Long Bone Histology of Sauropterygia from the Lower Muschelkalk of the Germanic Basin Provides Unexpected Implications for Phylogeny

Background

Sauropterygia is an abundant and successful group of Triassic marine reptiles. Phylogenetic relationships of Triassic Sauropterygia have always been unstable and recently questioned. Although specimens occur in high numbers, the main problems are rareness of diagnostic material from the Germanic Basin and uniformity of postcranial morphology of eosauropterygians. In the current paper, morphotypes of humeri along with their corresponding bone histologies for Lower to Middle Muschelkalk sauropterygians are described and interpreted for the first time in a phylogenetic context.

Methodology/Principal Findings

Nothosaurus shows a typical plesiomorphic lamellar-zonal bone type, but varying growth patterns and the occurrence of a new humerus morphotype point to a higher taxonomic diversity than was known. In contrast to the enormous morphological variability of eosauropterygian humeri not assigned to Nothosaurus, their long bone histology is relatively uniform and can be divided into two histotypes. Unexpectedly, both of these histotypes reveal abundant fibrolamellar bone throughout the cortex. This pushes the origin of fibrolamellar bone in Sauropterygia back from the Cretaceous to the early Middle Triassic (early Anisian). Histotype A is assigned to Cymatosaurus, a basal member of the Pistosauroidea, which includes the plesiosaurs as derived members. Histotype B is related to the pachypleurosaur Anarosaurus. Contrary to these new finds, the stratigraphically younger pachypleurosaur Neusticosaurus shows the plesiomorphic lamellar-zonal bone type and an incomplete endochondral ossification, like Nothosaurus.

Conclusions/Significance

Histological results hypothetically discussed in a phylogenetical context have a large impact on the current phylogenetic hypothesis of Sauropterygia, leaving the pachypleurosaurs polyphyletic. On the basis of histological data, Neusticosaurus would be related to Nothosaurus, whereas Anarosaurus would follow the pistosaur clade. Furthermore, the presence of fibrolamellar bone, which is accompanied with increased growth rates and presumably even with increased metabolic rates, already in Anarosaurus and Cymatosaurus can explain the success of the Pistosauroidea, the only sauropterygian group to survive into the Jurassic and give rise to the pelagic plesiosaur radiation.     

Life‐history strategies indicate live‐bearing in Nothosaurus (Sauropterygia)

In Sauropterygia, a diverse group of Mesozoic marine reptiles, fossil evidence of viviparity (live‐bearing) only exists for Pachypleurosauria and Plesiosauria, and was assumed to also be the case for nothosaurs. Previous studies have successfully applied an extant squamate model to sauropterygian life‐history traits. In extant squamates, oviparity and viviparity are associated with differences in life‐history trait combinations. We establish growth curves for Nothosaurus specimens based on their humeral histology. We then analyse life‐history traits derived from these curves and compare inferred traits to those of modern squamates and pachypleurosaurs to assess their reproduction mode. We show that birth to adult size ratios (i.e. birth size divided by the mother's size) provide good estimates of clutch sizes in extant squamates and in viviparous extinct marine reptiles, but these ratios cannot discriminate viviparous and oviparous squamates. Thus, large ratios do not indicate viviparity in fossil taxa to which the extant squamate model is applicable. Applying differences in birth size, age at maturation, and maximum longevity that are observed between extant viviparous and oviparous squamates to our Nothosaurus sample, we identified 7 out of 24 specimens as being potentially viviparous. Conversely, they suggested oviparity for many nothosaurs but also for many pachypleurosaur samples. Under the assumption that the entire clade Pachypleurosauria was viviparous, the majority of nothosaurs would also have been viviparous as they comprised trait combinations similar to those seen in pachypleurosaurs. Overall, this suggests that within nothosaurs and pachypleurosaurs both reproduction modes existed in different taxa.     

über Nothosauria (Sauropterygia,Reptilia) -ein Diskussionsbeitrag

The publication of K. Tschanz (1989):“Lariosaurus buzzii n. sp. from the Middle Triassic of Monte San Giorgio (Switzerland) with comments on the classification of nothosaurs” is reviewed. The results are as follows:

1. Lariosaurus buzzii n. sp. is not a member of the genusLariosaurus, as Tschanz pretends. Therefore a new genusSilvestrosaurus is proposed.
2. Lariosaurus andCeresiosaurus ar not closely related to each other.Ceresiosaurus is not a member of the Lariosauridae, but it requires a separate family, the Ceresiosauridae.
3. The contribution of Tschanz to the classification of the Nothosauria provides no new insight into this group; on the contrary it culminates in an unacceptable diagnosis of the Lariosauridae.

     

The Evolution of Marine Reptiles

Reptiles have repeatedly invaded marine environments despite their physiological constraints as air breathers. Marine reptiles were especially successful in the Mesozoic as major predators in the sea. There were more than a dozen groups of marine reptiles in the Mesozoic, of which four had more than 30 genera, namely sauropterygians (including plesiosaurs), ichthyopterygians, mosasaurs, and sea turtles. Medium-sized groups, such as Thalattosauria and Thalattosuchia, had about ten genera, whereas small groups, such as Hupehsuchia and Pleurosauridae, consisted of only two genera or less. Sauropterygia and Ichthyopterygia were the two longest surviving lineages, with 185 and 160 million years of stratigraphic spans, respectively. Mesozoic marine reptiles explored many different swimming styles and diets. Their diet included fish, cephalopods, other vertebrates, and hard-shelled invertebrates, whereas no herbivore is known at this point. Sauropterygians and ichthyopterygians gave rise to cruising forms that probably invaded outer seas. Intermediate forms that led to the cruising species are known in Ichthyopterygia but not as much in Sauropterygia. Discovery of new fossils should eventually reduce the gap in the fossil record.     

Postcranial morphology and growth of the pachypleurosaur Anarosaurus heterodontus (Sauropterygia) from the Lower Muschelkalk of Winterswijk, The Netherlands

Sauropterygia from the Muschelkalk are only found in lag deposits known as bone beds, and most of the material consists of isolated bones. Alpha taxonomy of Sauropterygia from the Germanic Basin which include Pachypleurosauria is thus based mainly on skull morphology of a few specimens. Articulated or associated postcranial material of pachypleurosaurs, associated with diagnostic skull material, is very rare in the Germanic Basin and currently occurs in larger numbers only in the Lower Muschelkalk of Winterswijk (Gelderland Province, The Netherlands), which continuously produces new material. For the first time, the morphology of several partially articulated skeletons of the pachypleurosaur Anarosaurus heterodontus is described and compared. Some of those specimens have skull material attached; others were identified as pachypleurosaurs on the basis of their long bone histology. The current study revealed that postcranial bones of A.?heterodontus feature a diverse morphology reflecting differences during ontogeny. Thus, A.?heterodontus specimens could be assigned to size classes (I?CIII). However, on the basis of morphology, histology, and maximal known size of isolated skulls and humeri, none of these specimens represent fully grown individuals. Growth mark counts of midshaft-femur samples, morphologically assigned to size class?III, document that this size class was reached within the first year of life. Size class?III continued into the second year of life, and then afterwards skeletal maturity was reached. Thus, a juvenile A.?heterodontus grew very fast, which is also indicated by its bone tissue type, composed of a high number of radial vascular canals and a fast-deposited bone matrix. The assignment of isolated bones from Lower to Middle Muschelkalk localities to A.?heterodontus is now possible with an extensive amended diagnosis of this taxon. This largely contributes to the understanding of taxonomical diversity and distribution. Morphological comparison of the postcranial skeleton of A.?heterodontus with that of the two other valid pachypleurosaurs from the Germanic Basin, Anarosaurus pumilio and Dactylosaurus, supports their close phylogenetic relationship. Furthermore, the skeleton of A.?heterodontus has no morphological or histological aquatic adaptation such as pachyostosis or pachyosteosclerosis and thus represents the least degree of aquatic adaptation within Pachypleurosauria.     

Reptile phylogeny and the interrelationships of turtles

A comprehensive analysis of amniote interrelationships is presented in an attempt to test turtle interrelationships. The results refute earlier hypotheses that turtles are related to parareptiles, i.e. to procolophonids or pareiasaurs. Instead, turtles are shown to be the sister-group of Sauropterygia, the two clades being nested within Sauria as sister-group of Lepidosauriformes. This scenario is also supported by several developmental and soft tissue characters which are shown to be congruent with the current phylogeny. The analysis strongly supports a monophyletic Parareptilia, sister-group of a monophylctic Eurcptilia. The Diapsida, however, is paraphyletic unless it includes turtles and sauropterygians. Additionally, the position of turtles within Diapsida has major implications for the evolutionary history and/or significance of many characters, i.e. temporal fenestration.     

Tooth implantation and replacement in Sauropterygia

Tooth replacement and implantation of Sauropterygia is described with special reference to the generaPlacodus andNothosaurus. Tooth replacement is horizontal, with the exception of the enlarged crushing tooth plates on the maxilla, palatine, and dentary ofPlacodus (placodonts), which are replaced vertically. Tooth implantation is thecodont, with variable ankylosis of the base of the root. Sauropterygia is unique compared to other reptiles in that replacement teeth are “alveolarized.” The alveolarization of replacement teeth adds to the evidence supporting of a monophyletic Sauropterygia (Placodontia plus Eosauropterygia).      

The status ofAnarosaurus multidentatus von Huene (Reptilia,Sauropterygia), from the lower anisian of the lechtaler alps (Arlberg,Austria)

The holotype ofAnarosaurus multidentatus von Huene (1958) from the lowermost Anisian of the Austrian Alps is redescribed and referred to the genusCymatosaurus. Eusauropterygian characters shared byA. multidentatus are the elongated and broadened Symphysis, and the contours of the lower jaw suggesting the presence of a constricted snout. Morphological details of the lower jaw Symphysis showA. multidentatus to be close toCymatosaurus among stem-group sauropterygians.Cymatosaurus multidentatus is the only representative of its genus known so far from the Alpine Triassic, and it represents the earliest known sauropterygian from the Alpine Triassic.     

Placement of attine ant-associated Pseudonocardia in a global Pseudonocardia phylogeny (Pseudonocardiaceae, Actinomycetales): a test of two symbiont-association models

We reconstruct the phylogenetic relationships within the bacterial genus Pseudonocardia to evaluate two models explaining how and why Pseudonocardia bacteria colonize the microbial communities on the integument of fungus-gardening ant species (Attini, Formicidae). The traditional Coevolution-Codivergence model views the integument-colonizing Pseudonocardia as mutualistic microbes that are largely vertically transmitted between ant generations and that supply antibiotics that specifically suppress the garden pathogen Escovopsis. The more recent Acquisition model views Pseudonocardia as part of a larger integumental microbe community that frequently colonizes the ant integument from environmental sources (e.g., soil, plant material). Under this latter model, ant-associated Pseudonocardia may have diverse ecological roles on the ant integument (possibly ranging from pathogenic, to commensal, to mutualistic) and are not necessarily related to Escovopsis suppression. We test distinct predictions of these two models regarding the phylogenetic proximity of ant-associated and environmental Pseudonocardia. We amassed 16S-rRNA gene sequence information for 87 attine-associated and 238 environmental Pseudonocardia, aligned the sequences with the help of RNA secondary structure modeling, and reconstructed phylogenetic relationships using a maximum-likelihood approach. We present 16S-rRNA secondary structure models of representative Pseudonocardia species to improve sequence alignments and identify sequencing errors. Our phylogenetic analyses reveal close affinities and even identical sequence matches between environmental Pseudonocardia and ant-associated Pseudonocardia, as well as nesting of environmental Pseudonocardia in subgroups that were previously thought to be specialized to associate only with attine ants. The great majority of ant-associated Pseudonocardia are closely related to autotrophic Pseudonocardia and are placed in a large subgroup of Pseudonocardia that is known essentially only from cultured isolates (rather than cloned 16S sequences). The preponderance of the known ant-associated Pseudonocardia in this latter clade of culturable lineages may not necessarily reflect abundance of these Pseudonocardia types on the ants, but isolation biases when screening for Pseudonocardia (e.g., preferential isolation of autotrophic Pseudonocardia with minimum-nutrient media). The accumulated phylogenetic patterns and the possibility of isolation biases in previous work further erode support for the traditional Coevolution-Codivergence model and calls for continued revision of our understanding how and why Pseudonocardia colonize the microbial communities on the integument of fungus-gardening ant species.     

Postcranial morphology of a basal Pistosauroidea (Sauropterygia) from the Lower Muschelkalk of Winterswijk,The Netherlands

Two partial postcranial skeletons from the Lower Muschelkalk (early Anisian) of Winterswijk, The Netherlands, are described in detail. The specimens were assigned to basal Pistosauroidea, presumably to cf. Cymatosaurus or a closely related taxon. Cymatosaurus is currently the earliest member of the Pistosauroidea and is only known from skull material. Taxonomical assignment is based on humerus morphology and histology, and on morphological differences from other Sauropterygia (Nothosauria and Pachypleurosauria).     

Cryptic Genetic Diversity Is Paramount in Small-Bodied Amphibians of the Genus Euparkerella (Anura: Craugastoridae) Endemic to the Brazilian Atlantic Forest

Morphological similarity associated to restricted distributions and low dispersal abilities make the direct developing “Terrarana” frogs of the genus Euparkerella a good model for examining diversification processes. We here infer phylogenetic relationships within the genus Euparkerella, using DNA sequence data from one mitochondrial and four nuclear genes coupled with traditional Bayesian phylogenetic reconstruction approaches and more recent coalescent methods of species tree inference. We also used Bayesian clustering analysis and a recent Bayesian coalescent-based approach specifically to infer species delimitation. The analysis of 39 individuals from the four known Euparkerella species uncovered high levels of genetic diversity, especially within the two previously morphologically-defined E. cochranae and E. brasiliensis. Within these species, the gene trees at five independent loci and trees from combined data (concatenated dataset and the species tree) uncovered six deeply diverged and geographically coherent evolutionary units, which may have diverged between the Miocene and the Pleistocene. These six units were also uncovered in the Bayesian clustering analysis, and supported by the Bayesian coalescent-based species delimitation (BPP), and Genealogical Sorting Index (GSI), providing thus strong evidence for underestimation of the current levels of diversity within Euparkerella. The cryptic diversity now uncovered opens new opportunities to examine the origins and maintenance of microendemism in the context of spatial heterogeneity and/or human induced fragmentation of the highly threatened Brazilian Atlantic forest hotspot.     

Late Bashkirian and early Moscovian conodonts from the Naqing section,Luodian, Guizhou,South China

Late Bashkirian and early Moscovian conodonts are abundant and diverse at the Naqing section, South China. All conodont genera known to have numerous species in the late Bashkirian–early Moscovian are recorded here, including Declinognathodus, Diplognathodus, Gondolella, Idiognathodus, Idiognathoides, Mesogondolella, Neognathodus, and Neolochriea. At Naqing, many species of these genera provide a succession of conodont chronomorphoclines throughout the Bashkirian–Moscovian boundary interval. They demonstrate that deposition was remarkably continuous through the boundary interval, a major criterion for selecting a Global Stratotype Section and Point (GSSP). This paper describes the current state of knowledge for several of these chronomorphoclines, and also provides an updated range chart of conodonts recovered from the Naqing section and their correlation with other regions.The taxon that best matches the current concept for the base of the Moscovian Stage in its type region is the phylogenetic first occurrence of Diplognathodus ellesmerensis. An ancestral form with most of the characteristics of D. ellesmerensis occurs at Naqing. More specimens are needed to completely document the chronomorphocline, but because D. ellesmerensis is found worldwide — including that close to the base of the type Moscovian — its evolutionary first occurrence would provide an almost ideal GSSP to define the base of the international Moscovian Stage.     

Advances in Taxonomy of Genus Phoma: Polyphyletic Nature and Role of Phenotypic Traits and Molecular Systematics

Phoma is a highly polyphyletic genus with its unclear species boundaries. The conventional system of identification is functional but it has its limitations. Besides morphological studies, chemotaxonomy, secondary metabolite and protein profiling have been assessed for the classification and identification of these fungi. Molecular datasets have provided a better outlook towards the phylogenetic and evolutionary trends of Phoma. Molecular markers such as ITS-rDNA, tubulin, actin, translation elongation factor have been widely used by the taxonomists to demarcate species. However, outcomes gained up till now represent preliminary step towards the study of Phoma systematics and a combined approach would be beneficial in the understanding of this polyphyletic group members. Lately, on the base of molecular phylogeny of the type species of the seven Phoma sections a new teleomorph family, Didymellaceae has been established, besides the Phaeosphaeriaceae related to sect. Paraphoma anamorphs, and the Leptosphaeriaceae to sect. Heterospora anamorphs. The estimated ratio is about 70 % of the recognized Phoma-like species can be associated with the Didymellaceae ascomycetous family.     

Genome assembly and annotation at the chromosomal level of first Pleuronectidae: Verasper variegatus provides a basis for phylogenetic study of Pleuronectiformes

High quality genome is of great significance for the mining of biological information resources of species. Up to now, the genomic information of several important economic flatfishes has been well explained. All these fishes are eyes on left side-type, and no high-quality genome of eyes on right side-type species has been reported. In this study, we applied a combined strategy involving stLFR and Hi-C technologies to generate sequencing data for constructing the chromosomal genome of Verasper variegates, which belongs to Pleuronectidae with characteristic of eyes on right side. The size of genome of V. variegatus is 556 Mb. More than 97.2% of BUSCO genes were detected, and N50 lengths of the contigs and scaffolds reached 79.8 Kb and 23.8 Mb, respectively, demonstrating the outstanding completeness and sequence continuity of the genome. A total of 22,199 protein-coding genes were predicted in the assembled genome, and more than 95% of those genes could be functionally annotated. Meanwhile, the genomic collinearity, gene family and phylogenetic analyses of similar species in Pleuronectiformes were also investigated and portrayed for metamorphosis and benthic adaptation. Sex related genes mapping has also been achieved at the chromosome level. This study is the first chromosomal level genome of a Pleuronectidae fish (V. variegatus). The chromosomal genome assembly constructed in this work will not only be valuable for conservation and aquaculture studies of the V. variegatus but will also be of general interest in the phylogenetic and taxonomic studies of Pleuronectiformes.     

The mitochondrial genomes of Amphiascoides atopus and Schizopera knabeni (Harpacticoida: Miraciidae) reveal similarities between the copepod orders Harpacticoida and Poecilostomatoida

Members of subclass Copepoda are abundant, diverse, and—as a result of their variety of ecological roles in marine and freshwater environments—important, but their phylogenetic interrelationships are unclear. Recent studies of arthropods have used gene arrangements in the mitochondrial (mt) genome to infer phylogenies, but for copepods, only seven complete mt genomes have been published. These data revealed several within-order and few among-order similarities. To increase the data available for comparisons, we sequenced the complete mt genome (13,831 base pairs) of Amphiascoides atopus and 10,649 base pairs of the mt genome of Schizopera knabeni (both in the family Miraciidae of the order Harpacticoida). Comparison of our data to those for Tigriopus japonicus (family Harpacticidae, order Harpacticoida) revealed similarities in gene arrangement among these three species that were consistent with those found within and among families of other copepod orders. Comparison of the mt genomes of our species with those known from other copepod orders revealed the arrangement of mt genes of our Harpacticoida species to be more similar to that of Sinergasilus polycolpus (order Poecilostomatoida) than to that of T. japonicus. The similarities between S. polycolpus and our species are the first to be noted across the boundaries of copepod orders and support the possibility that mt-gene arrangement might be used to infer copepod phylogenies. We also found that our two species had extremely truncated transfer RNAs and that gene overlaps occurred much more frequently than has been reported for other copepod mt genomes.     

Phylogenetic and molecular evolution of the ADAM (A Disintegrin And Metalloprotease) gene family from Xenopus tropicalis, to Mus musculus, Rattus norvegicus, and Homo sapiens

ADAM (a disintegrin and metalloprotease) genes have been identified in various tissues and species, and recently associated with several important human diseases such as tumor and asthma. Although various biological processes have been known for the ADAM family in different species including fertilization, neurogenesis, infection and inflammation, little is known about its detailed phylogenetic and molecular evolutionary history. In this study, the ADAMs of Xenopus (Silurana) tropicalis, Mus musculus, Rattus norvegicus, and Homo sapiens were collected and analyzed by using the Bayesian analysis and gene synteny analysis to establish a comprehensive phylogenetic relationship and evolutionary drive of this gene family. It was found that there were more ADAMs in the two rodents than in the amphibian, suggesting an expansion of the ADAM gene family during the early evolution of mammals. All ADAMs from this expansion were retained in both the rodents, but other duplication events occurred subsequently in the two rodents, respectively, leading to the classification of rodent ADAMs as classes I, II and III. Moreover, these duplicated ADAM genes in the rodents were found to be driven by positive selection, which might be the major force to retain them in the genome. Importantly, it was also found that orthologs of ADAM3 and 5 have been lost in humans. These results not only provide valuable information of the evolution of ADAM genes, but may also help in understanding the role of ADAM genes in the pathobiology of relevant diseases.     

Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

Molecular systematics of the genus Troglophilus (Rhaphidophoridae,Orthoptera) in Turkey: mitochondrial 16S rDNA evidences

This study focuses on the evolutionary relationships among Turkish species of the cave cricket genus Troglophilus.Fifteen populations were studied for sequence variation in a fragment (543 base pairs) of the mitochondrial DNA (mtDNA) 16S rDNA gene (16S) to reconstruct their phylogenetic relationships and biogeographic history. Genetic data retrieved three main clades and at least three divergent lineages that could not be attributed to any of the taxa known for the area. Molecular time estimates suggest that the diversification of the group took place between the Messinian and the Plio-Pleistocene.     

Automatic selection of partitioning schemes for phylogenetic analyses using iterative k-means clustering of site rates

Background

Model selection is a vital part of most phylogenetic analyses, and accounting for the heterogeneity in evolutionary patterns across sites is particularly important. Mixture models and partitioning are commonly used to account for this variation, and partitioning is the most popular approach. Most current partitioning methods require some a priori partitioning scheme to be defined, typically guided by known structural features of the sequences, such as gene boundaries or codon positions. Recent evidence suggests that these a priori boundaries often fail to adequately account for variation in rates and patterns of evolution among sites. Furthermore, new phylogenomic datasets such as those assembled from ultra-conserved elements lack obvious structural features on which to define a priori partitioning schemes. The upshot is that, for many phylogenetic datasets, partitioned models of molecular evolution may be inadequate, thus limiting the accuracy of downstream phylogenetic analyses.

Results

We present a new algorithm that automatically selects a partitioning scheme via the iterative division of the alignment into subsets of similar sites based on their rates of evolution. We compare this method to existing approaches using a wide range of empirical datasets, and show that it consistently leads to large increases in the fit of partitioned models of molecular evolution when measured using AICc and BIC scores. In doing so, we demonstrate that some related approaches to solving this problem may have been associated with a small but important bias.

Conclusions

Our method provides an alternative to traditional approaches to partitioning, such as dividing alignments by gene and codon position. Because our method is data-driven, it can be used to estimate partitioned models for all types of alignments, including those that are not amenable to traditional approaches to partitioning.     

栝楼属基于核糖体ITS序列的系统发育分析

栝楼属（Trichosanthes）是葫芦科(Cucurbitaceae)中一个种类较多、药用价值较大的属。本文基于ITS序列分析了栝楼属16个种的系统发育关系。聚类分析表明：在组的划分上,具有分类争议的截叶组(sect. Truncata)与大苞组(sect. Involucraria)形成一大支,处于一亚分枝位置,不支持独立成组。在属内分类地位上,在核酸水平不支持贵州栝楼(T.guizhouensis)为一独立种,结合形态特征建议并入双边栝楼(T.rosthornii)。大方油栝楼(T.dafangensis)与小苞组(sect. Trichosanthes)成为姊妹群,处于一个向叶苞组(sect. Foliobracteola)过渡位置,结合其形态特征,建议作为小苞组(sect. Trichosanthes)成员。