoniana, australis))), ( japonica, (uyii, (sumatrensis, kobiensis)), beka, (hardwickei, affinis)))), forbesi)), roperi), ocula), surinamensis), (Pickfordiateuthis pulchella, (Rossia, Euprymna))), Ctenopteryx), Moroteuthis),

632

16Se

16Ss

16Sg

COIe

COIs

COIg

DNAe

DNAs

DNAg DMFe

DMFg

DMRe

F. E. ANDERSON

(Helicocranchia, Taonius)), (( Joubiniteuthis, (Chiroteuthis, Mastigoteuthis)), Grimalditeuthis)), (Grimpoteuthis, Octopus)) (((duvauceli, etheridgei, chinensis, noctiluca, japonica, forbesi, (vulgaris, reynaudi), (opalescens, pealei, plei, gahi), bleekeri, brevis, (mercatoris, subulata), lessoniana, sepioidea, australis, Ctenopteryx, Moroteuthis, Helicocranchia, Grimalditeuthis), (Rossia, (Euprymna sp., Euprymna scolopes))), (Octopus, Grimpoteuthis)) ((((((((((((((duvauceli, ((etheridgei, chinensis), (noctiluca, japonica))), (mercatoris, subulata)), bleekeri), ((opalescens, brevis), (pealei, plei), gahi)), forbesi), (vulgaris, reynaudi)), lessoniana), (sepioidea, australis)), Moroteuthis), Helicocranchia), Grimalditeuthis), Ctenopteryx), (Rossia, (Euprymna sp., Euprymna scolopes))), (Octopus, Grimpoteuthis)) (((((((((((duvauceli, (mercatoris, subulata)), ((etheridgei, chinensis), (noctiluca, japonica))), bleekeri), ((opalescens, brevis), pealei, plei, gahi)), (forbesi, (vulgaris, reynaudi))), (lessoniana, Helicocranchia)), Moroteuthis), ((sepioidea, australis), Grimalditeuthis)), Ctenopteryx), (Rossia, (Euprymna sp., Euprymna scolopes))), (Octopus, Grimpoteuthis)) (duvauceli, ((((etheridgei, chinensis), ((forbesi, (vulgaris, reynaudi)), ((((((opalescens, brevis), ((pealei, gahi), plei)), (bleekeri, ((Rossia, Ctenopteryx), ((Moroteuthis, ((Chiroteuthis, Grimalditeuthis), Mastigoteuthis)), (Helicocranchia, Taonius))))), (sepioidea, (australis, Joubiniteuthis))), (lessoniana, (Euprymna scolopes, (Octopus, Grimpoteuthis)))), (mercatoris, subulata)))), noctiluca), japonica)) (((((((((duvauceli, japonica), noctiluca), (etheridgei, chinensis)), (forbesi, (vulgaris, reynaudi))), (mercatoris, subulata)), ((((opalescens, brevis), ((pealei, gahi), plei)), (bleekeri, ((Rossia, Ctenopteryx), ((Moroteuthis, ((Chiroteuthis, Grimalditeuthis), Mastigoteuthis)), (Helicocranchia, Taonius))))), (sepioidea, (australis, Joubiniteuthis)))), lessoniana), Euprymna scolopes), (Octopus, Grimpoteuthis)) ((((((((((duvauceli, ((etheridgei, japonica), chinensis)), noctiluca), (forbesi, (vulgaris, reynaudi))), (mercatoris, subulata)), (((((opalescens, pealei), gahi), plei), brevis), bleekeri)), sepioidea), (lessoniana, australis)), ((((Ctenopteryx, Helicocranchia), ((Chiroteuthis, Grimalditeuthis), ( Joubiniteuthis, Mastigoteuthis))), Taonius), Moroteuthis)), (Rossia, Euprymna scolopes)), (Octopus, Grimpoteuthis)) (((((((duvauceli, noctiluca), ((etheridgei, chinensis), japonica)), (forbesi, (vulgaris, reynaudi))), (((((opalescens, pealei), gahi), plei), brevis), bleekeri), ((mercatoris, subulata), (lessoniana, sepioidea))), (australis, Joubiniteuthis), ((Rossia, Ctenopteryx), Helicocranchia), Moroteuthis, Taonius, (Chiroteuthis, Grimalditeuthis, Mastigoteuthis)), (Euprymna sp., Euprymna scolopes)), (Octopus, Grimpoteuthis)) ((((((((((duvauceli, (etheridgei, chinensis)), japonica), noctiluca), (mercatoris, subulata)), (forbesi, (vulgaris, reynaudi))), (((((opalescens, pealei), gahi), plei), brevis), bleekeri)), (lessoniana, (sepioidea, australis))), ((((Ctenopteryx, Helicocranchia), ((Chiroteuthis, Grimalditeuthis), ( Joubiniteuthis, Mastigoteuthis))), Taonius), Moroteuthis)), (Rossia, (Euprymna, Euprymna scolopes))), (Octopus, Grimpoteuthis)) (same as DNAs) ((((((((((((((duvauceli, (noctiluca, (hardwickei, affinis))), ((etheridgei, chinensis), ( japonica, (uyii, (sumatrensis, kobiensis)), beka))), edulis), sibogae), singhalensis, reesi, bartschi), (vossi, pickfordae)), (forbesi, (vulgaris, reynaudi))), ((opalescens, pealei, plei, gahi, ocula, roperi, surinamensis, sanpaulensis, ((brevis, panamensis), diomedeae)), bleekeri)), argus, mercatoris, ((subulata, media), africana), lessoniana, sepioidea, australis, Pickfordiateuthis pulchella, (Rossia, (Euprymna, Euprymna scolopes))), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis))), Ctenopteryx), Helicocranchia), Taonius), (Octopus, Grimpoteuthis)) ((((((((((((duvauceli, ((((etheridgei, chinensis), bartschi), edulis, sibogae), reesi)), (noctiluca, ((vossi, pickfordae), singhalensis))), ( japonica, uyii, beka)), ((argus, mercatoris), ((subulata, media), africana))), (forbesi, (vulgaris, reynaudi))), (((opalescens, ((brevis, panamensis), diomedeae)), (((pealei, ocula, surinamensis), (gahi, sanpaulensis), roperi), plei)), bleekeri)), (lessoniana, (sepioidea, australis))), (sumatrensis, kobiensis, (hardwickei, affinis))), ((Ctenopteryx, (Helicocranchia, Taonius)), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis)))), Pickfordiateuthis pulchella), (Rossia, (Euprymna, Euprymna scolopes))), (Octopus, Grimpoteuthis)) ((((((((((duvauceli, (etheridgei, chinensis), noctiluca, japonica), (forbesi, (vulgaris, reynaudi)),

COMBINED-DATA PHYLOGENETICS OF LOLIGINID SQUIDS

DMRg

DMAFe

DMAFs

DMARe

DMARs

633

(((((opalescens, pealei), gahi), plei), brevis), bleekeri), (mercatoris, subulata)), lessoniana, sepioidea), (Rossia, (Euprymna, Euprymna scolopes))), australis), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis))), Ctenopteryx), Helicocranchia), Taonius), (Octopus, Grimpoteuthis)) ((((((((((duvauceli, (etheridgei, chinensis)), noctiluca), japonica), (mercatoris, subulata)), (forbesi, (vulgaris, reynaudi))), (((((opalescens, pealei), gahi), plei), brevis), bleekeri)), (lessoniana, (sepioidea, australis))), ((Ctenopteryx, (Helicocranchia, Taonius)), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis)))), (Rossia, (Euprymna sp., Euprymna scolopes))), (Octopus, Grimpoteuthis)) ((((((((((duvauceli, (etheridgei, chinensis)), noctiluca), japonica), (mercatoris, subulata)), (forbesi, (vulgaris, reynaudi))), (((((opalescens, pealei), gahi), plei), brevis), bleekeri)), ((lessoniana, sepioidea), australis)), ((Ctenopteryx, (Helicocranchia, Taonius)), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis)))), (Rossia, (Euprymna sp., Euprymna scolopes))), (Octopus, Grimpoteuthis)) ((((((((((((((duvauceli, (noctiluca, (hardwickei, affinis))), ((etheridgei, chinensis), ( japonica, (uyii, (sumatrensis, kobiensis)), beka))), edulis), sibogae), singhalensis, reesi, bartschi), (vossi, pickfordae)), (forbesi, (vulgaris, reynaudi))), ((opalescens, pealei, plei, gahi, ocula, roperi, surinamensis, sanpaulensis, ((brevis, panamensis), diomedeae)), bleekeri)), argus, mercatoris, ((subulata, media), africana), lessoniana, sepioidea, australis, Pickfordiateuthis pulchella, (Rossia, (Euprymna, Euprymna scolopes))), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis))), Ctenopteryx), Helicocranchia), Taonius), (Octopus, Grimpoteuthis)) ((((((((((((((duvauceli, (((etheridgei, chinensis), ( japonica, (uyii, (sumatrensis, kobiensis)), beka, (hardwickei, affinis))), edulis, sibogae), singhalensis, reesi, bartschi), noctiluca, (vossi, pickfordae)), (forbesi, (vulgaris, reynaudi))), (((((opalescens, pealei), (gahi, sanpaulensis), ocula, roperi, surinamensis), plei), ((brevis, panamensis), diomedeae)), bleekeri)), ((argus, mercatoris), ((subulata, media), africana))), lessoniana), sepioidea), australis), (Pickfordiateuthis pulchella, (Rossia, (Euprymna, Euprymna scolopes)))), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis))), Ctenopteryx), Helicocranchia), Taonius), (Octopus, Grimpoteuthis)) (((((((((((((duvauceli, noctiluca), ((etheridgei, chinensis), japonica)), (forbesi, (vulgaris, reynaudi))), (((((opalescens, pealei), gahi), plei), brevis), bleekeri)), (mercatoris, subulata)), lessoniana, sepioidea), (Rossia, (Euprymna sp., Euprymna scolopes))), australis), (Moroteuthis, (((Chiroteuthis, Grimalditeuthis), Mastigoteuthis), Joubiniteuthis))), Ctenopteryx), Helicocranchia), Taonius), (Octopus, Grimpoteuthis))