The mitochondrial genome of the water spider Argyroneta aquatica (Araneae: Cybaeidae)

We sequenced nearly the entire mitochondrial genome of Argyroneta aquatica, a wholly underwater‐living spider, thereby enhancing the available genomic information for Arachnida. The confirmed sequences contained the complete set of known genes present in other metazoan mitochondrial genomes. However, the mitochondrial gene order of A. aquatica was distinctly different from that of the most distant Chelicerata Limulus polyphemus (Xiphosura), probably because of a series of gene translocations and/or inversions. Comparison of arachnid mitochondrial gene orders for the purpose of phylogenetic inference is only minimally useful, but provides a strong signal in closely related lineages. To test the basal relationships and the evolutionary pattern of tRNA gene rearrangements among Arachnida, phylogenetic analyses using amino acid sequences of the 13 protein‐coding genes were performed. An interesting feature, the five 135‐bp tandem repeats and two 363‐bp tandem repeats, was identified in the putative control region. Although control region tandem repeats have been reported in many other arachnid and metazoan species, this is the first time it has been described in spiders.     

Elongation factor-2: a useful gene for arthropod phylogenetics.

Robust resolution of controversial higher-level groupings within Arthropoda requires additional sources of characters. Toward this end, elongation factor-2 sequences (1899 nucleotides) were generated from 17 arthropod taxa (5 chelicerates, 6 crustaceans, 3 hexapods, 3 myriapods) plus an onychophoran and a tardigrade as outgroups. Likelihood and parsimony analyses of nucleotide and amino acid data sets consistently recovered Myriapoda and major chelicerate groups with high bootstrap support. Crustacea + Hexapoda (= Pancrustacea) was recovered with moderate support, whereas the conflicting group Myriapoda + Hexapoda (= Atelocerata) was never recovered and bootstrap values were always <5%. With additional nonarthropod sequences included, one indel supports monophyly of Tardigrada, Onychophora, and Arthropoda relative to molluscan, annelidan, and mammalian outgroups. New and previously published sequences from RNA polymerase II (1038 nucleotides) and elongation factor-1alpha (1092 nucleotides) were analyzed for the same taxa. A comparison of bootstrap values from the three genes analyzed separately revealed widely varying values for some clades, although there was never strong support for conflicting groups. In combined analyses, there was strong bootstrap support for the generally accepted clades Arachnida, Arthropoda, Euchelicerata, Hexapoda, and Pycnogonida, and for Chelicerata, Myriapoda, and Pancrustacea, whose monophyly is more controversial. Recovery of some additional groups was fairly robust to method of analysis but bootstrap values were not high; these included Pancrustacea + Chelicerata, Hexapoda + Cephalocarida + Remipedia, Cephalocarida + Remipedia, and Malaocostraca + Cirripedia. Atelocerata (= Myriapoda + Hexapoda) was never recovered. Elongation factor-2 is now the second protein-encoding, nuclear gene (in addition to RNA polymerase II) to support Pancrustacea over Atelocerata. Atelocerata is widely cited in morphology-based analyses, and the discrepancy between results derived from molecular and morphological data deserves greater attention.     

The book lungs of Scorpiones and Tetrapulmonata (Chelicerata, Arachnida): evidence for homology and a single terrestrialisation event of a common arachnid ancestor

The question of whether Arachnida (Chelicerata) conquered terrestrial habitats only once or several times is controversial. The key group in this respect is the Scorpiones. Several authors claim that they became terrestrial independently of other arachnid lineages. This argumentation uses two lines of evidence. One is that book lungs of scorpions and other arachnids are considered non-homologous because they occur on different segments. The other line is based on fossil evidence which suggests that early scorpions were aquatic, together with a putative sister group relationship between scorpions and the aquatic Eurypterida. To address this problem we undertook a comparative scanning electron microscopical and histological study of the book lungs of scorpions, amblypygids, uropygids, and mesothelid spiders. In addition, we included the book gills of a xiphosuran. We found several detailed similarities in the book lungs shared by all arachnid taxa studied. Based on these findings we conclude that arachnid book lungs are homologous. Furthermore, we suggest that the apomorphic book lungs of arachnids indicate a single terrestrialisation event in the stem lineage leading to Arachnida.     

Phylogeny of the arachnid order Opiliones (Arthropoda) inferred from a combined approach of complete 18S and partial 28S ribosomal DNA sequences and morphology

The phylogenetic relationships among the main evolutionary lines of the arachnid order Opiliones were investigated by means of molecular (complete 18S rDNA and the D3 region of the 28S rDNA genes) and morphological data sets. Equally and differentially weighted parsimony analyses of independent and combined data sets provide evidence for the monophyly of the Opiliones. In all the analyses, the internal relationships of the group coincide in the monophyly of the following main groups: Cyphophthalmi, Eupnoi Palpatores, Dyspnoi Palpatores, and Laniatores. The Cyphophthalmi are monophyletic and sister to a clade that includes all the remaining opilionid taxa (=Phalangida). Within the Phalangida the most supported hypothesis suggests that Palpatores are paraphyletic, as follows: (Eupnoi (Dyspnoi + Laniatores)), but the alternative hypothesis (Laniatores (Eupnoi + Dyspnoi)) is more parsimonious in some molecular data analyses. Relationships within the four main clades are also addressed. Evolution of some morphological characters is discussed, and plesiomorphic states of these characters are evaluated using molecular data outgroup polarization. Finally, Martens' hypothesis of opilionid evolution is assessed in relation to our results.     

A phylogenetic analysis of the arachnid orders based on morphological characters

Morphological evidence for resolving relationships among arachnid orders was surveyed and assembled in a matrix comprising 59 euchelicerate genera (41 extant, 18 fossil) and 202 binary and unordered multistate characters. Parsimony analysis of extant genera recovered a monophyletic Arachnida with the topology (Palpigradi (Acaromorpha (Tetrapulmonata (Haplocnemata, Stomothecata nom. nov. )))), with Acaromorpha containing Ricinulei and Acari, Tetrapulmonata containing Araneae and Pedipalpi (Amblypygi, Uropygi), Haplocnemata (Pseudoscorpiones, Solifugae) and Stomothecata (Scorpiones, Opiliones). However, nodal support and results from exploratory implied weights analysis indicated that relationships among the five clades were effectively unresolved. Analysis of extant and fossil genera recovered a clade, Pantetrapulmonata nom nov. , with the topology (Trigonotarbida (Araneae (Haptopoda (Pedipalpi)))). Arachnida was recovered as monophyletic with the internal relationships (Stomothecata (Palpigradi, Acaromorpha (Haplocnemata, Pantetrapulmonata))). Nodal support and exploratory implied weights indicated that relationships among these five clades were effectively unresolved. Thus, some interordinal relationships were strongly and/or consistently supported by morphology, but arachnid phylogeny is unresolved at its deepest levels. Alternative hypotheses proposed in the recent literature were evaluated by constraining analyses to recover hypothesized clades, an exercise that often resulted in the collapse of otherwise well-supported clades. These results suggest that attempts to resolve specific nodes based on individual characters, lists of similarities, evolutionary scenarios, etc., are problematic, as they ignore broader impacts on homoplasy and analytical effects on non-target nodes.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 150 , 221–265.     

REVIEW Evolution and systematics of the Chelicerata

After approximately 40 years of discussion about the question of whether the Arthropoda are a monophyletic or a paraphyletic group or even a polyphyletic assemblage of unrelated taxa, most morphologists, palaeontologists and molecular taxonomists agree that the Arthropoda are a monophylum. The Euarthropoda are composed of the Arachnomorpha and Mandibulata. Myriapods are usually considered to be mandibulates; however, new molecular data as well as some morphological characters show similarities which the Myriapoda share with the Chelicerata, suggesting that there is no taxon Antennata or Atelocerata. Chelicerata are usually considered to be the sister group of Trilobita or, more correctly, Trilobita branch off from the chelicerate stem line. The first adaptive radiation of the Chelicerata took place in the Cambrian. All extant and some extinct orders were present during the Carboniferous. Two systems are compared. It is suggested that the Chelicerata contain the Pantopoda and Euchelicerata. The Euchelicerata are divided into Xiphosura and terrestrial Arachnida. Scorpiones are considered to be the sister group of all other arachnids, the Lipoctena and these are further divided into the Megoperculata (Uropygi, Amblypygi, and Araneae) and Apulmonata (all other groups). The Acari are tentatively considered to be a monophylum and the sister group of the Ricinulei. However, the Actinotrichida and Anactinotrichida diverged early and therefore have had a long history of independent evolution.     

First molecular evidence for the existence of a Tardigrada + Arthropoda clade

The complete 18S rDNA gene sequence of Macrobiotus group hufelandi (Tardigrada) was obtained and aligned with 18S rDNA and rRNA gene sequences of 24 metazoans (mainly protostomes). Discrete character (maximum-parsimony) and distance (neighbor-joining) methods were used to infer their phylogeny. The evolution of bootstrap proportions with sequence length (pattern of resolved nodes, PRN) was studied to test the resolution of the nodes in neighbor-joining trees. The results show that arthropods are monophyletic. Tardigrades represent the sister group of arthropods (in parsimony analyses) or they are related with crustaceans (distance analysis and PRN). Arthropoda are divided into two main evolutionary lines, the Hexapoda + Crustacea line (weakly supported), and the Myriapoda + Chelicerata line. The Hexapoda + Crustacea line includes Pentastomida, but the internal resolution is far from clear. The Insecta (Ectognatha) are monophyletic, but no evidence for the monophyly of Hexapoda is found. The Chelicerata are a monophyletic group and the Myriapoda cluster close to Arachnida. Overall, the results obtained represent the first molecular evidence for a Tardigrada + Arthropoda clade. In addition, the congruence between molecular phylogenies of the Arthropoda from other authors and this obtained here indicates the need to review those obtained solely on morphological characters.      

Complete Mitochondrial Genomes of Carcinoscorpius rotundicauda and Tachypleus tridentatus (Xiphosura,Arthropoda) and Implications for Chelicerate Phylogenetic Studies

Horseshoe crabs (order Xiphosura) are often referred to as an ancient order of marine chelicerates and have been considered as keystone taxa for the understanding of chelicerate evolution. However, the mitochondrial genome of this order is only available from a single species, Limulus polyphemus. In the present study, we analyzed the complete mitochondrial genomes from two Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus tridentatus to offer novel data for the evolutionary relationship within Xiphosura and their position in the chelicerate phylogeny. The mitochondrial genomes of C. rotundicauda (15,033 bp) and T. tridentatus (15,006 bp) encode 13 protein-coding genes, two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. Overall sequences and genome structure of two Asian species were highly similar to that of Limulus polyphemus, though clear differences among three were found in the stem-loop structure of the putative control region. In the phylogenetic analysis with complete mitochondrial genomes of 43 chelicerate species, C. rotundicauda and T. tridentatus were recovered as a monophyly, while L. polyphemus solely formed an independent clade. Xiphosuran species were placed at the basal root of the tree, and major other chelicerate taxa were clustered in a single monophyly, clearly confirming that horseshoe crabs composed an ancestral taxon among chelicerates. By contrast, the phylogenetic tree without the information of Asian horseshoe crabs did not support monophyletic clustering of other chelicerates. In conclusion, our analyses may provide more robust and reliable perspective on the study of evolutionary history for chelicerates than earlier analyses with a single Atlantic species.     

Failure of the ILD to determine data combinability for slow loris phylogeny

Tests for incongruence as an indicator of among-data partition conflict have played an important role in conditional data combination. When such tests reveal significant incongruence, this has been interpreted as a rationale for not combining data into a single phylogenetic analysis. In this study of lorisiform phylogeny, we use the incongruence length difference (ILD) test to assess conflict among three independent data sets. A large morphological data set and two unlinked molecular data sets--the mitochondrial cytochrome b gene and the nuclear interphotoreceptor retinoid binding protein (exon 1)--are analyzed with various optimality criteria and weighting mechanisms to determine the phylogenetic relationships among slow lorises (Primates, Loridae). When analyzed separately, the morphological data show impressive statistical support for a monophyletic Loridae. Both molecular data sets resolve the Loridae as paraphyletic, though with different branching orders depending on the optimality criterion or character weighting used. When the three data partitions are analyzed in various combinations, an inverse relationship between congruence and phylogenetic accuracy is observed. Nearly all combined analyses that recover monophyly indicate strong data partition incongruence (P = 0.00005 in the most extreme case), whereas all analyses that recover paraphyly indicate lack of significant incongruence. Numerous lines of evidence verify that monophyly is the accurate phylogenetic result. Therefore, this study contributes to a growing body of information affirming that measures of incongruence should not be used as indicators of data set combinability.     

Morphology of locomotor appendages in Arachnida: evolutionary trends and phylogenetic implications

The morphological diversity of locomotor appendages in Arachnida is surveyed lo reconstruct phylogenetic relationships and discover evolutionary trends in form and function. The appendicular skeleton and musculature of representatives from the ten living arachnid orders ate described, and a system of homology is proposed. Character polarities are established through comparison with an outgroup. Limulus polyphemus Xiphosura). Cladistic analysis suggests that Arachnida is monophyletic and that absence of extensor muscles is a primitive condition. Extensors are primitively absent in Araneae. Amblypygi, Uropygi, Palpigradi, Ricinulei and Acari. Most similarities in the appendages of these orders are symplesiomorphic so that phylogenetic relationships among the 'extensorless' groups cannot be resolved solely on the basis of appendicular characters. Extensor muscles appear to have evolved once, and their presence is considered a synapomorphic feature of Opiliones, Scorpiones, Pseudoscorpiones and Solifugae. Solifugae lack extensors, but a parsimonious interpretation of other characters indicates that this is a secondary, derived condition. The phylogenetic relationships among these four orders are clarified by modifications of the patellotibial joint. Cladistic analysis indicates that Opiliones may be the sister group of the other three orders and that Scorpiones is the sister group of Pseudoscorpiones and Solifugae. Conclusions concerning phylogenetic relationships and evolutionary morphology presented here differ substantially from those of earlier studies on the locomotor appendages of Arachnida.     

Morphology of locomotor appendages in Arachnida: evolutionary trends and phylogenetic implications

The morphological diversity of locomotor appendages in Arachnida is surveyed lo reconstruct phylogenetic relationships and discover evolutionary trends in form and function. The appendicular skeleton and musculature of representatives from the ten living arachnid orders ate described, and a system of homology is proposed. Character polarities are established through comparison with an outgroup. Limulus polyphemus Xiphosura). Cladistic analysis suggests that Arachnida is monophyletic and that absence of extensor muscles is a primitive condition. Extensors are primitively absent in Araneae. Amblypygi, Uropygi, Palpigradi, Ricinulei and Acari. Most similarities in the appendages of these orders are symplesiomorphic so that phylogenetic relationships among the ‘extensorless’ groups cannot be resolved solely on the basis of appendicular characters. Extensor muscles appear to have evolved once, and their presence is considered a synapomorphic feature of Opiliones, Scorpiones, Pseudoscorpiones and Solifugae. Solifugae lack extensors, but a parsimonious interpretation of other characters indicates that this is a secondary, derived condition. The phylogenetic relationships among these four orders are clarified by modifications of the patellotibial joint. Cladistic analysis indicates that Opiliones may be the sister group of the other three orders and that Scorpiones is the sister group of Pseudoscorpiones and Solifugae. Conclusions concerning phylogenetic relationships and evolutionary morphology presented here differ substantially from those of earlier studies on the locomotor appendages of Arachnida.     

Molecular phylogeny of the major arthropod groups indicates polyphyly of crustaceans and a new hypothesis for the origin of hexapods

A phylogeny of the arthropods was inferred from analyses of amino acid sequences derived from the nuclear genes encoding elongation factor-1 alpha and the largest subunit of RNA polymerase II using maximum- parsimony, neighbor-joining, and maximum-likelihood methods. Analyses of elongation factor-1 alpha from 17 arthropods and 4 outgroup taxa recovered many arthropod clades supported by previous morphological studies, including Diplopoda, Myriapoda, Insecta, Hexapoda, Branchiopoda (Crustacea), Araneae, Tetrapulmonata, Arachnida, Chelicerata, and Malacostraca (Crustacea). However, counter to previous studies, elongation factor-1 alpha placed Malacostraca as sister group to the other arthropods. Branchiopod crustaceans were found to be more closely related to hexapods and myriapods than to malacostracan crustaceans. Sequences for RNA polymerase II were obtained from 11 arthropod taxa and were analyzed separately and in combination with elongation factor-1 alpha. Results from these analyses were concordant with those derived from elongation factor-1 alpha alone and provided support for a Hexapoda/Branchiopoda clade, thus arguing against the monophyly of the traditionally defined Atelocerata (Hexapoda + Myriapoda).      

An enigmatic, solifuge-like fossil arachnid from the Lower Carboniferous of Kamienna Göra (Intra-Sudetic Basin), Poland

A new arachnid (Chelicerata: Arachnida) from the Lower Carboniferous (Upper Viséan) Szczawno Formation of Kamienna Göra, Poland, is described asSchneidarachne saganii n. gen. et n. sp. Early Carboniferous arachnids are generally rare and this new fossil cannot be easily assigned to any of the known arachnid Orders. It shares a number of features with some members of the arachnid order Solifugae (camel spiders, sun spiders): namely large, forward-projecting, chelate chelicerae with dentate fixed and free fingers, a distinct median sulcus on the carapace and an interrupted ridge of tubercles on the dorsal opisthosoma set into a loosely-defïned median field. However, it lacks unequivocal autapomorphies of Solifugae. This fossil may be one of a growing number of stem-group Palaeozoic arachnids which lack the füll set of diagnostic characters seen in crown-group members of the various orders. Thus,Schneidarachne saganii could represent a basai member of the lineage leading up to modem solifuges.     

Comparative genomic study of arachnid immune systems indicates loss of beta‐1,3‐glucanase‐related proteins and the immune deficiency pathway

Analyses of arthropod genomes have shown that the genes in the different innate humoral immune responses are conserved. These genes encode proteins that are involved in immune signalling pathways that recognize pathogens and activate immune responses. These immune responses include phagocytosis, encapsulation of the pathogen and production of effector molecules for pathogen elimination. So far, most studies have focused on insects leaving other major arthropod groups largely unexplored. Here, we annotate the immune‐related genes of six arachnid genomes and present evidence for a conserved pattern of some immune genes, but also evolutionary changes in the arachnid immune system. Specifically, our results suggest that the family of recognition molecules of beta‐1,3‐glucanase‐related proteins (βGRPs) and the genes from the immune deficiency (IMD) signalling pathway have been lost in a common ancestor of arachnids. These findings are consistent with previous work suggesting that the humoral immune effector proteins are constitutively produced in arachnids in contrast to insects, where these have to be induced. Further functional studies are needed to verify this. We further show that the full haemolymph clotting cascade found in the horseshoe crab is retrieved in most arachnid genomes. Tetranychus lacks at least one major component, although it is possible that this cascade could still function through recruitment of a different protein. The gel‐forming protein in horseshoe crabs, coagulogen, was not recovered in any of the arachnid genomes; however, it is possible that the arachnid clot consists of a related protein, spätzle, that is present in all of the genomes.     

Phylogeny of eriphioid crabs (Brachyura,Eriphioidea) inferred from molecular and morphological studies

The evolutionary relationships of the brachyuran crab superfamily Eriphioidea, commonly known as stone or rubble crabs, are examined. Analysis of three mitochondrial (12S, 16S and COI) and two nuclear loci (18S and Histone 3) was carried out for 51 taxa representing the Carpilioidea, Dairoidea, Eriphioidea, Goneplacoidea, Parthenopoidea, Pilumnoidea, Portunoidea, Pseudozioidea and Xanthoidea. Phylogenetic analyses of molecular data used three methods of inference that recovered similar topologies with minor differences. Maximum parsimony analysis of 20 morphological characters taken from first zoeas of 11 species yielded two equally parsimonious trees and generally supported the molecular analyses. None of the analyses recovered Eriphioidea as monophyletic, and each of the eriphioid families represented by two or more taxa was shown to be polyphyletic in both molecular and larval analyses. This study indicates that the present classification based on adult morphology is incongruent with phylogenetic relationships and that the diagnostic characters the result of convergence (particularly in feeding morphology) rather than shared ancestry.     

A Congruence Study of Molecular and Morphological Data for Eutherian Mammals

The four orders of eutherian mammals which are traditionally placed in the superorder Archonta [Chiroptera (microbats and megabats), Dermoptera (flying lemurs), Primates (primates), and Scandentia (tree shrews)] are among the best-studied taxa of their infraclass from both the molecular and morphological perspectives. Nevertheless, the ordinal relationships of archontans remain unresolved. While morphological studies favor their monophyly, molecular investigations do not. To evaluate these opposing conclusions, parsimony analyses were conducted with three separate sets of DNA sequences from both the nuclear and mitochondrial genomes and one file of morphological data for archontans and other eutherian mammals. Statistical tests of character support and ordinal branching pattern differences documented that the three sets of DNA sequences and their results were homogeneous and congruent, thereby allowing for the combination of these data into one large matrix for further phylogenetic analysis. In contrast, these same tests revealed that the combined sequence and morphological files and their topologies were in strong conflict. Archontan monophyly was supported by the morphological evidence, but this arrangement was strongly rejected by the combined DNA sequences that favored instead a grouping of Dermoptera, Primates, and Scandentia with Lagomorpha (rabbits) and Rodentia (rodents). Resolution of these significant differences will require further evaluations about the homologies and evolutionary properties of the molecular and morphological characters and about the appropriateness of the chosen phylogenetic methods, as well as the incorporation of new comparative data from both sources.     

ARTHROPOD PHYLOGENY: A COMBINED APPROACH

Abstract— Ribosomal and ubiquitin protein coding sequence data are generated from 20 arthropods and five close relatives. These molecular data are combined with morphological characters derived from the literature to approach arthropod phylogenetics from the perspective of total evidence. Trilobita were included in the analysis through morphological comparison alone. The overall data strongly support arthropod monophyly. Recent molecular analyses which have yield different results are shown to have been based on few characters, few taxa or both. The most parsimonious explanation of the data is (Annelida + (Onychophora + (Trilobita + Chelicerata) + (Crustacea + (Myriapoda + Hexapoda))))). The data are largely concordant both internally among data sets and externally with previous cladistic anatomical analyses.     

The disunity of "Mysidacea" (Crustacea)

New studies on malacostracan relationships have drawn attention to issues concerning monophyly of the order Mysidacea, manifested in recent crustacean classifications that treat the taxon as two separate orders, Lophogastrida and Mysida. We present molecular phylogenies of these orders based on complete sequences of nuclear small-subunit ribosomal DNA (18S rRNA), and morphological evidence is used to revise the classification of the order Mysida to better reflect evolutionary history. A secondary structure model for 18S rRNA was constructed and used to assign putative stem and loop regions to two groups of partitions for phylogenetic analyses. Phylogenies were estimated by maximum-likelihood, Bayesian inference, and maximum-parsimony. The analyses gave strong support for three independently derived lineages, represented by three monophyletic groups, Lophogastrida, Stygiomysida, and Mysida. The family Petalophthalmidae is considered as sister group to the family Mysidae, and Boreomysinae and Rhopalophthalminae are the most early derived of the Mysidae. The tribes contained in the current classification of the subfamily Mysinae are not well-supported by either molecular data or morphology.     

A morphometrics‐based phylogeny of the temperate Gondwanan mite harvestmen (Opiliones,Cyphophthalmi, Pettalidae)

A phylogenetic estimation of the temperate Gondwanan mite harvestman family Pettalidae (Arachnida, Opiliones, Cyphophthalmi) was conducted using 143 morphological variables (59 raw and 84 scaled measurements) from 37 ingroup and 15 outgroup terminals. We used custom algorithms to do pairwise comparisons between characters and identify sets of dependent characters, which were collapsed using principal components analysis. We analysed the resulting data without discretization under the parsimony criterion. Monophyly or paraphyly of most groups suspected from previous molecular and morphological phylogenetic studies were recovered. Trees were optimized for monophyly of 20 different focus clades by varying character phylogenetic independence. This yielded a final tree with monophyly of 15 out of 20 focus clades, including the South African pettalids, which contains the troglomorphic species Speleosiro argasiformis Lawrence, 1931. Two of the remaining five clades were found paraphyletic, with the genera Aoraki, Rakaia, and Siro always being found polyphyeletic.     

The position of Cetacea within mammalia: phylogenetic analysis of morphological data from extinct and extant taxa

Knowledge of the phylogenetic position of the order Cetacea (whales, dolphins, and porpoises) within Mammalia is of central importance to evolutionary biologists studying the transformations of biological form and function that accompanied the shift from fully terrestrial to fully aquatic life in this clade. Phylogenies based on molecular data and those based on morphological data both place cetaceans among ungulates but are incongruent in other respects. Morphologists argue that cetaceans are most closely related to mesonychians, an extinct group of terrestrial ungulates. They have disagreed, however, as to whether Perissodactyla (odd-toed ungulates) or Artiodactyla (even-toed ungulates) is the extant clade most closely related to Cetacea, and have long maintained that each of these orders is monophyletic. The great majority of molecule-based phylogenies show, by contrast, not only that artiodactyls are the closest extant relatives of Cetacea, but also that Artiodactyla is paraphyletic unless cetaceans are nested within it, often as the sister group of hippopotamids. We tested morphological evidence for several hypotheses concerning the sister taxon relationships of Cetacea in a maximum parsimony analysis of 123 morphological characters from 10 extant and 30 extinct taxa. We advocate treating certain multistate characters as ordered because such a procedure incorporates information about hierarchical morphological transformation. In all most-parsimonious trees, whether multistate characters are ordered or unordered, Artiodactyla is the extant sister taxon of Cetacea. With certain multistate characters ordered, the extinct clade Mesonychia (Mesonychidae + Hapalodectidae) is the sister taxon of Cetacea, and Artiodactyla is monophyletic. When all fossils are removed from the analysis, Artiodactyla is paraphyletic with Cetacea nested inside, indicating that inclusion of mesonychians and other extinct stem taxa in a phylogenetic analysis of the ungulate clade is integral to the recovery of artiodactyl monophyly. Phylogenies derived from molecular data alone may risk recovering inconsistent branches because of an inability to sample extinct clades, which by a conservative estimate, amount to 89% of the ingroup. Addition of data from recently described astragali attributed to cetaceans does not overturn artiodactyl monophyly.