Metformin Restrains the Proliferation of CD4+ T Lymphocytes by Inducing Cell Cycle Arrest in Normo- and Hyperglycemic Conditions.

CD4+ T lymphocytes play a key role in the modulation of the immune response by orchestrating both effector and regulatory functions. The effect of metformin on the immunometabolism of CD4+ T lymphocytes has been scarcely studied, and its impact under high glucose conditions, particularly concerning effector responses and glucose metabolism, remains unknown. This study aims to evaluate the effect of metformin on the modulation of the effector functions and glucose metabolism of CD4+ T lymphocytes under normo- and hyperglycemic conditions. CD4+ T lymphocytes, obtained from peripheral blood from healthy volunteers, were anti-CD3/CD28-activated and cultured for 4 days with three concentrations of metformin (0.1 mM, 1 mM, and 5 mM) under normoglycemic (5.5 mM) and hyperglycemic (25 mM) conditions. Effector functions such as proliferation, cell count, cell cycle analysis, activation markers and cytokine secretion were analyzed by flow cytometry. Glucose uptake was determined using the 2-NBDG assay, and levels of glucose, lactate, and phosphofructokinase (PFK) activity were assessed by colorimetric assays. Metformin at 5 mM restrained the cell counts and proliferation of CD4+ T lymphocytes by arresting the cell cycle in the S/G2 phase at the beginning of the cell culture, without affecting cell activation, cytokine production, and glucose metabolism. In fact, CD69 expression and IL4 secretion by CD4+ T lymphocytes was higher in the presence of 5 mM than the untreated cells in both glucose conditions. Overall, metformin inhibited proliferation through mechanisms associated with cell cycle arrest, leading to an increase in the S/G2 phases at the expense of G1 in activated CD4+ T lymphocytes in normo- and hyperglycemic conditions. Despite the cell cycle arrest, activated CD4+ T lymphocytes remained metabolically, functionally, and phenotypically activated.

Incidencia de diabetes gestacional en Chile durante el periodo 2001-2022 / Incidence of gestational diabetes in Chile during the period 2001-2022

Introducción: La diabetes mellitus gestacional (DG) se define como una hiperglucemia que se diagnostica por primera vez durante la gestación. Objetivo: Describir la incidencia de diabetes gestacional (DG) durante el periodo 2001-2022 en Chile. Método: Estudio observacional, descriptivo, ecológico y longitudinal. Se incluyeron los egresos hospitalarios consignados como diabetes durante el embarazo y DG en el periodo 2001-2022, de la base de datos del Departamento de Estadística e Información en Salud. Se determinó la incidencia de DG por la cantidad de partos institucionalizados, para cada año. Se analizaron la tendencia en el periodo y las diferencias entre regiones. Resultados: Se determinó un aumento de 2,615 casos de DG por 1000 partos atendidos por año en el periodo 2001-2022. En particular, en el periodo 2016-2022 la incidencia aumentó hasta 6,746 casos de DG por 1000 partos por año. En el año 2022, la región de La Araucanía presentó una incidencia de 284,4 casos por 1000 partos, lo que representa un aumento del 503% en relación con la incidencia media nacional (56,5 casos por 1000 partos). Conclusiones: Se demuestra un aumento significativo de la DG, en especial desde 2016. La situación en La Araucanía podría relacionarse con los niveles de pobreza multidimensional.

Introduction: Gestational diabetes mellitus (GDM) is defined as hyperglycemia first diagnosed during pregnancy. Objetive: To describe the incidence of gestational diabetes (GD) during the period 2001-2022 in Chile. Method: Observational, descriptive, ecological, longitudinal study. Hospital records of diabetes during pregnancy and GD in the period 2001-2022 were included, from the database of the Department of Statistics and Health Information. The incidence of GD was determined by the number of births, for each year. Trends in the period and differences between regions were analysed. Results: The results show an increase of 2.615 GD cases per 1000 births per year in the period 2001-2022. Particularly, in the period 2016-2022 the incidence increased to 6.746 cases of GD per 1000 births per year. In 2022, La Araucanía region presented an incidence of 284.4 cases per 1000 births, which represents an increase of 503% in relation to the mean national incidence (56.5 cases per 1000 births). Conclusions: A significant increase in DG is demonstrated, especially since 2016. The situation in La Araucanía could be related to the levels of multidimensional poverty.

Flickering of fetal erythrocytes membrane under gestational diabetes observed with dual time resolved membrane fluctuation spectroscopy.

The membrane flickering of human fetal red blood cells (RBCs) affected by gestational diabetes mellitus (GDM) was studied with dual time resolved membrane fluctuation spectroscopy (D-TRMFS). This new technique is a modified version of the dual optical tweezers method that has been adapted to measure the mechanical properties of RBCs at two distant membrane points simultaneously. The micro-rheological parameters were obtained from direct membrane flickering measurements, followed by Fourier decomposition and cell membrane model adjustment. Our results show a significant decrease of 6.01 ± 1.19 nm in membrane fluctuations amplitude in healthy fetal, compared with healthy adult RBCs, meanwhile the amplitude in GDM cells increased 3.22 ± 1.10 nm compared with healthy fetal RBCs. Between GDM and healthy fetal RBCs, there are significant differences, especially in the bending modulus. Considering the mean of the two membrane points measured, the tension for GDM RBCs increased by 6.431 ± 3.57 (10-7 [N/m]) compared with healthy fetal RBCs, meanwhile, the bending was increased by 2.483 ± 0.58 (10-19 [J]) in GDM compared with healthy fetal RBCs. These results showed significant increment of 1.23 ± 0.07-fold and 3.29 ± 0.36-fold in tension and bending modulus in GDM, respectively. The strong impact of GDM on bending modulus could be associated with oxidative stress and lipid peroxidation, previously reported in fetal plasma of GDM cases.

Physiological and Pathophysiological Role of Large-Conductance Calcium-Activated Potassium Channels (BKCa) in HUVECs and Placenta.

BKCa channels (large-conductance Ca2+-activated K+ channels) play a critical role in regulating vascular tone and blood pressure. These channels are present in the smooth muscle cells of blood vessels and are activated by voltage and increased intracellular Ca2+ concentration. More recently, the expression and activity of BKCa have been proposed to be relevant in endothelial cells, too, specifically in human umbilical vein endothelial cells (HUVECs), the more studied cell type in the fetoplacental circulation. The role of BKCa in endothelial cells is not well understood, but in HUVECs or placental endothelium, these channels could be crucial for vascular tone regulation during pregnancy as part of endothelium-derived hyperpolarization (EDH), a key mechanism for an organ that lacks nervous system innervation like the placenta.In this review, we will discuss the evidence about the role of BKCa (and other Ca2+-activated K+ channels) in HUVECs and the placenta to propose a physiological mechanism for fetoplacental vascular regulation and a pathophysiological role of BKCa, mainly associated with pregnancy pathologies that present maternal hypertension and/or placental hypoxia, like preeclampsia.

Pathophysiology of Preeclampsia and L-Arginine/L-Citrulline Supplementation as a Potential Strategy to Improve Birth Outcomes.

In preeclampsia, the shallow invasion of cytotrophoblast cells to uterine spiral arteries, leading to a reduction in placental blood flow, is associated with an imbalance of proangiogenic/antiangiogenic factors to impaired nitric oxide (NO) production. Proangiogenic factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), require NO to induce angiogenesis through antioxidant regulation mechanisms. At the same time, there are increases in antiangiogenic factors in preeclampsia, such as soluble fms-like tyrosine kinase type 1 receptor (sFIt1) and toll-like receptor 9 (TLR9), which are mechanism derivates in the reduction of NO bioavailability and oxidative stress in placenta.Different strategies have been proposed to prevent or alleviate the detrimental effects of preeclampsia. However, the only intervention to avoid the severe consequences of the disease is the interruption of pregnancy. In this scenario, different approaches have been analysed to treat preeclamptic pregnant women safely. The supplementation with amino acids is one of them, especially those associated with NO synthesis. In this review, we discuss emerging concepts in the pathogenesis of preeclampsia to highlight L-arginine and L-citrulline supplementation as potential strategies to improve birth outcomes. Clinical and experimental data concerning L-arginine and L-citrulline supplementation have shown benefits in improving NO availability in the placenta and uterine-placental circulation, prolonging pregnancy in patients with gestational hypertension and decreasing maternal blood pressure.

Development of the Placenta and Brain Are Affected by Selective Serotonin Reuptake Inhibitor Exposure During Critical Periods.

Selective serotonin reuptake inhibitors (SSRIs) are usually prescribed to treat major depression and anxiety disorders. Fetal brain development exhibits dependency on serotonin (5-hydroxytryptamine, 5-HT) from maternal, placental, and fetal brain sources. At very early fetal stages, fetal serotonin is provided by maternal and placental sources. However, in later fetal stages, brain sources are indispensable for the appropriate development of neural circuitry and the rise of emergent functions implied in behavior acquisition. Thus, susceptible serotonin-related critical periods are recognized, involving the early maternal and placental 5-HT synthesis and the later endogenous 5-HT synthesis in the fetal brain. Acute and chronic exposure to SSRIs during these critical periods may result in short- and long-term placental and brain dysfunctions affecting intrauterine and postnatal life. Maternal and fetal cells express serotonin receptors which make them susceptible to changes in serotonin levels influenced by SSRIs. SSRIs block the serotonin transporter (SERT), which is required for 5-HT reuptake from the synaptic cleft into the presynaptic neuron. Chronic SSRI administration leads to pre- and postsynaptic 5-HT receptor rearrangement. In this review, we focus on the effects of SSRIs administered during critical periods upon placentation and brain development to be considered in evaluating the risk-safety balance in the clinical use of SSRIs.

Effects of Prenatal Cannabinoids Exposure upon Placenta and Development of Respiratory Neural Circuits.

Cannabis use has risen dangerously during pregnancy in the face of incipient therapeutic use and a growing perception of safety. The main psychoactive compound of the Cannabis sativa plant is the phytocannabinoid delta-9-tetrahydrocannabinol (A-9 THC), and its status as a teratogen is controversial. THC and its endogenous analogues, anandamide (AEA) and 2-AG, exert their actions through specific receptors (eCBr) that activate intracellular signaling pathways. CB1r and CB2r, also called classic cannabinoid receptors, together with their endogenous ligands and the enzymes that synthesize and degrade them, constitute the endocannabinoid system. This system is distributed ubiquitously in various central and peripheral tissues. Although the endocannabinoid system's most studied role is controlling the release of neurotransmitters in the central nervous system, the study of long-term exposure to cannabinoids on fetal development is not well known and is vital for understanding environmental or pathological embryo-fetal or postnatal conditions. Prenatal exposure to cannabinoids in animal models has induced changes in placental and embryo-fetal organs. Particularly, cannabinoids could influence both neural and nonneural tissues and induce embryo-fetal pathological conditions in critical processes such as neural respiratory control. This review aims at the acute and chronic effects of prenatal exposure to cannabinoids on placental function and the embryo-fetal neurodevelopment of the respiratory pattern. The information provided here will serve as a theoretical framework to critically evaluate the teratogen effects of the consumption of cannabis during pregnancy.

COVID-19 on Pregnancy Outcomes, Mental Health and Placenta: Focus in Latin America.

The COVID-19 pandemic has impacted many aspects of health and society worldwide. One vulnerable group that faced SARS-CoV-2 infection is pregnant women, who were considered to have potentiated risk factors. In physiological pregnancy, maternal systems have several changes and adaptations to support fetal development. These changes involve regulations of cardiovascular, respiratory, and immunologic systems, among others, which SARS-CoV-2 could severely alter. Furthermore, the systemic effects of viral infection could be associated with placental dysfunction and adverse pregnancy outcomes, which have been studied from the start of the pandemic to date. Additionally, pregnancy is a condition of more significant mental health vulnerability, especially when faced with highly stressful situations. In this chapter, we have collected information on the effect of COVID-19 on maternal mortality, the SARS-CoV-2 infection rate in pregnancy, and the impact on pregnancy outcomes, maternal mental health, and placental function, with a particular focus on studies that consider the Latin American population.

The Placental Function Beyond Pregnancy: Insights from Latin America.

Currently, more than 100,000 papers had been published studying the placenta in both physiological and pathological contexts. However, relevant health conditions affecting placental function, mostly found in low-income countries, should be evaluated deeper. This review will raise some - of what we think necessary - points of discussion regarding challenging topics not fully understood, including the paternal versus maternal contribution on placental genes imprinting, placenta-brain communication, and some environmental conditions affecting the placenta. The discussions are parts of an international effort to fulfil some gaps observed in this area, and Latin-American research groups currently evaluate that.

Timing and delay discounting in attention-deficit/hyperactivity disorder: A translational approach.

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that often presents with abnormal time perception and increased impulsive choice behavior. The spontaneously hypertensive rat (SHR) is the most widely used preclinical model of the ADHD-Combined and ADHD-Hyperactive/Impulsive subtypes of the disorder. However, when testing the spontaneously hypertensive rat from Charles River (SHR/NCrl) on timing and impulsive choice tasks, the appropriate control strain is not clear, and it is possible that one of the possible control strains, the Wistar Kyoto from Charles River (WKY/NCrl), is an appropriate model for ADHD-Predominately Inattentive. Our goals were to test the SHR/NCrl, WKY/NCrl, and Wistar (WI; the progenitor strain for the SHR/NCrl and WKY/NCrl) strains on time perception and impulsive choice tasks to assess the validity of SHR/NCrl and WKY/NCrl as models of ADHD, and the validity of the WI strain as a control. We also sought to assess impulsive choice behavior in humans diagnosed with the three subtypes of ADHD and compare them with our findings from the preclinical models. We found SHR/NCrl rats timed faster and were more impulsive than WKY/NCrl and WI rats, and human participants diagnosed with ADHD were more impulsive compared to controls, but there were no differences between the three ADHD subtypes.

Tipos de partos y niveles de oxitocina, cortisol y hormonas tiroideas en plasma del cordón umbilical / Types of deliveries and levels of oxytocin, cortisol and thyroid hormones in umbilical cord plasma

Introducción: La placenta sintetiza y secreta varias hormonas que permiten la regulación del embarazo, el trabajo de parto y la adaptación metabólica materno-fetal. Su comportamiento asociado al tipo de parto puede dar información relevante sobre efectos epigenéticos. Objetivo: Describir el tipo de parto con los niveles de oxitocina, cortisol y hormonas tiroideas en plasma de cordón umbilical al nacer. Método: A 50 mujeres con embarazos principalmente normales se les cuantificaron los niveles neurohormonales en plasma de cordón umbilical, obtenido inmediatamente tras el periodo expulsivo. Los resultados se incorporaron a la base de datos clínicos de cada participante y se analizaron con Stata v.14.0. El protocolo fue aprobado por el comité de ética. Resultados: Hubo 33 partos vaginales (12 espontáneos, 13 acelerados y 8 inducidos) y 17 cesáreas (7 electivas y 10 de urgencia). Se observaron mayores niveles de cortisol en los partos vaginales acelerados; las cesáreas tuvieron menores niveles de cortisol y hormona estimulante de la tiroides. Las intervenciones clínicas, con altos o bajos niveles hormonales, están en directa relación con el tipo de parto. Conclusiones: El cortisol y la hormona estimulante de la tiroides medidos en plasma de cordón umbilical variaron según el tipo de parto. Esto es una primera cuantificación de hormonas en plasma de cordón umbilical y su posible regulación placentaria a propósito del tipo de parto.

Introduction: The placenta synthesizes and secretes several hormones allowing the regulation of pregnancy, labor and maternal-fetal metabolic adaptation. Their behavior associated with the type of delivery, may provide relevant information on epigenetic effects. Objective: To describe the type of delivery with the levels of oxytocin, cortisol and thyroid hormones in umbilical cord plasma at birth. Method: Neurohormonal levels from umbilical cord plasma obtained immediately post expulsion, were quantified in 50 women with mainly normal pregnancies. Results incorporated into the clinical database of each participant, statistically analyzed in Stata v.14.0. Protocol approved by ethics committee. Results: 33 were vaginal deliveries (12 spontaneous, 13 accelerated, 8 induced) and 17 cesarean sections (7 elective and 10 emergency). Higher cortisol levels were observed in accelerated vaginal deliveries, cesarean sections had lower cortisol and thyroid stimulating hormone levels. While clinical interventions, with high or low hormone levels, were related to the type of delivery. Conclusions: Cortisol and thyroid stimulating hormone measured in umbilical cord plasma varied according to the type of delivery. This is a first quantification of hormones in umbilical cord plasma and their possible placental regulation in relation to the type of delivery.

The South American moth Rheumapteramochica (Dognin, 1904) (Lepidoptera, Geometridae, Larentiinae) rediscovered after more than a century of anonymity.

Rheumapteramochica (Dognin, 1904) (Lepidoptera, Geometridae, Larentiinae) is reported from Chile for the first time. It was described from the western slopes of the Andes of southern Peru more than 100 years ago, and was recently rediscovered in Chile after larvae were collected and reared on the shrub Sennabirostrisvar.arequipensis (Meyen ex Vogel) H.S. Irwin & Barneby (Fabaceae). This discovery expands the known distribution of this moth and provides its first host plant record. The genitalia of R.mochica are described and illustrated for the first time and compared to those of R.affirmata (Guenée, [1858]). A maximum likelihood analysis based on mitochondrial DNA sequences clustered R.mochica as sister to R.affirmata with 3.6-3.8% divergence (K2P). A lectotype is designated for Calocalpemochica Dognin, 1904.

Cryptic Diversity in the Monotypic Neotropical Micromoth Genus Angelabella (Lepidoptera: Gracillariidae) in the Peru-Chile Desert.

Angelabella (Lepidoptera: Gracillariidae: Oecophyllembiinae) is considered a monotypic Neotropical genus of leaf miner micromoths known only from a few valleys of the arid environments of the Peru-Chile desert, particularly the southernmost part of Peru and northernmost part of Chile (type locality), where natural populations of its primary host plant occur. The geographic distribution of potential host plants provides a scenario for a wider range for this micromoth genus. The aims of this study were to explore the geographic range of Angelabella, determine the spatial distribution of mitochondrial lineages, and test lineage conspecificity hypotheses. The spatial distribution of genetic diversity indicated the presence of four spatial clusters, three of which are north of the previously known geographic range. Genetic distances were 0.2-0.8% and 3.6-8.3% (K2P) between haplotypes of the same and different spatial clusters, respectively. Phylogenetic relationships indicated reciprocal monophyly among the four spatial clusters, suggesting that allopatric differentiation processes have governed the recent history of Angelabella in these arid environments. These groups were defined as different species by four species delimitation methods, suggesting that Angelabella is not a monotypic genus, but harbors at least four morphologically cryptic allopatric species with restricted geographic ranges, including the type species and three candidate species.

A new species of Ithome Chambers (Lepidoptera, Cosmopterigidae, Chrysopeleiinae) from the Atacama Desert revealed by morphology and DNA barcodes.

Morphology and DNA barcode sequences were used to assess the taxonomic status of a micro-moth of the genus Ithome Chambers, 1875 (Lepidoptera, Cosmopterigidae, Chrysopeleiinae), whose larvae feed on inflorescences of Prosopis tamarugo Phil. (Fabaceae), a tree native to the Pampa del Tamarugal, Atacama Desert, northern Chile. As a result, Ithome tamarugensis Vargas, sp. nov. is described and illustrated. Its genitalia are remarkably similar to those of Ithome tiaynai Vargas, 2004 from coastal valleys of the Atacama Desert. However, the two species can be recognized by the shape of the phallus in males and the shape of the antrum and ductus bursae in females. The genetic distance between DNA barcodes of I. tamarugensis and I. tiaynai was 3.0-3.3% (K2P), and a maximum likelihood analysis indicated that they are in reciprocally monophyletic clusters, providing additional support for the heterospecific status suggested by morphology.

Fractura de Hoffa en el cóndilo femoral. Reporte de caso / Femur Hoffa fracture. A case report

Las fracturas de tercio distal de fémur unicondilares en el plano coronal son conocidas como fracturas de Hoffa, son poco frecuentes y muy raras. Se han reportado pocos casos este tipo de fracturas, por definición son fracturas inestables y por lo tanto requieren de resolución quirúrgica. El mecanismo de trauma en este tipo de fracturas es directo con la rodilla en flexión. Muchas veces pueden pasar desapercibidas en las proyección radiográficas anteroposterior (AP), y por eso es importante evaluar la proyección lateral. Cuando se sospechan o se evidencian es necesario tomar una tomografía axial computarizada (TAC) con el fin de definir claramente su su resolución quirúrgica, puesto que el abordaje y el método de fijación es controversial. Aunque hoy en día ha aumentado la disponibilidad y las características delos diferentes tipos de implantes, no hay un consenso en la literatura debido principalmente a la falta de experticia en estos poco frecuentes casos.

Unicondylar coronal plane fractures of the distal third of the femur are known as Hoffa fractures, are uncommon and rather rare. Only few cases of this type of fracture have been reported worldwide. By definition they are unstable fractures and therefore require surgical resolution. The mechanism of trauma in this type of fracture is an anterior to posterior direct trauma with a flexed knee. They can often go unnoticed on the anterior-posterior (AP) radiographic projection, so it is important to carefully evaluate the lateral projection. When they are suspected or perhaps evident, it is necessary to perform a computerized axial tomography (CT) in order to clearly define both, the characteristics of the fracture itself as the surgical resolution options, since the approach and the fixation method are controversial. Although nowadays the availability and characteristics of the different types of implants have increased, there is no consensus about the way to manage these fractures in medical literature, mainly due to the lack of expertise in these rare cases.

A new species of Stenoptilia Hübner (Lepidoptera: Pterophoridae) associated with Neobartsia peruviana (Orobanchaceae) in the Andes of northern Chile

Abstract The plume moth genus Stenoptilia Hübner, [1825] (Lepidoptera: Pterophoridae) is recorded for the first time from Chile. Adults of Stenoptilia socoromaensis Vargas & Gielis sp. nov. from the northernmost part of the Chilean Andes are described and illustrated. The larvae of S. socoromaensis feed on buds, flowers and unripe fruits of the hemiparasitic plant Neobartsia peruviana (Walp.) Uribe-Convers & Tank (Orobanchaceae). Pairwise distances of a DNA barcode sequence of S. socoromaensis with congeneric species ranged from 9.1 to 12.6% (K2P).

First record of the sedge feeder Bactra verutana Zeller (Lepidoptera: Tortricidae) in Chile based on morphology and DNA barcodes

Abstract The sedge-feeding moth Bactra verutana Zeller, 1875 (Lepidoptera: Tortricidae: Olethreutinae: Bactrini), described from Dallas, Texas, USA, is widespread, recorded throughout much North America, Central and South America, including the Caribbean, and Africa. The species is recorded for the first time from Chile based on specimens collected in the coastal valleys of the Atacama Desert, where its larvae feed on Cyperus corymbosus Rottb. var. subnodosus (Nees & Meyen) Kük. (Cyperaceae). A single DNA barcode haplotype, which is widespread in USA, was found in two Chilean specimens sequenced.

Morphology of the immature stages and notes on the natural history of the little-known plume moth Stenoptilodes juanfernandicus Gielis (Lepidoptera: Pterophoridae).

Stenoptilodes juanfernandicus Gielis, 1991 (Lepidoptera: Pterophoridae) was described from the Juan Fernandez Islands, Chile; it was subsequently recorded from the Galápagos Islands and mainland Ecuador. Its larva and pupa are described and illustrated for the first time with the aid of light and scanning electron microscopy. Descriptions are based on specimens collected as larvae displaying cryptic coloration feeding on inflorescences of Verbena hispida Ruiz Pav. (Verbenaceae) in the Andes of northern Chile. Several differences were found with the larva and pupa of congeneric representatives, suggesting that immature morphology should be further explored to assess its value in the taxonomy and systematics of Stenoptilodes Zimmerman, 1958. Verbena hispida is the first host plant recorded for S. juanfernandicus, a finding that will be helpful to further explore different Neotropical environments to better characterize the geographic range of this plume moth.

A new species of Strepsicrates Meyrick (Lepidoptera: Tortricidae) from the Atacama Desert of northern Chile previously misidentified as S. smithiana Walsingham.

The adult, larva, and pupa of Strepsicrates gattii Vargas-Ortiz Vargas, sp. n. (Lepidoptera: Tortricidae: Olethreutinae: Eucosmini), are described and illustrated from the Atacama Desert of northern Chile. The larvae are leaf-tiers on the vulnerable native tree Morella pavonis (Myricaceae). As S. gattii was previously misidentified as S. smithiana Walsingham, morphological differences that enable the separation of the two species are highlighted. Sequences of the DNA barcode fragment of the cytochrome oxidase subunit I mitochondrial gene of the new species are provided and used in a Bayesian analysis with congeneric representatives to assess their relationships preliminarily. The divergence (K2P) with S. smithiana was 6.4-7.4%, providing additional support for separating the two species.