An integrative taxonomic revision of slug-eating snakes (Squamata: Pareidae: Pareineae) reveals unprecedented diversity in Indochina

Slug-eating snakes of the subfamily Pareinae are an insufficiently studied group of snakes specialized in feeding on terrestrial mollusks. Currently Pareinae encompass three genera with 34 species distributed across the Oriental biogeographic region. Despite the recent significant progress in understanding of Pareinae diversity, the subfamily remains taxonomically challenging. Here we present an updated phylogeny of the subfamily with a comprehensive taxon sampling including 30 currently recognized Pareinae species and several previously unknown candidate species and lineages. Phylogenetic analyses of mtDNA and nuDNA data supported the monophyly of the three genera Asthenodipsas, Aplopeltura, and Pareas. Within both Asthenodipsas and Pareas our analyses recovered deep differentiation with each genus being represented by two morphologically diagnosable clades, which we treat as subgenera. We further apply an integrative taxonomic approach, including analyses of molecular and morphological data, along with examination of available type materials, to address the longstanding taxonomic questions of the subgenus Pareas, and reveal the high level of hidden diversity of these snakes in Indochina. We restrict the distribution of P. carinatus to southern Southeast Asia, and recognize two subspecies within it, including one new subspecies proposed for the populations from Thailand and Myanmar. We further revalidate P. berdmorei, synonymize P. menglaensis with P. berdmorei, and recognize three subspecies within this taxon, including the new subspecies erected for the populations from Laos and Vietnam. Furthermore, we describe two new species of Pareas from Vietnam: one belonging to the P. carinatus group from southern Vietnam, and a new member of the P. nuchalis group from the central Vietnam. We provide new data on P. temporalis, and report on a significant range extension for P. nuchalis. Our phylogeny, along with molecular clock and ancestral area analyses, reveal a complex diversification pattern of Pareinae involving a high degree of sympatry of widespread and endemic species. Our analyses support the “upstream” colonization hypothesis and, thus, the Pareinae appears to have originated in Sundaland during the middle Eocene and then colonized mainland Asia in early Oligocene. Sundaland and Eastern Indochina appear to have played the key roles as the centers of Pareinae diversification. Our results reveal that both vicariance and dispersal are responsible for current distribution patterns of Pareinae, with tectonic movements, orogeny and paleoclimatic shifts being the probable drivers of diversification. Our study brings the total number of Pareidae species to 41 and further highlights the importance of comprehensive taxonomic revisions not only for the better understanding of biodiversity and its evolution, but also for the elaboration of adequate conservation actions.

A philosophical perspective on the prenatal in utero microbiome debate

Within the last 6 years, a research field has emerged that focuses on the characterization of microbial communities in the prenatal intrauterine environment of humans and their putative role in human health. However, there is considerable controversy around the existence of such microbial populations. The often contentious debate is primarily focused on technical aspects of the research, such as difficulties to assure aseptic sampling and to differentiate legitimate signals in the data from contamination. Although such discussions are clearly important, we feel that the problems with the prenatal microbiome field go deeper. In this commentary, we apply a philosophical framework to evaluate the foundations, experimental approaches, and interpretations used by scientists on both sides of the debate. We argue that the evidence for a “sterile womb” is based on a scientific approach that aligns well with important principles of the philosophy of science as genuine tests of the hypothesis and multiple angles of explanatory considerations were applied. In contrast, research in support of the “in utero colonization hypothesis” is solely based on descriptive verifications that do not provide explanatory insight, which weakens the evidence for a prenatal intrauterine microbiome. We propose that a reflection on philosophical principles can inform not only the debate on the prenatal intrauterine microbiome but also other disciplines that attempt to study low-biomass microbial communities.

No Consistent Evidence for Microbiota in Murine Placental and Fetal Tissues

ABSTRACT The existence of a placental microbiota and in utero colonization of the fetus have been the subjects of recent debate. The objective of this study was to determine whether the placental and fetal tissues of mice harbor bacterial communities. Bacterial profiles of the placenta and fetal brain, lung, liver, and intestine samples were characterized through culture, quantitative real-time PCR (qPCR), and 16S rRNA gene sequencing. These profiles were compared to those of the maternal mouth, lung, liver, uterus, cervix, vagina, and intestine, as well as to background technical controls. Positive bacterial cultures from placental and fetal tissue samples were rare; of the 165 total bacterial cultures of placental tissue samples from the 11 mice included in this study, only nine yielded at least a single colony, and five of those nine positive cultures came from a single mouse. Cultures of fetal intestinal tissue samples yielded just a single bacterial isolate, Staphylococcus hominis, a common skin bacterium. Bacterial loads of placental and fetal brain, lung, liver, and intestinal tissues were not higher than those of DNA contamination controls and did not yield substantive 16S rRNA gene sequencing libraries. From all placental or fetal tissue samples (n = 51), there was only a single bacterial isolate that came from a fetal brain sample having a bacterial load higher than that of contamination controls and that was identified in sequence-based surveys of at least one of its corresponding maternal samples. Therefore, using multiple modes of microbiological inquiry, there was not consistent evidence of bacterial communities in the placental and fetal tissues of mice. IMPORTANCE The prevailing paradigm in obstetrics has been the sterile womb hypothesis, which posits that fetuses are first colonized by microorganisms during the delivery process. However, some are now suggesting that fetuses are consistently colonized in utero by microorganisms from microbial communities that inhabit the placenta and intra-amniotic environment. Given the established causal role of microbial invasion of the amniotic cavity (i.e., intra-amniotic infection) in pregnancy complications, especially preterm birth, if the in utero colonization hypothesis were true, there are several aspects of current understanding that will need to be reconsidered; these aspects include the magnitude of intra-amniotic microbial load required to cause disease and its potential influence on the ontogeny of the immune system. However, acceptance of the in utero colonization hypothesis is premature. Herein, we do not find consistent evidence for placental and fetal microbiota in mice using culture, qPCR, and DNA sequencing.

No consistent evidence for microbiota in murine placental and fetal tissues

ABSTRACTThe existence of a placental microbiota and in utero colonization of the fetus has been the subject of recent debate. The objective of this study was to determine whether the placental and fetal tissues of mice harbor bacterial communities. Bacterial profiles of the placenta and fetal brain, lung, liver, and intestine were characterized through culture, qPCR, and 16S rRNA gene sequencing. These profiles were compared to those of the maternal mouth, lung, liver, uterus, cervix, vagina, and intestine, as well as to background technical controls. Positive bacterial cultures from placental and fetal tissues were rare; of the 165 total bacterial cultures of placental tissues from the 11 mice included in this study, only nine yielded at least a single colony, and five of those nine positive cultures came from a single mouse. Cultures of fetal intestinal tissues yielded just a single bacterial isolate: Staphylococcus hominis, a common skin bacterium. Bacterial loads of placental and fetal brain, lung, liver, and intestinal tissues were not higher than those of DNA contamination controls and did not yield substantive 16S rRNA gene sequencing libraries. From all placental or fetal tissues (N = 49), there was only a single bacterial isolate that came from a fetal brain sample having a bacterial load higher than that of contamination controls and that was identified in sequence-based surveys of at least one of its corresponding maternal samples. Therefore, using multiple modes of microbiologic inquiry, there was not consistent evidence of bacterial communities in the placental and fetal tissues of mice.IMPORTANCEThe prevailing paradigm in obstetrics has been the sterile womb hypothesis, which posits that fetuses are first colonized by microorganisms during the delivery process. However, some are now suggesting that fetuses are consistently colonized by microorganisms in utero by microbial communities that inhabit the placenta and intra-amniotic environment. Given the established causal role of microbial invasion of the amniotic cavity (i.e. intra-amniotic infection) in pregnancy complications, especially preterm birth, if the in utero colonization hypothesis were true, there are several aspects of current understanding that will need to be reconsidered including the magnitude of intra-amniotic microbial load required to cause disease and their potential influence on the ontogeny of the immune system. However, acceptance of the in utero colonization hypothesis is premature. Herein, we do not find consistent evidence for placental and fetal microbiota in mice using culture, qPCR, and DNA sequencing.

Cisco diversity in a historical drainage of glacial Lake Algonquin

Cisco (Coregonus artedi (sensu lato) Lesueur, 1818) forms matching in appearance to Blackfin Cisco from the Laurentian Great Lakes occur in four lakes in Algonquin Park, Ontario, Canada, a historical drainage of glacial Lake Algonquin (precursor of lakes Michigan and Huron). Their occurrence may represent colonization from glacial Lake Algonquin drainage patterns 13 000 calibrated years BP or independent evolution within each lake. Gill-raker numbers, temperature at capture depth during lake stratification, and hurdle models of habitat distribution are summarized. Blackfin (nigripinnis-like) in the four lakes had higher gill-raker numbers than artedi-like cisco captured in nearby lakes or within the same lake. Two lakes have a bimodal gill-raker distribution that indicate co-occurrence of two forms. Blackfin occupied the hypolimnion with a peak depth distribution at 20–25 m. Maximum depth for blackfin was 35–40 m. The presence of the opossum shrimp (Mysis diluviana Audzijonyte and Väinölä, 2005) appears necessary for the occurrence of cisco diversity in lakes but not sufficient in all cases. The presence of two forms of cisco in at least two lakes points to the possibility of the colonization hypothesis or the ecological speciation hypothesis as accounting for this phenomenon. Genetic analysis is needed to determine which of these hypotheses best accounts for the occurrence of blackfin in Algonquin Park.

Contamination Is Not Linked to the Gestational Microbiome

ABSTRACT Differentiating between contamination and the genuine presence of 16S rRNA genes in gestational tissue samples is the gold standard for supporting the in utero colonization hypothesis. During gestation, the fetus undergoes significant physiological changes that may be directly affected by maternal colonization of key bacterial genera. In this study, lab benches, necropsy tables, and air ducts were swabbed at the same time as clinical sampling. The relative and absolute abundance of bacteria present in sheep samples was determined by culture-independent and culture-dependent means. Of 14 healthy pregnant ewes, there was no evidence of any bacteria in the fetal liver, spleen, or brain cortex using culture-independent techniques despite evidence of the presence of bacteria in various locations of the necropsy room used for 11 of these 14 sheep. Of the 336 bacterial genera found in the room swabs, only 12 (5%) were also found in the saliva and vaginal swabs among the three ewes for which bacteria were detected. These 12 taxa represent 1.32% of the relative abundance and approximately 393 16S rRNA copies/swab in these three ewes. Using careful necropsy protocols, bacterial contamination of sheep tissues was avoided. Contamination of saliva and vaginal samples was limited to less than 2% of the bacterial population. IMPORTANCE Recent evidence for a gestational microbiome suggests that active transfer between mother and fetus in utero is possible, and, therefore, actions must be taken to clarify the presence versus absence of these organisms in their respected sources. The value of this study is the differentiation between bacterial DNA identified in the necropsy rooms of animals and bacterial DNA whose origin is purely clinical in nature. We do not know the extent to which microorganisms traverse maternal tissues and infiltrate fetal circulation, so measures taken to control for contamination during sample processing are vital for addressing these concerns.

The role of gut microbiota in the effects of maternal obesity during pregnancy on offspring metabolism

Obesity is considered a global epidemic. Specifically, obesity during pregnancy programs an increased risk of the offspring developing metabolic disorders in addition to the adverse effects on the mother per se. Large numbers of human and animal studies have demonstrated that the gut microbiota plays a pivotal role in obesity and metabolic diseases. Similarly, maternal obesity during pregnancy is associated with alterations in the composition and diversity of the intestine microbial community. Recently, the microbiota in the placenta, amniotic fluid, and meconium in healthy gestations has been investigated, and the results supported the “in utero colonization hypothesis” and challenged the traditional “sterile womb” that has been acknowledged worldwide for more than a century. Thus, the offspring microbiota, which is crucial for the immune and metabolic function and further health in the offspring, might be established prior to birth. As a detrimental intrauterine environment, maternal obesity influences the microbial colonization and increases the risk of metabolic diseases in offspring. This review discusses the role of the microbiota in the impact of maternal obesity during pregnancy on offspring metabolism and further analyzes related probiotic or prebiotic interventions to prevent and treat obesity and metabolic diseases.

Evaluating existing movement hypotheses in linear systems using larval stream salamanders

Because of their linear nature, streams provide a restrictive framework to understand the movement ecology of many animals. Stream movements have been characterized under two competing hypotheses. The colonization hypothesis dictates that small individuals experience passive drift, but concurrent, upstream movement by larger individuals replaces the loss of small individuals. Alternatively, the production hypothesis suggests that downstream movements are a consequence of limited resource availability. Previous research suggests that large larvae should move upstream and vice versa for small larvae, which should therefore be found downstream more often. We conducted a mark–recapture study of larval red salamanders ( Pseudotriton ruber (Sonnini de Manoncourt and Latreille, 1801)) to assess the validity of these hypotheses. We found that no larvae exhibited downstream movement (skew = 0.361, p = 0.019; biased upstream), and large larvae were the only size cohort to exhibit directional movement upstream (skew = 0.901, p = 0.035). Contrary to predictions under the colonization hypothesis, small larvae were found upstream more frequently than large larvae (N = 871, H = 16.29, df = 2, p < 0.001). Our results suggest that larval movements are related to abiotic stream conditions, and we conclude that neither hypothesis fully explains stream movement. In the absence of drift, new movement hypotheses are necessary to describe persistent upstream movement in streams. These hypotheses should consider individual causes of movement and the direction of movements that will improve the fitness of the organism.