The complete plastome of Panax stipuleanatus: Comparative and phylogenetic analyses of the genus Panax (Araliaceae)

Panax stipuleanatus (Araliaceae) is an endangered and medicinally important plant endemic to China. However, phylogenetic relationships within the genus Panax have remained unclear. In this study, we sequenced the complete plastome of P. stipuleanatus and included previously reported Panax plastomes to better understand the relationships between species and plastome evolution within the genus Panax. The plastome of P. stipuleanatus is 156,069 base pairs (bp) in length, consisting of a pair of inverted repeats (IRs, each 25,887 bp) that divide the plastome into a large single copy region (LSC, 86,126 bp) and a small single copy region (SSC, 8169 bp). The plastome contains 114 unigenes (80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes). Comparative analyses indicated that the plastome gene content and order, as well as the expansion/contraction of the IR regions, are all highly conserved within Panax. No significant positive selection in the plastid protein-coding genes was observed across the eight Panax species, suggesting the Panax plastomes may have undergone a strong purifying selection. Our phylogenomic analyses resulted in a phylogeny with high resolution and supports for Panax. Nine proteincoding genes and 10 non-coding regions presented high sequence divergence, which could be useful for identifying different Panax species.     

Chloroplast phylogenomic analyses maternal relationships among sections in the genus Populus

The genus Populus L. has been divided into five sections based on morphological characters, but the phylogenetic relationships among sections remain uncertain. Topological discrepancies have been reported between trees obtained using nuclear and plastid sequences. We selected nine chloroplast genomes from all five sections, including two new sequenced species in this study for analyses of maternal phylogenetic relationships in the genus Populus at the sectional level. Phylogenetic analyses were performed using various subsets of data, coding sequences, noncoding sequences, and different districts of the genome, yielding contradictory outcomes for various subsets. According to our phylogenetic analyses, (1) a robust maternal phylogenetic relationship among sections based on complete chloroplast genomes was obtained; (2) Sect. Tacamahaca can be divided into two clades based on maternally inherited loci, i.e. cladeⅠ, distributed in North America and northeast China, and cladeⅡ, distributed in southwest China; (3) SSC-noncoding regions revealed an inconsistent topology compared with all other subsets; (4) this discrepancy may be resulted from incomplete lineage sorting between species of Populus. We tested multiple partitioning schemes to resolve deep-level phylogenetic relationships in Populus, and complete noncoding subset is most recommended.     

The first complete plastome sequence of the basal asterid family Styracaceae (Ericales) reveals a large inversion

Plastome sequences are rich sources of information for resolving difficult phylogenetic relationships and provide genomic data for conservation studies. Here, the complete plastome sequence of Alniphyllum eberhardtii Guillaumin is reported, representing the first plastome of the basal asterid family Styracaceae (Ericales). The plastome is 155,384 bp in length and contains 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes, totaling 113 unique genes with 19 genes in the inverted repeat region. Unusual features of the plastome include the presence a large 20-kb inversion in the Large Single-Copy region, the pseudogenization of the accD gene, and the loss of the second intron from clpP. The 20-kb inversion includes 14 genes and has not been previously reported in other Ericales plastomes. Thirty-nine plastid simple sequence repeats (SSRs) that may provide genetic resources for the conservation of this economically import timber plant are characterized. Phylogenetic results inferred from ML and MP analyses of 66 plastid genes and 26 taxa reveal that the Styracaceae are sister to a clade including Actinidiaceae and Ericaceae and suggest that complete plastomes are likely to be very helpful in resolving the basal relationships among Ericales families, which have resisted resolution in smaller phylogenetic data sets.     

The first complete plastid genome of Burmannia disticha L. from the mycoheterotrophic monocot family Burmanniaceae

Burmanniaceae is one major group within the monocot order Dioscoreales that has not had its plastome sequenced. Members of Burmanniaceae are mostly achlorophyllous, although the genus Burmannia also includes autotrophs. Here, we report sequencing and analysis of the first Burmanniaceae plastid genome from Burmannia disticha L.. This plastome is 157,480 bp and was assembled as a circular sequence with the typical quadripartite structure of plant plastid genomes. This plastome has a regular number of potentially functional genes with a total of 111, including 78 protein coding genes, 4 ribosomal RNA (rRNA) genes, and 29 tRNA genes. The ratio of the total length of genic:intergenic DNA is 1.58:1, and the mean length of intergenic regions is 398 bp, the longest being 1918 bp. The overall GC content of the B. disticha plastome is 34.90%, and the IR regions in B. disticha are more GC rich (39.50%) than the LSC (32.30%) and SSC (28.80%) regions. Phylogenetic analysis of protein-coding sequences from plastomes of related species in the order Dioscoreales support a clade comprising Burmanniaceae and Dioscoreaceae. This phylogenetic placement is congruent with previous findings based on nuclear and mitochondrial evidence.     

Comparative analysis of plastomes in Oxalidaceae: Phylogenetic relationships and potential molecular markers

The wood sorrel family, Oxalidaceae, is mainly composed of annual or perennial herbs, a few shrubs, and trees distributed from temperate to tropical zones. Members of Oxalidaceae are of high medicinal, ornamental, and economic value. Despite the rich diversity and value of Oxalidaceae, few molecular markers or plastomes are available for phylogenetic analysis of the family. Here, we reported four new whole plastomes of Oxalidaceae and compared them with plastomes of three species in the family, as well as the plastome of Rourea microphylla in the closely related family Connaraceae. The eight plastomes ranged in length from 150,673 bp (Biophytum sensitivum) to 156,609 bp (R. microphylla). Genome annotations revealed a total of 129–131 genes, including 83–84 protein-coding genes, eight rRNA genes, 37 tRNA genes, and two to three pseudogenes. Comparative analyses showed that the plastomes of these species have minor variations at the gene level. The smaller plastomes of herbs B. sensitivum and three Oxalis species are associated with variations in IR region sizes, intergenic region variation, and gene or intron loss. We identified sequences with high variation that may serve as molecular markers in taxonomic studies of Oxalidaceae. The phylogenetic trees of selected superrosid representatives based on 76 protein-coding genes corroborated the Oxalidaceae position in Oxalidales and supported it as a sister to Connaraceae. Our research also supported the monophyly of the COM (Celastrales, Oxalidales, and Malpighiales) clade.     

Complete Plastid Genome Sequence of the Basal Asterid Ardisia polysticta Miq. and Comparative Analyses of Asterid Plastid Genomes

Ardisia is a basal asterid genus well known for its medicinal values and has the potential for development of novel phytopharmaceuticals. In this genus of nearly 500 species, many ornamental species are commonly grown worldwide and some have become invasive species that caused ecological problems. As there is no completed plastid genome (plastome) sequence in related taxa, we sequenced and characterized the plastome of Ardisia polysticta to find plastid markers of potential utility for phylogenetic analyses at low taxonomic levels. The complete A. polysticta plastome is 156,506 bp in length and has gene content and organization typical of most asterids and other angiosperms. We identified seven intergenic regions as potentially informative markers with resolution for interspecific relationships. Additionally, we characterized the diversity of asterid plastomes with respect to GC content, plastome organization, gene content, and repetitive sequences through comparative analyses. The results demonstrated that the genome organizations near the boundaries between inverted repeats (IRs) and single-copy regions (SCs) are polymorphic. The boundary organization found in Ardisia appears to be the most common type among asterids, while six other types are also found in various asterid lineages. In general, the repetitive sequences in genic regions tend to be more conserved, whereas those in noncoding regions are usually lineage-specific. Finally, we inferred the whole-plastome phylogeny with the available asterid sequences. With the improvement in taxon sampling of asterid orders and families, our result highlights the uncertainty of the position of Gentianales within euasterids I.     

Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication

The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has been implicated as an origin of replication in several species of plants. In the evening primrose, Oenothera, this site was found to vary greatly in size, with plastid genomes (plastomes) being readily distinguished. To determine whether plastome "strength" in transmission could be correlated with variation at oriB, the 16S rRNA-trnI spacer was sequenced from five plastomes. The size variation was found to be due to differential amplification (and deletion) of combinations of sequences belonging to seven families of direct repeats. From these comparisons, one short series of direct repeats and one region capable of forming a hairpin structure were identified as candidates for the factor that could be responsible for the differences between strong and weak plastome types. Ample sequence variation allowed phylogenetic inferences to be made about the relationships among the plastomes. Phylogenetic trees also could be constructed for most of the families of direct repeats. The amplifications and deletions of repeats that account for the size variation at oriB are proposed to have occurred through extensive replication slippage at this site.      

Phylogenomics and biogeography of Wisteria: Implications on plastome evolution among inverted repeat-lacking clade (IRLC) legumes

The genus Wisteria (Fabaceae) is disjunctly distributed in eastern Asian and eastern North American temperate deciduous forests, and it is widely cultivated around the world as spectacular garden plants. It is a member of inverted repeat-lacking clade (IRLC). The IRLC Species are characterized by the loss of an IR region in their plastomes, which has long been of great interest. In this research, we report whole plastome sequences from all four Wisteria species and a Wisteriopsis japonica, combining these with existing data to explore phylogenetic relationships and biogeography of Wisteria, as well as plastome evolution of IRLC species. Phylogenetic analyses recognized a clade containing Glycyrrhiza–Wisteriopsis–Wisteria as sister to the remaining genera of IRLC. North American Wisteria frutescens and the three Asian species formed reciprocal clades, and Wisteria brachybotrys was sister to Wisteria floribunda and Wisteria sinensis. Wisteria may have originated in Japan near the boundary of the Oligocene and Miocene. The disappearance of Bering Land Bridge in the late Miocene might lead to the Eastern Asian–Eastern North American disjunction of Wisteria. Allopatric speciation of Wisteria between the Japanese archipelago and the Asian continent in the Quaternary increased the species richness of eastern Asia in comparison with eastern North America. Synonymous substitution rates (d_S) of protein-coding genes in the IRLC species were around 2-fold (SC genes) or 11-fold (IR genes) higher than those of non-IRLC species. For both SC and IR genes, herbaceous legumes have around 3-fold higher d_S than woody ones. Both loss of one IR region and herbaceous habit elevated substitution rates of the plastomes.     

Complete sequencing and comparative analyses of the pepper (Capsicum annuum L.) plastome revealed high frequency of tandem repeats and large insertion/deletions on pepper plastome

Plants in the family Solanaceae are used as model systems in comparative and evolutionary genomics. The complete chloroplast genomes of seven solanaceous species have been sequenced, including tobacco, potato and tomato, but not peppers. We analyzed the complete chloroplast genome sequence of the hot pepper, Capsicum annuum. The pepper chloroplast genome was 156,781 bp in length, including a pair of inverted repeats (IR) of 25,783 bp. The content and the order of 133 genes in the pepper chloroplast genome were identical to those of other solanaceous plastomes. To characterize pepper plastome sequence, we performed comparative analysis using complete plastome sequences of pepper and seven solanaceous plastomes. Frequency and contents of large indels and tandem repeat sequences and distribution pattern of genome-wide sequence variations were investigated. In addition, a phylogenetic analysis using concatenated alignments of coding sequences was performed to determine evolutionary position of pepper in Solanaceae. Our results revealed two distinct features of pepper plastome compared to other solanaceous plastomes. Firstly, large indels, including insertions on accD and rpl20 gene sequences, were predominantly detected in the pepper plastome compared to other solanaceous plastomes. Secondly, tandem repeat sequences were particularly frequent in the pepper plastome. Taken together, our study represents unique features of evolution of pepper plastome among solanaceous plastomes.     

Molecular phylogenetics of Trypanosomatidae: contrasting results from 18S rRNA and protein phylogenies

Phylogenetic analyses of the family Trypanosomatidae have been conducted using both 18S rRNA gene sequences and a variety of protein sequences. Using a variety of phylogenetic methods, 18S rRNA phylogenies indicate that the genus Trypanosoma is not monophyletic. Rather, they suggest that the American and African trypanosomes constitute distinct clades. By contrast, phylogenetic analyses of available sequences in 42 protein families gene generally supported monophyly of the genus Trypanosoma. One possible explanation for these conflicting results is poor taxon sampling in the case of protein coding genes, most of which have been sequenced for only a few species of Trypanosomatidae.     

The complete chloroplast genome of tall fescue (Lolium arundinaceum; Poaceae) and comparison of whole plastomes from the family Poaceae

In this paper, we describe the complete chloroplast genome of Lolium arundinaceum. This sequence is the culmination of a long-term project completed by >400 undergraduates who took general genetics at Middle Tennessee State University from 2004-2007. It was undertaken in an attempt to introduce these students to an open-ended experiential/exploratory lesson to produce and analyze novel data. The data they produced should provide the necessary information for both phylogenetic comparisons and plastome engineering of tall fescue. The fescue plastome (GenBank FJ466687) is 136048 bp with a typical quadripartite structure and a gene order similar to other grasses; 56% of the plastome is coding region comprised of 75 protein-coding genes, 29 tRNAs, four rRNAs, and one hypothetical coding region (ycf). Comparisons of Poaceae plastomes reveal size differences between the PACC (subfamilies Panicoideae, Arundinoideae, Centothecoideae, and Chloridoideae) and BOP (subfamilies Bambusoideae, Oryzoideae, and Pooideae) clades. Alignment analysis suggests that several potentially conserved large deletions in previously identified intergenic length polymorphic regions are responsible for the majority of the size discrepancy. Phylogenetic analysis using whole plastome data suggests that fescue closely aligns with Lolium perenne. Some unique features as well as phylogenetic branch length calculations, however, suggest that a number of changes have occurred since these species diverged.     

质体系统发育基因组解析旋花科系统发育关系

旋花科是一个世界广布的类群,具有丰富的形态特征和重要的经济价值。然而,目前该科主要分支或族间的系统发育关系问题一直未解决。为解析旋花科内系统发育关系,该研究代表性选取旋花科内8个族40个物种,基于质体全基因组数据,使用最大似然法和贝叶斯推论进行系统发育分析。结果表明：(1)旋花科质体基因组均为四分体结构,质体基因组大小为113 273～164 112 bp,蛋白质编码基因数目为66～79个。(2)基于五种DNA矩阵(即WCG、CDS、LSC、IR、SSC)的系统发育分析结果显示,WCG矩阵和CDS矩阵的拓扑结构基本一致,仅少数分支的支持率略有差异;LSC矩阵和WCG矩阵的拓扑结构差异在于菟丝子族、马蹄金族和盐帚花族的系统位置;AU检验和SH检验结果显示,WCG矩阵和SSC矩阵与IR矩阵的拓扑结构有显著冲突。(3)所有系统发育分析结果均显示,菟丝子属和马蹄金族都包括在旋花亚科内,应处理为族等级。(4)基于WCG矩阵和CDS矩阵较好地解决了旋花科8个族之间的系统发育关系,即心被藤族和丁公藤族聚为一支,最先从旋花亚科分化出来,随后是菟丝子族,剩下的5个族分成2个分支。(5)系统发育基因组分析...     

Phylogeny of Malpighiaceae: evidence from chloroplast ndhF and trnl-F nucleotide sequences

The Malpighiaceae are a family of ～1250 species of predominantly New World tropical flowering plants. Infrafamilial classification has long been based on fruit characters. Phylogenetic analyses of chloroplast DNA nucleotide sequences were analyzed to help resolve the phylogeny of Malpighiaceae. A total of 79 species, representing 58 of the 65 currently recognized genera, were studied. The 3' region of the gene ndhF was sequenced for 77 species and the noncoding intergenic spacer region trnL-F was sequenced for 65 species; both sequences were obtained for the outgroup, Humiria (Humiriaceae). Phylogenetic relationships inferred from these data sets are largely congruent with one another and with results from combined analyses. The family is divided into two major clades, recognized here as the subfamilies Byrsonimoideae (New World only) and Malpighioideae (New World and Old World). Niedenzu's tribes are all polyphyletic, suggesting extensive convergence on similar fruit types; only de Jussieu's tribe Gaudichaudieae and Anderson's tribes Acmanthereae and Galphimieae are monophyletic. Fleshy fruits evolved three times in the family and bristly fruits at least three times. Among the wing-fruited vines, which constitute more than half the diversity in the family, genera with dorsal-winged samaras are fairly well resolved, while the resolution of taxa with lateral-winged samaras is poor. The trees suggest a shift from radially symmetrical pollen arrangement to globally symmetrical pollen at the base of one of the clades within the Malpighioideae. The Old World taxa fall into at least six and as many as nine clades.     

Complete chloroplast genome sequence of Magnolia kwangsiensis (Magnoliaceae): implication for DNA barcoding and population genetics

Here, we report a completely sequenced plastome using Illumina/Solexa sequencing-by-synthesis (SBS) technology. The plastome of Magnolia kwangsiensis Figlar & Noot. is 159?667 bp in length with a typical quadripartite structure: 88?030 bp large single-copy (LSC) and 18?669 bp small single-copy (SSC) regions, separated by two 26?484 bp inverted repeat (IR) regions. The overall predicted gene number is 129, among which 17 genes are duplicated in IR regions. The plastome of M. kwangsiensis is identical in its gene order to previously published plastomes of magnoliids. Furthermore, the C-to-U type RNA editing frequency of 114 seed plants is positively correlated with plastome GC content and plastome length, whereas plastome length is not correlated with GC content. A total of 16 potential putative barcoding or low taxonomic level phylogenetic study markers in Magnoliaceae were detected by comparing the coding and noncoding regions of the plastome of M. kwangsiensis with that of Liriodendron tulipifera L. At least eight markers might be applied not only to Magnoliaceae but also to other taxa. The 86 mononucleotide cpSSRs that distributed in single-copy noncoding regions are highly valuable to study population genetics and conservation genetics of this endangered rare species.     

Plastome phylogenomic analysis of Torreya (Taxaceae)

Torreya Arn., a small genus of Taxaceae, consists of six species occurring in North America and eastern Asia. Several phylogenetic studies have previously been undertaken to reveal relationships within this genus, although only a few DNA segments or species were used. In the present study, we sequenced five Torreya plastomes and combined these with two existing plastomes from the genus to investigate plastome evolution and phylogenetic relationships within Torreya. All sequenced Torreya plastomes shared the same complement of 82 protein‐coding genes, 4 ribosomal RNA genes, and 31 transfer RNA genes. Phylogenetic inference using a maximum likelihood framework consisted of an 82‐gene, 17‐taxon dataset, including all species of Torreya, resolved Torreya as a monophyletic clade. Strongly supported relationships within the genus include the position of the early diverging T. jackii Chun, the two sister pairs T. fargesii Franch.–T. nucifera (L.) Siebold & Zucc. and T. grandis Fortune ex Lindl.–T. californica Torr., and the monophyly of the clade including T. fargesii var. yunnanensis, T. fargesii, and T. nucifera. In addition to the inference of species relationships, divergence time estimation and biogeographical analysis were carried out. The diversification of Torreya was estimated to be approximately 8.9 Ma. Ancestral state reconstruction of the geographical area suggested China/eastern North America as the most likely ancestral region for the six extant Torreya species.     

Testing and using complete plastomes and ribosomal DNA sequences as the next generation DNA barcodes in Panax (Araliaceae)

Complete plastid genome (plastome) sequences and nuclear ribosomal DNA (nrDNA) regions have been proposed as candidates for the next generation of DNA barcodes for plant species discrimination. However, the efficacy of this approach still lacks comprehensive evaluation. We carried out a case study in the economically important but phylogenetically and taxonomically difficult genus Panax (Araliaceae). We generated a large data set of plastomes and nrDNA sequences from multiple accessions per species. Our data improved the phylogenetic resolution and levels of species discrimination in Panax, compared to any previous studies using standard DNA barcodes. This provides new insights into the speciation, lineage diversification and biogeography of the genus. However, both plastome and nrDNA failed to completely resolve the phylogenetic relationships in the Panax bipinnatifidus species complex, and only half of the species within it were recovered as monophyletic units. The results suggest that complete plastome and ribosomal DNA sequences can substantially increase species discriminatory power in plants, but they are not powerful enough to fully resolve phylogenetic relationships and discriminate all species, particularly in evolutionarily young and complex plant groups. To gain further resolving power for closely related species, the addition of substantial numbers of nuclear markers is likely to be required.     

The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera, subsection Oenothera: I. sequence evaluation and plastome evolution

The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome–genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163 365 bp (plastome IV) to 165 728 bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsection-specific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I–III in one clade, while plastome IV appears to be closest to the common ancestor.     

Molecular markers for phylogenetic applications derived from comparative plastome analysis of Prunus species

Interspecific and intergeneric relationships of Prunus s.l. are still unclear due to low levels of genetic variation among species, and resulting partially unresolved phylogenetic inferences. Here we sequenced and compared six complete plastomes from two subgenera of Prunus in order to choose molecular markers to increase the amount of genetic variation suitable for inference of Prunus phylogeny. The plastomes range between 157 817 and 158 995 bp in length, and we found different levels of inverted repeat (IR) contraction among the three sampled subgenera of Prunus s.l. Most regions in Prunus plastomes considered individually provide low phylogenetic resolution at the subgenus or species level compared to a tree constructed using all 78 coding regions combined. We compared levels of variation among 206 coding regions and noncoding (intergenic and intron) plastid regions and inferred phylogenies from each region considered individually. We then chose using two regions together for future studies of relationships in Prunus, ycf1 and trnT-L, that display high to moderate levels of variation among coding and intergenic regions, respectively, and that individually permit inference of resolved species-level trees in Prunus with moderate to strong branch support. Considered together, these two regions allow inference of the same topology of Prunus inferred using all coding plastid regions combined, with comparable levels of tree support to the full plastome set. These two loci should therefore be useful as a plastid phylogenetic marker set for further inference of relationships within Prunus s.l.     

The Complete Chloroplast Genome of <Emphasis Type="Italic">Coix lacryma-jobi</Emphasis> and a Comparative Molecular Evolutionary Analysis of Plastomes in Cereals

Graminoid molecular evolution was investigated by chloroplast genome (plastome) scale analyses. A complete plastome from Coix lacryma-jobi (Poaceae) and a draft plastome from Joinvillea plicata (Joinvilleaceae) were sequenced and analyzed. The draft plastome included conserved protein-coding loci routinely analyzed in previous studies plus one additional locus of demonstrated phylogenetic utility. The methodological approach was to directly sequence overlapping amplicons from known plastome regions. Over 100 pairs of amplification and sequencing primers were designed and positioned to flank overlapping 1,200-base pair fragments around the entire plastome. Newly determined sequences were analyzed with published plastomes from representatives of Panicoideae, Ehrhartoideae, and Pooideae. Considerable variation was found for studies within the family and even within Andropogoneae. Readily interpreted mutation patterns were observed, such as small inversions in hairpin-loop regions and indels, which were common in intergenic spacers. Maximum or near-maximum bootstrap support was observed in all analyses resolving relationships between subfamilies. However, the addition of characters from noncoding regions increased the number of parsimony-informative characters and lengthened short internal branches (Andropogoneae), better defining intergeneric relationships. Thus, characters in complete plastomes can be used over a wide scope of phylogenetic studies.     

Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data

Entomopathogenic nematodes of the genus Steinernema are lethal parasites of insects that are used as biological control agents of several lepidopteran, dipteran and coleopteran pests. Phylogenetic relationships among 25 Steinernema species were estimated using nucleotide sequences from three genes and 22 morphological characters. Parsimony analysis of 28S (LSU) sequences yielded a well-resolved phylogenetic hypothesis with reliable bootstrap support for 13 clades. Parsimony analysis of mitochondrial DNA sequences (12S rDNA and cox 1 genes) yielded phylogenetic trees with a lower consistency index than for LSU sequences, and with fewer reliably supported clades. Combined phylogenetic analysis of the 3-gene dataset by parsimony and Bayesian methods yielded well-resolved and highly similar trees. Bayesian posterior probabilities were high for most clades; bootstrap (parsimony) support was reliable for approximately half of the internal nodes. Parsimony analysis of the morphological dataset yielded a poorly resolved tree, whereas total evidence analysis (molecular plus morphological data) yielded a phylogenetic hypothesis consistent with, but less resolved than trees inferred from combined molecular data. Parsimony mapping of morphological characters on the 3-gene trees showed that most structural features of steinernematids are highly homoplastic. The distribution of nematode foraging strategies on these trees predicts that S. hermaphroditum, S. diaprepesi and S. longicaudum (US isolate) have cruise forager behaviours.