INITIAL CHARACTERIZATION OF MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) CLASS IIB EXON 2 IN AN ENDANGERED RATTLESNAKE, THE EASTERN MASSASAUGA (SISTRURUS CATENATUS)

Genes of the major histocompatibility complex (MHC) play an important role in the vertebrate immune system and exhibit remarkably high levels of polymorphism, maintained by strong balancing selection. While the conservation implications of MHC variation have been explored in a variety of vertebrates, non-avian reptiles (most notably snakes) have received less attention. To address this gap and take the first steps toward more extensive population-level analyses, we cloned and sequenced MHC IIB exon 2 in an endangered rattlesnake, the Eastern Massasauga (Sistrurus catenatus). Based on three individuals, we found evidence of at least four putatively functional loci. These sequences exhibited relatively high levels of variation and significantly higher rates of nonsynonymous to synonymous substitutions, especially within the antigen-binding sites, indicating strong positive selection. Phylogenetic analysis revealed a pattern of trans-species polymorphism, also suggesting positive selection. These results contribute to our understanding of MHC variation in non-avian reptiles and form a basis for more studies of MHC variation in snakes of conservation concern

The Chloroplast Genome of Anomochloa Marantoidea (Anomochlooideae; Poaceae) Comprises a Mixture of Grass-like and Unique Features

Features in the complete plastome of Anomochloa marantoidea (Poaceae) were investigated. This species is one of four of Anomochlooideae, the crown node of which diverged before those of any other grass subfamily. The plastome was sequenced from overlapping amplicons using previously designed primers. The plastome of A. marantoidea is 138 412 bp long with a typical gene content for Poaceae. Five regions were examined in detail because of prior surveys that identifi ed structural alterations among graminoid Poales. Anomochloa marantoidea was found to have an intron in rpoC1 , unlike other Poaceae. The insertion region of rpoC2 is unusually short in A. marantoidea compared with those of other grasses, but with atypically long subrepeats. Both ycf1 and ycf2 are nonfunctional as is typical in grasses, but A. marantoidea has a uniquely long ψ ycf1. Finally, the rbcL - psaI spacer in A. marantoidea is atypically short with no evidence of the ψ rpl23 locus found in all other Poaceae. Some of these features are of noteworthy dissimilarity between A . marantoidea and those crown grasses for which entire plastomes have been sequenced. Complete plastome sequences of other Anomochlooideae and outgroups will further advance our understanding of the evolutionary events in the plastome that accompanied graminoid diversifi cation

Phylogenetics of Paniceae (Poaceae)

Paniceae demonstrate unique variability of photosynthetic physiology and anatomy, including both non-Kranz and Kranz species and all subtypes of the latter. This variability suggests hypotheses of independent origin or reversals (e.g., from C4 to C3). These hypotheses can be tested by phylogenetic analysis of independent molecular characters. The molecular phylogeny of 57 species of Paniceae was explored using sequences from the grass-specific insert found in the plastid locus rpoC2. Phylogenetic analyses confirmed some long-recognized alliances in Paniceae, some recent molecular phylogenetic results, and suggested new relationships. Broadly, Paniceae were found to be paraphyletic with Andropogoneae, Panicum was found to be polyphyletic, and Oplismenus hirtellus was resolved as the sister group to the remaining ingroup species. A particularly well-supported clade in the rpoC2 tree included four genera with non-Kranz species and three with distinctively keeled paleas. As previously suggested, the PCK (phosphoenol pyruvate carboxykinase) C4 subtype arose once within Paniceae. All clades with non-Kranz species had Kranz ancestors or sister taxa suggesting repeated loss of the Kranz syndrome

Phylogenetic Analyses of Two Mitochondrial Metabolic Genes Sampled in Parallel from Angiosperms Find Fundamental Interlocus Incongruence

Plant molecular phylogeneticists have supported an analytical approach of combining loci from different genomes, but the combination of mitochondrial sequences with chloroplast and nuclear sequences is potentially problematic. Low substitution rates in mitochondrial genes should decrease saturation, which is especially useful for the study of deep divergences. However, individual mitochondrial loci are insuffi ciently informative, so that combining congruent loci is necessary. For this study atp1 and cox1 were selected, which are of similar lengths, encode components of the respiratory pathway, and generally lack introns. Thus, these genes might be expected to have similar functional constraints, selection pressures, and evolutionary histories. Strictly parallel sampling of 52 species was achieved as well as six additional composite terminals with representatives from the major angiosperm clades. However, analyses of the separate loci produced strongly incongruent topologies. The source of the incongruence was investigated by validating sequences with questionable affi nities, excluding RNA-edited nucleotides, deleting taxa with unexpected phylogenetic associations, and comparing different phylogenetic methods. However, even after potential artifacts were addressed and sites and taxa putatively associated with confl ict were excluded, the resulting gene trees for the two mitochondrial loci were still substantially incongruent by all measures examined. Therefore, combining these loci in phylogenetic analysis may be counterproductive to the goal of fully resolving the angiosperm phylogeny

Placing the Monocots: Conflicting Signal from Trigenomic Analyses

Despite recent significant advances in understanding angiosperm phylogeny, the position of monocots remains uncertain. We present here a phylogeny inferred from four genes that unambiguously unite monocots with eumagnoliids. A well-supported position for the monocots was obtained only after we replaced the available nuclear 18S rDNA sequence data with data from phytochrome C in a matrix that also included plastid rbcL and ndhF and mitochondrial atp1. Over 5000 base pairs of sequence data from 42 taxa were analyzed using Bayesian inference. The results of these analyses united monocots with the eumagnoliids in a well-supported clade. Although the substitution of phytochrome C for 18S data led to a highly supported position for the monocots, comparison with more densely sampled single-gene studies revealed conflict among data sets. This indicates that larger data sets from each genome should be explored explicitly to evaluate the position of the monocots, and that each of these larger data sets also should be investigated for insight into potential sources of conflict

The floral transcriptomes of four bamboo species (Bambusoideae; Poaceae): support for common ancestry among woody bamboos

Background Next-generation sequencing now allows for total RNA extracts to be sequenced in non-model organisms such as bamboos, an economically and ecologically important group of grasses. Bamboos are divided into three lineages, two of which are woody perennials with bisexual flowers, which undergo gregarious monocarpy. The third lineage, which are herbaceous perennials, possesses unisexual flowers that undergo annual flowering events. Results Transcriptomes were assembled using both reference-based and de novo methods. These two methods were tested by characterizing transcriptome content using sequence alignment to previously characterized reference proteomes and by identifying Pfam domains. Because of the striking differences in floral morphology and phenology between the herbaceous and woody bamboo lineages, MADS-box genes, transcription factors that control floral development and timing, were characterized and analyzed in this study. Transcripts were identified using phylogenetic methods and categorized as A, B, C, D or E-class genes, which control floral development, or SOC or SVP-like genes, which control the timing of flowering events. Putative nuclear orthologues were also identified in bamboos to use as phylogenetic markers. Conclusions Instances of gene copies exhibiting topological patterns that correspond to shared phenotypes were observed in several gene families including floral development and timing genes. Alignments and phylogenetic trees were generated for 3,878 genes and for all genes in a concatenated analysis. Both the concatenated analysis and those of 2,412 separate gene trees supported monophyly among the woody bamboos, which is incongruent with previous phylogenetic studies using plastid markers.We thank R. Macias for access to his collection at the El Riscal bamboo plantation and A. Hernandez for aid in preparing samples for RNA sequencing. Sequencing and library preparation were supported by the Northern Illinois University Department of Biological Sciences and the Plant Molecular Biology Center. This project was also funded by NSF/NASA grant DEB1342782 to MRD. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. This article is made openly accessible in part by an award from the Northern Illinois University Libraries’ Open Access Publishing Fund

Complex Transitions Between C3 and C4 Photosynthesis During the Evolution of Paniceae: A Phylogenetic Case Study Emphasizing the Position of Steinchisma Hians (Poaceae), A C3-C4 Intermediate

A two-tiered, nested molecular phylogenetic study of panicoid grasses to explore character state transitions between the C3 and C4 adaptive syndromes is presented. A broad survey of 92 panicoid species was sampled for the grass-specific insert sequence in the chloroplast RNA polymerase locus (rpoC2), combining published and unpublished sequences. This portion of the study also included an intensive phylogenetic investigation of one clade of seven species that included Steinchisma hians, which is notable for exhibiting intermediacy between the C3 and C4 photosynthetic types. Both rpoC2 data and previously published sequences of the F subunit of an NADH-dependent dehydrogenase were analyzed together for this small group. A rigorous phylogenetic investigation of S. hians and 13 other species of Panicoideae included in the broad survey was then performed with sequences of both rpoC2 and the externally transcribed spacer region of the nuclear ribosomal repeat. These 14 species were selected to maximize representation among photosynthetic subtypes. Combined analysis resolved single origins of two photosynthetic subtypes. A reversion of C4 to C3 photosynthesis during the evolution of the lineage that includes S. hians is identified. These and other recent results indicate that repeated reversions from C4 to C3 have occurred. The C3 species Panicum laxum has a strongly supported sister group relationship to S. hians (C3-C4). The most parsimonious interpretation is that S. hians represents an incipient reversal from C3 to C4 photosynthesis, beginning with the capacity to compartmentalize photorespiratory metabolism in the bundle sheath tissue

Phylogenetically Widespread Multiple Paternity in New World Natricine Snakes

We used microsatellite DNA markers to identify the extent to which multiple paternity within litters occurs among species of New World natricine snakes. We selected seven species to represent the three major clades of Natricinae and all three subclades of the gartersnake clade. Microsatellite DNA genotyping of dams and litters confirmed multiple paternity within litters of six species, including Thamnophis radix, T. sauritus, Storeria dekayi, S. occipitomaculata, Nerodia rhombifer, and Regina septemvittata. Multiple paternity was not evident in one litter of nine Thamnophis melanogaster. Together with published data documenting multiple paternity in T. bulteri, T. elegans, T. sirtalis, and N. sipedon, these results confirm the phylogenetically widespread occurrence of multiple paternity among New World natricines, emphasizing the need to consider phylogenetic (historical) explanations when analyzing snake mating systems

Interaction Study of MADS-Domain Proteins in Tomato

MADS-domain proteins are important transcription factors involved in many biological processes of plants. Interactions between MADS-domain proteins are essential for their functions. In tomato (Solanum lycopersicum), the number of MIKCc-type MADSdomain proteins identified has totalled 36, but a largescale interaction assay is lacking. In this study, 22 tomato MADS-domain proteins were selected from six functionally important subfamilies of the MADS-box gene family, to create the first large-scale tomato protein interaction network. Compared with Arabidopsis and petunia (Petunia hybrida), protein interaction patterns in tomato displayed both conservation and divergence. The majority of proteins that can be identified as putative orthologues exhibited conserved interaction patterns, and modifications were mostly found in genes underlining traits unique to tomato. JOINTLESS and RIN, characterized for their roles in abscission zone development and fruit ripening, respectively, showed enlarged interaction networks in comparison with their Arabidopsis and petunia counterparts. Novel interactions were also found for members of the expanded subfamilies, such as those represented by AP1/FUL and AP3/PI MADS-domain proteins. In search for higher order complexes, TM5 was found to be the preferred bridge among the five SEP-like proteins. Additionally, 16 proteins with the MADS-domain removed were used to assess the role of the MADS-domain in protein–protein interactions. The current work provides important knowledge for further functional and evolutionary study of the MADSbox genes in tomato

Cell Wall Enzymes in Zygnema circumcarinatum UTEX 1559 Respond to Osmotic Stress in a Plant-Like Fashion

Fitzek E, Orton L, Entwistle S, et al. Cell Wall Enzymes in Zygnema circumcarinatum UTEX 1559 Respond to Osmotic Stress in a Plant-Like Fashion. FRONTIERS IN PLANT SCIENCE. 2019;10: 732.Previous analysis of charophyte green algal (CGA) genomes and transcriptomes for specific protein families revealed that numerous land plant characteristics had already evolved in CGA. In this study, we have sequenced and assembled the transcriptome of Zygnema circumcarinatum UTEX 1559, and combined its predicted protein sequences with those of 13 additional species [five embryophytes (Emb), eight charophytes (Cha), and two chlorophytes (Chl) as the outgroup] for a comprehensive comparative genomics analysis. In total 25,485 orthologous gene clusters (OGCs, equivalent to protein families) of the 14 species were classified into nine OGC groups. For example, the Cha+Emb group contains 4,174 OGCs found in both Cha and Emb but not Chl species, representing protein families that have evolved in the common ancestor of Cha and Emb. Different OGC groups were subjected to a Gene Ontology (GO) enrichment analysis with the Chl+Cha+Emb group (including 5,031 OGCs found in Chl and Cha and Emb) as the control. Interestingly, nine of the 20 top enriched GO terms in the Cha+Emb group are cell wall-related, such as biological processes involving celluloses, pectins, lignins, and xyloglucans. Furthermore, three glycosyltransferase families (GT2, 8, 43) were selected for in-depth phylogenetic analyses, which confirmed their presence in UTEX 1559. More importantly, of different CGA groups, only Zygnematophyceae has land plant cellulose synthase (CesA) orthologs, while other charophyte CesAs form a CGA-specific CesA-like (Csl) subfamily (likely also carries cellulose synthesis activity). Quantitative real-time-PCR experiments were performed on selected GT family genes in UTEX 1559. After osmotic stress treatment, significantly elevated expression was found for GT2 family genes ZcCesA, ZcCslC and ZcCslA-like (possibly mannan and xyloglucan synthases, respectively), as well as for GT8 family genes (possibly pectin synthases). All these suggest that the UTEX 1559 cell wall polysaccharide synthesis-related genes respond to osmotic stress in a manner that is similar to land plants