Moving towards a better understanding of iterative evolution: an example from the late Silurian Monograptidae (Graptolithina) of the Baltic Basin

Iterative evolution has proved a difficult evolutionary phenomenon to study and interpret. Inferences of causality vary from study to study and quantitatively based phylogenetic reconstruction has never been attempted. In an effort to better understand iterative evolution we employed stratocladistics, gap analysis and disparity analysis to study the case of the Monograptidae in the aftermath of the late Silurian Cyrtograptus lundgreni extinction event. Our combination of gap analytical and stratocladistic techniques allowed us to elucidate the evolutionary relationships between the studied taxa. Based on our stratocladistic results we recommend the generic reassignment of five monograptid taxa. The stratocladistic results, in conjunction with morphological disparity analysis suggest the presence of a persistent developmental potential for the emergence of iteratively evolving characters. This persistent potential appears to be limited by extrinsic ecological constraints, which would have relaxed in the aftermath of the C. lundgreni extinction event. Our findings indicate that iterative evolution in the late Silurian Monograptidae is a product of the interaction of both intrinsic and extrinsic constraints on the acquisition of the iteratively evolving character, with the exact causality being dependent on the particular character.     

Molecular phylogeny of the stromateoid fishes (Teleostei: Perciformes) inferred from mitochondrial DNA sequences and compared with morphology-based hypotheses

The phylogenetic relationships among 21 species of stromateoid fishes, representing five families and 13 genera, were reconstructed using 3263bp of mitochondrial DNA sequences, including the posterior half of the 16S rRNA and entire COI and Cytb genes. The resultant molecular phylogenies were compared with previous phylogenetic hypotheses inferred from morphological characters. Molecular phylogenetic trees were constructed using the maximum parsimony, maximum likelihood, and Bayesian methods. All three methods resulted in well-resolved trees with most nodes being supported by moderate to high support values. In contrast to previous morphological analyses, which resulted in non-monophyly of Centrolophidae, all three methods utilized for the present molecular analyses supported the monophyly of Centrolophidae, as well as the reciprocal monophyly of the other stromateoid families, previous morphological hypotheses being rejected by the Templeton and Shimodaira-Hasegawa tests. In addition, the three methods indicated a sister-group relationship between Ariommatidae and Nomeidae. The position of Tetragonuridae was, however, incongruent between the MP method and the ML and Bayesian methods, being placed in the most basal position of Stromateoidei in the former, but occupying a sister relationship to Stromateidae in the latter. Comparison of the molecular phylogenies to previous morphological hypotheses suggested that evolutionary changes in morphological characters have not occurred equally among the stromateoid lineages, the evolution of the centrolophids not having been accompanied by appreciable morphological changes, whereas other stromateoids have undergone considerable morphological changes during their evolutionary history. The molecular phylogenies also shed some light on the evolutionary pattern of the pharyngeal sac, two of the four types of sac corresponding to two main lineages of Stromateoidei. Some taxonomic implications were also discussed.     

Laying the foundations of evolutionary and systematic studies in crickets (Insecta,Orthoptera): a multilocus phylogenetic analysis

Orthoptera have been used for decades for numerous evolutionary questions but several of its constituent groups, notably crickets, still suffer from a lack of a robust phylogenetic hypothesis. We propose the first phylogenetic hypothesis for the evolution of crickets sensu lato, based on analysis of 205 species, representing 88% of the subfamilies and 71% tribes currently listed in the database Orthoptera Species File (OSF). We reconstructed parsimony, maximum likelihood and Bayesian phylogenies using fragments of 18S, 28SA, 28SD, H3, 12S, 16S, and cytb (~3600 bp). Our results support the monophyly of the cricket clade, and its subdivision into two clades: mole crickets and ant‐loving crickets on the one hand, and all the other crickets on the other (i.e. crickets sensu stricto). Crickets sensu stricto form seven monophyletic clades, which support part of the OSF families, “subfamily groups”, or subfamilies: the mole crickets (OSF Gryllotalpidae), the scaly crickets (OSF Mogoplistidae), and the true crickets (OSF Gryllidae) are recovered as monophyletic. Among the 22 sampled subfamilies, only six are monophyletic: Gryllotalpinae, Trigonidiinae, Pteroplistinae, Euscyrtinae, Oecanthinae, and Phaloriinae. Most of the 37 tribes sampled are para‐ or polyphyletic. We propose the best‐supported clades as backbones for future definitions of familial groups, validating some taxonomic hypotheses proposed in the past. These clades fit variously with the morphological characters used today to identify crickets. Our study emphasizes the utility of a classificatory system that accommodates diagnostic characters and monophyletic units of evolution. Moreover, the phylogenetic hypotheses proposed by the present study open new perspectives for further evolutionary research, especially on acoustic communication and biogeography.     

Phylogeny and evolution of calcareous sponges: monophyly of calcinea and calcaronea,high level of morphological homoplasy,and the primitive nature of axial symmetry

Because calcareous sponges are triggering renewed interest with respect to basal metazoan evolution, a phylogenetic framework of their internal relationships is needed to clarify the evolutionary history of key morphological characters. Morphological variation was scored at the suprageneric level within Calcispongia, but little phylogenetic information could be retrieved from morphological characters. For the main subdivision of Calcispongia, the analysis of morphological data weakly supports a classification based upon cytological and embryological characters (Calcinea/Calcaronea) rather than the older classification scheme based upon the aquiferous system (Homocoela/Heterocoela). The 18S ribosomal RNA data were then analyzed, both alone and in combination with morphological characters. The monophyly of Calcispongia is highly supported, but the position of this group with respect to other sponge lineages and to eumetazoan taxa is not resolved. The monophyly of both Calcinea and Calcaronea is retrieved, and the data strongly rejected the competing Homocoela/Heterocoela hypothesis. The phylogeny implies that characters of the skeleton architecture are highly homoplastic, as are characters of the aquiferous system. However, axial symmetry seems to be primitive for all Calcispongia, a conclusion that has potentially far-reaching implications for hypotheses of early body plan evolution in Metazoa.     

On the status of the Serranid fish genus Epinephelus: evidence for paraphyly based upon 16S rDNA sequence

Historically, attempts to elucidate evolutionary relationships among members of the genus Epinephelus (Teleostei: Serranidae), commonly known as groupers, have been hindered by the overwhelming number of species (98, sensu stricto), a pan global distribution, and the lack of morphological specializations traditionally used in ichthyological classification. To date, no comprehensive phylogenetic study, morphological or molecular, to evaluate the monophyly of this genus has been presented. In this study, previous hypotheses regarding the relationships among the American grouper species and the allied genera were evaluated by examination of mitochondrial DNA sequences of the 16S ribosomal DNA region. A 590-bp region of the 16S rDNA gene was amplified using a universal primer pair for 42 serranid species, including members of the genera Epinephelus, Mycteroperca, and Paranthias from the New World and selected Indo-Pacific congeners. Maximum-parsimony criteria and neighbor-joining analysis dispute the monophyly of the American Epinephelus species as previously hypothesized. The data support the monophyly of Cephalopholis only with the inclusion of the morphologically distinct Paranthias and the monophyly of Mycteroperca with the inclusion of the Indo-Pacific Anyperodon leucogrammicus.     

Phylogenetic Relationships Between the Orders Artiodactyla and Cetacea: A Combined Assessment of Morphological and Molecular Evidence

A character analysis of selected conservative morphological traits from extant and fossil artiodactyls and cetaceans was combined with a similar analysis of conservative nucleotide positions from the complete mitochondrial cytochrome b sequences of available extant artiodactyls, cetaceans, sirenians, perissodactyls, and other mammals. This combined analysis focuses on the evidence that supports conflicting hypotheses of artiodactyl monophyly, including the affinities of hippopotamids and the monophyly or paraphyly of odontocete cetaceans. Highly conserved morphological traits of the astragalus and deciduous dentition provide strong corroboration of artiodactyl monophyly, including extant and fossil hippopotamids. In contrast, cytochrome b gene sequences are incapable of confirming this monophyly, due to excessive homoplasy of nucleotide and amino acid traits within extant Eutheria. In like manner, highly conserved and uniquely derived morphological features of the skull and auditory regions provide robust corroboration of Odontoceti monophyly, including extant and fossil physeteroids. Several nucleotide similarities do exist between physeteroids and mysticetes; however, most are either silent third-position transversions or occur also in two or more odontocete families. We suggest that increased taxon sampling, combined with functional considerations of amino acids and their secondary structure in protein-coding genes, are essential requirements for the phylogenetic interpretations of molecules at higher taxonomic levels, especially when they conflict with well-supported hypotheses of mammalian phylogeny, corroborated by uniquely derived morphological traits from extant and fossil taxa.     

Probing the Monophyly of the Sphaeropleales (Chlorophyceae) Using Data From Five Genes

Molecular phylogenetic analyses have had a major impact on the classification of the green algal class Chlorophyceae, corroborating some previous evolutionary hypotheses, but primarily promoting new interpretations of morphological evolution. One set of morphological traits that feature prominently in green algal systematics is the absolute orientation of the flagellar apparatus in motile cells, which correlates strongly with taxonomic classes and orders. The order Sphaeropleales includes diverse green algae sharing the directly opposite (DO) flagellar apparatus orientation of their biflagellate motile cells. However, algae across sphaeroplealean families differ in specific components of the DO flagellar apparatus, and molecular phylogenetic studies often have failed to provide strong support for the monophyly of the order. To test the monophyly of Sphaeropleales and of taxa with the DO flagellar apparatus, we conducted a molecular phylogenetic study of 16 accessions representing all known families and diverse affiliated lineages within the order, with data from four plastid genes (psaA, psaB, psbC, rbcL) and one nuclear ribosomal gene (18S). Although single‐gene analyses varied in topology and support values, analysis of combined data strongly supported a monophyletic Sphaeropleales. Our results also corroborated previous phylogenetic hypotheses that were based on chloroplast genome data from relatively few taxa. Specifically, our data resolved Volvocales, algae possessing predominantly biflagellate motile cells with clockwise (CW) flagellar orientation, as the monophyletic sister lineage to Sphaeropleales, and an alliance of Chaetopeltidales, Chaetophorales, and Oedogoniales, orders having multiflagellate motile cells with distinct flagellar orientations involving the DO and CW forms.     

PHYLOGENETIC ANALYSIS OF THE MARINE AND FRESHWATER THALAS-SIOSIROID DIATOMS

The thalassiosiroid centric diatoms are distinguished by at least one synapomorphy, the strutted process or fultoportula. Variously classified as a family (Thalassiosiraceae) or an order (Thalassiosirales) among centric diatoms, it is generally conceded that the group of several hundred fossil and living species is monophyletic as a whole. There are two ecological groups of thalassiosiroids, marine and freshwater. It has been hypothesized, based on an ecletic, non-rigorous, evolutionary taxonomy perspective that both the marine and freshwater ecological groups are also monophyletic, but this hypothesis has never been tested in a rigorous framework. Likewise, the freshwater thalassiosiroid species have been grouped into several genera and subgenera using an evolutionary taxonomic approach, but these hypotheses have not fully been tested using cladistic analysis. Focusing mainly on freshwater species, but including at least one representative of each marine genus and one representative from each of several proposed subgeneric groupings of the genus Thalassiosira , we scored morphological characters for fossil and living marine and freshwater Thalassiosiraceae to test these hypotheses. Our cladistic results provide strong support for monophyly for the freshwater group, but it seems unlikely that the marine group is monophyletic. The cladistic results are corroborated to greater or lesser degrees by the fossil record. The implications for evolution in the group and for taxon sampling in molecular studies we are conducting will be discussed.     

EVOLUTION OF PARASITISM AMONG CLOSELY RELATED SPECIES: PHYLOGENETIC RELATIONSHIPS AND THE ORIGIN OF INQUILINISM IN GALL WASPS (HYMENOPTERA,CYNIPIDAE)

A new term, agastoparasitism, is proposed for parasitism among closely related species. Cynipid inquilines are typical agastoparasites. They cannot induce galls; instead their larvae live inside the galls formed by other cynipids. As in many other groups of agastoparasites, there are two competing hypotheses for the evolutionary origin of cynipid inquilines: either they arose from one of their cynipid hosts, and later radiated to exploit other gall-inducing cynipids (monophyletic origin), or they arose repeatedly, each inquiline from its host (polyphyletic origin). These hypotheses for the origin of cynipid inquilines were tested by a phylogenetic analysis of representative species of cynipid gall inducers and inquilines based on adult morphological characters. The analysis supported the monophyly of the inquilines and indicated an origin from gall inducers related to the genus Diastrophus, one of the current host groups. To examine whether the result of the analysis was influenced by convergent similarities among inquilines because of their similar mode of life, all putative apomorphies shared by some or all of the inquilines but not occurring in any of the gall inducers were removed. Despite this, the phylogenetic conclusions essentially remained the same, that is, the support for inquiline monophyly was not caused by convergent evolution. Based on these results, adaptive aspects of the evolutionary origin and maintenance of cynipid inquilinism are discussed, as well as general patterns in the evolution of agastoparasitism.     

Phylogeny and morphological evolution of the so‐called bougainvilliids (Hydrozoa,Hydroidolina)

We present phylogenetic analyses (parsimony, maximum likelihood and Bayesian inference) for 69 lineages of anthoathecate hydroids based on 18 morphological characters (12 proposed for the first time) plus mitochondrial (16S and COI) and nuclear (18S and 28S) molecular markers. This study aims to test the monophyly of the present concept of the family Bougainvilliidae, assessing its phylogenetic position within Hydroidolina. Our working hypothesis is used as a context for inferring the evolution of certain morphological characters, focusing on the exoskeleton. Our results shed light on some phylogenetic uncertainties within Hydroidolina, delimiting eight well‐supported linages, viz. Hydroidolina, Siphonophorae, Leptothecata, Aplanulata, Filifera II, Filifera III, Capitata and Pseudothecata taxon novum, the latter supported by four morphological synapomorphies. The monophyly of several families was not supported, viz. Bougainvilliidae, Cordylophoridae, Oceaniidae, Rathkeidae and Pandeidae. Some of the genera typically considered in Bougainvilliidae, including Bougainvillia, fell into the clade Pseudothecata, which is consistently reconstructed as the sister group of Leptothecata. We formally suggest that Dicoryne be removed from Bougainvilliidae and placed in the resurrected family Dicorynidae. The exoskeleton was a key feature in the diversification of Hydroidolina, especially with the transition from the bare hydranth to one completely enveloped within the exoskeleton. In this context, bougainvilliids exhibit several intermediate states in the development of the exosarc. Although the concatenated analysis unravels some interesting hypotheses, taxon sampling is still deficient and therefore more data are necessary for achieving a more complete understanding of the evolution and ecology of bougainvilliids and their allies.     

Testing Phylogenetic Hypotheses of the Subgenera of the Freshwater Crayfish Genus Cambarus (Decapoda: Cambaridae)

Background

The genus Cambarus is one of three most species rich crayfish genera in the Northern Hemisphere. The genus has its center of diversity in the Southern Appalachians of the United States and has been divided into 12 subgenera. Using Cambarus we test the correspondence of subgeneric designations based on morphology used in traditional crayfish taxonomy to the underlying evolutionary history for these crayfish. We further test for significant correlation and explanatory power of geographic distance, taxonomic model, and a habitat model to estimated phylogenetic distance with multiple variable regression.

Methodology/Principal Findings

We use three mitochondrial and one nuclear gene regions to estimate the phylogenetic relationships for species within the genus Cambarus and test evolutionary hypotheses of relationships and associated morphological and biogeographical hypotheses. Our resulting phylogeny indicates that the genus Cambarus is polyphyletic, however we fail to reject the monophyly of Cambarus with a topology test. The majority of the Cambarus subgenera are rejected as monophyletic, suggesting the morphological characters used to define those taxa are subject to convergent evolution. While we found incongruence between taxonomy and estimated phylogenetic relationships, a multiple model regression analysis indicates that taxonomy had more explanatory power of genetic relationships than either habitat or geographic distance.

Conclusions

We find convergent evolution has impacted the morphological features used to delimit Cambarus subgenera. Studies of the crayfish genus Orconectes have shown gonopod morphology used to delimit subgenera is also affected by convergent evolution. This suggests that morphological diagnoses based on traditional crayfish taxonomy might be confounded by convergent evolution across the cambarids and has little utility in diagnosing relationships or defining natural groups. We further suggest that convergent morphological evolution appears to be a common occurrence in invertebrates suggesting the need for careful phylogenetically based interpretations of morphological evolution in invertebrate systematics.     

A multi-locus analysis of phylogenetic relationships within cheilostome bryozoans supports multiple origins of ascophoran frontal shields

Phylogenetic relationships within the bryozoan order Cheilostomata are currently uncertain, with many morphological hypotheses proposed but scarcely tested by independent means of molecular analysis. This research uses DNA sequence data across five loci of both mitochondrial and nuclear origin from 91 species of cheilostome Bryozoa (34 species newly sequenced). This vastly improved the taxonomic coverage and number of loci used in a molecular analysis of this order and allowed a more in-depth look into the evolutionary history of Cheilostomata. Maximum likelihood and Bayesian analyses of individual loci were carried out along with a partitioned multi-locus approach, plus a range of topology tests based on morphological hypotheses. Together, these provide a comprehensive set of phylogenetic analyses of the order Cheilostomata. From these results inferences are made about the evolutionary history of this order and proposed morphological hypotheses are discussed in light of the independent evidence gained from the molecular data.Infraorder Ascophorina was demonstrated to be non-monophyletic, and there appears to be multiple origins of the ascus and associated structures involved in lophophore extension. This was further supported by the lack of monophyly within each of the four ascophoran grades (acanthostegomorph/spinocystal, hippothoomorph/gymnocystal, umbonulomorph/umbonuloid, lepraliomorph/lepralioid) defined by frontal-shield morphology. Chorizopora, currently classified in the ascophoran grade Hippothoomorpha, is phylogenetically distinct from Hippothoidae, providing strong evidence for multiple origins of the gymnocystal frontal shield type. Further evidence is produced to support the morphological hypothesis of multiple umbonuloid origins of lepralioid frontal shields, using a step-wise set of topological hypothesis tests combined with examination of multi-locus phylogenies.     

Molecular dating and phylogenetic relationships among Teiidae (Squamata) inferred by molecular and morphological data

We present a phylogenetic analysis of teiid lizards based on partitioned and combined analyses of 12S and 16S mitochondrial DNA sequences, and morphological and ultrastructural characters. There were some divergences between 12S and 16S cladograms, but phylogenetic analyses of the combined molecular data corroborated the monophyly of Tupinambinae, Teiinae, and "cnemidophorines", with high support values. The total combined analysis (molecules+morphology) produced similar results, with well-supported Teiinae and "cnemidophorines". We present an evolutionary scenario for the evolution of Teiidae, based on molecular dating of evolutionary events using Bayesian methods, ancestral areas analysis, the fossil record, the geographic distribution of genera, and environmental and geologic changes during the Tertiary. According to this scenario, (1) all current teiid genera, except Aspidoscelis, originated in isolation in South America; (2) most teiid genera originated during the Eocene, a period characterized by savanna expansion in South America; and (3) Cnemidophorus originated in South America, after which some populations dispersed to Central America during the Late Miocene.     

Insights into the phylogenetic relationships within Cixiidae (Hemiptera: Fulgoromorpha): cladistic analysis of a morphological dataset

Abstract.  According to the most recent classifications proposed, the planthopper family Cixiidae comprises three subfamilies, namely Borystheninae, Bothriocerinae and Cixiinae, the latter with 16 tribes. Here we examine morphological characters to present the first phylogenetic reconstructions within Cixiidae derived from a cladistic analysis. We scored 85 characters of the head, thorax, and male and female genitalia for 50 taxa representative of all cixiid subfamilies and tribes and for six outgroup taxa. Analyses were based on maximum parsimony – using both equally weighted and successive weighting procedures – and Bayesian inferences. The monophyly of most currently accepted tribes and subfamilies was investigated through Templeton statistical tests of alternative phylogenetic hypotheses. The cladistic analyses recover the monophyly of Cixiidae, the subfamily Bothriocerinae, and the tribes Pentastirini, Mnemosynini, and Eucarpiini. Successive weighting and Bayesian inference recover the monophyly of the tribe Gelastocephalini, but only Bayesian inference supports the monophyly of Semoniini. The relationships recovered support the groups [Stenophlepsini (Borystheninae + Bothriocerinae)] arising from the tribe Oecleini, and [Andini + Brixiidini + Brixiini (polyphyletic) + Bennini]. Templeton tests reject the alternative hypothesis of a monophyletic condition for the tribe Pintaliini as presently defined.     

Structural alignment of 18S and 28S rDNA sequences provides insights into phylogeny of Phytophaga (Coleoptera: Curculionoidea and Chrysomeloidea)

We performed a comparative study of partial rDNA sequences from a variety of Coleoptera taxa to construct an annotated alignment based on secondary structure information, which in turn, provides improved rRNA structure models useful for phylogenetic reconstruction. Subsequent phylogenetic analysis was performed to test monophyly and interfamilial relationships of the megadiverse plant feeding beetle group known as ‘Phytophaga’ (Curculionoidea and Chrysomeloidea), as well as to discover their closest relatives among the Cucujiformia. Parsimony and Bayesian analyses were performed based on the structural alignment of segments of 18S rRNA (variable regions V4‐V5, V7‐V9) and 28S rRNA (expansion segment D2). A total of 104 terminal taxa of Coleoptera were included: 96 species of Cucujiformia beetles, representing the families and most ‘subfamilies’ of weevils and chrysomeloids (Phytophaga), as well as several families of Cleroidea, Tenebrionoidea and Cucujoidea, and eight outgroups from three other polyphagan series: Scarabaeiformia, Elateriformia and Bostrichiformia. The results from the different methods of analysis agree — recovering the monophyly of the ‘Phytophaga’, including Curculionoidea and Chrysomeloidea as sister groups. The curculionoid and chrysomeloid phylogeny recovered from the aligned 18S and 28S rDNA segments, which is independent of morphological data, is in agreement with recent hypotheses or concepts based on morphological evidence, particularly with respect to familial relationships. Our results provide clues about the evolutionary origin of the phytophagan beetles within the megaclade Cucujiformia, suggesting that the sister group of ‘Curculionoidea + Chrysomeloidea’ is a clade of the ‘Cucujoidea’, represented in this study by species in Boganiidae, Erotylidae, Nitidulidae, Cucujidae and Silvanidae. The Coccinellidae and Endomychidae are not grouped with the latter, and the remaining terminal taxa are nested in Tenebrionoidea and Cleroidea. We propose that the combination of structurally aligned ribosomal RNA gene regions 18S (V4‐V5, V7‐V9) and 28S (D2) are useful in testing monophyly and resolving relationships among beetle superfamilies and families.     

Testing the monophyly of the New Zealand and Australian endemic family Conoesucidae Ross based on combined molecular and morphological data (Insecta: Trichoptera: Sericostomatoidea)

Conoesucidae (Trichoptera, Insecta) are restricted to SE Australia, Tasmania and New Zealand. The family includes 42 described species in 12 genera, and each genus is endemic to either New Zealand or Australia. Although monophyly has been previously assumed, no morphological characters have been proposed to represent synapomorphies for the group. We collected molecular data from two mitochondrial genes (16S and cytochrome oxidase I), one nuclear gene (elongation factor 1-α) (2237–2277 bp in total), and 12 morphological characters to produce the first phylogeny of the family. We combined the molecular and morphological characters and performed both a maximum parsimony analysis and a Bayesian analysis to test the monophyly of the family, and to hypothesize the phylogeny among its genera. The parsimony analysis revealed a single most parsimonious tree with Conoesucidae being a monophyletic taxon and sistergroup to the Calocidae. The Bayesian inference produced a distribution of trees, the consensus of which is supported with posterior probabilities of 100% for 15 out of 22 possible ingroup clades including the most basal branch of the family, indicating strong support for a monophyletic Conoesucidae. The most parsimonious tree and the tree from the Bayesian analysis were identical except that the ingroup genus Pycnocentria changed position by jumping to a neighbouring clade. Based on the assumption that the ancestral conoesucid species was present on both New Zealand and Australia, a biogeographical analysis using the dispersal-vicariance criteria demonstrated that one or two (depending on which of the two phylogenetic reconstructions were applied) sympatric speciation events took place on New Zealand prior to a single, late dispersal from New Zealand to Australia.     

Phylogenetics of the woodrat genus Neotoma (Rodentia: Muridae): implications for the evolution of phenotypic variation in male external genitalia

Interspecific morphological variation in animal genitalia has long attracted the attention of evolutionary biologists because of the role genital form may play in the generation and/or maintenance of species boundaries. Here we examine the origin and evolution of genital variation in rodents of the muroid genus Neotoma. We test the hypothesis that a relatively rare genital form has evolved only once in Neotoma. We use four mitochondrial and four nuclear markers to evaluate this hypothesis by establishing a phylogenetic framework in which to examine genital evolution. We find intron seven of the beta-fibrinogen gene to be a highly informative nuclear marker for the levels of differentiation that characterize Neotoma with this locus evolving at a rate slower than cytochrome b but faster than 12S. We estimate phylogenetic relationships within Neotoma using both maximum parsimony and maximum likelihood-based Bayesian methods. Our Bayesian and parsimony reconstructions differ in significant ways, but we show that our parsimony analysis may be influenced by long-branch attraction. Furthermore, our estimate of Neotoma phylogeny remains consistent across various data partitioning strategies in the Bayesian analyses. Using ancestral state reconstruction, we find support for the monophyly of taxa that possess the relatively rare genital form. However, we also find support for the independent evolution of the common genital form and discuss possible underlying developmental shifts that may have contributed to our observed patterns of morphological evolution.